Isoxazole sulfonamide compound with BRD4 inhibitory activity and preparation method and application thereof
阅读说明：本技术 具有brd4抑制活性的异噁唑磺酰胺类化合物及其制备方法和应用 (Isoxazole sulfonamide compound with BRD4 inhibitory activity and preparation method and application thereof ) 是由 张茂风 于 2021-09-15 设计创作，主要内容包括：本发明提供了一种具有BRD4抑制活性的异噁唑磺酰胺类化合物或其药学上可接受的盐,所述化合物具有如通式(I)所示的结构式。本发明还提供了所述的化合物或其药学上可接受的盐或所述的药物组合物在制备用于预防或治疗与BRD4相关的癌症药物中的应用,所述的癌症包括白血病、淋巴瘤、骨髓瘤、肺癌、前列腺癌、胰腺癌、结肠癌、乳腺癌、肝癌和胃癌等。本发明提供了一种新型BRD4抑制剂,并通过实验证明该类具有3-乙基苯并[d]异噁唑磺酰胺结构的新化合物,具有很好的BRD4蛋白抑制效果和体外抑制肿瘤细胞的效果,尤其是针对白血病细胞,部分化合物的抑制活性达到或优于阳性对照化合物。化合物还可以有效抑制c-Myc癌基因的表达。(The invention provides an isoxazole sulfonamide compound with BRD4 inhibitory activity or a pharmaceutically acceptable salt thereof, wherein the compound has a structural formula shown as a general formula (I). The invention also provides application of the compound or the pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparing a medicament for preventing or treating BRD 4-related cancers, wherein the cancers comprise leukemia, lymphoma, myeloma, lung cancer, prostate cancer, pancreatic cancer, colon cancer, breast cancer, liver cancer, gastric cancer and the like. The invention provides a novel BRD4 inhibitor, and experiments prove that the novel compound with a 3-ethylbenzo [ d ] isoxazole sulfonamide structure has good BRD4 protein inhibition effect and in-vitro tumor cell inhibition effect, and especially aiming at leukemia cells, the inhibition activity of part of compounds reaches or is superior to that of a positive control compound. The compounds are also effective in inhibiting the expression of the c-Myc oncogene.)
1. Isoxazole sulfonamides or pharmaceutically acceptable salts thereof having BRD4 inhibitory activity, wherein the compound has the formula shown in general formula (I):
wherein R is selected fromHeterocyclyl and C1-C6Any one of alkyl groups;
wherein R is1-R5Selected from H, halogen, methoxy, nitro, trifluoromethyl, trifluoromethoxy or 2, 3-dihydrobenzofuranyl; the heterocyclic group is selected from thiophene or pyridine;
alkyl refers to a fully saturated hydrocarbon chain, which may be straight or branched.
2. The isoxazole sulfonamide compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the isoxazole sulfonamide compound is selected from the group consisting of:
n- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) ethanesulfonamide
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) propane-1-sulfonamide
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) butane-1-sulfonamide
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) benzenesulfonamide
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2-fluorobenzenesulphonamide
2-chloro-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) benzenesulfonamide
2-bromo-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) benzenesulfonamide
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2-methoxybenzenesulphonamide
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2- (trifluoromethoxy) benzenesulfonamide
3-chloro-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) benzenesulfonamide
3-bromo-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) benzenesulfonamide
5-chloro-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2-methoxybenzenesulphonamide
5-bromo-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2-methoxybenzenesulphonamide
5-bromo-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2, 3-dihydrobenzofuran-7-sulphonamide
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -4-fluorobenzenesulphonamide
4-chloro-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) benzenesulfonamide
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -4-nitrobenzenesulfonamide
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -4-methoxybenzenesulphonamide
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -4- (trifluoromethoxy) benzenesulfonamide
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2, 4-dimethoxybenzenesulfonamide
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2-methoxy-4-nitrobenzenesulfonamide
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -3-fluoro-4-methoxybenzenesulphonamide
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -3, 4-dimethoxybenzenesulfonamide
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2, 3-dihydrobenzofuran-5-sulphonamide
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) thiophene-2-sulphonamides
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) pyridine-3-sulphonamide.
3. The isoxazole sulfonamide compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the isoxazole sulfonamide compound is:
n- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -4-methoxybenzenesulphonamide.
4. The isoxazole sulfonamide compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the isoxazole sulfonamide compound is:
n- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2-methoxybenzenesulphonamide.
5. The isoxazole sulfonamide compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the isoxazole sulfonamide compound is:
n- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2, 4-dimethoxybenzenesulfonamide.
6. The isoxazole sulfonamide compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the isoxazole sulfonamide compound is:
n- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2-methoxy-4-nitrobenzenesulfonamide.
7. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.
8. Use of a compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 7, for the manufacture of a medicament for the prevention or treatment of BRD 4-related cancers, including leukemia, lymphoma, myeloma, lung, prostate, pancreatic, colon, breast, liver and stomach cancers.
9. The use of claim 8, wherein the cancer is leukemia.
10. The process for the preparation of the compound of formula (I) according to claim 1, wherein the synthetic route is as follows:
wherein R is selected fromHeterocyclyl and C1-C6An alkyl group; wherein R is1-R5Selected from H, halogen, methoxy, nitro, trifluoromethyl, trifluoromethoxy, 2, 3-dihydrobenzofuranyl; the heterocyclic radical is selected from thiophene and pyridine;
the preparation method of the compound with the general formula (I) specifically comprises the following steps:
1) taking 2, 4-dimethoxyaniline, acetic anhydride and triethylamine to carry out N-acetylation reaction to obtain a compound 2; the mol ratio of the 2, 4-dimethoxyaniline to the acetic anhydride to the triethylamine is 1: 1.5-2: 1.5-3, taking dichloromethane as a solvent, and reacting at room temperature for 4-12 h;
2) taking the compound 2, propionyl chloride and anhydrous aluminum trichloride to carry out Friedel-crafts reaction to obtain a compound 3; the mol ratio of the compound 2 to the propionyl chloride to the anhydrous aluminum trichloride is 1: 3: 3.5-4, taking dichloromethane as a solvent, controlling the reaction temperature to be 40-43 ℃, and controlling the reaction time to be 2-3 h;
3) carrying out oximation reaction on the compound 3, hydroxylamine hydrochloride and anhydrous sodium acetate to obtain a compound 4; the mol ratio of the compound 3 to the hydroxylamine hydrochloride to the anhydrous sodium acetate is 1: 1.5-2: 1.5-2, wherein the solvent is an ethanol-water mixed solvent, the reaction temperature is 80 ℃, and the reaction time is 1.5-3 h;
4) taking the compound 4 and N, N-dimethylformamide dimethyl acetal to carry out dehydration cyclization reaction to obtain a compound 5; the mol ratio of the compound 4 and the N, N-dimethylformamide dimethyl acetal is 1: 4-5, wherein the solvent is 1, 4-dioxane, the reaction temperature is 100 ℃, and the reaction time is 10 min;
5) taking the compound 5 and dilute hydrochloric acid to carry out hydrolysis reaction to obtain a compound 6; the weight g/volume ml ratio of the compound 5 to 3mol/L diluted hydrochloric acid is 1: 20-30 ℃, the reaction temperature is 90 ℃, and the reaction time is 3-4 h;
6) taking the compound 6 and different sulfonyl chlorides to carry out N-sulfonylation reaction to obtain a final product; the molar ratio of compound 6 to the different sulfonyl chlorides was 1: 1.1-2, the addition of pyridine is 0.005ml per mg of compound 6, the solvent is dichloromethane, the reaction temperature is 40-43 ℃, and the reaction time is 1.5-3 h.
Technical Field
The invention relates to the field of medicinal chemistry, in particular to a preparation method of isoxazole sulfonamide compounds with BRD4 inhibition effect, a composition containing the compounds and application of the compounds.
