A highly efficient hpk1 degrader compound and a preparation method and application thereof

Abstract

Provided are HPK1 degradation agent compounds, and preparation methods and applications thereof. Specifically, provided are applications of a compound having a structure of Formula (I) and a pharmaceutical composition thereof in preventing and / or treating HPK1 related diseases (e.g., cancer or an immunological disease).

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a new HPK1 degradation agent compound, a pharmaceutical composition containing the same, a preparation method thereof and application thereof in prevention and / or treatment of HPK1 related diseases. BACKGROUND

[0002] Targeted protein degradation is one of the emerging directions in the field of drug research and development. As a new drug discovery technology, a PROTAC (PROTAC) can connect a target protein with an E3 ligase, thereby inducing ubiquitination of the target protein, so that the proteasome can recognize and degrade the target protein. Unlike the occupation-driven mechanism of traditional small molecule inhibitors, the protein degradation mechanism of the PROTAC molecule does not require a high binding force with the active site of the target protein, and it can maintain efficacy at a lower concentration and be more persistent.

[0003] Hematopoietic progenitor kinase 1 (HPK1, also known as MAP4K1) is a serine / threonine kinase mainly expressed in hematopoietic cells, including their progenitor cells, especially in T cells, B cells and dendritic cells. HPK1 is an important immunosuppressive regulatory kinase, and its function in T cells has been well studied. It inhibits the activity of the T cell receptor (TCR) signaling pathway by phosphorylating the receptor protein SLP76, affecting downstream T cell activation and proliferation. HPK1 can bind to many linker proteins, such as the SLP76 family, Gads, HIP-55, GRB2 family, LAT, CRK family, etc., activate the SAPK / JNK signaling pathway of hematopoietic stem cells, and thus negatively regulate the TCR pathway. Studies have found that the expression of HPK1 in different cancer patients is positively correlated with T cell exhaustion signals. Inhibition of the HPK1 pathway has application potential in improving T cell function, antigen presentation and combating immunosuppressive tumor microenvironments. In addition to the above-mentioned main role of HPK1 through kinase activity, there are also reports that HPK1 has a scaffold function, and HPK1 knockout mice have no abnormalities in the resting state, which indicates that the development of HPK1 degradation agents can more completely inhibit HPK1 related functions and have good safety.

[0004] Many studies have shown that HPK1 is a potential target for tumor immunotherapy, and there is currently no drug targeting HPK1 on the market, so it is necessary to develop more efficient drugs targeting HPK1 to meet the clinical needs. SUMMARY

[0005] The present application provides a compound having HPK1 degradation activity and a pharmaceutically acceptable salt, a prodrug, an isotopic derivative, a metabolite, a stereoisomer, a N-oxide or a prodrug having the following table structure:

[0006]

[0007]

[0008]

[0009]

[0010]

[0011]

[0012]

[0013]

[0014]

[0015]

[0016]

[0017] In one aspect, the present application also provides a pharmaceutical composition comprising a compound as described above, or a stereoisomer, a tautomer, a solvate, a pharmaceutically acceptable salt, a metabolite, an isotopic derivative, a N-oxide or a prodrug thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

[0018] In yet another aspect, the present application also provides use of a compound as described above, or a stereoisomer, a tautomer, a solvate, a pharmaceutically acceptable salt, a metabolite, an isotopic derivative, a N-oxide or a prodrug thereof, or a pharmaceutical composition for manufacturing a medicament for preventing and / or treating a disease related to HPK1 activity. The disease related to HPK1 activity includes a cancer or an immunological 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, hematoma and lymphoma; the immunological disease is preferably lupus erythematosus, psoriasis, inflammatory bowel disease and rheumatoid arthritis.

[0019] In one aspect, the present application also provides a method for preventing and / or treating a disease associated with HPK1 activity, comprising administering to a patient in need thereof a therapeutically effective amount of a compound as described above, or a stereoisomer, a tautomer, a solvate, a pharmaceutically acceptable salt, a metabolite, an isotopic derivative, an N-oxide, or a prodrug thereof, or a pharmaceutical composition. The disease associated with HPK1 activity includes a cancer or an immunological 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, cytoma, glioma, hematoma, and lymphoma; the immunological disease is preferably lupus erythematosus, psoriasis, inflammatory bowel disease, and rheumatoid arthritis.

[0020] In another aspect, in the above uses or methods, the above compound as described above, or a stereoisomer, a tautomer, a solvate, a pharmaceutically acceptable salt, a metabolite, an isotopic derivative, an N-oxide, or a prodrug thereof, or a pharmaceutical composition can be used alone, or in combination with other kinds of pharmaceutical preparations and / or treatment methods.

[0021] It is particularly noted that, herein, when referring to a "compound" having a specific structural formula, it generally also encompasses its stereoisomers, diastereoisomers, enantiomers, racemic mixtures, and isotopic derivatives.

[0022] It is well known to those skilled in the art that a salt, a solvate, a hydrate of a compound is an alternative existing form of the compound, which can be converted into the compound under certain conditions, and therefore, it is particularly noted that, herein, when referring to a compound, it generally also includes its pharmaceutically acceptable salt, and further includes its solvate and hydrate.

[0023] Similarly, herein, when referring to a compound, it generally also includes its prodrug, metabolite, and N-oxide.

[0024] The "stereoisomers" of the compound of formula (I) of the present application refer to enantiomers when there is an asymmetric carbon atom in the compound of formula (I); to syn and anti isomers when there is a carbon-carbon double bond or a cyclic structure in the compound; to tautomers when there is a ketone or oxime, etc. in the compound, and all enantiomers, diastereoisomers, racemic isomers, syn and anti isomers, tautomers, geometric isomers, epimers, and mixtures thereof of the compound of formula (I) are included in the scope of the present application.

[0025] The "pharmaceutically acceptable salt" according to the present application refers to a pharmaceutically acceptable addition salt of an acid and a base or a solvate thereof. Such a pharmaceutically acceptable salt includes a salt of the following acid: 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, alkanecarboxylic acid (such as acetic acid, HOOC-(CH2)n-COOH (where n is 0-4)), and the like. A salt of a base: sodium salt, potassium salt, calcium salt, ammonium salt, and the like. A variety of non-toxic pharmaceutically acceptable addition salt known to those skilled in the art.

[0026] The pharmaceutically acceptable salt according to the present application can be prepared by a conventional method, for example, by dissolving the compound of the present application in a water-miscible organic solvent (for example, acetone, methanol, ethanol, and acetonitrile), adding an excess of an aqueous solution of an organic acid or an inorganic acid thereto, so that the salt is precipitated from the resulting mixture, removing the solvent and the remaining free acid therefrom, and then isolating the precipitated salt.

[0027] The precursor or metabolite according to the present application can be a precursor or metabolite known in the art, as long as the precursor or metabolite is converted by in vivo metabolism to form the compound. For example, "prodrug" refers to those prodrugs of the compound of the present application, which are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use within the scope of sound medical judgment. The term "prodrug" refers to compounds that rapidly undergo conversion by in vivo metabolism to yield the parent compound of the above formula, for example, by metabolism in vivo.

[0028] The present application provides a compound of the specific structure of formula (I) and pharmaceutically acceptable salts, solvates, stereoisomers, prodrugs, metabolites thereof for use in the prevention and / or treatment of HPK1 related diseases.

[0029] The HPK1 related diseases according to the present application include cancer, inflammation, and immunological diseases, and the like.

[0030] The compound of the present application according to the present application can be used alone or in combination with other kinds of pharmaceutical preparations (such as PD-1 / PD-L1 antagonists, and the like) and / or treatment methods (such as radiotherapy / chemotherapy, and the like) when used in the prevention and / or treatment of HPK1 related diseases.

[0031] The present application also provides the use of the compound of the present application in the preparation of a medicament for the prevention and / or treatment of HPK1 related cancer, inflammation, and immunological diseases.

[0032] In addition, the present application provides a pharmaceutical composition for the prevention and / or treatment of HPK1 related cancer, inflammation, and immunological diseases, which comprises the compound of the present application as an active ingredient. DETAILED DESCRIPTION

[0033] Definitions

[0034] If not otherwise specified, the definitions of the terms used in the present application (including the specification and claims) are as follows. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. If not otherwise specified, conventional methods of mass spectroscopy, nuclear magnetic, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology are employed. In the present application, "or" or "and" as used by their normal meaning means "and / or" unless otherwise stated.

[0035] In the specification and claims, a given chemical formula or name shall encompass all stereo and optical isomers and racemates thereof unless otherwise indicated. All chiral (enantiomeric and diastereomeric) and racemic forms are within the scope of the present application unless otherwise indicated. Many geometric isomers of C=C double bonds, C=N double bonds, ring systems, etc. can also be present in the compounds, and all such isomers are contemplated in the present application. The present application describes cis- and trans- (or E- and Z-) geometric isomers of the compounds of the present application and they can be isolated as a mixture of isomers or as separate isomers. The compounds of the present application can be isolated in optically active or racemic forms. All processes used to prepare compounds of the application and intermediates used therein are considered to be part of the present application. When preparing enantiomeric or diastereomeric products, they can be separated by conventional methods, e.g., by chromatography or fractional crystallization. The end products of the present application are obtained in either free (neutral) or salt form. Both the free form and the salts of these end products are within the scope of the present application. If desired, one form of a compound can be converted into another form. A free base or acid can be converted into a salt; a salt can be converted into the free compound or another salt; a mixture of isomeric compounds of the present application can be separated into the individual isomers. The compounds of the present application, free forms and salts, can exist in a variety of tautomeric forms, in which hydrogen atoms are transposed to other parts of the molecule and the chemical bonds between the atoms of the molecule are accordingly rearranged. It is to be understood that all tautomeric forms are included in the present application.

[0036] In cases wherein there are nitrogen atoms on the compounds of the present application (e.g., amines), these nitrogen atoms can be converted to N-oxides by treatment with an oxidizing agent (e.g., mCPBA and / or hydrogen peroxide) to give other compounds of the present application. Thus, shown and claimed nitrogen atoms are considered to cover both the shown nitrogen and its N-oxide to give derivatives of the present application.

[0037] In the present application, the term "patient" refers to an organism that is treated by the methods of the present application. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians / monkeys, equines, bovines, porcines, canines, felines, etc.) and most preferably refers to humans.

[0038] In the present application, the term "effective amount" means that amount of a drug or pharmaceutical agent, i.e. a compound of the present application, that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought, for instance, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means an amount of a compound effective to achieve a therapeutic, healing, prophylactic, or palliative effect on a disease, condition, or adverse effect, or to decrease the rate of progress of a disease or condition, as compared to not receiving the above amount of the respective subject. An effective amount can be given in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or route of administration. The term also includes within its scope an effective amount to enhance normal physiological function.

[0039] In the present application, the term "treatment" includes its broadest meaning as used in the art, and encompasses therapeutic and / or prophylactic treatment of a subject. In particular, the "treatment" includes any treatment which results in the alleviation, inhibition, elimination and amelioration and / or prevention of a condition, disease, disorder, etc., such as reducing, decreasing, modulating, ameliorating, eliminating, preventing, obviating or improving symptoms thereof. The therapeutic treatment includes alleviating, inhibiting or ameliorating symptoms or conditions of a disease; inhibiting the development of a complication; ameliorating an underlying metabolic syndrome; inhibiting the development of a disease or symptom, such as controlling the progression of a disease or condition; reducing a disease or symptom; causing regression of a disease or symptom; reducing complications resulting from a disease or symptom, or treating an indication resulting from a disease or symptom. The prophylactic treatment includes prior treatment to prevent, block or delay, slow down the occurrence or development or to attenuate the severity of a disease or condition.

[0040] Likewise, a "therapeutic agent" also includes an agent or reagent which has therapeutic and / or prophylactic treatment of a subject.

[0041] In the present application, the term "pharmaceutical" or "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, and / or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0042] In the present application, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable vehicle" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium stearate or zinc stearate, or steric acid), or solvent / capsule material, involved in carrying or transporting the subject compound from one organ or portion of the body to another organ or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which have been used to form pharmaceutical preparations include gelatin, sugars, kaolin, diluent, excipient, manufacturing aid, lubricant, talc, magnesium stearate, calcium stearate or zinc stearate, or steric acid.

[0043] In the present application, the term "pharmaceutical composition" means a composition comprising a compound of the present application in combination with at least one other pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to a medium generally accepted in the art for the delivery of biologically active agents to animals, particularly mammals, and more specifically humans. It includes, i.e., an adjuvant, excipient, or vehicle, such as diluents, preserving agents, fillers, flow regulating agents, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents, and dispersing agents, depending on the nature of the dosage form and mode of delivery.

[0044] Specific Pharmaceutical and Medical Terms

[0045] In the present application, the term "acceptable", as used herein, means no undue harmful effect on the general health of the treated subject.

[0046] In the present application, the term "cancer", as used herein, refers to an abnormal growth of cells that is uncontrolled and, under certain conditions, is capable of metastasizing (spreading). This type of cancer includes, but is not limited to, solid tumors (e.g., bladder, bowel, brain, breast, uterine, cardiac, kidney, lung, lymphatic tissue (lymphoma), ovarian, pancreatic or other endocrine organ (e.g., thyroid), prostate, skin (melanoma), or blood tumors (e.g., non-leukemic leukemia).

