CN111888346A

CN111888346A - Application of aurantio-obtusin

Info

Publication number: CN111888346A
Application number: CN201910366642.6A
Authority: CN
Inventors: 安泳潼; 王辉俊; 吕兴; 吴若铭; 叶冠
Original assignee: Shanghai Phaarmaceuticals Holding Co ltd
Current assignee: Shanghai Phaarmaceuticals Holding Co ltd; Shanghai Pharmaceuticals Holding Co Ltd
Priority date: 2019-05-05
Filing date: 2019-05-05
Publication date: 2020-11-06

Abstract

The invention relates to application of aurantio-obtusin. The invention discloses application of aurantio-obtusin shown as a formula I or pharmaceutically acceptable salt thereof in preparing a BLK inhibitor. The invention also discloses application of the aurantio-obtusin shown as the formula I or pharmaceutically acceptable salt thereof in preparing a medicament for treating autoimmune diseases. The aurantio-obtusin of the invention can effectively inhibit BLK, has good treatment effect on autoimmune diseases, especially ankylosing spondylitis, has small toxic and side effects,the safety is high.

Description

Application of aurantio-obtusin

Technical Field

The invention relates to application of aurantio-obtusin.

Background

Ankylosing Spondylitis (AS) is a disease with inflammation of the sacroiliac joints and spinal attachment points AS the main symptom. It is mainly characterized by chronic inflammatory arthritis involving the spine and pelvis, causing progressive new bone formation and fusion in the painful, stiff and affected areas. Meanwhile, the eye, lung, muscle and bone diseases of different degrees are caused, and the autoimmune function is also disturbed, so the medicine belongs to the autoimmune disease. Generally, the disease is relatively hidden, no clinical symptoms exist in the early stage, some patients can show mild general symptoms in the early stage, such as hypodynamia, emaciation, long-term or intermittent low fever, anorexia, mild anemia and the like, and with the occurrence and development of the disease, a considerable part of patients with ankylosing spondylitis can not take care of life, the pain is great, and the diseases are frequently and repeatedly attacked, and the harm is not small.

The incidence of AS is related to various factors, genetic factors and immune factors are the most important, the incidence is strongly related to HLA-B27, the serum complement of 60 percent of AS patients is increased, most cases have IgA type wet factors, and the serum C4 and IgA levels are obviously increased. Trauma, endocrine, metabolic disorders and allergies are also suspected as causative agents. Several studies have shown a correlation between mutations and expression of immunomodulatory genes, suggesting that immunomodulatory mechanisms may prevent or exacerbate disease progression. The research finds that the B cells and the subgroup thereof in the peripheral blood of the population with various autoimmune diseases have imbalance disorder. The most important of the B cells is probably to activate the immune response of T cells by exerting an antigen presenting function, and also to induce the differentiation of Th2 as antigen presenting cells by secretion of cytokines and by deriving from cell surface-specific receptors. B-cell lymphocyte kinase (BLK) is a Src family protein tyrosine kinase named for its high expression in B cell lines, and encodes a non-receptor protein tyrosine kinase that forms the Src kinase family with Lyn, Fyn (Hum Mol gene.2012, 21(17): 3918-25.). src family kinases can promote phosphorylation, activation of the BCR signaling elements Ig- α, Ig- β and initiation of downstream signaling cascades, i.e., transduction of BCR signals downstream, by driving recruitment of another tyrosine kinase Syk. Research has shown that risk alleles of BLK are associated with reduced expression of BLK mRNA in B cell line transformation, and that BLK protein levels after mutation affect B cell tolerance mechanisms and induce immune diseases (N Engl J Med.2008,358(9): 900-9.; science.1990,247(4940): 332-6). Different kinds of autoimmune diseases share common immune pathways and genetic susceptibility factors. The significant manifestation of the BLK haplotypes associated with these diseases is associated with reduced levels of BLK transcription in the B lymphoblastoid cell line, a common risk gene underlying different autoimmune diseases.

