Azo compound with anti-inflammatory activity as well as preparation method and application thereof

Abstract

The invention discloses an azo compound with anti-inflammatory activity and a preparation method and application thereof, and is characterized in that the structural formula of the compound is as shown in formula I. The preparation method comprises the following steps: firstly, culturing actinomycetes through fermentation to obtain an azo fermentation product, and then extracting the fermentation product with ethyl acetate to obtain a crude extract; and then separating and purifying the obtained crude extract by adopting normal-phase silica gel column chromatography, reverse medium-pressure column chromatography and reversed-phase semi-preparative high performance liquid chromatography to obtain the azo compound, and the azo compound has the advantage of remarkable anti-inflammatory activity, so that the azo compound has potential in the development of medicines for preventing and treating inflammation-related diseases.

Description

Technical Field

[0001] The present invention relates to an azo compound, in particular to an azo compound extracted from marine actinomycetes, a preparation method and application thereof. Background Art

[0002] Natural products containing nitrogen-nitrogen bonds have always been extremely attractive secondary metabolites in natural product chemistry. Among them, compounds containing azoxy groups (RN = N+-O-) belong to a rare and highly promising subclass. In addition to their unique electronic and steric effects, this type of azoxy compound has shown significant pharmacological potential, especially in terms of anticancer, antibacterial, and anti-inflammatory biological activities. For example, compounds such as valinomycin, azoxymycin, and elastomycin have been shown to have biological significance since the 1950s. However, the structural diversity of naturally occurring azoxy compounds remains very limited, and to date, only 15 different chemical scaffolds have been successfully characterized.

[0003] While azoxy compounds are gaining increasing industrial importance, particularly in dyes, polymer stabilizers, electrochemical materials, and agrochemical precursors, traditional synthesis methods such as Hofmann elimination and Wallach rearrangement face challenges such as harsh reaction conditions, poor selectivity, and environmental toxicity. Therefore, naturally derived azoxy compounds, as a more environmentally friendly alternative with complex structures and synthesized by microorganisms, have attracted widespread attention.

[0004] Marine invertebrates, particularly marine sponges, are a significant source of novel nitrogen metabolites due to their unique symbiotic microbiota and chemical defense systems. Actinomycetes in sponges harbor inactive gene clusters for azoxy metabolites, demonstrating significant potential for development, particularly in the biosynthesis of azoxy natural products. With their unique metabolic pathways, marine actinomycetes represent a rich resource for the discovery of novel lead compounds, playing a crucial role in microbial drug discovery.

[0005] Inflammation is the body's protective response to infection, trauma, or other harmful stimuli. However, prolonged or excessive inflammation can lead to various chronic diseases, such as rheumatoid arthritis, cardiovascular disease, and cancer. Therefore, the search for natural compounds with anti-inflammatory properties is of great clinical significance. The present inventors discovered a new azo natural product while investigating the chemical composition of the ethyl acetate extract of marine actinomycete NBUD211 fermented in liquid medium A. Currently, there are no reports on the chemical structure or anti-inflammatory activity of this compound, and therefore, no related drugs are available on the market. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an azo compound with anti-inflammatory activity and a preparation method and use thereof.

[0007] The technical solution adopted by the present invention to solve the above technical problems is:

[0008] 1. An azo compound, the structural formula of which is shown in (I);

[0009]

[0010] 2. The preparation method of the above-mentioned azo compound comprises the following steps:

[0011] (1) Fermentation production

[0012] Streptomyces sp., a marine actinomycete with a deposit number of CGMCC No. 24623, was streaked onto a plate of Gao's solid medium No. 1 and incubated upside down in an incubator at 25-30°C for 5 days. A single colony was then picked and inoculated into liquid medium A. The culture was then placed on a shaker at 25-30°C and 220 rpm / min for 14 days, and the fermentation broth was collected.

