Coumarin compounds, methods of making and using the same

Abstract

The application provides a coumarin compound and a preparation method and application thereof. The coumarin compound is separated from Zanthoxylum bungeanum Maxim, raw materials are abundant, and an extraction and separation process is simple. The coumarin compound prepared by the application has a significant inhibitory effect on HFLS-RA (rheumatoid arthritis fibroblast-like synoviocyte) proliferation. The anti-inflammatory activity of the compound is screened by using a RAW 264.7 cell model induced by LPS, and the compound significantly inhibits the production of IL-6 at a non-toxic concentration of 40 umol / L.

Description

Technical Field

[0001] This invention relates to the field of compound technology, and in particular to a coumarin compound, its preparation method, and its application. Background Technology

[0002] Coumarins, also known as benzopyrene, are a class of compounds with the benzo-α-protopyranone structure. They are widely found in various plants in nature, and also in a few fungi and bacteria, as secondary metabolites of plants. Clinically, coumarin drugs are used as oral anticoagulants. Their basic structure is 4-hydroxycoumarin, and representative drugs include the newer anticoagulants acenitroprusside and warfarin.

[0003] Rheumatoid arthritis (RA) is an autoimmune inflammatory arthritis affecting approximately 1% of the world's population. It is a potentially debilitating disease, with women being two to three times more likely to be affected than men. Clinical manifestations of RA include joint swelling and pain, joint deformities, and osteoporosis. The disease is prone to relapse and has a relatively slow onset. As joint inflammation progresses, varying degrees of bone and cartilage destruction occur, leading to joint deformities and even loss of limb function. In severe cases, it can cause loss of earning capacity, significantly impacting the patient's physical and mental health. Traditional Chinese medicine and its effective components have shown significant efficacy in treating rheumatoid arthritis and have become a hot topic in the field of autoimmune diseases.

[0004] IL-6 is a highly pleiotropic cytokine. As a typical pro-inflammatory cytokine, it can be produced by various cells and participates in the pathogenesis of inflammatory diseases. IL-6 and its receptor play a protective role by promoting B cell proliferation and downregulating T cells to improve immune efficiency. However, excessive production of cytokines can also increase the probability of developing autoimmune diseases. For example, IL-6 / IL6R levels are elevated in the serum and synovial fluid of rheumatoid arthritis (RA) patients, and the elevated concentration is correlated with the severity of the patient's clinical manifestations. Tocilizumab is the first approved antirheumatic drug that directly targets IL-6R and inhibits the IL-6 signaling pathway.

[0005] Most currently used medications for treating rheumatoid arthritis have side effects. For example, the most common side effect of nonsteroidal anti-inflammatory drugs (NSAIDs) is gastrointestinal reactions, such as nausea, vomiting, abdominal pain, and diarrhea; in severe cases, it may cause gastrointestinal ulcers and bleeding. Glucocorticoids commonly cause high blood pressure, high blood sugar, infections, and osteoporosis. Disease-modifying antirheumatic drugs (DMARDs) often experience gastrointestinal reactions during use, and occasionally cause skin redness, itching, and rashes; high doses may damage liver and kidney function. Therefore, developing novel, highly effective, and low-toxicity antirheumatic drugs derived from natural sources and acting on specific targets is a major direction for future treatment of rheumatoid arthritis.

[0006] Zanthoxylum dimorphophyllum var. spinifolium is an evergreen shrub or small tree belonging to the genus Zanthoxylum in the Rutaceae family. The root is used medicinally, known as "Jianxuefei," also called "Sanxuefei," "Cisanjia," etc. It has the effects of dispelling wind and cold, promoting blood circulation and relaxing muscles and tendons, and relieving pain. In Guizhou, the Miao and Buyi ethnic groups mainly use it to treat rheumatoid arthritis, traumatic injuries, coughs due to wind-cold, carbuncles, and other ailments. The inventors of this invention intend to isolate coumarin compounds from Zanthoxylum dimorphophyllum. Summary of the Invention

[0007] The technical problem solved by this invention is to provide a coumarin compound, its preparation method and application, which has a significant inhibitory effect on the proliferation of HFLS-RA cells and a significant inhibitory activity on IL-6 expression. It is naturally derived and the preparation method is simple.

