A glutarimide compound and its preparation method and application

Abstract

The present invention relates to a glutarimide compound, the chemical structure of which is shown in the following formula (I): wherein L is selected from D and a glutarimide compound is selected from the present invention, which can simultaneously degrade BDR4 and GSPT1. The compound shows a good killing effect on blood system tumor cells (including multiple myeloma, lymphoma and leukemia), which is better than the clinical candidate drug CC-90009.

Description

Technical Field

[0001] The present invention relates to the technical field of compounds, and more specifically, to a glutarimide compound and a preparation method and application thereof. Background Art

[0002] Leukemia, malignant lymphoma and multiple myeloma are the three major malignant tumors of the blood system. According to GLOBOCAN2020 and the latest statistics from China, non-Hodgkin lymphoma (NHL) and leukemia are the two types of malignant tumors with the highest incidence and mortality in the blood system. In 2020, the incidence and mortality of NHL were 2.8% and 2.6% respectively, while the incidence and mortality of leukemia were 2.5% and 3.1% respectively. In 2020, there were 97,788 and 88,249 new NHL and leukemia patients in China. Thanks to the widespread use of new targeted drugs, the 5-year survival rate of various blood system tumors has increased significantly by more than 20% compared with 50 years ago. On the other hand, the survival rate of patients with acute myeloid leukemia (AML) is still at a relatively low level. Although the existing tumor targeted drugs for NHL and AML have reduced the side effects of drugs to a certain extent, they are prone to drug resistance. At the same time, most drugs are developed by foreign institutions, some drugs are too expensive, and the burden on patients is heavy, and there is a shortage of locally developed drugs. AML and NHL are characterized by low survival rates and high recurrence rates. Long-term disease control is still poor in most patients with relapsed or refractory AML. Therefore, there is a huge clinical need that has not been met. There is an urgent need to develop drugs with independent intellectual property rights in my country for the treatment of AML and NHL to break the foreign technology monopoly.

[0003] G1 to S phase transition 1 (GSPT1) was initially identified as an essential gene for the transition from G1 to S phase in the cell cycle, and was later renamed eukaryotic peptide release factor 3a (eRF3a). As a key member of peptide release factors, eRF3a participates in a variety of biological processes, including terminating protein translation, regulating intracellular mRNA degradation, regulating cell growth cycle and apoptosis, and participating in cytoskeleton formation. GSPT1 dysfunction is closely related to the occurrence and evolution of a variety of common malignant tumors, such as NHL and AML. CC-90009 is the first clinical candidate drug specifically targeting GSPT1 degradation. It is a Phase I clinical candidate drug for the treatment of acute leukemia AML (clinical trial number: NCT04336982) and myelodysplastic syndrome MDS (clinical trial number: NCT02848001). However, CC-90009 has a single structure and its inhibitory effect on hematological tumors needs to be improved.

[0004]

[0005] As an epigenetic regulator and transcriptional cofactor, BRD4 is closely related to gene transcription, cell cycle and apoptosis, invasion and metastasis. Abnormal expression of BRD4 can lead to dysregulation of multiple gene expressions, affect the functions of related genes, and play an important role in tumor occurrence, development, infiltration and metastasis of tumor cells. BRD4 inhibitors show good anti-tumor effects and have good application prospects in various tumors, especially hematological tumors. However, whether it is BRD4 inhibitors or GSPT1 PROTAC molecules, it is difficult to achieve good therapeutic effects on refractory NHL and AML. In order to overcome this problem, the applicant discovered through scientific research that the design of compounds to degrade BRD4 and GSPT1 at the same time can significantly enhance the killing effect of compounds on NHL and AML tumor cells. At present, there is no compound that can degrade BRD4 and GSPT1 at the same time, and it is difficult to take both into account. The development of compounds that can degrade BRD4 and GSPT1 at the same time for the treatment of NHL and AML tumor patients has important research significance and a broad pre-clinical application period. Summary of the invention

[0006] The inventors found through research that the designed compound degrades BRD4 and GSPT1 simultaneously, showing stronger inhibitory activity than the clinical phase I drug CC-90009. The purpose of the present disclosure is to provide a bifunctional compound that degrades BDR4 and GSPT1 proteins simultaneously, and a preparation method and application thereof. The compound, as a bifunctional degrader of BRD4 and GSPT1 proteins, can significantly inhibit the growth of NHL and AML tumor cells.

[0007] A first aspect of the present invention provides a glutarimide compound.

[0008] Specifically, a glutarimide compound has a molecular structure shown in the following formula (I):

[0009]

[0010] Among them, L is selected from D is selected from

[0011] Preferably, the glutarimide compound is characterized by having any one of the following structural formulas Ⅰa, Ⅰb or Ⅰc:

[0012]

[0013] Among them, L is selected from

[0014] Further preferably, the glutarimide compound has any one of the structural formulas shown in the following formulas 1-18:

[0015]

[0016] The second aspect of the present invention provides a method for preparing a glutarimide compound, comprising the following steps:

[0017] The compound represented by formula (II) and the compound represented by formula (III) are subjected to condensation reaction in the presence of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholinium chloride and nitrogen methylmorpholine to obtain a compound represented by formula (1):

[0018]

[0019] Among them, L1 is selected from

[0020] D is selected from

[0021] The temperature of the condensation reaction is 20-30° C., the time is 0.5-3 hours, the amount of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholinium chloride is 1.5 equivalents, the amount of nitrogen methylmorpholine is 2 equivalents, and the solvent is DMF.

[0022] In a third aspect, the present invention also relates to a pharmaceutical composition, which comprises the above-mentioned compound or its tautomer, enantiomer, diastereomer, mixture of enantiomers and diastereomers, racemate, mesomer, mixture of racemate and mesomer, pharmaceutically acceptable hydrate, pharmaceutically acceptable salt, solvate, polymorph or nitrogen oxide in a physiologically acceptable medium. Preferably, the drug also includes a pharmaceutically acceptable carrier and excipients.

[0023] In a fourth aspect, the present invention also relates to the compounds described above or their tautomers, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, racemates, mesomers, mixtures of racemates and mesomers, pharmaceutically acceptable hydrates, pharmaceutically acceptable salts, solvates, polymorphs or nitrogen oxides, and the use of the pharmaceutical compositions in the preparation of drugs for BRD4 degradation or GSPT1 degradation.

