WO2021147699A1 - Pyridoheterocyclic compound, and preparation method therefor and use thereof - Google Patents

Abstract

The present invention relates to a pyridoheterocyclic compound, and a preparation method therefor and the use thereof. Specifically, the present invention relates to a compound or a pharmaceutically acceptable salt, ester, solvate (e.g. a hydrate), stereoisomer, tautomer, polymorph, metabolite or prodrug thereof, a pharmaceutical composition and kit containing same, a preparation method therefor, and the use thereof in the preparation of a drug for preventing or treating cancers.

Description

Pyridoheterocyclic compounds, preparation method and application thereof Technical field

The present invention relates to pyridoheterocyclic compounds or their pharmaceutically acceptable salts, esters, solvates (such as hydrates), stereoisomers, tautomers, polymorphs, metabolites or prodrugs, including The pharmaceutical composition and the kit, the preparation method and the use thereof in the preparation of a medicine for preventing or treating diseases mediated by TGFβR1 (especially cancer, such as liver cancer).

Background technique

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that regulates various cellular responses, such as cell proliferation, differentiation, migration, and apoptosis. The TGF-β superfamily includes TGF-β1, TGF-β2, TGF-β3, activin, inhibin, bone morphogenetic protein and so on. TGF-β transduces signals through two highly conserved single-transmembrane serine/threonine kinases, namely TGFβR1 and TGFβR2 (ACS Med Chem Lett. 2018, 9, 1117).

Smads are important TGF-β signal transduction and regulatory molecules in cells, which can directly transduce TGF-β signals from the cell membrane to the nucleus. The TGF-β/Smads signaling pathway plays an important role in the occurrence and development of tumors. . In TGF-β/Smads signal transduction, activated TGF-β first binds to TGFβR2 on the cell membrane surface to form a heterodimeric complex, and TGFβR1 recognizes and binds to the binary complex. The activated TGFβR1 further phosphorylates the Smad2/Smad3 protein, which further combines with Smad4 to form a heterotrimeric complex, which enters the nucleus and cooperates with auxiliary activation/repressor factors to regulate the transcription of target genes (Nature. 2003, 425, 577). Changes in any link of the TGF-β/Smads signaling pathway will cause abnormalities in the signal transduction pathway (PNAS.2019, 116, 9166).

The TGF-β signaling pathway is dysregulated in many diseases including cancer. TGFβR1 is found in gastric cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, liver cancer, lung cancer, cervical cancer, and head and neck cancer cell lines and tumor tissues. The protein level increased significantly. The activation of TGF-β signaling pathway triggers obvious pathological effects in tumor stroma, including immunosuppression, angiogenesis and connective tissue hyperplasia. In addition, the TGF-β signaling pathway can enhance the invasiveness of tumor cells, promote the transformation of epithelial cells to mesenchyme, and enhance the tolerance of tumor epithelial cell therapy (Nat Neurosci.2014,17,943).

At present, the development of TGFβR1 inhibitors, a key target of the TGF-β signaling pathway, has gradually gained attention in the pharmaceutical industry. Published patent applications include WO2002094833A1, WO2009150547A1, WO2017035118A1, WO2018019106A1, etc. However, the art still urgently needs new TGFβR1 inhibitors, especially TGFβR1 inhibitors with novel structure, high activity and high selectivity.

Summary of the invention

The present invention is based on the structure of TGFβ protein, and through screening and research of a large number of compounds, novel structural TGFβR1 inhibitors have been discovered, and these compounds can inhibit the activity of TGFβR1. As a TGFβR1 inhibitor, it can be used to treat proliferative disorders and apoptotic disorders caused by the TGF-β signaling pathway, especially diseases mediated by TGFβR1, such as cancer, such as liver cancer.

One aspect of the present invention provides a compound having a structure represented by formula I, a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, polymorph, metabolite or prodrug of the compound :

Where R _{1 is} selected from

Another aspect of the present invention provides a compound having a structure represented by Formula II, a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, polymorph, metabolite, or precursor of the compound. Medicine:

Where R _{2 is} selected from

In another aspect, the present invention provides a pharmaceutical composition comprising a preventive or therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt, ester, solvate (e.g., hydrate), stereoisomer, Tautomers, polymorphs, metabolites or prodrugs, and one or more pharmaceutically acceptable carriers.

In another aspect, the present invention provides a medicine kit comprising:

a) The compound of the present invention or a pharmaceutically acceptable salt, ester, solvate (such as hydrate), stereoisomer, tautomer, polymorph, metabolite or prodrug thereof, or the present invention ’S pharmaceutical composition;

b) Optional packaging and/or instructions.

In another aspect, the present invention provides a compound of the present invention or a pharmaceutically acceptable salt, ester, solvate (e.g., hydrate), stereoisomer, tautomer, polymorph, metabolite or The prodrug, or the pharmaceutical composition of the present invention, or the kit of the present invention is used to treat diseases or disorders mediated by TGFβR1, and the diseases or disorders mediated by TGFβR1 are cancer, such as lung cancer, Colorectal cancer, multiple myeloma, acute myelogenous leukemia, T-acute lymphoblastic leukemia, pancreatic cancer, liver cancer, neuroblastoma, breast cancer, ovarian cancer, melanoma.

In another aspect, the present invention provides a compound of the present invention or a pharmaceutically acceptable salt, ester, solvate (e.g., hydrate), stereoisomer, tautomer, polymorph, metabolite or Prodrug, or the pharmaceutical composition of the present invention, or the kit of the present invention, its application in the preparation of a medicament for the treatment of diseases or disorders mediated by TGFβR1, said diseases or related diseases mediated by TGFβR1 The condition is cancer, such as lung cancer, colorectal cancer, multiple myeloma, acute myelogenous leukemia, T-acute lymphoblastic leukemia, pancreatic cancer, liver cancer, neuroblastoma, breast cancer, ovarian cancer, melanoma.

In another aspect, the present invention provides a method for preventing or treating a disease or disorder mediated by TGFβR1, which comprises administering a preventive or therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof to an individual in need thereof , Esters, solvates (such as hydrates), stereoisomers, tautomers, polymorphs, metabolites or prodrugs, or the pharmaceutical composition of the present invention, or the kit of the present invention; preferably Specifically, the disease or disorder mediated by TGFβR1 is cancer, such as lung cancer, colorectal cancer, multiple myeloma, acute myelogenous leukemia, T-acute lymphoblastic leukemia, pancreatic cancer, liver cancer, neuroblastoma Cell tumor, breast cancer, ovarian cancer, melanoma.

Definition of Terms

Unless otherwise defined below, the meanings of all technical and scientific terms used herein are intended to be the same as those commonly understood by those skilled in the art. The reference to the technology used herein is intended to refer to the technology generally understood in the art, including those technical changes or equivalent technology substitutions that are obvious to those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention.

As used herein, the terms "include", "include", "have", "include" or "relevant" and other variants herein are inclusive or open-ended, and do not Exclude other unlisted elements or method steps.

The present invention also includes all pharmaceutically acceptable isotope-labeled compounds, which are the same as the compounds of the present invention, except that one or more atoms have the same atomic number but the atomic mass or mass number is different from the predominant atomic mass in nature. Or atomic substitution of mass number. Examples of isotopes suitable for inclusion in the compounds of the present invention include, but are not limited to, isotopes of hydrogen (e.g. ² H, ³ H, deuterium D, tritium T); isotopes of carbon (e.g. ¹¹ C, ¹³ C, and ¹⁴ C); chlorine isotopes (e.g., ³⁷ Cl); fluorine isotope (e.g. ¹⁸ F); iodine isotopes (e.g., ¹²³ I and ¹²⁵ I); nitrogen isotope (e.g., ¹³ N and ¹⁵ N); isotopes of oxygen (e.g., ¹⁵ O, ¹⁷ O and ¹⁸ O); and sulfur isotopes (such as ³⁵ S).

The present invention covers all possible crystalline forms or polymorphs of the compounds of the present invention, which can be a single polymorph or a mixture of more than one polymorph in any ratio.

It should also be understood that certain compounds of the present invention may exist in free form for treatment, or, when appropriate, in the form of their pharmaceutically acceptable derivatives. In the present invention, pharmaceutically acceptable derivatives include but are not limited to: pharmaceutically acceptable salts, esters, solvates, metabolites or prodrugs, which can be directly administered to patients in need thereof. Or indirectly provide the compound of the present invention or its metabolites or residues. Therefore, when the "compound of the present invention" is referred to herein, it is also intended to encompass the above-mentioned various derivative forms of the compound.

The term "pharmaceutically acceptable" as used in the present invention means that the substance or composition must be chemically and/or toxicologically compatible with the other components constituting the formulation and/or the mammal to be treated with it.

The pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts and base addition salts thereof. Suitable acid addition salts are formed from acids that form pharmaceutically acceptable salts. Suitable base addition salts are formed from bases that form pharmaceutically acceptable salts. For a review of suitable salts, see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

The compound of the present invention may exist in the form of a solvate (preferably a hydrate), wherein the compound of the present invention contains a polar solvent as a structural element of the compound crystal lattice, in particular, for example, water, methanol or ethanol. The amount of polar solvent, especially water, can be present in a stoichiometric or non-stoichiometric ratio.

Also included within the scope of the present invention are metabolites of the compounds of the present invention, that is, substances formed in the body when the compounds of the present invention are administered. Such products can be produced by, for example, oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the administered compound. Therefore, the present invention includes metabolites of the compounds of the present invention, including compounds prepared by a method in which the compounds of the present invention are contacted with a mammal for a time sufficient to produce their metabolites.

The present invention further includes within its scope the prodrugs of the compounds of the present invention, which are certain derivatives of the compounds of the present invention that may themselves have less pharmacological activity or no pharmacological activity, when administered to the body or The above can be converted into the compound of the present invention having the desired activity by, for example, hydrolytic cleavage. Usually such prodrugs will be functional group derivatives of the compound, which are easily converted into the desired therapeutically active compound in vivo. For other information about the use of prodrugs, please refer to "Pro-drugs as Novel Delivery Systems", Volume 14, ACS Symposium Series (T. Higuchi and V. Stella) and "Bioreversible Carriers in Drug Design," Pergamon Press, 1987 ( Edited by EBRoche, American Pharmaceutical Association). The prodrugs of the present invention can be used, for example, by using certain parts known to those skilled in the art as "pro-moiety (for example, "Design of Prodrugs", described in H. Bundgaard (Elsevier, 1985))" It can be prepared by substituting appropriate functional groups present in the compounds of the present invention.

The present invention also encompasses compounds of the present invention containing protecting groups. In any process of preparing the compounds of the present invention, protection of sensitive groups or reactive groups on any relevant molecule may be necessary and/or desirable, thereby forming a chemically protected form of the compounds of the present invention. This can be achieved through conventional protective groups, such as those described in Protective Groups in Organic Chemistry, ed. JFW McOmie, Plenum Press, 1973; and TW Greene & P. GMWuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991 Protecting groups, these references are incorporated herein by reference. Using methods known in the art, the protecting group can be removed at an appropriate subsequent stage.

In the present invention

The bond at represents the bond connected to the rest of the compound structure.

