阅读说明：本技术 一种基于激活jwa基因和降解her2的抗肿瘤化合物、其制备方法及其用途 (Anti-tumor compound based on JWA gene activation and HER2 degradation, preparation method and application thereof ) 是由 周建伟 任彦霖 陈冬寅 黄叶飞 李爱萍 于 2019-08-12 设计创作，主要内容包括：本发明涉及一种基于激活JWA基因和降解HER2的抗肿瘤化合物、其制备方法及其用途。该抗肿瘤化合物为式I化合物。该制备方法为采用两化合物经一步反应合成式I化合物。该用途为用于制备JWA基因激活剂或抗肿瘤药物。本发明的抗肿瘤化合物及其药用盐能够有效激活JWA蛋白的表达,并进一步通过级联激活E3泛素化酶等特异性降解过表达的HER2蛋白从而抑制肿瘤细胞的增殖和体内生长。(The invention relates to an anti-tumor compound based on JWA gene activation and HER2 degradation, a preparation method and application thereof. The antitumor compound is a compound shown in a formula I. The preparation method adopts two compounds to synthesize the compound of the formula I through one-step reaction. The application is used for preparing JWA gene activator or antitumor drug. The anti-tumor compound and the medicinal salt thereof can effectively activate the expression of JWA protein, and further specifically degrade over-expressed HER2 protein by cascade activation of E3 ubiquitinase and the like, thereby inhibiting the proliferation and in-vivo growth of tumor cells.)

1. An anti-tumor compound based on activation of the JWA gene and degradation of HER2, which is a compound of formula I:

wherein R is¹Selected from OH and CH₃、CH₂CH₃、OCH₃、OCH₂CH₃；

R²Selected from H, OH, CH₃、CH₂CH₃、CH₂CH₂CH₃、OCH₃、OCH₂CH₂CH₃；

R³Selected from F, Cl, Br, I, CF₃；

R⁴Selected from H, F, Cl, Br, I, CF₃；

R⁵Selected from H, CH₃、CH₂CH₃。

2. The anti-neoplastic compound of claim 1, wherein R¹Is OCH₃；R²Is H or OCH₃；R³Is F or CF₃；R⁴Is H or F; r⁵Is H.

3. The anti-neoplastic compound of claim 1, wherein the compound of formula I is one of the following:

4-fluoro-N- (4-methoxybenzo [ d ] thiazol-2-yl) benzo [ d ] thiazol-2-amine; 4, 7-difluoro-N- (4-methoxybenzo [ d ] thiazol-2-yl) benzo [ d ] thiazol-2-amine; 4, 6-difluoro-N- (4-methoxybenzo [ d ] thiazol-2-yl) benzo [ d ] thiazol-2-amine; 4-methoxy-N- (4- (trifluoromethyl) benzo [ d ] thiazol-2-yl) benzo [ d ] thiazol-2-amine; n- (4-fluorobenzo [ d ] thiazol-2-yl) -4, 7-dimethoxybenzo [ d ] thiazol-2-amine; n- (4-fluorobenzo [ d ] thiazol-2-yl) -4, 6-dimethoxybenzo [ d ] thiazol-2-amine; 4, 7-dimethoxy-N- (4- (trifluoromethyl) benzo [ d ] thiazol-2-yl) benzo [ d ] thiazol-2-amine; 4, 6-dimethoxy-N- (4- (trifluoromethyl) benzo [ d ] thiazol-2-yl) benzo [ d ] thiazol-2-amine;

the structural formulas are respectively as follows:

4. a process for the preparation of an anti-tumor compound according to any one of claims 1 to 3, characterized in that it comprises the following steps:

wherein X is one of Cl, Br and I; the copper salt is cupric oxide, cuprous oxide, cupric chloride, cuprous chloride, cupric bromide, cuprous bromide or cuprous iodide; the alkali is sodium hydroxide, potassium carbonate, sodium carbonate, potassium tert-butoxide or sodium hydride; the solvent is tetrahydrofuran, dimethyl sulfoxide or N, N-dimethylformamide; the heating temperature is 80-120 ℃.

5. The process according to claim 4, wherein X is Cl; the copper salt is cuprous iodide; the alkali is potassium carbonate; the solvent is dimethyl sulfoxide; the heating temperature was 100 ℃.

6. A pharmaceutically acceptable salt of the anti-neoplastic compound of any one of claims 1 to 3.

7. The pharmaceutical salt of claim 6, wherein the pharmaceutical salt is a salt of the compound of formula I with hydrochloric acid, sulfuric acid, benzenesulfonic acid, p-toluenesulfonic acid, phosphoric acid, hydrobromic acid, maleic acid, fumaric acid, or malic acid.

8. A pharmaceutical composition comprising an anti-neoplastic compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt according to claim 6 or 7.

9. Use of the anti-tumor compound of any one of claims 1 to 3, or the pharmaceutically acceptable salt of claim 6 or 7, or the pharmaceutical composition of claim 8 for the preparation of a JWA gene activator or an anti-tumor drug.

10. The use of claim 9, wherein the tumor is related tumor with low JWA gene expression and over-expression of HER2, and the tumor comprises breast cancer, gastric cancer, lung cancer, brain glioma; the anti-tumor drug is a drug which realizes the anti-tumor effect by degrading HER2 protein after activating the expression of JWA protein.

Technical Field

The invention relates to an anti-tumor compound, which plays an anti-tumor role based on the activation of JWA gene and the degradation of HER2, and also relates to a preparation method and application of the compound, belonging to the field of anti-tumor drugs.

