PROTAC compound for degrading HPK1 protein and application thereof

Abstract

The invention discloses a PROTAC compound with a structure shown in a formula I and capable of degrading HPK1 protein and application of the PROTAC compound in preparation of a medicinal preparation for preventing or treating diseases caused by overexpression of HPK1, such as malignant tumors.

Description

Technical Field

[0001] The present invention relates to the field of chemical synthesis, and in particular to a PROTAC compound for degrading HPK1 protein and applications thereof. Background Art

[0002] T cell-based cell therapy is a promising new approach in adoptive immunotherapy for cancer. Although chimeric antigen receptors (CARs) targeting CD19 mediate effective anti-tumor responses in hematological malignancies, in many respects, CAR-T cells and other forms of adoptive T cell transfer (ACT) are still in their early stages. Clinical trials have fully demonstrated that early T cell dysfunction is the main factor limiting the anti-tumor efficacy of T cell therapy. Therefore, finding the key signaling pathways that regulate T cell dysfunction in the tumor microenvironment (TME) and improving T cell function by regulating the signaling pathways that affect T cell dysfunction, thereby enhancing its anti-tumor effect, is the key to improving the anti-tumor efficacy of T cell therapy.

[0003] Endogenous or adoptively transferred cytotoxic T cells are important mediators of antitumor immunity. Continuous antigen exposure leads to a gradual loss of specific effector functions and proliferation capacity, as well as significant transcriptional, epigenetic, and metabolic changes, resulting in T cell dysfunction. Understanding how to modulate T cell dysfunction and enhance its effector functions is a hot topic in cancer immunotherapy.

[0004] In the clinic, cancer immunotherapies based on restoring the activity of dysfunctional T cells, such as checkpoint blockade, have achieved some therapeutic benefits. Despite this, many patients still do not achieve sustained therapeutic responses. Some scholars have proposed that this treatment failure may be due to the fact that the stable epigenetic state in cells with dysfunctional T cells limits their ability to recover. Furthermore, multiple transcription factors (TFs) have been shown to regulate T cell dysfunction in the tumor microenvironment, including NFAT, Eomes, T-bet, Blimp-1, BATF, FoxO1, VHL, c-Maf, TOX, NR4A1, and TCF1. However, strategies to improve T cell function by regulating TFs are complex, and the mechanisms of action are not yet clearly defined. However, improving T cell function through the use of small molecule chemotherapies that specifically target druggable targets of T cell dysfunction is an ideal immunotherapy-based cancer treatment strategy.

[0005] Hematopoietic progenitor kinase 1 (HPK1, also known as MAP4K1) is a serine / threonine kinase, an immunosuppressive regulatory kinase encoded by the MAP4K1 gene and restrictedly expressed in hematopoietic cells such as T cells, B cells and dendritic cells (DC). It is a currently known negative regulator of T cell receptors and is believed to play a key role in the activation of T cells. The latest studies have shown that HPK1 regulates T cell dysfunction through the HPK1-NFκB-Blimp1 signaling axis. After the T cell surface receptor (TCR) is activated, HPK1 in the cytoplasm is recruited to the cell membrane surface and binds to different adapter proteins, thereby participating in the regulation of different signaling pathways and transcription factors. A recent study analysis showed that HPK1 kinase activity inhibits the expression of proteins including CD4 + T cells, CD8 + HPK1 is a key molecule involved in the immune function of various cells, including T cells and dendritic cells (DCs). Inhibition of its kinase domain has been shown to trigger anti-tumor immune responses. Multiple studies have shown that HPK1 is a promising drug target in cancer immunotherapy.

[0006] Currently, no drugs selectively targeting HPK1 are available. Only Treadwell Therapeutics (US) initiated Phase I and II clinical trials of CFI-402411 (NCT04521413) in the fourth quarter of 2020, and BeiGene (my country) initiated a Phase I clinical trial of the pyrrolo[2,3-b]pyridine derivative BGB-15025 (NCT04649385) in the first quarter of 2021 (neither company's chemical structure has been published). Furthermore, the importance of HPK1 in T cell-based immune cancer therapy and the molecular mechanisms by which it influences T cell function require further investigation.

[0007] The latest research shows that compared with PDCD1 KO CAR-T cells, MAP4K1 KO CAR-T cells significantly enhance anti-tumor activity, demonstrating that HPK1 kinase knockout is an effective strategy for anti-tumor activity. Experiments in HPK1 knockout mouse models have shown that improving T cell function can inhibit tumor growth without inducing a significant autoimmune response, avoiding the lethal inflammatory effects of knockout of other negative regulatory factors (such as CTLA4 and Cbl-b). Therefore, selective HPK1 kinase knockout is a safe and effective therapeutic strategy in tumor immunotherapy.

[0008] PROteolytic Targeting Chimera (PROTAC) technology is an emerging technology that chemically induces polyubiquitination of target proteins and degrades them through the ubiquitin-proteasome system. PROTAC consists of three parts: an E3 ubiquitin ligase ligand, a target protein ligand, and a connecting chain. Unlike the "occupancy-driven" mode of action of traditional small molecule inhibitors, PROTAC is an "event-driven" mode of action that selectively recruits E3 ligases and specifically degrades target proteins through the 26s proteasome. It is a new pharmacological mode of action and brings new strategies to new drug research and development. Unlike the emerging CRISPR / Cas9 gene knockout technology, PROTAC technology is a new technology for rapid, reversible, economical, and simple post-translational processing of proteins, providing new methods for the treatment of diseases.

[0009] Since the PROTAC concept was first proposed in 2001, PROTACs have achieved exciting results. In particular, the results of the Phase II I clinical trial of ARV-471 demonstrated its clinical applicability, greatly stimulating research interest in PROTACs. Due to its unique pharmacological mode of action, PROTACs have broad application prospects and potential for development in specifically targeting disease-causing proteins, particularly undruggable targets. They have been hailed as the arrival of the fourth pharmaceutical revolution.

