CN110343109B

CN110343109B - Dihydropyridone-sulfonamide derivative, pharmaceutically acceptable salt thereof, preparation method and application thereof

Info

Publication number: CN110343109B
Application number: CN201910771932.9A
Authority: CN
Inventors: 刘宏民; 孙凯; 郑一超; 李雯; 赵兵; 冯雪建; 符运栋; 宋启梦
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2019-08-21
Filing date: 2019-08-21
Publication date: 2021-11-23
Anticipated expiration: 2039-08-21
Also published as: CN110343109A

Abstract

Aiming at the problems in the prior art, the invention provides a dihydropteridinone-sulfonamide derivative, a preparation method and application thereof, and provides a new choice for research and development of an anti-tumor drug in the aspect of inhibiting bromodomain-containing protein BRD 4. Activity experiments prove that the novel compound with the dihydropteridinone structure has good BRD4 protein inhibition effect, can inhibit tumor cell proliferation, and particularly aims at gastric cancer cells, and the inhibition activity of part of compounds reaches or is superior to that of a positive control compound JQ 1. The invention provides a new drug choice for treating BRD4 inhibitor, tumor and other diseases.

Description

Dihydropyridone-sulfonamide derivative, pharmaceutically acceptable salt thereof, preparation method and application thereof

Technical Field

The invention relates to the field of medicinal chemistry, in particular to a dihydropteridinone-sulfonamide derivative, a pharmaceutically acceptable salt thereof, a preparation method and application thereof.

Background

Malignant tumor is the first major disease threatening the health of people in China. The national cancer center data shows that at present, 7.5 people per minute in China are diagnosed with cancer. Therefore, the research of the antitumor drug and the related target inhibitors thereof has important clinical significance and social value.

Brd (bromodomain), also known as bromodomain protein, is a conserved protein domain that specifically recognizes acetylated lysine residues, and plays a key role in regulation of chromatin assembly and gene transcription. Multiple studies prove that BRD is a highly adaptable drug target, and small-molecule inhibitors acting on BRD have wide prospects in tumor treatment.

BRD4 is a member of the bromodomain protein family, and its biological function is most widely and extensively studied. BRD4 recognizes not only promoter regions, but also intergenic and intragenic regions, which are involved in gene expression. The BRD4 protein performs a transcriptional regulatory function in physiological conditions, regulating normal cellular processes; while in abnormal states, they recruit various proteins to chromatin and transcription sites, regulating the transcription of genes closely related to gene expression, such as c-Myc and BCL-2. BRD4 recruits proteins at the intercellular level in a positive transcription elongation factor (P-TEFb) dependent manner, and regulates the transcription process by coupling with RNA polymerase II, thereby promoting abnormal expression of disease-related genes.

A great deal of research shows that the BRD4 protein has a close relationship with the occurrence and development of tumors, for example, the Bromodomain coding region of BRD4 and NUT (nucleoprotein in testis) gene are ectopic to form BRD-NUT fusion type proto-oncogene, which can cause midline cancer; inhibition of BRD4 expression in melanoma significantly slows the growth of melanoma cells; inhibiting expression of BRD4 in malignant peripheral nerve sheath tumor cells, resulting in death of the tumor cells; inhibition of BRD4 can inhibit proliferation of hepatocarcinoma cells; by interfering with the binding of BRD4 to the oncogene MYC, silencing of the oncogene MYC can be directly caused in models of various hematopoietic tumors, such as AML, Burkitt's lymphoma, multiple myeloma, and B-cell acute lymphocytic leukemia. Bromodomain proteins can regulate the assembly of histone acetylation-dependent chromatin complexes expressed by inflammatory genes and regulate transcription of inflammatory genes. Research has shown that BRD4 can act as a transcriptional co-activator of NF-Kb to promote inflammation. In the field of cardiovascular diseases, BRD4 has also been found to play a key co-activator of pathological gene transactivation during cardiomyocyte hypertrophy, playing an important role in heart failure; the BRD4 small molecule inhibitor JQ1 can be a potential drug for treating heart failure and improve the cardiac function.

The potential value of the bromodomain protein family in anti-inflammatory and anti-tumor aspects has attracted high attention of various pharmaceutical companies, colleges and scientific research institutions all over the world, and BRD4 also becomes an important target in related fields. The BRD4 is taken as a target, and a small molecule inhibitor with high activity and high selectivity is found to become a research hotspot for treating diseases such as tumor, inflammation, cardiovascular disease, AIDS and the like at present, so that the BRD4 has wide development and application prospects.

At present, various BRD4 small molecule inhibitors represented by JQ1 are reported, and related compounds OTX-015, I-BET762, TEN-010, CPI-0610, RVX-208 and the like of the inhibitors are researched in clinical stage and are used for treating acute leukemia, other blood malignant tumors, advanced solid tumors, glioblastoma, lymphoma, myeloma, midline cancer, breast cancer, coronary heart disease, arteriosclerosis, non-small cell lung cancer and other tumors and malignant diseases. However, the number of BRD4 small-molecule inhibitors currently entering clinical research is few, and there is an urgent need to find more novel and efficient BRD4 inhibitors, which provide new options for treatment of diseases such as tumors.

The applicant reports in Chinese patent CN109503586A that a novel dihydropteridinone BRD4 protein inhibitor has the structural characteristic of hydrazone bond, and the inhibitory activity on BRD4 protein can reach IC₅₀0.24. mu.M. In order to further improve the activity of the BRD4 inhibitor and provide a new choice of antitumor drugs, the applicant carries out further research on the compounds.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a dihydropteridinone-sulfonamide derivative, a preparation method and application thereof, and provides a new choice for research and development of an anti-tumor drug in the aspect of inhibiting bromodomain-containing protein BRD 4.

The technical scheme adopted by the invention is as follows:

a dihydropteridinone-sulfonamide derivative has a structure shown in a general formula (I):

wherein R is¹Is a saturated hydrocarbonA group, aryl or heterocyclic group;

the saturated hydrocarbon group is a straight chain or branched chain saturated hydrocarbon group with 1-6 carbon atoms, a cyclic saturated hydrocarbon group with 3-6 carbon atoms, or a cyclic saturated hydrocarbon group with 3-6 carbon atoms, wherein the straight chain or branched chain saturated hydrocarbon group with 1-6 carbon atoms is connected;

the aryl is any one of pure phenyl, benzyl and naphthyl which are substituted by 1, 2 or 3 first substituents;

the heterocyclic group is any one of piperidyl, pyrrolyl, pyrazolyl, imidazolyl, furyl, indolyl, morpholinyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, piperazinyl, substituted piperazinyl, pyrazinyl and pyridazinyl which are pure and substituted by 1, 2 or 3 first substituents;

R²is hydrogen, alkoxy, aryloxy or heterocyclyl;

the alkyl group in the alkoxy group is a straight or branched chain saturated hydrocarbon group of 1 to 6 carbon atoms, a cyclic saturated hydrocarbon group of 3 to 6 carbon atoms, or a cyclic saturated hydrocarbon group of 3 to 6 carbon atoms to which a straight or branched chain saturated hydrocarbon group of 1 to 6 carbon atoms is attached, and wherein each carbon atom is optionally substituted with oxygen;

the aryl in the aryloxy is any one of pure phenyl, benzyl and naphthyl which are substituted by 1, 2 or 3 first substituents;

the first substituent is independently selected from the group consisting of halogen, haloalkyl, alkyl, cyano, hydroxy, nitro, mercapto, alkoxy, alkylthio, aralkyl, diarylalkyl;

R³is heteroalkyl, aryl, heterocyclic;

the heteroalkyl group is a straight or branched chain saturated hydrocarbon group of 1 to 8 carbon atoms, a cyclic saturated hydrocarbon group of 3 to 8 carbon atoms, or a cyclic saturated hydrocarbon group of 3 to 8 carbon atoms to which a straight or branched chain saturated hydrocarbon group of 1 to 8 carbon atoms is attached, each carbon atom being optionally substituted with an oxygen atom or a nitrogen atom;

the aryl is any one of phenyl, benzyl and naphthyl which are pure or substituted by 1, 2 or 3 second substituent groups;

the heterocyclic group is any one of piperidyl, pyrrolyl, pyrazolyl, imidazolyl, furyl, morpholinyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, piperazinyl, substituted piperazinyl, pyrazinyl, pyridazinyl, quinolyl, quinoxalinyl, indolyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothienyl, benzisothiazolyl, benzofuranyl, benzodioxanyl, benzodioxolanyl, and benzodioxolanyl which are pure or substituted by 1, 2, or 3 second substituents;

the second substituent is independently selected from the group consisting of halogen, haloalkyl, alkyl, cyano, hydroxy, mercapto, amino, nitro, alkoxy, alkylthio, aralkyl, diarylalkyl, tetrahydropyrrolyl, morpholinyl, alkoxymorpholinyl, piperazinyl, piperidinyl, alkylaminopiperidinyl.

Preferably, said R is¹Is any one of methyl, ethyl, propyl, isopropyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, benzyl, naphthyl, thienyl, furyl, phenylpropenyl, indolyl, pyrrolyl and pyridyl;

the R is²Is any one of hydrogen, methoxy, ethoxy, propoxy, isopropoxy, butoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, phenoxy, benzyloxy, methylamino, ethylamino, propylamino, isopropylamino, cyclopropylamino, substituted cyclopropylamino, piperidinyl, pyrrolyl, pyrazolyl, imidazolyl, indolyl, morpholinyl, piperazinyl, substituted piperazinyl, pyrazinyl, pyridazinyl;

the R is³Is methyl, ethyl, propyl, isopropyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexylPhenyl, benzyl, naphthyl, indolyl, pyridyl, alkyl containing hetero atoms, methyl and/or halogen monosubstituted, disubstituted cyclopropyl, methyl monosubstituted phenyl, tert-butyl monosubstituted phenyl, halogen monosubstituted phenyl, hydroxy and methoxy disubstituted phenyl, halogen disubstituted phenyl, dimethoxy substituted phenyl, hydroxy and halogen disubstituted phenyl, halogen and nitro disubstituted phenyl, ethoxy and hydroxy disubstituted phenyl, diethylamino and hydroxy disubstituted phenyl, acetonitrile monosubstituted phenyl, naphthyl, indolyl, hydroxy monosubstituted naphthyl, pyridyl, methyl monosubstituted pyridyl, methoxy monosubstituted pyridyl, amino monosubstituted pyridyl, halogen disubstituted pyridyl, nitro monosubstituted pyridyl, halogen trisubstituted pyridyl.

