Application of diphenyl ether compound in preparation of medicine for inhibiting activity of cryptococcus
By isolating and purifying the fermentation product of Aspergillus strain ZJU5-1, the diphenyl ether compound formula (I) obtained exhibits moderate inhibitory activity against multiple strains of Cryptococcus, solving the problem of the lack of effective drugs to inhibit cryptococcal infection in clinical practice and providing a potential new approach to treating cryptococcal infection.
Patent Information
- Application Number
- CN202410272990.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-03-11
- Publication Date
- 2025-09-12
AI Technical Summary
Currently, there is a lack of effective drugs to inhibit cryptococcal infection in clinical practice, especially for the treatment of central nervous system infections. Existing drugs such as fluconazole have limited effects.
A diphenyl ether compound of formula (I) was obtained by isolating and purifying an Aspergillus strain ZJU5-1 and extracting and isolating its fermentation product. The compound exhibited moderate inhibitory activity against multiple strains of Cryptococcus and was suitable for the research of anti-cryptococcal drugs.
The invention provides a compound with moderate inhibitory activity against multiple strains of Cryptococcus, which is suitable for developing anti-cryptococcal drugs, fills the gap of lack of effective drugs in clinic, and provides a potential new way to treat cryptococcal infection.
Smart Images
Figure CN120617239A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to diphenyl ether compounds having anti-cryptococcal activity. Background Art
[0002] Fungal infection is a relatively common problem in the field of human health. Cryptococcus sp. is a type of human pathogenic fungus with a round yeast cell-like morphology, some of which have visible budding structures and capsule tissues. It is an opportunistic pathogen, and cryptococcal infection is distributed worldwide. Its infection can be divided into epidermal infection and life-threatening invasive fungal infection. It usually causes lung infection after inhalation through the respiratory tract and then spreads to other parts of the body. It is especially prone to central nervous system infection, leading to chronic meningitis and increasing the patient's mortality rate. At present, there is no specific drug to deal with cryptococcal infection in clinical practice, so the development of drug lead compounds that inhibit the activity of cryptococci is an urgent problem to be solved. Summary of the Invention
[0003] The first object of the present invention is to provide a compound having anti-cryptococcal activity, the structural formula of which is shown in formula (I):
[0004]
[0005] The second object of the present invention is to provide an Aspergillus sp. strain ZJU5-1, which was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on November 6, 2019, with the deposit number CGMCC No. 18829.
[0006] The third object of the present invention is to provide a method for preparing the compound of formula (I), comprising the following steps:
[0007] PDA plate activated Aspergillus sp. ZJU5-1;
[0008] Rice solid medium fermentation strain ZJU5-1;
[0009] Ultrasonic extraction of the fermentation product with ethyl acetate yields a crude extract.
[0010] The crude extract was eluted by silica gel column chromatography under reduced pressure to obtain eluate A, which was concentrated under reduced pressure to obtain extract A;
[0011] Elution of extract A by normal phase silica gel column chromatography yielded eluate B, which was concentrated under reduced pressure to yield extract B;
[0012] Extract B was eluted by ODS reverse phase silica gel column chromatography to obtain eluate C, which was concentrated under reduced pressure to obtain extract C;
[0013] Extract C was separated and purified by reverse phase semi-preparative HPLC to obtain the compound of formula (I).
[0014] The compound of formula (I) provided by the present invention has moderate inhibitory activity against 6 strains of Cryptococcus gattii (C. gattii 3271G1 / 3284G14 / R265 / R272 / WM276 / 3291) and 1 strain of Cryptococcus neoformans (C. neoformans H99), and is suitable for the research of anti-cryptococcal active lead compounds or the preparation of anti-cryptococcal drugs. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 The compound of formula (I) 1 H NMR spectrum.
[0016] Figure 2 The compound of formula (I) 13 C NMR spectrum. DETAILED DESCRIPTION
[0017] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.
[0018] Example 1
[0019] Isolation and identification of strain ZJU5-1
[0020] 1. Isolation of strain ZJU5-1
[0021] A strain of bacteria was isolated from sediments in the South Atlantic in July 2012 and named strain ZJU5-1.