[0008] In view of this, the present invention provides a coumarin compound having the following structural formula:

[0009]

[0010] Wherein, R1 is hydrogen (-H) or methyl (-CH3); R2 is isopentenyl. Or 2-methyl-2-methoxy-3-pentenyl

[0011] Preferably, the structural formula is shown in formula (2):

[0012]

[0013] Preferably, the structural formula is shown in formula (3):

[0014]

[0015] Accordingly, the present invention provides a method for preparing coumarin compounds, comprising the following steps: heating and refluxing methanol on branches and leaves of Zanthoxylum bungeanum, extracting, filtering, and obtaining a total extract; loading the total extract dry onto a silica gel column, using a petroleum ether-ethyl acetate and dichloromethane-methanol system as eluents, performing gradient elution, combining the eluent fractions with a volume ratio of 100:50, removing the solvent, and obtaining product A; loading product A onto an MCI chromatography column, using a methanol-water system as eluents, performing gradient elution, combining the eluent fractions with a volume ratio of 60:40, removing the solvent, and obtaining product A1; performing silica gel column chromatography, using petroleum ether / ethyl acetate as eluents, performing gradient elution, combining the eluent fractions with a volume ratio of 90:10, removing the solvent, and obtaining product A1-1; separating product A1-1 by gel column chromatography, eluting with methanol, and then separating by RP-18 column chromatography to obtain the first compound shown in formula (2).

[0016]

[0017] Preferably, in the step of obtaining product A, the silica gel column is 40-80 mesh.

[0018] Preferably, the gel column is a Sephadex LH-20 gel column.

[0019] Preferably, the RP-18 column uses a methanol-water system with a volume ratio of 75:25 as the eluent.

[0020] Accordingly, the present invention also provides a method for preparing coumarin compounds, comprising the following steps: heating and refluxing methanol on branches and leaves of Zanthoxylum bungeanum, extracting, filtering, and obtaining a total extract; loading the total extract dry into a silica gel column, using a petroleum ether-ethyl acetate and dichloromethane-methanol system as eluents, performing gradient elution, combining fractions with a volume ratio of 100:20, removing the solvent, and obtaining product A2; separating product A2 by RP-18 column chromatography, combining fractions with a methanol / water ratio of 50:50, drying, and obtaining product A2-1; separating product A2-1 by silica gel column chromatography, using chloroform / methanol as eluents, performing gradient elution, combining fractions with a volume ratio of 100:5, drying, and obtaining product A2-1a; separating product A2-1a by gel column chromatography, eluting with methanol, and obtaining the second compound shown in formula (3).

[0021]

[0022] Preferably, in the step of obtaining product A, the silica gel column is 40-80 mesh.

[0023] Preferably, the gel column is a Sephadex LH-20 gel column.

[0024] As can be seen from the above scheme, this invention provides a coumarin compound, its preparation method, and its application. The coumarin compounds isolated from *Zanthoxylum bungeanum* are abundant and the extraction and separation process is simple. The coumarin compounds prepared by this invention have a significant inhibitory effect on the proliferation of HFLS-RA (fibroblast-like synovial cells of rheumatoid arthritis), and their inhibitory activity is higher than that of the positive control drug dexamethasone. The anti-inflammatory activity of the compounds was screened using an LPS-induced RAW 264.7 cell model; at a non-toxic concentration of 40 μmol / L, the compounds significantly inhibited the production of IL-6. Detailed Implementation

[0025] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims of the present invention.

