KIF18a inhibitor and use thereof

Abstract

Provided is a KIF18A inhibitor, which is a compound represented by formula (I) or a pharmaceutically acceptable salt, a solvate, an ester, an acid, a metabolite or a prodrug thereof. Also provided is a use of the KIF18A inhibitor in selectively inhibiting KIF18A activity, or for treating or preventing diseases, disorders or conditions which are regulated or affected by KIF18A activity or in which KIF18A activity or overexpression is involved, particularly cancer, and more particularly cancer with chromosomal instability.

Description

KIF18A inhibitors and uses thereof TECHNICAL FIELD

[0001] The present application relates to the field of pharmacy, in particular to deuterated small molecule compounds composed of a benzamide structural unit and a fused heterobicyclic structural unit, and methods and uses thereof for treating and / or preventing diseases. BACKGROUND

[0002] Chromosomal instability (CIN) refers to ongoing genomic alterations, including amplification or deletion of chromosomal copy number or structure, ranging from point mutations to small-scale genomic changes or even whole chromosome number changes. CIN is caused by persistent errors in chromosome segregation during mitosis, and is one of the important characteristics of cancer cells, and can be an important mechanism of tumor formation. CIN is widespread in various types of cancer, especially occurring at a very high frequency in high-grade serous ovarian cancer (HGSOC), triple-negative breast cancer (TNBC), colorectal cancer (CRC) and other tumors, and is closely related to tumor metastasis, immune escape and treatment resistance, etc.

[0003] The key to correct chromosome segregation is the coordinated regulation of mitotic spindle shape and function by various kinases and kinesins. KIF18A is a member of the kinesin-8 family, an enzyme that integrates microtubule motility and depolymerization activity, can reversibly bind to microtubules, affect kinetochore microtubule dynamics to control correct chromosome positioning and spindle tension, and plays a key role in cell division. KIF18A is lowly expressed in human normal tissues, but is significantly overexpressed and functionally abnormal in many cancers, and KIF18A overexpression is associated with tumor grade, metastasis and low survival rate.

[0004] Functional analysis shows that overexpression of KIF18A in human breast cancer cells can lead to cell multinucleation, and inhibition of KIF18A can inhibit the growth of cancer cells in vivo and in vitro. In addition to its effects on tumor cell mitosis, attenuation of KIF18A function also inhibits tumor cell migration and induces apoptosis. Therefore, KIF18A can serve as a potential prognostic marker and new target for cancer therapy.

[0005] KIF18A inhibitors can selectively kill chromosomally unstable cancer cells, and have broad prospects as a potential tumor treatment strategy. However, there are relatively few reported KIF18A inhibitors. Patents US2022 / 0372018A1, US2022 / 0106293A1, CN115594664A, CN115772159A, CN115785068A, WO2023 / 028564A1, etc. disclose several aromatic heterocyclic compounds that can inhibit the enzyme activity of KIF18A.

[0006] Despite some advances in this field, there remains a great need in the art to develop potent, specific, safe KIF18A small molecule inhibitors for the treatment of cancers with chromosomal instability. SUMMARY

[0007] The present invention aims to provide a deuterated compound capable of selectively inhibiting KIF18A activity, which can provide additional advantages in improving drug safety, solubility, and liver microsomal stability, improving pharmacokinetics, etc., while providing comparable KIF18A inhibitory activity compared to the corresponding non-deuterated small molecule compound.

[0008] According to one aspect of the present invention, there is provided a KIF18A inhibitor, which is a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, ester, acid, metabolite, or prodrug thereof,

[0009] wherein,

[0010] X1and X2are each independently selected from N and CH;

[0011] R 1 selected from hydrogen, -P(=O)(OH)2, and -S(=O)2(OH);

[0012] R 2a , R 2b , R 3a , and R 3b are each independently selected from hydrogen or deuterium, provided that at least one of R 2a , R 2b , R 3a , and R 3b is deuterium;

[0013] m is an integer selected from 1, 2, or 3;

[0014] n is an integer selected from 1, 2, or 3;

[0015] q is an integer selected from 0, 1, or 2, and when q is not 0, R 4 are each independently selected from halogen.

[0016] In further embodiments, m and n are each independently 1.

[0017] In other preferred embodiments, q is 2.

[0018] In yet another preferred embodiment, R 4 are each independently fluorine.

[0019] Preferably, m is 1, q is 2, and R 4 The substitution position on the nitrogen heterocycle is para to the nitrogen atom.

[0020] In another embodiment, X1is CH.

[0021] In another embodiment, X2is CH.

[0022] Preferably, X1and X2are both CH.

[0023] In other preferred embodiments, at least two of R 2a , R 2b , R 3a , and R 3b are deuterium. More preferably, wherein R 2a and R 2b are deuterium. Further preferably, R 2a , R 2b , R 3a , and R 3b are all deuterium.

[0024] In other preferred embodiments, R 1 is hydrogen.

[0025] The KIF18A inhibitor according to the present application is preferably a compound selected from the following structures, or a pharmaceutically acceptable salt, solvate, ester, acid, metabolite, or prodrug thereof:

[0026] In another aspect of the present application, there is provided a pharmaceutical composition comprising a compound of the present application, or a pharmaceutically acceptable salt, solvate, ester, acid, metabolite, or prodrug thereof, and a pharmaceutically acceptable carrier or excipient, and optionally, another therapeutic agent.