[0047] In the present application, the term "co-administration" or its grammatical equivalents, as used herein, means the administration of two or more selected therapeutic agents to a single patient at the same time by the same or different routes of administration.

[0048] In the present application, the term "enhance" or "enhancing", as used herein, means an intended result that can be an increase or prolongation in potency or duration. Thus, in the context of enhancing the therapeutic effect of a drug, the term "enhancing" means the ability of a drug to increase or prolong the potency or duration of action in a system. As used herein, "enhancing value" means the ability to maximize the enhancement of another therapeutic agent in an ideal system.

[0049] In the present application, the term "immune disorder" refers to a disease or condition that results from an adverse or deleterious reaction to an endogenous or exogenous antigen. The result is usually a dysfunction of cells, or destruction and dysfunction as a result, or destruction of organs or tissues that can produce the immunological symptoms.

[0050] In the present application, the terms "kit" and "product package" are synonymous.

[0051] As used herein, the terms "subject," "recipient," "patient," or "host" include mammals and non-mammals. Mammals include, but are not limited to, primates, humans, non-human primates, agricultural animals such as cows, horses, goats, sheep, pigs, and the like, domestic animals such as rabbits, dogs, and the like, and laboratory animals including rodents such as rats, mice, and guinea pigs and the like. Non-mammals include, but are not limited to, birds, fish, and the like. In a preferred embodiment, the mammal is a human.

[0052] As used herein, a compound or pharmaceutical composition is "administered" to a subject to effect an improvement in a disease, condition, or disorder, particularly an improvement in the severity thereof, a delay of onset, a slowing of progression, or a reduction in the duration thereof. The improvement can be attributed to or associated with the administration, whether fixed or temporal, continuous or intermittent. DETAILED DESCRIPTION

[0053] The present application can be better understood with reference to the following examples, which are intended for the purpose of illustration only and are not intended to limit the scope of the application.

[0054] In the present application, when no preparation route is mentioned, the relevant starting materials and intermediates are purchased from commercial reagents (e.g., from Aldrich, Sigma, etc.).

[0055] The abbreviations used in the present application have the following meanings:

[0056]

[0057]

[0058] In the following examples, the reaction temperature is room temperature (15-30°C) unless otherwise specified.

[0059] The compounds of the present application are isolated and purified by preparative TLC, silica gel column chromatography, Prep-HPLC and / or flash column chromatography. The structure of the compounds is confirmed by 1 H NMR and / or MS. The reaction is monitored by TLC or LC-MS.

[0060] 1H-NMR spectra were recorded on a Bruker instrument at 500 MHz. Chemical shift values are expressed in parts per million, i.e. delta values. The following abbreviations are used for multiplicity in NMR signals: s = singlet, br s = broad singlet, d = doublet, t = triplet, m = multiplet. Coupling constants are listed as J values, measured in Hz. LC-MS experimental conditions: Instrument: Thermo U3000, ALLtech ELSD, MSQ, UV detector combined with ELSD and MSD (flow ratio of 4: 1). Column: Waters X-Bridge C-18, 3.5 μm, 4.6 x 50 mm; column temperature: 30 °C. Gradient [time (min) / solvent B in A (%)] : 0.00 / 5.0, 1.40 / 95, 2.80 / 95, 2.82 / 5, 3.00 / 5. (Solvent A = 0.01% trifluoroacetic acid in water; solvent B = 0.01% trifluoroacetic acid in acetonitrile). UV detection: 214 / 254 / 280 / 300 nm; DAD detection: 210-350 nm; flow rate: 2 mL / min; MS: ESI, 100-1500 m / z.

[0061] Preparative HPLC used basic method or acidic method (basic method mobile phase: acetonitrile / 0.05% ammonium bicarbonate aqueous solution, acidic method mobile phase: acetonitrile / 0.05% formic acid aqueous solution); instrument: Thermo U3000 AFC-3000; column: Globalsil C-18 12 nm, 250 x 20 mm, 10 μm, or equivalent; flow rate: 20 mL / min, gradient elution separation was performed.

[0062] Example 1

[0063]

[0064] First step: 5-Fluoro-2-nitroaniline (5.0 g, 32.03 mmol) and 1-tert-butoxycarbonyl piperazine (6.3 g, 33.63 mmol) were dissolved in NMP (30 mL), DIPEA (11.2 mL, 64.06 mmol) was added, and the reaction was carried out at 120 °C overnight. The reaction solution was diluted with ethyl acetate, washed with water and saturated brine in turn, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give yellow solid 1a (9.6 g, yield 93%). ESI-MS (m / z): 323.7 [M+H] + .

[0065] Second step: 1a (9.6 g, 29.78 mmol) was dissolved in anhydrous ethanol (40 mL), 10% palladium-carbon (0.361 g, 2.98 mmol) was added, and the reaction system was replaced with hydrogen gas by a hydrogen balloon. The system was stirred at 40 °C overnight under the pressure of the hydrogen balloon. Then 3-ethoxy-3-iminopropionic acid ethyl ester hydrochloride (8.7 g, 44.63 mmol) was added to the reaction solution, and the temperature was raised to 80 °C. The stirring was continued for 4 hours. After the reaction was completed, the system was filtered with diatomite, washed with dichloromethane, and the filtrate was concentrated to obtain yellow solid 1b (10 g, yield 87%). ESI-MS (m / z): 389.5 [M+H] + .

[0066] Third step: 1b (10 g, 25.74 mmol) and 2-aminothiophene-3-carbonitrile (3.20 g, 25.74 mmol) were dissolved in tetrahydrofuran (50 mL), and 2M LDA THF solution (64 mL, 128.71 mmol) was added under nitrogen protection. The system was raised to 40 °C and stirred for 2 hours. After the reaction was completed, the system was quenched with saturated ammonium chloride, extracted with dichloromethane three times, and the organic phase was washed with water and saturated sodium chloride solution successively. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol) to obtain brown solid 1c (6.8 g, yield 56%). ESI-MS (m / z): 467.3 [M+H] + .

[0067] Fourth step: 1c (6.8 g, 14.6 mmol) was dissolved in dioxane (30 mL) and dichloromethane (10 mL), and 4M HCl dioxane solution (20 mL) was added. The stirring was continued at room temperature for 2 hours. The reaction system was concentrated to obtain 1d (5.2 g, yield 98%). ESI-MS (m / z): 367.7 [M+H] + .

[0068] Fifth step: 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyiso-1,3-dione (500 mg, 2 mmol), potassium iodide (30 mg, 0.15 mmol) and sodium bicarbonate (305 mg, 2 mmol) were dissolved in DMF (5 mL), and 8-bromo-1-octanol (502 mg, 2.4 mmol) was added to the reaction system. Then the reaction system was heated to 80 °C and stirred overnight. The reaction system was reduced to room temperature, diluted with ethyl acetate, and extracted with saturated brine three times. The organic phase was dried with anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol) to obtain the final product 1e (425 mg, yield 44%). ESI-MS (m / z): 403.1 [M+H] + .

[0069] Step 6: Dissolve le (100 mg, 0.25 mmol) in dichloromethane (4 mL), add Dess-Martin Oxidizing reagent (106 mg, 0.25 mmol) to the reaction, then stir the reaction at room temperature for 1 hour, check the reaction by LCMS, the reaction is complete. Filter the reaction, wash with dichloromethane, dry the organic phase with saturated sodium bicarbonate solution, saturated brine, then dry over anhydrous sodium sulfate, filter, concentrate to give the final product If (91 mg, yield 86%), which is used directly in the next step. ESI-MS (m / z): 401.3 [M+H] + .

[0070] Step 7: Dissolve Id (50 mg, 0.14 mmol), If (56 mg, 0.14 mmol) in dichloroethane (4 mL) and methanol (2 mL) under ice bath condition, add sodium acetate (19 mg, 0.14 mmol) to the reaction, then stir the reaction for 0.5 hour, then add sodium triacetoxyborohydride (59 mg, 0.28 mmol). Stir the reaction at room temperature overnight, check the reaction by LCMS, the reaction is complete. Concentrate the reaction, dissolve the residue in methanol, purify by HPLC to give compound 1 (23 mg, yield 29%). ESI-MS (m / z): 751.4 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.60 (d, J = 6.2 Hz, 1H), 12.08 (s, 1H), 11.11 (s, 1H), 10.85 - 10.53 (m, 1H), 8.25 (s, 1H), 7.98 (s, 1H), 7.81 (dd, J = 8.5, 7.3 Hz, 1H), 7.59 (d, J = 5.8 Hz, 1H), 7.54 - 7.42 (m, 2H), 7.19 - 7.08 (m, 2H), 6.91 - 6.86 (m, 1H), 5.08 (dd, J = 12.8, 5.5 Hz, 1H), 4.21 (t, J = 6.4 Hz, 2H), 3.09 (t, J = 4.9 Hz, 4H), 2.96 - 2.80 (m, 2H), 2.32 (t, J = 7.4 Hz, 2H), 2.06 - 1.95 (m, 2H), 1.77 (p, J = 6.6 Hz, 2H), 1.53 - 1.28 (m, 12H), 1.26 - 1.14 (m, 2H).

[0071] Example 2

[0072]

[0073] Compound 2 can be obtained by replacing 8-bromo-1-octanol in the fifth step of Example 1 with 9-bromo-1-nonanol using similar method and reaction procedures. ESI-MS (m / z): 765.9 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.59 (d, J = 6.3 Hz, 1H), 12.07 (s, 1H), 11.10 (s, 1H), 10.86 - 10.53 (m, 1H), 8.07 - 7.90 (m, 1H), 7.79 (t, J = 7.9 Hz, 1H), 7.57 (d, J = 5.8 Hz, 1H), 7.54 - 7.40 (m, 3H), 7.23 - 7.02 (m, 2H), 6.92 - 6.83 (m, 1H), 5.07 (dd, J = 12.8, 5.4 Hz, 1H), 4.19 (t, J = 6.4 Hz, 2H), 3.08 (t, J = 4.7 Hz, 4H), 2.91 - 2.82 (m, 1H), 2.66 - 2.52 (m, 3H), 2.30 (t, J = 7.4 Hz, 2H), 2.06 - 1.98 (m, 1H), 1.75 (t, J = 7.3 Hz, 3H), 1.51 - 1.18 (m, 14H).

[0074] Example 3

[0075]

[0076] Compound 3 can be obtained by replacing 8-bromo-1-octanol in the fifth step of Example 1 with 11-bromo-1-undecanol using similar method and reaction procedures. ESI-MS (m / z): 793.5 [M+H] + ; 1H NMR (500 MHz, DMSO-d6) δ 12.59 (d, J = 5.9 Hz, 1H), 12.07 (s, 1H), 11.09 (s, 1H), 10.83 - 10.45 (m, 1H), 8.05 - 7.89 (m, 1H), 7.79 (t, J = 7.9 Hz, 1H), 7.57 (d, J = 5.8 Hz, 1H), 7.36 - 7.29 (m, 3H), 7.25 - 7.03 (m, 2H), 6.88 (td, J = 7.0, 5.8, 2.4 Hz, 1H), 5.07 (dd, J = 12.8, 5.4 Hz, 1H), 4.18 (t, J = 6.5 Hz, 2H), 3.09 (s, 4H), 2.95 - 2.77 (m, 1H), 2.78 - 2.21 (m, 3H), 2.30 (t, J = 7.0 Hz, 2H), 2.05 - 1.98 (m, 1H), 1.96 - 1.51 (m, 2H), 1.49 - 1.41 (m, 5H), 1.35 - 1.20 (m, 14H).

[0077] Example 4

[0078]

[0079] Compound 4 was obtained by replacing 8-bromo-1-octanol with 7-bromo-1- heptanol in the fifth step of Example 1, using similar procedures and reaction steps. ESI-MS (m / z): 737.8 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.63 (s, 1H), 12.06 (s, 1H), 11.09 (s, 1H), 10.95 - 10.35 (m, 1H), 7.95 (s, 1H), 7.80 (t, J = 7.9 Hz, 1H), 7.57 (d, J = 5.7 Hz, 1H), 7.53 - 7.41 (m, 3H), 7.21 - 7.06 (m, 2H), 6.87 (t, J = 7.5 Hz, 1H), 5.07 (dd, J = 12.7, 5.4 Hz, 1H), 4.20 (t, J = 6.5 Hz, 2H), 3.08 (t, J = 4.7 Hz, 4H), 2.91 - 2.82 (m, 1H), 2.65 - 2.53 (m, 2H), 2.31 (t, J = 7.3 Hz, 2H), 2.05 - 1.99 (m, 1H), 1.80 - 1.73 (m, 2H), 1.56 - 1.13 (m, 12H).

[0080] Example 5

[0081]

[0082] Compound 5 was obtained by replacing 8-bromo-1-octanol in the fifth step of Example 1 with 5-bromo-1-pentanol using similar method and reaction procedure. ESI-MS (m / z): 709.2 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.59 (d, J = 6.0 Hz, 1H), 12.07 (s, 1H), 11.09 (s, 1H), 10.85 - 10.44 (m, 1H), 7.97 (s, 1H), 7.80 (t, J = 7.8 Hz, 1H), 7.58 (d, J = 5.8 Hz, 1H), 7.54 - 7.41 (m, 3H), 7.20 - 7.06 (m, 2H), 6.92 - 6.83 (m, 1H), 5.07 (dd, J = 12.6, 5.5 Hz, 1H), 4.22 (t, J = 6.5 Hz, 2H), 3.11 - 3.05 (m, 4H), 2.89 - 2.83 (m, 1H), 2.54 - 2.53 (m, 4H), 2.38 - 2.32 (m, 2H), 2.07 - 1.75 (m, 4H), 1.60 - 1.43 (m, 5H).