Further preferably, said R¹Is cyclopentyl or benzyl;

the R is²Is hydrogen, ethoxy, morpholinyl, phenoxy, heteroatom substituted phenoxy;

the R is³Is hydrogen atom, benzyl, methoxy substituted benzyl, halogen substituted phenyl, methoxy substituted phenyl, halogen substituted phenyl, thiophene substituted methylene, cyclopentyl, cyclohexyl, cyclopropyl, methyl, ethyl, propyl, isopropyl, dimethyl, butyl, diethyl, pentyl, hydroxypropyl, hydroxyethyl, oxobutyl, hexyl, methylpiperazinyl, methyl, ethyl, methyl, propyl, isopropyl, butyl, pentyl, hexyl, pentyl, hexyl, pentyl, hexyl, pentyl, hexyl, pentyl, hexyl, pentyl, hexyl, pentyl, hexyl, pentyl, hexyl, pentyl, hexyl, pentyl, hexyl, pentyl, hexyl, pentyl, hexyl, pentyl, hexyl, pentyl, hexyl, pentyl, or hexyl, or the like,

The specific structure is shown in formulas 1 to 35:

pharmaceutically acceptable salts of the dihydropteridinone-sulfonamide derivatives are the acid addition salts thereof with: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, borate, methanesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, benzenesulfonic acid, citric acid, lactic acid, pyruvic acid, tartaric acid, acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, salicylic acid, or phenylacetic acid; the dihydropteridinone-sulfonamide derivative is used as a basic compound, and can be conveniently added into a solvent (common organic solvent or aqueous solvent) to react with the acid to obtain a corresponding salt, which is common and conventional acid-base reaction in the field, and the specific method is not described any more.

The invention provides a preparation method of a dihydropteridinone-sulfonamide derivative, which comprises the following steps:

a. taking o-nitrofluorobenzene 36 as a raw material, and reacting with chlorosulfonic acid for 5-12 hours at 80-120 ℃ under stirring, wherein the molar ratio of the o-nitrofluorobenzene 36 to the chlorosulfonic acid is 1: 1-40; after the reaction is finished, slowly dropwise adding the system into ice water to quench redundant chlorosulfonic acid, extracting the reaction system for three times by using an extracting agent, combining, washing by using a saturated sodium bicarbonate solution, separating, drying an organic phase, and spin-drying to obtain an intermediate 37. The extractant may be ethyl acetate, dichloromethane, trichloromethane or dichloroethane. The yield was 80%.

b. In the presence of organic base, at 0-10 ℃, stirring the intermediate 37 and the corresponding amine compound in a molar ratio of 1:1-2 for reaction, and after the TLC detection reaction is finished, performing column chromatography to obtain an intermediate 38. The organic base can be triethylamine, pyridine, triethylene diamine, N-methyl morpholine, potassium tert-butoxide, sodium tert-butoxide or butyl lithium. The corresponding amine compound is the substituent R³Corresponding amine compounds. The yield was 60%.

c. Dissolving the intermediate 38 in an organic solvent, adding a corresponding reagent, and stirring at 10-100 ℃, wherein the molar ratio of the intermediate 38 to the corresponding reagent is 1: 1-5; completion of the reactionThen, washing with weak acid, extracting the aqueous phase with ethyl acetate for three times, combining the organic phases, drying, and spin-drying to obtain an intermediate 39. The organic solvent may be dimethyl sulfoxide or N, N-dimethylformamide. The corresponding reagent is the substituent R²The corresponding compound. The yield is 50-80%.

d. Dissolving the intermediate 39 in a solvent, adding a catalytic amount of palladium-carbon catalyst, heating to 20-80 ℃ in a hydrogen atmosphere, stirring for 4-8 hours, filtering to remove palladium-carbon after TLC detection reaction is finished, and spin-drying the filtrate to obtain an intermediate 40. The organic solvent may be tetrahydrofuran, diethyl ether, methanol, ethanol or acetic acid. The yield is 50-90%.

e. Dissolving a compound 41 in methanol, dropwise adding thionyl chloride in an ice bath, and carrying out reflux reaction, wherein the molar ratio of the compound 41 to the thionyl chloride is 1: 1-10; after the reaction is finished, the reaction system is decompressed and concentrated, a product is separated out and filtered to obtain a product 42, and the crude product is subjected to the next reaction without purification. The yield was about 90%.

f. Dissolving the intermediate 42 in chloroform, dichloromethane or dichloroethane, adding corresponding carbonyl compound, adding appropriate amount of alkaline compound and reducing agent under ice bath, and stirring at 0-30 deg.C until the raw materials disappear. After the reaction is finished, quenching reaction, extracting, combining organic phases, drying, filtering and concentrating to obtain an intermediate 43, and directly carrying out the next reaction without purification. The yield was 70%. The corresponding carbonyl compound is a substituent R¹The corresponding compound; the basic compound may be Na₂CO₃、K₂CO₃、CsCO₃NaOAc or KOAc; the reducing agent may be NaBH₄、KBH₄、NaBH(OAc)₃Or NaBH₃CN; the molar ratio of the intermediate 42 to the corresponding carbonyl compound is 1:1-2, the molar ratio of the intermediate 42 to the basic compound is 1:1 to 1:3, and the molar ratio of the intermediate 42 to the reducing agent is 1: 0.25-10;

g. will be intermediateDissolving the body 43 in chloroform, dichloromethane or dichloroethane, adding 2, 4-dichloro-5-nitropyrimidine and the alkaline compound under ice bath, and stirring at 0-30 ℃ until the raw materials disappear. After the reaction, the solvent was removed by concentration under reduced pressure, and after dilution, washing and drying of the organic layer, intermediate 44 was obtained by silica gel column chromatography. The yield was 85%. The basic compound may be Na₂CO₃、K₂CO₃、CsCO₃、NaOAc、KOAc；

The molar ratio of the intermediate 43, the 2, 4-dichloro-5-nitropyrimidine and the basic compound is 1:1-3: 1-3;

h. dissolving the intermediate 44 in acetic acid, heating to 50-70 ℃, adding reduced iron powder, stirring for reaction, and heating to 100-110 ℃ for continuous reaction. After the reaction is finished, filtering, washing residues, concentrating the filtrate, and performing column chromatography to separate the intermediate 45. The yield is about 60%;

the molar ratio of the intermediate 44 to the reduced iron powder is 1: 2-20;

i. adding the intermediate 45 into anhydrous DMF, adding methyl iodide (1.3 equivalent relative to the intermediate 45) and NaH (1.3 equivalent relative to the intermediate 45) under ice bath, stirring for reaction at 0-30 ℃, quenching a reaction system after the reaction is finished, extracting, washing an organic phase with water, drying and concentrating to obtain an intermediate 46, and directly using the intermediate for the next reaction without purification. The yield is 90%;

j. dissolving compounds shown in formula 40 and formula 46 in a molar ratio of 1:0.5-1 in an organic solvent, adding hydrochloric acid with a mass fraction of 20-38% relative to 2-10 times equivalent (based on HCl content) of the compound 40, and heating at 60-120 ℃ for reaction for 24-72 hours; after the reaction is finished, spin-drying the solvent, washing with saturated sodium bicarbonate solution, extracting with ethyl acetate, combining ethyl acetate phases, concentrating, and carrying out column chromatography to obtain the final product 1-35. The yield is 53-78%;

the organic solvent can be a single solvent or a mixed solvent of methanol, ethanol, isopropanol, tetrahydrofuran and 1, 4-dioxane.

The dihydropteridinone-sulfonamide derivative is used as an application of a BRD4 protein inhibitor in preparing antitumor drugs, and is used for preventing or treating various parenchymal organ tumors mediated by BRD4, including but not limited to gastric cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, prostate cancer, leukemia, lymphoma and the like.

The total yield of the target compound can reach 15-22% (calculated by raw material 41), and activity experiments prove that the novel compound with the dihydropteridinone structure has good BRD4 protein inhibition effect, can inhibit tumor cell proliferation, and particularly aims at gastric cancer cells, and the inhibition activity of part of the compound reaches or is superior to that of a positive control compound JQ 1. The invention provides a new drug choice for treating BRD4 inhibitor, tumor and other diseases.

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.

In the following examples, the structure of the compounds was determined by Nuclear Magnetic Resonance (NMR) and High Resolution Mass Spectrometry (HRMS). The nuclear magnetic resonance apparatus is a Bruker DPX-400 type superconducting nuclear magnetic resonance apparatus in Sweden, and Tetramethylsilane (TMS) is used as an internal standard; the high resolution mass spectrometer used was a Waters-Micromass Q-Tof mass spectrometer.

The synthesis of intermediates 40 and 46 is illustrated below by way of example of the preparation of intermediates 40a and 46a required for the synthesis of compound 29.

Synthesis of intermediate 37

Adding chlorosulfonic acid (2.48g,21.26mmol) and 2-fluoronitrobenzene (1.00g,7.09mmol) into a round-bottom flask, heating to 100 ℃, stirring for 12h, cooling the reaction system to room temperature after TLC detection reaction is finished, slowly dropping the system solution into ice water to quench redundant chlorosulfonic acid, extracting the reaction system with ethyl acetate (30mL multiplied by 3) for three times, combining organic phases, washing the organic phases with saturated sodium bicarbonate solution (30mL), separating the organic phases, removing water from the organic phases, and spin-drying to obtain the product. Yield 80%, light yellow oily liquid.¹H NMR(400MHz,CDCl₃)δ8.93–8.67(m,1H),8.44–8.24(m,1H),7.62(t,J＝9.3Hz,1H).¹³C NMR(100MHz,CDCl₃)δ160.34,157.59,140.66,140.61,133.98,133.88,126.17,126.16,120.85,120.62.