[0026] This invention discloses a coumarin compound having the following structural formula:

[0027]

[0028] Wherein, R1 is hydrogen (-H) or methyl (-CH3); R2 is isopentenyl. Or 2-methyl-2-methoxy-3-pentenyl

[0029] As a preferred option, the structure is shown in equation (2):

[0030]

[0031] As a preferred option, the structural formula is shown in equation (3):

[0032]

[0033] Accordingly, the present invention provides a method for preparing coumarin compounds, comprising the following steps: heating and refluxing methanol on branches and leaves of Zanthoxylum bungeanum, extracting, filtering, and obtaining a total extract; loading the total extract dry onto a silica gel column, using a petroleum ether-ethyl acetate and dichloromethane-methanol system as eluents, performing gradient elution, combining the eluent fractions with a volume ratio of 100:50, removing the solvent, and obtaining product A; loading product A onto an MCI chromatography column, using a methanol-water system as eluents, performing gradient elution, combining the eluent fractions with a volume ratio of 60:40, removing the solvent, and obtaining product A1; performing silica gel column chromatography, using petroleum ether / ethyl acetate as eluents, performing gradient elution, combining the eluent fractions with a volume ratio of 90:10, removing the solvent, and obtaining product A1-1; separating product A1-1 by gel column chromatography, eluting with methanol, and then separating by RP-18 column chromatography to obtain the first compound shown in formula (2), i.e., compound 1.

[0034]

[0035] As a preferred embodiment, the dried branches and leaves of *Zanthoxylum bungeanum* are used in this invention. In the step of obtaining product A, the silica gel column is preferably 40-80 mesh; the gradient elution is preferably performed sequentially at a volume ratio of (100:0) to (0:100). In the step of obtaining product A1, the gradient elution is preferably performed using a methanol-water system as the eluent at a volume ratio of (100:1) to (0:100). In the step of obtaining product A1-1, 300-400 mesh silica gel is preferably used, and the volume ratio of petroleum ether to ethyl acetate is preferably 90:10 to 10:10. In the step of obtaining the first compound, the gel column is preferably a Sephadex LH-20 gel column. The RP-18 column preferably uses a methanol-water system with a volume ratio of 75:25 as the eluent.

[0036] Accordingly, the present invention also provides a method for preparing coumarin compounds, comprising the following steps: heating and refluxing methanol on branches and leaves of Zanthoxylum bungeanum, extracting, filtering, and obtaining a total extract; loading the total extract dry into a silica gel column, using a petroleum ether-ethyl acetate and dichloromethane-methanol system as eluents, performing gradient elution, combining fractions with a volume ratio of 100:20, removing the solvent, and obtaining product A2; separating product A2 by RP-18 column chromatography, combining fractions with a methanol / water ratio of 50:50, drying, and obtaining product A2-1; separating product A2-1 by silica gel column chromatography, using chloroform / methanol as eluents, performing gradient elution, combining fractions with a volume ratio of 100:5, drying, and obtaining product A2-1a; separating product A2-1a by gel column chromatography, eluting with methanol, and obtaining the second compound shown in formula (3), i.e., compound 2.

[0037]

[0038] As a preferred embodiment, the branches and leaves of *Zanthoxylum bungeanum* used in this invention are dried branches and leaves. In the step of obtaining product A2, the silica gel column is preferably 40-80 mesh; the gradient elution is preferably performed sequentially at a volume ratio of (100:0) to (0:100). In the step of obtaining product A2-1, the methanol / water eluent is preferably 20:80 to 100:0. In the step of obtaining product A2-1a, 300-400 mesh silica gel is preferably used, and the chloroform / methanol ratio is preferably 100:0 to 0:100. In the step of obtaining the second compound, the gel column is preferably a Sephadex LH-20 gel column.

[0039] The present invention also provides the application of the coumarin compounds described in the above-mentioned technical solution in the preparation of anti-rheumatoid arthritis drugs.

[0040] As can be seen from the above technical solutions, the coumarin compounds isolated from Zanthoxylum bungeanum of this invention are derived from abundant raw materials and the extraction and separation process is simple. The compounds exhibit significant inhibitory effects on the proliferation of fibroblast-like synovial cells in HFLS-RA rheumatoid arthritis, with inhibitory activity significantly superior to the positive control drug dexamethasone. The anti-inflammatory activity of the compounds was screened using an LPS-induced RAW264.7 cell model. At a non-toxic concentration of 40 μmol / L, the compounds significantly inhibited IL-6 production, and their inhibitory activity against IL-6 was significantly higher than that of the positive control drug dexamethasone.

