CN116375713A - A DCLK1 protein degradation targeting chimera and its application - Google Patents

Abstract

The invention discloses a compound or a pharmaceutically acceptable salt thereof, the general structural formula is as follows:

The compounds of the present invention exhibit good DCLK1 protein degradation activity, and the test results of DCLK1 protein degradation activity show that compounds P‑11, P‑12 and P‑14 with pomalidomide as E3 ligase ligands have better degradation activity, and is better than the compound with VHL as E3 ligase ligand.

Description

A DCLK1 protein degradation targeting chimera and its application

technical field

The invention belongs to the technical field of medicine, and in particular relates to a DCLK1 protein degradation targeting chimera and its application.

Background technique

As can be seen from the history of drug development, the focus of drug development has always been to exert therapeutic effects by blocking receptors or modulating protein binding sites. However, this mode of drug action usually requires high doses of drugs to maintain high target occupancy, while possibly causing side effects due to off-targets. Kinase inhibitors also have this limitation in the mode of action. To solve this problem, proteolysis-targeting chimeras (PROTACs) technology emerged as the times require, using the cell's own degradation mechanism to degrade target proteins, which can Solve the defect of low selectivity of kinase inhibitors.

PROTAC molecules generally include two key domains: a specific domain that binds to a target protein (protein of interest, POI) and a domain that binds to E3 ubiquitination ligase, which are connected by a specific linker to induce the binding of POI. Ubiquitinated and degraded by the proteasomal pathway. Importantly, PROTAC molecules can still be recycled after POI ubiquitination, and then participate in new reactions. From a pharmacodynamic point of view, this catalytic degradation method is more meaningful than increasing the occupancy of the target protein. In addition, depending on the stability of PROTAC molecules in the cell and the rate of POI resynthesis, the duration of action of PROTAC molecules varies. As long as there is a small-molecule ligand with an appropriate affinity for POI and can place the target protein and E3 ligase in a suitable space to form a target protein-PROTAC-E3 ligase ternary complex, PROTAC molecules can theoretically be targeted for degradation. any type of protein.

At present, common E3 ligase ligands include CRBN, VHL, MDM2, clAP1, KEAP1 and RNF114, among which CRBN and VHL are the most commonly used. The distribution of different E3 ligases in tissues, the affinity and selectivity to target proteins all show diversity, which has an important impact on the safety, druggability and efficacy of PROTACs. Therefore, the synthesis of PROTACs needs to be guided by rational medicinal chemistry and pharmacology means.

After Gleevec was launched as the first protein kinase inhibitor in 2001, protein kinase inhibitors have received extensive attention and research. Currently, 48 protein kinase inhibitors have been launched. However, the selectivity of protein kinase inhibitors is poor, mainly because one-third of protein kinases may be phosphorylated, and the binding domains of most kinases are very similar, so it is very difficult to achieve selective inhibition. In addition, the "off-target effect" of kinase inhibitors often causes toxic side effects in clinical practice, so improving the selectivity of kinase inhibitors and reducing their toxicity is of great significance to the optimization of kinase drugs.

Since PROTAC needs to place the target protein and E3 ligase in a suitable space, a stable target protein-PROTAC-E3 ligase ternary complex is formed, and there are suitable ubiquitination sites (such as lysine) on the surface of the target protein. ), so even if the selectivity of the kinase inhibitor is poor, the design of PROTAC can also improve the selectivity, so PROTAC technology is one of the important strategies to improve selectivity.

Double cortin-like kinase 1 (DCLK1) is a microtubule-associated serine/threonine kinase that structurally belongs to the calcium/calmodulin-dependent protein kinase family (calcium/calmodulin- dependent protein kinases, CaMK), mainly expressed in the cytoplasm. DCLK1 consists of 729 amino acid molecules, which are divided into three parts: DCX domain, PEST sequence and kinase domain, and its N-terminal region has two microtubule-binding domains (DCX1 and DCX2, respectively) to drive microtubule-related functions ; The C-terminal region contains a serine/threonine kinase domain that is highly similar to the calcium/calmodulin-dependent protein 1 (CaMK 1) kinase domain.

DCLK1 has many biological functions. At present, the research work using it as a drug target is mainly focused on anti-tumor, but no significant progress has been made. Recent studies have confirmed that DCLK1 is closely related to inflammation. Therefore, it is speculated that DCLK1 is very likely to be a potential anti-tumor Inflammatory drug targets.

Contents of the invention

The first object of the present invention is to provide a compound capable of preparing DCLK1 protein degradation targeting chimera.

The second object of the present invention is to provide an application of the compound in the preparation of DCLK1 protein degradation targeting chimeras.

In order to achieve the above object, the technical scheme adopted in the present invention is as follows:

The first aspect of the present invention provides a compound or a pharmaceutically acceptable salt thereof, the general structural formula is as follows:

Wherein, X is selected from one of the following structures: bond,

m is selected from positive integers from 1 to 10 (such as 1, 2, 3, 4, 5, 6, 7);

n is selected from positive integers from 1 to 10 (such as 1, 2, 3, 4, 5);

r is a positive integer selected from 1 to 10 (such as 1, 2, 3, 4, 5, 6, 7);

p is selected from positive integers from 1 to 10 (such as 1, 2, 3, 4);

q is a positive integer selected from 1 to 10 (such as 1, 2, 3);

s is selected from positive integers from 1 to 10 (such as 1, 2, 3, 4, 5, 6, 7);

R is selected from one of the following structures:

R ₁ is selected from hydrogen, methyl.

