Heterocyclic compounds and uses thereof

Abstract

The present application relates to a kind of heterocyclic compounds and its application, the structure of the heterocyclic compound is as shown in formula (I) or formula (II), it is proved by experiment that the heterocyclic compounds can be degraded CK1 alpha by molecular glue mechanism, thereby affecting p53 pathway, Wnt / β-catenin pathway and ATM pathway etc., can be used for preventing or treating the disease caused by CK1 alpha abnormal high expression, can be used for preparing the drug for treating hematological malignancies and solid tumor, especially for preparing the drug for treating acute myeloid leukemia.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and more particularly to a heterocyclic compound and application thereof. BACKGROUND

[0002] Molecular glue and proteolysis targeting chimeras (PROTACs) are two rapidly developing technologies for targeted protein degradation in recent years. Among them, molecular glue acts as a glue molecule between target protein and E3 ubiquitin ligase, induces the formation of ternary complex and promotes the protein-protein interaction (PPI) between target protein and E3 ubiquitin ligase, thereby promoting the ubiquitination and degradation of target protein. Compared with PROTACs molecules, molecular glue does not require a specific linker or a high-affinity ligand for the target protein. However, the discovery of molecular glue is highly accidental, and there is still a lack of systematic discovery means and reasonable design strategies. The existing molecular glue approved for clinical application is mainly an immunomodulator, also known as an immunomodulatory imide drug (IMiDs), such as thalidomide, lenalidomide and pomalidomide, which is used for the treatment of multiple myeloma, myelodysplastic syndrome and the like.

[0003] The reported molecular glue degraders target IKZF1 / 2 / 3, RBM39, GSPT1, CK1a, BCL6, Cyclin K, NEK7, etc. Among them, casein kinase CK1a 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 / beta-catenin pathway and ATM pathway. It has been reported that CK1a can negatively regulate the activity of p53 protein by forming an active complex with ubiquitin ligase MDM family proteins (doi:10.1016 / j.tips.2018.10.009). Inhibition of CK1a by using shRNA genetic knockout or small molecule inhibitor D4476 (a CK1a kinase inhibitor) can effectively slow down the proliferation of AML cells (doi:10.1084 / jem.20131033); and in AML patients, it was also found that the abnormally high expression of CK1a was 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 CK1a can inhibit tumor growth by inducing autophagy and enhancing the stability of tumor suppressor PTEN (doi:10.1038 / s41556-018-0065-8). In addition, CK1a 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). As can be seen, existing research shows that CK1a can serve as a potential target for the treatment of various cancers, especially as a potential therapeutic target for hematological malignancies and solid tumors.

[0004] However, so far, there are only a few molecular glue degraders capable of targeting and degrading CK1a protein, such as a class of isoindolinone compounds in patent CN117561244A, CK1a degrader SJ3149 with anti-proliferative activity discovered by Rankovic et al. (doi:10.1038 / s41467-024-44698-1), etc. Currently, lenalidomide is the only drug approved by FDA to degrade CK1a, but its efficacy is weak, and other molecular glue degraders targeting CK1a protein are mostly in the preclinical research stage. As can be seen, there is a lack of effective therapeutic drugs for CK1a, a potential target for cancer treatment. Therefore, it is of great significance to develop novel and highly selective CK1a degraders for the treatment of cancers with CK1a as a potential target. SUMMARY

[0005] In view of the above defects or improvement needs of the prior art, the present application provides a new heterocyclic compound and its application, which aims to find that the heterocyclic compound can efficiently degrade CK1a through the mechanism of molecular glue, thereby affecting the p53 pathway, Wnt / β-catenin pathway and ATM pathway, etc., and can be used for preventing or treating diseases caused by abnormal high expression of CK1a, thereby solving the technical problem that there is a lack of effective therapeutic drugs for CK1a as a potential target for cancer treatment.

[0006] To achieve the above-mentioned purpose, according to one aspect of the present application, a heterocyclic compound is provided, and the structure of the compound 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 by one or more halogens, a six-membered heterocycloalkyl group containing oxygen and / or nitrogen, or an aromatic group substituted by halogen, methyl and / or methoxy;

[0010] The structure of formula (II) is as follows:

[0011]

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

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

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

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

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

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

[0018]

[0019]

[0020] According to another aspect of the present application, there is further provided an enantiomeric, stereoisomeric, solvate, deuterated, polymorph or pharmaceutically acceptable salt of the heterocyclic compound as described herein.

[0021] According to another aspect of the present application, there is further provided a use of the heterocyclic compound as described herein, and an enantiomeric, stereoisomeric, solvate, deuterated, polymorph or pharmaceutically acceptable salt thereof, in the preparation of a molecular glue degrader targeting CK1a.

[0022] According to another aspect of the present application, there is further provided a use of the heterocyclic compound as described herein, and an enantiomeric, stereoisomeric, solvate, deuterated, polymorph or pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating or preventing a disease associated with high expression of CK1a, wherein the disease associated with high expression of CK1a includes a hematological malignancy and a solid tumor associated with p53 signaling pathway.

[0023] Preferably, in the use, the hematological malignancy includes acute myeloid leukemia; the solid tumor includes non-small cell lung cancer, prostate cancer.

[0024] Preferably, in the use, the medicament is for treating or preventing acute myeloid leukemia.

[0025] According to another aspect of the present application, there is further provided a medicament for treating acute myeloid leukemia, wherein the pharmaceutical effective component of the medicament includes one or more of the heterocyclic compound as described herein, an enantiomeric, stereoisomeric, solvate, deuterated, polymorph or pharmaceutically acceptable salt thereof.

[0026] Preferably, in the medicament, the pharmaceutical effective component includes one or more of the heterocyclic compound as described herein, an enantiomeric, stereoisomeric, solvate, deuterated, polymorph or pharmaceutically acceptable salt thereof.

[0027] Preferably, in the medicament, the pharmaceutical effective component includes dCK1a-1 and / or dCK1a-2.

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

[0029] The heterocyclic compound provided by the present application is a compound of formula (I) or formula (II), experiments prove that the compound can target degradation of CK1a to activate p53-related transcription regulation events, and can be used as a molecular glue degrader targeting CK1a, and has the advantages of good selectivity, high activity, high oral availability, etc. compared with existing molecular glue degraders, and can be used for preventing or treating CK1a high expression related tumor diseases, and can realize high activity and high selectivity of targeted degradation of CK1a protein in human acute myeloid leukemia cells Molt-13, and can be used for preparing a drug for treating acute myeloid leukemia. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Figure 6 is the degradation effect of different concentrations of the compound on CK1a in MOLM 13 cells in vitro.

[0031] Figure 2 Figure 7 is the degradation effect of compound dCK1a-1 on CK1a in cells under different action times.

[0032] Figure 3 Figure 8 is that dCK1a-1 cannot degrade CK1a in CRBN knockout cells.

[0033] Figure 4 Figure 9 is the quantitative proteomics experiment proving that the compound dCK1a-1 has good specificity of target point.

[0034] Figure 5 Figure 10 is that the compound dCK1a-1 causes cell apoptosis.

[0035] Figure 6 Figure 11 is that the compound dCK1a-1 causes cell cycle arrest.

[0036] Figure 7 Figure 12 is the degradation effect of compound dCK1a-2 on CK1a in mice. DETAILED DESCRIPTION

[0037] In order to further illustrate the technical means and effects adopted by the present application to achieve the predetermined object, the specific embodiments, structures, features and effects of the present application are described in detail below in combination with the drawings and preferred embodiments.

