CN115124590A - PROTAC compound for targeted degradation of FLT3-ITD mutant protein and preparation method and application thereof - Google Patents

PROTAC compound for targeted degradation of FLT3-ITD mutant protein and preparation method and application thereof Download PDF

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CN115124590A
CN115124590A CN202210794029.6A CN202210794029A CN115124590A CN 115124590 A CN115124590 A CN 115124590A CN 202210794029 A CN202210794029 A CN 202210794029A CN 115124590 A CN115124590 A CN 115124590A
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protac
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ligand
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周芙玲
周海兵
辛丽兰
许荣
谭御心
张楠
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Zhongnan Hospital of Wuhan University
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Abstract

The invention discloses a PROTAC compound for targeted degradation of FLT3-ITD mutant protein and a preparation method and application thereof, wherein the PROTAC compound for targeted degradation of FLT3-ITD mutant protein has a structure shown in the following formula I or formula IIStructural formula (xvi): linker is a linking group selected from the group consisting of-alkylene, -alkoxy, and-heterocyclyl; e3 ligand is an E3 ligase ligand. The PROTAC compound for targeted degradation of FLT3-ITD mutant protein has AML resistance activity, has obvious inhibitory activity on human acute myelogenous leukemia cell MOLM-13 cell line, can be developed as a novel AML resistance drug, and has wide application prospect.
Figure DDA0003731451940000011

Description

PROTAC compound for targeted degradation of FLT3-ITD mutant protein and preparation method and application thereof
Technical Field
The invention relates to the technical field of medicines, and in particular relates to a PROTAC compound for targeted degradation of FLT3-ITD mutant protein, and a preparation method and application thereof.
Background
Acute Myeloid Leukemia (AML) is one of the most lethal hematological malignancies worldwide. Among all subtypes of leukemia, AML accounts for the highest percentage of leukemic deaths, approximately as high as 62%, and patients have a poor prognosis, short survival, and less than 30% overall five-year survival. The most common mutant form in newly diagnosed AML patients is the FLT3-ITD mutation, which is closely associated with poor prognosis, high mortality, and high relapse rates in patients.
The main drugs aiming at FLT3-ITD mutation are small molecule inhibitors, which can be divided into the first generation and the second generation according to the specificity of FLT3 protein kinase. The first generation of inhibitors are non-selective and multi-target inhibitors, such as midostaurin and the like. As single-drug therapy, the medicines have poor curative effect and more adverse reactions, and part of the medicines are further researched as combined therapy. The second generation inhibitors were more selective and showed stronger inhibition and less off-target toxicity to the FLT3-ITD mutation, with gillitinib receiving FDA approval as a single drug in 2018 for treatment of relapsed or refractory AML patients. However, despite the clinical efficacy of the second generation inhibitors, they still face some unexpected safety and drug resistance problems, which are urgently needed to be overcome due to the change of microenvironment or secondary mutation.
Compared with the action mechanism that the traditional small molecule inhibitor inhibits the function of the target protein by combining the active site of the target protein, the PROTAC (proteolytic targeting chimera) can degrade the target protein by hijacking the inherent ubiquitin-proteasome system in vivo, thereby achieving the purpose of treatment. PROTAC is a bifunctional molecule consisting of a small molecule ligand that recognizes a target protein, an E3 ubiquitin protein ligase recruiter, and a linker chain joining the two. Because the requirement on the affinity of the targeting protein micromolecule ligand is not high, and the catalytic property of the targeting protein micromolecule ligand ensures that a small amount of the targeting protein micromolecule ligand can play a protein degradation function, the technology can well overcome the problem of serious drug resistance of the existing drugs.
Therefore, there is a need to develop a ProTAC compound for targeted degradation of FLT3-ITD mutant protein and a preparation method thereof.
Disclosure of Invention
The PROTAC compound for targeted degradation of FLT3-ITD mutant protein has AML resistance activity, has obvious inhibition activity on human acute myelogenous leukemia cell MOLM-13 cell line, can be developed as a novel AML resistance drug, and has wide application prospect.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, there is provided a procac compound targeted for degrading an FLT3-ITD mutein, the procac compound targeted for degrading an FLT3-ITD mutein having the structural formula I or II below:
Figure BDA0003731451920000021
in the formula I or the formula II, the reaction solution,
linker is a linking group selected from one or more of the following linear or branched alkylene, alkoxy or heterocyclic groups, - (CH) 2 ) n -、-(CH 2 ) n CO-、-NR 1 (CH 2 ) n CO-、-NR 2 (CH 2 ) n -、-(OCH 2 CH 2 O) n -、-(CH 2 CH 2 O) n -、-(OCH 2 CH 2 OCH 2 ) n -、-(CH 2 CH 2 OCH 2 ) n -、-(CH 2 CH 2 OCH 2 CH 2 ) n -, alkenylene, alkynylene, cycloalkylene, heteroarylene group or any combination thereof, wherein n represents a natural number from 1 to 20, R 1 、R 2 Each independently of the other being H or C 1-10 An alkyl group;
e3 ligand is an E3 ligase ligand selected from one of the following ligands:
Figure BDA0003731451920000022
further, the PROTAC compound for targeted degradation of the FLT3-ITD mutein comprises one of the following compounds X1-X22, wherein the compounds X1-X22 are shown in Table 1.
Table 1: PROTAC overview for Targeted degradation of FLT3-ITD muteins
Figure BDA0003731451920000023
Figure BDA0003731451920000031
Figure BDA0003731451920000041
Figure BDA0003731451920000051
Figure BDA0003731451920000061
Figure BDA0003731451920000071
The compounds numbered as X-3, X-4, X-5, X-6, X-7, X-8, X-21 and X-22 in the table 1 are PROTAC compounds with excellent activity and targeted degradation of FLT3-ITD mutant protein.
In a second aspect of the invention, a pharmaceutical composition against AML is provided, comprising the PROTAC-like compound or a pharmacologically or physiologically acceptable salt thereof targeted to degrade the FLT3-ITD mutein, and a pharmaceutically acceptable carrier or excipient.
In a third aspect of the invention, a method for preparing the PROTAC compounds for targeted degradation of FLT3-ITD muteins is provided, the method comprising:
the PROTAC compounds for targeted degradation of FLT3-ITD mutant protein are divided into compounds of series I, series II, series III and series IV,
the series I PROTAC compound is a compound shown as a general formula (I), wherein Linker is n ═ 1-20-alkylene, E3 ligand is the VHL ligand, the methyl-bearing VHL ligand, pomalidomide, lenalidomide or 4-hydroxythalidomide;
the PROTAC compound in the series II is a compound shown as a general formula (II), Linker is n-alkylene of 1-20, and E3 ligand is the methyl-bearing VHL ligand;
the series III PROTAC compound is a compound shown as a general formula (I), Linker is n-alkoxy of 1-20, and E3 ligand is the methyl-bearing VHL ligand;
the series IV PROTAC compounds are shown in a general formula (I), Linker is a heterocyclic group, and E3 ligand is the VHL ligand with methyl;
the preparation of the PROTAC compound of the series I comprises two aspects:
(1) the Gilitinib derivative I reacts with a VHL ligand derivative substituted by a terminal halogen atom or a VHL ligand derivative with methyl in DMF (N, N-dimethylformamide) under the conditions of potassium carbonate and potassium iodide to obtain the PROTAC compound I (1). The reaction is carried out at 90 ℃ and the preferred time for the reaction is 12 h.
Figure BDA0003731451920000081
Synthetic route of PROTAC compounds in series I (1)
(2) The Gilitinib derivative I reacts with an alkyl chain CRBN ligand derivative substituted by a terminal halogen atom in DMF under the condition of sodium bicarbonate to obtain a series I (2) PROTAC compound. The reaction is carried out at 90 ℃ and the preferred time for the reaction is 5 h.
Figure BDA0003731451920000082
Figure BDA0003731451920000091
Synthetic route of PROTAC compounds in series I (2)
The preparation of the series II PROTAC compound comprises the following steps: the VHL ligand derivative with the terminal N-Boc (tert-butyloxycarbonyl) substituent and the connecting chain as the alkyl chain firstly removes the Boc protective group under the acidic condition, and then reacts with the Gilitinib derivative II in DMF under the conditions of HATU (O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate) and DIPEA (N, N-diisopropylethylamine) to obtain a series II PROTAC compound. The reaction is carried out at room temperature, preferably for 0.5 h.
Figure BDA0003731451920000092
Synthetic route of series II PROTAC compounds
The preparation of series III PROTAC compounds comprises the following steps: the geritinib derivative I with the terminal substituted by tert-butyl ester and the connecting chain as an alkoxy chain is subjected to tert-butyl ester removal under an acidic condition, and then reacts with a VHL ligand with methyl in DMF under the conditions of HATU and DIPEA to obtain a series III PROTAC compound. The reaction is carried out at room temperature, preferably for 0.5 h.
Figure BDA0003731451920000093
Synthesis route of series III PROTAC compounds
The preparation method of the series IV PROTAC compound comprises the following steps: the geritinib derivative I with the terminal substituted by tert-butyl ester and the connecting chain as a heterocyclic chain firstly removes the tert-butyl ester under an acidic condition, and then reacts with a VHL ligand with a methyl group in DMF under the conditions of HATU and DIPEA to obtain a series of IV PROTAC compounds. The reaction is carried out at room temperature, preferably for 0.5 h.
Figure BDA0003731451920000094
Synthetic route of series IV PROTAC compounds
Wherein the linking chain is an-alkylene, -alkoxy chain or-heterocyclic group of n ═ 1 to 20; r is any one of H atom or CH 3; x is CO or CH; y is N or C.
Further, the ratio of the amounts of the gelitinib derivative i, the VHL ligand derivative (or the methyl-bearing VHL ligand derivative), the potassium carbonate and the potassium iodide in the series i is 1: 1.2: 4: 1.4;
gelitinib derivative I, CRBN ligand derivative and NaHCO in series II 3 The ratio of the amounts of substances (1): 1.2: 2; the ratio of the amount of the Gelitinib derivative II, the methyl-bearing VHL ligand derivative, HATU and DIPEA in the series II is 1: 1.2: 1.1: 6.0;
the ratio of the amounts of the substance of the Gilitinib derivative I with alkoxy side chain in the series III, the substance with methyl VHL ligand, HATU and DIPEA is 1: 1.2: 1.1: 6.0;
the ratio of the amount of substances of the Gilitinib derivative I with a heterocyclic side chain, the substance with a methyl VHL ligand, HATU and DIPEA in the series IV is 1: 1.2: 1.1: 6.0.
in a third aspect of the invention, the PROTAC compound for targeted degradation of FLT3-ITD mutant protein or the anti-AML pharmaceutical composition is provided for application as a FLT3-ITD degrading agent in resisting AML.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
the PROTAC compound for targeted degradation of FLT3-ITD mutant protein can effectively inhibit MOLM-13 cell activity, has better FLT3-ITD protein degradation capability and better activity on AML mutant cells, and can be used for preparing AML-resistant drugs.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 shows the results of the proteolytic activity of a portion of the compounds synthesized according to the invention.
Detailed Description
The present invention will be specifically explained below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented thereby. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be obtained by an existing method.
The ProTAC compound for targeted degradation of FLT3-ITD mutant protein, the preparation method and the application thereof in resisting acute myeloid leukemia will be described in detail in the following examples, comparative examples and experimental data.
Example 1, 6-Ethyl-3- (3-methoxy-4- (piperazin-1-yl) phenyl) amino) -5- (tetrahydro-2H-pyran-4-yl) amino) pyrazine-2-carboxamide
This preparation provides two synthetic routes in the present invention, one of which is shown below.
Figure BDA0003731451920000111
Step 1: preparation of tert-butyl 4- (2-methoxy-4-nitrophenyl) piperazine-1-carboxylate
Figure BDA0003731451920000112
The compound 2-fluoro-5-nitrobenzyl ether 1(1.0g, 1.0eq) and the compound 1-tert-butoxycarbonylpiperazine 2(1.2g, 1.1eq) were weighed into a 100mL round-bottomed flask, dissolved by adding DMF (10mL), followed by addition of anhydrous K 2 CO 3 (2.4g, 3.0eq), heated to 95 ℃ and stirred for 4 h. And (3) after TLC monitoring reaction is completed, cooling the reaction solution to room temperature, adding a proper amount of water, separating out a solid, performing suction filtration, rinsing the filter cake twice with PE, and drying at 65 ℃ to obtain a yellow solid product 3, 1.8g, wherein the reaction yield is 91.32%. 1 H NMR(400MHz,Chloroform-d)δ7.88(dd,J＝8.8,2.5Hz,1H),7.74(d,J＝2.5Hz,1H),6.90(d,J＝8.8Hz,1H),3.98(s,3H),3.63(t,J＝5.0Hz,4H),3.18(t,J＝5.0Hz,4H),1.50(s,9H).