The pharmaceutically acceptable salt is an acid addition salt formed by the above compound and 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, pyruvic, acetic, maleic, succinic, fumaric, salicylic, phenylacetic, or mandelic acids.

The compound is selected from one of the following structures:

The second aspect of the present invention provides an application of the compound or a pharmaceutically acceptable salt thereof in the preparation of a DCLK1 protein degradation targeting chimera molecule, the DCLK1 protein targeting degradation chimera molecule consists of three parts, and the E3 ligase ligand ligands (VHL ligands, pomalidomide ligands and lenalidomide ligands), target protein ligands and linker linkers. E3 ligase ligands are responsible for the specific recruitment of E3 ligases. Target protein ligands are used to target and capture proteins of interest. Linker is used to bind these two ligands to form a stable ternary complex. Therefore, PROTAC molecules can recruit E3 ligase to the vicinity of the target protein, and label the target protein with "ubiquitin", and the protein labeled with ubiquitin on the cell will be sent to the proteasome for degradation. In this way, PROTAC molecules can specifically promote the degradation of disease-causing proteins, thereby achieving the purpose of treating diseases.

The third aspect of the present invention provides a use of the compound or a pharmaceutically acceptable salt thereof in the preparation of anti-inflammatory drugs.

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

Owing to adopting above-mentioned technical scheme, the present invention has following advantage and beneficial effect:

The compound of the present invention shows good DCLK1 protein degrading activity, and the DCLK1 protein degrading activity test result shows, under the single concentration of 10 μ M, in the DCLK1 protein degradation targeting chimera (compound P-1) that uses VHL as E3 ligase ligand ~P-6, P-9), the compounds P-1 and P-9 of the carbon-oxa chain and the aliphatic chain containing three carbon atoms have certain degradation activity, and the aliphatic chain compound P of 4-8 carbon atoms From -2 to P-6, as the chain length increased, the degradation activity gradually weakened; the compound P-9 with the best anti-inflammatory activity was tested in a time-dependent manner, and the degradation rate of DCLK1 decreased with time from 0.5h to 3h after administration. The growth gradually increases, and the degradation effect does not change after 3 hours, indicating that the PROTAC molecule can reach the maximum degradation at 3 hours, so the subsequent drug action time is 3 hours; for some DCLK1 protein degradation targets that use pomalidomide as the E3 ligase ligand A 10μM single-concentration degradation activity screening was performed on the chimera, and it was found that compounds P-11, P-12 and P-14 had good degradation activities, and their corresponding compounds were no linker, aliphatic linker with 2 carbon atoms, and aliphatic linker with 5 carbon atoms. Carbon-atom adipose linker targeting chimeras for DCLK1 protein degradation. By comparing the degradation activities of two different types of E3 ligase ligands, it was found that the PROTAC molecule whose E3 ligase ligand is CRBN has better degradation activity. The compound P-14 with better degradation activity was selected for concentration-dependent degradation experiments. It was found that in the three concentrations of 0.1-10 μM, as the concentration increased, the degradation efficiency decreased instead. The reason for the analysis may be that PROTAC molecules produced HOOK effect, the specific degradation efficiency needs to be further verified.

As the first reported DCLK1 protein degradation targeting chimera, the compound of the present invention has further development and research value.

Description of drawings

Fig. 1 is a schematic diagram of the in vitro anti-inflammatory activity test results of the compounds of the present invention.

Fig. 2 is a schematic diagram of the degradation activity test results of DCLK1-PROTAC.

Detailed ways

In order to illustrate the present invention more clearly, the present invention will be further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not limit the protection scope of the present invention.

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 LRRK2-IN-1, a DCLK1 inhibitor with excellent activity, is shown below:

Example 1

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

Reagents and conditions: a) SOCl ₂ , MeOH, 60°C, 2h, yield 92%; b) DIPEA, 1,4-dioxane, 50°C, 5h, yield 82%; c) Fe, AcOH, 60°C, overnight, yield 93%; d) MeI, NaH, DMF, 0℃, 5h, yield 80%; e) DCM, EDCI, HOBT, DIPEA, rt, 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)

Put compound 1 (2-(methylamino)benzoic acid) (5.00g, 33.08mmol) in a 250mL eggplant-shaped bottle, add methanol (100mL) to dissolve, slowly add thionyl chloride (19.70g, 165.38 mmol), react at 60°C for 2h. TLC detects that the reaction is complete, the reaction solution is poured into ice water (500mL), stirred for 5min, a large amount of white solid is precipitated, filtered by suction, dried, without further purification, compound 2 (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 (1.00g, 6.06mmol) and compound 3 (2,4-dichloro-5-nitropyrimidine, 1.40g, 7.26mmol) were dissolved in 1,4-dioxane (50mL), stirred DIPEA (1.60 g, 12.12 mmol) was slowly added dropwise, and stirred at 50° C. for more than 5 h. After the reaction was complete, 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=15:1-10:1) to obtain compound 4 (yellow solid, 1.6 g, 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: 2-Chloro-11-methyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (5) Synthesis