Synthesis of intermediate 38a

Adding the intermediate 37(100mg,0.417mmol) into a round-bottom flask, adding 10mL of anhydrous THF for dissolving, cooling for 10min under ice bath, adding cyclopropylamine (23.83mg,0.417mmol) and triethylamine (105.49mg,1.04mmol), stirring for 20min under ice bath, performing TLC detection on the reaction, then performing spin-drying on the solvent, diluting the system with ethyl acetate (20mL) and saturated saline (20mL), adjusting the pH to about 7, performing liquid separation, discarding the aqueous phase, spin-drying the organic phase, and purifying by using silica gel column chromatography (petroleum ether PE/ethyl acetate EA is 4:1) to obtain the product with the yield of 60%. Pale yellow solid, melting point 143-144 ℃.¹H NMR(400MHz,CDCl₃)δ8.84(d,J＝2.2Hz,1H),8.59(s,1H),8.02–7.89(m,1H),7.50(d,J＝9.2Hz,1H),2.75–2.66(m,1H),1.11–1.01(m,2H),0.79–0.72(m,2H).¹³C NMR(100MHz,CDCl₃)δ149.93,133.80,129.29,116.58,24.91,8.05.HR-MS(ESI):Calcd.C₉H₉FN₂O₄S,[M+H]⁺m/z:409.2352,found:409.2351.

Synthesis of intermediate 39a

Adding the intermediate 38a (50mg,0.19mmol) and phenol (21.70mg,0.228mmol) into a round-bottom flask, adding 8mL of dimethyl sulfoxide as a solvent, heating to 100 ℃, stirring for 4-5h, adding ethyl acetate (15mL) to dilute the reaction system after TLC detection reaction is finished, washing with saturated saline (10mL multiplied by 3), separating, discarding the aqueous phase, taking the organic phase, drying the organic phase, and spin-drying to obtain the product. The yield thereof was found to be 78%. White solid, melting point 153-.¹H NMR(400MHz,DMSO-d₆)δ8.55(s,1H),8.30(d,J＝2.3Hz,1H),7.99–7.84(m,1H),7.55(d,J＝9.2Hz,1H),7.46–7.38(m,2H),7.34(t,J＝7.3Hz,1H),7.15–7.06(m,2H),2.83–2.65(m,1H),0.99–0.88(m,2H),0.76–0.67(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ148.96,148.88,133.72,130.45,130.09,127.74,127.55,122.18,119.67,117.16,25.07,7.50.HR-MS(ESI):Calcd.C₁₅H₁₄N₂O₅S,[M+H]⁺m/z:335.0696,found:335.0695.

Synthesis of intermediate 40a

Adding the intermediate 39a (50mg, 0.15mmol, 1eq.) into a round-bottom flask, adding 10mL tetrahydrofuran as a solvent, adding a catalytic amount of palladium-carbon, heating to 50 ℃ under a hydrogen atmosphere, stirring for 6-8h, filtering to remove the palladium-carbon after TLC detection reaction is finished, and spin-drying the filtrate to obtain an intermediate 40 a. The yield was 80%, pale yellow solid, melting point 156 ℃ 157 ℃.¹H NMR(400MHz,CDCl₃)δ7.32–7.24(m,3H),7.24–7.19(m,1H),7.12(d,J＝2.1Hz,1H),7.05–6.93(m,3H),4.52(s,1H),3.27(s,2H),2.52–2.43(m,1H),0.85–0.78(m,2H),0.60–0.53(m,2H).¹³C NMR(100MHz,CDCl₃)δ149.94,144.19,132.54,129.44,126.76,123.11,122.61,122.56,116.11,110.64,24.92,7.38.HR-MS(ESI):Calcd.C₁₅H₁₆N₂O₃S,[M+H]⁺m/z:305.0955,found:305.0956.

Synthesis of intermediate 42

2-aminobutyric acid (1.00g, 1.0eq.) was put in a 50mL eggplant-shaped bottle, anhydrous methanol (15mL) was added, and SOCl was slowly added at 0 deg.C₂(2.31g, 2.0eq.) TLC showed the reaction was complete after heating to reflux for 1.5 hours. Evaporation of the volatiles gave a white solid, which was washed with Et₂And O, grinding the solid, and performing suction filtration to obtain a crude product of the intermediate 42, wherein the crude product can be directly subjected to the next reaction without purification, and the yield is 90%. White solidMelting point 120-122 ℃.¹H NMR(400MHz,D₂O)δ4.04(t,J＝6.2Hz,1H),3.76(s,3H),1.99–1.82(m,2H),0.97–0.87(m,3H).¹³C NMR(101MHz,D₂O)δ170.79,53.99,53.46,23.21,8.42.HR-MS(ESI):Calcd.C₅H₁₂ClNO₂,[M+H]⁺m/z:118.0863,found:118.0867.

Synthesis of intermediate 43a

Intermediate 42(1.00g, 1.0eq.) and cyclopentanone (548mg, 1.0eq.) were ice-cooled for 10 minutes, and NaOAc (547mg, 1.0eq.) and nabh (oac) were added₃(2.07g, 1.5 eq.). The reaction mixture was stirred at room temperature overnight. TLC shows that after the reaction is finished, saturated NaHCO is added into the reaction system₃The reaction was quenched in solution and then with CH₂Cl₂(20 mL. times.2) the aqueous layer was extracted, the organic layers were combined and washed with anhydrous Na₂SO₄Drying, filtration and concentration gave intermediate 43a in 65% yield. A colorless oily liquid.¹H NMR(400MHz,DMSO-d⁶)δ3.63(s,3H),3.10(t,J＝6.6Hz,1H),2.98–2.84(m,1H),1.80(s,1H),1.67–1.54(m,4H),1.54–1.48(m,2H),1.47–1.39(m,2H),1.32–1.21(m,2H),0.84(t,J＝7.4Hz,3H).¹³C NMR(101MHz,DMSO-d⁶)δ175.71,60.88,57.32,51.19,33.17,32.10,26.25,23.38,23.33,10.21.HR-MS(ESI):Calcd.C₁₀H₁₉NO₂,[M+H]⁺m/z:186.1494,found:186.1495.

Synthesis of intermediate 44a

Adding intermediate 43a (1.00g, 1.0eq.) into acetone 15mL, adding 2, 4-dichloro-5-nitropyrimidine (1.05g, 1.0eq.) after ice bath for 10 minutes, and then adding K₂CO₃(750mg, 1.0eq.), and stirred at room temperature overnight. After completion of the reaction, the solvent was removed, and the residue was dissolved with ethyl acetate and washed with waterThe organic layers were collected, dried over anhydrous sodium sulfate, and purified by silica gel flash column chromatography (PE/EA ═ 10:1) to give intermediate 44a in 89% yield. Yellow solid, melting point 110-111 ℃.¹H NMR(400MHz,DMSO-d⁶)δ8.84(s,1H),4.29–4.14(m,1H),3.64(s,3H),3.52–3.48(m,1H),2.28–2.15(m,1H),2.04–1.90(m,2H),1.85–1.77(m,2H),1.72–1.61(m,3H),1.51–1.35(m,2H),0.94(t,J＝7.8Hz,3H).¹³C NMR(101MHz,DMSO-d⁶)δ164.21,151.73,151.14,137.83,119.27,61.06,59.38,28.30,28.28,26.36,23.88,23.86,8.33.HR-MS(ESI):Calcd.C₁₄H₁₉ClN₄O₄,[M+Na]⁺m/z:365.0993,found:365.0992.

Synthesis of intermediate 45a

Intermediate 44a (1.00g, 1.0eq.) was added to 20mL of glacial acetic acid, heated to 70 ℃, and iron powder (325mg, 2.0eq.) was added portionwise over 5 minutes. The reaction was stirred at 70 ℃ for 1 hour and then warmed to 100 ℃ for 4-5 hours, after completion of the reaction, the mixture was filtered through celite, the residue was washed with appropriate methanol, the volatiles were evaporated and purified by flash column chromatography (PE/EA ═ 1.5:1) to give intermediate 45a in 50% yield. Yellow solid, melting point 180-182 ℃.¹H NMR(400MHz,DMSO-d⁶)δ10.85(s,1H),7.57(s,1H),4.30–4.20(m,1H),4.18–4.04(m,1H),2.00–1.76(m,7H),1.76–1.65(m,1H),1.62–1.42(m,2H),0.92–0.68(m,3H).¹³C NMR(101MHz,DMSO-d⁶)δ164.21,151.73,151.14,137.83,119.27,61.06,59.38,28.30,28.28,26.36,23.88,23.86,8.33.HR-MS(ESI):Calcd.C₁₃H₁₇ClN₄O,[M+Na]⁺m/z:303.0989,found:303.0975.

Synthesis of intermediate 46a

Intermediate 45a (1.00g, 1.0 e)q.) adding anhydrous DMF, carrying out ice bath for 10 minutes, adding methyl iodide (657mg, 1.3eq.) and NaH (185mg, 1.3eq.) with the mass percent of 60% for stirring at room temperature for 3 hours, quenching the reaction system with ice water after the reaction is finished, extracting the product with EtOAc, washing the organic layer twice with water, drying with anhydrous sodium sulfate, and carrying out spin drying to obtain the intermediate 46a, wherein the intermediate is directly used for the next step without purification and the yield is 90%. Yellow solid, melting point 182 ℃ and 183 ℃.¹H NMR(400MHz,DMSO-d⁶)δ7.87(s,1H),4.39–4.31(m,1H),4.17(p,J＝8.3Hz,1H),3.24(s,3H),1.97–1.77(m,7H),1.74–1.65(m,1H),1.61–1.48(m,2H),0.74(t,J＝7.5Hz,3H).¹³C NMR(101MHz,DMSO)δ163.16,152.13,151.53,138.49,121.24,60.76,59.32,28.38,28.33,27.91,26.61,23.85,23.81,8.48.HR-MS(ESI):Calcd.C₁₄H₁₉ClN₄O,[M+H]⁺m/z:295.1325,found:295.1322.

