Heterocyclic compound and application thereof

Abstract

The invention relates to a heterocyclic compound and application thereof.The structure of the heterocyclic compound is shown as a formula (I) or a formula (II), experiments prove that the heterocyclic compound can efficiently degrade CK1alpha through a molecular glue mechanism, so that a p53 pathway, a Wnt / beta-catenin pathway, an ATM pathway and the like are affected, and the heterocyclic compound can be used for preventing or treating diseases caused by abnormally high expression of CK1alpha, and can be used for treating diseases such as diseases caused by abnormally high expression of CK1alpha. The compound can be used for preparing medicines for treating malignant tumors and solid tumors of a blood system, especially medicines for treating acute myeloid leukemia.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically to a heterocyclic compound and application thereof. Background Art

[0002] Molecular glue and protein degradation targeting chimeras (PROTACs) are two targeted protein degradation technologies that have developed rapidly in recent years. Among them, molecular glue acts as a glue molecule between the target protein and the E3 ubiquitin ligase, inducing the formation of a ternary complex and promoting the protein-protein interaction (PPI) between the target protein and the E3 ubiquitin ligase, thereby promoting the ubiquitination and degradation of the target protein. Compared with PROTACs molecules, molecular glues do not require specific linkers or high-affinity ligands for the targeted protein. However, the discovery of molecular glues is highly accidental, and there is still a lack of systematic discovery methods and reasonable design strategies. The existing molecular glues that have been approved for clinical use are mainly immunomodulators, also known as immunomodulatory imine drugs (IMiDs), such as thalidomide, lenalidomide and pomalidomide, which are used to treat multiple myeloma, myelodysplastic syndrome, etc.

[0003] The targets of molecular glue degraders reported so far include IKZF1 / 2 / 3, RBM39, GSPT1, CK1α, BCL6, Cyclin K, NEK7, etc. Among them, casein kinase CK1α belongs to the highly conserved serine / threonine kinase family, phosphorylates a variety of substrate proteins involved in survival pathways, and participates in various physiological and pathological processes of cells through different signal transduction pathways such as p53 pathway, Wnt / β-catenin pathway and ATM pathway. It has been reported that CK1α can negatively regulate p53 protein activity by forming an active complex with ubiquitin ligase MDM family proteins (doi:10.1016 / j.tips.2018.10.009). Inhibition of CK1α by genetic knockdown using shRNA or the small molecule inhibitor D4476 (a CK1α kinase inhibitor) can effectively slow down AML cell proliferation (doi:10.1084 / jem.20131033); and in AML patients, abnormally high expression of CK1α was also found to be highly correlated with shorter overall survival (doi:10.3892 / or.2020.7760). In solid tumors, such as non-small cell lung cancer (NSCLC), depletion of CK1α can inhibit tumor growth by inducing autophagy and enhancing the stability of the tumor suppressor PTEN (doi:10.1038 / s41556-018-0065-8). In addition, CK1α has also been reported to be involved in enzalutamide drug resistance in prostate cancer (doi:10.1016 / j.xcrm.2023.101015) and erlotinib drug resistance in non-small cell lung cancer (doi:10.1158 / 0008-5472.CAN-15-1113). It can be seen that existing studies have shown that CK1α can be used as a potential target for the treatment of various cancers, especially as a potential therapeutic target for hematological malignancies and solid tumors.

[0004] However, only a few molecular glue degraders that can target and degrade CK1α protein have been discovered so far, such as a class of isoindolinone compounds in patent CN117561244A, and the CK1α degrader SJ3149 (doi: 10.1038 / s41467-024-44698-1) with anti-proliferative activity discovered by Rankovic et al. At present, lenalidomide is the only drug approved by the FDA to degrade CK1α, but its efficacy is weak, and most other molecular glue degraders targeting CK1α protein are in the preclinical research stage. It can be seen that there is a lack of effective therapeutic drugs for CK1α, a potential target for cancer treatment. Therefore, the development of efficient CK1α degraders with novel structure and good selectivity is of great significance for the treatment of cancers with CK1α as a potential target. Summary of the invention

[0005] In view of the above defects or improvement needs of the prior art, the present invention provides a new heterocyclic compound and its application, the purpose of which is to find that this type of heterocyclic compound can efficiently degrade CK1α through the mechanism of molecular glue, thereby affecting the p53 pathway, Wnt / β-catenin pathway and ATM pathway, etc., and can be used to prevent or treat diseases caused by abnormally high expression of CK1α, thereby solving the technical problem of the lack of effective therapeutic drugs for CK1α, a potential target for cancer treatment.

[0006] To achieve the above object, according to one aspect of the present invention, a heterocyclic compound is provided, the structure of which is shown in formula (I) or formula (II); wherein the structure of formula (I) is as follows:

[0007]

[0008] In formula (I), R is selected from halogen, Ra-NH,

[0009] wherein Ra in Ra-NH is selected from a six-membered cycloalkyl group which is unsubstituted or substituted with one or more halogens, a six-membered heterocycloalkyl group containing oxygen and / or nitrogen, or an aromatic group substituted with halogen, methyl and / or methoxy;

[0010] The structure of formula (II) is shown below:

[0011]

[0012] In formula (II), R1 is selected from H, halogen, -CH3, -NH2 or -CN; R2 is selected from H, -CH3, or

[0013] Preferably, the heterocyclic compound, wherein R in formula (I) is Cl, Br,

[0014] Preferably, the heterocyclic compound, in formula (II), R2 is H, and R1 is selected from H, Br, Cl, -NH2, -CN, or -CH3;

[0015] R2 is -CH3, R1 is selected from H, Br or Cl;

[0016] R2 is R1 is selected from Br or Cl.

[0017] Preferably, the heterocyclic compound includes a compound having the following structure:

[0018]

[0019]

[0020] According to another aspect of the present invention, there is also provided an enantiomer, stereoisomer, solvate, deuterated substance, polymorph or pharmaceutically acceptable salt of the heterocyclic compound of the present invention.

[0021] According to another aspect of the present invention, there is also provided a use of the heterocyclic compound as described in the present invention and its enantiomers, stereoisomers, solvates, deuterated compounds, polymorphs or pharmaceutically acceptable salts in the preparation of a molecular glue degradation agent targeting CK1α.

[0022] According to another aspect of the present invention, there is also provided a use of the heterocyclic compound as described in the present invention and its enantiomers, stereoisomers, solvates, deuterated compounds, polymorphs or pharmaceutically acceptable salts in the preparation of drugs for treating or preventing diseases associated with high expression of CK1α, wherein the diseases associated with high expression of CK1α include hematological malignancies and solid tumors associated with the p53 signaling pathway.

[0023] Preferably, in the application, the hematological malignancies include acute myeloid leukemia; the solid tumors include non-small cell lung cancer and prostate cancer.

[0024] Preferably, the application is used for preparing a drug for treating or preventing acute myeloid leukemia.

[0025] According to another aspect of the present invention, a drug for treating acute myeloid leukemia is provided, wherein the active ingredients of the drug include one or more of the heterocyclic compound as described in the present invention, its enantiomers, stereoisomers, solvates, deuterated substances, polymorphs or pharmaceutically acceptable salts.

[0026] Preferably, the drug, its active ingredient comprises one or more of the heterocyclic compound as described in the present invention, its enantiomers, stereoisomers, solvates, deuterated substances, polymorphs or pharmaceutically acceptable salts.

[0027] Preferably, the pharmaceutical active ingredients of the drug include dCK1α-1 and / or dCK1α-2.

[0028] In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects compared with the prior art due to the series of new heterocyclic compounds synthesized by the present invention:

[0029] The heterocyclic compounds with structures such as formula (I) or formula (II) provided by the present invention have been experimentally confirmed to be able to target and degrade CK1α to activate p53-related transcriptional regulatory events, and can be used as molecular glue degraders targeting CK1α. Compared with existing molecular glue degraders, they have the advantages of good selectivity, high activity, and high oral availability. They can be used to prevent or treat tumor diseases related to high expression of CK1α, and in particular, can achieve highly active and highly selective targeted degradation of CK1α protein in human acute myeloid leukemia cells Molt-13, and are used to prepare drugs for treating acute myeloid leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The degradation effect of different concentrations of compounds on CK1α in MOLM 13 cells in vitro.

[0031] Figure 2 It is the degradation effect of compound dCK1α-1 on CK1α in cells under different action time.

[0032] Figure 3 It is that dCK1α-1 in CRBN knockout cells cannot degrade CK1α.

[0033] Figure 4 Quantitative proteomics experiments have proven that the compound dCK1α-1 has good target specificity.

[0034] Figure 5 It is the compound dCK1α-1 that causes cell apoptosis.

[0035] Figure 6 It is the compound dCK1α-1 that causes cell cycle arrest.

[0036] Figure 7 It is the degradation effect of compound dCK1α-2 on CK1α in mice. DETAILED DESCRIPTION

[0037] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0038] the term:

[0039] In the present invention, "solvate" refers to a compound containing a crystallization solvent molecule in a drug crystal, and is usually called a hydrate when the solvent molecule is a water molecule. "Deuterated compound" refers to a compound in which hydrogen (H) is partially or completely replaced by deuterium (D) element in a drug molecule, wherein deuterium is an isotope of hydrogen, has a heavier atomic mass, and has a chemical symbol of 2H or D. "Polymorph" refers to the formation of different polymorphs of the same drug due to its different crystal structures. "Pharmaceutically acceptable salt" generally refers to the preparation of a drug into a salt form, including organic or inorganic salts, in order to increase the solubility of the drug or to enhance the stability of the drug. "Halogen" means chlorine, bromine, fluorine and / or iodine; "halogen substitution" refers to chloro, bromo, fluoro and / or iodo.