[0022] Strain ZJU5-1 is a fungus of marine origin.
[0023] 2. Identification of strain ZJU5-1
[0024] 1. Molecular identification
[0025] The partial sequence of 18S ribosomal RNA of strain ZJU5-1 is shown in SEQ ID NO. 1 in the sequence listing, and has the highest sequence similarity of 99.81% to GENBANK ACCESSION NO. KR611594.1. Based on the above identification results, strain ZJU5-1 belongs to Aspergillus sp.
[0026] 3. Deposit of strain ZJU5-1
[0027] Strain ZJU5-1, belonging to Aspergillus sp., was deposited in the General Microbiology Center of the China Culture Collection Administration on November 6, 2019. The center is abbreviated as CGMCC. The address of the center is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit number of strain ZJU5-1 is CGMCC No. 18829.
[0028] Example 2
[0029] Preparation of compounds
[0030] PDA medium: Mix 200 g of potato, 20 g of glucose, 15 g of agar, and 1000 mL of water, and sterilize by high-pressure steam at 121°C for 30 min.
[0031] Seed culture medium: Mix 4.0 g of glucose, 4.0 g of yeast extract, 10.0 g of maltose extract, and 1000 mL of water, and sterilize by high-pressure steam at 121°C for 30 min.
[0032] Rice culture medium: Add 80 g of rice and 120 mL of water into a 500 mL Erlenmeyer flask, mix well, and sterilize by high-pressure steam at 121°C for 30 min.
[0033] 1. Solid fermentation of strain ZJU5-1
[0034] 1.1 Activation of strain ZJU5-1
[0035] The strain was cultured on the PDA plate for 7 days. After the colonies filled the plate, 2 to 3 1 cm 2 The bacterial blocks of different sizes were inoculated into 500 mL Erlenmeyer flasks and cultured at a constant temperature on a shaking table for 4 days as seed culture medium.
[0036] 1.2 Solid-state fermentation of strain ZJU5-1
[0037] Take 10 mL of the seed culture solution from step 1.1 and inoculate it into a flask containing rice culture medium and culture it at a constant temperature for 30 days.
[0038] 2. Extraction of compounds
[0039] 2.1 Add an equal volume of ethyl acetate to the flask (containing the solid fermentation product) obtained in step 1.2 for extraction, ultrasonically extract at room temperature for 24 hours, and collect the first supernatant.
[0040] 2.2 Add an equal volume of ethyl acetate to the conical flask in step 2.1 for extraction, ultrasonically extract for 24 hours, and collect the second supernatant.
[0041] 2.3 Add an equal volume of ethyl acetate to the conical flask in step 2.2 for extraction, ultrasonically extract for 24 hours, and collect the supernatant for the third time.
[0042] 2.4 Add an equal volume of ethyl acetate to the conical flask in step 2.3 for extraction, ultrasonically extract for 24 hours, and collect the fourth supernatant.
[0043] 2.5 The four supernatants obtained in steps 2.1 to 2.4 were combined and distilled to dryness under reduced pressure to obtain 65.0 g of crude extract paste.
[0044] 3. Separation and purification of compounds
[0045] 3.1 Vacuum silica gel column chromatography
[0046] The crude extract (65.0 g) obtained in step 2.5 was subjected to vacuum silica gel column chromatography. The parameters for the vacuum silica gel column chromatography separation of the compound of formula (I) are as follows:
[0047] The column model is: 8×40 cm; the filling material is: thin layer chromatography silica gel (200-300 mesh); the process of obtaining eluent A is: eluting with 2000 mL eluent (composed of 2 parts by volume of petroleum ether and 1 part by volume of ethyl acetate), and collecting the eluent A.