The main difference between the synthesis of intermediates 40 and 46 for the remaining compounds and the synthesis of intermediates 40a and 46a for compound 29 described above is that the substituent R¹、R²、R³The differences of the corresponding raw materials, except for the replacement of the raw materials, can be obtained by those skilled in the art by performing conventional adjustment according to the above scheme to obtain the synthetic processes of intermediates 40 and 46 corresponding to the remaining compounds, which are not described herein again, and the following raw materials are listed as the following table one:

in one of the compounds 1 to 35, the substituent R¹、R²、R³Corresponding raw materials

Example 29: synthesis of Compound 29

The preparation of compounds 1-35 is the same, taking the preparation of compound 29 as an example.

Dissolving the prepared 40a and 46a in a solvent by taking absolute ethyl alcohol and 1, 4-dioxane as solvents according to a molar ratio of 1:1, adding 2.5 equivalents (relative to 40a) of concentrated hydrochloric acid, heating and refluxing for 48 hours, after the reaction is finished, spin-drying the solvent, washing with a saturated sodium bicarbonate solution, extracting with ethyl acetate, combining ethyl acetate phases, concentrating, and carrying out column chromatography to obtain a final product 29. The yield thereof was 76%, which was a pale yellow solid having a melting point of 146-.¹H NMR(400MHz,DMSO-d₆)δ9.52(s,1H),8.44(s,1H),7.87(s,1H),7.83(d,J＝8.0Hz,1H),7.77(d,J＝1.8Hz,1H),7.44–7.31(m,5H),7.27(d,J＝7.2Hz,2H),5.48(d,J＝15.3Hz,1H),4.36(d,J＝15.3Hz,1H),4.09(t,J＝4.8Hz,1H),3.28(s,3H),2.07(s,1H),1.86–1.74(m,2H),0.75(t,J＝7.3Hz,3H),0.46–0.39(m,2H),0.37(s,2H).¹³C NMR(100MHz,DMSO-d₆)δ162.53,155.03,151.23,141.69,140.49,138.56,136.87,129.06,128.55,127.80,127.36,121.28,118.12,116.02,114.95,60.33,46.94,27.75,24.24,24.09,8.62,5.14,5.10.HR-MS(ESI):Calcd.C₂₅H₂₈N₆O₃S,[M+H]⁺m/z:493.2017,found:493.2016.

Example 1: synthesis of Compound 1

The procedure is as in example 29. Yield 63% as pale yellow solid, melting point 179-.¹H NMR(400MHz,DMSO-d₆)δ9.41(s,1H),8.45(s,1H),8.04(t,J＝6.3Hz,1H),7.87–7.80(m,2H),7.43(t,J＝8.0Hz,1H),7.32–7.21(m,6H),4.62–4.51(m,1H),4.23–4.17(m,1H),3.99(d,J＝6.3Hz,2H),3.25(s,3H),2.03(d,J＝6.2Hz,1H),1.93(d,J＝6.4Hz,1H),1.71(d,J＝10.8Hz,5H),1.64–1.55(m,3H),0.78(t,J＝7.5Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.83,154.92,151.58,141.88,140.90,138.53,137.81,129.11,128.17,127.50,127.07,121.30,117.82,115.73,115.60,58.84,57.42,46.15,28.94,28.74,27.78,26.46,23.06,22.66,9.05.HR-MS(ESI):Calcd.C₂₇H₃₂N₆O₃S,[M+H]⁺m/z:521.2335,found:521.2334.

Example 2: synthesis of Compound 2

The procedure is as in example 29. The yield was 63% as pale yellow solid, melting point 155-.¹H NMR(400MHz,DMSO-d₆)δ10.22(s,1H),9.40(s,1H),8.44(s,1H),7.82(s,1H),7.78(d,J＝9.5Hz,1H),7.37(t,J＝8.0Hz,1H),7.25–7.18(m,3H),7.10(d,J＝7.6Hz,2H),7.00(t,J＝7.3Hz,1H),4.60–4.48(m,1H),4.24–4.18(m,1H),3.25(s,3H),2.03(d,J＝5.9Hz,1H),1.91(d,J＝9.3Hz,1H),1.78–1.65(m,5H),1.65–1.55(m,3H),0.78(t,J＝7.5Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.84,154.80,151.57,141.86,139.93,138.42,137.88,129.11,129.02,123.77,121.66,119.80,117.86,115.79,58.83,57.40,28.93,28.71,27.78,26.46,23.05,22.64,9.04.HR-MS(ESI):Calcd.C₂₆H₃₀N₆O₃S,[M+H]⁺m/z:507.2178,found:507.2179.

Example 3: synthesis of Compound 3

The procedure is as in example 29. Yield 65%, pale yellow solid, melting point 145-146 ℃.¹H NMR(400MHz,DMSO-d₆)δ9.81(s,1H),9.35(s,1H),8.32(t,J＝1.9Hz,1H),7.80(s,1H),7.78–7.74(m,1H),7.35(t,J＝8.0Hz,1H),7.17(d,J＝7.7Hz,1H),7.01–6.95(m,2H),6.81–6.75(m,2H),4.56–4.45(m,1H),4.23–4.12(m,1H),3.64(d,J＝7.4Hz,3H),3.22(d,J＝9.3Hz,3H),2.06–1.96(m,1H),1.94–1.87(m,1H),1.79–1.65(m,5H),1.64–1.52(m,3H),0.76(t,J＝7.5Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.84,156.36,154.81,151.56,141.76,139.89,138.40,130.30,128.95,123.32,121.51,117.98,115.84,115.76,114.16,58.87,57.41,55.07,28.92,28.72,27.76,26.45,23.06,22.66,9.03.HR-MS(ESI):Calcd.C₂₇H₃₂N₆O₄S,[M+H]⁺m/z:537.2284,found:537.2285.

Example 4: synthesis of Compound 4

The procedure is as in example 29. Yield 61%, pale yellow solid, melting point 147-.¹H NMR(400MHz,DMSO-d₆)δ9.39(s,1H),8.44(s,1H),8.03(t,J＝6.3Hz,1H),7.84(d,J＝8.5Hz,2H),7.43(t,J＝8.0Hz,1H),7.32–7.21(m,6H),4.61–4.52(m,1H),4.22–4.16(m,1H),3.99(d,J＝6.3Hz,2H),3.25(s,3H),2.03(d,J＝6.2Hz,1H),1.98–1.90(m,1H),1.71(d,J＝10.9Hz,5H),1.66–1.56(m,3H),0.78(t,J＝7.5Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.84,154.93,151.59,141.88,140.91,138.52,137.80,129.10,128.16,127.49,127.07,121.31,117.83,115.74,115.62,58.87,57.44,46.16,28.95,28.75,27.78,26.46,23.07,22.67,9.04.HR-MS(ESI):Calcd.C₂₇H₃₁BrN₆O₃S,[M+H]⁺m/z:599.1440,found:599.1440.

Example 5: synthesis of Compound 5

The procedure is as in example 29. Yield 68% of pale yellow solid, melting point 157-.¹H NMR(400MHz,DMSO-d₆)δ9.41(s,1H),8.43(s,1H),8.07(t,J＝6.3Hz,1H),7.88–7.79(m,2H),7.42(t,J＝8.0Hz,1H),7.32–7.24(m,3H),4.57(d,J＝7.4Hz,1H),4.23–4.16(m,1H),3.98(d,J＝6.2Hz,2H),3.25(s,3H),2.06–1.99(m,1H),1.92(s,1H),1.72(s,5H),1.65–1.54(m,3H),0.78(t,J＝7.4Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.83,154.90,151.57,141.87,140.86,138.53,134.04,129.52,129.43,129.11,128.11,125.03,121.29,117.79,115.72,115.56,114.99,114.78,58.83,57.40,54.88,45.38,28.93,28.72,27.78,26.45,23.05,22.64.HR-MS(ESI):Calcd.C₂₇H₃₁FN₆O₃S,[M+H]⁺m/z:539.2240,found:539.2239.

Example 6: synthesis of Compound 6

The procedure is as in example 29. Yield 63% as pale yellow solid, melting point 178-.¹H NMR(400MHz,DMSO-d₆)δ9.40(s,1H),8.43(s,1H),7.95(t,J＝6.2Hz,1H),7.84(d,J＝10.2Hz,2H),7.43(t,J＝7.9Hz,1H),7.29(d,J＝7.7Hz,1H),7.16(d,J＝8.3Hz,2H),6.83(d,J＝8.4Hz,2H),4.66–4.49(m,1H),4.26–4.14(m,1H),3.92(d,J＝6.1Hz,2H),3.71(s,3H),3.25(s,3H),2.03(s,1H),1.93(s,1H),1.73(s,5H),1.66–1.55(m,3H),0.78(t,J＝7.4Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.82,158.39,154.92,151.57,141.85,140.94,138.54,129.59,129.07,128.86,121.23,117.82,115.71,115.61,113.56,58.84,57.41,55.00,45.69,28.94,28.73,27.77,26.45,23.06,22.66,9.04.HR-MS(ESI):Calcd.C₂₈H₃₄N₆O₄S,[M+H]⁺m/z:551.2440,found:551.2441.

Example 7: synthesis of Compound 7

The procedure is as in example 29. Yield 67% as pale yellow solid, melting point 191-192 ℃.¹H NMR(400MHz,DMSO-d₆)δ9.54(s,1H),7.95–7.84(m,4H),7.66(d,J＝8.9Hz,2H),7.19(t,J＝7.9Hz,1H),6.84–6.76(m,3H),4.48–4.40(m,1H),4.26–4.21(m,1H),3.93(d,J＝6.3Hz,2H),3.69(s,3H),3.26(s,3H),2.09–2.01(m,1H),1.93(d,J＝8.7Hz,1H),1.85–1.72(m,5H),1.67–1.57(m,3H),0.78(t,J＝7.4Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.90,159.15,154.62,151.53,144.88,139.36,138.32,131.06,129.21,127.38,119.65,117.10,116.03,112.89,112.65,59.27,57.96,54.90,46.03,28.77,28.36,27.76,26.47,22.87,22.53,8.93.HR-MS(ESI):Calcd.C₂₈H₃₄N₆O₄S,[M+H]⁺m/z:551.2440,found:551.2441.