[0027] Other aspects of the present application also relate to a method or use of a compound of the present application, or a pharmaceutically acceptable salt, solvate, ester, acid, metabolite, or prodrug thereof, for selectively inhibiting KIF18A activity, or use of a compound of the present application, or a pharmaceutically acceptable salt, solvate, ester, acid, metabolite, or prodrug thereof, in the manufacture of a medicament for selectively inhibiting KIF18A activity.

[0028] Further aspects of the present application also relate to a method or use of a compound of the present application, or a pharmaceutically acceptable salt, solvate, ester, acid, metabolite, or prodrug thereof, for treating or preventing a disease, disorder, or condition modulated by, or affected by, or involving KIF18A activity, or use of a compound of the present application, or a pharmaceutically acceptable salt, solvate, ester, acid, metabolite, or prodrug thereof, in the manufacture of a medicament for treating or preventing a disease, disorder, or condition modulated by, or affected by, or involving KIF18A activity or overexpression.

[0029] Preferably, the disease, disorder or condition modulated by or affected by KIF18A activity or in which KIF18A activity or overexpression is implicated is a cancer.

[0030] In a more preferred aspect, the disease, disorder or condition is selected from one or more of the following cancers with chromosomal instability: lung squamous carcinoma, lung adenocarcinoma, non-small cell lung cancer, small cell lung cancer, head and neck squamous carcinoma, breast cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, ovarian cancer, esophageal squamous carcinoma, gastric cancer, liver cancer, oral cancer, urothelial carcinoma, prostate cancer, bladder cancer, renal cell carcinoma, gastrointestinal stromal tumor, cervical cancer, endometrial cancer, rhabdomyosarcoma, fibrosarcoma, neuroendocrine tumor, mesothelioma, brain cancer, and malignant glioma. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 shows the proliferation inhibition curves of the compounds of the application and control compounds on human embryonic kidney cells HEK293. DETAILED DESCRIPTION

[0032] DEFINITIONS

[0033] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this application belongs. In the specification, the singular forms also include the plural unless the context clearly dictates otherwise. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. In case of a conflict in terminology, the present specification, including the definitions, controls. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0034] Unless otherwise indicated, the present application employs conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Unless specific definitions are provided, the nomenclature and laboratory procedures in analytical chemistry, synthetic organic and medicinal chemistry, and pharmacology described herein are those well-known and

[0035] The term "pharmaceutically acceptable salt" herein refers to salts that retain the desired biological activity of the subject compounds and exhibit minimal undesired toxicological effects. These pharmaceutically acceptable salts can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid or free base form with the appropriate base or acid, respectively.

[0036] "Solvate" or "solvates" refers to a solvent addition form of a compound that typically contains either stoichiometric or non-stoichiometric amounts of a solvent. Some compounds have a tendency to trap a fixed or stoichiometric ratio of solvent molecules in the crystalline solid state, forming a solvate. If the solvent is water, the solvate is a hydrate; if the solvent is alcohol, the solvate is an alcoholate. Hydrates are formed by the combination of water molecules with molecules of the substance, in which the water retains its molecular state as H2O.

[0037] A "metabolite" of a compound disclosed herein is a derivative of a compound formed when the compound is metabolized. The term "active metabolite" refers to a biologically active derivative of a compound formed when the compound is metabolized. The term "metabolized" as used herein refers to the sum of the processes by which a particular substance is changed by an organism (including, but not limited to, hydrolysis reactions and reactions catalyzed by enzymes, such as oxidation reactions). Thus, enzymes can produce a particular structural transformation of a compound. For example, cytochrome P450 catalyzes a variety of oxidation and reduction reactions, while glucuronyl transferases catalyze the conjugation of activated glucuronic acid molecules to aromatic alcohols, aliphatic alcohols, carboxylic acids, amines, and free thiols. Further information on metabolism can be found in The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill (1996). Metabolites of compounds disclosed herein can be identified by administering the compound to a host and analyzing tissue samples from the host, or by incubating the compound with hepatic cells in vitro and analyzing the resultant compounds. Both of these methods are known in the art. In some embodiments, a metabolite of a compound is formed by an oxidation process and corresponds to the corresponding hydroxyl-containing compound. In some embodiments, a compound is metabolized to a pharmaceutically active metabolite.

[0038] The term "modulate" as used herein refers to interacting directly or indirectly with a target to alter the activity of the target, including, by way of example only, enhancing the activity of the target, inhibiting the activity of the target, limiting the activity of the target, or prolonging the activity of the target.

[0039] The term "prodrug" or "prodrug" refers to a derivative that can not be pharmacologically active, but under certain circumstances is orally or parenterally administrable and thereafter metabolized in the body to form a compound of the application that is pharmacologically active. Non-limiting examples of prodrugs include esters, carbonates, hemiesters, phosphates, nitroesters, sulfates, sulfoxides, amides, carbamates, nitrogen-containing compounds, phosphoramides, glycosides, ethers, acetals, and ketals, and the like.

[0040] "Effective amount" means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought by a researcher or clinician. In addition, the term "therapeutically effective amount" means any amount of a compound that, when administered to a subject in need, will cure, heal, prevent, or partially arrest the progress of a disease, condition, or disorder, or to relieve one or more symptoms of the disease, disorder, or condition, or to reduce the rate of development of a disease or disorder. Also included within the scope of this term are amounts effective to enhance normal physiological function.