Background
Acetylation modification of histone lysine is one of the important post-translational modifications. The bromodomain is a protein domain, can recognize acetylated lysine on histone, further opens a chromatin structure, promotes transcription activation, and is closely related to the occurrence of diseases such as tumor or inflammation. At present, it has been found that 61 bromodomains, present in 46 proteins, can be divided into eight families. BRD4 is one of the BET (Bromodomain and Extra-Terminal domain) family members, contains two bromodomains, BD1 and BD2, respectively, and binds acetylated lysine via BD1 and BD 2.
BRD4 can recruit positive transcriptional elongation factor b (P-TEFb) to the promoter and activate RNA polymerase II. In addition, BRD4 can promote the transcription of key genes (Bcl-2, c-Myc and CDK6), and plays a crucial role in the proliferation and cell cycle progression of tumor cells. Thus, expression of key oncogenes may be suppressed by displacing BRD4 from chromatin. BRD4 protein is associated with a variety of human diseases and conditions, including cancer, such as Acute Myeloid Leukemia (AML), MLL, gastrointestinal stromal tumor (GIST), triple negative breast cancer, prostate cancer, neuroblastoma, pancreatic cancer, cholangiocarcinoma, colon cancer, and inflammation.
BRD4 is found to be overexpressed in a variety of tumors and functions to promote the expression of a variety of oncogenes. The small molecular compound is combined with the bromodomain pocket of BRD4, so that the function of BRD4 can be effectively inhibited, the expression of oncogene can be inhibited, and the anticancer effect can be achieved. There is a large body of research literature in the field that demonstrates this mechanism, and some review literature follows:
[1]Filippakopoulos,P.,Knapp,S.Targeting bromodomains:epigenetic readers of lysine acetylation.Nat Rev Drug Discov,13:337–356(2014).
[2]Fujisawa T,Filippakopoulos P.Functions of bromodomain-containing proteins and their roles in homeostasis and cancer.Nat Rev Mol Cell Biol,18(4):246-262(2017).
in addition, there are several BRD4 inhibitors that are being studied clinically as new antitumor agents.
Chinese patent application CN105085427A specifically discloses a benzo [ d ] benzene represented by general formula (A)]Isoxazole compounds and application thereof. The compound can effectively inhibit bromodomains (bromodomain) of BET family proteins, so that the interaction between the BET family proteins and chromatin histones is blocked, gene transcription is further regulated, downstream signal path changes are caused, and important influences are generated on various diseases. Therefore, the compound and the composition provided by the invention can be used for preparing medicines for treating or preventing diseases such as tumor formation, inflammation, virus infection, cell proliferation disorder, autoimmune disease, septicemia and the like. In addition, the optimum compound in this patent is also reported in the paper (Journal of Medicinal Chemistry,2018,61(7):3037-3058), and in the evaluation of antitumor activity at the cellular level, the compound has weak selective inhibition on specific tumor cells and has inhibitory effect on various tumor cells except leukemia cells (IC)500.69 μm), has certain effect (IC) on prostate cancer cells and mammary gland cells50 1-2μΜ)。
Since BRD4 is associated with the occurrence and progression of various tumors, inhibition of BRD4 is expected to inhibit the proliferation of various tumors. However, although different inhibitors of BRD4 have good binding activity to BRD4 at the in vitro protein level, differences in physicochemical properties (e.g., solubility, membrane permeability, metabolic properties) of the compounds themselves may result in differences in activity at the cell level from that at the protein level.
Therefore, the above prior patents have the following technical problems:
firstly, the selective inhibition effect on tumor cells is not high, and potential side effects can be brought when a single tumor is treated in a targeted mode, namely, the inhibition effect on other tissue cells can be generated while specific tumor cells are killed.
Secondly, the activity of the optimized compound of the above prior patent in combination with BRD4 needs to be further improved. If the activity of the compound at the target level in vitro is not high enough (i.e., the binding activity to BRD4 is not high enough), the effect of inhibiting tumor proliferation by binding and inhibiting the target, and thus the relevant pathway, is reduced after entering the more complex cells of the environment. In this case, to achieve the same effect, the dosage needs to be increased, with high dosages presenting a high risk of toxic side effects. In addition, if the cytotoxicity screening is directly carried out without screening based on the target, the target of the compound is unclear, and the safety risk is higher.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a novel isoxazole sulfonamide compound with BRD4 inhibition effect and a preparation method thereof, and also aims to provide the application of the isoxazole sulfonamide compound as an anti-tumor drug.
The specific technical scheme of the invention is as follows:
the invention provides an isoxazole sulfonamide compound or a pharmaceutically acceptable salt thereof, wherein the compound has a structural formula shown as a general formula (I):
wherein R is selected fromHeterocyclyl and C1-C6Any one of alkyl groups;
wherein R is1-R5Selected from H, halogen, methoxy, nitro, trifluoromethyl, trifluoromethoxy or 2, 3-dihydrobenzofuranyl; the heterocyclic group is selected from thiophene or pyridine.
"alkyl" refers to a fully saturated hydrocarbon chain, which may be straight or branched.
Preferred compounds of the general formula (I) according to the invention are those of the following structure:
n- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) ethanesulfonamide (7a)
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) propane-1-sulfonamide (7b)
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) butane-1-sulfonamide (7c)
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) benzenesulfonamide (7d)
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2-fluorobenzenesulfonamide (7e)
2-chloro-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) benzenesulfonamide (7f)
2-bromo-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) benzenesulfonamide (7g)
N- (3-Ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2-methoxybenzenesulphonamide (7h)
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2- (trifluoromethoxy) benzenesulfonamide (7i)
3-chloro-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) benzenesulfonamide (7j)
3-bromo-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) benzenesulfonamide (7k)
5-chloro-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2-methoxybenzenesulphonamide (7l)
5-bromo-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2-methoxybenzenesulphonamide (7m)
5-bromo-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2, 3-dihydrobenzofuran-7-sulfonamide (7N)
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -4-fluorobenzenesulfonamide (7o)
4-chloro-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) benzenesulfonamide (7p)
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -4-nitrobenzenesulfonamide (7q)
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -4-methoxybenzenesulphonamide (7r)
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -4- (trifluoromethoxy) benzenesulfonamide (7s)
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2, 4-dimethoxybenzenesulfonamide (7t)
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2-methoxy-4-nitrobenzenesulfonamide (7u)
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -3-fluoro-4-methoxybenzenesulphonamide (7v)
N- (3-Ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -3, 4-dimethoxybenzenesulfonamide (7w)
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2, 3-dihydrobenzofuran-5-sulfonamide (7x)
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) thiophene-2-sulphonamide (7y)
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) pyridine-3-sulfonamide (7z)
The specific structures of the individual compounds are shown in table 1:
table 1 preferred compound structures
More preferably still, the first and second liquid crystal compositions are,
the isoxazole sulfonamide compound is any one of the following compounds:
n- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -4-methoxybenzenesulphonamide (7 r).
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2-methoxybenzenesulphonamide (7 h).
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2, 4-dimethoxybenzenesulfonamide (7 t).
N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2-methoxy-4-nitrobenzenesulfonamide (7 u).
In the present invention, the pharmaceutically acceptable salts may include acid addition salts of the compounds of formula (I) with the following acids: hydrobromic acid, hydrochloric acid, sulphuric acid, phosphoric acid, borates, methanesulphonic acid, p-toluenesulphonic acid, naphthalenesulphonic acid, benzenesulphonic acid, citric acid, lactic acid, pyruvic acid, tartaric acid, acetic acid, maleic or succinic acid, mandelic acid, fumaric acid, salicylic acid or phenylacetic acid. In addition, acid salts of inorganic bases are also included, such as: containing a basic metal cation, an alkaline earth metal cation or an ammonium cation salt.