[0010] In 2021, Nurix disclosed the first degrader targeting HPK1. Among the 97 compounds involved in the patent, 22 compounds have good degradation activity (D max >65%); In 2023, the company submitted another patent application, and many analogs of the 303 compounds showed strong degradation ability (DC 50 >100nM; D max >85%); other similar degraders have also been reported [Yuan YH, et al. Expert Opin Ther Pat. 2025;`35(4):387-408.]

[0011] Although the above studies have obtained HPK1 degraders with obvious degradation effects, no drugs targeting HPK1 degradation have been approved for marketing so far. Therefore, discovering more HPK1-targeting degraders with better performance remains a problem to be solved. Summary of the Invention

[0012] The present invention provides a new class of PROTAC compounds targeting HPK1 protein degradation, as well as a preparation method and pharmaceutical application thereof.

[0013] In order to achieve the above objectives, the present invention provides a PROTAC compound for degrading HPK1 protein with the following structure:

[0014]

[0015] Wherein, L is a connecting chain connected to the 4- or 5-position of the isoindole-1,3-dione structure on the right side, and its structure is:

[0016]

[0017] Furthermore, the PROTAC compound for degrading HPK1 protein provided by the present invention has the following structure:

[0018]

[0019]

[0020]

[0021]

[0022] The present invention also provides a method for preparing the PROTAC compound for degrading HPK1 protein, comprising the following steps:

[0023] Step 1: According to the method published in the literature [European Journal of Medicinal Chemistry, 2024, 267: 116-206], a small molecule ligand targeting HPK1 (9) was synthesized:

[0024]

[0025] Step 2: Prepare key intermediates 3, 6, 9, 12, 18, 21, and their analogs according to the methods and reaction conditions shown in S1-S6 below, wherein n is an integer of 1-10:

[0026]

[0027] S1. Reagents and conditions: (a) tert-butyl 2-bromoacetate, K2CO3, KI, DMF, 60°C, 6h, 65; (b)

[0028] TFA, DCM, 85%;

[0029]

[0030] S2. Reagents and conditions: (a) tert-butyl 2-bromoacetate, K2CO3, KI, DMF, 60°C, 6h, 65; (b)

[0031] TFA, DCM, 85%;

[0032]

[0033] S3. Reagents and conditions: (a) tert-Butylpiperidine-4-carboxylate, DIPEA, ACN, 80°C, 8h, 75%; (b)

[0034] TFA, DCM, 80%;

[0035]

[0036] S4. Reagents and conditions: (a) DIPEA, DMSO, 150°C, 8 h, 60-75%; (b) TFA, DCM, 85%;

[0037]

[0038] S5. Reagents and conditions: (a) K2CO3, DMF, 120°C, 8 h, 65%; (b) TFA, DCM, 80%;

[0039]

[0040] S6. Reagents and conditions: (a) DIPEA, DMF, 100°C, 8h, 55-75%; (b) TFA, DCM, 80%.

[0041] Step 3: The small molecule ligand (9) targeting HPK1 obtained in step 1 and the key intermediate obtained in step 2 are subjected to a condensation reaction in THF at 50° C. in the presence of HATU and DIPEA to obtain the target product.

[0042] In another aspect, the present invention also provides a pharmaceutical composition comprising the compound as described above, or its stereoisomers, tautomers, solvates, pharmaceutically acceptable salts, metabolites, isotopic derivatives, N-oxides or prodrugs, and a pharmaceutically acceptable carrier, diluent or excipient.

[0043] In yet another aspect, the present invention further provides the use of the above-described compound, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, metabolite, isotopic derivative, N-oxide, or prodrug thereof, or a pharmaceutical composition thereof, in a medicament for preventing and / or treating a disease associated with HPK1 activity. The HPK1 activity-associated disease includes cancer or an immune disease; the cancer is preferably lung cancer, thyroid cancer, liver cancer, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, squamous cell carcinoma, head and neck cancer, oral cancer, thyroid cancer, nasopharyngeal cancer, breast cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, endometrial cancer, bladder cancer, bone cancer, brain cancer, skin cancer, melanoma, sarcoma, cell tumor, glioma, hematologic tumor, and lymphoma; and the immune disease is preferably lupus erythematosus, psoriasis, inflammatory bowel disease, and rheumatoid arthritis.

[0044] In one aspect, the present invention also provides a method for preventing and / or treating diseases related to HPK1 activity, comprising administering to a patient in need thereof a therapeutically effective amount of the compound as described above, or its stereoisomers, tautomers, solvates, pharmaceutically acceptable salts, metabolites, isotopic derivatives, N-oxides or prodrugs, or pharmaceutical compositions.

[0045] The HPK1 activity-related diseases include cancer or immune diseases; the cancer is preferably lung cancer, thyroid cancer, liver cancer, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, squamous cell carcinoma, head and neck cancer, oral cancer, thyroid cancer, nasopharyngeal cancer, breast cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, endometrial cancer, bladder cancer, bone cancer, brain cancer, skin cancer, melanoma, sarcoma, cell tumor, glioma, blood tumor and lymphoma; the immune disease is preferably lupus erythematosus, psoriasis, inflammatory bowel disease and rheumatoid arthritis.

[0046] In another aspect, in the above-mentioned uses or methods, the above-mentioned compounds, or their stereoisomers, tautomers, solvates, pharmaceutically acceptable salts, metabolites, isotopic derivatives, N-oxides or prodrugs, or pharmaceutical compositions can be used alone or in combination with other types of pharmaceutical preparations and / or treatment methods.

[0047] It is important to note that, herein, when reference is made to a "compound" having a particular structural formula, it generally also encompasses stereoisomers, diastereomers, enantiomers, racemic mixtures and isotopic derivatives thereof.

[0048] It is well known to those skilled in the art that the salts, solvates and hydrates of a compound are alternative forms of existence of the compound, and they can all be converted into the compound under certain conditions. Therefore, it is particularly noted that when a compound is mentioned in this article, it generally also includes its pharmaceutically acceptable salts, and further includes its solvates and hydrates.