Step 2: preparation of tert-butyl 4- (4-amino-2-methoxyphenyl) piperazine-1-carboxylate
Figure BDA0003731451920000121
Weighing Compound 3(1.0g, 1.0 e)q) in a 100mL round bottom flask, add solvent EtOH (25mL) to dissolve, then add Fe powder (1.3g, 8.0eq), weigh NH 4 Cl (1.6g, 10.0eq) was added as an aqueous solution, warmed to 90 ℃ and stirred at this temperature for 1 h. After TLC to monitor completion of the reaction, the reaction was filtered through celite, the filter cake was washed with EtOH (25mL), the filtrate was collected and concentrated under reduced pressure, the concentrated product was dissolved with EA (50mL), washed twice with water (2X 50mL), anhydrous Na 2 SO 4 The organic phase was dried, EA was removed by distillation under the reduced pressure, and purified by column chromatography to give the product 4 as a light brown solid in a yield of 65.85 mg, 600.0 mg. 1 H NMR(400MHz,Chloroform-d)δ6.76(d,J＝8.2Hz,1H),6.29(d,J＝2.5Hz,1H),6.26(dd,J＝8.2,2.5Hz,1H),3.83(s,3H),3.60(t,J＝5.0Hz,4H),3.56(s,2H),2.91(t,J＝5.0Hz,4H),1.50(s,9H).
And step 3: preparation of tert-butyl 4- (3-carbamoyl-6-chloro-5-ethylpyrazin-2-yl) amino) -2-methoxyphenyl) piperazine-1-carboxylate
Figure BDA0003731451920000122
Compound 4(200.0mg, 1.0eq) and compound 3, 5-dichloro-6-ethylpyrazine-2-carboxamide 5(171.8mg, 1.2eq) were weighed into a sealed tube of 40mL volume, dissolved by the addition of NMP (2mL), followed by the addition of DIPEA (252.3mg, 2.0eq), warmed to 170 ℃ and stirred for 2 h. TLC to monitor completion of the reaction, the reaction mixture was extracted with EA (3X 30mL) and water (40mL), and the organic layers were combined and washed with saturated brine (2X 40mL), anhydrous Na 2 SO 4 Drying, distilling under reduced pressure to remove EA, and purifying by column chromatography to obtain yellow solid product 6, 304.0mg, reaction yield 95.16%. 1 H NMR(400MHz,Chloroform-d)δ10.73(s,1H),7.77(d,J＝4.3Hz,1H),7.41(d,J＝2.4Hz,1H),7.18(dd,J＝8.6,2.4Hz,1H),6.91(d,J＝8.5Hz,1H),5.65(d,J＝4.0Hz,1H),3.93(s,3H),3.64(t,J＝4.9Hz,4H),3.00(t,J＝5.0Hz,4H),2.88(q,J＝7.5Hz,2H),1.51(s,9H),1.33–1.30(m,3H).
And 4, step 4: preparation of tert-butyl 4- (4- (3-carbamoyl-5-ethyl-6- (tetrahydro-2H-pyran-4-yl) amino) pyrazin-2-yl) amino) -2-methoxyphenyl) piperazine-1-carboxylate
Figure BDA0003731451920000123
Compound 6(230mg, 1.0eq) and compound 4-aminotetrahydropyran 7(189.5mg, 4.0eq) were weighed into a 40mL sealed tube, dissolved by the addition of NMP (1.5mL), followed by the addition of DIPEA (242.3mg, 4.0eq), and the sealed tube placed in a microwave reactor and stirred at 120 ℃ for 2 h. TLC to monitor completion of the reaction, the reaction mixture was extracted with EA (3X 30mL) and water (40mL), and the organic layers were combined and washed with saturated brine (2X 40mL), anhydrous Na 2 SO 4 Drying, distilling off EA under reduced pressure, and purifying by column chromatography to obtain yellow solid product 8, 247.1mg, reaction yield 94.93%. 1 H NMR(400MHz,Chloroform-d)δ10.78(s,1H),7.55(d,J＝2.4Hz,1H),7.52(d,J＝2.3Hz,1H),6.92(d,J＝2.3Hz,1H),6.85(d,J＝8.7Hz,1H),5.28(d,J＝4.6Hz,1H),4.67(d,J＝7.3Hz,1H),4.27–4.18(m,1H),4.08–4.02(m,2H),3.90(s,3H),3.64(t,J＝5.0Hz,4H),3.59–3.51(m,2H),3.04–2.96(m,4H),2.54(q,J＝7.4Hz,2H),2.14–2.06(m,2H),1.64–1.54(m,2H),1.51(s,9H),1.32(t,J＝7.4Hz,3H).
And 5: preparation of 6-ethyl-3- (3-methoxy-4- (piperazin-1-yl) phenyl) amino) -5- (tetrahydro-2H-pyran-4-yl) amino) pyrazine-2-carboxamide
Figure BDA0003731451920000131
Compound 8(385.1mg, 1.0eq) was weighed into a 50mL round bottom flask, dissolved by addition of DCM (4mL), and stirred at rt for 1h with addition of excess TFA (4 mL). After TLC monitoring the reaction was complete, DCM was removed by distillation under reduced pressure and saturated NaHCO was used 3 Adjusting pH of the solution to weak alkalinity, extracting the reaction solution with EA (3X 30mL), combining the organic layers, anhydrous Na 2 SO 4 Drying, removing the solvent by distillation under the reduced pressure, and purifying by column chromatography to obtain yellow solid product 9, 262.5mg, reaction yield 83.14%. 1 H NMR(400MHz,Chloroform-d)δ10.75(s,1H),7.54(d,J＝4.4Hz,1H),7.50(dd,J＝8.6,2.4Hz,1H),6.92(d,J＝2.3Hz,1H),6.87(d,J＝8.7Hz,1H),5.38(d,J＝4.5Hz,1H),4.67(d,J＝7.3Hz,1H),4.27–4.17(m,1H),4.07–4.00(m,2H),3.89(s,3H),3.59–3.50(m,2H),3.12–3.06(m,4H),3.06–2.98(m,4H),2.53(q,J＝7.4Hz,2H),2.14–2.06(m,2H),1.64–1.52(m,2H),1.31(t,J＝7.4Hz,3H).
The second synthetic route is shown below.
Figure BDA0003731451920000141
Step 1: preparation of 3, 5-dichloro-6-ethylpyrazine-2-carboxylic acid methyl ester
Figure BDA0003731451920000142
The compound 3, 5-dichloro-6-ethylpyrazine-2-carboxamide (600mg, 1.0eq) was weighed into a 50mL round bottom flask, dissolved by adding 2mol/L of hydrochloric acid methanol solution (14mL, excess), and the round bottom flask was placed in a 70 ℃ oil bath for reflux reaction for 24 h. After TLC detection reaction is completed, reduced pressure distillation is used to remove solvent, and saturated NaHCO is added 3 The solution (40mL) was dissolved, extracted with DCM (3X 30mL), the organic layers were combined and washed with saturated brine (2X 40mL), anhydrous Na 2 SO 4 Drying, removal of the solvent by distillation under reduced pressure gave the crude product which was purified by column chromatography to give 446mg of a colorless transparent oily liquid 10 in 69.59% yield. 1 H NMR(400MHz,Chloroform-d)δ4.01(s,3H),3.00(q,J＝7.5Hz,2H),1.32(t,J＝7.5Hz,3H).
And 2, step: preparation of methyl 3-chloro-6-ethyl-5- (tetrahydro-2H-pyran-4-yl) amino) pyrazine-2-carboxylate
Figure BDA0003731451920000143
Compound 10(446mg, 1.0eq) was weighed into a 50mL single vial, 4-aminotetrahydropyran (383.8mg, 2.0eq) was added followed by DMF (3mL) and triethylamine (576.0mg, 3.0eq) in that order, and stirred at room temperature for 2 h. TLC to monitor completion of the reaction, dilute the solution with water (30mL), extract the reaction with EA (3X 30mL), combine the organic layers and brineDMF was removed by washing with water (2X 40mL), anhydrous Na 2 SO 4 Drying, removal of the solvent by distillation under the reduced pressure gave a crude product which was purified by column chromatography to give 469mg of pure white solid 11 in 82.40% yield. 1 H NMR(400MHz,Chloroform-d)δ5.02(d,J＝7.5Hz,1H),4.28–4.17(m,1H),4.02–3.95(m,2H),3.92(s,3H),3.59–3.50(m,2H),2.65(q,J＝7.5Hz,2H),2.07–2.00(m,2H),1.64–1.49(m,2H),1.27(t,J＝7.5Hz,3H).
And step 3: preparation of methyl 3- (4- (tert-butoxycarbonyl) piperazin-1-yl) -3-methoxyphenyl) amino) -6-ethyl-5- ((tetrahydro-2H-pyran-4-yl) amino) pyrazine-2-carboxylate
Figure BDA0003731451920000151
A40 mL sealed tube was taken, compound 11(214.5mg, 1.1eq), compound 4(200.0mg, 1.0eq), 4, 5-bis (diphenylphosphino) -9, 9-dimethylxanthene (xanth-phos, 113.0mg, 0.3eq), cesium carbonate (636.0mg, 3.0eq) and palladium acetate (43.8mg, 0.3eq) were weighed in order, and THF (1.5mL) was added as a solvent under argon protection. The reaction system is reacted for 1h in a microwave reactor at 100 ℃. After completion of the reaction, the reaction mixture was returned to room temperature, about 40mL of water was added, the reaction mixture was extracted with EA (3X 30mL), the organic layers were combined, and saturated brine (40mL) and saturated NaHCO were used for the organic layer in this order 3 Washing with anhydrous Na 2 SO 4 Drying, removal of the solvent by distillation under the reduced pressure gave the crude product which was purified by column chromatography to give 306mg of yellow solid 12 in 82.41% yield. 1 H NMR(400MHz,Chloroform-d)δ10.78(s,1H),7.53(t,J＝3.7Hz,1H),7.50(d,J＝2.4Hz,1H),6.92(d,J＝2.3Hz,1H),6.84(d,J＝8.6Hz,1H),5.41(d,J＝4.6Hz,1H),4.72(d,J＝7.3Hz,1H),4.24–4.15(m,1H),4.07–4.00(m,2H),3.89(s,3H),3.63(t,J＝5.0Hz,4H),3.56–3.47(m,2H),2.98(dd,J＝8.2,3.5Hz,4H),2.53(q,J＝7.4Hz,2H),2.11–2.04(m,2H),1.63–1.55(m,2H),1.50(s,9H),1.30(t,J＝7.4Hz,3H).
And 4, step 4: preparation of 3- (4- (tert-butoxycarbonyl) piperazin-1-yl) -3-methoxyphenyl) amino) -6-ethyl-5- ((tetrahydro-2H-pyran-4-yl) amino) pyrazine-2-carboxylic acid
Figure BDA0003731451920000152
Compound 12(306mg, 1.0eq) was weighed into a 50mL single-neck flask, dissolved in methanol (8mL), and LiOH. H was added 2 O (101.2mg, 4.0eq) was reacted at 80 ℃ for 18 h. TLC monitored reaction completion using EA (3X 30mL) with saturated NH 4 Extraction was performed with a Cl (50mL) solution, and the organic layers were combined and washed with saturated brine (2X 40mL), anhydrous Na 2 SO 4 Drying and distillation under reduced pressure removed EA to give crude brown solid 13(315.5mg) which was used in the next reaction without further purification.
And 5: preparation of tert-butyl 4- (4- (3-carbamoyl-5-ethyl-6- (tetrahydro-2H-pyran-4-yl) amino) pyrazin-2-yl) amino) -2-methoxyphenyl) piperazine-1-carboxylate
Figure BDA0003731451920000161
Compound 13(315.5mg, 1.0eq) was weighed into a 50ml single-neck flask, dissolved by adding solvent DMF (5ml), followed by dropwise addition of DIPEA (293.0mg, 4.0eq) and stirring for several minutes, followed by sequential addition of NH 4 Cl (45.5mg, 1.5eq) and HATU (237.1mg, 1.1eq) were reacted and the reaction was stirred at room temperature overnight. TLC showed complete reaction, the reaction mixture was extracted with EA (3X 30mL) and water (40mL), and the organic layers were combined and washed with saturated brine (2X 40mL) and anhydrous Na 2 SO 4 Drying, distillation under reduced pressure to remove EA, and column chromatography purification gave 300.0mg of yellow solid 8 in 95.40% yield. 1 H NMR(400MHz,Chloroform-d)δ10.78(s,1H),7.53(t,J＝3.7Hz,1H),7.50(d,J＝2.4Hz,1H),6.92(d,J＝2.3Hz,1H),6.84(d,J＝8.6Hz,1H),5.41(d,J＝4.6Hz,1H),4.72(d,J＝7.3Hz,1H),4.26–4.16(m,1H),4.07–4.00(m,2H),3.89(s,3H),3.63(t,J＝5.0Hz,4H),3.58–3.49(m,2H),2.98(dd,J＝8.2,3.5Hz,4H),2.53(q,J＝7.4Hz,2H),2.12–2.06(m,2H),1.63–1.55(m,2H),1.50(s,9H),1.30(t,J＝7.4Hz,3H).
