CN116375713A

CN116375713A - DCLK1 protein degradation targeting chimeric and application thereof

Info

Publication number: CN116375713A
Application number: CN202310209418.2A
Authority: CN
Inventors: 盛春泉; 武善超; 李柯良; 刘杨; 董国强; 陈玉平; 孙驭航; 王磊; 白学鑫; 夏哲炜
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2023-03-07
Filing date: 2023-03-07
Publication date: 2023-07-04

Abstract

The invention discloses a compound or a medicinal salt thereof, and the structural general formula is shown as follows:

the compound of the invention shows good DCLK1 protein degradation activity, and the test result of the DCLK1 protein degradation activity shows that pomalidomide is used as E3 connectionThe compounds P-11, P-12 and P-14 of the ligase ligand have better degradation activity and are superior to the compounds taking VHL as the E3 ligase ligand.

Description

DCLK1 protein degradation targeting chimeric and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a DCLK1 protein degradation targeting chimeric body and application thereof.

Background

From the history of drug development, it has been seen that the focus of drug development has been on therapeutic effects by blocking receptors or modulating protein binding sites. However, this mode of drug action generally requires high doses of drug to maintain a high target occupancy, and may cause side effects due to off-target. The kinase inhibitor also has the limitation on the action mode, so that the protein degradation targeting chimeric (PROTACs) technology is developed to solve the problem, and the defect of low selectivity of the kinase inhibitor can be solved by degrading the target protein by utilizing the degradation mechanism of the cell.

The PROTAC molecule generally comprises two key domains: specific domains that bind to the target protein (protein ofinterest, POI) and domains that bind to the E3 ubiquitination ligase, both linked by a specific linker, induce ubiquitination of the POI and degrade by the proteasome pathway. Importantly, the PROTAC molecule can still be recovered after POI ubiquitination, and then participate in a new reaction. From the pharmacodynamics point of view, this catalytic degradation mode is more significant than increasing the target protein occupancy. Furthermore, the duration of action of the PROTAC molecule varies depending on the stability of the PROTAC molecule in the cell and the rate of POI resynthesis. The PROTAC molecule can theoretically target degradation of any type of protein, provided that it has a suitable small molecule ligand with an affinity for the POI and is able to place the target protein in a suitable spatial position with the E3 ligase, forming a target protein-PROTAC-E3 ligase ternary complex.

Currently, the common E3 ligase ligands are CRBN, VHL, MDM, clAP1, KEAP1 and RNF114, with CRBN and VHL being the most common. The distribution of different E3 ligases in tissues, affinity to target proteins and selectivity all exhibit diversity, which has an important impact on the safety, patentability and efficacy of PROTAC. Thus, there is a need to direct the synthesis of PROTAC by rational pharmaceutical chemistry and pharmacological means.

After the market of glibenches as the first protein kinase inhibitor in 2001, protein kinase inhibitors have received extensive attention and research, and 48 protein kinase inhibitors have been marketed. However, protein kinase inhibitors are less selective, mainly because one third of the protein kinases may be phosphorylated and the binding domains of most kinases are very similar, making selective inhibition very difficult. In addition, the off-target effect of the kinase inhibitor often causes toxic and side effects clinically, so that the selectivity of the kinase inhibitor is improved, and the toxicity of the kinase inhibitor is reduced, thereby having great significance for optimizing the kinase drug.

Since the target protein and the E3 ligase need to be placed at a proper spatial position to form a stable target protein-PROTAC-E3 ligase ternary complex, and the surface of the target protein has a proper ubiquitination site (such as lysine), the selectivity can be well improved after the PROTAC is designed even though the selectivity of the kinase inhibitor is poor, so the PROTAC technology is one of important strategies for improving the selectivity.

Bisadrenocortical hormone-like kinase 1 (dclk1) is a microtubule-associated serine/threonine kinase that structurally belongs to the calmodulin-dependent protein kinase family (calcium/calmodulin-dependent protein kinases, caMK) and is predominantly expressed in the cytosol. DCLK1 is composed of 729 amino acid molecules, and is divided into three parts of DCX domain, PEST sequence and kinase domain, and its N-terminal region has two microtubule binding domains (DCX 1 and DCX2 respectively) for driving microtubule-related functions; the C-terminal region comprises a serine/threonine kinase domain, which is highly similar to the calcium/calmodulin-dependent protein 1 (CaMK 1) kinase domain.

DCLK1 has many biological functions, and the research work using the DCLK1 as a drug target is mainly focused on anti-tumor aspects, but no remarkable progress is made, and recent researches prove that the DCLK1 is closely related to inflammation, so that the DCLK1 is presumed to be a potential anti-inflammatory drug target.

Disclosure of Invention

It is a first object of the present invention to provide a compound that can prepare DCLK1 protein degradation targeting chimeras.

The second object of the invention is to provide an application of the compound in preparing DCLK1 protein degradation targeting chimeric.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

the first aspect of the invention provides a compound or a pharmaceutically acceptable salt thereof, which has the following structural formula:

wherein X is selected from one of the following structures: a key(s),

m is selected from positive integers of 1 to 10 (e.g., 1,2, 3, 4, 5, 6, 7);

n is selected from positive integers of 1 to 10 (e.g., 1,2, 3, 4, 5);

r is selected from positive integers of 1 to 10 (e.g., 1,2, 3, 4, 5, 6, 7);

p is selected from positive integers of 1 to 10 (e.g., 1,2, 3, 4);

q is selected from positive integers of 1 to 10 (e.g., 1,2, 3);

s is selected from positive integers of 1 to 10 (e.g., 1,2, 3, 4, 5, 6, 7);

r is selected from one of the following structures:

R ₁ selected from hydrogen, methyl.

The pharmaceutically acceptable salts are acid addition salts of the above compounds with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

The compound is selected from one of the following structures:

in a second aspect, the invention provides an application of the compound or the pharmaceutically acceptable salt thereof in preparing DCLK1 protein degradation targeting chimeric, wherein the DCLK1 protein targeting degradation chimeric molecule consists of three parts, an E3 ligase ligand (VHL ligand, pomalidomide ligand and lenalidomide ligand), a target protein ligand and a linker. The E3 ligase ligand is responsible for specific recruitment of E3 ligase. Target protein ligands are used to target and capture target proteins. Linker was used to combine these two ligands to form a stable ternary complex. Thus, the protoc molecule is able to recruit the E3 ligase to the vicinity of the target protein, labeling the target protein with a "ubiquitin" tag, whereas the ubiquitin tagged protein on the cell will be sent to the proteasome for degradation. Thus, the PROTAC molecule can specifically promote the degradation of pathogenic proteins, thereby achieving the purpose of treating diseases.

In a third aspect, the invention provides the use of the compound or a pharmaceutically acceptable salt thereof in the manufacture of an anti-inflammatory medicament.

The inflammation in the anti-inflammatory drug refers to inflammation related to DCLK1 expression, and the inflammation related to DCLK1 expression can be sepsis, septicemia, acute lung injury or diabetic complications.

By adopting the technical scheme, the invention has the following advantages and beneficial effects:

the compound of the invention shows good DCLK1 protein degradation activity, and the test result of the DCLK1 protein degradation activity shows that in DCLK1 protein degradation targeting chimera (compounds P-1 to P-6 and P-9) taking VHL as an E3 ligase ligand, the compounds P-1 and P-9 of a carbon oxygen heterochain and a fatty chain containing three carbon atoms have certain degradation activity, and the degradation activity gradually weakens with the increase of chain length in the fatty chain compounds P-2 to P-6 of 4 to 8 carbon atoms; the compound P-9 with the best anti-inflammatory activity is subjected to a time-dependent experiment, after 0.5 to 3 hours of administration, the degradation rate of DCLK1 is gradually increased along with the time increase, and the degradation effect is not changed after 3 hours, which indicates that the PROTAC molecule can reach the maximum degradation at 3 hours, so that the subsequent drug action time is 3 hours; the DCLK1 protein degradation targeting chimeric body with the pomalidomide as the E3 ligase ligand is subjected to 10 mu M single-concentration degradation activity screening, and the compounds P-11, P-12 and P-14 are found to have better degradation activity, and the corresponding DCLK1 protein degradation targeting chimeric body respectively comprises a non-linker, a fatty linker containing 2 carbon atoms and a fatty linker containing 5 carbon atoms. By comparing the degradation activities of two different types of E3 ligase ligands, the PROTAC molecule with the E3 ligase ligand of CRBN has better degradation activity. The concentration-dependent degradation experiment is preferably performed on the compound P-14 with better degradation activity, and the fact that the degradation efficiency is reduced along with the increase of the concentration in three concentrations of 0.1-10 mu M is found, and the analysis reason probably is that the PROTAC molecule generates a HOOK effect at high concentration, and the specific degradation efficiency is yet to be further verified.

The compound provided by the invention has further development and research values as a DCLK1 protein degradation targeting chimeric body reported for the first time.

Drawings

FIG. 1 is a graphical representation of the results of in vitro anti-inflammatory activity assays of compounds of the present invention.

FIG. 2 is a schematic diagram showing the results of the degradation activity test of DCLK 1-PROTAC.

Detailed Description

In order to more clearly illustrate the present invention, the present invention will be further described with reference to preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.

In the present invention, EDCI is 1-ethyl-3 (3-dimethylpropylamine) carbodiimide, HOBT is 1-hydroxybenzotriazole, DIPEA is N, N-diisopropylethylamine, DCM is dichloromethane, meOH is methanol, and THF is tetrahydrofuran.

The structure of DCLK1 inhibitor LRRK2-IN-1 with excellent activity is shown as follows:

example 1

Synthesis of 4- (((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl [5,4-b ] [1,4] diazepin-2-yl) amino) -3-methoxy-N- (piperidin-4-yl) benzamide (8 g)

Reagents and conditions: a) SOCl ₂ ,MeOH,60℃,2h,yield 92％；b)DIPEA,1,4-dioxane,50℃,5h,yield 82％；c)Fe,AcOH,60℃,overnight,yield 93％；d)MeI,NaH,DMF,0℃,5h,yield 80％；e)DCM,EDCI,HOBT,DIPEA,r.t.,overnight,yield 84.3％；f)X-Phos,Pd ₂ (dba) ₃ ,1,4-dioxane,100℃,5h,yield 37.1％；g)DCM,TFA,4h,yield 39％.

Step a: synthesis of methyl 2- (methylamino) benzoate (2)

Compound 1 (2- (methylamino) benzoic acid) (5.00 g,33.08 mmol) was placed in a 250mL eggplant-shaped bottle, methanol (100 mL) was added for dissolution, thionyl chloride (19.70 g,165.38 mmol) was slowly added dropwise with stirring, and the reaction was carried out at 60℃for 2h. TLC detection was complete, and the reaction solution was poured into ice water (500 mL), stirred for 5min, and a large amount of white solid was precipitated, suction-filtered, dried, and without further purification, compound 2 was obtained (white solid, 5g, yield: 92%). ¹ H NMR(300MHz,DMSO-d ₆ )δ:7.73(s,1H),7.40(s,1H),6.93(s,1H),6.82(s,1H),3.95(s,3H),2.64(s,3H)。

Step b: synthesis of methyl 2- ((2-chloro-5-nitropyrimidin-4-yl) (methyl) amino) benzoate (4)

Compound 2 (1.00 g,6.06 mmol) and compound 3 (2, 4-dichloro-5-nitropyrimidine, 1.40g,7.26 mmol) were dissolved in 1,4-dioxane (50 mL), DIPEA (1.60 g,12.12 mmol) was slowly added dropwise with stirring, and stirring was continued for more than 5h at 50 ℃. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain a crude product, which was then separated and purified by silica gel column chromatography (hexane: etoac=15:1 to 10:1) to obtain compound 4 (yellow solid, 1.6g, yield: 82%). ¹ H NMR(300MHz,DMSO-d ₆ )δ:8.72(s,1H),7.93(dd,J＝8.3,1.6Hz,1H),7.65-7.76(m,1H),7.46-7.59(m,2H),4.03-4.26(m,2H),3.47(s,3H),1.19(t,J＝7.1Hz,3H)。

Step c: synthesis of 2-chloro-11-methyl-5, 11-dihydro-6H-benzo [ e ] pyrimido [5,4-b ] [1,4] diazepin-6-one (5)

Compound 4 (1.00 g,3.10 mmol) was dissolved in 20mL of acetic acid, iron powder (0.87 g,15.49 mmol) was added sequentially with stirring, DIPEA (0.80 g,6.20 mmol) and stirred overnight at 60 ℃. After completion of TLC detection, the remaining iron powder was filtered, and the filtrate was added to 500mL of ice water, a large amount of white solid was precipitated, suction-filtered, and the cake was dried at 50℃and directly subjected to the next step without purification to give Compound 5 (white solid, 0.7g, yield: 93%). ¹ H NMR(300MHz,DMSO-d6)δ:10.48(s,1H),8.15(s,1H),7.72(d,J＝7.5Hz,1H),7.59(t,J＝7.5Hz,1H),7.28(d,J＝8.0Hz,1H),7.22(t,J＝7.5Hz,1H),3.34(s,3H)。

