CN115417858A - Intracellular self-assembly ALK degradation agent based on bioorthogonal strategy and preparation method and application thereof - Google Patents

Intracellular self-assembly ALK degradation agent based on bioorthogonal strategy and preparation method and application thereof Download PDF

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CN115417858A
CN115417858A CN202211207796.9A CN202211207796A CN115417858A CN 115417858 A CN115417858 A CN 115417858A CN 202211207796 A CN202211207796 A CN 202211207796A CN 115417858 A CN115417858 A CN 115417858A
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徐盛涛
徐进宜
占飞雁
姚鸿
谢绍文
朱静杰
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Abstract

The invention discloses an intracellular self-assembly ALK degradation agent based on a bioorthogonal strategy, and a preparation method and application thereof, and belongs to the field of biological medicines. The compound of the invention prepares two parts of precursor molecules of PROTAC by connecting a click chemical fragment which generates a bio-orthogonal reaction with a protein ligand or an E3 ubiquitin ligase ligand, and spontaneously reacts after mixing to generate the PROTAC molecules. The protein degradation agent is beneficial to overcoming the problems of poor membrane permeability and poor solubility caused by the large molecular weight of the existing ALK-targeting protein degradation agent, and is expected to be applied to the preparation of antitumor drugs.

Description

Intracellular self-assembly ALK degradation agent based on bioorthogonal strategy and preparation method and application thereof
Technical Field
The invention belongs to the field of biomedicine, particularly relates to an ALK (anaplastic lymphoma kinase) degrading agent as well as a preparation method and application thereof, and particularly relates to an intracellular self-assembly ALK degrading agent based on a bioorthogonal strategy as well as a preparation method and application thereof.
Background
Anaplastic Lymphoma Kinase (ALK), a receptor tyrosine kinase of the Insulin Receptor (IR) subfamily, was originally found in Anaplastic Large Cell Lymphoma (ALCL) cell lines. Chromosomal rearrangements are the most common genetic aberrations of ALK, leading to the fusion of multiple ALK, enhancing their oncogenic potential, and ALK has become an attractive therapeutic target. Thus, therapeutic strategies that inhibit ALK kinase activity may produce fewer side effects. At present, five small-molecule inhibitors of ALK, including Crizotinib (Crizotinib, 2011, pfize), ceritinib (Ceritinib, 2014, novartis), aletinib (aletinib, 2015, roche), bugatinib (Brigatinib, (2017, aria) and loratinib (lrlatinib, 2018, pfizer), have been approved by the FDA for clinical application to show good therapeutic effects, but the drug resistance of these drugs still remains a serious challenge.
In recent years, targeted protein-targeting chimeras (PROTACs) technology has become one of the most promising cancer treatment strategies. The PROTACs consist of three parts, a ligand binding to a target, an E3 ubiquitin ligase ligand and a linker connecting the two parts, so that a target protein is ubiquitinated and degraded through the ubiquitin-proteasome system. PROTACs technology has been reported in ALK fusion protein degradation field, but its purpose isThe properties of the former PROTACs may limit their potential as therapeutic drugs. PROTACs require binding of a target protein ligand and an E3 ligase ligand, typically having a high molecular weight of 800-1000Da and a molecular weight of-200
Figure BDA0003874766470000011
Polar surface area of (a). The combination of these properties limits cell permeability and solubility and affects the bioavailability and pharmacokinetics of the drug, particularly in the central nervous system.
The bioorthogonal reaction is widely applied in the fields of cell marking, imaging, delivery and the like by virtue of high reaction rate and good orthogonality, but is less applied in the field of protein degradation and needs to be further deeply researched.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide an intracellular self-assembly ALK degrading agent based on a bioorthogonal strategy, which is beneficial to solving the problems of poor membrane permeability and poor solubility caused by the large molecular weight of the existing ALK-targeting protein degrading agent; another objective of the present invention is to provide a preparation method of the ALK-targeting intracellular self-assembly degradation agent based on the bioorthogonal strategy; the invention also aims to provide an application of the ALK-targeting intracellular self-assembly degradation agent based on the bioorthogonal strategy in preparing anti-tumor and cancer drugs.
Based on the existing PROTACs design concept, the invention connects bioorthogonal fragments with CRBN ligand pomalidomide and ALK inhibitor Alletinib derivatives through connecting chains respectively, and obtains the ALK protein degradation agent based on the PROTACs technology through bioorthogonal Click reaction.
The technical scheme is as follows: the intracellular self-assembly ALK degrading agent based on the bioorthogonal strategy is obtained by bioorthogonal reaction of two parts of precursor molecules, wherein the precursor molecules are P and Q parts shown as a general formula I, or an optically active body or a racemate thereof, a diastereoisomer mixture and pharmaceutically acceptable salts;
Figure BDA0003874766470000021
wherein, the A group is a group with alkyne, alkene, phosphine or boric acid;
the B group is a group with azide, triazine, tetrazine, amino acid hydrazide or cyclopropane;
<xnotran> C , , ,1,4- 1,3- , , -O-, -CONH-, NHCO-, -NHCONH-, -NH-, -S-, , , , , , , , ; </xnotran>
The D group is an aromatic ring, an aromatic heterocyclic ring, a spiro ring, a bridged ring, a spiro ring, a piperazine ring or a piperazine-piperidine ring.
Preferably, the a group is one of the following:
Figure BDA0003874766470000022
the B group is one of the following groups:
Figure BDA0003874766470000031
the C group is one of the following groups:
Figure BDA0003874766470000032
wherein m is any integer from 1 to 10, and n is any integer from 0 to 10;
the D group is one of the following groups:
Figure BDA0003874766470000033
wherein, the A1 group and the B1 group generate bioorthogonal reaction, and the A2 group and the B2 group generate bioorthogonal reaction. Preferably, the P and Q molecules are:
Figure BDA0003874766470000041
wherein m is any integer from 1 to 10, and n is any integer from 0 to 10.
Preferably, the P molecule
Figure BDA0003874766470000051
The preparation process comprises the following steps:
Figure BDA0003874766470000052
preferably, the P molecule
Figure BDA0003874766470000053
The preparation process comprises the following steps:
Figure BDA0003874766470000054
preferably, the Q molecule
Figure BDA0003874766470000055
The preparation process of n =0 is as follows:
Figure BDA0003874766470000056
preferably, the Q molecule
Figure BDA0003874766470000061
The preparation process of n =0 is as follows:
Figure BDA0003874766470000062
the intracellular self-assembly ALK degrading agent based on the bioorthogonal strategy can be applied to the preparation of antitumor drugs.