Background

Malignant tumors (cancers) are a major public health problem that seriously threatens the health of the Chinese population. Statistical data published by the national cancer center in 2019 show that the death of malignant tumors accounts for 23.91% of all the causes of death of residents, the morbidity and the mortality of the malignant tumors are in a continuously rising state in recent decades, the medical cost caused by the malignant tumors exceeds 2200 hundred million every year, and the prevention and control situation is severe. The cancer burden is in a continuously rising state compared to historical data. Over the last 10 years, the incidence of malignancy has remained on the order of 3.9% and mortality has remained 2.5% per year. Lung cancer, liver cancer, upper digestive system tumor, colorectal cancer, female breast cancer and the like are still main malignant tumors in China. Lung cancer is the first disease in men and breast cancer is the first disease in women. From the age distribution, the incidence of malignant tumor increases with the increase of age, the incidence of malignant tumor in the young people under 40 years old is at a lower level, the incidence rapidly increases from 40 years old, the incidence distribution is mainly concentrated above 60 years old, and the peak is reached by the age group of 80 years old; the age distribution of different malignant tumors is different. The survival rate of malignant tumors is gradually increased in the last 10 years, the relative survival rate of the malignant tumors in China in 5 years is about 40.5 percent at present, and the relative survival rate is improved by about 10 percent compared with the relative survival rate before 10 years, but the survival rate is far away from the developed countries. The malignant tumors are different in sex, the first malignant tumor of a male is lung cancer, about 52.0 ten thousand new cases occur every year, other high-incidence malignant tumors are gastric cancer, liver cancer, colorectal cancer, esophageal cancer and the like in sequence, and the incidence of the first 10 malignant tumors accounts for 82.20% of the incidence of all the malignant tumors of the male. The first disease of the female is breast cancer, the disease is about 30.4 ten thousand every year, other main high-incidence malignant tumors are lung cancer, colorectal cancer, thyroid cancer, gastric cancer and the like in turn, and the disease of the first 10 malignant tumors of the female accounts for 79.10% of the total malignant tumors of the female.

Since the identification of human epidermal growth factor receptor 2(ERBB2 or HER2) in 1985, the association of the human ERBB2(HER2) oncogene in oncology, particularly in Breast Cancer (BC), has been of widespread interest. The gene encodes 185-kD transmembrane human epidermal growth factor receptor 2(HER2), which belongs to members of receptor families such as HER1 (or EGFR), HER3 and HER 4. Activation of their tyrosine kinase domains is usually achieved by homodimerization and heterodimerization induced by a specific ligand. Once activated, cell signaling through the family receptors results in the proliferation and survival of cells. The activation mechanism of HER2 differs from other molecules in that it lacks a specific growth factor ligand, but is activated due to its fixed conformation, similar to the conditions under which the ligand activates. This property makes HER2 the first heterodimeric partner of the other receptors of this family.

HER2 positive breast cancer (HER2)⁺BC) accounts for 15-20% of all BCs. Over the last 20 years, the invention and use of monoclonal antibodies (MoAbs), Tyrosine Kinase Inhibitors (TKIs) and antibody-drug conjugates (adc) against HER2 significantly improved the prognosis of patients. However, only about 50% of HER2 positive patients respond to targeted therapy, for HER2⁺Metastatic BC (mBC) was almost ineffective. The reason for most treatment failures is primary or acquired resistance to anti-HER 2 treatment. In recent years, various resistance mechanisms have been discovered, such as impaired drug binding to HER2, constitutive activation of signaling pathways parallel or downstream to HER2, metabolic reprogramming, or reduced immune system activity, among others. Therefore, for HER2 positive patients, the search for therapeutic approaches to degrade the over-expressed HER2 protein is currently in urgent need.

The inventor of the present invention has focused on exploring the structure and function of the JWA (also known as ARL6IP5) gene discovered and named by himself, particularly the relationship between the gene and human serious diseases, for more than 20 years, and has achieved a series of research results. The JWA gene was subsequently found to be a key molecule involved in the regulation of cell differentiation (Chinese Sci Bull, 2001; Chinese Sci Bull, 2002; biochem Biophys Res Commu, 2006); JWA proteins are cytoskeletal binding proteins that have the function of regulating cytoskeletal assembly and Cell migration (Chinese Sci Bull, 2003; Chinese Sci Bull.2004; Cell Signal.2007); JWA is an environmental response gene and DNA repair protein for normal cell activity in body tissues (J Biomed Sci, 2005; FreeRadic Biol Med, 2007; Nucleic Acids Res, 2009); the JWA gene expression level in various cancer tissues such as gastric cancer, melanoma, esophageal cancer, liver cancer and the like is obviously lower than that in paracancer normal tissues; cancer tissue JWA expression levels are positively correlated with patient survival (Oncogene, 2010; Clin Cancer Res, 2012; J Gastroenterol, 2013; Carcinogenesis, 2014); in the research on cisplatin resistance, the fact that the expression level of JWA of cisplatin-resistant gastric cancer cells is improved can be found that the sensitivity of the cancer cells to the cisplatin is restored by inhibiting the activity of CK2 so as to inhibit the phosphorylation level of DNA repair protein XRCC1 and the tolerance of the gastric cancer cells to the cisplatin (Cell Death Dis, 2014). It has also been discovered recently that JWA inhibits HER2 expression levels in gastric cancer cells through a transcriptional and post-translational ubiquitination modification mechanism, which is associated with JWA inhibiting the malignant phenotype of gastric cancer cells (Oncotarget,2016a,2016 b). In summary, the JWA gene can be produced by a variety of different molecular machines to exert the effect of a cancer suppressor gene at multiple targets.

Meanwhile, based on this series of research results, the present inventors have applied for a plurality of inventions including: patent No. CN98111422.9, publication No. CN1065876C, patent invention entitled "protein-specific antibody encoding cytoskeleton-like gene"; patent number CN201310178099.X, publication number CN103239710B, patent invention named "polypeptide with antitumor activity and its use"; patent No. CN201610164491.2, publication No. CN105820230B, patent invention named "an anti-tumor active polypeptide and its use"; patent No. CN201610857409.4, publication No. CN106632299B, patent invention named "antitumor compound, its preparation method and its use".

