Protac class compound targeting cbfssmmhc protein and preparation method and application thereof

Abstract

The application discloses a PROTAC compound targeting CBFbeta-SMMHC protein and a preparation method and application thereof. The degradation agent takes a CRBN ligand as an E3 ubiquitin ligase ligand part, connects a CBFbeta-SMMHC protein ligand through alkyl side chains of different lengths, and synthesizes a series of target product PROTAC (target-induced protein degradation complex) molecules. The compound can effectively degrade CBFbeta-SMMHC protein, thereby inhibiting the proliferation of M4Eo type acute myeloid leukemia cells. The compound can be used for preparing a drug for treating acute myeloid leukemia.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a PROTAC-type compound targeting CBFβ-SMMHC protein, its preparation method, and its application. Background Technology

[0002] Acute myeloid leukemia (AML) is a malignant hematologic malignancy caused by the arrest and abnormal proliferation of hematopoietic cells, characterized by high invasiveness and heterogeneity. Causes of AML include gene mutations, deletions, or chromosomal translocations. Chromosomal translocations lead to gene rearrangements, resulting in the partial fusion and expression of two genes. The abnormally produced fusion protein disrupts normal cellular physiological functions and causes malignant transformation of hematopoietic cells. Inversion or translocation of chromosome 16 can produce the CBFβ-SMMHC fusion gene, involving core binding factor B (CBFβ) and smooth muscle myosin heavy chain (SMMHC). CBFβ-SMMHC is a major cause of M4Eo leukemia; this protein contains the 165 amino acids at the N-terminus of CBFβ and the C-terminal portion of SMMHC.

[0003] CBFβ-SMMHC leukemia accounts for approximately 10% of new AML cases annually. Due to the unclear molecular pathological mechanisms of this type of leukemia, current clinical treatment lacks effective targeted therapies and primarily relies on chemotherapy combined with cytarabine and daunorubicin. This treatment strategy achieves a five-year survival rate of approximately 45-65% in younger patients, but only about 20% in patients over 60 years of age. Furthermore, CBFβ-SMMHC leukemia has a high relapse rate, with a 3-year relapse rate of approximately 29% in young patients and as high as 55% in elderly patients. Therefore, in-depth research on CBFβ-SMMHC leukemia and the exploration of more effective targeted therapy strategies are imperative and of significant scientific and clinical importance.

[0004] In recent years, the development of PROTAC technology, which targets protein degradation, has provided new insights into disease treatment. Unlike traditional small molecule inhibitors that inhibit the function of target proteins by binding to their active sites, PROTACs can specifically bind to target proteins and induce their degradation, thereby achieving therapeutic goals. PROTACs consist of three parts: a small molecule ligand that recognizes the target protein, an intermediate linker chain, and an E3 ubiquitin ligase ligand. This bifunctional molecule can simultaneously bind to both the target protein and the E3 ubiquitin ligase, thereby inducing the degradation of the target protein via the proteasome pathway. Compared to traditional small molecule inhibitors, PROTACs require lower concentrations to function and exhibit better specificity. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the primary objective of this invention is to provide a PROTAC-type compound that targets the CBFβ-SMMHC protein.

[0006] A second objective of this invention is to provide a method for preparing PROTAC-type compounds that target the CBFβ-SMMHC protein.

[0007] A third objective of this invention is to provide the application of targeting CBFβ-SMMHC protein in the treatment of acute myeloid leukemia. The described PROTAC compounds can effectively degrade CBFβ-SMMHC protein, thereby inhibiting the proliferation of leukemia cells, and can be developed as novel drugs for the treatment of acute myeloid leukemia, showing broad application prospects.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the PROTAC-type compounds provided by the present invention have the following general structural formula:

[0010]

[0011] In the general formula, E3 ligase is a small protease molecule with ubiquitination function encoded by genes such as VHL (von Hippel-Lindau) or CRBN (Cereblon), and its ligands are specifically manifested as follows: (VHL ligand) (with methyl VHL ligand) (Pomalidamine) (4-hydroxythalidomide) and (Lenalidomide). The linker is a connecting group, representing an alkylene group, an alkoxy group, or a heterocyclic group, wherein the alkylene group, alkoxy group, or heterocyclic group may be selected from one or more straight-chain or branched alkylene groups, alkoxy groups, such as -(CH2). n -、-(CH2) n CO-, -NR1(CH2) n CO-, -NR2(CH2) n -、-(OCH2CH2O) n -、-(CH2CH2O) n -、-(OCH2CH2OCH2) n -、-(CH2CH2OCH2) n -、-(CH2CH2OCH2CH2) n- an alkenyl group, an alkyne group, a cycloalkyl group, a heteroaryl group, or any combination thereof, where n represents a natural number from 1 to 20, and R1 and R2 are each independently chosen as H or C. 1-10 alkyl.