[0024] In a fifth aspect, the present invention also relates to the use of the above-mentioned compound or its tautomer, enantiomer, diastereomer, mixture of enantiomers and diastereomers, racemate, mesomer, mixture of racemate and mesomer, pharmaceutically acceptable hydrate, pharmaceutically acceptable salt, solvate, polymorph or nitrogen oxide, and the pharmaceutical composition in the preparation of drugs for treating tumor diseases related to BRD4 and GSPT1 targets. Preferably, the tumor diseases include non-Hodgkin's lymphoma, chronic lymphocytic leukemia, B-cell lymphoma, mantle cell lymphoma, and acute myeloid leukemia.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] The present invention provides a glutarimide compound having a molecular structure shown in formula (I), which can significantly degrade BRD4 and GSPT1, and is a BRD4 / GSPT1 dual-target degradation compound with significant effects, showing stronger inhibitory activity than the clinical phase I drug CC-90009. Therefore, the present invention provides a glutarimide compound for the treatment of blood system NHL and AML tumors, and has good application prospects. DETAILED DESCRIPTION

[0027] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0028] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0029] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0030] The room temperature described in the examples is the conventional room temperature in the art, preferably 20-30°C.

[0031] The progress of the reaction of the present invention is monitored by conventional monitoring methods (such as TLC (thin layer chromatography), LCMS (liquid chromatography-mass spectrometry) or NMR (nuclear magnetic resonance)), and the disappearance of the reaction substrate is generally regarded as the reaction endpoint.

[0032] In the following specific examples, the liquid chromatography conditions used for the detection and identification of the prepared compounds are: Shimadzu LCMS2020, G1322A degasser, G1312 binary high pressure pump, G1329A autosampler, G1316A column oven, G4212B diode array detector. The chromatographic column is XbridgeC18 (50mm×4.6mm, 5.0μm), deionized water is used as mobile phase A, acetonitrile containing 0.1% trifluoroacetic acid is used as mobile phase B, and gradient elution is performed. The specific gradient elution procedure is as follows:

[0033]

[0034]

[0035] The flow rate was 1.5 mL / min, the column temperature was 40 °C, and the detection wavelength was 254 nm.

[0036] the term

[0037] Unless otherwise specified, the following terms appearing in the specification and claims of the present invention have the following meanings:

[0038] The present invention also relates to useful forms of the compounds disclosed herein, for example, metabolites, hydrates, solvates, prodrugs, salts, especially pharmaceutically acceptable salts, and coprecipitates.

[0039] Further, the compounds of the present invention may exist in free form, for example, as a free base or free acid or zwitterion, or may exist in salt form. The salt may be any salt commonly used in pharmacy, an organic or inorganic addition salt, in particular any pharmaceutically acceptable organic or inorganic addition salt.

[0040] The pharmaceutically acceptable salts of the compounds of the present invention may be, for example, acid addition salts of the compounds of the present invention carrying nitrogen atoms in the chain or ring, for example, acid addition salts of sufficiently basic compounds of the present invention, for example, acid addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, bisulfuric acid, phosphoric acid or nitric acid, or acid addition salts formed with organic acids, for example, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamic acid, cyclopentane acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid or thiocyanic acid.

[0041] The compounds of the present invention may contain unnatural proportions of isotopes at one or more of the atoms constituting the compound, for example, replacement of hydrogen with heavy hydrogen to form a deuterated drug.

[0042] As used herein, the term "stereoisomer" refers to compounds that have identical chemical constitution but differ in the arrangement of the atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformational isomers, and the like.

[0043] As used herein, the term "enantiomers" refers to two stereoisomers of a compound that are non-superimposable mirror images of one another.

[0044] As used herein, the term "diastereomer" refers to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties or biological activities. Mixtures of diastereomers can be separated using chiral HPLC.

[0045] Drug sensitivity experiments were used to detect the inhibitory effect of the compound on the activity of NHL and AML tumor cells, and WB experiments were used to detect the degradation effect of the compound on GSPT1 and BRD4. The results showed that the compound had a good killing effect on NHL and AML tumor cells, and could efficiently degrade GSPT1 and BRD4, which was better than the clinical candidate drug CC-90009.

[0046] It will be appreciated by those skilled in the art that some compounds of formula (I) may contain one or more chiral centers and therefore may exist as two or more stereoisomers. Therefore, the compounds of the present invention may exist as single stereoisomers (e.g. enantiomers, diastereomers) and mixtures thereof in any proportion, such as racemates, and, where appropriate, as tautomers and geometric isomers.

[0047] Example 1

[0048] The specific operation method of the glutarimide compound route of the present invention is as follows: the target product is synthesized by route 1

[0049] Route 1:

[0050]

[0051] The compound represented by formula (II) and the compound represented by formula (III) (II:III equivalent ratio = 1:1) are subjected to condensation reaction under the conditions of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholinium chloride (1.5 equivalents), nitrogen methylmorpholine (2 equivalents), and DMF (20 equivalents). The temperature of the condensation reaction is 20-30°C, the time is 0.5-3 hours, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholinium chloride is 1.5 equivalents, nitrogen methylmorpholine is 2 equivalents, and the solvent is DMF. After the reaction is completed by LCMS monitoring, the reaction mixture is poured into water, extracted with ethyl acetate, the organic phase is washed with brine 3 times, and dried over anhydrous sodium sulfate, and purified by Prep-TLC to obtain the final product, namely compound 1-18.

[0052] The identification results of target compound 1 are as follows:

[0053] [4-(4-(1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-hydro-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetyl)piperidin-4-yl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione](1). 1HNMR(400MHz,DMSO)δ11.10(s,1H),7.72(d,J=6.4Hz,1H),7.48(dd,J=15.9,7.5Hz,4H ),7.38(s,2H),5.10(dd,J=12.7,5.4Hz,1H),4.59(t,J=6.7Hz,1H),4.48–4.39(m,1H) ,4.24(s,1H),3.63(dd,J=14.8,5.4Hz,2H),3.48(d,J=29.8Hz,5H),3.22–3.09(m,2H) ,2.96–2.83(m,2H),2.80–2.52(m,10H),2.42(s,3H),2.10–1.81(m,3H),1.64(s,3H). 13 C NMR (101MHz, DMSO) δ173.13,170.44,168.38,167.51,166.84,163.33,163.18,155.56,150.16,144.50,142.60,137.11,136.50,135.59, 134.09,132.48,131.10,130.56,130.28,129.98,128.89,124.19,54 .65,54.12,49.35,48.79,35.16,31.35,22.31,14.31,13.10,11.64.