[0048] 3.2 Normal phase silica gel column chromatography
[0049] The eluate A obtained in step 3.1 was concentrated to dryness under reduced pressure to obtain extract A (6.4 g), which was subjected to normal phase silica gel column chromatography. The parameters for the normal phase silica gel column chromatography separation of the compound of formula (I) are as follows:
[0050] The column model is: 6×30 cm; the filling material is: thin layer chromatography silica gel (200-300 mesh); the process of obtaining eluent B is: eluting with 1000 mL of eluent (composed of 4 parts by volume of petroleum ether and 1 part by volume of ethyl acetate) to obtain eluent 1, eluting with 500 mL of eluent (composed of 3 parts by volume of petroleum ether and 1 part by volume of ethyl acetate) to obtain eluent 2, eluting with 250 mL of eluent (composed of 2 parts by volume of petroleum ether and 1 part by volume of ethyl acetate) to obtain eluent 3, and collecting and combining eluents 1 to 3 to obtain eluent B.
[0051] 3.3 Reversed-phase silica gel column chromatography
[0052] The eluate B obtained in step 3.2 was concentrated to dryness under reduced pressure to obtain extract B (2.6 g), which was subjected to reverse-phase silica gel column chromatography. The parameters for the reverse-phase silica gel column chromatography separation of the compound of formula (I) are as follows:
[0053] The column model is: 4×20 cm; the filling material is: YMC ODS filler; the mobile phase is methanol and ddH2O (wherein the methanol content percentage is 70%); 500 mL of eluent is collected to obtain eluent C.
[0054] 3.4 Reversed-phase HPLC column separation
[0055] The eluate C obtained in step 3.3 was concentrated to dryness under reduced pressure to obtain extract C (60.5 mg), which was then subjected to reverse-phase HPLC column separation. The parameters of the column used for the reverse-phase HPLC separation of the compound of formula (I) are as follows:
[0056] The column model is: xbridge C18 column (5μm; 10×250mm); the process for obtaining the compound of formula (I) is: elution with 60% by volume methanol / acid water for 90.5min at a flow rate of 2.0mL / min. The collection retention time is 65.0~68.0min (the peak retention time t R =66.5min) of the eluent.
[0057] 3.5 The HPLC eluate collected in step 3.4 was distilled to dryness under reduced pressure to obtain 6.0 mg of the compound of formula (I).
[0058] 4. Characterization of compounds
[0059] The product of step 3.5 was analyzed by organic mass spectrometry and nuclear magnetic resonance spectroscopy.
[0060] The characterization data of the product are as follows:
[0061] Compound: brown powder; molecular formula: C 14 H 12 O5; molecular weight: 260.07; 1 H NMR spectrum see Figure 1 , 13 C NMR spectrum see Figure 2 .
[0062] Based on the physicochemical data and spectrum of the above compound, the product of step 3 is the compound described by formula (I).
[0063]
[0064] Example 3
[0065] Inhibitory effect of the compound of formula (I) on Cryptococcus sp.
[0066] The microdilution method was used to detect the minimum inhibitory concentration (MIC, 200 μL system) of the compound of formula (I) against Cryptococcus sp.
[0067] Fluconazole was used as a positive control for the compound and parallel experiments were performed.
[0068] The fungi used in this embodiment are:
[0069] 6 strains of Cryptococcus gattii: C.gattii 3271G1, C.gattii 3284G14, C.gattii R265, C.gattiiR272, C.gattii WM276, C.gattii 3291.
[0070] 1 strain of Cryptococcus neoformans: C. neoformans H99.
[0071] The specific steps of the experiment are as follows:
[0072] 1. Prepare MIC board
[0073] The compound was prepared as a 3.2 mg / mL stock solution in dimethyl sulfoxide (DMSO). 42.0, 21.0, 10.5, 5.25, 2.63, and 1.31 μL of the stock solution were then added to 2100 μL of RPMI 1640 medium (10.4 g / L RPMI 1640 dry powder, 34.53 g / L MOPS buffer powder, pH 7.0, filter-sterilized) to prepare test solutions of 64, 32, 16, 8, 4, and 2 μg / mL, respectively. Fluconazole (positive control) and DMSO (negative control) were prepared using the same method. 100 μL of each of the compound, fluconazole, and DMSO solutions was added to wells 1 through 6 of a 96-well plate. 200 μL of RPMI 1640 medium was added to well 7 as a blank control.