[0083] Example 6

[0084]

[0085] Compound 6 was obtained by replacing 8-bromo-1-octanol in the fifth step of Example 1 with 6-bromo-1-hexanol using similar method and reaction procedure. ESI-MS (m / z): 723.3 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.59 (d, J = 5.6 Hz, 1H), 11.09 (s, 1H), 10.81 - 10.54 (m, 1H), 8.03 - 7.75 (m, 2H), 7.58 (d, J = 5.8 Hz, 1H), 7.55 - 7.41 (m, 3H), 7.23 - 7.05 (m, 2H), 6.90 - 6.55 (m, 2H), 5.07 (dd, J = 12.6, 5.5 Hz, 1H), 4.21 (t, J = 6.3 Hz, 2H), 3.28 - 2.86 (m, 6H), 2.39 - 2.29 (m, 2H), 2.05 - 1.95 (m, 2H), 1.77 (s, 2H), 1.54 - 1.13 (m, 10H).

[0086] Example 7

[0087]

[0088] First step: 2-(2,6-dioxopiperidin-3-yl)-4-fluoroiso-1,3-dione 7a (100 mg, 0.36 mmol) and 4-hydroxymethylpiperidine (42 mg, 0.36 mmol) were dissolved in DMF (5 mL), N,N-diisopropylethylamine (93 mg, 0.72 mmol) was added to the reaction system, then the reaction system was heated to 80 °C and stirred overnight. The reaction system was reduced to room temperature, diluted with ethyl acetate and extracted with saturated brine 3 times, the organic phase was dried with anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol) to obtain the final product 7b (130 mg, yield 97%). ESI-MS (m / z): 372.8 [M+H] + .

[0089] Second step: 7b (100 mg, 0.27 mmol) was dissolved in dichloromethane (4 mL), and then des-martin periodinane (125 mg, 0.29 mmol) was added to the reaction system. The reaction system was stirred at room temperature for 1 hour, and LCMS detection showed that the substrate reaction was complete. The reaction system was filtered, washed with dichloromethane, and the organic phase was washed with saturated sodium bicarbonate solution, saturated brine, and then dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the final product 7c (76 mg, yield 77%), which was directly used in the next step reaction. ESI-MS (m / z): 370.4 [M+H] + .

[0090] Third step: 1d (50 mg, 0.14 mmol) and 7c (52 mg, 0.14 mmol) were dissolved in dichloroethane (4 mL) and methanol (2 mL) under ice bath conditions, and then sodium acetate (19 mg, 0.14 mmol) was added to the reaction system. After stirring for 0.5 hours, sodium triacetoxyborohydride (59 mg, 0.28 mmol) was added. The system was stirred at room temperature overnight, and LCMS monitoring showed that the reaction was complete. The reaction liquid was concentrated, the residue was dissolved in methanol, and HPLC preparation purification was performed to obtain compound 7 (19 mg, yield 14%). ESI-MS (m / z): 720.5 [M+H] + ; 1H NMR (500 MHz, DMSO-d6) δ 12.61 (d, J = 4.5 Hz, 1H), 12.10 (s, 1H), 11.09 (s, 1H), 10.89 - 10.46 (m, 1H), 7.98 (s, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.59 (d, J = 5.7 Hz, 1H), 7.31 - 7.16 (m, 1H), 7.33 (dd, J = 12.0, 7.8 Hz, 2H), 7.23 - 7.09 (m, 2H), 6.98 - 6.84 (m, 1H), 5.09 (dd, J = 12.7, 5.6 Hz, 1H), 3.71 (d, J = 11.7 Hz, 2H), 3.12 (d, J = 6.1 Hz, 4H), 2.94 - 2.83 (m, 3H), 2.65 - 2.55 (m, 5H), 2.27 (d, J = 7.0 Hz, 2H), 2.10 - 1.70 (m, 5H), 1.38 - 1.28 (m, 2H).

[0091] Example 8

[0092]

[0093] Replacing 2-(2,6-dioxopiperidin-3-yl)-4-fluoroiso-1,3-dione in the first step of Example 7 with 2-(2,6-dioxopiperidin-3-yl)-5-fluoroiso-1,3-dione, Compound 8 can be obtained in a similar manner and reaction procedure. ESI-MS (m / z): 720.8 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.61 (s, 1H), 12.05 (s, 1H), 11.08 (s, 1H), 10.79 - 10.38 (m, 1H), 7.98 (s, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.59 (d, J = 5.7 Hz, 1H), 7.52 - 7.40 (m, 1H), 7.32 (s, 1H), 7.27 - 7.08 (m, 3H), 6.96 - 6.83 (m, 1H), 5.07 (dd, J = 13.1, 5.3 Hz, 1H), 4.06 (d, J = 12.9 Hz, 2H), 3.12 (s, 4H), 3.05 - 2.83 (m, 4H), 2.66 - 2.58 (m, 1H), 2.22 (d, J = 7.0 Hz, 2H), 2.07 - 1.79 (m, 5H), 1.37 - 1.09 (m, 5H).

[0094] Example 9

[0095]

[0096] Compound 9 was obtained by replacing 8-bromo-l-octanol in the fifth step of Example 1 with bromoethanol, using similar methods and reaction procedures. ESI-MS (m / z): 667.9 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.60 (d, J = 5.6 Hz, 1H), 12.09 (s, 1H), 11.11 (s, 1H), 10.74 - 10.59 (m, 1H), 7.97 (d, J = 13.6 Hz, 1H), 7.87 - 7.79 (m, 1H), 7.62 - 7.40 (m, 4H), 7.19 (dd, J = 12.4, 4.0 Hz, 1H), 7.13 - 6.85 (m, 2H), 5.10 (dd, J = 12.8, 5.4 Hz, 1H), 4.39 (t, J = 5.7 Hz, 2H), 3.11 (t, J = 4.9 Hz, 5H), 2.90 - 2.83 (m, 3H), 2.75 (t, J = 4.9 Hz, 4H), 2.09 - 1.94 (m, 2H).

[0097] Example 10

[0098]

[0099] Compound 10 was obtained by replacing 4-hydroxymethylpiperidine in the first step of Example 7 with (S)-pyrrolidin-3-methanol, using similar methods and reaction procedures. ESI-MS (m / z): 706.3 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.62 (s, 1H), 12.08 (s, 1H), 11.05 (s, 1H), 10.83 - 10.50 (m, 1H), 7.95 (s, 1H), 7.61 - 7.54 (m, 2H), 7.52 - 7.43 (m, 1H), 7.32 - 6.97 (m, 4H), 6.90 (d, J = 8.3 Hz, 1H), 5.09 - 5.04 (m, 1H), 3.69 - 3.54 (m, 3H), 3.12 (s, 3H), 2.65 - 2.56 (m, 6H), 2.42 (d, J = 7.3 Hz, 2H), 2.14 - 1.94 (m, 4H), 1.78 - 1.40 (m, 3H).

[0100] Example 11

[0101]

[0102] Compound 11 was obtained by replacing 8-bromo-1-octanol in the fifth step of Example 1 with 1-bromo-1-butanol, using similar procedures and reaction steps. ESI-MS (m / z): 695.8 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.60 (s, 1H), 12.08 (s, 1H), 11.11 (s, 1H), 10.95 - 10.29 (m, 1H), 7.98 (d, J = 11.2 Hz, 1H), 7.81 (dd, J = 8.5, 7.3 Hz, 1H), 7.61 - 7.43 (m, 4H), 7.23 - 7.04 (m, 2H), 6.93 - 6.85 (m, 1H), 5.09 (dd, J = 12.7, 5.4 Hz, 1H), 4.27 (t, J = 6.3 Hz, 2H), 3.10 (s, 4H), 2.93 - 2.82 (m, 1H), 2.61 - 2.53 (m, 6H), 2.42 (t, J = 7.0 Hz, 2H), 2.07 - 1.99 (m, 1H), 1.87 - 1.76 (m, 2H), 1.74 - 1.61 (m, 2H).

[0103] Example 12

[0104]

[0105] First step: 2-(2,6-dioxopiperidin-3-yl)-4-fluoroiso-1,3-dione 7a (100 mg, 0.36 mmol) and (R)-pyrrolidin-3-methanol (40 mg, 0.36 mmol) were dissolved in DMF (5 mL), N,N-diisopropylethylamine (93 mg, 0.72 mmol) was added to the reaction system, then the reaction system was heated to 80 °C and stirred overnight. The reaction system was reduced to room temperature, diluted with ethyl acetate and extracted with saturated brine 3 times, the organic phase was dried with anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol) to obtain the final product 12a (126 mg, yield 95%). ESI-MS (m / z): 358.6 [M+H] + .

[0106] Second step: 12a (100 mg, 0.30 mmol) was dissolved in dichloromethane (4 mL), after adding Dess-Martin Oxidizing agent (125 mg, 0.30 mmol) to the reaction system, the reaction system was stirred at room temperature for 2 hours. The reaction system was filtered, washed with dichloromethane, and the organic phase was washed with saturated sodium bicarbonate solution, saturated brine, then dried over anhydrous sodium sulfate, filtered, concentrated to give the final product 12b (73 mg, yield 78%), which was directly used in the next step reaction. ESI-MS (m / z): 356.4 [M+H] + .

[0107] Third step: 1d (50 mg, 0.14 mmol) and 12b (48 mg, 0.14 mmol) were dissolved in dichloroethane (4 mL) and methanol (2 mL) under ice bath conditions, sodium acetate (19 mg, 0.14 mmol) was added to the reaction system, then the reaction system was stirred for 0.5 hours, then sodium triacetoxyborohydride (59 mg, 0.28 mmol) was added. The system was stirred at room temperature overnight. The reaction liquid was concentrated, the residue was dissolved in DMF, and HPLC preparation purification gave compound 12 (14 mg, yield 18%). ESI-MS (m / z): 706.1 [M+H] + ; 1 HNMR (500 MHz, DMSO-d6) δ 12.61 (d, J = 4.7 Hz, 1H), 12.10 (s, 1H), 11.05 (s, 1H), 10.88 - 10.57 (m, 1H), 7.97 (s, 1H), 7.61 - 7.53 (m, 2H), 7.50 - 7.36 (m, 1H), 7.22 - 7.09 (m, 4H), 6.93 - 6.87 (m, 1H), 5.07 (ddd, J = 12.7, 5.4, 1.5 Hz, 1H), 3.67 - 3.62 (m, 2H), 3.59 - 3.53 (m, 2H), 3.12 (t, J = 5.3 Hz, 5H), 2.91 - 2.83 (m, 1H), 2.66 - 2.55 (m, 6H), 2.44 - 2.41 (m, 2H), 2.11 - 2.06 (m, 1H), 2.03 - 1.97 (m, 1H), 1.74 - 1.68 (m, 1H).

[0108] Example 17

[0109]

[0110] Step 1 : Compound 7c (70 mg, 0.19 mmol), oxone (116 mg, 0.19 mmol) were dissolved in DMF (3 mL) and stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate and extracted with water and saturated brine three times. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give the final product 17a (45 mg, yield 62%). ESI-MS (m / z): 386.8 [M+H] + .

[0111] Step 2: Compound 17a (35 mg, 0.09 mmol) was dissolved in DMF (2 mL) and stirred at 0 °C. HATU (68 mg, 0.18 mmol) was added, followed by DIPEA (57 mg, 0.45 mmol) and compound 1d (43 mg, 0.09 mmol) after 10 min. The reaction mixture was stirred at room temperature overnight. The reaction mixture was filtered and purified by HPLC to give compound 17 (12 mg, yield 18%). ESI-MS (m / z): 734.8 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.65 (s, 1H), 12.32 - 11.78 (br, 1H), 11.09 (s, 1H), 10.84 - 10.38 (m, 1H), 8.11 - 7.89 (m, 1H), 7.69 (dd, J = 8.5, 7.1 Hz, 1H), 7.59 (d, J = 5.8 Hz, 1H), 7.550 - 7.39 (m, 1H), 7.35 (dd, J = 11.6, 7.8 Hz, 2H), 7.27 - 7.15 (m, 2H), 6.98 - 6.91 (m, 1H), 5.10 (dd, J = 12.8, 5.4 Hz, 1H), 3.78 - 3.65 (m, 6H), 3.16 - 3.06 (m, 4H), 3.03 - 2.85 (m, 4H), 2.65 - 2.54 (m, 2H), 2.03 (ddd, J = 10.7, 5.6, 3.1 Hz, 1H), 1.88 - 1.77 (m, 4H).

