WO2013127345A1

WO2013127345A1 - Compounds as protein kinase inhibitors and use thereof

Info

Publication number: WO2013127345A1
Application number: PCT/CN2013/071967
Authority: WO
Inventors: 邹强; 刘阳; 马正伟; 阳泰; 李丽梅; 李敏惠; 杨淑霞; 王衍堂; 李华; 郑静; 刘进; 罗兴燕; 胡松; 唐媚; 唐信威
Original assignee: 成都医学院
Priority date: 2012-02-29
Filing date: 2013-02-28
Publication date: 2013-09-06
Also published as: CN102603727B; CN102603727A

Abstract

Provided are compounds as protein kinase inhibitors and use thereof. Preparation methods of the compounds are also provided. The compounds can effectively inhibit the activities of protein kinases and proliferation of abnormal activated T-cell, and the compounds can be used to treat diseases such as autoimmune diseases and malignant tumors, especially lymphoma, multiple myeloma, breast cancer, liver cancer, transplant rejection, allergic arthritis and rheumatoid arthritis, and are suitable for industrial applications.

Description

Compound for inhibiting protein kinase and use thereof

The present invention relates to a compound which inhibits protein kinase and uses thereof.

Background technique

Protein kinases, also known as protein phosphakinase. A class of enzymes that catalyze the phosphorylation of proteins. It can transfer the γ-phosphate on adenosine triphosphate (ATP) to the hydroxyl group of certain serine, threonine or tyrosine residues on the amino acid residue of the protein molecule, thereby changing the conformation and activity of the protein and enzyme. . Phosphorylation of proteins is an important link in many signaling pathways. Most important life processes in cells are inseparable from protein phosphorylation. These enzymes are key factors in regulating cellular signaling, including cell proliferation and cell differentiation.

Protein kinases are classified into five classes: protein serine/threonine kinase, protein tyrosine kinase, proteomic kinase, protein tryptophan kinase, and protein aspartyl/glutamyl kinase. Protein kinases play an important role in the regulation and maintenance of cellular processes, and abnormalities in kinase activity have been observed in many disease states, including: malignant tumors, immune diseases, cardiovascular diseases, diabetes, infectious diseases, joints. Inflammation and other immune disorders, nervous systems such as Alzheimer's disease, Alzheimer's disease AD, etc., have now been found to be associated with protein kinases in more than 400 human diseases.

Protein kinase inhibitors are a class of compounds that inhibit protein kinase activity. According to the type of protein kinase, it can be divided into serine/threonine protein kinase inhibitor or tyrosine kinase inhibitor. The former can be divided into three groups according to the action site, one group acts on the catalytic region, and one group acts on the regulation. The zone, the other group has an effect on both the regulatory zone and the catalytic zone. There are various research reports on protein kinase inhibitors, such as patent applications: CN201110310167, CN200810028982 and the like. Numerous studies have reported that protein kinase inhibitors can be used to treat diseases such as malignant tumors and arthritis caused by protein kinase activation, and at the same time, it can prevent the activation of sputum lymphocytes and have a therapeutic effect on various immune diseases (Zhou Yiming, Et al., Research progress in tyrosine protein kinase inhibitors, 2000, Vol. 6, No. 3). Because of the significant medical use of protein kinase inhibitors, research on such drugs is particularly important.

Summary of the invention

It is an object of the present invention to provide a novel class of compounds which inhibit protein kinases. Another object of the invention is to provide new uses for such compounds.

In particular, the present invention provides the use of a compound of formula I for the preparation of a protein kinase inhibitor,

_H, fluorenyl, =0, —O—C—R? or — ₀ R ₇ , wherein R ₇ is a C1-C5 fluorenyl group;

R ₂ is -H, fluorenyl, _R ₅ -NH-R ₆ or -R ₈ 0H, wherein is a fluorenyl or alkenyl group, an indenyl group, a cyclodecyl group, and a C1-C5 fluorenyl group;

R ₃ is -H, fluorenyl, C6-10 aryl, substituted fluorenyl or substituted C6-10 aryl; or, together with the carbon atom to which they are attached, form a cyclodecyl group, a cyclononanone group or Substituted cyclodecyl, cyclononanone;

Or, together with the carbon atom to which they are attached, form a cyclodecyl group, a cyclofluorenone group or a substituted cyclodecyl group, a cyclononanone group;

Is 0 or S;

For -H or thiol, . An aromatic group of C6-10;

Or, , . Together with the carbon atom to which they are attached, an aromatic group of C6-10 or an aromatic group of substituted C6-10 is formed.

Further, the protein kinase inhibitor is a serine/threonine protein kinase inhibitor or a tyrosine kinase inhibitor.

Wherein the protein kinase inhibitor is a medicament for treating a malignant tumor, an autoimmune disease, a heart disease, a diabetes, an infectious disease, an arthritis, an immune disorder or Alzheimer's disease.

Further, the malignant tumor is multiple myeloma, lymphocytic leukemia, granulocyte leukemia, lymphoma, liver cancer, lung cancer, pancreatic cancer, colon cancer or breast cancer; the autoimmune disease is rheumatoid arthritis , myeloproliferative disorders, transplant rejection, asthma, lupus erythematosus, psoriasis, allergies or contact dermatitis.

Wherein the compound is a compound of formula II, 2-(4-phenyl-2-thiazolyl)-4,5,6,7-tetrahydro-2H-carbazol-3-ol, or 2-(6- Methoxy-2-benzothiazolyl) -4,5,6,7-tetrahydro-2H-indazol-3-ol; wherein, the structural formula of the compound of formula II is as follows

Formula II, o

II

Is an -H, anthracenyl, C6-10 aryl, substituted fluorenyl or substituted C6-10 aryl; or, together with the carbon atom to which they are attached, form a cyclodecyl, cyclofluorenyl or substituted Cyclodecyl, cyclononanone;

It is 0 or 5.

