A pyrrolo-cycloquinol compound and its use in the preparation of anti-inflammatory drugs

Abstract

The application discloses a pyrroloepoxyquinol compound or a pharmaceutical salt thereof, and a chemical structure of the pyrroloepoxyquinol compound is shown as formula (I). The pyrroloepoxyquinol compound is obtained by extraction, separation and purification from a D-1 fungus of the genus Arenicella in the Arctic soil. The compound is proved to have a significant effect of inhibiting NO release of LPS-induced RAW 264.7 cells by in-vitro activity test, and the effect is not realized by inhibiting cell proliferation or toxicity, which indicates that the compound has anti-inflammatory activity and can be used for developing a new type of anti-inflammatory drug.

Description

Technical Field

[0001] This invention belongs to the field of marine biology and pharmaceutical technology. Specifically, it relates to a pyrroloepoxyquinone alcohol compound obtained from the Arctic soil fungus D-1 through fermentation on a solid culture medium, extraction, separation and purification, and its application in the preparation of anti-inflammatory drugs. Background Technology

[0002] Marine natural products are an important source and new breakthrough for innovative drugs, and have always been a focus of attention in the drug development community. Currently, researchers mainly focus on subtropical regions, but as research deepens, attention is turning to the largely unexplored polar regions of the North and South Poles. The unique climatic conditions of polar regions, such as strong winds, freeze-thaw cycles, low temperatures, dryness, low nutrition, and high ultraviolet radiation, have led to the evolution of unique physiological and biochemical characteristics in polar microorganisms. Therefore, compared with compounds isolated from terrestrial regions, compounds derived from polar regions exhibit uniqueness in terms of diversity, structural characteristics, and biological functions. The drug material Eutypella sp. D-1 was isolated from the high latitudes of the Arctic (78°55'N), belonging to the phylum Ascomycetes, order Xylariales, family Diatrypaceae, and genus Eutypella. Currently, literature reports that the secondary metabolites of this genus mainly include sesquiterpenes, diterpenes, steroids, benzopyran derivatives, lactones, and cytochalasin compounds. These compounds exhibit a wide variety of biological activities, such as antitumor, neurotoxicity, antimalarial, and antibacterial effects. For example, eutypellenone A, a diterpene compound isolated from fungi of this genus, has very good anti-inflammatory activity. It can inhibit NF-κB activity in a dose-dependent manner and can significantly inhibit lipopolysaccharide-induced nitric oxide production (see reference: YU HB, WANG XL, ZHANG YX, et al. Libertellenones O–S and Eutypellenones A and B, Pimarane Diterpene Derivatives from the Arctic Fungus Eutypella sp. D-1[J]. Journal of Natural Products, 2018, 81(7): 1553-60.).

[0003] To date, there have been no reports of pyrrolocycloquinone alcohols with anti-inflammatory activity isolated from the fungus D-1 of the genus Polypodiaceae found in Arctic soil. Summary of the Invention

[0004] The purpose of this invention is to provide a novel pyrroloepoxyquinone alcohol compound obtained by extraction, separation and purification from the genus *Curvulopterus* D-1 in Arctic soil.

[0005] Another object of the present invention is to provide a method for extracting the pyrroloepoxyquinone alcohol compounds.

[0006] Another object of the present invention is to provide the use of the pyrroloepoxyquinone alcohol compound in the preparation of anti-inflammatory drugs.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a pyrroloepoxyquinone alcohol compound or a pharmaceutical salt thereof, the chemical structure of which is shown in formula (I):

[0009]

[0010] The pyrrolocyclooxyquinone alcohols were obtained by extraction, separation and purification from the genus *Curvulopterus* D-1 in Arctic soil.

[0011] A second aspect of the present invention provides a method for extracting the pyrroloepoxyquinone alcohol compound, comprising the following steps:

[0012] Step 1: Preparation of total extract:

[0013] A small amount of mycelium of *Cyclocarya spp.* D-1 was picked from the preservation plate and inoculated into a bottle containing PDB medium for seed culture. The bottle was incubated at 25–30°C (preferably 28°C) on a shaker for at least 3 days to obtain the seed culture. The seed culture was then inoculated into a petri dish containing solid fermentation medium for scale-up culture and placed at 18–22°C (preferably 20°C) for static culture for 30–60 days (preferably 45 days) to obtain the solid culture medium.