Compound

One aspect of the present invention provides a compound having a structure represented by formula I, a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, polymorph, metabolite or prodrug of the compound :

Where R _{1 is} selected from

Another aspect of the present invention provides a compound having a structure represented by Formula II, a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, polymorph, metabolite, or precursor of the compound. Medicine:

Where R _{2 is} selected from

In certain embodiments of the invention, R _{2 is} selected from

In certain embodiments of the invention, the compounds of the invention include, but are not limited to:

In certain embodiments of the invention, the compounds of the invention include, but are not limited to:

Pharmaceutical composition and kit

Another object of the present invention is to provide a pharmaceutical composition comprising a preventive or therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt, ester, solvate (e.g., hydrate), stereoisomer, Tautomers, polymorphs, metabolites or prodrugs, and one or more pharmaceutically acceptable carriers.

Another object of the present invention is to provide a medicine kit comprising:

a) The compound of the present invention or a pharmaceutically acceptable salt, ester, solvate (such as hydrate), stereoisomer, tautomer, polymorph, metabolite or prodrug thereof, or the present invention Pharmaceutical composition; and

b) Optional packaging and/or instructions.

In the present invention, "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient or vehicle administered together with a therapeutic agent, and it is suitable for contact with humans and/or other substances within the scope of reasonable medical judgment. Animal tissue without excessive toxicity, irritation, allergic reactions, or other problems or complications corresponding to a reasonable benefit/risk ratio.

The pharmaceutical composition of the present invention can act systemically and/or locally. For this purpose, they can be administered by suitable routes, such as parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intradermal, transdermal, rectal, intracranial, intraperitoneal, intranasal, and intramuscular routes Or administered as an inhalant.

For these administration routes, the pharmaceutical composition of the present invention can be administered in a suitable dosage form. The dosage form includes but not limited to tablet, capsule, lozenge, hard candy, powder, spray, cream, ointment, suppository, gel, paste, lotion, ointment, aqueous suspension , Injectable solutions, elixirs, syrups, etc.

When administered orally, the pharmaceutical composition of the present invention can be prepared into any orally acceptable preparation form, including but not limited to tablets, capsules, aqueous solutions, aqueous suspensions, and the like. The pharmaceutical composition of the present invention can also be administered in the form of a sterile injection.

The pharmaceutical composition of the present invention may contain 0.01 mg to 1000 mg of the compound of the present invention.

In some embodiments, the present invention provides a method for preparing the pharmaceutical composition or pharmaceutical formulation of the present invention, the method comprising combining the compound of the present invention or a pharmaceutically acceptable salt, ester, solvate (e.g., hydrate), Stereoisomers, tautomers, polymorphs, metabolites or prodrugs are combined with one or more pharmaceutically acceptable carriers.

The pharmaceutical composition of the present invention can be optionally administered in combination with other agents that have at least a certain effect in the treatment of various diseases. In some embodiments, the present invention provides a combined preparation of a compound of the present invention and an additional therapeutic agent for simultaneous, separate or sequential use in treatment.

Treatment methods and uses

Another object of the present invention is to provide the compound of the present invention or a pharmaceutically acceptable salt, ester, solvate (e.g., hydrate), stereoisomer, tautomer, polymorph, metabolite or The prodrug, or the pharmaceutical composition of the present invention, or the kit of the present invention, is used to treat diseases or disorders mediated by TGFβR1.

Another object of the present invention is to provide the compound of the present invention or a pharmaceutically acceptable salt, ester, solvate (e.g., hydrate), stereoisomer, tautomer, polymorph, metabolite or The use of the prodrug, or the pharmaceutical composition of the present invention, or the kit of the present invention in the preparation of a medicament for treating diseases or disorders mediated by TGFβR1.

Another object of the present invention is to provide a method for preventing or treating a disease or disorder mediated by TGFβR1, which comprises administering to an individual in need thereof a preventive or therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof , Esters, solvates (such as hydrates), stereoisomers, tautomers, polymorphs, metabolites or prodrugs, or the pharmaceutical composition of the present invention, or the kit of the present invention.

According to some embodiments of the present invention, the disease or disorder mediated by TGFβR1 is cancer, such as lung cancer, colorectal cancer, multiple myeloma (MM), acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), pancreatic cancer, liver cancer, neuroblastoma, breast cancer, ovarian cancer, melanoma.

The term "effective amount" as used herein refers to an amount sufficient to achieve a desired preventive or therapeutic effect, for example, an amount that achieves alleviation of one or more symptoms associated with the disease to be treated.

The dosage regimen can be adjusted to provide the best desired response. For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the urgent need of the treatment situation. It should be noted that the dose value may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It should be further understood that for any particular individual, the specific dosing regimen should be adjusted over time according to the individual's needs and the professional judgment of the person administering the composition or supervising the administration of the composition.

The amount of the compound of the present invention administered will depend on the individual being treated, the severity of the disorder or condition, the rate of administration, the treatment of the compound, and the judgment of the prescribing physician. Generally, the effective dose is about 0.001 mg/kg body weight/day to about 10000 mg/kg body weight/day. Where appropriate, the effective dose is about 0.01 mg/kg body weight/day to about 1000 mg/kg body weight/day. About 0.01 to 1000 mg/kg of the subject's body weight can be administered every day, every two days, or every three days, usually 0.1 to 500 mg/kg of the subject's body weight. An exemplary treatment regimen is one or more times a day or one or more times a week or one or more times a month. The formulation is usually administered multiple times, and the interval between single doses can be daily, weekly, monthly or yearly. Alternatively, the preparation may be administered in the form of a sustained-release preparation, in which case a lower frequency of administration is required. The dosage and frequency vary according to the half-life of the preparation in the subject. It can also be different depending on whether it is a preventive treatment or a therapeutic treatment. In prophylactic applications, relatively low doses are given over a long period of time at relatively low frequency intervals. In therapeutic applications, it is sometimes necessary to administer relatively high doses at relatively short intervals until the progression of the disease is delayed or stopped, and preferably until the individual shows partial or complete amelioration of disease symptoms, after which the patient can be administered Prevention program.

The amount of the compound of the present invention administered will depend on the individual being treated, the severity of the disorder or condition, the rate of administration, the treatment of the compound, and the judgment of the prescribing physician.

The purpose of the term "treatment" used in the present invention is to reduce or eliminate the targeted disease state or condition. If a subject receives a therapeutic amount of a compound, an optical isomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof according to the method described herein, the subject exhibits one or more signs and symptoms Observable and/or detectable decrease or improvement, then the subject is successfully "treated". It should also be understood that the treatment of the disease state or condition includes not only complete treatment, but also includes not achieving complete treatment, but achieving some biological or medical related results.

"Treatment" means any administration of a compound of the invention, including:

(1) Prevent the occurrence of disease in animals that may have disease tendency but have not experienced or displayed disease pathology or symptomology;

(2) Suppress the disease in an animal that is experiencing or showing disease pathology or symptomology (that is, preventing the further development of pathology and/or symptomology); or

(3) Improving the disease (that is, reversing the pathology and/or symptomology) in animals that are experiencing or showing disease pathology or symptomology.

"Individual" as used herein includes human or non-human animals. Exemplary human individuals include human individuals (referred to as patients) or normal individuals suffering from diseases such as those described herein. In the present invention, "non-human animals" include all vertebrates, such as non-mammals (such as birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and/or domesticated animals (such as sheep, dogs). , Cats, cows, pigs, etc.).

In order to make the objectives and technical solutions of the present invention clearer, the following describes the embodiments of the present invention in detail with reference to examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention.

The reagents or instruments used in the examples are all conventional products that can be purchased on the market. If no specific conditions are specified, the normal conditions or the conditions recommended by the manufacturer shall be used. The term "room temperature" used in the present invention means 20°C ± 5°C. When used to modify a certain numerical value or numerical range, the term "about" used in the present invention refers to the numerical value or numerical range and the error range acceptable to those skilled in the art of the numerical value or numerical range, for example, the error The range is ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, etc.

Beneficial effect

The compound of the present invention has a novel structure, has obvious inhibitory activity on TGF-β and its signaling pathway, and can be used for the treatment of proliferative disorders and cell apoptosis disorders caused by TGF-β signaling pathway.

Example

The present invention is further described below in conjunction with examples, but these examples are not intended to limit the scope of the present invention.

In the conventional synthesis method of the present invention and the examples and intermediate synthesis examples, the meaning of each abbreviation is shown in the table below.

The structures of the compounds described in the following examples were confirmed by nuclear magnetic resonance ( ¹ H-NMR) and/or mass spectrometry (MS).

The measuring instrument of nuclear magnetic resonance ( ¹ H-NMR) uses Bruker 400MHz nuclear magnetic resonance instrument, and the measuring solvent is deuterated methanol (CD ₃ OD), deuterated chloroform (CDCl ₃ ), hexadeuterated dimethyl sulfoxide (DMSO-d) ₆ ), the internal standard substance is tetramethylsilane (TMS).

The meanings of the abbreviations in the nuclear magnetic resonance (NMR) data in the following examples are as follows:

s: single, d: doublet, t: triplet, q: quartet, dd: double doublet, qd: quadruple Peak (quartet doublet), ddd: double double doublet, ddt: double double triplet, dddd: double double doublet, m: multiplet , Br: broad, J: coupling constant, Hz: Hertz, δ: chemical shift.

All chemical shift (δ) values are given in units of parts per million (ppm).

The measuring instrument for mass spectrometry (MS) uses Agilent 6120B mass spectrometer, and the ion source is an electrospray ion source (ESI).

The compound of the present invention can be separated and purified by preparative high performance liquid chromatography (Prep-HPLC) and silica gel column chromatography.

Column chromatography generally uses Claricep Flash Silica silica gel column (Bona Aigel) stationary phase. Eluent system A: acetonitrile and water system; eluent system B: dichloromethane and methanol system; the volume ratio of the solvent is adjusted according to the polarity of the compound.

Preparative high performance liquid chromatography (Prep-HPLC) purification, the method used is as follows:

Method A:

Column: Waters SunFire Prep C18 OBD 5μm 19x150mm

Mobile phase A: 10%-90% acetonitrile; Mobile phase B: 90%-10% formic acid aqueous solution (containing 0.05% formic acid, v/v), 0-20min, flow rate: 28mL/min;

Method B:

Column: Waters SunFire Prep C18 OBD 5μm 19x250mm

Mobile phase A: 30%-90% acetonitrile; Mobile phase B: 70%-10% trifluoroacetic acid aqueous solution (containing 0.05% trifluoroacetic acid, v/v), 0-16min, flow rate: 28mL/min;

Method C:

Column: XBridge Prep C18 OBD 5μm 19x150mm

Mobile phase A: 10%-70% acetonitrile; Mobile phase B: 90%-30% ammonium bicarbonate aqueous solution (containing 0.05% ammonium bicarbonate, m/v), 0-18min, flow rate: 30mL/min;

Method D:

Column: SunFire Prep C18 OBD 5μm 19x150mm

Mobile phase A: 20%-80% acetonitrile; mobile phase B: 80%-20% ammonium bicarbonate aqueous solution (containing 0.05% ammonium bicarbonate, m/v), 0-16min, flow rate: 28mL/min;

Method E:

Column: XBridge Prep C18 OBD 5μm 19x150mm

Mobile phase A: 30%-90% acetonitrile; Mobile phase B: 70%-10% ammonium bicarbonate aqueous solution (containing 0.05% ammonium bicarbonate, m/v), 0-16min, flow rate: 30mL/min;

Method F:

Column: SunFire Prep C18 OBD 5μm 19x150mm

Mobile phase A: 15%-70% acetonitrile; Mobile phase B: 85%-30% ammonium bicarbonate aqueous solution (containing 0.05% ammonium bicarbonate, m/v), 0-16min, flow rate: 28mL/min;