[0041] The term "treatment" as used herein covers alleviating a disease, disorder, or condition. The term includes the administration and / or application of one or more compounds described herein to a subject to provide management or treatment of a condition. "Treatment" for purposes of the present disclosure can but need not provide cure; rather, "treatment" can be palliative in that it can provide management of a condition. When the compounds described herein are used to address unwanted proliferative cells, including cancer, "treatment" includes partial or complete destruction of the unwanted proliferative cells, but with minimal damage to normal cells. The desired mechanism of treatment of unwanted rapidly proliferating cells, including cancer cells, is apoptosis at the cellular level.

[0042] The term "prevention" as used herein includes co-prevention or slowing the onset of clinically significant disease or prevention or slowing the onset of pre-clinically significant disease stage in an at-risk individual. This includes prophylactic treatment of individuals at risk of developing a disease.

[0043] The term "subject" or "patient" includes an organism, such as a human or non-human animal, that can have a condition or a condition associated with reduced or insufficient programmed cell death (apoptosis) or that can otherwise benefit from administration of a compound of the application. Preferred humans include human patients that have or are predisposed to a condition or associated condition as described herein. The term "non-human animal" includes vertebrates, such as mammals, e.g., non-human primates, sheep, cows, dogs, cats, and rodents such as mice, and non-mammals, such as chickens, amphibians, reptiles, etc.

[0044] GI 50 refers to the concentration of a drug required to inhibit the growth of 50% of cells, i.e., the concentration of a drug at which 50% of the cells, such as cancer cells, have their growth inhibited or controlled.

[0045] IC 50 refers to the amount, concentration or dosage of a particular test compound that achieves 50% inhibition of the maximum effect in an assay measuring the effect.

[0046] EC 50 refers to the dosage, concentration or amount of a compound that elicits 50% of the maximum expression of a particular response induced, stimulated or potentiated by a particular assay compound in a dose-dependent response.

[0047] KIF18A inhibitors of the invention

[0048] The present invention relates to a KIF18A inhibitor which is a compound of formula (I) or a pharmaceutically acceptable salt, solvate, ester, acid, metabolite or prodrug thereof,

[0049] wherein,

[0050] X1and X2are each independently selected from N and CH;

[0051] R 1 is selected from hydrogen, -P(=O)(OH)2, and -S(=O)2(OH);

[0052] R 2a , R 2b , R 3a , and R 3b are each independently selected from hydrogen or deuterium, provided that at least one of R 2a , R 2b , R 3a , and R 3b is deuterium;

[0053] m is an integer selected from 1, 2, or 3;

[0054] n is an integer selected from 1, 2, or 3;

[0055] q is an integer selected from 0, 1, or 2, and when q is not 0, R 4 are each independently selected from halogen.

[0056] In further embodiments, m and n are each independently 1.

[0057] In other preferred embodiments, q is 2.

[0058] In yet further preferred embodiments, R 4 are each independently fluorine.

[0059] Preferably, m is 1, q is 2, and R 4 The substitution position on the nitrogen heterocycle is para to the nitrogen atom.

[0060] In further embodiments, X1is CH.

[0061] In yet further embodiments, X2is CH.

[0062] Preferably, X1and X2are both CH.

[0063] In other preferred embodiments, R 2a , R 2b , R 3a , and R3b at least two of R 2a and R 2b are deuterium. Further preferred, R 2a , R 2b , R 3a and R 3b are deuterium.

[0064] In other preferred embodiments, R 1 is hydrogen.

[0065] Further preferred, the present application relates to the compounds shown in the following table, or a pharmaceutically acceptable salt, solvate, ester, acid, metabolite or prodrug thereof.

[0066] Described herein are novel KIF18A inhibitors. Also described herein are pharmaceutically acceptable salts, solvates, esters, acids, metabolites and prodrugs of such compounds.

[0067] The compounds of the present application can exist in free form, e.g. as a free base or a free acid, or in zwitterionic form, or can exist as salts. The salts can be any salt, either organic or inorganic, and particularly any physiologically acceptable organic or inorganic addition salt, which is commonly used in pharmacy, e.g. a salt of a pharmaceutically acceptable cation or a salt of a pharmaceutically acceptable anion.

[0068] Salts which are preferred for the purposes of the present application are physiologically acceptable salts of the compounds according to the application. However, salts which are not themselves suitable for pharmaceutical applications but which, for example, can be employed for the isolation or purification of the compounds according to the application are also included.

[0069] The term "pharmaceutically acceptable salts" refers to relatively non-toxic, inorganic or organic acid addition salts of the compounds of the present application, e.g. see S.M. Berge et al., "Pharmaceutical Salts, J. Pharm. Sci. 1977, 66, 1-19.

[0070] Pharmaceutically acceptable salts of the compounds of the present application include acid addition salts with inorganic acids, carboxylic acids and sulfonic acids, for example salts of hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid, nitric acid; or with organic acids, for example formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxyphenyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, tertiary butyl acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfaminic acid, trifluoromethanesulfonic acid, dodecylsulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalene- disulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptanoic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid or thiocyanic acid.

[0071] Pharmaceutically acceptable salts of the compounds of the present application also include salts of customary bases, such as and preferably alkali metal salts (for example sodium and potassium salts), alkaline earth metal salts (for example calcium and magnesium salts) and ammonium salts derived from ammonia or organic amines having 1 to 16 carbon atoms, such as and preferably ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, mono-, di- and triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-methylmorpholine, arginine, lysine, ethylenediamine, N-methylpiperidine, N-methylglucamine, dimethylglucamine, ethylglucamine, 1,6-hexanediamine, glucosamine, sarcosine, serinol, tris(hydroxymethyl)aminomethane, aminopropanediol, a Sovak base and 1-amino-2,3,4-butantriol.

