Thiazole compounds, deSUMO and / or deubiquitinase inhibitors, their preparation methods and applications

Abstract

This invention discloses a thiazole compound, a deSUMO and / or deubiquitinase inhibitor, its preparation method, and its application, belonging to the technical fields of molecular biology and biomedicine. The thiazole compound has the structure shown in Formula E, wherein R is selected from one of C1-5 alkyl, heterocyclic alkyl, benzoheterocyclic, substituted piperidine, substituted piperazine, phenyl, benzyl, or H, and A is selected from one of a benzene ring, piperidine, or hydrogen. Furthermore, this invention also provides a method for synthesizing the above-mentioned thiazole compound. Additionally, this invention provides a deSUMO and / or deubiquitinase inhibitor, which is one or more of the above-mentioned thiazole compounds. Furthermore, this invention provides the application of the above-mentioned deSUMO and / or deubiquitinase inhibitor in the preparation of a drug that inhibits the activity of deSUMO and / or deubiquitinase. This thiazole compound can effectively inhibit the activity of DESI2 enzyme.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and biomedicine, specifically to a thiazole compound, a deSUMO and / or deubiquitinase inhibitor, its preparation method, and its application. Background Technology

[0002] Abnormalities in the ubiquitin-proteasome pathway are a significant factor leading to the dysregulation of protein homeostasis. In this process, deubiquitinating enzymes, responsible for removing ubiquitin chains from protein substrates, are crucial. Abnormal activity or expression of these enzymes can cause functional changes in key oncogenic / tumor suppressor proteins, directly or indirectly leading to tumor development and malignant progression. Therefore, the discovery and research of small molecule inhibitors targeting deubiquitinating enzymes has become a hot topic in anti-tumor drug candidate research. SUMOylation is a post-translational modification process involving the attachment of a small protein called SUMC (Small Ubiquitin-like Modifier) ​​to a lysine residue of a target protein. This process regulates various important functions in cells, such as protein localization, stability, activity, and participation in signal transduction pathways. SUMOylation is similar to ubiquitination, and it can be reversible; SUMOylation or ubiquitination of proteins can be removed by specific deSUMOylating or deubiquitinating enzymes, restoring the protein to its unmodified state. DESI2 (desumoylating isopeptidase 2) is a novel gene obtained through large-scale sequencing, possessing both deSUMOylating and deubiquitinating enzyme activities. Specifically, DESI2 has been shown to achieve Lys48 and Lys63-specific deubiquitinating enzyme activity. Reports indicate that the DESI2 gene interacts with the AKT / mTOR signaling pathway, further influencing tumor cell proliferation; however, its mechanism of action remains unclear.

[0003] Janus kinase 2 (JAK2) plays a crucial role in hematopoietic regulation and is essential for the signaling of hematopoietic receptors. The JAK2-V617F mutation, leading to constitutive kinase activation, is the most common genetic event in myeloporotic neoplasms (MPNs). It is present in over 95% of polycythemia vera (PV) and approximately 50% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) cases. A percentage of MPN patients progress to secondary acute myeloid leukemia (sAML), characterized by significant genetic heterogeneity. This heterogeneity makes sAML insensitive to conventional chemotherapy, lacking effective treatment options and associated with poor prognosis and high relapse rates. Currently, despite advances in understanding pathogenesis and the availability of new therapies (e.g., hematopoietic stem cell transplantation), the 5-year overall survival rate for patients with secondary AML remains below 30%. JAK1 / 2 kinase inhibitors (e.g., ruxolitinib) have shown clinical benefit as monotherapy or in combination with chemotherapy. However, their clinical application is limited by their cytotoxicity to normal cells due to inhibition of wild-type (wt) JAK2. Furthermore, long-term treatment with JAK2 kinase inhibitors can lead to inhibitor-resistant cells, as the JAK2 pathway is reactivated via a JAK2-dependent heterodimer complex, which resists inhibitors and leads to the accumulation of phosphorylated JAK2, further activating downstream proliferation signaling. Therefore, developing a novel strategy to target and degrade mutant JAK2 using intracellular degradation mechanisms could provide greater clinical benefit to patients.

[0004] This patented technology research revealed that the de-SUMOylating and deubiquitinating enzyme DESI2 is a key factor regulating the stability of the JAK2-V617F protein. DESI2 is highly expressed in hematologic malignancies such as diffuse large B-cell lymphoma (DLBCL), MPN, and AML, and also highly expressed in various malignant solid tumors such as glioblastoma multiforme (GBM), low-grade glioma (LGG), pancreatic cancer (PAAD), and renal cancer. However, it is not expressed or is expressed at low levels in major organs, suggesting the therapeutic potential of DESI2 inhibitors and the possibility that they may not cause serious complications. Currently, there are no marketed drugs targeting DESI2. Summary of the Invention

[0005] The research in this invention revealed that the deSUMOylating and deubiquitinating enzyme DESI2 is a key factor regulating the stability of the JAK2-V617F protein. DESI2 is highly expressed in hematologic malignancies such as DLBCL, MPN, and AML, and also highly expressed in various malignant solid tumors such as GBM, LGG, and PAAD, while it is not expressed or is expressed at low levels in major organs. This suggests the therapeutic potential of DESI2 inhibitors and that they may not cause serious complications. Currently, there are no marketed drugs targeting DESI2.

[0006] The purpose of this invention is to overcome the above-mentioned technical deficiencies and provide a thiazole compound, a deSUMO and / or deubiquitinating enzyme inhibitor, its preparation method and application, thereby solving the technical problem of how to effectively inhibit DESI2 enzyme in the prior art.

[0007] To achieve the above-mentioned technical objectives, the present invention provides a thiazole compound having the structure shown in Formula E. Or its tautomers, stereoisomers, enantiomers, diastereomers, pharmaceutically acceptable salts, prodrugs, or N-oxides;

[0008] Wherein, R is selected from one of C1-5 alkyl, heterocyclic alkyl, benzoheterocyclic, substituted piperidine, substituted piperazine, phenyl, benzyl or H, and A is selected from one of benzene ring, piperidine or hydrogen.

[0009] In any embodiment, R is selected from 1,2-methylenedioxyphenyl, 1-methylindolyl, 1-methyl-1,2,3,6-tetrahydropyridin-4-yl, 1-acetyl-1,2,3,6-tetrahydropyridin-4-yl, 1-ethanesulfonyl-1,2,3,6-tetrahydropyridin-4-yl, 1-(1-methylpiperidin-4-yl)-1H-pyrazole-4-yl, 1-(1-acetylpiperidin-4-yl)-1H-pyrazole-4-yl, 1-(1-ethanesulfonylpiperidin-4-yl)-1H-pyrazole-4-yl, 3,5-dimethyl-1H-pyrazole-4-yl, 2-morpholinoacetyl, benzyl, 4-(2-morpholinoacetyl)piperazin-1-yl, or 4-benzylpiperazin-1-yl.

[0010] In any embodiment, the compound is selected from one of the following structural formulas:

[0011]

[0012]

[0013] Furthermore, the present invention also proposes a method for synthesizing the above-mentioned thiazole compounds by means of one of methods A, B, C, and D;

[0014] Method A includes the following steps:

[0015] Step S1, A is obtained through a condensation reaction. b

[0016] Step S2, The boron ester or boric acid with an R group is obtained through a coupling reaction.

[0017] Step S3, A is obtained by removing the tert-butyloxycarbonyl group under acidic conditions. d

[0018] Step S4, A is obtained by reacting with cyanogen bromide under alkaline conditions. e

[0019]

[0020] Step S5, A is obtained through a condensation reaction under alkaline conditions.

[0021]

[0022] Method B includes the following steps:

[0023] Step T1, will B is obtained through a condensation reaction. b

[0024]

[0025] Step T2, will B is obtained by removing the tert-butyloxycarbonyl group under acidic conditions. c Boc is an abbreviation for tert-butyloxycarbonyl.

[0026] Step T3, B is obtained by coupling reaction with boron esters or boric acids containing R groups. d

[0027]

[0028] Step T4, B is obtained by reacting with cyanogen bromide under alkaline conditions. e

[0029]

[0030] Step T5, The product is obtained by removing the tert-butyloxycarbonyl group under acidic conditions and then proceeding to the next step via substitution or condensation under basic conditions.

[0031] Method C includes the following steps:

[0032] Step U1: Piperazine is obtained under alkaline conditions through substitution or condensation reactions.

[0033] Step U2, will and It is obtained through a substitution reaction under alkaline conditions.

[0034] Step U3, will and Obtained through a condensation reaction

[0035] Step U4, Under acidic conditions, the tert-butyloxycarbonyl group is removed and reacted with cyanogen bromide to give C. d

[0036]

[0037] The method D includes the following steps:

[0038] Step V1, will The tert-butyloxycarbonyl group was removed under acidic conditions to obtain...

[0039] Step V2, will It is obtained through substitution or condensation reactions under alkaline conditions.

[0040] Step V3, will and Obtained through a condensation reaction

[0041] Step V4, will The tert-butyloxycarbonyl group was removed under acidic conditions to obtain...

[0042] Step V5, will It reacts with cyanogen bromide under alkaline conditions to obtain

[0043] In addition, the present invention also proposes a deSUMO and / or deubiquitinase inhibitor, which is one or more of the above-mentioned thiazole compounds, or one or more of the thiazole compounds obtained by the above-mentioned synthetic methods.

[0044] In addition, the present invention also proposes the use of the above-mentioned deSUMO and / or deubiquitinase inhibitor in the preparation of a drug for inhibiting deSUMO and / or deubiquitinase activity.

[0045] In any embodiment, the deubiquitination and / or deSUMOylation enzyme is one or more of SENP1, SENP2, SENP3, SENP5, SENP6, SENP7, DESI1, and DESI2.

[0046] In any embodiment, the drug is a drug that reduces the stability of JAK2; and / or, the drug is a drug that targets whether the gene for JAK2 is mutated or the type of mutation in the organism; and / or, the drug is a drug that regulates the level of ubiquitination or SUMOylation in the organism; and / or, the drug is a drug that treats and / or prevents diseases caused by deubiquitination or deSUMOylation.

[0047] In addition, the present invention also proposes the use of one or more of the above-mentioned thiazole compounds, or one or more of the thiazole compounds obtained by the above-mentioned synthetic methods, in the preparation of medicaments for treating and / or preventing diseases and related conditions mediated by JAK2 enzyme.

[0048] In addition, the present invention also proposes the use of one or more of the above-mentioned thiazole compounds, or one or more of the thiazole compounds obtained by the above-mentioned synthetic methods, in the preparation of drugs for inhibiting tumor cells.