[0012] The PROTAC compounds targeting CBFβ-SMMHC fusion protein provided by this invention include, but are not limited to, the compounds shown in Table 1 below:

[0013] Table 1: Overview of PROTAC compounds targeting the degradation of CBFβ-SMMHC fusion protein

[0014]

[0015] Secondly, methods for preparing PROTAC-type compounds targeting CBFβ-SMMHC protein are provided, including:

[0016] The preparation method of PROTAC compounds that target the degradation of CBFβ-SMMHC fusion protein as shown in general formula (I) is divided into series I: CBFβ-SMMHC inhibitors and their derivatives, series II: CRBN ligand derivatives, and series III: synthetic routes of PROTAC compounds.

[0017] In Series I, the compounds represented by CBFβ-SMMHC inhibitors and their derivatives have a linker of n=1-20 alkylene groups. In Series II, the compounds are CRBN ligand derivatives with a linker of n=1-20 alkylene groups. In Series III, the synthetic routes for PROTAC-type compounds are shown in general formula (I), with a linker of n=1-20 alkylene groups, and the E3 ligand being the aforementioned VHL ligand, a methylated VHL ligand, pomalidomide, lenalidomide, or 4-hydroxythalidomide.

[0018] The preparation methods of CBFβ-SMMHC inhibitors and their derivatives in Series I include:

[0019] 5-Hydroxypyridinaldehyde reacts with tert-butyl bromoacetate in DMF (N,N-dimethylformamide) under potassium carbonate conditions to give compound 3. Compound 3 is then reacted with 2-nitro-4-(trifluoromethoxy)aniline in an ethanol solution of 5 mol / L NaS₂O₄ at 70 °C for 4 h to give compound 5. Compound 5 is then reacted with TFA (trifluoroacetic acid) in DCM (dichloromethane) solution at room temperature for 1 h to give compound 6. Compound 6 then undergoes a condensation reaction in DMF solution under HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate) and DIPEA (diisopropylethylamine) conditions to give 11d-e.

[0020] a Reagents and conditions: (a) K2CO3, DMF, rt, 12h; (b) 5mol / L NaS2O4, EtOH, 70℃, 4h. (c) TFA, DCM, rt, 1h; (d) HATU, DIPEA, DMF, rt, 4h.

[0021] Synthetic routes for Series I CBFβ-SMMHC inhibitors and their derivatives are as follows: Figure 1 As shown;

[0022] The preparation methods for CRBN ligand derivatives in Series II include:

[0023] Lenalidomide reacted with tert-butyl bromoacetate in DMF under potassium carbonate conditions to obtain compound 8. Then, it was reacted in DCM under TFA conditions at room temperature for 1 hour to obtain compound 9. Then, it was condensed with tert-butyl carbamates of different chain lengths in DMF solution under HATU and DIPEA conditions to obtain 11a-c.

[0024] a Reagents and conditions: (a) K2CO3,DMF,rt,12h; (c) TFA,DCM,rt,1h; (d) HATU,DIPEA,DMF,rt,4h.

[0025] The synthetic route for series II CRBN ligand derivatives is as follows: Figure 2 As shown;

[0026] The preparation methods of PROTAC compounds in Series III include: reacting CBFβ-SMMHC inhibitors with CRBN ligand derivatives of alkyl chains of different lengths, or CBFβ-SMMHC inhibitors of different lengths with CRBN ligand derivatives, in DMF under HATU and DIPEA conditions to obtain Series II PROTAC compounds.

[0027] a Reagents and conditions: (d)HATU,DIPEA,DMF,rt,4h.

[0028] Synthetic routes for Series III PROTAC compounds are as follows: Figure 3 As shown;

[0029] Thirdly, the application of any of the aforementioned PROTAC compounds targeting CBFβ-SMMHC protein in the preparation of CBFβ-SMMHC protein degrading agents is provided.

[0030] Fourthly, the application of any of the aforementioned PROTAC compounds that target and degrade CBFβ-SMMHC protein in the preparation of drugs for treating acute myeloid leukemia is provided. The pharmaceutical compound for treating acute myeloid leukemia comprises the aforementioned PROTAC compound of CBFβ-SMMHC protein or a pharmacologically or physiologically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. The PROTAC compound or a pharmacologically or physiologically acceptable salt thereof serves as a CBFβ-SMMHC degrading agent.

[0031] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0032] This invention provides a method for synthesizing PROTAC-like compounds targeting CBFβ-SMMHC protein. A series of targeted PROTAC-like compounds were synthesized by linking CBFβ protein ligands and CRBN ligands with side chains of different lengths. These compounds can effectively degrade CBFβ-SMMHC protein, thereby inhibiting the proliferation of leukemia cells. They have broad application prospects and can be developed as novel drugs for the treatment of acute myeloid leukemia. Attached Figure Description

[0033] Figure 1 This is a synthetic route diagram for the series I CBFβ-SMMHC inhibitors and their derivatives of the present invention.

[0034] Figure 2 This is a synthetic route diagram for the series II CRBN ligand derivatives of this invention.

[0035] Figure 3 This is a synthetic route diagram for the series III PROTAC compounds of this invention.