[0040] More than 600 E3 ligases have been reported, but only five are used for molecular glue-mediated degradation, namely CRBN, DDB1, β-TrCP, DCAF15, and SIAH1. Among them, CRBN (cereblon) is a substrate receptor protein of the Cullin 4-gene (RING) E3 ubiquitin ligase complex CRL4 CRBN, which contains specific binding domains (substrate binding domain and ligand binding domain) in its structure, which are essential for its function. Among them, the substrate binding domain of CRBN interacts with specific substrate proteins, while its ligand binding domain can bind to certain small molecule ligands. These ligands bind to CRBN and can change the conformation of CRBN, thereby affecting its binding to the substrate, and then affecting the ubiquitination and degradation of the substrate. It has been proven that the glutarimide moiety shared by the three immunomodulatory drugs (IMiDs), lenalidomide, thalidomide and pomalidomide, contributes the most to the interaction with the CRBN binding pocket, while the differences in the parts outside the pocket can give CRBN different protein interfaces, thereby recruiting different substrates through protein interaction mechanisms.

[0041] The present invention synthesizes a series of heterocyclic compounds based on the glutarimide skeleton, and finds that the heterocyclic compounds having the structure shown in formula (I) or formula (II) bind to the E3 ligase ligand CRBN, can specifically recruit the target protein CK1α, and then promote the ubiquitination and degradation of the CK1α protein, and can be used as a molecular glue degrader for targeted degradation of the CK1α protein, and can be used to treat hematological malignancies and solid tumors. The structure of the heterocyclic compound is shown in formula (I) or formula (II):

[0042]

[0043] Wherein R in formula (I) is selected from halogen, Ra-NH,

[0044] Wherein Ra in Ra-NH is selected from a six-membered cycloalkyl group which is unsubstituted or substituted with one or more halogens, a six-membered heterocycloalkyl group containing oxygen and / or nitrogen, or an aromatic group substituted with halogen, methyl and / or methoxy.

[0045] In formula (II), R1 is selected from H, halogen, -CH3 (methyl, Me), -NH2 (amino) or -CN (cyano); R2 is selected from H, Me or wait.

[0046] Based on this, the present invention provides a heterocyclic compound having a structure as shown in formula (I) or formula (II), and an enantiomer, stereoisomer, solvate, deuterated compound, polymorph or pharmaceutically acceptable salt thereof, wherein the structure of the heterocyclic compound is as shown in formula (I):

[0047]

[0048] In formula (I), R is selected from halogen, Ra-NH,

[0049] Wherein Ra in Ra-NH is selected from a six-membered cycloalkyl group which is unsubstituted or substituted with one or more halogens, a six-membered heterocycloalkyl group containing oxygen and / or nitrogen, or an aromatic group substituted with halogen, methyl (-CH3), and / or methoxy (-O-CH3).

[0050] In some embodiments, R in formula (I) is halogen, such as Cl or Br.

[0051] In some embodiments, R in formula (I) is Ra-NH, wherein Ra is selected from a six-membered cycloalkyl group which is unsubstituted or substituted with two halogen fluorines, specifically That is, the specific structure of R is as follows:

[0052]

[0053] In some embodiments, R in formula (I) is Ra-NH, wherein Ra is selected from a six-membered heterocyclic alkyl group containing oxygen and / or nitrogen, specifically That is, the specific structure of R is as follows:

[0054]

[0055] In some embodiments, R in formula (I) is Ra-NH, wherein Ra is selected from aromatic groups substituted by halogen and methyl (-CH3) or methoxy (-O-CH3), specifically That is, the specific structure of R is as follows:

[0056]

[0057] Preferably, the structure of the heterocyclic compound is as follows:

[0058]

[0059] Alternatively, the structure of the heterocyclic compound is as shown in formula (II):

[0060]

[0061] In the structure shown in formula (II), R1 is selected from H, halogen, -CH3 (methyl, Me), -NH2 (amino) or -CN (cyano); R2 is selected from H, Me or Wherein Me represents methyl group (-CH3).

[0062] In some embodiments, in formula (II), R2 is H, and R1 is selected from H, Br, Cl, -NH2, -CN, or -CH3; preferably R1 is Cl or -CH3.

[0063] In some embodiments, in formula (II), R2 is -CH3, and R1 is selected from H, Br or Cl.

[0064] In some embodiments, R2 in formula (II) is R1 is selected from Br or Cl.

[0065] Preferably, the structure of the heterocyclic compound is as follows:

[0066]

[0067] In addition, the present invention also provides an enantiomer, stereoisomer, solvate, deuterated substance, polymorph or pharmaceutically acceptable salt of the heterocyclic compound of the present invention.

[0068] The present invention also provides a use of the heterocyclic compound as described in the present invention, its enantiomer, stereoisomer, solvate, deuterated product, polymorph or pharmaceutically acceptable salt in the preparation of a molecular glue degrader targeting CK1α; the molecular glue degrader is used to promote the binding of the E3 ligase ligand CRBN to the target protein CK1α, promote the ubiquitination and degradation of the target protein, thereby achieving targeted selective degradation of the CK1α protein and activating transcriptional regulation related to the p53 signaling pathway.

[0069] In addition, the present invention also provides a use of the heterocyclic compound as described in the present invention, its enantiomer, stereoisomer, solvate, deuterated substance, polymorph or pharmaceutically acceptable salt in the preparation of a drug for treating or preventing diseases related to abnormal CK1α protein function.

[0070] The diseases associated with abnormal CK1α protein function are diseases associated with high expression of CK1α, including tumors associated with the p53 signaling pathway, the tumors including hematological malignancies and solid tumors, wherein hematological malignancies include acute myeloid leukemia; solid tumors include non-small cell lung cancer and prostate cancer; and are preferably used in the preparation of drugs for the treatment or prevention of acute myeloid leukemia.

[0071] The present invention also provides a drug for treating acute myeloid leukemia, wherein the active ingredients of the drug include one or more of the heterocyclic compound as described in the present invention, its enantiomers, stereoisomers, solvates, deuterated compounds, polymorphs or pharmaceutically acceptable salts; preferably, the active ingredients of the drug include dCK1α-1 and / or dCK1α-2.

[0072] In some embodiments, the drug further comprises a pharmaceutically acceptable carrier; its administration method includes but is not limited to injection and oral administration; oral administration is preferred.

[0073] The following are examples

[0074] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. The molecular weight and structure of the compounds were determined by mass spectrometry and nuclear magnetic resonance, respectively.

[0075] Example 1: Synthesis of (S)-3-(5-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-047-P1) and (S)-3-(5-(5-chloropyrazolo[1,5-a]pyrimidin-3-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-047-P2)

[0076] The synthetic routes of compounds LC-02-047-P1 and LC-02-047-P2 are as follows:

[0077]

[0078] Step 1: Synthesis of (S)-5-amino-4-(5-bromo-1-oxoisoindolin-2-yl)-5-oxopentanoic acid tert-butyl ester (LC-01-151): 4-bromo-2-bromomethylbenzoic acid methyl ester (1.3 eq.) and (S)-4,5-diamino-5-oxopentanoic acid tert-butyl ester hydrochloride (1 eq.) were dissolved in acetonitrile, and then N,N-diisopropylethylamine (3 eq.) was added. The reaction solution was heated to 80°C and stirred for 12 hours. The reaction was completed by LCMS, and the reaction solution was cooled to room temperature. Water was added to the reaction solution, extracted with ethyl acetate, and the organic phase was washed with saturated brine, combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain (S)-5-amino-4-(5-bromo-1-oxoisoindolin-2-yl)-5-oxopentanoic acid tert-butyl ester as a white solid. (14.52 g, yield 87.25%). LC-MS (ESI) C 17 H 22 BrN2O4 + [M+H] + , calculated values: 397.08 and 399.07; measured values: 397.11 and 399.12.

[0079] Step 2: Synthesis of (S)-5-amino-5-oxo-4-(1-oxo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-2-yl)pentanoic acid tert-butyl ester (LC-01-156): (S)-5-amino-4-(5-bromo-1-oxoisoindolin-2-yl)-5-oxopentanoic acid tert-butyl ester (1 eq.) and biboronic acid pinacol ester (1.5 eq.) were dissolved in 1,4-dioxane, and potassium acetate (3 eq.) was added. The atmosphere was replaced with nitrogen three times, Pd(dppf)Cl2 (0.03 eq.) was added, and the atmosphere was replaced with nitrogen three times again. The reaction solution was heated to 100°C and stirred for 12 hours. The reaction was completed by LCMS, and the reaction solution was cooled to room temperature. Water was added to the reaction solution, extracted with ethyl acetate, the organic phase was washed with saturated brine, the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain (S)-5-amino-5-oxo-4-(1-oxo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-2-yl)pentanoic acid tert-butyl ester as a white solid. (14.83 g, yield 89.97%). LC-MS (ESI) C 23 H 34 BN2O6 + [M+H] + , calculated value: 445.25; measured value: 445.30.

[0080] Step 3: Synthesis of tert-butyl (S)-5-amino-4-(5-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)-1-oxoisoindolin-2-yl)-5-oxopentanoate (LC-02-003-P1) and tert-butyl (S)-5-amino-4-(5-(5-chloropyrazolo[1,5-a]pyrimidin-3-yl)-1-oxoisoindolin-2-yl)-5-oxopentanoate (LC -02-003-P2): (S)-5-amino-5-oxo-4-(1-oxo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-2-yl)pentanoic acid tert-butyl ester (1 eq.) and 3-bromo-5-chloropyrazolo[1,5-a]pyrimidine (1.5 eq.) were dissolved in 1,4-dioxane, and 3M potassium phosphate solution (3 eq.) was added. The atmosphere was replaced with nitrogen three times, Pd(dtbpf)Cl2 (0.1 eq.) was added, and the atmosphere was replaced with nitrogen three times again. The reaction solution was heated to 100°C and stirred for 3 hours. The reaction was detected to be complete by LCMS, and the reaction solution was cooled to room temperature. Water and ethyl acetate were added to the reaction solution for extraction, and the extraction was repeated three times until the extraction was complete. The organic phase was washed with saturated brine, the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by high performance liquid chromatography to give yellow solid tert-butyl (S)-5-amino-4-(5-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)-1-oxoisoindolin-2-yl)-5-oxopentanoate (LC-02-003-P1, 71.00 mg, yield 33.20%) and white solid tert-butyl (S)-5-amino-4-(5-(5-chloropyrazolo[1,5-a]pyrimidin-3-yl)-1-oxoisoindolin-2-yl)-5-oxopentanoate (LC-02-003-P2, 59.00 mg, 27.89%). LC-02-003-P1: LC-MS (ESI): C 23 H 25 BrN5O4 + [M+H] + , calculated value: 515.38; measured value: 515.40.