[0014] The PDB culture medium formula is: 10g potato extract powder, 20g glucose, and 1000mL distilled water.

[0015] The solid fermentation medium formula is as follows: sucrose 51.4 g / L, sodium nitrate 3.3 g / L, urea 2.5 g / L, yeast extract 0.7 g / L, dipotassium hydrogen phosphate trihydrate 0.07 g / L, magnesium sulfate heptahydrate 0.4 g / L, potassium chloride 0.625 g / L, ferrous sulfate heptahydrate 18.75 mg / L, anhydrous calcium chloride 6.5 g / L, cobalt chloride hexahydrate 3.125 mg / L, and agar 20 g / L;

[0016] After fermentation, the solid culture medium was crushed and extracted with an equal volume of dichloromethane:methanol in a volume ratio of 1:1 at least three times for at least 30 minutes each time to obtain the extract.

[0017] The above extract was concentrated to obtain the total extract;

[0018] The second step is separation and purification:

[0019] ① The total extract was subjected to reduced pressure liquid column chromatography (VLC) with gradient elution using petroleum ether:ethyl acetate ratios of 100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, and 1:1 v / v. Similar fractions were combined based on thin-layer chromatography (TLC) to obtain 20 fractions, Fr.AT.

[0020] ② The fraction Fr.Q was subjected to reversed-phase medium-pressure column chromatography with gradient elution using methanol / water as solvent. The methanol ratio ranged from 60% to 100%, and the elution time was 180 minutes. Based on the colorimetric analysis of TLC thin-layer chromatography, similar fractions were combined to obtain 6 fractions Fr.Q1-Q6.

[0021] ③ The component Fr.Q3 was purified by reversed-phase high-performance liquid chromatography under the following conditions: 35% acetonitrile / water, flow rate 2 mL / min, detection wavelength 260 nm, and retention time 25 min, to obtain the compound shown in formula (I).

[0022] The inoculation amount for scaling up the culture in a petri dish containing solid fermentation medium is: the seed culture is inoculated at an inoculation amount of 5% v / v.

[0023] A third aspect of the present invention provides the use of the pyrroloepoxyquinone alcohol compound or its pharmaceutical salt in the preparation of an anti-inflammatory drug.

[0024] The anti-inflammatory drug refers to an anti-inflammatory drug that uses the pyrroloepoxyquinone alcohol compound or its pharmaceutical salt as a single active ingredient, or that combines the pyrroloepoxyquinone alcohol compound or its pharmaceutical salt with other drug carriers and other anti-inflammatory drugs.

[0025] The application mentioned is in the preparation of anti-inflammatory drugs, specifically in the preparation of drugs for the prevention or treatment of inflammation.

[0026] The inflammation described is an inflammatory response involving nitric oxide (NO).

[0027] In a fourth aspect, the present invention provides the use of the pyrroloepoxyquinone alcohol compound or a pharmaceutical salt thereof in the preparation of a medicament for inhibiting the release of nitric oxide (NO) from macrophages.

[0028] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0029] The pyrroloepoxyquinone alcohol compounds obtained in this invention contain a rare pyrroloepoxyquinone alcohol structural core. In vitro activity assays demonstrated that the compounds, at a concentration of 5 μM, significantly inhibited LPS-induced nitric oxide (NO) release from RAW264.7 cells, with an inhibition rate of 76.18%. CCK-8 assays showed that the compounds, at a concentration of 5 μM, did not exhibit cytotoxicity or inhibitory activity against RAW264.7 cells, with a cell proliferation rate of 143.35%. These experimental results indicate that the compounds possess significant anti-inflammatory activity, and this anti-inflammatory activity is not achieved through inhibition of cell proliferation or toxicity. Therefore, they can be used to develop novel anti-inflammatory drugs for the treatment of inflammatory diseases.