Method G:

Column: Waters SunFire Prep C18 OBD 5μm 19x150mm

Mobile phase A: 20%-90% acetonitrile; Mobile phase B: 80%-10% formic acid aqueous solution (containing 0.05% formic acid, v/v), 0-18min, flow rate: 28mL/min;

Method H:

Column: XBridge Prep C18 OBD 5μm 19x150mm

Mobile phase A: 10%-90% acetonitrile; Mobile phase B: 90%-10% ammonium bicarbonate aqueous solution (containing 0.05% ammonium bicarbonate, m/v), 0-16min, flow rate: 28mL/min;

Method I:

Column: Waters SunFire Prep C18 OBD 5μm 19x150mm

Mobile phase A: 30%-70% acetonitrile; Mobile phase B: 70%-30% formic acid aqueous solution (containing 0.05% formic acid, v/v), 0-16min, flow rate: 28mL/min;

Method J:

Column: Waters XBridge Prep C18 OBD 5μm 19x150mm

Mobile phase A: 15%-85% acetonitrile; Mobile phase B: 85%-15% formic acid aqueous solution (containing 0.05% formic acid, v/v), 0-16min, flow rate: 28mL/min;

Method K:

Column: Waters SunFire Prep C18 OBD 5μm 19x150mm

Mobile phase A: 10%-90% acetonitrile; Mobile phase B: 90%-10% formic acid aqueous solution (containing 0.05% formic acid, v/v), 0-16min, flow rate: 28mL/min;

Method L:

Column: Waters XBridge Prep C18 OBD 5μm 19x150mm

Mobile phase A: 10%-90% acetonitrile; Mobile phase B: 90%-10% formic acid aqueous solution (containing 0.05% formic acid, v/v), 0-18min, flow rate: 28mL/min;

Method M:

Column: Waters SunFire Prep C18 OBD 5μm 19x150mm

Mobile phase A: 10%-90% acetonitrile; Mobile phase B: 90%-10% trifluoroacetic acid aqueous solution (containing 0.05% trifluoroacetic acid, v/v), 0-18min, flow rate: 28mL/min;

Method N:

Column: Waters XBridge Prep C18 OBD 5μm 19x150mm

Mobile phase A: 10%-90% acetonitrile; Mobile phase B: 90%-10% ammonium bicarbonate aqueous solution (containing 0.05% ammonium bicarbonate, m/v), 0-18min, flow rate: 28mL/min;

Method O

Column: Waters XBridge Prep C18 OBD 5μm 19x150mm

Mobile phase A: 10%-90% acetonitrile; mobile phase B: 90%-10% ammonium bicarbonate aqueous solution (containing 0.05% ammonium bicarbonate, m/v), 0-20 min, flow rate: 28 mL/min.

The reagents used in this application were purchased from companies such as Shanghai Beide Pharmaceutical, Jiangsu Aikang Biomedicine, Chengdu Jiante, or Terbo Chemical.

Example 1: 2-(2-(5-(isoquinolin-4-ylamino)pyrazolo[1,5-a]pyridine-3-carbonyl)-2-azaspiro[3.3]heptane-6 Synthesis of -yl)-N-methylacetamide (Compound 1).

Step 1: Synthesis of 2-(2-(tert-butoxycarbonyl)-2-azaspiro[3.3]hept-6-yl)acetic acid (compound 1-2)

At 25°C, 6-(2-ethoxy-2-oxoethyl)-2-azaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester (2.0g, 7.06mmol) was dissolved in tetrahydrofuran ( 20 mL), water (10 mL), lithium hydroxide monohydrate (1.48 g, 35.29 mmol) was added all at once, and the reaction was stirred at 25° C. for 16 h. The reaction solution was evaporated to dry tetrahydrofuran under reduced pressure, diluted with 30mL of water, adjusted to pH=3 with 2N citric acid, added with ethyl acetate (50mL*3) for extraction, combined the organic phases, washed with water (50mL*3), dried with anhydrous sodium sulfate After filtering, the organic phase was evaporated to dryness under reduced pressure to obtain the title compound (1.5 g). It was directly used in the next reaction without further purification.

ESI-MS(m/z): 200.1[M+H] ⁺ .

Step 2: Synthesis of 6-(2-(methylamino)-2-oxoethyl)-2-azaspiro[3.3]heptan-2-carboxylic acid tert-butyl ester (Compound 1-3)

At 25°C, 2-(2-(tert-butoxycarbonyl)-2-azaspiro[3.3]hepta-6-yl)acetic acid (1.0g, 3.92mmol), methylamine hydrochloride (396.68mg, 5.88mmol) ) Was dissolved in N,N-dimethylformamide (15 mL), and diisopropylethylamine (1.01 g, 7.83 mmol) and HATU (2.23 g, 5.88 mmol) were added. The temperature was raised to 25° C. and the reaction was stirred for 16 h. The reaction solution was diluted with water (50mL), extracted with ethyl acetate (50mL*3), combined the organic phases, washed with water (50mL*3), dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain the title compound (1.05g) ). It was directly used in the next reaction without further purification.

ESI-MS(m/z): 213.2[M-56+H] ⁺ .

Step 3: Synthesis of N-methyl-2-(2-azaspiro[3.3]hept-6-yl)acetamide (Compound 1-4)

At 25°C, 6-(2-(methylamino)-2-oxoethyl)-2-azaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester (1.05g, 3.91mmol) was dissolved in two Trifluoroacetic acid (3mL) was added to methyl chloride (15mL), and the reaction was stirred at 25°C for 2h. The reaction solution was evaporated to dryness under reduced pressure to obtain the title compound (658 mg), which was directly used in the next reaction without further purification.

ESI-MS(m/z): 169.3[M+H] ⁺ .

Step 4: 2-(2-(5-Bromopyrazolo[1,5-a]pyridine-3-carbonyl)-2-azaspiro[3.3]hept-6-yl)-N-methylacetamide (Compound 1-5) Synthesis

At 25°C, the 5-bromopyrazolo[1,5-a]pyridine-3-carboxylic acid (500mg, 2.07mmol), N-methyl-2-(2-azaspiro[3.3]heptane-6 -Yl)acetamide (509mg, 2.49mmol) was dissolved in N,N-dimethylformamide (10mL), HATU (1.18g, 3.11mmol) and diisopropylethylamine (536mg, 4.15mmol) were added sequentially , The reaction was stirred at 25°C for 2h. The reaction solution was filtered and purified by chromatography (acetonitrile/water/0.05% trifluoroacetic acid=70/30/0.5), and the preparation solution was lyophilized to obtain the title compound (400 mg).

ESI-MS(m/z): 391.1[M+H] ⁺ .

Step 5: 2-(2-(5-(isoquinolin-4-ylamino)pyrazolo[1,5-a]pyridine-3-carbonyl)-2-azaspiro[3.3]hepta-6- Synthesis of yl)-N-methylacetamide (Compound 1)

At 25℃, isoquinolin-4-amine (18mg, 127.79μmol), 2-(2-(5-bromopyrazolo[1,5-a]pyridine-3-carbonyl)-2-azaspiro [3.3]Heptan-6-yl)-N-methylacetamide (50mg, 127.79μmol), Pd ₂ (dba) ₃ (12mg, 12.78μmol), BINAP (16mg, 25.56μmol), t-BuONa (25mg, 255.58μmol) was dissolved in toluene (2mL), and heated to 90°C under the protection of nitrogen to react for 16h. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure, and purified by preparative high performance liquid chromatography (Method A). The preparation solution was lyophilized to obtain the title compound (13.85 mg).

Its structure is characterized as follows:

¹ H NMR(400MHz,DMSO-d ₆ )δ9.34(d,J=6.8Hz,2H), 8.66(d,J=7.5Hz,1H), 8.57(s,1H), 8.37(d,J= 8.2Hz, 1H), 8.27 (d, J = 8.5Hz, 1H), 8.12 (s, 1H), 8.10-7.98 (m, 1H), 7.97-7.87 (m, 1H), 7.78-7.67 (m, 1H) ), 7.58(d,J=2.5Hz,1H),7.00(dd,J=7.5,2.6Hz,1H),4.56-3.65(m,4H),2.54(d,J=4.6Hz,3H),2.47 -2.34(m,1H),2.32-2.22(m,2H), 2.15(d,J=7.5Hz,2H),1.92-1.77(m,2H).

ESI-MS(m/z): 455.0[M+H] ⁺ .

Example 2: N-methyl-2-(2-(5-((1,4-dimethyl-1H-pyrazol-5-yl)amino)pyrazolo[1,5-a]pyridine Synthesis of -3-carbonyl)2-azaspiro[3.3]hepta-6-yl)-acetamide (compound 2)

1,4-Dimethyl-1H-pyrazole-5-amine (22.73mg, 0.20mmol), 2-(2-(5-bromopyrazolo[1,5-a]pyridine-3-carbonyl) 2-Azaspiro[3.3]heptan-6-yl)-N-methylacetamide (80mg, 0.20mmol), tris(dibenzylideneacetone) two palladium (18.72mg, 0.02mmol), (±)- 2,2'-bis-(diphenylphosphino)-1,1'-binaphthyl (25.46mg, 0.04mmol), sodium tert-butoxide (39.3mg, 0.41mmol) dissolved in toluene (5mL), protected by nitrogen The temperature was raised to 90°C and reacted for 12h. The reaction solution was cooled to room temperature, the reaction solution was concentrated, and the concentrated solution was purified by preparative high performance liquid chromatography (method C). The preparation solution was lyophilized to obtain the title compound (10 mg).

Its structure is characterized as follows:

¹ H NMR(400MHz,DMSO-d ₆ )δ8.53(d,J=7.4Hz,1H),8.50(s,1H),8.03(s,1H),7.70(d,J=4.4Hz,1H) ,7.35(s,1H),6.90(d,J=2.2Hz,1H),6.59(dd,J=7.4,2.5Hz,1H),4.44-4.22(m,2H),4.09-3.83(m,2H ),3.59(s,2H),2.53(d,J=4.6Hz,2H),2.45-2.37(m,1H),2.30-2.23(m,2H),2.14(d,J=7.5Hz,2H) ,1.88-1.81(m,4H).

ESI-MS(m/z): 422.2[M+H] ⁺ .

Example 3: 2-(2-(5-((3-hydroxy-5-methylphenyl)amino)pyrazolo[1,5-a]pyridine-3-carbonyl)2-azaspiro[ 3.3] Synthesis of Hept-6-yl)-N-methylacetamide

Combine 3-amino-5-methylphenol (25.18mg, 0.20mmol), 2-(2-(5-bromopyrazolo[1,5-a]pyridine-3-carbonyl)2-azaspiro[ 3.3) Hept-6-yl)-N-methylacetamide (80mg, 0.20mmol), tris(dibenzylideneacetone) two palladium (18.72mg, 0.02mmol), (±)-2,2'-bis -(Diphenylphosphino)-1,1'-binaphthyl (25.46mg, 0.04mmol), sodium tert-butoxide (39.3mg, 0.41mmol) dissolved in toluene (5mL), heated to 90℃ under nitrogen protection 12h. The reaction solution was cooled to room temperature, the reaction solution was concentrated, and the concentrated solution was purified by preparative high performance liquid chromatography (Method F). The preparation solution was lyophilized to obtain the title compound (15 mg).