Disclosure of Invention

The main purposes of the invention are: based on the prior art and according to the latest research results of the inventors, an anti-tumor compound based on the activation of a JWA gene and the degradation of HER2 is provided, which can activate the expression of the JWA gene and then specifically degrade HER2 through a cascade activation of an E3 ubiquitinase and other mechanisms, thereby realizing the inhibition or treatment effect on HER2 over-expression malignant tumors. Also, a preparation method and use of the compound are provided.

The main technical concept of the invention is as follows: the JWA shown by the inventor in the previous series of research results has the outstanding biological function of the cancer suppressor gene and is verified in experimental intervention models for the occurrence and development of various human malignant tumors. As the expression level of JWA protein in most malignant tumor tissue cells is obviously lower than that of paracancer normal tissues, JWA protein has the function of inhibiting HER2 overexpression. Therefore, the inventor designs and constructs a reporter gene cell model containing a JWA gene promoter regulatory region, and carries out high-throughput screening to find a small molecule compound capable of activating JWA gene expression; if the expected small molecule compound is obtained, the potential anti-tumor biological effect of the compound is verified in cancer cells with high expression of HER 2.

The inventor takes the technical idea as the main technical idea, carries out high-throughput screening by cooperating with organizations such as national compound libraries and the like, further carries out targeted compound molecular structure modification on the basis of some lead compounds obtained after screening, and synthesizes a series of bis-benzothiazole amine compounds; and then, the JWA gene expression of cells and tissues and organs in a mouse body can be effectively activated by the series of compounds through the research verification of various cell models and animal models.

The anti-tumor compound provided by the invention comprises the following components:

an anti-tumor compound based on activation of the JWA gene and degradation of HER2, which is a compound of formula I:

wherein R is¹Selected from OH and CH₃、CH₂CH₃、OCH₃、OCH₂CH₃；

R²Selected from H, OH, CH₃、CH₂CH₃、CH₂CH₂CH₃、OCH₃、OCH₂CH₂CH₃；

R³Selected from F, Cl, Br, I, CF₃；

R⁴Selected from H, F, Cl, Br, I, CF₃；

R⁵Selected from H, CH₃、CH₂CH₃。

The compounds are bis-benzothiazolamines.

Preferably, R¹Is OCH₃；R²Is H or OCH₃；R³Is F or CF₃；R⁴Is H or F; r⁵Is H.

Preferably, the compound of formula I is one of the following compounds:

4-fluoro-N- (4-methoxybenzo [ d ] thiazol-2-yl) benzo [ d ] thiazol-2-amine; 4, 7-difluoro-N- (4-methoxybenzo [ d ] thiazol-2-yl) benzo [ d ] thiazol-2-amine; 4, 6-difluoro-N- (4-methoxybenzo [ d ] thiazol-2-yl) benzo [ d ] thiazol-2-amine; 4-methoxy-N- (4- (trifluoromethyl) benzo [ d ] thiazol-2-yl) benzo [ d ] thiazol-2-amine; n- (4-fluorobenzo [ d ] thiazol-2-yl) -4, 7-dimethoxybenzo [ d ] thiazol-2-amine; n- (4-fluorobenzo [ d ] thiazol-2-yl) -4, 6-dimethoxybenzo [ d ] thiazol-2-amine; 4, 7-dimethoxy-N- (4- (trifluoromethyl) benzo [ d ] thiazol-2-yl) benzo [ d ] thiazol-2-amine; 4, 6-dimethoxy-N- (4- (trifluoromethyl) benzo [ d ] thiazol-2-yl) benzo [ d ] thiazol-2-amine;

the structural formulas are respectively as follows:

experiments prove that the anti-tumor compound can effectively activate JWA gene expression of cells and tissues and organs in a mouse body. Particularly, the research of the subject group of the inventors reveals that the anti-tumor compound specifically degrades HER2 protein overexpressed in tumor cells through a mechanism of cascade activation of E3 ubiquitinase and the like, and has the functions of inhibiting the proliferation, the growth and the like of the tumor cells. Meanwhile, the remarkable tumor inhibition effect of the representative compounds in the anti-tumor compounds is also verified in a human breast cancer cell subcutaneous tumor-bearing nude mouse model which over-expresses HER 2; particularly, the representative compound also shows a certain protective effect on the functions of main organs such as heart, liver and kidney of mice while exerting a remarkable tumor inhibition effect (details are shown in the following specific examples).

The present invention also provides:

the method for preparing the antitumor compound is characterized by comprising the following steps:

wherein X is one of Cl, Br and I; the copper salt is cupric oxide, cuprous oxide, cupric chloride, cuprous chloride, cupric bromide, cuprous bromide or cuprous iodide; the alkali is sodium hydroxide, potassium carbonate, sodium carbonate, potassium tert-butoxide or sodium hydride; the solvent is tetrahydrofuran, dimethyl sulfoxide or N, N-dimethylformamide; the heating temperature is 80-120 ℃. (Note: R)¹、R²、R³、R⁴And R⁵The definitions are as described above and will not be described in detail here)

Preferably, X is Cl; the copper salt is cuprous iodide; the alkali is potassium carbonate; the solvent is dimethyl sulfoxide; the heating temperature was 100 ℃.

The present invention also provides:

pharmaceutically acceptable salts of the anti-neoplastic compounds described supra.

Preferably, the pharmaceutically acceptable salt is a salt of the compound of formula I with hydrochloric acid, sulfuric acid, benzenesulfonic acid, p-toluenesulfonic acid, phosphoric acid, hydrobromic acid, maleic acid, fumaric acid, or malic acid.

The present invention also provides:

a pharmaceutical composition comprising an anti-neoplastic compound as described above, or a pharmaceutically acceptable salt thereof as described above.

The present invention also provides:

use of the anti-tumor compound or the pharmaceutically acceptable salt or the pharmaceutical composition for preparing a JWA gene activator or an anti-tumor drug.