[0112] Example 18

[0113]

[0114] First step: 2-(2,6-dioxopiperidin-3-yl)-5-fluoroiso-1,3-dione (100 mg, 0.36 mmol) and 4-hydroxymethylpiperidine (42 mg, 0.36 mmol) were dissolved in DMF (5 mL), N,N- diisopropylethylamine (93 mg, 0.72 mmol) was added to the reaction system, then the reaction system was heated to 80 °C and stirred overnight. The reaction system was cooled to room temperature, diluted with ethyl acetate and extracted with saturated brine 3 times, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel column chromatography to obtain the final product 18a (124 mg, yield 95%). ESI-MS (m / z): 372.7 [M+H] + .

[0115] Second step: 18a (70 mg, 0.19 mmol) and oxone (116 mg, 0.19 mmol) were dissolved in DMF (3 mL) and stirred at room temperature overnight, the reaction system was diluted with ethyl acetate and extracted with water and saturated brine 3 times, the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the final product 18b (52 mg, yield 71%). ESI-MS (m / z): 370.5 [M+H] + .

[0116] Third step: 18b (35 mg, 0.09 mmol) was dissolved in DMF (2 mL) and stirred at 0 °C, 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (68 mg, 0.18 mmol) was added, after 10 minutes, DIPEA (57 mg, 0.45 mmol) and compound 1d (43 mg, 0.09 mmol) were added in turn, the temperature was raised to room temperature and stirred overnight. The reaction system was filtered and purified by HPLC preparation to obtain compound 18 (16 mg, yield 24%). ESI-MS (m / z): 734.7 [M+H] + ; 1H NMR (500 MHz, DMSO-d6) δ 12.60 (s, 1H), 12.31 - 11.60 (m, 1H), 11.01 (s, 1H), 10.87 - 10.30 (m, 1H), 8.10 - 7.77 (m, 1H), 7.60 (d, J = 8.5 Hz, 1H), 7.52 (d, J = 5.7 Hz, 1H), 7.49 - 7.39 (m, 1H), 7.27 (d, J = 2.3 Hz, 1H), 7.21 - 7.07 (m, 3H), 6.91 - 6.84 (m, 1H), 5.00 (dd, J = 12.8, 5.5 Hz, 1H), 4.02 (d, J = 13.1 Hz, 2H), 3.90 - 3.25 (m, 4H), 3.11 - 2.97 (m, 7H), 2.86 - 2.78 (m, 1H), 2.59 - 2.47 (m, 2H), 1.98 - 1.91 (m, 1H), 1.72 - 1.54 (m, 4H).

[0117] Example 19

[0118]

[0119] First step: 19a (1 g, 5.79 mmol) and 19a-1 (1.97 g, 6.37 mmol) were added to a mixed solvent of 1,4-dioxane (16 mL) and water (4 mL), and then potassium carbonate solid (2.40 g, 17.38 mmol) and Pd(dppf)Cl2(424.01 mg, 0.58 mmol) were added. The reaction system was replaced with nitrogen three times with a nitrogen balloon, and reacted at 90 °C for 3 hours under the protection of a nitrogen balloon. After the reaction was completed, the reaction solution was cooled to room temperature. The reaction solution was diluted with ethyl acetate, and washed with water and saturated brine in turn. The organic phase was dried over anhydrous sodium sulfate. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol) to obtain yellow solid 19b (1.41 g, yield 76.3%). ESI-MS (m / z): 320.6 [M+H] + .

[0120] Second step: 19b (1.41 g, 4.42 mmol) was dissolved in anhydrous methanol (30 mL), 10% palladium-carbon (0.56 g, 5.30 mmol) was added, the reaction system was replaced with hydrogen by a hydrogen balloon, and stirred at 25 °C for 3 hours under the pressure of the hydrogen balloon. Then 3-ethoxy-3-iminopropionic acid ethyl ester hydrochloride (1.29 g, 4.43 mmol) was added, and the reaction was continued to stir at 70 °C for 4 hours. The system was filtered with diatomite, washed with dichloromethane, and the filtrate was concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol) to obtain brown solid 19c (1.72 g, yield 100%). ESI-MS (m / z): 388.6 [M+H] + .

[0121] Third step: 19c (0.5 g, 1.29 mmol) and 2-aminothiophene-3-carbonitrile (160 mg, 1.29 mmol) were dissolved in tetrahydrofuran (10 mL), and 1M LiHMDS THF solution (12.9 mL, 12.9 mmol) was added under nitrogen protection. The reaction system was stirred at 35 °C for 2 hours. The system was quenched with saturated ammonium chloride, extracted with ethyl acetate three times, and the organic phase was washed with water and saturated sodium chloride solution successively. The organic phase was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol) to obtain brown solid 19d (457 mg, yield 76.1%). ESI-MS (m / z): 466.7 [M+H] + .

[0122] Fourth step: 19d (457 mg, 0.98 mmol) was dissolved in dioxane (5 mL) and dichloromethane (5 mL), and 4M HCl dioxane solution (5 mL) was added. The reaction was stirred at room temperature for 3 hours. The reaction system was concentrated to obtain compound 19e (429 mg, yield 99%). ESI-MS (m / z): 366.2 [M+H] + .

[0123] Fifth step: 19e (100 mg, 0.27 mmol) and 7c (101 mg, 0.27 mmol) were dissolved in dichloroethane (4 mL) and methanol (2 mL) under ice bath conditions. Sodium acetate (37 mg, 0.27 mmol) was added to the reaction system, followed by stirring for 0.5 hours. Then sodium triacetoxyborohydride (35 mg, 0.55 mmol) was added, and the system was stirred at room temperature overnight. The reaction was concentrated, and the residue was dissolved in DMF. Compound 19 (21 mg, yield 11%) was obtained by HPLC preparation purification. ESI-MS (m / z): 719.4 [M+H] + ; 1H NMR (500 MHz, DMSO-d6) δ 12.72 (s, 1H), 12.11 (s, 1H), 11.09 (s, 1H), 10.85 - 10.56 (m, 1H), 8.19 (s, 1H), 7.68 (dd, J = 8.5, 7.1 Hz, 1H), 7.62 - 7.53 (m, 2H), 7.52 - 7.44 (m, 1H), 7.33 (dd, J = 13.1, 7.8 Hz, 2H), 7.19 (d, J = 5.7 Hz, 1H), 7.05 (d, J = 8.5 Hz, 1H), 5.10 (dd, J = 12.7, 5.5 Hz, 1H), 3.71 (d, J = 11.6 Hz, 2H), 3.02 (d, J = 10.7 Hz, 2H), 2.93 - 2.86 (m, 3H), 2.63 - 2.56 (m, 2H), 2.27 (d, J = 7.0 Hz, 2H), 2.10 - 2.01 (m, 3H), 1.89 - 1.71 (m, 8H), 1.40 - 1.31 (m, 2H).

[0124] Example 20

[0125]

[0126] Compound 20 was obtained by replacing 4-hydroxymethylpiperidine in the first step of Example 7 with trans-4-aminocyclohexylmethanol in a similar manner and reaction procedure. ESI-MS (m / z): 734.8 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.61 (s, 1H), 12.10 (s, 1H), 11.10 (s, 1H), 10.80 - 10.51 (m, 1H), 7.98 (s, 1H), 7.67 - 7.41 (m, 3H), 7.26 - 6.99 (m, 4H), 6.95 - 6.86 (m, 1H), 6.19 (d, J = 8.2 Hz, 1H), 5.05 (dd, J = 12.8, 5.5 Hz, 1H), 3.11 (t, J = 4.7 Hz, 6H), 2.94 - 2.79 (m, 2H), 2.64 - 2.55 (m, 2H), 2.54 - 2.53 (m, 2H), 2.19 (d, J = 7.1 Hz, 2H), 2.07 - 1.99 (m, 3H), 1.87 (d, J = 12.8 Hz, 2H), 1.64 - 1.52 (m, 1H), 1.32 - 1.22 (m, 2H), 1.07 (q, J = 12.1 Hz, 2H).

[0127] Example 21

[0128]

[0129] Compound 21 was obtained by replacing 4-hydroxymethylpiperidine in the first step of Example 7 with 4-piperidinethanol using similar method and reaction procedure. ESI-MS (m / z): 734.5 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.60 (s, 1H), 12.10 (s, 1H), 11.09 (s, 1H), 10.71 - 10.54 (m, 1H), 7.98 (s, 1H), 7.71 - 7.56 (m, 2H), 7.44 (d, J = 8.7 Hz, 1H), 7.33 (t, J = 8.5 Hz, 2H), 7.23 - 7.05 (m, 2H), 6.93 - 6.85 (m, 1H), 5.09 (dd, J = 12.7, 5.5 Hz, 1H), 3.69 (d, J = 11.6 Hz, 3H), 3.12 (t, J = 4.9 Hz, 5H), 2.91 - 2.83 (m, 3H), 2.60 - 2.55 (m, 4H), 2.45 - 2.40 (m, 2H), 2.06 - 1.96 (m, 1H), 1.81 (d, J = 12.0 Hz, 2H), 1.53 - 1.33 (m, 5H).

[0130] Example 22

[0131]

[0132] Compound 22 was obtained by replacing 4-hydroxymethylpiperidine in the first step of Example 7 with (R)-3-piperidinemethanol using similar method and reaction procedure. ESI-MS (m / z): 720.4 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.69 - 12.53 (m, 1H), 12.09 (s, 1H), 11.14 - 11.04 (m, 1H), 10.81 - 10.50 (m, 1H), 8.05 - 7.92 (m, 1H), 7.74 - 7.65 (m, 1H), 7.63 - 7.56 (m, 1H), 7.54 - 7.41 (m, 1H), 7.38 - 7.29 (m, 2H), 7.21 - 7.03 (m, 2H), 6.96 - 6.81 (m, 1H), 5.17 - 5.03 (m, 1H), 3.84 - 3.55 (m, 2H), 3.09 (s, 4H), 2.96 - 2.84 (m, 2H), 2.72 - 2.54 (m, 4H), 2.33 - 2.16 (m, 2H), 2.08 - 1.96 (m, 6H), 1.87 - 1.66 (m, 3H).

[0133] Example 23

[0134]

[0135] Compound 23 was obtained by replacing 4-hydroxymethylpiperidine with (S)-3-piperidinemethanol in the first step of Example 7, using similar method and reaction procedures. ESI-MS (m / z): 720.7 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.60 (d, J = 8.6 Hz, 1H), 12.08 (s, 1H), 11.21 - 11.00 (m, 1H), 10.83 - 10.53 (m, 1H), 8.10 - 7.91 (m, 1H), 7.73 - 7.65 (m, 1H), 7.60 (dd, J = 7.9, 5.8 Hz, 1H), 7.54 - 7.41 (m, 1H), 7.39 - 7.27 (m, 2H), 7.21 - 7.02 (m, 2H), 6.93 - 6.83 (m, 1H), 5.18 - 5.01 (m, 1H), 3.84 - 3.56 (m, 2H), 3.14 - 3.00 (m, 4H), 2.93 - 2.84 (m, 2H), 2.70 - 2.55 (m, 4H), 2.48 - 2.39 (m, 2H), 2.02 - 1.92 (m, 6H), 1.88 - 1.64 (m, 3H).

[0136] Example 24

[0137]

[0138] Compound 24 was obtained by replacing 8-bromo-1-octanol with 9-bromo-1- nonanol in the fifth step of Example 1, and replacing 2-(2,6-dioxopiperidin-3-yl)-4- hydroxyiso-1,3-dione with (2,6-dioxopiperidin-3-yl)-5-hydroxyiso-1,3-dione in the fifth step of Example 1, using similar method and reaction procedures. ESI-MS (m / z): 765.9 [M+H] + ; 1H NMR (500 MHz, DMSO-d6) δ 12.60 (d, J = 6.1 Hz, 1H), 12.09 (s, 1H), 11.11 (s, 1H), 10.79 - 10.53 (m, 1H), 7.97 (d, J = 13.0 Hz, 1H), 7.82 (d, J = 8.3 Hz, 1H), 7.59 (d, J = 5.7 Hz, 1H), 7.53 - 7.39 (m, 2H), 7.34 (dd, J = 8.3, 2.3 Hz, 1H), 7.21 - 7.07 (m, 2H), 6.92 - 6.82 (m, 1H), 5.12 (dd, J = 12.8, 5.4 Hz, 1H), 4.17 (t, J = 6.5 Hz, 2H), 3.09 (t, J = 4.9 Hz, 4H), 2.92 - 2.84 (m, 1H), 2.66 - 2.53 (m, 3H), 2.31 (t, J = 7.4 Hz, 2H), 2.11 - 1.96 (m, 2H), 1.76 (p, J = 6.8 Hz, 2H), 1.48 - 1.26 (m, 14H).

[0139] Example 25

[0140]

[0141] Compound 25 was obtained by replacing 7c in the fifth step of Example 19 with 12b, using similar methods and reaction procedures. ESI-MS (m / z): 705.9 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.75 (s, 1H), 12.12 (d, J = 8.7 Hz, 1H), 11.07 (s, 1H), 10.78 - 10.58 (m, 1H), 8.06 (s, 1H), 7.62 - 7.45 (m, 4H), 7.19 (d, J = 5.6 Hz, 1H), 7.14 (t, J = 8.3 Hz, 2H), 7.06 (d, J = 8.3 Hz, 1H), 5.11 - 5.04 (m, 1H), 3.86 - 3.40 (m, 5H), 2.95 - 2.81 (m, 2H), 2.70 - 2.54 (m, 3H), 2.29 - 1.65 (m, 10H), 1.27 - 1.20 (m, 2H).