[0054] The preparation method of the glutarimide compound 2-18 of the present invention is the same as the preparation method of the above-mentioned compound 1. The identification results of the target product 2-18 are as follows:

[0055] [4-(4-((1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-hydro-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetyl)piperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione](2). 1HNMR (400MHz, DMSO) δ11.09(s,1H),7.72(s,1H),7.50(d,J=8.3Hz,2H),7.45(d,J=3.9Hz,2H),7.37 (s,2H),5.10(dd,J=12.5,5.3Hz,1H),4.59(t,J=6.7Hz,1H),4.37(d,J=12.7Hz,1H),4.15(d,J=12.4 Hz,1H),3.81(s,1H),3.67–3.53(m,2H),3.41(s,4H),3.21–3.08(m,2H),2.89(t,J=13.1Hz,1H),2.6 8–2.54(m,9H),2.42(s,3H),2.23(s,2H),2.10–1.93(m,2H),1.86(s,2H),1.75(s,1H),1.64(s,3H). 13 CNMR(101MHz,DMSO)δ177.53,173.18,170.32,168.12,167.53,155.78,150.05,137.13,136.34,135.47,132.67,131.01,130.56,130.30,130.1 3,128.89,124.18,120.12,115.40,113.73,61.82,61.38,54.55,53.28 ,49.19,41.59,36.54,35.27,31.25,27.18,22.37,14.27,13.18,11.74.

[0056] [4-(4-(1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-hydro-thieno[3,2-f][1,2,4]s-triazolo[4,3-a][1,4]diazepin-6-yl)acetyl)azetidin-3-yl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione](3). 1HNMR(400MHz,DMSO)δ11.09(s,1H),7.72(td,J=7.8,4.0Hz,1H),7.47(ddd,J=19.0,8.6,4.5Hz,4H),7.40 –7.30(m,2H),5.10(dd,J=12.8,5.4Hz,1H),4.49(t,J=6.6Hz,1H),4.41(dt,J=20.1,7.7Hz,1H),4.30–4.1 9(m,1H),3.95(dd,J=14.8,7.5Hz,1H),3.78(d,J=26.8Hz,1H),3.27(dd,J=17.0,7.0Hz,6H),3.20–3.13( m,1H),2.89(dd,J=14.4,11.5Hz,1H),2.59(d,J=9.8Hz,9H),2.41(s,3H),2.09–2.01(m,1H),1.63(s,3H). 13 C NMR(101MHz,DMSO)δ173.15,170.39,170.22,170.08,167.46,166.73,163.58 ,155.40,150.40,149.98,137.03,136.33,135.54,134.07,132.63,131.17,13 0.60,130.28,130.02,128.91,124.18,117.12,115.50,54.20,53.99,53.59,51.59,50.65,49.45,49.28,33.78,33.74,31.28,22.48,14.48,13.10,11.64.

[0057] [4-(4-((1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-hydro-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetyl)azetidin-3-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione](4). 1HNMR(400MHz,DMSO)δ11.09(s,1H),7.72(t,J=7.7Hz,1H),7.50(dd,J=8.7,2.1Hz,2H), 7.46–7.43(m,2H),7.37(t,J=6.9Hz,2H),5.10(dd,J=12.9,5.3Hz,1H),4.51–4.42(m,2 H),4.02(dd,J=31.1,18.9Hz,3H),3.59(s,2H),3.29–3.19(m,3H),3.19–3.09(m,2H),2 .94–2.85(m,2H),2.58(d,J=19.3Hz,10H),2.41(s,3H),2.05–2.00(m,1H),1.63(s,3H). 13 C NMR (101MHz, DMSO) δ173.20,170.38,170.12,170.07,167.44,166.70,163.60,163.53,155.40,150.31,137.13,136.30,135.65,134.00,132.62 ,131.16,130.57,130.28,129.97,128.92,127.06,124.18,54.47,54.19 ,52.08,49.22,33.59,31.36,29.33,26.09,22.38,14.44,13.04,11.75.

[0058] [4-(3-(4-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-hydro-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetyl)piperazin-1-yl)azetidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione](5). 1HNMR (400MHz, DMSO) δ11.08(s,1H),7.61–7.55(m,1H),7.49(d,J=8.7Hz,2H),7.43(d,J=8.6Hz,2H),7.13(d, J=7.0Hz,1H),6.81(d,J=8.6Hz,1H),5.05(dd,J=12.7,5.4Hz,1H),4.58(t,J=6.7Hz,1H),4.29(s,2H),4.01( s,2H),3.69(s,2H),3.60(dd,J=16.3,7.1Hz,1H),3.51(d,J=5.7Hz,4H),3.30–3.23(m,1H),2.94–2.84(m,1H ),2.60(s,3H),2.57(s,1H),2.45(d,J=4.2Hz,2H),2.40(s,3H),2.33(s,2H),2.04–1.97(m,1H),1.62(s,3H). 13 C NMR (101MHz, DMSO) δ173.23,170.42,168.58,167.62,166.88,163.30,155 .66,150.19,148.17,137.16,135.61,135.41,133.70,132.57,131.11,13 0.54,130.29,130.04,128.89,120.30,112.24,110.60,54.56,54.16,49. 71,49.33,49.06,45.21,41.41,35.07,31.29,22.54,14.35,13.09,11.67.

[0059] [4-(3-((4-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-hydro-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetyl)piperazin-1-yl)methyl)azetidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione](6). 1HNMR (400MHz, DMSO) δ11.10 (s, 1H), 7.62 (t, J = 7.8Hz, 1H), 7.51–7.42 (m, 4H), 7.21 (dd, J = 15. 8,8.7Hz,1H),7.06(t,J=6.6Hz,1H),6.96(s,1H),5.07(dd,J=12.7,5.3Hz,1H),4.58(dd,J=1 2.3,5.9Hz,1H),4.38(d,J=6.6Hz,1H),3.96(s,1H),3.73(d,J=20.4Hz,4H),3.65–3.48(m,4H ),3.40(s,2H),2.89(s,1H),2.66–2.55(m,5H),2.41(s,7H),2.08–1.99(m,1H),1.63(s,3H). 13 C NMR (101MHz, DMSO) δ173.21,170.48,168.48,167.71,163.29,155.68,150. 16,146.81,137.17,136.72,135.60,132.65,132.58,131.09,130.55,130.2 9,130.05,128.88,117.56,110.97,109.82,58.89,54.58,48.97,46.79,45.33,43.61,41.56,37.24,36.10,35.14,31.44,22.57,14.42,13.09,11.71.