Step d: synthesis of 2-chloro-5, 11-dimethyl-5, 11-dihydro-6H-benzo [ e ] pyrimido [5,4-b ] [1,4] diazepin-6-one (6)

Compound 5 (1.00 g,3.84 mmol) was dissolved in dry DMF (20 mL), naH (120.00 mg,4.60 mmol) was added in portions at-10℃and after 0.5h reaction, methyl iodide (660.00 mg,4.60 mmol) was added dropwise slowly, and the reaction was transferred to room temperature and stirring continued for 4h. After the starting materials were completely reacted, the reaction solution was poured into ice water (400 mL), stirred for 5min, a large amount of white solid was precipitated, suction-filtered, and the cake was dried at 50 ℃ to constant weight without further purification to give compound 6 (white solid, 0.8g, yield 80%). ¹ H NMR(300MHz,DMSO-d ₆ )δ:8.60(s,1H),7.71(dd,J＝7.8,1.5Hz,1H),7.51-7.62(m,1H),7.29(d,J＝8.3Hz,1H),7.22(t,J＝7.7Hz,1H),3.42(s,3H),3.34(s,3H)。

Step e: synthesis of 4- (4-amino-3-methoxybenzamide) piperidine-1-carboxylic acid tert-butyl ester (9)

Compound 7 (2.00 g,12.00 mmol), compound 8 (2.88 g,14.36 mmol), EDCI (2.75 g,14.36 mmol), HOBT (1.94 g,14.36 mmol) were weighed into a 100mL round bottom flask, dried dichloromethane (40 mL) was added and stirred to dissolve thoroughly, DIPEA (5.93 mL,35.89 mmol) was slowly added dropwise while stirring and the reaction was stirred overnight at room temperature, monitored by TLC plate (developer condition, DCM: meoh=100:10) and was complete. Pouring the reaction solution into a 250mL separating funnel, adding 100mL of water, adding 30mL of dichloromethane, shaking fully, standing until the liquid is thoroughly layered, taking the lower layer of liquid out of the 250mL round bottom flask, extracting repeatedly for 4-5 times, merging the extracts, spin-drying, stirring for column chromatography, eluting with an eluent: DCM: meoh=100:1, gradually increasing in polarity to DCM: meoh=100:5, and the eluate was concentrated under reduced pressure to give compound 9 (light yellow solid, 3.52g, yield 84.3%).

Step f: synthesis of 4- (((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl [5,4-b ] [1,4] diazepin-2-yl) amino) -3-methoxy-N- (piperidin-4-yl) benzamide (8 g)

Compound 6 (250 mg,0.90 mmol), compound 9 (34 mg,0.98 mmol), X-Phos (39 mg,0.08 mmol), pd were weighed out ₂ (dba) ₃ (74 mg,0.08 mmol) and potassium carbonate (375)mg,2.68 mmol) was placed in a 100mL round bottom flask, dried 1,4-Dioxane (30 mL) was added to dissolve thoroughly, and the flask was evacuated to N ₂ The reaction was refluxed at 106 ℃ for 4.5h under protection, the whole reaction process was avoided as much as possible from contacting air and water, the spherical condenser tube used was dried thoroughly in an oven in advance, the TLC plate was monitored (developer condition, DCM: meoh=100:10), and the reaction was complete. Filtering the reaction solution by using a triangular funnel, removing inorganic salt solids as far as possible, flushing filter residues for 3 times by using a little methanol, pouring the filtrate into a 100mL separating funnel, adding 40mL of water, adding 15mL of ethyl acetate, fully shaking, standing until the liquid is thoroughly layered, taking the upper liquid into a 100mL round bottom flask, repeatedly extracting for 4-5 times, merging the extracts, spin-drying, then mixing with a sample column for chromatography, eluting with an eluent: pe:ea=50:1, polarity gradually increases to: PE: ea=73:27, and the eluate was concentrated under reduced pressure to give product 10 (yellow solid, 195mg, yield 37.1%).

Compound 10 (195 mg,0.33 mmol) was weighed into a 50mL round bottom flask, dried dichloromethane (8 mL) was added for complete dissolution, trifluoroacetic acid (2 mL) was added with stirring, the reaction was stirred at room temperature overnight, monitored by TLC plates (developer conditions, DCM: meoh=100:10) and the reaction was complete. Spin-drying the reaction solution, adding a little methanol for full dissolution, spin-drying again, repeating for 5-6 times, and finally fully dissolving with dichloromethane, directly stirring for column chromatography, eluting with an eluent: DCM: meoh=100:1, gradually increasing in polarity to DCM: meoh=100:7, and the eluate was concentrated under reduced pressure to give 8g of product (white solid, 63mg, 39% yield). ¹ H NMR(600MHz,CDCl ₃ )δ:8.51(d,J＝8.4Hz,1H),8.15(s,1H),7.78-7.86(m,2H),7.45(d,J＝1.6Hz,1H),7.38-7.43(m,1H),7.31(dd,J＝8.4,1.6Hz,1H),7.12(t,J＝7.8Hz,1H),7.07(d,J＝8.4Hz,1H),6.06-6.17(m,1H),4.02-4.13(m,1H),3.96(s,3H),3.48(s,3H),3.41(s,3H),2.77(t,J＝12.1Hz,1H),2.1-2.3(m,2H),2.04(d,J＝11.1Hz,2H),1.40-1.48(m,2H)； ¹³ C NMR(150MHz,CDCl ₃ )δ:168.22,166.35,163.67,155.38,151.30,149.28,147.47,132.46,132.35,132.28,127.53,126.49,123.84,122.04,118.97,117.39,116.45,109.31,56.00,47.33,45.44,38.24,35.96,33.48.

Example 2

Synthesis of (2S, 4R) -1- ((S) -2- (3- (4- (4- (5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl) [5,4-b ] [1,4] diaza-2- (yl) amino) -3-methoxybenzamido) piperidin-1-yl) -3-oxopropanamido) -3, 3-dimethylbutyryl) -4-hydroxy-N- ((S) -1- (4- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide (P-1)

Reagents and conditions: a) EDCI, HOBT, DIPEA, DCM, rt,1h, yield 80-90%; b) MeOH, THF, H ₂ O,LiOH,rt,50℃,5h,yield 80-87％；c)EDCI,HOBT,DIPEA,DCM,rt,2h,yield 50-65％.

Step a: synthesis of Compound 12

8g (0.70 g,1.44 mmol) of compound and 11 (0.20 g,1.72 mmol) were dissolved in 20mL of dry DCM, EDCI (0.30 g,1.72 mmol), HOBT (0.20 g,1.72 mmol) and DIPEA (0.40 g,3.59 mmol) were added sequentially and stirred at room temperature for 2h. After TLC monitoring the completion of the reaction, the reaction solution was washed 3 times with 60mL of saturated sodium chloride solution, and the organic layer was concentrated under reduced pressure, and purified by silica gel column chromatography (hexane/etoac=3:1-1:1) to give compound 12 (white solid, 698mg, yield 85%). ¹ H NMR(300MHz,CDCl ₃ )δ:8.50(d,J＝8.3Hz,1H),8.14(s,1H),7.84(dd,J＝7.8,1.5Hz,1H),7.40-7.45(m,2H),7.30(dd,J＝8.4,1.5Hz,1H),7.14(t,J＝7.4Hz,1H),7.08(d,J＝8.3Hz,1H),6.08(d,J＝7.8Hz,1H),4.62(d,J＝13.9Hz,1H),4.18-4.26(m,1H),3.97(s,3H),3.72-3.77(m,1H),3.70(q,J＝7.1Hz,1H),3.49(s,3H),3.42(s,3H),3.21-3.26(m,1H),2.78-2.85(m,1H),2.11-2.16(m,1H),2.03-2.08(m,1H),1.48-1.57(m,4H),1.46(s,3H).

Step b: synthesis of (P-1)

Compound 12 (500 mg,0.85 mmol) was dissolved in THF, meOH, H ₂ To a mixed solvent (100 mL) of o=3:2:1, liOH (96 mg,4.25 mmol) was added in portions, and the reaction was allowed to proceed for 5h at room temperature, and was complete by tlc. Distilling the reaction solution under reduced pressure to remove organic solvent, transferring to ice bath, adjusting pH to neutrality with 1mol/L HCl, precipitating a large amount of white solid, vacuum filtering, oven drying, and no further purificationThe crude product of compound 13 was 400mg in 87% yield.

Compound 13 (100 mg,0.17 mmol) and VHL ligand 14 (133.2 mg,0.26 mmol) were dissolved in dry DCM (20 mL) and EDCI (58 mg,0.26 mmol), HOBT (42 mg,0.26 mmol), DIPEA (77 mg,0.52 mmol) were added sequentially and reacted at room temperature for 2h. After the completion of the reaction, the reaction mixture was washed three times with saturated sodium chloride solution (150 ml×3), dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (DCM: meoh=100:1 to 100:3) to give compound P-1 (white solid, 300mg, yield 37%). ¹ H NMR(600MHz,CDCl ₃ )δ:8.64(d,J＝7.5Hz,1H),8.50(dd,J＝8.5,4.5Hz,1H),8.37(d,J＝8.2Hz,1H),8.13(s,1H),7.81-7.86(m,2H),7.38-7.50(m,3H),7.29-7.36(m,4H),7.10-7.15(m,1H),7.06(d,J＝8.4Hz,1H),6.94(dd,J＝102.9,8.0Hz,1H),5.00-5.07(m,1H),4.73(t,J＝8.4Hz,1H),4.63(t,J＝8.0Hz,1H),4.54-4.61(m,2H),4.47(s,1H),4.13-4.23(m,1H),4.07(dd,J＝31.0,13.0Hz,1H),3.90-3.95(m,3H),3.76-3.89(m,1H),3.56-3.63(m,1H),3.46-3.49(m,3H),3.39(s,3H),3.31-3.37(m.1H),3.11-3.18(m,1H),2.68-2.76(m,2H),2.46-2.50(m,3H),2.22-2.32(m,1H),1.91-2.15(m,3H),1.45(d,J＝6.9Hz,3H),1.40(d,J＝6.1Hz,2H),1.07(s,9H)； ¹³ C NMR(150MHz,CDCl ₃ )δ:172.63,171.47,169.59,168.86,168.43,168.05,167.73,167.32,165.03,156.66,152.58,151.69,150.57,149.73,148.66,144.93,144.78,133.88,133.68,133.00,132.01,130.80,128.41,128.20,127.75,125.21,123.47,121.14,118.81,117.77,110.68,71.50,60.19,59.98,59.70,59.44,58.38,57.36,55.49,50.23,48.31,46.75,43.67,42.85,42.01,41.03,39.62,37.68,37.45,37.29,36.77,36.41,33.59,32.99,27.92,23.68,19.94,18.68,17.42,13.46；HRMS(ESI,positive)m/z calcd for C ₅₂ H ₆₂ N ₁₁ O ₈ S[M+H] ⁺ :1000.4498；found 1000.4487.

Example 3

Synthesis of (2R, 4S) -1- ((R) -2- (4- (4- (4- ((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl ] [5,4-b ] [1,4] diaza-2- (amino) -3-methoxybenzamido) piperidin-1-yl) -4-oxapentanamido) -3, 3-dimethylbutyryl) -4-hydroxy-N- ((R) -1- (4- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide (P-2)

Step a: synthesis of Compound 12-1

The synthesis method refers to step a in example 2, substituting compound 11 with compound 4-methoxy-4-oxobutanoic acid. White solid 600mg, 86% yield. ¹ H NMR(300MHz,CDCl ₃ )δ:8.51(d,J＝8.3Hz,1H),8.15(s,1H),7.85(dd,J＝7.8,1.6Hz,1H),7.39-7.46(m,2H),7.29(dd,J＝8.3,1.6Hz,1H),7.15(t,J＝7.6Hz,1H),7.10(d,J＝8.4Hz,1H),6.09(d,J＝7.9Hz,1H),4.61(d,J＝14.0Hz,1H),4.19-4.27(m,1H),3.98(s,3H),3.70-3.78(m,1H),3.71(q,J＝7.2Hz,1H),3.50(s,3H),3.44(s,3H),3.20-3.28(m,1H),2.79-2.86(m,1H),2.09-2.17(m,1H),2.02-2.09(m,1H),1.49-1.58(m,6H),1.46(s,3H).