Further, the tumor is lung cancer, lung adenocarcinoma, anaplastic Lymphoma Kinase (ALK) mutation-positive non-small cell lung cancer, anaplastic large cell lymphoma, colorectal cancer, astrocytoma, acute myelogenous leukemia, or ovarian cancer.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages: the compound of the invention prepares two parts of precursor molecules of PROTACs by connecting the click chemical fragment which generates the bio-orthogonal reaction with a protein ligand or an E3 ubiquitin ligase ligand, and the two parts of precursor molecules are mixed and then spontaneously react to generate the PROTACs molecules. The preparation method is favorable for overcoming the problems of poor membrane permeability and poor solubility caused by the large molecular weight of the existing targeting ALK protein degradation agent, and is expected to be applied to the preparation of antitumor drugs.
Drawings
FIG. 1 shows the degradation of ALK protein when P1 and Q1 were administered alone in Karpas299 cells and P1 (1, 5, 10. Mu.M) was administered separately with Q1 (1. Mu.M) unchanged.
Detailed Description
The technical scheme of the invention is further explained by combining the attached drawings.
Example 1
Preparation of (R, E) -cyclooct-4-en-1-yl (2- (4- (3-cyano-9-ethyl-6, 6-dimethyl-11-oxo-6, 11-dihydro-5H-benzo [ b ] carbazol-8-yl)) piperazin-1-yl) -2-oxoethyl) carbamate (P1), of the formula:
Figure BDA0003874766470000071
step 1: preparation of (Z) - (cyclooct-4-en-1-yloxy) trimethylsilane (1 a)
Figure BDA0003874766470000072
(Z) -cyclooct-4-en-1-ol (5 g, 39mmol), imidazole (6.7g, 99mmol) and 100ml chloroform were mixed, stirred at 0 ℃ and trimethylchlorosilane (10.9g, 7.94mmol) was added. The reaction solution was allowed to move to room temperature, during which time a white precipitate was formed and the reaction was allowed to proceed for about 3 hours. Cooling to 0 deg.C, extracting with dichloromethane, washing with saturated brine, collecting organic layer, drying with anhydrous sodium sulfate, distilling off organic solvent under reduced pressure, and purifying the residue by silica gel column chromatography, eluting with petroleum ether to obtain colorless oily liquid 1.779g, with yield of 98.32%.
And 2, step: preparation of ((9-oxabicyclo [6.1.0] non-4-yl) oxy) trimethylsilane (1 b)
Figure BDA0003874766470000073
1a (12.92g, 53mmol), 70m dichloromethane and 70ml saturated sodium bicarbonate solution were mixed, peracetic acid (12g, 59mmol) was added in portions at 0 ℃ to change the reaction solution from a clear transparent solution to a white turbid solution, and stirred at room temperature. Extraction with a dichloromethane solution was performed, an organic layer was collected, dried over anhydrous sodium sulfate, and the organic solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography using petroleum ether/ethyl acetate (V/V = 100) to give a colorless oily liquid weighing 8.53g, yield 51.92%.
And step 3: preparation of 5- ((trimethylsilyl) oxy) -2- (triphenyl-L5-phosphonyl) cycloocta-1-ol (1 c)
Figure BDA0003874766470000074
1b (6.5g, 25mmol) and triphenylphosphine (5.7g, 30mmol) were dissolved in 80ml anhydrous DMF and tert-butyllithium (1.97g, 30mmol) was added dropwise over 15min at-78 ℃. The reaction solution was allowed to warm to room temperature and stirred for 20h. After the reaction was completed, ethyl acetate was added for extraction, the organic layer was collected, dried over anhydrous sodium sulfate, the organic solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography using dichloromethane/methanol (V/V = 100) to give a colorless oily liquid weighing 11.56g with a yield of 60.23%.
And 4, step 4: preparation of (S, E) - (cyclooct-4-en-1-oxy) trimethylsilane (1 d)
Figure BDA0003874766470000081
1c (5.31g, 11.58mmol) was dissolved in 100ml DMF and sodium hydride (60% dispersion in mineral oil) (1.62g, 40mmol) was added at 0 deg.C, allowed to warm to room temperature and stirred for 5h. The reaction was cooled to 0 ℃ and quenched by addition of ethyl acetate and water. The organic layer was collected by extraction with ethyl acetate, dried over anhydrous sodium sulfate, and the organic solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography using petroleum ether/ethyl acetate (V/V = 100) to give a colorless oily liquid weighing 1.98g with a yield of 33.23%.
And 5: preparation of (S, E) -cyclooct-4-en-1-ol (1E)
Figure BDA0003874766470000082
35ml of TBAF was added to 1d (500mg, 207mmol) and reacted at 70 ℃. The reaction solution was cooled to 0 ℃ and quenched by the addition of dichloromethane and water. Dichloromethane extraction, organic layer was collected, dried over anhydrous sodium sulfate, organic solvent was evaporated under reduced pressure, and residue was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (V/V = 1) to give colorless oily liquid, weight 100mg, yield 21.55%.
The reaction process of the steps is as follows:
Figure BDA0003874766470000083
step 6: preparation of (S, E) -cyclooctyl-4-en-1-yl (4-nitrophenyl) carbonate (1 f)
Figure BDA0003874766470000084
DMAP (418mg, 3.4 mmol) was added to 4ml of an anhydrous DMF solution of 1e (216mg, 1.7 mmol), and after stirring for 5min, 4-nitrophenyl chloroformate (540mg, 2.5 mmol) was added at room temperature under protection of Ar, and the mixture was stirred at room temperature for about 3 to 4 hours. After completion of the reaction, the reaction mixture was quenched with 1N diluted hydrochloric acid, extracted with dichloromethane, the organic layer was collected, dried over anhydrous sodium sulfate, and the organic solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography using petroleum ether/ethyl acetate (V/V = 50) to give a colorless oily liquid weighing 200mg with a yield of 71.35%.
And 7: preparation of (S, E) - ((cyclooctyl-4-en-1-yloxy)) carbonyl) Glycine (1 g)
Figure BDA0003874766470000091
Glycine (40mg, 531 μmol) was dissolved in 3ml thf, DIPEA (221mg, 2mmol) was added thereto, 1f (100mg, 312 μmol) was added after stirring, stirring was performed at ordinary temperature, quenching was performed with water, PH =3-4 was adjusted with 1N diluted hydrochloric acid, dichloromethane was extracted, an organic layer was collected, dried over anhydrous sodium sulfate, the organic solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography, eluted with dichloromethane/methanol (V/V = 10) to obtain a colorless oily liquid, weighing 10mg, and yield 30.41%.