Preferably, the tumor is related tumor with low JWA gene expression and over-expression of HER2, and the tumor comprises breast cancer, gastric cancer, lung cancer, brain glioma and the like; the anti-tumor drug is a drug which realizes the anti-tumor effect by degrading HER2 protein after activating the expression of JWA protein.

In addition, as for the specific dosage forms of the above anti-tumor compound, the pharmaceutically acceptable salt and the pharmaceutical composition, the specific dosage forms can be selected according to the actual needs according to the prior art, such as tablets, powders, pills, capsules, suppositories, granules, suspensions, oral liquids, injections and other pharmaceutical dosage forms; among them, tablets and capsules for oral administration contain conventional excipients such as: fillers, lubricants, dispersants, diluents, and binders.

Compared with the prior art, the anti-tumor compound and the medicinal salt thereof can effectively activate the expression of JWA protein, and further specifically degrade over-expressed HER2 protein by cascade activation of E3 ubiquitinase and the like, thereby inhibiting the proliferation and in-vivo growth of tumor cells.

The anti-tumor compound and the medicinal salt thereof have the outstanding advantage of degrading HER2 protein overexpressed in cancer cells, which is completely different from various anti-HER 2 antibodies or inhibitors for inhibiting HER2 protein kinase activity which are universally used internationally so far. The molecular cell biology theory shows that the anti-tumor compound and the medicinal salt thereof are obviously superior to the prior anti-cancer treatment means aiming at HER2 over-expression cancer, because the anti-tumor compound has the function of finally degrading HER2 protein, thus fundamentally eliminating the material basis of the drug resistance phenomenon caused by HER protein adaptive transformation after the anti-HER 2 antibody or inhibitor is used at present. The anti-tumor compound and the medicinal salt thereof have obvious activity of treating tumors under lower dosage, have no toxicity to normal tissue cells, have certain protection effect on important organs such as heart, liver, kidney and the like, and have wide application prospect.

Drawings

FIG. 1 is a sequence diagram of a JWA gene cDNA, amino acids encoding a protein, and a promoter for constructing a reporter gene in example 11 of the present invention.

FIG. 2 is a structural diagram of a reporter gene including a JWA gene promoter in example 11 of the present invention.

FIG. 3 is a graph showing the effect of JAC1 on the formation of two human breast cancer cell clones in example 12 of the present invention.

FIG. 4 is a graph showing the results of the effect of JAC1 on HER2 expression and cell localization in two human breast cancer cells in example 13 of the present invention.

FIG. 5 is a graph showing the results of JAC1 activating JWA expression in two human breast cancer cells while inhibiting HER2 expression in example 14 of the present invention.

FIG. 6 is a graph showing the results of JAC1 inhibiting migration of BT474 and SKBR3 human breast cancer cells in example 15 of the present invention.

FIG. 7 is a graph showing the weight gain of mice bearing subcutaneous tumors of JAC1 treated human breast cancer BT474 cells in example 16.

FIG. 8 is a graph of the subcutaneous tumor volume of JAC 1-treated human breast cancer BT474 cells in example 16 of the present invention.

FIG. 9 is a graph showing the results of JAC1 treatment of subcutaneous tumor-bearing weight in mice with human breast cancer BT474 cells in example 16.

FIG. 10 is a graph showing the results of the tumor weight/body weight ratio of the subcutaneous tumor-bearing mice treated with JAC1 in example 16.

FIG. 11 is a graph of the results of the subcutaneous tumor-bearing tissue of human breast cancer BT474 isolated from each treatment group in example 16 of the present invention.

FIG. 12 is a graph showing the results of the subcutaneous tumor-bearing rate of each group of mice with human breast cancer BT474 cells in example 16.

FIG. 13 is the related molecular expression pattern of subcutaneous tumor-bearing tissue of each group of human breast cancer BT474 mice in example 16 of the present invention.

FIG. 14 is a photograph of the HE staining of the major organ of a mouse with subcutaneous tumor of JAC1 treated human breast cancer cells in example 17 of the present invention.

FIG. 15 is a graph showing the biochemical results of blood treatment of human breast cancer cell subcutaneous tumor-bearing mice by JAC1 in example 18 of the present invention.

FIG. 16 is a graph showing the results of verifying the off-target effect that JAC1 activates JWA expression and inhibits HER2 expression in example 19 of the present invention.

Detailed Description

The invention is described in further detail below with reference to embodiments and with reference to the drawings. The invention is not limited to the examples given.

Example 1

Preparation of 4-fluoro-N- (4-methoxybenzo [ d ] thiazol-2-yl) benzo [ d ] thiazol-2-amine:

2-chloro-4-fluorobenzo [ d ] is added into a 50mL dry eggplant-shaped bottle with a spherical reflux condenser tube and magnetons]Thiazole (1g,5.3mmol), 4-methoxybenzo [ d]Thiazol-2-amine (0.96g,5.3mmol), cuprous iodide (0.31g,0.3mmol), potassium carbonate (1.47g,10.6mmol), and dimethyl sulfoxide (20ml) were heated to 100 ℃ and reacted for 6 hours. After the reaction, the reaction mixture was cooled to room temperature, water (60mL) and ethyl acetate (30mL) were added thereto, extraction was carried out 3 times, washing was carried out with water, washing was carried out with saturated brine, drying was carried out with anhydrous sodium sulfate, filtration was carried out, the solvent was distilled off under reduced pressure to obtain a black solid, crude product, silica gel powder, sand and silica gel column purification were carried out [ eluent: V ] to obtain a crude product_PE:V_EA＝5:1]To obtain a black solid, and after beating several times with ethyl acetate or methanol, the compound was obtained as a white solid (0.7g, yield: 39%).