[0049] Similarly, reference herein to a compound generally also includes prodrugs, metabolites, and N-oxides thereof.

[0050] The "pharmaceutically acceptable salts" of the present invention refer to pharmaceutically acceptable acid and base addition salts or solvates thereof. Such pharmaceutically acceptable salts include salts of the following acids: hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, sulfurous acid, formic acid, toluenesulfonic acid, methanesulfonic acid, nitric acid, benzoic acid, citric acid, tartaric acid, maleic acid, hydroiodic acid, alkanoic acids (such as acetic acid, HOOC-(CH2)n-COOH (wherein n is 0 to 4)), or mixed salts thereof. Salts of bases: sodium salts, potassium salts, calcium salts, ammonium salts, etc. Various non-toxic pharmaceutically acceptable addition salts are known to those skilled in the art.

[0051] The pharmaceutically acceptable salts of the present invention can be prepared by conventional methods, for example, by dissolving the compound of the present invention in a water-miscible organic solvent (e.g., acetone, methanol, ethanol and acetonitrile), adding an excess of an organic acid or an aqueous inorganic acid solution thereto to precipitate the salt from the resulting mixture, removing the solvent and the remaining free acid therefrom, and then isolating the precipitated salt.

[0052] The precursors or metabolites described herein may be any known precursors or metabolites in the art, as long as the precursors or metabolites are converted to compounds through in vivo metabolism. For example, "prodrugs" refer to those prodrugs of the compounds of the present invention that, within the scope of sound medical judgment, are suitable for contact with tissues of humans and lower animals without undue toxicity, irritation, allergic reactions, etc., and are effective for their intended use at a reasonable benefit / risk ratio. The term "prodrug" refers to a compound that is rapidly converted in vivo to produce the parent compound of the above formula, for example, through in vivo metabolism.

[0053] Compared with the prior art, the present invention has the following advantages:

[0054] Based on the PROTAC (Proteolysis-targeting chimaeras) technology that has developed rapidly in recent years, the present invention solves the problem of drug resistance and insufficient activity of small molecule inhibitors in the form of protein degradation. A PROTACs compound provided by the present invention does not need to occupy the active site for a long time like an inhibitor, but only needs to bind to the target protein briefly to induce protein degradation. Since the protein needs to be resynthesized to restore its function after being degraded, this greatly delays the development of drug resistance; it is still effective for proteins that have already developed drug resistance. Therefore, a degrader targeting a protein related to the T cell receptor signaling pathway provided by the present invention can be used in the preparation of drugs for preventing or treating diseases mediated by the protein, such as various malignant tumors or immune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is the degradation result diagram of three concentrations of PROTAC compound;

[0056] Figure 2 This is the result of the XZP-16 cell proliferation inhibition experiment;

[0057] Figure 3 The drug concentration-time curves of XZP-1, 2, 3, and 16 are shown;

[0058] Figure 4 This is the drug concentration-time curve of XZP-14 injection / gavage. DETAILED DESCRIPTION

[0059] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0060] Example 1: Synthesis of target head

[0061]

[0062] 5-Fluoro-2-nitroanisole reacts with Boc-piperazine to produce compound 3, which is then reduced to the nitro group to produce compound 4. This is then condensed with compound 5 to produce compound 6, which is then subjected to Suzuki coupling with compound 7. Finally, the Boc protecting group is removed to obtain the target compound 9, which is used in subsequent reactions. For specific reaction conditions, see reference (Zeng Shenzhao, et al. European Journal of Medicinal Chemistry, 2024, 267:116-206).

[0063] Example 2: Synthesis of intermediates

[0064] Doxamines are commonly used ligands for targeting ubiquitin E3 ligases in PROTAC technology. Their preparation technology has been published in many literatures. Common attachment sites and preparation methods are as follows:

[0065]

[0066] S1. Reagents and conditions: (a) tert-butyl 2-bromoacetate, K2CO3, KI, DMF, 60°C, 6h, 65; (b)

[0067] TFA, DCM, 85%.

[0068]

[0069] S2. Reagents and conditions: (a) tert-butyl 2-bromoacetate, K2CO3, KI, DMF, 60°C, 6h, 65; (b)

[0070] TFA, DCM, 85%.

[0071]

[0072] S3. Reagents and conditions: (a) tert-Butylpiperidine-4-carboxylate, DIPEA, ACN, 80°C, 8h, 75%; (b)

[0073] TFA, DCM, 80%.

[0074]

[0075] S4. Reagents and conditions: (a) DIPEA, DMSO, 150°C, 8h, 60-75%; (b) TFA, DCM, 85%.

[0076]

[0077] S5. Reagents and conditions: (a) K2CO3, DMF, 120℃, 8h, 65%; (b) TFA, DCM, 80%.

[0078]

[0079] S6. Reagents and conditions: (a) DIPEA, DMF, 100°C, 8h, 55-75%; (b) TFA, DCM, 80%.

[0080] Example 3: Synthesis of PROTAC compounds XZP-1 to XZP-9

[0081]

[0082] The XZP series are all composed of different carboxyl-terminated linkers + E3 ligands and target heads through condensation reaction. The condensation agent is HATU, the organic base is DIPEA, the solvent is THF, and the reaction temperature is 50℃.

[0083] XZP-1~XZP-9 NMR data:

[0084] XZP-1:1H NMR (400MHz, CHLOROFORM-D) δ10.30(s,1H),9.11(d,J=5.0Hz,1H),8.60(d,J=4.4Hz,1H),8.40(d,J=8.7Hz,1H),8.11(d,J=5. 0Hz,1H),7.46(t,J=7.8Hz,1H),7.40(td,J=8.4,6.5Hz,1H),7.05(d,J=7.0Hz,1H),6.96(d,J=8.5Hz,1H),6.87–6.83(m,1H),6 .82(s,1H),6.55(d,J=9.0Hz,1H),6.32(t,J=5.6Hz,1H),4.89(dd,J=11.8,5.4Hz,1H),3.84(s,3H),3.81(s,3H),3.62(s,2H) ,3.37(q,J=6.1Hz,2H),3.14(t,J=4.9Hz,4H),2.90–2.64(m,4H),2.46(t,J=6.7Hz,2H),2.11–2.02(m,2H),2.02–1.96(m,2H).