[0013] (2) Extraction

[0014] Adding an equal volume of ethyl acetate to the fermentation broth obtained in step (1), repeatedly extracting 2-5 times, and then vacuum evaporating the ethyl acetate extract to obtain a crude extract;

[0015] (3) Isolation, purification and preparation of compounds

[0016] The crude extract obtained in step (2) was first dissolved in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and then 200-300 mesh silica gel was added to mix the sample, and normal phase medium pressure column chromatography was performed, and gradient elution was performed using a petroleum ether-ethyl acetate solution containing 85-100% by volume of ethyl acetate as an eluent at a flow rate of 50 mL / min. The eluate was collected, and 100-200 mesh reversed phase silica gel was added to the collected eluate to mix the sample and perform reverse phase medium pressure column chromatography, and linear gradient elution was performed using methanol-water with a volume percentage of 25-100% by volume of methanol as an eluent, and the elution time was 120 min. Finally, after elution with 100% by volume of methanol for 30 min, the fractions were arranged in descending order of polarity and combined to obtain 7 components; the obtained 6th component was separated and purified by semi-preparative reverse phase high performance liquid chromatography using a mixed solution of acetonitrile and water with a volume ratio of 65:35 as a mobile phase to obtain a compound, whose structure is shown in (I):

[0017]

[0018] Furthermore, the Gao's solid culture medium No. 1 described in step (1) is prepared as follows: 20 g of soluble starch, 1 g of KNO3, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.5 g of NaCl, 0.01 g of FeSO4·7H2O and 20 g of agar are added to 1000 mL of distilled water, adjusted to pH = 7.4-7.6, sterilized at 121°C for 20 min, cooled to 60°C and poured into a plate.

[0019] Furthermore, the preparation method of the liquid culture medium A described in step (1) is as follows: 20g starch, 2g yeast extract, 6g peptone, 1g calcium carbonate, 100mg potassium bromide, 40mg ferrous sulfate tetrahydrate and 30g sea salt are dissolved in 1000mL water.

[0020] Furthermore, the separation column used in the normal phase medium pressure column chromatography in step (3) is Silica Flash Column 330g; the separation column used in the reverse phase medium pressure column chromatography is SEPAFLASH SW040.

[0021] Furthermore, the flow rate of the compound separation and preparation by semi-preparative reverse-phase high performance liquid chromatography described in step (3) is 2 mL / min.

[0022] 3. Use of the above-mentioned azo compounds in the preparation of drugs with anti-inflammatory activity.

[0023] Compared with the prior art, the advantages of the present invention are: the present invention discloses an azo compound and its preparation method and use, the fermentation product is obtained by microbial fermentation culture, and then the fermentation product is soaked and extracted with ethyl acetate to obtain a crude extract, and then the crude extract is separated and purified by medium-pressure normal-phase column chromatography, medium-pressure reversed-phase column chromatography, and semi-preparative high-performance liquid chromatography to obtain the compound, the compound structure belongs to the uncommon azo oxide class, it has significant anti-inflammatory activity, and can be used for the development of drugs to inhibit related diseases caused by inflammation.

[0024] The above-mentioned marine actinomycetes (Streptomyces sp.) is an H-1 strain, classified as Streptomyces (Streptomyces sp.), with a preservation number of CGMCC No. 24623. It was deposited on March 31, 2022 at the General Microbiology Center of the China Culture Collection Administration, with the preservation address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the mass spectrum data diagram of compound 1 of the present invention;

[0026] Figure 2 is the ultraviolet absorption pattern of compound 1 of the present invention;

[0027] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of compound 1 of the present invention;

[0028] Figure 4 is the carbon NMR spectrum of compound 1 of the present invention;

[0029] Figure 5 is the DEPT-135 nuclear magnetic resonance spectrum of compound 1 of the present invention;

[0030] Figure 6 The nuclear magnetic resonance of compound 1 of the present invention is 1 H- 1 H COSY spectrum;

[0031] Figure 7 is the nuclear magnetic resonance HSQC spectrum of compound 1 of the present invention;

[0032] Figure 8 is the nuclear magnetic resonance HSBC spectrum of compound 1 of the present invention;

[0033] Figure 9 is the COX-2 inhibition curve of compound 1 of the present invention;

[0034] Figure 10 The effect of compound 1 of the present invention at 2 μM, 10 μM and 30 μM on the production of nitric oxide (NO) in RAW264.7 macrophages induced by LPS;

[0035] Figure 11 The graph shows the effects of compound 1 of the present invention at 2 μM, 10 μM and 30 μM on the generation of reactive oxygen species (ROS) in RAW264.7 macrophages induced by LPS. DETAILED DESCRIPTION

[0036] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[0037] Example 1: The azo compound extracted from marine actinomycetes has the formula (I):

[0038]

[0039] Example 2: The method for preparing the azo compound shown in Example 1 above specifically comprises the following steps:

[0040] Step 1, fermentation production: Marine actinomycetes (Streptomyces sp.) with a preservation number of CGMCC No. 24623 were streaked and cultured on Gao's solid medium No. 1 (prepared as follows: 20 g of soluble starch, 1 g of KNO3, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.5 g of NaCl, 0.01 g of FeSO4·7H2O, and 20 g of agar in 1000 mL of distilled water, adjusted to pH = 7.4-7.6). After 5 days, a single colony was selected and inoculated into liquid medium A (the formula of liquid medium A is: 20 g of starch, 2 g of yeast extract, 6 g of peptone, 1 g of calcium carbonate, 100 mg of potassium bromide, 40 mg of ferrous sulfate tetrahydrate, and 30 g of sea salt dissolved in 1000 mL of water). Subsequently, the culture was placed on a shaker at 28°C and 220 rpm / min for 11 days, during which time the fermentation broth was collected for further analysis.