Compound 4 (1.00g, 3.10mmol) was dissolved in 20mL of acetic acid, iron powder (0.87g, 15.49mmol) and DIPEA (0.80g, 6.20mmol) were added successively under stirring, and stirred overnight at 60°C. After the TLC detection reaction is complete, filter the remaining iron powder, add the filtrate to 500mL ice water, a large amount of white solids are precipitated, filter with suction, dry the filter cake at 50°C, and proceed directly to the next step without purification to obtain compound 5 (white Solid, 0.7 g, 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: 2-Chloro-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one Synthesis of (6)

Compound 5 (1.00g, 3.84mmol) was dissolved in dry DMF (20mL), and NaH (120.00mg, 4.60mmol) was added in batches at -10°C. After reacting for 0.5h, iodomethane (660.00mg , 4.60mmol), and then the reaction was transferred to room temperature and continued to stir for 4h. After the raw materials were completely reacted, the reaction solution was poured into ice water (400mL), stirred for 5min, a large amount of white solids were precipitated, filtered with suction, and the filter cake was dried at 50°C to constant weight without further purification to obtain compound 6 (white Solid, 0.8 g, 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=8.3 Hz, 1H), 7.22(t, J=7.7Hz, 1H), 3.42(s, 3H), 3.34(s, 3H).

Step e: Synthesis of tert-butyl 4-(4-amino-3-methoxybenzamido)piperidine-1-carboxylate (9)

Weigh compound 7 (2.00g, 12.00mmol), compound 8 (2.88g, 14.36mmol), EDCI (2.75g, 14.36mmol), HOBT (1.94g, 14.36mmol) in a 100mL round bottom flask, add dry dichloro Methane (40mL) was fully dissolved, and DIPEA (5.93mL, 35.89mmol) was slowly added dropwise while stirring, and the reaction was stirred overnight at room temperature, monitored by TLC (developer conditions, DCM:MeOH=100:10), and the reaction was complete. Pour the reaction solution into a 250mL separating funnel, add 100mL of water, add 30mL of dichloromethane, shake fully, let stand until the liquid is completely separated, take the lower layer of liquid into a 250mL round bottom flask, and repeat the extraction 4-5 times, The extracts were combined and spin-dried, then mixed sample column chromatography, eluent: DCM:MeOH=100:1, the polarity gradually increased to DCM:MeOH=100:5, and the eluent was concentrated under reduced pressure to obtain compound 9 ( Pale yellow solid, 3.52g, yield 84.3%).

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

Weigh compound 6 (250mg, 0.90mmol), compound 9 (344mg, 0.98mmol), X-Phos (39mg, 0.08mmol), Pd ₂ (dba) ₃ (74mg, 0.08mmol) and potassium carbonate (375mg, 2.68mmol ) into a 100mL round bottom flask, add dry 1,4-Dioxane (30mL) to fully dissolve, vacuum _N2 protection, reflux reaction at 106°C for 4.5h, try to avoid contact with air and water during the whole reaction process, use spherical condensation The tube should be fully dried in an oven in advance, and monitored by TLC (developer conditions, DCM:MeOH=100:10), the reaction is complete. The reaction solution was suction-filtered with a triangular funnel to remove inorganic salt solids as much as possible, and the filter residue was washed with a little methanol for 3 times, the filtrate was poured into a 100mL separating funnel, 40mL of water was added, and finally 15mL of ethyl acetate was added, shaken fully, and allowed to stand After the liquid is completely separated, take the upper liquid in a 100mL round bottom flask, repeat the extraction 4-5 times, combine the extracts and spin dry, then mix the sample column chromatography, eluent: PE:EA=50:1, polar Gradually increase to: PE:EA=73:27, the eluate was concentrated under reduced pressure to obtain product 10 (yellow solid, 195 mg, yield 37.1%).

Weigh compound 10 (195mg, 0.33mmol) in a 50mL round bottom flask, add dry dichloromethane (8mL) to fully dissolve, add trifluoroacetic acid (2mL) while stirring, stir and react at room temperature overnight, TLC plate monitoring ( Developing agent conditions, DCM:MeOH=100:10), the reaction is complete. Spin the reaction solution to dryness, add a little methanol to fully dissolve, then spin dry again, repeat 5-6 times, finally fully dissolve with dichloromethane and directly mix the sample for column chromatography, eluent: DCM:MeOH=100:1, extremely The stability was gradually increased to DCM:MeOH=100:7, and the eluent was concentrated under reduced pressure to obtain 8 g of the product (white solid, 63 mg, yield 39%). 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

(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-oxo Propionamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-(4-methylthiazol-5-yl)phenyl) Synthesis of 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

Compound 8g (0.70g, 1.44mmol) and compound 11 (0.20g, 1.72mmol) were dissolved in 20mL dry DCM, then EDCI (0.30g, 1.72mmol), HOBT (0.20g, 1.72mmol), DIPEA ( 0.40g, 3.59mmol), stirred at room temperature for 2h. After the reaction was completely completed as monitored by TLC, the reaction solution was washed 3 times with 60 mL of saturated sodium chloride solution, the organic layer was concentrated under reduced pressure, and the compound was separated and purified by silica gel column chromatography (hexane/EtOAc=3:1-1:1) 12 (white solid, 698 mg, 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 a mixed solvent (100 mL) of THF:MeOH:H ₂ O = 3:2:1, and LiOH (96 mg, 4.25 mmol) was added in batches, and reacted at room temperature for 5 h, TLC detects that the reaction is complete. The reaction solution was distilled under reduced pressure to remove the organic solvent. After transferring to an ice bath, the pH was adjusted to neutral with 1mol/L HCl. A large amount of white solids were precipitated. They were suction filtered and dried without further purification to obtain the crude compound 13. Product 400mg, yield 87%.