[0085] XZP-2:1H NMR(400MHz,CHLOROFORM-D)δ10.31(s,1H),9.11(d,J=5.0Hz,1H),8.62(d,J=3.6Hz,1H),8.42(d,J=8.7Hz,1H),8.11(d,J=5.0Hz,1H),7.45(t,J=7.8Hz,1H),7.41–7.36(m,1H),7.04(d,J=7.0Hz,1H),6.86(s,1H),6.84(d,J=2.2Hz,1H),6.82(d,J=2.7Hz,1H),6.59(s,1H),6.21(t,J=5.5Hz,1H),4.88(dd,J=11.8,5.3Hz,1H),3.85(s,3H),3.81(s,3H),3.26(q,J=6.1Hz,2H),3.16(s,4H),2.93–2.61(m,4H),2.37(t,J=7.3Hz,2H),2.02(s,2H),1.71(d,J=7.2Hz,2H),1.67(d,J=7.3Hz,2H),1.47(q,J=7.6Hz,2H),1.25(d,J=4.1Hz,2H).

[0086] XZP-3:1H NMR(400MHz,CHLOROFORM-D)δ10.42(s,1H),9.12(d,J=4.9Hz,1H),8.81(s,1H),8.55(s,1H),8.10(d,J=4.9Hz,1H),7.48–7.43(m,1H),7.39(dd,J=8.4,6.4Hz,1H),7.03(d,J=6.7Hz,1H),6.86(s,1H),6.84(d,J=3.6Hz,1H),6.82(d,J=3.8Hz,1H),6.19(s,1H),4.95–4.85(m,1H),3.90(s,3H),3.81(s,3H),3.35(s,4H),3.22(s,2H),2.94–2.61(m,4H),2.31(d,J=31.7Hz,2H),2.01(s,2H),1.38–1.33(m,4H),1.27–1.18(m,6H),1.04–0.68(m,2H).

[0087] XZP-4:1H NMR(400MHz,DMSO-D6)δ11.07(s,1H),10.07(s,1H),9.20(d,J=5.0Hz,1H),8.13(d,J=8.8Hz,1H),8.06(d,J=5.0Hz,1H),7.52(d,J=1.6Hz,1H),7.49(d,J=8.3Hz,1H),7.03(t,J=8.6Hz,2H),6.97(d,J=3.2Hz,1H),6.95(d,J=1.4Hz,1H),6.68(d,J=2.5Hz,1H),6.53(dd,J=8.9,2.4Hz,1H),6.46(t,J=5.9Hz,1H),5.01(dd,J=12.9,5.4Hz,1H),3.79(s,3H),3.76(s,3H),3.54(q,J=5.3Hz,4H),3.24–3.19(m,2H),3.14–3.06(m,4H),2.47–2.46(m,6H),2.28(t,J=7.4Hz,2H),1.22–1.17(m,14H),0.83–0.79(m,2H).

[0088] XZP-5:1H NMR(400MHz,CHLOROFORM-D)δ10.28(s,1H),9.10(d,J=4.9Hz,1H),8.39(d,J=8.8Hz,1H),8.11(d,J=5.1Hz,1H),7.74(d,J=8.4Hz,1H),7.40(q,J=7.9Hz,1H),7.33(s,1H),7.19(d,J=8.5Hz,1H),6.84(d,J=8.2Hz,2H),6.55–6.49(m,2H),4.92(dd,J=12.1,5.3Hz,1H),4.22(t,J=4.6Hz,2H),3.85(s,3H),3.82(s,3H),3.78(d,J=6.9Hz,2H),3.69(t,J=4.5Hz,4H),3.64–3.56(m,12H),3.13(dt,J=10.9,5.0Hz,6H),2.85–2.73(m,2H),2.66(t,J=6.4Hz,2H),1.25(d,J=9.7Hz,2H).

[0089] XZP-6:1H NMR(400MHz,CHLOROFORM-D)δ10.27(s,1H),9.10(dd,J=5.0,1.6Hz,1H),8.78(s,1H),8.37(d,J=9.1Hz,1H),8.11(dd,J=5.0,1.5Hz,1H),7.68–7.61(m,1H),7.46(dd,J=7.2,1.8Hz,1H),7.43–7.34(m,1H),7.29(dd,J=8.4,1.8Hz,1H),6.87–6.80(m,2H),6.52(d,J=8.9Hz,1H),4.99(s,2H),4.94(dd,J=11.6,5.4Hz,1H),3.82(s,3H),3.80(s,3H),3.79–3.69(m,4H),3.24–3.06(m,4H),2.87–2.70(m,3H),2.10(dt,J=11.5,4.3Hz,1H).

[0090] XZP-7:1H NMR(400MHz,CHLOROFORM-D)δ10.29(s,1H),9.10(d,J=5.0Hz,1H),8.69(s,1H),8.40(d,J=8.4Hz,1H),8.11(d,J=5.0Hz,1H),7.76(d,J=7.6Hz,1H),7.44–7.38(m,1H),7.35(d,J=9.4Hz,1H),6.83(dt,J=8.6,4.5Hz,2H),6.53(s,2H),5.27(s,1H),4.90(d,J=17.7Hz,2H),3.84(s,3H),3.81(s,3H),3.80–3.60(m,4H),3.29–3.10(m,4H),2.80–2.69(m,2H),1.23(s,2H).

[0091] XZP-8:1H NMR (400MHz, CHLOROFORM-D) δ10.29(s,1H),9.11(d,J=5.0Hz,1H),8.62(d,J=4.7Hz,1H),8.40(d,J=8.9Hz,1H),8.1 2(d,J=5.0Hz,1H),7.64(d,J=8.5Hz,1H),7.39(td,J=8.4,6.5Hz,1H),7.03(dd,J=8.6,2.3Hz,1H),6.83(dt,J=8.7,4 .5Hz,2H),6.54(d,J=7.8Hz,2H),4.92(dd,J=12.1,5.4Hz,1H),3.85(s,3H),3.81(s,3H),3.69(s,2H),3.15(dd,J=1 4.1,8.7Hz,4H),3.07–2.99(m,2H),2.84–2.68(m,4H),2.12–2.06(m,1H),1.96–1.81(m,4H),1.24(d,J=11.5Hz,4H).