[0081] LC-02-003-P2: LC-MS(ESI)C 23 H 25 ClN5O4 + [M+H] + , calculated value: 469.93; measured value: 469.90.

[0082] Step 4: Synthesis of (S)-3-(5-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-047-P1) and (S)-3-(5-(5-chloropyrazolo[1,5-a]pyrimidin-3-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-047-P2): Tert-butyl (S)-5-amino-4-(5-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)-1-oxoisoindolin-2-yl)-5-oxopentanoate (1 eq.) was dissolved in acetonitrile, and benzenesulfonic acid (3 eq.) was added. The reaction solution was heated to 100°C and stirred for 12 hours. The reaction was completed by LCMS, and the reaction solution was cooled to room temperature. The solvent was concentrated under reduced pressure, ethyl acetate and petroleum ether were added to the residue for slurrying, and (S)-3-(5-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-047-P1, 23.00 mg, yield 37.85%) was obtained as a white solid by filtration. LC-MS (ESI) C 19 H 15 BrN5O3 + [M+H] + , calculated value: 442.04; measured value: 442.11.1H NMR (400MHz, DMSO-d6) δ11.06 (s, 1H), 9.29 (d, J = 4.0Hz, 1H), 8.47 (s, 1H), 8.4 2(s,1H),8.40(d,J=8.0Hz,1H),7.91(d,J=8.0Hz,1H),7.82(d,J=8.0Hz,1H),5 .17(dd,J=12.0,4.0Hz,1H),4.59(d,J=16.0Hz,1H),4.47(d,J=16.0Hz,1H),2 .98-2.89(m,1H),2.65-2.60(m,1H),2.46-2.39(m,1H),2.07-2.04(m,1H).13C NMR(150MHz,DMSO-d6)δ172.92,170.96,167.50,155.68,145.29,144.41,142.87,139.1 7,137.36,133.66,127.35,123.61,122.53,106.71,83.59,51.81,47.49,31.22,22.47.

[0083] Tert-butyl (S)-5-amino-4-(5-(5-chloropyrazolo[1,5-a]pyrimidin-3-yl)-1-oxoisoindolin-2-yl)-5-oxopentanoate (1 eq.) was dissolved in acetonitrile and benzenesulfonic acid (3 eq.) was added. The reaction solution was heated to 100°C and stirred for 12 hours. The reaction was completed by LCMS, and the reaction solution was cooled to room temperature. The solvent was concentrated under reduced pressure, ethyl acetate and petroleum ether were added to the residue for slurrying, and S)-3-(5-(5-chloropyrazolo[1,5-a]pyrimidin-3-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-047-P2, 15.00 mg, yield 38.18%) was obtained by filtration as a white solid. LC-MS (ESI) C 19 H 15 ClN5O3 + [M+H] + , calculated value: 396.09; found value: 396.17.1H NMR (400MHz, DMSO-d6) δ11.01 (s, 1H), 9.26 (d, J = 8.0Hz, 1H), 8.95 (s, 1H), 8.2 7(s,1H),8.24(d,J=8.0Hz,1H),7.81(d,J=8.0Hz,1H),7.26(d,J=8.0Hz,1H),5 .14(dd,J=12.0,4.0Hz,1H),4.55(d,J=16.0Hz,1H),4.39(d,J=16.0Hz,1H),2 .98-2.88(m,1H),2.63-2.59(m,1H),2.47-2.38(m,1H),2.04-2.01(m,1H).13C NMR(150MHz,DMSO-d6)δ172.96,171.11,167.99,150.83,144.49,143.31,142.88,138.9 7,134.75,129.51,125.31,123.54,120.02,109.78,108.43,51.67,47.31,31.26,22.52.

[0084] Example 2: Synthesis of (S)-3-(5-(5-(benzo[d][1,3]dioxol-5-ylamino)pyrazolo[1,5-a]pyrimidin-3-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-104)

[0085]

[0086] Step 1: Synthesis of tert-butyl (S)-5-amino-4-(5-(5-(benzo[D][1,3]dioxol-5-ylamino)pyrazolo[1,5-a]pyrimidin-3-yl)-1-oxoisoindolin-2-yl)-5-oxopentanoate (LC-02-101): tert-butyl (S)-5-amino-5-oxo-4-(1-oxo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-2-yl)pentanoate (1 eq.) and N-(benzo[d][1,3]dioxol-5-yl)-3-bromopyrazolo[1,5-a]pyrimidin-5-amine (1.2 eq.) were dissolved in 1,4-dioxane and 3 M potassium phosphate solution (3 eq.) was added. The gas was replaced with nitrogen three times, Pd(dtbpf)Cl2 (0.1eq.) was added, and the gas was replaced with nitrogen three times again. The reaction solution was heated to 100°C and stirred for 3 hours. The reaction was detected to be complete by LCMS, and the reaction solution was cooled to room temperature. Water and ethyl acetate were added to the reaction solution for extraction, and the extraction was repeated three times until the extraction was complete. The organic phase was washed with saturated brine, combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by high performance liquid chromatography to obtain tert-butyl (S)-5-amino-4-(5-(5-(benzo[D][1,3]dioxol-5-ylamino)pyrazolo[1,5-a]pyrimidin-3-yl)-1-oxoisoindolin-2-yl)-5-oxopentanoate as a white solid. (45.00 mg, yield 68.68%). LC-MS(ESI)C30H31N6O6 + [M+H] + , calculated value: 571.23; measured value: 571.21.

[0087] Step 2: Synthesis of (S)-3-(5-(5-(benzo[d][1,3]dioxol-5-ylamino)pyrazolo[1,5-a]pyrimidin-3-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-104)

[0088] Tert-butyl (S)-5-amino-4-(5-(5-(benzo[d][1,3]dioxol-5-ylamino)pyrazolo[1,5-a]pyrimidin-3-yl)-1-oxoisoindolin-2-yl)-5-oxopentanoate (1 eq.) was dissolved in acetonitrile and benzenesulfonic acid (3 eq.) was added. The reaction solution was heated to 100°C and stirred for 12 hours. The reaction was completed by LCMS and the reaction solution was cooled to room temperature. The solvent was concentrated under reduced pressure, ethyl acetate and petroleum ether were added to the residue for slurrying, and (S)-3-(5-(5-(benzo[d][1,3]dioxol-5-ylamino)pyrazolo[1,5-a]pyrimidin-3-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-104: 22.00 mg, yield 56.19%) was obtained as a white solid by filtration.

[0089] LC-MS(ESI)C26H21N6O5 + [M+H] + , calculated value: 497.16; found value: 497.21. 1H NMR (400MHz, DMSO-d6) δ11.02 (s, 1H), 9.89 (s, 1H), 8.71 (d, J = 4.0 Hz, 1H), 8.55 (s, 1H), 8.40 (s, 1H), 8.13 (d, J = 8.0 Hz, 1H), 7.81 (s, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.12 (d, J = 8.0 Hz, 1H), 6.99 (d, J = 8.0 Hz, 1H) ,6.53(d,J=8.0Hz,1H),6.06(d,J=4.0Hz,2H),5.14(dd,J=12.0,4.0Hz,1H),4.50(d,J=16.0Hz,1H),4 .39(d,J=16.0Hz,1H),2.98-2.89(m,1H),2.65-2.60(m,1H),2.45-2.37(m,1H),2.04-2.02(m,1H).13C NMR(150MHz,DMSO-d6)δ172.99,171.18,168.22,153.18,147.21,144.13,142.82,142.63,142.43,136.74,136.28,13 4.15,127.98,124.27,123.14,119.03,112.30,108.23,104.88,101.90,101.27,101.13,54.93,51.61,31.30,22.57.

[0090] Example 3: Synthesis of (S)-3-(5-(5-((3-chloro-4-methylphenyl)amino)pyrazolo[1,5-a]pyrimidin-3-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-105)

[0091]

[0092] Referring to the synthesis method of Example 2, N-(benzo[d][1,3]dioxol-5-yl)-3-bromopyrazolo[1,5-a]pyrimidin-5-amine was replaced with 3-bromo-N-(3-chloro-4-methylphenyl)pyrazolo[1,5-a]pyrimidin-5-amine (LC-02-093) to synthesize the target product LC-02-105. LCMS (ESI) C26H22ClN6O3 + [M+H] + , calculated value: 501.15; found value: 501.61. 1HNMR (400MHz, DMSO-d6) δ11.03 (s, 1H), 10.07 (s, 1H), 8.74 (d, J = 8.0 Hz, 1H), 8.58 (s, 1H), 8.47 (s, 1H), 8.26 (d, J = 8.0 Hz, 1H), 8.23 ​​(s, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.38-7.34 (m,2H),6.58(d,J=8.0Hz,1H),5.14(dd,J=12.0,4.0Hz,1H),4.51(d,J=16.0Hz,1H),4.41(d,J=16.0 Hz,1H),2.98-2.89(m,1H),2.64-2.60(m,1H),2.48-2.37(m,1H),2.32(s,3H),2.05-2.02(m,1H).13C NMR(150MHz,DMSO-d6)δ172.97,171.20,168.19,153.08,143.79,142.80,142.74,138.91,136.66,136.52,133.1 3,131.23,128.99,128.18,124.43,123.19,118.91,117.94,105.33,101.51,51.66,47.33,31.30,22.57,19.02.