[0049] Compared with the prior art, the beneficial effects of the present invention include: we designed and synthesized a series of new thiazole compounds that can effectively inhibit the activity of DESI2 enzyme and achieve selective degradation of mutant JAK2 enzyme. Attached Figure Description

[0050] Figure 1 This is the LC-MS analysis result of Example 1 of the present invention; it confirms that DESI2 is a novel epigenetic regulator of JAK2-V617F; wherein, Figure 1 A: IP-JAK2, qualitative analysis of the protein was performed using LC-MS, and DESI2 was enriched most strongly, ranking Top 1; Figure 1 B: Co-IP and WB experiments were used to detect the interaction between DESI2 and JAK2. Figure 1 C: In human JAK2-V617F + The interaction between DESI2 and JAK2 was detected in AML cell lines using endogenous Co-IP and Western blot experiments. Figure 1 D: In human JAK2-V617F +Endogenous IP and WB experiments were performed in (HEL) and JAK2-WT(K562) AML cell lines to detect the binding of DESI2 to JAK2, showing that the binding was mutation-selective.

[0051] Figure 2 and Figure 3 Example 2 of this invention, based on comprehensive clinical sample library analysis, shows that DESI2 is a poor prognostic marker for various malignant tumors, especially AML. Figure 2 Comprehensive analysis of clinical sample databases shows that DESI2 is highly expressed in a variety of malignant tumors and is a poor prognostic marker for AML. Figure 2 A: Comparison of survival probabilities of AML patients based on DESI2 expression levels in Kaplan-Meier; Figure 2 B: Using the cBioPortal database, compare the survival probability of AML patients based on DESI2 expression levels; Figure 2 C: Using the TCGA database, compare the survival probability of AML patients based on DESI2 expression levels; Figure 2 D: Analysis using the GEPIA database showed that DESI2 is highly expressed in various malignant solid tumors. DLBC: Diffuse large B-cell lymphoma; GBM: Glioblastoma multiforme; ESCA: Esophageal cancer; THYM: Thymoma; LGG: Low-grade glioma; PAAD: Pancreatic cancer. Figure 3 Comprehensive clinical sample bank analysis shows that DESI2 is a poor prognostic marker for various malignant tumors. Figure 3 A-3D: Analysis using the GEPIA and Human Protein Atlas databases, and comparison of survival probabilities of patients with various solid tumors based on DESI2 expression levels in the Kaplan-Meier dataset, showed that high DESI2 expression is closely related to prognosis, survival, and metastasis in various malignant solid tumors. PAAD: pancreatic cancer; LGG: low-grade glioma; Colon Cancer: colon cancer; Renal Cancer: kidney cancer. Figure 3 E: The experiment tested the anti-proliferative effect of the representative inhibitor 03-012 on various DESI2-overexpressing and prognostic solid tumors, including DLBCL, COAD, and PAAD.

[0052] Figure 4 In Example 3 of this invention, DESI2 knockout and knockdown affect leukemia cell proliferation by degrading JAK2 protein. Figure 4 A: The effects of DESI2 knockdown or knockout on JAK2 protein levels and downstream signaling pathways; Figure 4 B-4D: Effect of DESI2 knockdown or elimination on the proliferation of JAK2-V617F positive cells; Figure 4E: Detection and quantification of background fluorescence values ​​of HEL-Luc, HEL-SCR-Luc and two HEL-shDESI2-Luc cell lines; Figure 4 F-4I: DESI2 knockdown significantly improved HEL cell proliferation and mouse survival in vivo, but had no significant effect on mouse body weight (n=6).

[0053] Figure 5 In Example 4 of this invention, DESI2 stabilizes the JAK2-V617F protein through de-SUMOylation and de-ubiquitination. Figure 5 A: The effect of DESI2 knockdown on JAK2 transcription levels; Figure 5 B: The effect of knocking down DESI2 on the half-life of JAK2 protein; Figure 5 C: The proteasome inhibitor MG132 can rescue the effect of DESI2 knockout on JAK2 protein levels; Figure 5 D: The effect of knocking down DESI2 on JAK2 ubiquitination and SUMOylation levels; Figure 5 E: Effects of SUMOylase inhibitors on JAK2 ubiquitination and SUMOylation levels in DESI2 knocked-down HEL cells; Figure 5 F-5G: Detects the level of JAK2 ubiquitination in cells after DESI2 knockdown following a mutation at the K962 site of JAK2. DM: double mutant.

[0054] Figure 6 This is an example of the enzyme activity detection and in vitro functional verification of DESI2 by WWQ-03-12, a representative DESI2 inhibitor of this invention, as described in Example 25 of the present invention. Figure 6 A: Detection of DESI2 enzyme activity inhibition by WWQ-03-012; Figure 6 B: The antiproliferative activity of WWQ-03-012 against JAK2-V617F positive cells compared to other derivatives; Figure 6 The effects of C-6D:WWQ-03-012 on the degradation activity of JAK2 and downstream pathways; Figure 6 E: Effects of WWQ-03-012 on endogenous JAK2-V617F(HEL) and JAK2-WT(K562) protein levels in leukemia cells; Figure 6 F: The effect of WWQ-03-012 on JAK2 ubiquitination levels; Figure 6 G:WWQ-03-012's degradation activity against JAK2 depends on DESI2.

[0055] Figure 7 This describes the efficacy of WWQ-03-12 from Example 26 of this invention in a preclinical model of leukemia patient samples. Figure 7A-7B: Effects of WWQ-03-012 on the proliferation of two JAK2-V617F-driven cell lines (HEL and Ba / F3-JAK2-V617F) and PBMCs; Figure 7 C: The therapeutic effect of WWQ-03-012 on three MPN (MPN#1-#3) patient samples after 72h treatment; Figure 7 D: WWQ-03-012 Comparison of the effect of XL-106C on the proliferation of two MPN patient samples (MPN#4 and #5) that are JAK2-V617F positive.

[0056] Figure 8 This describes the therapeutic effect of WWQ-03-12 in an animal model of leukemia, as described in Example 27 of this invention. Figure 8 A: Animal experiment flowchart; Figure 8 B-8C: The DESI2 inhibitor 03-012 showed comparable efficacy to the marketed drug Ruxolitinib in its effect on tumor cell proliferation in vivo (n=4 or 5). Figure 8 D. Mouse weight records after transplantation of HEL-lluc cells showed no significant effect on body weight.

[0057] Figure 9 The graph shows the degradation detection results of the compounds proposed in this invention on the JAK2-V617F protein, with 03-012 (i.e., WWQ-03-12) being a representative compound. The vertical axis represents the relative protein expression level of JAK2-V617F. Detailed Implementation

[0058] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60–120 and 80–110 are listed for a specific parameter, it is understood that ranges of 60–110 and 80–120 are also expected. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1–3, 1–4, 1–5, 2–3, 2–4, and 2–5. In this application, unless otherwise stated, the numerical range "a–b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0059] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0060] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0061] This specific embodiment provides a thiazole compound having the structure shown in Formula E. Or its tautomers, stereoisomers, enantiomers, diastereomers, pharmaceutically acceptable salts, prodrugs, or N-oxides;

[0062] Wherein, R is selected from C1-5 alkyl, heterocyclic alkyl, benzoheterocyclic, substituted piperidine, substituted piperazine, phenyl, benzyl or H, and A is selected from benzene ring, piperidine or hydrogen.

[0063] In some embodiments, R is selected from 1,2-methylenedioxyphenyl, 1-methylindolyl, 1-methyl-1,2,3,6-tetrahydropyridin-4-yl, 1-acetyl-1,2,3,6-tetrahydropyridin-4-yl, 1-ethanesulfonyl-1,2,3,6-tetrahydropyridin-4-yl, 1-(1-methylpiperidin-4-yl)-1H-pyrazole-4-yl, 1-(1-acetylpiperidin-4-yl)-1H-pyrazole-4-yl, 1-(1-ethanesulfonylpiperidin-4-yl)-1H-pyrazole-4-yl, 3,5-dimethyl-1H-pyrazole-4-yl, 2-morpholinoacetyl, benzyl, 4-(2-morpholinoacetyl)piperazin-1-yl, or 4-benzylpiperazin-1-yl.

[0064] In some embodiments, the compound is selected from one of the following structural formulas:

[0065]

[0066]

[0067]

[0068] This specific embodiment also proposes a method for synthesizing the above-mentioned thiazole compounds, which is synthesized by one of methods A, B, C, and D; these methods are described in the invention description and will not be repeated here.

[0069] This specific embodiment also proposes a DESI2 inhibitor, which is one or more of the above-mentioned thiazole compounds, or one or more of the thiazole compounds prepared by the above-mentioned synthesis method.

[0070] Furthermore, this specific embodiment also proposes the application of the above-mentioned DESI2 inhibitor in the preparation of a drug that inhibits DESI2 activity.

[0071] Furthermore, this specific embodiment also proposes the use of one or more of the above-mentioned thiazole compounds, or one or more of the thiazole compounds obtained by the above-mentioned synthesis method, in the preparation of medicaments for treating and / or preventing diseases and related conditions mediated by JAK2 enzyme.

[0072] Furthermore, this specific embodiment also proposes the use of one or more of the above-mentioned thiazole compounds, or one or more of the thiazole compounds obtained by the above-mentioned synthetic method, in the preparation of a drug for inhibiting deubiquitination and / or deSUMOylation enzymes.

[0073] In some embodiments, the deubiquitination and / or deSUMOylation enzyme is one or more of SENP1, SENP2, SENP3, SENP5, SENP6, SENP7, DESI1, and DESI2.

[0074] In some embodiments, the drug is a drug that reduces the stability of JAK2; and / or, the drug is a drug that targets whether the gene for JAK2 in an organism is mutated or the type of mutation; and / or, the drug is a drug that regulates the level of ubiquitination or SUMOylation in an organism; and / or, the drug is a drug that treats and / or prevents diseases caused by deubiquitination or deSUMOylation.

[0075] This specific embodiment also proposes the use of one or more of the above-mentioned thiazole compounds, or one or more of the thiazole compounds obtained by the above-mentioned synthesis method, in the preparation of drugs for inhibiting tumor cells.

[0076] This invention, through extensive and in-depth research, has discovered that deSUMOylases and deubiquitinates significantly affect the stability of JAK2 protein and its V617F mutant form, which are highly expressed in hematological malignancies. Biochemical, cell biological, clinical database analyses, primary patient sample testing, and various in vivo animal model experiments were conducted on DESI2 deSUMOylases and their inhibitors, and this invention was completed based on these findings. In the following examples, unless otherwise stated, reagents were obtained from commercial suppliers (e.g., Aldrich Chemical, Biotech, or TCI) and used without further purification.