And step 8: preparation of (R, E) -Cyclooct-4-en-1-yl (2- (4- (3-cyano-9-ethyl-6, 6-dimethyl-11-oxo-6, 11-dihydro-5H-benzo [ b ] carbazol-8-yl)) piperazin-1-yl) -2-oxoethyl) carbamate (P1)
1g (58mg, 255. Mu. Mol), HATU (146mg, 382. Mu. Mol), DIPEA (106mg, 382. Mu. Mol) were dissolved in 2ml of anhydrous DMF and stirred for 10min, then 9-ethyl-6, 6-dimethyl-11-oxo-8- (piperazin-1-yl) -611-dihydro-5H-benzo [ B ] was added]Carbazole-3-carbonitrile (106mg, 267. Mu. Mol) was stirred at room temperature for 2 hours. Quenched with water, extracted with ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, and the organic solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography, eluting with dichloromethane/methanol (V/V = 20) to give a white solid, weighing 40mg, and yield 45.36%. 1 H NMR(500MHz,Chloroform-d)δ8.87(s,1H),8.20(d,J＝7.0Hz,1H),7.77–7.71(m,2H),7.69(dd,J＝7.0,1.9Hz,1H),6.81(s,1H),5.59–5.45(m,3H),4.72(p,J＝6.0Hz,1H),3.93–3.85(m,1H),3.85(dd,J＝15.6,5.3Hz,1H),3.67(dd,J＝6.2,3.5Hz,2H),3.63(dd,J＝6.2,3.5Hz,2H),3.08(ddd,J＝13.2,6.2,3.5Hz,4H),2.62(qt,J＝7.5,1.1Hz,2H),2.12–1.72(m,7H),1.69–1.56(m,4H),1.57–1.44(m,1H),1.20(t,J＝7.4Hz,3H)； 13 C NMR(125MHz,Chloroform-d)δ182.83,169.70,155.89,152.97,151.89,146.40,133.03,130.60,130.00,129.78,129.77,128.78,126.68,125.78,122.86,117.79,115.00,111.61,109.29,109.24,77.42,49.89,45.10,43.19,35.73,33.63,33.07,28.27,27.75,27.00,25.39,25.12,14.39；HRMS(ESI)C 36 H 42 N 5 O 4 + [M+H] + (ii) a Exact Mass:608.3231, found 608.3227.
The reaction process of the steps comprises:
Figure BDA0003874766470000092
example 2
Preparation of (R, E) -cyclooct-4-en-1-yl (3- (4- (3-cyano-9-ethyl-6, 6-dimethyl-11-oxo-6, 11-dihydro-5H-benzo [ b ] carbazol-8-yl)) piperazin-1-yl) -3-propionyl) carbamate (P2), of the formula:
Figure BDA0003874766470000101
the synthesis method is the same as that of example 1 by changing glycine to beta-alanine in step 7 of example 1.HRMS (ESI) C 37 H 44 N 5 O 4 + [M+H] + (ii) a Exact Mass:622.3388, found 622.3356.
Example 3
Preparation of (R, E) -Cyclooct-4-en-1-ol (4- (4- (3-cyano-9-ethyl-6, 6-dimethyl-11-oxo-6, 11-dihydro-5H-benzo [ b ] carbazol-8-yl)) piperazin-1-yl) -4-oxobutyl) carbamate (P3)
Figure BDA0003874766470000102
The glycine in step 7 of example 1 was replaced with gamma-aminobutyric acid, and the synthesis method was the same as in example 1.HRMS (ESI) C 38 H 46 N 5 O 4 + [M+H] + (ii) a Exact Mass:636.3544, found 636.3587.
Example 4
Preparation of (R, E) -Cyclooct-4-en-1-ol (5- (4- (3-cyano-9-ethyl-6, 6-dimethyl-11-oxo-6, 11-dihydro-5H-benzo [ b ] carbazol-8-yl)) piperazin-1-yl) -5-oxopentyl) carbamate (P4)
Figure BDA0003874766470000103
The glycine in step 7 of example 1 was changed to 5-aminovaleric acid and the synthesis was performed as in example 1.HRMS (ESI) C 39 H 48 N 5 O 4 + [M+H] + (ii) a Exact Mass:650.3701, found 650.3718.
Example 5
Preparation of (R, E) -Cyclooct-4-en-1-ol (6- (4- (3-cyano-9-ethyl-6, 6-dimethyl-11-oxo-6, 11-dihydro-5H-benzo [ b ] carbazol-8-yl)) piperazin-1-yl) -6-oxohexyl) carbamate (P5)
Figure BDA0003874766470000104
The synthesis was performed in the same manner as in example 1 except that glycine in step 7 of example 1 was changed to 6-aminocaproic acid. HRMS (ESI) C 40 H 50 N 5 O 4 + [M+H] + (ii) a Exact masses 664.3857, found 664.3813.
Example 6
Preparation of (R, E) -cyclooct-4-en-1-ol (7- (4- (3-cyano-9-ethyl-6, 6-dimethyl-11-oxo-6, 11-dihydro-5H-benzo [ b ] carbazol-8-yl)) piperazin-1-yl) -7-oxoheptyl) carbamate (P6)
Figure BDA0003874766470000111
The synthesis was performed in the same manner as in example 1 except that glycine in step 7 of example 1 was changed to 7-aminoheptanoic acid. HRMS (ESI) C 41 H 52 N 5 O 4 + [M+H] + (ii) a Exact Mass:678.4014, found 678.4056.
Example 7
Preparation of (R, E) -Cyclooct-4-en-1-yl 2- (4- (3-cyano-9-ethyl-6, 6-dimethyl-11-oxo-6, 11-dihydro-5H-benzo [ b ] carbazol-8-yl)) piperazin-1-yl) acetate (P7)
Figure BDA0003874766470000112
Step 1 preparation of (S, E) -Cyclooct-4-en-1-yl 2-bromoacetate (7 a)
Figure BDA0003874766470000113
EDCI (723mg, 3.4mmol) and DMAP (418mg, 3.4mmol) were added to 4ml of anhydrous DMF solution of bromoacetic acid, and 1e (216mg, 1.7mmol) was further added thereto, and the reaction was carried out at room temperature in a dry tube. Quenching with water, extraction with ethyl acetate, collection of the organic layer, drying over anhydrous sodium sulfate, evaporation of the organic solvent under reduced pressure, and purification of the residue by chromatography on silica gel column using petroleum ether/ethyl acetate (V/V = 50).