The invention further provides application of the compound shown in the general formula (I) or the pharmaceutically acceptable salt thereof or the pharmaceutical composition containing the compound in preparing a medicament for inhibiting BRD 4.
The invention further relates to application of the compound shown in the general formula (I) or the pharmaceutically acceptable salt thereof or the pharmaceutical composition containing the compound in preparation of medicines for preventing or treating BRD 4-related cancers, wherein the cancers are selected from leukemia, lymphoma, myeloma, lung cancer, prostatic cancer, pancreatic cancer, colon cancer, breast cancer, liver cancer, gastric cancer and the like. More preferably, the cancer is leukemia.
To accomplish the synthetic objectives of the present invention, the present invention employs the following synthetic schemes to prepare compounds of formula (I):
wherein R is selected fromHeterocyclyl and C1-C6Any one of alkyl groups;
wherein R is1-R5Selected from H, halogen, methoxy, nitro, trifluoromethyl, trifluoromethoxy or 2, 3-dihydrobenzofuranyl; the heterocyclic group is selected from thiophene or pyridine.
"alkyl" refers to a fully saturated hydrocarbon chain, which may be straight or branched.
The preparation method of the compound with the general formula (I) specifically comprises the following steps:
1) taking 2, 4-dimethoxyaniline, acetic anhydride and triethylamine to carry out N-acetylation reaction to obtain a compound 2; the mol ratio of the 2, 4-dimethoxyaniline to the acetic anhydride to the triethylamine is 1: (1.5-2): (1.5-3), the solvent is dichloromethane, the reaction temperature is room temperature, and the reaction time is 4-12 h;
2) taking the compound 2, propionyl chloride and anhydrous aluminum trichloride to carry out Friedel-crafts reaction to obtain a compound 3; the mol ratio of the compound 2 to the propionyl chloride to the anhydrous aluminum trichloride is 1: 3: (3.5-4), the solvent is dichloromethane, the reaction temperature is 40-43 ℃, and the reaction time is 2-3 h;
3) carrying out oximation reaction on the compound 3, hydroxylamine hydrochloride and anhydrous sodium acetate to obtain a compound 4; the mol ratio of the compound 3 to the hydroxylamine hydrochloride to the anhydrous sodium acetate is 1: (1.5-2): (1.5-2), the solvent is an ethanol-water mixed solvent, the reaction temperature is 80 ℃, and the reaction time is 1.5-3 h;
4) taking the compound 4 and N, N-dimethylformamide dimethyl acetal to carry out dehydration cyclization reaction to obtain a compound 5; the mol ratio of the compound 4 and the N, N-dimethylformamide dimethyl acetal is 1: (4-5), wherein the solvent is 1, 4-dioxane, the reaction temperature is 100 ℃, and the reaction time is 10 min;
5) taking the compound 5 and dilute hydrochloric acid to carry out hydrolysis reaction to obtain a compound 6; the weight/volume ratio (g/ml) of compound 5 to dilute hydrochloric acid (3mol/L) was 1: 20-30 ℃, the reaction temperature is 90 ℃, and the reaction time is 3-4 h;
6) taking the compound 6 and different sulfonyl chlorides to carry out N-sulfonylation reaction to obtain a final product; the molar ratio of compound 6 to sulfonyl chloride is 1: (1.1-2), the pyridine is added in an amount of 0.005ml per mg of the compound 6, the solvent is dichloromethane, the reaction temperature is 40-43 ℃, and the reaction time is 1.5-3 h.
Preferably, the first and second electrodes are formed of a metal,
in the step 1), the molar ratio of the 2, 4-dimethoxyaniline to the acetic anhydride to the triethylamine is 1: 1.8: 1.6.
in the step 2), the mol ratio of the compound 2, propionyl chloride and anhydrous aluminum trichloride is 1: 3: 4.
in the step 3), the molar ratio of the compound 3, hydroxylamine hydrochloride and anhydrous sodium acetate is 1: 2: 1.6.
in the step 4), the molar ratio of the compound 4 to the N, N-dimethylformamide dimethyl acetal is 1: 4.6.
in the step 5), the weight/volume ratio (g/ml) of the compound 5 to the diluted hydrochloric acid (3mol/L) is 1: 26.
in the step 6), the molar ratio of the compound 6 to sulfonyl chloride is 1: 1.2.
the invention has the technical effects that:
1) according to the invention, the ethyl is introduced into the 3-position of the compound skeleton, so that the binding effect of the position and the protein is improved, and the upper substituent is screened, so that the activity of the obtained optimized compound is higher than that of the compound in the prior art. In addition, in the aspect of tumor cell activity evaluation, the novel compound has better selectivity on leukemia cells and has poor activity on other cells such as prostate cancer, breast cancer and the like (>20 mu m).
2) Experiments prove that the novel BRD4 inhibitor has good BRD4 protein inhibition effect and in-vitro tumor cell inhibition effect, and especially has the inhibition activity of partial compounds reaching or being superior to that of positive control compounds for leukemia cells. The compounds are also effective in inhibiting the expression of the c-Myc oncogene. As a novel BRD4 inhibitor, the derivative provides a new candidate drug for treating diseases such as tumors.
3) The invention also provides a synthesis method of the BRD4 inhibitor, and the target compound is efficiently prepared by designing a synthesis route.
Drawings
FIG. 1 is an inhibition curve of a portion of representative compounds of the present invention.
FIG. 2 shows the results of the test of the oncogene inhibitory activity of the compound 7r of the present invention.
Detailed Description
The invention is further described below with reference to examples, which are intended to be illustrative only and not to limit the scope of the invention.
EXAMPLE 1N- (3-Ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) ethanesulfonamide (7a)
Step 1 preparation of N- (2, 4-dimethoxyphenyl) acetamide (2) 2, 4-dimethoxyaniline (15g,97.9mmol) and dichloromethane (40mL) were added sequentially to a dry 250mL reaction flask, cooled to 0 deg.C, acetic anhydride (18.0g,176.3mmol) was added with stirring, triethylamine (15.9g,156.7mmol) was slowly added dropwise, and after stirring at room temperature for 4h, the reaction was detected by TLC and ended. The solvent was evaporated off under reduced pressure, diluted hydrochloric acid was added, filtration was carried out, the filter cake was washed with diluted hydrochloric acid for 2 times, and drying was carried out to obtain 16.5g of a white solid with a yield of 86.3%.1H NMR(400MHz,DMSO-d6),δ:8.99(s,1H),7.64(d,J＝8.7Hz,1H),6.59(s,1H),6.45(d,J＝8.8Hz,1H),3.79(s,3H),3.73(s,3H),2.01(s,3H)；MS-ESI,m/z:196.1(M+H)+。.