[0077] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0078] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0079] Hematologic malignancies are among the most common types of cancer. Despite significant advancements in modern medicine, the survival rate and quality of life for patients with hematologic malignancies remain poor. The V617F mutation, leading to constitutive activation of the non-receptor tyrosine kinase JAK2, is the most common malignant event in multiple myeloma (MPN). AML secondary to MPN is genetically heterogeneous and insensitive to traditional chemotherapy drugs, resulting in a long-term lack of effective treatments and a poor prognosis. Existing JAK1 / 2 kinase inhibitors such as ruxolitinib offer clinical benefits, but the toxicity of inhibiting wild-type JAK2 to normal cells limits their clinical application, and small-molecule inhibition of JAK2 kinase activity easily leads to drug resistance. In recent years, increasing research has shown that epigenetic modifications such as ubiquitination and SUMOylation are important factors regulating protein stability. Therefore, discovering new epigenetic modifiers and utilizing the body's own protein degradation system to target and degrade mutant-activated JAK2 is a novel strategy to improve safety and overcome inhibitor resistance. Based on this, the following series of studies are proposed.

[0080] Due to limited existing research on the mechanism of DESI2, we first conducted an in-depth study on its mechanism of action, determining that DESI2 maintains the stability of the JAK2-V617F protein through de-SUMOylation and deubiquitination. Furthermore, by constructing DESI2 knockout cell lines and xenograft tumor models, we verified the effect of DESI2 on the in vivo proliferation of JAK2-V617F-driven leukemia cells. Based on this, we first designed and synthesized a series of novel DESI2 inhibitors and tested their degradation ability against JAK2-V617F. Based on this degradation effect, we continuously modified the compounds to obtain those with optimal activity.

[0081] Next, we used pure enzyme activity assays to test the effects of a series of thiazole derivative DESI2 inhibitors and the optimal compound on DESI2 enzyme activity and IC50. 50 We also compared the degradation effect of the optimal compound on JAK2-V617F in cell lines before and after DESI2 knockout. Including this assay helps eliminate false positive results due to off-target effects or toxicity. More importantly, we also tested the anti-proliferative effects of representative DESI2 inhibitors, 03-012, on various DESI2-overexpressing and prognostic solid tumors, demonstrating the wide range of applications for DESI2 inhibitors.

[0082] Furthermore, we examined the therapeutic effects of the optimal DESI2 inhibitor compound on primary cells from clinical patients and mouse xenograft tumor models. This allows for preliminary assessment of the compound's clinical potential before formal clinical trials. This newly introduced technique not only addresses the issue of differences between cell lines and real-world clinical settings but also, combined with the aforementioned improvements and adjustments, further enhances the likelihood of the compound's clinical application.

[0083] Example 1. DESI2 is a novel epigenetic modifier of JAK2-V617F that directly interacts with JAK2.

[0084] To identify epigenetic modifiers affecting the stability of JAK2-V617F protein, we first overexpressed JAK2-V617F in HEK293T cells. After 48 hours, cells were collected, and JAK2 antibody was added to the cell lysis buffer, followed by overnight incubation at 4°C. Protein A / G magnetic beads were then added to the cell lysis buffer to enrich JAK2 protein. 2× protein loading was added, and the mixture was incubated at 95°C for 8 minutes to dissociate the protein from the magnetic beads. The prepared samples were separated by SDS-PAGE, and silver staining was used to preliminarily determine whether the target band was successfully enriched. After excising the target band, the protein was detected by liquid chromatography-mass spectrometry (LC-MS). The results are as follows: Figure 1 As shown in Figure A, after enriching JAK2 protein using immunoprecipitation (IP), DESI2 was simultaneously enriched as the most abundant peptide (Top 1) with high strength and confidence. This result indicates a direct binding between DESI2 and JAK2, potentially representing a novel component of the JAK2 complex.

[0085] To further confirm this binding, we performed Western blot (WB) assays on the IP-treated samples. The results are as follows: Figure 1 As shown in Figure B: In cell lines overexpressing JAK2-V617F, endogenous DESI2 was significantly enriched after IP administration, and conversely, IP administration of DESI2 also significantly enriched JAK2-V617F protein. Further... Figure 1 In C, in human JAK2-V617F + Endogenous Co-IP experiments were performed in AML cell lines, demonstrating a significant interaction between endogenous DESI2 and JAK2. These results further indicate a binding interaction between DESI2 and JAK2. More importantly, we observed this interaction in human JAK2-V617F... + Endogenous IP and Western blot experiments were performed in (HEL) and JAK2-WT(K562) AML cell lines to detect the binding of DESI2 to JAK2. The results showed that the binding had significant mutation selectivity. Figure 1 D).

[0086] Example 2. DESI2 is highly expressed in hematologic malignancies and various malignant solid tumors, and affects patient prognosis and survival.

[0087] DESI2 is a de-SUMOylating and deubiquitinating enzyme involved in epigenetic modifications, but research on it is currently limited. We first analyzed public clinical bioinformatics databases, revealing high DESI2 expression in various cancer types, particularly hematologic malignancies such as AML, MPN, and DLBCL. A comprehensive analysis of multiple clinical sample databases showed that high DESI2 expression often leads to poorer prognostic survival. Figure 2 and 3 This suggests the potential of DESI2 as a therapeutic target for hematologic malignancies and various solid tumors. Furthermore, we designed experiments to examine the anti-proliferative effects of 03-012 on various DESI2-overexpressing and prognostic-related solid tumors (such as DLBCL, COAD, and PAAD), demonstrating the broad application scenarios of DESI2 inhibitors. Figure 3 E).

[0088] Example 3. Knocking out DESI2 disrupts the proliferation of leukemia cells in vitro and in vivo by degrading JAK2 protein.

[0089] DESI2 possesses both SUMOylase and deubiquitinase activities. To elucidate the effects of DESI2 binding to JAK2, we first knocked down (KD) or knocked out (KO) DESI2 in JAK2-V617F mutant-driven HEL and UKE-1 cell lines. Then, Western blotting was used to detect JAK2 protein levels and proteins related to downstream JAK2 signaling pathways. The results are shown in the figure: both KD and KO-DESI2 significantly downregulated JAK2 protein levels, further affecting the activation of its downstream STAT3 / 5 signaling pathway and significantly inhibiting cell proliferation. Figure 4 A-4D).

[0090] Considering the important role of JAK2 in hematological malignancies, we overexpressed Luciferase in HEL, HEL-SCR, and DESI2 KD HEL cells. The background fluorescence values ​​of the four cell lines showed no significant difference. Figure 4 E). Next, the four cell lines were respectively injected with 1×10 6 Cells were injected into NSG mice via tail vein (n=6), and the body weight, fluorescence value, and survival time of each mouse were recorded regularly. Results showed that the fluorescence value (corresponding to tumor burden) of the KD group was significantly lower than that of the control group, with a significantly prolonged survival time and more stable body weight. Figure 4(F-4I). The above experiments confirm that DESI2 plays an important role in tumor cell proliferation by affecting the stability of JAK2 protein, which further suggests the potential of DESI2 as a therapeutic target for hematological malignancies.

[0091] Example 4. DESI2 mediates the stability of the JAK2 protein by de-SUMOylating and deubiquitinizing the K962 site.

[0092] Next, to investigate the mechanism by which DESI2 stabilizes JAK2 protein, given that DESI2 knockdown significantly reduces JAK2 protein levels, we assessed the JAK2 mRNA level in HEL cells after DESI2 knockdown using q-PCR. The results showed that the JAK2 mRNA level was almost unaffected. Figure 5 A). We further investigated the changes in the half-life of JAK2 protein in the presence of cyclohexylimide (CHX, a protein synthesis inhibitor in eukaryotes). We found that in the KD-DESI2 cell line, CHX treatment significantly shortened the half-life of JAK2 protein. Figure 5 B) This indicates that the DESI2-induced degradation of JAK2 occurs through a post-translational pathway.

[0093] To further investigate the specific stages at which DESI2 affects JAK2 protein stability, the proteasome inhibitor MG132 was applied to DESI2-knockdown cells, and JAK2 protein levels were found to be restored. Figure 5 C). Furthermore, we observed that in DESI2 knockdown cells, both JAK2 polyubiquitination and SUMOylation levels were significantly increased (C). Figure 5 D). To rule out off-target effects, we treated cells with the SUMOylation inhibitor ML-792. The results showed that ML-792 significantly reduced the SUMOylation and ubiquitination levels of JAK2 in SCR cell lines, but had no effect on the SUMOylation and ubiquitination levels of JAK2 after DESI2 knockdown. Figure 5 E).

[0094] Furthermore, the public database (SUMOplot) was modified using LC-MS and SUMOylation. TM Analysis showed that DESI2 may function by de-SUMOylating and deubiquitinizing lysine residues at K962 and K970 sites of JAK2. Figure 5F). We first constructed JAK2 K962A, K970A, and Double Mutant (DM) overexpression plasmids, transfected HEK-293T SCR and DESI2-KD cells, and performed ubiquitin-pulldown assays. The results showed that when the K962 site of JAK2 was mutated, DESI2 knockdown could no longer increase the ubiquitination level of JAK2. Figure 5 These results indicate that DESI2 mediates the stability of the JAK2 protein by SUMOylating the K962 site of JAK2.

[0095] Preparation of M1:

[0096]

[0097] (S)-3-((5-bromobenzo[d]thiazo-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0098] Compound (S)-1-Boc-3-carboxypyrrolidine (1.13 g, 5.24 mmol) was dissolved in DMF (5 mL). HATU (2.5 g, 6.55 mmol) and TEA (2.91 mL, 21.83 mmol) were added to the solution. After stirring at room temperature for 15 minutes, 2-amino-5-bromobenzothiazole (1 g, 4.37 mmol) was added, and the reaction mixture was stirred at room temperature for 5 hours. After the reaction was confirmed by LC-MS, the mixture was diluted with water and extracted three times with ethyl acetate. The organic phases were combined, washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (ethyl acetate / PE = 1 / 2, V / V), yield 85.7%. LC-MS (ESI) m / z: 426 [M+H] + .