Step 6: preparation of 6-ethyl-3- (3-methoxy-4- (piperazin-1-yl) phenyl) amino) -5- (tetrahydro-2H-pyran-4-yl) amino) pyrazine-2-carboxamide
Figure BDA0003731451920000162
Compound 8(300.0mg, 1.0eq) was weighed into a 50ml single-necked flask, dissolved in DCM (1.5ml) and added TFA (1.5ml, excess) dropwise and stirred at room temperature for 30 min. TLC monitored the reaction complete and the solvent and TFA were distilled off under reduced pressure. Purification by column chromatography gave 9 as a yellow solid, 230.0mg, 93.52% yield. 1 H NMR(400MHz,Chloroform-d)δ10.76(s,1H),7.57–7.52(m,1H),7.49(dd,J＝8.6,2.3Hz,1H),6.92(d,J＝2.3Hz,1H),6.86(d,J＝8.7Hz,1H),5.47(s,1H),4.69(d,J＝7.3Hz,1H),4.27–4.17(m,1H),4.06–4.01(m,2H),3.88(s,3H),3.58–3.50(m,2H),3.15–3.11(m,4H),3.09–3.02(m,4H),2.53(q,J＝7.4Hz,2H),2.13–2.06(m,2H),1.64–1.52(m,2H),1.32(t,J＝7.4Hz,3H).
The synthesis method of the Gilitinib derivative II is the same as that of the compound 13 in the first synthesis route.
Example 2, (2S, 4R) -1- (S) -2-amino-3, 3-dimethylbutyryl) -4-hydroxy-N- (S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide
Figure BDA0003731451920000171
Step 1: preparation of tert-butyl (S) - (1- (4-bromophenyl) ethyl) carbamate
Figure BDA0003731451920000172
(S) - (-) -1- (4-bromobenzene) ethylamine 14(5g, 1.0eq) was weighed into a 100mL single-necked flask, dissolved in DCM (15mL) as a solvent, and Et was added immediately 3 N (2.8g, 1.1eq), after a few minutes in an ice bath, a solution of tert-butyl dicarbonate (6.0g, 1.1eq) in DCM (5mL) was added slowly with stirring. After the addition, the ice bath was removed and the reaction was carried out at room temperature for 2 hours. TLC monitored completion of reaction, and extraction with water and DCM (3X 30mL)Take the run, combine the organic layers and backwash twice with water (2X 40mL), anhydrous Na 2 SO 4 Drying, and distilling under reduced pressure to remove DCM gave 15, 7.57g as a crude white solid product in 100% yield.
And 2, step: preparation of tert-butyl (S) - (1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamate
Figure BDA0003731451920000173
Compound 15(6g, 1.0eq), 4-methylthiazole (4.0g, 2.0eq), palladium acetate (44.8mg, 0.01eq) and potassium acetate (3.9g, 2.0eq) were weighed out separately in a 150mL single vial, argon was replaced, solvent DMAC (15mL) was added under inert gas protection to dissolve, and the mixture was heated to 90 ℃ for reaction for 18 h. And after the reaction is finished, the funnel is paved with diatomite and filtered, filter residues are washed by DMAC (20mL), filtrate is taken, water with the volume being 4-5 times that of the filtrate is added into the filtrate under the ice bath condition, the mixture is stirred for 1 hour, and offwhite solid is separated out. And filtering to obtain filter residues, washing with PE (polyethylene), drying, and performing column chromatography purification operation to obtain a pure white product 17, 3.3g with the yield of 52%. 1 H NMR(400MHz,Chloroform-d)δ8.68(s,1H),7.42(d,J＝8.4Hz,2H),7.37(d,J＝8.1Hz,2H),4.94(s,1H),2.54(s,3H),1.48(d,J＝6.8Hz,3H),1.44(s,9H).
And 3, step 3: preparation of tert-butyl (2S, 4R) -4-hydroxy-2- (S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidine-1-carboxy
Figure BDA0003731451920000181
Compound 17(6.0g, 1.0eq) was weighed into a 50mL single vial, dissolved by the addition of DCM (6mL), followed by the dropwise addition of TFA (12mL) and stirring at room temperature for 30 min. After TLC monitoring the reaction was complete, the solvent and TFA were distilled off under reduced pressure to give brown oily liquid 18 which was used directly in the next reaction. The deprotected intermediate 18 was dissolved in DMF (10mL), followed by dropwise addition of DIPEA (9.7g, 4.0eq), stirring at room temperature for several minutes, followed by addition of Boc-L-hydroxyproline (4.8g, 1.1eq) and HATU (7.9g, 1.1eq) in that order, and reaction at room temperature for 30 min. TLC monitoringAfter completion of the reaction, the reaction mixture was extracted with EA (3X 40mL) and water, the organic phases were combined, washed with saturated brine (2X 40mL), and then dried over anhydrous Na 2 SO 4 Drying, distillation under reduced pressure to remove EA, and further column chromatography purification yielded 19, 5.8g of white solid product. The overall yield of the two steps is 71%.
And 4, step 4: preparation of tert-butyl ((S) -1- ((2S, 4R) -4-hydroxy-2- ((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3, 3-dimethyl-1-oxobutan-2-yl) carbamate
Figure BDA0003731451920000182
Compound 19(5.8g, 1.0eq) was weighed into a 50mL one-neck flask, dissolved by the addition of DCM (10mL), followed by the dropwise addition of TFA (10mL) and stirred at room temperature for 30 min. After TLC monitoring the reaction was complete, the solvent and TFA were distilled off under reduced pressure to give a brown oily liquid 20 which was used directly for the next reaction. The deprotected intermediate 20 was dissolved in DMF (6mL), followed by dropwise addition of DIPEA (10.4g, 6.0eq), stirring at room temperature for several minutes, followed by addition of Boc-L-tert-leucine (4.7g, 1.5eq) and HATU (5.6g, 1.1eq) in that order, and reaction at room temperature for 30 min. After completion of the TLC monitoring, the reaction mixture was extracted with EA (3X 40mL) and water, the organic phases were combined, washed with saturated brine (2X 40mL), and then anhydrous Na was added 2 SO 4 Drying, distilling under reduced pressure to remove EA, and further purifying by column chromatography to obtain 21, 2.6g of white solid product. The overall yield of the two steps is 36%.
And 5: preparation of (2S, 4R) -1- (S) -2-amino-3, 3-dimethylbutyryl) -4-hydroxy-N- (S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide
Figure BDA0003731451920000191
Compound 21(3.0g, 1.0eq) was weighed into a 50mL one-neck flask, dissolved by the addition of DCM (6mL), followed by the dropwise addition of TFA (6mL) and stirred at room temperature for 30 min. After TLC monitoring of the reaction completion, the solvent and most of the TFA were distilled off under reduced pressure and saturated NaHCO was used 3 The pH of the solution was adjusted to 9-10, solvent extraction (3X 30mL) with DCM: MeOH 10:1 and organic phase was combined with anhydrous Na 2 SO 4 Drying, removing the solvent by distillation under the reduced pressure, and further purifying by column chromatography to obtain 22 as a white solid, 2.0g, in 82% yield. 1 H NMR(400MHz,Chloroform-d)δ8.66(s,1H),8.22(d,J＝7.3Hz,1H),7.37(s,4H),5.07(p,J＝7.0Hz,1H),4.78(t,J＝8.3Hz,1H),4.41(d,J＝3.7Hz,1H),3.79(d,J＝11.3Hz,1H),3.62–3.55(m,1H),3.38(s,1H),2.51(s,3H),2.22–2.12(m,2H),1.50(d,J＝6.9Hz,3H),0.99(s,9H). 13 C NMR(100MHz,Chloroform-d)δ173.26,170.77,150.40,148.28,143.97,131.66,130.52,129.46,126.27,69.84,60.33,58.77,56.87,48.87,37.13,35.67,26.13,22.42,16.08.
VHL ligands without methyl group were prepared as above.
Example 3 preparation of an alkyl chain VHL ligand substituted with a halogen atom at the end
By way of example for the preparation of (2S, 4R) -1- (S) -2- (5-bromopentylamino) -3, 3-dimethylbutyryl) -4-hydroxy-N- (S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide (24), it was obtained by the following reaction.
Figure BDA0003731451920000192
Compound 22(150.0mg, 1.0eq) and acid 23(73.3mg, 1.2eq) of the corresponding chain length were weighed into a 50mL single vial, dissolved by adding solvent DMF, followed by dropwise addition of DIPEA (261.6mg, 6.0eq), stirring for several minutes, followed by addition of condensing agent HATU (141.4mg, 1.1eq) and reaction at room temperature for 30 min. After TLC to monitor the reaction completion, the reaction mixture was extracted with EA (3X 30mL) and water, the organic phases were combined and washed with saturated brine (2X 40mL) to remove excess DMF, followed by anhydrous Na 2 SO 4 Drying, distilling under reduced pressure to remove EA, and further purifying by column chromatography to obtain the corresponding white solid product 24, 105mg, reaction yield 62%. 1 H NMR(400MHz,Chloroform-d)δ8.70(s,1H),7.51(d,J＝7.9Hz,1H),7.41(d,J＝8.4Hz,2H),7.38(d,J＝8.5Hz,2H),6.39(d,J＝8.9Hz,1H),5.10(p,J＝7.0Hz,1H),4.68(t,J＝7.9Hz,1H),4.59(d,J＝8.9Hz,1H),4.51(s,1H),4.03(d,J＝11.3Hz,1H),3.64(dd,J＝11.3,3.8Hz,1H),3.39(t,J＝6.4Hz,2H),2.53(s,3H),2.44–2.36(m,1H),2.25–2.17(m,2H),2.12–2.03(m,1H),1.90–1.81(m,2H),1.79–1.70(m,2H),1.50–1.48(m,3H),1.05(s,9H).
Other alkyl chain VHL ligands substituted with halogen at the end were prepared as described above.
Example 4 preparation of alkyl chain CRBN ligand substituted with halogen atom at end
Taking the preparation of 8-bromo-N- (2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisooctanol-4-yl) octanoyl amide (29) as an example, it was obtained by the following reaction.
Figure BDA0003731451920000201
Weighing the compound 25(1.5g, 1.0eq) and placing the mixture in a single-mouth bottle, replacing the mixture with argon, adding a solvent DCM (5mL) to dissolve the mixture under the protection of inert gas, slowly adding the compound 26, namely oxalyl chloride (1.7g, 2.0eq) dropwise in ice bath, then adding a drop of DMF dropwise, stirring the mixture for a plurality of minutes in ice bath, and reacting the mixture for 2 hours at room temperature. After completion of the reaction, DCM was removed by concentration under reduced pressure to give compound 27, which was used in the next step without purification. The mixture was dissolved in THF (5ml), and then compound 28(0.92g, 0.5eq) was added and reacted at room temperature overnight. After the reaction, THF was removed by distillation under reduced pressure, and the product was purified by column chromatography to give 29 as a white solid, 850mg, in a total yield of 26% in two steps. 1 H NMR(400MHz,Chloroform-d)δ9.42(s,1H),8.83(d,J＝8.9Hz,2H),7.72(dd,J＝8.5,7.3Hz,1H),7.55(d,J＝7.3Hz,1H),5.03–4.94(m,1H),3.41(t,J＝6.8Hz,2H),2.94–2.85(m,1H),2.85–2.72(m,2H),2.47(t,J＝7.5Hz,2H),2.21–2.14(m,1H),1.90–1.82(m,2H),1.80–1.71(m,2H),1.47–1.33(m,6H).
Taking the preparation of 4- (8-bromooctyl) oxy) -2- (2, 6-dioxopiperidin-3-yl) isoindole-1, 3-dione (32) as an example, it was obtained by the following reaction.
Figure BDA0003731451920000211
Compound 30(300mg, 1.0eq) and compound 31(357.1mg, 1.2eq) were weighed into a 50mL single vial, dissolved in DMF (5mL) and then DIPEA (424.2mg, 3.0eq) was added dropwise and heated to 90 ℃ for 3 h. After TLC to monitor the reaction completion, the reaction mixture was extracted with EA (3X 30mL) and water, the organic phases were combined and washed with saturated brine (2X 40mL) to remove excess DMF, followed by anhydrous Na 2 SO 4 Drying, distilling under reduced pressure to remove EA, and further purifying by column chromatography to give compound 32 as a white solid, 165.0mg, in 32% yield. 1 H NMR(400MHz,Chloroform-d)δ9.12(d,J＝5.7Hz,1H),7.60(t,J＝7.9Hz,1H),7.34(dd,J＝7.5,2.6Hz,1H),7.16(d,J＝8.5Hz,1H),4.50–4.92(m,1H),4.10(t,J＝6.6Hz,2H),3.36(t,J＝6.8Hz,2H),2.83–2.68(m,3H),2.12–2.01(m,1H),1.85–1.75(m,4H),1.50–1.41(m,2H),1.40–1.26(m,6H).
The preparation method of other alkyl chain CRBN ligand with halogen atom at the tail end is the same as that of the CRBN ligand.
EXAMPLE 5 preparation of PROTAC Compounds of series I (1)
Taking the preparation of 6-ethyl-3- ((4- (4- (5- ((S) -1- ((2S, 4R) -4-hydroxy-2- ((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3, 3-dimethyl-1-oxobutan-2-yl) amino) -5-oxopentyl) piperazin-1-yl) -3-methoxyphenyl) amino) -5- ((tetrahydro-2H-pyran-4-yl) amino) pyrazine-2-carboxamide (X-1) as an example, it was obtained by the following reaction.