Further, R1 is -0H, -H, sulfhydryl, =0;

R2 is -H, fluorenyl, -R5-NH-R6, wherein R5 is a fluorenyl or alkenyl group; R6 is a fluorenyl group or a cyclic fluorenyl group;

R3 is -H, sulfhydryl;

Or, R1, R2 together with the carbon atom to which they are attached form a cyclodecyl group, a cyclofluorenone group or a substituted cyclodecyl group or a cyclofluorenone group;

Or R2, R3 together with the carbon atom to which they are attached form a cyclodecyl group, a cyclofluorenone group or a substituted cyclodecyl group or a cyclononanone group;

R4 is N or S.

further,

〇

! ^ is -OH, -H, -〇 - c - ^R ₇ or - 0R ₇ , and R ₇ is a C1-C3 fluorenyl group;

R ₂ is -H, a C1-C5 sulfhydryl group, or a -R muscle as a C1-C3 sulfhydryl group;

R ₃ is -H, a C1-C3 fluorenyl group, a phenyl group, or a halogen-substituted C1-C3 fluorenyl group;

Or, together with the carbon atom to which they are attached, form a cycloalkyl group of C3-8;

It is 0 or 5.

go a step further,

0

II

! ^ is -OH, _ ^Q - ^C _ ^R7 or -0R ₇ , the R7 is a methyl group;

Is -H, or -R ₈ 0H, the ethyl group;

a methyl group substituted with methyl, phenyl, or trifluoro;

Or, together with the carbon atoms to which they are attached, form a C5-C6 cyclodecyl group;

It is 0 or 5.

Further preferably, the compound is:

2-(1H-benzothiazolyl-2-yl)-4,5,6,7-tetrahydro-2hydro-indole-f--3-ol, 2-(l-hydrobenzimidazolyl-2-yl)-4,5,6,7-tetrahydro-2hydro-indole-cono-3-ol,

1-(1,3-benzothiazolyl-2yl)-1,5,6,7-tetrahydro-4H-indazole-4-one,

1-(1,3-benzothiazolyl-2(yl)-3,4-dimethyl-1hydro-pyrazole-5-ol,

2-(2-benzothiazolyl)-2,4,5,6-tetrahydrocyclopentadiazole-3-ol,

1-(2-benzothiazolyl)-3-(trifluoromethyl)pyrazole-5-ol,

1-(2-benzothiazolyl)-4-(2-hydroxyethyl)-3-methylpyrazole-5-ol,

2-(2-benzothiazolyl)-4,5,6,7-tetrahydro-2H-indazole-3-acetate,

2-(2-benzoxazolyl)-4,5,6,7-tetrahydro-2H-indazole-3-ol, or

1-(2-Benzothiazolyl)-3-phenylpyrazole-5-ol.

The invention also provides a compound of formula I which has the structural formula:

Formula I

0

II

_H, fluorenyl, =0, —0—C—R? or — ₀ R ₇ , wherein R ₇ is a C1-C5 fluorenyl group;

Is 0 or S;

For -H or thiol, . An aromatic group of C6-10; or, . Together with the carbon atom to which they are attached, an aromatic group of C6-10 or an aromatic group of substituted C6-10 is formed.

Further, the compound is a compound of formula II, 2-(4-phenyl-2-thiazolyl)-4,5,6,7-tetrahydro-2H-indazol-3-ol, or 2-(6 -Methoxy-2-benzothiazolyl) -4,5,6,7-tetrahydro-2H-indazol-3-ol; wherein, the structural formula of the compound of formula II is as follows

Formula II, o

II

Is 0 or S;

When R ₂ , together with the carbon atom to which they are attached, form a cycloalkyl group of C6, does R ₄ not be? >.

Further, R1 is -0H, -H, sulfhydryl, =0;

R3 is -H, sulfhydryl;

R4 is N or S;

When R ₂ , together with the carbon atom to which they are attached, forms a cycloalkyl group of C6, R _{4 is} not

Further preferably, the compound is:

2-(1H-benzimidazolyl-2-yl)-4,5,6,7-tetrahydro-2hydrogen-indole frustum-3-ol,

1-(1,3-benzothiazolyl-2yl)-1,5,6,7-tetrahydro-4H-indazole-4-one,

1-(1,3-benzothiazolyl-2(yl)-3,4-dimethyl-1hydro-pyrazole-5-ol,

2-(2-benzothiazolyl)-2,4,5,6-tetrahydrocyclopentadiazole-3-ol,

1-(2-benzothiazolyl)-3-(trifluoromethyl)pyrazole-5-ol,

1-(2-benzothiazolyl)-4-(2-hydroxyethyl)-3-methylpyrazole-5-ol,

2-(2-benzothiazolyl)-4,5,6,7-tetrahydro-2H-indazole-3-acetate,

2-(2-benzoxazolyl)-4,5,6,7-tetrahydro-2H-indazole-3-ol, or

1-(2-Benzothiazolyl)-3-phenylpyrazole-5-ol.

The present invention also provides 2-(1H-benzothiazolyl-2yl)-4,5,6,7-tetrahydro-2hydro-indole-f--3-ol The compound of the formula III, 2-mercaptobenzothiazole, is subjected to a condensation reaction to obtain a target compound; wherein the structural formula of the compound of the formula III is as follows

Among them, (^ is the base of ^-^.

Further, the compound of the formula III is one or a mixture of two or more of ethyl 2-cyclohexanonecarboxylate, methyl 2-cyclohexanonecarboxylate or propyl 2-cyclohexanonecarboxylate; The molar ratio of 2-mercaptobenzothiazole is (0.5-1.5): 1; the organic acid or inorganic acid is used as a catalyst in the reaction, and the molar ratio of the catalyst to the compound of formula III is (0.01-0.1): 1; Select toluene, ethanol, methanol; reaction temperature is 20-200 ° C.

Further, the molar ratio of the compound of the formula III to 2-cyclohexanone ethyl ester, the compound of the formula III and the 2-mercaptobenzothiazole is (0.7-1): 1.

Further, the catalyst is selected from the group consisting of acetic acid, p-toluenesulfonic acid, formic acid, or non-oxidizing inorganic acid.

Preferably, the catalyst is selected from the group consisting of acetic acid, and the molar ratio of acetic acid to the compound of formula III is (0.012-0.014): 1; or

The catalyst is selected from p-toluenesulfonic acid, and the molar ratio of p-toluenesulfonic acid to the compound of formula III is (0.056-0.057): 1.

Further, the solvent is toluene.

Further, the reaction temperature is from 120 to 125 °C.

Further, after the condensation reaction, it was recrystallized from ethanol.

Experiments have shown that the compounds of the present invention can effectively inhibit protein kinase activity and inhibit abnormally activated T cell proliferation, and can be used for treating diseases such as autoimmune diseases and malignant tumors, especially for lymphoma, multiple myeloma, breast cancer, liver cancer, Transplant rejection, allergy and rheumatoid arthritis have significant therapeutic effects and have good industrial application prospects.

