A compound extracted and separated from Tianping grass and application of the compound in preparing anti-inflammatory drugs

Abstract

The present invention relates to the technical field of pharmaceutical compounds, and specifically discloses a new compound 11 extracted and separated from Tian Ping grass β ,13-(6-amino-purine-9-yl)-dihydrolactate, and the use of the new compound in the preparation of anti-inflammatory drugs. The present invention uses modern spectral techniques such as 1D-NMR, 2D-NMR, high-resolution mass spectrometry, etc. to structurally identify the separated monomer compounds and deduce the molecular structure of the compound. The results of enzyme activity experiments show that the new compound has good inhibitory activity against BTK. The results of molecular docking show that the new compound has good binding activity with the target protein, and the interaction force is mainly hydrogen bonding and hydrophobic forces. By detecting the inhibitory effect of the new compound on the NO release of the mouse macrophage cell line RAW264.7, it is shown that the new compound has good anti-inflammatory effect and has good application prospects in the treatment of inflammation.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal compounds, and in particular to a compound 11β,13-(6-amino-purine-9-yl)-dihydrolactogenin extracted and separated from Herba Lycopersici and application of the compound in preparing anti-inflammatory drugs. Background Art

[0002] Lactuca tatarica is the dried whole herb of Lactuca tatarica, a plant of the Asteraceae family. Lactuca tatarica is also known as milk lettuce, bitter lettuce, bitter lettuce, purple-flowered mountain lettuce, and Mengshan lettuce. The whole herb can be used as medicine for red eyes, leucorrhea, carbuncle, erysipelas, intestinal carbuncle, etc. It is also picked by the people as a wild vegetable for consumption, and has anti-tumor, anti-inflammatory, antibacterial, and analgesic effects.

[0003] Bruton's Tyrosine Kinase (BTK) is a key member of the B cell receptor (BCR) signaling pathway and the first Tec family tyrosine kinase. It has been identified as a dual-function regulator of apoptosis, promoting radiation-induced apoptosis but inhibiting apoptosis of fas-activated B cells. BTK is a cytoplasmic, non-receptor tyrosine kinase (nRTK) that transmits signals through a variety of cell surface molecules and is expressed by all cells of the hematopoietic lineage except T, NK, and plasma cells. In 1952, American pediatrician Ogdon Bruton discovered X-linked agammaglobulinemia (XLA), a genetic disease that causes recurrent bacterial infections and sepsis in early childhood. Studies have shown that BTK not only signals the B cell receptor (BCR) antigen to participate in the response, but also stimulates CD40, toll-like receptors (TLRs), Fc receptors (FCRs), and chemokine receptors. In addition, BTK can regulate signal transduction and overexpression, leading to autoimmunity, and reducing BTK levels can improve the prognosis of autoimmune diseases. BTK is also involved in other cell signaling pathways, including the NF-κβ, NFAT (nuclear factor of activated T cells), and MAPK (mitogen-activated protein kinase) pathways. BTK inhibition leads to the inactivation of downstream cellular pathways, including ERK (extracellular signal-regulated kinase), PKB (protein kinase B, also known as Akt), and NF-κβ, disrupting intercellular communication through chemotaxis and adhesion disruption signaling, ultimately leading to disruption of DNA synthesis and apoptosis in B cells. Therefore, BTK is considered a viable therapeutic target for the treatment of inflammatory diseases and tumors.

[0004] In the Chinese patent publications, the invention patent application with publication number CN109528798A discloses the use of lactic acid alcohol extract in psoriasis treatment drugs, so that the lactic acid alcohol extract can significantly improve the situation of psoriasis pruritus and skin lesions, and the discovery of this therapeutic effect shows that lactic acid has a good drug prospect, which can provide a certain basis for the research and development of psoriasis treatment drugs and clinical application. The invention disclosure with publication number CN109602775A applied by South-Central University for Nationalities discloses the application of lactic acid alcohol extract in anti-breast cancer, and this invention can provide a new selection for treating breast cancer. Lactococcus is a plant of the genus Lactococcus of the Compositae family that is both medicinal and edible, and the alcohol extract of lactic acid can significantly inhibit the growth of breast cancer MCF-7 cells, and can significantly inhibit the growth of mouse model mammary tumors, and the discovery of these anti-cancer effects proves that lactic acid has a good drug prospect, which can provide new ideas for the research and development of prevention, health care and treatment of breast cancer drugs and clinical application. The biological activities of Herba Lycopodii reported in the above patent documents and other documents are mainly concentrated on the research of its extracts, while there are few reports on the anti-inflammatory effects of its chemical components. Therefore, it is of great significance to extract and separate chemical components from Herba Lycopodii and study its pharmacological activity. Summary of the invention