[0030] This invention discloses a pyrroloepoxyquinone alcohol compound obtained by extraction, separation and purification from the genus *Cyclocarya* D-1 in Arctic soil. In vitro activity tests have demonstrated that this compound significantly inhibits LPS-induced NO release from RAW 264.7 cells, and this effect is not achieved through inhibition of cell proliferation or toxicity, indicating that the compound possesses anti-inflammatory activity and can be used to develop novel anti-inflammatory drugs.

[0031] This invention provides new lead compounds for the research and development of new anti-inflammatory drugs and provides a scientific basis for the development and utilization of polar marine medicinal resources.

[0032] Preservation information for biological material samples:

[0033] Depository Institution: China Center for Type Culture Collection

[0034] Address: Wuhan University, Wuhan, China

[0035] Date of deposit: April 12, 2013

[0036] Accession number: CCTCC NO: M2013144

[0037] Classification and nomenclature: Eutypella sp. D-1 Detailed Implementation

[0038] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0039] The Arctic soil genus *Curvulopterus* D-1 used in this invention was isolated from a high-latitude region in the Arctic (78°55'N).

[0040] Example 1: Preparation of the compound of the present invention

[0041] Step 1: Preparation of total extract:

[0042] A small amount of mycelium of *C. curvaturesporum* D-1 with preservation number CCTCC NO: M 2013144 was picked from the preservation plate and inoculated into 250 mL Erlenmeyer flasks for seed culture. Each Erlenmeyer flask contained 100 mL of PDB medium and was cultured at 28°C on a shaker at 180 r / min for 3 days to obtain the seed culture. Then, the seed culture was inoculated into 20 mm × 20 cm petri dishes containing 200 mL of solid fermentation medium at a 5% v / v inoculation rate for scale-up culture. The culture was then placed at 20°C and incubated statically for 45 days to obtain the solid culture medium.

[0043] The PDB culture medium formula is: 10g potato extract powder, 20g glucose, and 1000mL distilled water.

[0044] The solid fermentation medium formula is as follows: sucrose 51.4 g / L, sodium nitrate 3.3 g / L, urea 2.5 g / L, yeast extract 0.7 g / L, dipotassium hydrogen phosphate trihydrate 0.07 g / L, magnesium sulfate heptahydrate 0.4 g / L, potassium chloride 0.625 g / L, ferrous sulfate heptahydrate 18.75 mg / L, anhydrous calcium chloride 6.5 g / L, cobalt chloride hexahydrate 3.125 mg / L, and agar 20 g / L;

[0045] After fermentation, the solid culture medium was crushed and extracted three times with an equal volume of dichloromethane:methanol = 1:1 v / v using ultrasound for 30 min each time to obtain the extract.

[0046] The above extract was concentrated to obtain the total extract;

[0047] The second step is separation and purification:

[0048] ① The total extract was subjected to reduced pressure liquid column chromatography (VLC) with gradient elution using petroleum ether:ethyl acetate ratios of 100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, and 1:1 v / v. Similar fractions were combined based on thin-layer chromatography (TLC) to obtain 20 fractions, Fr.AT.

[0049] ② The fraction Fr.Q was subjected to reversed-phase medium-pressure column chromatography with gradient elution using methanol / water as solvent. The methanol ratio ranged from 60% to 100%, and the elution time was 180 minutes. Based on the colorimetric analysis of TLC thin-layer chromatography, similar fractions were combined to obtain 6 fractions Fr.Q1-Q6.

[0050] ③ The component Fr.Q3 was purified by reversed-phase high-performance liquid chromatography under the following conditions: 35% acetonitrile / water, flow rate 2 mL / min, detection wavelength 260 nm, and retention time 25 min, to obtain the compound shown in formula (I).

[0051] Step 3, structural identification

[0052] The chemical structure of the compound shown in formula (I) was determined using various modern spectroscopic techniques, including NMR, HRESIMS, IR, and UV. The structural formula is shown below:

[0053]

[0054] The compound shown in formula (I) has the molecular formula C 19 H 27 NO5, brown oily; (c 0.10,MeOH); UV(MeOH)(logε)λ max 255(3.79),300(3.53);IR(KBr)v max 3313,2930,2859,1649,1582,1535,1460,1370,1154,1063,1022,954,893,751cm –1 ; m / z 372.1783[M+Na] + (Cacld forC 19 H 27 NO5Na, 372.1781); 1 H and 13 The C10 NMR spectral data are shown in Table 1.