[0072] The present application includes all possible salts of the compounds of the present application, which can be single salts or any mixture of said salts in any ratio.

[0073] Solvates are for the purposes of the present application those forms of the compounds of the present application which form a complex with solvent molecules by coordination, either in the solid state or in the liquid state. Hydrates are special solvate forms in which water is the solvent which coordinates. Hydrates are preferred as solvates within the scope of the present application.

[0074] Furthermore, the present application also includes prodrugs of the compounds of the present application. The term "prodrug" includes compounds which can themselves be biologically active or inert but which are converted (for example by metabolism or hydrolysis) into a compound of the present application in the course of their residence time in the body.

[0075] In addition, the present application includes all possible crystalline forms or polymorphs of the compounds of the present application, either as individual polymorphs, or as a mixture of more than one polymorph in any ratio.

[0076] In the present specification, in some instances for the sake of convenience, the structural formula of a compound represents a particular isomer, but the present application includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbon atoms, stereoisomers, tautomers, and the like.

[0077] The compounds of the present application can exist in any configuration or as a mixture of racemates. When the compounds used according to the present application contain more than one chiral center, they can exist as diastereomers. The diastereomeric isomer compounds can be separated by methods known to those skilled in the art (e.g., chromatography or crystallization), and the individual enantiomers can be separated as described above. The present application includes the use of each diastereomeric compound and mixtures thereof used according to the present application. The compounds used according to the present application can exist in different tautomeric forms or in different geometric isomeric forms, and the present application includes the use of each tautomeric and / or geometric isomer of the compounds used according to the present application and mixtures thereof. The compounds used according to the present application can exist as zwitterions. The present application includes the use of each zwitterionic form of the compounds used according to the present application and mixtures thereof.

[0078] Screening and characterization of pharmaceutically acceptable salts, polymorphs, and / or solvates can be accomplished using a variety of techniques, including but not limited to thermal analysis, X-ray diffraction, spectroscopy, microscopy methods, elemental analysis. Various spectroscopic techniques used include but are not limited to Raman, FTIR, UVIS, and NMR (liquid and solid state). Various microscopy techniques include but are not limited to IR microscopy and Raman microscopy.

[0079] Pharmaceutical uses of the present application

[0080] The compounds of formula (I) of the present application, or a pharmaceutically acceptable salt, solvate, ester, acid, metabolite or prodrug thereof, are capable of selectively inhibiting the enzymatic activity of KIF18A and are therefore useful in the treatment or prevention of a disease, disorder or condition modulated by or affected by KIF18A activity or in which KIF18A activity or overexpression is implicated.

[0081] In preferred aspects, the disease, disorder or condition modulated by or affected by KIF18A activity or in which KIF18A activity or overexpression is implicated is a cancer, particularly a cancer with chromosomal instability, including but not limited to one or more of: lung squamous carcinoma, lung adenocarcinoma, non-small cell lung cancer, small cell lung cancer, head and neck squamous carcinoma, breast cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, ovarian cancer, esophageal squamous carcinoma, gastric cancer, liver cancer, oral cancer, urothelial cancer, prostate cancer, bladder cancer, renal cell carcinoma, gastrointestinal stromal tumor, cervical cancer, endometrial cancer, rhabdomyosarcoma, fibrosarcoma, neuroendocrine tumor, mesothelioma, brain cancer, or malignant glioma.

[0082] The compounds of the application can act systemically and / or topically. To this end, they can be administered in a suitable manner, for example by the oral route, parenteral route, pulmonary route, nasal route, sublingual route, lingual route, buccal route, rectal route, cutaneous route, transdermal route, conjunctival route or auricular route, or in the form of an implant or stent.

[0083] Preferably, in the embodiments of the application, the medicament comprising a compound of the application can be administered to a patient by at least one of injection, orally, inhalation, rectally and transdermally.

[0084] Regardless of the route of administration selected, the KIF18A inhibitors of the application, and / or the pharmaceutical compositions of the application, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

[0085] In treating a patient according to the present application, the amount of a given medicament depends on a number of factors, such as the particular dosing regimen, the type and severity of the disease or condition, the unique characteristics of the subject or host being treated, e.g., body weight, but, in general, the dosage administered is commonly in the range of 0.02-5000 mg / day, e.g., about 1-1500 mg / day, for therapeutic use in humans. The desired dose can be conveniently presented in divided or sustained release dosage forms, e.g., two, three, four or more sub-doses administered at appropriate intervals as described above. It will be appreciated by one of ordinary skill in the art that the actual dose level and timing of administration of the active ingredients will be selected in accordance with the dosage and the severity of the condition being treated, and the particular patient's circumstances as well as the judgment of the attending physician.

[0086] Actual dosage levels and time course of administration of the compounds of the present application can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, without being toxic to the patient.

[0087] Pharmaceutical compositions

[0088] Another aspect of the application relates to pharmaceutical compositions comprising a compound of Formula (I) of the application, or a pharmaceutically acceptable salt, solvate, ester, acid, metabolite, or prodrug thereof, and a pharmaceutically acceptable diluent, carrier, or excipient, and optionally one or more other therapeutic agents.

[0089] The compounds of the application can be administered as the sole pharmaceutical agent or in combination with one or more other therapeutic agents, where the combination causes no unacceptable adverse effects. The pharmaceutical compositions include administration of a single pharmaceutical dosage formulation comprising a compound of the application and one or more other therapeutic agents, as well as administration of a compound of the application and each of the various other therapeutic agents, in its own separate pharmaceutical dosage formulation. For example, a compound of Formula (I) and other therapeutic agents can be administered together in a single oral dosage composition, such as a tablet or capsule, to a patient, or each agent can be administered in separate dosage formulations.