[0099] Preparation of M2:

[0100]

[0101] (S)-3-((5-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazo-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0102] Intermediate M1 (0.75 g, 2.3 mmol) was dissolved in 1,4-dioxane (10 mL). Benzo[d][1,3]dioxanepenten-5-ylboronic acid (382 mg, 2.3 mmol), sodium carbonate (500 mg, 4.6 mmol), and water (1 mL) were added to the solution, and the mixture was purged with nitrogen. Then, [1,1-bis(diphenylphosphine)ferrocene]palladium dichloride (260 mg, 0.115 mmol) was added to the mixture, and the mixture was purged with nitrogen again. The reaction was carried out at 85 °C for 3 h. After LC-MS analysis confirmed the reaction was complete, the reaction mixture was cooled to room temperature, concentrated, and the residue was dissolved in ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (ethyl acetate / PE = 1 / 2, V / V), yield 65.3%. LC-MS (ESI) m / z: 468 [M+H]+.

[0103] Preparation of M3:

[0104]

[0105] (S)-3-((5-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazo-2-yl)pyrrolidine-3-carboxamide

[0106] Intermediate M2 (400 mg, 1.1 mmol) was dissolved in dichloromethane (5 mL), and then ethyl acetate hydrochloride solution (4 M, 0.5 mL) was added to the solution. The mixture was stirred at room temperature for 1 hour. LC-MS analysis confirmed the completion of the reaction. The solution was then concentrated, and the product was used directly in the next reaction without further purification, with a yield of 100%. LC-MS (ESI) m / z: 368 [M+H]+.

[0107] Preparation of compound 1:

[0108]

[0109] (S)-N-(5-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazo-2-yl)-1-cyanopyrrolidine-3-carboxamide

[0110] Intermediate M3 (100 mg, 0.27 mmol) was dissolved in anhydrous dichloromethane (10 mL). TEA (0.19 mL, 1.35 mmol), potassium carbonate (0.2 g, 1.35 mmol), and cyanogen bromide (29 mg, 0.27 mmol) were added to the solution, and the mixture was stirred at room temperature for 10 minutes. The reaction mixture was then quenched by rapidly adding water (20 mL), and the mixture was separated. The aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1, V / V), yield 71.3%.

[0111] The proton and carbon spectral data of compound 1 are as follows:

[0112] 1 H NMR (400MHz, DMSO-d6) δ12.63(s,1H),8.02(d,J=8.3Hz,1H),7.94(d,J=1.3Hz,1H),7.56(dd ,J=8.3,1.6Hz,1H),7.35(d,J=1.6Hz,1H),7.23(dd,J=8.1,1.7Hz,1H),7.02(d,J=8.1Hz,1H) ,6.08(s,2H),3.69–3.62(m,1H),3.59(dd,J=9.6,6.1Hz,1H),3.48(d,J=7.7Hz,1H),3.46–3. 43(m,1H),3.40(d,J=6.8Hz,1H),2.23(dt,J=13.3,6.5Hz,1H),2.11(td,J=14.2,7.1Hz,1H).

[0113] 13 C NMR(101MHz,DMSO-d6)172.07,158.82,149.73,148.48,147.33,138.90,134.79,130.71,122.52,121.05 ,118.68,117.58,109.16,107.95,107.91,101.64,52.49,50.37,43.81,29.62.LC-MS(ESI)m / z:393[M+H] + .

[0114] Example 6:

[0115] Preparation of compound 2:

[0116]

[0117] (S)-1-cyano-N-(5-(1-methyl-1H-indol-5-yl)benzo[d]thiazo-2-yl)pyrrolidine-3-carboxamide

[0118] Step A: (S)-3-((5-(1-methyl-1H-indol-5-yl)benzo[d]thiazo-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester was prepared using the method for preparing M2, replacing benzo[d][1,3]dioxane-5-ylboronic acid with 1-methylindol-5-boronic acid pinacol ester.

[0119] Step B: (S)-1-cyano-N-(5-(1-methyl-1H-indol-5-yl)benzo[d]thiazolyl-2-yl)pyrrolidine-3-carboxamide (400 mg, 0.84 mmol) was dissolved in dichloromethane (5 mL), and then ethyl acetate hydrochloride solution (4 M, 0.5 mL) was added to the solution. The mixture was stirred at room temperature for 1 hour. LC-MS was used to confirm the completion of the reaction. The solution was then concentrated, and the product was used directly in the next reaction without further purification, with a yield of 100%. LC-MS (ESI) m / z: 377 [M+H]+.

[0120] Step C: (S)-N-(5-(1-methyl-1H-indol-5-yl)benzo[d]thiazolyl-2-yl)pyrrolidine-3-carboxamide (100 mg, 0.27 mmol) was dissolved in anhydrous dichloromethane (10 mL). DMF (0.5 mL), potassium carbonate (0.2 g, 1.35 mmol), and cyanogen bromide (29 mg, 0.27 mmol) were added to the solution, and the mixture was stirred at room temperature for 10 minutes. The reaction mixture was then quickly quenched by adding water (20 mL). The mixture was separated, and the aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1, V / V), yield 51.1%. LC-MS (ESI) m / z: 402 [M+H] + . :

[0121] Example 7:

[0122] Preparation of compound 3

[0123]

[0124] (S)-1-cyano-N-(5-(3,5-dimethyl-1H-pyrazol-4-yl)benzo[d]thiazo-2-yl)pyrrolidine-3-carboxamide

[0125] Step A: (S)-3-((5-(3,5-dimethyl-1H-pyrazole-4-yl)benzo[d]thiazo-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester was prepared using the method for preparing M2, replacing benzo[d][1,3]dioxane-5-ylboronic acid with 3,5-dimethylpyrazole-4-boronic acid pinacol ester.

[0126] Step B: Dissolve (S)-1-cyano-N-(5-(3,5-dimethyl-1H-pyrazol-4-yl)benzo[d]thiazolyl)pyrrolidine-3-carboxamide (400 mg, 0.91 mmol) in dichloromethane (5 mL), then add ethyl acetate hydrochloride solution (4 M, 0.5 mL) to the solution and stir at room temperature for 1 hour. After confirming the completion of the reaction by LC-MS, concentrate the solution. The product can be used directly in the next reaction without further purification, with a yield of 100%. LC-MS (ESI) m / z: 442 [M+H] + .

[0127] Step C: Dissolve (S)-N-(5-(3,5-dimethyl-1H-pyrazol-4-yl)benzo[d]thiazolyl)pyrrolidine-3-carboxamide (100 mg, 0.23 mmol) in anhydrous dichloromethane (10 mL). Add TEA (0.16 mL, 1.15 mmol), potassium carbonate (0.17 g, 1.15 mmol), and cyanogen bromide (25 mg, 0.23 mmol) to the solution and stir the mixture at room temperature for 10 minutes. Then, quickly pour water (20 mL) into the reaction solution to quench the reaction, separate the layers, and extract the aqueous phase with dichloromethane. Combine the organic phases, wash with water, dry to anhydrous sodium sulfate, filter, and concentrate. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1, V / V), yield 61.3%.

[0128] The proton NMR data for compound 3 are as follows:

[0129] 1 H NMR (400MHz, DMSO-d6) δ12.73(s,1H),12.66–12.09(m,1H),8.00(d,J=8.2Hz,1H),7.62(d,J=1.2Hz,1H),7.25(dd,J=8.2,1.5Hz,1H),3.68(dd,J=9.4 ,7.8Hz,1H),3.58(dd,J=9.5,6.0Hz,1H),3.54–3.46(m,3H),2.31–2.25(m ,1H),2.23(s,6H),2.11(td,J=14.3,7.1Hz,1H).LC-MS(ESI)m / z:367[M+H] + .

[0130] Example 8:

[0131] Preparation of N1:

[0132]

[0133] (S)-3-((6-bromobenzo[d]thiazo-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0134] Compound (S)-1-Boc-3-carboxypyrrolidine (1.13 g, 5.24 mmol) and 2-amino-6-bromobenzothiazole (1 g, 4.37 mmol) were dissolved in DMF (5 mL). HATU (2.5 g, 6.55 mmol) and TEA (2.91 mL, 21.83 mmol) were added to the solution. After stirring at room temperature for 15 minutes, 2-amino-6-bromobenzothiazole (1 g, 4.37 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 3 hours. After the reaction was confirmed by LC-MS, the mixture was diluted with water and extracted three times with ethyl acetate. The organic phases were combined, washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (ethyl acetate / PE = 1 / 2, V / V), yield 91.2%. LC-MS (ESI) m / z: 426 [M+H] + .

[0135] Preparation of N2:

[0136]

[0137] (S)-3-((6-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazo-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0138] Intermediate N1 (0.75 g, 2.3 mmol) was dissolved in 1,4-dioxane (10 mL). Benzo[d][1,3]dioxanepenten-5-ylboronic acid (382 mg, 2.3 mmol), sodium carbonate (500 mg, 4.6 mmol), and water (1 mL) were added to the solution, and the mixture was purged with nitrogen. Then, [1,1-bis(diphenylphosphine)ferrocene]palladium dichloride (260 mg, 0.115 mmol) was added to the mixture, and the mixture was purged with nitrogen again. The reaction was carried out at 85 °C for 3 h. After LC-MS analysis confirmed the reaction was complete, the reaction mixture was cooled to room temperature, concentrated, and the residue was dissolved in ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (ethyl acetate / PE = 1 / 2, V / V), yield 74.3%. LC-MS (ESI) m / z: 468 [M+H] + .

[0139] Preparation of N3:

[0140]

[0141] (S)-3-((6-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazo-2-yl)pyrrolidine-3-carboxamide

[0142] Intermediate N2 (400 mg, 1.1 mmol) was dissolved in dichloromethane (5 mL), and then ethyl acetate hydrochloride solution (4 M, 0.5 mL) was added to the solution. The mixture was stirred at room temperature for 1 hour. After LC-MS detection confirmed the reaction was complete, the solution was concentrated. The product was used directly in the next reaction without further purification, with a yield of 100%. LC-MS (ESI) m / z: 368 [M+H] + .

[0143] Preparation of compound 4:

[0144]

[0145] (S)-N-(5-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazo-2-yl)-1-cyanopyrrolidine-3-carboxamide

[0146] Intermediate N3 (100 mg, 0.27 mmol) was dissolved in anhydrous dichloromethane (10 mL). DMF (0.5 mL), potassium carbonate (0.2 g, 1.35 mmol), and cyanogen bromide (29 mg, 0.27 mmol) were added to the solution, and the mixture was stirred at room temperature for 45 minutes. The reaction mixture was then quenched by rapidly adding water (20 mL), and the mixture was separated. The aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1, V / V) in 90.1% yield. LC-MS (ESI) m / z: 393 [M+H] + .

[0147] Example 9:

[0148] Preparation of compound 5:

[0149]

[0150] Step A: (S)-3-((6-(1-methyl-1H-indol-5-yl)benzo[d]thiazo-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester was prepared using the method for preparing N2, replacing benzo[d][1,3]dioxane-5-ylboronic acid with 1-methylindol-5-boronic acid pinacol ester.