Although there are many traditional methods of treating AS, it is still only symptomatic relief and not completely curative at present. Nonsteroidal anti-inflammatory drugs (NSAIDs) can reduce pain and stiffness in patients, but it has not been demonstrated how they affect the progression of bony rigidity. Sulfasalazine (SSZ) has proven effective in treating AS, and is primarily indicated for the amelioration of peripheral arthritis in patients. A significant proportion of AS patients have difficulty controlling their symptoms with conventional therapies and lack of evidence that these therapies can inhibit the progression of axial arthropathy and improve the prognosis of the disease. Other existing medicines have the defects that the circulation is slow, and the liver, the kidney, the stomach and the like can be damaged after long-term administration. As traditional Chinese medicine in China, the Chinese traditional medicine has the advantages of strong effect, quick response, lasting drug effect and the like, has relatively small dependence, drug resistance and toxic and side effects, and can be deeply researched. However, the existing traditional Chinese medicine treatment mainly stays on the aspect of relieving symptoms, targets are not clear, and the traditional Chinese medicine treatment is effective after long-term administration. In view of the defects of the existing treatment method, the development of a monomer medicament which has a definite target point, can exactly treat AS and can avoid serious side effects and is relatively safe from the traditional Chinese medicine has very important clinical significance.

The AURANTIO-OBTUSIN (AURANTIO-OBTUSIN, 1,3, 7-trihydroxy-2, 8-dimethoxy-6-methylanthracene-9, 10-dione) is a yellow acerola anthraquinone compound, and has the effects of reducing blood sugar and blood fat, resisting adenosine diphosphate, resisting estrogen activity and inhibiting platelet aggregation induced by estrogen, arachidonic acid and collagen. The prior patents show that the treatment field of the aurantio-obtusin is distributed in aspects of lipid reduction, anti-inflammation, anti-infection and the like. The aurantio-obtusin is a special component in the cassia seed, and the cassia seed has the main pharmacological actions of improving eyesight, reducing blood fat, reducing blood pressure, protecting liver, inhibiting bacteria, resisting oxidation, reducing diarrhea and the like. Is clinically used for treating diseases such as hyperlipoidemia, hypertension, constipation and the like, and has good treatment effect. The cassia seed related medicaments comprise: jiangzhining is collected in Qianjin Fang (Qianjin prescription), is collected in the thirteenth book of 1993 Ministry of public health of the people's republic of China (traditional Chinese medicine), consists of four traditional Chinese medicines, namely hawthorn (with cores removed), prepared fleece-flower root, cassia seed and lotus leaf, and is indicated by modern pharmacological research to be used for reducing blood fat, softening blood vessels, enhancing coronary blood circulation and resisting arrhythmia and hyperlipidemia, and has obvious curative effect when being clinically applied to hyperlipidemia.

Disclosure of Invention

The invention aims to overcome the defect that the existing medicines for treating autoimmune diseases, particularly ankylosing spondylitis, are single in type, and provides application of aurantio-obtusin. The aurantio-obtusin can effectively inhibit BLK, has good treatment effect on autoimmune diseases, particularly ankylosing spondylitis, and has small toxic and side effects and high safety.

The invention solves the technical problems through the following technical scheme.

The invention provides application of aurantio-obtusin shown as a formula I or pharmaceutically acceptable salt thereof in preparing a BLK inhibitor;

the aurantio-obtusin shown in the formula I can also be used as a lead compound to be applied to preparation of BLK inhibitors.

The invention also provides application of the aurantio-obtusin shown as the formula I or pharmaceutically acceptable salt thereof in preparing a medicament for treating autoimmune diseases;

wherein the autoimmune disease can be common autoimmune disease, including ankylosing spondylitis, rheumatoid arthritis, psoriasis, systemic lupus erythematosus or asthma, preferably ankylosing spondylitis.

The aurantio-obtusin shown in the formula I can also be used as a lead compound and applied to preparation of medicaments for treating autoimmune diseases.

The purity of the aurantio-obtusin is preferably more than or equal to 98 percent.

The above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention without departing from the common general knowledge in the art.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: the aurantio-obtusin can effectively inhibit BLK, has good treatment effect on autoimmune diseases, particularly mandatory spondylitis, and has small toxic and side effects and high safety.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Example 1: enzyme activity inhibition test of aurantio-obtusin on BLK target

1. Experimental background:

the inhibition effect of the test compound on BLK kinase was examined by using Caliper mobility shift assay (Caliper mobility shift assay).

2. Sample information

Preparing the aurantio-obtusin and the positive medicine staurosporine into 10mM stock solution by DMSO, subpackaging a small amount of the stock solution and storing the stock solution at-80 ℃ for later use.

3. Materials and reagents

4. Test method

4.1. 1 Xkinase base buffer and stop buffer were prepared to test for kinase

1)1 Xkinase base buffer

Comprises 50mM HEPES, pH 7.5 and 0.0015% Brij-35

2) Stop buffer

100mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3 and 50mM EDTA

2. Preparation of Compounds

1) Compounds were diluted with 100% DMSO to 50-fold the highest concentration in the reaction (50 × compounds). For example, if a maximum inhibitory concentration of 20. mu.M is desired, a 1mM compound DMSO solution is prepared at this step. 10 μ L of 10mM compound was transferred to one well of a 96-well plate and 90 μ L of 100% DMSO was added.