Step 2 preparation of (R, E) -cyclooct-4-en-1-yl-2- (4- (3-cyano-9-ethyl-6, 6-dimethyl-11-oxo-6, 11-dihydro-5H-benzo [ b ] carbazol-8-yl)) piperazin-1-yl) acetate
7a (69mg, 255. Mu. Mol), 9-ethyl-6, 6-dimethyl-11-oxo-8- (piperazin-1-yl) -6, 11-dihydro-5H-benzo [ B ]]Carbazole-3-carbonitrile (106mg, 267. Mu. Mol) and DIPEA (31mg, 255. Mu. Mol) were dissolved in DMSO, and stirred with heating at 90 ℃ for 2 hours. Quenching with water, extraction with ethyl acetate, collection of the organic layer, drying over anhydrous sodium sulfate, evaporation of the organic solvent under reduced pressure, and purification of the residue by chromatography on a silica gel column, eluting with dichloromethane/methanol (V/V = 20) gave a pale yellow solid, weighing 30mg, yield 53.52%. 1 H NMR(500MHz,Chloroform-d)δ8.87(s,1H),8.20(d,J＝7.0Hz,1H),7.77–7.71(m,2H),7.69(dd,J＝7.0,1.9Hz,1H),6.81(s,1H),5.59–5.45(m,2H),4.72(p,J＝6.2Hz,1H),3.35(s,2H),3.17(t,J＝4.6Hz,4H),2.71(dt,J＝13.0,4.6Hz,4H),2.62(qt,J＝7.5,1.1Hz,2H),2.12–1.95(m,2H),1.98–1.91(m,1H),1.94–1.85(m,1H),1.88–1.82(m,1H),1.85–1.74(m,1H),1.77–1.66(m,6H),1.68–1.60(m,1H),1.63–1.54(m,1H),1.56–1.44(m,1H),1.20(t,J＝7.4Hz,3H)； 13 C NMR(125MHz,Chloroform-d)δ182.83,169.43,152.97,151.89,146.40,133.03,130.63,130.00,129.77,128.78,126.68,125.78,122.86,117.79,115.00,111.61,109.29,109.20,76.92,57.35,52.44,50.18,35.73,33.88,33.32,28.27,27.74,26.99,25.39,25.12,14.39；HRMS(ESI)C 35 H 41 N 4 O 3 + [M+H] + (ii) a Exact Mass:565.3173, found 565.3151.
The reaction process of the steps comprises:
Figure BDA0003874766470000121
example 8
Preparation of (R, E) Cyclooct-4-en-1-yl-3- (4- (3-cyano-9-ethyl-6, 6-dimethyl-11-oxo-6, 11-dihydro-5H-benzo [ b ] carbazol-8-yl)) piperazin-1-yl) propanoate (P8)
Figure BDA0003874766470000122
The synthesis was performed in the same manner as in example 7 except that the bromoacetic acid in step 1 of example 7 was changed to 3-bromopropionic acid. HRMS (ESI) C 36 H 43 N 4 O 3 + [M+H] + (ii) a Exact masses 579.3330, found 579.3321.
Example 9
Preparation of (R, E) Cyclooct-4-en-1-yl-4- (4- (3-cyano-9-ethyl-6, 6-dimethyl-11-oxo-6, 11-dihydro-5H-benzo [ b ] carbazol-8-yl)) piperazin-1-yl) propionate (P9)
Figure BDA0003874766470000123
The synthesis was performed in the same manner as in example 7 except that the bromoacetic acid in step 1 of example 7 was changed to 4-bromobutyric acid. HRMS (ESI) C 37 H 45 N 4 O 3 + [M+H] + (ii) a Exact Mass:593.3486, found 593.3472.
Example 10
Preparation of (R, E) Cyclooct-4-en-1-yl-5- (4- (3-cyano-9-ethyl-6, 6-dimethyl-11-oxo-6, 11-dihydro-5H-benzo [ b ] carbazol-8-yl)) piperazin-1-yl) propionate (P10)
Figure BDA0003874766470000131
The synthesis was performed in the same manner as in example 7 except that the bromoacetic acid in step 1 of example 7 was changed to 4-bromobutyric acid. HRMS (ESI) C 38 H 47 N 4 O 3 + [M+H] + (ii) a Exact Mass:607.3643, found 607.3678.
Example 11
Preparation of (R, E) Cyclooct-4-en-1-yl-6- (4- (3-cyano-9-ethyl-6, 6-dimethyl-11-oxo-6, 1-dihydro-5H-benzo [ b ] carbazol-8-yl)) piperazin-1-yl) propionate (P11)
Figure BDA0003874766470000132
The synthesis was the same as in example 7, except that the bromoacetic acid in step 1 of example 7 was changed to 5-bromovaleric acid. HRMS (ESI) C 39 H 49 N 4 O 3 + [M+H] + (ii) a Exact masses 621.3799, found 621.3721.
Example 12
Preparation of (R, E) Cyclooct-4-en-1-yl-7- (4- (3-cyano-9-ethyl-6, 6-dimethyl-11-oxo-6, 11-dihydro-5H-benzo [ b ] carbazol-8-yl)) piperazin-1-yl) propionate (P12)
Figure BDA0003874766470000133
The synthesis was performed in the same manner as in example 7 except that the bromoacetic acid in step 1 of example 7 was changed to 6-bromohexanoic acid. HRMS (ESI) C 40 H 51 N 4 O 3 + [M+H] + (ii) a Exact Mass:635.3956, found 635.3972.
Example 13
Preparation of bicyclo [6.1.0] non-4-yn-9-yl-methyl (2- (4- (3-cyano-9-ethyl-6, 6-dimethyl-11-oxo-6, 11-dihydro-5H-benzo [ b ] carbazol-8-yl)) piperazin-1-yl) -2-oxoethyl) carbamate (P13)
Figure BDA0003874766470000141
Step 1: preparation of (1R, 8S, Z) -bicyclo [6.1.0] non-4-ene-9-carboxylic acid ethyl ester (13 a)
Figure BDA0003874766470000142
1, 5-cyclooctadiene (2.5 ml, 20mmol) and Rh2 (OAc) 4 (33.79mg, 67. Mu. Mol) were dissolved in 2ml of DMF, and a solution of ethyl diazoacetate (400. Mu.l) in DMF was added dropwise to the reaction solution at 0 ℃ over 3 hours. Reacting for 15h at normal temperature. After completion of the reaction, quenching with water was performed, extraction was performed with ethyl acetate, the organic layer was collected, dried over anhydrous sodium sulfate, the organic solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography using petroleum ether/ethyl acetate (V/V = 20) to give a pale yellow solid weighing 60mg, yield 53.72%.