Its structure is characterized as follows:

¹ H NMR(400MHz,DMSO-d ₆ )δ9.38(s,1H),8.68(s,1H),8.51(d,J=7.5Hz,1H),8.04(s,1H),7.70(d, J = 4.6Hz, 1H), 7.65 (d, J = 2.4 Hz, 1H), 6.73 (dd, J = 7.5, 2.6 Hz, 1H), 6.45 (s, 2H), 6.28 (s, 1H), 4.47- 4.25(m,2H),4.08-3.82(m,3H),2.54(d,J=4.6Hz,3H),2.46-2.38(m,1H),2.32-2.25(m,2H), 2.20(s, 2H), 2.15 (d, J = 7.5Hz, 2H), 1.89-1.83 (m, 2H).

ESI-MS(m/z): 434.2[M+H] ⁺ .

Example 4: N-methyl-2-(2-(5-((1-methyl-3-(m-tolyl)-1H-pyrazol-5-yl)amino)pyrazolo[1,5 -a] Synthesis of pyridine-3-carbonyl)-2-azaspiro[3.3]hept-6-yl)acetamide (compound 4).

At 25°C, 2-methyl-5-(m-tolyl)pyrazol-3-amine (24mg, 127.79μmol), 2-(2-(5-bromopyrazolo[1,5-a]pyridine -3-carbonyl)-2-azaspiro[3.3]heptan-6-yl)-N-methylacetamide (50mg, 127.79μmol), Pd ₂ (dba) ₃ (12mg, 12.78μmol), BINAP (16mg , 25.56μmol), t-BuONa (25mg, 255.58μmol) were dissolved in toluene (2mL), and heated to 90℃ for 16h under the protection of nitrogen. Cool to room temperature, filter, concentrate the filtrate, and purify by preparative high performance liquid chromatography (Method B). The preparation is lyophilized to obtain the title compound (10 mg).

Its structure is characterized as follows:

¹ H NMR(400MHz,DMSO-d ₆ )δ8.81(s,1H), 8.58(d,J=7.5Hz,1H), 8.07(s,1H), 7.74-7.66(m,1H), 7.63( d, J = 1.8 Hz, 1H), 7.58 (dt, J = 7.6, 1.5 Hz, 1H), 7.35 (d, J = 2.6 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.12 ( d,J=8.2Hz,1H),6.77(dd,J=7.5,2.6Hz,1H),6.62(s,1H),4.42-3.97(m,4H),3.72(s,3H),2.53(d ,J=4.6Hz,3H), 2.46–2.31(m,4H), 2.31–2.22(m,2H), 2.14(d,J=7.5Hz,2H), 1.90–1.79(m,2H).

ESI-MS(m/z):498.3[M+H] ⁺ .

Example 5: 2-(2-(5-(Benzo[d]oxazol-4-ylamino)pyrazolo[1,5-a]pyridine-3-carbonyl)-2-azaspiro[3.3 ]Hept-6-yl)-N-methylacetamide (Compound 5)

At 25℃, benzo[d]oxazole-4-amine (10mg, 0.077mmol), 2-(2-(5-bromopyrazolo[1,5-a]pyridine-3-carbonyl)-2 -Azaspiro[3.3]heptan-6-yl)-N-methylacetamide (30mg, 0.077mmol), Pd ₂ (dba) ₃ (7mg, 0.077mmol), BINAP (10mg, 0.015mmol), t- BuONa (15mg, 0.15mmol) was dissolved in toluene (2mL) and heated to 90°C under the protection of nitrogen to react for 16h. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated. The crude product was dissolved in acetonitrile, and then purified by preparative high performance liquid chromatography (Method N), and the preparation was lyophilized to obtain the title compound (2.43 mg).

Its structure is characterized as follows:

¹ H NMR(400MHz,DMSO-d ₆ )δ9.21(s,1H), 8.74(s,1H), 8.58(d,J=7.5Hz,1H), 8.08(s,1H), 7.74(d, J = 2.6Hz, 1H), 7.73–7.65 (m, 1H), 7.50–7.39 (m, 2H), 7.34 (dd, J = 7.0, 1.9 Hz, 1H), 6.96 (dd, J = 7.6, 2.6 Hz ,1H), 4.45–3.87(m,4H), 2.54(d,J=4.6Hz,3H), 2.46–2.37(m,1H),2.34–2.23(m,2H), 2.15(d,J=7.5 Hz, 2H), 1.93-1.80 (m, 2H).

ESI-MS(m/z): 445.0[M+H] ⁺ .

Example 6: N-methyl-2-(2-(5-((1,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)amino)pyrazolo[1,5 -a] Synthesis of pyridine-3-carbonyl)-2-azaspiro[3.3]hept-6-yl)acetamide (compound 6).

At 25℃, 1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-amine (9mg, 76.68μmol), 2-(2-(5-bromopyrazolo[1,5- a]Pyridin-3-carbonyl)-2-azaspiro[3.3]hept-6-yl)-N-methylacetamide (30mg, 76.68μmol), Pd ₂ (dba) ₃ (7mg, 7.67μmol), BINAP (10mg, 15.34μmol) and t-BuONa (15mg, 153.35μmol) were dissolved in toluene (2mL), nitrogen gas was bubbled for 2min, and microwave heating to 110℃to react for 30min. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated. The crude product was dissolved in acetonitrile and purified by preparative high performance liquid chromatography (Method O), and the preparation was lyophilized to obtain the title compound (2.41 mg).

Its structure is characterized as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ 11.84 (s, 1H), 8.89 (s, 1H), 8.46 (d, J = 7.5 Hz, 1H), 8.01 (s, 1H), 7.95 (s, 1H), 7.76–7.65(m,1H), 6.96–6.79(m,1H), 4.40–3.88(m,4H), 2.72–2.60(m,2H), 2.54(d,J=4.6Hz,3H) ,2.52–2.37(m,5H), 2.35–2.23(m,2H), 2.15(d,J=7.4Hz,2H), 1.92–1.79(m,2H).

ESI-MS(m/z): 434.1[M+H] ⁺ .

Example 7: N-methyl-2-(2-(5-(1-methyl-3-(2-methylphenyl)-1H-pyrazol-5-amino)pyrazolo[1, Synthesis of 5-a]pyridine-3-carbonyl)2-azaspiro[3.3]hept-6-yl)-acetamide (compound 7)

The 1-methyl-3-(2-methylphenyl)-1H-pyrazol-5-amine (38.28mg, 0.20mmol), 2-(2-(5-bromopyrazolo[1,5- a]Pyridin-3-carbonyl)2-azaspiro[3.3]hept-6-yl)-N-methylacetamide (80mg, 0.20mmol), tris(dibenzylideneacetone)dipalladium (18.72mg, 0.02mmol), BINAP (25.46mg, 0.04mmol), and sodium tert-butoxide (39.3mg, 0.41mmol) were dissolved in toluene (5mL), and heated to 90°C for 12h under nitrogen protection. The reaction solution was cooled to room temperature, the reaction solution was concentrated, and the concentrate was purified by preparative high performance liquid chromatography (Method D). The preparation solution was lyophilized to obtain the title compound (30 mg).

Its structure is characterized as follows:

¹ H NMR(400MHz,DMSO-d ₆ )δ8.83(s,1H), 8.58(d,J=7.6Hz,1H), 8.07(s,1H), 7.69(s,1H), 7.60–7.54( m,1H),7.41(d,J=2.2Hz,1H),7.29–7.21(m,3H),6.77(dd,J=7.5,2.6Hz,1H),6.44(s,1H),4.47–4.26 (m,2H),4.09–3.89(m,2H),3.74(s,3H),2.53(d,J=4.6Hz,3H), 2.49(s,3H), 2.46–2.37(m,1H), 2.30–2.23(m,2H), 2.14(d,J=7.5Hz,2H), 1.88–1.81(m,2H).

ESI-MS(m/z):498.3[M+H] ⁺ .

Example 8: N-methyl-2-(2-(5-((2-methyl-2,4,5,6-tetrahydrocyclopentane[c]pyrazol-3-yl)amino) Synthesis of pyrazole[1,5-a]pyridine-3-carbonyl)-2-azaspiro[3.3]hept-6-yl)-acetamide (compound 8)

Step 1: N-methyl-2-(2-(5-((2-methyl-2,4,5,6-tetrahydrocyclopentane[c]pyrazol-3-yl)amino)pyridine Synthesis of azole[1,5-a]pyridine-3-carbonyl)-2-spiro[3.3]hept-6-yl)-acetamide (compound 8)

The 2-methyl-5,6-dihydro-4H cyclopentan[c]pyrazol-3-amine (10.52mg, 76.67μmol), 2-(2-(5-bromopyrazolo[1,5- a)Pyridin-3-carbonyl)2-azaspiro[3.3]hept-6-yl)-N-methylacetamide (30mg, 76.67μmol), Pd ₂ (dba) ₃ (7.02mg, 7.67μmol), BINAP (9.55mg, 15.33μmol) and sodium tert-butoxide (14.74mg, 153.35μmol) were dissolved in toluene (2mL), and heated to 90℃ for 16h under nitrogen protection. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated. The concentrate was purified by preparative high performance liquid chromatography (Method G). The preparation was lyophilized to obtain the title compound (4.70 mg).

Its structure is characterized as follows:

¹ H NMR(400MHz,Methanol-d ₄ )δ8.39(d,J＝7.5Hz,1H), 8.07(s,1H), 7.29(d,J＝2.5Hz,1H), 6.70(dd,J＝ 7.5, 2.6Hz, 1H), 4.61-4.30 (m, 2H), 4.22-3.93 (m, 2H), 3.67 (s, 3H), 2.71-2.65 (m, 5H), 2.60-2.51 (m, 3H) , 2.48-2.35 (m, 4H), 2.27 (d, J = 7.6 Hz, 2H), 2.06-1.88 (m, 2H).

ESI-MS(m/z): 448.1[M+H] ⁺ .

Example 9: N-methyl-2-(2-(5-(5-methoxy-2-(1H-pyrrol-1-yl)anilino)pyrazolo[1,5-a]pyridine- Synthesis of 3-carbonyl)2-azaspiro[3.3]hept-6-yl)-acetamide (compound 9)

The 5-methoxy-2-(1H-pyrrol-1-yl)aniline (38.49mg, 0.20mmol), 2-(2-(5-bromopyrazolo[1,5-a]pyridine-3- Carbonyl) 2-azaspiro[3.3]heptan-6-yl)-N-methylacetamide (80mg, 0.20mmol), Pd ₂ (dba) ₃ (18.72mg, 0.02mmol), BINAP (25.46mg, 0.04 mmol), sodium tert-butoxide (39.3 mg, 0.41 mmol) were dissolved in toluene (5 mL), and the temperature was raised to 90° C. for reaction for 12 h under nitrogen protection. The reaction solution was cooled to room temperature, the reaction solution was concentrated, and the concentrate was purified by preparative high performance liquid chromatography (Method E). The preparation solution was lyophilized to obtain the title compound (20 mg).

Its structure is characterized as follows:

¹ H NMR(400MHz,DMSO-d ₆ )δ8.42(d,J=7.6Hz,1H),8.30(s,1H),8.00(s,1H),7.69(d,J=4.4Hz,1H) ,7.33(d,J=8.8Hz,1H), 7.26(d,J=2.2Hz,1H), 6.98(d,J=2.8Hz,1H), 6.90(dd,J=8.8,2.8Hz, 1H) ,6.85(t,J=2.1Hz,2H),6.62(dd,J=7.5,2.6Hz,1H),6.08(t,J=2.1Hz,2H),4.47–4.20(m,2H),4.11– 3.88(m,2H), 3.79(s,3H), 2.54(d,J=4.6Hz,3H), 2.46–2.38(m,1H), 2.31–2.23(m,2H), 2.14(d,J= 7.6Hz, 2H), 1.88-1.81 (m, 2H).