[0005] In view of the problems and shortcomings in the above-mentioned prior art, the purpose of the present invention is to extract a new compound from Tianpingcao and provide its anti-inflammatory pharmaceutical use.

[0006] In order to achieve the above-mentioned purpose of the present invention, the present invention extracts a new compound from Tianping grass:

[0007] Formula (1): Chemical formula is C 20 H 21 N 5 O 5 , molecular weight is 412.1611, name is 11β,13-(6-amino-purine-9-yl)-dihydrolactate, structural formula is as follows:

[0008]

[0009] The method for extracting the above-mentioned new compound (1) from Tian Ping grass comprises the following steps:

[0010] The dried whole herb of Tian Ping Cao was taken and crushed, and then extracted and concentrated with 90 v / v% ethanol and 60 v / v% ethanol by percolation method, and then extracted with petroleum ether, ethyl acetate and water-saturated n-butanol in sequence. The obtained n-butanol layer extract was separated and purified by macroporous resin column chromatography, silica gel column chromatography, Sephadex LH-20, medium-pressure reverse-phase silica gel chromatography and ODS-HPLC to obtain a new compound.

[0011] In addition, the present invention evaluates the anti-inflammatory activity of the new compound from three aspects: enzyme activity experiment, molecular docking and NO test. The new compound has good inhibitory activity against BTK, with an IC50 value of 2.75±0.98μmol / L, and has a strong inhibitory effect on the NO release of RAW264.7 cells. The active new compound extracted and separated by the present invention can be used to prepare anti-inflammatory drugs and health products, preferably as a BTK inhibitor.

[0012] The beneficial effects of the present invention are:

[0013] The present invention extracts and separates a new compound from Tian Ping grass. The new compound belongs to sesquiterpenes and has good inhibitory activity on BTK enzyme, so it can be used to prepare anti-inflammatory medicines and health products. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the HR-ESI-MS spectrum of the new compound of the present invention;

[0015] Figure 2 is the IR spectrum of the new compound of the present invention;

[0016] Figure 3 The new compound of the present invention 1 H-NMR spectrum;

[0017] Figure 4 The new compound of the present invention 13 C-NMR spectrum;

[0018] Figure 5 DEPT135°NMR spectrum of the new compound of the present invention;

[0019] Figure 6 is the nuclear magnetic resonance HMQC spectrum of the new compound of the present invention;

[0020] Figure 7 is the nuclear magnetic resonance HMBC spectrum of the new compound of the present invention;

[0021] Figure 8 is the COSEY nuclear magnetic resonance spectrum of the new compound of the present invention;

[0022] Fig. 9 is the nuclear magnetic resonance NOSEY spectrum of the new compound of the present invention;

[0023] Fig.10a and Fig.10b They are two-dimensional and three-dimensional effect diagrams of the new compound of the present invention and the target protein BTK, respectively. DETAILED DESCRIPTION

[0024] The applicant will now provide a clear and complete description of the technical solution of the present invention in conjunction with the embodiments of the present invention and the accompanying drawings.

[0025] The reagents used in the following examples are all common commercial reagents with analytical purity.