[0060] [4-(4-((4-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-hydro-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetyl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione](7). 1HNMR (400MHz, DMSO) δ11.10 (s, 1H), 7.70–7.65 (m, 1H), 7.49 (d, J = 8.7Hz, 2H), 7.44 ( d,J=8.6Hz,2H),7.32(t,J=7.5Hz,2H),5.09(dd,J=12.8,5.4Hz,1H),4.58(t,J=6.7 Hz,1H),3.80–3.48(m,8H),2.88(t,J=10.7Hz,3H),2.59(d,J=7.0Hz,6H),2.41(s,8 H),2.07–2.01(m,1H),1.82(d,J=26.0Hz,3H),1.63(s,3H),1.35(d,J=11.2Hz,2H). 13 C NMR (101MHz, DMSO) δ173.24,170.44,168.54,167.51,166.70,163.31,155.66,150.55,150.19,137.15,136.16,135.61,134.07,132.57, 131.12,130.56,130.29,130.04,128.89,124.32,116.67,114.77,54 .56,51.31,49.18,35.12,31.37,30.81,22.50,14.42,13.10,11.68.

[0061] [4-(4-(4-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-hydro-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetyl)piperazin-1-yl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione](8). 1 HNMR (400MHz, DMSO) δ11.10(s,1H),7.68(t,J=7.8Hz,1H),7.48(t,J=9.5Hz,4H),7.34( d,J=6.9Hz,2H),5.10(dd,J=12.8,5.4Hz,1H),4.59(t,J=6.7Hz,1H),3.77(d,J=11.0Hz ,4H),3.61(dd,J=16.3,7.0Hz,4H),2.89(t,J=11.8Hz,3H),2.67(d,J=51.8Hz,10H),2. 41(s,3H),2.07–2.01(m,1H),1.92(d,J=3.9Hz,2H),1.70(d,J=9.5Hz,2H),1.63(s,3H).13 CNMR(101MHz,DMSO)δ173.23,170.42,168.53,167.49,166.73,163.33,1 55.66,150.20,150.08,137.16,136.19,135.62,134.03,132.58,131.12 ,130.55,130.29,130.05,128.89,124.35,116.83,115.04,61.03,54.57 ,50.69,49.08,48.82,35.03,31.37,27.94,22.49,14.42,13.09,11.67.

[0062] [5-(4-(1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-hydro-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetyl)piperidin-4-yl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione](9). 1 HNMR(400MHz,DMSO)δ11.09(s,1H),7.67(dd,J=8.3,2.0Hz,1H),7.50–7.40(m,4H),7.32(s,1H), 7.25(d,J=8.5Hz,1H),5.06(dd,J=12.7,5.3Hz,1H),4.57(t,J=6.7Hz,1H),4.39(d,J=11.2Hz,1H) ,4.18(d,J=12.3Hz,1H),3.40(s,4H),3.20–3.04(m,2H),2.88(t,J=15.5Hz,2H),2.64(d,J=40.9H z,12H),2.39(s,3H),2.03(s,1H),1.92(d,J=11.5Hz,1H),1.83(s,1H),1.61(s,3H),1.55(s,1H). 13C NMR (101MHz, DMSO) δ173.30,170.49,168.33,167.96,167.44,163.32,155.72,155.52,150.28,137.08,135.67,134.19,132.49,131.17,130 .53,130.30,130.06,128.88,125.33,118.77,118.25,108.26,61.15, 54.60,49.17,48.60,47.34,35.06,31.35,22.64,14.39,13.13,11.63.

[0063] [5-(4-((1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-hydro-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetyl)azetidin-3-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione](10). 1 HNMR(400MHz,DMSO)δ11.08(s,1H),7.69(d,J=8.5Hz,1H),7.52–7.48(m,2H),7.44(d,J=8 .5Hz,2H),7.36(s,1H),7.28(d,J=8.7Hz,1H),5.08(dd,J=12.9,5.3Hz,1H),4.48(td,J=7 .0,3.0Hz,2H),4.00(dd,J=37.8,23.7Hz,2H),3.46(s,4H),3.31–3.05(m,4H),2.94–2.83 (m,2H),2.59(d,J=13.2Hz,8H),2.41(s,3H),2.05–1.98(m,1H),1.63(s,3H),1.24(s,2H). 13 C NMR(101MHz,DMSO)δ173.21,170.47,167.95,167.38,163.63,155.35,150.41,137.13,135.68,134.40,132.63,131.16,1 30.56,130.27,130.01,128.92,125.30,118.31,108.46,54.12,52.12,49.10,33.56,31.23,22.70,14.53,12.95,11.70.

[0064] [5-(4-((1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-hydro-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetyl)piperidin-4-yl)methyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione](11). 1 HNMR (400MHz, DMSO) δ11.08(s,1H),7.68(d,J=8.5Hz,1H),7.49(d,J=8.4Hz,2H),7.46–7.41(m,2H),7.34(s,1H),7 .27(d,J=8.3Hz,1H),5.07(dd,J=12.9,5.3Hz,1H),4.58(t,J=6.6Hz,1H),4.36(d,J=12.2Hz,1H),4.13(d,J=13.0H z,1H),3.65–3.58(m,1H),3.43(s,6H),3.20–3.08(m,2H),2.88(dd,J=22.5,8.6Hz,1H),2.60(s,8H),2.41(s,3H), 2.21(s,2H),2.06–2.00(m,1H),1.86(d,J=11.1Hz,2H),1.74(d,J=13.6Hz,1H),1.63(s,3H),1.24(d,J=9.4Hz,2H). 13 C NMR (101MHz, DMSO) δ173.24,170.48,168.18,167.97,167.39,155.74,155.63,150.16,137.16,135.58,134.24,132.56,131.11, 130.55,130.07,128.88,125.32,118.69,118.21,108.28,54.62,53.09,49.17,35.20,31.37,30.51,22.57,14.41,13.09,11.66.

[0065] [5-(4-(1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-hydro-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetyl)azetidin-3-yl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione](12). 1HNMR (400MHz, DMSO) δ11.08(s,1H),7.70(dd,J=8.5,3.1Hz,1H),7.47(dd,J=11.0,5.8Hz,4H ),7.38(s,1H),7.30(d,J=7.8Hz,1H),5.08(dd,J=12.9,5.3Hz,1H),4.51–4.38(m,2H),4.30– 4.21(m,1H),4.00–3.88(m,1H),3.82–3.71(m,1H),3.50(s,4H),3.24(dd,J=17.5,10.5Hz,4H ),2.95–2.83(m,1H),2.60(s,6H),2.41(s,3H),2.08–1.97(m,1H),1.63(s,3H),1.23(s,2H). 13 C NMR (101MHz, DMSO) δ173.22,170.47,167.95,167.39,155.63,155.44,150.34,137.14,135.63,134.18,132.62,131.17,130.58, 130.27,129.95,128.90,125.26,118.34,108.42,53.44,49.33,49.08,46.95,33.67,31.35,29.41,22.58,14.52,13.17,11.81.