Figure BDA0003731451920000212
Weighing corresponding compound 24(64mg, 1.2eq), K 2 CO 3 (48.5mg, 4.0eq), KI (21.9mg, 1.4eq) in a single-neck flask, dissolved in DMF, stirred for several minutes, then compound 9(40mg, 1.0eq) was added and heated to 90 ℃ overnight. After TLC monitoring of the reaction completion, the reaction was extracted with EA (3X 30mL) and water, the organic phases combined and washed with saturated brine (2X 40mL) to remove excess DMF, followed by anhydrous Na 2 SO 4 Drying, distilling under reduced pressure to remove EA, and further purifying by column chromatography to obtainTo the corresponding target final product X-1 as a yellow solid, 26.6mg, the reaction yield was 31%. 1 H NMR(400MHz,Chloroform-d)δ10.74(s,1H),8.67(s,1H),7.59(d,J＝7.9Hz,1H),7.54(d,J＝4.8Hz,1H),7.46(dd,J＝8.6,2.3Hz,1H),7.38(d,J＝1.4Hz,4H),6.91(d,J＝2.3Hz,1H),6.85(d,J＝8.6Hz,1H),6.60(d,J＝8.9Hz,1H),5.60(d,J＝4.6Hz,1H),5.10(t,J＝7.2Hz,1H),4.75–4.69(m,2H),4.64(d,J＝9.0Hz,1H),4.51(s,1H),4.23–4.15(m,1H),4.07(d,J＝11.3Hz,1H),4.05–3.97(m,2H),3.85(s,3H),3.66–3.62(m,1H),3.55–3.47(m,4H),3.16–3.02(m,4H),3.68–2.79(m,4H),2.53–2.50(m,5H),2.43–2.37(m,1H),2.30–2.23(m,2H),2.16–2.11(m,1H),2.07(dd,J＝11.4,3.2Hz,2H),1.65(d,J＝8.4Hz,2H),1.60–1.54(m,4H),1.49(d,J＝6.9Hz,3H),1.33–1.29(m,3H),1.06(s,9H). 13 C NMR(100MHz,Chloroform-d)δ173.50,171.95,170.37,170.00,152.37,151.92,151.03,150.39,148.39,143.28,136.00,135.90,131.62,130.78,130.72,129.53,126.45,117.94,112.62,112.51,105.03,69.81,66.89,58.74,57.97,57.55,55.66,53.25,50.54,48.81,36.00,35.34,33.12,31.93,29.70,29.37,26.56,25.82,24.55,23.47,22.70,16.09,14.16,10.51.HRMS-ESI(m/z):[M+H] + calcd for[C 51 H 72 N 11 O 7 S] + 982.5331；found 982.5368.
The preparation of the other compounds of the series I (1) is as above.
EXAMPLE 6 preparation of series I (2) PROTAC Compounds
Taking the preparation of 3- (4- (8- (2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisooctanol-4-yl) amino) -8-oxooctyl) piperazin-1-yl) -3-methoxyphenyl) amino) -6-ethyl-5- (tetrahydro-2H-pyran-4-yl) amino) pyrazine-2-carboxamide (X-14) as an example, it was obtained by the following reaction.
Figure BDA0003731451920000221
Weighing compound 29(100.8mg, 1.2eq) and compound 9(80mg, 1.0eq) in a 50mL single-neck flask, adding solvent DMF (3mL) for dissolution, and then adding NaHCO 3 The solid (29.5mg, 2.0eq) was heated to 90 ℃ for 3 h. TLC monitoring reactionAfter completion, the reaction was extracted with EA (3X 30mL) and water, the organic phases were combined and washed with saturated brine (2X 40mL) to remove excess DMF, followed by anhydrous Na 2 SO 4 Drying, distillation under reduced pressure to remove EA, and further purification by column chromatography gave the final product X-14 as a yellow solid, 62.4mg, 42% yield. 1 H NMR(400MHz,Chloroform-d)δ10.71(d,J＝5.4Hz,1H),9.45(s,1H),8.78(d,J＝8.5Hz,1H),7.72–7.63(m,1H),7.50(dd,J＝9.6,5.6Hz,2H),7.44(dd,J＝8.6,2.3Hz,1H),6.89(d,J＝2.3Hz,1H),6.84(d,J＝8.7Hz,1H),5.66(d,J＝4.6Hz,1H),4.97–4.91(m,1H),4.75(d,J＝7.3Hz,1H),4.21–4.13(m,1H),4.03–3.96(m,2H),3.83(s,3H),3.54–3.46(m,2H),3.13–3.02(m,4H),2.85–2.82(m,1H),2.80–2.58(m,6H),2.52–2.44(m,2H),2.43–2.37(m,2H),2.18–2.12(m,1H),2.08–2.02(m,2H),1.78–1.68(m,2H),1.60–1.50(m,4H),1.45–1.28(m,8H),1.28–1.26(m,3H). 13 C NMR(100MHz,Chloroform-d)δ172.46,171.88,170.45,169.28,168.89,166.80,152.36,151.92,151.01,137.71,136.35,136.26,135.73,131.13,130.70,125.17,118.37,117.82,115.34,112.47,104.99,66.88,58.72,55.61,53.32,50.67,49.34,47.67,45.86,37.80,33.06,31.47,29.67,29.21,28.89,27.59,26.51,25.29,24.50,22.78,14.83,14.15,10.49,9.62.HRMS-ESI(m/z):[M+H] + calcd for[C 44 H 57 N 10 O 8 ] + 853.4355；found 853.4352.
The preparation of the other compounds of the series I (2) is as above.
Example 7 preparation of VHL ligand terminated by an N-Boc substituted linker chain as the alkyl chain
Taking the preparation of tert-butyl (6- ((S) -1- ((2S, 4R) -4-hydroxy-2- ((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3, 3-dimethyl-1-oxobutan-2-yl) amino) -6-oxohexyl) carbamate (34) as an example, it was obtained by the following reaction.
Figure BDA0003731451920000231
Compound 22(100mg, 1.0eq) was weighed out together with the corresponding compound 33(57.2mg,1.1eq) and a single-neck flask, add solvent DMF to dissolve, then add DIPEA (116.3mg, 4.0eq) dropwise, stir for several minutes, add HATU (94.1mg, 1.1eq) and react at room temperature for 1 h. After TLC monitoring of the reaction completion, the reaction was extracted with EA (3X 30mL) and water, the organic phases combined and washed with saturated brine (2X 40mL) to remove excess DMF, followed by anhydrous Na 2 SO 4 Drying, distilling under reduced pressure to remove EA, and further purifying by column chromatography to obtain the corresponding intermediate white solid 34, 140mg, with a reaction yield of 97%. 1 H NMR(400MHz,Chloroform-d)δ8.64(s,1H),7.63(d,J＝7.7Hz,1H),7.33(d,J＝2.3Hz,4H),6.69(d,J＝8.3Hz,1H),5.08–5.01(m,1H),4.65(t,J＝8.0Hz,1H),4.59(d,J＝9.2Hz,1H),4.45(s,1H),3.96(d,J＝11.0Hz,1H),3.67–3.58(m,1H),3.01(d,J＝6.7Hz,2H),2.46(s,3H),2.33–2.20(m,1H),2.20–2.10(m,2H),2.06(dd,J＝16.9,8.6Hz,1H),1.59–1.49(m,2H),1.42(d,J＝7.0Hz,3H),1.38(s,9H),1.25–1.20(m,2H),0.99(s,9H).
Other VHL ligands with N-Boc substituted linker chain at the end were prepared as described above.
EXAMPLE 8 preparation of Serial II PROTAC Compounds
Taking the preparation of 6-ethyl-N- (6- (S) -1- (2S, 4R) -4-hydroxy-2- (S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3, 3-dimethyl-1-oxobutan-2-yl) amino) -6-oxohexyl) -3- (3-methoxy-4- (4-methylpiperazin-1-yl) phenyl) amino) -5- (tetrahydro-2H-pyran-4-yl) amino) pyrazine-2-carboxamide (42a) as an example, it was obtained by the following reaction.
Figure BDA0003731451920000241
The corresponding compound 34(70.0mg, 1.0eq) was weighed out, dissolved by addition of DCM and reacted at room temperature for 30min with dropwise addition of excess TFA. TLC monitored the reaction was complete and DCM and TFA were distilled off under reduced pressure to give brown oily liquid 35 which was carried on to the next reaction without further purification. First, an appropriate amount of DMF was added to dissolve it, DIPEA (100mg, 8.0eq) was added dropwise with stirring for several minutes, and then Compound 36(45.5mg, 1.1eq) and HA were added sequentiallyTU (40.5mg, 1.1eq) was reacted at room temperature for 30 min. After TLC monitoring of the reaction completion, the reaction was extracted with EA (3X 30mL) and water, the organic phases combined and washed with saturated brine (2X 40mL) to remove excess DMF, followed by anhydrous Na 2 SO 4 Drying, distilling under reduced pressure to remove EA, and further purifying by column chromatography to obtain yellow solid X-9 of corresponding target PROTAC molecule, 77.5mg, with reaction yield of 67%. 1 H NMR(400MHz,Chloroform-d)δ10.89(s,1H),8.65(s,1H),7.79–7.71(m,1H),7.53(d,J＝8.7Hz,1H),7.52–7.46(m,1H),7.34(d,J＝7.3Hz,4H),6.86(t,J＝2.8Hz,1H),6.82(dd,J＝9.3,5.1Hz,1H),6.46–6.35(m,1H),5.05(q,J＝6.8Hz,1H),4.76–4.69(m,1H),4.67(d,J＝7.4Hz,1H),4.59(d,J＝8.8Hz,1H),4.48(s,1H),4.20–4.16(m,1H),4.03–3.93(m,3H),3.83(s,3H),3.62(dd,J＝11.0,3.8Hz,1H),3.55–3.46(m,2H),3.38–3.31(m,2H),3.18–3.00(m,4H),2.75–2.65(m,4H),2.53–2.46(m,5H),2.42–2.30(m,4H),2.20–2.13(m,2H),2.10–2.00(m,3H),1.67–1.50(m,6H),1.45(d,J＝6.7Hz,3H),1.28–1.21(m,5H),1.01(s,9H). 13 C NMR(100MHz,Chloroform-d)δ173.41,171.73,170.07,167.55,152.42,151.60,150.51,150.41,148.33,143.29,136.01,135.95,131.60,130.73,130.33,129.49,126.46,117.92,113.13,111.98,104.68,69.77,66.93,58.80,57.39,56.78,55.63,55.30,50.83,48.77,47.67,46.07,45.86,38.81,38.66,36.29,36.02,35.41,33.12,30.31,29.72,28.89,26.56,26.50,25.36,24.60,23.69,22.97,22.21,16.07,14.08,10.71,9.63.HRMS-ESI(m/z):[M+H] + calcd for[C 53 H 76 N 11 O 7 S] + 1010.5644；found 1010.5658.
The preparation method of other series II PROTAC compounds is the same as above.
Example 9 preparation of Gilitinib derivative I having a Tert-butyl ester substituent linking chain as the alkoxy chain
Taking the preparation of tert-butyl 2- (2- (4- (4- (3-carbamoyl-5-ethyl-6- (tetrahydro-2H-pyran-4-yl) amino) pyrazin-2-yl) amino) -2-methoxyphenyl) piperazin-1-yl) ethoxy) acetate (41) as an example, it was obtained by the following reaction.
Figure BDA0003731451920000251
Step 1: preparation of tert-butyl acetate 2- (2- (2-hydroxyethoxy) ethoxy)
Figure BDA0003731451920000252
Weighing the corresponding compound 37(6.0g, 1.0eq) in a double-mouth bottle, replacing with argon to maintain an inert gas protection environment, adding a solvent DMF for dissolution, slowly adding NaH (2.7g, 1.2eq) in portions in an ice bath, and stirring at room temperature for 1 h. Compound 38 t-butyl bromoacetate (11.0g, 1.0eq) was then added under ice-bath and, after addition was complete, the ice-bath was removed and the reaction was allowed to proceed overnight at room temperature. After the reaction is finished, water is slowly added to quench the reaction, then EA and water are used for extracting the reaction solution, organic phases are combined, and anhydrous Na is used for 2 SO 4 Drying, distilling under reduced pressure to remove EA, and purifying by column chromatography to obtain compound yellow oily liquid 39, 0.7g, reaction yield 6%. 1 H NMR(400MHz,Chloroform-d)δ3.99(s,2H),3.72–3.67(m,6H),3.61–3.57(m,2H),1.44(s,9H).
And 2, step: preparation of tert-butyl 2- (2- (p-tolyloxy) ethoxy) acetate
Figure BDA0003731451920000253
The corresponding compound 39(400mg, 1.0eq) was weighed into a single vial, dissolved by addition of solvent DCM, followed by sequential addition of p-toluenesulfonyl chloride (380.8mg, 1.1eq), Et 3 N (643.8mg, 3.5eq) was reacted with DMAP (13.3mg, 0.06eq) at room temperature for 2 h. After TLC monitoring the reaction, the reaction solution was extracted with DCM and water, the organic layers were combined and Na anhydrous 2 SO 4 Drying and further purification by column chromatography gave 40 as a colourless liquid, 328.8mg, in 48% reaction yield. 1 H NMR(400MHz,Chloroform-d)δ7.81–7.74(m,2H),7.33(d,J＝8.1Hz,2H),4.19–4.12(m,2H),3.97(s,2H),3.72–3.65(m,2H),3.66–3.62(m,2H),3.62–3.59(m,2H),2.43(s,3H),1.46(s,9H).