[0093] Example 4: Synthesis of (S)-3-(5-(5-(benzylamino)pyrazolo[1,5-a]pyrimidin-3-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-116)

[0094]

[0095] Referring to the synthesis method of Example 2, N-(benzo[d][1,3]dioxol-5-yl)-3-bromopyrazolo[1,5-a]pyrimidin-5-amine was replaced with N-benzyl-3-bromopyrazolo[1,5-a]pyrimidin-5-amine (LC-02-097) to synthesize the target product LC-02-116. LCMS (ESI) C26H23N6O3+[M+H]+, calculated value: 467.19; found value: 467.36.1H NMR (400MHz, DMSO-d6) δ11.02(s,1H),8.55(d,J=8.0Hz,1H),8.45(s,1H),8.40(t,J=4.0Hz,1H),8.25(s,1 H),8.12(d,J=8.0Hz,1H),7.66(d,J=8.0Hz,1H),7.46(d,J=4.0Hz,2H),7.36(t,J=8.0Hz,2H),7.24(t,J=8 .0Hz,1H),6.42(d,J=8.0Hz,1H),5.12(dd,J=12.0,4.0Hz,1H),4.63(d,J=8.0Hz,2H),4.46(d,J=16.0Hz,1 H),4.33(d,J=16.0Hz,1H),2.97-2.88(m,1H),2.63-2.59(m,1H),2.48-2.40(m,1H),2.03-2.00(m,1H).13C NMR(150MHz,DMSO-d6)δ172.99,171.24,168.31,145.07,142.63,142.10,139.49,137.13,135.67,1 28.39,127.53,127.47,126.85,124.03,123.01,118.61,103.76,51.58,47.22,44.40,31.30,22.54.

[0096] Example 5: Synthesis of (S)-3-(1-oxo-5-(5-(phenylethylamino)pyrazolo[1,5-a]pyrimidin-3-yl)isoindolphen-2-yl)piperidine-2,6-dione (LC-02-117)

[0097]

[0098] Referring to the synthesis method of Example 2, N-(benzo[d][1,3]dioxol-5-yl)-3-bromopyrazolo[1,5-a]pyrimidin-5-amine was replaced with 3-bromo-N-phenethylpyrazolo[1,5-a]pyrimidin-5-amine (LC-02-098) to synthesize the target product LC-02-117. LCMS (ESI) C27H25N6O3+[M+H]+, calculated value: 481.20; found value: 481.23.1H NMR (400MHz, DMSO-d6) δ11.05(s,1H),8.53(d,J=4.0Hz,1H),8.47(s,1H),8.38(s,1H),8.25(d,J=8.0Hz ,1H),7.98(t,J=4.0Hz,1H),7.68(d,J=8.0Hz,1H),7.35-7.32(m,4H),7.26-7.20(m,1H),6.34(d,J=8.0H z,1H),5.14(dd,J=12.0,4.0Hz,1H),4.43(d,J=16.0Hz,1H),4.29(d,J=16.0Hz,1H),3.67(q,J=8.0Hz,2H ),2.97(t,J=8.0Hz,2H),2.94-2.89(m,1H),2.64-2.60(m,1H),2.44-2.33(m,1H),2.04-2.01(m,1H).13C NMR (150MHz, DMSO-d6) δ172.99,171.25,168.30,156.02,145.28,142.65,142.13,139.60,137.39,135.57,128. 65,128.46,127.53,126.22,124.05,123.01,118.61,103.70,100.49,51.56,47.12,42.17,34.46,31.28,22.65.

[0099] Example 6: Synthesis of (S)-3-(5-(5-(cyclohexylamino)pyrazolo[1,5-a]pyrimidin-3-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-119)

[0100]

[0101] Referring to the synthesis method of Example 2, N-(benzo[d][1,3]dioxol-5-yl)-3-bromopyrazolo[1,5-a]pyrimidin-5-amine was replaced with 3-bromo-N-cyclohexylpyrazolo[1,5-a]pyrimidin-5-amine (LC-02-106) to synthesize the target product LC-02-119. LCMS (ESI) C25H27N6O3+[M+H]+, calculated value: 459.22; found value: 459.29.1H NMR (400MHz, DMSO-d6) δ11.01(s,1H),8.49(d,J=8.0Hz,1H),8.45(s,1H),8.38(s,1H),8.19(d,J=8.0Hz, 1H),7.70-7.66(m,2H),6.32(d,J=4.0Hz,1H),5.11(dd,J=12.0,4.0Hz,1H),4.46(d,J=16.0Hz,1H),4.33( d,J=16.0Hz,1H),3.94-3.83(m,1H),2.96-2.87(m,1H),2.62-2.58(m,1H),2.47-2.39(m,1H),2.10-2.08( m,2H),2.02-1.99(m,1H),1.79-1.76(m,2H),1.67-1.63(m,1H),1.47-1.38(m,2H),1.33-1.21(m,3H).13C NMR (150MHz, DMSO-d6) δ172.98,171.24,168.30,155.24,145.31,142.67,141.99,137.43,135.44,127.44 ,123.84,123.01,118.41,103.40,100.50,54.93,51.62,49.82,47.21,31.79,31.28,25.54,24.61,22.51.

[0102] Example 7: Synthesis of (S)-3-(1-oxo-5-(5-(tetrahydro-2H-pyran-4-yl)amino)pyrazolo[1,5-a]pyrimidin-3-yl)isoindolphen-2-yl)piperidine-2,6-dione (LC-02-120)

[0103]

[0104] Referring to the synthesis method of Example 2, N-(benzo[d][1,3]dioxol-5-yl)-3-bromopyrazolo[1,5-a]pyrimidin-5-amine was replaced with 3-bromo-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[1,5-a]pyrimidin-5-amine (LC-02-107) to synthesize the target product LC-02-120. LCMS (ESI) C24H25N6O4+[M+H]+, calculated value: 460.20; found value: 460.26. 1HNMR (400MHz, DMSO-d6) δ11.01(s,1H),8.53(d,J=8.0Hz,1H),8.47(s,1H),8.39(s,1H),8.17(d,J=8.0Hz,1H),7.81(d,J=8.0Hz,1H),7.69(d,J=8.0Hz,1H),6.32(d,J=8.20Hz,1H),5 .12(dd,J=12.0,4.0Hz,1H),4.46(d,J=16.0Hz,1H),4.34(d,J=16.0Hz,1H),4.18-4.05(m,1H),3.93(d,J=12.0Hz,2H),3 .52(t,J=12.0Hz,2H),2.97-2.88(m,1H),2.63-2.59(m,1H),2.48-2.40(m,1H),2.07-1.99(m,3H),1.57-1.47(m,2H).13C NMR(150MHz,DMSO-d6)δ172.98,171.23,168.31,155.20,145.17,142.72,142.07,137.29,135.65,127 .51,123.86,123.09,118.49,103.57,100.41,65.99,54.93,51.59,47.31,47.18,32.04,31.28,22.52.

[0105] Example 8: Synthesis of (S)-3-(5-(5-((3-chloro-4-methoxyphenyl)amino)pyrazolo[1,5-a]pyrimidin-3-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-121)

[0106]

[0107] Referring to the synthesis method of Example 2, N-(benzo[d][1,3]dioxol-5-yl)-3-bromopyrazolo[1,5-a]pyrimidin-5-amine was replaced with 3-bromo-N-(3-chloro-4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-amine (LC-02-103) to synthesize the target product LC-02-121. LCMS (ESI) C26H22ClN6O4+[M+H]+, calculated value: 517.14; found value: 517.19.1H NMR (400MHz, DMSO-d6) δ11.03(s,1H),9.98(s,1H),8.71(d,J=8.0Hz,1H),8.56(s,1H),8.43(d,J=4 .0Hz,1H),8.24-8.22(m,2H),7.74(d,J=8.0Hz,1H),7.45(dd,J=8.0,4.0Hz,1H),7.21(d,J=8.0Hz, 1H),6.54(d,J=8.0Hz,1H),5.14(dd,J=12.0,4.0Hz,1H),4.50(d,J=16.0Hz,1H),4.40(d,J=16.0Hz ,1H),3.86(s,3H),2.98-2.89(m,1H),2.64-2.60(m,1H),2.46-2.35(m,1H),2.04-2.01(m,1H).13C NMR(150MHz,DMSO-d6)δ172.97,171.19,168.21,153.08,150.06,143.94,142.80,142.64,136.73,136.38,133.53,1 28.08,124.35,123.21,120.72,120.63,119.25,118.86,113.22,105.09,101.37,56.28,51.63,47.26,31.29,22.59.

[0108] Example 9: Synthesis of (S)-3-(5-(5-((3-chloro-4-methoxyphenyl)amino)pyrazolo[1,5-a]pyrimidin-3-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-135)

[0109]

[0110] Referring to the synthesis method of Example 2, N-(benzo[d][1,3]dioxol-5-yl)-3-bromopyrazolo[1,5-a]pyrimidine-5-amine was replaced with 3-bromo-N-(4,4-difluorocyclohexyl)pyrazolo[1,5-a]pyrimidine-5-amine (LC-02-122) to synthesize the target product LC-02-135. LCMS (ESI) C25H25F2N6O3+[M+H]+, calculated value: 495.20; found value: 495.52. 1HNMR (400MHz, DMSO-d6) δ11.02(s,1H),8.54(d,J=8.0Hz,1H),8.48(s,1H),8.34(s,1H),8.23(d,J=8.0Hz,1H),7.80(d,J=4.0Hz,1H),7.71(d,J=8.0Hz,1H),6.34(d,J =8.0Hz,1H),5.11(dd,J=12.0,4.0Hz,1H),4.48(d,J=16.0Hz,1H),4.36(d,J=16.0Hz,1H),4.15-4.04(m,1H),2.9 6-2.87(m,1H),2.63-2.59(m,1H),2.47-2.37(m,1H),2.20-2.05(m,6H),2.04-1.96(m,2H),1.72-1.60(m,2H).13C NMR(150MHz,DMSO-d6)δ172.97,171.23,168.32,155.42,145.12,142.73,142.14,137.24,135.66,1 27.56,123.99,123.18,122.27,118.52,103.70,100.49,54.93,51.64,47.27,31.27,27.31,22.57.