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), and DIPEA (77mg, 0.52mmol), reacted at room temperature for 2h. After the reaction was monitored by TLC, the reaction liquid was washed three times with saturated sodium chloride solution (150mL×3), dried over anhydrous sodium sulfate, and separated and purified by silica gel column chromatography (DCM:MeOH=100:1-100:3) Compound P-1 (white solid, 300 mg, yield 37%) was obtained. 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 ,151.69,150.57,149.73,148.66,144.93,144.78,133.88,133.68,133.00,132.01,130.80,128.41,128.20,127.75,125.21,123.47,121.14,1 18.81, 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

(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-oxa Pentamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((R)-1-(4-(4-(4-methylthiazol-5-yl)phenyl)ethyl base) synthesis of pyrrolidine-2-carboxamide (P-2)

Step a: Synthesis of compound 12-1

Compound 11 was replaced by compound 4-methoxy-4-oxobutanoic acid, and the synthesis method was referred to step a in Example 2. White solid 600mg, yield 86%. 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

For the synthesis method, refer to step b in 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:174.53,173.3,171.83,169.59,167.93,165.06,156.66,152.54,151.78,150.57,149.72,148.77,144.73,133.8 7,133.72,133.02,132.10 . 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

(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]diazepin-2-yl)amino)-3-methoxybenzamido)piperidin-1-yl)-5-oxo Pentamide)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-(4-methylthiazol-5-yl)phenyl)ethyl ) Synthesis of pyrrolidine-2-carboxamide (P-3)

Step a: Synthesis of compound 12-2

Compound 11 was replaced by compound 5-methoxy-5-oxopentanoic acid, and the synthesis method was referred to step a in Example 2. 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

For the synthesis method, refer to step b in Example 2. White solid 106 mg, yield 36%. 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; found1028.4836.

Example 5

(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-oxohexa Amino)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-(4-methylthiazol-5-yl)phenyl)ethyl) Synthesis of Pyrrolidine-2-Carboxamide (P-4)

Step a: Synthesis of compound 12-3

Replace compound 11 with compound 6-methoxy-6-oxohexanoic acid, and refer to step a in Example 2 for the synthesis method. 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

For the synthesis method, refer to step b in 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, 12 7.80, 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

(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]diazepin-2-yl(amino)-3-methoxybenzamido)piperidin-1-yl)-7-oxoheptyl Amino)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-(4-methylthiazol-5-yl)phenyl)ethyl ) Synthesis of pyrrolidine-2-carboxamide (P-5)

Step a: Synthesis of Compound 12-4

Replace compound 11 with compound 7-methoxy-7-oxoheptanoic acid, and refer to step a in Example 2 for the synthesis method. White solid 600mg, yield 86%. 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

For the synthesis method, refer to step b in Example 2. White solid 120mg, yield 33%. 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

(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-oxoctyl Amino)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-(4-methylthiazol-5-yl)phenyl)ethyl) Synthesis of Pyrrolidine-2-Carboxamide (P-6)

Step a: Synthesis of compound 12-5

Replace compound 11 with compound 8-methoxy-8-oxoctanoic acid, and refer to step a in Example 2 for the synthesis method. 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

For the synthesis method, refer to step b in Example 2. White solid 39 mg, yield 28%. 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=3.2 Hz, 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, 14 4.58, 133.84, 133.74, 132.94,132.25,130.91,128.45,127.84,127.80,125.24,123.52,120.66,118.76,117.77,110.70,71.86,71.27,59.98,58.88,58.19,57.39,5 0.19, 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/zcalcd for C ₅₇ H ₇₂ N ₁₁ O ₈ S[M+H] ⁺ : 1070.5281; found 1070.5285.

Example 8

(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)benzene Synthesis of (yl)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%.

Step: Synthesis of Compound P-7

Dissolve compound 8g (500mg, 1.03mmol), compound 15 (165mg, 1.23mmol) in dry DCM (20mL), then add EDCI (287mg, 1.54mmol), HOBT (209mg, 1.54mmol), DIPEA (387mg , 3.08mmol), stirred at room temperature for 0.5h. After the reaction was completed, the reaction liquid was distilled under reduced pressure, and purified by silica gel column chromatography (DCM:MeOH=100:1) to obtain 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), react at room temperature for 1 h. After the reaction was monitored by TLC, the reaction solution 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 obtain 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, 12 6.41, 123.89, 122.13, 119.54, 117.47, 116.51, 109.36, 70.10, 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

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

Replace compound 15 with 3,6-octandioxadioic acid, and refer to Example 8 for other synthesis methods. White solid 90.6 mg, yield 40%. 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

(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-methoxybenzamido)piperidin-1-yl)- 4,14-Dioxo-6,9,12-trioxa-3-azidomercaptoalkyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazole Synthesis of )-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (P-9)

Replace compound 15 with 2,2'-((oxybis(ethane-2,1-diyl))bis(oxy))diacetic acid, and refer to Example 8 for other synthesis methods. White solid 84mg, yield 34%. 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, 11 0.75,72.78,72.50,71.94,71.34,60.22,58.32,57.36, HRMS (ESI, positive) m/z calcd for C ₅₇ H ₇₂ N ₁₁ O ₁₁ S[M+H] ⁺ :1118.5128; found1118.5101.