[0041] Step 2, extract extraction: adding an equal volume of ethyl acetate to the fermentation broth obtained in step (1), repeatedly extracting three times, and then vacuum evaporating the ethyl acetate extract to obtain a crude extract;

[0042] Step 3, separation and preparation of the compound: The crude extract obtained in step 2 was first dissolved in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and then 200-300 mesh silica gel was added to mix the sample and subjected to normal phase medium pressure column chromatography (the separation column used was Silica Flash Column 330g), and a petroleum ether-ethyl acetate solution with a volume fraction ratio of 85-100% was used as the eluent for gradient elution at a flow rate of 50 mL / min, and the eluate was collected; 100-200 mesh reversed-phase silica gel was added to the collected eluate and mixed with the sample for reversed-phase medium pressure column chromatography (the separation column used was SEPAFLASH SW040), using methanol-water with a methanol volume percentage of 25-100% as the eluent for linear gradient elution, the elution time is 120 min, and finally eluting with 100% methanol for 30 min. The eluted fractions were collected and arranged in descending order of polarity, and combined to obtain 7 components; the obtained 6th component was separated and purified by semi-preparative reversed-phase high performance liquid chromatography using a mixed solution consisting of acetonitrile and water in a volume ratio of 65:35 as the mobile phase at a flow rate of 2 mL / min to obtain a compound, whose structure is shown in (I):

[0043]

[0044] Example 3: Structural analysis of the azo compound prepared in Example 2 above.

[0045] The azo compound I prepared in Example 2 is a white powder. Figure 1The HR-ESI-MS spectrum of the compound of the present invention shows a quasi-molecular ion peak m / z 241.1900 [M+H] + (The calculated molecular weight is 241.1916, C 13 H 26 N2O3), and its molecular formula is determined to be C 13 H 26 N2O3, indicating that its unsaturation is 2. Figure 2 As shown, the UV spectrum of the compound shows a characteristic absorption at 238 nm, and the infrared spectrum of compound I shows a characteristic absorption at 1457 cm -1 A characteristic band is shown at , indicating that the compound has an azoxy functional group (N=NO). Figure 3 and the compounds in Table 1 1 H NMR data showed that the two methyl groups (δ H 0.86 and 1.01), five methylene groups, one methoxy group (δ H 3.19) and two olefinic hydrogen atoms (δ H 7.11 and 6.87) corresponding signals, suggesting the presence of a double bond in the molecule. Figure 4 and Figure 5 It can be seen that further 13 C NMR and DEPT-13 spectral analysis showed 13 carbon signals, including a methoxy group (δ C 58.3), 6 methylene groups, two olefin carbons (δ C 138.1 and 134.4), two methine carbons (δ C 67.9 and 64.6) and two methyl carbons (δ C 20.1 and 14.0). The type of carbon in the compound structure is determined by Figure 7 The HSQC spectral correlation in is further supported by Figure 6 It can be seen that 1 H- 1 H COSY spectrum showed that H-6(δ H 7.11) and H-7(δ H 6.87), H-8(δ H 2.20), H-9(δ H 1.43), H-10(δ H 1.25), H-11(δ H 1.29) and H-12(δ H 1.27) there is a correlation signal between them.