Step b: synthesis of Compound P-2

The synthesis method is described in step b of example 2. White solid 100mg, yield 30%. ¹ H NMR(600MHz,CDCl ₃ )δ:8.67(s,1H),8.51(d,J＝8.4Hz,1H),8.16(s,1H),7.86(d,J＝4.5Hz,1H),7.84(dd,J＝7.9,1.7Hz,1H),7.64-7.69(m,1H),7.46-7.50(m,1H),7.32-7.45(m,6H),7.13(t,J＝7.5Hz,1H),7.09(t,J＝8.4Hz,1H),6.86-6.94(m,1H),5.01-5.12(m,1H),4.72-4.81(m,1H),4.49-4.60(m,2H),4.46(s,1H),4.18-4.27(m,1H),4.08(t,J＝12.1Hz,1H),3.94(s,3H),3.84-3.91(m,1H),3.57-3.61(m,1H),3.49(s,3H),3.41(s,3H),3.10-3.16(m,1H),2.63-2.75(m,3H),2.50(s,3H),2.44-2.48(m,1H),2.35-2.42(m,1H),2.10-2.17(m,1H),2.02-2.08(m,1H),1.47(d,J＝6.7Hz,3H),1.23-1.34(m,4H),1.06(s,9H)； ¹³ C NMR(150MHz,CDCl ₃ )δ:174.53,173.3,171.83,169.59,167.93,165.06,156.66,152.54,151.78,150.57,149.72,148.77,144.73,133.87,133.72,133.02,132.10,130.87,128.48,127.79,125.22,123.45,120.47,118.79,117.75,110.75,71.55,59.83,59.48,58.20,57.37,50.20,48.28,46.28,43.01,39.64,37.54,37.31,36.46,33.83,33.11,32.94,32.27,30.37,29.31,27.92,24.00,23.56,15.58,12.78；HRMS(ESI,positive)m/z calcd for C ₅₃ H ₆₄ N ₁₁ O ₈ S[M+H] ⁺ :1014.4655；found 1014.4554.

Example 4

Synthesis of (2S, 4R) -1- ((S) -2- (5- (4- (4- ((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl ] [5,4-b ] [1,4] diaza-2-yl) amino) -3-methoxybenzamido) piperidin-1-yl) -5-oxopentanoamide) -3, 3-dimethylbutyryl) -4-hydroxy-N- ((S) -1- (4- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide (P-3)

Step a: synthesis of Compound 12-2

The synthesis method refers to step a in example 2 by substituting compound 11 with compound 5-methoxy-5-oxopentanoic acid. White solid 500mg, yield 80%. ¹ H NMR(300MHz,CDCl ₃ )δ:8.50(d,J＝8.5Hz,1H),8.15(s,1H),7.95(s,1H),7.84(d,J＝7.8Hz,1H),7.40-7.49(m,2H),7.29(d,J＝8.6Hz,1H),7.14(t,J＝7.7Hz,1H),7.07(d,J＝8.3Hz,1H),6.03(d,J＝7.7Hz,1H),4.60(d,J＝13.6Hz,1H),4.16-4.25(m,1H),3.97(s,3H),3.89(d,J＝13.8Hz,1H),3.67(s,3H),3.49(s,3H),3.42(s,3H),3.19(t,J＝11.7Hz,1H),2.78(t,J＝11.9.Hz,1H),2.38-2.43(m,4H),2.15-2.19(m,1H),2.03-2.08(m,1H),1.93-2.00(m,4H).

Step b: synthesis of Compound P-3

The synthesis method is described in step b of example 2. 106mg of white solid was obtained in 36% yield. ¹ H NMR(600MHz,CDCl ₃ )δ:8.68(s,1H),8.53(dd,J＝8.3,5.0Hz,1H),7.88(s,1H),7.86(dd,J＝7.9,1.2Hz,1H),7.48(s,1H),7.32-7.47(m,6H),7.15(t,J＝7.4Hz,1H),7.09(d,J＝8.7Hz,1H),6.67-6.91(m,1H),6.51-6.61(m,1H),5.01-5.13(m,1H),4.73(q,J＝7.9Hz,1H),4.64-4.57(m,1H),4.52-4.56(m,1H),4.44-4.52(m,1H),4.26-4.19(m,1H),4.09-4.18(m,1H),3.96(s,3H),3.82-3.88(m,1H),3.56-3.63(m,1H),3.51(s,3H),3.43(s,3H),3.12-3.21(m,1H),2.73-2.80(m,1H),2.53(s,3H),2.19-2.49(m,6H),2.09-2.16(m,2H),1.91-2.05(m,4H),1.46(d,J＝22.8Hz,3H),1.08(s,9H)； ¹³ C NMR(150MHz,CDCl ₃ )δ:174.72,173.42,172.33,171.10,169.56,167.89,163.91,156.64,152.51,150.56,149.83,148.83,14.68,133.86,132.20,130.90,127.78,127.75,120.71,118.79,117.81,110.70,71.43,59.79,59.39,58.18,57.38,50.18,48.47,46.18,42.43,39.63,37.34,36.37,34.22,33.26,27.98,23.65,17.46；HRMS(ESI,positive)m/z calcd for C ₅₄ H ₆₆ N ₁₁ O ₈ S[M+H] ⁺ :1028.4211；found 1028.4836.

Example 5

Synthesis of (2S, 4R) -1- ((S) -2- (6- (4- (4- ((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl ] [5,4-b ] [1,4] diaza-2- (amino) -3-methoxybenzamido) piperidin-1-yl) -6-oxa-amino) -3, 3-dimethylbutyryl) -4-hydroxy-N- ((S) -1- (4- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide (P-4)

Step a: synthesis of Compound 12-3

The synthesis method refers to step a in example 2 by substituting compound 11 with compound 6-methoxy-6-oxohexanoic acid. White solid 400mg, yield 83%. ¹ H NMR(300MHz,CDCl ₃ )δ:8.49(d,J＝8.4Hz,1H),8.14(s,1H),7.93(s,1H),7.83(dd,J＝7.8,1.5Hz,1H),7.44(d,J＝1.7Hz,1H),7.39-7.43(m,1H),7.30(dd,J＝8.4,1.6Hz,1H),7.13(t,J＝7.6Hz,1H),7.06(d,J＝8.4Hz,1H),7.15(d,J＝7.9Hz,1H),4.60(d,J＝14.0Hz,1H),4.16-4.25(m,1H),3.96(s,3H),3.85(d,J＝13.7Hz,1H),3.64(s,3H),3.48(s,3H),3.41(s 3H),3.14-3.21(m,1H),2.72-2.80(m,1H),2.28-2.36(m,6H),2.12-2.18(m,1H),1.2-1.68(m,6H).

Step b: synthesis of Compound P-4

The synthesis method is described in step b of example 2. White solid 110mg, yield 36%. ¹ H NMR(600MHz,CDCl ₃ )δ:8.68(s,1H),8.51(t,J＝7.3Hz,1H),8.16(s,1H),7.98(s,1H),7.86(dd,J＝7.6,1.4Hz,1H),7.34-7.59(m,9H),7.15(t,J＝7.6Hz,1H),7.09(d,J＝8.3Hz,1H),6.46-6.62(m,2H),5.02-5.13(m,1H),4.70-4.79(m,2H),4.48(s,1H),4.21(s,1H),4.08(d,J＝11.1Hz,1H),3.96(s,3H),3.84(d,J＝12.2Hz,1H),3.56-3.63(m,1H),3.50(s,3H),3.43(s,3H),3.17(t,J＝12.5Hz,1H),2.71-2.80(m,1H),2.53(s,3H),213-2.43(m,8H),1.59-1.72(m,4H),1.47(d,J＝4.5Hz,3H),1.06(s,9H)； ¹³ C NMR(150MHz,CDCl ₃ )δ:174.72,173.37,172.67,171.20,169.48,167.96,165.21,151.69,149.97,148.93,144.63,133.89,132.63,130.90,127.80,125.34,123.42,120.70,118.86,117.98,110.73,71.40,59.91,59.11,58.29,57.40,50.18,48.52,46.10,42.36,39.69,37.42,37.23,36.57,34.12,33.20,27.92,26.51,25.87,24.01,23.63,17.41；HRMS(ESI,positive)m/z calcd for C ₅₅ H ₆₈ N ₁₁ O ₈ S[M+H] ⁺ :1042.4968；found 1042.4967.

Example 6

Synthesis of (2S, 4R) -1- ((S) -2- (7- (4- (4- ((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl [5,4-b ] [1,4] diaza-2-yl (amino) -3-methoxybenzamido) piperidin-1-yl) -7-oxoheptanamido) -3, 3-dimethylbutyryl) -4-hydroxy-N- ((S) -1- (4- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide (P-5)

Step a: synthesis of Compound 12-4

The synthesis was carried out by substituting compound 11 with compound 7-methoxy-7-oxoheptanoic acid, step a of example 2. White solid 600mg, 86% yield. ¹ H NMR(300MHz,DMSO-d ₆ )δ:8.43(s,1H),8.32(d,J＝8.4Hz,1H),8.22(d,J＝8.0Hz,1H),8.13(s,1H),7.68(dd,J＝7.6,1.5Hz,1H),7.54(dd,J＝8.4,1.5Hz,1H),7.47-7.52(m,2H),7.24(d,J＝8.4Hz,1H),7.16(t,J＝7.3Hz,1H),4.38(d,J＝12.4Hz,1H),4.00-4.07(m,1H),3.92(s,3H),3.89(s,1H),3.80(s,3H)3.39(s,3H),3.34(s,3H),3.09(t,J＝12.4Hz,1H),2.64(t,J＝12.1Hz,1H),2.25-2.37(m,2H),2.13(t,J＝7.1Hz,2H),1.76-1.88(m,2H),1.65-1.71(m,2H),1.34-1.51(m,2H).

Step b: synthesis of Compound P-5

The synthesis method is described in step b of example 2. White solid 120mg, 33% yield. ¹ H NMR(600MHz,CDCl ₃ )δ:8.67(s,1H),8.50(t,J＝7.8Hz,1H),8.15(d,J＝1.5Hz,1H),7.87(s,1H),7.84(dd,J＝8.0,1.5Hz,1H),7.49-7.53(m,1H),7.48(s,1H),7.34-7.43(m,6H),7.13(t,J＝7.6Hz,1H),7.07(d,J＝8.3Hz,1H),6.63-6.81(m,1H),6.43-6.60(m,1H),5.04-5.12(m,1H),4.67-4.75(m,1H),4.53-4.64(m,2H),4.49(s,1H),4.22(s,1H),4.02(dd,J＝20.0,11.4Hz,1H),3.95(s,3H),3.85(d,J＝12.9Hz,1H),3.58-3.63(m,1H),3.49(s,3H),3.41(s,3H),3.06-3.20(m,2H),2.68-2.76(m,1H),2.51(s,3H),2.22-2.42(m,4H),2.08-2.19(m,1H),1.57-1.65(m,4H),1.46(d,J＝6.8Hz,3H),1.29-1.37(m,6H),1.03(s,9H)； ¹³ C NMR(150MHz,CDCl ₃ )δ:174.99,173.26,172.97,171.34,169.56,167.97,165.08,156.62,152.46,151.74,150.54,149.73,148.79,144.70,133.72,133.01,132.13,130.88,128.49,127.79,125.24,123.45,120.83,118.79,117.83,110.73,71.26,60.12,58.98,58.32,57.38,55.09,50.17,48.50,46.20,42.40,39.64,37.34,36.80,34.23,33.20,31.05,29.90,27.92,26.48,26.16,24.05,23.58,17.40；HRMS(ESI,positive)m/z calcd for C ₅₆ H ₇₀ N ₁₁ O ₈ S[M+H] ⁺ :1056.5124；found 1056.5131.

Example 7

Synthesis of (2S, 4R) -1- ((S) -2- (8- (4- (4- ((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl [5,4-b ] [1,4] diaza-2- (amino) -3-methoxybenzamido) piperidin-1-yl) -8-oxooctylamino) -3, 3-dimethylbutyryl) -4-hydroxy-N- ((S) -1- (4- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide (P-6)

Step a: synthesis of Compound 12-5

The synthesis method refers to step a in example 2, substituting compound 11 with compound 8-methoxy-8-oxooctanoic acid. White solid 400mg, yield 80%. ¹ H NMR(300MHz,CDCl ₃ )δ:8.49(d,J＝8.2Hz,1H),8.14(s,1H),7.88(s,1H),7.82(d,J＝7.5Hz,1H),7.44(d,J＝1.4Hz,1H),7.40(t,J＝7.3Hz,1H),7.32(dd,J＝8.6,1.1Hz,1H),7.12(t,J＝7.6Hz,1H),7.05(d,J＝8.3Hz,1H),6.26(d,J＝7.9Hz,1H),4.60(d,J＝13.0Hz,1H),4.15-4.23(m,1H),3.95(s,3H),3.86(d,J＝14.2Hz,1H),3.62(s,3H),3.47(s,3H),3.40(s,3H),3.17(t,J＝12.1Hz,1H),2.71-2.80(m,1H),2.11-2.17(m,1H),1.98-2.03(m,1H),1.56-1.63(m,10H),1.42-1.44(m,2H),1.35-1.39(m,2H).