And 3, step 3: preparation of tert-butyl 2- (2- (4- (4- (3-carbamoyl-5-ethyl-6- (tetrahydro-2H-pyran-4-yl) amino) pyrazin-2-yl) amino) -2-methoxyphenyl) piperazin-1-yl) ethoxy) acetate
Figure BDA0003731451920000261
Weighing corresponding compound 40(45.2mg, 1.1eq), K 2 CO 3 (60.7mg, 4.0eq), KI (23.4mg, 1.3eq) in a single vial, dissolved in DMF and stirred for several minutes, followed by addition of Compound 9(50mg, 1.0eq) and heating to 85 ℃ for reaction overnight. After TLC to monitor the reaction completion, the reaction mixture was extracted with EA (3X 30mL) and water, the organic phases were combined and washed with saturated brine (2X 40mL) to remove excess DMF, followed by anhydrous Na 2 SO 4 Drying, distillation under reduced pressure to remove EA, and further purification by column chromatography gave 41 as a yellow solid, 49.4mg, reaction yield 68%. 1 H NMR(400MHz,Chloroform-d)δ10.74(s,1H),7.49(d,J＝4.5Hz,1H),7.45(dd,J＝8.6,2.3Hz,1H),6.86(d,J＝2.3Hz,1H),6.82(d,J＝8.6Hz,1H),5.58(s,1H),4.78(d,J＝7.3Hz,1H),4.20–4.13(m,1H),4.02–3.98(m,2H),3.97–3.94(m,2H),3.82(s,3H),3.76(t,J＝5.5Hz,2H),3.70–3.67(m,2H),3.67–3.64(m,2H),3.52–3.45(m,2H),3.19–3.11(m,4H),2.96–2.85(m,4H),2.83(t,J＝5.6Hz,2H),2.49(q,J＝7.3Hz,2H),2.07–2.01(m,2H),1.60–1.52(m,2H),1.43(s,9H),1.35(t,J＝7.4Hz,3H).
The preparation method of the Gilitinib derivative I with the other tert-butyl ester substituted connecting chain as the alkoxy chain is the same as that of the Gilitinib derivative I.
EXAMPLE 10 preparation of series III PROTAC Compounds
Taking the preparation of 6-ethyl-3- (4- (2- (2- ((S) -1- (2S, 4R) -4-hydroxy-2- ((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3, 3-dimethyl-1-oxobutan-2-yl) amino) -2-oxoethoxy) ethyl) piperazin-1-yl) -3-methoxyphenyl) amino) -5- (tetrahydro-2H-pyran-4-yl) amino) pyrazine-2-carboxamide (X-19) as an example, it was obtained by the following reaction.
Figure BDA0003731451920000262
The corresponding compound 41(49.3mg, 1.0eq) was weighed out, dissolved in DCM, and reacted at rt with an excess of TFA for 2 h. After the reaction was completed, distillation under reduced pressure gave brown oily liquid 42 which was used in the next step without further purification. Compound 42 was dissolved by adding DMF as a solvent, DIPEA (58.1mg, 6.0eq) was added dropwise and stirred for several minutes, followed by the sequential addition of Compound 22(40.0mg, 1.2eq) and HATU (31.3mg, 1.1eq) and reaction at room temperature for 30 min. After TLC monitoring of the reaction completion, the reaction was extracted with EA (3X 30mL) and water, the organic phases combined and washed with saturated brine (2X 40mL) to remove excess DMF, followed by anhydrous Na 2 SO 4 Drying, distillation under reduced pressure to remove EA, and further purification by column chromatography gave X-19 as a yellow solid, 34mg, with a reaction yield of 44%. 1 H NMR(400MHz,Chloroform-d)δ10.74(s,1H),8.67(s,1H),7.57(d,J＝7.7Hz,1H),7.53(d,J＝4.6Hz,1H),7.46(dd,J＝8.6,2.3Hz,1H),7.38(d,J＝2.6Hz,4H),7.35(s,1H),6.91(d,J＝2.3Hz,1H),6.85(d,J＝8.6Hz,1H),5.52(d,J＝4.5Hz,1H),5.12–5.04(m,1H),4.78–4.74(m,1H),4.70(d,J＝7.3Hz,1H),4.60(d,J＝8.8Hz,1H),4.24–4.14(m,1H),4.06–3.98(m,5H),3.85(s,3H),3.73–3.69(m,2H),3.68–3.62(m,6H),3.55–3.47(m,2H),3.13–3.04(m,4H),2.76–2.67(m,6H),2.55–2.49(m,5H),2.48–2.42(m,1H),2.10–2.03(m,3H),1.61–1.53(m,2H),1.48(d,J＝6.9Hz,3H),1.29(t,J＝7.4Hz,3H),1.08(s,9H). 13 C NMR(100MHz,CDCl 3 )δ171.31,170.35,170.29,169.97,152.39,151.91,151.02,150.35,148.37,143.34,136.24,135.78,131.65,130.71,130.67,129.50,126.42,117.84,112.64,112.52,105.06,71.20,70.47,70.31,69.86,68.92,66.91,58.64,57.72,57.03,56.78,55.66,53.89,50.84,48.85,47.66,45.95,35.84,35.47,33.12,29.69,26.52,24.54,22.35,16.10,10.53.HRMS-ESI(m/z):[M+H] + calcd for[C 52 H 74 N 11 O 9 S] + 1028.5386；found 1028.5391.
The preparation method of the PROTAC compounds of other series III and series IV is the same as that of the PROTAC compounds of other series III and series IV.
Example 11 preparation of Gilitinib derivative I having Tert-butyl ester as the terminal substituent linking chain as the heterocyclic chain
Obtained by the following reaction, taking the preparation of tert-butyl 2- (4- (4- (3-carbamoyl-5-ethyl-6- (tetrahydro-2H-pyran-4-yl) amino) pyrazin-2-yl) amino) -2-methoxyphenyl) piperazin-1-yl) ethyl) piperazin-1-yl) acetate (44) as an example.
Figure BDA0003731451920000271
Compound 43 was prepared by the method in reference example 9. Compound 43(122.7mg) was weighed into a single-necked flask, dissolved in DCM, added with excess TFA and reacted at room temperature for 30 min. After the reaction is finished, the excess TFA and the solvent are removed by distillation under reduced pressure, and NaHCO is used 3 Dissolving and adjusting pH to 9-10, extracting reaction solution with DCM, combining organic phases and using anhydrous Na 2 SO 4 Drying, distilling under reduced pressure to remove DCM to obtain the corresponding crude compound, and further purifying by column chromatography to obtain the de-Boc intermediate 44, 52.4mg, with a reaction yield of 50%. 1 H NMR (400MHz, Chloroform-d) δ 10.73(s,1H),7.51(d, J ═ 4.5Hz,1H),7.46(dd, J ═ 8.6,2.3Hz,1H),6.87(d, J ═ 2.3Hz,1H),6.83(d, J ═ 8.6Hz,1H),5.62(d, J ═ 4.6Hz,1H),4.67(d, J ═ 7.3Hz,1H), 4.23-4.12 (m,1H), 4.03-3.96 (m,2H),3.85(s,3H), 3.54-3.45 m,2H), 3.16-2.99 (m,4H),2.89(t, J ═ 4.9, 4H), 2.65 (s, 2.65, 2.75-3.45 m,2H), 2.50 mg (t, 2.50H), 2.50 mg-2.52H, 50H, 49 mg (t, 2.50H), 2.50H, 50mg, 50H, 49 (H), 2.50H, 50mg, 50H, 49 mg, 2H, 50, 2H, 49, 2H, and 5H, 1.0eq) in a single-neck bottle, adding solvent DMF to dissolve, and adding K 2 CO 3 The solid (51.0mg, 4.0eq) was heated to 50 ℃ for 2 h. After TLC to monitor the reaction completion, the reaction mixture was extracted with EA (3X 30mL) and water, the organic phases were combined and washed with saturated brine (2X 40mL) to remove excess DMF, followed by anhydrous Na 2 SO 4 Drying, distillation under reduced pressure to remove EA, and further purification by column chromatography gave 45 as a yellow solid, 16mg, reaction yield 26%. 1 H NMR(400MHz,Chloroform-d)δ10.75(s,1H),7.54–7.47(m,2H),6.90(d,J＝2.3Hz,1H),6.86(d,J＝8.6Hz,1H),5.30(s,1H),4.65(d,J＝7.3Hz,1H),4.25–4.16(m,1H),4.06–4.00(m,2H),3.87(s,3H),3.57–3.49(m,2H),3.13–3.05(m,6H),2.82–2.73(m,4H),2.71–2.57(m,12H),2.56–2.49(m,2H),2.12–2.06(m,2H),1.62–1.53(m,2H),1.47(s,9H),1.31(t,J＝7.3Hz,3H).
Example 12 preparation and characterization of ProTAC Compounds Targeted degradation of FLT3-ITD muteins
(1) Compounds numbered X-2 in Table 1
The title compound was prepared in 46% yield as a yellow solid according to the procedure in example 5.
1 H NMR(400MHz,Chloroform-d)δ10.73(s,1H),8.67(s,1H),7.56(d,J＝7.8Hz,1H),7.53(d,J＝4.6Hz,1H),7.45(dd,J＝8.6,2.3Hz,1H),7.40–7.33(m,4H),6.91(d,J＝2.3Hz,1H),6.84(d,J＝8.6Hz,1H),6.60(d,J＝9.0Hz,1H),5.70(d,J＝4.5Hz,1H),5.12–5.06(m,1H),4.76–4.70(m,2H),4.64(d,J＝9.0Hz,1H),4.51(s,1H),4.23–4.14(m,1H),4.08–3.96(m,5H),3.85(s,3H),3.63(dd,J＝11.2,3.6Hz,1H),3.55–3.46(m,2H),3.16–3.30(m,4H),2.78–2.62(m,4H),2.54–2.47(m,5H),2.45–2.37(m,3H),2.26–2.17(m,2H),2.11–2.03(m,3H),1.67–1.61(m,2H),1.50–1.46(m,4H),1.48(d,J＝6.9Hz,3H),1.32–1.29(m,3H),1.05(s,9H). 13 C NMR(100MHz,Chloroform-d)δ173.58,171.95,170.41,170.05,152.37,151.92,151.01,150.40,148.36,143.30,136.07,135.86,131.62,130.75,129.51,126.46,117.90,112.59,112.49,105.02,69.72,66.90,58.79,58.34,57.45,56.95,55.66,53.29,50.61,48.79,47.65,45.68,36.18,35.99,35.46,34.46,33.09,31.91,29.68,29.35,27.06,26.56,26.07,25.39,24.53,22.69,22.25,16.09,14.83,14.15,10.51,9.32.HRMS-ESI(m/z):[M+H] + calcd for[C 52 H 74 N 11 O 7 S] + 986.5488；found 986.5491.
(2) Compounds numbered X-3 in Table 1
The title compound was prepared in 60% yield as a yellow solid according to the procedure in example 5.
1 H NMR(400MHz,Chloroform-d)δ10.74(s,1H),8.67(s,1H),7.63(d,J＝7.9Hz,1H),7.53(d,J＝4.5Hz,1H),7.46(dd,J＝8.6,2.3Hz,1H),7.38(s,4H),6.90(d,J＝2.3Hz,1H),6.85(d,J＝8.6Hz,1H),6.51(d,J＝9.0Hz,1H),5.68(d,J＝4.5Hz,1H),5.12–5.07(m,1H),4.77–4.70(m,2H),4.64(d,J＝9.0Hz,1H),4.51(s,1H),4.23–4.14(m,1H),4.08–3.98(m,3H),3.85(s,3H),3.65–3.61(m,1H),3.57–3.47(m,4H),3.18–3.04(m,4H),2.79–3.69(m,4H),2.55–2.48(m,5H),2.41–2.35(m,1H),2.24–2.18(m,2H),2.06(dd,J＝13.1,4.2Hz,3H),1.61(d,J＝6.4Hz,2H),1.59–1.52(m,4H),1.49(d,J＝6.9Hz,3H),1.43(dd,J＝8.3,6.3Hz,2H),1.33–1.29(m,5H),1.05(s,9H). 13 C NMR(100MHz,Chloroform-d)δ173.64,171.76,170.40,170.17,152.36,151.93,151.00,150.38,148.34,143.43,135.98,135.86,131.66,130.80,130.69,129.49,126.47,118.01,112.60,112.45,104.98,69.80,66.89,58.84,58.35,57.40,56.94,55.66,53.20,52.83,50.35,48.79,47.66,36.31,36.19,35.46,34.46,33.09,31.91,31.50,30.12,29.68,29.36,28.87,27.02,26.54,25.96,25.48,24.54,22.28,16.08,14.82,14.15,7.92.HRMS-ESI(m/z):[M+H] + calcd for[C 53 H 76 N 11 O 7 S] + 1010.5644；found 1010.5665.
(3) Compounds numbered X-4 in Table 1
The title compound was prepared in 46% yield as a yellow solid by the method of reference example 5.