[0038] Terms:

[0039] In the present application, "solvate" refers to a compound containing solvent molecules in the crystal of the drug, usually when the solvent molecules are water molecules, it is called hydrate. "Deuterated compound" refers to a compound in which deuterium (D) element partially or completely replaces hydrogen (H) in the drug molecule, where deuterium is an isotope of hydrogen with a heavier atomic mass, and the chemical symbol is 2H or D. "Polymorph" refers to different polymorphs of the same drug due to different crystal structures. "Pharmaceutically acceptable salt" usually refers to the preparation of a drug into a salt form in order to increase the solubility of the drug or to enhance the stability of the drug, including organic or inorganic salts. "Halogen" means chlorine, bromine, fluorine and / or iodine; "halogen substitution" means chlorination, bromination, fluorination and / or iodination.

[0040] Currently, more than 600 E3 ligases have been reported, but only 5 have been used for molecular glue-mediated degradation, namely CRBN, DDB1, β-TrCP, DCAF15 and SIAH1. Among them, CRBN (cereblon) is a substrate receptor protein of Cullin 4-based (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. The substrate binding domain of CRBN interacts with specific substrate proteins, while the ligand binding domain can bind to certain small molecule ligands, which bind to CRBN and change the conformation of CRBN, thereby affecting its binding to substrates, and further affecting the ubiquitination and degradation of substrates. It has been proven that the interaction of the glutarimide moiety common to the three immunomodulatory drugs (IMiDs) lenalidomide, thalidomide and pomalidomide with the CRBN binding pocket contributes the most, while the differences in the part outside the pocket can make CRBN have different protein interfaces, thereby recruiting different substrates through protein interaction mechanisms.

[0041] The present application is based on the synthesis of a series of heterocyclic compounds with a glutarimide skeleton, and it is found that the heterocyclic compounds with structures as shown in formula (I) or formula (II) bind to E3 ligand CRBN, can specifically recruit target protein CK1α, and further promote the ubiquitination and degradation of CK1α protein, and can be used as a molecular glue degrader targeting CK1α protein, which can be used for the treatment of hematological malignancies and solid tumors. The structure of the heterocyclic compound is as shown in formula (I) or formula (II):

[0042]

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

[0044] In Ra-NH, Ra is selected from unsubstituted or substituted six-membered cycloalkyl groups, oxygen- and / or nitrogen-containing six-membered heterocycloalkyl groups, or aromatic groups substituted with halogens, methyl groups, and / or methoxy groups.

[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 with a structure as shown in formula (I) or formula (II), and its enantiomers, stereoisomers, solvates, deuterates, polymorphs, or pharmaceutically acceptable salts, wherein the structure of the heterocyclic compound is shown in formula (I):

[0047]

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

[0049] In Ra-NH, Ra is selected from unsubstituted or substituted six-membered cycloalkyl groups, oxygen- and / or nitrogen-containing six-membered heterocycloalkyl groups, or aromatic groups substituted with halogens, methyl (-CH3), and / or methoxy (-O-CH3).

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

[0051] In some embodiments, R in formula (I) is Ra-NH, wherein Ra is selected from unsubstituted or substituted six-membered cycloalkyl groups, specifically as follows: 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 oxygen- and / or nitrogen-containing six-membered heterocyclic alkyl groups, specifically as follows: 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 with halogens and methyl (-CH3) or methoxy (-O-CH3), specifically as follows: The specific structure of R is as follows:

[0056]

[0057] Preferably, the heterocyclic compound has the structure as shown below:

[0058]

[0059] Alternatively, the heterocyclic compound has the structure 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 (-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, in formula (II), R2 is R1 is selected from Br or Cl.

[0065] Preferably, the heterocyclic compound has the structure as shown below:

[0066]

[0067] In addition, the present application also provides an enantiomer, stereoisomer, solvate, deuteride, polymorph or pharmaceutically acceptable salt of the heterocyclic compound as described in the present application.

[0068] The present application also provides a use of the heterocyclic compound as described in the present application, an enantiomer, stereoisomer, solvate, deuteride, polymorph or pharmaceutically acceptable salt thereof in the preparation of a molecular glue degrader targeting CK1α; the molecular glue degrader is used to promote the binding of E3 ligase ligand CRBN to target protein CK1α, promote the ubiquitination and degradation of the target protein, and then realize the targeted selective degradation of CK1α protein, and activate the transcriptional regulation related to the p53 signaling pathway.

[0069] In addition, the present application also provides a use of the heterocyclic compound as described in the present application, an enantiomer, stereoisomer, solvate, deuteride, polymorph or pharmaceutically acceptable salt thereof in the preparation of a drug for treating or preventing a disease related to the abnormal function of CK1α protein.

[0070] The disease associated with the abnormal function of CK1a protein is a disease associated with high expression of CK1a, including tumors associated with p53 signaling pathway, including hematological malignancies and solid tumors, wherein the hematological malignancies include acute myeloid leukemia; the solid tumors include non-small cell lung cancer and prostate cancer; preferably for preparing a medicament for treating or preventing acute myeloid leukemia.

[0071] The present application also provides a medicament for treating acute myeloid leukemia, wherein the effective component of the medicament comprises one or more of the heterocyclic compounds, enantiomers, stereoisomers, solvates, deuterium compounds, polymorphs or pharmaceutically acceptable salts thereof as described in the present application; preferably the effective component of the medicament comprises dCK1a-1 and / or dCK1a-2.

[0072] In some embodiments, the medicament further comprises a pharmaceutically acceptable carrier; and the administration mode comprises, but is not limited to, injection and oral administration; preferably oral administration.

[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 are 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 synthesis route of compounds LC-02-047-P1 and LC-02-047-P2 is as follows:

[0077]

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

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

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

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

[0082] Step 4: Synthesis of (S)-3-(5-(3-bromopyrazolo[l,5-a]pyrimidin-5-yl)-l- oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-047-P1) and (S)-3-(5-(5- chloropyrazolo[l,5-a]pyrimidin-3-yl)-l-oxoisoindolin-2-yl)piperidine-2,6-dione (LC- 02-047-P2): (S)-5-amino-4-(5-(3-bromopyrazolo[l,5-a]pyrimidin-5-yl)-l- oxoisoindolin-2-yl)-5-oxopentanoate (1 eq.) was dissolved in acetonitrile, and benzenesulfonic acid (3 eq.) was added. The reaction was warmed to 100 °C and stirred for 12 h. The reaction was checked by LCMS and cooled to room temperature. The solvent was removed under reduced pressure, and the residue was slurried with ethyl acetate and petroleum ether to give (S)-3-(5-(3-bromopyrazolo[l,5-a]pyrimidin-5-yl)-l- oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-047-P1, 23.00 mg, 37.85% yield) as a white solid. LC-MS (ESI) C 19 H 15 BrN5O3 + [M+H] + , calc: 442.04; found: 442.11. 1H NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H), 9.29 (d, J = 4.0 Hz, 1H), 8.47 (s, 1H), 8.42 (s, 1H), 8.40 (d, J = 8.0 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 5.17 (dd, J = 12.0, 4.0 Hz, 1H), 4.59 (d, J = 16.0 Hz, 1H), 4.47 (d, J = 16.0 Hz, 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 (150 MHz, DMSO-d6) δ 172.92, 170.96, 167.50, 155.68, 145.29, 144.41, 142.87, 139.17, 137.36, 133.66, 127.35, 123.61, 122.53, 106.71, 83.59, 51.81, 47.49, 31.22, 22.47.