[0111] Example 10: Synthesis of (S)-3-(5-(5-((2-morpholinethyl)amino)pyrazolo[1,5-a]pyrimidin-3-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-148)

[0112]

[0113] Referring to the synthesis method of Example 2, N-(benzo[d][1,3]dioxol-5-yl)-3-bromopyrazolo[1,5-a]pyrimidin-5-amine was replaced with 3-bromo-N-(2-morpholinoethyl)pyrazolo[1,5-a]pyrimidin-5-amine (LC-02-123) to synthesize the target product LC-02-148. LCMS (ESI) C25H28N7O4+[M+H]+, calculated value: 490.22; found value: 490.32.1H NMR (400MHz, DMSO-d6) δ11.03(s,1H),8.61(d,J=8.0Hz,1H),8.50(s,1H),8.26(s,1H),8.24(d,J= 8.0Hz,1H),8.08(t,J=8.0Hz,1H),7.72(d,J=8.0Hz,1H),6.38(d,J=8.0Hz,1H),5.13(dd,J=12.0, 4.0Hz,1H),4.48(d,J=16.0Hz,1H),4.35(d,J=16.0Hz,1H),3.98-3.82(m,4H),3.77-3.55(m,5H), 3.34-3.05(m,3H),2.98-2.89(m,1H),2.64-2.60(m,1H),2.44-2.33(m,1H),2.04-2.01(m,1H).13C NMR(150MHz,DMSO-d6)δ172.97,171.22,168.29,155.98,144.82,142.72,142.39,137.02,136.02,127 .77,124.38,123.21,118.96,104.21,63.32,54.93,54.60,51.58,51.41,47.20,35.04,31.28,22.64.

[0114] Example 11: Synthesis of (S)-3-(1-oxo-5-(pyrazolo[1,5-a]pyrimidin-5-yl)isoindolin-2-yl)piperidine-2,6-dione (LC-04-075)

[0115] The synthetic route of LC-04-075 is as follows:

[0116]

[0117] Step 1: Synthesis of tert-butyl (S)-5-amino-5-oxo-4-(1-oxo-5-(pyrazolo[1,5-a]pyrimidin-5-yl)isoindolin-2-yl)pentanoate (LC-02-070): tert-butyl (S)-5-amino-5-oxo-4-(1-oxo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-2-yl)pentanoate (1 eq.) and 5-chloropyrazolo[1,5-a]pyrimidine (1 eq.) were dissolved in ethylene glycol dimethyl ether, and saturated sodium carbonate solution was added. The atmosphere was replaced with nitrogen three times, Pd(PPh3)2Cl2 (0.05 eq.) was added, and the atmosphere was replaced with nitrogen three times again. The reaction solution was heated to 80°C and stirred for 3 minutes. The reaction was completed by LCMS, and the reaction solution was cooled to room temperature. Water and ethyl acetate were added to the reaction solution for extraction, and the extraction was repeated three times until the extraction was complete. The organic phase was washed with saturated brine, combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain tert-butyl (S)-5-amino-5-oxo-4-(1-oxo-5-(pyrazolo[1,5-a]pyrimidin-5-yl)isoindolin-2-yl)pentanoate as a yellow solid. (150.00 mg, yield 76.52%). LC-MS (ESI) C23H26N5O4 + [M+H] + , calculated value: 436.20; measured value: 436.29.

[0118] Step 2: Synthesis of (S)-3-(1-oxo-5-(pyrazolo[1,5-a]pyrimidin-5-yl)isoindolin-2-yl)piperidine-2,6-dione (LC-04-075): Dissolve tert-butyl (S)-5-amino-5-oxo-4-(1-oxo-5-(pyrazolo[1,5-a]pyrimidin-5-yl)isoindolin-2-yl)pentanoate (1 eq.) in acetonitrile and add benzenesulfonic acid (3 eq.). Heat the reaction solution to 100°C and stir for 12 hours. The reaction is completed by LCMS and the reaction solution is cooled to room temperature. The solvent was concentrated under reduced pressure, ethyl acetate and petroleum ether were added to the residue for slurrying, and filtered to obtain (S)-3-(1-oxo-5-(pyrazolo[1,5-a]pyrimidin-5-yl)isoindolin-2-yl)piperidine-2,6-dione (LC-04-075: 56.00 mg, yield 44.99%) as a yellow solid. LC-MS (ESI) C19H16N5O3 + [M+H] +, calculated value: 362.13; found value: 362.19. 1H NMR (400MHz, DMSO-d6) δ11.05 (s, 1H), 9.25 (d, J = 8.0 Hz, 1H), 8.46 (s, 1H), 8.36 (d, J = 8.0 Hz, 1H), 8.28 (d, J = 4.0 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 6.81 (d ,J=4.0Hz,1H),5.17(dd,J=12.0,4.0Hz,1H),4.58(d,J=16.0Hz,1H),4.45(d,J=16.0H z,1H),3.01-2.85(m,1H),2.67-2.58(m,1H),2.48-2.36(m,1H),2.11-1.97(m,1H).13C NMR(150MHz,DMSO-d6)δ172.90,170.97,167.54,154.50,147.72,145.70,142.83,139.7 3,136.48,133.31,127.17,123.47,122.40,105.79,96.69,51.77,47.43,31.22,22.47.

[0119] Example 12: Synthesis of (S)-3-(5-(3-chloropyrazolo[1,5-a]pyrimidin-5-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-04-087)

[0120] The structural formula of compound LC-04-087 (dCK1α-1) is as follows:

[0121]

[0122] Referring to the synthesis method of Example 11, 5-chloropyrazolo[1,5-a]pyrimidine was replaced with 3,5-dichloropyrazolo[1,5-a]pyrimidine to synthesize the target product LC-04-087 (dCK1α-1). LCMS (ESI) C19H15ClN5O3+[M+H]+, calculated value: 396.09; found value: 396.18.1H NMR (400MHz, DMSO-d6) δ11.06 (s, 1H), 9.27 (d, J = 4.0Hz, 1H), 8.48 (s, 1H), 8.4 3(s,1H),8.40(d,J=8.0Hz,1H),7.91(d,J=8.0Hz,1H),7.82(d,J=8.0Hz,1H),5 .17(dd,J=12.0,4.0Hz,1H),4.59(d,J=16.0Hz,1H),4.47(d,J=16.0Hz,1H),2 .98-2.89(m,1H),2.65-2.60(m,1H),2.47-2.39(m,1H),2.07-2.04(m,1H).13C NMR(150MHz,DMSO-d6)δ172.92,170.96,167.49,155.47,143.30,143.03,142.87,139.14,137.3 3,133.67,127.64,127.36,125.50,123.59,122.55,106.69,98.51,51.82,47.49,31.22,22.47.

[0123] Example 13: Synthesis of (S)-3-(5-(3-aminopyrazolo[1,5-a]pyrimidin-5-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-04-081)

[0124] The structural formula of compound LC-04-081 is as follows:

[0125]

[0126] Referring to the synthesis method of Example 11, 5-chloropyrazolo[1,5-a]pyrimidine was replaced with 5-chloropyrazolo[1,5-a]pyrimidin-3-amine to synthesize the target product LC-04-081: LCMS (ESI) C19H17N6O3+[M+H]+, calculated value: 377.14; found value: 377.72.1H NMR (400MHz, DMSO-d6) δ11.06 (s, 1H), 9.20 (d, J = 8.0Hz, 1H), 8.49 (s, 1H), 8.4 5(d,J=8.0Hz,1H),8.18(s,1H),7.93(d,J=8.0Hz,1H),7.76(d,J=8.0Hz,1H),5 .17(dd,J=12.0,4.0Hz,1H),4.59(d,J=16.0Hz,1H),4.46(d,J=16.0Hz,1H),2 .99-2.90(m,1H),2.65-2.61(m,1H),2.47-2.40(m,1H),2.06-2.02(m,1H).13C NMR (150MHz, DMSO-d6) δ172.93,170.97,167.53,142.85,139.37,136.61,133.46,127.16,123.57,122.35,106.06,54.93,51.82,31.22,22.45.

[0127] Example 14: Synthesis of (S)-5-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)pyrazolo[1,5-a]pyrimidine-3-cyano (LC-04-077)

[0128] The structural formula of compound LC-04-077 is as follows:

[0129]

[0130] Referring to the synthesis method of Example 11, 5-chloropyrazolo[1,5-a]pyrimidine was replaced with 5-chloropyrazolo[1,5-a]pyrimidine-3-cyano to synthesize the target product LC-04-077: LCMS (ESI) C20H15N6O3+[M+H]+, calculated value: 387.12; found value: 387.49. 1H NMR (400MHz, DMSO-d6) δ11.06 (s, 1H), 9.48 (d, J = 8.0Hz, 1H), 8.85 (s, 1H), 8. 53(s,1H),8.43(d,J=8.0Hz,1H),8.06(d,J=8.0Hz,1H),7.92(d,J=8.0Hz,1H) ,5.18(dd,J=12.0,4.0Hz,1H),4.60(d,J=16.0Hz,1H),4.48(d,J=16.0Hz,1H) ,2,98-2.89(m,1H),2.65-2.61(m,1H),2.47-2.39(m,1H),2.07-2.05(m,1H). 13 C NMR(150MHz,DMSO-d6)δ172.91,170.94,167.37,158.52,149.52,148.46,142.93,138.4 2,134.26,127.78,123.70,122.99,113.52,108.72,81.19,51.85,47.53,31.22,22.46.