[0066] [5-(3-(4-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-hydro-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetyl)piperazin-1-yl)azetidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione](13). 1HNMR (400MHz, DMSO) δ11.08(s,1H),7.66(d,J=8.3Hz,1H),7.50(d,J=8.5Hz,2H),7.44(d,J=8.5Hz,2H),6 .81(s,1H),6.68(d,J=8.4Hz,1H),5.06(dd,J=12.9,5.2Hz,1H),4.58(t,J=6.6Hz,1H),4.14(t,J=7.4Hz, 2H),3.91(s,2H),3.71(s,2H),3.62(dd,J=16.2,7.2Hz,2H),3.52(d,J=4.3Hz,3H),3.44(d,J=6.6Hz,2H) ,2.92–2.85(m,1H),2.60(s,3H),2.42(s,3H),2.36(s,2H),2.04–1.98(m,1H),1.63(s,3H),1.23(s,2H). 13 C NMR (101MHz, DMSO) δ173.22,170.58,168.73,167.98,167.79,163.50,155.70,155.37,150.18,137.17,135.52,134.23,132.59,131.10,130 .62,130.29,130.00,128.93,125.18,117.29,114.66,104.98,55.41, 54.70,54.40,49.18,35.32,31.39,29.28,22.55,14.57,13.03,11.68.

[0067] [5-(3-((4-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-hydro-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetyl)piperazin-1-yl)methyl)azetidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione](14). 1HNMR(400MHz,DMSO)δ11.06(s,1H),7.59(t,J=7.3Hz,1H),7.46(dd,J=19.6,8.3Hz ,4H),7.09(s,1H),6.97(d,J=8.4Hz,1H),5.04(dd,J=12.4,4.6Hz,1H),4.58(t,J=6 .6Hz,1H),4.40(dd,J=32.8,23.9Hz,1H),4.05–3.45(m,10H),3.32(s,2H),2.87(dd ,J=17.0,8.2Hz,1H),2.60(s,5H),2.41(s,7H),2.01(d,J=5.3Hz,1H),1.63(s,3H). 13 C NMR (101MHz, DMSO) δ173.29,170.57,168.50,168.10,167.56,155.73,154.89,150.17,137.15,135.60,134.69,132.57,131 .21,130.55,130.28,130.08,128.88,125.41,54.60,49.04,46.67,41.80,37.56,35.17,31.39,22.64,14.34,13.15,11.68.

[0068] [5-(4-((4-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-hydro-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetyl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione](15). 1HNMR (400MHz, DMSO) δ11.08(s,1H),7.65(d,J=8.5Hz,1H),7.49(d,J=8.6Hz,2H),7.44(d,J=8.5H z,2H),7.31(s,1H),7.24(d,J=8.6Hz,1H),5.07(dd,J=12.8,5.3Hz,1H),4.58(t,J=6.6Hz,1H),4 .04(d,J=12.4Hz,2H),3.83–3.46(m,6H),3.01–2.86(m,3H),2.60(s,5H),2.41(s,6H),2.33(s,2 H),2.18(s,2H),2.05–1.99(m,1H),1.83(d,J=11.2Hz,2H),1.63(s,3H),1.17(d,J=11.3Hz,2H). 13 C NMR (101MHz, DMSO) δ173.28,170.58,168.05,167.30,163.28,155.66,155.39,150.17,137.02,135.51,134.44,132.54,131.17,130. 56,130.28,129.93,128.95,125.42,118.01,117.66,108.12,54.55,49.14,47.53,35.24,31.52,30.05,22.60,14.36,13.13,11.84.

[0069] [5-(4-(4-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-hydro-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetyl)piperazin-1-yl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione](16). 1HNMR (400MHz, DMSO) δ11.08(s,1H),7.66(d,J=8.5Hz,1H),7.49(d,J=8.6Hz,2H),7.43(d,J= 8.5Hz,2H),7.33(s,1H),7.27(d,J=8.6Hz,1H),5.06(dd,J=12.9,5.3Hz,1H),4.57(t,J=6.7 Hz,1H),4.10(d,J=12.0Hz,2H),3.75–3.55(m,4H),2.92(ddd,J=31.6,21.4,8.8Hz,4H),2.6 0(s,8H),2.41(s,3H),2.08–1.79(m,4H),1.63(s,3H),1.50(d,J=10.0Hz,2H),1.23(s,2H). 13 C NMR (101MHz, DMSO) δ173.36,170.44,168.06,167.41,163.37,155.66,155.24,150.15,137.13,135.74,134.26,132.50,131.12, 130.55,130.20,130.10,128.89,125.30,118.05,108.22,54.54,49.14,46.98,35.09,31.37,27.47,22.53,14.60,13.09,11.67.

[0070] [3-(5-(4-(4-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-hydro-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetyl)piperazin-1-yl)piperidin-1-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidine-2,6-dione](17). 1HNMR (400MHz, DMSO) δ11.06(s,1H),7.59(d,J=8.2Hz,1H),7.46(d,J=8.2Hz,2H),7.39(d,J=8. 2Hz,2H),7.31(s,1H),7.29(d,J=8.5Hz,1H),5.11(dd,J=12.4,4.6Hz,1H),4.54(t,J=6.6Hz,1H ),4.08(d,J=12.3Hz,2H),3.89(s,3H),3.71–3.61(m,4H),3.01(ddd,J=30.2,22.18.7Hz,4H), 2.59(s,8H),2.40(s,3H),2.06–1.89(m,4H),1.62(s,3H),1.50(d,J=10.0Hz,2H),1.24(s,2H). 13 CNMR(101MHz,DMSO)δ173.38,170.41,167.41,163.38,155.62,155.44,150.05,138.16,135.74,134.26,131.97,131.02,130.45 ,130.30,130.11,128.99,125.40,118.15,108.24,54.60,49.04,46.92,35.29,30.81,31.27,27.45,22.59,14.34,13.15,11.68.