ESI-MS(m/z):499.2[M+H] ⁺ .

Example 10: N-[(3S,5R)-1-tert-butoxycarbonyl-5-(carbamoyl)pyrrolidin-3-yl]-3-[3-(3,5-dimethyl-1H -Pyrazol-1-yl)phenyl]imidazo[1,2-a]pyridine-7-carboxamide (Compound 10)

Step 1: 3-(3-(3,5-Dimethyl-1H-pyrazol-1-yl)phenyl)imidazo[1,2-a]pyridine-7-carboxylic acid methyl ester (Compound 10-2 )Synthesis

Combine 3-iodoimidazo[1,2-a]pyridine-7-methyl ester (850mg, 2.81mmol) and 3,5-dimethyl-1-(3-(4,4,5,5-tetra Methyl-1,3,2-dioxaborolan-2-yl)phenyl)-1H-pyrazole (910mg, 3.05mmol) dissolved in a mixture of dioxane (12mL) and water (3mL) In the solvent, potassium carbonate (777mg, 5.63mmol) and dichlorodi-tert-butyl-(4-dimethylaminophenyl) phosphopalladium(II) (199mg, 281.39μmol) were added. After the addition, the reaction was replaced with nitrogen. The system was 3 times, and under the protection of nitrogen, the temperature was raised to 95° C. and reacted for 2 hours. After the reaction solution was cooled to room temperature, dichloromethane (100 mL) was added, and then washed with water (30 mL) and saturated brine (30 mL) each time. The organic phase was dried over anhydrous sodium sulfate, the desiccant was filtered off, and the filtrate was evaporated under reduced pressure. The crude product was obtained, which was purified by column chromatography (dichloromethane/methanol=50/1) to obtain the title compound (905 mg).

MS(ESI): m/z 347.2[M+H] ⁺ .

Step 2: 3-(3-(3,5-Dimethyl-1H-pyrazol-1-yl)phenyl)imidazo[1,2-a]pyridine-7-carboxylic acid (compound 10-3) synthesis

Dissolve 3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)imidazo[1,2-a]pyridine-7-carboxylic acid methyl ester (905mg, 2.61mmol) In the mixed solvent of tetrahydrofuran (12 mL) and water (4 mL), lithium hydroxide monohydrate (548 mg, 13.06 mmol) was added, and after the addition, the reaction was carried out at room temperature for 2 hours. The pH of the aqueous phase of the reaction solution was adjusted to 7 with 2N aqueous hydrochloric acid solution, the solvent was evaporated under reduced pressure, a mixed solvent of DCM and MeOH (v/v=5/1) was added, the insoluble matter was filtered off, and the filtrate was evaporated under reduced pressure to obtain the title compound The crude product was 725 mg, and the product was directly used in the next reaction without purification.

¹ H-NMR (400MHz, DMSO-d ₆ ): δ8.48 (dd, J = 7.1, 1.0Hz, 1H), 7.99 (s, 1H), 7.84 (s, 1H), 7.74 (s, 1H), 7.71–7.62 (m, 2H), 7.55 (dt, J = 7.4, 1.9 Hz, 1H), 7.46 (dd, J = 7.1, 1.6 Hz, 1H), 6.11 (s, 1H), 2.38 (s, 3H) , 2.20(s,3H).

MS(ESI): m/z 333.2[M+H] ⁺ .

Step 3: N-[(3S,5R)-1-tert-butoxycarbonyl-5-(methoxycarbonyl)pyrrolidin-3-yl]-3-[3-(3,5-dimethyl-1H- Synthesis of Pyrazol-1-yl)phenyl]imidazo[1,2-a]pyridine-7-carboxamide (Compound 10-4)

Combine 3-[3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl]imidazo[1,2-a]pyridine-7-carboxylic acid (80mg, 0.24mmol), (2R ,4S)-1-tert-butoxycarbonyl-4-aminopyrrolidine-2-carboxylic acid methyl ester (64.68mg, 0.26mmol) was dissolved in DMF (5mL), then HOBt (48.79mg, 0.36mmol), EDCI (55.37mg, 0.29mmol), DIPEA (62.22mg, 0.48mmol), placed at room temperature and reacted for 12h. The reaction solution was poured into water and extracted with ethyl acetate (15 mL*3). The organic phases were combined, dried and concentrated to obtain the title compound (120 mg). The product was directly used in the next reaction without purification.

ESI-MS(m/z): 559.3[M+H] ⁺ .

Step 4: N-[(3S,5R)-1-tert-butoxycarbonyl-5-(carbamoyl)pyrrolidin-3-yl]-3-[3-(3,5-dimethyl-1H- Synthesis of Pyrazol-1-yl)phenyl]imidazo[1,2-a]pyridine-7-carboxamide (Compound 10-5)

N-[(3S,5R)-1-tert-butoxycarbonyl-5-(methoxycarbonyl)pyrrolidin-3-yl]-3-[3-(3,5-dimethyl-1H-pyrazole -1-yl)phenyl]imidazo[1,2-a]pyridine-7-carboxamide (120mg, 0.21mmol) was dissolved in methylamine ethanol solution (5mL, 33%), and the temperature was raised to 40°C to react for 12h. The reaction solution was concentrated, water was added, and the mixture was extracted with ethyl acetate (15 mL*3). The organic phases were combined, dried and concentrated to obtain the title compound (110 mg). The product was directly used in the next reaction without purification.

ESI-MS(m/z): 558.3[M+H] ⁺ .

Step 5: N-[(3S,5R)-5-(carbamoyl)pyrrolidin-3-yl]-3-[3-(3,5-dimethyl-1H-pyrazol-1-yl) Synthesis of Phenyl]imidazo[1,2-a]pyridine-7-carboxamide (Compound 10-6)

N-[(3S,5R)-1-tert-butoxycarbonyl-5-(carbamoyl)pyrrolidin-3-yl]-3-[3-(3,5-dimethyl-1H-pyrazole -1-yl)phenyl]imidazo[1,2-a]pyridine-7-carboxamide (110mg, 0.20mmol) was dissolved in dichloromethane (5mL), trifluoroacetic acid (2mL) was added, and the reaction was kept at room temperature 5h. The reaction solution was concentrated and added to water, adjusted to pH=8 with sodium carbonate, extracted with ethyl acetate (15 mL*3), combined the organic phases, dried and concentrated to obtain the title compound (80 mg). The product was directly used in the next reaction without purification.

ESI-MS(m/z): 458.2[M+H] ⁺ .

Step 6: N-[(3S,5R)-1-benzoyl-5-(carbamoyl)pyrrolidin-3-yl]-3-[3-(3,5-dimethyl-1H-pyridine) Synthesis of azol-1-yl)phenyl]imidazo[1,2-a]pyridine-7-carboxamide (compound 10)

N-[(3S,5R)-5-(carbamoyl)pyrrolidin-3-yl]-3-[3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl ]Imidazo[1,2-a]pyridine-7-carboxamide (80mg, 0.17mmol), potassium benzoate (30.81mg, 0.19mmol) were dissolved in DMF (5mL), and HOBt (35.44mg, 0.26mmol) ), EDCI (40.22mg, 0.21mmol), DIPEA (45.20mg, 0.35mmol), and react at room temperature for 6h. The reaction solution was poured into water, extracted with ethyl acetate, the organic phases were combined, dried and concentrated, and the concentrated solution was purified by preparative high performance liquid chromatography (Method E). The preparation solution was lyophilized to obtain the title compound (30 mg).

Its structure is characterized as follows:

¹ H NMR(400MHz,DMSO-d ₆ )δ8.82(d,J=6.3Hz,1H), 8.63(d,J=7.2Hz,1H), 8.25(s,1H), 8.02(s,1H) ,7.95(d,J=4.6Hz,1H),7.77(s,1H),7.69(t,J=6.7Hz,2H),7.62-7.56(m,3H),7.46(d,J=7.5Hz, 3H),7.36-7.32(m,1H),6.11(s,1H),4.70-4.63(m,1H),4.51(d,J=4.5Hz,1H),3.93(dd,J=10.6,5.7Hz ,1H), 3.47(dd,J＝10.6,4.3Hz,1H), 2.64(d,J＝4.5Hz,2H),2.38(s,3H),2.33(d,J＝1.9Hz,1H),2.19 (s, 3H), 2.15 (dd, J = 12.9, 6.4 Hz, 1H).

ESI-MS(m/z):562.3[M+H] ⁺ .

Example eleven: (S)-3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-N-(1-(3-methoxyphenyl)-3- Synthesis of (Methylamino)-3-oxopropyl)imidazo[1,2-a]pyridine-7-carboxamide (Compound 11)

Step 1: (S)-3-(3-(3,5-Dimethyl-1H-pyrazol-1-yl)phenyl)imidazole[1,2-a]pyridine-7-carboxamide)-3 Synthesis of -(3-Methoxyphenyl) Methyl Propionate (Compound 11-1)

Combine 3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)imidazo[1,2-a]pyridine-7-carboxylic acid (40mg, 0.12mmol) and (S )-3-Amino-3-(3-methoxyphenyl)propionic acid methyl ester hydrochloride (32.5mg, 0.13mmol) was dissolved in DMF (3mL), HOBt (24.4mg, 0.18mmol) and EDCI were added (27.7mg, 0.14mg), DIPEA (31mg, 0.24mmol) was added dropwise and the reaction was stirred for 12h. Add 10 mL of water and ethyl acetate to the reaction solution, stir, stand still for liquid separation, extract the aqueous phase with ethyl acetate (15 mL*3), combine the organic phases and wash with water (10 mL*3), dry, and concentrate under reduced pressure to obtain 62 mg. Used directly in the next reaction.

ESI-MS(m/z): 524.2[M+H] ⁺

Step 2: (S)-3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-N-(1-(3-methoxyphenyl)-3-(form Synthesis of Amino)-3-oxopropyl)imidazo[1,2-a]pyridine-7-carboxamide (Compound 11)

(S)-3-(3-(3,5-Dimethyl-1H-pyrazol-1-yl)phenyl)imidazole[1,2-a]pyridine-7-carboxamide)-3-( Methyl 3-methoxyphenyl)propionate (62 mg, 0.12 mmol) was dissolved in methylamine ethanol solution (5 mL, 33%), and reacted at 40° C. for 12 h. The solvent was evaporated under reduced pressure, the concentrate was purified by preparative high performance liquid chromatography (Method K), and the preparation was lyophilized to obtain the title compound (22.8 mg).

Its structure is characterized as follows:

¹ H NMR (400MHz, DMSO-d ₆ ): δ9.13 (d, J = 8.2Hz, 1H), 8.66 (dd, J = 7.3, 0.9Hz, 1H), 8.29 (dd, J = 1.8, 0.9Hz ,1H), 8.02 (s, 1H), 7.86 (d, J = 5.1 Hz, 1H), 7.79 (t, J = 1.9 Hz, 1H), 7.74-7.65 (m, 2H), 7.59 (dt, J = 7.3,2.0Hz,1H),7.38(dd,J=7.2,1.8Hz,1H),7.24(t,J=7.8Hz,1H),7.03-6.92(m,2H),6.82-6.79(m,1H ), 6.11 (s, 1H), 5.44 (q, J = 7.7 Hz, 1H), 3.74 (s, 3H), 2.68 (t, J = 7.6 Hz, 2H), 2.54 (d, J = 4.6 Hz, 3H ), 2.39 (d, J = 0.8Hz, 3H), 2.19 (s, 3H).