¹H NMR(400MHz,DMSO-d₆)δ7.75(d,J＝5.8Hz,1H),7.49(d,J＝7.9Hz,1H),7.28-7.17(m,3H),7.00(d,J＝8.0Hz,1H),3.92(s,3H)。

ESI-MS m/z:332.1{[M+H]⁺}。

Example 2

Preparation of 4, 7-difluoro-N- (4-methoxybenzo [ d ] thiazol-2-yl) benzo [ d ] thiazol-2-amine:

referring to the procedure shown in example 1, wherein "2-chloro-4-fluorobenzo [ d ] thiazole" was replaced with "2-chloro-4, 7-difluorobenzo [ d ] thiazole", off-white solid was obtained (1.1g, yield: 57%).

¹H NMR(400MHz,DMSO-d₆)δ7.73(d,J＝5.4Hz,1H),7.28-7.17(m,3H),7.02(d,J＝8.0Hz,1H),3.92(s,3H)。

ESI-MS m/z:350.1{[M+H]⁺}。

Example 3

Preparation of 4, 6-difluoro-N- (4-methoxybenzo [ d ] thiazol-2-yl) benzo [ d ] thiazol-2-amine:

referring to the procedure shown in example 1, wherein "2-chloro-4-fluorobenzo [ d ] thiazole" was replaced with "2-chloro-4, 6-difluorobenzo [ d ] thiazole", an off-white solid was obtained (0.8g, yield: 45%).

¹H NMR(400MHz,DMSO-d₆)δ7.65(d,J＝5.8Hz,1H),7.35(d,J＝7.9Hz,1H),7.28-7.17(m,2H),7.03(d,J＝8.0Hz,1H),3.92(s,3H)。

ESI-MS m/z:350.1{[M+H]⁺}。

Example 4

Preparation of 4-methoxy-N- (4- (trifluoromethyl) benzo [ d ] thiazol-2-yl) benzo [ d ] thiazol-2-amine:

referring to the procedure shown in example 1, wherein "2-chloro-4-fluoropheno [ d ] thiazole" was replaced with "2-chloro-4- (trifluoromethyl) benzo [ d ] thiazole", white solid was obtained (1.1g, yield: 67%).

¹H NMR(400MHz,DMSO-d₆)δ7.55(d,J＝5.8Hz,1H),7.39(d,J＝7.9Hz,1H),7.15-7.07(m,3H),7.00(d,J＝8.0Hz,1H),3.92(s,3H)。

ESI-MS m/z:382.1{[M+H]⁺}。

Example 5

Preparation of N- (4-fluorobenzo [ d ] thiazol-2-yl) -4, 7-dimethoxybenzo [ d ] thiazol-2-amine:

referring to the procedure shown in example 1, in which "4-methoxybenzo [ d ] thiazol-2-amine" was replaced with "4, 7-methoxybenzo [ d ] thiazol-2-amine", white solid was obtained (1.4g, yield: 73%).

¹H NMR(400MHz,DMSO-d₆)δ7.74(d,J＝5.8Hz,1H),7.36(d,J＝7.9Hz,1H),7.27-7.15(m,1H),7.00(d,J＝8.0Hz,2H),3.82(s,3H),3.76(s,3H)。

ESI-MS m/z:362.1{[M+H]⁺}。

Example 6

Preparation of N- (4-fluorobenzo [ d ] thiazol-2-yl) -4, 6-dimethoxybenzo [ d ] thiazol-2-amine:

referring to the procedure shown in example 1, in which "4-methoxybenzo [ d ] thiazol-2-amine" was replaced with "4, 6-methoxybenzo [ d ] thiazol-2-amine", white solid was obtained (1.2g, yield: 64%).

¹H NMR(400MHz,DMSO-d₆)δ7.75(d,J＝5.8Hz,1H),7.28-7.17(m,2H),7.21(s,1H),6.79(s,1H),3.88(s,3H),3.83(s,3H)。

ESI-MS m/z:332.1{[M+H]⁺}。

Example 7

Preparation of 4, 7-dimethoxy-N- (4- (trifluoromethyl) benzo [ d ] thiazol-2-yl) benzo [ d ] thiazol-2-amine:

referring to the procedure shown in example 1, wherein "2-chloro-4-fluorobenzo [ d ] thiazole" was replaced with "2-chloro-4- (trifluoromethyl) benzo [ d ] thiazole" and "4-methoxybenzo [ d ] thiazol-2-amine" was replaced with "4, 7-methoxybenzo [ d ] thiazol-2-amine", a white solid was obtained (0.9g, yield: 53%).

¹H NMR(400MHz,DMSO-d₆)δ7.75(d,J＝5.8Hz,1H),7.49(d,J＝7.9Hz,1H),7.28-7.17(m,1H),7.00(d,J＝8.0Hz,2H),3.92(s,3H)。

ESI-MS m/z:412.1{[M+H]⁺}。

Example 8

Preparation of 4, 6-dimethoxy-N- (4- (trifluoromethyl) benzo [ d ] thiazol-2-yl) benzo [ d ] thiazol-2-amine:

referring to the procedure shown in example 1, wherein "2-chloro-4-fluorobenzo [ d ] thiazole" was replaced with "2-chloro-4- (trifluoromethyl) benzo [ d ] thiazole" and "4-methoxybenzo [ d ] thiazol-2-amine" was replaced with "4, 6-methoxybenzo [ d ] thiazol-2-amine", a white solid was obtained (0.8g, yield: 47%).

¹H NMR(400MHz,DMSO-d₆)δ7.75(d,J＝5.8Hz,1H),7.49(d,J＝7.9Hz,1H),7.28-7.17(m,1H),7.21(s,1H),7.69(s,1H),3.92(s,3H)。

ESI-MS m/z:412.1{[M+H]⁺}。

It should be noted that examples 1 to 8 only exemplify some compounds screened at high throughput according to the present invention, and do not exemplify all compounds according to the present invention.

Example 9

Preparation of the pharmaceutical composition: tablet formulation

A compound of formula I or a pharmaceutically acceptable salt thereof (1g) was mixed with lactose (23g) and microcrystalline cellulose (5.7g) using a mixer. The resulting mixture was press-molded with a roller compactor to obtain a sheet-like pressed material. The flaky pressed material was ground into powder with a hammer mill, and the resulting powdery material was sieved through a 20-mesh sieve. A portion of light silica (0.3g) and magnesium stearate (0.3g) was added to the sieved material and mixed. The resulting mixed product was tableted with a tableting machine to prepare tablets.