[0071] [3-(5-(4-(4-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6-hydro-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetyl)piperazin-1-yl)piperidin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione](18). 1HNMR (400MHz, DMSO) δ11.08(s,1H),7.61(d,J=8.5Hz,1H),7.48(d,J=8.6Hz,2H),7.41(d,J=8. 5Hz,2H),7.31(s,1H),7.27(d,J=8.6Hz,1H),5.09(dd,J=12.6,5.1Hz,1H),4.55(t,J=6.7Hz,1H ),4.40(s,2H),4.09(d,J=12.6Hz,2H),3.71–3.51(m,4H),2.94(ddd,J=31.2,21.4,8.7Hz,4H), 2.61(s,8H),2.42(s,3H),2.08–1.90(m,4H),1.61(s,3H),1.51(d,J=10.0Hz,2H),1.22(s,2H). 13 C NMR (101MHz, DMSO) δ173.35,170.43,167.42,163.37,155.61,155.24,150.15,137.13,135.75,133.26,132.59,131.12,130.55, 130.10,129.50,128.89,125.41,118.15,108.22,55.54,49.19,48.15,46.99,35.17,31.27,27.47,24.53,14.60,13.19,11.68.

[0072] Comparative Example 1

[0073] The reference compound C2 was synthesized by referring to the preparation method of patent CN117143114 A, and the structural formula is as follows: 1 H NMR (400MHz, DMSO): δ9.85(s,1H),8.20(s,1H),7.45(d,J=8.5Hz,2H),7.39(dd,J=7.5,1.5Hz ,1H),7.36(d,J=8.5Hz,2H),7.29-7.22(m,2H),6.37(s,2H),6.01(s,1H),4.64(t,J=6.8Hz,1H ),4.45-4.28(m,1H),4.28-4.07(m,2H),3.82-3.57(m,2H),3.43(m,4H),2.76(s,3H),2.73(m ,4H),2.61(m,2H),2.45(s,3H),2.39(s,3H),2.23(s,3H),1.73(s,3H),1.36(t,J=6.1Hz,3H); 13C NMR(DMSO-d6)δ173.2,172.1,171.0,170.1,165.1,162.6,162.5,159.9,156.9,149.5,140.8,138.8,137.5,137.1,136.1,133.5,13 2.4,131.4,130.6,129.5,129.0,128.1,127.0,125.6,120.3,82.4,55.1,51.2,45.3,44.8,42.1,25.6,18.3,17.4,13.4,12.2,11.7.

[0074] Comparative Example 2

[0075] The reference compound 165 was synthesized according to the preparation method of patent US20230348418A1, and the structural formula is as follows: 1 H NMR (400MHz, DMSO): δ10.96(s,1H),9.87(s,1H),7.79(s,1H),7.71-7.63(m,2H),7.45(s,1H),7.40-7.30(m,2H),5.28(s,2H),5.12-5.04( m,1H),4.51-4.45(m,1H),4.36-4.35(m,1H),3.00–2.86(m,1H),2.61(d,J=17.2Hz,1H),2.47–2.34(m,1H),2.28(s,3H),2.10–1.95(m,1H). 13 C NMR (101 MHz, DMSO) δ 173.31, 171.41, 169.32, 153.85, 134.74, 132.51, 130.92, 130.71, 130.64, 129.38, 129.38, 129.32, 127.65, 123.47, 122.68, 120.27, 70.05, 68.12, 52.00, 31.61, 22.90. Comparative Example 3

[0076] Paper Nat Chem Biol.2018,14(7):706-714.doi:10.1038 / s41589-018-0055-y. Title: Plasticity inbinding confers selectivity in ligand-induced protein degradation. A series of CRBN-BRD4 degraders were reported, among which the compound, named dBET6 in the literature, has the following structural formula: The compound has been commercialized and purchased directly from Targetmol (CAS No.: 1950634-92-0).

[0077] Comparative Example 4

[0078] Paper JMed Chem.2021,64(4):1835-1843.doi:10.1021 / acs.jmedchem.0c01489. Title: CC-90009: A Cereblon E3 Ligase Modulating Drug That Promotes Selective Degradation of GSPT1 for the Treatment of Acute Myeloid Leukemia. The discovery process of CC-90009 is reported. The structural formula is as follows:

[0079] The compound was commercially available and purchased directly from InvivoChem (CAS No.: 1860875-51-9).

[0080] Product effect testing

[0081] Degradation of BRD4 and GSPT1 proteins (Western blot)

[0082] 1. Cell culture: The culture medium of MOLM13 cells was RMPI1640+10% FBS+1% penicillin-streptomycin solution, and the cells were cultured at 37°C and 5% CO2.

[0083] 2. Plate laying: One day in advance, add 2 ml of 1×10 6 The cells were cultured overnight and the cell density reached 2×10 6 When the concentration of the test compound was 1 μM, each well was added with the test compound solution and the cells were placed in a 37°C, 5% CO2 incubator for 24 h.

[0084] 3. Extraction of Cellular Protein

[0085] (1) Mix RIPA lysis buffer and protease inhibitors.

[0086] (2) Centrifuge the cells and discard the culture medium, resuspend them in PBS, centrifuge them, and keep the precipitate.

[0087] (3) Add RIPA mixed lysis buffer to each well according to the cell number, and transfer to a 1.5 ml EP tube after pipetting.

[0088] (4) Place the 1.5 ml EP tube on ice for 10 min, centrifuge at 12,000 g for 10 min at 4°C, and transfer the protein supernatant to a new 1.5 ml EP tube.

[0089] 4. Bradford assay for total protein concentration

[0090] (1) Dilute the supernatant protein 20 times and transfer 3 μl of dilution solution + 100 μl of Bradford to a new tube.

[0091] (2) After mixing, take 90 μl into a 96-well plate and let it stand for 5 min.

[0092] (3) The absorbance was measured by ultraviolet spectrophotometer at a wavelength of 595 nm.

[0093] 5. SDS-polyacrylamide gel electrophoresis

[0094] (1) Add SDS loading buffer to the protein sample and denature at 95°C for 5 min.

[0095] (2) Carefully remove the comb from the gel and mount the glass plate containing the SDS-PAGE gel on the electrophoresis stand with the short plate facing inward. Place the plate in the electrophoresis tank and pour 1× Tris-Gly electrophoresis buffer that has been pre-cooled at 4°C overnight into the tank.

[0096] (3) Add protein sample to each lane according to the principle of equal mass and equal volume, add 3 μl protein marker on both sides, and add an equal volume of 1× loading buffer to the lane without sample. The sample volume in each well should be 15-20 μl.

[0097] (4) Cover the electrophoresis tank, turn on the power supply, and perform electrophoresis at a constant voltage of 80 V for 20-30 min. After the protein sample is compressed into a line, increase the voltage to 120 V and perform electrophoresis for about 40-60 min. Continue electrophoresis until bromophenol blue reaches the bottom of the separation gel.