[0055] Table 1 shows the NMR spectral data of the compounds represented by formula (I).

[0056]

[0057]

[0058] a Measured at 125 MHz in CDCl3; b Measured at 500 MHz in CDCl3.

[0059] Example 2

[0060] In vitro anti-inflammatory activity assay of the compound represented by formula (I) of this invention:

[0061] In vitro anti-inflammatory experiments were conducted on the compound shown in formula (I) of this invention. The mouse macrophages RAW264.7 used were purchased from ATCC, ATCC number TIB-71.

[0062] 1. Detection of the effect of the compound shown in formula (I) on lipopolysaccharide (LPS)-induced NO release in RAW264.7 cells.

[0063] The inhibitory effect of the compound of formula (I) on LPS-induced NO release in mouse RAW 264.7 macrophages was detected using the Griess method. RAW 264.7 cells were prepared into 5 × 10⁶ cells... 4 100 μL of cell suspension per well was seeded into each well of a 96-well plate. After cell attachment, LPS (1 μg / mL) was added to the LPS group, 5 μM of the compound shown in formula (I) was added to the experimental group, and 0.1 μL of DMSO was added to each well of the blank control group. After incubation at 37°C for 1 h, LPS (1 μg / mL) was added, and the culture was continued for 24 h. The cell supernatant was collected, and the absorbance (OD value) of each well was recorded using a NO detection kit. The Inhibition Rate (IR)% was calculated according to the formula: Inhibition Rate (IR)% = (OD value) / (OD value) / (OD value). 样品 –OD 空白 ) / (OD LPS –OD 空白 )×100%, calculate the NO generation inhibition rate (IR%).

[0064] 2. Effect of the compound shown in formula (I) on the viability of Raw264.7 cells.

[0065] The toxicity of the compound shown in formula (I) to Raw 264.7 cells was detected using the CCK-8 assay. 100 μL of the supernatant was used for NO detection, and 10 μL of the prepared CCK-8 solution was added to each well. The cells were incubated at 37°C for 1 h. When a significant change was observed, the OD value at 450 nm was measured in each well. The result was calculated using the formula: Proliferation Rate (PR)% = (OD...) / (PR...) 样品 -OD 空白 ) / (OD LPS -OD 空白 )×100%, calculate the effect on cell proliferation (PR%).

[0066] 3. Experimental Results

[0067] Griess's experimental results showed that the compound shown in formula (I) significantly inhibited LPS-induced NO release from RAW264.7 cells at a concentration of 5 μM, with an inhibition rate (IR%) of 76.18%. CCK-8 assay results showed that the compound of this invention did not exhibit cytotoxicity or inhibitory effects on RAW264.7 cells at a concentration of 10 μM, with a cell proliferation rate (PR%) of 143.35%.

[0068] 4. Conclusion

[0069] The compound shown in formula (I) significantly inhibited the release of NO from LPS-induced RAW 264.7 cells at a concentration of 5 μM, and this effect was not achieved by inhibiting cell proliferation or toxicity, indicating that the compound has anti-inflammatory activity. Therefore, the compound of the present invention can be used as a lead compound for anti-inflammatory drugs.

[0070] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A pyrroloepoxyquinone alcohol compound or a pharmaceutical salt thereof, characterized in that, Its chemical structure is shown in formula (I):

2. The pyrroloepoxyquinone alcohol compound or its pharmaceutical salt according to claim 1, characterized in that, The pyrrolocyclooxyquinone alcohol compounds were obtained by extraction, separation and purification from the genus *Eutypella* D-1 of Arctic soil. The preservation number of *Eutypella* D-1 is CCTCC NO: M2013144, and its classification name is *Eutypella* sp. D-1.