[0142] Example 26

[0143]

[0144] First step: Dissolve tert-butyl 2,6-diazaspiro[3.4]octane-6-carboxylate (1.0 g, 4.7 mmol) and 5-fluoro-2-nitroaniline (0.74 g, 4.7 mmol) in NMP (10 mL), add DIPEA (1.64 mL, 9.4 mmol), and react at 120 °C overnight. Dilute the reaction with ethyl acetate, wash sequentially with water and saturated brine, dry the organic phase over anhydrous sodium sulfate, filter, and concentrate to give yellow solid 26a (1.58 g, 96% yield). ESI-MS (m / z): 349.6 [M+H] + .

[0145] Second step: Dissolve 26a (1.6 g, 4.6 mmol) in anhydrous ethanol (20 mL), add 10% palladium on carbon (50 mg, 0.46 mmol), and stir the reaction system under hydrogen balloon pressure at 40 °C overnight. Monitor the reaction for complete consumption of the substrate by LCMS. Then add 3-ethoxy-3-iminopropionic acid ethyl ester hydrochloride (1.35 g, 6.9 mmol) to the reaction, and continue stirring at 80 °C for 4 hours. Filter the system over celite, wash with dichloromethane, and concentrate the filtrate to give yellow solid 26b (1.47 g, 77% yield). ESI-MS (m / z): 415.5 [M+H] + .

[0146] Third step: Dissolve 26b (1.47 g, 3.55 mmol) and 2-aminothiophene-3-carbonitrile (0.44 g, 3.55 mmol) in tetrahydrofuran (10 mL), and add 2M LDA in THF (14 mL, 28.4 mmol) under nitrogen protection. Stir the system at 40 °C for 2 hours. Quench the reaction system with saturated ammonium chloride, extract three times with dichloromethane, and wash the organic phase sequentially with water and saturated sodium chloride solution. Concentrate the organic phase, and purify the residue by silica gel column chromatography (dichloromethane / methanol) to give brown solid 26c (381 mg, 22% yield). ESI-MS (m / z): 493.7 [M+H] + .

[0147] Fourth step: Dissolve 26c (300 mg, 0.6 mmol) in TFA (2 mL) and dichloromethane (4 mL), and stir at ice bath temperature for 2 hours. Concentrate the reaction system to give 26d (300 mg, 97% yield). ESI-MS (m / z): 393.6 [M+H] + .

[0148] Step 5: 26d (70 mg, 0.14 mmol) and 12b (49 mg, 0.14 mmol) were dissolved in dichloroethane (4 mL) and methanol (2 mL) under ice-bath condition, sodium acetate (37 mg, 0.28 mmol) was added to the reaction system, then the reaction system was stirred for 0.5 h, then sodium triacetoxyborohydride (88 mg, 0.42 mmol) was added, the system was stirred at room temperature overnight. After the reaction was completed, the reaction liquid was concentrated, the residue was dissolved in DMF, and compound 26 (10 mg, 10% yield) was obtained by HPLC preparation purification. ESI-MS (m / z): 732.4 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.64 - 12.40 (m, 1H), 12.09 (s, 1H), 11.07 (s, 1H), 10.84 - 10.41 (m, 1H), 8.21 (s, 2H), 7.95 (s, 1H), 7.63 - 7.51 (m, 2H), 7.50 - 7.36 (m, 1H), 7.18 (d, J = 5.6 Hz, 1H), 7.16 - 7.04 (m, 2H), 6.72 - 6.58 (m, 1H), 6.35 (d, J = 8.4 Hz, 1H), 5.12 - 5.01 (m, 1H), 3.81 - 3.67 (m, 9H), 2.95 - 2.75 (m, 4H), 2.64 - 2.53 (m, 4H), 2.18 - 1.91 (m, 6H).

[0149] Example 27

[0150]

[0151] Compound 27 was obtained by replacing 12b in Step 5 of Example 26 with 15b, using similar methods and reaction procedures. ESI-MS (m / z): 718.4 [M+H] + ; 1H NMR (500 MHz, DMSO-d6) δ 12.63 - 12.30 (m, 1H), 12.07 (s, 1H), 11.07 (s, 1H), 10.87 - 10.52 (m, 1H), 8.19 (s, 2H), 7.96 (d, J = 13.0 Hz, 1H), 7.62 - 7.52 (m, 2H), 7.49 - 7.28 (m, 1H), 7.18 (d, J = 5.7 Hz, 1H), 7.10 (d, J = 6.9 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H), 6.72 - 6.59 (m, 1H), 6.36 (d, J = 8.7 Hz, 1H), 5.05 (dd, J = 12.7, 5.5 Hz, 1H), 4.35 - 4.28 (m, 2H), 3.90 - 3.67 (m, 6H), 2.98 - 2.35 (m, 2H), 2.78 (s, 2H), 2.70 (d, J = 7.5 Hz, 2H), 2.63 - 2.50 (m, 4H), 2.08 - 1.97 (m, 3H).

[0152] Example 28

[0153]

[0154] Compound 28 was obtained by replacing tert-butyl 2,6-diazaspiro[3.4]octane-6- carboxylate with tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate in the first step of Example 26, in a similar manner and reaction procedure. ESI-MS (m / z): 774.4 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.58 (d, J = 10.3 Hz, 1H), 12.08 (s, 1H), 11.06 (s, 1H), 10.80 - 10.53 (m, 1H), 7.97 (s, 1H), 7.63 - 7.52 (m, 2H), 7.50 - 7.40 (m, 1H), 7.23 - 7.14 (m, 2H), 7.13 - 7.10 (m, 2H), 6.93 - 6.86 (m, 1H), 5.07 (dd, J = 12.8, 5.5 Hz, 1H), 3.63 - 3.54 (m, 2H), 3.08 (s, 4H), 2.92 - 2.84 (m, 1H), 2.42 - 2.34 (m, 6H), 2.06 - 1.97 (m, 4H), 1.73 - 1.56 (m, 6H), 1.53 - 1.43 (m, 6H).

[0155] Example 29

[0156]

[0157] Using tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate instead of tert-butyl 2,6-diazaspiro[3.4]octane-6-carboxylate in the first step of Example 26, and using 15b instead of 12b in the fifth step of Example 26, compound 29 can be obtained in a similar manner and reaction procedure. ESI-MS (m / z): 760.4 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.60 (s, 1H), 12.07 (s, 1H), 11.07 (s, 1H), 10.62 (s, 1H), 8.32 (s, 1H), 7.60 - 7.53 (m, 1H), 7.51 - 7.40 (m, 1H), 7.26 - 7.04 (m, 4H), 6.91 - 6.63 (m, 2H), 5.05 (dd, J = 12.8, 5.5 Hz, 1H), 4.29 (s, 2H), 3.93 - 3.62 (m, 1H), 3.29 - 2.99 (m, 4H), 2.92 - 2.83 (m, 2H), 2.39 - 2.36 (m, 4H), 2.02 - 1.97 (m, 4H), 1.67 - 1.45 (m, 10H).

[0158] Example 30

[0159]

[0160] Using tert-butyl 2,8-diazaspiro[4.5]decane-2-carboxylate instead of tert-butyl 2,6-diazaspiro[3.4]octane-6-carboxylate in the first step of Example 26, compound 30 can be obtained in a similar manner and reaction procedure. ESI-MS (m / z): 760.8 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.59 (d, J = 8.2 Hz, 1H), 12.08 (s, 1H), 11.07 (s, 1H), 10.83 - 10.54 (m, 1H), 8.05 - 7.91 (m, 1H), 7.61 - 7.53 (m, 2H), 7.51 - 7.41 (m, 1H), 7.26 - 7.09 (m, 3H), 6.94 - 6.77 (m, 2H), 5.34 - 5.02 (m, 1H), 4.51 - 4.21 (m, 2H), 3.88 (s, 2H), 3.29 - 3.01 (m, 4H), 2.94 - 2.53 (m, 8H), 2.21 - 1.96 (m, 2H), 187 - 1.46 (m, 6H), 1.44 - 1.12 (m, 3H).

[0161] Example 31

[0162]

[0163] Compound 31 was obtained by replacing tert-butyl 2,6-diazaspiro[3.4]octane-6- carboxylate in the first step of Example 26 with tert-butyl 2,8-diazaspiro[4.5]decane-2- carboxylate, while replacing 12b in the fifth step of Example 26 with 15b, in a similar manner and reaction procedure. ESI-MS (m / z): 746.9 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.58 (d, J = 9.0 Hz, 1H), 12.08 (s, 1H), 11.06 (s, 1H), 10.80 - 10.56 (m, 1H), 8.16 - 7.91 (m, 1H), 7.60 - 7.54 (m, 2H), 7.50 - 7.41 (m, 1H), 7.21 - 7.10 (m, 4H), 6.91 - 6.86 (m, 1H), 5.06 (dd, J = 12.9, 5.5 Hz, 1H), 3.67 - 3.52 (m, 3H), 3.15 - 2.82 (m, 6H), 2.70 - 2.53 (m, 4H), 2.12 - 1.97 (m, 3H), 1.76 - 1.41 (m, 9H).

[0164] Example 32

[0165]

[0166] Compound 32 was obtained by replacing tert-butyl 2,6-diazaspiro[3.4]octane-6- carboxylate in the first step of Example 26 with 2-tert-butoxycarbonyl-2,7- diazaspiro[3.5]nonane, while replacing 12b in the fifth step of Example 26 with 15b, in a similar manner and reaction procedure. ESI-MS (m / z): 731.7 [M+H] + ; 1H NMR (500 MHz, DMSO-d6) δ 12.60 (s, 1H), 12.13 (s, 1H), 11.09 (s, 1H), 10.89 - 10.52 (m, 1H), 7.98 (s, 1H), 7.60 - 7.53 (m, 2H), 7.50 - 7.36 (m, 1H), 7.23 - 7.08 (m, 3H), 6.88 (d, J = 8.5 Hz, 1H), 6.77 (d, J = 8.4 Hz, 1H), 5.04 (dd, J = 12.8, 5.5 Hz, 1H), 4.25 (t, 2H), 3.84 (t, 3H), 3.03 (s, 7H), 2.93 - 2.80 (m, 2H), 2.69 (s, 3H), 2.60 - 2.55 (m, 1H), 2.02 - 1.95 (m, 1H), 1.82 (t, J = 5.4 Hz, 4H).

[0167] Example 33

[0168]

[0169] Compound 33 was obtained by replacing tert-butyl 2,6-diazaspiro[3.4]octane-6- carboxylate in the first step of Example 26 with 2-tert-butoxycarbonyl-2,7- diazaspiro[3.5]nonane, in a similar manner and procedure. ESI-MS (m / z): 746.6 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.59 (d, J = 8.8 Hz, 1H), 12.07 (s, 1H), 11.06 (s, 1H), 10.62 (s, 1H), 7.97 (s, 1H), 7.61 - 7.53 (m, 2H), 7.50 - 7.30 (m, 1H), 7.22 - 7.08 (m, 4H), 6.91 - 6.85 (m, 1H), 5.06 (ddd, J = 12.5, 5.5, 1.9 Hz, 1H), 3.66 - 3.47 (m, 3H), 3.35 (s, 4H), 3.05 - 2.99 (m, 7H), 2.90 - 2.84 (m, 1H), 2.61 - 2.55 (m, 1H), 2.48 - 2.47 (m, 1H), 2.27 - 2.21 (m, 1H), 2.06 - 1.98 (m, 2H), 1.85 - 1.76 (m, 4H), 1.68 - 1.61 (m, 1H).

[0170] Example 34

[0171]

[0172] Using tert-butyl 2,6-diazaspiro[3.4]octane-2-carboxylate instead of tert-butyl 2,6-diazaspiro[3.4]octane-6-carboxylate in the first step of Example 26, and using 15b instead of 12b in the fifth step of Example 26, Compound 35 can be obtained in a similar manner and reaction procedure. ESI-MS (m / z): 718.2 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.60 - 12.42 (m, 1H), 12.10 (s, 1H), 11.06 (s, 1H), 10.72 - 10.53 (m, 1H), 7.95 (s, 1H), 7.67 - 7.52 (m, 2H), 7.40 (dd, J = 8.5 Hz, 1H), 7.22 - 7.10 (m, 3H), 6.80 - 6.45 (m, 1H), 6.35 (d, J = 8.1 Hz, 1H), 5.07 (dd, J = 12.8, 5.5 Hz, 1H), 3.64 - 3.59 (m, 3H), 3.56 - 3.51 (m, 6H), 3.02 - 2.80 (m, 2H), 2.61 - 2.54 (m, 2H), 2.42 - 2.28 (m, 5H), 2.09 - 1.98 (m, 2H), 1.81 - 1.63 (m, 5H).