[0131] Example 15: Synthesis of (S)-3-(5-(2-methylpyrazolo[1,5-a]pyrimidin-5-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-04-155)

[0132] The structural formula of compound LC-04-155 is as follows:

[0133]

[0134] Referring to the synthesis method of Example 11, 5-chloropyrazolo[1,5-a]pyrimidine was replaced with 5-chloro-2-methylpyrazolo[1,5-a]pyrimidine (LC-04-147) to synthesize the target product LC-04-155: LCMS (ESI) C20H18N5O3+[M+H]+, calculated value: 376.14; found value: 376.37. 1H NMR (400MHz, DMSO-d6) δ11.05 (s, 1H), 9.11 (d, J = 4.0Hz, 1H), 8.43 (s, 1H), 8. 33(d,J=8.0Hz,1H),7.87(d,J=8.0Hz,1H),7.63(d,J=8.0Hz,1H),6.58(s,1H) ,5.17(dd,J=12.0,4.0Hz,1H),4.56(d,J=16.0Hz,1H),4.44(d,J=16.0Hz,1H) ,2.98-2.89(m,1H),2.64-2.60(m,1H),2.44-2.38(m,4H),2.05-2.03(m,1H). 13 C NMR(150MHz,DMSO-d6)δ172.91,170.98,167.57,155.20,154.09,148.40,142.80,139.86,1 35.78,133.18,127.06,123.43,122.26,104.87,95.83,51.77,47.43,31.22,22.47,14.32.

[0135] Example 16: Synthesis of (S)-3-(5-(3-chloro-2-methylpyrazolo[1,5-a]pyrimidin-5-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-04-162)

[0136] The structural formula of compound LC-04-162 is as follows:

[0137]

[0138] Referring to the synthesis method of Example 11, 5-chloropyrazolo[1,5-a]pyrimidine was replaced with 3,5-dichloro-2-methylpyrazolo[1,5-a]pyrimidine (LC-04-150) to synthesize the target product LC-04-162: LCMS (ESI) C20H17ClN5O3+[M+H]+, calculated value: 410.10; found value: 410.73. 1H NMR (400MHz, DMSO-d6) δ11.05(s,1H),9.15(d,J=8.0Hz,1H),8.44(s,1H),8.36(d,J=8.0Hz,1H),7.89(d,J=8.0Hz,1H),7.73(d,J=8.0Hz,1H),5.17( dd,J=12.0,4.0Hz,1H),4.58(d,J=16.0Hz,1H),4.46(d,J=16.0Hz,1H),2. 98-2.89(m,1H),2.64-2.60(m,1H),2.46-2.39(m,4H),2.07-2.03(m,1H). 13 C NMR(150MHz,DMSO-d6)δ172.90,170.94,167.50,155.10,151.64,143.36,136.70,133.56,1 28.36,127.63,127.27,125.49,123.56,122.44,105.95,51.80,47.48,31.21,22.45,11.99.

[0139] Example 17: Synthesis of (S)-3-(5-(3-chloro-2-(morpholinomethyl)pyrazolo[1,5-a]pyrimidin-5-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (dCK1α-2)

[0140] The structural formula of compound dCK1α-2 is as follows:

[0141]

[0142] Referring to the synthesis method of Example 11, 5-chloropyrazolo[1,5-a]pyrimidine was replaced with 4-((3,5-dichloropyrazolo[1,5-a]pyrimidin-2-yl)methyl)morpholine (LC-04-179) to synthesize the target product dCK1α-2: LCMS (ESI) C24H24ClN6O4+[M+H]+, calculated value: 495.16; found value: 495.31. 1H NMR (400MHz, DMSO-d6) δ11.06 (s, 1H), 9.23 (d, J = 8.0Hz, 1H), 8.47 (s, 1H), 8.39 (d, J=8.0Hz,1H),7.91(d,J=8.0Hz,1H),7.80(d,J=8.0Hz,1H),5.17(dd,J=12.0,4.0Hz ,1H),4.59(d,J=16.0Hz,1H),4.47(d,J=16.0Hz,1H),3.72(s,2H),3.56(d,J=4.0H z,4H),2.98-2.89(m,1H),2.65-2.60(m,1H),2.48-2.39(m,5H),2.06-2.04(m,1H). 13 C NMR(150MHz,DMSO-d6)δ172.91,170.95,167.50,155.27,150.79,143.38,142.87,139.21,136.90, 133.63,127.32,123.59,122.48,106.61,98.53,66.15,53.15,53.05,51.81,47.48,31.22,22.46.

[0143] Example 18: Synthesis of (S)-3-(5-(3-methylpyrazolo[1,5-a]pyrimidin-5-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-04-113)

[0144]

[0145] Step 1: Synthesis of tert-butyl (S)-5-amino-4-(5-(3-methylpyrazolo[1,5-a]pyrimidin-5-yl)-1-oxoisoindolin-2-yl)-5-oxopentanoate (LC-04-097): tert-butyl (S)-5-amino-4-(5-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)-1-oxoisoindolin-2-yl)-5-oxopentanoate (1 eq.) and 2,4,4,5,5-pentamethyl-1,3,2-dioxaborane (1 eq.) were dissolved in a mixed solvent of 1,4-dioxane and water, and potassium carbonate (2.5 eq.) was added. The atmosphere was replaced with nitrogen three times, Pd(dppf)Cl2 (0.05 eq.) was added, and the atmosphere was replaced with nitrogen three times again. The reaction solution was heated to 90°C and stirred for 16 hours. The reaction was completed by LCMS detection, and the reaction solution was cooled to room temperature. Water and ethyl acetate were added to the reaction solution for extraction, and the extraction was repeated three times until the extraction was complete. The organic phase was washed with saturated brine, combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain tert-butyl (S)-5-amino-4-(5-(3-methylpyrazolo[1,5-a]pyrimidin-5-yl)-1-oxoisoindolin-2-yl)-5-oxopentanoate as a yellow solid. (98.00 mg, yield 44.86%). LC-MS (ESI) C24H28N5O4 + [M+H] + , calculated value: 450.22; measured value: 450.29.

[0146] Step 2: Synthesis of (S)-3-(5-(3-methylpyrazolo[1,5-a]pyrimidin-5-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-04-113): Dissolve tert-butyl (S)-5-amino-4-(5-(3-methylpyrazolo[1,5-a]pyrimidin-5-yl)-1-oxoisoindolin-2-yl)-5-oxopentanoate (1 eq.) in acetonitrile and add benzenesulfonic acid (3 eq.). The reaction solution was heated to 100°C and stirred for 12 hours. The reaction was completed by LCMS and the reaction solution was cooled to room temperature. The solvent was removed by concentration under reduced pressure, ethyl acetate and petroleum ether were added to the residue for slurrying, and (S)-3-(5-(3-methylpyrazolo[1,5-a]pyrimidin-5-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-04-113: 43.00 mg, yield 52.54%) was obtained as a yellow solid by filtration. LC-MS (ESI) C20H18N5O3 + [M+H] +, calculated value: 376.14; found value: 376.47. 1HNMR (400MHz, DMSO-d6) δ11.05 (s, 1H), 9.14 (d, J = 8.0 Hz, 1H), 8.45 (s, 1H), 8.38 (d, J = 8.0 Hz, 1H), 8.12 (s, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H z,1H),5.17(dd,J=12.0,4.0Hz,1H),4.58(d,J=16.0Hz,1H),4.45(d,J=16.0Hz,1H),3.0 0-2.86(m,1H),2.67-2.59(m,1H),2.48-2.38(m,1H),2.36(s,3H),2.07-2.00(m,1H).13C NMR(150MHz,DMSO-d6)δ172.93,170.99,167.61,152.98,145.40,145.31,142.83,139.90,1 36.18,133.14,127.03,123.49,122.19,105.25,105.13,51.79,47.46,31.23,22.48,7.49.

[0147] Scheme 1: Synthesis of intermediate N-(benzo[d][1,3]dioxol-5-yl)-3-bromopyrazolo[1,5-a]pyrimidin-5-amine (LC-02-092)

[0148]

[0149] 3-Bromo-5-chloropyrazolo[1,5-a]pyrimidine (1 eq.) and benzo[d][1,3]dioxol-5-amine (1.5 eq.) were dissolved in n-butanol, and DIEA (3 eq.) was added. The reaction solution was heated to 125°C and stirred for 16 hours. The reaction was detected to be complete by LCMS, and the reaction solution was cooled to room temperature. The solvent was removed by concentration under reduced pressure, and water and ethyl acetate were added for extraction. The extraction was repeated three times until the extraction was complete, and the organic phase was washed with saturated brine, combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain N-(benzo[d][1,3]dioxol-5-yl)-3-bromopyrazolo[1,5-a]pyrimidine-5-amine as a white solid. (215.00 mg, yield 75.01%). LC-MS (ESI) C13H10BrN4O2+[M+H]+, calculated value: 332.99 and 334.99; found value: 333.11 and 335.08.

[0150] Synthesis of intermediate 3-bromo-N-(3-chloro-4-methylphenyl)pyrazolo[1,5-a]pyrimidin-5-amine (LC-02-093): Referring to the synthesis method of LC-02-092, benzo[d][1,3]dioxol-5-amine was replaced with 3-chloro-4-methylaniline to synthesize the target product. LCMS (ESI) C13H11BrClN4+[M+H]+, calculated values: 336.99 and 338.99; found values: 337.05 and 339.03.

[0151] Synthesis of intermediate N-benzyl-3-bromopyrazolo[1,5-a]pyrimidin-5-amine (LC-02-097): Referring to the synthesis method of LC-02-092, benzo[d][1,3]dioxol-5-amine was replaced with benzylamine to synthesize the target product. LCMS (ESI) C13H12BrN4+[M+H]+, calculated values: 303.03 and 305.02; found values: 303.07 and 305.04.

[0152] Synthesis of intermediate 3-bromo-N-phenethylpyrazolo[1,5-a]pyrimidin-5-amine (LC-02-098): Referring to the synthesis method of LC-02-092, benzo[d][1,3]dioxol-5-amine was replaced with phenethylamine to synthesize the target product. LCMS (ESI) C14H14BrN4+[M+H]+, calculated values: 317.04 and 319.04;

[0153] Measured values: 317.14 and 319.09.

[0154] Synthesis of intermediate 3-bromo-N-cyclohexylpyrazolo[1,5-a]pyrimidin-5-amine (LC-02-106): Referring to the synthesis method of LC-02-092, benzo[d][1,3]dioxol-5-amine was replaced with cyclohexylamine to synthesize the target product. LCMS (ESI) C12H16BrN4+[M+H]+, calculated values: 295.06 and 297.06;

[0155] Measured values: 295.27 and 297.21.