Example 11

(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-tetraoxo-3-azaheptadecanoyl)-4-hydroxy-N-((S)-1-(4-(4-methyl Synthesis of thiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (P-10)

Replace compound 15 with 3,6,9,12-tetraoxatetradecane-1,14-dicarboxylic acid, and refer to Example 8 for other synthesis methods. 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, _{found _ _ _} ^_ 1162.5424.

Example 12

4-(((5,11-Dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimidinyl[5,4-b][1,4]diazepine- 2-yl)amino)-N-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidine-4- base)-3-methoxybenzamide (P-11) synthesis

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

Step: Synthesis of Compound P-11

Compound 8g (0.5g, 1.03mmol) and pomalidomide (331mg, 1.23mmol) were dissolved in dry DCM (20mL), and EDCI (229mg, 1.23mmol), HOBT (167mg, 1.23mmol) and DIPEA ( 258mg, 2.05mmol), reacted at room temperature for 2h. After the reaction was completed, the reaction solution was washed with saturated sodium chloride solution (20mL×3), dried over anhydrous sodium sulfate, and then separated and purified by silica gel column chromatography (DCM/MeOH=100:1) to obtain compound P -11 (yellow solid, 100 mg, 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 ,170.50,167.56,167.49,166.81,165.51,163.45,155.62,152.80,150.31,149.57,148.14,136.22,134.16,133.04,132.20,131.73,128.50,1 26.59, 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

4-(((5,11-Dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimidinyl[5,4-b][1,4]diazepine- 2-yl)amino)-N-(1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)glycyl) Synthesis of 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 (500mg, 1.03mmol) and Boc-glycine (210mg, 1.23mmol) were dissolved in dry DCM (20mL), and EDCI (287mg, 1.54mmol), HOBT (209mg, 1.54mmol) and DIPEA ( 387mg, 1.54mmol), react at room temperature for 1h. After the reaction was detected by TLC, the reaction solution was washed with saturated sodium chloride (20mL×3), the organic layer was dried over anhydrous sodium sulfate, distilled under reduced pressure, and silica gel column chromatography (hexane:EtOAc=4:1-1:1 ) separation and purification to obtain compound 19 (yellow solid, 527 mg, 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 (500mg, 0.78mmol) was placed in a 25mL reaction flask, and 10mL DCM was added to fully dissolve it, TFA (273.6mg, 2.33mmol) was slowly added dropwise, and reacted at room temperature for 2h. After the reaction was monitored by TLC, the reaction solution was distilled under reduced pressure without further purification to obtain a crude product of compound 20. Then compound 20 and pomalidomide (248.4mg, 0.93mmol) obtained in the previous step were dissolved in dry DCM (20mL), and EDCI (229.2mg, 1.16mmol), HOBT (166.8mg, 1.16mmol) and DIPEA were added successively (309.6mg, 2.33mmol), react at room temperature for 2h. After the reaction was completed, the reaction solution (20mL×3) was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and after vacuum distillation, it was separated and purified by silica gel column chromatography (DCM/MeOH=100:1) to obtain compound P- 12 (yellow solid, 600 mg, 37% yield). 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, 12 4.06, 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

4-(((5,11-Dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimidinyl[5,4-b][1,4]diazepine- 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) synthesis

Step a: Synthesis of compound 19-1

Replace compound 18 with Boc-β-alanine, and refer to Example 13 for other synthesis methods. White solid 286mg, yield 88%. 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

For the synthetic method, refer to 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

4-((5,11-Dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimidine[5,4-b][1,4]diazepine-2- Base)-amino)-N-(1-(5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino ) pentanoyl) piperidin-4-yl) -3-methoxybenzamide (P-14) synthesis

Step a: Synthesis of compound 19-2

Replace compound 18 with Boc-5-aminovaleric acid, and refer to Example 13 for other synthesis methods. White solid 396 mg, yield 90%. 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, 1.6 Hz, 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

For the synthetic method, refer to Step b of Example 13. 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/zcalcd for C ₄₄ H ₄₇ N ₁₀ O ₈ [M+H] ⁺ :843.3573; found 843.3575.

Example 16

4-(((5,11-Dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimidinyl[5,4-b][1,4]diazepine- 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) synthesis

Step a: Synthesis of compound 19-3

Replace compound 18 with 6-((tert-butoxycarbonyl)amino)hexanoic acid, and refer to Example 13 for other synthesis methods. White solid 256mg, yield 86%. 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

For the synthetic method, refer to Step b of Example 13. Yellow solid 90 mg, 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; found3757.