[0083] (S)-5-amino-4-(5-(5-chloropyrazolo[l,5-a]pyrimidin-3-yl)-l-oxoisoindolin-2-yl)- 5-oxopentanoic acid tert-butyl ester (1 eq.) was dissolved in acetonitrile, and benzenesulfonic acid (3 eq.) was added. The reaction was warmed to 100 °C and stirred for 12 h. The reaction was checked by LCMS for completion, and the reaction was cooled to room temperature. The solvent was removed under reduced pressure, and the residue was slurried with ethyl acetate and petroleum ether to give (S)-3-(5-(5-chloropyrazolo[l,5-a]pyrimidin-3-yl)-l-oxoisoindolin-2-yl)piperidine- 2,6-dione (LC-02-047-P2, 15.00 mg, 38.18% yield) as a white solid. LC-MS (ESI) C 19 H 15 ClN5O3 + [M+H] + 396.09; found: 396.17. ¾ NMR (400 MHz, DMSO-d6) d 11.01 (s, 1H), 9.26 (d, J = 8.0 Hz, 1H), 8.95 (s, 1H), 8.27 (s, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.26 (d, J = 8.0 Hz, 1H), 5.14 (dd, J = 12.0, 4.0 Hz, 1H), 4.55 (d, J = 16.0 Hz, 1H), 4.39 (d, J = 16.0 Hz, 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 (150 MHz, DMSO-d6) d 172.96, 171.11, 167.99, 150.83, 144.49, 143.31, 142.88, 138.97, 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][l,3]dioxol-5-ylamino)pyrazolo[l,5- a]pyrimidin-3-yl)-l-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][l,3]dioxol-5- ylamino)pyrazolo[l,5-a]pyrimidin-3-yl)-l-oxoisoindolin-2-yl)-5-oxopentanoate (LC-02-101): (S)-tert-butyl 5-amino-5-oxo-4-(l-oxo-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)isoindolin-2- yl)pentanoate (1 eq.) and N-(benzo[d][l,3]dioxol-5-yl)-3-bromopyrazolo[l,5-a]pyrimidin-5- amine (1.2 eq.) were dissolved in 1,4-dioxane, and 3 M potassium phosphate solution (3 eq.) was added. Nitrogen was bubbled through three times, Pd(dtbpf)Cl2(0.1 eq.) was added, and nitrogen was bubbled through again three times. The reaction was heated to 100 °C and stirred for 3 h. The reaction was checked by LCMS, and the reaction was cooled to room temperature. Water and ethyl acetate were added to the reaction, 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 give tert-butyl (S)-5-amino-4-(5-(5-(benzo[d][l,3]dioxol-5-ylamino)pyrazolo[l,5-a]pyrimidin-3-yl)-l- oxoisoindolin-2-yl)-5-oxopentanoate as a white solid. (45.00 mg, 68.68% yield). LC-MS (ESI) C30H31N6O6 + [M+H] + Calcd: 571.23; Found: 571.21.

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

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

[0089] LC-MS (ESI) C26H21N6O5 + [M+H] + Calcd: 497.16; Found: 497.21. 1H NMR (400 MHz, DMSO-d6) δ 11.02 (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 = 8.0 Hz, 1H), 6.06 (d, J = 4.0 Hz, 2H), 5.14 (dd, J = 12.0, 4.0 Hz, 1H), 4.50 (d, J = 16.0 Hz, 1H), 4.39 (d, J = 16.0 Hz, 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 (150 MHz, DMSO-d6) δ 172.99, 171.18, 168.22, 153.18, 147.21, 144.13, 142.82, 142.63, 142.43, 136.74, 136.28, 134.15, 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[l,5- a]pyrimidin-3-yl)-l-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-105)

[0091]

[0092] The target product LC-02-105 was synthesized by replacing N-(benzo[d][l,3]dioxol-5-yl)-3- bromopyrazolo[l,5-a]pyrimidin-5-amine with 3-bromo-N-(3-chloro-4-methylphenyl)pyrazolo[l,5- a]pyrimidin-5-amine (LC-02-093) in the synthetic method of Reference Example 2. LCMS (ESI) C26H22ClN6O3 + [M+H] + , calculated: 501.15; found: 501.61. 1H NMR (400 MHz, DMSO-d6) δ 11.03 (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 = 8.0 Hz, 1H), 5.14 (dd, J = 12.0, 4.0 Hz, 1H), 4.51 (d, J = 16.0 Hz, 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 (150 MHz, DMSO-d6) δ 172.97, 171.20, 168.19, 153.08, 143.79, 142.80, 142.74, 138.91, 136.66, 136.52, 133.13, 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[l,5-a]pyrimidin-3-yl)-l- oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-116)

[0094]

[0095] The target product LC-02-116 was synthesized by replacing N-(benzo[d][l,3]dioxol-5-yl)-3- bromopyrazolo[l,5-a]pyrimidin-5-amine with N-benzyl-3-bromopyrazolo[l,5-a]pyrimidin-5-amine (LC-02-097) according to the synthetic method of Reference Example 2. LCMS (ESI) C26H23N6O3+[M+H]+, calculated: 467.19; found: 467.36. 1H NMR (400 MHz, DMSO-d6) δ 11.02 (s, 1H), 8.55 (d, J = 8.0 Hz, 1H), 8.45 (s, 1H), 8.40 (t, J = 4.0 Hz, 1H), 8.25 (s, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 4.0 Hz, 2H), 7.36 (t, J = 8.0 Hz, 2H), 7.24 (t, J = 8.0 Hz, 1H), 6.42 (d, J = 8.0 Hz, 1H), 5.12 (dd, J = 12.0, 4.0 Hz, 1H), 4.63 (d, J = 8.0 Hz, 2H), 4.46 (d, J = 16.0 Hz, 1H), 4.33 (d, J = 16.0 Hz, 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 (150 MHz, DMSO-d6) δ 172.99, 171.24, 168.31, 145.07, 142.63, 142.10, 139.49, 137.13, 135.67, 128.39, 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-(l-oxo-5-(5-(phenethylamino)pyrazolo[l,5-a]pyrimidin-3- yl)isoindolin-2-yl)piperidine-2,6-dione (LC-02-117)

[0097]

[0098] Reference to the synthetic method of Example 2, replace N-(benzo[d][l,3]dioxol-5-yl)-3- bromopyrazolo[l,5-a]pyrimidin-5-amine with 3-bromo-N-phenethylpyrazolo[l,5-a]pyrimidin-5- amine (LC-02-098) to synthesize the target product LC-02-117. LCMS (ESI) C27H25N6O3+[M+H]+, calculated: 481.20; found: 481.23. 1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 8.53 (d, J = 4.0 Hz, 1H), 8.47 (s, 1H), 8.38 (s, 1H), 8.25 (d, J = 8.0 Hz, 1H), 7.98 (t, J = 4.0 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.35 - 7.32 (m, 4H), 7.26 - 7.20 (m, 1H), 6.34 (d, J = 8.0 Hz, 1H), 5.14 (dd, J = 12.0, 4.0 Hz, 1H), 4.43 (d, J = 16.0 Hz, 1H), 4.29 (d, J = 16.0 Hz, 1H), 3.67 (q, J = 8.0 Hz, 2H), 2.97 (t, J = 8.0 Hz, 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 (150 MHz, DMSO-d6) δ 172.99, 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[l,5-a]pyrimidin-3-yl)-l- oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-119)

[0100]