[0036] Figure 4 This invention provides an immunoblotting assay to detect the degradation of CBFβ-SMMHC protein by PROTAC compounds.

[0037] Figure 5This invention provides a flow cytometry assay for detecting the degradation of CBFβ-SMMHC protein by PROTAC compounds.

[0038] Figure 6 This invention relates to the effects of PROTAC compounds on the proliferation of leukemia cells. Detailed Implementation

[0039] To clarify the purpose, technical solution, and advantages of this invention, the technical solution of this invention will be clearly and completely described in conjunction with the following embodiments and accompanying drawings.

[0040] Example 1: Preparation of tert-butyl 2-((6-formylpyridin-3-yl)oxy)acetate (3):

[0041]

[0042] 5-Hydroxypyridinaldehyde 2 (1.0 equiv.) and K₂CO₃ (2.0 equiv.) were weighed separately and placed in a 100 mL single-necked flask, dissolved in DMF, and stirred for about 10 minutes. Then, tert-butyl 2-bromoacetate 1 (1.1 equiv.) was added, and the mixture was reacted overnight at room temperature. After the reaction was complete, the mixture was quenched with water, extracted with ethyl acetate and water, concentrated the organic phase, evaporated to dryness, and purified by silica gel column chromatography to obtain compound 3. A pale yellow solid was given in 75% yield. 1 H NMR (400MHz, Chloroform-d) δ10.00(s,1H),8.45(s,1H),7.97(d,J=8.6Hz,1H),7.28(d,J=4.7Hz,1H),4.67(s,2H),1.50(s,9H).

[0043] Example 2: Preparation of tert-butyl acetate (5) of 2-((6-(5-(trifluoromethoxy)-1Hbenzimidazol-2-yl)pyridin-3-yl)oxy)acetate:

[0044]

[0045] 2-Nitro-4-(trifluoromethoxy)aniline 3 (1.0 equiv.) and 2-((6-formylpyridin-3-yl)oxy)tert-butyl acetate 4 (1.0 equiv.) were weighed separately and placed in a 100 mL single-necked flask. They were dissolved in ethanol, and then 5 mol / L Na2S2O4 solution was added. The mixture was refluxed at 70 °C for 4 hours. After the reaction was completed by TLC monitoring, the mixture was extracted with ethyl acetate and water. The organic phase was concentrated and evaporated to dryness. The compound was then purified by silica gel column chromatography to obtain a pale yellow solid 5 with a yield of 67%. 1H NMR(400MHz,Chloroform-d)δ11.03(s,1H),8.38(d,J=8.8Hz,1H),8.33(d,J=2.8Hz,1H),7.7 4(d,J=36.4Hz,1H),7.34(dd,J=8.7,2.9Hz,1H),7.21–7.14(m,1H),4.65(s,2H),1.51(s,9H).

[0046] Example 3: Preparation of 2-((6-(5-(trifluoromethoxy)-1Hbenzimidazol-2-yl)pyridin-3-yl)oxy)acetic acid (6):

[0047]

[0048] 1.0 equiv. of 2-((6-(5-(trifluoromethoxy)-1Hbenzimidazol-2-yl)pyridin-3-yl)oxy)tert-butyl acetate was weighed into a 50 mL single-necked flask, and 1 mL of trifluoroacetic acid was added. The reaction was carried out at room temperature for 1 h, monitored by TLC. After the reaction was complete, the mixture was extracted with ethyl acetate and water. The organic phase was concentrated and evaporated to dryness, and then purified by silica gel column chromatography. A yellow solid 6 was obtained in 95% yield.

[0049] General procedure for amide condensation (11a-c):

[0050] 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)acetic acid 9 (1.0 equiv.) was used to dissolve tert-butylcarbamates 10a-c with different side chains and HATU (3.0 equiv.) in 2 mL of dry DMF. The mixture was stirred at room temperature for 10 min, and finally DIPEA (1.2 equiv.) was added and the reaction was carried out at room temperature for 3-4 h. After complete reaction of 11a-c by TLC, the residue was extracted three times with ethyl acetate and water. The organic phases were combined and concentrated, and the crude product 11a-c was purified by column chromatography (dichloromethane:methanol, 150:1-10:1). The crude product was a yellow solid with a yield of 69-85%.

[0051] Example 4: Preparation of tert-butyl carbamate (11a):

[0052]

[0053] 9g of 2-((2-(2,6-dioxadiazin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)acetic acid (1.0 equiv.) was weighed. Tert-butyl (4-aminobutyl)carbamate 10a and HATU (3.0 equiv.) were dissolved in a dry 2ml LDM solution. The mixture was stirred at room temperature for 10 min, and finally, DIPEA (1.2 equiv.) was added, and the reaction was allowed to proceed at room temperature for 3-4 h. After the reaction was complete as monitored by TLC, the mixture was extracted with ethyl acetate and water. The organic phase was concentrated and evaporated to dryness, and then purified by silica gel column chromatography. A yellow solid 11a was obtained, with a yield of 69%. 1 H NMR(400MHz,Chloroform-d)δ9.39(s,1H),7.73(t,J=7.9Hz,1H),7.51(d,J=7.3Hz,1H),7.23(d,J=8.3Hz,1H),4.96(s,1H),4 .64(q,J=14.1Hz,2H),3.19(s,1H),3.09(d,J=6.4Hz,2H),2.80(dd,J=24.5,12.6Hz,2H),1.51(d,J=7.1Hz,4H),1.40(s,9H).