[0041] To further understand the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments. The scope of protection of the present invention is not limited by the following embodiments.

[0042] The raw materials and chemical reagents used in the embodiments of this invention are all commercially available.

[0043] Example 1

[0044] 1. Take a number of dried branches and leaves of Zanthoxylum bungeanum, extract them several times by heating and refluxing with methanol, filter, and obtain the total extract.

[0045] 2. The total extract was loaded into a 40-80 mesh silica gel column by dry loading. The petroleum ether-ethyl acetate and dichloromethane-methanol system were used as eluents. Gradient elution was performed sequentially at volume ratios of (100:0) to (0:100). The eluent was analyzed. The fractions with a volume ratio of 100:50 were combined, the solvent was removed, and product A was obtained.

[0046] 3. Load product A onto an MCI chromatography column and use a methanol-water system as the eluent. Perform gradient elution at a volume ratio of (100:1) to (0:100). Combine the fractions with a volume ratio of 60:40, remove the solvent, and obtain product A1.

[0047] 4. The product A1 was subjected to silica gel column chromatography (300-400 mesh silica gel; petroleum ether / ethyl acetate, 90:10 to 10:10) gradient elution. The eluent fractions with a volume ratio of 90:10 were combined, the solvent was removed, and the product A1-1 was obtained.

[0048] 5. Separate product A1-1 through a Sephadex LH-20 gel column and elute with methanol to obtain product A1-1a;

[0049] 6. The product A1-1a was separated by RP-18 column chromatography (elution system: methanol / water, 75:25) to obtain compound 1.

[0050] Compound 1 was detected as a pale yellow powder. [α] 25 D = +71.4 (c = 0.001, MeOH). HR-ESI-MS gives a quasi-molecular ion peak at m / z: 383.1839 [M+Na]. + (calcd.for C 21 H 28 O5Na 383.1829). (Combined) 1 H-NMR spectrum, 13 C1-NMR spectrum, determining the molecular formula C 21 H 28 O5, with an unsaturation degree of 8. Simultaneously, by measuring the two-dimensional nuclear magnetic resonance spectrum... 1 H- 1 H COSY, HSQC, HMBC, NOESY, and circular dichroism (CD) were used to determine the signal assignments of all hydrogen and carbon atoms and the chemical structure of the compound. 1 H NMR and 13 The C NMR data are shown in Tables 1 and 2.

[0051] Example 2

[0052] 1. Take a number of dried branches and leaves of Zanthoxylum bungeanum, extract them several times by heating and refluxing with methanol, filter, and obtain the total extract.

[0053] 2. The total extract was loaded into a 40-80 mesh silica gel column by dry loading. The petroleum ether-ethyl acetate and dichloromethane-methanol system were used as eluents. Gradient elution was performed sequentially at volume ratios of (100:0) to (0:100). The eluent was analyzed. The fractions with a volume ratio of 100:20 were combined, the solvent was removed, and product A2 was obtained.

[0054] 3. Product A2 was separated by RP-18 column chromatography (methanol / water, 20:80 to 100:0), and the methanol / water fractions (50:50) were combined and dried to obtain product A2-1.

[0055] 4. Separate product A2-1 by silica gel column chromatography (300-400 mesh silica gel; elution system chloroform / methanol 100:0 to 0:100); combine the chloroform / methanol elution fractions at a ratio of 100:5 and dry to obtain product A2-1a;

[0056] 5. The product A2-1a was separated by Sephadex LH-20 gel column chromatography with 100% pure methanol as the elution system to obtain compound 2.