[0151] Step B: (S)-3-((6-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)pyrrolidine-3-carboxamide was prepared using the method for preparing N3, replacing (S)-3-((5-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylate with (S)-3-((6-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylate.

[0152] Step C: Dissolve (S)-3-((6-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazolyl-2-yl)pyrrolidine-3-carboxamide (100 mg, 0.27 mmol) in anhydrous dichloromethane (10 mL). Add DMF (0.5 mL), potassium carbonate (0.2 g, 1.35 mmol), and cyanogen bromide (29 mg, 0.27 mmol) to the solution and stir the mixture at room temperature for 45 minutes. Then, quickly pour water (20 mL) into the reaction solution to quench the reaction, separate the phases, and extract the aqueous phase with dichloromethane. Combine the organic phases, wash with water, dry with anhydrous sodium sulfate, filter, and concentrate. The crude product is purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1, V / V) in 90.1% yield.

[0153] The proton NMR data for compound 5 are as follows:

[0154] 1 H NMR (400MHz, DMSO-d6) δ12.60(s,1H),8.31(d,J=1.3Hz,1H),7.90(s,1H),7.79(dt,J=8.5 ,5.1Hz,2H),7.54(s,2H),7.38(d,J=3.0Hz,1H),6.50(d,J=3.1Hz,1H),3.83(s,3H),3.67( dd,J=9.6,7.8Hz,1H),3.61(dd,J=9.6,6.0Hz,1H),3.48(ddd,J=10.9,9.1,2.5Hz,2H),3.4 3–3.40(m,1H),2.25(td,J=13.2,7.4Hz,1H),2.17–2.06(m,1H).LC-MS(ESI)m / z:402[M+H] + .

[0155] Example 10:

[0156] Preparation of O1:

[0157]

[0158] (S)-N-(6-bromobenzo[d]thiazo-2-yl)pyrrolidine-3-carboxamide

[0159] (S)-3-((6-bromobenzo[d]thiazolyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester (1 g, 0.94 mmol) was dissolved in dichloromethane (5 mL), and then ethyl acetate hydrochloride solution (4 M, 0.5 mL) was added to the solution. The mixture was stirred at room temperature for 1 hour. After LC-MS detection confirmed the completion of the reaction, the solution was concentrated. The product was used directly in the next reaction without further purification, with a yield of 100%. LC-MS (ESI) m / z: 326 [M+H] + .

[0160] Preparation of O2:

[0161]

[0162] (S)-4-(2-(pyrrolidine-3-carboxamido)benzo[d]thiazo-6-yl)-3,6-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester

[0163] Intermediate O1 (920 mg, 2.3 mmol) was dissolved in 1,4-dioxane (10 mL). N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester (710 mg, 2.3 mmol), sodium carbonate (500 mg, 4.6 mmol), and water (1 mL) were added to the solution, and the mixture was purged with nitrogen. Then, [1,1-bis(diphenylphosphine)ferrocene]palladium dichloride (260 mg, 0.115 mmol) was added to the mixture, and the mixture was purged with nitrogen again. The reaction was carried out at 85 °C for 3 h. After LC-MS analysis confirmed the reaction was complete, the reaction mixture was cooled to room temperature, concentrated, and the residue was dissolved in ethyl acetate and washed with saturated brine. The residue was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1, V / V), yielding 67.3%. LC-MS(ESI) m / z: 429 [M+H] + .

[0164] Preparation of O3:

[0165]

[0166] (S)-4-(2-(1-cyanopyrrolidine-3-carboxamido)benzo[d]thiazo-6-yl)-3,6-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester

[0167] Intermediate O2 (122 mg, 0.27 mmol) was dissolved in anhydrous dichloromethane (10 mL). Potassium carbonate (0.2 g, 1.35 mmol) and cyanogen bromide (29 mg, 0.27 mmol) were added to the solution, and the mixture was stirred at room temperature for 15 minutes. The reaction mixture was then quenched by rapidly adding water (20 mL). The mixture was separated, and the aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1, V / V) in 83.2% yield. LC-MS (ESI) m / z: 454 [M+H] + .

[0168] Preparation of compound 6:

[0169]

[0170] (S)-1-cyano-N-(6-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)benzo[d]thiazolyl)pyrrolidine-3-carboxamide: Intermediate O3 (100 mg, 0.22 mmol) was dissolved in dichloromethane (5 mL), and then ethyl acetate hydrochloride solution (4 M, 0.5 mL) was added to the solution. The mixture was stirred at room temperature for 1 hour. LC-MS was used to confirm the completion of the reaction. The solution was concentrated, and the product was dissolved in anhydrous THF (5 mL). TEA (0.092 mL, 0.66 mmol), methyl iodoform (156 mg, 1.1 mmol), and potassium carbonate (153 mg, 1.1 mmol) were added. The mixture was stirred at room temperature for 2 hours, and then quenched with dimethylamine. The reaction solution was diluted with dichloromethane, filtered, washed, dried over anhydrous sodium sulfate, and concentrated. After purification by silica gel column chromatography (dichloromethane / methanol = 10 / 1, V / V), the yield was 53.1%. LC-MS (ESI) m / z: 368 [M+H] + .

[0171] Example 11:

[0172] Preparation of compound 7

[0173]

[0174] (S)-N-(6-(1-acetyl-1,2,3,6-tetrahydropyridin-4-yl)benzo[d]thiazolyl)-1-cyanopyrrolidine-3-carboxamide

[0175] Step A: (S)-N-(6-(1-acetyl-1,2,3,6-tetrahydropyridin-4-yl)benzo[d]thiazolyl-2-yl)pyrrolidine-3-carboxamide was prepared using the method for preparing O2, replacing N-Boc-1,2,5,6-tetrahydropyridin-4-boronic acid pinacol ester with 1-acetyl-5,6-dihydro-2H-pyridin-4-boronic acid pinacol ester.

[0176] Step B: (S)-N-(6-(1-acetyl-1,2,3,6-tetrahydropyridin-4-yl)benzo[d]thiazolyl)pyrrolidine-3-carboxamide (100 mg, 0.27 mmol) was dissolved in anhydrous dichloromethane (10 mL). DMF (1 mL), potassium carbonate (0.2 g, 1.35 mmol), and cyanogen bromide (29 mg, 0.27 mmol) were added to the solution, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was then quickly quenched by adding water (20 mL), and the mixture was separated. The aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1, V / V) in 79.2% yield. 1H NMR (400MHz, CDCl3) δ11.35–10.58(m,1H),7.83(dd,J=6.8,1.4Hz,1H),7.71(dd,J=8.5,2.2 Hz,1H),7.49(d,J=8.5Hz,1H),6.14(d,J=19.2Hz,1H),4.31(d,J=2.7Hz,1H),4.21(d,J=2.8 Hz,1H),3.89(t,J=5.7Hz,1H),3.83–3.64(m,4H),3.57–3.47(m,1H),3.37–3.24(m,1H),2.6 6(d,J=26.0Hz,2H),2.40–2.25(m,2H),2.23(d,J=12.8Hz,3H).LC-MS(ESI)m / z:396[M+H]+.

[0177] Example 12:

[0178] Preparation of compound 12

[0179]

[0180] The target compound obtained in this embodiment is referred to in this invention as Example 12, WWQ-03-012, or 03-012:

[0181] (S)-N-(7-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazo-2-yl)-1-cyanopyrrolidine-3-carboxamide

[0182] Step A: (S)-3-((7-bromobenzo[d]thiazolyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0183] The method for preparing M1 was adopted, in which 2-amino-7-bromobenzothiazole was used to replace benzo[d][1,3]dioxane-5-ylboronic acid.

[0184] Step B: ((S)-3-((7-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester was prepared using the method for preparing M2, replacing (S)-3-((5-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester with (S)-3-((7-bromobenzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester.

[0185] Step C: (S)-3-((7-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)pyrrolidine-3-carboxamide was prepared using the method for preparing M3, replacing (S)-3-((5-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylate with tert-butyl pyrrolidine.

[0186] Step D: (S)-N-(7-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)-1-cyanopyrrolidine-3-carboxamide is prepared by replacing (S)-3-((5-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester with (S)-3-((7-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester with (S)-3-((7-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester with (S)-3-((7-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester.

[0187] Example 13:

[0188] Preparation of compound 8

[0189]

[0190] (S)-N-(6-(1-ethanesulfonyl-1,2,3,6-tetrahydropyridin-4-yl)benzo[d]thiazolyl)-1-cyanopyrrolidine-3-carboxamide

[0191] Step A: (S)-N-(6-(1-ethanesulfonyl-1,2,3,6-tetrahydropyridin-4-yl)benzo[d]thiazolyl)pyrrolidine-3-carboxamide was prepared using the method for preparing O2, replacing N-Boc-1,2,5,6-tetrahydropyridin-4-boronic acid pinacol ester with 1-ethanesulfonyl-5,6-dihydro-2H-pyridin-4-boronic acid pinacol ester.

[0192] Step B: (S)-N-(6-(1-ethanesulfonyl-1,2,3,6-tetrahydropyridin-4-yl)benzo[d]thiazolyl)pyrrolidine-3-carboxamide (120 mg, 0.27 mmol) was dissolved in anhydrous dichloromethane (10 mL). DMF (1 mL), potassium carbonate (0.2 g, 1.35 mmol), and cyanogen bromide (29 mg, 0.27 mmol) were added to the solution, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was then quickly quenched by adding water (20 mL), and the mixture was separated. The aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1, V / V), yield 73.7%.

[0193] The proton NMR data for compound 8 are as follows:

[0194] 1 H NMR (400MHz, DMSO-d6) δ12.60(s,1H),8.09(d,J=1.6Hz,1H),7.72(d,J=8.5Hz,1 H),7.58(dd,J=8.5,1.8Hz,1H),6.27(s,1H),3.89(d,J=2.9Hz,2H),3.62(ddd,J= 15.6,9.6,6.9Hz,2H),3.52–3.45(m,2H),3.45–3.40(m,3H),2.96(s,3H),2.68(s ,2H),2.24(td,J=13.2,7.3Hz,1H),2.17–2.05(m,1H).LC-MS(ESI)m / z:446[M+H] + .