[0090] When separate dosage formulations are used, the compounds of the application and one or more other therapeutic agents can be administered at essentially the same time (e.g., concurrently) or at separately staggered times (e.g., sequentially).

[0091] In particular, the compounds of the application can be used in fixed combination or separate combination with other antineoplastic agents, such as alkylating agents, antimetabolites, plant-derived antineoplastic agents, hormonal therapy agents, topoisomerase inhibitors, camptothecin derivatives, kinase inhibitors, targeted drugs, antibodies, interferons and / or biological response modifiers, anti-angiogenic compounds, and other antineoplastic drugs.

[0092] The compounds of the application can also be used in cancer treatment in conjunction with radiotherapy and / or surgical intervention.

[0093] Preparation of compounds

[0094] The compounds of the application can be synthesized using standard synthetic techniques known to those skilled in the art or using methods known in the art in combination with the methods described herein. Additionally, the solvents, temperatures, and other reaction conditions given herein can be varied by one skilled in the art. As further guidance, the following synthetic methods can also be utilized.

[0095] The reactions can be used in sequence to provide the compounds described herein; or they can be used to synthesize fragments which are subsequently incorporated by the methods described herein and / or methods known in the art.

[0096] The starting materials used to synthesize the compounds described herein can be synthesized or can be obtained from commercial sources. The compounds described herein and other related compounds having different substituents can be synthesized using techniques and starting materials known to those skilled in the art. The general methods for preparing the compounds disclosed herein can be derived from reactions known in the art, and the reactions can be modified by reagents and conditions deemed appropriate by those skilled in the art to introduce the various moieties in the molecules provided herein.

[0097] If desired, the reaction products can be isolated and purified using conventional techniques, including but not limited to filtration, distillation, crystallization, chromatography, and the like. The products can be characterized using conventional methods, including physical constants and spectral data.

[0098] Non-limiting examples of synthetic schemes for preparing compounds of Formula (I) are described below.

[0099] Examples

[0100] The following specific, non-limiting examples are to be construed as illustrative only and without resting on any theory of the invention. While further detailed description is not necessary, it is believed that one skilled in the art can, based on the description herein, fully utilize the present disclosure.

[0101] The structure of the compounds is determined by nuclear magnetic resonance (NMR) or / and mass spectrometry (MS). The determination solvent of NMR is deuterium dimethyl sulfoxide (DMSO-d 6 ), deuterium chloroform (CDCl3) or deuterium methanol (CD3OD).

[0102] In the examples, unless otherwise specified, the solution refers to an aqueous solution.

[0103] In the examples, unless otherwise specified, the temperature of the reaction is room temperature, for example 20-30°C.

[0104] English abbreviations:

[0105] ACN: acetonitrile;

[0106] Brettphos: 2-(dicyclohexylphosphino)-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl;

[0107] Brettphos Pd G3: methane sulfonic acid (2-dicyclohexylphosphino)-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II);

[0108] DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene;

[0109] DCM: dichloromethane;

[0110] DIPEA: N,N-diisopropylethylamine;

[0111] DMF: N,N-dimethylformamide;

[0112] HATU: 2-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate;

[0113] LiHMDS: lithium bis(trimethylsilyl)amide;

[0114] mCPBA: meta-chloroperoxybenzoic acid;

[0115] NMP: N-methylpyrrolidone;

[0116] Ruphos: 2-dicyclohexylphospho-2',6'-diisopropoxy-l,l'-biphenyl;

[0117] Ruphos Pd G2: chloro(2-dicyclohexylphosphino-2',6'-diisopropoxy-l,l'- biphenyl)(2-amino-l,l'-biphenyl-2-yl)palladium(II);

[0118] TEA: triethylamine;

[0119] THF: tetrahydrofuran.

[0120] Example 1: Synthesis of intermediates

[0121] 1.1 Synthesis of intermediate A

[0122] 4-nitro-2-(6-azaspiro[2.5]oct-6-yl)benzamide

[0123] Step A: Preparation of 2-fluoro-4-nitrobenzamide

[0124] To a solution of 2-fluoro-4-nitrobenzoic acid (24.06 g, 130.00 mmol, 1.0 eq.) in N,N-dimethylformamide (250 mL) was added N,N-diisopropylethylamine (50.41 g, 390.00 mmol, 3.0 eq.), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (54.37 g, 143.00 mmol, 1.1 eq.) and ammonium chloride (9.04 g, 169.00 mmol, 1.3 eq.) successively. The reaction was stirred at 20 °C for 3 h. The reaction was quenched with water (1.2 L) and extracted with ethyl acetate (4 x 550 mL). The combined organic phase was washed with saturated aqueous ammonium chloride solution (2 x 600 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to about 250 mL volume. The solid product was obtained by filtration. 2-Fluoro-4-nitrobenzamide (21.00 g, yellow solid) was obtained in 87.7% yield.

[0125] LCMS: (ESI) [M+H] = 185.0. +

[0126] 1 H NMR: (400 MHz, DMSO-d6) δ 8.20 (dd, J = 10.0, 2.0 Hz, 1H), 8.15-8.11 (m, 1H), 8.07 (s, 1H), 7.93 (s, 1H), 7.87 (dd, J = 8.4, 6.8 Hz, 1H).