[0054] Example 5: Preparation of (6-(2-((2-(2,6-dioxopiridine-3-yl)-1,3-dioxoisoindol-4-yl)oxy)acetamyl)hexyl)tert-butyl carbamate (11b):

[0055]

[0056] 9-((2-(2,6-dioxadiazin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)acetic acid (1.0 equiv.) was weighed. Tert-butyl (6-aminohexyl)carbamate 10b and HATU (3.0 equiv.) were dissolved in a dry 2 mL LDM solution. The mixture was stirred at room temperature for 10 min, and finally, DIPEA (1.2 equiv.) was added, and the reaction was carried out at room temperature for 3–4 h. After the reaction was complete as monitored by TLC, the mixture was extracted with ethyl acetate and water. The organic phase was concentrated and evaporated to dryness, and then purified by silica gel column chromatography. A yellow solid 11b was obtained, with a yield of 74%. 1H NMR(400MHz,Chloroform-d)δ9.37(s,1H),7.73(t,J=7.9Hz,1H),7.55–7.43(m,2H),7.21(d,J=8.4Hz,1H),5.05–4.93(m,1H),4.79( s,1H),4.63(s,2H),3.45–3.27(m,2H),3.07(s,2H),2.92–2.74(m,2H),2.26(d,J=94.5Hz,2H),1.60–1.53(m,9H),1.35-1.24(m,6H).

[0057] Example 6: Preparation of (8-(2-((2-(2,6-dioxopiridine-3-yl)-1,3-dioxoisoindol-4-yl)oxy)acetamyl)octyl)tert-butyl carbamate (11c):

[0058]

[0059] 9g of 2-((2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)acetic acid (1.0 equiv.) was weighed. 10c of tert-butyl(8-aminooctyl)carbamate and HATU (3.0 equiv.) were dissolved in a dry 2ml LDM solution. The mixture was stirred at room temperature for 10 min, and finally, 1.2 equiv. of DIPEA was added. The reaction was allowed to proceed for 3-4 h at room temperature. After the reaction was complete as monitored by TLC, the mixture was extracted with ethyl acetate and water. The organic phase was concentrated and evaporated to dryness, then purified by silica gel column chromatography. A yellow solid, 11c, was given in 85% yield. 1 H NMR(400MHz,Chloroform-d)δ9.47(s,1H),7.69(t,J=8.0Hz,1H),7.48(d,J=6.8Hz,1H ),7.40(t,J=5.8Hz,1H),7.18(d,J=8.4Hz,1H),5.05–4.91(m,1H),4.77(s,1H),4.61( s,2H),3.30(d,J=7.3Hz,2H),3.03(d,J=7.1Hz,2H),2.93(d,J=12.5Hz,1H),2.86–2.7 4(m,J=23.0,11.9Hz,3H),1.52(d,J=8.0Hz,2H),1.38(s,9H),1.22(d,J=14.1Hz,10H).

[0060] Example 7: Preparation of (10-(2-((6-(5-(trifluoromethoxy)-1Hbenzimidazol-2-yl)pyridin-3-yl)oxy)acetamyl)decyl)tert-butyl carbamate (11d):

[0061]

[0062] 1.0 equiv. of compound 2-((6-(5-(trifluoromethoxy)-1Hbenzimidazol-2-yl)pyridin-3-yl)oxy)acetic acid 6 was weighed. 10 d of tert-butyl (10-aminodecyl)carbamate and HATU (3.0 equiv.) were dissolved in a dry 2 mL LDM solution. The mixture was stirred at room temperature for 10 min, and finally, DIPEA (1.2 equiv.) was added, and the reaction was allowed to proceed at room temperature for 3–4 h. After the reaction was complete as monitored by TLC, the mixture was extracted with ethyl acetate and water. The organic phase was concentrated and evaporated to dryness, and then purified by silica gel column chromatography. 11 d of a yellow solid was obtained, with a yield of 85%. 1 H NMR (400MHz, DMSO-d6) δ8.47(d,J=2.8Hz,1H),8.29(d,J=8.8Hz,1H),8.23(t,J=5 .8Hz,1H),7.67(d,J=8.7Hz,1H),7.61–7.54(m,2H),7.20(dd,J=8.6,2.3Hz,1H), 6.75(q,J=5.9,4.3Hz,1H),4.70(s,2H),3.13(q,J=6.6Hz,2H),2.92–2.85(m,2H) ,1.42(dd,J=14.7,7.4Hz,2H),1.36(s,9H),1.35–1.30(m,2H),1.36-1.16(m,8H).