Step 2 preparation of N- (4-hydroxy-2-methoxy-5-propionylphenyl) acetamide (3) N- (2, 4-dimethoxyphenyl) acetamide (2) (15.0g,76.8mmol), dichloromethane (40mL) were added to a dry 250mL reaction flask, placed in an ice bath, propionyl chloride (21.3g,230.5mmol) was added, stirred vigorously, and then anhydrous aluminum trichloride (41.0g,307.4mmol) was added in portions. The temperature is raised to 43 ℃, the mixture is stirred for 3 hours, and the TLC detection reaction is finished. The mixture was quenched by dropping into crushed ice, the organic layer was separated, concentrated under reduced pressure, dilute hydrochloric acid (80mL) was added dropwise to the residue, stirred for 0.5h, filtered, the filter cake was washed with water, and dried to give the desired compound as a pale green solid 14.5g with a yield of 79.5%.1H NMR(400MHz,DMSO-d6),δ:12.57(s,1H),9.19(brs,1H),8.30(s,1H),6.58(s,1H),3.87(s,3H),2.95(q,J＝7.2Hz,2H),2.05(s,3H),1.09(t,J＝7.2Hz,3H)；MS-ESI,m/z:238.1(M+H)+。
Step 3 preparation of N- (4-hydroxy-5- (1- (hydroxyimino) propyl) -2-methoxyphenyl) acetamide (4) compound (3) (12.0g,50.6mmol), an ethanol-water mixed solvent (135mL, anhydrous ethanol: water ═ 7:3, v/v), hydroxylamine hydrochloride (7.0g,101.2mmol) and anhydrous sodium acetate (6.6g,80.9mmol) were added to a dry 250mL reaction flask, and stirred at 80 ℃ for 2 h. After the TLC detection reaction is finished, cooling is carried out, the solvent is concentrated under reduced pressure to one third of the residual volume, water (125mL) is added, the precipitated solid is filtered, washed and dried, and the target compound which is 10.5g of light green solid is obtained with the yield of 82.3%.1H NMR(400MHz,DMSO-d6),δ:11.80(s,1H),11.31(s,1H),9.07(brs,1H),7.87(s,1H),6.55(s,1H),3.80(s,3H),2.70(q,J＝7.5Hz,2H),2.03(s,3H),1.08(t,J＝7.5Hz,3H)；MS-ESI,m/z:253.1(M+H)+。
Step 4N- (3-Ethyl-6-methoxybenzo [ d ]]Preparation of isoxazol-5-yl) acetamide (5) Compound (4) (10.0g,39.6mmol) and 1, 4-dioxane (50mL) were added to a dry 250mL reaction flask and N, N-dimethylformamide dimethyl acetal (DMF-DMA) (24mL) was added dropwise with vigorous stirring. The temperature is raised to 100 ℃ and the reaction is carried out for 10 min. After the TLC detection reaction, diluted hydrochloric acid (80mL) and water (200mL) were added, the mixture was stirred, and the precipitated solid was filtered by suction, washed with water, and dried to obtain 5.1g of the objective compound as a pale green solid with a yield of 54.9%.1H NMR(400MHz,DMSO-d6),δ:9.29(brs,1H),8.27(s,1H),7.37(s,1H),3.93(s,3H),2.91(q,J＝7.6Hz,2H),2.11(s,3H),1.30(t,J＝7.6Hz,3H)；MS-ESI,m/z:235.1(M+H)+。
Step 53-Ethyl-6-methoxybenzo [ d]Preparation of isoxazol-5-amine (6) Compound (5) (3.7g,15.8mmol) and hydrochloric acid (3mol/L,95mL) were added to a 250mL reaction flask, heated and stirred at 90 ℃ for 3h, and the reaction was complete by TLC. And (3) adjusting the pH value to 7-9 by using a sodium hydroxide solution, carrying out suction filtration on the precipitated precipitate, washing with water, and drying to obtain a target compound which is 2.9g of a tawny solid, wherein the yield is 95.5%.1H NMR(400MHz,CDCl3),δ:6.92(s,1H),6.82(s,1H),3.92(s,3H),3.85(brs,2H),2.89(q,J＝7.6Hz,2H),1.38(t,J＝7.6Hz,3H)；13C NMR(101MHz,CDCl3)δ159.00,158.75,151.19,133.75,113.77,102.82,91.52,77.38,77.06,76.74,55.91,18.90,12.19；MS-ESI,m/z:193.1(M+H)+。
Step 6N- (3-Ethyl-6-methoxybenzo [ d ]]Preparation of isoxazol-5-yl) ethanesulfonamide (7a) in a dry 100mL reaction flask, dichloromethane (6mL), compound 6(60mg,0.31mmol) and ethylsulfonyl chloride (47.6mg,0.37mmol) were added, and pyridine (0.3mL) was added dropwise. The mixture was heated to reflux and stirred for 2h and the reaction was monitored by TLC. Adding diluted hydrochloric acid (10mL) and water (20mL), extracting with ethyl acetate (15 mL. times.2), washing the organic layer with water, saturated brine, drying over anhydrous sodium sulfate, concentrating under reduced pressure, and passing the residue through silicaPurifying by gel column chromatography to obtain the target compound. White solid; 54.9 percent of yield; m.p. 122-123 ℃;1H NMR(400MHz,CDCl3)δ7.77(s,1H,7-ArH),7.05(s,1H,4-ArH),6.78(brs,1H,SO2NH),3.99(s,3H,OCH3),3.04(q,J＝7.4Hz,2H,SO2CH 2CH3),2.96(q,J＝7.6Hz,2H,3-CH 2CH3),1.41(t,J＝7.6Hz,3H,3-CH2CH 3),1.34(t,J＝7.4Hz,3H,SO2CH2CH 3)；13C NMR(101MHz,CDCl3)δ161.35,159.85,152.37,123.45,114.51,112.75,92.22,56.51,45.51,18.84,12.11,8.12；LC-MS(ESI)m/z calcd for C12H16N2O4S[M+H]+:285.0831；found:285.0.
EXAMPLE 2N- (3-Ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) propane-1-sulphonamide (7b)
Prepared according to the method of example 1, using compound 6 and propylsulfonyl chloride as reactants. White solid; 39.4 percent of yield; m.p. 108-;1H NMR(400MHz,CDCl3)δ7.76(s,1H,7-ArH),7.05(s,1H,4-ArH),6.76(s,1H,SO2NH),3.99(s,3H,OCH3),3.05–2.90(m,4H,SO2CH 2,3-CH 2CH3),1.88–1.76(m,2H,SO2CH2CH 2),1.41(t,J＝7.5Hz,3H,3-CH2CH 3),0.99(t,J＝7.4Hz,3H,CH2CH2CH 3)；13C NMR(101MHz,CDCl3)δ161.35,159.87,152.40,123.49,114.53,112.75,92.22,56.52,52.94,18.85,17.20,12.92,12.11；LC-MS(ESI)m/z calcd for C13H18N2O4S[M+H]+:299.0987；found:299.1.
EXAMPLE 3N- (3-Ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) butane-1-sulphonamide (7c)
Prepared according to the method of example 1, using compound 6 and 1-butanesulfonyl chloride as reactants. White solid; 48 percent of yield; m.p. 100-;1H NMR(400MHz,CDCl3)δ7.76(s,1H,7-ArH),7.05(s,1H,4-ArH),6.76(s,1H,SO2NH),3.99(s,3H,OCH3),3.10–2.85(m,4H,SO2CH 2,3-CH 2CH3),1.85–1.75(m,2H,SO2CH2CH 2),1.51–1.28(m,5H,3-CH2CH 3,SO2CH2CH2CH 2),0.88(t,J＝7.0Hz,3H,CH2CH2CH 3)；13C NMR(101MHz,CDCl3)δ161.36,159.88,152.39,123.49,114.53,112.79,92.21,56.48,50.94,25.41,21.44,18.85,13.56,12.11；LC-MS(ESI)m/z calcd for C14H20N2O4S[M+H]+:313.1144；found:313.1.
example 4N- (3-Ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) benzenesulfonamide (7d)
Prepared according to the method of example 1, using compound 6 as a reactant with benzenesulfonyl chloride. White solid; 47.8 percent of yield; 169-170 ℃ in m.p.;1H NMR(400MHz,CDCl3)δ7.78(s,1H,ArH),7.74–7.56(m,2H,phenyl ArH),7.56–7.30(m,3H,phenyl ArH),6.91(brs,1H,SO2NH),6.81(s,1H,4-ArH),3.62(s,3H,OCH3),2.97(d,J＝5.5Hz,2H,3-CH 2CH3),1.42(s,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ161.74,159.90,153.16,138.80,133.05,128.77(2×C),127.22(2×C),122.92,114.87,114.36,91.94,56.12,18.88,12.121；LC-MS(ESI)m/z calcd for C16H16N2O4S[M+H]+:333.0831；found:333.0.