[0101] The target product LC-02-119 was synthesized by replacing N-(benzo[d][l,3]dioxol-5-yl)-3-bromopyrazolo[l,5-a]pyrimidin-5-amine with 3-bromo-N-cyclohexylpyrazolo[l,5-a]pyrimidin-5-amine (LC-02-106) according to the synthetic method of Reference Example 2. LCMS (ESI) C25H27N6O3+[M+H]+, calc. 459.22; found 459.29. 1H NMR (400 MHz, DMSO-d6) δ 11.01 (s, 1H), 8.49 (d, J = 8.0 Hz, 1H), 8.45 (s, 1H), 8.38 (s, 1H), 8.19 (d, J = 8.0 Hz, 1H), 7.70-7.66 (m, 2H), 6.32 (d, J = 4.0 Hz, 1H), 5.11 (dd, J = 12.0, 4.0 Hz, 1H), 4.46 (d, J = 16.0 Hz, 1H), 4.33 (d, J = 16.0 Hz, 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 (150 MHz, DMSO-d6) δ 172.98, 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-(l-oxo-5-(5-(tetrahydro-2H-pyran-4-yl)amino)pyrazolo[l,5-a]pyrimidin-3-yl)isoindolin-2-yl)piperidine-2,6-dione (LC-02-120)

[0103]

[0104] The target product LC-02-120 was synthesized by replacing N-(benzo[d][l,3]dioxol-5-yl)-3-bromopyrazolo[l,5-a]pyrimidin-5-amine with 3-bromo-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[l,5-a]pyrimidin-5-amine (LC-02-107) in the synthetic method of Reference Example 2. LCMS (ESI) C24H25N6O4+[M+H]+, calc. 460.20; found 460.26. 1H NMR (400 MHz, DMSO-d6) δ 11.01 (s, 1H), 8.53 (d, J = 8.0 Hz, 1H), 8.47 (s, 1H), 8.39 (s, 1H), 8.17 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H), 6.32 (d, J = 8.20 Hz, 1H), 5.12 (dd, J = 12.0, 4.0 Hz, 1H), 4.46 (d, J = 16.0 Hz, 1H), 4.34 (d, J = 16.0 Hz, 1H), 4.18 - 4.05 (m, 1H), 3.93 (d, J = 12.0 Hz, 2H), 3.52 (t, J = 12.0 Hz, 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 (150 MHz, DMSO-d6) δ 172.98, 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[l,5-a]pyrimidin-3-yl)-l- oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-121)

[0106]

[0107] The target product LC-02-121 was synthesized by replacing N-(benzo[d][l,3]dioxol-5-yl)-3- bromopyrazolo[l,5-a]pyrimidin-5-amine with 3-bromo-N-(3-chloro-4-methoxyphenyl)pyrazolo[l,5- a]pyrimidin-5-amine (LC-02-103) in the synthetic method of Reference Example 2. LCMS (ESI) C26H22ClN6O4+[M+H]+, calculated: 517.14; found: 517.19. 1H NMR (400 MHz, DMSO-d6) δ 11.03 (s, 1H), 9.98 (s, 1H), 8.71 (d, J = 8.0 Hz, 1H), 8.56 (s, 1H), 8.43 (d, J = 4.0 Hz, 1H), 8.24 - 8.22 (m, 2H), 7.74 (d, J = 8.0 Hz, 1H), 7.45 (dd, J = 8.0, 4.0 Hz, 1H), 7.21 (d, J = 8.0 Hz, 1H), 6.54 (d, J = 8.0 Hz, 1H), 5.14 (dd, J = 12.0, 4.0 Hz, 1H), 4.50 (d, J = 16.0 Hz, 1H), 4.40 (d, J = 16.0 Hz, 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 (150 MHz, DMSO-d6) δ 172.97, 171.19, 168.21, 153.08, 150.06, 143.94, 142.80, 142.64, 136.73, 136.38, 133.53, 128.08, 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[l,5- a]pyrimidin-3-yl)-l-oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-135)

[0109]

[0110] Reference to the synthetic method of Example 2, replace N-(benzo[d][l,3]dioxol-5-yl)-3- bromopyrazolo[l,5-a]pyrimidin-5-amine with 3-bromo-N-(4,4-difluorocyclohexyl)pyrazolo[l,5- a]pyrimidin-5-amine (LC-02-122) to synthesize the target product LC-02-135. LCMS (ESI) C25H25F2N6O3+[M+H]+, calc. 495.20; found 495.52. 1H NMR (400 MHz, DMSO-d6) δ 11.02 (s, 1H), 8.54 (d, J = 8.0 Hz, 1H), 8.48 (s, 1H), 8.34 (s, 1H), 8.23 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 4.0 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 6.34 (d, J = 8.0 Hz, 1H), 5.11 (dd, J = 12.0, 4.0 Hz, 1H), 4.48 (d, J = 16.0 Hz, 1H), 4.36 (d, J = 16.0 Hz, 1H), 4.15 - 4.04 (m, 1H), 2.96 - 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 (150 MHz, DMSO-d6) δ 172.97, 171.23, 168.32, 155.42, 145.12, 142.73, 142.14, 137.24, 135.66, 127.56, 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-morpholinoethyl)amino)pyrazolo[l,5-a]pyrimidin-3-yl)-l- oxoisoindolin-2-yl)piperidine-2,6-dione (LC-02-148)

[0112]

[0113] Reference to the synthetic method of Example 2, replace N-(benzo[d][l,3]dioxol-5-yl)-3- bromopyrazolo[l,5-a]pyrimidin-5-amine with 3-bromo-N-(2-morpholinoethyl)pyrazolo[l,5- a]pyrimidin-5-amine (LC-02-123) to synthesize the target product LC-02-148. LCMS (ESI) C25H28N7O4+[M+H]+, calculated: 490.22; found: 490.32. 1H NMR (400 MHz, DMSO-d6) δ 11.03 (s, 1H), 8.61 (d, J = 8.0 Hz, 1H), 8.50 (s, 1H), 8.26 (s, 1H), 8.24 (d, J = 8.0 Hz, 1H), 8.08 (t, J = 8.0 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H), 6.38 (d, J = 8.0 Hz, 1H), 5.13 (dd, J = 12.0, 4.0 Hz, 1H), 4.48 (d, J = 16.0 Hz, 1H), 4.35 (d, J = 16.0 Hz, 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 (150 MHz, DMSO-d6) δ 172.97, 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-(l-oxo-5-(pyrazolo[l,5-a]pyrimidin-5-yl)isoindolin-2- yl)piperidine-2,6-dione (LC-04-075)

[0115] LC-04-075 synthesis route is as follows:

[0116]

[0117] Step 1: Synthesis of tert-butyl (S)-5-amino-5-oxo-4-(l-oxo-5-(pyrazolo[l,5- a]pyrimidin-5-yl)isoindolin-2-yl)pentanoate (LC-02-070): tert-butyl (S)-5-amino-5- oxo-4-(l-oxo-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)isoindolin-2-yl)pentanoate (1 eq.) and 5-chloropyrazolo[l,5-a]pyrimidine (1 eq.) were dissolved in ethylene glycol dimethyl ether, and saturated sodium carbonate solution was added. Nitrogen was bubbled through three times, and Pd(PPh3)2Cl2(0.05 eq.) was added. Nitrogen was bubbled through again three times, and the reaction was heated to 80 °C and stirred for 3 min. The reaction was monitored by LCMS until completion. The reaction was cooled to room temperature. Water and ethyl acetate were added to the reaction, and the extraction was repeated three times until 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 give tert-butyl (S)-5-amino-5-oxo-4-(l-oxo-5-(pyrazolo[l,5-a]pyrimidin-5-yl)isoindolin-2- yl)pentanoate as a yellow solid. (150.00 mg, 76.52% yield). LC-MS (ESI) C23H26N5O4 + [M+H] + Calculated: 436.20; Found: 436.29.

