Compound extracted and separated from anacyclus pyrethrum root and application of compound in preparation of anti-inflammatory drugs

Abstract

The invention relates to the technical field of medicinal compounds, and particularly discloses a novel compound (S, E)-5-hydroxy-N-isobutyl-4-oxo-2-decenamide extracted and separated from anacyclus pyrethrum roots, and application of the novel compound in preparation of anti-inflammatory drugs. Modern spectrum technologies such as 1D-NMR (1D-Nuclear Magnetic Resonance), 2D-NMR (2D-Nuclear Magnetic Resonance), high-resolution mass spectrum and the like are used for carrying out structure identification on a monomeric compound obtained through separation, and the molecular structure of the compound is deduced. An enzyme activity experiment result shows that the novel compound has relatively good activity of inhibiting TYK2. Molecular docking results show that the novel compound and the target protein have good binding activity, and the interaction force is mainly hydrogen bonds and hydrophobic force. By detecting the inhibiting effect of the novel compound on the NO release amount of a mouse macrophage line RAW264.7, the novel compound has a good anti-inflammatory effect and has a good application prospect in the aspect of treating inflammation.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal compounds, and in particular to a compound (S,E)-5-hydroxy-N-isobutyl-4-oxo-2-decenamide extracted and separated from the root of Anachia chinensis, and application of the compound in the preparation of anti-inflammatory drugs. Background Art

[0002] Anachyra root is the dried root of Anacyclus pyrethrum (L) DC, a plant in the Asteraceae family. It is primarily distributed in northern Africa and parts of Europe, with some distribution in my country's Xinjiang Uyghur Autonomous Region. Its properties are classified as dry and hot at the end of the third and first four levels, and it has the effects of clearing abnormal mucus, relieving stagnation, and alleviating pain.

[0003] TYK2 (tyrosine kinase 2), a member of the Janus kinase family, plays a crucial role in the regulation of cytokines such as IL-12, IL-23, and type I interferons (IFNs). Like other JAK family members, TYK2 possesses seven distinct homology regions, which can be divided into four domains: the kinase domain (JH1), the pseudokinase domain (JH2), the Src homology domains (JH3 and JH4), and the FERM domains (JH5, JH6, and JH7). The first two (JH1 and JH2) are the domains where TYK2 exerts its primary functions. The kinase domain (JH1) serves as the binding site for adenosine triphosphate (ATP) and is primarily responsible for substrate phosphorylation. The pseudokinase domain (JH2), similar to the JH1 domain, does not catalyze substrate phosphorylation but rather regulates the catalytic activity of JH1. When cytokines bind to extracellular cytokine receptors, they induce phosphorylation of JAKs in the cytoplasm. Phosphorylated JAKs further activate phosphorylation of STATs. Once phosphorylated, STATs dimerize and migrate to the nucleus to regulate gene transcription, thereby controlling immune cell proliferation, differentiation, and apoptosis. Studies have found that chronic inflammation in Crohn's disease is characterized by responses of T helper cell subtypes 1 (Th1) and 17 (Th17), as well as deficient regulatory T cell activity. In contrast, chronic inflammation in ulcerative colitis (UC) is often characterized by a Th2 response. Many cytokines involved in these T cell responses signal through JAK receptors to promote inflammation. TYK2, an intracellular tyrosine kinase widely expressed in the human body, plays a key role in mediating signaling and functional responses downstream of IL-12, IL-23, and type I interferon receptors. The IL-12 and IL-23 signaling pathways are involved in the initiation and maintenance of chronic inflammation in inflammatory bowel disease (IBD). Therefore, selectively inhibiting TYK2 kinase activity is an important option for treating inflammation.