3. A method for extracting pyrroloepoxyquinone alcohols as described in claim 1 or 2, characterized in that, Includes the following steps: Step 1: Preparation of total extract: A small amount of mycelium of *Cyclocarya spp.* D-1 was picked from the preservation plate and inoculated into a bottle containing PDB medium for seed culture. The bottle was incubated at 25–30°C in a shaker for at least 3 days to obtain the seed culture. The seed culture was then inoculated into a petri dish containing solid fermentation medium for scale-up culture and placed at 18–22°C for static culture for 30–60 days to obtain the solid culture medium. The preservation number of *Polychaete flexures* D-1 is CCTCC NO: M2013144, and its classification name is *Polychaete flexures* D-1 Eutypella sp. D-1. The PDB culture medium formula is: 10g potato extract powder, 20g glucose, and 1000mL distilled water. The solid fermentation medium formula is as follows: sucrose 51.4 g / L, sodium nitrate 3.3 g / L, urea 2.5 g / L, yeast extract 0.7 g / L, dipotassium hydrogen phosphate trihydrate 0.07 g / L, magnesium sulfate heptahydrate 0.4 g / L, potassium chloride 0.625 g / L, ferrous sulfate heptahydrate 18.75 mg / L, anhydrous calcium chloride 6.5 g / L, cobalt chloride hexahydrate 3.125 mg / L, and agar 20 g / L; After fermentation, the solid culture medium was crushed and extracted with an equal volume of dichloromethane:methanol in a volume ratio of 1:1 at least three times for at least 30 minutes each time to obtain the extract. The above extract was concentrated to obtain the total extract; The second step is separation and purification: ① The total extract was subjected to reduced pressure liquid column chromatography (VLC) with gradient elution using petroleum ether:ethyl acetate ratios of 100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, and 1:1 v / v. Similar fractions were combined based on thin-layer chromatography (TLC) to obtain 20 fractions, Fr.AT. ② The fraction Fr.Q was subjected to reversed-phase medium-pressure column chromatography with gradient elution using methanol / water as solvent. The methanol ratio ranged from 60% to 100%, and the elution time was 180 minutes. Based on the colorimetric analysis of TLC thin-layer chromatography, similar fractions were combined to obtain 6 fractions Fr.Q1-Q6. ③ The component Fr.Q3 was purified by reversed-phase high-performance liquid chromatography under the following conditions: 35% acetonitrile / water, flow rate 2 mL / min, detection wavelength 260 nm, and retention time 25 min, to obtain the compound shown in formula (I).

4. The extraction method for pyrroloepoxyquinone alcohols according to claim 3, characterized in that, The inoculation amount for scaling up the culture in a petri dish containing solid fermentation medium is: the seed culture is inoculated at an inoculation amount of 5% v / v.

5. The use of a pyrroloepoxyquinone alcohol compound as described in claim 1 or 2, or a pharmaceutical salt thereof, in the preparation of an anti-inflammatory drug.

6. The use of the pyrroloepoxyquinone alcohol compound or its pharmaceutical salt according to claim 5 in the preparation of anti-inflammatory drugs, characterized in that, The anti-inflammatory drug refers to an anti-inflammatory drug that uses the pyrroloepoxyquinone alcohol compound or its pharmaceutical salt as a single active ingredient, or that combines the pyrroloepoxyquinone alcohol compound or its pharmaceutical salt with other drug carriers and other anti-inflammatory drugs.

7. The use of the pyrroloepoxyquinone alcohol compound or its pharmaceutical salt according to claim 6 in the preparation of anti-inflammatory drugs, characterized in that, The application mentioned is in the preparation of anti-inflammatory drugs, specifically in the preparation of drugs for the prevention or treatment of inflammation.

8. The use of the pyrroloepoxyquinone alcohol compound or its pharmaceutical salt according to claim 7 in the preparation of anti-inflammatory drugs, characterized in that, The inflammation described is an inflammatory response involving nitric oxide.

9. The use of a pyrroloepoxyquinone alcohol compound as described in claim 1 or 2, or a pharmaceutical salt thereof, in the preparation of a medicament for inhibiting the release of nitric oxide from macrophages.

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