Step 2: preparation of (1R, 8S, Z) -bicyclo [6.1.0] non-4-ene-9-methanol (13 b)
Figure BDA0003874766470000143
Sodium borohydride (59mg, 6 mmol) was placed in a two-necked flask through Ar, 2ml of anhydrous dichloromethane solution was added dropwise under ice-bath, 13a (100mg, 5.2mmol) was dissolved in 1ml of anhydrous dichloromethane solution and added dropwise at 0 ℃. The reaction solution was stirred at room temperature for 2h. Cooled to 0 ℃ and quenched by addition of water. Suction filtration and evaporation of the organic solvent under reduced pressure were carried out, and the residue was purified by silica gel column chromatography, eluting with petroleum ether/ethyl acetate (V/V = 20).
And step 3: preparation of ((1R, 8S)) -4, 5-dibromobicyclo [6.1.0] nonan-9-yl) methanol (13 c)
Figure BDA0003874766470000144
13b (100mg, 700. Mu. Mol) was dissolved in 3ml of THF and stored in ice bath. Br2 (70. Mu.l, 1 mmol) was dissolved in CH2Cl2 solution and added dropwise, the ice bath was removed and the reaction was carried out at room temperature. After extraction with 3ml of 10% (sodium thiosulfate solution; dichloromethane extraction, evaporation of the organic solvent under reduced pressure, the residue was purified by column chromatography on silica gel, eluting with petroleum ether/ethyl acetate (V/V = 20) to give an oily liquid weighing 77mg, yield 56.41%.
And 4, step 4: preparation of ((1R, 8S)) -bicyclo [6.1.0] non-4-yn-9-yl) methanol (13 d)
Figure BDA0003874766470000151
13c (50mg, 560. Mu. Mol) was dissolved in 2ml of chloroform. KotBu (1ml, 1mmol) was added dropwise at 0 ℃ for 10min, the reaction was heated at 80 ℃ under reflux for about 3h after cooling to room temperature, quenched with a saturated NH4Cl solution, chloroform was removed by rotary evaporation, ethyl acetate was extracted, the organic solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (V/V = 100) to give an oily liquid weighing 34mg, yield 87.41%.
The reaction process of the steps comprises:
Figure BDA0003874766470000152
and 5: preparation of bicyclo [6.1.0] non-4-yn-9-yl-methyl (2- (4- (3-cyano-9-ethyl-6, 6-dimethyl-11-oxo-6, 11-dihydro-5H-benzo [ b ] carbazol-8-yl)) piperazin-1-yl) -2-oxoethyl) carbamate (P13)
The synthesis method was the same as example 1 except that 1e in step 7 of example 1 was changed to 13 d. 1 H NMR(500MHz,Chloroform-d)δ8.87(s,1H),8.20(d,J＝7.0Hz,1H),7.77–7.71(m,2H),7.69(dd,J＝7.0,2.2Hz,1H),6.81(s,1H),5.71(t,J＝6.0Hz,1H),4.12(d,J＝4.5Hz,2H),3.90(d,J＝6.0Hz,2H),3.67(dd,J＝6.2,3.5Hz,2H),3.63(dd,J＝6.2,3.5Hz,2H),3.08(ddd,J＝13.2,6.2,3.5Hz,4H),2.62(qd,J＝7.4,1.0Hz,2H),2.41–2.29(m,4H),1.93–1.84(m,1H),1.79–1.69(m,3H),1.68(dd,J＝8.3,4.3Hz,1H),1.63–1.52(m,2H),1.20(t,J＝7.4Hz,3H)； 13 CNMR(125MHz,Chloroform-d)δ182.58,169.70,156.77,156.64,151.66,145.87,133.23,130.58,129.05,128.78,126.68,125.92,122.84,117.84,114.99,112.77,109.40,109.23,86.16,63.95,49.89,45.10,43.13,38.62,29.47,29.35,28.30,27.98,17.94,17.86,17.13,14.39；HRMS(ESI)C 38 H 42 N 5 O 4 + [M+H] + (ii) a Exact Mass:632.3231, found 632.3256.
Example 14
Preparation of N- (bicyclo [2.2.1] hept-5-en-2-ylmethyl) -2- (4- (3-cyano-9-ethyl-6, 6-dimethyl-11-oxo-6, 11-dihydro-5H-benzo [ b ] carbazol-8-yl)) piperazin-1-yl) acetamide (P14)
Figure BDA0003874766470000161
Example 7 was repeated except that 1e in step 1 of example 7 was changed to norbornene-2-methanamine, and the synthesis was performed in the same manner as in example 7. 1 H NMR(500MHz,Chloroform-d)δ8.87(s,1H),8.20(d,J＝7.0Hz,1H),7.77–7.71(m,2H),7.69(dd,J＝7.1,1.9Hz,1H),6.83–6.76(m,2H),5.97(ddd,J＝7.3,5.5,1.7Hz,1H),5.86(ddd,J＝7.3,4.5,1.8Hz,1H),3.22–3.07(m,9H),2.81–2.70(m,6H),2.62(qt,J＝7.5,1.1Hz,2H),2.13(dddp,J＝8.4,5.1,3.3,1.6Hz,1H),1.73(s,2H),1.60(dt,J＝12.5,4.9Hz,1H),1.46(dt,J＝12.4,4.9Hz,1H),1.40–1.33(m,1H),1.25–1.17(m,4H)； 13 C NMR(125MHz,Chloroform-d)δ182.58,172.21,156.77,151.67,145.87,137.82,134.16,133.23,130.61,129.05,128.78,126.68,125.92,122.84,117.84,114.99,112.77,109.40,109.20,60.24,52.44,50.19,47.43,46.14,44.13,42.05,38.62,37.66,33.09,28.30,27.98,14.39；HRMS(ESI)C 35 H 40 N 5 O 2 + [M+H] + (ii) a Exact Mass:562.3177, found 562.3154.