[0004] In the Chinese patent publications, the invention with publication number CN105902722B discloses a Chinese medicine composition for treating vitiligo (with Anaki root as one of the components) and its preparation method and application. This invention can provide a new option for treating vitiligo. The invention with publication number CN106822208A discloses the use of Anaki root in the preparation of antidepressant drugs. By analyzing the effects of Anaki root extract on endogenous and exogenous depression models, it was shown that Anaki root extract has a certain antidepressant effect on chronic depressed rats and drug-induced depressed mice. The discovery of these antidepressant effects proves that Anaki root can provide new ideas for the research and development of drugs for the prevention, health care and treatment of depression, as well as clinical applications. The biological activities related to Anaki root reported in the above patent documents and other documents are mainly concentrated on the research of its extracts, and there are few reports on the anti-inflammatory effects of the chemical components contained therein. Therefore, it is of great significance to extract and separate chemical components from Anaki root 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 the root of Anachia chinensis and provide its anti-inflammatory pharmaceutical use.

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

[0007] Formula (1): Chemical formula is C 14 H 25 NO3, molecular weight is 254.1752, the name is (S, E)-5-hydroxy-N-isobutyl-4-oxo-2-decenamide, the structural formula is as follows:

[0008]

[0009] The method for extracting the above-mentioned novel compound (1) from the root of Anachia chinensis comprises the following steps:

[0010] Dried Anacardia root was crushed and extracted with 90 v / v% ethanol and 60 v / v% ethanol by percolation, respectively. The extracts were combined and concentrated, and then extracted with petroleum ether, chloroform, ethyl acetate, and water-saturated n-butanol, respectively. The chloroform and ethyl acetate extracts were combined and separated and purified by normal phase silica gel column chromatography, Sephadex LH-20, medium pressure reversed phase silica gel chromatography, and ODS-HPLC, respectively, to obtain a new compound.

[0011] In addition, the present invention carries out the anti-inflammatory activity evaluation of the new compound from three aspects: enzyme activity test, molecular docking and NO test. The new compound has good inhibitory activity against TYK2, and its IC 50The value was 2.65±0.68 μM, and it had 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, preferably as a TYK2 inhibitor.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention extracts and separates a new compound from the root of Anachia chinensis. The new compound belongs to the amide alkaloids, has good inhibitory activity on the TYK2 enzyme, and has a strong inhibitory effect on the NO release of RAW264.7 cells, so it can be used to prepare anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0015] Figure 2 IR spectrum of the new compound obtained in Example 1 of the present invention;

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

[0017] Figure 4 The new compound obtained in Example 1 of the present invention 13 C-NMR spectrum;

[0018] Figure 5 DEPT135° NMR spectrum of the new compound obtained in Example 1 of the present invention;

[0019] Figure 6 DEPT90° NMR spectrum of the new compound obtained in Example 1 of the present invention;

[0020] Figure 7 This is the HMQC NMR spectrum of the new compound obtained in Example 1 of the present invention;

[0021] Figure 8 This is the HMBC nuclear magnetic resonance spectrum of the new compound obtained in Example 1 of the present invention;

[0022] Figure 9 This is the COSEY NMR spectrum of the new compound obtained in Example 1 of the present invention;

[0023] Figure 10 The experimental and calculated ECD spectra of the new compound obtained in Example 1 of the present invention;

[0024] Figure 11a and Figure 11bThey are respectively two-dimensional and three-dimensional effect diagrams of the new compound obtained in Example 1 of the present invention and the target protein TYK2. DETAILED DESCRIPTION

[0025] 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.

[0026] The reagents used in the following examples are all common commercial reagents with analytical purity.

[0027] Example 1: Preparation of the compound of formula (1)

[0028] Step 1: 7.0 kg of dried Anacardiaceae root was crushed and passed through a 20-mesh sieve, and then extracted with 90 v / v% ethanol (230 L) and 60 v / v% ethanol (195 L) by percolation. The extracts were combined and concentrated to obtain 1150 g of a total extract.