[0057] Compound 2 was detected as a pale yellow powder. [α] 25 D = +81.8 (c = 0.003, MeOH). HR-ESI-MS gives a quasi-molecular ion peak at m / z: 399.1789 [M+Na]. + (calcd.for C 21 H 28 O6Na 399.1778). (Combined) 1 H-NMR spectrum, 13 C1-NMR spectrum, determining the molecular formula C 21 H 28 O6, with an unsaturation degree of 8. Simultaneously, two-dimensional nuclear magnetic resonance spectroscopy was measured. 1 H- 1 H COSY, HSQC, HMBC, NOESY, and circular dichroism (CD) were used to determine the signal assignments of all hydrogen and carbon atoms and the chemical structure of the compound. 1 H NMR and 13 C1NMR data are shown in Tables 1 and 2.

[0058] Table 1. Compound 1 and Compound 2 1 H-NMR (600MHz, CD3OD) data

[0059]

[0060]

[0061] Table 2 shows compounds 1 and 2. 13 C-NMR (150MHz, CD3OD) data

[0062]

[0063] Example 3

[0064] The CCK-8 assay was used to determine the inhibitory activity of compounds 1 and 2 prepared in the embodiments of the present invention on the proliferation of HFLS-RA cells (human rheumatoid arthritis fibroblast synovial cells).

[0065] Cell culture:

[0066] HFLS-RA cells were cultured in DMEM high-glucose medium (containing 10% fetal bovine serum and 1% penicillin-dextrose antibodies). Cells were passaged in T25 cell culture flasks and then incubated at 37°C in a 5% CO2 incubator. Cells used in the experiment were passaged three times after resuscitation.

[0067] Cell proliferation inhibition activity test:

[0068] HFLS-RA cells in logarithmic growth phase were harvested and processed at a concentration of 1×10⁻⁶ cells / cells. 4 Each well was seeded with a specific concentration of dexamethasone (positive control) and the test compound. After 24 hours of incubation, different concentrations of dexamethasone (positive control) and the test compound were added. After another 48 hours of incubation, 10 μL of CCK-8 was added to each well, and the plates were incubated for another 2 hours. The absorbance of each well was then measured at 450 nm using a microplate reader. The cell inhibition rate was calculated. The experiment was repeated three times. The results are shown in Table 3.

[0069] Table 3. Inhibitory activity of coumarin compounds on the proliferation of HFLS-RA cells (IC50) 50 )

[0070]

[0071] Example 4

[0072] The effect of the compound on LPS-induced IL-6 expression in RAW 264.7 cells was determined by ELISA.

[0073] Grouping:

[0074] RAW 264.7 cells from normal culture were harvested, the original culture medium was aspirated, and the cells were washed with PBS, followed by trypsin digestion. The cells were then pipetted to form single cells and seeded at 10,000 cells / well (200 μl per well) in a 96-well plate. A control group was also included; 100 μl of sterile PBS was added around each well, and the plates were incubated overnight at 37°C with 5% CO2. The next day, when the cells reached approximately 80% confluence, drug treatment was initiated. The drug was prepared at the concentrations required for the following experimental groups and added. The cells were then incubated again at 37°C with 5% CO2 for 48 hours, and the cell supernatant was collected for subsequent assays.

[0075] Experimental Groups:

[0076] (1) RAW 264.7 comparison;

[0077] (2) RAW 264.7 + LPS 10ug / ml;

[0078] (3) RAW 264.7 + LPS 10ug / ml + Dexamethasone 50ug / ml;

[0079] (4) RAW 264.7 + LPS 10ug / ml + compound 140umol / l;

[0080] (5) RAW 264.7 + LPS 10ug / ml + compound 240umol / l;

[0081] ELISA testing

[0082] Before the experiment begins, all reagents should be brought to room temperature; when preparing reagents or samples, they should be thoroughly mixed and foaming should be avoided as much as possible.

[0083] (1) Washing the plate: Add 300 μl of wash buffer to each well, let stand for 40 seconds, shake dry, wash the plate 3 times, and gently pat the liquid in the wells on absorbent paper to dry them.