[0074] 2. Preparation of bacterial solution
[0075] Take Cryptococcus sp. in the logarithmic growth phase and make a bacterial suspension with RPMI 1640 medium. Count the cells under a hemocytometer and adjust the bacterial concentration in the test solution to 5×10 6 CFU / mL, and then diluted 1000 times to obtain a concentration of 5×10 3 CFU / mL of the test bacterial solution.
[0076] 3. Inoculation of bacterial solution
[0077] Add 100 μL of bacterial solution to wells 1-6 of the MIC plate (500 cells / well). After inoculation, incubate the plate in a 37°C incubator for 3 days and assess the results. The total volume is 200 μL. The concentrations of the compound and the positive control, fluconazole, in wells 1-6 are 32, 16, 8, 4, 2, and 1 μg / mL, respectively.
[0078] 4. Result judgment
[0079] The lowest concentration of the compound of formula (I) that completely inhibited bacterial growth in the wells was taken as the MIC value.
[0080] The test data are meaningful only when Cryptococcus grows significantly in all wells of the negative control group and the wells of the blank control group remain clear.
[0081] When a single jump well occurs in the microdilution method, the highest concentration of compound that inhibits bacterial growth should be recorded. If multiple jump wells occur, the result should not be reported and the test should be repeated.
[0082] The inhibitory effect of the compound of formula (I) on the growth of Cryptococcus required three biological replicates.
[0083] The MIC values of the compounds described in formula (I) against various strains of Cryptococcus are:
[0084] C. gattii 3271G1: 8 μg / mL (stronger than positive);
[0085] C.gattii 3284G14: 16μg / mL;
[0086] C. gattii R265: 8 μg / mL;
[0087] C. gattii R272: 8 μg / mL;
[0088] C. gattii WM276: 16 μg / mL;
[0089] C. gattii 3291: 32 μg / mL;
[0090] C.neoformans H99: 8 μg / mL.
[0091] The MIC values of the positive control drug fluconazole against each strain of Cryptococcus are:
[0092] C. gattii 3271G1: 16 μg / mL;
[0093] C.gattii 3284G14: 16μg / mL;
[0094] C.value R265:8µg / mL.
[0095] C.value R272:8µg / mL.
[0096] C.value of WM276:2µg / mL.
[0097] C.value 3291:8μg / mL.
[0098] C.neoformans H99:4µg / mL.
Claims
1. The use of a diphenyl ether compound in the preparation of a drug active in inhibiting cryptococcus, characterized in that: The structural formula of the diphenyl ether compound is shown in Formula I:
2. The use of the diphenyl ether compound according to claim 1 in the preparation of a drug active in inhibiting cryptococci, characterized in that: The preparation method of the diphenyl ether compound comprises the following steps: PDA plate activated Aspergillus sp. ZJU5-1; Rice solid medium fermentation strain ZJU5-1; Ultrasonic extraction of the fermentation product with ethyl acetate yields a crude extract. The crude extract was eluted by silica gel column chromatography under reduced pressure to obtain eluate A; Elution of extract A by normal phase silica gel column chromatography yields eluate B; Extract B was eluted by ODS reverse phase silica gel column chromatography to obtain eluate C; Reverse-phase semi-preparative HPLC was used to separate and purify extract C to obtain the diphenyl ether compound.
3. The use of the diphenyl ether compound according to claim 1 in the preparation of a drug active in inhibiting cryptococci, characterized in that: The deposit number of the Aspergillus sp. ZJU5-1 is CGMCC No.18829.
4. The use of the diphenyl ether compound according to claim 1 in the preparation of a drug active in inhibiting cryptococci, characterized in that: The cryptococcus is Cryptococcus gattii or Cryptococcus neoformans.
5. Use of the diphenyl ether compound according to claim 1 in the preparation of a drug active in inhibiting cryptococci, characterized in that: The Cryptococcus gattii is C. gattii 3271G1, C. gattii 3284G14, C. gattii R265, C. gattii R272, C. gattii WM276 or C. gattii 3291.
6. Use of the diphenyl ether compound according to claim 1 in the preparation of a drug active in inhibiting cryptococci, characterized in that: The Cryptococcus neoformans is C. neoformans H99.