MS(ESI): m/z 523.3[M+H] ⁺ .

Example 12: 3-(3-(3,5-Dimethyl-1H-pyrazol-1-yl)phenyl)-N-(4-2-(methylamino)-2-ethoxy )Benzyl)imidazo[1,2-a]pyridine-7-carboxamide (Compound 12)

Step 1: 2-(4-((3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)imidazo[1,2-a]pyridine-7-methan Synthesis of amide) methyl) phenyl) methyl acetate (compound 12-1)

Combine 3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)imidazo[1,2-a]pyridine-7-carboxylic acid (30mg, 90.26μmol) and 2- (4-(Aminomethyl)phenyl) methyl acetate hydrochloride (21mg, 97.40μmol) was dissolved in dimethylformamide (1mL), then HATU (41mg, 108.32μmol) and diisopropyl were added Ethylamine (46mg, 361.06μmol), after the addition, the reaction was stirred at room temperature for 15h. The reaction solution was slowly poured into 10 mL of clean water, and then extracted with ethyl acetate (10 mL*3). The organic phases were combined, washed with saturated brine (10 mL), and the organic phases were dried with anhydrous sodium sulfate and evaporated to dryness under reduced pressure. The title compound (41 mg) was obtained.

MS(ESI): m/z 494.2[M+H] ⁺ .

Step 2: 3-(3-(3,5-Dimethyl-1H-pyrazol-1-yl)phenyl)-N-(4-2-(methylamino)-2-ethoxy)benzyl Yl)imidazo[1,2-a]pyridine-7-carboxamide (Compound 12)

2-(4-((3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)imidazo[1,2-a]pyridine-7-carboxamide) Methyl)phenyl)acetate (41mg, 83.07μmol) was dissolved in methylamine ethanol solution (3mL, 33%), and then the temperature was raised to 45°C for 24h. The reaction solution was directly evaporated to dryness under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (acetonitrile/water/trifluoroacetic acid=27/73/0.05) to obtain the trifluoroacetate salt of the title compound (36mg).

Its structure is characterized as follows:

¹ H NMR (400MHz, DMSO-d ₆ ): δ9.50 (t, J = 5.9Hz, 1H), 8.84-8.77 (m, 1H), 8.38 (s, 1H), 8.31 (s, 1H), 7.96 (d,J=4.4Hz,1H),7.84(s,1H),7.73(d,J=5.1Hz,2H),7.71-7.64(m,2H),7.28(d,J=8.1Hz,2H) ,7.22(d,J=8.2Hz,2H),6.13(s,1H),4.50(d,J=5.8Hz,2H),2.55(d,J=4.6Hz,3H),2.39(d,J= 0.8Hz, 3H), 2.20 (s, 3H).

¹⁹ F NMR (400MHz, DMSO-d ₆ ): δ-74.18.

MS(ESI): m/z 493.2[M+H] ⁺ .

Example 13: 3-(3-(3,5-Dimethyl-1H-pyrazol-1-yl)phenyl)-N-((4-(carbamoyl)cyclohexyl)methyl)imidazole Synthesis of [1,2-a]pyridine-7-carboxamide (Compound 13)

Step 1: 4-((3-(3,5-Dimethyl-1H-pyrazol-1-yl)phenyl)imidazole[1,2-a]pyridine-7-carboxamide)methyl)cyclohexane Synthesis of Methyl Carboxylate (Compound 13-1)

Combine 3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)imidazo[1,2-a]pyridine-7-carboxylic acid (40mg, 0.12mmol) and 4- Methyl aminomethylcyclohexanecarboxylate hydrochloride (27.5mg, 0.13mmol) was dissolved in DMF (3mL), HOBt (24.3mg, 0.18mmol) and EDCI (27.7mg, 0.14mg) were added, and DIPEA ( 31 mg, 0.24 mmol) and the reaction was stirred for 12 h. Add 10 mL each of water and ethyl acetate to the reaction solution, stir and let stand for liquid separation. The aqueous phase is extracted with ethyl acetate (15mL*2), the organic phases are combined and washed with water (10mL*3), dried, concentrated under reduced pressure, and used directly In the next step.

MS(ESI): m/z 486.2[M+H] ⁺ .

Step 2: 3-(3,5-Dimethyl-1H-pyrazol-1-yl)phenyl)-N-((4-(carbamoyl)cyclohexyl)methyl)imidazo[1,2 -a] Synthesis of pyridine-7-carboxamide (Compound 13)

The 4-((3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)imidazole[1,2-a]pyridine-7-carboxamide)methyl)cyclohexanecarboxylic acid The methyl ester (58mg, 0.12mmol) was dissolved in methylamine ethanol solution (5mL, 33%), and the reaction was heated at 50°C for 48h. The solvent was evaporated under reduced pressure, the concentrate was purified by preparative high performance liquid chromatography (Method J), and the preparation was lyophilized to obtain the title compound (15.1 mg).

Its structure is characterized as follows:

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.70 (t, J = 5.7Hz, 1H), 8.65 (dd, J = 7.2, 0.9Hz, 1H), 8.24 (dd, J = 1.8, 0.9Hz ,1H),8.01(s,1H),7.78(t,J=2.0Hz,1H),7.73-7.58(m,4H),7.42(dd,J=7.3,1.8Hz,1H),6.12(s, 1H), 3.15 (t, J = 6.3 Hz, 2H), 2.54 (d, J = 4.6 Hz, 3H), 2.39 (d, J = 0.7 Hz, 3H), 2.20 (s, 3H), 2.02 (d, J = 12.1Hz, 1H), 1.83-1.70 (m, 4H), 1.54 (s, 1H), 1.39-1.27 (m, 2H), 0.96 (t, J = 11.3Hz, 2H).

MS(ESI): m/z 485.3[M+H] ⁺ .

Example fourteen: (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-N-(3-(methylamino)-3-oxy Synthesis of 1-(pyridin-3-yl)propyl)imidazo[1,2-a]pyridine-7-amide (compound 14)

Step 1: (S)-3-(3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)imidazo[1,2-a]pyridine-7-carboxy Synthesis of Acylamino)-3-(Pyridin-3-yl) Methyl Propionate (Compound 14-1)

At 25°C, (S)-3-amino-3-(pyridin-3-yl) methyl propionate (33mg, 0.18mmol), 3-(3-(3,5-dimethyl-1H-pyridine) (Azol-1-yl)phenyl)imidazo[1,2-a]pyridine-7-carboxylic acid (50mg, 0.15mmol) dissolved in N,N-dimethylformamide (3mL), and then added 1-hydroxyl Benzotriazole (30mg, 0.23mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (35mg, 0.18mmol), N,N-diisopropylethyl Amine (39mg, 0.mmol), the reaction was stirred at 25°C for 16h. Water (30mL) was added to the reaction solution, extracted with ethyl acetate (30mL*3), the organic phases were combined, washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain the title compound (80mg) , Used directly in the next reaction without further purification.

ESI-MS(m/z):495.1[M+H] ⁺ .

Step 2: (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-N-(3-(methylamino)-3-oxo- Synthesis of 1-(pyridin-3-yl)propyl)imidazo[1,2-a]pyridine-7-amide (compound 14)

At 25°C, (S)-3-(3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)imidazo[1,2-a]pyridine-7 -Carboxamido)-3-(pyridin-3-yl)propionic acid methyl ester (80mg, 0.16mmol) was dissolved in methylamine ethanol solution (4mL), and the temperature was raised to 40°C and the reaction was stirred for 16h. The reaction solution was filtered, and the filtrate was purified by preparative high performance liquid chromatography (Method M), and the preparation solution was freeze-dried to obtain the trifluoroacetate (28.29 mg) of the title compound.

Its structure is characterized as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ 9.50 (d, J = 7.6 Hz, 1H), 8.85-8.77 (m, 2H), 8.66 (d, J = 5.1, 1.5 Hz, 1H), 8.39 ( s, 1H), 8.34 (s, 1H), 8.26-8.18 (m, 1H), 8.02-7.93 (m, 1H), 7.89-7.82 (m, 1H), 7.79-7.65 (m, 4H), 7.60 ( dd,J=7.2,1.8Hz,1H),6.13(s,1H),5.55(q,J=7.5Hz,1H),2.91-2.76(m,2H),2.54(d,J=4.5Hz,3H ), 2.40(s, 3H), 2.19(s, 3H).

¹⁹ F NMR (400MHz, DMSO-d ₆ ): δ-74.46.

ESI-MS(m/z):493.9[M+H] ⁺ .

Example 15: 3-(3-(3,5-Dimethyl-1H-pyrazol-1-yl)phenyl)-N-((1S,3S)-3-(carbamoyl methyl ester) Synthesis of Cyclohexyl)imidazo[1,2-a]pyridine-7-carboxamide (Compound 15)

Step 1: (1S,3S)-3-(3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)imidazo[1,2-a]pyridine-7 -Formamide) Synthesis of methyl cyclohexylcarboxylate (Compound 15-1)

Combine 3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)imidazo[1,2-a]pyridine-7-carboxylic acid (30mg, 90.26μmol) and (1S ,3S) Methyl-3-aminocyclohexylcarboxylate (21mg, 108.32μmol) was dissolved in dimethylformamide (1mL), and then EDCI (21mg, 108.32μmol), HOBT (18mg, 135.40μmol) and diisopropyl were added. Propylethylamine (46mg, 361.06μmol), after the addition, the reaction was stirred at room temperature for 15h. The reaction solution was slowly poured into 10 mL of water, and then extracted with ethyl acetate (10 mL*3). The organic phases were combined, washed with saturated brine (10 mL), and the organic phases were dried with anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The filtrate was crude. The crude product was purified by column chromatography (acetonitrile/water/trifluoroacetic acid=27/73/0.05) to obtain 28 mg of the trifluoroacetate salt of the title compound.

MS(ESI): m/z 472.3[M+H] ⁺ .

Step 2: 3-(3-(3,5-Dimethyl-1H-pyrazol-1-yl)phenyl)-N-((1S,3S)-3-(carbamoylmethyl)cyclohexyl ) Synthesis of imidazo[1,2-a]pyridine-7-carboxamide (Compound 15)

Add (1S,3S)-3-(3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)imidazo[1,2-a]pyridine-7-methyl Amide) trifluoroacetate (28mg, 47.82μmol) of methylcyclohexylcarboxylate was dissolved in methylamine ethanol solution (3mL, 33%), and then heated to 45°C for 24h. The reaction solution was directly evaporated to dryness under reduced pressure to obtain a crude product. The crude product was preparatively purified by high performance liquid chromatography (Method K), and the preparation was lyophilized to obtain the title compound (6 mg).

Its structure is characterized as follows:

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.65(d,J=7.9Hz,1H), 8.31(d,J=9.9Hz,2H), 8.02(s,1H), 7.78(s,1H) ),7.75-7.66(m,2H),7.66-7.57(m,2H),7.41(dd,J=7.3,1.8Hz,1H),6.12(s,1H),4.18(s,1H),2.58( s, 4H), 2.39 (s, 3H), 2.20 (s, 3H), 1.78 (t, J = 5.8 Hz, 2H), 1.72-1.51 (m, 6H).