[0092] XZP-9:1H NMR(400MHz,CHLOROFORM-D)δ10.30(s,1H),8.63(s,1H),8.41(d,J=8.8Hz,1H),8.11(d,J=4.9 Hz,1H),7.60(d,J=8.2Hz,1H),7.40(td,J=8.4,6.5Hz,1H),6.86–6.81(m,2H),6.76(d,J=2.0Hz ,1H),6.59–6.48(m,3H),4.90(dd,J=12.2,5.2Hz,1H),4.22(dt,J=23.4,7.6Hz,4H),3.85(s,3H ),3.81(s,3H),3.15(t,J=5.0Hz,4H),2.87–2.63(m,4H),2.11–2.05(m,1H),1.24–1.22(m,4H).

[0093] Example 4: Synthesis of PROTAC compounds XZP-11 to XZP-13

[0094]

[0095] XZP-11 to XZP-13 were synthesized via azide-alkyne cycloaddition click reactions. First, pomalidomide was substituted with PEG chains of varying lengths to yield compound 19. Subsequently, an azide group was added to the bromine-substituted position, and finally, a click reaction with an alkyne-containing target head was performed to yield XZP-11 to XZP-13.

[0096] 1H NMR data of XZP-11 to XZP-13:

[0097] XZP-11: 1H NMR (400 MHz, CHLOROFORM-D) δ 10.26 (s, 1H), 9.09 (d, J = 5.0 Hz, 1H), 8.36 (d, J = 8.7 Hz, 1H), 8.11 (d, J = 5.0 Hz, 1H), 7.57 (s, 1H), 7.41–7.35 (m, 2H), 7.08 (d, J = 7.1 Hz, 1H), 6.86–6.81 (m, 3H), 6.54–6.48 (m, 2H), 5.28 (d, J = 9.9 Hz, 4H), 4.98–4.91 (m, 1H), 4.49 (td, J = 4.8, 2.4 Hz, 2H), 4.09–3.95 (m, 2H), 3.83 (s, 3H), 3.81 (s, 3H), 3.55 (d, J = 3.4 Hz, 4H), 3.20 (t, J = 4.9 Hz, 4H), 2.79–2.72 (m, 4H), 2.69 (t, J = 4.9 Hz, 4H), 1.24–1.22 (m, 4H).

[0098] XZP-12: 1H NMR (400 MHz, CHLOROFORM-D) δ 10.26 (s, 1H), 9.09 (d, J = 5.1 Hz, 1H), 8.35 (d, J = 8.8 Hz, 1H), 8.11 (d, J = 5.0 Hz, 1H), 7.55 (s, 1H), 7.41–7.35 (m, 2H), 7.10 (d, J = 7.0 Hz, 1H), 6.87–6.80 (m, 4H), 6.54–6.49 (m, 2H), 5.30 (s, 2H), 4.92 (dd, J = 12.5, 5.3 Hz, 1H), 4.48 (t, J = 5.0 Hz, 2H), 4.07 (dt, J = 12.8, 6.3 Hz, 1H), 4.02–3.96 (m, 1H), 3.83 (s, 3H), 3.81 (s, 3H), 3.58 (d, J = 7.0 Hz, 11H), 3.18 (dd, J = 6.5, 3.5 Hz, 4H), 2.97–2.85 (m, 4H), 2.76–2.70 (m, 4H), 2.68–2.64 (m, 4H).

[0099] XZP-13:1H NMR (400MHz, CHLOROFORM-D) δ10.26(s,1H),9.09(d,J=5.0Hz,1H),8.36(d,J=8.8Hz,1H),8.11(d,J=5.0Hz,1H ),7.58(s,1H),7.41–7.36(m,2H),7.09(d,J=7.1Hz,1H),6.85(d,J=2.3Hz,1H),6.83(dd,J=2.6,1.6Hz,2H),6. 82(d,J=1.8Hz,1H),6.55–6.49(m,2H),5.33(s,2H),5.27(s,1H),4.48(t,J=5.1Hz,2H),4.08–3.88(m,4H),3.8 3(s,3H),3.81(s,3H),3.59–3.57(m,12H),3.22(d,J=5.1Hz,4H),2.99–2.89(m,4H),2.74(hept,J=3.4Hz,8H).

[0100] Example 5: Synthesis of PROTAC compounds XZP-14 to XZP-16

[0101]

[0102] The synthetic routes of XZP-14 to XZP-16 are similar. The target head undergoes reductive amination with compound 21, followed by removal of the Boc protecting group to obtain compound 23. Pomalidomide undergoes substitution reaction with compound 18 to obtain compound 19, which then undergoes substitution reaction with compound 23 under alkaline conditions to obtain the final product.

[0103] XZP-14~XZP-16 NMR data:

[0104] XZP-14:1H NMR(400MHz,CHLOROFORM-D)δ11.19(s,1H),10.27(s,1H),9.10(d,J=5.0Hz,1H),8.88(dd,J=8.5,0.7Hz,1H),8.37(d,J=8.8Hz,1H),8.12(d,J=5.0Hz,1H),7.68(dd,J=8.5,7.3Hz,1H),7.52(dd,J=7.4,0.8Hz,1H),7.40(td,J=8.4,6.5Hz,1H),6.87–6.82(m,2H),6.56–6.49(m,2H),4.95(dd,J=11.8,5.4Hz,1H),3.84(s,3H),3.81(s,3H),3.20(t,J=4.9Hz,4H),3.16(s,2H),2.93–2.85(m,3H),2.83–2.73(m,2H),2.61(s,4H),2.36–2.24(m,4H),2.16(dt,J=8.3,2.8Hz,1H),1.78(t,J=10.7Hz,2H),1.27–1.21(m,1H),1.09(s,1H),0.86–0.82(m,1H).