[0195] Example 14:

[0196] Preparation of P1:

[0197]

[0198] (S)-4-(4-(2-(pyrrolidine-3-carboxamido)benzo[d]thiazo-6-yl)-1H-pyrazole-1-yl)piperidine-1-carboxylic acid tert-butyl ester

[0199] Intermediate O1 (920 mg, 2.3 mmol) was dissolved in 1,4-dioxane (10 mL). 4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylic acid tert-butyl ester (867 mg, 2.3 mmol), sodium carbonate (500 mg, 4.6 mmol), and water (1 mL) were added to the solution, and the mixture was purged with nitrogen. Then, [1,1-bis(diphenylphosphine)ferrocene]palladium dichloride (260 mg, 0.115 mmol) was added to the mixture, and the mixture was again purged with nitrogen. The reaction was carried out at 85 °C for 3 h. After LC-MS analysis confirmed the reaction was complete, the reaction mixture was cooled to room temperature, concentrated, and the residue was dissolved in ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1, V / V), yield 57.5%. LC-MS (ESI) m / z: 497 [M+H]+.

[0200] Preparation of P2:

[0201]

[0202] (S)-4-(4-(2-(1-cyanopyrrolidine-3-carboxamido)benzo[d]thiazo-6-yl)-1H-pyrazole-1-yl)piperidine-1-carboxylic acid tert-butyl ester

[0203] Intermediate P1 (140 mg, 0.27 mmol) was dissolved in anhydrous dichloromethane (10 mL). DMF (0.5 mL), potassium carbonate (0.2 g, 1.35 mmol), and cyanogen bromide (29 mg, 0.27 mmol) were added to the solution, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was then quenched by rapidly adding water (20 mL), and the mixture was separated. The aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1, V / V), yield 63.3%. LC-MS (ESI) m / z: 522 [M+H] + .

[0204] Preparation of compound 9:

[0205]

[0206] (S)-1-cyano-N-(6-(1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl)benzo[d]thiazo-2-yl)pyrrolidine-3-carboxamide

[0207] Intermediate P2 (115 mg, 0.22 mmol) was dissolved in dichloromethane (5 mL), and then ethyl acetate hydrochloride solution (4 M, 0.5 mL) was added. The mixture was stirred at room temperature for 1 hour. After LC-MS analysis confirmed the reaction was complete, the solution was concentrated, and the product was dissolved in anhydrous THF (5 mL). TEA (0.092 mL, 0.66 mmol), methyl iodoform (156 mg, 1.1 mmol), and potassium carbonate (153 mg, 1.1 mmol) were added. The mixture was stirred at room temperature for 2 hours, and then quenched with dimethylamine. The reaction solution was diluted with dichloromethane, filtered, washed, dried over anhydrous sodium sulfate, and concentrated. After purification by silica gel column chromatography (dichloromethane / methanol = 10 / 1, V / V), the yield was 47.3%. LC-MS (ESI) m / z: 436 [M+H] + .

[0208] Example 15:

[0209] Preparation of compound 10

[0210]

[0211] (S)-N-(6-(1-(1-acetylpiperidin-4-yl)-1H-pyrazol-4-yl)benzo[d]thiazo-2-yl)-1-cyanopyrrolidine-3-carboxamide

[0212] Intermediate P2 (115 mg, 0.22 mmol) was dissolved in dichloromethane (5 mL), and then ethyl acetate hydrochloride solution (4 M, 0.5 mL) was added. The mixture was stirred at room temperature for 1 hour. After LC-MS analysis confirmed the reaction was complete, the solution was concentrated, and the product was dissolved in anhydrous dichloromethane (5 mL). TEA (0.092 mL, 0.66 mmol) and acetyl chloride (32 μL, 0.44 mmol) were added. The mixture was stirred at room temperature for 2 hours. The reaction solution was diluted with dichloromethane, filtered, washed, dried over anhydrous sodium sulfate, and concentrated. After purification by silica gel column chromatography (dichloromethane / methanol = 20 / 1, V / V), the yield was 89.1%. LC-MS (ESI) m / z: 464 [M+H] + .

[0213] Example 16:

[0214] Preparation of compound 11

[0215]

[0216] (S)-1-cyano-N-(6-(1-(1-(ethylsulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)benzo[d]thiazo-2-yl)pyrrolidine-3-carboxamide

[0217] Intermediate P2 (115 mg, 0.22 mmol) was dissolved in dichloromethane (5 mL), and then ethyl acetate hydrochloride solution (4 M, 0.5 mL) was added. The mixture was stirred at room temperature for 1 hour. After LC-MS analysis confirmed the reaction was complete, the solution was concentrated, and the product was dissolved in anhydrous dichloromethane (5 mL). TEA (0.092 mL, 0.66 mmol) and ethanesulfonyl chloride (40 μL, 0.44 mmol) were added. The mixture was stirred at room temperature for 2 hours. The reaction solution was diluted with dichloromethane, filtered, washed, dried over anhydrous sodium sulfate, and concentrated. After purification by silica gel column chromatography (dichloromethane / methanol = 20 / 1, V / V), the yield was 91.3%. LC-MS (ESI) m / z: 514 [M+H] + .

[0218] Example 17:

[0219] Preparation of compound 13

[0220]

[0221] (S)-N-(4-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazo-2-yl)-1-cyanopyrrolidine-3-carboxamide

[0222] Step A: (S)-3-((4-bromobenzo[d]thiazolyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0223] The method for preparing M1 was adopted, in which 2-amino-4-bromobenzothiazole was used to replace benzo[d][1,3]dioxane-5-ylboronic acid.

[0224] Step B: (S)-3-((4-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester was prepared using the method for preparing M2, replacing (S)-3-((5-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester with (S)-3-((4-bromobenzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester.

[0225] Step C: (S)-3-((4-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)pyrrolidine-3-carboxamide was prepared using the method for preparing M3, replacing (S)-3-((5-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylate with ((S)-3-((4-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylate.

[0226] Step D: (S)-N-(4-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)-1-cyanopyrrolidine-3-carboxamide is prepared by replacing (S)-3-((5-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester with (S)-3-((4-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester with (S)-3-((4-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester with (S)-3-((4-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester.

[0227] The proton NMR data for compound 13 are as follows:

[0228] 1 H NMR (400MHz, DMSO-d6) δ12.63(s,1H),7.95(dd,J=7.8,0.9Hz,1H),7.47(dd,J=7. 5,1.0Hz,1H),7.40(d,J=1.6Hz,1H),7.36(t,J=7.7Hz,1H),7.23(dd,J=8.1,1.7Hz ,1H),7.02(d,J=8.1Hz,1H),6.09(s,2H),3.62(ddd,J=15.6,9.6,7.0Hz,2H),3.52 –3.46(m,1H),3.46–3.40(m,2H),2.22(td,J=13.2,7.3Hz,1H),2.17–2.04(m,1H).

[0229] Example 18:

[0230] Preparation of compound 14

[0231]

[0232] (R)-1-cyano-N-(5-(3,5-dimethyl-1H-pyrazol-4-yl)benzo[d]thiazo-2-yl)pyrrolidine-3-carboxamide

[0233] Step A: (R)-3-((5-bromobenzo[d]thiazolyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester was prepared using the method for preparing M1, replacing (S)-1-Boc-3-carboxypyrrolidine with (R)-1-Boc-3-carboxypyrrolidine.

[0234] Step B: (R)-3-((5-(3,5-dimethyl-1H-pyrazole-4-yl)benzo[d]thiazo-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester was prepared using the method for preparing M2, replacing benzo[d][1,3]dioxane-5-ylboronic acid with 3,5-dimethylpyrazole-4-boronic acid pinacol ester.

[0235] Step C: (R)-1-cyano-N-(5-(3,5-dimethyl-1H-pyrazol-4-yl)benzo[d]thiazolyl)pyrrolidine-3-carboxamide (400 mg, 0.91 mmol) was dissolved in dichloromethane (5 mL), and then ethyl acetate hydrochloride solution (4 M, 0.5 mL) was added to the solution. The mixture was stirred at room temperature for 1 hour. LC-MS analysis confirmed the reaction was complete. The solution was then concentrated, and the product was used directly in the next reaction without further purification, with a yield of 100%. LC-MS (ESI) m / z: 442 [M+H] + .

[0236] Step D: (R)-N-(5-(3,5-dimethyl-1H-pyrazol-4-yl)benzo[d]thiazolyl)pyrrolidine-3-carboxamide (100 mg, 0.23 mmol) was dissolved in anhydrous dichloromethane (10 mL). TEA (0.16 mL, 1.15 mmol), potassium carbonate (0.17 g, 1.15 mmol), and cyanogen bromide (25 mg, 0.23 mmol) were added to the solution, and the mixture was stirred at room temperature for 10 minutes. The reaction mixture was then quickly quenched by adding water (20 mL), and the mixture was separated. The aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1, V / V), yield 57.2%.

[0237] Example 19:

[0238]

[0239] (S)-1-cyano-N-(6-(3,5-dimethyl-1H-pyrazol-4-yl)benzo[d]thiazo-2-yl)pyrrolidine-3-carboxamide

[0240] Step A: (S)-3-((6-(3,5-dimethyl-1H-pyrazol-4-yl)benzo[d]thiazo-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester was prepared using the method for preparing N2, replacing benzo[d][1,3]dioxane-5-ylboronic acid with 3,5-dimethylpyrazol-4-boronic acid pinacol ester.

[0241] Step B: (S)-N-(6-(3,5-dimethyl-1H-pyrazol-4-yl)benzo[d]thiazol-2-yl)pyrrolidine-3-carboxamide was prepared using the method for preparing N3, replacing (S)-3-((6-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylate with (S)-3-((6-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)carbamoyl)pyrrolidine-1-carboxylate.

[0242] Step C: (S)-1-cyano-N-(6-(3,5-dimethyl-1H-pyrazol-4-yl)benzo[d]thiazol-2-yl)pyrrolidine-3-carboxamide was prepared by replacing (S)-3-((6-(benzo[d][1,3]dioxacyclopenten-5-yl)benzo[d]thiazol-2-yl)pyrrolidine-3-carboxamide with (S)-N-(6-(3,5-dimethyl-1H-pyrazol-4-yl)benzo[d]thiazol-2-yl)pyrrolidine-3-carboxamide using the method of compound 4.

[0243] Example 20:

[0244] Preparation of compound 16

[0245] Preparation of Q1:

[0246]

[0247] 1-Benzylpiperazine

[0248] Piperazine (3.58 g, 42 mmol) was dissolved in dichloromethane (15 mL), and then a dichloromethane solution of benzyl bromide (1 mL, 8.4 mmol) (5 mL) was slowly added dropwise to the reaction solution at 0 °C. After the addition was complete, the reaction was continued at 0 °C for 1 hour. The reaction was confirmed by LC-MS. The solution was washed repeatedly with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1, V / V), with a yield of 97.5%. LC-MS (ESI) m / z: 177 [M+H] + .