[0127] Step B: Preparation of 4-nitro-2-(6-azaspiro[2.5]oct-6-yl)benzamide (Intermediate A)

[0128] To a solution of 2-fluoro-4-nitrobenzoic acid (24.06 g, 130.00 mmol, 1.0 eq.) in N,N-dimethylformamide (250 mL) was added N,N-diisopropylethylamine (50.41 g, 390.00 mmol, 3.0 eq.), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (54.37 g, 143.00 mmol, 1.1 eq.) and ammonium chloride (9.04 g, 169.00 mmol, 1.3 eq.) successively. The reaction was stirred at 20 °C for 3 h. The reaction was quenched with water (1.2 L) and extracted with ethyl acetate (4 x 550 mL). The combined organic phase was washed with saturated aqueous ammonium chloride solution (2 x 600 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to about 250 mL volume. The solid product was obtained by filtration. 2-Fluoro-4-nitrobenzamide (21.00 g, yellow solid) was obtained in 87.7% yield.

[0129] LCMS: (ESI) [M+H] = 185.0. +

[0126] 1 H NMR: (400 MHz, DMSO-d6) δ 8.20 (dd, J = 10.0, 2.0 Hz, 1H), 8.15-8.11 (m, 1H), 8.07 (s, 1H), 7.93 (s, 1H), 7.87 (dd, J = 8.4, 6.8 Hz, 1H).

[0127] Step B: Preparation of 4-nitro-2-(6-azaspiro[2.5]oct-6-yl)benzamide (Intermediate A)

[0128] To a solution of 2-fluoro-4-nitrobenzoic acid (24.06 g, 130.00 mmol, 1.0 eq.) in N,N-dimethylformamide (250 mL) was added N,N-diisopropylethylamine (50.41 g, 390.00 mmol, 3.0 eq.), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (54.37 g, 143.00 mmol, 1.1 eq.) and ammonium chloride (9.04 g, 169.00 mmol, 1.3 eq.) successively. The reaction was stirred at 20 °C for 3 h. The reaction was quenched with water (1.2 L) and extracted with ethyl acetate (4 x 550 mL). The combined organic phase was washed with saturated aqueous ammonium chloride solution (2 x 600 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to about 250 mL volume. The solid product was obtained by filtration. 2-Fluoro-4-nitrobenzamide (21.00 g, yellow solid) was obtained in 87.7% yield.

[0129] LCMS: (ESI) [M+H] = 185.0. +

[0126] 1 H NMR: (400 MHz, DMSO-d6) δ 8.20 (dd, J = 10.0, 2.0 Hz, 1H), 8.15-8.11 (m, 1H), 8.07 (s, 1H), 7.93 (s, 1H), 7.87 (dd, J = 8.4, 6.8 Hz, 1H).

[0127] Step B: Preparation of 4-nitro-2-(6-azaspiro[2.5]oct-6-yl)benzamide (Intermediate A)

[0128] To a solution of 2-fluoro-4-nitrobenzoic acid (24.06 g, 130.00 mmol, 1.0 eq.) in N,N-dimethylformamide (250 mL) was added N,N-diisopropylethylamine (50.41 g, 390.00 mmol, 3.0 eq.), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (54.37 g, 143.00 mmol, 1.1 eq.) and ammonium chloride (9.04 g, 169.00 mmol, 1.3 eq.) successively. The reaction was stirred at 20 °C for 3 h. The reaction was quenched with water (1.2 L) and extracted with ethyl acetate (4 x 550 mL). The combined organic phase was washed with saturated aqueous ammonium chloride solution (2 x 600 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to about 250 mL volume. The solid product was obtained by filtration. 2-Fluoro-4-nitrobenzamide (21.00 g, yellow solid) was obtained in 87.7% yield.

[0130] 1 H NMR:(400MHz,DMSO-d6)δ8.19(s,1H),7.87-7.81(m,2H),7.78(s,1H),7.75(d,J=8.4Hz,1H),3.11-3.03(m,4H),1.56-1.46(m,4H),0.35(s,4H)。

[0131] 1.2 Synthesis of Intermediate B

[0132] 2-(N-(4-((7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridin-5-yl)carbamoyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)acetic acid ethyl ester

[0133] Step A: Preparation of 5-bromo-7-chloropyrazolo[1,5-a]pyridine

[0134] Dissolve 5-bromopyrazolo[1,5-a]pyridine (10.00 g, 50.75 mmol, 1.0 eq.) in tetrahydrofuran (100 mL) and cool to -78 °C under nitrogen. Add lithium bis(trimethylsilyl)amide (1 M, 55.8 mL, 55.8 mmol, 1.1 eq.) dropwise. Continue to stir at -78 °C for 0.5 h. Add a solution of hexachloroethane (13.22 g, 55.83 mmol, 1.1 eq.) in tetrahydrofuran (20 mL) dropwise. Continue to stir at -78 °C for 1 h. Quench the reaction with saturated aqueous ammonium chloride solution (120 mL) and extract with ethyl acetate (2 x 100 mL). Dry the combined organic phases over anhydrous sodium sulfate and concentrate. Purify the crude product by column chromatography (petroleum ether / ethyl acetate = 20 / 1; V / V) to give 5-bromo-7-chloropyrazolo[1,5-a]pyridine (9.68 g, yellow solid) in 82.4% yield.

[0135] LCMS: (ESI) [M+H] + = 231.0.

[0136] 1 H NMR:(400MHz,CDCl3)δ8.04(d,J=2.4Hz,1H),7.69(d,J=1.6Hz,1H),7.01(d,J=1.6Hz,1H),6.59(d,J=2.0Hz,1H).