[0173] Example 35

[0174]

[0175] Using tert-butyl 2,6-diazaspiro[3.4]octane-2-carboxylate instead of tert-butyl 2,6-diazaspiro[3.4]octane-6-carboxylate in the first step of Example 26, and using 15b instead of 12b in the fifth step of Example 26, Compound 35 can be obtained in a similar manner and reaction procedure. ESI-MS (m / z): 718.2 [M+H] + ; 1H NMR (500 MHz, DMSO-d6) δ 12.53 - 12.38 (m, 1H), 12.07 (s, 1H), 11.07 (s, 1H), 10.80 - 10.59 (m, 1H), 8.20 (s, 1H), 8.00 - 7.88 (m, 1H), 7.59 - 7.53 (m, 2H), 7.48 - 7.38 (m, 1H), 7.17 (d, J = 5.6 Hz, 1H), 7.10 (d, J = 6.9 Hz, 1H), 6.80 - 6.75 (m, 2H), 6.54 - 6.47 (m, 1H), 5.04 (dd, J = 12.7, 5.5 Hz, 1H), 4.25 (s, 2H), 3.86 (s, 2H), 3.23 - 3.18 (m, 7H), 2.92 - 2.82 (m, 2H), 2.74 - 2.54 (m, 5H), 2.15 (t, J = 6.8 Hz, 2H), 2.06 - 1.95 (m, 2H).

[0176] Example 36

[0177]

[0178] Compound 36 was obtained by replacing tert-butyl 2,6-diazaspiro[3.4]octane-6- carboxylate in the first step of Example 26 with tert-butyl 2,6-diaza-spiro[3.4]octane-2- carbonate, in a similar manner and procedure. ESI-MS (m / z): 732.4 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.52 (s, 1H), 12.08 (s, 1H), 11.06 (s, 1H), 10.83 - 10.50 (m, 1H), 8.02 - 7.78 (m, 1H), 7.59 - 7.53 (m, 2H), 7.47 - 7.38 (m, 1H), 7.17 - 7.10 (m, 3H), 6.78 - 6.67 (m, 1H), 6.50 (d, J = 8.4 Hz, 1H), 5.06 (dd, J = 12.8, 5.3 Hz, 1H), 3.64 - 3.48 (m, 4H), 3.23 - 3.16 (m, 6H), 2.91 - 2.84 (m, 1H), 2.65 - 2.55 (m, 2H), 2.32 - 2.21 (m, 2H), 2.15 (t, J = 6.3 Hz, 2H), 2.07 - 1.97 (m, 4H), 1.75 - 1.58 (m, 2H).

[0179] Example 37

[0180]

[0181] First Step: 2-(2,6-dioxopiperidin-3-yl)-5-fluoroiso-1,3-dione (100 mg, 0.36 mmol) and azetidin-3-ylmethanol (31 mg, 0.36 mmol) were dissolved in DMF (5 mL), DIPEA (93 mg, 0.72 mmol) was added to the reaction system, then the reaction system was heated to 80 °C and stirred overnight. The reaction system was reduced to room temperature, diluted with ethyl acetate and extracted with saturated brine 3 times, the organic phase was dried with anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol) to obtain the final product 37a (120 mg, yield 97%). ESI-MS (m / z): 343.8 [M+H] + .

[0182] Second Step: 37a (120 mg, 0.34 mmol) was dissolved in dichloromethane (4 mL), and Dess-Martin oxidant (158 mg, 0.37 mmol) was added to the reaction system, then the reaction system was stirred at room temperature for 1 hour. The reaction system was filtered, washed with dichloromethane, and the organic phase was washed with saturated sodium bicarbonate solution and saturated brine successively, then dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the final product 37b (98 mg, yield 84%), which was directly used in the next step. ESI-MS (m / z): 341.2 [M+H] + .

[0183] Third Step: 26d (80 mg, 0.16 mmol) and 37b (54 mg, 0.16 mmol) were dissolved in 1,2-dichloroethane (4 mL) and methanol (2 mL) under ice bath conditions, sodium acetate (43 mg, 0.32 mmol) was added to the reaction system, then the reaction system was stirred for 0.5 hours, and then sodium triacetoxyborohydride (100 mg, 0.47 mmol) was added, and the system was stirred at room temperature overnight. The reaction liquid was concentrated, the residue was dissolved in methanol, and HPLC preparation purification was performed to obtain compound 37 (9 mg, yield 8%). ESI-MS (m / z): 718.4 [M+H] + ; 1H NMR (500 MHz, DMSO-d6) δ 12.62 - 12.41 (m, 1H), 12.09 (s, 1H), 11.07 (s, 1H), 10.79 - 10.53 (m, 1H), 8.30 (s, 2H), 7.96 (s, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.58 (dd, J = 5.9, 2.2 Hz, 1H), 7.49 - 7.37 (m, 1H), 7.18 (d, J = 5.6 Hz, 1H), 6.78 (d, J = 2.1 Hz, 1H), 6.71 - 6.61 (m, 2H), 6.40 - 6.32 (m, 1H), 5.05 (dd, J = 12.7, 5.5 Hz, 1H), 4.15 (t, J = 8.2 Hz, 2H), 3.78 - 3.69 (m, 6H), 2.98 - 2.85 (m, 3H), 2.78 - 2.58 (m, 6H), 2.06 - 1.98 (m, 4H).

[0184] Example 38

[0185]

[0186] Using tert-butyl 2,6-diazaspiro[3.4]octane-6-carboxylate in place of tert-butyl 2,6-diazaspiro[3.4]octane-2-carboxylate in the first step of Example 26, and 37b in place of 12b in the fifth step of Example 26, compound 38 can be obtained in a similar manner and reaction procedure. ESI-MS (m / z): 718.4 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.54 - 12.42 (m, 1H), 12.06 (s, 1H), 11.07 (s, 1H), 10.81 - 10.55 (m, 1H), 8.31 (s, 1H), 8.01 - 7.84 (m, 1H), 7.63 (d, J = 8.3 Hz, 1H), 7.61 - 7.56 (m, 1H), 7.49 - 7.37 (m, 1H), 7.17 (d, J = 5.7 Hz, 1H), 6.79 - 6.68 (m, 2H), 6.63 (dd, J = 8.4, 2.1 Hz, 1H), 6.55 - 6.47 (m, 1H), 5.05 (dd, J = 12.8, 5.5 Hz, 1H), 4.09 (t, J = 8.0 Hz, 2H), 3.70 (dd, J = 8.4, 5.2 Hz, 2H), 3.27 - 3.18 (m, 7H), 2.91 - 2.84 (m, 1H), 2.77 - 2.54 (m, 5H), 2.18 - 2.12 (m, 2H), 2.06 - 1.94 (m, 2H).

[0187] Example 39

[0188]

[0189] Compound 39 was obtained by replacing tert-butyl 2,6-diazaspiro[3.4]octane-6- carboxylate with tert-butyl 2,8-diazaspiro[4.5]decane-2-carboxylate in the first step of Example 26, while replacing 12b with 37b in the fifth step of Example 26, in a similar manner and reaction procedure. ESI-MS (m / z): 746.3 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.59 (d, J = 9.0 Hz, 1H), 12.07 (s, 1H), 11.07 (s, 1H), 10.80 - 10.54 (m, 1H), 8.44 - 7.84 (m, 1H), 7.74 - 7.60 (m, 2H), 7.58 - 7.35 (m, 1H), 7.22 - 7.10 (m, 2H), 6.89 (dt, J = 9.0, 3.0 Hz, 1H), 6.78 (d, J = 2.2 Hz, 1H), 6.64 (dd, J = 8.3, 2.1 Hz, 1H), 5.35 - 5.00 (m, 1H), 4.13 (t, J = 8.1 Hz, 2H), 3.70 (dd, J = 8.4, 5.4 Hz, 2H), 3.17 - 2.83 (m, 8H), 2.71 - 2.62 (m, 2H), 2.61 - 2.54 (m, 3H), 2.43 - 2.39 (m, 2H), 1.72 - 1.59 (m, 6H).

[0190] Example 40

[0191]

[0192] Compound 40 was obtained by replacing tert-butyl 2,6-diazaspiro[3.4]octane-6- carboxylate with tert-butyl 2,7-diazaspiro[3.5]nonane-7-carboxylate in the first step of Example 26, while replacing 12b with 15b in the fifth step of Example 26, in a similar manner and reaction procedure. ESI-MS (m / z): 732.9 [M+H] + ; 1H NMR (500 MHz, DMSO-d6) δ 12.62 - 12.38 (m, 1H), 12.09 (s, 1H), 11.07 (s, 1H), 10.52 - 10.39 (m, 1H), 7.95 (s, 1H), 7.60 - 7.52 (m, 2H), 7.50 - 7.38 (m, 1H), 7.34 - 7.02 (m, 2H), 6.83 - 6.55 (m, 2H), 6.35 (dd, J = 8.3, 2.1 Hz, 1H), 5.05 (dd, J = 12.7, 5.5 Hz, 1H), 4.30 (s, 2H), 3.83 (d, J = 8.5 Hz, 2H), 3.54 (s, 4H), 3.48 (s, 1H), 2.93 - 2.83 (m, 2H), 2.58 (dd, J = 14.0, 5.1 Hz, 4H), 2.41 - 2.26 (m, 3H), 2.05 - 1.92 (m, 1H), 1.81 - 1.67 (m, 4H).

[0193] Example 41

[0194]

[0195] Using tert-butyl 2,7-diazaspiro[3.5]nonane-7-carboxylate instead of tert-butyl 2,6-diazaspiro[3.4]octane-6-carboxylate in the first step of Example 26, and using 37b instead of 12b in the fifth step of Example 26, compound 41 can be obtained in a similar manner and reaction procedure. ESI-MS (m / z): 732.6 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.52 (d, J = 26.0 Hz, 1H), 12.10 (s, 1H), 11.07 (s, 1H), 10.79 - 10.56 (m, 1H), 7.96 (s, 1H), 7.85 - 7.52 (m, 2H), 7.56 - 7.33 (m, 1H), 7.17 (d, J = 5.7 Hz, 1H), 6.83 - 6.55 (m, 3H), 6.39 - 6.29 (m, 1H), 5.05 (dd, J = 12.8, 5.5 Hz, 1H), 4.13 (t, J = 8.1 Hz, 2H), 3.69 (dd, J = 8.3, 5.4 Hz, 3H), 3.54 (s, 4H), 3.06 - 2.80 (m, 3H), 2.62 - 2.55 (m, 3H), 2.42 - 2.32 (m, 3H), 2.06 - 1.95 (m, 1H), 1.76 (t, J = 5.3 Hz, 4H).

[0196] Example 43

[0197]

[0198] Replacing tert-butyl 2,6-diazaspiro[3.4]octane-6-carboxylate with tert-butyl 2,6- diazaspiro[3.3]heptane-2-carboxylate in the first step of Example 26, while replacing 12b with 15b in the fifth step of Example 26, compound 43 can be obtained using similar methods and reaction procedures. ESI-MS (m / z): 704.7 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.64 - 12.43 (m, 1H), 12.07 (s, 1H), 11.07 (s, 1H), 10.70 - 10.48 (m, 1H), 7.95 (s, 1H), 7.61 - 7.50 (m, 2H), 7.47 - 7.34 (m, 1H), 7.21 - 7.05 (m, 2H), 6.79 - 6.58 (m, 2H), 6.36 - 6.31 (m, 1H), 5.04 (dd, J = 12.7, 5.5 Hz, 1H), 4.25 (s, 2H), 3.85 (d, J = 4.4 Hz, 6H), 3.51 - 3.41 (m, 4H), 2.90 - 2.83 (m, 1H), 2.69 - 2.52 (m, 5H), 2.04 - 1.92 (m, 1H).

[0199] Example 44

[0200]

[0201] Replacing tert-butyl 2,6-diazaspiro[3.4]octane-6-carboxylate with tert-butyl 2,6- diazaspiro[3.3]heptane-2-carboxylate in the first step of Example 26, while replacing 12b with 37b in the fifth step of Example 26, compound 44 can be obtained using similar methods and reaction procedures. ESI-MS (m / z): 704.5 [M+H] + ; 1H NMR (500 MHz, DMSO-d6) δ 12.64 - 12.42 (m, 1H), 12.08 (s, 1H), 11.07 (s, 1H), 10.72 - 10.38 (m, 1H), 7.96 (s, 1H), 7.66 - 7.55 (m, 2H), 7.50 - 7.30 (m, 1H), 7.17 (dd, J = 5.8, 1.7 Hz, 1H), 6.79 - 6.59 (m, 3H), 6.39 - 6.29 (m, 1H), 5.05 (dd, J = 12.8, 5.4 Hz, 1H), 4.08 (t, J = 8.0 Hz, 2H), 3.85 (d, J = 3.5 Hz, 4H), 3.72 - 3.61 (m, 3H), 3.56 - 3.39 (m, 3H), 2.92 - 2.85 (m, 1H), 2.76 - 2.53 (m, 5H), 2.05 - 1.94 (m, 1H).

[0202] Example 45

[0203]

[0204] Compound 45 was obtained by using tert-butyl 2,8-diaza-spiro[4.5]decane-2- carboxylate to replace tert-butyl 2,6-diazaspiro[3.4]octane-6-carboxylate in the first step of Example 26, and using 14b to replace 12b in the fifth step of Example 26, with similar method and procedure. ESI-MS (m / z): 760.8 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.58 (d, J = 9.5 Hz, 1H), 12.08 (s, 1H), 11.07 (s, 1H), 10.84 - 10.52 (m, 1H), 8.30 - 7.94 (m, 1H), 7.68 - 7.56 (m, 2H), 7.51 - 7.40 (m, 1H), 7.21 - 7.10 (m, 2H), 6.89 (dd, J = 9.1, 2.4 Hz, 2H), 6.81 (dd, J = 8.6, 2.2 Hz, 1H), 5.05 (dd, J = 12.9, 5.4 Hz, 1H), 3.61 - 3.38 (m, 4H), 3.19 - 2.99 (m, 6H), 2.92 - 2.84 (m, 1H), 2.65 - 2.54 (m, 4H), 2.47 - 2.39 (m, 4H), 2.17 - 2.12 (m, 1H), 2.04 - 1.96 (m, 2H), 1.82 - 1.65 (m, 5H).