[0249] Preparation of Q2:

[0250]

[0251] 5-(4-Benzylpiperazine-1-yl)thiazolyl-2-amine

[0252] Intermediate Q1 (1.76 g, 10 mmol) was dissolved in acetonitrile (15 mL), and then 5-bromothiazol-2-amine (1.79 g, 10 mmol) and cesium carbonate (6.53 g, 20 mmol) were added to the reaction solution. The reaction solution was stirred at room temperature for 3 h. The reaction was confirmed by LC-MS, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1, V / V), with a yield of 37.1%. LC-MS (ESI) m / z: 275 [M+H] + .

[0253] Preparation of Q3:

[0254]

[0255] (S)-3-((5-(4-benzylpiperazin-1-yl)thiazolyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0256] (S)-1-Boc-3-carboxypyrrolidine (238 mg, 1.08 mmol) was dissolved in DMF (2 mL). HCTU (455 mg, 1.08 mmol) and DIEA (0.39 mL, 2.16 mmol) were added to the solution. After stirring at room temperature for 15 minutes, intermediate Q2 (198 mg, 0.72 mmol) was added, and the reaction mixture was stirred at room temperature for 5 hours. LC-MS analysis confirmed the reaction was complete. The mixture was diluted with water and extracted three times with ethyl acetate. The organic phases were combined, washed with 1 M hydrochloric acid, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1, V / V), yield 89.1%. LC-MS (ESI) m / z: 472 [M+H] + .

[0257] Preparation of Q4:

[0258]

[0259] (S)-N-(5-(4-benzylpiperazin-1-yl)thiazo-2-yl)pyrrolidine-3-carboxamide

[0260] Intermediate Q3 (190 mg, 0.51 mmol) was dissolved in dichloromethane (5 mL), and then ethyl acetate hydrochloride solution (4 M, 0.5 mL) was added to the solution. The mixture was stirred at room temperature for 1 hour. After LC-MS detection confirmed the reaction was complete, the solution was concentrated. The product was used directly in the next reaction without further purification, with a yield of 100%. LC-MS (ESI) m / z: 372 [M+H] + .

[0261] Preparation of compound 15:

[0262]

[0263] (S)-N-(5-(4-benzylpiperazin-1-yl)thiazo-2-yl)-1-cyanopyrrolidine-3-carboxamide

[0264] Intermediate Q4 (100 mg, 0.27 mmol) was dissolved in acetonitrile (2 mL), and the solution was cooled to 0 °C. Potassium carbonate (0.2 g, 1.35 mmol) and cyanogen bromide (29 mg, 0.27 mmol) were added to the solution, and the mixture was stirred at 0 °C for 1 hour. The reaction mixture was then quenched by rapidly adding water (20 mL), and the mixture was separated. The aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1, V / V) in 64.2% yield.

[0265] The proton NMR data for compound 15 are as follows:

[0266] 1 H NMR (400MHz, DMSO) δ12.04(s,1H),7.35(d,J=4.8Hz,4H),7.28(d,J=4.5Hz,1H),6.64(s,1H),3.60(dd,J=9.4,7.9Hz,1H),3.56-3.50 (m,2H),3.50-3.45(m,2H),3.48(s,4H),3.35-3.24(m,2H),3.02(s,4H),2.16(td,J=13.0,7.2Hz,1H),2.02(td,J=14.4,7.2Hz,1H).

[0267] Example 21:

[0268] Preparation of R1:

[0269]

[0270] 2-Morphyrin-1-(piperazin-1-yl)ethyl-1-one

[0271] 2-Morpholine acetic acid (0.47 g, 3.24 mmol) was dissolved in DMF (2 mL). HCTU (1.7 g, 4.86 mmol) and TEA (1.12 mL, 2.16 mmol) were added to the solution. After stirring at room temperature for 15 minutes, piperazine (1.38 g, 16.2 mmol) was added, and the reaction mixture was stirred at room temperature for 4 hours. After LC-MS analysis confirmed the reaction was complete, the mixture was diluted with water and extracted three times with ethyl acetate. The organic phases were combined, washed with 1 M hydrochloric acid, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1, V / V), yield 76.1%. LC-MS (ESI) m / z: 214 [M+H] + .

[0272] R2 was prepared by replacing intermediate Q1 with intermediate R1, using the same method as the preparation of Q2.

[0273] R3: (S)-3-((5-(4-(2-morpholinoacetyl)piperazin-1-yl)thiazolyl-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester was prepared using method Q3, with intermediate R2 replacing Q2.

[0274] R4: (S)-N-(5-(4-(2-morpholinoacetyl)piperazin-1-yl)thiazo-2-yl)pyrrolidine-3-carboxamide was prepared using the Q4 method, with intermediate R3 replacing Q3.

[0275] Preparation of compound 17

[0276]

[0277] Intermediate R4 (100 mg, 0.2 mmol) was dissolved in methanol (2 mL). Sodium acetate (50 mg, 0.6 mmol) and cyanogen bromide (15 mg, 0.12 mmol) were added to the solution, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was then quenched by rapidly adding water (20 mL), and the mixture was separated. The aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1, V / V), yield 37.1%. LC-MS (ESI) m / z: 434 [M+H] + .

[0278] Example 22:

[0279] Preparation of S1:

[0280]

[0281] 4,5,6,7-Tetrahydrothiazo[4,5-c]pyridine-2-amine

[0282] 0.51 g (2 mmol) of 2-amino-6,7-dihydrothiazo[4,5-c]pyridine-5(4H)-carboxylic acid tert-butyl ester was dissolved in tetrahydrofuran (5 mL), and then ethyl acetate hydrochloride solution (4 M, 0.5 mL) was added. The mixture was stirred at room temperature for 1 hour. After LC-MS analysis confirmed the reaction was complete, the solution was concentrated. The product was used directly in the next reaction without further purification, with a yield of 100%. LC-MS (ESI) m / z: 156 [M+H] + .

[0283] Preparation of S2:

[0284]

[0285] 5-Benzyl-4,5,6,7-Tetrahydrothiazo[4,5-c]pyridine-2-amine

[0286] S1 (0.42 g, 1.71 mmol) was dissolved in DMF (2 mL). Benzyl bromide (0.34 g, 1.71 mmol) and cesium carbonate (1.82 g, 3.42 mmol) were added to the solution, and the mixture was stirred overnight at room temperature. After the reaction was confirmed by LC-MS, the mixture was filtered. The filtrate was diluted with water and extracted with ethyl acetate. The organic phases were combined, washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1, V / V), with a yield of 84.1%.

[0287] Preparation of S3:

[0288]

[0289] (S)-3-((5-benzyl-4,5,6,7-tetrahydrothiazo[4,5-c]pyridin-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0290] (S)-1-Boc-3-carboxypyrrolidine (217 mg, 0.98 mmol) was dissolved in DMF (5 mL). HCTU (414 mg, 0.98 mmol) and DIEA (0.35 mL, 1.95 mmol) were added to the solution. After stirring at room temperature for 15 minutes, intermediate S2 (160 mg, 0.65 mmol) was added, and the reaction mixture was stirred at room temperature for 5 hours. LC-MS analysis confirmed the reaction was complete. The mixture was diluted with water and extracted three times with ethyl acetate. The organic phases were combined, washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1, V / V), yield 70.7%. LC-MS (ESI) m / z: 443 [M+H] + .

[0291] Preparation of S4:

[0292]

[0293] (S)-N-(5-benzyl-4,5,6,7-tetrahydrothiazo[4,5-c]pyridin-2-yl)pyrrolidine-3-carboxamide

[0294] Intermediate S4 (163 mg, 0.37 mmol) was dissolved in dichloromethane (5 mL), and then ethyl acetate hydrochloride solution (4 M, 0.5 mL) was added to the solution. The mixture was stirred at room temperature for 1 hour. After LC-MS analysis confirmed the reaction was complete, the solution was concentrated. The product was used directly in the next reaction without further purification, with a yield of 100%. LC-MS (ESI) m / z: 343 [M+H] + .

[0295] Preparation of compound 18:

[0296]

[0297] (S)-N-(5-benzyl-4,5,6,7-tetrahydrothiazo[4,5-c]pyridin-2-yl)-1-cyanopyrrolidine-3-carboxamide

[0298] Intermediate S4 (127 mg, 0.37 mmol) was dissolved in acetonitrile (2 mL), and the solution was cooled to 0 °C. Potassium carbonate (0.2 g, 1.35 mmol) and cyanogen bromide (120 mg, 1.11 mmol) were added to the solution, and the mixture was stirred at 0 °C for 1 hour. The reaction mixture was then quenched by rapidly adding water (20 mL), and the mixture was separated. The aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1, V / V) in 73.1% yield.

[0299] The proton NMR data for compound 17 are as follows:

[0300] 1 H NMR (400MHz, DMSO) δ12.32(s,1H),7.36(d,J=16.8Hz,5H),3.71(s,2H),3.62(dd,J=9.4,7.8Hz,2H),3.56–3.48(m,2H),3 .47–3.43(m,2H),3.38–3.25(m,2H),2.80(d,J=82.0Hz,3H),2.19(td,J=13.0,7.3Hz,1H),2.03(dt,J=19.8,7.1Hz,1H).

[0301] Example 23:

[0302] Preparation of T1:

[0303]

[0304] 1-(2-amino-6,7-dihydrothiazo[4,5-c]pyridin-5(4H)-yl)-2-morpholinoethane-1-one

[0305] 2-Morpholine acetic acid (0.47 g, 3.24 mmol) was dissolved in DMF (2 mL). HCTU (1.7 g, 4.86 mmol) and TEA (1.12 mL, 2.16 mmol) were added to the solution. After stirring at room temperature for 15 minutes, S1 (2.51 g, 16.2 mmol) was added, and the reaction mixture was stirred at room temperature for 4 hours. LC-MS analysis confirmed the reaction was complete. The mixture was diluted with water and extracted three times with ethyl acetate. The organic phases were combined, washed with 1 M hydrochloric acid, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1, V / V), yield 81.3%. LC-MS (ESI) m / z: 283 [M+H] + .

[0306] T2: (S)-3-((5-(2-morpholinoacetyl)-4,5,6,7-tetrahydrothiazo[4,5-c]pyridin-2-yl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester was prepared using the same method as for S2, with intermediate T1 replacing intermediate S1.

[0307] T3: (S)-N-(5-(2-morpholinoacetyl)-4,5,6,7-tetrahydrothiazo[4,5-c]pyridin-2-yl)pyrrolidine-3-carboxamide was prepared using the S3 method, with intermediate T2 replacing S2.