[0026] Example 1: Preparation of the compound of formula (1)

[0027] Step 1, taking 16.0 kg of the whole herb of Lactuca tatarica, crushing it through a 20-mesh sieve, and extracting it with 90 v / v% ethanol (194 L) and 60 v / v% ethanol (171 L) by percolation method, combining the extracts, and concentrating to obtain 4500 g of the total extract;

[0028] Step 2, after suspending the total extract obtained in step 1 with water, extracting with petroleum ether, ethyl acetate, and water-saturated n-butanol in sequence to obtain a petroleum ether extract (590.0 g), an ethyl acetate extract (161.0 g), and a n-butanol extract (712.0 g), respectively;

[0029] Step 3, the n-butanol extract (712.0 g) obtained in step 2 was separated by macroporous resin chromatography, and gradient eluted with purified water, 30 v / v% ethanol, 50 v / v% ethanol, 70 v / v% ethanol, and 95 v / v% ethanol in sequence, and the eluate with 30 v / v% ethanol was collected;

[0030] Step 4: The eluate obtained in step 3 was separated by silica gel column chromatography, and then 2 Cl 2 :CH 3 OH was gradient eluted at a volume ratio of 95:5, 93:7, 90:10, 85:15, and 80:20, and the eluate with a volume ratio of dichloromethane to methanol of 90:10 was collected, numbered as Fr.3, and concentrated to dryness under reduced pressure; then separated by Sephadex LH-20, chloroform-methanol (volume ratio 1:1) was used as the eluent, and the amount of the eluent was 1.8 column volumes, and the eluate of the 0.8-1.1 column volume part was collected, numbered as Fr.3.3, and concentrated to dryness under reduced pressure; then separated by medium-pressure reverse-phase silica gel chromatography, gradient eluted with 15v / v%, 24v / v%, 30v / v%, 50v / v% and 70v / v% methanol in turn, and the eluate with 30v / v% methanol was collected; finally purified by ODS-HPLC, and the eluent was MeCN-H 2 O(MeCN:H 2O volume ratio is 15:85), the chromatographic column is a C-18 column (5μm, 250mm×10mm), the flow rate is 3.0mL / min, the eluate from 22 to 23.5 minutes is collected, concentrated and dried under reduced pressure to obtain a new compound (3.6mg).

[0031] Structural identification: Using modern spectroscopic techniques such as 1 H NMR spectroscopy, 13 The structure of the new compound obtained in step 4 was identified by C NMR, DEP T135° NMR, two-dimensional NMR (HMQC, HMBC, COSEY, NOSEY), high-resolution mass spectrometry (HR-ESI-MS), and IR. Figure 1-9 ;

[0032] After identification, the molecular weight of the new compound obtained in step 4 is 412.1611 and the chemical formula is C 20 H 21 N 5 O 5 , named 11β,13-(6-amino-purine-9-yl)-dihydrolactate, with a structural formula as shown in the following formula (1); its nuclear magnetic resonance spectrum data are shown in Table 1.

[0033] Table 1 Compounds of formula (1) 1 H and 13 C NMR data ( 1 H NMR, 500 MHz, 13 C NMR, 126MHz, inDMSO, δppm, J Hz)

[0034]

[0035]

[0036] The structure of the resulting compound is shown below:

[0037]

[0038]

[0039] The HMBC spectrum of the obtained compound is shown in the following formula (2):

[0040]

[0041] Example 2: To detect the inhibitory activity of the novel compound of the present invention prepared in Example 1 on Bruton's tyrosine kinase (BTK), the following enzyme activity experiment was performed using the product of BPS Bioscience (Cat. No.: #79568):

[0042] Step 1: Thaw 5× kinase assay buffer, ATP and Poly-Glu, Tyr at low temperature, and add DTT to the 5× kinase assay buffer to a concentration of 10 mM.

[0043] Step 2: Add 6 μL of 5× kinase assay buffer (containing 10 mM DTT), 1 μL of 500 uM ATP, 1 μL of 10 mg / mL Poly-Glu, Tyr, and 17 μL of ultrapure water to each well.

[0044] Step 3: Add 5 μL of the prepared inhibitor solution to the inhibitor group, and add buffer solution without inhibitor to the blank group and the test group.