As an illustration, the compounds of formula I can be used from example 1 to example 8.

As examples, the acid moiety of the pharmaceutically acceptable salt may be hydrochloric acid, sulfuric acid, benzenesulfonic acid, p-toluenesulfonic acid, phosphoric acid, hydrobromic acid, maleic acid, fumaric acid, or malic acid.

Example 10

Preparation of the pharmaceutical composition: gelatin capsule

A compound of formula I or a pharmaceutically acceptable salt thereof (1g) is granulated with microcrystalline cellulose (0.35g) and lactose (0.15g) in water and the granules are then mixed with sodium gemfibrozil (0.04g) and magnesium stearate (0.01 g). The obtained mixed product was filled into gelatin capsules to prepare gelatin capsules. (the gelatin capsule of this example was manufactured by Suzhou capsules, Inc., China, and the gelatin capsule conforms to the pharmaceutical standards)

As an illustration, the compounds of formula I can be used from example 1 to example 8.

As examples, the acid moiety of the pharmaceutically acceptable salt may be hydrochloric acid, sulfuric acid, benzenesulfonic acid, p-toluenesulfonic acid, phosphoric acid, hydrobromic acid, maleic acid, fumaric acid, or malic acid.

Example 11

Comparison of biological Activity of bis-benzothiazoles:

the compounds synthesized in examples 1 to 8 were named JAC1, JAC1011, JAC1012, JAC1013, JAC1014, JAC1015, JAC1016, JAC1017 in this order.

Adopting human bronchial epithelium HBE cells stably transfected with reporter genes containing JWA gene 2000bp promoter sequences and luciferase indication functions to carry out high-throughput screening, and measuring the reporter gene fluorescence values of the 8 representative compounds as shown in Table 1 after two rounds of high-throughput screening, wherein the concentration range of the compounds is 0.00-3.30 mu g/mL, and the time for treating the cells by the compounds is 48 hours; the English-Chinese comparison of 8 representative compounds is shown in Table 2.

The results in table 1 show that all 8 compounds can activate the expression of the JWA gene in cells and have a good dose-effect relationship, wherein the activation of the JWA gene by JAC1 is particularly significant.

The full-length cDNA sequence of JWA gene, amino acid sequence of encoded protein and DNA sequence of 2000bp promoter of JWA gene for constructing luciferase reporter gene are shown in figure 1. The molecular structure of luciferase reporter gene containing JWA gene promoter sequence is shown in figure 2.

TABLE 1 fluorescence values of bis-benzothiazoloamines and reporter gene thereof

TABLE 2 numbering of bis-benzothiazoles and names in English and Chinese

Example 12

Effect of JAC1 on clonogenic (malignant proliferation) capacity of HER2 positive breast cancer cells:

this example aims at testing the effect of small molecule compound JAC1 treatment on clonogenic (malignant proliferation) capacity of HER2 overexpressed human breast cancer cells BT474 and SKBR 3.

Two human breast cancer cell lines BT474 and SKBR3 are cultured in DMEM medium containing fetal calf serum 20% and 10% respectively, conventional double antibody containing 5% CO at 37 deg.C₂. Taking BT in logarithmic growth phase474 and SKBR3 cells were evenly seeded in six-well plates at a density of 800 cells/well, and after the cells were attached to the wall, they were treated separately by changing to JAC 1-containing medium at different concentrations (0, 1, 10. mu.M). According to the growth condition of the cell clone clusters, the cell culture is stopped after 10 to 15 days of culture, the culture dish is removed, the cell clone clusters growing are stained by crystal violet after being fixed by methanol, the number of the clone clusters (containing more than 50 cells) is counted by ImageJ software after photographing, and the counting result is analyzed by statistical software SPSS.

The experimental results are shown in fig. 3, compared with the control group, the BT474 and SKBR3 treated by different doses of the small molecule compound JAC1 significantly inhibited the cell clonogenic capacity and showed a dose-effect relationship.

It should be noted that, after the remaining compounds of examples 1 to 8 were tested according to the method of this example, the results were substantially consistent with JAC1, and the cell clonogenic capacities of BT474 and SKBR3 were all significantly inhibited in a dose-response relationship.

Example 13

Effect of JAC1 on cell localization of HER2 positive breast cancer cell HER2 molecule:

this example aims to observe whether treatment with the small molecule compound JAC1 has an effect on HER2 localization in cells.

The cells were inoculated in 35mm dedicated laser confocal dishes, treated with JAC1 at different concentrations (0, 1, 10 μ M) for 24 hours, fixed with methanol for 15 minutes, washed three times with PBST, blocked with normal sheep blood for 1 hour, incubated overnight at 4 ℃ with primary antibody, washed three times with PBST the next day, added with fluorescent secondary antibody and DAPI, and photographed under laser confocal conditions.

The experimental result is shown in fig. 4, with the increase of the concentration of JAC1, the JWA content is increased, the HER2 expression level is reduced, but the weakened HER2 is still positioned on the cell membrane, namely JAC1 treatment has no influence on the cell membrane positioning property of HER 2.

It should be noted that, after the remaining compounds of examples 1 to 8 were tested according to the method of this example, the results were substantially consistent with those of JAC1, i.e., the JWA content increased and the HER2 expression level decreased with the increase of the compound concentration, but the attenuated HER2 was still localized on the cell membrane, i.e., the treatment did not affect the cell membrane localization property of HER 2.

Example 14

Effect of JAC1 on expression levels of JWA and HER2 proteins in HER2 positive breast cancer cells:

the purpose of this example is to demonstrate the effect of JAC1 treatment on increasing JWA and decreasing HER2 protein expression levels using a cell model.