[0118] Step 2: Synthesis of (S)-3-(l-oxo-5-(pyrazolo[l,5-a]pyrimidin-5-yl)isoindolin-2- yl)piperidine-2,6-dione (LC-04-075): tert-butyl (S)-5-amino-5-oxo-4-(l-oxo-5-(pyrazolo[l,5- a]pyrimidin-5-yl)isoindolin-2-yl)pentanoate (1 eq.) was dissolved in acetonitrile, and benzenesulfonic acid (3 eq.) was added. The reaction was heated to 100 °C and stirred for 12 h. The reaction was monitored by LCMS until completion. The reaction was cooled to room temperature. The solvent was removed by concentration under reduced pressure. The residue was slurried with ethyl acetate and petroleum ether, and (S)-3-(l-oxo-5-(pyrazolo[l,5-a]pyrimidin-5-yl)isoindolin-2-yl)piperidine-2,6-dione (LC-04-075) was obtained as a yellow solid. (56.00 mg, 44.99% yield). LC-MS (ESI) C19H16N5O3 + [M+H] +Calculated: 362.13; Found: 362.19. 1H NMR (400 MHz, DMSO-d6) δ 11.05 (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 = 4.0 Hz, 1H), 5.17 (dd, J = 12.0, 4.0 Hz, 1H), 4.58 (d, J = 16.0 Hz, 1H), 4.45 (d, J = 16.0 Hz, 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 (150 MHz, DMSO-d6) δ 172.90, 170.97, 167.54, 154.50, 147.72, 145.70, 142.83, 139.73, 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[l,5-a]pyrimidin-5-yl)-l- oxoisoindolin-2-yl)piperidine-2,6-dione (LC-04-087)

[0120] The compound LC-04-087 (dCK1a-1) has the following structural formula:

[0121]

[0122] Reference to the synthetic method of Example 11, 5-chloropyrazolo[l,5- a]pyrimidine was replaced by 3,5-dichloropyrazolo[l,5-a]pyrimidine to synthesize the target product LC-04-087 (dCK1a-1). LCMS (ESI) C19H15ClN5O3+[M+H]+, calculated: 396.09; found: 396.18. 1H NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H), 9.27 (d, J = 4.0 Hz, 1H), 8.48 (s, 1H), 8.43 (s, 1H), 8.40 (d, J = 8.0 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 5.17 (dd, J = 12.0, 4.0 Hz, 1H), 4.59 (d, J = 16.0 Hz, 1H), 4.47 (d, J = 16.0 Hz, 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 (150 MHz, DMSO-d6) δ 172.92, 170.96, 167.49, 155.47, 143.30, 143.03, 142.87, 139.14, 137.33, 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[l,5-a]pyrimidin-5-yl)-l- oxoisoindolin-2-yl)piperidine-2,6-dione (LC-04-081)

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

[0125]

[0126] The target product LC-04-081 was synthesized according to the synthetic method of Reference Example 11, replacing 5-chloropyrazolo[1,5-a]pyrimidine with 5-chloropyrazolo[1,5-a]pyrimidine-3-amine: LCMS (ESI) C19H17N6O3+[M+H]+, calc. mass: 377.14; found: 377.72.1H NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H), 9.20 (d, J = 8.0 Hz, 1H), 8.49 (s, 1H), 8.45 (d, J = 8.0 Hz, 1H), 8.18 (s, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 5.17 (dd, J = 12.0, 4.0 Hz, 1H), 4.59 (d, J = 16.0 Hz, 1H), 4.46 (d, J = 16.0 Hz, 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 (150 MHz, DMSO-d6) δ 172.93, 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-carbonitrile (LC-04-077)

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

[0129]

[0130] The target product LC-04-077 was synthesized according to the synthetic method of Reference Example 11, replacing 5-chloropyrazolo[1,5-a]pyrimidine with 5-chloropyrazolo[1,5-a]pyrimidine-3-carbonitrile: LCMS (ESI) C20H15N6O3+[M+H]+, calc. mass: 387.12; found: 387.49. 1H NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H), 9.48 (d, J = 8.0 Hz, 1H), 8.85 (s, 1H), 8.53 (s, 1H), 8.43 (d, J = 8.0 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 5.18 (dd, J = 12.0, 4.0 Hz, 1H), 4.60 (d, J = 16.0 Hz, 1H), 4.48 (d, J = 16.0 Hz, 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 (150 MHz, DMSO-d6) δ 172.91, 170.94, 167.37, 158.52, 149.52, 148.46, 142.93, 138.42, 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[l,5-a]pyrimidin-5-yl)-l- oxoisoindolin-2-yl)piperidine-2,6-dione (LC-04-155)

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

[0133]

[0134] The target product LC-04-155 was synthesized according to the synthetic method of Reference Example 11, replacing 5-chloropyrazolo[l,5-a]pyrimidine with 5-chloro-2- methylpyrazolo[l,5-a]pyrimidine (LC-04-147): LCMS (ESI) C20H18N5O3+ [M+H]+, calc 376.14; found 376.37. 1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 9.11 (d, J = 4.0 Hz, 1H), 8.43 (s, 1H), 8.33 (d, J = 8.0 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 6.58 (s, 1H), 5.17 (dd, J = 12.0, 4.0 Hz, 1H), 4.56 (d, J = 16.0 Hz, 1H), 4.44 (d, J = 16.0 Hz, 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 (150 MHz, DMSO-d6) δ 172.91, 170.98, 167.57, 155.20, 154.09, 148.40, 142.80, 139.86, 135.78, 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[l,5-a]pyrimidin-5-yl)-l- oxoisoindolin-2-yl)piperidine-2,6-dione (LC-04-162)

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

[0137]

[0138] The target product LC-04-162 was synthesized by replacing 5-chloropyrazolo[l,5-a]pyrimidine with 3,5-dichloro-2-methylpyrazolo[l,5-a]pyrimidine (LC-04-150) according to the synthetic method of Reference Example 11: LCMS (ESI) C20H17ClN5O3+ [M+H]+, calc 410.10; found 410.73. 1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 9.15 (d, J = 8.0 Hz, 1H), 8.44 (s, 1H), 8.36 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 5.17 (dd, J = 12.0, 4.0 Hz, 1H), 4.58 (d, J = 16.0 Hz, 1H), 4.46 (d, J = 16.0 Hz, 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 (150 MHz, DMSO-d6) δ 172.90, 170.94, 167.50, 155.10, 151.64, 143.36, 136.70, 133.56, 128.36, 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[l,5- a]pyrimidin-5-yl)-l-oxoisoindolin-2-yl)piperidine-2,6-dione (dCK1a-2)

[0140] The structural formula of compound dCK1a-2 is as follows:

[0141]