[0156] Synthesis of intermediate 3-bromo-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[1,5-a]pyrimidin-5-amine (LC-02-107): Referring to the synthesis method of LC-02-092, benzo[d][1,3]dioxol-5-amine was replaced with tetrahydro-2H-pyran-4-amine to synthesize the target product. LCMS (ESI) C11H14BrN4O+[M+H]+, calculated values: 297.04 and 299.04; found values: 297.21 and 299.54.

[0157] Synthesis of intermediate 3-bromo-N-(3-chloro-4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-amine (LC-02-103): Referring to the synthesis method of LC-02-092, benzo[d][1,3]dioxol-5-amine was replaced with 3-chloro-4-methoxyaniline to synthesize the target product. LCMS (ESI) C13H11BrClN4O+[M+H]+, calculated values: 352.98 and 354.98; found values: 353.43 and 355.56.

[0158] Synthesis of intermediate 3-bromo-N-(4,4-difluorocyclohexyl)pyrazolo[1,5-a]pyrimidin-5-amine (LC-02-122): Referring to the synthesis method of LC-02-092, benzo[d][1,3]dioxol-5-amine was replaced with 4,4-difluorocyclohexane-1-amine to synthesize the target product. LCMS (ESI) C12H14BrF2N4+[M+H]+, calculated values: 331.04 and 333.04; found values: 331.18 and 333.75.

[0159] Synthesis of intermediate 3-bromo-N-(2-morpholinoethyl)pyrazolo[1,5-a]pyrimidin-5-amine (LC-02-123): Referring to the synthesis method of LC-02-092, benzo[d][1,3]dioxol-5-amine was replaced with 2-morpholinoethane-1-amine to synthesize the target product. LCMS (ESI) C12H17BrN5O+[M+H]+, calculated values: 326.06 and 328.06; found values: 326.32 and 328.46.

[0160] Scheme 2: Synthesis of intermediate 4-((3,5-dichloropyrazolo[1,5-a]pyrimidin-2-yl)methyl)morpholine (LC-04-179)

[0161]

[0162] Step 1: Synthesis of 2-methylpyrazolo[1,5-a]pyrimidin-5(4H)-one (LC-04-134)

[0163] 1,3-Dimethylpyrimidine-2,4(1H,3H)-dione (1eq.) and 5-methyl-1H-pyrazol-3-amine (1eq.) were dissolved in ethanol, and sodium ethoxide (3eq.) was added at 0°C. The reaction solution was heated to 80°C and stirred for 12 hours. The reaction was detected to be complete by LCMS, and the reaction solution was cooled to room temperature. The pH of the reaction solution was adjusted to pH=3 with 3M hydrochloric acid. The organic solvent was removed by concentration under reduced pressure, and water and ethyl acetate were added for extraction. The extraction was repeated three times until the extraction was complete, and the organic phase was washed with saturated brine, combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain 2-methylpyrazolo[1,5-a]pyrimidin-5(4H)-one as a white solid. (2.80g, yield 91.16%). LC-MS (ESI) C7H8N3O+[M+H]+, calculated value: 150.07; found value: 150.13.

[0164] Step 2: Synthesis of 5-chloro-2-methylpyrazolo[1,5-a]pyrimidine (LC-04-147)

[0165] 2-Methylpyrazolo[1,5-a]pyrimidin-5(4H)-one (1 eq.) was dissolved in DMF, and NCS (1.2 eq.) was added at 25°C. The reaction solution was heated to 60°C and stirred for 12 hours. The reaction was detected to be complete by LCMS, and the reaction solution was cooled to room temperature. The organic solvent was removed by vacuum concentration, and water and ethyl acetate were added for extraction. The extraction was repeated three times until the extraction was complete. The organic phase was washed with saturated brine, combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain 5-chloro-2-methylpyrazolo[1,5-a]pyrimidine as a white solid. (2.90 g, yield 92.17%). LC-MS (ESI) C7H7ClN3+[M+H]+, calculated value: 168.04; found value: 168.23.

[0166] Step 3: Synthesis of 3,5-dichloro-2-methylpyrazolo[1,5-a]pyrimidine (LC-04-150)

[0167] 5-Chloro-2-methylpyrazolo[1,5-a]pyrimidine (1 eq.) was dissolved in acetonitrile, and POCl3 (2.7 eq.) was added at 25°C. The reaction solution was heated to 80°C and stirred for 3 hours. The reaction was detected to be complete by LCMS, and the reaction solution was cooled to room temperature. The organic solvent was removed by vacuum concentration, and water and ethyl acetate were added for extraction. The extraction was repeated three times until the extraction was complete. The organic phase was washed with saturated brine, combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain 3,5-dichloro-2-methylpyrazolo[1,5-a]pyrimidine as a white solid. (3.00 g, yield 85.81%). LC-MS (ESI) C7H6Cl2N3+[M+H]+, calculated value: 202.00; found value: 202.11.

[0168] Step 4: Synthesis of 2-(bromomethyl)-3,5-dichloropyrazolo[1,5-a]pyrimidine (LC-04-181)

[0169] 3,5-dichloro-2-methylpyrazolo[1,5-a]pyrimidine (1 eq.) was dissolved in CCl4, and NBS (1.5 eq.) and BPO (0.05 eq.) were added at 25°C. The reaction solution was heated to 80°C and stirred for 12 hours. The reaction was detected to be complete by LCMS, and the reaction solution was cooled to room temperature. The organic solvent was removed by concentration under reduced pressure, and water and ethyl acetate were added for extraction. The extraction was repeated three times until the extraction was complete, and the organic phase was washed with saturated brine, combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain 2-(bromomethyl)-3,5-dichloropyrazolo[1,5-a]pyrimidine as a white solid. (525.00 mg, yield 37.76%). LC-MS (ESI) C7H5BrCl2N3+[M+H]+, calculated values: 279.90 and 281.90; found values: 280.14 and 282.17.

[0170] Step 5: Synthesis of 4-((3,5-dichloropyrazolo[1,5-a]pyrimidin-2-yl)methyl)morpholine (LC-04-179)

[0171] 2-(Bromomethyl)-3,5-dichloropyrazolo[1,5-a]pyrimidine (1 eq.) and morpholine (0.9 eq.) were dissolved in THF, and K2CO3 (3.0 eq.) was added. Stir at 25°C for 1 hour. The reaction was detected to be complete by LCMS, and the reaction solution was cooled to room temperature. Water and ethyl acetate were added to the reaction system for extraction, and the extraction was repeated three times until the extraction was complete. The organic phase was washed with saturated brine, combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by pre-TLC to obtain 4-((3,5-dichloropyrazolo[1,5-a]pyrimidin-2-yl)methyl)morpholine as a white solid. (91.00 mg, yield 59.35%). LC-MS (ESI) C11H13Cl2N4O+[M+H]+, calculated value: 287.05; found value: 287.05.

[0172] Example 19: Effects of different concentrations of compounds on the degradation of CK1α in MOLM 13 cells in vitro

[0173] 1. Cell Culture and Drug Treatment

[0174] The human acute myeloid leukemia cell line MOLM 13 cell line was inoculated into a six-well plate for culture, and the cells were treated with the specified compounds and their concentrations (the concentrations of compounds LC-04-075, LC-02-047-P1, LC-04-113, LC-04-087, LC-04-081, LC-04-077, LC-04-155 and LC-04-162 were 100 nM and 20 nM) for 6 hours, and the DMSO group was set as the control.

[0175] 2. Cell Collection and Protein Extraction

[0176] Collect the cells into a 1.5 mL centrifuge tube, centrifuge at 5000 rpm for 1 minute, remove the supernatant, and obtain a cell pellet. Wash the cell pellet three times with pre-cooled PBS, add RIPA lysis buffer and protease inhibitors to the cell pellet, break the cells with a pipette, and centrifuge at 13000 rpm for 20 minutes at 4°C to obtain an extracted protein solution. Determine the protein concentration in the extract by the BCA method.

[0177] 3. Polyacrylamide gel electrophoresis (SDS-PAGE)

[0178] Take the cell lysate and mix it with SDS-PAGE loading buffer, and boil the sample at 95℃ for 5 minutes. Spot the boiled sample on the 12.5% ​​SDS-PAGE gel, and spot the protein marker in one of the sample wells to indicate the relative molecular mass of the protein. Put the gel plate into the electrophoresis tank and add enough electrophoresis buffer to cover the gel. Connect the power supply, set the voltage and perform electrophoresis until the sample runs to the bottom of the gel.

[0179] 4. Western Blot

[0180] The proteins were transferred from the gel to a PVDF membrane, and the corresponding primary antibodies were used to incubate the CK1α protein and β-actin protein in the corresponding areas on the membrane. The secondary antibodies labeled with near-infrared fluorescence were used to bind to the primary antibodies and the color was developed using a near-infrared laser imager.

[0181] like Figure 1 As shown, under the condition of incubation of 100nM and 20nM compounds for 6 hours, the compounds have a degradation effect on CK1α, indicating the potential of the compounds for the treatment of acute myeloid leukemia. Among them, the effective dose of compounds LC-02-047-P1, LC-04-113, LC-04-087, LC-04-155 and LC-04-162 can be as low as 20nM, and the degradation effect of compound LC-04-087 (dCK1α-1) is the best.

[0182] Example 20: Degradation of CK1α in cells by compound dCK1α-1 at different action times

[0183] The experimental process is similar to that of Example 19, except that Figure 2 The incubation conditions of cells and compounds in the results were adjusted: with or without the addition of compound dCK1α-1 (LC-04-087), the incubation time was set to 0h, 2h, 4h, 6h, 16h and 24h. After each time point of incubation, the cells were collected and frozen in a -20℃ refrigerator. After all time points were collected, they were processed together for subsequent experimental steps. Another difference is Figure 3 The cells used in the results are CRBN knockout MOLM 13 cell line and wild-type MOLM 13 cell line.