Example 17

4-(((5,11-Dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimidinyl[5,4-b][1,4]diazepine- 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) synthesis

Step a: Synthesis of Compound 19-4

Replace compound 18 with 7-(N-tert-butoxycarbonylamino)heptanoic acid, and refer to Example 13 for other synthesis methods. White solid 240mg, yield 82%. 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

For the synthetic method, refer to Step b of Example 13. Yellow solid 90 mg, yield 36%. 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 ,117.95,117.63,110.84,109.96,109.51,56.51,55.36,49.02,47.10,44.59,42.28,40.74,37.93,36.07,32.73,31.81,31.44,31.41,29.06,2 9.00, 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

4-(((5,11-Dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimidinyl[5,4-b][1,4]diazepine- 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) synthesis

Step a: Synthesis of Compound 19-5

Replace compound 18 with Boc-8-aminooctanoic acid, and refer to Example 13 for other synthesis methods. White solid 251 mg, yield 85%. 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

For the synthetic method, refer to Step b of Example 13. 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

4-(((5,11-Dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimidinyl[5,4-b][1,4]diazepine- 2-yl)amino)-N-(1-(2-(2-(2-(2-((2-(2-2,6-dioxopiperidin-3-yl)-1,3- Dioxoisoindoline-4-yl)amino)ethoxy)ethoxy)ethoxy)acetyl)piperidin-4-yl)-3-methoxybenzamide (P-18) synthesis

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 (1.0g, 2.05mmol) and compound 21 (662mg, 2.46mmol) were dissolved in dry DCM (50mL), then EDCI (458mg, 2.46mmol), HOBT (333mg, 2.46mmol), DIPEA (516mg , 4.10mmol), reacted at room temperature for 2h. After the reaction was monitored by TLC, the reaction liquid was distilled under reduced pressure, and then separated and purified by silica gel column chromatography (DCM/MeOH=100:0.5) to obtain compound 22 (white solid, 963 mg, 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 0.1 g of palladium on carbon was added to react at room temperature under hydrogen overnight. After the reaction was monitored by TLC, the reaction solution was filtered with diatomaceous earth, and the filtrate was distilled under reduced pressure without further purification to obtain 400 mg of a crude product of compound 23. Then compound 23 (300 mg, 0.44 mmol), pomalidomide (138 mg, 0.53 mmol) were dissolved in dry DCM (15 mL), and then EDCI (115 mg, 0.66 mmol), HOBT (83 mg, 0.66 mmol), DIPEA (194mg, 1.50mmol) was reacted at room temperature for 2h. After the reaction was completed, the reaction solution was washed with saturated sodium chloride solution (20mL×3), the organic layer was dried over anhydrous sodium sulfate, distilled under reduced pressure, and separated and purified by silica gel column chromatography (DCM/MeOH=100:1) , to obtain compound P-18 (yellow solid, 508.9 mg, 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=7.8 Hz, 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, 5.6 Hz, 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, 167.48,165.52,163.44,155.59,152.80,149.56,148.12,148.86,136.67,133.04,132.55,132.20,131.74,128.42,126.59,124.05,121.83,12 0.59, 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} ; + H] ⁺ :933.3890; found933.3867.

Example 20

4-(((5,11-Dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimidinyl[5,4-b][1,4]diazepine- 2-yl)amino)-N-(1-(2-((2-(2,6-dioxapiperidin-3-yl)-1,3-dioxoisoindol-4-yloxy ) acetyl) piperidin-4-yl)-3-methoxybenzamide (P-19) synthesis

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%.

Step: Synthesis of Compound P-19

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

Compound 26 (90mg, 0.23mmol) was placed in a 25mL reaction flask, 10mL DCM was added to fully dissolve, TFA (2mL) was slowly added dropwise, and reacted at room temperature for 2h. After the reaction was monitored by TLC, the reaction liquid was distilled under reduced pressure without further purification to obtain a crude product of compound 27. Compound 8g (113mg, 0.23mmol) and compound 27 (92mg, 0.28mmol) were dissolved in dry DCM (10mL), and EDCI (53mg, 0.28mmol), HOBT (38mg, 0.28mmol), DIPEA (60mg, 0.46mmol), reacted at room temperature for 2h. After the reaction was monitored by TLC, the reaction solution was distilled under reduced pressure, and then separated and purified by silica gel column chromatography (DCM/MeOH=100:1) to obtain compound P-19 (yellow solid, 70 mg, 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, 152.80,149.56,148.14,136.97,133.60,133.04,132.20,1321.78,128.44,126.58,124.06,121.84,120.62,118.30,117.99,116.65,115.92,1 10.02, 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

4-(((5,11-Dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimidinyl[5,4-b][1,4]diazepine- 2-yl)amino)-N-(1-(6-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)hexa-5- Synthesis of Alkynyl)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

The synthesis method refers to the literature (Wang, B., et al., EurJMed Chem, 2021, 219: 113425.).

Step b: Synthesis of Compound P-20

Compound 8g (83mg, 0.17mmol) and compound 30 (67mg, 0.21mmol) were dissolved in dry DCM (10mL), and EDCI (40mg, 0.21mmol), HOBT (28mg, 0.21mmol), DIPEA (27mg, 0.34mmol), reacted at room temperature for 2h. After the reaction was monitored by TLC, the reaction solution was distilled under reduced pressure, and then separated and purified by silica gel column chromatography (DCM/MeOH=100:1) to obtain compound P-20 (yellow solid, 50 mg, 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

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

Step a: Synthesis of compound 30-1

Replace compound 29 with 6-heptynoic acid, and refer to Step a of Example 21 for other synthesis methods. 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

For the synthetic method, refer to step b of Example 21. Yellow solid 90 mg, 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:173.30,171.42,168.12,167.46,165.50,163.43,155.58,152.78,149.54,148.13,144.30,1 34.37, 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, 55.5 0,52.04,47.42,47.07,44.52,40.73,37.92,36.05, 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

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

Step a: Synthesis of compound 30-2

Replace compound 29 with 7-octynoic acid, and refer to step a of Example 21 for other synthesis methods. The light blue solid was 371 mg, and the yield was 51%. 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, 34.0 6, 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

For the synthetic method, refer to step b of Example 21. 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 [MH] ^- :850.3682; found850.3685.