[0142] The target product dCK1a-2 was synthesized by replacing 5-chloropyrazolo[l,5- a]pyrimidine with 4-((3,5-dichloropyrazolo[l,5-a]pyrimidin-2-yl)methyl)morpholine (LC- 04-179) according to the synthetic method of Reference Example 11: LCMS (ESI) C24H24ClN6O4+[M+H]+, calc 495.16; found 495.31. 1H NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H), 9.23 (d, J = 8.0 Hz, 1H), 8.47 (s, 1H), 8.39 (d, J = 8.0 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 5.17 (dd, J = 12.0, 4.0 Hz, 1H), 4.59 (d, J = 16.0 Hz, 1H), 4.47 (d, J = 16.0 Hz, 1H), 3.72 (s, 2H), 3.56 (d, J = 4.0 Hz, 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 (150 MHz, DMSO-d6) δ 172.91, 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[l,5-a]pyrimidin-5-yl)-l- 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[l,5- a]pyrimidin-5-yl)-l-oxoisoindolin-2-yl)-5-oxopentanoate (LC-04-097): tert-butyl (S)-5-amino-4-(5-(3-bromopyrazolo[l,5-a]pyrimidin-5-yl)-l-oxoisoindolin- 2-yl)-5-oxopentanoate (1 eq.) and 2,4,4,5,5-pentamethyl-l,3,2-dioxaborinane (1 eq.) were dissolved in a mixed solvent of 1,4-dioxane and water, and potassium carbonate (2.5 eq.) was added. Nitrogen was replaced three times, Pd(dppf)Cl2(0.05 eq.) was added, and nitrogen was replaced again three times. The reaction solution was warmed to 90 °C and stirred for 16 hours. The reaction was detected by LCMS to be completed, 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 it 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[l,5-a]pyrimidin-5-yl)-l-oxoisoindolin- 2-yl)-5-oxopentanoate as a yellow solid. (98.00 mg, yield 44.86 %). LC-MS (ESI) C24H28N5O4 + [M+H] + Calcd: 450.22; Found: 450.29.

[0146] Step 2: Synthesis of (S)-3-(5-(3-methylpyrazolo[l,5-a]pyrimidin-5-yl)-l-oxoisoindolin- 2-yl)piperidine-2,6-dione (LC-04-113): tert-butyl (S)-5-amino-4-(5-(3-methylpyrazolo[l,5- a]pyrimidin-5-yl)-l-oxoisoindolin-2-yl)-5-oxopentanoate (1 eq.) was dissolved in acetonitrile, and benzenesulfonic acid (3 eq.) was added. The reaction solution was warmed to 100 °C and stirred for 12 hours. The reaction was detected by LCMS to be completed, and the reaction solution was cooled to room temperature. The solvent was removed by concentration under reduced pressure, and the residue was slurried with ethyl acetate and petroleum ether. Filtration yielded (S)-3-(5-(3-methylpyrazolo[l,5-a]pyrimidin-5-yl)-l-oxoisoindolin-2- yl)piperidine-2,6-dione (LC-04-113: 43.00 mg, yield 52.54 %) as a yellow solid. LC-MS (ESI) C20H18N5O3 + [M+H] +, Calc. 376.14; Found 376.47. 1H NMR (400 MHz, DMSO-d6) δ 11.05 (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), 5.17 (dd, J = 12.0, 4.0 Hz, 1H), 4.58 (d, J = 16.0 Hz, 1H), 4.45 (d, J = 16.0 Hz, 1H), 3.00 - 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 (150 MHz, DMSO-d6) δ 172.93, 170.99, 167.61, 152.98, 145.40, 145.31, 142.83, 139.90, 136.18, 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][l,3]dioxol-5-yl)-3-bromo pyrazolo[l,5-a]pyrimidine-5-amine (LC-02-092)

[0148]

[0149] Dissolve 3-bromo-5-chloropyrazolo[l,5-a]pyrimidine (1 eq.) and benzo[d][l,3]dioxol-5-amine (1.5 eq.) in n-butanol, add DIEA (3 eq.). The reaction solution is heated to 125 °C and stirred for 16 hours. The reaction is monitored by LCMS until completion. The reaction solution is cooled to room temperature. The solvent is removed by concentration under reduced pressure. The residue is extracted with water and ethyl acetate, and the extraction is repeated three times until completion. The organic phase is washed with saturated brine, combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue is purified by silica gel column chromatography to give N-(benzo[d][l,3]dioxol-5-yl)-3-bromo pyrazolo[l,5-a]pyrimidine-5-amine as a white solid. (215.00 mg, yield 75.01 %). LC-MS (ESI) C13H10BrN4O2+[M+H]+, Calc. 332.99 and 334.99; Found 333.11 and 335.08.

[0150] Synthesis of intermediate 3-bromo-N-(3-chloro-4-methylphenyl)pyrazolo[l,5- a]pyrimidin-5-amine (LC-02-093): Refer to the synthetic procedure of LC-02-092, replace benz[d][l,3]dioxol-5-amine with 3-chloro-4-methylaniline to synthesize the target product. LCMS (ESI) C13H11BrClN4+[M+H]+calcd: 336.99 and 338.99; found: 337.05 and 339.03.

[0151] Synthesis of intermediate N-benzyl-3-bromopyrazolo[l,5-a]pyrimidin-5-amine (LC-02-097): Refer to the synthetic procedure of LC-02-092, replace benz[d][l,3]dioxol-5-amine with benzylamine to synthesize the target product. LCMS (ESI) C13H12BrN4+[M+H]+calcd: 303.03 and 305.02; found: 303.07 and 305.04.

[0152] Synthesis of intermediate 3-bromo-N-phenethylpyrazolo[l,5-a]pyrimidin-5-amine (LC-02-098): Refer to the synthetic procedure of LC-02-092, replace benz[d][l,3]dioxol-5-amine with phenethylamine to synthesize the target product. LCMS (ESI) C14H14BrN4+[M+H]+calcd: 317.04 and 319.04;

[0153] found: 317.14 and 319.09.

[0154] Synthesis of intermediate 3-bromo-N-cyclohexylpyrazolo[l,5-a]pyrimidin-5-amine (LC-02-106): Refer to the synthetic procedure of LC-02-092, replace benz[d][l,3]dioxol-5-amine with cyclohexylamine to synthesize the target product. LCMS (ESI) C12H16BrN4+[M+H]+calcd: 295.06 and 297.06;

[0155] found: 295.27 and 297.21.

[0156] Synthesis of intermediate 3-bromo-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[l,5- a]pyrimidin-5-amine (LC-02-107): Refer to the synthetic procedure of LC-02-092, replace benz[d][l,3]dioxol-5-amine with tetrahydro-2H-pyran-4-amine to synthesize the target product. LCMS (ESI) C11H14BrN4O+[M+H]+calcd: 297.04 and 299.04; found: 297.21 and 299.54.

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

[0158] Synthesis of intermediate 3-bromo-N-(4,4-difluorocyclohexyl)pyrazolo[l,5- a]pyrimidin-5-amine (LC-02-122): Refer to the synthetic method of LC-02-092, replace benz[d][l,3]dioxol-5-amine with 4,4-difluorocyclohexan-l -amine to synthesize the target product. LCMS (ESI) C12H14BrF2N4+[M+H]+, calculated: 331.04 and 333.04; found: 331.18 and 333.75.

[0159] Synthesis of intermediate 3-bromo-N-(2-morpholinoethyl)pyrazolo[l,5-a]pyrimidin-5- amine (LC-02-123): Refer to the synthetic method of LC-02-092, replace benz[d][l,3]dioxol-5-amine with 2-morpholinoethan-l -amine to synthesize the target product. LCMS (ESI) C12H17BrN5O+[M+H]+, calculated: 326.06 and 328.06; found: 326.32 and 328.46.