Step b: synthesis of Compound P-6

The synthesis method is described in step b of example 2. 39mg of white solid was obtained in 28% yield. ¹ H NMR(600MHz,DMSO-d ₆ )δ:8.67(s,1H),8.54(dd,J＝8.4,5.1Hz,1H),8.18(d,J＝1.8Hz,1H),7.84-7.87(m,2H),7.34-7.50(m,9H),7.15(t,J＝7.2Hz,1H),7.08(d,J＝8.1Hz,1H),6.47(dd,J＝8.1,69.1Hz,1H),6.30(dd,J＝8.6,31.0Hz,1H),5.06-5.11(m,1H),4.72(q,J＝8.6Hz,1H),4.57-4.64(m,2H),4.50(s,1H),4.19-4.27(m,1H),4.03(t,J＝11.2Hz,1H),3.98(s,3H),3.85-3.90(m,1H),3.65(s,1H),3.56-3.60(m,H),3.51(s,3H),3.43(s,3H),3.16-3.21(m,1H),2.72-7.78(m,1H),2.53(s,3H),2.29-2.40(m,2H),1.98-2.25(m,8H),1.59-1.64(m,4H),1.48(d,J＝6.9Hz,1H),1.31-1.35(m,4H),1.03(d,J＝3.2Hz,1H)； ¹³ C NMR(150MHz,DMSO-d ₆ )δ:175.05,173.40,173.04,171.19,169.57,167.91,165.06,156.70,152.62,151.66,150.60,149.86,148.78,144.58,133.84,133.74,132.94,132.25,130.91,128.45,127.84,127.80,125.24,123.52,120.66,118.76,117.77,110.70,71.86,71.27,59.98,58.88,58.19,57.39,50.19,48.50,46.16,42.32,39.63,37.62,37.32,36.60,34.45,34.38,33.26,30.01,29.87,27.89,26.55,26.42,23.60,17.46；HRMS(ESI,positive)m/z calcd for C ₅₇ H ₇₂ N ₁₁ O ₈ S[M+H] ⁺ :1070.5281；found 1070.5285.

Example 8

Synthesis of (2S, 4R) -1- ((S) -2- (2- (2- (4- (4- ((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimido [5,4-b ] [1,4] diazepin-2-yl) amino) -3-methoxybenzamido) piperidin-1-yl) -2-oxoethoxy) acetamido) -3, 3-dimethylbutyryl) -4-hydroxy-N- ((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide (P-7)

Reagents and conditions: a) EDCI, HOBT, DIPEA, DCM, rt,0.5h; b) EDCI, HOBT, DIPEA, DCM, rt,1h,two steps yield 30-40%.

The steps are as follows: synthesis of Compound P-7

8g (500 mg,1.03 mmol) of compound, 15 (165 mg,1.23 mmol) were dissolved in dry DCM (20 mL), EDCI (287 mg,1.54 mmol), HOBT (209 mg,1.54 mmol) and DIPEA (387 mg,3.08 mmol) were added sequentially and stirred at room temperature for 0.5h. After the reaction was completed, the reaction solution was distilled under reduced pressure, and purified by silica gel column chromatography (DCM: meoh=100:1) to give compound 16. Compound 16 (300 mg,0.50 mmol), intermediate 8 (266 mg,0.60 mmol), EDCI (133 mg,0.70 mmol), HOBT (97 mg,0.70 mmol) and DIPEA (258 mg,2.00 mmol) were then dissolved in dry DCM (20 mL) and reacted for 1h at room temperature. After the completion of the TLC monitoring, the reaction mixture was washed 3 times with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (DCM: meoh=100:1) to give compound P-7 (white solid, 154mg, yield 30%). ¹ H NMR(600MHz,CDCl ₃ )δ:8.68(s,1H),8.55(d,J＝8.1Hz,1H),8.19(s,1H),7.73-7.79(m,1H),7.90(s,1H),7.87(d,J＝7.5Hz,1H),7.32-7.57(m,9H),7.16(t,J＝7.5Hz,1H),7.10(d,J＝7.9Hz,1H),6.69(d,J＝46.8Hz,1H),5.01-5.13(m,1H),4.48-4.78(m,4H),4.08-4.28(m,4H),3.96(s,3H),3.64-3.80(m,2H),3.52(s,3H),3.45(s,3H),3.14-3.21(m,1H),2.80-2.87(m,1H),2.53(s,3H),2.33-2.43(m,2H),2.01-2.20(m,4H),1.45-1.48(m,3H),1.31-1.37(m,2H),1.11(s,9H)； ¹³ C NMR(150MHz,CDCl ₃ )δ:171.43,170.09,169.88,168.22,166.64,163.74,155.24,151.11,150.32,149.19,148.41,147.45,146.43,132.50,132.36,131.64,130.73,129.48,126.95,126.41,123.89,122.13,119.54,117.47,116.51,109.36,70.10,58.79,57.38,56.86,56.02,48.85,46.99,41.36,38.29,35.99,35.37,32.60,31.82,31.58,29.69,26.54,22.62,22.27,16.07；HRMS(ESI,positive)m/z calcd for C ₅₃ H ₆₄ N ₁₁ O ₉ S[M+H] ⁺ :1030.4604；found 1030.4634.

Example 9

Synthesis of (2S, 4R) -1- ((S) -2- (2- (2- (2- (4- (4- ((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl [5,4-b ] [1,4] diaza-2-yl) amino) -3-methoxybenzamido) piperidin-1-yl) -2-oxoethoxy) ethoxy) acetamido) -3, 3-dimethylbutyryl) -4-hydroxy-N- ((S) -1- (4- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide (P-8)

The compound 15 is replaced by 3, 6-octadioxirane, and other synthetic methods are described in example 8. 90.6mg of white solid was obtained in 40% yield. ¹ H NMR(600MHz,CDCl ₃ )δ:8.68(s,1H),8.55(d,J＝8.6Hz,1H),8.18(s,1H),7.85-7.90(m,2H),7.38-7.51(m,5H),7.30-7.35(m,3H),7.17(t,J＝7.6Hz,1H),7.04-7.14(m,2H),6.78-6.84(m,1H),5.06(t,J＝7.3Hz,1H),4.57-4.63(m,2H),4.45(t,J＝23.2Hz,2H),4.17-4.23(m,2H),3.73-3.86(m,4H),3.67(s,3H),3.52(s,3H),3.44(s,3H),3.11-3.23(m,1H),2.76-2.87(m,1H),2.52(s,3H),2.30-2.37(m,1H),1.75-2.01(m,8H),1.48(d,J＝7.1Hz,1H),9.01(s,9H)；HRMS(ESI,positive)m/z calcd for C ₅₅ H ₆₈ N ₁₁ O ₁₀ S[M+H] ⁺ :1074.4866；found 1074.4873.

Example 10

Synthesis of (2S, 4R) -1- ((S) -2- (tert-butyl) -14- (4- (4- ((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl [5,4-b ] [1,4] diaza-2- (amino) -3-methoxybenzoylamino) piperidin-1-yl) -4, 14-dioxo-6, 9, 12-trioxa-3-azidothioalkyl) -4-hydroxy-N- ((S) -1- (4- (4-methylthiazol) -5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide (P-9)

Other synthetic methods refer to example 8, substituting compound 15 with 2,2' - ((oxybis (ethane-2, 1-diyl)) bis (oxy)) diacetic acid. White solid 84mg, 34% yield. ¹ H NMR(600MHz,CDCl ₃ )δ:8.65(s,H),8.48(dd,J＝27.7,8.2Hz,1H),8.14(s,1H),7.85(s,1H),7.83(d,J＝7.6Hz,1H),7.32-7.49(m,9H),7.12(t,J＝7.3Hz,1H),7.06(d,J＝8.2Hz,1H),5.01-5.11(m,1H),4.46-4.68(m,4H),4.15-4.23(m,2H),3.91-3.96(m,4H),3.62-3.71(m,8H),3.48(s,3H),3.40(s,3H),3.04-3.19(m,2H),2.67-2.83(m,4H),2.51(s,3H),2.33-2.41(m,1H),2.05-2.13(m,2H),1.43-1.46(m,4H),1.38(d,J＝6.5Hz,3H),1.04(s,9H)； ¹³ C NMR(150MHz,CDCl ₃ )δ:172.47,171.88,171.39,169.57,169.29,167.88,165.09,155.64,152.47,151.68,150.56,149.77,148.72,144.70,133.85,133.72,133.01,132.12,130.87,127.60,127.80,125.23,123.42,121.05,118.80,117.83,110.75,72.78,72.50,71.94,71.34,60.22,58.32,57.36,54.94,50.19,48.19,45.86,43.28,42.89,39.63,37.33,37.24,34.17,32.99,27.85,23.58,19.94,18.80,17.42；HRMS(ESI,positive)m/z calcd for C ₅₇ H ₇₂ N ₁₁ O ₁₁ S[M+H] ⁺ :1118.5128；found 1118.5101.

Example 11

Synthesis of (2S, 4R) -1- ((S) -2- (tert-butyl) -17- (4- (4- ((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl [5,4-b ] [1,4] diaza-2- (yl) amino) -3-methoxybenzamido) piperidin-1-yl) -4, 17-dioxo-6, 9,12, 15-tetraoxy-3-azaheptadecanoyl) -4-hydroxy-N- ((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide (P-10)

The compound 15 is replaced by 3,6,9, 12-tetraoxatetradecane-1, 14-dicarboxylic acid, and other synthetic methods are described in example 8. White solid 57mg, yield 32%. ¹ H NMR(600MHz,DMSO-d ₆ )δ:8.63(s,1H),8.48(d,J＝8.3Hz,1H),8.13(s,1H),7.82(s,1H),7.80(d,J＝7.9Hz,1H),7.54(d,J＝7.3Hz,1H),7.45(s,1H),7.28-7.43(m,8H),7.10(t,J＝7.6Hz,1H),7.04(d,J＝8.3Hz,1H),6.76(s,1H),4.99-5.07(m,1H),4.69(t,J＝7.9Hz,1H),4.57(d,J＝9.0Hz,1H),4.51(d,J＝12.8Hz,1H),4.45(s,1H),4.10-4.28(m,4H),3.95-4.00(m,2H),3.92(s,3H),3.83-3.92(m,2H),3.60-3.67(m,12H),9.45(s,3H),3.38(s,3H),3.06-3.16(m,1H),2.74(t,J＝12.09Hz,1H),2.62(s,2H),2.48(s,3H),2.31-2.38(m,1H),2.03-2.11(m,2H),1.45-1.55(m,2H),1.43(d,J＝6.9Hz,3H),1.01(s,9H)； ¹³ C NMR(150MHz,DMSO-d ₆ )δ:171.43,170.30,169.88,168.20,167.80,166.56,163.70,155.30,151.17,150.28,149.21,148.46,147.41,132.48,132.37,130.80,129.52,127.12,126.43,123.87,122.11,119.38,117.41,116.42,109.34,71.05,70.56,70.45,70.39,70.02,58.52,57.04,56.83,56.04,48.83,47.05,44.23,41.29,38.26,35.97,35.79,35.41,32.74,31.87,31.57,29.68,26.50,22.22,16.08,14.10；HRMS(ESI,positive)m/z calcd for C ₅₉ H ₇₆ N ₁₁ O ₁₂ S[M+H] ⁺ :1162.5390；found 1162.5424.

Example 12

Synthesis of 4- (((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl [5,4-b ] [1,4] diazepin-2-yl) amino) -N- (1- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindol-4-yl) piperidin-4-yl) -3-methoxybenzamide (P-11)

Reagents and conditions: a) EDCI, HOBT, DIPEA, DCM, r.t.,2h, yield 63%.

The steps are as follows: synthesis of Compound P-11

8g (0.5 g,1.03 mmol) of the compound and pomalidomide (331 mg,1.23 mmol) were dissolved in dry DCM (20 mL), EDCI (229 mg,1.23 mmol), HOBT (167 mg,1.23 mmol) and DIPEA (258 mg,2.05 mmol) were added sequentially and reacted at room temperature for 2h. After the completion of the reaction, the reaction mixture was washed with a saturated sodium chloride solution (20 ml×3), dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (DCM/meoh=100:1) to give compound P-11 (yellow solid, 100mg, yield 36%). ¹ H NMR(600MHz,DMSO-d ₆ )δ:11.09(s,1H),8.45(s,1H),8.34(d,J＝8.7Hz,1H),8.25(d,J＝7.9Hz,1H),8.15(s,1H),7.66-7.73(m,2H),7.58(dd,J＝1.8,8.3Hz,1H),7.48-7.55(m,2H),7.35(d,J＝7.1Hz,1H),7.26(d,J＝7.9Hz,3H),7.16-7.20(m,1H),5.10(dd,J＝12.7,5.5Hz,1H),4.00-4.10(m,1H),3.94(s,3H),3.75(d,J＝11.9Hz,3H),3.41(s,3H),3.36(s,3H),3.03(t,J＝12.8Hz,1H),2.84-2.93(m,1H),2.52-2.64(m,2H),2.00-2.09(m,1H),1.90-1.97(m,2H),1.78-1.89(m,2H)； ¹³ C NMR(150MHz,DMSO-d ₆ )δ:173.27,170.50,167.56,167.49,166.81,165.51,163.45,155.62,152.80,150.31,149.57,148.14,136.22,134.16,133.04,132.20,131.73,128.50,126.59,124.50,124.06,121.83,120.65,118.31,118.04,116.96,115.02,109.98,56.53,50.71,49.27,46.73,37.94,36.08,32.06,31.43,26.81,22.57；HRMS(ESI,positive)m/z calcd for C ₃₉ H ₃₈ N ₉ O ₇ [M+H] ⁺ :744.2889；found 744.2904.