In the synthesis method of the present invention (2-(1H-benzothiazolyl-2yl)-4,5,6,7-tetrahydro-2hydro-indole-f--3-ol), the reaction raw material is easily available, The reaction operation is simple, the product yield can reach more than 40%, the purity is above 95%, and can be as high as 98%, the yield and purity are high, and the repeatability is good, which provides a practicality for the compound. Synthetic methods provide a reliable source for further study of such compounds.

DRAWINGS

Figure 1 Effect on protein kinase activity Figure 2 Effect of transplant rejection

Figure 3 Effect on delayed allergic reaction

Figure 4 NMR identification of compound 1

Figure 5 Figure 4 is an enlarged view of Part A

Figure 6 Figure 4 is an enlarged view of part B

Figure 7 Mass spectrum of compound 1

Figure 8 HPLC diagram of Compound 1

detailed description

Example 1 Preparation of 2-(1H-benzothiazolyl-2yl)-4,5,6,7-tetrahydro-2hydro-carbazole-3-ol (Compound 1)

N

: just

Mixing the raw material methyl 2-cyclohexanonecarboxylate (0.264 mol), 2-mercaptobenzothiazole (0.333 mol), 700 mL of toluene, and taking 1 ml of acetic acid (the molar ratio of acetic acid to the raw material 2-cyclohexanone methyl ester is about 0.07). : 1), in a lOOOOmL reaction flask, heated at 120-125 ° C for 5 h (TLC monitoring reaction end point). The reaction liquid was evaporated to dryness, and the residue was crystallized from EtOAc (EtOAc). :

ESI-MS: m/z 270 (M-l)-UV (MeOH) ληιαχ = 218 nm;

lH NMR (DMSO-d6, 600MHz): δ 8.01 (d, lH, J = 7.86 Hz), 7.78 (d, lH, J = 7.80 Hz), 7.46 (t, lH, J = 7.6 Hz), 7.33 ( t, lH, J = 7.5 Hz), 2.50 (m, 2H), 2.20 (m, 2H), 1.73 (m, 2H), 1.67 (m, 2H).

13C NMR (DMSO-d6, 150 MHz); δ 162.3, 154.6, 153.3, 148.9, 132.2, 126.9, 124.3, 122.6, 121.0, 102.5, 22.3, 22.2, 21.7, 18.7; ESI-MS: m/z 272 [M + 1]+; HRESIMS m/z calcd for C14H14N30S [M + 1]+ 272.0852, found 272.0849. Example 2 Synthesis of Compound 1

Methyl 2-cyclohexanonecarboxylate (0.264 mol), 2-mercaptobenzothiazole (0.333 mol), 700 mL of toluene, 1 mL of acetic acid in a 1000 mL reaction flask were weighed, and the reaction was monitored by TLC, and the reaction was carried out at room temperature for 3 days to reach the end point. The reaction mixture was evaporated to dryness. The residue was evaporated, evaporated, evaporated, evaporated. The obtained compound is identified as a compound Matter 1.

Example 3 Synthesis Method of Compound 1

5毫升 (The molar ratio of formic acid to raw material cyclohexanone ester is about 0. The molar ratio of methyl formate to cyclohexanone is about 0. 05: 1), in a lOOOOmL reaction flask, externally heated at 120_125 ° C reflux reaction for 5 h (TLC monitoring reaction end point). The reaction mixture was evaporated to dryness. EtOAc m. The obtained compound was identified as Compound 1.

Example 4 Synthesis Method of Compound 1

The mixed materials were ethyl 2-cyclohexanonecarboxylate (0.264 mol), 2-mercaptobenzothiazole (0.333 mol), ethanol 700 mL, 1 mL of acetic acid in a lOOOOmL reaction flask, and refluxed for 36 h (TLC monitoring reaction end point). The reaction mixture was evaporated to dryness vacuoljjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj The obtained compound was identified as Compound 1.

Example 5 Synthesis Method of Compound 1

The mixed materials were ethyl 2-cyclohexanonecarboxylate (0.264 mol), 2-mercaptobenzothiazole (0.333 mol), 700 mL of toluene, 1 mL of acetic acid in a lOOOOmL reaction flask, and refluxed for 42 h (TLC monitoring reaction end point). The reaction mixture was evaporated to dryness crystals crystals crystals crystals The obtained compound was identified as Compound 1.

Example 6 Synthesis Method of Compound 1

Add methyl 2-cyclohexanonecarboxylate (0.0132 mol), 2-mercaptobenzothiazole (0.0133 mol), 20 ml of toluene, 0.01 ml of acetic acid in a 50 ml reaction flask (the molar ratio of acetic acid to cyclohexanone ester is about 0.013). : 1), external heating 120-125 ° C reflux reaction for 5h; the reaction liquid is evaporated to dryness under reduced pressure; the residue is recrystallized from 20mL of ethanol, filtered and dried to give an orange-yellow solid as 2-(1H-benzothiazolyl) -2 base)-4,5,6,7-tetrahydro-2hydro-indazol-3-ol. The yield was 58% and the purity was higher than 95%. The obtained compound was identified as Compound 1.

Example 7 Synthesis Method of Compound 1

In a 250 ml reaction flask, add 2-cyclohexanone methyl ester (0.0132 mol), 2-mercaptobenzothiazole (0.0133 mol), toluene 100 ml, p-toluenesulfonic acid 0.45 g (p-toluenesulfonic acid and The molar ratio of cyclohexanone ester is about 0.0568: 1 ), and the external reaction is heated at 120-125 ° C for 5 h; the reaction liquid is evaporated to dryness under reduced pressure; ethyl acetate is extracted, washed with water, dried, concentrated, and the residue is recrystallized with 200 mL of ethanol. Filtration, drying, and an orange-yellow solid is 2-(1H-benzothiazolyl-2-yl)-4,5,6,7-tetrahydro-2hydro-hydrazine-decane-3-ol. The yield is 59% and the purity is higher than 95%. The obtained compound was identified as Compound 1.

Example 8 Preparation of 2-(2-benzothiazolyl)-2,4,5,6-tetrahydrocyclopentpyrazol-3-ol (abbreviated as Compound 5)

The same procedure as in Example 1 was carried out except that the starting material cyclopentanone ester was used in place of the cyclohexanone ester in the above reaction. Example 9 Preparation of 1-(2-benzothiazolyl)-3-(trifluoromethyl)pyrazole-5-ol (abbreviated as Compound 6)

According to the reaction formula, the raw material ester (2g, O.Ol lmol), raw material hydrazine (2.2g, 0.013mol), toluene 70mL, acetic acid O. lmL in 150mL reaction flask, external heating 120-125 °C reflux reaction for 5h (TLC monitoring Reaction end point). The reaction mixture was evaporated to dryness crystals crystals crystals crystals The structural identification data of this compound are as follows:

1H NMR (DMS0-d6, 600MHz): δ 8.08 (d, lH, J = 6.0 Hz), 7.94 (d, lH, J = 6.0 Hz), 7.52 (t, lH, J = 6.0 Hz), 7.43 (t , lH, J = 6.0 Hz), 5.95 (s, 1H), 3.43 (s, lH).