Example 15
Preparation of N- (2- ((2- (2, 6-dioxopiperidin-3-yl)) -1, 3-dioxoisoindolin-4-yl) amino) ethyl) -4-6-methyl-1, 2,4, 5-tetrazin-3-yl) benzamide (Q1)
Figure BDA0003874766470000162
Step 1: preparation of 4- ((2-aminoethyl) amino) -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione (14 a)
Figure BDA0003874766470000163
Glycine tert-butyl ester (3ml, 5.2mmol) was dissolved in DMSO, 2- (2, 6-dioxopiperidin-3-yl) -4-fluoroisoindoline-1, 3-dione (300mg, 3mmol) was added, DIPEA (560. Mu.l, 5 mmol) was added, and the mixture was heated at 90 ℃. Adding water for quenching, extracting by ethyl acetate, evaporating the organic solvent under reduced pressure, adding trifluoroacetic acid and dichloromethane solution, and reacting at normal temperature by a drying tube. Dried and purified by silica gel column chromatography eluting with dichloromethane/methanol (V/V = 100) to give a yellow solid weighing 150mg, yield 56.41%.
Step 2: preparation of 4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzoic acid (14 b)
Figure BDA0003874766470000171
Acetonitrile (710. Mu.l, 2 mmol) was added to a mixed solution of p-cyanobenzoic acid (200mg, 1mmol) and molecular sieve, hydrazine hydrate (3.3ml, 5 mmol) was added dropwise, and the mixture was sealed under Ar protection at 60 ℃ for 24h. After cooling to room temperature, 6ml of water was dissolved with NaNO2 (1.88g, 5 mmol) and slowly added dropwise to the mixture under ice-bath, 1M diluted hydrochloric acid was added until PH =3 ceased to outgas, extracted with ethyl acetate, evaporated under reduced pressure to remove the organic solvent, and purified by silica gel column chromatography eluting with dichloromethane/methanol (V/V = 100) to give a magenta solid weighing 110mg, yield 46.59%.
And step 3: preparation of N- (2- ((2- (2, 6-dioxopiperidin-3-yl)) -1, 3-dioxoisoindolin-4-yl) amino) ethyl) -4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzamide (Q1)
14b (50mg, 50mmol), HATU (123.87mg, 100mmol) were dissolved in anhydrous DMF and DIPEA (107. Mu.l, 150 mmol) was added and stirred for 10min, 14a (72.79mg, 50mmol) was added and the reaction was quenched with water at ambient temperature, extracted with ethyl acetate and purified by column chromatography on silica gel eluting with dichloromethane/methanol (V/V = 100) to give an orange solid weighing 25mg, 34.41% yield. 1 H NMR(500MHz,Chloroform-d)δ9.80(s,1H),8.83–8.77(m,1H),8.25–8.19(m,2H),8.05–7.99(m,2H),7.88–7.82(m,1H),7.54(dd,J＝7.9,7.0Hz,1H),7.39(dd,J＝6.9,1.2Hz,1H),7.17(dd,J＝7.8,1.2Hz,1H),5.48(t,J＝3.6Hz,1H),3.61–3.48(m,4H),2.75(s,2H),2.66–2.51(m,2H),2.17(dddd,J＝12.5,7.0,4.4,3.6Hz,1H),1.77(dddd,J＝12.5,7.0,4.5,3.7Hz,1H)； 13 C NMR(125MHz,Chloroform-d)δ172.08,169.62,168.26,167.91,166.70,166.53,165.89,144.50,137.65,132.38,132.22,131.06,129.52,128.99,120.69,120.40,114.15,51.34,43.50,39.36,29.89,24.67,21.82；HRMS(ESI)C 25 H 23 N 8 O 5 + [M+H] + (ii) a Exact Mass:515.1786, found 515.1791.
Example 16
Preparation of N- (3- ((2- (2, 6-dioxopiperidin-3-yl)) -1, 3-dioxoisoindol-4-yl) amino) propyl) -4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzamide (Q2)
Figure BDA0003874766470000181
Change of tert-butyl glycinate from step 1 of example 15 to tert-butyl 3-amino propionate and synthesisThe procedure is as in example 15.HRMS (ESI) C 26 H 25 N 8 O 5 + [M+H] + (ii) a Exact Mass:529.1942, found 529.1931.
Example 17
Preparation of N- (4- ((2- (2, 6-dioxopiperidin-3-yl)) -1, 3-dioxoisoindol-4-yl) amino) propyl) -4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzamide (Q3)
Figure BDA0003874766470000182
Tert-butyl glycinate in step 1 of example 15 was replaced by tert-butyl 4-aminobutyrate, and the synthesis was the same as in example 15.HRMS (ESI) C 27 H 27 N 8 O 5 + [M+H] + (ii) a Exact masses: 543.2099, found 543.2077.
Example 18
Preparation of N- (5- ((2- (2, 6-dioxopiperidin-3-yl)) -1, 3-dioxoisoindol-4-yl) amino) propyl) -4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzamide (Q4)
Figure BDA0003874766470000183
The synthesis was performed in the same manner as in example 15 except that tert-butyl glycinate in step 1 of example 15 was changed to tert-butyl 5-aminovalerate. HRMS (ESI) C 28 H 29 N 8 O 5 + [M+H] + (ii) a Exact masses: 557.2255, found 557.2223.
Example 19
Preparation of N- (6- ((2- (2, 6-dioxopiperidin-3-yl)) -1, 3-dioxoisoindol-4-yl) amino) propyl) -4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzamide (Q5)
Figure BDA0003874766470000191
Replacement of Glycine tert-butyl ester in step 1 of example 15 by 6-aminoTert-butyl hexanoate was synthesized as in example 15.HRMS (ESI) C 29 H 31 N 8 O 5 + [M+H] + (ii) a Exact Mass:571.2412, found 571.2408.
Example 20
Preparation of N- (7- ((2- (2, 6-dioxopiperidin-3-yl)) -1, 3-dioxoisoindol-4-yl) amino) propyl) -4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzamide (Q6)
Figure BDA0003874766470000192
Tert-butyl glycinate in step 1 of example 15 was replaced by tert-butyl 7-aminoheptanoate, and the synthesis was the same as in example 15.HRMS (ESI) C 30 H 33 N 8 O 5 + [M+H] + (ii) a Exact masses: 585.2568, found 585.2570.