1 H NMR(400MHz,Chloroform-d)δ10.74(s,1H),8.66(s,1H),7.57(d,J＝7.8Hz,1H),7.52(d,J＝4.6Hz,1H),7.44(dd,J＝8.6,2.3Hz,1H),7.37(s,4H),6.90(d,J＝2.3Hz,1H),6.84(d,J＝8.7Hz,1H),6.48(d,J＝9.0Hz,1H),5.79(d,J＝4.5Hz,1H),5.12–5.05(m,1H),4.78–4.69(m,2H),4.64(d,J＝9.0Hz,1H),4.51(s,1H),4.23–4.14(m,1H),4.05–3.96(m,3H),3.85(s,3H),3.63(dd,J＝11.1,3.9Hz,1H),3.53–3.46(m,2H),3.18–3.00(m,4H),2.79–2.64(m,4H),2.54–2.46(m,5H),2.45–2.38(m,3H),2.22–2.15(m,2H),2.09–2.02(m,3H),1.62–1.50(m,6H),1.47(d,J＝6.9Hz,3H),1.33–1.25(m,9H),1.04(s,9H). 13 C NMR(100MHz,Chloroform-d)δ173.56,171.87,170.43,170.14,152.37,151.92,151.00,150.40,148.35,143.33,136.07,135.87,131.62,130.76,129.49,126.47,117.91,112.59,112.50,105.01,69.71,66.90,58.82,58.64,57.33,56.89,55.65,53.35,50.61,48.78,47.66,45.83,36.38,35.51,33.08,31.90,29.68,29.34,29.01,28.90,27.20,26.54,26.23,25.50,24.53,22.68,16.08,14.83,14.16,10.51.HRMS-ESI(m/z):[M+H] + calcd for[C 54 H 78 N 11 O 7 S] + 1024.5801；found 1024.5806.
(4) Compound numbered X-5 in Table 1
The title compound was prepared in 66% yield as a yellow solid by the method of reference example 5.
1 H NMR(400MHz,Chloroform-d)δ10.72(s,1H),8.67(s,1H),7.60(d,J＝7.9Hz,1H),7.52(d,J＝4.6Hz,1H),7.44(dd,J＝8.5,2.3Hz,1H),7.38(s,4H),6.90(d,J＝2.3Hz,1H),6.85(d,J＝8.7Hz,1H),6.48(d,J＝8.9Hz,1H),5.78–5.72(m,1H),5.14–5.05(m,1H),4.75(d,J＝7.3Hz,1H),4.70(t,J＝7.9Hz,1H),4.63(d,J＝9.0Hz,1H),4.49(d,J＝5.5Hz,1H),4.23–4.14(m,1H),4.07–3.98(m,3H),3.85(s,3H),3.63(dd,J＝11.2,3.7Hz,1H),3.55–3.46(m,2H),3.17–2.99(m,6H),2.74–2.58(s,4H),2.54–2.46(m 5H),2.43–2.35(m,3H),2.17(t,J＝7.6Hz,2H),2.10–2.03(m,3H),1.56(dd,J＝12.0,4.3Hz,4H),1.48(d,J＝6.9Hz,3H),1.30–1.27(m,5H),1.26–1.23(m,6H),1.04(s,9H). 13 C NMR(100MHz,Chloroform-d)δ173.73,171.84,170.42,170.09,152.38,151.93,151.03,150.42,148.37,143.31,136.18,135.81,131.62,129.51,126.49,117.91,112.60,112.54,105.05,69.81,66.91,58.81,58.74,57.38,56.84,55.66,53.50,53.38,50.70,48.79,47.66,36.45,35.99,35.45,33.10,31.92,31.51,30.12,29.69,29.29,29.13,29.09,27.42,26.54,25.64,24.53,22.69,22.23,16.09,14.83,14.15,10.52.HRMS-ESI(m/z):[M+H] + calcd for[C 55 H 80 N 11 O 7 S] + 1038.5957；found 1038.5986.
(5) Compounds numbered X-6 in Table 1
The title compound was prepared in 63% yield as a yellow solid according to the procedure in example 5.
1 H NMR(400MHz,Chloroform-d)δ10.73(s,1H),8.67(s,1H),7.58(d,J＝7.8Hz,1H),7.52(d,J＝4.5Hz,1H),7.44(dd,J＝8.6,2.3Hz,1H),7.37(s,4H),6.90(d,J＝2.3Hz,1H),6.85(d,J＝8.6Hz,1H),6.48(d,J＝8.9Hz,1H),5.84(d,J＝5.3Hz,1H),5.09(t,J＝7.2Hz,1H),4.77(d,J＝7.2Hz,1H),4.70(t,J＝7.8Hz,1H),4.62(d,J＝8.9Hz,1H),4.51(s,1H),4.23–4.14(m,1H),4.05–3.96(m,3H),3.85(s,3H),3.64(dd,J＝11.2,4.0Hz,1H),3.53–3.47(m,2H),3.16–2.96(m,4H),2.77–2.63(m,4H),2.53–2.46(m,5H),2.45–2.37(m,3H),2.17(t,J＝7.6Hz,2H),2.05(dd,J＝13.7,3.8Hz,3H),1.60–1.51(m,6H),1.47(d,J＝6.9Hz,3H),1.31–1.25(m,13H),1.04(s,9H). 13 C NMR(100MHz,Chloroform-d)δ173.65,171.84,170.45,170.14,152.38,151.93,151.01,150.43,148.34,143.33,136.14,135.84,131.62,130.78,130.75,126.49,117.92,112.58,105.03,69.79,66.91,58.87,58.75,57.37,56.83,55.65,53.38,50.67,48.77,47.66,38.84,36.07,35.50,33.07,29.67,29.41,29.33,29.20,28.95,27.49,25.70,24.52,23.95,22.96,22.23,16.09,14.08,10.52.HRMS-ESI(m/z):[M+H] + calcd for[C 56 H 82 N 11 O 7 S] + 1052.6114；found 1052.6117.
(6) Compounds numbered X-7 in Table 1
The title compound was prepared in 44% yield as a yellow solid according to the procedure in example 5.
1 H NMR(400MHz,Chloroform-d)δ10.71(s,1H),8.67(s,1H),7.56(d,J＝7.8Hz,1H),7.52(d,J＝4.7Hz,1H),7.45(dd,J＝8.6,2.3Hz,1H),7.38(d,J＝1.7Hz,4H),6.90(d,J＝2.3Hz,1H),6.85(d,J＝8.6Hz,1H),6.41(d,J＝9.1Hz,1H),5.65(d,J＝4.6Hz,1H),5.12–5.07(m,1H),4.75–4.68(m,2H),4.62(d,J＝9.0Hz,1H),4.52–4.49(m,1H),4.25–4.14(m,1H),4.07–3.97(m,3H),3.86(s,3H),3.63(dd,J＝11.2,3.7Hz,1H),3.55–3.47(m,2H),3.16–2.99(m,6H),2.73–2.65(m,4H),2.54–2.47(m,5H),2.42–2.37(m,3H),2.17(t,J＝7.6Hz,2H),2.10–2.04(m,3H),1.56(dd,J＝12.5,4.4Hz,4H),1.48(d,J＝6.9Hz,3H),1.31–1.25(m,15H),1.04(s,9H). 13 C NMR(100MHz,Chloroform-d)δ173.79,171.89,170.39,170.02,152.39,151.93,151.04,150.40,148.40,143.28,136.08,135.88,131.61,130.80,130.72,129.53,126.48,117.97,112.63,112.53,105.06,69.85,66.90,58.75,57.39,56.81,55.66,53.49,53.34,50.56,48.80,47.65,36.51,35.87,33.12,31.92,29.69,29.46,29.41,29.35,29.24,29.19,26.53,26.41,25.65,24.54,22.69,22.22.HRMS-ESI(m/z):[M+H] + calcd for[C 57 H 83 N 11 O 7 S] + 1066.6270；found 1066.6302.
(7) Compound numbered X-8 in Table 1
The title compound was prepared in 52% yield as a yellow solid according to the procedure in example 5.
1 H NMR(400MHz,Chloroform-d)δ10.72(s,1H),8.68(s,1H),7.59(d,J＝7.9Hz,1H),7.53(d,J＝4.5Hz,1H),7.45(dd,J＝8.5,2.3Hz,1H),7.39(s,4H),6.91(d,J＝2.3Hz,1H),6.86(d,J＝8.6Hz,1H),6.44(d,J＝8.9Hz,1H),5.71(d,J＝4.5Hz,1H),5.10(p,J＝7.0Hz,1H),4.74(d,J＝7.3Hz,1H),4.68(d,J＝8.0Hz,1H),4.62(d,J＝8.9Hz,1H),4.50(s,1H),4.24–4.15(m,1H),4.08–3.99(m,3H),3.86(s,3H),3.63(dd,J＝11.3,3.6Hz,1H),3.55–3.47(m,2H),3.16–3.00(m,4H),2.76–2.64(m,4H),2.54–2.47(m,5H),2.45–2.36(m,3H),2.17(t,J＝7.6Hz,2H),2.12–2.03(m,3H),1.62–1.52(m,6H),1.49(d,J＝6.9Hz,3H),1.31–1.23(m,17H),1.05(s,9H). 13 C NMR(100MHz,Chloroform-d)δ173.87,171.84,170.40,170.06,152.39,151.92,151.04,150.43,148.38,143.27,136.23,135.78,131.60,130.80,130.69,129.52,126.49,117.90,112.59,112.54,105.04,69.86,66.91,58.80,57.39,56.83,55.65,53.41,50.76,48.80,47.65,36.52,35.90,35.38,33.11,29.69,29.54,29.52,29.49,29.43,29.28,29.24,27.60,26.64,26.53,25.68,24.54,22.21,16.09,10.51.HRMS-ESI(m/z):[M+H] + calcd for[C 58 H 85 N 11 O 7 S] + 1080.6427；found 1080.6427.
(8) Compounds numbered X-10 in Table 1
The title compound was prepared in 44% yield as a yellow solid according to the procedure in example 8.
1 H NMR(400MHz,Chloroform-d)δ10.94(s,1H),8.67(s,1H),7.77(t,J＝6.1Hz,1H),7.58–7.50(m,2H),7.41–7.35(m,4H),6.88(d,J＝2.3Hz,1H),6.85(d,J＝8.6Hz,1H),6.28(d,J＝8.8Hz,1H),5.08(q,J＝7.2Hz,1H),4.72–4.64(m,2H),4.59(d,J＝8.8Hz,1H),4.50(s,1H),4.25–4.15(m,1H),4.05–3.98(m,3H),3.85(s,3H),3.62(dd,J＝11.3,3.8Hz,1H),3.58–3.49(m,2H),3.38(q,J＝6.9Hz,2H),3.18–3.01(m,4H),2.75–2.63(m,4H),2.51(d,J＝4.3Hz,5H),2.43(t,J＝6.9Hz,1H),2.39(s,3H),2.16(t,J＝7.5Hz,2H),2.11–2.05(d,J＝15.3Hz,3H),1.64–1.53(m,6H),1.48(d,J＝6.9Hz,3H),1.30–1.24(m,9H),1.03(s,9H). 13 C NMR(100MHz,Chloroform-d)δ173.60,171.71,170.08,167.51,152.42,151.58,150.50,150.43,148.35,143.29,136.18,135.73,131.60,130.77,130.26,129.50,126.49,118.01,113.25,111.89,104.62,69.80,66.94,58.82,57.36,56.77,55.64,55.22,50.64,48.77,47.68,45.90,38.97,36.43,36.02,35.43,33.14,31.91,29.88,29.68,29.09,28.98,26.86,26.51,25.56,24.58,22.69,22.20,16.08,14.16,10.67.HRMS-ESI(m/z):[M+H] + calcd for[C 55 H 80 N 11 O 7 S] + 1038.5957；found 1038.5945.
(9) Compounds numbered X-11 in Table 1
The title compound was prepared in 42% yield as a yellow solid according to the procedure in example 8.
1 H NMR(400MHz,Chloroform-d)δ10.99(s,1H),8.66(s,1H),7.76(t,J＝6.0Hz,1H),7.58(dd,J＝8.6,2.3Hz,1H),7.50(d,J＝7.8Hz,1H),7.37(d,J＝2.8Hz,4H),6.86–6.82(m,2H),6.26(d,J＝8.7Hz,1H),5.10–5.05(m,1H),4.73–4.66(m,2H),4.58(d,J＝8.8Hz,1H),4.51–4.48(m,1H),4.24–4.15(m,1H),4.03–3.98(m,3H),3.84(s,3H),3.62(dd,J＝11.1,3.9Hz,1H),3.57–3.49(m,2H),3.40–3.35(m,2H),3.23–3.16(m,4H),2.83–2.97(m,4H),2.55–2.51(m,5H),2.50(s,3H),2.46–2.37(m,1H),2.14(t,J＝7.6Hz,2H),2.10–2.04(m,3H),1.64–1.51(m,6H),1.47(d,J＝6.9Hz,3H),1.29–1.24(m,13H),1.02(s,9H). 13 C NMR(100MHz,Chloroform-d)δ173.52,171.81,169.97,167.47,152.40,151.57,150.41,148.37,143.26,136.67,134.86,131.59,130.77,130.37,129.50,126.46,118.32,113.33,111.65,104.42,77.48,77.16,69.80,66.93,58.75,57.34,56.69,55.66,54.88,53.51,49.76,48.77,47.69,45.85,45.18,39.00,36.51,35.91,35.40,33.14,29.89,29.67,29.37,29.21,26.99,26.51,25.64,24.58,22.23,16.09,10.63,8.67.HRMS-ESI(m/z):[M+H] + calcd for[C 57 H 84 N 11 O 7 S] + 1066.6270；found 1066.6302.