ESI-MS: m/z 284 (Ml) - Example 10 l-(2-benzothiazolyl)-4-(2-hydroxyethyl)-3-methyl-IH-pyrazol-5-ol ( Preparation of compound 7)

After the reaction material was replaced by the preparation method of Example 3, a white solid powder (yield: </RTI> y. The structural identification data of this compound are as follows:

1H NMR (DMS0-d6, 600MHz): δ 8.02 (d, lH, J = 12.0 Hz), 7.80 (d, lH, J = 6.0 Hz), 7.47 (t, lH, J = 12.0 Hz), 7.33 (t , lH, J = 6.0 Hz), 3.47 (t, 2H, J = 6.0 Hz), 2.37 (t, 2H, J = 6.0 Hz), 2.20 (s, 3H).

ESI-MS: m/z 274 (Ml) - Example 11 2-(6-methoxy-2-benzothiazolyl) -4,5,6,7-tetrahydro-2H-indazole-3- Alcohol -丄^骈璨°%Γ8*

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'(HZ'^LVZ ^ζ (ΗΖ'^)££'ί '(ΗΖ'^9'ί 9 ¹ (ZHW009 ' 9P-0SWa)3⁄4W HI

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^:丄^骈璨W3⁄4

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01

L96UO/£lOZ l3/13d lH NMR (DMSO-d6, 600MHz): δ 8.05 (d, lH, J = 6.0 Hz), 7.89 (m, 3H), 7.50 (m, 4H), 7.38 (t, lH, J = 6.0 Hz), 6.10 (s, lH).

ESI-MS: m/z 292 (Ml) - Example 15 2-(2-benzothiazolyl) -4,5,6,7-tetrahydro-2H-indazole-3-acetate (referred to as compound 9 for short) Preparation

2-(1H-benzothiazolyl-2-yl)-4,5,6,7-tetrahydro-2hydro-indole frustum-3-ol (1.Og, 0.0037 mol) prepared in Example 1. , Ac ₂ 0 20 mL in a lOOmL reaction flask, refluxing for 1 h (TLC monitoring reaction end point). The reaction mixture was evaporated to dryness crystals crystals crystals crystals crystals The structural identification data of this compound are as follows:

1H NMR (DMSO-d6, 600MHz): δ 8.02 (d, lH, J = 6.0 Hz), 7.85 (d, lH, J = 6.0 Hz), 7.48 (t, lH, J = 6.0 Hz), 7.38 ( t,lH,J=6.0Hz), 2.64(t,2H,J=6.0Hz), 2.47(s,3H), 2.41(t,2H,J=6.0Hz), 1.76(d,2H,J=6.0 Hz), 1.68 (d, 2H, J = 6.0 Hz).

ESI-MS: m/z 314(M+l)+

In summary, in the synthesis method of the compound of the present invention, the reaction raw material is easy to obtain, the reaction operation is simple, the product yield can reach 40% or more, the purity is high, and the repeatability is good, which provides a practical compound for the present invention. Good synthesis methods provide a reliable source for further study of such compounds. The beneficial effects of the present invention will be specifically described below by way of test examples.

Test Example 1 Effect of the compound of the present invention on protein kinase activity

CTLL-2 cells were plated at a level of 1 X 10 ⁷ cells/well in a 6-well plate and Compound 1 of the present invention was added. After 6 hours of IL-2 starvation, IL-2 was added to a final concentration of 100 U/ml and incubated for 10 min. The cells were collected, centrifuged at 600 G for 5 min, the supernatant was removed by centrifugation, washed once with PBS, and the cell pellet was taken, and the cells were lysed by cell lysate. The protein was collected for Western-blot detection, and the kinase to be tested was phosphorylated Akt, phosphorylated JAK3, and phosphorylated STAT5.

Experimental results: The results are shown in Figure 1.

The results indicate that the compound of the present invention 1 (prepared in Example 1) can inhibit phosphorylation of the protein kinase Akt (serine/threonine protein kinase), JAK3 (tyrosine kinase), and STAT5 (signaling and transcriptional activator), indicating The compounds of the invention are effective in inhibiting protein kinase activity.

Test Example 2 Effect of the compound of the present invention on T cells

1. To rest T cell toxicity

method: 1. Preparation of human peripheral blood mononuclear cells (PBMC): Blood donation by healthy people, the blood is diluted with PBS, and 3 ml of PBMC separation solution is added to a 10 ml sharp-bottomed glass centrifuge tube, and the temperature is inclined at 45 ° C. 6 ml of blood-PBS mixture was added, centrifuged at 600 g for 20 min, and the intermediate layer rich in PBMC was aspirated, washed twice with PBS, and then resuspended in cell culture medium.

2. Purification of T cells from PBMC: T cells were isolated from PBMC using the Miltenyl T Cell Magnetic Bead Purification Kit as follows: The PBMC cell suspension prepared above was centrifuged at 500 G for 5 minutes to remove the supernatant; PBS buffer 1 ( pH=7.2, containing 0.5% BSA, 2mM EDTA) After resuspending 45 μΐ, add biotin-labeled anti-CD14, CD 16, CD 19, CD36, CD56, CD123, and Glycophorin A antibody 30μ1, mix and incubate at 4°C 20 minute. Add the above buffer lml 500G, wash the cells for 5 minutes, and remove the supernatant. Resuspend the cells, install the adsorption column, let the cell suspension pass through the adsorption column, and adsorb non-T cells to obtain purified T cells.

3. The T cell was plated in a 96-well plate at a volume of 200 μl according to 2 x ^{10 5} cells/?L. The highest concentration of the compound was 50 μM, and the mixture was diluted by a 5-fold gradient. After 72 hours of culture, the drug was tested for cytotoxicity against sputum using CCK-8 staining. Cell viability = drug group cell OD / no drug group cell number xOD. The survival rate was 95%. The drug concentration was considered to be non-toxic to T cells. IC50 is the concentration at which a drug inhibits half of T cell activity. See Table 1 for the results.