[0029] Step 2: After suspending the total extract obtained in step 1 with water, extracting with petroleum ether, chloroform, ethyl acetate, and water-saturated n-butanol in sequence to obtain a petroleum ether extract (57.0 g), a chloroform extract (128.0 g), an ethyl acetate extract (35.0 g), and a n-butanol extract (252.0 g), respectively;

[0030] Step 3, the chloroform extract (128.0 g) and the ethyl acetate extract (35.0 g) obtained in step 2 were combined and separated by silica gel column chromatography, and gradient eluted with PE-EtOAc in a volume ratio of 10:1, 8:1, 6:1, 4:1, 3:1, 2:1, 1:1, 1:2, and 1:3, and the eluate with a petroleum ether-ethyl acetate volume ratio of 8:1 was collected, numbered Fr.3, and concentrated to dryness under reduced pressure; then filtered through Sephadex The product was separated by LH-20 chromatography using chloroform-methanol (volume ratio 1:1) as the eluent and 1.5 column volumes of the eluent. The eluate from 0.1 to 0.8 column volumes was collected, designated as Fr.3.1, and concentrated to dryness under reduced pressure. The product was then separated by medium-pressure reverse-phase silica gel chromatography using gradient elution with 50 v / v%, 70 v / v%, and 90 v / v% methanol, respectively. The eluate with 70 v / v% methanol was collected and concentrated to dryness under reduced pressure. The product was finally purified by ODS-HPLC using MeOH-H2O (MeOH:H2O volume ratio 60:40) as the eluent and a C-18 column (5 μm, 250 mm × 10 mm) at a flow rate of 3.0 mL / min and a detection wavelength of 210 nm. The eluate from 30 to 32 minutes was collected and concentrated to dryness under reduced pressure to obtain a new compound (20.6 mg).

[0031] Structural identification: Using modern spectroscopy techniques such as 1H NMR nuclear magnetic spectrum, 13 C NMR nuclear magnetic spectrum, DEPT135° NMR nuclear magnetic spectrum, DEPT90° NMR nuclear magnetic spectrum, two-dimensional nuclear magnetic spectrum (HMQC, HMBC, COSY), high resolution mass spectrum (HR-ESI-MS), infrared spectrum (IR) were used to identify the structure of the new compound obtained in step 3, and the results are shown in Table 1. Figure 1-9 ;

[0032] It was identified that the molecular weight of the new compound obtained in step 3 was 254.1752, and the chemical formula was C 14 H 25 NO3, named (S, E)-5-hydroxy-N-isobutyl-4-oxo dec-2-enamide, and the structural formula is as follows (1); the nuclear magnetic resonance spectrum data is shown in Table 1.

[0033] Table 1: Nuclear magnetic resonance spectrum data of the compound of formula (1) 1 H and 13 C NMR nuclear magnetic data 1 H NMR, 500MHz, 13 C NMR, 125MHz, in CD3OD, δ ppm, J Hz)

[0034]

[0035]

[0036] The structure of the obtained compound is shown in the following formula (1):

[0037]

[0038] The results obtained from the HMBC spectrum of the obtained compound are shown in the following formula (2):

[0039]

[0040] In order to detect the inhibitory activity of the new compound of the application prepared in Example 1 on tyrosine kinase 2 (TYK2), the following enzyme activity experiment was performed using the product of BPS Bioscience Company (item number: #79527):

[0041] Step 1, thaw 5x kinase assay buffer, ATP and 10X IRS1-tide at low temperature, and add DTT to the 5x kinase assay buffer to make its concentration 10mM.

[0042] Step 2: Add 6 μL of 5× kinase assay buffer (containing 10 mM DTT), 1 μL of 500 μM ATP, 5 μL of 10× IRS1-tide, and 13 μL of ultrapure water to each well, for a final volume of 25 μL per well.

[0043] Step 3: 5 μL of the prepared inhibitor (new compound obtained in Example 1, Abr ocitinib) solution was added to the inhibitor group, and 5 μL of buffer solution without inhibitor was added to the blank group and the test group.

[0044] Step 4: Dilute 5× kinase assay buffer (containing 10 mM DTT) to 1× kinase assay buffer (containing 2 mM DTT) with ultrapure water. Add 20 μL of 1× kinase assay buffer (containing 2 mM DTT) to each well of the blank group.

[0045] Step 5: Thaw TYK2 kinase on ice and dilute it to 0.5 ng / μL with 1× kinase assay buffer (containing 2 mM DTT). Add 20 μL of the diluted TYK2 enzyme to the test group and inhibitor group, respectively, to initiate the reaction. Incubate each group at 30°C for 40 minutes.