[0160] Scheme II, synthesis of intermediate 4-((3,5-dichloropyrazolo[l,5-a]pyrimidin-2- yl)methyl)morpholine (LC-04-179)

[0161]

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

[0163] Dissolve 1,3-dimethylpyrimidine-2,4(lH,3H)-dione (1 eq.) and 5-methyl-lH-pyrazol-3-amine (1 eq.) in ethanol, add sodium ethoxide (3 eq.) at 0 °C. Warm the reaction solution to 80 °C, stir for 12 hours. Detect the completion of the reaction by LCMS, cool the reaction solution to room temperature. Adjust the pH of the reaction solution to pH = 3 with 3M hydrochloric acid. Concentrate the organic solvent under reduced pressure, extract with water and ethyl acetate, repeat the extraction three times until the extraction is complete, wash the organic phase with saturated brine, combine the organic phase, dry over anhydrous sodium sulfate, filter and concentrate under reduced pressure. Purify the residue by silica gel column chromatography to obtain 2-methylpyrazolo[l,5-a]pyrimidin-5(4H)-one as a white solid. (2.80 g, yield 91.16 %). LC-MS (ESI) C7H8N3O+[M+H]+, calc. 150.07; found 150.13.

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

[0165] Dissolve 2-methylpyrazolo[l,5-a]pyrimidin-5(4H)-one (1 eq.) in DMF, add NCS (1.2 eq.) at 25 °C. Warm the reaction solution to 60 °C, stir for 12 hours. Detect the completion of the reaction by LCMS, cool the reaction solution to room temperature. Concentrate the organic solvent under reduced pressure, extract with water and ethyl acetate, repeat the extraction three times until the extraction is complete, wash the organic phase with saturated brine, combine the organic phase, dry over anhydrous sodium sulfate, filter and concentrate under reduced pressure. Purify the residue by silica gel column chromatography to obtain 5-chloro-2-methylpyrazolo[l,5-a]pyrimidine as a white solid. (2.90 g, yield 92.17 %). LC-MS (ESI) C7H7ClN3+[M+H]+, calc. 168.04; found 168.23.

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

[0167] Dissolve 5-chloro-2-methylpyrazolo[1,5-a]pyrimidine (1 eq.) in acetonitrile, add POCl3(2.7 eq.) at 25 °C. Warm the reaction solution to 80 °C, stir for 3 hours. Detect the completion of the reaction by LCMS, cool the reaction solution to room temperature. Concentrate the organic solvent under reduced pressure, extract with water and ethyl acetate, repeat the extraction three times until the extraction is complete, wash the organic phase with saturated brine, combine the organic phase, dry over anhydrous sodium sulfate, filter and concentrate under reduced pressure. Purify the residue 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: 202.00; found: 202.11.

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

[0169] Dissolve 3,5-dichloro-2-methylpyrazolo[1,5-a]pyrimidine (1 eq.) in CCl4, add NBS (1.5 eq.) and BPO (0.05 eq.) at 25 °C. Warm the reaction solution to 80 °C, stir for 12 hours. Detect the completion of the reaction by LCMS, cool the reaction solution to room temperature. Concentrate the organic solvent under reduced pressure, extract with water and ethyl acetate, repeat the extraction three times until the extraction is complete, wash the organic phase with saturated brine, combine the organic phase, dry over anhydrous sodium sulfate, filter and concentrate under reduced pressure. Purify the residue 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: 279.90 and 281.90; found: 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] Dissolve 2-(bromomethyl)-3,5-dichloropyrazolo[1,5-a]pyrimidine (1 eq.) and morpholine (0.9 eq.) in THF, and add K2CO3 (3.0 eq.). Stir at 25°C for 1 hour. Detect the completion of the reaction by LCMS, and cool the reaction solution to room temperature. Add water and ethyl acetate to the reaction system to extract, repeat the extraction three times until the extraction is complete, wash the organic phase with saturated brine, combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Purify the residue 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: 287.05; found: 287.05.

[0172] Example 19: Degradation of CK1a in MOLM 13 cells in vitro by different concentrations of compounds

[0173] 1. Cell culture and drug treatment

[0174] Inoculate the human acute myeloid leukemia cell line MOLM 13 cell line into a six-well plate for culture, and treat the cells 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 are 100 nM, 20 nM) for 6 hours, and set up a DMSO group as a 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 the cell pellet. Wash the cell pellet three times with pre-cooled PBS, then add RIPA lysis buffer and protease inhibitors to the cell pellet, blow and break the cells with a pipette gun, and centrifuge at 13000 rpm at 4°C for 20 minutes to obtain the extracted protein solution. Determine the protein concentration in the extract by the BCA method.

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

[0178] Mix cell lysis buffer with SDS-PAGE loading buffer and boil at 95°C for 5 minutes. Spot the boiled sample onto a 12.5% ​​SDS-PAGE gel, and simultaneously spot a protein marker onto one of the wells to indicate the relative molecular mass of the protein. Place the gel plate in the electrophoresis tank and add enough electrophoresis buffer to cover the gel. Connect the power supply and set the voltage for electrophoresis until the sample reaches the bottom of the gel.

[0179] 4. Western Blot Detection

[0180] The proteins were transferred from the gel to a PVDF membrane, and the corresponding regions of CK1α and β-actin proteins on the membrane were incubated with the appropriate primary antibodies. The primary antibodies were then combined with near-infrared fluorescently labeled secondary antibodies, and the proteins were developed using a near-infrared laser imager.

[0181] like Figure 1 As shown, under incubation conditions of 100 nM and 20 nM concentrations for 6 hours, the compounds exhibited degradative activity against CK1α, demonstrating their potential for treating acute myeloid leukemia. Among the compounds, LC-02-047-P1, LC-04-113, LC-04-087, LC-04-155, and LC-04-162 showed effective doses as low as 20 nM, with compound LC-04-087 (dCK1α-1) exhibiting the best degradation effect.

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

[0183] The experimental procedure is similar to that of Example 19, except that... Figure 2 The results showed adjustments to the incubation conditions between cells and the compound: with or without the addition of compound dCK1α-1 (LC-04-087), incubation times were set to 0 h, 2 h, 4 h, 6 h, 16 h, and 24 h, respectively. After incubation at each time point, cells were harvested and cryopreserved at -20°C. Once all time points were collected, cells were processed together for subsequent experimental steps. Another difference lies in... Figure 3 The cells used in the results were the CRBN knockout MOLM 13 cell line and the wild-type MOLM 13 cell line.

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

[0185] like Figure 3 As shown, dCK1α-1 in CRBN knockout cells cannot degrade CK1α, compared to Figure 2The results demonstrate that dCK1 a-1 degrades CK1 a through CRBN.

[0186] Example 21 : Validation of compound dCK1 a-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 grew to confluence, compound dCK1 a-1 was added for treatment, and the same amount of DMSO solvent was added to the control group. The cells were incubated at 37°C for 6 hours. After the cells were washed with pre-cooled PBS, the cell precipitate was obtained by centrifugation.

[0189] 2. Protein extraction and enzymolysis

[0190] Lysis solution with a composition 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. The cell lysate was obtained by centrifugation at 12000g for 15 minutes. The protein concentration of the lysate was determined using the BCA method. 50 micrograms of protein was taken, and reduction and alkylation reactions were performed by adding TCEP and CAA. Then the Urea concentration in the lysate was reduced to below 2M by adding 100mM Tris-HCl solution. 1 microgram of trypsin was added to the reaction solution, and the enzyme was cut at 37°C overnight. The next day, TFA was added to reduce the pH of the solution to 6.0 to terminate the enzyme cutting. The sample was desalted using an SDB-RPS desalting column. The eluted peptide sample was vacuum dried and stored in a -20°C refrigerator.