(10) Compounds numbered X-12 in Table 1
The title compound was prepared in 47% yield as a yellow solid according to the procedure in example 8.
1 H NMR(400MHz,Chloroform-d)δ10.99(s,1H),8.65(s,1H),7.75(t,J＝6.0Hz,1H),7.58(dd,J＝8.6,2.3Hz,1H),7.51(d,J＝7.9Hz,1H),7.36(d,J＝1.9Hz,4H),6.85–6.81(m,2H),6.28(d,J＝8.8Hz,1H),5.10–5.03(m,1H),4.75(d,J＝7.3Hz,1H),4.66(t,J＝7.8Hz,1H),4.57(d,J＝8.8Hz,1H),4.50–4.45(m,1H),4.22–4.13(m,1H),4.03–3.93(m,3H),3.82(s,3H),3.62(dd,J＝11.2,4.1Hz,1H),3.55–3.48(m,2H),3.39–3.34(m,2H),3.24–3.16(m,4H),3.00–2.84(m,4H),2.56–2.51(m,5H),2.49(s,3H),2.42–2.33(m,1H),2.12(t,J＝7.6Hz,2H),2.08–2.02(m,3H),1.63–1.50(m,6H),1.46(d,J＝6.9Hz,3H),1.28–1.21(m,17H),1.01(s,9H). 13 C NMR(100MHz,Chloroform-d)δ173.47,171.74,170.04,167.45,152.38,151.56,150.38,148.33,143.30,136.76,134.68,131.58,130.74,130.42,129.48,126.45,118.36,113.31,111.58,104.37,77.55,77.23,76.91,69.78,66.93,58.79,57.33,56.69,55.65,54.82,49.59,48.75,47.69,45.89,45.05,38.98,36.52,35.44,33.11,29.88,29.50,29.47,29.43,29.29,29.24,27.00,26.51,25.68,24.56,22.22,16.08,10.61,8.65.HRMS-ESI(m/z):[M+H] + calcd for[C 59 H 88 N 11 O 7 S] + 1094.6583；found 1092.6616.
(11) Compounds numbered X-13 in Table 1
The title compound was prepared in 37% yield as a yellow solid according to the procedure in example 5.
1 H NMR(400MHz,Chloroform-d)δ10.72(s,1H),8.67(s,1H),7.52(d,J＝4.6Hz,1H),7.48(t,J＝6.0Hz,1H),7.44(dd,J＝8.6,2.3Hz,1H),7.35(s,4H),6.92(d,J＝2.3Hz,1H),6.85(d,J＝8.7Hz,1H),6.41(d,J＝9.0Hz,1H),5.63(d,J＝4.6Hz,1H),4.74–4.69(m,2H),4.60(d,J＝8.9Hz,1H),4.57–4.51(m,2H),4.34(dd,J＝15.0,5.3Hz,1H),4.24–4.15(m,1H),4.07–3.98(m,3H),3.86(s,3H),3.64(dd,J＝11.2,3.6Hz,1H),3.55–3.48(m,2H),3.14–3.00(m,4H),2.72–2.60(m,4H),2.52–2.49(m,5H),2.48–2.43(m,1H),2.37(t,J＝7.9Hz,2H),2.23–2.13(m,3H),2.10–2.04(m,2H),1.63–1.54(m,4H),1.50(d,J＝10.3Hz,2H),1.31–1.27(m,9H),0.95(s,9H). 13 C NMR(100MHz,Chloroform-d)δ173.66,171.74,171.07,170.36,152.39,151.91,151.04,150.37,148.38,138.18,136.31,135.74,131.64,130.84,130.65,129.46,128.04,117.82,112.63,112.56,105.07,69.80,66.91,58.79,57.30,56.93,55.66,53.48,50.89,47.65,46.02,43.17,36.41,36.29,35.31,33.13,29.69,29.00,28.85,27.23,26.43,25.45,24.54,16.07,10.51.HRMS-ESI(m/z):[M+H] + calcd for[C 53 H 76 N 11 O 7 S] + 1010.5644；found 1010.5645.
(12) Compounds numbered X-15 in Table 1
The title compound was prepared in 29% yield as a yellow solid according to the procedure in example 6.
1 H NMR(400MHz,Chloroform-d)δ10.73(s,1H),7.65(dd,J＝8.5,7.3Hz,1H),7.50(d,J＝4.6Hz,1H),7.46(dd,J＝8.6,2.3Hz,1H),7.42(d,J＝7.2Hz,1H),7.19(d,J＝8.5Hz,1H),6.89(d,J＝2.3Hz,1H),6.85(d,J＝8.6Hz,1H),5.60(d,J＝4.6Hz,1H),4.97–4.92(m,1H),4.74(d,J＝7.2Hz,1H),4.22–4.10(m,3H),4.04–3.97(m,2H),3.84(s,3H),3.55–3.46(m,2H),3.17–3.01(m,4H),2.89–2.64(m,7H),2.53–2.47(m,2H),2.46–2.41(m,2H),2.14–2.03(m,3H),1.91–1.82(m,2H),1.59–1.46(m,6H),1.39–1.31(m,6H),1.29–1.27(m,3H). 13 C NMR(100MHz,Chloroform-d)δ171.89,170.45,168.94,167.18,165.78,156.64,152.37,151.92,151.02,136.48,136.16,135.81,133.76,130.72,118.94,117.86,117.03,115.62,112.50,112.39,104.93,69.51,66.89,58.73,55.62,53.29,50.69,49.12,47.68,45.87,33.05,31.49,29.68,29.39,29.11,28.77,27.51,26.44,25.88,24.50,22.69,14.83,10.49,9.32.HRMS-ESI(m/z):[M+H] + calcd for[C 44 H 58 N 9 O 8 ] + 840.4403；found 840.4427.
(13) Compounds numbered X-16 in Table 1
The title compound was prepared in 37% yield as a yellow solid according to the procedure in example 6.
1 H NMR(400MHz,Chloroform-d)δ10.73(s,1H),8.86(s,1H),7.78(d,J＝7.9Hz,1H),7.63(d,J＝7.4Hz,1H),7.53(d,J＝4.8Hz,1H),7.46–7.39(m,2H),6.92(d,J＝2.3Hz,1H),6.83(d,J＝8.7Hz,1H),5.59(d,J＝4.6Hz,1H),4.99(dd,J＝12.7,5.4Hz,1H),4.74(d,J＝7.2Hz,1H),4.40–4.31(s,2H),4.23–4.13(m,1H),4.04–3.96(m,2H),3.84(s,3H),3.55–3.46(m,2H),3.17–2.96(m,4H),2.76–2.61(m,5H),2.53–2.39(m,6H),2.11–2.03(m,3H),1.80–1.69(m,2H),1.62–1.51(m,4H),1.41–1.34(m,2H),1.32–1.24(m,5H). 13 C NMR(100MHz,Chloroform-d)δ172.30,172.00,170.42,169.15,152.38,151.95,151.04,136.15,135.80,134.11,133.29,132.58,130.82,126.26,117.88,112.54,105.07,66.88,58.25,55.67,53.28,52.07,50.61,47.68,36.43,34.50,33.09,31.92,29.70,26.93,26.05,25.34,24.53,23.11,10.50.HRMS-ESI(m/z):[M+H] + calcd for[C 42 H 55 N 10 O 7 ] + 811.4250；found 811.4273.
(14) Compounds numbered X-17 in Table 1
The title compound was prepared in 43% yield as a yellow solid according to the procedure in example 6.
1 H NMR(400MHz,Chloroform-d)δ10.72(s,1H),9.14(s,1H),7.74(d,J＝7.6Hz,1H),7.59(d,J＝7.1Hz,1H),7.52(s,1H),7.40(d,J＝8.9Hz,2H),6.90(s,1H),6.80(d,J＝8.6Hz,1H),5.83(s,1H),5.10–5.01(m,1H),4.87(d,J＝6.8Hz,1H),4.50–4.25(m,2H),4.14(s,1H),4.02–3.93(m,2H),3.80(s,3H),3.47(t,J＝10.9Hz,2H),3.19–3.02(m,4H),2.97(q,J＝7.3Hz,1H),2.92–2.70(m,4H),2.69–2.60(m,1H),2.53–2.41(m,5H),2.07–1.96(m,3H),1.72–1.61(m,2H),1.59–1.46(m,4H),1.33–1.18(m,11H). 13 C NMR(100MHz,Chloroform-d)δ172.67,172.51,170.69,170.53,169.28,152.34,151.97,150.95,136.13,135.50,134.28,133.41,132.47,131.12,118.10,112.35,104.97,66.86,55.69,53.55,53.01,49.90,47.68,45.98,32.94,29.68,28.96,27.07,25.56,24.50,10.50,8.86.HRMS-ESI(m/z):[M+H] + calcd for[C 44 H 59 N 10 O 7 ] + 839.4563；found 839.4581.
(15) Compounds numbered X-18 in Table 1
The title compound was prepared in 38% yield as a yellow solid according to the procedure in example 6.
1 H NMR(400MHz,Chloroform-d)δ10.73(s,1H),8.62(s,1H),7.68(t,J＝6.1Hz,2H),7.56–7.50(m,1H),7.49–7.43(m,2H),6.90(s,1H),6.85(d,J＝8.7Hz,1H),5.43–5.37(m,1H),5.17(dd,J＝13.5,5.5Hz,1H),4.72(d,J＝7.3Hz,1H),4.50(d,J＝16.8Hz,1H),4.35(d,J＝16.6Hz,1H),4.24–4.15(m,1H),4.03(d,J＝11.3Hz,2H),3.86(s,3H),3.53(t,J＝11.5Hz,2H),3.17–3.05(m,5H),2.88–2.71(m,4H),2.57–2.44(m,6H),2.18–2.07(m,3H),1.73(p,J＝7.4Hz,2H),1.62–1.52(m,4H),1.42–1.29(m,15H). 13 C NMR(100MHz,Chloroform-d)δ172.48,172.36,170.59,170.41,169.23,152.34,151.96,151.04,136.03,135.84,134.44,133.22,132.54,130.84,128.93,126.42,120.58,117.92,112.58,112.49,105.04,66.88,62.51,58.45,55.66,53.14,51.76,50.45,47.65,46.72,45.84,36.74,33.06,32.58,31.61,29.70,29.32,29.24,29.18,27.35,26.11,25.68,24.54,23.33,10.50,9.40.HRMS-ESI(m/z):[M+H] + calcd for[C 46 H 63 N 10 O 7 ] + 867.4876；found 867.4902.
(16) Compounds numbered X-20 in Table 1
The title compound was prepared in 49% yield as a yellow solid by the method of reference example 10.
1 H NMR(400MHz,Chloroform-d)δ10.72(s,1H),8.65(s,1H),7.67(d,J＝7.7Hz,1H),7.50(d,J＝4.4Hz,1H),7.42(dd,J＝8.6,2.3Hz,1H),7.38(d,J＝4.5Hz,1H),7.37–7.32(m,4H),6.89(d,J＝2.3Hz,1H),6.82(d,J＝8.6Hz,1H),5.77(d,J＝4.4Hz,1H),5.06(p,J＝7.0Hz,1H),4.77–4.69(m,2H),4.60(d,J＝8.9Hz,1H),4.21–4.12(m,1H),4.03–3.95(m,5H),3.83(s,3H),3.74–3.58(m,13H),3.52–3.45(m,2H),3.25–3.16(m,1H),3.09–3.01(m,4H),2.74–2.65(m,4H),2.53–2.47(m,6H),2.42–2.34(m,1H),2.05–2.01(m,2H),1.60–1.50(m,2H),1.46(d,J＝6.9Hz,3H),1.26(t,J＝7.4Hz,3H),1.04(s,9H). 13 C NMR(100MHz,CDCl 3 )δ171.17,170.38,170.20,170.12,152.37,151.90,151.01,150.36,148.33,143.41,136.27,135.74,131.65,130.67,130.65,129.46,127.84,126.40,117.85,112.61,112.53,105.07,76.92,71.11,70.60,70.43,70.32,69.81,68.72,66.89,58.75,57.81,56.95,56.77,55.64,53.81,50.81,48.80,47.64,46.09,36.02,35.50,33.08,26.49,24.51,22.33,16.07,11.36,10.53.HRMS-ESI(m/z):[M+H] + calcd for[C 54 H 78 N 11 O 10 S] + 1072.5648；found 1072.6564.
(17) Compounds numbered X-21 in Table 1
The title compound was prepared in 16% yield as a yellow solid according to the procedure in example 10.