[0205] Example 46

[0206]

[0207] Compound 46 was obtained by replacing tert-butyl 2,6-diazaspiro[3.4]octane-6- carboxylate with tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate in the first step of Example 26, and replacing 12b with 14b in the fifth step of Example 26, using similar methods and reaction procedures. ESI-MS (m / z): 718.9 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.52 (s, 1H), 12.08 (s, 1H), 11.07 (s, 1H), 10.74 - 10.56 (m, 1H), 7.96 (d, J = 15.9 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.60 - 7.57 (m, 1H), 7.52 - 7.29 (m, 1H), 7.21 - 7.14 (m, 1H), 6.91 - 6.77 (m, 2H), 6.75 - 6.50 (m, 1H), 6.37 - 6.29 (m, 1H), 5.05 (dd, J = 12.9, 5.3 Hz, 1H), 3.86 (d, J = 4.5 Hz, 4H), 3.56 - 3.44 (m, 4H), 3.41 - 3.37 (m, 4H), 3.13 - 3.07 (m, 1H), 2.93 - 2.81 (m, 1H), 2.63 - 2.54 (m, 2H), 2.46 - 2.44 (m, 1H), 2.38 - 2.29 (m, 1H), 2.16 - 2.07 (m, 1H), 2.04 - 1.95 (m, 1H), 1.77 - 1.65 (m, 1H).

[0208] Example 47

[0209]

[0210] First Step: 2-(2,6-dioxo-piperidin-3-yl)-5-methyl-isoindole-l,3-dione (300 mg, 1.10 mmol) and N-bromosuccinimide (294.18 mg, 1.65 mmol) were dissolved in acetonitrile (20 mL), and azobisisobutyronitrile (18.09 mg, 0.11 mmol) was added. The reaction system was replaced with nitrogen three times with a nitrogen balloon, and reacted at 75 °C for 8 hours under the protection of a nitrogen balloon. The reaction solution was reduced to room temperature, diluted with ethyl acetate, washed with water and saturated brine in turn, and the organic phase was dried over anhydrous sodium sulfate. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol) to obtain compound 47a (300 mg, yield 78%) ESI-MS (m / z): 351.2 [M+H] + .

[0211] Second step: Dissolve 1d (200 mg, 0.55 mmol) and 1-Boc-3-azetidinone (93.44 mg, 0.55 mmol) in a mixed solvent of MeOH (5 mL) and DCE (5 mL), add sodium acetate (74.27 mg, 0.55 mmol), stir at room temperature for 30 min. Then add sodium cyanoborohydride (68.60 mg, 1.09 mmol) to the reaction solution, continue to stir at room temperature for 6 h. Dilute the reaction solution with dichloromethane, wash successively with saturated sodium bicarbonate and saturated brine, dry the organic phase with anhydrous sodium sulfate. Concentrate the filtrate, purify the residue by silica gel column chromatography (dichloromethane / methanol) to obtain compound 47b (52 mg, yield 18%). ESI-MS (m / z): 522.8 [M+H] +

[0212] Third step: Dissolve 47b (52 mg, 0.1 mmol) in dichloromethane (4 mL), add TFA (1 mL), stir at room temperature for 30 min. Concentrate the reaction system to obtain compound 47c (40 mg, yield 95%). ESI-MS (m / z): 422.6 [M+H] +

[0213] Fourth step: Dissolve 47c (40 mg, 0.1 mmol) and 47a (83.31 mg, 0.14 mmol) in DMF (1 mL), add DIPEA (36.79 mg, 0.28 mmol), heat to 90 °C for 16 h. Purify the reaction solution by HPLC preparation to obtain compound 47 (12 mg, yield 18%). ESI-MS (m / z): 692.7 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.61 (s, 1H), 12.11 (s, 1H), 11.13 (s, 1H), 10.70-10.59 (m, 1H), 7.99 (s, 1H), 7.88 (d, J = 7.6 Hz, 1H), 7.84-7.76 (m, 2H), 7.59 (d, J = 5.7 Hz, 1H), 7.52-7.30 (m, 1H), 7.21-7.09 (m, 2H), 6.91-6.85 (m, 1H), 5.15 (dd, J = 12.8, 5.4 Hz, 1H), 3.79 (s, 2H), 3.47-3.43 (m, 3H), 3.15-3.06 (m, 5H), 2.91-2.85 (m, 1H), 2.74-2.52 (m, 3H), 2.46-2.40 (m, 4H), 2.17-1.92 (m, 1H).

[0214] Example 48

[0215]

[0216] Using tert-butyl 2,6-diazaspiro[3.3]octane-6-carboxylate in place of tert-butyl 2,6-diazaspiro[3.4]octane-6-carboxylate in the first step of Example 26, Compound 48 can be obtained in a similar manner and reaction sequence. ESI-MS (m / z): 718.6 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.60 - 12.45 (m, 1H), 12.08 (s, 1H), 11.06 (s, 1H), 10.80 - 10.52 (m, 1H), 7.96 (d, J = 7.1 Hz, 1H), 7.61 - 7.52 (m, 2H), 7.49 - 7.38 (m, 1H), 7.21 - 7.05 (m, 3H), 6.74 - 6.42 (m, 1H), 6.34 (d, J = 8.3 Hz, 1H), 5.27 - 4.81 (m, 1H), 3.85 (d, J = 3.8 Hz, 4H), 3.64 - 3.46 (m, 5H), 3.45 - 3.36 (m, 4H), 2.93 - 2.85 (m, 1H), 2.65 - 2.55 (m, 1H), 2.48 - 2.43 (m, 2H), 2.28 - 2.19 (m, 1H), 2.07 - 1.95 (m, 2H), 1.69 - 1.58 (m, 1H).

[0217] Example 49

[0218]

[0219] Using tert-butyl 3-formylazetidine-1-carboxylate in place of 1-Boc-3-azetidinone in the second step of Example 47, Compound 49 can be obtained in a similar manner and reaction sequence. ESI-MS (m / z): 706.8 [M+H] + ; 1H NMR (500 MHz, DMSO-de) δ 12.60 (d, J = 6.5 Hz, 1H), 12.09 (s, 1H), 11.13 (s, 1H), 10.77 - 10.54 (m, 1H), 8.19 (s, 2H), 7.98 (s, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.82 - 7.75 (m, 2H), 7.58 (d, J = 5.7 Hz, 1H), 7.52 - 7.40 (m, 1H), 7.20 - 7.06 (m, 2H), 6.93 - 6.83 (m, 1H), 5.15 (dd, J = 12.8, 5.4 Hz, 1H), 3.75 (s, 2H), 3.43 - 3.40 (m, 6H), 3.07 (t, J = 4.9 Hz, 4H), 2.94 - 2.86 (m, 3H), 2.71 - 2.61 (m, 2H), 2.59 - 2.54 (m, 3H), 2.10 - 2.03 (m, 1H).

[0220] Example 50

[0221]

[0222] Using 1-tert-butoxycarbonylpiperidine-4-carboxaldehyde instead of 1-Boc-3- azetidinone in the second step of Example 47, compound 50 was obtained in a similar manner and reaction procedure. ESI-MS (m / z): 734.9 [M+H] + ; 1 H NMR (500 MHz, DMSO-de) δ 12.60 (d, J = 6.5 Hz, 1H), 12.09 (s, 1H), 11.13 (s, 1H), 10.77 - 10.54 (m, 1H), 8.19 (s, 2H), 7.98 (s, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.82 - 7.75 (m, 2H), 7.58 (d, J = 5.7 Hz, 1H), 7.52 - 7.40 (m, 1H), 7.20 - 7.06 (m, 2H), 6.93 - 6.83 (m, 1H), 5.15 (dd, J = 12.8, 5.4 Hz, 1H), 3.75 (s, 2H), 3.43 - 3.40 (m, 6H), 3.07 (t, J = 4.9 Hz, 4H), 2.94 - 2.86 (m, 3H), 2.71 - 2.61 (m, 2H), 2.59 - 2.54 (m, 3H), 2.10 - 2.03 (m, 1H).

[0223] Example 51

[0224]

[0225] Compound 51 was obtained by replacing tert-butyl 2,6-diazaspiro[3.4]octane-6- carboxylate in the first step of Example 26 with tert-butyl 2,6-diaza-spiro[3.4]octane-2- carbonate, and replacing 12b in the fifth step of Example 26 with 18b, in a similar manner and sequence of reactions. ESI-MS (m / z): 746.5 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.47 - 12.34 (m, 1H), 11.99 (s, 1H), 11.00 (s, 1H), 10.77 - 10.47 (m, 1H), 8.12 - 7.75 (m, 1H), 7.58 (d, J = 8.6 Hz, 1H), 7.54 - 7.48 (m, 1H), 7.43 - 7.30 (m, 1H), 7.23 (d, J = 2.4 Hz, 1H), 7.15 (dd, J = 8.8, 2.4 Hz, 1H), 7.11 (dd, J = 5.7, 1.6 Hz, 1H), 6.73 - 6.60 (m, 1H), 6.51 - 6.38 (m, 1H), 4.99 (dd, J = 12.8, 5.5 Hz, 1H), 3.95 (d, J = 13.0 Hz, 2H), 3.24 - 3.16 (m, 4H), 3.15 - 3.05 (m, 4H), 2.90 - 2.77 (m, 3H), 2.56 - 2.46 (m, 2H), 2.25 (d, J = 6.8 Hz, 2H), 2.08 (t, J = 6.8 Hz, 2H), 1.99 - 1.91 (m, 1H), 1.71 (d, J = 12.8 Hz, 2H), 1.59 - 1.46 (m, 1H), 1.15 - 1.06 (m, 2H).

[0226] Example 52

[0227]

[0228] Compound 52 was obtained by replacing tert-butyl 2,6-diazaspiro[3.4]octane-6- carboxylate in the first step of Example 37 with tert-butyl 1,4-diazepane-1-carboxylate, and replacing 12b in the fifth step of Example 26 with 18b, in a similar manner and sequence of reactions. ESI-MS (m / z): 734.5 [M+H] + ; 1H NMR (500 MHz, DMSO-d6) δ 12.49 (s, 1H), 12.07 (s, 1H), 11.07 (s, 1H), 10.63 (s, 1H), 7.91 (s, 1H), 7.64 - 7.56 (m, 2H), 7.45 - 7.36 (m, 1H), 7.31 - 7.14 (m, 3H), 7.05 - 6.76 (m, 1H), 6.67 (d, J = 8.7 Hz, 1H), 5.06 (dd, J = 12.7, 5.4 Hz, 1H), 4.00 (d, J = 12.3 Hz, 2H), 3.57 - 3.49 (m, 4H), 2.97 - 2.73 (m, 6H), 2.61 - 2.52 (m, 4H), 2.31 - 2.29 (m, 1H), 1.99 (s, 1H), 1.93 - 1.88 (m, 2H), 1.80 - 1.70 (m, 3H), 1.16 - 1.05 (m, 2H).

[0229] Example 53

[0230]

[0231] Using tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate instead of tert-butyl 2,6-diazaspiro[3.4]octane-6-carboxylate in the first step of Example 26, and using 18b instead of 12b in the fifth step of Example 26, compound 53 can be obtained in a similar manner and reaction procedure. ESI-MS (m / z): 788.7 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.94 (s, 1H), 11.07 (s, 1H), 10.51 (s, 1H), 7.65 (d, J = 8.7 Hz, 1H), 7.53 (d, J = 5.7 Hz, 1H), 7.48 - 7.38 (m, 1H), 7.30 (d, J = 2.3 Hz, 1H), 7.27 - 7.06 (m, 3H), 6.87 (d, J = 8.7 Hz, 1H), 5.06 (dd, J = 12.8, 5.4 Hz, 1H), 4.03 (d, J = 12.8 Hz, 2H), 3.13 - 3.05 (m, 4H), 3.03 - 2.82 (m, 4H), 2.65 - 2.53 (m, 3H), 2.39 - 2.31 (m, 4H), 2.19 - 2.12 (m, 2H), 2.04 - 1.99 (m, 1H), 1.83 - 1.74 (m, 3H), 1.63 - 1.47 (m, 8H).

[0232] Example 54

[0233]

[0234] Replacing tert-butyl 2,6-diazaspiro[3.4]octane-6-carboxylate in the first step of Example 26 with 2-Boc-octahydropyrrolo[3,4-c]pyrrole, while replacing 12b in the fifth step of Example 26 with 18b, compound 54 can be obtained using similar methods and reaction procedures. ESI-MS (m / z): 746.9 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 12.60 (s, 1H), 12.09 (s, 1H), 11.06 (s, 1H), 10.78 - 10.58 (m, 1H), 7.94 (s, 1H), 7.78 - 7.52 (m, 2H), 7.50 - 7.35 (m, 1H), 7.31 - 7.13 (m, 3H), 6.94 - 6.82 (m, 1H), 6.70 - 6.59 (m, 1H), 5.05 (dd, J = 12.7, 5.4 Hz, 1H), 4.02 (d, J = 13.1 Hz, 2H), 3.47 - 3.41 (m, 2H), 3.03 - 2.84 (m, 7H), 2.62 - 2.54 (m, 4H), 2.48 - 2.43 (m, 2H), 2.29 - 2.18 (m, 2H), 2.05 - 1.94 (m, 1H), 1.84 - 1.71 (m, 3H), 1.18 - 1.09 (m, 2H).