[0063] Example 8: Preparation of (12-(2-((6-(5-(trifluoromethoxy)-1Hbenzimidazol-2-yl)pyridin-3-yl)oxy)acetamido)dodecyl)tert-butyl carbamate (11e):

[0064]

[0065] 1.0 equiv. of compound 2-((6-(5-(trifluoromethoxy)-1Hbenzimidazol-2-yl)pyridin-3-yl)oxy)acetic acid 6 was weighed. Tert-butyl (12-aminododecyl)carbamate 10e and HATU (3.0 equiv.) were dissolved in a dry 2 mL LDM solution. The mixture was stirred at room temperature for 10 min, and finally, DIPEA (1.2 equiv.) was added, and the reaction was carried out at room temperature for 3–4 h. After the reaction was complete as monitored by TLC, the mixture was extracted with ethyl acetate and water. The organic phase was concentrated and evaporated to dryness, and then purified by silica gel column chromatography. A yellow solid 11e was given, with a yield of 85%. 1H NMR(400MHz,DMSO-d6)δ8.92(s,1H),8.48(d,J=2.8Hz,1H),8.29(d,J=8.8Hz,1H),8 .22(t,J=5.8Hz,1H),7.67(d,J=8.7Hz,1H),7.61–7.54(m,2H),7.22–7.19(m,J=8.8, 2.4,1.0Hz,1H),6.74(t,J=5.8Hz,1H),4.70(s,2H),2.87(q,J=6.6Hz,2H),2.54–2.4 6(m,2H),1.44–1.37(m,2H),1.36(s,9H),1.34–1.27(m,2H),1.13(d,J=7.5Hz,16H).

[0066] The general procedure for the amide condensation of 12a-c is as follows: 2-((6-(5-(trifluoromethoxy)-1Hbenzimidazol-2-yl)pyridin-3-yl)oxy)acetic acid 6 (1.0 equiv.), 2-((2-(2,6-dioxopyridin-3-yl)-1,3-dioxoisoindole-4-yl)oxy)acetamide derivative 11a-c (1.1 equiv.), and HATU (3.0 equiv.) are dissolved in 2 mL of dry DMF, stirred at room temperature for 10 min, and finally DIPEA (1.2 equiv.) is added and reacted at room temperature for 3–4 h. After complete reaction of 12a-c by TLC, the residue is extracted three times with ethyl acetate and water. The organic phases are combined and concentrated, and the crude product is purified by column chromatography (dichloromethane:methanol, 150:1–10:1) to provide product 12a-c.

[0067] The general procedure for amide condensation of 12d-e is as follows: 2-((2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)acetic acid 9 (1.0 equiv.), 2-((6-(5-(trifluoromethoxy)-1Hbenzimidazol-2-yl)pyridin-3-yl)oxy)acetamide derivative 11d-e (1.1 equiv.), and HATU (3.0 equiv.) are dissolved in 2 mL of dry DMF, stirred at room temperature for 10 min, and finally DIPEA (1.2 equiv.) is added and reacted at room temperature for 3–4 h. Complete reaction of 12d-e is detected by TLC, and the residue is extracted three times with ethyl acetate and water. The organic phases are combined and concentrated, and the crude product is purified by column chromatography (dichloromethane:methanol, 200:1–10:1) to provide the product (12d-e).

[0068] Example 9: Preparation of 2-((2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindole-4-yl)oxy)-N-(4-(2-((6-(5-(trifluoromethoxy)-1H-benzimidazol-2-yl)pyridin-3-yl)oxy)acetamido)butyl)acetamide (12a):

[0069]

[0070] 2-((6-(5-(trifluoromethoxy)-1Hbenzimidazol-2-yl)pyridin-3-yl)oxy)acetic acid 6 (1.0 equiv.), (4-(2-((2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindole-4-yl)oxy)acetamyl)tert-butylcarbamate 11a (1.1 equiv.) and HATU (3.0 equiv.) were dissolved in 2 mL of dry DMF, stirred at room temperature for 10 min, and finally DIPEA (1.2 equiv.) was added and reacted at room temperature for 3–4 h. The complete reaction of 12a was detected by TLC, and the residue was extracted three times with ethyl acetate and water. The organic phases were combined and concentrated, and the crude product was purified by column chromatography (dichloromethane:methanol, 150:1–10:1) to give a white solid 12a in 57% yield, mp 139.7–140.8 °C. 1 H NMR (400MHz, DMSO-d6) δ13.27(s,1H),11.17(s,1H),8.49(d,J=2.9Hz,1H),8.30(d,J=9.0Hz,2H),8.0 4(t,J=5.9Hz,1H),7.81(t,J=7.9Hz,1H),7.69–7.56(m,2H),7.50(d,J=7.3Hz,1H),7.39(d,J=8.5Hz, 1H),7.23(d,J=10.3Hz,1H),5.14(dd,J=13.0,5.4Hz,1H),4.80(s,2H),4.71(s,2H),3.17(d,J=6.3Hz ,4H),2.96–2.86(m,J=18.8,14.4,5.4Hz,1H),2.61(d,J=17.2Hz,2H),2.10–2.01(m,1H),1.46(s,4H). 13C NMR(100MHz,DMSO-d6)δ173.39,170.43,167.41,167.30,167.23,165.99,155.72,155.51,153.04,144.36,143.87,142.60,140.97,139 .78,138.46,123.20,122.83,120.76,118.40,117.09,116.48,67.92,67.47,49.24,38.47,31.38,31.11,26.84,22.44.HRMS(ESI)calcd for C 34 H 30 F3N7O9[M+H]+,738.2130; found 738.2131.