[0045] Step 4: Dilute 5× kinase assay buffer (containing 10 mM DTT) to 1× kinase assay buffer (containing 2 mM DTT) with ultrapure water, and add 20 μL of 1× kinase assay buffer (containing 2 mM DTT) to each well of the blank group.

[0046] Step 5. After thawing on ice, BTK kinase was diluted to 10 ng / mL with 1× kinase assay buffer (containing 2 mM DTT). 20 μL of the diluted BTK enzyme was added to the test group and inhibitor group to initiate the reaction, and the reaction was incubated at 30°C for 60 minutes.

[0047] Step 6. After the solution in the wells reacted for 60 minutes, 50 μL of Kinase-Glo Max reagent was added to each well, and the plate was wrapped with aluminum foil and incubated at room temperature for 15 minutes. The reagents and amounts added to the blank group, inhibitor group, and test group are shown in Table 2.

[0048] Step 7. Immediately read the absorbance value of the sample in a microplate reader capable of reading chemiluminescence.

[0049] Table 2: Reagents added to the blank group, inhibitor group, and test group

[0050]

[0051]

[0052] Table 3 Inhibitory activity of new compounds on BTK enzyme

[0053] Inhibitor Group No. IC50(μM) New compounds 2.75±0.98 LFM-A13 3.20±0.90

[0054] Among them, IC50 (μM) is the concentration of the drug when the enzyme activity inhibition rate is 50%, which is used to indicate the inhibitory activity against BTK enzyme; LFM-A13 is the positive control drug for BTK enzyme. As shown in Table 3, the new compound has good inhibitory activity against BTK enzyme.

[0055] Example 3: In order to better understand the binding mode of the new compound to BTK, a molecular docking method is used to verify and illustrate:

[0056] Step 1. Databases include UniProt database (https: / / www.uniprot.org / ) and PDB database (http: / / www.rcsb.org / ); software include ChemOffice2010 (PerkinElmer, USA), S YBYL 1.0 software (Tripos, USA), Discovery Studio 2017R2 Client (DS, developed by Accelrys, USA), and PyMOL.

[0057] Step 2: Use ChemDraw software to draw the structural formula of the new compound and save it in mol2 format. Import the mol2 format structural formula of the new compound into SYBYL 2.0 software, use the molecular mechanics program Mini mize to optimize the structure, assign the Tripos force field and load the Gasteiger-Huckel charge, save the optimized stable conformation in mol2 format, and establish a ligand small molecule compound library to prepare for molecular docking.

[0058] Step 3: Download the crystal structure of the target protein Bruton's tyrosine kinase (PDB ID: 5P9J) from the PDB database (http: / / www.rcsb.org), modify, hydrogenate and load AMBER FF99 charges on the target protein using the Docking module in the Application, determine the active site of docking based on the ligand in the target protein complex, and save the processed protein to prepare for subsequent molecular docking studies.

[0059] Step 4: Use the Surflex-dock module of SYBYL 2.0 software to carry out molecular docking between the ligand small molecule compound library and the target protein. The docking result is given by the scoring function Total Score and saved in mol2 format. The ligand molecules are screened using the Total Score scoring function of the SYBYL molecular docking module. The Total Score scoring function comprehensively considers factors such as polarity, hydrophobicity, enthalpy and solvation. The larger the value, the more stable the docking complex, indicating that the matching and binding effect between the small molecule compound and the macromolecular protein is better.

[0060] Step 5: Use the receptor-ligand interactions module in the Discovery Studio software to analyze the molecular docking results and produce three-dimensional and two-dimensional renderings.

[0061] Table 4 Docking scores of new compounds and target proteins

[0062]

[0063] Bruton's tyrosine kinase (BTK) proteins have high amino acid sequence homology and similar protein structures. They all have a polyprotein homology (PH) domain at the amino terminus, as well as four structural functional domains: a Tec homology (TH) domain, a Src homology (SH3) domain, an SH2 domain, and a C-terminal region with kinase activity.