Example 21
Preparation of N- (8- ((2- (2, 6-dioxopiperidin-3-yl)) -1, 3-dioxoisoindol-4-yl) amino) propyl) -4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzamide (Q7)
Figure BDA0003874766470000193
Tert-butyl glycinate in step 1 of example 15 was replaced with tert-butyl 8-aminocaprylate and the synthesis was the same as in example 15.HRMS (ESI) C 31 H 35 N 8 O 5 + [M+H] + (ii) a Exact Mass:599.2725, found 599.2765.
Example 22
Preparation of N- (2- ((2- (2, 6-dioxopiperidin-3-yl)) -1, 3-dioxoisoindolin-4-yl) amino) ethyl) -6- (pyrimidin-2-yl) -1,2,4, 5-tetrazine-3-carboxamide (Q8)
Tert-butyl glycinate in step 1 of example 15 was replaced with tert-butyl 8-aminocaprylate and the synthesis was the same as in example 15.HRMS (ESI) C 31 H 35 N 8 O 5 + [M+H] + (ii) a Exact masses 599.2725, trueFound 599.2765.
Figure BDA0003874766470000201
The procedure of example 15 was repeated except that the acetonitrile and p-cyanobenzoic acid in step 2 of example 15 were changed to 2-cyanopyrimidine and cyanoacetic acid. 1 H NMR(500MHz,Chloroform-d)δ9.80(s,1H),8.88(d,J＝4.0Hz,2H),8.79(t,J＝4.9Hz,1H),8.51(t,J＝5.8Hz,1H),7.54(dd,J＝7.9,7.0Hz,1H),7.42–7.33(m,2H),7.20–7.15(m,1H),5.48(t,J＝3.6Hz,1H),3.70–3.50(m,4H),2.66–2.51(m,2H),2.17(dddd,J＝12.5,7.0,4.4,3.6Hz,1H),1.77(dddd,J＝12.5,7.0,4.5,3.7Hz,1H)； 13 CNMR(125MHz,Chloroform-d)δ172.08,169.62,166.70,166.53,163.90,163.16,162.11,159.00,158.88,144.51,132.38,132.22,121.15,120.69,120.40,114.15,51.34,43.50,39.26,29.89,24.67；HRMS(ESI)C 22 H 19 N 10 O 5 + [M+H] + (ii) a Exact Mass:503.1534, found 503.1576.
Example 23
Preparation of N- (4- ((2- (2, 6-dioxopiperidin-3-yl)) -1, 3-dioxoisoindol-4-yl) amino) butyl) -6- (pyrimidin-2-yl) -1,2,4, 5-tetrazine-3-amide (Q9)
Figure BDA0003874766470000202
The synthesis was the same as example 15 except that tert-butyl glycinate in step 1 of example 15 was changed to tert-butyl 4-aminobutyrate and that acetonitrile and p-cyanobenzoic acid in step 2 were changed to 2-cyanopyrimidine and cyanoacetic acid. HRMS (ESI) C 24 H 23 N 10 O 5 + [M+H] + (ii) a Exact masses: 531.1847, found 531.1851.
Example 24
Preparation of N- (5- ((2- (2, 6-dioxopiperidin-3-yl)) -1, 3-dioxoisoindolin-4-yl) amino) ethyl) -6- (pyrimidin-2-yl) -1,2,4, 5-tetrazine-3-carboxamide (Q10)
Figure BDA0003874766470000211
The synthesis was the same as in example 15 except that tert-butyl glycinate in step 1 of example 15 was changed to tert-butyl 5-aminopentanoate and that acetonitrile and p-cyanobenzoic acid in step 2 were changed to 2-cyanopyrimidine and cyanoacetic acid. HRMS (ESI) C 25 H 25 N 10 O 5 + [M+H] + (ii) a Exact Mass:545.2004, found 545.2016.
Example 25
Preparation of N- (6- ((2- (2, 6-dioxopiperidin-3-yl)) -1, 3-dioxoisoindolin-4-yl) amino) ethyl) -6- (pyrimidin-2-yl) -1,2,4, 5-tetrazine-3-carboxamide (Q11)
Figure BDA0003874766470000212
The synthesis was the same as in example 15 except that tert-butyl glycinate in step 1 of example 15 was changed to tert-butyl 6-aminocaproate and that acetonitrile and p-cyanobenzoic acid in step 2 were changed to 2-cyanopyrimidine and cyanoacetic acid. HRMS (ESI) C 26 H 27 N 10 O 5 + [M+H] + (ii) a Exact Mass:559.2160, found 559.2135.
The following are some of the biological activity test results for the compounds of the invention:
the compound of the invention can not only promote the degradation of ALK protein, but also inhibit the activity of ALK kinase, and can play a role in inhibiting the proliferation of certain ALK mutation positive cells.
Western immunoblot (Western Blot): the tumor cells are 8 x 10 5 Cells/well were seeded in 6-well plates containing 2 ml. After 24 hours, cells were treated with different concentrations of drug. After 24 hours the medium was removed and the cells were washed with PBS. Cells were placed on ice and treated with RIPA protein lysate containing Halt protease and phosphatase inhibitors. After the lysate was centrifuged at 14000RPM for 20 minutes at 4 ℃, the supernatant was collected. The equivalent amount of protein is added into 4XSDS loading buffer solution for denaturation treatment at 95 ℃ for 5 minutesFreezing to-20 deg.C or directly performing protein electrophoresis. The electrophoresis gel is prepared by adopting Biyunshi quick gel preparation kit. The electrophoresis tank and related components were purchased from Berkox (Bio-rad) under the same pressure for 105v 1 hours. The membrane transfer was performed using a PVDF membrane, and the membrane transfer system was placed on ice for 1.5 hours using an equal current of 400 mA. After membrane transfer, block with TBST +5% milk powder for 1 hour. Specific steps of immunoblotting refer to the antibody specification of Cell Signaling Technology.
semi-Inhibitory Concentration (IC) of compound 50 ) And (3) determination: compound IC of the present invention 50 The measurement was performed using WST reagent from Promega corporation. The method comprises the following specific steps: cells were seeded at 3000 cells/well in RPMI medium containing 100 microliters of serum. The next day, the drug and the compound of the invention were serially diluted and added to the cells. After 72 hours of treatment with the compound of the present invention, the cell activity assay reagent described above was added to the culture medium according to the instructions to perform cell activity assay. The negative control is DMSO, the positive control is the original drug, and the cells are treated by the same treatment method as the compound. Inhibition of cell growth by Compounds of the invention was plotted by Prism Graphpad software and from this the statistics of Compounds of the invention IC 50 The results are shown in table 1 below.