[0071] Example 10: Preparation of 2-((2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindole-4-yl)oxy)-N-(6-(2-((6-(5-(trifluoromethoxy)-1H-benzimidazol-2-yl)pyridin-3-yl)oxy)hexyl)acetamide (12b):

[0072]

[0073] 2-((6-(5-(trifluoromethoxy)-1Hbenzimidazol-2-yl)pyridin-3-yl)oxy)acetic acid 6 (1.0 equiv.), tert-butyl(6-(2-((2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindole-4-yl)oxy)acetamyl)hexyl)carbamate 11b (1.1 equiv.), and HATU (3.0 equiv.) were dissolved in 2 mL of dry DMF, stirred at room temperature for 10 min, and finally DIPEA (1.2 equiv.) was added and reacted at room temperature for 3–4 h. The complete reaction of 12b was detected by TLC, and the residue was extracted three times with ethyl acetate and water. The organic phases were combined and concentrated, and the crude product was purified by column chromatography (dichloromethane:methanol, 150:1–10:1) to give a white solid 12b in 67% yield, mp 152.5–153.4 °C. 1H NMR (400MHz, DMSO-d6) δ11.18(s,1H),9.15(s,1H),8.48(s,1H),8.35–8.22(m,2H),7.98(t,J=5.8Hz,1H), 7.81(t,J=7.9Hz,1H),7.67(d,J=8.7Hz,1H),7.62–7.57(m,2H),7.50(d,J=7.3Hz,1H),7.39(d,J=8.5Hz,1H ),7.21(d,J=10.9Hz,1H),5.14(dd,J=12.9,5.4Hz,1H),4.78(s,2H),4.71(s,2H),2.96–2.87(m,J=16.7,13 .7,5.3Hz,1H),2.60(dd,J=17.0,12.7Hz,2H),2.11–2.01(m,1H),1.43(q,J=6.5Hz,4H),1.30–1.23(m,4H). 13 C NMR(100MHz,DMSO-d6)δ173.32,170.40,167.27,167.22,167.14,166.00, 164.81,155.65,155.45,153.32,141.40,138.38,137.41,133.47,122.99 ,122.86,120.77,120.43,117.24,116.53,113.12,112.09,68.04,67.57, 49.26,38.72,31.42,29.48,29.40,26.47,26.42,22.45.HRMS(ESI)calcd for C 36 H 34 F3N7O9[M+H]+,766.2443; found 766.2443.

[0074] Example 11: Preparation of 2-((2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindole-4-yl)oxy)-N-(8-(2-((6-(5-(trifluoromethoxy)-1H-benzimidazol-2-yl)pyridin-3-yl)oxy)acetamyl)octyl)acetamide (12c):

[0075]

[0076] 2-((6-(5-(trifluoromethoxy)-1Hbenzimidazol-2-yl)pyridin-3-yl)oxy)acetic acid 6 (1.0 equiv.), (8-(2-((2-(2,6-dioxopiridine-3-yl)-1,3-dioxoisoindole-4-yl)oxy)acetamyl)octyl)tert-butyl carbamate 11c (1.1 equiv.) and HATU (3.0 equiv.) were dissolved in 2 mL of dry DMF, stirred at room temperature for 10 min, and finally DIPEA (1.2 equiv.) was added and reacted at room temperature for 3–4 h. The complete reaction of 12c was detected by TLC, and the residue was extracted three times with ethyl acetate and water. The organic phases were combined and concentrated, and the crude product was purified by column chromatography (dichloromethane:methanol, 150:1–10:1) to give a white solid 12c in 48% yield, mp 132.5–133.4 °C. 1 H NMR (400MHz, DMSO-d6) δ13.25(s,1H),11.17(s,1H),8.48(d,J=2.8Hz,1H),8.29(d,J=8.8Hz,1H),8.23(t,J= 5.8Hz,1H),7.95(t,J=5.8Hz,1H),7.81(dd,J=8.5,7.3Hz,1H),7.59(dd,J=8.8,2.9Hz,1H),7.51(d,J=7.2Hz, 1H),7.39(d,J=8.5Hz,1H),7.21(d,J=8.8Hz,1H),5.14(dd,J=12.9,5.3Hz,1H),4.78(s,2H),4.71(s,2H),3.1 9–3.07(m,4H),2.99–2.83(m,1H),2.65–2.57(m,1H),2.07–2.03(m,J=7.6,2.8Hz,2H),1.41(p,J=6.4Hz,4H). 13 C NMR(100MHz,DMSO-d6)δ173.30,170.38,167.25,167.21,167.11,165.99,155 .61,155.44,153.31,144.42,141.42,138.34,137.38,134.27,133.46,123.0 8,122.84,120.76,117.25,116.52,113.09,112.06,105.13,68.05,67.59,49 .27,38.78,31.41,29.51,29.40,29.17,29.14,26.76,22.46.HRMS(ESI)calcd for C 38 H 38F3N7O9[M+H]+,794.2756; found794.2761.