[0064] By studying the structural model of new compounds and BTK ( Fig.10a and Fig.10b ). The compound forms hydrogen bond interactions with THR410, GLY411, LYS430, GLU475, and MET477, and the compound forms hydrophobic interactions with residues LEU408, VAL416, ALA428, and LEU528 to stabilize. These hydrogen bonds and hydrophobic interactions play an important role in the binding of the new compound to BTK. Therefore, combined with the docking score of the new compound with BTK, it is shown that the new compound of the present invention has good binding activity with the target protein, and the new compound may be a potential inhibitor of BTK enzyme.

[0065] Example 4: In order to further verify the anti-inflammatory activity of the new compound, the effect of the new compound on the NO release of RAW264.7 cells was detected.

[0066] The following reagents and instruments were used: RAW264.7 cell line (Cell Collection Center, Wuhan University); DMEM medium (Gibco), lipopolysaccharide (Sigma); fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd.); NO detection kit (Shanghai Biyuntian Biotechnology Co., Ltd.); sterile CO 2 Incubator (Heal Force Biomedical Technology Holdings Co., Ltd.); full-wavelength microplate reader (Thermo Fisher Scientific Inc.).

[0067] Step 1: Use DMEM medium containing 10% fetal bovine serum in 5% CO 2 RAW264.7 cells were cultured at 37°C to the logarithmic phase.

[0068] Step 2: RAW264.7 cell suspension (1×10 4After culturing for 24 hours, the cytotoxicity of compounds at different concentrations was determined by MTT colorimetry. The compound at a concentration of 50 μM had no toxic effect on RAW264.7 cells. Specifically, take out the culture plate, add 20 μL of MTT solution (5 mg / mL) to each well, continue incubation at 37°C for 4 hours, and then terminate the culture. Carefully remove the culture supernatant in the well, add 150 μL DMSO to each well, and shake for 10 minutes. Select a wavelength of 490 nm, and measure the light absorption value (OD) of each well on an automatic microplate reader.

[0069] Step 3: Take RAW264.7 cells (1×10 4 / well) were inoculated in a 96-well plate, and a blank group, a model group (1μg / mL LPS), a drug group (1μg / mL LPS+new compound) and a positive control group (1μg / mL LPS+SMT) were set up, with 5 replicate wells in each group. After continuing to culture for 24 hours, 50μL of supernatant was taken, and 50μL of solution A and solution B were added respectively according to the Griess method. The absorbance value was detected on an automatic microplate reader (measured at 540nm), and the IC50 value was calculated.

[0070] Table 5 In vitro anti-inflammatory activity of new compounds

[0071] Compound No. IC50(μmol / L) New compounds 7.09±0.38 S-Methylisothiourea sulfate 8.24±0.45

[0072] The results showed that the new compound could significantly downregulate the NO release of inflammatory cells, and its IC50 value was lower than that of the positive drug S-methylisothiourea sulfate, indicating that the new compound had good anti-inflammatory activity.

Claims

1. Compound 11β,13-(6-amino-purine-9-yl)-dihydrolactate, chemical formula C 20 H 21 N5O5, the structural formula is as follows:

2. Use of the compound 11β,13-(6-amino-purine-9-yl)-dihydrolactate according to claim 1 in the preparation of BTK inhibitors.

3. Use of the compound 11β,13-(6-amino-purine-9-yl)-dihydrolactate according to claim 1 in the preparation of a drug for inhibiting NO release.

4. Use of the compound 11β,13-(6-amino-purine-9-yl)-dihydrolactate according to claim 1 in the preparation of anti-inflammatory drugs.

5. A method for extracting the compound 11β,13-(6-amino-purine-9-yl)-dihydrolactate according to claim 1, characterized in that: The extraction steps are as follows: taking the whole dried herb of Tian Ping Cao, crushing it, extracting and concentrating it with 90v / v% ethanol and 60v / v% ethanol in turn by percolation, extracting it with petroleum ether, ethyl acetate and water-saturated n-butanol in turn, and separating and purifying the obtained n-butanol layer extract by macroporous resin column chromatography, silica gel column chromatography, Sephadex LH-20, medium-pressure reversed-phase silica gel chromatography and ODS-HPLC in turn to obtain the compound.

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