[0191] 3. TMT labeling

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

[0193] 4. Mass spectrometry detection

[0194] The mass spectrometry detection of the sample used an UltiMate 3000 RSLCnano nanoliter liquid chromatograph coupled with a QExactive HF mass spectrometer of Thermo. The peptide sample was injected by an automatic injector, combined to a C18 trapping column (75μm*2cm, 3μm particle size, 120A pore size, Thermo), and then eluted by a C18 analytical column (75μm*15cm, 2μm particle size, 120A pore size, Thermo). The eluted sample was ionized by a nanoelectrospray ion source, and then detected by the mass spectrometer. The pore size (Thermo) was then analyzed on a self-made analytical column (75 μm * 25 cm, 1.9 μm particle size). Separation was performed within the pore size range. An analytical gradient was established using mobile phase A (0.1% formalic acid / 3% DMSO / 97% H2O) and mobile phase B (0.1% formalic acid / 3% DMSO / 97% ACN). The flow rate was set to 300 nL / min. Mass spectrometry was performed in DDA mode for data acquisition. MS1 full scan parameters were set as follows: resolution 60 K @ 200 m / z, scan range 350-1800 m / z, AGC target 3E6, and maximum injection time 50 ms. The precursor ion selection window was set to 1.2 Da, selecting the top 15 precursor ions for fragmentation, and the HCD collision energy was set to 32%. MS2 scan parameters were set as follows: resolution 45 K @ 200 m / z, AGC target 1E5, and maximum injection time 100 ms. The dynamic exclusion time was set to 30 seconds.

[0195] 5. Proteomics data analysis

[0196] The raw mass spectrometry data was retrieved using the TMT workflow built into the FragPipe software. The main search parameters were as follows: the database used was human protein sequence data downloaded from the UniProt database from SwissProt; the digestion parameter was set to Trypsin, allowing a maximum of two missed cleavage sites; the mass tolerance for primary mass spectrometry was set to 10 ppm, and the mass tolerance for fragment ions was set to 20 ppm; variable modifications were set to TMTpro (K / peptide N-term), Oxidation (M), and Acetylation (protein N-term), and fixed modifications were set to Carbamidomethylation (C); search results were filtered using 1% FDR; protein quantification was calculated from the intensity of the TMT reporter ions using the IonQuant algorithm. Differentially expressed proteins were calculated using ratios and the p-value of the T-test. The volcano plot was generated using the R package EnhancedVolcano.

[0197] like Figure 4 As shown, quantitative proteomics results revealed that, compared to the control group (DMSO), the protein with the largest downregulation in the dCK1α-1 treatment group was CSNK1A1, i.e., CK1α. This result indicates that dCK1α-1 exhibits excellent selectivity in degrading CK1α.

[0198] Example 22: Effect of compound dCK1a-1 on cell apoptosis and cell cycle by flow cytometry

[0199] After treating cells with DMSO, 40 nM dCK1a-1, 200 nM dCK1a-1 for 24 hours, the cells were detected using an Aurora flow cytometer (Cytek Biosciences). The kit used for detecting cell apoptosis was the Annexin V-FITC Apoptosis Detection Kit (Biouniquer, #C1062M); the proportion of cell apoptosis was counted by the Annexin V+ channel, and the data picture was processed by FlowJo 10.9.0 software.

[0200] The sample and instrument used for cell cycle detection were the same as those for apoptosis detection, and the kit used was the Cell Cycle Analysis Kit (Biouniquer, #C1052M). Cell cycle data analysis was processed by FlowJo 10.9.0 and GraphPad PRISM 9.5 software.

[0201] As shown in Figure 5 , compared with the DMSO control, the proportion of cell apoptosis gradually increased with the increase of the concentration of dCK1a-1 compound-treated cells.

[0202] As shown in Figure 6 , compared with the DMSO control, the cell cycle of dCK1a-1 compound-treated cells was arrested with the increase of the concentration.

[0203] Example 23: Effect of compound dCK1a-2 on CK1a protein and P53-related target genes in mouse models

[0204] 1. Construction of mouse model

[0205] 4x10 6 Human acute myeloid leukemia cells MOLM 13 were injected subcutaneously into 6-week-old female nude mice. After the mice were fed until the tumors grew, the mice were randomly divided into three groups, with three replicates in each group.

[0206] 2. Compound treatment of tumor-bearing mice

[0207] Different doses of dCK1a-2 compound were fed orally, including control, 20 mg / kg, and 40 mg / kg, with 3 mice in each group. After three days of feeding, the mice were sacrificed.

[0208] 3. Sample collection and detection

[0209] The tumor samples were collected from the subcutaneous tumors, and WB was used to detect CK1a protein, and qPCR was used to detect the mRNA expression of CK1a, P21, MDM2, Bax, Bbc3 and other P53 related genes.

[0210] As shown in Figure 7 Figure 6, compound dCK1a-2 can significantly reduce the content of CK1a 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 CK1a did not change significantly, which also indicates that the effect of compound dCK1a-2 on CK1a is post-transcriptional regulation, which is consistent with the mechanism of targeted degradation. The results show that dCK1a-2 can significantly degrade CK1a in the tumor of the mouse model through oral administration (oral dose of 20 mg / kg-3~40 mg / kg-1), which shows the potential for treating acute myeloid leukemia.

[0211] The above is only a preferred embodiment of the present application, not any form of limitation on the present application, although the present application has been disclosed as above with a preferred embodiment, however, it is not intended to limit the present application, any person skilled in the art, without departing from the scope of the technical scheme of the present application, can make some changes or modifications of the above disclosed technical content as equivalent embodiments, but as long as it does not deviate from the technical scheme of the present application, any modification, equivalent change and modification of the above embodiments according to the technical essence of the present application, all still belong to the scope of the technical scheme of the present application.

Claims

1. A heterocyclic compound, characterized in that, The structures of the compounds are shown in formula (I) or formula (II), wherein the structure of formula (I) is as follows: In formula (I), R is selected from halogens, Ra-NH, In Ra-NH, Ra is selected from unsubstituted or substituted six-membered cycloalkyl groups, or six-membered heterocycloalkyl groups containing oxygen and / or nitrogen. The structure of formula (II) is as follows: In formula (II), R1 is selected from H, halogens, -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, and R1 is selected from H, Br, Cl, -NH2, -CN, or -CH3; Alternatively, R2 may be -CH3, and R1 may be selected from H, Br, or Cl; Or R2 is R1 is selected from Br or Cl.

4. A heterocyclic compound, characterized in that, The heterocyclic compound has the following structure:

5. An enantiomer, stereoisomer, or pharmaceutically acceptable salt of the heterocyclic compound as described in any one of claims 1 to 4.

6. The use of a heterocyclic compound as described in any one of claims 1 to 4, and its enantiomers, stereoisomers, or pharmaceutically acceptable salts, in the preparation of a molecular gel degrading agent targeting CK1α, characterized in that, The molecular glue degrader targeting CK1α is used in the preparation of drugs for the treatment of acute myeloid leukemia.

7. The use of a heterocyclic compound as described in any one of claims 1 to 4, and its enantiomers, stereoisomers, or pharmaceutically acceptable salts thereof, in the preparation of medicaments for the treatment or prevention of diseases associated with CK1α overexpression, characterized in that, The diseases associated with high CK1α expression are hematologic malignancies related to the p53 signaling pathway; the hematologic malignancies are acute myeloid leukemia.

8. A drug for treating acute myeloid leukemia, characterized in that, The active ingredient of the drug comprises one or more of the heterocyclic compound, its enantiomer, stereoisomer, or pharmaceutically acceptable salt as described in any one of claims 1 to 4.

9. The medicament as described in claim 8, characterized in that, The active ingredient of the drug comprises one or more of the heterocyclic compound, its enantiomer, stereoisomer, or pharmaceutically acceptable salt as described in claim 4.

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