Example 13

Synthesis of 4- (((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl [5,4-b ] [1,4] diazepin-2-yl) amino) -N- (1- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindol-4-yl) glycyl) piperidin-4-yl) -3-methoxybenzamide (P-12)

Reagents and conditions: a) EDCI, HOBT, DIPEA, DCM, r.t.,1h, yield 80-90%; b) DCM, TFA, r.t.,2h, yield 90-95%; c) EDCI, HOBT, DIPEA, DCM, r.t.,2h, yield 60-65%.

Step a: synthesis of Compound 19

Compound 8g (500 mg,1.03 mmol) and Boc-glycine (210 mg,1.23 mmol) were dissolved in dry DCM (20 mL), EDCI (287 mg,1.54 mmol), HOBT (209 mg,1.54 mmol) and DIPEA (387 mg,1.54 mmol) were added sequentially with stirring and reacted at room temperature for 1h. After the completion of the TLC detection reaction, the reaction mixture was washed with saturated sodium chloride (20 ml×3), and the organic layer was dried over anhydrous sodium sulfate, distilled under reduced pressure, and purified by silica gel column chromatography (hexane: etoac=4:1-1:1) to give compound 19 (yellow solid, 527mg, yield 80%). ¹ H NMR(600MHz,DMSO-d ₆ )δ:8.46(s,1H),8.34(s,J＝8.4Hz,1H),8.24(d,J＝7.7Hz,1H),8.16(s,1H),7.70(dd,J＝7.7,1.6Hz,1H),7.56(dd,J＝8.4,1.7Hz,1H),7.49-7.53(m,2H),7.26(d,J＝8.1Hz,1H),7.16-7.20(m,1H),6.80-8.87(m,1H),4.39(d,J＝13.1Hz,1H),3.99-4.09(m,1H),3.94(s,3H),3.88(d,J＝13.4Hz,1H),3.41(s,3H),3.35(s,3H),3.12(t,J＝12.9Hz,1H),2.95(q,J＝6.5Hz,1H),2.64-2.71(m,1H),2.32(t,J＝6.9Hz,2H),1.84(dd,J＝35.1,10.5Hz,2H),1.38(s,9H).

Step b: synthesis of Compound P-12

Compound 19 (500 mg,0.78 mmol) was placed in a 25mL reaction flask, 10mL of LDCM was added for complete dissolution, TFA (273.6 mg,2.33 mmol) was slowly added dropwise, and the reaction was carried out at room temperature for 2h. After TLC monitoring the reaction, the reaction solution was distilled under reduced pressure, and no further purification was required to obtain a crude product of compound 20. Then compound 20 obtained in the previous step and pomalidomide (248.4 mg,0.93 mmol) were dissolved in dry DCM (20 mL), EDCI (229.2 mg,1.16 mmol), HOBT (166.8 mg,1.16 mmol) and DIPEA (309.6 mg,2.33 mmol) were added in this order and reacted at room temperature for 2h. After the reaction was completed, the reaction mixture was washed with a saturated sodium chloride solution (20 ml×3), dried over anhydrous sodium sulfate, and distilled under reduced pressure, followed by separation and purification by silica gel column chromatography (DCM/meoh=100:1) to give compound P-12 (yellow solid, 600mg, yield 37%). ¹ H NMR(600MHz,DMSO-d ₆ )δ:11.11(s,1H),8.45(s,1H),8.34(d,J＝8.3Hz,1H),8.22(d,J＝7.9Hz,1H),8.16(s,1H),7.69(dd,J＝7.7,1.5Hz,1H),7.62(t,J＝8.1Hz,1H),7.55(d,J＝7.5Hz,1H),7.48-7.53(m,2H),7.25(d,J＝8.5Hz,1H),7.18(t,J＝7.2Hz,1H),7.06-7.15(m,2H),5.08(dd,J＝12.9,5.3Hz,1H),4.34-4.48(m,1H),4.15-4.33(m,2H),4.06-4.14(m,1H),3.94-4.00(m,1H),3.93(s,3H),3.41(s,3H),3.36(s,3H),3.12-3.27(m,1H),2.69-2.96(m,2H),2.50-2.65(m,2H),1.98-2.01(m,1H),1.86-1.96(m,2H),2.69-2.96(m,2H),1.41-1.63(m,1H),1.13-1.35(m,1H)； ¹³ C NMR(150MHz,DMSO-d ₆ )δ:173.26,170.53,169.28,167.82,167.48,166.60,165.61,163.44,155.60,152.81,149.56,148.15,145.90,136.64,133.04,132.50,132.20,131.77,128.44,126.58,124.06,121.83,120.59,118.76,118.31,118.01,111.28,110.02,56.53,49.07,46.96,44.14,43.31,41.30,37.94,36.07,32.32,31.64,31.46,22.62.

Example 14

Synthesis of 4- (((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl [5,4-b ] [1,4] diazepin-2-yl) amino) -N- (1- (3- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindol-4-yl) amino) propionyl) piperidin-4-yl) -3-methoxybenzamide (P-13)

Step a: synthesis of Compound 19-1

The compound 18 was replaced with Boc-beta-alanine and the other synthesis was as described in example 13. 286mg of white solid was obtained in 88% yield. ¹ H NMR(600MHz,DMSO-d ₆ )δ:8.45(s,1H),8.33(d,J＝7.9Hz,1H),8.23(d,J＝7.6Hz,1H),8.14(s,1H),7.69(dd,J＝7.8,1.6Hz,1H),7.55(dd,J＝8.3,1.6Hz,1H),7.49-7.54(m,2H),7.25(d,J＝8.1Hz,1H),7.17(t,J＝7.0Hz,1H),6.79-6.87(m,1H),4.38(d,J＝13.2Hz,1H),4.00-4.09(m,1H),3.93(s,3H),3.87(d,J＝13.3Hz,1H),3.40(s,3H),3.34(s,3H),3.11(t,J＝13.0Hz,1H),2.94(q,J＝6.5Hz,2H),2.64-2.70(m,1H),2.31(t,J＝7.0Hz,1H),1.75-1.90(m,2H),1.58-1.62(m,2H),1.37(s,9H).

Step b: synthesis of Compound P-13

Synthetic method reference example 13. Yellow solid 80mg, yield 29%. ¹ H NMR(600MHz,DMSO-d ₆ )δ:8.54(dd,J＝8.7,3.1Hz,1H),8.42(s,1H),8.34(s,1H),8.20(s,1H),7.81-7.94(m,2H),7.49-7.58(m,1H),7.40-7.49(m,2H),2.49(dd,J＝8.6,1.9Hz,1H),7.29(s,1H),7.15-7.19(m,1H),7.08-7.15(m,2H),7.00(dd,J＝8.7,3.1Hz,1H),6.48-6.65(m,1H),6.39(dd,J＝36.7,8.3Hz,1H),4.87-4.96(m,1H),4.63-4.72(m,1H),4.15-4.26(m,1H),3.99(d,J＝1.8Hz,3H),3.72-3.88(m,2H),3.63-3.72(m,1H),3.52(s,3H),3.49(s,3H),3.18(t,J＝12.4Hz,1H),2.69-2.91(m,4H),2.54-2.64(m,1H),2.00-2.14(m,3H)； ¹³ C NMR(150MHz,DMSO-d ₆ )δ:170.97,169.35,169.05,168.71,167.61,166.70,163.70,155.31,151.23,149.23,147.46,146.55,136.28,132.59,132.48,127.17,126.47,123.86,122.14,119.24,117.42,116.70,116.59,116.37,111.77,110.37,109.38,56.03,48.99,47.12,44.79,41.10,38.72,35.95,32.67,32.24,31.79,31.44,29.69,22.66.

Example 15

Synthesis of 4- ((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidine [5,4-b ] [1,4] diazepin-2-yl) -amino) -N- (1- (5- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) amino) pentanoyl) piperidin-4-yl) -3-methoxybenzamide (P-14)

Step a: synthesis of Compound 19-2

The compound 18 was replaced with Boc-5-aminopentanoic acid and other synthetic methods were described in example 13. 396mg of white solid was obtained in 90% yield. ¹ H NMR(600MHz,DMSO-d ₆ )δ:8.45(s,1H),8.33(d,J＝7.6Hz,1H),8.10-8.21(m,1H),7.69(dd,J＝7.7,1.6Hz,1H),7.54(dd,J＝8.4,1.6Hz,1H),7.47-7.53(m,2H),7.25(d,J＝8.4Hz,1H),7.18(t,J＝7.3Hz,1H),6.79(t,J＝5.4Hz,1H),4.38(d,J＝13.2Hz,1H),3.99-4.10(m,1H),3.93(s,3H),3.86-3.92(m,1H),3.41(s,3H),3.35(s,3H),3.11(t,J＝12.3Hz,1H),2.92(q,J＝6.7Hz,1H),2.67(t,J＝11.7Hz,1H),2.31(t,J＝7.3Hz,2H),1.84(dd,J＝37.0,11.0Hz,2H),1.44-1.52(m,3H),1.37(s,9H).

Step b: synthesis of Compound P-14

Synthetic method reference is made to example 13, step b. Yellow solid 200mg, yield 41%. ¹ H NMR(600MHz,DMSO-d ₆ )δ:11.09(s,1H),8.45(s,1H),8.33(d,J＝8.7Hz,1H),8.17(s,1H),8.15(s,1H),7.69(dd,J＝7.8,1.7Hz,1H),7.59(t,J＝8.1Hz,1H),7.48-7.56(m,3H),7.25(d,J＝8.4Hz,1H),7.18(t,J＝7.5Hz,1H),7.12(d,J＝8.6Hz,1H),7.02(d,J＝7.0Hz,1H),6.57(t,J＝5.6Hz,1H),5.05(dd,J＝12.8,5.5Hz,1H),4.38(d,J＝12.9Hz,1H),3.99-4.99(m,1H),3.93(s,3H),3.86-3.92(m,1H),3.41(s,3H),3.35(s,3H),3.08-3.18(m,2H),2.83-2.95(m,1H),2.64-2.71(m,1H),2.54-2.61(m,1H),2.51-2.54(m,1H),2.39(t,J＝7.3,1H),2.00-2.05(m,1H),1.78-1.89(m,2H),1.56-1.68(m,4H),1.33-1.53(m,3H)；HRMS(ESI,positive)m/z calcd for C ₄₄ H ₄₇ N ₁₀ O ₈ [M+H] ⁺ :843.3573；found 843.3575.

Example 16

Synthesis of 4- (((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl [5,4-b ] [1,4] diazepin-2-yl) amino) -N- (1- (6- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindol-4-yl) amino) hexanoyl) piperidin-4-yl) -3-methoxybenzamide (P-15)

Step a: synthesis of Compound 19-3

The compound 18 was replaced with 6- ((tert-butoxycarbonyl) amino) hexanoic acid and other synthetic methods were as described in example 13. 256mg of white solid was obtained in 86% yield. ¹ H NMR(600MHz,DMSO-d ₆ )δ:8.45(s,1H),8.33(d,J＝8.3Hz,1H),8.19(d,J＝7.5Hz,1H),8.15(s,1H),7.69(dd,J＝7.8,1.7Hz,1H),7.55(d,J＝8.4Hz,1H),7.45-7.53(m,2H),7.25(d,J＝8.3Hz,1H),7.18(t,J＝7.3Hz,1H),6.77(t,J＝5.6Hz,1H),4.38(d,J＝13.2Hz,1H),4.00-4.08(m,1H),3.93(s,3H),3.89(d,J＝13.7Hz,1H),3.41(s,3H),3.35(s,3H),3.11(t,J＝12.1Hz,1H),2.90(t,J＝6.8Hz,2H),2.66(t,J＝11.9Hz,1H),2.30(t,J＝7.3Hz,2H),1.83(dd,J＝36.4,10.4Hz,2H),1.44-1.53(m,4H),1.37(s,9H),1.23-1.29(m,4H).

Step b: synthesis of Compound P-15

Synthetic method reference is made to example 13, step b. Yellow solid 90mg, yield 28%. ¹ H NMR(600MHz,DMSO-d ₆ )δ:11.09(s,1H),8.45(s,1H),8.33(d,J＝8.5Hz,1H),8.13-8.20(m,2H),6.89(dd,J＝7.8,1.8Hz,1H),7.56-7.61(m,1H),7.53-7.56(m,1H),7.47-7.53(m,2H),7.25(d,J＝8.3Hz,1H),7.18(t,J＝7.2Hz,1H),7.10(d,J＝8.7Hz,1H),7.02(d,J＝6.9Hz,1H),6.54(t,J＝5.8Hz,1H),6.05(dd,J＝5.6,13.0Hz,1H),4.38(d,J＝13.2Hz,1H),3.99-4.08(m,1H),3.93(s,3H),3.87-3.92(m,1H),3.41(s,3H),3.35(s,3H),3.27-3.32(m,2H),3.11(t,J＝12.1Hz,1H),2.83-2.92(m,1H),2.56-2.62(m,1H),2.51-2.56(m,1H),2.29-2.37(m,2H),2.00-2.06(m,1H),1.77-1.90(m,2H),1.50-1.64(m,4H),1.24-1.41(m,4H)； ¹³ C NMR(150MHz,DMSO-d ₆ )δ:173.25,170.83,170.55,169.42,167.76,167.48,165.50,163.43,155.60,152.80,149.56,148.14,146.91,136.75,133.04,132.67,132.20,131.75,128.45,126.59,124.05,121.83,120.57,118.31,118.00,117.66,110.85,109.97,109.45,56.52,49.02,47.09,44.58,42.26,40.75,37.94,36.07,32.72,31.80,31.44,29.04,26.61,25.16,22.63；HRMS(ESI,positive)m/z calcd for C ₄₄ H ₄₇ N ₁₀ O ₈ [M+H] ⁺ :857.3429；found 3757.