[0137] Step B: Preparation of 5-bromo-7-(3,3-difluoroazetidin-l-yl)pyrazolo[l,5- a]pyridine

[0138] To a solution of 5-bromo-7-chloropyrazolo[l,5-a]pyridine (2.88 g, 12.44 mmol, 1.0 eq.) in N-methylpyrrolidine (20 mL) was added 3,3-difluoroazetidine hydrochloride (4.83 g, 37.33 mmol, 3.0 eq.) followed by 1,8-diazabicyclo[5.4.0]undec-7-ene (9.47 g, 62.21 mmol, 5.0 eq.). The reaction was heated at 135 °C for 20 h. The reaction was allowed to cool to room temperature and diluted with saturated aqueous ammonium chloride solution (150 mL) and extracted with ethyl acetate (2 x 200 mL). The organic phases were combined, washed with saturated brine (2 x 150 mL), dried over anhydrous sodium sulfate and concentrated. The crude product obtained was purified by column chromatography (petroleum ether / ethyl acetate = 35 / 1; V / V) to give 5-bromo-7-(3,3-difluoroazetidin-l-yl)pyrazolo[l,5-a]pyridine (2.32 g, yellowish solid) in 64.7% yield.

[0139] LCMS: (ESI) [M+H] = 288.0. +

[0140] 1 H NMR: (400 MHz, CDC13) δ 7.87 (d, J = 2.0 Hz, 1H), 7.23 (d, J = 2.0 Hz, 1H), 6.38 (d, J = 2.4 Hz, 1H), 5.80 (d, J = 1.6 Hz, 1H), 4.65 (t, J = 12.0 Hz, 4H).

[0141] Step C: Preparation of N-(7-(3,3-difluoroazetidin-l-yl)pyrazolo[l,5-a]pyridin-5-yl)-4- nitro-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0142] ​To a solution of 5-bromo-7-(3,3-difluoroazetidin-l-yl)pyrazolo[l,5- a]pyridine (800 mg, 2.78 mmol, 1.0 eq.) in dioxane (15 mL) was added 4-nitro-2-(6- azaspiro[2.5]octan-6-yl)benzamide (764 mg, 2.78 mmol, 1.0 eq.), 2-(dicyclohexylphosphino)-3,6-dimethoxy-2',4',6'-triisopropyl-l,r-biphenyl (89 mg, 0.17 mmol, 0.06 eq.), methanesulfonic acid (2-dicyclohexylphosphino)-3,6-dimethoxy-2',4',6'- triisopropyl-l,r-biphenyl (2'-amino-l,r-biphenyl-2-yl)palladium(II) (76 mg, 0.08 mmol, 0.03 eq.) and cesium carbonate (2.71 g, 8.33 mmol, 3.0 eq.). The reaction was heated at 120 °C for 6 h under nitrogen atmosphere. The reaction was allowed to cool to room temperature and diluted with water (40 mL) and extracted with dichloromethane (2 x 80 mL). The organic layers were combined, dried over anhydrous sodium sulfate and concentrated. The crude obtained was slushed in ethyl acetate (40 mL) and filtered. The filter cake was dried under vacuum to obtain N-(7-(3,3-difluoroazetidin-l-yl)pyrazolo[l,5-a]pyridin-5-yl)-4-nitro-2-(6- azaspiro[2.5]octan-6-yl)benzamide (840 mg, yellow solid) in 62.7% yield.

[0143] LCMS: (ESI) [M+H] = 483.2. + = 483.2.

[0144] 1 H NMR: (400 MHz, DMSO-d6) δ 10.90 (s, 1H), 7.95-7.88 (m, 3H), 7.80 (dd, J = 7.6, 1.2 Hz, 1H), 7.75 (d, J = 1.6 Hz, 1H), 6.51 (d, J = 2.4 Hz, 1H), 6.28 (d, J = 2.0 Hz, 1H), 4.66 (t, J = 12.0 Hz, 4H), 3.16-3.09 (m, 4H), 1.51-1.40 (m, 4H), 0.31 (s, 4H).

[0145] Step D: Preparation of 4-amino-N-(7-(3,3-difluoroazetidin-l-yl)pyrazolo[l,5- a]pyridin-5-yl)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0146] N-(7-(3,3-difluoroazetidin-l-yl)pyrazolo[l,5-a]pyridin-5-yl)-4-nitro-2-(6- azaspiro[2.5]octan-6-yl)benzamide (1.06 g, 2.20 mmol, 1.0 eq.) was dissolved in acetic acid (10 mL) and zinc powder (1.15 g, 17.60 mmol, 8.0 eq.) was added. The reaction was stirred at 30 °C for 1 h. The reaction was diluted with dichloromethane (150 mL) and filtered through celite. The filtrate was neutralized with saturated sodium carbonate and acetic acid. The organic phase was dried over anhydrous sodium sulfate and concentrated. The resulting crude product was separated and purified by column chromatography (petroleum ether / ethyl acetate = 3 / 2; V / V) to give 4-amino-N-(7-(3,3-difluoroazetidin-l-yl)pyrazolo[l,5-a]pyridin-5-yl)-2-(6- azaspiro[2.5]octan-6-yl)benzamide (957 mg, yellow solid) in 96.1% yield.