Conventionally digesting cells in logarithmic growth phase, spreading the cells on a 6-well plate, treating the cells with JAC1 at a cell density of 40-50% for 24 hours, adding 0.1mL of RIPA (containing 0.5% PMSF) cell lysate to collect protein, centrifuging at 12000 Xg for 15 minutes, taking supernatant, measuring the protein concentration, and boiling the protein. And (3) selecting 10% polyacrylamide gel for protein electrophoresis, adding 40 mu g of protein into each hole, and selecting 60V, 30 min, 90V and 1.5-2 h as electrophoresis conditions. After the electrophoresis is finished, the membrane is transferred by a wet transfer method, so that the protein is transferred from the gel to the PVDF membrane. After the membrane transfer is finished, 5% skim milk is sealed for 1-2 hours at normal temperature, the membrane is washed for 3 times by TBST (containing 0.1% Tween20), 5 minutes each time, and the corresponding primary antibody is incubated at 4 ℃ overnight. The following day, TBST (with 0.1% Tween20) washed membranes 3 times for 5 minutes each, incubated secondary antibody for 1-2 hours at room temperature, and TBST (with 0.1% Tween20) washed membranes 8 times for 5 minutes each. And dripping ECL luminous liquid on the film, and exposing.

The results of the experiment are shown in figure 5, JWA protein level increased and HER2 protein level decreased with increasing concentration of JAC 1.

It should be noted that, for each of the remaining compounds of examples 1 to 8, the results obtained by the method of this example were substantially identical to those of JAC1, i.e., the level of JWA protein was increased and the level of HER2 protein was decreased with the increase of the compound concentration.

Example 15

Effect of JAC1 on migratory capacity of HER2 positive breast cancer cells:

the purpose of this example is to examine the effect of JAC1 on the cell migration ability of BT474 and SKBR3 breast cancer cells by the Transwell migration assay.

BT474 and SKBR3 cells in logarithmic growth phase are taken and spread on a 6-well plate, the cell density is 40-50%, and after the cells adhere to the wall, the medicine is added, and the concentration of JAC1 is 0, 1 and 10 mu M respectively. Spreading glue in a Transwell chamber: the day before the experiment, 24-well plates, several Transwell chambers were prepared. FN was diluted to 1mg/mL stock solution, 10-fold to a final concentration of 100. mu.g/mL. The bottom of each chamber was coated with FN 50. mu.L, air dried in a clean bench for 2 hours, and left overnight in a cell culture chamber.

On the day of the experiment, cells were digested with 0.25% pancreatin, suspended in serum-free medium, counted and adjusted to a cell density of 3X 10⁵One/ml, seed 100. mu.L to the upper layer of the Transwell chamber. The lower chamber of the chamber was filled with 600. mu.L of medium containing 10% FBS and 100ng/ml EGF. After 12 hours of incubation in the incubator, the Transwell chamber was removed and fixed with 95% methanol for 20 minutes. Washed 3 times with PBS and stained with crystal violet for 30 minutes. The cells were washed 3 more times with PBS and the upper chamber was gently wiped off with a cotton swab. Finally, the chambers are placed on a glass slide, observed and photographed under an inverted microscope, a visual field is respectively photographed at the upper part, the lower part, the left part, the right part and the middle part of each chamber, and the number of the cells passing through the upper chamber is counted and statistically analyzed.

The experimental result is shown in fig. 6, JAC1 can obviously inhibit the migration capacity of breast cancer cells BT474 and SKBR3, and is in a dose-effect relationship.

It should be noted that, after the remaining compounds of examples 1 to 8 were tested according to the method of this example, the results were substantially consistent with JAC1, and the migration ability of BT474 and SKBR3 of breast cancer cells was significantly inhibited, and the dose-response relationship was observed.

Example 16

Effect of JAC1 on tumor growth in HER2 positive breast cancer cells in subcutaneous tumor-bearing nude mice:

the purpose of this example is to establish a subcutaneous tumor-bearing model of human breast cancer cell immunodeficiency mice, and observe the effect of JAC1 on the subcutaneous growth of breast cancer cells after prognosis.

Preparing 5 × 10 human breast cancer cell BT474 cells in logarithmic growth phase under aseptic condition⁶100 mu L of cell suspension, and carrying out subcutaneous tumor bearing on BALB/c nude mice until the tumor volume is 75-125mm³When the temperature of the water is higher than the set temperature,mice were randomly assigned to groups of blank control (Mock), solvent control (vehicle), 50mg/kg JAC1 treatment, 100mg/kg JAC1, and positive control herceptin in combination with paclitaxel (TH). Wherein JAC 1: daily administration, intraperitoneal injection; herceptin: 10mg/kg, twice weekly for intraperitoneal injection; paclitaxel: 20mg/kg, once a week, and injected intraperitoneally. From the day of administration, the body weight of the mice was weighed every two days while observing and measuring the tumor diameter of the mice, and the tumor volume was calculated. According to the requirements of animal ethics, the volume of the tumor-bearing tumor in the model mouse reaches 2500-³And ending the model, euthanizing the treated experimental animal after anesthesia, and carrying out corresponding detection and analysis.

As a result, it was found that: the body weights of the mice in each group showed a slow rising trend during the experiment (fig. 7).

Compared with the control group, the tumor volume increase rate of the 50mg/kg JAC1 treated group, the 100mg/kg JAC1 group and the herceptin combined with paclitaxel (TH) treated group is obviously reduced (figure 8), the tumor weight is reduced (P <0.05), and the average tumor size of each group is sequentially TH group <100mg/kg JAC1 group <50mg/kg JAC1 group (figure 9); tumor weight ratio was decreased (P <0.05) and TH group tumor weight ratio was lowest (fig. 10).

FIG. 11 is a photograph of tumor tissues isolated from each group of mice.