2. Inhibition of CD3, CD28 double-antibody stimulation in human T cell proliferation

Method:

1. Preparation of medium containing anti-human CD3, CD28 double antibody: 2 g/ml anti-human CD3 antibody was coated with PBS for 12 hours, washed twice with PBS, and then added with medium containing lg/ml anti-human CD28 antibody 100 μl .

2. Carboxyfluorescein acetoacetate succinimidyl ester (CFSE): CFSE was added to the purified T cells, wherein the final concentration of CFSE was 2.5 μΜ, and the concentration of sputum cells was lxlO ⁶ cells/ml. Incubate at 37 ° C for 10 minutes, centrifuge at 300 G for 5 minutes, discard the supernatant, wash the medium twice, and then resuspend with lg/ml anti-human CD28 antibody medium and place a 96-well plate. The compound was added after dilution with a gradient, cultured for 72 hours, and observed by an upflow cytometer, and the IC50 for inhibiting T cell proliferation was calculated. Experimental results: See Table 1 for results.

Table 1

Note: "ΝΑ" means no inhibitory activity or very low activity.

The abnormal proliferation of sputum cells has an important influence on the production of autoimmune diseases and malignant tumors. The experimental results show that the compound of the present invention can significantly inhibit human activated tau cells, but is not toxic to resting sputum cells. The compounds of the present invention have a certain preventive or therapeutic effect on autoimmune diseases and malignant tumors caused by abnormal proliferation of tau cells.

Test Example 3 The compound of the present invention inhibits proliferation of human lymphocyte tumor cells

METHODS: Jurkat cells were plated at 1500 cells/well in 96-well plates. In the culture system, the highest concentration of the compound was 50 μM, and the drug concentration was diluted according to a 5-fold gradient. After 48 hours, the compound was added to l(^l CCk-8, and after incubation for 6 hours, the absorbance at 450 nM was measured by a microplate reader.

The method for calculating the growth inhibition rate of the compound against the tumor cells was carried out according to the National Cancer Institute (NCI) standard method: when Ti (drug culture for 48 h, CCK-8 colorimetric absorption OD value) ≥ Tz (without drug group) CCK-8 color absorbing OD value at the start of culture), tumor cell survival rate = [ΓΠ-Τζ) / (^-Τζ)] χ100, where C is CCK-8 color absorbing OD after 48 hours without drug group Value; when Ti < Tz, tumor cell survival rate = [0^2) 2: ^ 100.

Experimental results: See Table 2 for the results.

Table 2

Compound name

Inhibition of Jurkat cell proliferation 50 (μΜ)

2-(1 Hydrogen-benzothiazolyl-2yl)-4,5,6,7-tetrahydro-2hydrogen-

1 2.1

Carbazole-3-ol

2-(1 Hydrogen-benzimidazolyl-2yl)-4,5,6,7-tetrahydro-2 Hydrogen-

2 2.3

Carbazole-3-ol

1-(1,3-benzothiazolyl-2yl)-1,5,6,7-tetrahydro-4H-indole

3 4

Azole-4-one

1-(1,3-benzothiazolyl-2yl)-3,4-dimethyl-l-hydrogen-pyridyl

4 23.3

Oxazole-5-ol Nitrogen-(6-methyl-1,3-benzothiazolyl-2yl)-4,5,6,7-tetra

Comparative example 1 NA

Hydrogen-1-benzothiophene-3-carboxamide

Nitrogen-1,3-benzothiazolyl-2,p-methyl-l-hydrogen-pyrazole-5-ratio 2 NA

Formamide

5-[(1,3-Benzothiazolyl-2methyl)amino]-1-ethyl-nitrogen - Comparative Example 3 NA

Methyl-4,5,6,7-tetrahydro-1hydrogen-carbazole-3-carboxamide

Note: "NA" means no inhibitory activity or very low activity. For the compound number, see Test Example 2. The experimental results show that the compound of the present invention has an obvious inhibitory effect on lymphoma cells, and the inhibitory effects of the compounds 1, 2, and 3 are superior.

Test Example 4 The compound of the present invention inhibits proliferation of human multiple osteosarcoma cells

METHODS: Multiple myeloma cells RPMI8226 cells were plated at 5,000 cells/well in 96-well plates. In the culture system, the highest concentration of the compound was 50 μΜ, and the drug concentration was diluted according to a 5-fold gradient. After 48 hours, the compound was added to ΙΟμΙ CCk-8, and after incubation for 6 hours, the absorbance at 450 nM was measured by a microplate reader. The method for calculating the tumor cell growth inhibition rate according to the drug is carried out according to the National Cancer Institute (NCI) standard method: when Ti (drug group culture for 48 h, CCK-8 colorimetric absorption OD value) ≥ Tz (without drug group) CCK-8 color absorbing OD value at the start of culture), tumor cell survival rate = [Cn-Tz) / (; C-Tz)] xl00, where C is CCK-8 colorimetric absorption after 48 hours without drug group OD value; when Ti < Tz, tumor cell survival rate = [0^2) 2: ^100.

Experimental results: See Table 3 for the results.

Compound Name Inhibits proliferation of multiple myeloma cells

ΙΟ50 (μΜ)

2-(1 Hydrogen-benzothiazolyl-2yl)-4,5,6,7-tetrahydro-2hydrogen-

1 1.1

Carbazole-3-ol

2-(1 Hydrogen-benzimidazolyl-2yl)-4,5,6,7-tetrahydro-2 Hydrogen-

2 1.5

Carbazole-3-ol

1-(1,3-benzothiazolyl-2yl)-1,5,6,7-tetrahydro-4H-indole

3 3.4

Azole-4-one

1-(1,3-benzothiazolyl-2yl)-3,4-dimethyl-l-hydrogen-pyridyl

4 6.1

Azole-5-ol

2-(2-benzothiazolyl)-2,4,5,6-tetrahydrocyclopentpyrazol-3-ol

5 1.2

1-(2-benzothiazolyl)-3-(trifluoromethyl)-Iff-pyrazole-5-

6 Alcohol 2.3

1-(2-benzothiazolyl)-4-(2-hydroxyethyl)-3-methyl

7 pyrazole-5-ol 4.6

2-(6-methoxy-2-benzothiazolyl)-4,5,6,7-tetrahydro-2ί -

8 carbazole-3-ol 5.7

9 2-(2-benzothiazolyl)-4,5,6,7-tetrahydro-2ί-carbazole-3-vinegar 6.1 Acid ester

2-(2-benzoxazolyl)-4,5,6,7-tetrahydrocarbazole -3-

10 alcohol 7.2

2-(4-phenyl-2-thiazolyl)-4,5,6,7-tetrahydro-22-carbazole-3-

11 Alcohol 7.6

1-(2-benzothiazolyl)-3-phenyl-Ιί-pyrazole-5-ol

12 8.1

For the compound number, see Test Example 2.