2) To two blank wells 100 μ L of 100% DMSO was added as no compound control well and no kinase control well in the same 96-well plate.

3) Preparing a middle concentration reaction hole:

taking 50 times of the compound in the 1), putting the compound in a 96-well compound plate, and then taking the compound at the concentration as a first concentration, performing 3-fold gradient dilution by using DMSO, and diluting 10 concentrations in total.

The middle plate was placed on a shaker and mixed for 10 minutes.

3. Preparation of assay plates

1) Transfer 5 μ L per well from 96 well intermediate plate to 384 well plate and make 2 replicates.

4. Kinase reaction

1) Kinase was added to 1 × kinase base buffer to prepare a 2.5 × enzyme solution, and FAM-labeled fluorophore and ATP were added to 1 × kinase base buffer to prepare a 2.5 × substrate solution.

2) The 2.5 Xenzyme solution was transferred to assay plates already containing 5. mu.L of compound 10% DMSO. To each well of a 384 well assay plate was added 10. mu.L of a 2.5 Xenzyme solution. Incubate at room temperature for 10 minutes.

3) To each well of a 384 well assay plate was added 10. mu.L of a 2.5 Xsubstrate solution.

4) Incubate at 28 ℃ for 60 minutes.

25 μ L of stop buffer was added to stop the kinase reaction.

Caliper reading

Data was collected on the Caliper. (Instrument: Caliper EZ reader, supplier: Caliper Life sciences, USA)

6. Fitting of curves

1) Conversion data was copied from the Caliper program.

2) Conversion of conversion to inhibition

Inhibition rate ═ (conversion rate of DMSO control-conversion rate of compound group)/(conversion rate of DMSO control-conversion rate of no enzyme control) × 100

3) Adjusting data in Xlfit entry version 5.4.0.8 to obtain IC₅₀The value is obtained.

IC₅₀The calculation formula is as follows: y ═ down + (up-down)/(1 + (IC)₅₀/X) ^ gradient)

The experimental results are as follows: IC of aurantio-obtusin₅₀At a rate of 5.955 μ M,

IC of positive medicine staurosporine₅₀It was 4.6 nM.

Table 1 shows the inhibition of BLK by aurantio-obtusin at different concentrations.

TABLE 1

Concentration of orange Cassia tora extract (nM)	Inhibition ratio (%)
		20000	76.7
6667	51.6
		2222	26.0
741	19.2
		247	4.8
82	2.3
		27	-1.9
9	8.1
		3	-3.1
1	-2.4

Example 2: enzymatic assay for selective inhibition of kinases by aurantio-obtusin

A 2.5 x enzyme solution of the following kinases was prepared according to the method in example 1: the kinases MUSK, SGK, TNIK, CK1, NEK2, CAMK2a, PKN1, AMPKa1 and EphA 2.

The inhibition rate of aurantio-obtusin against the above kinase at 20 μ M was measured by the method described in example 1.

The experimental results are as follows: the inhibition of the kinases MUSK, SGK, TNIK, BLK, CK1, NEK2, CAMK2a, PKN1, AMPKa1 and EphA2 at a concentration of 20 μ M is shown in Table 2 below:

TABLE 2

Kinase enzymes	Inhibition ratio (%)	Kinase enzymes	Inhibition ratio (%)
				MUSK	42.1	NEK2	8.9
SGK	7.4	CAMK2a	1.3
				TNIK	64.3	PKN1	8.2
BLK	76.7	AMPKa1	-9.4
				CK1δ	20.5	EphA2	48

From the results in table 2, it is known that at 20 μ M, aurantio-obtusin has the highest inhibitory activity on BLK, and the inhibition rate can reach 76.7%, and has good selectivity compared with other kinases.

Claims

1. Application of aurantio-obtusin shown in formula I or its pharmaceutically acceptable salt in preparing BLK inhibitor is provided;

2. the use according to claim 1, wherein the aurantio-obtusin of formula I is used as a lead compound.

3. Application of aurantio-obtusin shown as formula I or its pharmaceutically acceptable salt in preparing medicine for treating autoimmune disease is provided;

4. the use of claim 3, wherein the autoimmune disease is ankylosing spondylitis, rheumatoid arthritis, psoriasis, systemic lupus erythematosus or asthma.

5. The use of claim 4, wherein the autoimmune disease is ankylosing spondylitis.

6. Use according to claim 3, wherein the aurantio-obtusin of formula I is used as a lead compound.