Example 8: synthesis of Compound 8

The procedure is as in example 29. Yield 74% light yellow solid, melting point 167-.¹H NMR(400MHz,DMSO-d₆)δ9.41(s,1H),8.44(s,1H),7.93–7.78(m,3H),7.43(t,J＝8.0Hz,1H),7.29(t,J＝7.7Hz,2H),7.21(t,J＝7.7Hz,1H),6.95–6.81(m,2H),4.64–4.49(m,1H),4.23–4.17(m,1H),3.95(d,J＝6.1Hz,2H),3.69(s,3H),3.25(s,3H),2.02(s,1H),1.92(s,1H),1.72(s,5H),1.64–1.52(m,3H),0.78(t,J＝7.3Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.82,156.32,154.93,151.57,141.80,140.83,138.55,129.06,128.35,128.19,125.28,121.25,119.99,117.85,115.70,115.64,110.29,58.82,57.40,55.13,28.93,28.73,27.77,26.46,23.05,22.64,9.05.HR-MS(ESI):Calcd.C₂₈H₃₄N₆O₄S,[M+H]⁺m/z:551.244,found:551.244.

Example 9: synthesis of Compound 9

The procedure is as in example 29. Yield 61%, pale yellow solid, melting point 178-.¹H NMR(400MHz,DMSO-d₆)δ9.41(s,1H),8.45(d,J＝1.8Hz,1H),8.16(t,J＝6.2Hz,1H),7.84(d,J＝8.0Hz,2H),7.43(t,J＝8.0Hz,1H),7.41–7.38(m,1H),7.29(d,J＝7.8Hz,1H),6.94–6.90(m,2H),4.63–4.53(m,1H),4.22–4.14(m,3H),3.25(s,3H),2.04(d,J＝5.8Hz,1H),1.97–1.90(m,1H),1.78–1.67(m,5H),1.66–1.57(m,3H),0.78(t,J＝7.5Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.83,154.91,151.58,141.89,140.86,140.73,138.54,129.13,126.64,125.83,125.55,121.38,117.85,115.74,115.57,58.84,57.42,41.42,28.95,28.76,27.78,26.46,23.08,22.67,9.05.HR-MS(ESI):Calcd.C₂₅H₃₀N₆O₃S₂,[M+H]⁺m/z:,found:527.1898.

Example 10: synthesis of Compound 10

The procedure is as in example 29. The yield was 60% as a pale yellow solid with a melting point of 156 ℃ and 157 ℃.¹H NMR(400MHz,DMSO-d₆)δ9.46(s,1H),8.34(s,1H),7.96(d,J＝9.6Hz,1H),7.86(s,1H),7.51(t,J＝8.0Hz,1H),7.20(d,J＝8.3Hz,1H),4.59–4.52(m,1H),4.22–4.18(m,1H),3.65–3.62(m,4H),3.25(s,3H),2.89–2.85(m,4H),2.03(d,J＝7.9Hz,1H),1.92(s,1H),1.73(d,J＝11.4Hz,5H),1.65–1.57(m,3H),0.78(t,J＝7.5Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.84,154.82,151.59,142.17,138.50,134.53,129.29,122.22,118.92,116.19,115.88,65.25,58.82,57.42,45.93,28.92,28.70,27.81,26.45,23.09,22.69,9.05.HR-MS(ESI):Calcd.C₂₄H₃₂N₆O₄S,[M+H]⁺m/z:501.2284,found:501.2285.

Example 11: synthesis of Compound 11

The procedure is as in example 29. The yield was 68% as a pale yellow solid with a melting point of 156 ℃ and 157 ℃.¹H NMR(400MHz,DMSO-d₆)δ9.39(s,1H),8.42(d,J＝1.8Hz,1H),7.84(s,1H),7.80(d,J＝8.2Hz,1H),7.53(d,J＝7.4Hz,1H),7.42(t,J＝8.0Hz,1H),7.30(d,J＝7.7Hz,1H),4.57(d,J＝7.8Hz,1H),4.23–4.18(m,1H),3.25(s,3H),2.94(s,1H),2.04(d,J＝5.0Hz,1H),1.97–1.90(m,1H),1.79–1.69(m,5H),1.65–1.55(m,7H),1.44(d,J＝10.8Hz,1H),1.17–1.04(m,5H),0.78(t,J＝7.4Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.83,154.93,151.58,142.51,141.70,138.50,129.01,121.07,117.71,115.70,115.42,58.87,57.43,52.10,33.22,28.94,28.74,27.77,26.45,24.84,24.45,23.08,22.68,9.03.HR-MS(ESI):Calcd.C₂₆H₃₆N₆O₃S,[M+H]⁺m/z:513.2648,found:513.2649.

Example 12: synthesis of Compound 12

The procedure is as in example 29. Yield 78%, pale yellow solid, melting point 151-.¹H NMR(400MHz,DMSO-d₆)δ9.45(s,1H),8.35(s,1H),7.92(d,J＝8.2Hz,1H),7.86(s,1H),7.47(t,J＝8.0Hz,1H),7.18(d,J＝7.6Hz,1H),4.65–4.51(m,1H),4.23–4.16(m,1H),3.25(s,3H),2.94–2.83(m,4H),2.03(d,J＝7.4Hz,1H),1.96–1.89(m,1H),1.81–1.66(m,5H),1.65–1.51(m,7H),1.36(s,2H),0.78(t,J＝7.4Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.82,154.87,151.59,142.08,138.57,135.69,129.14,121.80,118.70,115.92,115.82,58.75,57.36,46.61,28.94,28.75,27.80,26.46,24.67,23.08,22.85,22.67,9.06.HR-MS(ESI):Calcd.C₂₅H₃₄N₆O₃S,[M+H]⁺m/z:499.2491,found:499.2492.

Example 13: synthesis of Compound 13

The procedure is as in example 29. Yield 73% as pale yellow solid, melting point 158-.¹H NMR(400MHz,DMSO-d₆)δ9.40(s,1H),8.42(s,1H),7.84(s,1H),7.80(d,J＝8.5Hz,1H),7.59(d,J＝7.2Hz,1H),7.42(t,J＝7.9Hz,1H),7.29(d,J＝7.8Hz,1H),4.62–4.50(m,1H),4.24–4.18(m,1H),3.25(s,3H),2.91(d,J＝6.7Hz,1H),2.60(d,J＝11.2Hz,2H),2.07(s,4H),1.92(d,J＝9.7Hz,1H),1.83–1.68(m,7H),1.65–1.51(m,5H),1.37(d,J＝9.5Hz,2H),0.78(t,J＝7.4Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.83,154.92,151.59,142.26,141.75,138.50,129.08,121.17,117.71,115.72,115.39,58.86,57.42,53.91,50.17,45.71,32.25,28.94,28.75,27.77,26.46,23.08,22.67,9.05.HR-MS(ESI):Calcd.C₂₆H₃₇N₇O₃S,[M+H]⁺m/z:528.2757,found:528.2758.

Example 14: synthesis of Compound 14

The procedure is as in example 29. The yield was 53%, pale yellow solid, melting point 166-.¹H NMR(400MHz,DMSO-d₆)δ9.40(s,1H),8.41(s,1H),7.82(d,J＝12.7Hz,2H),7.53(d,J＝7.1Hz,1H),7.43(t,J＝8.0Hz,1H),7.29(d,J＝7.8Hz,1H),4.63–4.50(m,1H),4.26–4.13(m,1H),3.45–3.39(m,1H),3.25(s,3H),2.03(d,J＝6.3Hz,1H),1.98–1.89(m,1H),1.82–1.67(m,5H),1.66–1.50(m,7H),1.43–1.22(m,5H),0.78(t,J＝7.4Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.83,154.93,151.58,141.78,141.73,138.51,129.01,121.16,117.91,115.72,58.87,57.43,54.47,32.40,28.93,28.72,27.77,26.45,23.07,22.79,22.67,9.04.HR-MS(ESI):Calcd.C₂₅H₃₄N₆O₃S,[M+H]⁺m/z:499.2491,found:499.2493.

Example 15: synthesis of Compound 15

The procedure is as in example 29. Yield 63% as pale yellow solid, melting point 178-.¹H NMR(400MHz,DMSO-d₆)δ9.41(s,1H),8.44(s,1H),7.89–7.83(m,2H),7.81(d,J＝2.3Hz,1H),7.45(t,J＝8.0Hz,1H),7.30(d,J＝7.8Hz,1H),4.63–4.48(m,1H),4.25–4.16(m,1H),3.25(s,3H),2.16–2.09(m,1H),2.08–2.01(m,1H),1.98–1.89(m,1H),1.81–1.68(m,5H),1.67–1.56(m,3H),0.78(t,J＝7.4Hz,3H),0.51–0.45(m,2H),0.44–0.37(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ162.85,154.92,151.59,141.80,140.52,138.47,128.99,121.40,118.12,115.97,115.74,58.90,57.46,28.92,28.72,27.78,26.45,24.11,23.08,22.69,9.03,5.15.HR-MS(ESI):Calcd.C₂₃H₃₀N₆O₃S,[M+H]⁺m/z:471.2178,found:471.2177.