Example 17

Synthesis of 4- (((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl [5,4-b ] [1,4] diazepin-2-yl) amino) -N- (1- (7- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindol-4-yl) amino) heptanoyl) piperidin-4-yl) -3-methoxybenzamide (P-16)

Step a: synthesis of Compound 19-4

The compound 18 was replaced with 7- (N-t-butoxycarbonylamino) heptanoic acid and other synthetic methods were as described in example 13. 240mg of white solid was obtained in 82% yield. ¹ H NMR(600MHz,CDCl ₃ )δ:8.44(s,1H),8.32(d,J＝8.3Hz,1H),8.23(d,J＝7.7Hz,1H),7.69(dd,J＝7.7,1.2Hz,1H),7.55(d,J＝8.5Hz,1H),7.48-7.54(m,2H),7.25(d,J＝8.4Hz,1H),7.17(t,J＝7.5Hz,1H),4.39(d,J＝12.8Hz,1H),4.01-4.10(m,1H),3.93(s,3H),3.89(d,J＝12.8Hz,1H),3.54-3.69(m,4H),3.40(s,3H),3.35(s,3H),3.10(t,J＝12.5Hz,1H),2.66(t,J＝12.0Hz,1H),2.55-2.63(m,2H),2.31(t,J＝7.5Hz,2H),1.83(dd,J＝36.4,11.5Hz,2H),1.34-1.54(m,6H),1.28(s,9H).

Step b: synthesis of Compound P-16

Synthetic method reference is made to example 13, step b. Yellow solid 90mg, 36% yield. ¹ H NMR(600MHz,DMSO-d ₆ )δ:11.09(s,1H),8.44(s,1H),8.33(d,J＝8.5Hz,1H),8.16(d,J＝8.1Hz,1H),8.14(s,1H),7.69(dd,J＝7.8,1.6Hz,1H),7.55-7.59(m,1H),7.54(dd,J＝8.6,1.8Hz,1H),7.47-7.52(m,2H),7.25(d,J＝8.3Hz,1H),7.17(t,J＝7.8Hz,1H),7.01(d,J＝6.9Hz,1H),6.53(t,J＝5.7Hz,1H),5.05(dd,J＝12.9,5.4Hz,1H),4.39(d,J＝12.5Hz,1H),4.00-4.99(m,1H),3.94(s,3H),3.88(d,J＝12.8Hz,1H),3.41(s,3H),3.35(s,3H),3.29(q,J＝6.5Hz,1H),3.11(t,J＝12.4Hz,1H),2.82-2.93(m,1H),2.66(t,J＝11.6Hz,1H),2.50-2.62(m,2H),2.31(t,J＝7.5Hz,1H),2.00-2.06(m,1H),1.79-1.88(m,2H),1.55-1.60(m,2H),1.49-1.53(m,2H),1.33-1.38(m,4H),1.21-1.26(m,2H)； ¹³ C NMR(150MHz,DMSO-d ₆ )δ:173.25,170.87,170.54,169.43,167.76,167.48,165.51,163.43,155.59,152.77,149.55,148.11,146.91,136.73,133.03,132.67,132.20,131.75,128.42,126.58,124.04,121.83,120.58,118.29,117.95,117.63,110.84,109.96,109.51,56.51,55.36,49.02,47.10,44.59,42.28,40.74,37.93,36.07,32.73,31.81,31.44,31.41,29.06,29.00,26.72,25.38,22.93,22.62,22.51,14.41；HRMS(ESI,positive)m/z calcd for C ₄₅ H ₄₉ N ₁₀ O ₈ [M+H] ⁺ :871.3886；found 871.3871.

Example 18

Synthesis of 4- (((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl [5,4-b ] [1,4] diazepin-2-yl) amino) -N- (1- (8- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindol-4-yl) amino) octanoyl) piperidin-4-yl) -3-methoxybenzamide (P-17)

Step a: synthesis of Compound 19-5

The compound 18 was replaced with Boc-8-aminocaprylic acid and other synthetic methods were as described in example 13. 251mg of white solid was obtained in 85% yield. ¹ H NMR(600MHz,CDCl ₃ )δ:8.50(d,J＝7.8Hz,1H),8.29(d,J＝7.1Hz,1H),8.16(s,1H),7.86-7.95(m,1H),7.84(dd,J＝7.9,1.4Hz,1H),7.47(t,J＝7.5Hz,1H),7.38-7.45(m,2H),7.29(d,J＝8.5Hz,1H),7.13(t,J＝7.6Hz,1H),7.07(s,1H),7.06(s,1H),6.86(d,J＝8.6Hz,1H),6.21(t,J＝5.7Hz,1H),6.07(d,J＝7.5Hz,1H),4.89(dd,J＝12.3,5.2Hz,1H),4.60(d,J＝12.7Hz,1H),4.15-4.25(m,1H),3.97(s,3H),3.86(d,J＝12.7Hz,1H),3.49(s,3H),3.42(s,3H),3.25(q,J＝6.6Hz,2H),3.19(t,J＝12.5Hz,1H),2.64-2.90(m,4H),2.33(dd,J＝12.7,7.5Hz,2H),1.96-2.18(m,3H),1.36(s,9H).

Step b: synthesis of Compound P-17

Reference implementation of the Synthesis methodExample 13 step b. Yellow solid 60mg, yield 28%. ¹ H NMR(600MHz,CDCl ₃ )δ:8.50(d,J＝7.8Hz,1H),8.29(d,J＝7.1Hz,1H),8.16(s,1H),7.86-7.95(m,1H),7.84(dd,J＝7.7,1.4Hz,1H),7.47(t,J＝7.5Hz,1H),7.39-7.45(m,2H),7.29(d,J＝8.6Hz,1H),7.13(t,J＝7.7Hz,1H),7.07(d,J＝7.3Hz,1H),6.86(d,J＝8.6Hz,1H),6.21(t,J＝5.6Hz,1H),6.07(d,J＝7.7Hz,1H),4.89(dd,J＝12.3,5.3Hz,1H),4.61(d,J＝12.8Hz,1H),4.15-4.25(m,1H),3.97(s,3H),3.82-3.91(m,1H),3.49(s,3H),3.42(s,3H),3.25(q,J＝6.8Hz,2H),3.19(t,J＝12.3Hz,1H),2.64-2.92(m,4H),2.26-2.41(m,2H),1.97-2.18(m,3H),1.52-1.70(m,10H),1.34-1.39(m,4H)； ¹³ C NMR(150MHz,CDCl ₃ )δ:172.93,172.35,170.92,169.74,169.55,169.00,167.90,165.14,148.95,148.40,137.50,133.86,133.77,127.84,125.29,120.36,118.80,118.03,117.89,112.78,111.26,110.69,57.41,50.27,48.63,45.96,43.98,42.12,39.66,39.66,37.37,34.70,34.41,33.33,32.79,30.66,30.50,30.42,28.12,26.60,24.20；HRMS(ESI,positive)m/z calcd for C ₄₇ H ₅₃ N ₁₀ O ₂ [M+H] ⁺ :885.4042；found 885.4048.

Example 19

Synthesis of 4- (((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl [5,4-b ] [1,4] diazepin-2-yl) amino) -N- (1- (2- (2- (2- ((2- (2-2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) amino) ethoxy) acetyl) piperidin-4-yl) -3-methoxybenzamide (P-18)

Reagents and conditions: a) EDCI, HOBT, DIPEA, DCM, rt,1h, yield 68%; b) DCM, H ₂ ,Pd/C,rt,overnight,yield 85％；c)EDCI,HOBT,DIPEA,DCM,rt,1h,yield 78％.

Step a: synthesis of Compound 22

Compound 8g (1.0 g,2.05 mmol) and compound 21 (662 mg,2.46 mmol) were dissolved in dry DCM (50 mL), EDCI (458 mg,2.46 mmol), HOBT (333 mg,2.46 mmol), DIPEA (516 mg,4.10 mmol) and reacted at room temperature for 2h. After TLC monitoring the end of the reaction, the reaction was distilled under reduced pressure and purified by silica gel column chromatography (DCM/meoh=100:0.5) to give compound 22 (white solid, 963mg, yield 68%). ¹ H NMR(600MHz,CDCl ₃ )δ:8.53(d,J＝8.2Hz,1H),8.17(s,1H),7.94(s,1H),7.85(dd,J＝7.8,1.7Hz,1H),7.43-7.49(m,2H),7.41(d,J＝1.8Hz,1H),7.17-7.14(m,1H),7.12(d,J＝8.3Hz,1H),6.61(s,1H),6.38(d,J＝8.2Hz,1H),4.52(d,J＝13.0Hz,1H),4.21-4.67(m,3H),3.99(s,3H),3.78-3.84(m,1H),3.70-3.77(m,10H),3.51(s,3H),3.46(s,3H),3.15-3.20(m,2H),2.75-2.81(m,1H),1.97-2.10(m,2H),1.61-1.74(m,2H).

Step b: synthesis of Compound P-18

Compound 22 (0.5 g,0.71 mmol) was dissolved in DCM (20 mL) and 0.1g palladium on carbon was added and reacted overnight at room temperature under hydrogen. After the completion of the TLC monitoring, the reaction solution was filtered through celite, and the filtrate was distilled under reduced pressure, without further purification, to give 400mg of crude compound 23. Compound 23 (300 mg,0.44 mmol) and pomalidomide (138 mg,0.53 mmol) were then dissolved in dry DCM (15 mL) and EDCI (115 mg,0.66 mmol), HOBT (83 mg,0.66 mmol) and DIPEA (194 mg,1.50 mmol) were added sequentially and reacted at room temperature for 2h. After the completion of the reaction, the reaction mixture was washed with saturated sodium chloride solution (20 ml×3), and the organic layer was dried over anhydrous sodium sulfate, distilled under reduced pressure, and purified by silica gel column chromatography (DCM/meoh=100:1) to give compound P-18 (yellow solid, 508.9mg, yield 78%). ¹ H NMR(600MHz,DMSO-d ₆ )δ:11.09(s,1H),8.45(s,1H),8.32(d,J＝8.5Hz,1H),8.17(d,J＝7.7Hz,1H),8.14(s,1H),7.69(dd,J＝7.8,1.7Hz,1H),7.47-7.58(m,4H),7.25(d,J＝8.4Hz,1H),7.18(t,J＝7.8Hz,1H),7.12(d,J＝8.6Hz,1H),7.02(d,J＝7.1Hz,1H),6.59(t,J＝5.8Hz,1H),5.05(dd,J＝12.9,5.6Hz,1H),4.32(d,J＝14.0Hz,1H),4.09-4.13(m,1H),4.00-4.07(m,1H),3.92(s,3H),3.83(d,J＝14.2Hz,1H),3.61(t,J＝5.6Hz,1H),3.51-3.59(m,7H),3.42-3.51(m,3H),3.41(s,3H),3.35(s,3H),3.16(d,J＝5.0Hz,1H),3.02-3.10(m,1H),2.82-2.91(m,1H),2.71(t,J＝12.5Hz,1H),2.55-2.64(m,1H),1.94-2.06(m,2H),1.84(t,J＝13.4Hz,1H),1.32-1.56(m,3H)； ¹³ C NMR(150MHz,DMSO-d ₆ )δ:173.25,170.52,169.40,167.74,167.48,165.52,163.44,155.59,152.80,149.56,148.12,148.86,136.67,133.04,132.55,132.20,131.74,128.42,126.59,124.05,121.83,120.59,118.31,117.98,117.86,111.12,109.97,109.72,70.25,70.14,70.07,69.36,49.03,46.98,43.93,42.15,40.87,37.94,36.07,32.57,31.71,31.44,22.61；HRMS(ESI,positive)m/z calcd for C ₄₇ H ₅₃ N ₁₀ O ₁₁ [M+H] ⁺ :933.3890；found933.3867.

Example 20

Synthesis of 4- (((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl [5,4-b ] [1,4] diazepin-2-yl) amino) -N- (1- (2- ((2- (2, 6-dioxapiperidin-3-yl) -1, 3-dioxaisoindol-4-yloxy) acetyl) piperidin-4-yl) -3-methoxybenzamide (P-19)

Reagents and conditions: a) DIPEA, DMF,80 ℃,8h, yield 58%; b) DCM, TFA, rt,2h, yield 90-95%; c) EDCI, HOBT, DIPEA, DCM, rt,1h, yield 38%.