[0308] Preparation of compound 19:

[0309]

[0310] (S)-1-cyano-N-(5-(2-morpholinoacetyl)-4,5,6,7-tetrahydrothiazo[4,5-c]pyridin-2-yl)pyrrolidine-3-carboxamide

[0311] Intermediate T3 (100 mg, 0.2 mmol) was dissolved in DMF (2 mL). Potassium carbonate (140 mg, 1 mmol) and cyanogen bromide (50 mg, 0.5 mmol) were added to the solution, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was then quenched by rapidly adding water (20 mL), and the mixture was separated. The aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1, V / V), yield 52.1%. LC-MS (ESI) m / z: 405 [M+H] + .

[0312] Example 24. Degradation level of JAK2 by DESI2 inhibitor

[0313] HEL cells were used at a rate of 5 × 10 5 Cells were seeded into each well of a 24-well plate and cultured using 1 ml of culture medium. The compounds were dissolved in pure dimethyl sulfoxide (DMSO) at a stock solution concentration of 10 mM. The DMSO solution was serially diluted three-fold from 10 μM to three different concentrations, with 1 μL added to each well. 1 μL of diluted DMSO solution was added to each negative control well. After 24 h of incubation, cells were collected, and the degradation effect of the compounds prepared in Examples 5-17 on JAK2 was assessed by Western blotting (WB). The results are shown in Table 1.

[0314] Table 1

[0315]

[0316] Where A represents EC50 value ≤ 10μM; B represents 20μM ≤ EC50 ≤ 50μM; and C represents 50μM ≤ IC value ≤ 100μM.

[0317] Example 25. Inhibitory level and in vitro functional verification of DESI2 inhibitor on enzyme activity.

[0318] The enzyme activity assay buffer was prepared by adding 40 mM Tris (pH 7.5), 20 mM magnesium chloride (MgCl2), 0.1 mg / ml bovine serum albumin (BSA), and 50 μM dithiothreitol (DTT). The compounds were dissolved in pure DMSO to a stock solution concentration of 10 mM. The DMSO solutions were serially diluted three-fold from 100 μM to a total of six concentrations. Each diluted compound was then diluted 1:20 with the enzyme activity assay buffer, and 1 μL was added to each well, with two replicates per concentration. 1 μL of the 1:20 diluted DMSO solution was added to both the negative and positive control wells. The 2.5× substrate / ATP working solution was prepared by adding 2 μL of the enzyme activity assay buffer and 0.5 μg / μL of the substrate peptide to each well. Prepare a 2.5× enzyme reaction working solution containing 2.5 ng / μl DESI2 recombinant protein enzyme activity test buffer. Add 2 μl of the 2.5× enzyme reaction working solution to each well, and add only 2 μl of enzyme activity test buffer to the negative control well. Seal the plate and centrifuge, then incubate at room temperature for 1 hour. After the reaction, add 5 μl of the detection reagent to each well and incubate at room temperature for 40 minutes. Measure the final luminescence signal. Calculate the average values ​​of the positive and negative wells as the positive control value (Signal) and negative control value (DOS). Calculate the inhibition rate of the working well signal value (Signaltest) according to the formula Inhibition rate = (Signalp.negDOSignal) / (Signal.-Signal.)×100%. Plot the concentration-inhibition rate curve using non-linear fitting in GraphPadtestDOSne8Prism software, and calculate the IC50. 50 and DC 50 value( Figure 6 Compared to its analogues, WWQ-03-012 inhibits DESI2 enzyme activity (IC50). 50 =47.3 nM), enhancing the polyubiquitination level of JAK2 and significantly promoting the degradation of mutant JAK2 protein (DC). 50 =543.6 nM) while having no effect on wild-type protein and blocking its downstream signaling pathways, thereby exerting a higher anti-proliferative efficacy in leukemia cells. Furthermore, our DESI2 knockout experiment ruled out the off-target effects of WWQ-03-012, meaning that its JAK2 degradation activity is dependent on DESI2.

[0319] Example 26. Antiproliferative efficacy of DESI2 inhibitors on primary cells from MPN patients

[0320] Next, we evaluated the growth-inhibitory effects on different preclinical cell models by testing the therapeutic potential of its primary DESI2 inhibitor, WWQ-03-012. Compared with the FDA-approved JAK1 / 2 inhibitor ruxolitinib, WWQ-03-012 significantly inhibited the proliferation of two JAK2-V617F-driven cell lines (HEL and Ba / F3-JAK2-V617F) after 24 or 72 hours of treatment, with little effect on normal human PBMCs. Figure 7 These results (A and 7B) also indicate that our compound is more effective against JAK2 mutant cells. We then compared the therapeutic effect of WWQ-03-012 on three MPN (MPN#1-#3) patient samples after 72 hours of treatment, and the results showed that WWQ-03-012 demonstrated higher potency than Ruxolitinib in preclinical samples. Figure 7 C). Furthermore, we found that WWQ-03-012 was more effective than our previously developed inhibitor XL-106C in two JAK2-V617F-positive MPN patient samples (MPN #4 and #5). These results suggest that blocking DESI2 may be a more effective treatment strategy. Figure 7 D).

[0321] Example 27. Therapeutic effect of DESI2 inhibitor on AML mouse model

[0322] Female NSG mice, approximately 6 weeks old, were selected (purchased from Southern Model Biotechnology). All animals were housed in a designated pathogen-free facility and used in accordance with Tongji University's animal care regulations. In vivo studies were approved by the Tongji University Ethics Committee. 3 × 10⁶ HEL-Luc cells were used. 6 / Injected into mice via tail vein only, when the fluorescence value reaches 5×10 6 Upon successful model establishment, the animals were randomly divided into a blank (solvent) control group, a treatment group, and a positive control group, with 5 mice in each group (n=4 or 5) for efficacy studies. The DESI2 inhibitor was administered via HKI suspension (0.5% methylcellulose / 0.4% Tween 80 dissolved in ddH2O) twice daily via intraperitoneal injection (ip) twice daily (BID). The control group received a specific dose of the solvent via injection. Ruxolitinib (a JAK1 / 2 inhibitor, ip, QD), a marketed drug, was used as the positive control group. Body weight and tumor growth were measured periodically after DESI2 inhibitor administration, with body weight measured every 2 days and fluorescence imaging every 3 days. Results showed that the DESI2 inhibitor 03-012 significantly inhibited tumor cell proliferation in vivo, with effects comparable to the marketed drug Ruxolitinib, and had no significant effect on animal body weight. Figure 8A-8D).

[0323] The compounds involved in this embodiment, including the degradation detection data of compounds in all embodiments, are as follows: Figure 9 As shown, some compounds exhibit good degradation effects on JAK2-V617F, DC 50 Below 1 μM, the ordinate represents the relative protein expression level of JAK2-V617F.

[0324] In summary, the technical solution of this invention first discovers DESI2, a novel regulator of JAK2-V617F protein stability, and verifies its function and mechanism in in vitro and in vivo models. Then, by designing, synthesizing, and improving the structure of DESI2 inhibitors, testing the degradation effect of these compounds on JAK2-V617F protein, and combining this with direct enzyme activity detection, several effective DESI2 inhibitors for degrading JAK2 mutant proteins are provided. The inclusion of primary clinical patient samples and mouse xenograft tumor treatment models indicates the potential of DESI2 inhibitors for clinical application. The combination and synergistic effect of these technical approaches give this invention significant technical advantages.

[0325] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A thiazole compound, characterized in that, The compound is selected from one of the following structural formulas: 。 2. A method for synthesizing the thiazole compound according to claim 1, characterized in that, Synthesize using one of methods A, B, C, or D; Method A includes the following steps: Step S1, place A a and A is obtained through a condensation reaction. b ; Step S2, place A b A is obtained by coupling reaction with boron esters or boric acids containing R groups. c ; Step S3, place A c A is obtained by removing the tert-butyloxycarbonyl group under acidic conditions. d ; Step S4, place A d A is obtained by reacting with cyanogen bromide under alkaline conditions. e ; Step S5, place A e A is obtained through a condensation reaction under alkaline conditions. ; Method B includes the following steps: Step T1, B a and B is obtained through a condensation reaction. b ; Step T2, B b B is obtained by removing the tert-butyloxycarbonyl group under acidic conditions. c ; Step T3, B c B is obtained by coupling reaction with boron esters or boric acids containing R groups. d ; Step T4, B d B is obtained by reacting with cyanogen bromide under alkaline conditions. e ; Step T5, B e B is obtained by removing the tert-butyloxycarbonyl group under acidic conditions and then reacting it with substitution or condensation under basic conditions. ; Method C includes the following steps: Step U1: Piperazine is reacted with a substitution reaction or a condensation reaction under alkaline conditions to obtain C. a ; Step U2, place C a and C is obtained through a substitution reaction under alkaline conditions. b ; Step U3, place C b and C is obtained through a condensation reaction. c ; Step U4, place C c Under acidic conditions, the tert-butyloxycarbonyl group is removed and reacted with cyanogen bromide to give C. d ； The method D includes the following steps: Step V1, D a D is obtained by removing the tert-butyloxycarbonyl group under acidic conditions. b ; Step V2, D b D is obtained under alkaline conditions through substitution or condensation reactions. c ； Step V3, D c and D is obtained through a condensation reaction. d ； Step V4, D d D is obtained by removing the tert-butyloxycarbonyl group under acidic conditions. e ； Step V5, D e D is obtained by reacting with cyanogen bromide under alkaline conditions. f .

3. A deSUMOylase and deubiquitinase inhibitor, characterized in that, It is one or more of the thiazole compounds described in claim 1, or one or more of the thiazole compounds prepared by the synthetic method described in claim 2.

4. The use of the deSUMO and deubiquitinase inhibitor of claim 3 in the preparation of a medicament for inhibiting deSUMO and deubiquitinase activity, characterized in that, The deubiquitination and deSUMOylation enzyme is DESI2.

5. The application according to claim 4, characterized in that, The drug is a drug that reduces the stability of JAK2; and / or, the drug is a drug that targets the genetic mutation type of JAK2 in an organism; and / or, the drug is a drug that regulates the levels of ubiquitination and SUMOylation in an organism; and / or, the drug is a drug that treats and / or prevents diseases caused by deubiquitination and deSUMOylation.

6. The use of one or more of the thiazole compounds of claim 1, or one or more of the thiazole compounds prepared by the synthetic method of claim 2, in the preparation of medicaments for treating and / or preventing diseases and related conditions mediated by JAK2 enzyme.

7. The application according to any one of claims 4 or 6, characterized in that, The drug is used to inhibit tumor cells.

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