[0184] like Figure 2 As shown, the dCK1α-1 compound showed a significant CK1α degradation effect after incubation for 2 hours at a concentration of 100 nM.

[0185] like Figure 3 As shown, dCK1α-1 in CRBN knockout cells cannot degrade CK1α. Figure 2The results demonstrated that dCK1α-1 degrades CK1α through CRBN.

[0186] Example 21: Verification of compound dCK1α-1 target specificity by quantitative proteomics technology

[0187] 1. Cell Sample Preparation

[0188] Human acute myeloid leukemia cell line MOLM13 was cultured in a six-well plate. After the cells were fully grown, compound dCK1α-1 was added for treatment. The control group was added with an equal amount of DMSO solvent and incubated at 37°C for 6 hours. After washing the cells with pre-cooled PBS, the cell pellet was obtained by centrifugation.

[0189] 2. Protein Extraction and Enzymatic Hydrolysis

[0190] A lysis buffer consisting of 8M Urea / 100mM Tris-HCl (pH 8.5) was added to the cell sample, and the cells were lysed using an ultrasonic cell disruptor in an ice-water bath, and centrifuged at 12000g for 15 minutes to obtain a cell lysate. The protein concentration of the lysate was determined using the BCA method. 50 micrograms of protein were taken each, and TCEP and CAA were added for reduction and alkylation reactions. The Urea concentration in the lysate was then reduced to below 2M by adding 100mM Tris-HCl solution. 1 microgram of trypsin was added to the reaction solution, and the enzyme digestion was carried out at 37°C overnight. The next day, TFA was added to reduce the pH of the solution to 6.0 to terminate the enzyme digestion. The sample was desalted using an SDB-RPS desalting column. The eluted peptide samples were vacuum dried and stored in a -20°C refrigerator.

[0191] 3. TMT labeling

[0192] The samples were labeled using the TMTpro-16plex kit (Thermo). The labeling steps were performed according to the instructions of the kit. After labeling, the samples were mixed in equal proportions. The samples were desalted using a C18 desalting column and then separated into 15 components by reverse phase-high pH liquid chromatography. The samples were vacuum dried and stored in a -20°C refrigerator.

[0193] 4. Mass spectrometry detection

[0194] The mass spectrometry of the samples was performed using the Thermo UltiMate 3000RSLCnano nanoliter liquid phase tandem QExactive HF mass spectrometer. The peptide samples were injected through an automatic sampler and bound to a C18 trapping column (75 μm*2 cm, 3 μm particle size, pore size, Thermo), and then on a homemade analytical column (75μm*25cm, 1.9μmparticle size, pore size). The analytical gradient was established using mobile phase A (0.1% formic acid / 3% DMSO / 97% H2O) and mobile phase B (0.1% formic acid / 3% DMSO / 97% ACN). The flow rate of the analysis was set to 300nL / min. The mass spectrometer was used for data acquisition in DDA mode. The parameters of the MS1 full scan were set as follows: resolution 60K@200m / z, scanning range 350-1800m / z, AGC target set to 3E6, and maximum injection time set to 50ms. The selection window of the precursor parent ion was set to 1.2Da, the top15 parent ions were selected for fragmentation, and the HCD collision energy was set to 32%. The parameters of the MS2 scan were set as follows: resolution was set to 45K@200m / z, AGC target was set to 1E5, and the maximum injection time was set to 100ms. The dynamic exclusion time was set to 30 seconds.

[0195] 5. Proteomics Data Analysis

[0196] The TMT process built into the FragPipe software was used to perform database retrieval on the mass spectrometry raw data. The main retrieval parameters were as follows: the database used the human protein sequence data of SwissProt downloaded from the UniProt database; the enzymatic parameters were set to Trypsin, allowing a maximum of 2 missed cleavage sites; the mass tolerance of the primary mass spectrometry was set to 10ppm, and the mass tolerance of the fragment ion was set to 20ppm; the variable modification was set to TMTpro (K / peptide N-term), Oxidation (M), Acetylation (protein N-term), and the fixed modification was set to Carbamidomethylation (C); the search results were screened by 1% FDR; the quantitative value of the protein was calculated from the intensity of the TMT reporter ion using the IonQuant algorithm. Differential proteins were calculated by ratios and T-test P values. The software used to draw the volcano map was the R package EnhancedVolcano.

[0197] like Figure 4 As shown in the figure, the quantitative proteomics results showed that compared with the control DMSO group, the protein with the largest down-regulation in the dCK1α-1 treatment group was CSNK1A1, i.e., CK1α. This result shows that dCK1α-1 has very good selectivity in degrading CK1α.

[0198] Example 22: Detection of the effect of compound dCK1α-1 on cell apoptosis and cell cycle by flow cytometry

[0199] After the cells were treated with DMSO, 40nM dCK1α-1, and 200nM dCK1α-1 for 24 hours, they were detected using an Aurora flow cytometer (Cytek Biosciences). The kit used to detect cell apoptosis was the Annexin V-FITC apoptosis detection kit (Biyuntian, #C1062M); the percentage of cell apoptosis was counted using the Annexin V+ channel, and the data images were processed using FlowJo 10.9.0 software.

[0200] The samples and instruments used for cell cycle detection were the same as those for apoptosis detection, and the kit used was the Cell Cycle Analysis Kit (#C1052M) from Bio-Tech. Cell cycle data analysis was processed using FlowJo 10.9.0 and GraphPad PRISM 9.5 software.

[0201] like Figure 5 As shown, compared with the DMSO control, the proportion of apoptotic cells in cells treated with dCK1α-1 compound gradually increased with the increase of concentration.

[0202] like Figure 6 As shown, compared with the DMSO control group, cells treated with dCK1α-1 compounds showed cell cycle arrest as the concentration increased.

[0203] Example 23: Effects of compound dCK1α-2 on CK1α protein and P53-related target genes in mouse models

[0204] 1. Construction of Mouse Model

[0205] 4×10 6 Human acute myeloid leukemia cells MOLM 13 were injected subcutaneously into 6-week-old female nude mice. After the mice were raised until tumors grew, they were randomly divided into 3 groups, with three replicates in each group.

[0206] 2. Compound Treatment of Tumor-bearing Mice

[0207] Different doses of dCK1α-2 compound were orally fed, namely control, 20 mg / kg, and 40 mg / kg, with 3 mice in each group. After three days of feeding, the mice were killed.

[0208] 3. Sample collection and testing

[0209] Subcutaneous tumor samples were collected and subjected to WB experiments to detect CK1α protein and qPCR to detect the mRNA expression of P53-related genes such as CK1α, P21, MDM2, Bax, and Bbc3.

[0210] like Figure 7 As shown, the compound dCK1α-2 can significantly reduce the content of CK1α in mouse tumors, and can also increase the mRNA levels of P53-related target genes P21, MDM2, Bax, and Bbc3. It is worth noting that the mRNA level of CK1α did not change significantly, which also shows that the effect of compound dCK1α-2 on CK1α is post-transcriptional regulation, which is consistent with the targeted degradation mechanism. This result shows that dCK1α-2 can significantly degrade CK1α in tumors in mouse models by oral administration (oral dose range of 20mg / kg-3 to 40mg / kg-1), showing its potential for the treatment of acute myeloid leukemia.

[0211] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A heterocyclic compound, characterized in that The structure of the compound is shown in formula (I) or formula (II), wherein the structure of formula (I) is as follows: In formula (I), R is selected from halogen, Ra-NH, wherein Ra in Ra-NH is selected from a six-membered cycloalkyl group which is unsubstituted or substituted with one or more halogens, a six-membered heterocycloalkyl group containing oxygen and / or nitrogen, or an aromatic group substituted with halogen, methyl and / or methoxy; The structure of formula (II) is shown below: In formula (II), R1 is selected from H, halogen, -CH3, -NH2 or -CN; R2 is selected from H, -CH3 or 2. The heterocyclic compound according to claim 1, characterized in that In formula (I), R is Cl, Br, 3. The heterocyclic compound according to claim 1, characterized in that In formula (II), R2 is H, R1 is selected from H, Br, Cl, -NH2, -CN, or -CH3; R2 is -CH3, R1 is selected from H, Br or Cl; R2 is R1 is selected from Br or Cl.

4. The heterocyclic compound according to any one of claims 1 to 3, characterized in that The heterocyclic compounds include compounds with the following structures:

5. An enantiomer, stereoisomer, solvate, deuterated substance, polymorph or pharmaceutically acceptable salt of the heterocyclic compound according to any one of claims 1 to 4.

6. Use of the heterocyclic compound according to any one of claims 1 to 4 and its enantiomers, stereoisomers, solvates, deuterated compounds, polymorphs or pharmaceutically acceptable salts in the preparation of a molecular glue degradation agent targeting CK1α.

7. Use of a heterocyclic compound according to any one of claims 1 to 4 and its enantiomers, stereoisomers, solvates, deuterated compounds, polymorphs or pharmaceutically acceptable salts in the preparation of a drug for treating or preventing a disease associated with high expression of CK1α, characterized in that: The diseases associated with high CK1α expression include hematological malignancies and solid tumors associated with the p53 signaling pathway.

8. The use according to claim 7, characterized in that The hematological malignancies include acute myeloid leukemia; the solid tumors include non-small cell lung cancer and prostate cancer; and the method is preferably used in the preparation of drugs for treating or preventing acute myeloid leukemia.

9. A drug for treating acute myeloid leukemia, characterized in that: The active ingredients of the drug include one or more of the heterocyclic compound according to any one of claims 1 to 4, its enantiomers, stereoisomers, solvates, deuterated substances, polymorphs or pharmaceutically acceptable salts.

10. The drug according to claim 9, characterized in that The active ingredients of the drug include one or more of the heterocyclic compound as described in claim 4, its enantiomers, stereoisomers, solvates, deuterated compounds, polymorphs or pharmaceutically acceptable salts; preferably, the active ingredients of the drug include dCK1α-1 and / or dCK1α-2.

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