The steps are as follows: synthesis of Compound P-19

Compound 24 (110 mg,0.40 mmol) and compound 25 (94 mg,0.48 mmol) were dissolved in dry DMF (10 mL), DIPEA (104 mg,0.80 mmol) was added and transferred to 80℃and stirred overnight. After the TLC detection reaction was completed, the reaction solution was poured into 300mL of water, extracted with ethyl acetate (20 ml×3), the organic layer was collected, washed 3 times with saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated, and the obtained crude product was separated and purified by silica gel column chromatography (DCM/meoh=100:1) to give compound 26 (yellow solid, 90mg, yield 58%).

Compound 26 (90 mg,0.23 mmol) was placed in a 25mL reaction flask and 10mL DCM was added to dissolve the compound slowly dropwise TFA (2 mL) and reacted at room temperature for 2h. After TLC monitoring the reaction, the reaction solution was distilled under reduced pressure, and no further purification was required to obtain a crude product of compound 27. Compound 8g (113 mg,0.23 mmol) and compound 27 (92 mg,0.28 mmol) were dissolved in dry DCM (10 mL)EDCI (53 mg,0.28 mmol), HOBT (38 mg,0.28 mmol) and DIPEA (60 mg,0.46 mmol) were added in this order, and the reaction was continued at room temperature for 2h. After TLC monitoring the end of the reaction, the reaction was distilled under reduced pressure and purified by silica gel column chromatography (DCM/meoh=100:1) to give compound P-19 (yellow solid, 70mg, yield 38%). ¹ H NMR(600MHz,DMSO-d ₆ )δ:11.12(s,1H),8.45(s,1H),8.34(d,J＝8.6Hz,1H),8.23(d,J＝7.6Hz,1H),8.16(s,1H),7.78(t,J＝8.1Hz,1H),7.69(dd,J＝7.8,1.4Hz,1H),7.56(d,J＝8.4Hz,1H),7.48-7.53(m,2H),7.46(d,J＝7.4Hz,1H),7.33(d,J＝8.7Hz,1H),7.26(d,J＝8.4Hz,1H),7.18(t,J＝7.3Hz,1H),5.21-5.34(m,1H),5.11(dd,J＝12.9,5.5Hz,1H),4.3(d,J＝11.3Hz,1H),4.04-4.14(m,1H),3.94(s,3H),3.86(d,J＝13.1Hz,1H),3.41(s,3H),3.36(s,3H),3.20(t,J＝11.9Hz,1H),2.85-2.94(m,1H),2.80(t,J＝11.6Hz,1H),2.53-2.64(m,1H),2.01-2.08(m,1H),1.82-1.95(m,2H),1.58-1.66(m,1H),1.41-1.52(m,1H),1.16-1.31(m,2H)； ¹³ C NMR(150MHz,DMSO-d ₆ )δ:170.24,170.39,167.49,167.27,165.71,165.62,165.28,163.44,155.16,155.60,152.80,149.56,148.14,136.97,133.60,133.04,132.20,1321.78,128.44,126.58,124.06,121.84,120.62,118.30,117.99,116.65,115.92,110.02,66.65,60.21,56.53,49.24,46.96,43.59,41.10,37.94,36.08,32.40,31.61,31.42,22.47,21.22,14.55；HRMS(ESI,positive)m/z calcd for C ₄₁ H ₄₀ N ₉ O ₉ [M+H] ⁺ :802.2944；found 802.2959.

Example 21

Synthesis of 4- (((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl [5,4-b ] [1,4] diazepin-2-yl) amino) -N- (1- (6- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindol-4-yl) hexa-5-ynyl) piperidin-4-yl) -3-methoxybenzamide (P-20)

Reagents and conditions: a) DIPEA, DMF,90 ℃,5h, yield 69%; b) EDCI, HOBT, DIPEA, DCM,2h, yield 85%.

Step a: synthesis of Compound 30

Synthetic methods are described in the literature (Wang, B., et al, eurJMed Chem,2021, 219:113425.).

Step b: synthesis of Compound P-20

8g (83 mg,0.17 mmol) of compound and 30 (67 mg,0.21 mmol) were dissolved in dry DCM (10 mL), EDCI (40 mg,0.21 mmol), HOBT (28 mg,0.21 mmol) and DIPEA (27 mg,0.34 mmol) were added sequentially and reacted at room temperature for 2h. After TLC monitoring the end of the reaction, the reaction was distilled under reduced pressure and purified by silica gel column chromatography (DCM/meoh=100:1) to give compound P-20 (yellow solid, 50mg, yield 37%). ¹ H NMR(600MHz,DMSO-d ₆ )δ:10.99(d,J＝4.5Hz,1H),8.45(s,1H),8.33(d,J＝8.5Hz,1H),8.11-8.19(m,2H),7.67-7.72(m,2H),7.63(d,J＝7.6Hz,1H),7.47-7.59(m,6H),7.26(d,J＝8.5Hz,1H),7.18(t,J＝7.8Hz,1H),5.16(dd,J＝13.2,5.3Hz,1H),4.47(d,J＝17.94Hz,1H),4.37(d,J＝13.8Hz,1H),4.32(d,J＝17.5Hz,1H),4.01-4.07(m,1H),3.93(s,3H),3.91(s,1H),3.41(s,3H),3.37(s,3H),3.09-3.16(m,1H),2.88-2.97(m,1H),2.64-2.71(m,1H),2.56-2.62(m,1H),2.40(t,J＝7.0Hz,1H),1.97-2.04(m,1H),1.78-1.89(m,1H),1.67-1.73(m,2H),1.58-1.64(m,2H),1.44-1.50(m,1H),1.34-1.41(m,1H),1.21-1.29(m,1H)； ¹³ C NMR(150MHz,DMSO-d ₆ )δ:173.31,171.43,170.70,168.12,167.46,165.49,163.43,155.59,152.80,149.55,148.13,144.31,134.37,133.02,132.43,132.18,131.73,131.16,129.04,128.44,126.57,124.04,123.06,121.81,120.55,119.28,118.30,118.00,109.96,96.67,56.50,52.03,47.42,47.07,44.52,37.92,36.05,32.69,32.14,31.78,31.69,28.09,24.55,22.76,19.04.

Example 22

Synthesis of 4- (((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl [5,4-b ] [1,4] diazepin-2-yl) amino) -N- (1- (7- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindol-4-yl) hept-6-ynyl) piperidin-4-yl) -3-methoxybenzamide (P-21)

Step a: synthesis of Compound 30-1

The compound 29 was replaced by 6-heptynoic acid and the other synthetic method was referred to in example 21, step a. Light blue solid 325mg, yield 55%. ¹ H NMR(600MHz,DMSO-d ₆ )δ:12.04(s,1H),10.98(s,1H),7.71(d,J＝7.3Hz,1H),7.63(d,J＝7.5Hz,1H),7.52(t,J＝7.5Hz,1H),7.37-7.43(m,2H),5.14(dd,J＝13.2,4.5Hz,1H),4.38(dd,J＝85.2,17.5Hz,2H),2.87-2.96(m,1H),2.57-2.64(m,1H),2.25-2.34(m,1H),1.94-2.06(m,1H),1.52-1.73(m,4H).

Step b: synthesis of Compound P-21

Synthetic method reference is made to example 21, step b. Yellow solid 90mg, yield 35%. ¹ H NMR(600MHz,DMSO-d ₆ )δ:10.99(d,J＝3.9Hz,1H),8.45(s,1H),8.33(d,J＝8.1Hz,1H),8.03-8.23(m,2H),7.70(t,J＝8.4Hz,2H),7.63(d,J＝7.6Hz,1H),7.44-7.58(m,4H),7.25(d,J＝8.4Hz,1H),7.18(t,J＝7.3Hz,1H),5.16(dd,J＝12.8,4.8Hz,1H),4.27-4.42(m,2H),4.01-4.06(m,1H),3.93(s,3H),3.90(s,1H),3.41(s,3H),3.36(s,3H),3.12(t,J＝12.9Hz,1H),2.85-2.97(m,1H),2.67(t,J＝11.8Hz,1H),2.59(d,J＝17.0Hz,1H),2.39(t,J＝7.1Hz,1H),1.99-2.04(m,1H),1.76-1.90(m,2H),1.55-1.75(m,4H),1.32-1.19(m,3H)； ¹³ C NMR(150MHz,DMSO-d ₆ )δ:173.30,171.42,168.12,167.46,165.50,163.43,155.58,152.78,149.54,148.13,144.30,134.37,133.02,132.43,132.18,131.73,129.04,128.44,126.57,124.04,123.05,121.81,120.55,119.28,118.29,118.00,109.97,96.67,77.07,55.50,52.04,47.42,47.07,44.52,40.73,37.92,36.05,32.70,32.15,31.78,31.68,28.12,124.55,22.77,19.05；HRMS(ESI,positive)m/z calcd for C ₄₆ H ₄₈ N ₉ O ₇ [M+H] ⁺ :838.3677；found 838.3657.

Example 23

Synthesis of 4- (((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl [5,4-b ] [1,4] diazepin-2-yl) amino) -N- (1- (8- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindol-4-yl) oct-7-ynyl) piperidin-4-yl) -3-methoxybenzamide (P-22)

Step a: synthesis of Compound 30-2

The compound 29 was replaced by 7-octynoic acid and other synthetic methods were referred to in example 21, step a. 371mg of pale blue solid was obtained in 51% yield. ¹ H NMR(600MHz,DMSO-d ₆ )δ12.00(s,1H),10.97(s,1H),7.75-7.67(m,1H),7.65-7.57(m,1H),7.51(t,J＝7.6Hz,1H),5.14(dd,J＝13.3,5.0Hz,1H),4.45(d,J＝17.7Hz,1H),4.32(d,J＝17.7Hz,1H),2.98-2.89(m,1H),2.62-2.56(m,1H),2.54-2.48(m,3H),2.25(t,J＝7.2Hz,2H),2.14-1.93(m,1H),1.77-1.26(m,6H)； ¹³ C NMR(150MHz,DMSO-d ₆ )δ174.87,173.34,171.46,168.16,144.24,134.54,132.45,129.07,123.07,119.34,96.74,76.92,52.15,47.48,34.06,31.69,28.31,28.24,24.51,22.84,19.16；HRMS(ESI,positive)m/z calcd for C ₂₁ H ₂₃ N ₂ O ₅ ⁺ 383.1597,found[M+H] ⁺ 383.2847

Step b: synthesis of Compound P-22

Synthetic method reference is made to example 21, step b. Yellow solid 80mg, yield 40%. ¹ H NMR(600MHz,DMSO-d ₆ )δ:11.01(s,1H),8.45(s,1H),8.33(d,J＝8.4Hz,1H),8.01-8.21(m,2H),7.66-7.73(m,2H),7.64(d,J＝7.7Hz,1H),7.47-7.58(m,4H),7.25(d,J＝8.4Hz,1H),7.17(t,J＝3.7Hz,1H),5.15(dd,J＝13.6,5.1Hz,1H),4.39(dd,J＝84.3,18.0Hz,2H),4.38(d,J＝12.5Hz,1H),4.00-4.12(m,1H),3.93(s,3H),3.90(d,J＝14.3Hz,1H),3.40(s,3H),3.35(s,3H),3.11(t,J＝12.8Hz,1H),2.87-2.96(m,1H),2.57-2.70(m,2H),2.44-2.50(m,2H),2.35(t,J＝7.3Hz,1H),1.97-2.05(m,1H),1.83(dd,J＝34.5,10.6Hz,2H),1.55-1.62(m,3H),1.43-1.50(m,3H),1.35-1.40(m,1H),1.22-1.27(m,1H)； ¹³ C NMR(150MHz,DMSO-d ₆ )δ:173.27,171.41,170.82,168.11,167.46,165.52,163.42,155.58,152.77,149.54,148.11,144.20,134.50,133.01,132.44,132.18,131.74,129.02,128.44,126.57,124.03,123.02,121.82,120.56,119.32,118.28,117.97,109.97,96.78,76.89,56.51,52.13,47.45,47.09,44.55,40.73,37.91,36.05,32.71,31.78,31.67,28.59,28.37,24.91,22.80,19.17；HRMS(ESI,positive)m/z calcd for C ₄₇ H ₄₈ N ₉ O ₇ [M-H] ^- :850.3682；found850.3685.

Example 24

Synthesis of 4- (((5, 11-dimethyl-6-oxo-6, 11-dihydro-5H-benzo [ e ] pyrimidinyl [5,4-b ] [1,4] diazepin-2-yl) amino) -N- (1- (10- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindol-4-yl) dec-9-ynyl) piperidin-4-yl) -3-methoxybenzamide (P-23)

Step a: synthesis of Compound 30-3

Alternative to replacing compound 29 with dec-9-ynoic acid, other synthetic methods refer to example 21, step a. Light blue solid 331mg, yield 59%. ¹ H NMR(600MHz,DMSO-d ₆ )δ:11.96(s,1H),10.99(s,1H),7.70(d,J＝7.6Hz,1H),7.63(d,J＝7.3Hz,1H),7.51(t,J＝7.6Hz,1H),7.29-7.41(m,1H),5.14(dd,J＝13.4,5.0Hz,1H),4.37(dd,J＝82.3,17.6Hz,1H),2.87-2.96(m,1H),2.56-2.63(m,1H),2.40-2.49(m,3H),2.19(t,J＝7.5Hz,2H),1.93-2.06(m,1H),1.53-1.60(m,2H),1.46-1.53(m,2H),1.38-1.56(m,2H),1.24-1.35(m,4H).