[0105] XZP-15:1H NMR(400MHz,CHLOROFORM-D)δ10.90(s,1H),10.28(s,1H),9.09(d,J=5.0Hz,1H),8.84(dd,J=8.5,0.8Hz,1H),8.38(d,J=8.8Hz,1H),8.10(d,J=5.0Hz,1H),7.69(dd,J=8.5,7.3Hz,1H),7.54(dd,J=7.4,0.8Hz,1H),7.39(td,J=8.4,6.5Hz,1H),6.86–6.82(m,2H),6.77(d,J=2.4Hz,1H),6.65(dd,J=8.8,2.5Hz,1H),4.91(dd,J=11.9,5.9Hz,1H),3.83(s,3H),3.81(s,3H),3.59(t,J=7.4Hz,1H),3.33–3.22(m,6H),3.18(d,J=4.7Hz,2H),3.03–2.96(m,1H),2.85–2.65(m,10H),2.18(q,J=3.2Hz,1H).

[0106] XZP-16:1H NMR (400MHz, CHLOROFORM-D) δ10.86 (s, 1H), 10.27 (s, 1H), 9.10 (d, J = 5.1Hz, 1H), 8.87–8.81 (m, 2H), 8. 38(d,J=8.7Hz,1H),8.11(d,J=5.0Hz,1H),7.68(dd,J=8.5,7.3Hz,1H),7.53(d,J=7.2Hz,1H),7.39(td, J=8.4,6.5Hz,1H),6.86–6.82(m,2H),6.56–6.50(m,2H),4.96(dd,J=12.1,5.3Hz,1H),3.83(s,3H),3. 81(s,3H),3.37(s,2H),3.30–3.17(m,8H),2.91–2.68(m,4H),2.55(t,J=4.9Hz,4H),2.17–2.11(m,1H).

[0107] Example 6: Synthesis of PROTAC compounds XZP-17 to XZP-19

[0108]

[0109] The synthetic routes of XZP-17 to XZP-19 are similar. The CRBN ligand first undergoes a substitution reaction with compound 24 to obtain compound 26. The hydroxyl group is then oxidized to a ketone using Dess-Martin periodinane, and then undergoes a reductive amination reaction with the target head to obtain the final product.

[0110] XZP-17~XZP-19 NMR data:

[0111] XZP-17:1H NMR(400MHz,CHLOROFORM-D)δ10.27(s,1H),9.10(d,J=5.0Hz,1H),8.38(d,J=8.8Hz,1H),8.11(d,J=5.0Hz,1H),7.64(d,J=8.6Hz,1H),7.40(td,J=8.4,6.4Hz,1H),7.27(d,J=2.3Hz,1H),7.01(ddd,J=7.5,5.0,2.3Hz,1H),6.84(t,J=7.8Hz,2H),6.56–6.50(m,2H),4.91(dd,J=12.1,5.4Hz,1H),3.84(s,3H),3.81(s,3H),3.18(t,J=4.9Hz,4H),2.99–2.85(m,4H),2.81–2.68(m,2H),2.58(t,J=4.9Hz,4H),2.26(d,J=6.9Hz,2H),2.13–2.06(m,1H),1.90(d,J=13.4Hz,2H),1.27(dd,J=18.1,6.8Hz,4H).

[0112] XZP-18:1H NMR(400MHz,CHLOROFORM-D)δ10.27(s,1H),9.09(d,J=5.0Hz,1H),8.37(d,J=8.7Hz,1H),8.10(d,J=5.0Hz,1H),7.64–7.58(m,1H),7.39(td,J=8.4,6.5Hz,1H),6.92(dd,J=6.7,2.3Hz,1H),6.87–6.81(m,2H),6.66(ddd,J=8.5,4.0,2.2Hz,1H),6.57–6.49(m,2H),4.83(ddd,J=48.1,10.1,6.1Hz,1H),3.84(s,3H),3.81(s,3H),3.59(s,2H),3.19(t,J=5.1Hz,4H),2.62(dtd,J=20.7,10.8,4.7Hz,6H),2.45(t,J=6.7Hz,2H),2.34(t,J=7.3Hz,2H),2.25–2.15(m,2H),1.82(dq,J=12.3,8.0Hz,1H),1.30–1.21(m,2H).

[0113] XZP-19:1H NMR (400MHz, DMSO-D6) δ11.06(s,1H),10.06(s,1H),9.19(d,J=5.0Hz,1H),8.12(dd,J=8.8,4.2Hz,1H),8.06(d,J=5.0Hz,1H) ,7.58(d,J=8.3Hz,1H),7.54–7.47(m,1H),7.04(d,J=8.5Hz,1H),6.96(t,J=8.9Hz,1H),6.72(d,J=2.1Hz,1H),6.63(d,J=2.4 Hz,1H),6.58(dd,J=8.4,2.1Hz,1H),6.50(dd,J=9.0,2.4Hz,1H),5.02(dd,J=12.9,5.3Hz,1H),4.09(t,J=8.1Hz,2H),3.79(s ,3H),3.76(s,3H),3.65(dd,J=8.5,5.3Hz,2H),3.39(s,8H),3.11(t,J=4.8Hz,4H),2.61(d,J=7.3Hz,2H),2.00–1.93(m,1H).

[0114] Example 7: Preparation of salt

[0115] The XZP-16 obtained in Example 5 was dissolved in ethyl acetate, hydrogen chloride gas was introduced, and the temperature was lowered to about 0 degrees. A solid was slowly precipitated, which was the hydrochloride of XZP-16.