The experimental results show that the compound of the present invention has an obvious inhibitory effect on multiple myeloma cells.

Test Example 5 The compound of the present invention inhibits proliferation of human breast cancer tumor cells

METHODS: Human breast cancer cells MCF-7 cells were plated at 96 cells/well in 96-well plates. In the culture system, the highest concentration of the drug was 50 μΜ, and the drug concentration was diluted according to a 5-fold gradient. After 48 hours of drug administration, l (^l CCk-8 was added, and after incubation for 6 hours, the absorbance of 450 nM wavelength was measured by a microplate reader. The calculation method of drug growth inhibition rate of tumor cells was in accordance with the National Cancer Institute (NCI). Standard method: When Ti (drug culture for 48h, CCK-8 colorimetric absorption OD value) ≥ Tz (excluding CCK-8 colorimetric absorption OD value at the start of drug group culture), tumor cell survival rate = [Cn- Tz) / (; C-Tz)] xl00, where C is the CCK-8 colorimetric absorption OD value after 48 hours without the drug group; when Ti < Tz, the tumor cell survival rate = [0^2) 2: ^ 100.

Experimental results: See Table 4 for the results.

Table 4

Compound Name Inhibits proliferation of multiple myeloma cells

ΙΟ50 (μΜ)

2-(1 Hydrogen-benzothiazolyl-2yl)-4,5,6,7-tetrahydro-2hydrogen-

1 3.1

Carbazole-3-ol

2-(1 Hydrogen-benzimidazolyl-2yl)-4,5,6,7-tetrahydro-2 Hydrogen-

2 2.7

Carbazole-3-ol

1-(1,3-benzothiazolyl-2yl)-1,5,6,7-tetrahydro-4H-indole

3 6.5

Azole-4-one

1-(1,3-benzothiazolyl-2yl)-3,4-dimethyl-l-hydrogen-pyridyl

4 4.3

Azole-5-ol

2-(2-benzothiazolyl)-2,4,5,6-tetrahydrocyclopentpyrazol-3-ol

5 2.1

1-(2-benzothiazolyl)-3-(trifluoromethyl)-Iff-pyrazole-5-

6 alcohol 6.8

1-(2-benzothiazolyl)-4-(2-hydroxyethyl)-3-methyl

7 pyrazole 5-alcohol 4.3

8 2-(6-Methoxy-2-benzothiazolyl)-4,5,6,7-tetrahydro-2ί - 6.7 Carbazole-3-ol

2-(2-benzothiazolyl)-4,5,6,7-tetrahydro-2ί-carbazole-3-vinegar

9 acid ester 2.4

2-(2-benzoxazolyl)-4,5,6,7-tetrahydrocarbazole -3-

10 Alcohol 3.1

2-(4-phenyl-2-thiazolyl)-4,5,6,7-tetrahydro-22-carbazole-3-

11 Alcohol 5.4

1-(2-benzothiazolyl)-3-phenyl-Ιί-pyrazole-5-ol

12 4.3

For the compound number, see Test Example 2.

The experimental results show that the compound of the present invention has an obvious inhibitory effect on human breast cancer tumor cells. Test Example 6 The compound of the present invention inhibits proliferation of human liver cancer tumor cells

METHODS: Hep3B, a multi-human liver cancer tumor cell, was plated at 96 cells in 2500 cells/well. In the culture system, the highest concentration of the drug was 50 μΜ, and the drug concentration was diluted according to a 5-fold gradient. After 48 hours of drug administration, l (^l CCk-8 was added, and after incubation for 6 hours, the absorbance of 450 nM wavelength was measured by a microplate reader. The calculation method of drug growth inhibition rate of tumor cells was in accordance with the National Cancer Institute (NCI). Standard method: when Ti (drug culture for 48h, CCK-8 color absorption OD value) ≥ Tz (excluding CCK-8 color absorption OD value at the beginning of drug group culture), swollen

100, wherein C is the CK value of CCK-8 color absorption after 48 hours without the drug group; when Ti<Tz, the tumor cell survival rate is [0^2) 2:^ 100.

Experimental results: See Table 5 for the results.

Compound Name Inhibits proliferation of multiple myeloma cells

ΙΟ50 (μΜ)

2-(1 Hydrogen-benzothiazolyl-2yl)-4,5,6,7-tetrahydro-2hydrogen-

1 2.7

Carbazole-3-ol

2-(1 Hydrogen-benzimidazolyl-2yl)-4,5,6,7-tetrahydro-2 Hydrogen-

2 2.1

Carbazole-3-ol

1-(1,3-benzothiazolyl-2yl)-1,5,6,7-tetrahydro-4H-indole

3 3.6

Azole-4-one

1-(1,3-benzothiazolyl-2yl)-3,4-dimethyl-l-hydrogen-pyridyl

4 3.7

Azole-5-ol

2-(2-benzothiazolyl)-2,4,5,6-tetrahydrocyclopentpyrazol-3-ol

5 2.5

1-(2-benzothiazolyl)-3-(trifluoromethyl)-Iff-pyrazole-5-

6 alcohol 6.3 L-(2-benzothiazolyl)-4-(2-hydroxyethyl)-3-methyl-lff-

7 pyrazole-5-ol 1.4

2-(6-methoxy-2-benzothiazolyl)-4,5,6,7-tetrahydro-2ί -

8 carbazole-3-ol 5.1

2-(2-benzothiazolyl)-4,5,6,7-tetrahydro-2ί-carbazole-3-vinegar

9 acid ester 6.2

2-(2-benzoxazolyl)-4,5,6,7-tetrahydrocarbazole -3-

10 alcohol 7.8

2-(4-phenyl-2-thiazolyl)-4,5,6,7-tetrahydro-22-carbazole-3-

11 Alcohol 3.1

1-(2-benzothiazolyl)-3-phenyl-Ιί-pyrazole-5-ol

12 5.4

For the compound number, see Test Example 2.

The experimental results show that the compound of the present invention has an obvious inhibitory effect on human liver cancer tumor cells.