EXAMPLE 5N- (3-Ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2-fluorobenzenesulfonamide (7e)
Prepared according to the method of example 1, using compound 6 and 2-fluorobenzenesulfonyl chloride as reactants. White solid; 54.4 percent of yield; m.p. 179-180 ℃;1H NMR(400MHz,CDCl3)δ7.73(d,J＝7.4Hz,2H,6'-ArH,7-ArH),7.58–7.43(m,1H,4'-ArH),7.29(brs,1H,SO2NH),7.20–7.07(m,2H,3',5'-ArH),6.85(s,1H,4-ArH),3.77(s,3H,OCH3),2.94(q,J＝7.5Hz,2H,3-CH 2CH3),1.39(t,J＝7.5Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ161.60,160.36(d,J＝257.6Hz,F-2'-ArC),159.83,157.81(d,J＝257.6Hz,F-2'-ArC),153.03,135.54(d,J＝9.1Hz,F-4'-ArC),135.45(d,J＝9.1Hz,F-4'-ArC),130.90,126.83(d,J＝13.1Hz,F-1'-ArC),126.70(d,J＝13.1Hz,F-1'-ArC),124.17(d,J＝4.0Hz,F-6'-ArC),124.13(d,J＝4.0Hz,F-6'-ArC),122.48,116.87(d,J＝21.2Hz,F-3'-ArC),116.66(d,J＝21.2Hz,F-3'-ArC),114.26,114.23,91.90,56.18,18.82,12.09；LC-MS(ESI)m/z calcd for C16H15FN2O4S[M+H]+:351.0737；found:351.0.
example 62-chloro-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) benzenesulfonamide (7f)
Prepared according to the method of example 1, using compound 6 and 2-chlorobenzenesulfonyl chloride as reactants. White solid; 54.4 percent of yield; m.p. 121-122 ℃;1H NMR(400MHz,CDCl3)δ7.93(dd,J＝7.9,1.3Hz,1H,6'-ArH,),7.70(s,1H,7-ArH),7.55(brs,1H,SO2NH),7.51–7.46(m,1H,3'-ArH),7.43(td,J＝7.7,1.4Hz,1H,4'-ArH),7.30–7.24(m,1H,5'-ArH),6.85(s,1H,4-ArH),3.78(s,3H,OCH3),2.93(q,J＝7.6Hz,2H,3-CH 2CH3),1.38(t,J＝7.6Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ161.45,159.78,152.83,136.24,134.10,132.06,131.81,131.61,126.81,122.62,114.19,113.57,91.93,56.13,18.81,12.10；LC-MS(ESI)m/z calcd for C16H15ClN2O4S[M+H]+:367.0441；found:367.0.
example 72-bromo-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) benzenesulfonamide (7g)
Prepared according to the method of example 1, using compound 6 and 2-bromobenzenesulfonyl chloride as reactants. White solid; 64.7 percent of yield; 141 ℃ in m.p. and 143 ℃;1H NMR(400MHz,CDCl3)δ8.00–7.93(m,1H,3'-ArH),7.73–7.67(m,2H,7-ArH,6'-ArH),7.64(brs,1H,SO2NH),7.38–7.28(m,2H,4',5'-ArH),6.85(s,1H,4-ArH),3.78(s,3H,OCH3),2.93(q,J＝7.6Hz,2H,3-CH 2CH3),1.38(t,J＝7.6Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ161.46,159.78,152.84,137.96,135.14,134.04,132.04,127.39,122.63,120.31,114.18,113.61,91.94,56.08,18.82,12.11；LC-MS(ESI)m/z calcd for C16H15BrN2O4S[M+H]+:410.9936；found:411.0.
EXAMPLE 8N- (3-Ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2-methoxybenzenesulphonamide (7h)
Prepared according to the method of example 1, using compound 6 as a reactant with 2-methoxybenzenesulfonyl chloride. White solid; 71.7 percent of yield; 142 ℃ and 143 ℃ in m.p.;1H NMR(400MHz,CDCl3)δ7.82(d,J＝7.6Hz,1H,6'-ArH),7.72(s,1H,7-ArH),7.59(brs,1H,SO2NH),7.45(t,J＝7.7Hz,1H,4'-ArH),7.00–6.90(m,2H,3',5'-ArH),6.86(s,1H,4-ArH),3.96(s,3H,2'-OCH3),3.83(s,3H,6-OCH3),2.91(q,J＝7.5Hz,2H,3-CH 2CH3),1.36(t,J＝7.5Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ160.91,159.81,156.45,152.21,135.05,130.94,126.20,123.72,120.27,114.21,111.97,111.80,91.81,56.35,56.10,18.79,12.10；LC-MS(ESI)m/z calcd for C17H18N2O5S[M+H]+:363.0936；found:363.1.
example 9N- (3-Ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2- (trifluoromethoxy) benzenesulfonamide (7i)
Prepared according to the method of example 1, using compound 6 and 2- (trifluoromethoxy) benzenesulfonyl chloride as reactants. White solid; 67.9 percent of yield; m.p. 92-93 ℃;1H NMR(400MHz,CDCl3)δ7.90(dd,J＝7.9,1.5Hz,1H,6'-ArH),7.73(s,1H,7-ArH),7.55(td,J＝8.4,1.6Hz,1H,4'-ArH),7.38–7.30(m,2H,SO2NH,3'-ArH),7.26(td,J＝8.0,0.8Hz,1H,5'-ArH),6.87(s,1H,4-ArH),3.79(s,3H,OCH3),2.93(q,J＝7.6Hz,2H,3-CH 2CH3),1.38(t,J＝7.6Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ161.43,159.82,152.61,146.22,134.92,131.69,130.09,125.90,124.16(q,J＝262.6Hz,OCF3),122.56,121.56(q,J＝262.6Hz,OCF3),118.97(q,J＝262.6Hz,OCF3),118.95,116.37(q,J＝262.6Hz,OCF3),114.24,113.24,91.89,56.13,18.81,12.10；LC-MS(ESI)m/z calcd for C17H15F3N2O5S[M+H]+:417.0654；found:417.0.
example 103-chloro-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) benzenesulfonamide (7j)
Prepared according to the method of example 1, using compound 6 and 3-chlorobenzenesulfonyl chloride as reactants. White solid; 53.5 percent of yield; 152-153 ℃ m.p.;1H NMR(400MHz,CDCl3)δ7.77(s,1H,7-ArH),7.73(s,1H,2'-ArH),7.51(d,J＝7.8Hz,1H,6'-ArH),7.47(d,J＝8.1Hz,1H,4'-ArH),7.30(t,J＝7.9Hz,1H,5'-ArH),6.95(brs,1H,SO2NH),6.85(s,1H,4-ArH),3.68(s,3H,OCH3),2.98(q,J＝7.6Hz,2H,3-CH 2CH3),1.43(t,J＝7.6Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ161.91,159.89,153.22,140.49,135.01,133.12,130.00,127.35,125.39,122.34,115.35,114.45,92.06,56.19,18.87,12.12；LC-MS(ESI)m/z calcd for C16H15ClN2O4S[M+H]+:367.0441；found:367.0.
example 113-bromo-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) benzenesulfonamide (7k)
Prepared according to the method of example 1, using compound 6 and 3-bromobenzenesulfonyl chloride as reactants. White solid; yield 70.7%; m.p. 160-;1H NMR(400MHz,CDCl3)δ7.89(s,1H,2'-ArH),7.77(s,1H,7-ArH),7.62(d,J＝7.8Hz,1H,6'-ArH),7.55(d,J＝7.7Hz,1H,4'-ArH),7.23(t,J＝8.0Hz,1H,5'-ArH),6.96(brs,1H,SO2NH),6.85(s,1H,4-ArH),3.68(s,3H,OCH3),2.98(q,J＝7.5Hz,2H,3-CH 2CH3),1.43(t,J＝7.5Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ161.93,159.88,153.25,140.61,136.00,130.20,130.19,125.82,122.71,122.31,115.45,114.44,92.06,56.21,18.87,12.13；LC-MS(ESI)m/z calcd for C16H15BrN2O4S[M+H]+:410.9936；found:411.0.