[0046] Step 6. After the solution in the wells has reacted for 40 minutes, add 50 μL of Kinase-Glo Max reagent to each well. Wrap the plate with aluminum foil and incubate at room temperature for 15 minutes. The reagents and amounts used in the blank, inhibitor, and test groups are shown in Table 2.

[0047] Step 7: Use a microplate reader to perform chemiluminescence detection.

[0048] Table 2: Reagents added to the blank group, inhibitor group, and experimental group

[0049] Blank group Inhibitor group experimental group 5×Kinase assay buffer (containing 10mM DTT) 6μL 6μL 6μL ATP (500 μM) 1 μL 1 μL 1 μL 10X IRS1-tide 5μL 5μL 5μL Water 13μL 13μL 13μL Test inhibitor - 5μL - Inhibitor Buffer(no inhibitor) 5μL - 5μL 1×Kinase assay buffer (contains 2mM DTT) 20 μL - - TYK2 (0.5 ng / μL) - 20 μL 20 μL Total 50 μL 50 μL 50 μL

[0050] Table 3: Inhibitory activity of new compounds against TYK2 enzyme

[0051] Inhibitor group number <![CDATA[IC 50 (μM)]]> New compounds 2.65±0.68 Abrocitinib (PF-04965842) 2.80±0.87

[0052] Among them, IC 50 (μM) is the drug concentration when the enzyme activity inhibition rate is 50%, which is used to indicate the inhibitory activity against TYK2 enzyme; Abrocitinib (PF-04965842) is the positive control drug for TYK2 enzyme. As shown in Table 3, the new compound has good inhibitory activity against TYK2 enzyme.

[0053] Example 3: To better understand the binding mode of the new compound obtained in Example 1 with TYK2, a molecular docking method was used to verify and illustrate the following:

[0054] Step 1. The databases include UniProt database (https: / / www.uniprot.org / ) and PDB database (http: / / www.rcsb.org / ); the software includes InDraw (Shanghai Eagle Valley Information Technology Co., Ltd., China), SYBYL 1.0 software (Tripos, USA), Discovery Studio 2017R2 Client (DS, developed by Accelrys, USA), and PyMOL.

[0055] Step 2: Use InDraw 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 1.0 software and use the molecular mechanics program Minimize to optimize the structure. Apply the Tripos force field and apply Gasteiger-Huckel charges. The optimized stable conformation is saved in mol2 format to establish a ligand small molecule compound library in preparation for molecular docking.

[0056] Step 3. Download the crystal structure of the target protein tyrosine kinase 2 (TYK2) (PDB ID: 7UYS) from the PDB database. Use the Docking module in the Application to modify, hydrogenate, and load AMBER-FF99 charges on the target protein. Determine the active site for docking based on the ligand in the target protein complex. Save the processed protein to prepare for subsequent molecular docking studies.

[0057] Step 4: Molecular docking of the ligand library with the target protein was performed using the Surflex-dock module in SYBYL 1.0 software. The docking results were given as a scoring function (Total Score) and saved in mol2 format. Ligand molecules were screened using the Total Score function of the SYBYL molecular docking module. The Total Score function comprehensively considers factors such as polarity, hydrophobicity, enthalpy, and solvation. A larger Total Score value indicates a more stable docked complex, indicating a better match between the small molecule compound and the macromolecule protein.

[0058] Step 5: Use the receptor-ligand interactions module in Discovery Studio software to analyze the molecular docking results and produce three-dimensional and two-dimensional renderings.

[0059] Table 4: Docking scores of new compounds and target proteins

[0060]

[0061] Study the structural model of the new compound and TYK2 ( Figure 11a and Figure 11b ). The compound is stabilized by hydrogen bond interactions with GLU979, VAL981, GLY984, and hydrophobic interactions with residues LYS930, VAL911, MET978, LEU903. These hydrogen bonds and hydrophobic interactions play an important role in the binding of the new compound to TYK2. Therefore, the docking score of the new compound binding to TYK2 indicates that the new compound of the present application has good binding activity to the target protein, and the new compound can be a potential inhibitor of the TYK2 enzyme.