Test Example 7 Investigation of the inhibition of transplant rejection by the compound of the present invention

To establish a skin graft model for mice of different strains: 8 to 12 weeks of C57 BL/6 and Bal b/c female mice were selected as experimental subjects, housed in an SPF barrier environment, irradiated with radiation, litter and drinking water. Autoclaved. Balb/c mice were used as recipients and C57/BL/6 mice were used as donors. After anesthesia, the donor was placed in a prone position. After disinfection, about 1 cm×1 cm of the back skin was taken, and the subcutaneous fat and blood vessels were scraped off and placed in sterile physiological saline for use. After being anesthetized, the recipient was placed in a prone position. After the back was disinfected, the skin slightly larger than the donor skin was removed and used as a transplant bed. The donor skin is placed on the transplant bed, aligned and secured with a band-aid.

The experiment was divided into 6 groups, 6 mice in each group: model group (normal saline), positive control group (cyclosporin CsA, 5 mg/kg/day), and compounds of the present invention 1, 2, 6, 11 (50 mg/kg/day) (See Figure 2 for the compound number). The administration was started one week before the transplantation, and the administration was continued for one week after the transplantation, and all were administered by intraperitoneal injection.

Transplant rejection test protocol: Remove the band-aid after 7 days of skin grafting. Observe the color, hardness, shedding and hair growth of the transplanted skin every day. If the skin is hard or the scar is detached, the graft is repelled. Or the rat hair grows, then the graft survives. The time from the transplantation to the occurrence of rejection was recorded, and the average survival time MST of the skin was calculated. See Figure 2 for the results.

The results showed that the compounds of the present invention 1, 2, 6, 11 can significantly prolong the survival time of the graft, and the effect is comparable to that of the positive control cyclosporin A. The compound of the present invention can effectively inhibit the transplant rejection; wherein, the compound 1 The effect is superior to other compounds.

Test Example 8: The inventive compound inhibits delayed type hypersensitivity (DTH). Establishment of a DTH model induced by dinitrofluorobenzene (DNFB): BALB/c mice were uniformly coated with 20μ1 0.5% (v/v) DNFB solution (dissolved in 4: 1 acetone-olive oil) to the left and right feet of the mice. Acetone/olive oil solvent was applied as a control alone, once a day for 2 consecutive days. The challenge test was carried out 9 days after the second sensitization: 10 μl of a 0.5% (v/v) DNFB solution was uniformly applied to both the inner and outer sides of the right ear of the mouse. After 72 h, use an 8 mm ear swollen ear device, pierce the ear with both ears, weigh and calculate the added weight of the right ear. Animal grouping: 35 BALB/c mice were randomly divided into groups A, B, C, D, E, and F, with 5 mice in each group. Group A was a positive group sensitized with DNFB, and group B was a positive control drug CsA. The groups C, D, E, and F are the treatment groups of the compound 1, 2, 6, 11 of the present invention (see the test example 2 for the compound number). From the first two days before the first DNFB sensitization until the ear piece was weighed, the compound of the present invention was intraperitoneally administered at a dose of 50 mg/kg/day, and the positive control drug CsA was intraperitoneally injected at a dose of 5 mg/kg/day. See Figure 3 for the results.

The results showed that the compounds of the present invention 1, 2, 6, and 11 were all effective in inhibiting delayed type hypersensitivity.

Test Example 9: The compound of the present invention inhibits rheumatoid arthritis

6-10 weeks of DBA/1J mice were selected and 50 ug of bovine type II collagen was completely emulsified with an equal volume of complete Freund's adjuvant (CFA) and injected subcutaneously. After 21 days, the vaccine was fully emulsified with 50 ug of the same antigen and incomplete Freund's adjuvant (IFA), and the immunization was boosted once. Observe the record from the 45th day. Using the 1-4 scoring method: 1 point, normal; 2 points, 1 joint swelling; 3 points, more than 1 joint swelling, but did not accumulate all joints; 4 points, the entire paw was severely swollen or stiff. The scores of each paw are added to obtain a total score of joint inflammation in mice. Mice with a total joint score greater than 1 were successfully established for the model.

Experimental group: 6 male Balb/C mice, 30 male DBA/1J mice with mouse rheumatoid arthritis model, 6 rats in each group, weighing 30±4.7g/only, grouped as follows: A Normal mouse control group; B. CIA: saline treatment group (negative control group:), cyclosporin CsA, 15 mg/kg/day), compound of the invention 1: 2-(1 hydrogen-benzothiazolyl group -2,4-,4,5,6,7-tetrahydro-2hydro-indazol-3-ol, compound 2 of the invention: 2-(1H-benzimidazolyl-2-yl)-4,5, 6,7-tetrahydro-2hydro-indole-trigestol-3-ol, compound 6 of the invention: 1-(2-benzothiazolyl: )-3-(trifluoromethyl)-m-pyrazole-5 - alcohol, compound 11 of the present invention: 2-(4-phenyl-2-thiazolyl)-4,5,6,7-tetrahydro-2H-indazol-3-ol administration group (50 mg/kg/day) ). On the 45th day, on the first day of treatment, the inside of the hind limbs of the mice was selected for intramuscular injection. Two weeks after the administration, the joint inflammation of each group was scored. See Table 6 for the results.

Table 6

Grouping

Control 1

0.9% NaCl (negative group) 4

Cyclosporin A (positive control) 2

Compound of the present invention 1 administration group 2

Compound 2 of the present invention is administered to group 2 Compound 6 of the present invention is administered to group 3

Compound of the present invention 11 administration group 1

For the compound number, see Test Example 2.

The experimental results show that the compounds 1, 2, 6, and 11 of the present invention can effectively treat rheumatoid arthritis, and the efficacy thereof is equivalent to the positive control cyclosporin A; among them, the compound 11 has the strongest pharmacological effect, even better than Positive drug.

In summary, the compound of the present invention can effectively inhibit protein kinase activity, can effectively inhibit abnormally activated T cell proliferation, and can be used for treating diseases such as autoimmune diseases and malignant tumors, especially for lymphoma, multiple myeloma, and mammary gland. Cancer, liver cancer, transplant rejection, allergy and rheumatoid arthritis have significant therapeutic effects and have good industrial application prospects.

Claims

Claim

1. The use of a compound of formula I,

0

_H, fluorenyl, =0, — 0—C—R? or — ₀ R ₇ , wherein R ₇ is a fluorenyl group of C1-C5;

Is 0 or S;

For -H or thiol, . An aromatic group of C6-10;

The use according to claim 1, wherein the protein kinase inhibitor is a serine/threonine protein kinase inhibitor or a tyrosine kinase inhibitor.