example 125-chloro-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2-methoxybenzenesulphonamide (7l)
Prepared according to the method of example 1, using compound 6 as a reactant with 2-methoxybenzenesulfonyl chloride. White solid; 71.4 percent of yield; 157 < 157- > 158 ℃;1H NMR(400MHz,CDCl3)δ7.80(d,J＝2.4Hz,1H,6'-ArH),7.72(s,1H,7-ArH),7.57(brs,1H,SO2NH),7.40(dd,J＝8.8,2.4Hz,1H,4'-ArH),6.94–6.84(m,2H,4-ArH,3'-ArH),3.95(s,3H,2'-OCH3),3.84(s,3H,6-OCH3),2.94(q,J＝7.6Hz,2H,3-CH 2CH3),1.38(t,J＝7.6Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ161.07,159.85,155.03,152.24,134.64,130.52,127.69,125.57,123.21,114.34,113.20,112.46,91.96,56.50,56.39,18.81,12.10；LC-MS(ESI)m/z calcd for C17H17ClN2O5S[M+H]+:397.0547；found:397.0.
example 135-bromo-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2-methoxybenzenesulphonamide (7m)
Prepared according to the method of example 1, using compound 6 and 5-bromo-2-methoxybenzenesulfonyl chloride as reactants. White solid; 43.5 percent of yield; 167-;1H NMR(400MHz,CDCl3)δ7.94(d,J＝2.4Hz,1H,6'-ArH),7.72(s,1H,7-ArH),7.60–7.50(m,2H,SO2NH,4'-ArH),6.89(s,1H,4-ArH),6.83(d,J＝8.8Hz,1H,3'-ArH),3.94(s,3H,2'-OCH3),3.84(s,3H,6-OCH3),2.94(q,J＝7.6Hz,2H,3-CH 2CH3),1.39(t,J＝7.6Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ161.07,159.87,155.53,152.21,137.59,133.31,128.05,123.21,114.38,113.63,112.43(2×C),91.98,56.46,56.40,18.84,12.15；LC-MS(ESI)m/z calcd for C17H17BrN2O5S[M+H]+:441.0042；found:441.0.
example 145-bromo-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2, 3-dihydrobenzofuran-7-sulphonamide (7N)
Prepared according to the method of example 1, using compound 6 and 5-bromobenzene-2, 3-dihydrobenzofuran-7-sulfonyl chloride as reactants. White solid; 84.0 percent of yield; 152-153 ℃ m.p.;1H NMR(400MHz,CDCl3)δ7.70(s,1H,7-ArH),7.64(d,J＝1.5Hz,1H,6'-ArH),7.41(brs,1H,SO2NH),7.39(d,J＝1.5Hz,1H,4'-ArH),6.90(s,1H,4-ArH),4.70(t,J＝8.8Hz,2H,OCH 2CH2),3.84(s,3H,OCH3),3.21(t,J＝8.8Hz,2H,OCH2CH 2),2.94(q,J＝7.6Hz,2H,3-CH 2CH3),1.39(t,J＝7.6Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ161.11,159.87,156.13,152.23,132.86,131.98,130.16,123.08,121.76,114.26,112.09,111.71,91.98,73.60,56.36,28.88,18.83,12.16；LC-MS(ESI)m/z calcd for C18H17BrN2O5S[M+H]+:453.0042；found:453.0.
EXAMPLE 15N- (3-Ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -4-fluorobenzenesulfonamide (7o)
Prepared according to the method of example 1, using compound 6 and 4-fluorobenzenesulfonyl chloride as reactants. White solid; 61.0 percent of yield; m.p. 140 ℃ and 142 ℃;1H NMR(400MHz,CDCl3)δ7.78(s,1H,7-ArH),7.73–7.64(m,2H,2',6'-ArH),7.10–7.00(m,2H,3',5'-ArH),6.91(brs,1H,SO2NH),6.85(s,1H,4-ArH),3.67(s,3H,OCH3),2.97(q,J＝7.6Hz,2H,3-CH 2CH3),1.42(t,J＝7.6Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ166.52,163.98,161.85(d,J＝199.0Hz,F-4'-ArC),159.88(d,J＝199.0Hz,F-4'-ArC),153.18,134.89(d,J＝4.0Hz,F-1'-ArC),134.85(d,J＝4.0Hz,F-1'-ArC),130.03(d,J＝9.1Hz,F-3',5'-ArC),129.94(d,J＝9.1Hz,F-2',6'-ArC),122.64,116.14(d,J＝22.2Hz,F-2',6'-ArC),115.92(d,J＝22.2Hz,F-3',5'-ArC),115.19,114.46,92.02,56.16,18.87,12.11；LC-MS(ESI)m/z calcd for C16H15FN2O4S[M+H]+:351.0737；found:351.0.
example 164-chloro-N- (3-ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) benzenesulfonamide (7p)
Prepared according to the method of example 1, using compound 6 and 4-chlorobenzenesulfonyl chloride as reactants. White solid; 55.3 percent of yield; 177 ℃ and 178 ℃ in m.p.;1H NMR(400MHz,CDCl3)δ7.78(s,1H,7-ArH),7.60(d,J＝7.4Hz,2H,2',6'-ArH),7.35(d,J＝7.4Hz,2H,3',5'-ArH),6.92(brs,1H,SO2NH),6.85(s,1H,4-ArH),3.66(s,3H,OCH3),2.98(d,J＝7.1Hz,2H,3-CH 2CH3),1.43(d,J＝6.7Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ161.84,159.86,153.15,139.62,137.31,129.07(2×C),128.66(2×C),122.51,115.18,114.45,92.07,56.18,18.88,12.11；LC-MS(ESI)m/z calcd for C16H15ClN2O4S[M+H]+:367.0441；found:367.0.
EXAMPLE 17N- (3-Ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -4-nitrobenzenesulfonamide (7q)
Prepared according to the method of example 1, using compound 6 and p-nitrobenzenesulfonyl chloride as reactants. White solid; 71.4 percent of yield; m.p. 168-169 ℃;1H NMR(400MHz,CDCl3)δ8.23(d,J＝8.8Hz,2H,3',5'-ArH),7.87(d,J＝8.8Hz,2H,2',6'-ArH),7.82(s,1H,7-ArH),7.02(brs,1H,SO2NH),6.86(s,1H,4-ArH),3.66(s,3H,OCH3),2.99(q,J＝7.6Hz,2H,3-CH 2CH3),1.44(t,J＝7.6Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ162.02,159.83,153.11,150.24,144.62,128.52(2×C),123.99(2×C),121.77,115.71,114.62,92.26,56.24,18.88,12.10；LC-MS(ESI)m/z calcd for C16H15N3O6S[M+H]+:378.0682；found:378.0.
EXAMPLE 18N- (3-Ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -4-methoxybenzenesulphonamide (7r)
Prepared according to the method of example 1, using compound 6 as a reactant with p-methoxybenzenesulfonyl chloride. White solid; 85.0 percent of yield; m.p. 145-146 ℃;1H NMR(400MHz,CDCl3)δ7.77(s,1H,7-ArH),7.61(d,J＝8.9Hz,2H,2',6'-ArH),6.92(brs,1H,SO2NH),6.85–6.79(m,3H,4-ArH,3',5'-ArH),3.79(s,3H,6-OCH3),3.68(s,3H,4'-OCH3),2.97(q,J＝7.6Hz,2H,3-CH 2CH3),1.42(t,J＝7.6Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ163.13,161.58,159.88,153.01,130.33,129.41(2×C),123.24,114.29,114.22,113.89(2×C),91.91,56.18,55.59,18.86,12.13；LC-MS(ESI)m/z calcd for C17H18N2O5S[M+H]+:363.0936；found:363.1.