Example 24

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

Step a: Synthesis of compound 30-3

Replace compound 29 with decan-9-ynoic acid, and refer to Step a of Example 21 for other synthesis methods. The light blue solid was 331 mg, and the yield was 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=7.7 Hz, 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, 11 8.27,117.94,109.94,96.83,76.83,56.49,52.10,47.41, _{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 ; 7 [} MH] ^- :878.3995; found 878.3932.

Example 25

In vitro anti-inflammatory activity test of the compounds of the present invention

Step a: ICR mouse peritoneal macrophage extraction and culture

Three days before the experiment, each mouse was injected with 5% broth (sodium thioglycolate) (2 mL) every day to obtain more peritoneal macrophages. Cell extraction follows the steps below:

1) The mice were killed, soaked in 75% ethanol for 2-3 minutes, and transferred to a clean bench. Lie supine and fix on the dissecting board, then wipe the abdominal skin with ethanol, cut the abdominal skin with surgical scissors, expose the abdominal muscle layer (be careful not to damage the abdominal muscle layer), and then disinfect the abdominal muscle layer with ethanol;

2) Use a 5mL syringe to extract RPMI1640 medium (5mL) containing 1% double antibody and inject it into the peritoneal cavity, gently shake the body of the mouse back and forth, and then use a syringe to extract the cell suspension and transfer it to a centrifuge tube. Repeat three more times;

3) Centrifuge the collected cell suspension at 1×10 ³ rpm for 5 min, discard the supernatant, blow the cells with RPMI1640 medium (10 mL) containing double antibody, and centrifuge at 1×10 3 rpm for 5 min;

4) Pipette evenly with RPMI1640 medium (5 mL) containing 10% fetal bovine serum and 1% double antibody, take the cell suspension (10 μL) and dilute it 10 times to 100 μL, and place it on a cell counting plate for counting;

5) Calculate the number of cells required for the experiment, dilute the cell suspension and transfer it directly to a 96-well plate or a 6-well plate, and incubate for 12 hours at 37°C in a cell culture incubator with a _CO2 concentration of 5%. The experiment can then be performed.

Step b: Enzyme-linked immunoassay (ELISA) to detect the content of inflammatory factors

Add 2 mL of cell suspension with a concentration of 8× ¹⁰ cells/mL to each well of a 6-well plate, incubate for 12 h at 37°C in a cell culture incubator with a CO _{concentration} of 5%, suck out the culture medium, and add Good compound 2mL. After further incubation for 1 h, 1 μL of 0.5 mg/mL LPS solution was added, and the incubation was continued for 72 h. The detection of inflammatory factors TNF-α and IL-6 by ELISA method can be carried out by referring to the method in the instruction manual of the ELISA kit, and the main steps are as follows.

1) Preparation of standard substance: Dilute the standard substance by 2 times with a maximum concentration of 2000pg/mL to 8 concentrations with the standard substance diluent;

2) Add 100 μL of the prepared standard substance and sample to be tested at each concentration to each well, and incubate at 37°C for 1 hour;

3) Aspirate the liquid in the wells, add 100 μL of detection antibody to each well except the blank, and incubate at 37°C for 1 hour;

4) Aspirate the liquid in the wells, add washing liquid to each well (the washing liquid is diluted 25 times with double distilled water, 200 μL) and let it stand for 2 minutes, absorb it, repeat 3 times and then dry it;

5) Add 100 μL of enzyme-labeled reagent to each well except the blank, and incubate at 37°C for 60 min;

6) Aspirate the liquid in the wells, add 200 μL of washing solution to each well and let it stand for 2 minutes, aspirate, repeat 3 times and then dry;

7) Add 90 μL each of chromogenic reagents A and B to each well, mix well, incubate at 37°C in the dark for 15 minutes, then add 50 μL of stop solution to terminate the reaction;

8) Immediately use a microplate reader to detect, and read the OD value of each well at a wavelength of 450nm. Use ELISACalc software to calculate the concentration of the measured inflammatory factors.

With LRRK2-IN-1 as a positive control, the mouse primary peritoneal macrophages induced by LPS were treated with 10 μM compounds P-1~P-6 and P-9 for 24 hours, and the inflammatory factor IL- 6 and changes in TNF-α release.

The experimental results are shown in Figure 1, which is a schematic diagram of the in vitro anti-inflammatory activity test results of the compounds of the present invention. Among them, the upper picture in Figure 1 is a schematic diagram of the change in the release of the inflammatory factor IL-6, and the lower picture in Figure 1 is a schematic diagram of the change in the release of the inflammatory factor TNF-α. It can be seen from the figure that, except compound P-9, other compounds have significantly lower anti-inflammatory activity in vitro than the positive drug LRRK2-IN-1, and compared with IL-6, the compound has a higher effect on TNF-α better inhibitory effect. Compound P-9 is a DCLK1 protein degradation targeting chimera with carboxychain as the linker. The reason for the better activity of molecular carboxychains may be that carboxychains have better fat solubility.