Step b: synthesis of Compound P-23

Yellow solid 60mg, yield 38%. ¹ H NMR(600MHz,DMSO-d ₆ )δ:11.02(s,1H),8.46(s,1H),8.34(d,J＝8.4Hz,1H),8.10-8.22(m,2H),7.71(t,J＝7.7Hz,1H),7.64(d,J＝8.4Hz,1H),7.47-7.60(m,4H),7.26(d,J＝8.4Hz,1H),7.19(t,J＝7.7Hz,1H),5.16(dd,J＝5.1,12.5Hz,1H),4.39(dd,J＝80.3,17.4Hz,2H),4.39(d,J＝12.2Hz,1H),4.00-4.10(m,1H),3.94(s,3H),3.89(d,J＝12.5Hz,1H),3.42(s,3H),3.36(s,3H),3.11(t,J＝13.2Hz,1H),2.88-2.98(m,1H),2.67(t,J＝12.5Hz,1H),2.61(d,J＝17.3Hz,1H),2.41-2.49(m,2H),2.32(t,J＝7.6Hz,2H),2.00-2.07(m,1H),1.85(dd,J＝11.8,35.3Hz,1H),1.55-1.63(m,2H),1.49-1.55(m,2H),1.42-1.46(m,2H),1.22-1.36(m,6H),0.81-0.90(m,1H)； ¹³ C NMR(150MHz,DMSO-d ₆ )δ:173.28,171.42,170.89,168.10,167.45,165.49,163.41,155.57,152.76,149.53,148.09,144.16,134.47,133.01,132.41,132.18,131.73,129.03,128.41,126.56,124.02,123.02,121.81,120.56,119.30,118.27,117.94,109.94,96.83,76.83,56.49,52.10,47.41,47.09,44.56,40.71,37.91,36.04,32.75,31.78,31.65,29.20,28.81,28.66,28.47,25.35,22.83,19.19；HRMS(ESI,positive)m/z calcd for C ₄₉ H ₅₂ N ₉ O ₇ [M-H] ^- :878.3995；found 878.3932.

Example 25

In vitro anti-inflammatory Activity test of Compounds of the invention

Step a: ICR mouse abdominal cavity macrophage extraction and culture

More peritoneal macrophages were obtained by daily injection of 5% broth (sodium thioglycolate) (2 mL) into each mouse 3 days prior to the experiment. Cell extraction was performed as follows:

1) Mice were sacrificed, soaked in 75% ethanol for 2-3min, and transferred to an ultra clean bench. Supine fixing on dissecting plate, wiping abdominal skin with ethanol, cutting abdominal skin with surgical scissors, exposing abdominal muscle layer (care should not destroy abdominal muscle layer), and sterilizing abdominal muscle layer with ethanol;

2) RPMI1640 medium (5 mL) containing 1% diabody was withdrawn with a 5mL syringe and injected into the abdominal cavity, the mouse body was gently shaken back and forth and left and right, and then the cell suspension was withdrawn with a syringe and transferred to a centrifuge tube. Repeating the above steps for three times;

3) The collected cell suspension was mixed at 1X 10 ³ Centrifuging at rpm for 5min, discarding supernatant, blowing cells with RPMI1640 medium (10 mL) containing double antibody, centrifuging at 1×103rpm for 5min;

4) Blowing uniformly with RPMI1640 medium (5 mL) containing 10% fetal bovine serum and 1% double antibody, taking cell suspension (10 mu L) and diluting 10 times to 100 mu L, and placing on a cell counting plate for counting;

5) The cell number required by the experiment is calculated, the cell suspension is directly transferred into a 96-well plate or a 6-well plate after being diluted, and the temperature is 37 ℃ and CO ₂ Experiments were performed after incubation for 12h in a 5% cell incubator.

Step b: enzyme-linked immunosorbent assay (ELISA) for detecting inflammatory factor content

The addition concentration of each well was 8X 10 in a 6-well plate ⁴ 2mL of cell suspension at 37℃and CO ₂ After incubation for 12h in a 5% cell incubator, the medium was aspirated and 2mL of the compound was added. After further incubation for 1h, 1. Mu.L of 0.5mg/mL LPS solution was added and incubation was continued for 72h. The ELISA method for detecting the inflammatory factors TNF-alpha and IL-6 can be carried out by referring to the method on the ELISA kit instruction, and the main steps are as follows.

1) Preparing a standard substance: diluting the standard substance to 8 concentrations according to the concentration 2 times of the maximum concentration of 2000pg/mL by using the standard substance diluent;

2) Adding 100 mu L of prepared standard substance and sample to be tested with each concentration into each hole, and incubating for 1h at 37 ℃;

3) Absorbing liquid in the holes, adding 100 mu L of detection antibody into each hole except the blank, and incubating for 1h at 37 ℃;

4) Sucking out the liquid in the holes, adding a washing liquid (the washing liquid is diluted by double distilled water for 25 times and used, 200 mu L) into each hole, standing for 2min, sucking out, repeating for 3 times, and then airing;

5) Except for blank, 100 mu L of enzyme-labeled reagent is added into each hole, and the mixture is incubated for 60min at 37 ℃;

6) Sucking out the liquid in the holes, adding 200 mu L of washing liquid into each hole, standing for 2min, sucking out, repeating for 3 times, and airing;

7) Adding 90 mu L of each of the color developing agents A and B into each hole, uniformly mixing, placing at 37 ℃ for light-shielding incubation for 15min, and adding 50 mu L of stop solution to stop the reaction;

8) Immediately, the OD value of each well was measured by using a microplate reader at a wavelength of 450 nm. The concentration of inflammatory factors measured was calculated using elidacalc software.

With LRRK2-IN-1 as a positive control, the change of the release amount of inflammatory factors IL-6 and TNF-alpha is detected by ELISA kit after the LPS-induced mouse primary peritoneal macrophages are reacted with 10 mu M of compounds P-1 to P-6 and P-9 for 24 hours.

The results of the experiment are shown in FIG. 1, and FIG. 1 is a schematic diagram showing the results of in vitro anti-inflammatory activity test of the compound of the present invention. The upper panel in FIG. 1 shows the variation of the amount of inflammatory factor IL-6 released, and the lower panel in FIG. 1 shows the variation of the amount of inflammatory factor TNF- α released. As can be seen from the figure, IN addition to compound P-9, the other compounds have significantly reduced anti-inflammatory activity IN vitro compared to the positive drug LRRK2-IN-1, and the inhibition of TNF- α by the compounds is better than that of IL-6. The compound P-9 is a DCLK1 protein degradation targeting chimeric taking a carbon-oxygen hybrid chain as a linker, and the reason for better activity of the molecular carbon-oxygen hybrid chain is probably that the carbon-oxygen hybrid chain has better fat solubility.

Example 26

Protein degradation experiment (Westernblot) of the compound of the invention on DCLK1

Step a: protein extraction

1) Collecting cells: discarding culture medium in 6-well plate or culture dish, washing cells twice with pre-cooled PBS, adding 100 μl of cell lysate (containing 1% protease inhibitor and 1% phosphatase inhibitor), standing at 4deg.C for 30min, scraping cells with cell scraper, collecting cell suspension into 1.5mL centrifuge tube, swirling for 1 time every 5min, and repeating for 2 times;

2) And (3) centrifuging: placing the cell suspension in a precooled centrifuge at 4deg.C, 1.2X10 ⁵ Centrifuging at rpm for 15min;

3) Protein denaturation: the supernatant after centrifugation was aspirated, added with a quarter volume of loadingbuffer, vortexed 1min, and denatured in a 95℃water bath for 20min.

Step b: protein concentration measurement (BCA method)

4) Preparing BCA working solution: preparing a solution A and a solution B according to a ratio of 50:1;

5) Adding 18 mu L of PBS, 2 mu L of protein supernatant and 200 mu L of BCA working solution into each well of a 96-well plate, placing into an enzyme-labeled instrument, vibrating the plate for 5min at 37 ℃ and incubating for 25min;

6) OD value at 562nm wavelength was measured, and the measured result was placed in a standard curve to measure protein concentration.

Step c: electrophoresis

1) Preparing glue;

2) Loading: and placing the gel into an electrophoresis tank, adding electrophoresis liquid, and pulling out the tooth comb. Adding a protein sample into the loading hole according to the calculated volume;

3) And running the gel at constant pressure, and electrifying for 80min at 100V.

Step d: transfer film

4) Cutting the glue into required segments, placing the segments on filter paper, cutting a PVDF film with proper size, placing the PVDF film in methanol for activating for 5min, and covering the PVDF film on the glue;

5) The film transfer clip is placed in a film transfer groove, and the constant current 270A is electrified for 100min.

Step e: antibody incubation

1) Closing: after the membrane transfer is completed, the PVDF membrane is washed once by TBST, put into 5% BSA and is shaken for 2 hours at room temperature;

2) Incubation resistance: slightly washing the membrane with TBST once, placing the membrane in the prepared primary antibody, and incubating the membrane at room temperature for 2 hours (or overnight at 4 ℃);

3) Secondary antibody incubation: after the incubation of the primary antibody, the membrane was washed 3 times (5 min each time) with TBST, then placed in a secondary antibody solution, and placed in a dark place at room temperature for 2h.

Step f: exposure to light

The membranes were washed three times (5 min each) with TBST in the dark, scanned with an LI-COR Odyssey membrane scanner, and then analyzed for protein bands with Image J software.

In the invention, PROTAC molecules P-1 to P-10 take VHL as an E3 ligase ligand, P-11 to P-19 take pomalidomide as an E3 ligase ligand, and P-20 to P-23 lenalidomide as an E3 ligase ligand.

The experimental results are shown in FIG. 2, and FIG. 2 is a schematic diagram of the degradation activity test results of DCLK 1-PROTAC. Wherein A is a schematic diagram of a degradation activity screening result of PROTAC molecules taking VHL as an E3 ligase ligand at 10 mu M; b is a schematic diagram of the experimental result of the time-dependent degradation of the compound 9 at 10 mu M; c is a schematic diagram of a degradation activity screening result of a PROTAC molecule taking pomalidomide as an E3 ligase ligand at 10 mu M; d is a schematic diagram of the experimental results of compound 14 dependent degradation. In DCLK1 protein degradation targeting chimeric (compounds P-1 to P-6, P-9) using VHL as E3 ligase ligand, the carbon-oxygen hybrid chain and the compounds P-1 and P-9 containing three carbon-atom fatty chain have certain degradation activity, and the degradation activity gradually decreases with the increase of chain length in the 4-8 carbon-atom fatty-chain compounds P-2 to P-6 (A in figure); the compound P-9 with the best anti-inflammatory activity is subjected to a time-dependent experiment, the degradation rate of DCLK1 is gradually increased with time from 0.5h to 3h after administration, and the degradation effect is not changed after 3h (B in figure 2), which shows that the PROTAC molecule can reach the maximum degradation at 3h, so that the subsequent drug action time is 3h; for DCLK1 protein degradation targeting chimeric body with pomalidomide as E3 ligase ligand, 10 mu M single concentration degradation activity screening is carried out, and the compounds P-11, P-12 and P-14 have better degradation activity (C in figure 2), which correspond to DCLK1 protein degradation targeting chimeric body without linker, aliphatic linker containing 2 carbon atoms and aliphatic linker containing 5 carbon atoms respectively. By comparing the degradation activities of two different types of E3 ligase ligands, the PROTAC molecule with the E3 ligase ligand of CRBN has better degradation activity. Concentration-dependent degradation experiments are preferably performed on the compound P-14 with better degradation activity, and it is found that in three concentrations of 0.1-10 mu M, the degradation efficiency is reduced with increasing concentration, and the analysis reason is probably that the PROTAC molecule generates a HOOK effect at high concentration, and the specific degradation efficiency is yet to be further verified (D in FIG. 2).

The foregoing description is only illustrative of the preferred embodiment of the present invention, and is not to be construed as limiting the invention, but is to be construed as limiting the invention to any and all simple modifications, equivalent variations and adaptations of the embodiments described above, which are within the scope of the invention, may be made by those skilled in the art without departing from the scope of the invention.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, which has the structural formula shown below:

wherein X is selected from one of the following structures: a key(s),

m is selected from positive integers of 1 to 10;

n is a positive integer from 1 to 10;

r is selected from positive integers of 1 to 10;

p is selected from positive integers of 1 to 10;

q is selected from positive integers of 1 to 10;

s is selected from positive integers of 1 to 10;

r is selected from one of the following structures:

R ₁ selected from hydrogen, methyl.

2. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from one of the following structures:

3. use of a compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, for the preparation of a DCLK1 protein degradation targeting chimera.

4. Use of a compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, in the manufacture of an anti-inflammatory medicament.