MS(ESI): m/z 471.3[M+H] ⁺ .

Example 16: (S)-N-(3-(5-hydroxy-1H-indol-3-yl)-1-(methylamino)-1-oxopropyl-2-yl)-3-( Synthesis of 3-(5-methyl-1H-pyrazol-1-yl)phenyl)imidazole[1,2-a]pyridine-7-amide (Compound 16)

Step 1: (S)-3-(5-hydroxy-1H-indol-3-yl)-2-(3-(3-(5-methyl-1H-pyrazol-1-yl)phenyl) Synthesis of imidazole [1,2-a]pyridine-7-amide) methyl propionate (compound 16-1)

(S)-2-amino-3-(5-hydroxy-1H-indol-3-yl)propionic acid methyl ester (32.58mg, 120.35μmol), 3-(3-(3,5-dimethyl -1H-pyrazol-1-yl)phenyl)imidazole[1,2-a]pyridine-7-carboxylic acid (40.00mg, 120.35μmol), 1-hydroxybenzotriazole (24.39mg, 180.53μmol), 1 -(3-Dimethylaminopropyl)-3-ethylcarbodiimide (27.69mg, 144.42μmol) and N,N'-diisopropylethylamine (31.11mg, 240.71μmol) were added to the single-mouth bottle Add N,N-dimethylformamide (3mL) and react at 25°C for 12h. The reaction solution was slowly poured into water (10mL), extracted with ethyl acetate (10mL*3), the combined organic phase was washed with saturated brine (10mL), the organic phase was dried, filtered, and the filtrate was evaporated under reduced pressure to obtain the title compound (20mg ), used directly in the next step without purification.

ESI-MS(m/z): 549.1[M+H] ⁺ .

Step 2: (S)-N-(3-(5-hydroxy-1H-indol-3-yl)-1-(methylamino)-1-oxopropyl-2-yl)-3-(3- Synthesis of (5-methyl-1H-pyrazol-1-yl)phenyl)imidazole[1,2-a]pyridine-7-amide (compound 16)

Add (S)-3-(5-hydroxy-1H-indol-3-yl)-2-(3-(3-(5-methyl-1H-pyrazol-1-yl)phenyl)imidazole[ 1,2-a]pyridine-7-amide) methyl propionate (20 mg, 36.46 μmol) was added to a single-necked flask, methylamine ethanol solution (3 mL, 33%) was added, and the reaction was carried out at 40° C. for 6 h. The reaction solution was concentrated, and the concentrated solution was prepared and purified by high performance liquid chromatography (Method K), and the preparation solution was lyophilized to obtain the title compound (10.05 mg).

Its structure is characterized as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ 10.45 (d, J = 2.0 Hz, 1H), 8.79 (d, J = 8.1 Hz, 1H), 8.63 (d, J = 7.3 Hz, 1H), 8.60 (s,1H),8.33-8.28(m,1H),8.07(q,J=4.5Hz,1H),8.01(s,1H),7.80-7.75(m,1H),7.73-7.65(m,2H) ), 7.59 (dt, J = 7.1, 2.0 Hz, 1H), 7.38 (dd, J = 7.2, 1.8 Hz, 1H), 7.11-7.08 (m, 1H), 7.07 (s, 1H), 6.96 (d, J = 2.3Hz, 1H), 6.58 (dd, J = 8.6, 2.3Hz, 1H), 6.11 (s, 1H), 4.70-4.62 (m, 1H), 3.19-3.00 (m, 2H), 2.62 (d ,J=4.5Hz,3H),2.38(s,3H),2.19(s,3H).

ESI-MS(m/z):548.1[M+H] ⁺ .

Example 17: (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-N-(1-(3-fluorophenyl)-3 -(Methylamino)-3-oxopropyl)imidazole[1,2-a]pyridine-7-amide (Compound 17)

Step 1: (S)-3-(3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)imidazo[1,2-a]pyridine-7-amide Synthesis of methyl)-3-(3-fluorophenyl)propionate (Compound 17-1)

At 25°C, (S)-3-amino-3-(3-fluorophenyl) methyl propionate (42mg, 0.18mmol), 3-(3-(3,5-dimethyl-1H-pyridine) (Azol-1-yl)phenyl)imidazo[1,2-a]pyridine-7-carboxylic acid (50mg, 0.15mmol) dissolved in N,N-dimethylformamide (3mL), and then added 1-hydroxyl Benzotriazole (30mg, 0.23mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (35mg, 0.18mmol), N,N-diisopropylethyl Amine (39mg, 0.3mmol), the reaction was stirred at 25°C for 16h. Water (30mL) was added to the reaction solution, extracted with ethyl acetate (30mL*3), the combined organic phases were washed with saturated brine (10mL*3), dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure to obtain the title compound ( 77mg), used directly in the next reaction without further purification.

ESI-MS(m/z): 512.3[M+H] ⁺ .

Step 2: (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-N-(1-(3-fluorophenyl)-3-( Synthesis of methylamino)-3-oxopropyl)imidazole[1,2-a]pyridine-7-amide (compound 17)

At 25°C, (S)-3-(3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)imidazo[1,2-a]pyridine-7 -Amido)-3-(3-fluorophenyl)propionic acid methyl ester (30mg, 0.059mmol) was dissolved in methylamine ethanol solution (2mL, 33%), heated to 40°C and stirred for 16h. The reaction solution was filtered, the filtrate was purified by preparative high performance liquid chromatography (Method I), and the preparation solution was freeze-dried to obtain the title compound (15 mg).

Its structure is characterized as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.18 (d, J = 8.1 Hz, 1H), 8.67 (d, J = 7.2, 0.9 Hz, 1H), 8.31 (s, 1H), 8.04 (s, 1H), 7.88(q,J=4.5Hz,1H),7.83-7.75(m,1H),7.75-7.64(m,2H),7.60(dt,J=7.3,1.9Hz,1H),7.44-7.33 (m,2H),7.30-7.18(m,2H),7.13-7.03(m,1H),6.12(s,1H),5.48(q,J=7.6Hz,1H),2.81-2.63(m,2H) ), 2.54(d,J=4.6Hz,3H), 2.39(s,3H), 2.20(s,3H).

ESI-MS(m/z): 511.3[M+H] ⁺ .

Example 18: (S)-N-(1-cyclopropyl-2-(methylamino)-2-oxoethyl)-3-(3-(3,5-dimethyl-1H-pyridine) Synthesis of azol-1-yl)phenyl)imidazole[1,2-a]pyridine-7-amide (compound 18)

Step 1: (S)-2-Cyclopropyl-2-(3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)imidazole [1,2-a] Synthesis of Methyl Pyridine-7-amide) Carboxylate (Compound 18-1)

Add (S)-2-amino-2-cyclopropaneacetate methyl ester hydrochloride (19.43mg, 117.32μmol), 3-(3-(3,5-dimethyl-1H-pyrazol-1-yl) Phenyl) imidazole[1,2-a]pyridine-7-carboxylic acid (50mg, 150.44μmol), 1-hydroxybenzotriazole (30.49mg, 225.66μmol), 1-(3-dimethylaminopropyl) -3-Ethylcarbodiimide (34.61mg, 180.53μmol) and N,N'-diisopropylethylamine (38.89mg, 300.88μmol) were added to the single-mouth bottle, and N,N-dimethylformaldehyde Amide (3mL), reacted at 25°C for 3h. The reaction solution was slowly poured into water (10mL), extracted with ethyl acetate (10mL*3), the combined organic phase was washed with saturated brine (10mL), the organic phase was dried, and then evaporated to dryness under reduced pressure to obtain the title compound (60mg). It was used directly in the next step without purification.

ESI-MS(m/z): 444.3[M+H] ⁺ .

Step 2: (S)-N-(1-cyclopropyl-2-(methylamino)-2-oxoethyl)-3-(3-(3,5-dimethyl-1H-pyrazole- Synthesis of 1-yl)phenyl)imidazole[1,2-a]pyridine-7-amide (Compound 18)

Add (S)-2-cyclopropyl-2-(3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)imidazole[1,2-a]pyridine- Methyl 7-amide) carboxylate (60 mg, 135.29 μmol) was added to a single-necked flask, methylamine ethanol solution (2 mL, 33%) was added, and the reaction was carried out at 40° C. for 16 h. The reaction solution was concentrated under reduced pressure, and the concentrated solution was prepared and purified by high performance liquid chromatography (Method L), and the preparation solution was lyophilized to obtain the title compound (23.5 mg).

Its structure is characterized as follows:

¹ H NMR(400MHz,DMSO-d ₆ )δ9.00(dd,J=7.2Hz,1H), 8.66(dd,J=7.2,0.9Hz,1H), 8.43-8.33(m,1H), 8.02( s,1H),7.98-7.90(m,1H),7.80(t,J=1.8Hz,1H),7.75-7.65(m,2H),7.60(dt,J=7.4,1.9Hz,1H),7.46 (dd,J=7.2,1.8Hz,1H),6.12(s,1H),3.75(t,J=8.4Hz,1H),2.63(d,J=4.5Hz,3H),2.39(s,3H) , 2.20 (s, 3H), 1.34-1.19 (m, 1H), 0.64-0.46 (m, 3H), 0.36-0.25 (m, 1H).

ESI-MS(m/z): 443.3[M+H] ⁺ .

Example 19: 3-[3-(3,5-Dimethyl-1H-pyrazol-1-yl)phenyl]-N-[5-(methylamino)-5-oxo-n-pentyl ] Imidazo[1,2-a]pyridine-7-carboxamide (Compound 19)

Step 1: 3-[3-(3,5-Dimethyl-1H-pyrazol-1-yl)phenyl]-N-[5-(methoxy)-5-oxo-n-pentyl]imidazole Synthesis of [1,2-a]pyridine-7-carboxamide (Compound 19-1)

Add 3-[3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl]imidazo[1,2-a]pyridine-7-carboxylic acid (80mg, 0.24mmol), 5- Methyl amino-valerate (34.73mg, 0.26mmol) was dissolved in DMF (5mL), and HOBt (48.79mg, 0.36mmol), EDCI (55.37mg, 0.29mmol), DIPEA (62.22mg, 0.48mmol) were added in sequence , Placed at room temperature for 12h. The reaction solution was poured into water, extracted with ethyl acetate (15mL*3), the organic phases were combined, washed with saturated brine (10mL*3), dried over anhydrous sodium sulfate, and concentrated to obtain the title compound (95mg), which was used directly without purification To the next step.

ESI-MS(m/z): 446.2[M+H] ⁺ .

Step 2: 3-[3-(3,5-Dimethyl-1H-pyrazol-1-yl)phenyl]-N-[5-(methylamino)-5-oxo-n-pentyl]imidazole Synthesis of and [1,2-a]pyridine-7-carboxamide (Compound 19)

Add 3-[3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl]-N-[5-(methoxy)-5-oxo-n-pentyl]imidazo[ 1,2-a]pyridine-7-carboxamide (80mg, 0.20mmol) was dissolved in methylamine ethanol solution (5mL, 33%), and the temperature was raised to 40°C for 12h. The reaction solution was concentrated, and the concentrated solution was purified by preparative high performance liquid chromatography (Method H) to obtain the title compound (40 mg).