[0116] Example 8: Kinase detection of some PROTACs

[0117] (1) Materials and methods

[0118] Prepare 2x ATP + substrate solution and 2x kinase + metal solution in assay buffer. Transfer 40 nL of compound to a 384 assay plate using an Echo 655. Add 2 μL of 2x kinase + metal solution, mix, and incubate in the 384 assay plate at 25°C for 10 minutes. Add 2 μL of 2x substrate + ATP solution to each well and incubate at 25°C for 60 minutes. Add 4 μL of ADP-Glo ​​reagent to each well and incubate at 25°C for 40 minutes. Add 8 μL of kinase assay reagent and incubate at 25°C for 40 minutes. Record the fluorescence signal on a microtiter reader.

[0119] (2) Experimental results

[0120] Table 1. Kinase inhibition rate of XZP-1 to XZP-9 on HPK1

[0121]

[0122] Kinase assay results at 100 nM revealed that, with the exception of XZP-4, all eight compounds inhibited HPK1 kinase, with XZP-7 showing the strongest inhibitory effect, reaching an inhibition rate of 89.68%. These inhibition rates indicate that XZP-1, XZP-6, and XZP-7 have high affinity for HPK1 kinase.

[0123] Example 9: Protein degradation effect of PROTACs

[0124] (1) Materials and methods

[0125] Cell culture: The cells used in this experiment were cultured in RPMI 1640 medium supplemented with 100 U / mL penicillin and 100 μg / mL streptomycin. When the cell density in the culture flask reached 80%-90%, the cells were harvested into a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the remaining cell pellet was added to 2 mL of culture medium and diluted 1:2 for subculture.

[0126] Compound Treatment and Dosing: When cells reach the logarithmic growth phase, count the cells. When the cell count reaches approximately 500,000 per mL of culture medium, seed the cells into 6-well plates. Prepare the compound into a 10 mM stock solution in 100% DMSO and store at -80°C. Treat the cells with the test compound at a concentration gradient of 0, 100, 1,000, and 10,000 nmol / L. After 24 hours, assay protein expression by Western blotting.

[0127] Western blotting assay: ① Protein extraction and sample preparation: After 24 hours of drug administration, cells were centrifuged at 5000 rpm for 10 minutes, the culture medium was discarded, and cells were washed twice with PBS. 100 μL of RIPA lysis buffer was added and transferred to an EP tube. Lysis was performed on ice with shaking for 30 minutes. Cells were centrifuged at 12000 rpm for 30 minutes in a pre-cooled 4°C centrifuge. The supernatant was transferred to a fresh EP tube. ② Protein concentration determination: According to the manufacturer's instructions, 20 mg / mL BSA was diluted proportionally to produce eight different concentrations of standard protein samples. 10 μL of each protein standard was added to a 96-well plate, with three replicates for each concentration. Subsequently, 200 μL of working solutions (Solutions A and B) were added to each well. The absorbance was measured at 575 nm, and the average value was used to construct a standard curve. Prepare samples with a protein loading volume of 30-50 μL per well. 10 μL of 5× Loading Buffer was added and mixed thoroughly. The plates were then boiled in a 100°C metal bath for 5 minutes. ③ Gel electrophoresis: Mount the precast gel in an electrophoresis tank and add freshly prepared electrophoresis buffer. Add 53 μL of marker and 10 μL of protein sample sequentially. After sample loading, adjust the running voltage to 160 V and terminate the electrophoresis after 45 minutes. Cut the PVDF membrane and activate it with methanol for at least 10 seconds before use. Place the sponge, filter paper, gel, and PVDF membrane in a "sandwich" configuration on a transfer apparatus and fill with 1× Transfer Buffer. Transfer the membrane at 200 mA on ice for 100 minutes. Blocking: Immerse the transferred PVDF membrane in prepared 5% skim milk and block on a shaker for at least 1 hour. Aspirate the blocking buffer and rinse three times with TBST, shaking for 10 minutes each time. Incubate at 4°C on a shaker with slow shaking overnight. Recover the primary antibody and rinse three times with TBST, shaking for 10 minutes each time. Add the secondary antibody and shake slowly for 1 hour at room temperature. Rinse again three times with TBST, shaking for 10 minutes each time. ④ Development: Place the strip face up on a white plate, add developer with a pipette to cover it, keep it away from light for 1 minute, cover it with plastic wrap, place it in a dark box, and take a picture.

[0128] (2) The results of the initial screening test are attached. Figure 1 The degradation rate results are summarized in Table 2:

[0129] Table 2. Degradation rate of HPK1 protein by XZP-1 to XZP-19 at different concentrations

[0130]

[0131] / indicates that activity was not performed or detected

[0132] In the initial screening of degradation activity, it can be seen that XZP-16 exhibits good degradation ability for HPK1 protein on Jurkat cells. The Jurkat T cell proliferation inhibition experiment was conducted on XZP-16 with good activity in the initial screening of degradation effect. The results are shown in the attached Figure 2 .

[0133] It can be seen that starting from 0.02 μM, even at a concentration as high as 5 μM, XZP-16 still has no inhibitory effect on Jurkat T cells, showing good safety.

[0134] Example 10: Pharmacokinetic evaluation of some PROTACs

[0135] (1) Materials and methods

[0136] In this experiment, triple quadrupole liquid chromatography-mass spectrometry was used to study the pharmacokinetic processes of XZP-1, XZP-2, XZP-3, XZP-10, XZP-14, and XZP-16, and to explore the absorption characteristics of the series of compounds in vivo, laying the foundation for future dosage form selection and drug development and application.

[0137] 1) Sample plasma preparation

[0138] Twenty-four ICR mice weighing 20 g (purchased from the Zhejiang Provincial Animal Experimental Center) were used as experimental animals. All mice were housed in a 12-hour light-dark cycle at a temperature of (22 ± 2)°C and a relative humidity of 50% to 60%. Food and water were available ad libitum. They were fasted for 12 hours before the experiment.

[0139] Mice were randomly divided into groups of 3. Blood (0.2-0.3 mL) was collected from the retro-orbital venous plexus at 15, 30, 60, 120, 240, 360, 480, and 720 minutes after oral administration. Anticoagulant was added and the cells were centrifuged at 13,000 rpm for 15 minutes. The upper plasma layer was collected and stored at -80°C until further use. For tail vein administration, 21 ICR mice weighing 20 g were randomly divided into groups of 3. Blood was collected at 5, 15, 30, 60, 120, 240, and 360 minutes after administration. All other experimental procedures were the same as for oral administration.