Tumor inhibition rate: 31.22% in the 50mg/kg JAC1 treatment group, 46.21% in the 100mg/kg JAC1 treatment group, and 52.36% in the herceptin and paclitaxel (TH) treatment group; 50mg/kg JAC1, 100mg/kg JAC1 and herceptin are effective in combination with paclitaxel, and the tumor inhibition rates of the treatment groups are as follows in sequence: TH >100mg/kg JAC1>50mg/kg JAC1 (FIG. 12).

Further performing immunoblotting detection on the expression level of tumor tissue related molecules of subcutaneous tumor-bearing breast cancer of each treatment group, wherein the result is shown in figure 13, the expression of HER2 of JAC1 treatment group is obviously reduced, and the expression level of JWA is obviously increased; group TH did not detect HER2 expression under the conditions of this experiment because herceptin is a humanized anti-HER 2 antibody that binds to an epitope of the cellular HER2 protein after use, rendering the primary antibody no longer able to bind HER2 in immunoblot experiments.

Example 17

Effect of JAC1 treatment on major visceral structure of tumor-bearing mice:

a nude mouse model of JAC1 for inhibiting the growth of subcutaneous tumor-bearing human breast cancer cells was constructed as in example 16. After the model is finished, in order to observe that the main organs of the experimental mouse have no damage under various treatment conditions, after the treated mouse is anesthetized and euthanized, the heart, the liver, the spleen, the lung, the kidney and the brain of the main organs of the mouse are taken and fixed by formalin solution in a conventional way, and are sliced in a conventional way after being embedded by paraffin, and the morphological structure of the main organs of the mouse is firstly detected to be normal by HE staining. The results showed that no significant abnormalities were observed in the major organs (heart, liver, spleen, lung, kidney, brain) of the mice of each treatment group under the microscope under the experimental conditions (fig. 14).

Example 18

Effect of JAC1 treatment on biochemical indicators of blood of tumor-bearing mice:

a nude mouse model of JAC1 for inhibiting the growth of subcutaneous tumor-bearing human breast cancer cells was constructed as in example 16. After the model is finished, in order to observe that the main organs of the experimental mouse have no damage under each treatment condition, whole blood is taken to separate serum after the mouse is anesthetized, and the biochemical index change of the serum is detected by a full-automatic biochemical analyzer. As shown in fig. 15, the results showed that, although no significant morphological changes were observed in the major organs of the mice in each treatment group, the liver functions ALT and AST, triglyceride TG, myocardial kinase CK and its isozyme CKMB were significantly reduced in the serum of the mice treated with JAC1, particularly 100mg/kg JAC1, and the level of superoxide dismutase SOD having an antioxidant effect was significantly increased, as compared to the control group. These indices also improved in the paclitaxel + herceptin treated group compared to the control group but none of the differences were statistically significant.

These results suggest that JAC1 not only can effectively inhibit the growth of subcutaneous tumor-bearing mice with human breast cancer BT474 cells, but also has a good intervention effect on in vivo biochemical dysfunction caused by tumor growth; although the tumor inhibition rate of the positive control drug treatment group is slightly higher than that of the JAC1 treatment group, the positive control drug treatment group has insignificant improvement effect on in vivo biochemical dysfunction caused by tumors.

Example 19

Verification of off-target effects of JAC1 activating JWA expression while inhibiting HER2 expression:

after observing that the small molecules of JAC1 act on the biological functions of HER2 overexpression cancer cells specifically, the off-target effect of JAC1 tumor inhibition is experimentally verified. Firstly, a human BGC823 gastric cancer cell which is positive for HER2 and has a knockout JWA gene is constructed by a Crisp/cas9 technology, a human BGC823 gastric cancer cell strain (which is proved to be positive for HER2 expression in the past) containing JWA (JWA WT) and a human BGC823 gastric cancer cell strain which lacks the JWA (JWA KO) gene is used for treating the cell for 48 hours by JAC 110 mu M and then protein is collected, and JWA wild and deletion BGC823 gastric cancer cell JWA and HER2 expression levels are detected by an immunoblotting method.

As shown in fig. 16, the results show: in JWA wild type (JWA WT) gastric cancer cells, JAC1 treatment can significantly activate JWA expression and inhibit HER2 expression level; however, in JWA deficient (JWA KO) gastric cancer cells, no significant effect was seen on JWA and HER2 expression after JAC1 treatment.

These results indicate that inhibition of HER2 by JAC1 is achieved by activating JWA, and that JAC1 has no significant off-target effect.

Example 20

The present invention obtained many compounds capable of activating the expression of JWA gene of cells when high throughput screening was performed according to the method of example 11, except the compounds of examples 1 to 8, as follows:

R¹＝OH，R²h or OH, R³Not being F or Cl, R⁴H or F, R⁵H or CH₃Or CH₂CH₃；

R¹＝CH₃，R²＝CH₃Or CH₂CH₃，R³Is I or CF₃，R⁴Br or CF₃，R⁵H or CH₃Or CH₂CH₃；

R¹＝CH₂CH₃，R²＝CH₂CH₂CH₃Or OCH₃，R³Br or Cl, R⁴Either Cl or I, R⁵H or CH₃Or CH₂CH₃；

R¹＝OCH₃，R²＝OCH₂CH₂CH₃Or OH, R³F or CF₃，R⁴H or CF₃，R⁵H or CH₃Or CH₂CH₃；

R¹＝OCH₂CH₃；R²＝CH₃Or OCH₃，R³Either Cl or I, R⁴Br or I, R⁵H or CH₃Or CH₂CH₃。

Note: when a substituent of the above compound may exist at a plurality of substitution positions, or a plurality of isomers of the compound exist, it is meant to include all possible compounds.

The compounds can activate the expression of JWA gene of cells, thereby having the prospect of preparing JWA gene activator or antitumor drugs, and therefore, the compounds are all included in the protection scope of the invention.

In addition, the compounds of the present invention may be used in combination with other drugs and other therapeutic means to enhance the effectiveness of the treatment of malignant tumors.

In addition to the above embodiments, the present invention may have other embodiments. All technical solutions formed by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the claims of the present invention.