[0098] 6. Transfer

[0099] (1) Prepare sponges, filter paper, and PVDF membranes. Pour 1× transfer buffer and methanol precooled at 4°C overnight into enamel dishes respectively. Soak the PVDF membrane in methanol for 10 min to activate it. Soak the sponges and filter paper in the transfer buffer.

[0100] (2) Remove the glass plate from the electrophoresis tank, separate the two layers of glass plates, cut off the concentrated gel, and according to the size of the target protein, cut off the excess separation gel and place it in the transfer buffer.

[0101] (3) Place the black gel and white membrane in the transfer holder in order. After each layer is placed, gently remove the bubbles and mark the PVDF membrane. After clamping, place it in the transfer electrophoresis tank with the black side facing the black side. Pour the transfer buffer and cover the lid.

[0102] (4) Place the transfer tank in a foam box filled with crushed ice. Turn on the power supply and transfer at a constant current of 260-280 mA for 60 minutes.

[0103] 7. Immunostaining

[0104] (1) Closed

[0105] 1) Prepare 5% skimmed milk powder, mix thoroughly and pour into the incubation box.

[0106] 2) After the transfer, remove the PVDF membrane, rinse it with TBST to remove the salt, put it in an incubation box containing 5% skimmed milk powder, and slowly shake it on a shaker to block it for 1 hour.

[0107] (2) Reaction of target protein with primary antibody (BRD4 and GSPT1):

[0108] 1) Prepare primary antibody reaction solution: prepare a 15 ml centrifuge tube, add 3 ml primary antibody diluent, add primary antibody according to the dilution ratio marked on the antibody manual, invert to mix, and place on ice.

[0109] 2) Transfer the PVDF membrane from the blocking solution to TBST and cut the bands according to the molecular weight of the target protein.

[0110] 3) Place the strip into a centrifuge tube containing the primary antibody reaction solution and incubate at 4°C with shaking overnight.

[0111] (3) Reaction of primary antibody and HRP-labeled secondary antibody:

[0112] 1) Wash with TBST 3 times, 10 min each time.

[0113] 2) Prepare secondary antibody reaction solution: prepare a 15 ml centrifuge tube, add 3 ml of 5% skim milk powder, then add goat anti-rabbit IgG-HRP (1:5000) or goat anti-mouse IgG-HRP (1:5000), and mix by inversion.

[0114] 3) Place the strips in a plastic box containing the secondary antibody reaction solution and incubate at room temperature with shaking for 1 hour.

[0115] (4) Exposure:

[0116] 1) Wash with TBST 3 times, 10 min each time.

[0117] 2) Mix equal volumes of Solution A and Solution B to prepare the color developing solution and protect from light.

[0118] 3) Place the strip on a black vinyl plate, wipe off excess water, add color developing solution to fully cover the strip, and expose to color.

[0119] Note: The development results were analyzed by grayscale analysis using Image J, and the degradation ratio was obtained by normalization, wherein the degradation effects of the compounds of the present application at a concentration of 1 μM are shown in Table 1. For the degradation effect, "++++" indicates that the degradation effect is between 100% and 90%; "+++" indicates that the degradation effect is between 90% and 70%; "++" indicates that the degradation effect is between 70% and 50%; "+" indicates that the degradation effect is between 50% and 30%; and "-" indicates that the degradation effect is between <30%.

[0120] Table 1 Degradation test results of each compound on BRD4 and GSPT1

[0121]

[0122] As shown in Table 1, compounds 1-18 can effectively degrade BDR4 and GSPT1 proteins in MOLM13 cells, and some of the compounds have significant degradation effects on BDR4 and GSPT1 proteins, and basically completely degrade BDR4 and GSPT1 proteins, which is better than the clinical drug CC-90009. Further, the positive control compound C2 is similar to the left end of the compound of the present invention, both of which have a similar mother core structure of JQ-1, but the control compound C2 cannot degrade BRD4 and GSPT1 proteins, indicating that the control compound C2 and the compound of the present invention have different mechanisms of action. At the same time, the structure of the positive control compound 165 is similar to the right end of the compound of the present invention, both of which have a CRBN-like structure, but compound 165 cannot effectively degrade BRD4 protein. In MOLM13 cells, the degradation effect of compound 165 on GSPT1 is weaker than that of some compounds of the present invention. The positive control dBET6 has a similar structure to the compound of the present invention, both of which are composed of JQ-1+Linker+CRBN. The main difference between the compound of the present invention and the present invention is that the Linker linker is different. The experimental results show that dBET6 can significantly degrade BRD4 protein, but the degradation effect on GSPT1 protein is much weaker than that of the compound of the present invention. In summary, since BDR4 and GSPT1 play a key regulatory role in the occurrence and development of NHL and AML tumors, designing compounds to degrade these two proteins at the same time is expected to further enhance the effect of degraders in the treatment of NHL and AML, thereby benefiting a large number of tumor patients.

[0123] Proliferation inhibitory activity against NHL and AML tumor cells

[0124] Reagents and consumables: sterile DMSO, cell culture medium (RMPI1640 + 10% FBS + 1% penicillin-streptomycin solution), 200μl 8 or 12 strip tubes, 0.2ml PCR tubes, 12-channel pipettes, small yellow plates, 10μl and 200μl pipettes, 96-well cell culture plates, 1.5ml EP tubes.

[0125] Core elements: cell density is 1-2×10 6 / ml, use strip tubes for operation, cell counting, the number of cells in a single well is about 10,000, and before dispensing the cells, use a 1ml pipette to gently mix twice to fully resuspend them.

[0126] Experimental steps:

[0127] 1. Subculture cells one day before the experiment so that the density is 1-2×10 6 / ml. If the culture medium has turned yellow, count the cells, centrifuge and change the medium, and adjust the concentration to 1×10 6 / ml or so;

[0128] 2. Use 30-100mM drug stock solution to prepare 5mM stock solution with DMSO (total volume greater than 50μl, then aliquot into 0.2ml PCR tubes, 10μl per tube, and store at -80 or -20℃). After using up the remaining aliquots, the stock solution can be temporarily stored at 4℃ for short-term storage for recent experiments;

[0129] 3. Take 2 μl of 5 mM stock solution and add it to 198 μl of complete medium. Cover and mark the lid and flick the tube about 20 times to mix well to obtain a temporary solution of 50 μM.