[0235] Example 55

[0236]

[0237] Replacing tert-butyl 2,6-diazaspiro[3.4]octane-6-carboxylate in the first step of Example 26 with tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate, while replacing 12b in the fifth step of Example 26 with 18b, compound 55 can be obtained using similar methods and reaction procedures. ESI-MS (m / z): 732.2 [M+H] + ; 1H NMR (500 MHz, DMSO-d6) δ 12.52 (s, 1H), 12.10 (s, 1H), 11.07 (s, 1H), 10.76 - 10.57 (m, 1H), 7.96 (s, 1H), 7.67 - 7.55 (m, 2H), 7.48 - 7.36 (m, 1H), 7.33 - 6.77 (m, 3H), 6.74 - 6.52 (m, 1H), 6.35 (d, J = 8.6 Hz, 1H), 5.06 (dt, J = 13.0, 4.4 Hz, 1H), 4.02 (d, J = 12.9 Hz, 2H), 3.85 (s, 4H), 3.61 - 3.56 (m, 4H), 3.05 - 2.81 (m, 5H), 2.64 - 2.54 (m, 3H), 2.47 - 2.43 (m, 1H), 2.36 - 2.26 (m, 1H), 2.04 - 1.98 (m, 1H), 1.79 - 1.65 (m, 1H), 1.46 - 1.43 (m, 1H).

[0238] Example 56

[0239]

[0240] First Step: Dissolve 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (1.0 g, 3.62 mmol) and 1-(tert-butoxycarbonyl)piperazine (846.60 mg, 3.80 mmol) in DMF (5 mL), add DIPEA (1.40 g, 10.86 mmol), and warm to 90 °C for 16 h. Cool the reaction to room temperature. Dilute the reaction with dichloromethane, wash sequentially with water and saturated brine, and dry the organic phase over anhydrous sodium sulfate. Concentrate the filtrate and purify the residue by silica gel column chromatography (dichloromethane / methanol) to give compound 56a (1.6 g, 99% yield). ESI-MS (m / z): 443.5 [M+H] + .

[0241] Second Step: Dissolve 56a (1.6 g, 3.62 mmol) in dioxane (10 mL), add 4 M HC1 in dioxane (5 mL), and stir at room temperature for 3 h. Concentrate the reaction to give 56b (1.2 g, 96% yield). ESI-MS (m / z): 343.6 [M+H] + .

[0242] Step 3: 1d (500 mg, 1.32 mmol) was dissolved in DMF (10 mL), DIPEA (852.94 mg, 6.60 mmol) was added. After stirring, bromoacetaldehyde diethyl acetal (390 mg, 1.98 mmol) was added, and the reaction was warmed to 80 °C for 16 h. The reaction was cooled to room temperature, diluted with ethyl acetate, washed with water and saturated brine successively, and the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol) to give 56c (128 mg, yield 21.2%). ESI-MS (m / z): 497.2 [M+H] + .

[0243] Step 4: To 56c (128 mg, 0.28 mmol) was added 4 M HC1 in dioxane (2 mL) and the reaction was stirred at 50 °C for 16 h. The reaction was concentrated to give 56d (100 mg, yield 93%). ESI-MS (m / z): 423.8 [M+H] + .

[0244] Step 5: 56d (50 mg, 0.13 mmol) and 56b (59.58 mg, 0.13 mmol) were dissolved in 1,2-dichloroethane (2 mL) and methanol (2 mL) under ice bath condition, sodium acetate (17.7 mg, 0.13 mmol) was added to the reaction system, then the reaction system was stirred for 30 min, and then sodium cyanoborohydride (16.35 mg, 0.26 mmol) was added. The system was stirred at room temperature for another 6 h. The reaction was diluted with dichloromethane, washed with saturated sodium bicarbonate and saturated brine successively, and the organic phase was dried over anhydrous sodium sulfate. The filtrate was concentrated, and the residue was dissolved in DMF. Compound 56 (5 mg, yield 5.2%) was obtained by HPLC preparation purification. ESI-MS (m / z): 735.8 [M+H] + ; 1HNMR (500 MHz, DMSO-d6) δ 12.79 (s, 1H), 12.07 (s, 1H), 11.08 (s, 1H), 10.57 (s, 1H), 7.88 (s, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.56 (d, J = 5.7 Hz, 1H), 7.51 - 7.39 (m, 1H), 7.35 (d, J = 2.3 Hz, 1H), 7.26 (dd, J = 8.7, 2.4 Hz, 1H), 7.21 - 7.06 (m, 2H), 6.91 - 6.85 (m, 1H), 5.07 (dd, J = 12.8, 5.4 Hz, 1H), 3.45 (t, J = 5.0 Hz, 4H), 3.13 - 3.08 (m, 4H), 2.92 - 2.79 (m, 1H), 2.63 - 2.56 (m, 10H), 2.55 - 2.53 (m, 4H), 2.04 - 2.00 (m, 1H).

[0245] Test Example 1: Detection of the ability of the compound to degrade HPK1 protein

[0246] The reagents required for use are as follows table:

[0247]

[0248]

[0249] Experimental procedure

[0250] The specific procedure is as follows: In Corning 24-well plates (catalog number 3524), Jurkat E6-1 (hereinafter referred to as Jurkat) cells were seeded at a volume of 1 mL / well in cell culture medium [RPMI 1640 (BI, 01-100-1ACS), 10% heat-inactivated FBS (BI, 04-002-1A), 1% Penicillin-Streptomycin (BI, 03-031-1B)] at a density of 2,500,000 cells / well. Jurkat cells were treated with a compound diluted in 0.2% DMSO according to the following dilution protocol: (1) 3 μL of the compound stock solution (concentration of 10 mM) was transferred to 1497 μL of culture medium to prepare a 20 μM compound solution; (2) starting from 20 μM, the compound was diluted 10-fold with 0.2% DMSO for a total of 6 doses; (3) 1 mL of each concentration of the compound solution was added to the cells (final compound concentrations of 0, 1, 10, 100, 1000, 10000 nM) and incubated for 20 h. Cells were collected in 15 mL centrifuge tubes and centrifuged at 300 g for 5 minutes. The culture medium was discarded, and 3 mL of pre-chilled PBS (4°C) was added to wash the cells. The washing buffer was then discarded. An appropriate amount of RIPA lysis buffer (containing a Protease and Phosphatase Inhibitor Cocktail) was added according to the cell volume. The mixture was thoroughly mixed and placed on ice for lysis for 30 minutes, with shaking every 10 minutes. The cells were then centrifuged at 20,000 rpm for 30 minutes at 4°C. The supernatant was transferred to a new centrifuge tube and centrifuged using Pierce chromatography. TMThe protein concentration was quantified by Rapid Gold BCA Protein Assay Kit (A53225). An appropriate amount of SDS-PAGE loading buffer was added to the protein sample, and the sample was heated in a boiling water bath for 5 minutes to denature the protein. The sample was stored at -20°C for later use. The precast gel (Genscript, M00655 or M00657) was used for electrophoresis at a voltage of 120V, and the membrane was transferred at 4°C at a voltage of 300mA for 100min. After the transfer was completed, the protein membrane was immediately placed in 5% skim milk diluted with TBST, and slowly shaken on a shaker at room temperature for 1h. According to the instructions for HPK1 Rabbit mAb (CST, 53453S) and GAPDH (D16H11) Rabbit Monoclonal antibody (CST, 5174S), the primary antibody was diluted according to the recommended ratio, and slowly shaken at 4°C overnight. Then, the membrane was washed with TBST for 3 times, 10min / time. According to the instructions for Goat Anti-Rabbit IgG (H&L)-HRP Conjugated (Thermofisher, G-21234), the secondary antibody was diluted according to the recommended ratio, and slowly shaken at room temperature for 1h. Then, the membrane was washed with TBST for 3 times, 10min / time. ECL luminescent liquid was added, and the results were observed by a developing instrument. The Western results were quantified by Image J software, and the degradation rate of HPK1 was calculated by comparing with the control group without the compound (degradation rate = 1 - drug group / control group). The half degradation concentration DC50 was calculated by the formula: DC50 = (1 - degradation rate) / degradation rate 50 The experimental results are shown in the following table:

[0251]

[0252]

[0253] From the results in the above table, it can be seen that the compound of the present application can significantly degrade the HPK1 protein in Jurkat cells.

[0254] Test Example 2: Detection of the agonistic ability of the compound to the cytokine interleukin-2 (IL-2) secreted by human PBMC cells and the effect of the compound on the activity of human PBMC cells

[0255] The reagents used are as follows

[0256]

[0257] Information on the source of experimental cells:

[0258] manufacturer lot product specification shanghai xianfeng biotech co., ltd. sc12132w / xcwbc6037w 10 / 25 million / vial

[0259] Experimental steps

[0260] The specific procedures are as follows: Human PBMCs were removed from liquid nitrogen according to standard procedures and thawed in a 37°C water bath. The cells were resuspended in RPMI 1640 medium (containing 10% FBS in this experiment), and washed twice by centrifugation. The human PBMCs were then resuspended in RPMI 1640 medium for later use. The compound powder was dissolved in DMSO to a concentration of 10 mM. 2 μL of the compound was added to 998 μL of RPMI 1640 medium and vortexed to obtain the highest concentration. The compound solution was gradually diluted 5-fold with 0.2% DMSO medium, resulting in 8 concentration points. RPMI 1640 medium containing 0.1% DMSO was used as a control. 90 μL of 0.5 × 10⁻⁶ FBS solution was added to each well of a Corning 96-well cell culture plate (catalog number: 3599). 5 Human PBMC cells were cultured, followed by the addition of an equal volume of compound dilution buffer. The control group received RPMI 1640 medium containing 0.2% DMSO. Cells were incubated at 37°C in a Thermo Fisher Scientific (model 3111) for 1 hour. Then, anti-human CD3 antibody (adjusted according to different batches of human PBMCs) and anti-human CD28 antibody at final concentrations of 0.37–1.1 μg / ml were added, and the cells were incubated at 37°C for 20 hours. The IL-2 content in the cell supernatant was detected using the Human IL-2 DuoSet ELISA kit, performed according to the kit's instructions. Data were presented as the fold-over ratio of the compound's stimulus signal to the signal from 0.1% DMSO and the half-maximal effective concentration (EC50). 50 Description. Cells were collected using CellTiter- The Luminescent Cell Viability Assay kit was used to detect cell viability. Cell viability data were obtained using the half-maximal inhibitory concentration (IC50) of the compound. 50 describe.

[0261] The results of the tests on the agonistic effect of the compound on the secretion of the human PBMC cytokine interleukin-2 (IL-2) and the effect of the compound on the viability of human PBMC cells are shown in the table below:

[0262]

[0263] As can be seen from the results in the table above, the compound described in this invention can significantly stimulate human PBMC cells to secrete the cytokine interleukin-2 (IL-2), and has little effect on the viability of human PBMC cells.

[0264] Test Example 3: Pharmacokinetic test of the compound in mice

[0265] Test animals: ICR mice (male, 25-35 g, 6-9 weeks old, SLAKE)

[0266] Test operation: ICR mice were randomly divided by weight, and the compound 7 of the application was administered by gavage, with a dose of 10 mg / kg, and the administration vehicle was 10% DMSO+10% PEG-400+10% Solutol+70% H2O. The mice were fasted for 1 h before gavage, and the food was restored 1 h after blood sampling, and the mice were allowed to drink water freely. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 24 h after administration. Before analysis, all samples were stored at -80°C, and the samples were quantitatively analyzed by LC-MS / MS.

[0267] The pharmacokinetic parameters of compound 7 in mice are shown in the following table:

[0268]

[0269] As shown in the above table, the compound of the application has high exposure in mice, high maximum blood concentration, and good oral absorption.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from any of the compounds shown in the table below:

2. A pharmaceutical composition comprising the compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

3. The use of the compound of claim 1, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2, in the preparation of a medicament for the prevention and / or treatment of diseases related to HPK1 activity.

4. The use as described in claim 3, wherein, The HPK1 activity-related diseases are cancer or immune diseases; the cancers are lung cancer, thyroid cancer, liver cancer, colon cancer, rectal cancer, pancreatic cancer, stomach cancer, esophageal cancer, squamous cell carcinoma, oral cancer, nasopharyngeal carcinoma, breast cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, endometrial cancer, bladder cancer, bone cancer, brain cancer, skin cancer, melanoma, sarcoma, glioma, and lymphoma; the immune diseases are lupus erythematosus, psoriasis, inflammatory bowel disease, and rheumatoid arthritis.

5. The use as described in claim 3, wherein, The compound of claim 1, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2, may be used alone or in combination with other types of pharmaceutical preparations.

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