3. Use according to claim 1 or 2, characterized in that the protein kinase inhibitor is for the treatment of malignant tumors, autoimmune diseases, cardiovascular diseases, diabetes, infectious diseases, arthritis, immune disorders or Drugs for Alzheimer's disease.

4. The use according to claim 3, wherein: the malignant tumor is multiple myeloma, lymphocytic leukemia, granulocyte leukemia, lymphoma, liver cancer, lung cancer, pancreatic cancer, colon cancer or breast cancer;

The autoimmune disease is rheumatoid arthritis, myeloproliferative disease, transplant rejection, asthma, lupus erythematosus, psoriasis, allergy or contact dermatitis.

5. Use according to claim 1 wherein the compound is a compound of formula II, 2-(4-phenyl-2-thiazolyl)-4,5,6,7-tetrahydro-2H- Oxazol-3-ol, or 2-(6-methoxy-2-benzothiazolyl:)-4,5,6,7-tetrahydro-2H-indazol-3-ol; wherein, formula II The structural formula of the compound is as follows

Formula II,

o

II

It is 0 or 5.

6. Use according to claim 1 or 5, characterized in that:

0

II

-H, — 0— ^c — ^R 7 or —OR ₇ , wherein R ₇ is a C1-C3 fluorenyl group;

It is 0 or 5.

7. Use according to claim 6, characterized in that:

0

II

! ^ is -OH, - ⁰ - ^C _ ^R ? or -0R ₇ , and R ₇ is a methyl group;

Is -H, or -R ₈ 0H, the ethyl group;

a methyl group substituted with methyl, phenyl, or trifluoro;

It is 0 or 5.

8. Use according to claim 7, characterized in that the compound is:

2-(1H-benzothiazolyl-2yl)-4,5,6,7-tetrahydro-2hydrogen-indole frustum-3-ol,

2-(1H-benzimidazolyl-2-yl)-4,5,6,7-tetrahydro-2hydrogen-indole frustum-3-ol,

1-(1,3-benzothiazolyl-2yl)-1,5,6,7-tetrahydro-4H-indazole-4-one,

1-(1,3-Benzothiazolyl-2-yl)-3,4-dimethyl-1hydro-pyrazol-5-ol, 2-(2-benzothiazolyl)-2,4,5,6-tetrahydrocyclopentadiazole-3-ol,

1-(2-benzothiazolyl)-3-(trifluoromethyl)pyrazole-5-ol,

1-(2-benzothiazolyl)-4-(2-hydroxyethyl)-3-methylpyrazole-5-ol,

2-(2-benzothiazolyl)-4,5,6,7-tetrahydro-2H-indazole-3-acetate,

2-(2-benzoxazolyl)-4,5,6,7-tetrahydro-2H-indazole-3-ol, or

1-(2-Benzothiazolyl)-3-phenylpyrazole-5-ol.

9. A compound of formula I having the structural formula:

Is 0 or S;

For -H or thiol, . An aromatic group of C6-10;

10. A compound according to claim 9 wherein: the compound is a compound of formula II, 2-(4-phenyl-2-thiazolyl)-4,5,6,7-tetrahydro-2H- Oxazol-3-ol, or 2-(6-methoxy-2-benzothiazolyl)-4,5,6,7-tetrahydro-2H-indazol-3-ol; wherein, a compound of formula II The structure is as follows

Formula II, _H, fluorenyl, =0, —O—C—R? or — ₀ R ₇ , wherein R ₇ is a C1-C5 fluorenyl group;

Is 0 or S;

When R ₂ , together with the carbon atom to which they are attached, forms a cycloalkyl group of C6, R _{4 is} not ^

11. A compound according to claim wherein: the compound is:

2-(1H-benzimidazolyl-2-yl)-4,5,6,7-tetrahydro-2hydrogen-indole frustum-3-ol,

1-(1,3-benzothiazolyl-2yl)-1,5,6,7-tetrahydro-4H-indazole-4-one,

1-(1,3-benzothiazolyl-2yl)-3,4-dimethyl-1hydro-pyrazole-5-ol,

2-(2-benzothiazolyl)-2,4,5,6-tetrahydrocyclopentadiazole-3-ol,

1-(2-benzothiazolyl)-3-(trifluoromethyl)pyrazole-5-ol,

1-(2-benzothiazolyl)-4-(2-hydroxyethyl)-3-methylpyrazole-5-ol,

2-(2-benzothiazolyl)-4,5,6,7-tetrahydro-2H-indazole-3-acetate,

2-(2-benzoxazolyl)-4,5,6,7-tetrahydro-2H-indazole-3-ol, or

1-(2-Benzothiazolyl)-3-phenylpyrazole-5-ol.

A method for synthesizing 2-(1H-benzothiazolyl-2yl)-4,5,6,7-tetrahydro-2hydro-indole frustum-3-alcohol according to claim 11, which is characterized in that : It includes the following steps:

The compound of the formula III, 2-mercaptobenzothiazole, is subjected to a condensation reaction to obtain a target compound; wherein the structural formula of the compound of the formula III is as follows:

Among them, (^ is the base of ^-^.

The synthesis method according to claim 12, wherein the compound of the formula III is ethyl 2-cyclohexanonecarboxylate, methyl 2-cyclohexanonecarboxylate or propyl 2-cyclohexanonecarboxylate. One or a mixture of two or more; the molar ratio of the compound of the formula III to 2-mercaptobenzothiazole is (0.5-1.5): 1; In the reaction, an organic acid or a mineral acid is used as a catalyst, and the molar ratio of the catalyst to the compound of the formula III is (0.01-0.1): 1; the solvent is selected from toluene, ethanol and methanol; and the reaction temperature is 20-200 °C.

The synthesis method according to claim 13, wherein the molar ratio of the compound of the formula III to 2-cyclohexanone ethyl ester, the compound of the formula III and the 2-mercaptobenzothiazole is (0.7-1). ): 1.

15. The method of synthesis according to claim 13, wherein the catalyst is selected from the group consisting of acetic acid, p-toluenesulfonic acid, formic acid, or a non-oxidizing inorganic acid.

The synthesis method according to claim 15, wherein the catalyst is selected from the group consisting of acetic acid, and the molar ratio of acetic acid to the compound of the formula III is (0.012-0.014): 1; or

The synthesis method according to claim 13, wherein the solvent is toluene.

The synthesis method according to claim 13, wherein the reaction temperature is

120-125 ° C.

The synthesis method according to claim 12, wherein after the condensation reaction, it is recrystallized from ethanol.