[0077] Example 12: Preparation of 2-((2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindole-4-yl)oxy)-N-(10-(2-((6-(5-(trifluoromethoxy)-1H-benzimidazol-2-yl)pyridin-3-yl)oxy)acetamyl)decyl)acetamide (12d):

[0078]

[0079] 2-((2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)acetic acid 9 (1.0 equiv.), (10-(2-((6-(5-(trifluoromethoxy)-1Hbenzimidazol-2-yl)pyridin-3-yl)oxy)acetamyl)decyl)tert-butyl carbamate 11d (1.1 equiv.), and HATU (3.0 equiv.) were dissolved in 2 mL of dry DMF, stirred at room temperature for 10 min, and finally DIPEA (1.2 equiv.) was added and reacted at room temperature for 3–4 h. The complete reaction of 12d was detected by TLC, and the residue was extracted three times with ethyl acetate and water. The organic phases were combined and concentrated, and the crude product was purified by column chromatography (dichloromethane:methanol, 200:1–10:1) to give a white solid 12d in 52% yield, mp 113.5–134.7 °C. 1 H NMR (400MHz, DMSO-d6) δ13.24(d,J=12.4Hz,1H),11.16(s,1H),8.47(d,J=2.8Hz,1H),8.31–8.23(m,2H),7.96 (q,J=5.6,4.7Hz,1H),7.85–7.72(m,2H),7.59(dd,J=8.7,3.1Hz,1H),7.53–7.42(m,2H),7.39(dd,J=8.6,3.3 Hz,1H),7.21(dd,J=15.5,8.6Hz,1H),5.18–5.10(m,1H),4.77(s,2H),4.70(s,2H),3.14(d,J=6.2Hz,2H),3.0 2(d,J=3.5Hz,2H),2.93–2.82(m,2H),2.63–2.54(m,J=15.5,4.2Hz,2H),1.46–1.33(m,6H),1.31–1.21(m,8H). 13CNMR(100MHz,DMSO-d6)δ173.27,170.37,167.22,167.08,165.98,155.63,155.51,141.44,138.34,137.39,133.48,122.87,120.7 9,117.24,116.51,110.00,109.05,105.47,68.05,67.60,49.26,31.42,29.44,29.25,29.17,26.84,26.76,22.46.HRMS(ESI)calcd forC 40 H 42 F3N7O9[M+H]+,844.2888; found 844.2880.

[0080] Example 13: Preparation of 2-((2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindole-4-yl)oxy)-N-(12-(2-((6-(5-(trifluoromethoxy)-1H-benzimidazol-2-yl)pyridin-3-yl)oxy)acetamido)dodecylacetamide (12e):

[0081]

[0082] 2-((2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)acetic acid 9 (1.0 equiv.), (12-(2-((6-(5-(trifluoromethoxy)-1Hbenzimidazol-2-yl)pyridin-3-yl)oxy)acetamyl)dodecyl)carbamate tert-butyl carbamate 11e (1.1 equiv.), and HATU (3.0 equiv.) were dissolved in 2 mL of dry DMF, stirred at room temperature for 10 min, and finally DIPEA (1.2 equiv.) was added and reacted at room temperature for 3–4 h. The complete reaction of 12e was detected by TLC, and the residue was extracted three times with ethyl acetate and water. The organic phases were combined and concentrated, and the crude product was purified by column chromatography (dichloromethane:methanol, 200:1–10:1) to give a white solid 12e in 65% yield, mp 122.3–123.1 °C. 1HNMR (400MHz, DMSO-d6) δ11.16(s,1H),8.96(s,1H),8.47(d,J=2.9Hz,1H),8.28(d,J=8.8Hz,1H),8.23(t,J= 5.8Hz,1H),7.95(t,J=5.7Hz,1H),7.83–7.78(m,1H),7.67(d,J=8.7Hz,1H),7.61–7.55(m,2H),7.50(d,J=7.3 Hz,1H),7.39(d,J=8.5Hz,1H),7.21(dd,J=8.7,2.3Hz,1H),5.18–5.06(m,1H),4.77(s,2H),4.70(s,2H),3.1 2(d,J=5.7Hz,4H),2.95–2.84(m,2H),2.70–2.54(m,2H),1.45–1.33(m,4H),1.31–1.20(m,4H),1.14(m,12H). 13 C NMR(100MHz,DMSO-d6)δ173.26,170.36,167.23,167.09,166.00,155.61,155.49,153.21,141.45,138.34,137.38,133.48,1 23.02,122.87,120.80,117.25,116.52,68.07,67.63,49.27,38.77,31.44,29.52,29.46,29.28,29.16,26.84,26.75,22.45. HRMS(ESI)calcdfor C 40 H 42 F3N7O9[M+H]+,872.3201; found 872.3196.