[0130] 4. Take an appropriate amount of 50μM drug and dilute it within 100 times to obtain the maximum working concentration of 1 (usually 0.5-10μM), and the volume cannot be less than 250μl;

[0131] 5. Place 12 tubes in a row on the small yellow plate. For each drug, set up 9 different concentration gradients (use 5-fold dilution for the initial experiment, then adjust, and use 3-fold dilution for the formal experiment) and 1 control well without drug. The following is an example of 3-fold dilution;

[0132] 6. Transfer 240 μl of working concentration 1 (note that the drug concentration is twice the actual working concentration because an equal volume of cells must be added) into the first column of the strip tubes, and add 160 μl of complete culture medium to the remaining wells using a spray gun;

[0133] 7. Take 80 μl of working solution 1 and add it to the next well. Pipette thoroughly for about 10 times and then take 80 μl and add it to the subsequent wells. Repeat this process to obtain a series of diluted drugs.

[0134] 8. Use a gun to transfer 50 μl of the diluted drug to a 96-well culture plate. Perform three replicates for each drug.

[0135] 9. Cultivate cells and count the cells. The density is 1-2×10 6 / ml is optimal, and then the cell density is diluted to 2×10 5 / ml (10,000 cells per well in the end), the total volume is calculated based on the total number of drugs to be used, and be sure to leave enough surplus;

[0136] 10. Transfer the cells into the dispensing tank, gently mix the cells twice with a 1ml pipette, and immediately use a dispenser to transfer 50μl of the cell suspension into the culture plate with the drug added. After all the drugs have been added to the single plate, gently rotate the culture plate to mix;

[0137] 11. Use a 1ml pipette to gently mix the cells twice again, and immediately use a dispenser to add cells from the subsequent culture plates. Gently rotate the culture plate to mix, and then place the cells in the incubator for 3 days.

[0138] 12.3 days later, CCK8 was detected and analyzed using Graphpad Prism software. Inhibition rate = (100-OD 样品 / OD 溶媒 )×100%, where OD 样品 OD is the absorbance value detected after adding each concentration of the test substance. 溶媒 The absorbance values ​​detected in the vehicle group (solvent was added but no test substance was added) are shown in Table 2. The cytotoxicity test results of each compound on NHL and AML tumor cells are shown in Table 2.

[0139] Note: IC 50 : Compound concentration that inhibits 50% tumor cell growth

[0140] IC 50 The size is divided into four levels:

[0141] "++++",IC 50 <100nM; "+++", IC 50 100-1000nM; "++", IC 50 1000-2000nM; “-” indicates IC 50 >2000nM.

[0142] Table 2 Cytotoxicity test results of each compound on NHL and AML tumor cells

[0143]

[0144]

[0145] As shown in Table 2, compounds 1-18 can significantly inhibit the growth of different NHL and AML tumor cell lines, and some of the compounds have significant inhibitory effects on NHL and AML tumor cell lines, and the effect is better than the clinical drug CC-90009. Further, the control compound C2 showed moderate or even weak inhibitory activity in different NHL and AML tumor cell tests, and the effect was inferior to the compound of the present invention. Although the control compound 165 was more effective than the control compound C2 in the test of some NHL and AML tumor cells, it was significantly inferior to the compound of the present invention in the THP-1 and Oci-AML3 cell tests. The control compound dBET6 was significantly inferior to the compound of the present invention in the Oci-AML3 cell test. From the above experimental data, it can be seen that the compounds of the present invention have significant effects in different NHL and AML tumor cell line tests by simultaneously degrading BDR4 and GSPT1 proteins, and are expected to treat NHL and AML tumor patients who are insensitive to existing drugs, and have strong application prospects.

[0146] Experiment on prevention and treatment of apoptosis in acute myeloid leukemia (AML)

[0147] 1. Drug preparation: The stock solution of the drug is 10 mM. It is diluted 10-fold with DMSO to prepare 2-8 gradient stock solutions of 1 mM, 0.1 mM, 0.01 mM, and 0.001 mM.

[0148] 2. Collect 5 mL of bone marrow from tumor patients (Note: the patient has signed the informed consent form and obtained the medical ethics review approval), centrifuge with Ficoll separation solution to separate the patient's mononuclear cells, wash twice with normal saline, adjust the cell concentration, and prepare a cell suspension with a concentration of 2 million / mL.

[0149] 3. Take the cell suspension prepared in step 2 and inoculate 200,000 cells / well into a 12-well plate for culture. The culture medium used is DMEM culture medium purchased from Thermo Company; the culture temperature is 37°C and the culture volume is 1 mL.

[0150] 4. Different concentrations of drug stock solutions (1:1000 dilution) were added to the treatment wells for treatment, and an equal volume of DMSO was added to the control group. The mixture was thoroughly mixed and the apoptotic ratio of leukemia cells was detected by flow cytometry after 48 hours. The experimental results are shown in Table 3.

[0151] The apoptosis ratio is divided into levels:

[0152] “+++”, apoptosis ratio>60%; “++”, 60%>apoptosis ratio>40%; “+” 40%>apoptosis ratio>20%; “-” indicates apoptosis ratio<20%.

[0153] Table 3 Test results of each compound on apoptosis of acute myeloid leukemia cells

[0154]

[0155] As shown in Table 3, compound 16 can significantly promote apoptosis of tumor cells in patients with acute myeloid leukemia, and its therapeutic effect is remarkable.

[0156] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A glutarimide compound, characterized in that: It has the molecular structure shown in the following formula (I): Formula (I); Among them, L is selected from , , ; D is selected from 、 、 ； The molecular structure represented by the formula (I) has any one of the following structural formulas: 、 、 ； Among them, L is selected from , , ; The formula Ia, Ib or Ic has any one of the following structural formulas: 。 2. The method for preparing a glutarimide compound according to claim 1, characterized in that: The steps include: The compound represented by formula (II) and the compound represented by formula (III) are subjected to condensation reaction in the presence of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholinium chloride and nitrogen methylmorpholine to obtain a compound represented by formula (1): Among them, L1 is selected from , , , , , ; D is selected from , , .

3. The preparation method according to claim 2, characterized in that: The condensation reaction temperature is 20-30° C., the time is 0.5-3 hours, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholinium chloride is 1.5 equivalents, nitrogen methylmorpholine is 2 equivalents, and the solvent is DMF.

4. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the compound according to claim 1 or a pharmaceutically acceptable salt thereof.

5. Use of the compound according to claim 1 in the preparation of a BRD4 degrader.

6. Use of the compound according to claim 1 in the preparation of a GSPT1 degrading agent.

7. Use of the compound according to claim 1 in the preparation of a drug for treating tumor diseases related to BRD4 and GSPT1 targets.

8. The use according to claim 7, characterized in that: The tumor diseases are non-Hodgkin's lymphoma, chronic lymphocytic leukemia, B-cell lymphoma, mantle cell lymphoma, and acute myeloid leukemia.

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