Example 16: synthesis of Compound 16

The procedure is as in example 29. Yield 68% as pale yellow solid, melting point 158-.¹H NMR(400MHz,DMSO-d₆)δ9.50(s,1H),7.96–7.80(m,3H),7.64(d,J＝8.8Hz,2H),7.41(d,J＝7.6Hz,1H),4.49–4.36(m,1H),4.26–4.19(m,1H),3.25(s,3H),2.03(d,J＝10.9Hz,1H),1.92(s,1H),1.75(d,J＝15.1Hz,5H),1.67–1.54(m,3H),0.95(d,J＝6.6Hz,3H),0.77(t,J＝7.5Hz,3H),0.37–0.28(m,1H),0.25–0.17(m,1H),0.14–0.07(m,1H),-0.02–-0.09(m,1H).¹³C NMR(100MHz,DMSO-d₆)δ162.90,154.62,151.52,144.58,138.33,132.64,127.23,117.01,115.98,59.34,58.02,53.02,28.74,28.35,27.76,26.45,22.86,22.54,20.98,17.47,8.91,3.23,2.70.HR-MS(ESI):Calcd.C₂₅H₃₄N₆O₃S,[M+H]⁺m/z:499.2486,found:499.2487.

Example 17: synthesis of Compound 17

The procedure is as in example 29. Yield 67% as pale yellow solid, melting point 167-.¹H NMR(400MHz,DMSO-d₆)δ9.39(s,1H),8.42(s,1H),7.84(s,1H),7.80(d,J＝8.2Hz,1H),7.41(t,J＝7.9Hz,1H),7.33(d,J＝7.8Hz,1H),7.24(s,2H),4.62–4.49(m,1H),4.23–4.15(m,1H),3.25(s,3H),2.04(d,J＝9.6Hz,1H),1.97–1.88(m,1H),1.81–1.55(m,8H),0.78(t,J＝7.4Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.83,154.97,151.57,144.37,141.62,138.51,128.80,120.82,117.32,115.66,114.74,58.87,57.42,28.93,28.70,27.77,26.46,23.02,22.62,9.05.HR-MS(ESI):Calcd.C₂₀H₂₆N₆O₃S,[M+H]⁺m/z:431.1865,found:431.1865.

Example 18: synthesis of Compound 18

The procedure is as in example 29. Yield 63% as pale yellow solid, melting point 156-.¹H NMR(400MHz,DMSO-d₆)δ9.42(s,1H),8.41(s,1H),7.84(d,J＝6.8Hz,2H),7.44(t,J＝7.9Hz,1H),7.32(d,J＝5.0Hz,1H),7.25(d,J＝7.7Hz,1H),4.64–4.47(m,1H),4.24–4.15(m,1H),3.25(s,3H),2.43(d,J＝4.9Hz,3H),2.03(s,1H),1.92(d,J＝7.0Hz,1H),1.73(s,5H),1.65–1.55(m,3H),0.78(t,J＝7.4Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.83,154.91,151.57,141.90,139.48,138.53,129.08,121.34,117.92,115.77,115.73,58.84,57.41,28.93,28.73,27.78,26.46,23.07,22.67,9.04.HR-MS(ESI):Calcd.C₂₁H₂₈N₆O₃S,[M+H]⁺m/z:445.2022,found:445.2021.

Example 19: synthesis of Compound 19

The procedure is as in example 29. Yield 67% as pale yellow solid, melting point 165-.¹H NMR(400MHz,DMSO-d₆)δ9.41(s,1H),8.41(t,J＝1.8Hz,1H),7.88–7.80(m,2H),7.48–7.41(m,2H),7.27(d,J＝7.7Hz,1H),4.62–4.51(m,1H),4.25–4.16(m,1H),3.25(s,3H),2.83–2.77(m,2H),2.04(d,J＝6.1Hz,1H),1.97–1.90(m,1H),1.80–1.68(m,5H),1.66–1.58(m,3H),0.78(t,J＝7.4Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.82,154.92,151.57,141.84,140.77,138.52,129.07,121.23,117.80,115.71,115.62,58.85,57.41,28.93,28.71,27.78,26.45,23.06,22.66,14.74,9.04.HR-MS(ESI):Calcd.C₂₂H₃₀N₆O₃S,[M+H]⁺m/z:459.2178,found:459.2179.

Example 20: synthesis of Compound 20

The procedure is as in example 29. Yield 65% as pale yellow solid, melting point 166-.¹H NMR(400MHz,DMSO-d₆)δ9.45(s,1H),8.36(s,1H),7.95(d,J＝8.1Hz,1H),7.86(s,1H),7.49(t,J＝8.0Hz,1H),7.21(d,J＝7.7Hz,1H),4.62–4.52(m,1H),4.24–4.17(m,1H),3.26(s,3H),2.62(s,6H),2.04(d,J＝7.4Hz,1H),1.98–1.89(m,1H),1.81–1.68(m,5H),1.67–1.56(m,3H),0.78(t,J＝7.4Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.83,154.86,151.59,142.10,138.52,134.84,129.17,121.86,118.82,116.08,115.83,58.79,57.39,37.62,28.92,28.72,27.79,26.45,23.08,22.68,9.04.HR-MS(ESI):Calcd.C₂₂H₃₀N6_O3S,[M+H]⁺m/z:459.2178,found:459.2179.

Example 21: synthesis of Compound 21

The procedure is as in example 29. Yield 65% as pale yellow solid, melting point 156 ℃ 156.¹H NMR(400MHz,DMSO-d₆)δ9.54(s,1H),7.94(s,1H),7.91(s,1H),7.86(s,1H),7.62(d,J＝8.9Hz,2H),7.18(q,J＝5.0Hz,1H),4.49–4.40(m,1H),4.27–4.21(m,1H),3.25(s,3H),2.38(d,J＝5.1Hz,3H),2.04(d,J＝16.9Hz,1H),1.92(s,1H),1.86–1.71(m,5H),1.67–1.57(m,3H),0.77(t,J＝7.5Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.83,154.93,151.57,142.08,141.75,138.50,129.03,121.09,117.79,115.71,115.55,58.88,57.43,45.22,28.93,28.71,27.77,26.44,23.20,23.17,23.07,22.68,9.04.HR-MS(ESI):Calcd.C₂₃H₃₂N₆O₃S,[M+H]⁺m/z:473.2335,found:473.2335.

Example 22: synthesis of Compound 22

The procedure is as in example 29. Yield 64% as pale yellow solid, melting point 145-146 ℃.¹H NMR(400MHz,DMSO-d₆)δ9.41(s,1H),8.40(s,1H),7.89–7.77(m,2H),7.50–7.40(m,2H),7.27(d,J＝7.7Hz,1H),4.67–4.46(m,1H),4.25–4.16(m,1H),3.25(s,3H),2.77–2.66(m,2H),2.09–2.01(m,1H),1.97–1.89(m,1H),1.80–1.67(m,5H),1.66–1.55(m,3H),1.42–1.35(m,2H),0.83–0.76(m,6H).¹³C NMR(100MHz,DMSO-d₆)δ162.83,154.92,151.57,141.81,140.86,138.52,129.06,121.20,117.78,115.71,115.60,58.84,57.41,44.42,28.93,28.72,27.77,26.45,23.06,22.66,22.39,11.14,9.04.HR-MS(ESI):Calcd.C₂₃H₃₂N₆O₃S,[M+H]⁺m/z:473.2335,found:473.2334.

Example 23: synthesis of Compound 23

The procedure is as in example 29. Yield 73% as pale yellow solid, melting point 158-.¹H NMR(400MHz,DMSO-d₆)δ9.41(s,1H),8.41(s,1H),7.89(d,J＝8.3Hz,1H),7.86(s,1H),7.43(t,J＝8.0Hz,1H),7.24(d,J＝7.7Hz,1H),4.65–4.49(m,1H),4.24–4.14(m,1H),3.25(s,3H),3.16(q,J＝7.1Hz,4H),2.03(d,J＝6.6Hz,1H),1.98–1.89(m,1H),1.83–1.67(m,5H),1.67–1.54(m,3H),1.05(t,J＝7.1Hz,6H),0.78(t,J＝7.4Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.83,154.87,151.60,142.09,139.93,138.52,129.20,121.42,117.99,115.80,115.33,58.78,57.38,41.82,28.94,28.74,27.80,26.45,23.09,22.68,14.06,9.05.HR-MS(ESI):Calcd.C₂₄H₃₄N₆O₃S,[M+H]⁺m/z:487.2491,found:487.2492.

Example 24: synthesis of Compound 24

The procedure is as in example 29. Yield 57% as pale yellow solid, melting point 145-146 ℃.¹H NMR(400MHz,DMSO-d₆)δ9.40(s,1H),8.40(s,1H),7.83(d,J＝11.7Hz,2H),7.48–7.38(m,2H),7.27(d,J＝7.7Hz,1H),4.64–4.47(m,1H),4.27–4.14(m,1H),3.25(s,3H),2.78–2.70(m,2H),2.08–2.00(m,1H),1.98–1.88(m,1H),1.81–1.66(m,5H),1.66–1.54(m,3H),1.39–1.31(m,2H),1.27–1.21(m,2H),0.83–0.75(m,6H).¹³C NMR(100MHz,DMSO-d₆)δ162.82,154.92,151.57,141.81,140.82,138.53,129.06,121.20,117.78,115.71,115.60,58.84,57.41,42.25,31.05,28.93,28.73,27.77,26.45,23.07,22.66,19.21,13.44,9.04.HR-MS(ESI):Calcd.C₂₄H₃₄N₆O₃S,[M+H]⁺m/z:487.2491,found:487.2489.

Example 25: synthesis of Compound 25

The procedure is as in example 29. Yield 63% as pale yellow solid, melting point 154-.¹H NMR(400MHz,DMSO-d₆)δ9.41(s,1H),8.41(s,1H),7.84(d,J＝7.8Hz,2H),7.49–7.38(m,2H),7.27(d,J＝7.8Hz,1H),4.63–4.50(m,1H),4.42(t,J＝5.0Hz,1H),4.25–4.16(m,1H),3.39–3.36(m,2H),3.26(s,3H),2.85–2.78(m,2H),2.10–2.01(m,1H),1.98–1.90(m,1H),1.79–1.67(m,5H),1.67–1.58(m,3H),1.57–1.51(m,2H),0.78(t,J＝7.4Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.83,154.92,151.57,141.83,140.67,138.51,129.07,121.22,117.81,115.72,115.65,58.85,58.12,57.41,32.34,28.94,28.71,27.77,26.45,23.07,22.66,9.04.HR-MS(ESI):Calcd.C₂₃H₃₂N₆O₄S,[M+H]⁺m/z:489.2284,found:489.2284.