[0062] Example 4: In order to further verify the anti-inflammatory activity of the new compound obtained in Example 1, the effect of the new compound on the NO release amount of RAW264.7 cells is detected.

[0063] The following reagents and instruments are used: RAW264.7 cell strain (China Typical Culture Collection Center); DMEM medium (Gibco Company), lipopolysaccharide (sigma company); fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd.); NO detection kit (Shanghai Biyun Tian Biotechnology Co., Ltd.), sterile CO2 incubator (Heal Force Biomedical Technology Holding Co., Ltd.); full-wavelength enzyme label instrument (Thermo Fisher Scientific).

[0064] Step 1: RAW264.7 cells are cultured to the logarithmic phase in DMEM medium containing 10% fetal bovine serum at 5% CO2, 37°C.

[0065] Step 2: The RAW264.7 cell suspension (1×10 4 cells / well) grown in the logarithmic phase in step 1 is uniformly inoculated in a 96-well plate, and after being cultured at 5% CO2, 37°C for 24 h, the cytotoxicity of different concentrations of compounds is determined by MTT colorimetry. The compound at a final concentration of 50 μM has no toxic effect on RAW264.7 cells. Specifically, the compound solution to be tested is added to the above 96-well plate, and a control group (without adding the compound) is set, and incubated at 5% CO2, 37°C for 48 h; the culture plate is taken out, 20 μL of MTT solution (5 mg / mL) is added to each well, and incubation is continued at 37°C for 4 h, and then the culture is terminated; the culture supernatant in the well is carefully aspirated, 150 μL of DMSO is added to each well, and oscillation is performed for 10 min, and the wavelength of 490 nm is selected, and the light absorption value (OD) of each well is determined on an automatic enzyme label instrument. Cell survival rate (%) = experimental group absorbance (OD 实验 ) / control group absorbance (OD 对照 )×100%, and a survival rate of ≥80% is generally considered to indicate that the compound has no obvious toxic effect.

[0066] 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-treated group (1μg / mL LPS + new compound) and a positive control group (1μg / mL LPS + dexamethasone) were set up, with 5 replicates in each group. Except for the blank group, the positive control group and the drug-treated group were incubated with drugs for 2 hours, and then LPS with a final concentration of 1μg / mL was added to stimulate the model group for 22 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), the content of nitric oxide in the supernatant was calculated, and the IC 50 value.

[0067] Table 5: In vitro anti-inflammatory activity of new compounds

[0068] Compound number IC 50 (μM) <!-- 6 -->]]> New compounds 7.99±1.64 Dexamethasone 8.36±0.58

[0069] The results showed that the new compound could significantly downregulate the NO release of inflammatory cells, and the IC 50 The value is smaller than that of the positive drug dexamethasone, indicating that the new compound has good anti-inflammatory activity.

Claims

1. Compound (S,E)-5-hydroxy-N-isobutyl-4-oxo-2-decenamide, chemical formula C 14 H 25 NO3, the structural formula is:

2. Use of the compound (S,E)-5-hydroxy-N-isobutyl-4-oxo-2-decenamide according to claim 1 in the preparation of anti-inflammatory drugs.

3. Use of the compound (S,E)-5-hydroxy-N-isobutyl-4-oxo-2-decenamide according to claim 1 in the preparation of a TYK2 inhibitor.

4. Use of the compound (S,E)-5-hydroxy-N-isobutyl-4-oxo-2-decenamide according to claim 1 in the preparation of a drug for inhibiting NO production.

5. A method for extracting the compound (S, E)-5-hydroxy-N-isobutyl-4-oxo-2-decenamide according to claim 1, characterized in that: The following steps are involved: Dried Anacardia root medicinal material is taken, crushed, and extracted with 90v / v% ethanol and 60v / v% ethanol by percolation, respectively. The extracts are combined, concentrated, and then extracted with petroleum ether, chloroform, ethyl acetate, and water-saturated n-butanol in sequence. The obtained chloroform and ethyl acetate layer extracts are combined and then separated and purified by normal phase silica gel column chromatography, Sephadex LH-20, medium pressure reverse phase silica gel chromatography, and ODS-HPLC in sequence to obtain the compound.

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