The P-series and Q-series compounds were dissolved in DMSO, respectively, and then the Q-series compound was administered for 24 hours first and then the P-series for 1 hour as described above. As shown in FIG. 1, the degradation of ALK protein when P1 and Q1 were administered alone in Karpas299 cells and P1 (1, 5, 10. Mu.M) was administered separately with Q1 (1. Mu.M) unchanged. In Karpas299 cells, the Q1 compound (1. Mu.M) was administered for 24 hours, the medium was replaced and P1 (1, 5, 10. Mu.M) was administered for 1 hour, followed by Western Blot analysis.
TABLE 1 Effect of partial Compounds on the growth of human cancer cells Karpas299 (containing NPM-ALK fusion Gene mutation) and SH-SY5Y (containing EML4-ALK fusion Gene mutation) IC 50 (nM)
Figure BDA0003874766470000221
Figure BDA0003874766470000231
DC 50 (concentration of drug corresponding to 50% protein degradation) calculation: the results of calculating the drug concentration range corresponding to half of the gray value by fitting the relationship curve between the drug concentration and the gray value according to the gray value corresponding to the western blotting band after the drug treatment are shown in table 2 below.
TABLE 2 influence of some compounds on ALK protein degradation ability of Karpas299 (containing NPM-ALK fusion gene mutation) and SH-SY5Y (containing EML4-ALK fusion gene mutation) human cancer cells DC 50 (nM)
Figure BDA0003874766470000232
Figure BDA0003874766470000241
As shown in FIG. 1, the degradation of ALK protein when P1 and Q1 were administered alone in Karpas299 cells and P1 (1, 5, 10. Mu.M) was administered separately with Q1 (1. Mu.M) unchanged. In Karpas299 cells, the Q1 compound (1. Mu.M) was administered for 24 hours, the medium was replaced and P1 (1, 5, 10. Mu.M) was administered for 1 hour, followed by Western Blot analysis.

Claims (9)

1. An intracellular self-assembly ALK degrading agent based on a bioorthogonal strategy is characterized in that the degrading agent is obtained by bioorthogonal reaction of two parts of precursor molecules, wherein the precursor molecules are P and Q parts shown as a general formula I, or an optically active body or a racemate, a diastereoisomer mixture and pharmaceutically acceptable salts thereof;
Figure FDA0003874766460000011
wherein, the A group is a group with alkyne, alkene, phosphine or boric acid;
the B group is a group with azide, triazine, tetrazine, amino acid hydrazide or cyclopropane;
<xnotran> C , , ,1,4- 1,3- , , -O-, -CONH-, NHCO-, -NHCONH-, -NH-, -S-, , , , , , , , ; </xnotran>
The D group is an aromatic ring, an aromatic heterocyclic ring, a spiro ring, a bridged ring, a spiro ring, a piperazine ring or a piperazine-piperidine ring.
2. The intracellular self-assembled ALK degrading agent based on the bio-orthogonal strategy according to claim 1, wherein the A group is one of the following groups:
Figure FDA0003874766460000012
the B group is one of the following groups:
Figure FDA0003874766460000021
the C group is one of the following groups:
Figure FDA0003874766460000022
wherein m is any integer of 1-10, and n is any integer of 0-10;
the D group is one of the following groups:
Figure FDA0003874766460000023
wherein, the A1 group and the B1 group generate bioorthogonal reaction, and the A2 group and the B2 group generate bioorthogonal reaction.
3. The intracellular self-assembled ALK degrader based on the bio-orthogonal strategy of claim 1, wherein the P and Q molecules are:
Figure FDA0003874766460000031
wherein m is any integer from 1 to 10, and n is any integer from 0 to 10.
4. The intracellular self-assembled ALK degrading agent based on the bioorthogonal strategy according to claim 3, wherein the P molecule
Figure FDA0003874766460000041
The preparation process comprises the following steps:
Figure FDA0003874766460000042
5. the intracellular self-assembled ALK degrading agent based on the bioorthogonal strategy according to claim 3, wherein the P molecule
Figure FDA0003874766460000043
The preparation process comprises the following steps:
Figure FDA0003874766460000044
6. the intracellular self-assembled ALK degrading agent based on the bioorthogonal strategy according to claim 3, wherein the agent is characterized in thatQ molecule
Figure FDA0003874766460000045
The preparation process of n =0 is as follows:
Figure FDA0003874766460000051
7. the intracellular self-assembled ALK degrading agent based on the bioorthogonal strategy according to claim 3, wherein the Q molecule
Figure FDA0003874766460000052
The preparation process of n =0 is as follows:
Figure FDA0003874766460000053
8. use of the intracellular self-assembled ALK degrader based on bio-orthogonal strategy as claimed in any one of claims 1-3 for the preparation of an anti-tumor drug.
9. The use of claim 8, wherein the tumor is lung cancer, lung adenocarcinoma, anaplastic lymphoma kinase mutation positive non-small cell lung cancer, anaplastic large cell lymphoma, colorectal cancer, astrocytoma, acute myelogenous leukemia, or ovarian cancer.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117700394A (en) * 2024-02-06 2024-03-15 南京大学 Tetrazine compound capable of performing rapid cycloaddition reaction on non-tensinogenic boric acid and biomedical application thereof
CN117700394B (en) * 2024-02-06 2024-05-28 南京大学 Tetrazine compound capable of performing rapid cycloaddition reaction on non-tensinogenic boric acid and biomedical application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190275161A1 (en) * 2016-06-10 2019-09-12 Otsuka Pharmaceutical Co., Ltd. Cliptac composition
CN112341436A (en) * 2020-11-20 2021-02-09 中国药科大学 Benzocarbazole proteolysis targeted chimeric molecule based on targeted inhibition and ALK degradation, preparation method and application

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190275161A1 (en) * 2016-06-10 2019-09-12 Otsuka Pharmaceutical Co., Ltd. Cliptac composition
CN112341436A (en) * 2020-11-20 2021-02-09 中国药科大学 Benzocarbazole proteolysis targeted chimeric molecule based on targeted inhibition and ALK degradation, preparation method and application

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117700394A (en) * 2024-02-06 2024-03-15 南京大学 Tetrazine compound capable of performing rapid cycloaddition reaction on non-tensinogenic boric acid and biomedical application thereof
CN117700394B (en) * 2024-02-06 2024-05-28 南京大学 Tetrazine compound capable of performing rapid cycloaddition reaction on non-tensinogenic boric acid and biomedical application thereof

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