[0140] 2) Sample method establishment

[0141] Prepare a 500ng / mL standard solution to determine the optimal detection method. First, select the full scan mass spectrometry scan mode and set the scan range according to the molecular weight of the compound. After determining the retention time, use the icon-production method to find the daughter ion. By setting different energies, determine an optimal energy value. Generally, the intensity of the parent ion is 1 / 4 of the daughter ion intensity. After determining the optimal energy and daughter ion, use the MRM method, input the daughter ion, parent ion, and energy into the selected method. After the scan is completed, check the results to confirm that there is no abnormality in the MRM spectrum, and then establish the analysis method for the sample based on the conditions explored above.

[0142] 3) Preparation of standard curve and treatment of plasma samples

[0143] Prepare nine concentrations of standard solutions (47.5 μL blank plasma + 2.5 μL working solution + 200 μL 5 ng / mL loratadine internal standard solution) 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 250 ng / mL, 500 ng / mL, 1000 ng / mL as well as a double blank solution (50 μL blank plasma + 200 μL acetonitrile) and a single blank solution (50 μL blank plasma + 200 μL loratadine internal standard solution), centrifuge them, and take the upper solution.

[0144] 50 μL of plasma sample at each time point was taken and added with 200 μL of loratadine internal standard solution to prepare analytical samples.

[0145] 4) Instrument analysis and data processing

[0146] Analyze the standard curve and analytical samples using the previously established sample analysis method. After the sample run is complete, use the Triple Quadrupole Quantitative Analysis software to add the sample, specify the target ion and internal standard compound for quantification, set the concentration level, enter the corresponding internal standard concentration and calibration concentration, check the qualifier ion settings, and save the analysis results.

[0147] (2) Experimental results

[0148] The drug concentration-time curves of XZP-1, XZP-2, XZP-3 and XZP-16 are attached. Figure 3 , XZP-14 injection / oral gavage drug concentration-time curve is attached Figure 4 The results of the pharmacokinetic experiments are shown in Table 3.

[0149] Table 3. Pharmacokinetic results of oral administration (PO) 10 mg / kg

[0150]

[0151] It can be seen that the pharmacokinetic properties of XZP-1, XZP-2, and XZP-3 flexible chain PROTACs are poor, and the area under the drug-time curve of 10 mg / kg is small after oral administration. In contrast, the pharmacokinetic properties of the rigid chain PROTAC XZP-14 are better, and the area under the drug-time curve of 10 mg / kg can reach 1500 ng / ml*h after oral administration. However, the pharmacokinetic parameters of XZP-16, which is also a rigid chain PROTAC, can show that not all rigid chain PROTACs can obtain good pharmacokinetic properties. A comparison of XZP-14 and XZP-16 shows that the six-membered ring structure of XZP-14 has more advantages in the pharmacokinetics of the compound, and its bioavailability is:

[0152]

[0153] As can be seen, the HPK1-targeted degradation compounds provided by the present invention exhibit potent tumor cell proliferation inhibition, excellent target protein degradation, and favorable pharmacokinetic properties. Therefore, they can be used in the preparation of pharmaceutical preparations for treating, preventing, and alleviating diseases caused by overexpression of HPK1. The diseases are lymphoma, blastoma, medulloblastoma, retinoblastoma, sarcoma, liposarcoma, synovial cell sarcoma, neuroendocrine tumor, carcinoid tumor, gastrinoma, islet cell carcinoma, mesothelioma, schwannoma, acoustic neuroma, meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignancy, squamous cell carcinoma, epithelial squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, adenocarcinoma lung cancer, squamous cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, intestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, metastatic breast cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, Merkel cell carcinoma, esophageal cancer, biliary tract tumors, head and neck cancer and hematological malignancies. The hematological malignancy is acute T-lymphocytic leukemia, chronic T-lymphocytic leukemia, acute B-lymphocytic leukemia, chronic B-lymphocytic leukemia, plasma cell tumor, multiple myeloma, macroglobulinemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, essential thrombocythemia or polycythemia vera.

Claims

1. A PROTAC compound for degrading HPK1 protein, characterized in that As shown in the following formula I structure: Wherein, L is a connecting chain, and its structure is one of the following:

2. The PROTAC compound for degrading HPK1 protein according to claim 1, characterized in that A compound of the following structure:

3. Stereoisomers, tautomers, deuterated forms, solvates, prodrugs, metabolites, and pharmaceutically acceptable salts thereof of the PROTAC compound for degrading HPK1 protein according to claim 1 or 2.

4. Use of the PROTAC compound for degrading HPK1 protein according to claim 1 or 2, or its stereoisomers, tautomers, deuterated forms, solvates, prodrugs, metabolites, and pharmaceutically acceptable salts, in the preparation of a medicament for treating, preventing, or alleviating diseases caused by excessive expression of HPK1 protein.

5. The use according to claim 4, characterized in that The diseases caused by excessive expression of HPK1 protein are lymphoma, blastoma, medulloblastoma, retinoblastoma, sarcoma, liposarcoma, synovial cell sarcoma, neuroendocrine tumor, carcinoid tumor, gastrinoma, islet cell carcinoma, mesothelioma, schwannoma, acoustic neuroma, meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignancy, squamous cell carcinoma, epithelial squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, adenocarcinoma lung cancer, squamous cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, intestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, Liver cancer, breast cancer, metastatic breast cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, anal cancer, penile cancer, Merkel cell carcinoma, esophageal cancer, biliary tract tumors, head and neck cancer, or hematological malignancies.

6. The use according to claim 5, characterized in that The hematological malignancy is acute T-lymphocytic leukemia, chronic T-lymphocytic leukemia, acute B-lymphocytic leukemia, chronic B-lymphocytic leukemia, plasma cell tumor, multiple myeloma, macroglobulinemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, essential thrombocythemia or polycythemia vera.

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