Example 26: synthesis of Compound 26

The procedure is as in example 29. Yield 53%, pale yellow solid, melting point 178-.¹H NMR(400MHz,DMSO-d₆)δ9.41(s,1H),8.41(t,J＝1.8Hz,1H),7.87–7.81(m,2H),7.50–7.41(m,2H),7.31–7.26(m,1H),4.68(t,J＝5.6Hz,1H),4.60–4.51(m,1H),4.23–4.16(m,1H),3.39(d,J＝6.4Hz,2H),3.25(s,3H),2.84–2.78(m,2H),2.08–2.01(m,1H),1.97–1.90(m,1H),1.78–1.67(m,5H),1.66–1.56(m,3H),0.78(t,J＝7.4Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.83,154.91,151.57,141.83,140.76,138.53,129.08,121.25,117.82,115.71,115.58,59.89,58.84,57.41,45.09,28.94,28.72,27.78,26.45,23.07,22.67,9.05.HR-MS(ESI):Calcd.C₂₂H₃₀N₆O₄S,[M+H]⁺m/z:475.2127,found:475.2126.

Example 27: synthesis of Compound 27

The procedure is as in example 29. Yield 60%, pale yellow solid, melting point 176-.¹H NMR(400MHz,DMSO-d₆)δ9.40(s,1H),8.40(s,1H),7.83(d,J＝10.5Hz,2H),7.59(t,J＝5.7Hz,1H),7.43(t,J＝7.9Hz,1H),7.28(d,J＝7.8Hz,1H),4.56(s,1H),4.20(d,J＝4.3Hz,1H),3.25(s,3H),3.17(s,3H),2.91(d,J＝5.7Hz,3H),2.10–1.99(m,1H),1.98–1.89(m,1H),1.84–1.66(m,5H),1.62(d,J＝6.8Hz,3H),0.78(t,J＝7.3Hz,3H).¹³C NMR(100MHz,DMSO-d₆)δ162.83,154.92,151.57,141.82,140.83,138.54,129.07,121.27,117.80,115.72,115.55,70.50,58.85,57.84,57.41,42.16,28.93,28.72,27.78,26.45,23.07,22.67,9.05.HR-MS(ESI):Calcd.C₂₃H₃₂N₆O₄S,[M+H]⁺m/z:449.2284,found:449.2285.

Example 28: synthesis of Compound 28

The procedure is as in example 29. The yield was 70% as pale yellow solid, melting point 156-.¹H NMR(400MHz,DMSO-d₆)δ9.41(s,1H),8.41(s,1H),7.92–7.74(m,2H),7.43(t,J＝7.9Hz,2H),7.26(d,J＝7.3Hz,1H),4.56(s,1H),4.20(d,J＝4.5Hz,1H),3.25(s,3H),2.80–2.69(m,2H),2.03(s,1H),1.93(s,1H),1.78–1.58(m,7H),1.34(d,J＝6.3Hz,2H),1.18(d,J＝7.6Hz,7H),0.87–0.71(m,6H).¹³C NMR(100MHz,DMSO-d₆)δ162.83,154.94,151.57,141.82,140.88,138.53,129.03,121.19,117.78,115.70,115.59,58.84,57.41,42.56,30.72,28.94,28.89,28.74,27.77,26.45,25.68,23.07,22.67,21.92,13.78,9.03.HR-MS(ESI):Calcd.C₂₆H₃₈N₆O₃S,[M+H]⁺m/z:515.2804,found:515.2803.

Example 30: synthesis of Compound 30

The procedure is as in example 29. The yield was 73% as a pale yellow solid with a melting point of 156 ℃ and 157 ℃.¹H NMR(400MHz,DMSO-d₆)δ8.21–8.06(m,2H),7.78(s,1H),7.39–7.27(m,4H),7.05–6.99(m,3H),6.63(s,1H),4.48–4.37(m,1H),4.18–4.11(m,1H),3.23(s,3H),2.45(s,1H),1.91–1.78(m,2H),1.71–1.52(m,6H),1.51–1.39(m,2H),0.77(q,J＝7.2Hz,5H),0.53–0.45(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ162.72,155.81,151.55,149.40,146.47,138.82,129.78,126.95,125.79,124.72,122.08,121.82,119.25,115.48,110.25,58.99,57.42,28.86,28.70,27.77,26.41,24.54,23.07,22.70,9.04,6.80.HR-MS(ESI):Calcd.C₂₉H₃₄N₆O₄S,[M+H]⁺m/z:563.2435,found:563.2437.

Example 31: synthesis of Compound 31

The procedure is as in example 29. Yield 63% as pale yellow solid, melting point 178-.¹H NMR(400MHz,DMSO-d₆)δ8.53–8.45(m,1H),8.22(d,J＝2.5Hz,1H),8.18–8.09(m,2H),7.79(s,1H),7.56–7.49(m,1H),7.48–7.40(m,1H),7.36–7.29(m,1H),7.04(d,J＝8.7Hz,1H),6.72(s,1H),4.48–4.36(m,1H),4.19–4.09(m,1H),3.23(s,3H),2.47(s,1H),1.92–1.77(m,2H),1.72–1.54(m,6H),1.50–1.39(m,2H),0.83–0.71(m,5H),0.55–0.44(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ162.73,155.78,151.56,148.04,146.79,146.23,143.53,138.83,130.04,125.93,124.89,124.71,121.80,118.15,115.54,110.33,58.96,57.40,28.87,28.69,27.77,26.41,24.53,23.08,22.69,9.05,6.79.HR-MS(ESI):Calcd.C₂₈H₃₃N₇O₄S,[M+H]⁺m/z:564.2388,found:564.2388.

Example 32: synthesis of Compound 32

The procedure is as in example 29. The yield thereof was found to be 53%. Pale yellow solid, melting point 181-182 ℃.¹H NMR(400MHz,DMSO-d⁶)δ9.40(s,1H),8.42(d,J＝1.8Hz,1H),7.93(s,1H),7.86–7.81(m,2H),7.42(t,J＝8.0Hz,1H),7.27(d,J＝7.8Hz,1H),4.62–4.49(m,1H),4.24–4.16(m,1H),3.25(s,3H),2.04(d,J＝6.1Hz,1H),1.97–1.90(m,1H),1.80–1.68(m,5H),1.65–1.56(m,3H),1.08(s,3H),0.78(t,J＝7.5Hz,3H),0.63(t,J＝5.1Hz,2H),0.36(q,J＝4.3Hz,2H).¹³C NMR(101MHz,DMSO-d⁶)δ162.84,154.92,143.07,141.72,138.47,128.95,121.17,118.00,115.74,115.69,58.93,57.47,30.76,28.91,28.71,27.78,26.44,23.89,23.08,22.70,12.96,9.04.HR-MS(ESI):Calcd.C₂₄H₃₂N₆O₃S,[M+H]⁺m/z:485.2330,found:485.2331

Example 33: synthesis of Compound 33

The procedure is as in example 29. The yield thereof was found to be 62%. Light yellow solid, melting point 167-.¹H NMR(400MHz,DMSO-d⁶)δ9.62(s,1H),8.44(s,1H),7.89(s,1H),7.84(d,J＝8.1Hz,1H),7.46(t,J＝8.0Hz,1H),7.42–7.25(m,5H),7.17(d,J＝7.7Hz,1H),5.50(d,J＝15.3Hz,1H),4.39(d,J＝15.4Hz,1H),4.06(t,J＝4.8Hz,1H),3.53(d,J＝3.7Hz,4H),3.28(s,3H),2.84–2.66(m,4H),1.89–1.74(m,2H),0.74(t,J＝7.4Hz,3H).¹³C NMR(101MHz,DMSO-d⁶)δ162.46,154.95,151.23,142.08,138.73,136.79,134.51,129.33,128.60,127.78,127.43,122.11,118.97,115.94,114.90,65.17,60.22,46.82,45.80,27.76,24.18,8.55.HR-MS(ESI):Calcd.C₂₆H₃₀N₆O₄S,[M+H]⁺m/z:523.2122,found:523.2121.

Example 34: synthesis of Compound 34

The procedure is as in example 29. The yield thereof was found to be 78%. Pale yellow solid, melting point 158-159 ℃.¹H NMR(400MHz,DMSO-d⁶)δ8.11(s,1H),8.07(d,J＝2.2Hz,1H),7.78(s,1H),7.49–7.41(m,1H),7.09(d,J＝8.7Hz,1H),6.50(s,1H),4.42–4.33(m,1H),4.18–4.13(m,1H),3.98(q,J＝7.1Hz,2H),3.23(s,3H),2.45(s,1H),1.91–1.85(m,1H),1.82–1.77(m,1H),1.73–1.64(m,3H),1.61–1.54(m,3H),1.52–1.45(m,2H),1.17(t,J＝7.1Hz,3H),0.82–0.71(m,6H),0.56–0.44(m,2H).¹³C NMR(101MHz,DMSO-d⁶)δ162.73,155.92,151.52,146.20,125.81,124.50,124.02,121.84,120.60,115.40,110.56,66.09,59.29,57.69,28.77,28.60,27.75,26.39,24.56,23.10,22.76,14.48,9.00,6.82.HR-MS(ESI):Calcd.C₂₅H₃₄N₆O₄S,[M+H]⁺m/z:515.2435,found:515.2436.

Example 35: synthesis of Compound 35

The procedure is as in example 29. The yield thereof was found to be 64%. Pale yellow solid, melting point 149-150 ℃.¹H NMR(400MHz,DMSO-d⁶)δ8.07(s,1H),7.76(d,J＝2.1Hz,1H),7.73(s,1H),7.37–7.27(m,1H),7.10(d,J＝8.6Hz,1H),6.32(s,1H),4.33–4.21(m,1H),4.18–4.09(m,1H),3.64–3.59(m,4H),3.22(s,3H),2.80(d,J＝4.1Hz,4H),2.44(s,1H),1.85–1.64(m,5H),1.60–1.50(m,3H),1.49–1.37(m,2H),0.80–0.72(m,5H),0.55–0.41(m,2H).¹³C NMR(101MHz,DMSO-d⁶)δ162.77,156.28,151.55,146.59,138.74,125.60,124.73,123.56,119.63,115.28,110.39,65.27,59.74,58.05,45.89,28.65,28.46,27.73,26.32,24.53,23.10,22.84,8.96,6.84.HR-MS(ESI):Calcd.C₂₇H₃₇N₇O₄S,[M+H]⁺m/z:556.2701,found:556.2702.