[0046] In addition, through Figure 8 The HMBC spectrum shows that H-8(δ H 2.20), and C-6 (δC 138.1) and C-10 (δ C 28.3), H-9(δ H 1.43) to C-7(δ C 134.4) and C-11(δ C The long-range correlation between H-6 (δ H 7.11) and H-7(δ H 6.87) has a coupling constant of 13.3 Hz, which further proves the trans double bond configuration between the two. Another part of the structure of compound I is shown by 1 H- 1 The H COSY spectrum revealed that H-2(δ H 3.87) and H-3(δ H 4.30), and the correlation between H-3(δ H 4.30) and H-4(δ H 3.51 and 3.57). In addition, the hydroxyl proton H-1 (δ H 1.01) and H-2(δ H 3.88) indicates that these protons belong to a tightly connected structural fragment. H 4.30) and C-1(δ C 20.1) and C-4(δ C 70.3) further supports the connectivity of the structure. H 3.51 and 3.57) to C-5 (δ C The HMBC correlation signal of 58.3) indicated that the fragment was connected to a methoxy group. The mass spectrometry data further showed that the compound contained two nitrogen atoms and one oxygen atom, which was consistent with its molecular formula (see Figure 1 ). Combined with the comparative analysis of spectral data and known compounds, it was speculated that the azoxy group was located between C-6 and C-3. Therefore, the planar structure of compound I was determined to be an azo type. The absolute configuration of compound 1 was determined by measuring circular dichroism (CD) spectral experimental data and ECD. The CD spectrum showed that compound 1 exhibited a positive Cotton effect at 235nm (Δε=+4.95) and a negative Cotton effect at 265nm (Δε=-2.25). Combined with ECD spectral analysis, the configuration of compound 1 was determined to be 2R,3R. Therefore, the compound was identified as a new type of azo oxide and named mariazomycin C.

[0047] Table 1. Compound I 1 H and 13C NMR data (DMSOd6)

[0048]

[0049]

[0050] Note 1: s - singlet, d - doublet, m - multiplet.

[0051] Note 2: 1 H was obtained at 600 MHz NMR; 13 C was obtained at 150 MHz NMR.

[0052] Example 4: Determination of the anti-inflammatory activity of the azo compounds shown in Example 1 above.

[0053] 1. Experimental samples

[0054] The experiments used RAW264.7 mouse macrophages (purchased from the American Type Culture Collection, ATCC, Rockville, MD, USA) and cultured in Dulbecco's Modified Eagle's Medium (DMEM, Nissui, Tokyo, Japan) containing high glucose. The medium was supplemented with 100 mg / mL streptomycin, 2.5 mg / L amphotericin B, and 10% heat-inactivated fetal bovine serum (FBS). The azo compound I described in Example 1 above was used as a test group, while the control group was treated with culture medium alone. During the experiments, the cells were stimulated with 25 ng / mL lipopolysaccharide (LPS) to evaluate the effects of the compounds.

[0055] 2. Experimental methods

[0056] (1) Cell culture and cell viability assay: RAW264.7 cells were cultured in a 37°C, 5% CO2 humidity-controlled incubator and maintained in high-glucose DMEM containing 100 mg / mL streptomycin, 2.5 mg / L amphotericin B, and 10% heat-inactivated fetal bovine serum. The cell suspension was plated at 1.0 × 10 cells per well. 4 Cells were seeded in 96-well plates and cultured for 12 hours. Compound I (at various dilutions, with a maximum concentration of 50 μM) was then treated for 24 hours. A control group was treated with culture medium alone. Cell viability was determined using the MTT assay, with absorbance measured at 450 nm using a SpectraMax Pro 7.1 reader.

[0057] (2) NO release level detection: RAW264.7 macrophages (about 1×10 4Cells were plated in 96-well plates (100 cells / well) and cultured for 12 hours. Cells were pretreated with different concentrations of drugs for 1 hour and then incubated with 25 ng / mL LPS for 24 hours. The NO concentration in the culture medium was determined using the Griess assay. Griess reagent (80 μL) was added to the culture supernatant (80 μL) and then incubated in the dark at 37°C for 15 minutes. The absorbance at 520 nm was measured using a SpectraMax Pro 7.1 reader (Molecular Devices, LLC, San Jose, California, USA). The NO concentration was calculated using a 0-100 μM sodium nitrite standard.

[0058] (3) Reactive oxygen species (ROS) assay: RAW264.7 macrophages (1×10 4 Cells were seeded in a dark-protected 96-well cell culture plate (100 cells / well) for 12 hours. Cells were pretreated with test compound I for 1 hour and then incubated with 1 μg / mL lipopolysaccharide (LPS) for 24 hours. 100 μL of a reactive oxygen species (ROS) fluorescent probe (abbreviated as DCFH-DA) was then added to each well, diluted to a final concentration of 100 μM in FBS-free DMEM medium, and incubated at 37°C for 30 minutes. The medium was removed, and 100 μL of PBS buffer was added to each well. Fluorescence was detected using a SpectraMax Pro 7.1 Reader (Molecular Devices, LLC, San Jose, CA, USA) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Diclofenac (Dic.) was used as a positive control (10 μM).