[0083] Example 14: Immunoblotting assay to detect the degradation of CBFβ-SMMHC protein by PROTAC compounds

[0084] U937 cells overexpressing CBFβ-SMMHC were used at 5 x 10 5 The particles were seeded at a density of 1 / mL in 6-well plates, and after 12 hours, DMSO or... Figure 4 The PROTAC compounds shown were used to treat cells for 6 hours, after which cells were harvested for Western blotting analysis. Figure 4 The CBFβ-SMMHC is abbreviated as CS. Experimental results show that the linker chain length is closely related to the degradation activity of the compound. 12a and 12b have poor degradation effects, while 12c-e effectively degrades the CS protein level to about 20%.

[0085] Example 15: Flow cytometry assay to detect the degradation of CBFβ-SMMHC protein by PROTAC compounds

[0086] U937 cells overexpressing HaloTag-CBFβ-SMMHC were used at 5 x 10 5 The particles were seeded at a density of 1 / mL in 6-well plates, and after 12 hours, DMSO or... Figure 5 The PROTAC compounds shown were treated with HaloTag staining agent for 6 hours, and the cells were harvested for flow cytometry analysis. The results showed that the linker chain length was closely related to the degradation activity of the compounds, and the 12-day cells showed the best degradation effect on CBFβ-SMMHC protein.

[0087] Example 16: Effects of PROTAC compounds on leukemia cell proliferation

[0088] Patient-derived CBFβ-SMMHC leukemia cells were seeded in 6-well plates and treated with DMSO or DMSO for 12 days, at concentrations as specified. Figure 6 As shown, cell counts were performed 24 hours later, and the experimental results showed that 12 days could inhibit the proliferation of leukemia cells in a concentration-dependent manner.

Claims

1. A PROTAC-like compound targeting CBFβ-SMMHC protein, characterized in that, The PROTAC compound is one of the following compounds: 2-((2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindole-4-yl)oxy)-N-(4-(2-((6-(5-(trifluoromethoxy)-1H-benzimidazol-2-yl)pyridin-3-yl)oxy)acetamyl)butyl)acetamide; 2-((2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindole-4-yl)oxy)-N-(6-(2-((6-(5-(trifluoromethoxy)-1H-benzimidazol-2-yl)pyridin-3-yl)oxy)acetamido)hexyl)acetamide; 2-((2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindole-4-yl)oxy)-N-(8-(2-((6-(5-(trifluoromethoxy)-1H-benzimidazol-2-yl)pyridin-3-yl)oxy)acetamyl)octyl)acetamide; 2-((2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindole-4-yl)oxy)-N-(10-(2-((6-(5-(trifluoromethoxy)-1H-benzimidazol-2-yl)pyridin-3-yl)oxy)acetamyl)decyl)acetamide; 2-((2-(2,6-dioxopiridine-3-yl)-1,3-dioxoisoindole-4-yl)oxy)-N-(12-(2-((6-(5-(trifluoromethoxy)-1H-benzimidazol-2-yl)pyridin-3-yl)oxy)acetamido)dodecyl)acetamide.

2. A method for preparing compound 12d according to claim 1, wherein compound 12d is: 2-((2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindole-4-yl)oxy)-N-(10-(2-((6-(5-(trifluoromethoxy)-1H-benzimidazol-2-yl)pyridin-3-yl)oxy)acetamyl)decyl)acetamide; Its features are: Includes the following steps: 2-((2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindole-4-yl)oxy)acetic acid was reacted with N-(10-aminodecyl)-2-((6-(5-(trifluoromethoxy)-1H-benzimidazol-2-yl)pyridin-3-yl)oxy)acetamide in anhydrous DMF at room temperature for 3–4 h in the presence of HATU and DIPEA to give the compound 12d; The molar ratio of 2-((2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindole-4-yl)oxy)acetic acid, N-(10-aminodecyl)-2-((6-(5-(trifluoromethoxy)-1H-benzimidazol-2-yl)pyridin-3-yl)oxy)acetamide, HATU and DIPEA is 1.0∶1.1∶3.0∶1.

2.

3. The use of a PROTAC compound targeting CBFβ-SMMHC protein as described in claim 1 in the preparation of a CBFβ-SMMHC protein degrading agent.

4. The use of a PROTAC compound as described in claim 1, which targets and degrades CBFβ-SMMHC protein, in the preparation of a drug for treating acute myeloid leukemia, characterized in that: The drug for treating acute myeloid leukemia comprises the PROTAC compound of claim 1, or a pharmacologically or physiologically acceptable salt of a PROTAC compound. The drug for treating acute myeloid leukemia also includes a pharmaceutically acceptable carrier or excipient.

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