BRD4 protein inhibitory Activity assay

1. Experimental methods

The HTRF combines two technologies of Fluorescence Resonance Energy Transfer (FRET) and time-resolved fluorescence (TRF), combines a homogeneous experiment mode of FRET and the low background characteristic of TRF, and has the characteristics of simple operation, high sensitivity, large flux and stable and reliable experiment data. With Anti-GST-Cryptate (Eu3+ Cryptate conjugated mouse monoclonal Anti-glutathione S-transferase) (Cisbio, 61GSTKLB) as an energy donor and Streptavidin-d2(d2-conjugated Streptavidin) (Cisbio, 610 GSTKLB) as an energy acceptor, 1.6nM GST-BRD4(BD1), 200nM Biotin-H4 (Gilbert chemical), 0.125 ng/. mu.L of Anti-GST-ti-Cryptate (Cisbio, 61GSTKLB), 2.5 ng/. mu.L of Streptavidin-d2(Cisbio, 610SADLB), Assay buffer (50mM Hepes,400mM, 50mM of sodium chloride, 0.1% of JOJJNJNJNJNJNJNJNJNJNJL), the final signal emission ratio of the compounds is judged by the interaction between the two kinds of compounds at room temperature, i.7%, 1.20 nM, 30 nM, and the final signal emission value of the biological molecules, when the inhibitor is added, the inhibitor can be combined with the active site of BRD4, so that the combination of BRD4 and a substrate is inhibited, when the interaction between two molecules is inhibited, the donor and the acceptor can not transfer energy due to long distance, so that the emission wavelength signal is reduced, the inhibition degree of the inhibitor on the interaction between BRD4 and histone can be evaluated, the inhibition activity of the small molecular compound of the invention on BRD4 protein can be tested, and the IC is calculated by SPSS or GraphPad in data processing₅₀. The experimental results are shown in the second table.

TABLE II results of BRD4 protein inhibitory Activity of the Compounds prepared in examples 1 to 35

Note: JQ1 is a positive control;

MTT method tumor cell inhibitory Activity test

1. Experimental methods

The partial compound of the invention and the positive control compound JQ1 are respectively prepared into solutions with concentration gradients of 50, 25, 12.5, 6.26, 3.125, 1.56, 0.78, 0.39 and 0.19 mu M by using 1640 culture medium, and are respectively co-cultured with gastric cancer cells SGC7901, BGC823, MGC803, MKN45 and leukemia cells THP1 in a 96-well plate for 5 days, wherein each well of the gastric cancer cells is paved with 1000 cells, and each well of the leukemia cells is paved with 5000 cells. After the compound stimulates gastric cancer cells for 5 days, adding MTT solution into the cells, wherein each hole is 20 mul, continuously culturing for 4 hours in an incubator, then slightly sucking out the original culture medium, adding 150 mul DMSO for dissolving, and slightly shaking for 10 minutes at room temperature to ensure that crystals are completely dissolved; and taking out the 96-well plate, and measuring the light absorption value of each well by a microplate reader at 570 nm. Adding MTT solution into the cells after the compound stimulates leukemia cells for 5 days, wherein each well has 20 mu L, continuously culturing for 4 hours in an incubator, adding 100 mu L of triple solution into each well, reacting for 24 hours, measuring the light absorption value of each well by an enzyme-labeling instrument at 570nm, and calculating the survival rate of the cells and IC according to the light absorption value₅₀The experimental results are shown in the third table.

TABLE III antitumor Activity of some Compounds of the invention in vitro

Note: JQ1 is a positive control; SGC7901, BGC823, MGC803, MKN45 are gastric cancer cells, THP1 is leukemia cells

The above test results show that the compounds provided in examples 1 to 35 have excellent inhibitory effects on BRD4 protein and tumor cells, and that some of the compounds have inhibitory effects on BRD4 protein that are superior or superior to those of compounds that have already entered clinical stage, such as OTX-015 (IC)₅₀ 240nM)、CPI-0610(IC₅₀ 120nM)、FT-010(IC₅₀ 100nM)、RVX-208(IC₅₀510 nM). The inhibitory activity of partial compounds on gastric cancer cells reaches or is better than that of a positive control compound JQ 1.

Finally, it should be noted that: the above embodiments are merely illustrative and not restrictive of the technical solutions of the present invention, and any equivalent substitutions and modifications or partial substitutions made without departing from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A dihydropteridinone-sulfonamide derivative characterized by: the structure is shown as the general formula (I):

wherein R is¹Is cyclopentyl;

R²is phenoxy, ethoxy, morpholinyl;

R³is cyclopropyl.

2. A dihydropteridinone-sulfonamide derivative characterized by: the concrete structure is shown in formulas 29-30 and 34-35:

3. a pharmaceutically acceptable salt of a dihydropteridinone-sulfonamide derivative of any of claims 1-2, characterized in that: the acid addition salt of the dihydropteridinone-sulfonamide derivative and the following acid: the acid is hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, boric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, benzenesulfonic acid, citric acid, lactic acid, pyruvic acid, tartaric acid, acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, salicylic acid or phenylacetic acid.

4. A process for the preparation of dihydropteridinone-sulphonamides derivatives, as claimed in any one of claims 1 to 2, characterized in that: dissolving compounds shown in formula 40 and formula 46 with a molar ratio of 1:0.5-1 in an organic solvent, adding hydrochloric acid, and heating at 60-120 ℃ for reaction for 24-72 hours;

5. a process for the preparation of dihydropteridinone-sulphonamides derivatives, according to claim 4, characterized in that: the preparation method of the compound shown in the formula 40 is as follows:

a. taking o-nitrofluorobenzene 36 as a raw material, reacting with chlorosulfonic acid at 80-120 ℃ for 5-12 hours under stirring, and performing post-treatment after the reaction to obtain an intermediate 37; the molar ratio of the o-nitrofluorobenzene 36 to the chlorosulfonic acid is 1: 1-40;

b. in the presence of organic base, stirring the intermediate 37 and the corresponding amine compound at a molar ratio of 1:1-2 at 0-10 ℃ for reaction, and after the reaction is finished, performing post-treatment to obtain an intermediate 38; the organic base is selected from triethylamine, pyridine, triethylene diamine, N-methylmorpholine, potassium tert-butoxide, sodium tert-butoxide or butyl lithium; the corresponding amine compound is an amine compound corresponding to a substituent R3;

c. dissolving the intermediate 38 in an organic solvent, adding a corresponding reagent, stirring and reacting at 10-100 ℃, and after the reaction is finished, carrying out post-treatment to obtain an intermediate 39; the organic solvent is dimethyl sulfoxide or N, N-dimethylformamide; the corresponding reagent is a compound corresponding to a substituent R2; the molar ratio of the intermediate 38 to the corresponding reagent is 1: 1-5;

d. dissolving the intermediate 39 in a solvent, adding a catalytic amount of palladium-carbon catalyst, heating to 20-80 ℃ in a hydrogen atmosphere, stirring for 4-8 hours, and obtaining an intermediate 40 after the reaction is finished;

the reaction route of the above steps is as follows:

6. a process for the preparation of dihydropteridinone-sulphonamides derivatives, according to claim 4, characterized in that: the preparation method of the compound shown in the formula 46 is as follows:

e. dissolving the compound 41 in an organic solvent, dropwise adding thionyl chloride under ice bath, carrying out reflux reaction, and carrying out post-treatment to obtain an intermediate 42 after the reaction is finished; the molar ratio of the compound 41 to the thionyl chloride is 1: 1-10;

f. dissolving the intermediate 42 in a solvent, adding a corresponding carbonyl compound, adding an alkaline compound and a reducing agent under ice bath, stirring at 0-30 ℃ until the raw materials disappear, and performing post-treatment after the reaction to obtain an intermediate 43; the corresponding carbonyl compound is a compound corresponding to a substituent R1; the reducing agent is selected from NaBH 4, KBH 4, NaBH (OAc)₃Or NaBH₃CN；

The molar ratio of the intermediate 42 to the corresponding carbonyl compound is 1:1-2, the molar ratio of the intermediate 42 to the basic compound is 1:1-3, and the molar ratio of the intermediate 42 to the reducing agent is 1: 0.25-10;

g. dissolving the intermediate 43 in a solvent, adding 2, 4-dichloro-5-nitropyrimidine and the alkaline compound under ice bath, stirring at room temperature until the raw materials disappear, and performing post-treatment after the reaction to obtain an intermediate 44;

the molar ratio of the intermediate 43, the 2, 4-dichloro-5-nitropyrimidine and the basic compound is 1: 1-3;

h. dissolving the intermediate 44 in acetic acid, heating to 50-70 ℃, adding reduced iron powder, stirring for reaction, heating to 100-110 ℃, continuing to react, and performing post-treatment to obtain an intermediate 45;

the molar ratio of the intermediate 44 to the reduced iron powder is 1: 2-20;

i. adding the intermediate 45 into anhydrous DMF, adding 1.3 equivalents of methyl iodide and 1.3 equivalents of NaH under ice bath, stirring at room temperature for reaction, and performing post-treatment after the reaction is finished to obtain an intermediate 46;

wherein the basic compound of steps f and g is selected from Na₂CO₃、K₂CO₃、CsCO₃NaOAc or KOAc.

7. Use of the dihydropteridinone-sulfonamide derivatives and pharmaceutically acceptable salts thereof as BRD4 protein inhibitors in the preparation of anti-tumor medicaments according to any of claims 1 to 3.