[0059] 3. Experimental results

[0060] The anti-inflammatory potential of compound 1 was pharmacologically evaluated using an in vitro inflammation model of RAW264.7 macrophages stimulated with LPS. Compound 1 exhibited anti-inflammatory activity in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS). Figure 9 The COX-2 inhibition curve of compound 1 of the present invention is shown in the COX-2 inhibition experiment. The COX-2 inhibition experiment shows that compound 1 inhibits cyclooxygenase-2 (COX-2) activity in a dose-dependent manner. Its IC 50 The value was 25.10 μM, showing a moderate enzyme inhibition effect, which was moderate compared with the control drug diclofenac.

[0061] Cells were pretreated with different concentrations of compound 1 for 1 hour and then treated with LPS. NO concentration in the culture medium was determined by the Griess method (LPS, 25 ng / mL, 24 hours). The fluorescence intensity of ROS (LPS, 1 μg / mL, 24 hours) was quantified using a fluorescence microplate reader. Diclofenac (Dic.) was used as a positive control (10 μM). The results shown are representative of three independent experiments. Figure 10 and Figure 11 As shown, the Griess test showed that compound I inhibited NO secretion in a concentration-dependent manner. At the same time, compound I also effectively inhibited the generation of ROS, further indicating that it has good anti-inflammatory activity. Further experiments showed that compound 1 significantly reduced the production of nitric oxide (NO) and reactive oxygen species (ROS) through a dual mechanism, alleviating the pathophysiological characteristics of chronic inflammation. Specifically, compound 1 inhibited the secretion of NO in a concentration-dependent manner and effectively reduced the generation of ROS. These results indicate that compound 1 has potential anti-inflammatory effects.

[0062] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by persons of ordinary skill in the art within the spirit and scope of the present invention shall also fall within the scope of protection of the present invention.

Claims

1. An azo compound, characterized in that The structural formula of the compound is shown in (I); 2. A method for preparing the azo compound according to claim 1, characterized in that The steps include: (1) Fermentation production Streptomyces sp., a marine actinomycete with the accession number CGMCC No. 24623, was streaked onto a plate of Gao's solid medium No. 1 and incubated upside down in an incubator at 28°C for 5 days. A single colony was then picked and inoculated into liquid medium A. The culture was then placed on a shaker at 28°C and 220 rpm / min for 11 days, and the fermentation broth was collected. (2) Extraction An equal volume of ethyl acetate was added to the fermented product obtained in step (1), and the extraction was repeated three times. The ethyl acetate extract was then vacuum evaporated to dryness to obtain a crude extract; (3) Isolation and preparation of compounds The crude extract obtained in step (2) was first dissolved in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and then 200-300 mesh silica gel was added to mix the sample, and normal phase medium pressure column chromatography was performed, using a petroleum ether-ethyl acetate solution with a volume ratio of 85-100% as an eluent for gradient elution, and the eluate was collected; the collected eluate was subjected to reverse phase medium pressure column chromatography, using methanol-water with a methanol volume percentage of 25-100% as an eluent, gradient elution for 120 minutes, and finally eluted with 100% methanol for 30 minutes. After collecting the eluted fractions, the fractions were arranged in descending order of polarity and combined to obtain 7 components; the obtained sixth component was separated and purified by preparative reverse phase high performance liquid chromatography using a mixed solution of acetonitrile and water with a volume ratio of 65:35 as a mobile phase to obtain a compound, whose structure is shown in (I):

3. The method for preparing an azo compound according to claim 2, wherein The Gao's No. 1 solid culture medium described in step (1) is prepared as follows: 20 g of soluble starch, 1 g of KNO3, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.5 g of NaCl, 0.01 g of FeSO4·7H2O and 20 g of agar are added to 1000 mL of distilled water and the pH is adjusted to 7.4-7.

6.

4. The method for preparing an azo compound according to claim 2, wherein The preparation method of the liquid culture medium A described in step (1) is as follows: 20g starch, 2g yeast extract, 6g peptone, 1g calcium carbonate, 100mg potassium bromide, 40mg ferrous sulfate tetrahydrate and 30g sea salt are dissolved in 1000mL water.

5. The method for preparing an azo compound according to claim 2, wherein: The flow rate for the compound separation and preparation by semi-preparative reversed-phase HPLC described in step (3) is 2 mL / min.

6. Use of the azo compound according to claim 1 in the preparation of anti-inflammatory drugs.

Images