1 H NMR(400MHz,Chloroform-d)δ10.73(s,1H),8.69(s,1H),7.92(d,J＝8.6Hz,1H),7.55–7.50(m,2H),7.50–7.47(m,1H),7.43–7.36(m,4H),6.91(d,J＝2.3Hz,1H),6.86(d,J＝8.6Hz,1H),5.42(d,J＝4.6Hz,1H),5.08(q,J＝7.2Hz,1H),4.76(t,J＝7.9Hz,1H),4.69(d,J＝7.3Hz,1H),4.50(d,J＝8.7Hz,2H),4.25–4.18(m,1H),4.14(d,J＝11.3Hz,1H),4.06–4.00(m,2H),3.88(s,3H),3.62(dd,J＝11.3,3.8Hz,1H),3.57–3.49(m,2H),3.15–3.01(m,6H),2.77–2.69(m,4H),2.67–2.55(m,12H),2.54–2.51(m,5H),2.15–2.02(m,4H),1.62–1.54(m,2H),1.50(d,J＝6.9Hz,3H),1.30(t,J＝7.4Hz,3H),1.08(s,9H). 13 C NMR(100MHz,CDCl 3 )δ171.53,170.98,170.32,169.88,152.40,151.92,151.05,150.37,148.44,143.25,136.21,135.80,131.64,130.79,130.65,129.55,126.43,117.83,112.64,112.51,105.03,69.97,66.93,61.12,58.39,57.22,55.69,53.90,53.78,53.44,50.89,48.87,47.68,35.55,34.92,33.15,29.71,26.56,24.55,22.32,16.12,10.52.HRMS-ESI(m/z):[M+H] + calcd for[C 54 H 78 N 13 O 7 S] + 1052.5862；found 1052.5852.
(18) Compounds numbered X-22 in Table 1
The title compound was prepared in 45% yield as a yellow solid according to the procedure in example 10.
1 H NMR(400MHz,Chloroform-d)δ10.73(s,1H),8.68(s,1H),7.97(d,J＝8.5Hz,1H),7.60(d,J＝7.8Hz,1H),7.52(d,J＝4.6Hz,1H),7.47(dd,J＝8.6,2.3Hz,1H),7.41–7.36(m,4H),6.91(d,J＝2.3Hz,1H),6.86(d,J＝8.7Hz,1H),5.54–5.48(m,1H),5.12–5.05(m,1H),4.77(t,J＝7.8Hz,1H),4.70(d,J＝7.4Hz,1H),4.49(d,J＝8.6Hz,1H),4.25–4.15(m,1H),4.15–4.10(m,1H),4.04–3.99(m,2H),3.87(s,3H),3.63(dd,J＝11.3,4.2Hz,1H),3.55–3.49(m,2H),3.14–3.03(m,4H),2.89–2.77(m,4H),2.71–2.62(m,4H),2.56–2.47(m,7H),2.44(t,J＝6.8Hz,2H),2.18(dd,J＝31.8,10.9Hz,2H),2.11–2.03(m,4H),1.74(t,J＝11.6Hz,2H),1.64–1.50(m,4H),1.49(d,J＝7.0Hz,3H),1.30(t,J＝7.3Hz,5H),1.08(s,9H). 13 C NMR(100MHz,CDCl 3 )δ171.61,171.39,170.36,169.89,152.38,151.93,151.04,150.44,148.41,143.33,135.99,135.87,131.67,130.71,129.53,126.44,117.98,112.64,112.47,105.01,69.97,66.91,58.36,57.24,55.95,55.68,54.29,53.10,50.33,48.86,47.67,45.14,34.89,33.63,33.15,32.68,26.54,24.55,22.33,16.10,10.51,8.70.HRMS-ESI(m/z):[M+H] + calcd for[C 55 H 78 N 12 O 7 S] + 1051.5910；found 1051.5922.
The chemical structure of the target compound of the present invention synthesized above is shown in table 1. Other compounds in table 1 can be prepared by the above method.
Example 13 testing of the biological Activity of ProTAC Compounds Targeted to degrade FLT3-ITD muteins
(1) Cell inhibitory activity test of ProTAC compounds targeting and degrading FLT3-ITD mutant protein:
detection of drug IC Using CCK-8 experiment 50 The value is obtained. Cells in logarithmic growth phase were first taken in the experiment. After centrifugation at 1000rpm for 5min, the supernatant was discarded. Cells were resuspended in complete medium containing 10% FBS. After cell counting and adjustment of cell concentration, the cells were seeded in a 96-well plate at a density of 10000 per well. The drug concentration gradient is adjusted to be 0, 0.1, 1, 5, 10, 20, 50, 100, 500 and 1000nM, the total volume of the culture medium in each well is 100 muL, at least 3 duplicate wells are arranged, the wells without cells use up the whole culture medium as blank control, and the periphery of the 96-well plate is filled with sterile PBS. At 72h of cell culture, to each wellAdding 10. mu.L of Cell Counting Kit 8(CCK-8) solution, mixing the cells uniformly, and then continuously putting the cells back into the Cell incubator for culture. Culturing for 1-4h, taking out cells (to avoid pollution), detecting absorbance (OD) value of each well at 450nm wavelength with multifunctional microplate reader, and calculating drug IC 50 The value is obtained.
(2) Cell degradation activity test of ProTAC compounds targeting degradation of FLT3-ITD mutant protein:
the expression of cell surface FLT3 protein was detected by flow cytometry. Cells in the logarithmic growth phase were taken in the experiment, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cells were resuspended in sterile PBS. After cell counting and adjustment of cell concentration, the cells were seeded in a six-well plate at a density of 5X 105 cells/well. Adjusting the drug concentration gradient to be 0, 1, 10, 100nM, the total volume of each hole to be 2mL, setting at least 3 multiple holes, and placing in an incubator for incubation for 12 h. The cells were pelleted by centrifugation at 1000rpm for 5min and the supernatant discarded. The cells were washed 1-2 times with PBS by centrifugation, the supernatant was discarded, the cells were resuspended in 50. mu.L PBS, and the appropriate concentration of the flow antibody CD135 was added and incubated at room temperature in the dark for 30 min. The cells were pelleted by centrifugation at 1000rpm for 5min and the supernatant discarded. Add 400. mu.L PBS per tube for resuspension. After filtering, the apoptosis rate is detected on a flow cytometry detector.
The invention uses the Gilitinib and the derivative 9 thereof as a contrast to carry out the cell inhibitory activity (IC) on the synthesized compound 50 ) And a protein degradation activity test, the results of the cell inhibitory activity are shown in table 2, and the protein degradation activity is shown in fig. 1. Wherein, the activity results of human acute myeloid leukemia cell strains carrying FLT3-ITD mutation in MOLM-13 cells are calculated in nM.
Table 2: results of the cytostatic Activity of the partial Compounds synthesized according to the invention
Figure BDA0003731451920000371
As can be seen from the above results of biological activity, most of the compounds had excellent cytostatic activity, IC 50 At a darnamo level and the activity of compound X-22 was comparable to the control drug, glicotinib. On the other hand in the determination of the degradation ActivityIn experiments, most compounds, especially ProTAC molecules based on VHL ligands with methyl groups, show excellent protein degradation capability, and can provide a new idea for treating AML as a novel degradation agent.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including the preferred embodiment and all changes and modifications that fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (6)

1. A PROTAC-like compound targeted to degrade FLT3-ITD muteins, wherein the PROTAC-like compound targeted to degrade FLT3-ITD muteins has a structural formula shown in formula I or formula II below:
Figure FDA0003731451910000011
in the formula I or the formula II,
linker is a linking group selected from one or more of the following linear or branched alkylene, alkoxy or heterocyclic groups, - (CH) 2 ) n -、-(CH 2 ) n CO-、-NR 1 (CH 2 ) n CO-、-NR 2 (CH 2 ) n -、-(OCH 2 CH 2 O) n -、-(CH 2 CH 2 O) n -、-(OCH 2 CH 2 OCH 2 ) n -、-(CH 2 CH 2 OCH 2 ) n -、-(CH 2 CH 2 OCH 2 CH 2 ) n -, alkenylene, alkynylene, cycloalkylene, heteroarylene group or any combination thereof, wherein n represents a natural number from 1 to 20, R 1 、R 2 Each independently of the other is selected from H or C 1-10 An alkyl group;
e3 ligand is an E3 ligase ligand selected from one of the following ligands:
Figure FDA0003731451910000012
2. the PROTAC compound for targeted degradation of FLT3-ITD mutein according to claim 1, wherein the PROTAC compound for targeted degradation of FLT3-ITD mutein comprises one of the following compounds X1-X22, wherein the structural formula of the compounds X1-X22 is sequentially:
Figure FDA0003731451910000013
Figure FDA0003731451910000021
Figure FDA0003731451910000031
3. a pharmaceutical composition against AML comprising a PROTAC compound or a pharmacologically or physiologically acceptable salt thereof targeted to degrade the FLT3-ITD mutein of claim 1 or 2, and a pharmaceutically acceptable carrier or excipient.
4. A method for preparing PROTAC compounds for targeted degradation of FLT3-ITD muteins according to claim 1 or 2, comprising:
the PROTAC compounds for targeted degradation of FLT3-ITD mutant protein are divided into compounds of series I, series II, series III and series IV,
the series I PROTAC compound is a compound shown as a general formula (I), Linker is alkylene with n being 1-20, and E3 ligand is the VHL ligand, the methyl-bearing VHL ligand, pomalidomide, lenalidomide or 4-hydroxy thalidomide;
the PROTAC compound in the series II is a compound shown as a general formula (II), Linker is n-alkylene of 1-20, and E3 ligand is the methyl-bearing VHL ligand;
the series III PROTAC compound is a compound shown as a general formula (I), Linker is n ═ 1-20-alkoxy, and E3 ligand is the methyl-bearing VHL ligand;
the series IV PROTAC compounds are shown in a general formula (I), Linker is a heterocyclic group, and E3 ligand is the VHL ligand with methyl;
the preparation method of the series I PROTAC compound comprises the following steps:
reacting the Gilitinib derivative I with a VHL ligand derivative substituted by a terminal halogen atom or a VHL ligand derivative with methyl in DMF (N, N-dimethylformamide) under the conditions of potassium carbonate and potassium iodide to obtain a series I (1) PROTAC compound;
the preparation method of the series II PROTAC compound comprises the following steps:
removing a Boc protective group from a VHL ligand derivative with an N-Boc (tert-butyloxycarbonyl) substituent at the tail end and an alkyl chain as a connecting chain under an acidic condition, and then reacting the VHL ligand derivative with a Gilitinib derivative II in DMF under the conditions of HATU (O- (7-azabenzotriazole-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate) and DIPEA (N, N-diisopropylethylamine) to obtain a series II PROTAC compound;
the preparation method of the series I II PROTAC compound comprises the following steps:
removing tert-butyl ester from a Gilitinib derivative I with a terminal substituted by tert-butyl ester and an alkoxy chain as a connecting chain under an acidic condition, and then reacting the Gilitinib derivative I with a VHL ligand with a methyl group in DMF under the conditions of HATU and DIPEA to obtain a PROTAC compound of series III;
the preparation method of the series of IV PROTAC compounds comprises the following steps:
the geritinib derivative I with the terminal substituted by tert-butyl ester and the connecting chain as a heterocyclic chain firstly removes the tert-butyl ester under an acidic condition, and then reacts with a VHL ligand with a methyl group in DMF under the conditions of HATU and DIPEA to obtain a series of IV PROTAC compounds.
5. The method for preparing PROTAC compounds for targeted degradation of FLT3-ITD mutant protein according to claim 4,
the ratio of the amounts of the Gilitinib derivative I, the VHL ligand derivative (or the methyl-bearing VHL ligand derivative), the potassium carbonate and the potassium iodide in the series I is 1: 1.2: 4: 1.4;
gelitinib derivative I, CRBN ligand derivative and NaHCO in series II 3 The ratio of the amounts of substances (1): 1.2: 2; the ratio of the amount of the Gelitinib derivative II, the methyl-bearing VHL ligand derivative, HATU and DIPEA in the series II is 1: 1.2: 1.1: 6.0;
the ratio of the amounts of the substance of the Gilitinib derivative I with alkoxy side chain in the series III, the substance with methyl VHL ligand, HATU and DIPEA is 1: 1.2: 1.1: 6.0;
the ratio of the Gelitinib derivative I with a heterocyclic side chain, a methyl VHL ligand, HATU and DIPEA in series IV is 1: 1.2: 1.1: 6.0.
6. the use of a PROTAC-like compound targeted to degrade FLT3-ITD muteins according to claim 1 or 2 or a pharmaceutical anti-AML composition according to claim 3 as a FLT3-ITD degrader against AML.
CN202210794029.6A 2022-07-05 2022-07-05 PROTAC compound for targeted degradation of FLT3-ITD mutant protein and preparation method and application thereof Pending CN115124590A (en)

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* Cited by examiner, † Cited by third party
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CN114573570A (en) * 2022-02-21 2022-06-03 中山大学 Alpha-synuclein targeted compound and preparation method and application thereof
CN115819423A (en) * 2022-11-29 2023-03-21 武汉大学 ProTAC compound of Reidesciclovir or intermediate thereof, preparation method thereof and application of anti-EV 71

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114573570A (en) * 2022-02-21 2022-06-03 中山大学 Alpha-synuclein targeted compound and preparation method and application thereof
CN114573570B (en) * 2022-02-21 2023-05-30 中山大学 Alpha-synuclein targeting compound, and preparation method and application thereof
CN115819423A (en) * 2022-11-29 2023-03-21 武汉大学 ProTAC compound of Reidesciclovir or intermediate thereof, preparation method thereof and application of anti-EV 71

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