[0084] (2) Sample addition: Set up blank wells, standard wells, and sample wells. Add 100 μl of sample diluent to the blank wells, and add 100 μl of standard or sample to the remaining wells. Cover the ELISA plate with a membrane and incubate at 37°C for 120 min. The order of sample addition and corresponding numbering are shown in Table 4.

[0085] (3) Discard the liquid, spin dry, wash the plate 3 times, soaking for 1-2 minutes each time, add 300μl wash buffer to each well, spin dry and gently pat the liquid in the well on absorbent paper;

[0086] (4) Add 100 μl of diluted Biotin-Conjugate antibody to each well, cover with a membrane, and incubate at 37°C for 1 hour.

[0087] (5) Discard the liquid in the hole, spin dry, wash the plate 3 times, and repeat step (3);

[0088] (6) Add 100 μl of Streptavidin-HRP diluted to 1× to each well and incubate at 37°C for 30 min;

[0089] (7) Discard the liquid in the hole, spin dry, wash the plate 3 times, and repeat step (3);

[0090] (8) Add 90 μl of TMB Substrate to each well and incubate at 37°C for 15 min for color development;

[0091] (9) Add 50 μl of Stop Solution to each well to terminate the reaction, mix well and immediately measure OD450.

[0092] Table 4 shows the effects of compounds 1 and 2 on LPS-induced IL-6 expression in RAW264.7 cells.

[0093]

[0094] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0095] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A coumarin compound, characterized in that, The structural formula is shown in equation (2):

2. A coumarin compound, characterized in that, The structural formula is shown in equation (3):

3. A method for preparing a coumarin compound, characterized in that, Includes the following steps: The branches and leaves of Zanthoxylum bungeanum were heated under reflux with methanol, extracted, filtered, and the total extract was obtained. The total extract was successively extracted with petroleum ether and ethyl acetate to obtain the petroleum ether ethyl acetate extract; The ethyl acetate extract was dry-loaded into a silica gel column and eluted using a dichloromethane-methanol system as the eluent. The eluent fractions with a volume ratio of 100:50 were combined, the solvent was removed, and product A was obtained. Product A was loaded onto an MCI chromatography column and eluted using a methanol-water system as the eluent. The eluent fractions with a volume ratio of 60:40 were combined and the solvent was removed to obtain product A1. Product A1 was subjected to silica gel column chromatography with petroleum ether / ethyl acetate as the eluent and gradient elution. The fractions with a volume ratio of 90:10 were combined and the solvent was removed to obtain product A1-1. Product A1-1 was separated by gel column chromatography, eluted with methanol, and then separated by RP-18 column chromatography to obtain the first compound shown in formula (2). In the step of obtaining product A, the silica gel column is 40-80 mesh. The gel column is a Sephadex LH-20 gel column. The RP-18 column uses a methanol-water system with a volume ratio of 75:25 as the eluent.

4. A method for preparing a coumarin compound, characterized in that, Includes the following steps: The branches and leaves of Zanthoxylum bungeanum were heated under reflux with methanol, extracted, filtered, and the total extract was obtained. The total extract was successively extracted with petroleum ether and ethyl acetate to obtain the petroleum ether ethyl acetate extract; The ethyl acetate extract was dry-loaded into a silica gel column and eluted using a petroleum ether-ethyl acetate and dichloromethane-methanol system as the eluent. The fractions with a volume ratio of 100:20 were combined, the solvent was removed, and product A2 was obtained. Product A2 was separated by RP-18 column chromatography. The fractions with a methanol / water ratio of 50:50 were combined and dried to obtain product A2-1. Product A2-1 was separated by silica gel column chromatography with chloroform / methanol as the eluent and gradient elution. The fractions with a volume ratio of 100:5 were combined and dried to obtain product A2-1a. The product A2-1a was separated by gel column chromatography and eluted with methanol to obtain the second compound shown in formula (3). In the step of obtaining product A, the silica gel column is 40-80 mesh.

5. The use of any one of the coumarin compounds of claims 1-2 in the preparation of an antirheumatoid arthritis drug.

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