Example 26

The protein degradation experiment (Westernblot) of the compound of the present invention to DCLK1

Step a: Protein extraction

1) Collect cells: Discard the medium in the 6-well plate or culture dish, wash the cells twice with pre-cooled PBS, add 100 μL of cell lysate (containing 1% protease inhibitor and 1% phosphatase inhibitor) , placed at 4°C for 30 minutes, scraped off the cells with a cell scraper, collected the cell suspension into a 1.5mL centrifuge tube, vortexed once every 5 minutes, and repeated 2 times;

2) Centrifugation: Place the cell suspension in a pre-cooled centrifuge and centrifuge at 4°C and 1.2×10 ⁵ rpm for 15 minutes;

3) Protein denaturation: absorb the centrifuged supernatant, add a quarter volume of loading buffer, vortex for 1 min, and denature in a 95°C water bath for 20 min.

Step b: Determination of protein concentration (BCA method)

4) Prepare BCA working solution: A solution and B solution are prepared according to 50:1;

5) Add 18 μL of PBS, 2 μL of protein supernatant and 200 μL of BCA working solution to each well of the 96-well plate, place in a microplate reader, shake the plate at 37°C for 5 min, and incubate for 25 min;

6) Measure the OD value at a wavelength of 562nm, place it in the standard music, and measure the protein concentration.

Step c: Electrophoresis

1) Compounding glue;

2) Sample loading: Put the gel into the electrophoresis tank, add the electrophoresis solution, and pull out the tooth comb. Add the protein sample to the sample well according to the calculated volume;

3) Running glue at constant voltage, energized at 100V for 80min.

Step d: transfer membrane

4) Cut the gel into required fragments and place them on filter paper, place PVDF membranes of suitable size in methanol for activation for 5 minutes, and then cover them on the gel;

5) Place the transfer clip in the transfer tank, and energize at a constant current of 270A for 100 minutes.

Step e: Antibody Incubation

1) Sealing: After the membrane transfer is completed, wash the PVDF membrane once with TBST, put it in 5% BSA, and shake it at room temperature for 2 hours;

2) Primary antibody incubation: Wash the membrane with TBST for a while, then place it in the prepared primary antibody, and incubate at room temperature for 2 hours (or overnight at 4°C);

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

Step f: Exposure

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

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

The experimental results are shown in Figure 2, which is a schematic diagram of the test results of the degradation activity of DCLK1-PROTAC. Among them, A is a schematic diagram of the degradation activity screening results of PROTAC molecules with VHL as the E3 ligase ligand at 10 μM; B is a schematic diagram of the time-dependent degradation experiment results of compound 9 at 10 μM; C is a schematic diagram of the results of the E3 ligase ligand with pomalidomide Schematic diagram of the screening results of the degradation activity of PROTAC molecules at 10 μM; D is a schematic diagram of the results of compound 14-dependent degradation experiments. At a single concentration of 10 μM, in the DCLK1 protein degradation targeting chimeras (compounds P-1~P-6, P-9) with VHL as the E3 ligase ligand, the carbon-oxa chain and the three-carbon atom Compounds P-1 and P-9 with aliphatic chains have a certain degradative activity, and in the aliphatic chain compounds P-2 to P-6 with 4-8 carbon atoms, the degradation activity gradually weakens as the chain length increases (Fig. 2 Middle A); Compound P-9 with the best anti-inflammatory activity was subjected to a time-dependent experiment. From 0.5h to 3h after administration, the degradation rate of DCLK1 gradually increased with time, and the degradation effect did not change after 3h (Fig. 2 Middle B), indicating that the PROTAC molecule can reach the maximum degradation in 3 hours, so the subsequent drug action time is 3 hours; for some DCLK1 protein degradation targeting chimeras using pomalidomide as the E3 ligase ligand, perform 10 μM single-concentration degradation activity After screening, it was found that compounds P-11, P-12, and P-14 had good degradation activity (C in Figure 2), and their corresponding compounds were no linker, fatty linker with 2 carbon atoms, and aliphatic linker with 5 carbon atoms. DCLK1 protein degradation targeting chimera of fat linker. By comparing the degradation activities of two different types of E3 ligase ligands, it was found that the PROTAC molecule whose E3 ligase ligand is CRBN has better degradation activity. The compound P-14 with better degradation activity was selected for concentration-dependent degradation experiments. It was found that in the three concentrations of 0.1-10 μM, as the concentration increased, the degradation efficiency decreased instead. The reason for the analysis may be that PROTAC molecules produced The HOOK effect and the specific degradation efficiency need to be further verified (D in Figure 2).

The above descriptions are only preferred embodiments of the present invention, and do not limit the present invention in any form. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with the technology of this patent Without departing from the scope of the technical solution of the present invention, personnel can use the technical content of the above prompts to make some changes or modify them into equivalent embodiments with equivalent changes. In essence, any simple modifications, equivalent changes and modifications made to the above embodiments still fall within the scope of the solutions of the present invention.

Claims (4)

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.

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