Its structure is characterized as follows:

1H NMR(400MHz,DMSO-d ₆ )δ8.73(t,J=5.5Hz,1H), 8.65(d,J=7.2Hz,1H), 8.23(s,1H), 8.00(s,1H), 7.78(s,1H),7.72(s,1H),7.70-7.66(m,2H),7.59(dt,J=7.2,1.9Hz,1H),7.41(dd,J=7.2,1.7Hz,1H) ,6.12(s,1H),3.32-3.25(m,2H),2.55(d,J=4.6Hz,2H),2.39(s,2H),2.20(s,2H),2.09(t,J=6.9 Hz, 2H), 1.53 (s, 3H).

ESI-MS(m/z): 445.2[M+H] ⁺ .

Example 20: 3-[3-(3,5-dimethylpyrazol-1-yl)phenyl]-N-[(1S)-1-(4-hydroxyphenyl)-3-(methyl Synthesis of Amino)-3-oxopropyl]imidazo[1,2-a]pyridine-7-carboxamide (Compound 20)

Step 1: ((S)-3-(3-(3,5-Dimethyl-1H-pyrazol-1-yl)phenyl)imidazole[1,2-a]pyridine-7-carboxamide)- Synthesis of Methyl 3-(4-Hydroxyphenyl) Propionate (Compound 20-1)

Combine 3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)imidazo[1,2-a]pyridine-7-carboxylic acid (25mg, 0.075mmol) and (3S )-3-Amino-3-(4-hydroxyphenyl) methyl propionate (16.2mg, 0.083mmol) was dissolved in DMF (1mL), HOBt (15.3mg, 0.11mmol) and EDCI (17.3mg, 0.09 mg), DIPEA (19.4 mg, 0.15 mmol) was added dropwise, and the reaction was stirred for 12 h. The reaction solution was mixed with 10 mL of water and ethyl acetate, stirred and allowed to stand for liquid separation. The aqueous phase was extracted twice with ethyl acetate. The combined organic phases were washed with water three times, dried, and concentrated under reduced pressure to obtain the title compound (34 mg). Purification is directly used in the next reaction.

MS(ESI): m/z 510.2[M+H] ⁺ .

Step 2: 3-[3-(3,5-Dimethylpyrazol-1-yl)phenyl]-N-[(1S)-1-(4-hydroxyphenyl)-3-(methylamino )-3-oxopropyl]imidazo[1,2-a]pyridine-7-carboxamide (Compound 20)

((S)-3-(3-(3,5-Dimethyl-1H-pyrazol-1-yl)phenyl)imidazole[1,2-a]pyridine-7-carboxamide)-3- (4-Hydroxyphenyl) methyl propionate (34mg, 0.067mmol) was dissolved in methylamine ethanol solution (5mL, 33%) and reacted at 50°C for 12h. The solvent was evaporated under reduced pressure, the concentrate was purified by preparative high performance liquid chromatography (Method L), and the preparation was lyophilized to obtain the title compound (10.3 mg).

Its structure is characterized as follows:

¹ H NMR (400MHz, DMSO-d ₆ ): δ9.30 (s, 1H), 9.04 (d, J = 8.3 Hz, 1H), 8.65 (dd, J = 7.2, 0.9 Hz, 1H), 8.29-8.26 (m,1H),8.02(s,1H),7.84-7.77(m,2H),7.73-7.66(m,2H),7.61-7.57(m,1H),7.38(dd,J=7.3,1.8Hz ,1H),7.24-7.16(m,2H),6.73-6.66(m,2H),6.11(s,1H),5.37(q,J=7.6Hz,1H),2.68-2.61(m,2H), 2.52(d,J=2.4Hz,3H), 2.39(s,3H), 2.19(s,3H).

MS(ESI): m/z 509.0[M+H] ⁺ .

Pharmacological activity test

Test Example 1: In vitro enzyme activity inhibition test (TGFβR1).

Experimental method: According to ^{the instructions of the ADP-Glo TM} Kinase Detection Kit (Promega), the inhibitory effect of the compound of the present invention on the enzyme activity of TGFβR1 is determined, and the steps are as follows:

After pre-incubating the TGFβR1 enzyme with different concentrations of test compounds (1000 nM, 100 nM, 10 nM) at 30°C for 30 min, TGFβR1 peptide and adenosine triphosphate (ATP) were added to initiate the reaction. After incubating at 30°C for 3 hours, ADP-Glo ^TM reagent was added, and after incubating at room temperature for 90 minutes, kinase detection reagent was added. After incubating for 30 min at room temperature, the chemiluminescence signal value was detected. Take the solvent group (DMSO) as the negative control, and the buffer group (without TGFβR1 enzyme) as the blank control, and calculate the percentage inhibition rate of different concentrations of compounds according to the following formula:

Percent inhibition rate=(1-(chemiluminescence signal value of different concentrations of compound-chemiluminescence signal value of blank control)/(chemiluminescence signal value of negative control-chemiluminescence signal value of blank control))×100%;

When the percentage inhibition rate is between 30-80%, calculate the compound's half inhibitory concentration (IC ₅₀ ) or range according to the following formula:

IC50 ₌ X×(1-percent inhibition rate (%))/percent inhibition rate (%), where: X is the test concentration of the compound.

The experimental results are shown in Table 1 below:

Table 1. The inhibitory activity of the compounds of the present invention on TGFβR1

Example number	IC 50 to TGFβR1
Example one	A
Example two	B
Example three	A
Example four	C
Example Seven	C
Example eight	A
Example 9	B
Example ten	B
Example 11	B
Example 12	B

Embodiment 13	B
Example Fourteen	B
Example 15	B
Example Sixteen	B
Example Seventeen	C
Embodiment 18	B
Example Nineteen	B
Embodiment Twenty	B

[Note] _{The range of IC 50} value is as follows: A≤100nM; 100nM＜B≤1000nM; C＞1000nM

It can be seen from Table 1 that the compounds of the present invention have a significant inhibitory effect on TGFβR1, especially the compounds of Examples 1, 3 and 8, whose IC ₅₀ for TGFβR1 are 13.05, 19.68 and 89.78 nM, respectively.

Test Example 2: In vitro enzyme activity inhibition test (TGFβR2).

Experimental method: According to ^{the instructions of ADP-Glo TM} Kinase Detection Kit (Promega), the inhibitory effect of the compound of the present invention on the enzyme activity of TGFβR2 was determined, and the steps were as follows:

After pre-incubating the TGFβR2 enzyme with different concentrations of test compounds (1000 nM, 100 nM, 10 nM) at 30° C. for 30 min, myelin basic protein (MBP) and adenosine triphosphate (ATP) were added to initiate the reaction. After incubating at 30°C for 3 hours, ADP-Glo ^TM reagent was added, and after incubating at room temperature for 90 minutes, kinase detection reagent was added. After incubating at room temperature for 30 min, the chemiluminescence signal value was detected. With the solvent group (DMSO) as the negative control, and the buffer group (without TGFβR2 enzyme) as the blank control, the percentage inhibition rates of different concentrations of compounds were calculated according to the following formula:

Percent inhibition rate=(1-(chemiluminescence signal value of different concentrations of compound-chemiluminescence signal value of blank control)/(chemiluminescence signal value of negative control-chemiluminescence signal value of blank control))×100%;

When the percentage inhibition rate is between 30-80%, calculate the compound's IC50 or range according to the following formula:

IC50=X×(1-percent inhibition rate (%))/percent inhibition rate (%), where: X is the concentration of the compound whose inhibition rate is between 30-80%.

The experimental results are shown in Table 2 below:

Table 2. Inhibition rate of the compound of the present invention on TGFβR2 enzyme activity

Example number	IC50 (nM) to TGFβR2
Example one	1084.88±128.53

It can be seen from Table 2 that the compounds of the present invention have weak inhibitory activity on TGFβR2.

It can be seen from Table 1 and Table 2 that the compound of the present invention has a selective inhibitory effect on TGFβR1.

Test Example 3: In vitro cell activity inhibition test.

Experimental method: Determine the inhibitory effect of the compound of the present invention on HEK293-SBE cells according to the instructions of ^{Bright-Glo TM Luciferase Detection Kit (Promega), the steps are as follows:}

HEK293-SBE cells (Bps bioscience) were added to a 96-well plate (10% FBS medium), 30,000/well, 37°C, 5% CO _{2 were} cultured overnight. The medium was changed to 0.5% FBS medium, and the test compound diluted in 0.5% FBS medium was added. The maximum final concentration of the test compound was 10 μM, and the compound was diluted by 4 times, with a total of 8 concentration gradients. After 4-5 hours of incubation, 10μl TGFβ was added. The final concentration of TGFβ is 0.5ng/ml. Add 10μl medium to replace TGFβ as a negative control group. No test compound was added to the blank control, and TGFβ was added. Add Bright Glo reagent to each well, and read the chemiluminescence signal value on the microplate reader.

Calculate the percentage inhibition rate of different concentrations of compounds according to the following formula:

Percent inhibition rate=(1-(chemiluminescence signal value of test compound-chemiluminescence signal value of blank control)/(chemiluminescence signal value of negative control-chemiluminescence signal value of blank control))×100%;

Plot the percentage inhibition rate of different concentrations of compounds with respect to the compound concentration, fit the curve according to the four-parameter model, and calculate the IC ₅₀ value by the following formula:

y=Min+(Max-Min)/(1+(x/IC ₅₀ )^(-Hillslope)), where: y is the percentage inhibition rate; Max and Min are the maximum and minimum values of the fitted curve respectively; x is The logarithmic concentration of the compound; and Hillslope is the slope of the curve.

The experimental results are shown in Table 3 below:

Table 3. The inhibitory effect of the compounds of the present invention on the luciferase reporter gene of HEK293-SBE cells

Example number	IC 50 (nM)
Example one	A
Example two	C
Example three	A
Example 9	B
Example 12	B
Example 15	B
Example Nineteen	B

[Note] _{The range of IC 50} value is as follows: A≤200nM; 200nM＜B≤1000nM; C＞1000nM

It can be seen from Table 3 that the compound of the present invention has an inhibitory effect on the luciferase reporter gene of HEK293-SBE cells induced by TGFβ.

Claims (7)

1. A compound having the structure of Formula I, a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, polymorph, metabolite or prodrug of the compound:

   

   Where R _{1 is} selected from

   

   
2. A compound having the structure of Formula II, a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, polymorph, metabolite or prodrug of the compound:

   

   Where R _{2 is} selected from

   

   
3. The compound of claim 1, which is a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, polymorph, metabolite, or prodrug of the compound:

   

   
4. The compound of claim 2, which is a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, polymorph, metabolite, or prodrug of the compound:

   

   
5. A pharmaceutical composition comprising a preventive or therapeutically effective amount of the compound according to any one of claims 1 to 4, and a pharmaceutically acceptable salt, ester, solvate, stereoisomer, or tautomer of the compound , Polymorphs, metabolites or prodrugs, and one or more pharmaceutically acceptable carriers.
6. A pill box, which contains:

   a) The compound of any one of claims 1-4, a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, polymorph, metabolite, or precursor of the compound Medicine, or the pharmaceutical composition of claim 5;

   b) Optional packaging and/or instructions.
7. The compound of any one of claims 1 to 4, a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, polymorph, metabolite, or prodrug of the compound, Or the pharmaceutical composition of claim 5, or the use of the kit of claim 6 in the preparation of a medicament for the treatment of diseases or disorders mediated by TGFβR1, the diseases or disorders mediated by TGFβR1 are Cancers such as lung cancer, colorectal cancer, multiple myeloma, acute myelogenous leukemia, T-acute lymphoblastic leukemia, pancreatic cancer, liver cancer, neuroblastoma, breast cancer, ovarian cancer, melanoma.