EXAMPLE 19N- (3-Ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -4- (trifluoromethoxy) benzenesulfonamide (7s)
Prepared according to the method of example 1, using compound 6 and 4- (trifluoromethoxy) benzenesulfonyl chloride as reactants. White solid; 83.1 percent of yield; m.p. 127-;1H NMR(400MHz,CDCl3)δ7.79(s,1H,7-ArH),7.71(d,J＝8.8Hz,2H,2',6'-ArH),7.21(d,J＝8.4Hz,2H,3',5'-ArH),6.88(brs,1H,SO2NH),6.84(s,1H,4-ArH),3.62(s,3H,OCH3),2.98(q,J＝7.6Hz,2H,3-CH 2CH3),1.43(t,J＝7.6Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ162.00,159.89,153.38,152.33,137.19,129.38(2×C),124.03(q,J＝260.6Hz,OCF3),122.33,121.45(q,J＝260.6Hz,OCF3),120.64(2×C),118.87(q,J＝260.6Hz,OCF3),116.29(q,J＝260.6Hz,OCF3),115.91,114.52,92.03,56.04,18.88,12.09；LC-MS(ESI)m/z calcd for C17H15F3N2O5S[M+H]+:417.0654；found:417.0.
EXAMPLE 20N- (3-Ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2, 4-dimethoxybenzenesulfonamide (7t)
Prepared by the method of reference example 1, using compound 6 and 2, 4-dimethoxybenzeneSulfonyl chloride as a reactant. White solid; 78.2 percent of yield; m.p. 151-;1H NMR(400MHz,CDCl3)δ7.80–7.72(m,1H,6'-ArH),7.70(s,1H,7-ArH),7.53(brs,1H,SO2NH),6.87(s,1H,4-ArH),6.45–6.35(m,2H,3',5'-ArH),3.90(s,3H,2'-OCH3),3.86(s,3H,6-OCH3),3.78(s,3H,4'-OCH3),2.91(q,J＝7.6Hz,2H,3-CH 2CH3),1.36(t,J＝7.6Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ165.12,160.80,159.82,157.94,152.08,132.80,124.03,118.28,114.19,111.47,103.99,99.21,91.78,56.37,56.05,55.68,18.80,12.13；LC-MS(ESI)m/z calcd for C18H20N2O6S[M+H]+:393.1042；found:393.1.
EXAMPLE 21N- (3-Ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2-methoxy-4-nitrobenzenesulfonamide (7u)
Prepared according to the method of example 1, using compound 6 and 2-methoxy-4-nitrobenzenesulfonyl chloride as reactants. White solid; 57.7 percent of yield; m.p. 215-;1H NMR(400MHz,CDCl3)δ7.99(d,J＝8.7Hz,1H,6'-ArH),7.85–7.77(m,2H,3',5'-ArH),7.74(s,1H,7-ArH),7.59(brs,1H,SO2NH),6.89(s,1H,4-ArH),4.10(s,3H,2'-OCH3),3.84(s,3H,6-OCH3),2.93(q,J＝7.6Hz,2H,3-CH 2CH3),1.38(t,J＝7.6Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ161.28,159.76,157.03,152.38,151.73,132.38,131.82,122.60,115.17,114.46,113.21,107.04,92.12,56.99,56.49,18.81,12.08；LC-MS(ESI)m/z calcd for C17H17N3O7S[M+H]+:408.0787；found:408.0.
EXAMPLE 22N- (3-Ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -3-fluoro-4-methoxybenzenesulphonamide (7v)
Prepared according to the method of example 1, using compound 6 and 3-fluoro-4-methoxybenzenesulfonyl chloride as reactants. White solid; 74.6 percent of yield; m.p. 203-;1H NMR(400MHz,CDCl3)δ7.76(s,1H,7-ArH),7.55–7.33(m,2H,2',6'-ArH),6.94(brs,1H,SO2NH),6.91–6.80(m,2H,4-ArH,5'-ArH),3.88(s,3H,4'-OCH3),3.73(s,3H,6-OCH3),2.97(q,J＝7.6Hz,2H,3-CH 2CH3),1.42(t,J＝7.4Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ161.68,159.88,152.96,152.70(d,J＝252.5Hz,F-3'-ArC),151.74(d,J＝10.1Hz,F-4'-ArC),151.64(d,J＝10.1Hz,F-4'-ArC),150.20(d,J＝252.5Hz,F-3'-ArC),130.65(d,J＝6.1Hz,F-1'-ArC),130.59(d,J＝6.1Hz,F-1'-ArC),124.73(d,J＝4.0Hz,F-5'-ArC),124.69(d,J＝4.0Hz,F-5'-ArC),122.82,115.39(d,J＝21.2Hz,F-2'-ArC),115.18(d,J＝21.2Hz,F-2'-ArC),114.40,112.38,112.36,92.05,56.40,56.26,18.88,12.13；LC-MS(ESI)m/z calcd for C17H17FN2O5S[M+H]+:381.0842；found:381.0.
EXAMPLE 23N- (3-Ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -3, 4-dimethoxybenzenesulfonamide (7w)
Prepared according to the method of example 1, using compound 6 and 3, 4-dimethoxybenzenesulfonyl chloride as reactants. White solid; 57.9 percent of yield; m.p. 144-145 ℃;1H NMR(400MHz,CDCl3)δ7.78(s,1H,7-ArH),7.31–7.26(m,1H,5'-ArH),7.14(d,J＝2.1Hz,1H,2'-ArH),6.95(brs,1H,SO2NH),6.84(s,1H,4-ArH),6.77(d,J＝8.5Hz,1H,6'-ArH),3.86(s,3H,6-OCH3),3.76(s,3H,3'-OCH3),3.69(s,3H,4'-OCH3),2.96(q,J＝7.6Hz,2H,3-CH 2CH3),1.41(t,J＝7.6Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ161.58,159.83,153.05,152.78,148.79,130.32,123.27,121.36,114.25,114.21,110.10,109.48,91.94,56.22,56.15,56.10,18.87,12.17；LC-MS(ESI)m/z calcd for C18H20N2O6S[M+H]+:393.1042；found:393.1.
EXAMPLE 24N- (3-Ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) -2, 3-dihydrobenzofuran-5-sulphonamide (7X)
Prepared by the method of reference example 1 using compound 6 and 2, 3-dihydrobenzofuran-5-sulfonylChlorine as a reactant. White solid; 58.3 percent of yield; m.p. 207 ℃ and 208 ℃;1H NMR(400MHz,CDCl3)δ7.75(s,1H,7-ArH),7.54–7.46(m,2H,2',6'-ArH),6.92(brs,1H,SO2NH),6.86(s,1H,4-ArH),6.70(d,J＝8.2Hz,1H,3'-ArH),4.61(t,J＝8.8Hz,2H,OCH 2CH2),3.15(t,J＝8.8Hz,2H,OCH2CH 2),2.97(q,J＝7.6Hz,2H,3-CH 2CH3),1.41(t,J＝7.6Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ164.09,161.47,159.87,152.87,130.32,128.94,128.00,124.57,123.39,114.29,113.72,109.28,91.93,72.28,56.23,28.93,18.88,12.15；LC-MS(ESI)m/z calcd for C18H18N2O5S[M+H]+:375.0936；found:375.1.
example 25N- (3-Ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) thiophene-2-sulfonamide (7y)
Prepared according to the method of example 1, using compound 6 as a reactant with thiophene-2-sulfonyl chloride. White solid; 71.4 percent of yield; 171 and 172 ℃ in m.p.;1H NMR(400MHz,CDCl3)δ7.82(s,1H,7-ArH),7.50(dd,J＝5.0,1.2Hz,1H,thiophen 5-H),7.40(dd,J＝3.7,1.2Hz,1H,thiophen 3-H),7.04(brs,1H,SO2NH),6.96(t,J＝4.0Hz,1H,thiophen 4-H),6.88(s,1H,4-ArH),3.72(s,3H,OCH3),2.98(q,J＝7.6Hz,2H,3-CH 2CH3),1.43(t,J＝7.6Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ161.79,159.93,153.21,139.10,132.70,132.51,127.16,122.79,114.66,114.35,91.98,56.24,18.87,12.14；LC-MS(ESI)m/z calcd for C14H14N2O4S2[M+H]+:339.0395；found:339.0.
example 26N- (3-Ethyl-6-methoxybenzo [ d ] isoxazol-5-yl) pyridine-3-sulphonamide (7z)
Prepared according to the method of example 1, using compound 6 as a reactant with pyridine-3-sulfonyl chloride. White solid; 69.2 percent of yield; m.p. 171-173 ℃;1H NMR(400MHz,CDCl3)δ8.91(s,1H,pyridine 2-H),8.72(d,J＝4.0Hz,1H,pyridine 4-H),7.92(d,J＝8.1Hz,1H,pyridine 6-H),7.81(s,1H,7-ArH),7.38–7.30(m,1H,pyridine 5-H),7.12(s,1H,SO2NH),6.84(s,1H,4-ArH),3.63(s,3H,OCH3),2.98(q,J＝7.6Hz,2H,3-CH 2CH3),1.43(t,J＝7.6Hz,3H,3-CH2CH 3)；13C NMR(101MHz,CDCl3)δ162.09,159.85,153.33,153.20(2×C),147.92,135.06,123.41,121.88,116.21,114.57,92.18,56.19,18.86,12.09；LC-MS(ESI)m/z calcd for C15H15N3O4S[M+H]+:334.0783；found:334.0.
example 27 BRD4 protein inhibitory Activity test (TSA experiment)
The live PCR system from Bio-Rad CFX96 was used for the thermomigration analysis. Add 10. mu.L of a biochemical reaction containing 1. mu.L BRD4 BD1 protein (100. mu.M), 1. mu.L of fluorescent dye (100 fold dilution, SYPRO Orange, ABI, Sigma) and 1. mu.L of buffer (100mM HEPES, 1500mM NaCl, 50% (v/v) glycerol, pH 7.5), 3. mu.L deionized water and 4. mu.L of compound (500. mu.M). The reaction mixture was added to a 96-well plate, filmed, centrifuged at 1000r/min for 1 min at room temperature, and then incubated on ice for 30 min in the dark. And (3) putting the incubated 96-well reaction plate into a real-time quantitative PCR instrument, setting excitation and emission wavelengths of SYPRO Orange dye at 465nm and 590nm respectively, setting a programmed temperature rise range at 30-80 ℃, setting a temperature change interval at 0.3 ℃, detecting once every 5 seconds, and storing a file. Melting temperature (T) was calculated by fitting melting curves using GraphPad Prismm)。ΔTmT representing test and blank reactionsmThe difference in value. All experiments were tested using three replicate wells. The results are shown in Table 2. Protein expression and purification were carried out according to the prior art, and the plasmid cDNA (pET24a-H6-Thr-BRD4(1) (residues N44-E168)) used was synthesized by Kinsrui Biotech, Inc., Nanjing.
TABLE 2 TSA method for determining the binding Activity of a Compound to BRD4(1) protein
Note: the control drug was selected from the optimal compounds in CN105085427A (example 37 compound), which is also compound 6i in Journal of Medicinal Chemistry,2018,61(7): 3037-3058.
Biological activity test results show that the compound of the general formula (I) provided by the invention has a good BRD4 protein binding effect (inhibition effect), and the binding activity of most compounds to BRD4 reaches or is superior to that of a positive control compound.
Example 28 CellTiter method tumor cell inhibitory Activity test
MV4-11 cells (ATCC, Rockville, Md., USA) in IMDM containing 10% FBS and 1% penicillin-streptomycin (Wikipedia Co., Ltd.) at 37 ℃ in the presence of 5% CO2The humidified incubator of (1) for cultivation. Cells were seeded in 384-well plates at 1500 cells per well, 20 μ L of medium was added and cultured for 12 hours. Different concentrations of diluted compounds or DMSO controls were added to the wells in a volume of 10 μ L, at a final concentration of 5nM to 100 μ M, and incubated for 96 hours. Then, 25. mu.L of CellTiter-GLO reagent (Promega) was added to each well and mixed on an orbital shaker for 10 minutes to induce cell lysis. The lysate was incubated for an additional 10 minutes and centrifuged for 1 minute. Luminescence was measured on a GLOMAX microplate luminometer (Promega) according to the manufacturer's instructions. Each concentration point was performed in triplicate. Fluorescence signals were normalized to DMSO-treated cells and inhibition curves and ICs were calculated by nonlinear regression and dose response inhibition equation analysis using GraphPad Prism 7 software50The value is obtained.
The results of the cell activity test showed that other compounds IC were present in addition to 7q and 7u50Are less than 10 mu M, and 7q and 7u are less than 35 mu M, so the compound of the general formula (I) provided by the invention has good leukemia tumor cell inhibition effect. The inhibition curves for a portion of representative compounds are shown in FIG. 1, where the IC for compound 7r is50The value was 0.39. mu.M, which is superior to the conversion in the control compound (Journal of Medicinal Chemistry,2018,61(7):3037-3058)Compound 6i, IC50The value was 0.69. mu.M).
Example 29 test for oncogene inhibitory Activity
MV4-11 cells in logarithmic growth phase were treated with different concentrations of compounds and cultured for 48 h. Total RNA was extracted by TRIzol kit (Beyotime). Before the experiment, the concentration of the sample was measured by Nanodrop 2000(Thermo fisher). A20. mu.L system was used for cDNA synthesis, comprising 2. mu.L of RNA, 4. mu.L of a 5 XiScript reaction mixture (Bio-Rad, Hercules, Calif., USA), 1. mu.L of iScript reverse transcriptase (Bio-Rad) and 13. mu.L of nuclease-free water. Using Applied BiosystemsReverse transcription was performed by a 96-well thermal cycler instrument (Life Technology), and the reaction was performed at 25 ℃ for 5 minutes, 42 ℃ for 30 minutes, and 85 ℃ for 5 minutes to obtain cDNA. Then, qRT-PCR detection was performed on the LightCycler 480Real-Time PCR System (Roche Applied Science) with a reaction System of 20. mu.L including 1. mu.L cDNA, 7.4. mu.L Nuclear-free water, 10. mu.L SsofgastaGreenSupermix (Bio-Rad, Hercules, Calif., USA), 0.8. mu.L forward primer, 0.8. mu.L reverse primer. Target mRNA expression was quantified using the 2- Δ Δ Ct method and normalized to GAPDH expression. The qPCR primers used (synthesized by seiry biotechnology limited, tokyo) had the following sequences:
c-Myc_fwd：5'-CACTAACATCCCACGCTCTGA-3'(SEQ No.1)
c-Myc_rev：5’-AAATCATCGCAGGCGGAACA-3’(SEQ No.2)
GAPDH_fwd：5'-AATGGGCAGCCGTTAGGAAA-3'(SEQ No.3)
GAPDH_rev：5'-GCGCCCAATACGACCAAATC-3'(SEQ No.4)
the test results are shown in fig. 2. As can be seen from FIG. 2, after treating leukemia MV4-11 cells with different concentrations of compound 7r (1. mu.M, 5. mu.M, 10. mu.M), compound 7r can effectively inhibit the transcription of oncogene c-Myc in the cells, and the inhibition effect is enhanced with the increase of compound concentration. Each administration group had significant differences compared to the blank control group. This experiment further elucidated the anti-tumor mechanism and effect of the compounds.
And (4) conclusion: the compound of the general formula (I), particularly the compound 7r, provided by the invention has a remarkable inhibition effect on an important oncogene c-Myc in a leukemia tumor cell MV 4-11.
Sequence listing
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