[0147] LCMS: (ESI) [M+H] = 453.2. +

[0148] 1 H NMR: (400 MHz, DMSO-d6) δ 12.48 (s, 1H), 7.90 (d, J = 2.0 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 1.6 Hz, 1H), 6.58 (d, J = 2.0 Hz, 1H), 6.47 - 6.40 (m, 2H), 6.22 (d, J = 2.0 Hz, 1H), 5.84 (s, 2H), 4.66 (t, J = 12.0 Hz, 4H), 2.95 (t, J = 5.2 Hz, 4H), 1.74 - 1.48 (m, 4H), 0.39 (s, 4H).

[0149] Step E: Preparation of ethyl 2-(N-(4-((7-(3,3-difluoroazetidin-l-yl)pyrazolo[l,5- a]pyridin-5-yl)carbamoyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)acetate (Intermediate B)

[0150] ​To a solution of 4-amino-N-(7-(3,3-difluoroazetidin-l-yl)pyrazolo[l,5- a]pyridin-5-yl)-2-(6-azaspiro[2.5]octan-6-yl)benzamide (905 mg, 2.00 mmol, 1.0 eq.) and triethylamine (708 mg, 7.00 mmol, 3.5 eq.) in tetrahydrofuran (22 mL) was added (chlorosulfonyl)acetic acid ethyl ester (933 mg, 5.00 mmol, 2.5 eq.) dropwise at 0 °C under nitrogen. The reaction was stirred at 0 °C for 2 h. The reaction was quenched by adding saturated ammonium chloride aqueous solution (80 mL) and extracted with ethyl acetate (2 x 80 mL). The combined organic phase was dried over anhydrous sodium sulfate and concentrated. The resulting crude product was purified by column chromatography (petroleum ether / ethyl acetate = 7 / 3; V / V) to give 2-(N-(4-((7-(3,3-difluoroazetidin-l-yl)pyrazolo[l,5-a]pyridin-5-yl)carbamoyl)-3-(6- azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)acetic acid ethyl ester (717 mg, yellow solid) with a yield of 59.5%.

[0151] LCMS: (ESI) [M+H] = 603.2. +

[0152] 1 H NMR: (400 MHz, DMSO-d6) δ 11.55 (s, 1H), 10.53 (s, 1H), 7.92 (d, J = 2.4 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 2.0 Hz, 1H), 7.16 (d, J = 2.0 Hz, 1H), 7.04 (dd, J = 8.8, 2.0 Hz, 1H), 6.48 (d, J = 2.4 Hz, 1H), 6.31 (d, J = 1.6 Hz, 1H), 4.66 (t, J = 12.4 Hz, 4H), 4.34 (s, 2H), 4.11 (q, J = 7.2 Hz, 2H), 3.02 - 2.96 (m, 4H), 1.59 - 1.51 (m, 4H), 1.18 (t, J = 7.2 Hz, 3H), 0.35 (s, 4H).

[0153] 1.3 Synthesis of Intermediate C

[0154] 2-(N-(4-((5-(3,3-difluoroazetidin-l-yl)-[l,2,4]triazolo[l,5-c]pyrimidin-7-yl)carbamoyl)-3-(6- azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)acetic acid ethyl ester

[0155] ​Step A: Preparation of 4-chloro-6-hydrazinyl-2-(methylthio)pyrimidine

[0156] To a solution of 4,6-dichloro-dimethylsulfanylpyrimidine (80.00 g, 410.13 mmol, 1.0 eq.) in tetrahydrofuran (400 mL) was cooled to 0 °C in ice bath, and hydrazine hydrate (80%, 20.51 g, 328.10 mmol, 0.8 eq.) dissolved in tetrahydrofuran (240 mL) and ethanol (80 mL) was added slowly. The reaction was stirred at 25 °C for 3 h. The reaction was concentrated to 100 mL volume, water (180 mL) was added and stirred at 25 °C for 15 min. The precipitated solid was collected by filtration, slurry with petroleum ether / ethyl acetate (19 / 1, V / V; 60 mL), filtered and the product dried under vacuum to give 4-chloro-6-hydrazinyl-2-(methylthio)pyrimidine (50.00 g, white solid), yield: 64.0%.

[0157] LCMS: (ESI) [M+H] = 191.0. +

[0158] 1 H NMR: (400 MHz, DMSO-d6) δ 8.85 (s, 1H), 6.51 (s, 1H), 4.60 (s, 2H), 2.42 (s, 3H).

[0159] Step B: Preparation of 7-chloro-5-(methylthio)-[1,2,4]triazolo[4,3-c]pyrimidine

[0160] To a solution of 4-chloro-6-hydrazinyl-2-(methylthio)pyrimidine (33.00 g, 173.09 mmol, 1.0 eq.) in formic acid (90 mL) was stirred at 105 °C for 18 h. The reaction was cooled to room temperature and concentrated. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 1 / 1; V / V) to give 7-chloro-5-(methylthio)-[1,2,4]triazolo[4,3-c]pyrimidine (3.70 g, yellow solid), yield: 10.7%.

[0161] LCMS: (ESI) [M+H] = 201.0. +

[0162] 1 H NMR: (400 MHz, DMSO-d6) δ 9.50 (d, J = 0.8 Hz, 1H), 7.81 (d, J = 0.8 Hz, 1H), 2.77 (s, 3H).

[0163] Step C: Preparation of 7-chloro-5-hydroxy-[1,2,4]triazolo[1,5-c]pyrimidine​​

[0164] To a solution of 7-chloro-5-(methylthio)-[l,2,4]triazolo[4,3-c]pyrimidine (3.70 g, 18.4 mmol, 1.0 eq.) in methanol (600 mL) was added aqueous potassium hydroxide (2 M, 50 mL). The reaction was stirred at 85 °C for 3 h. The reaction was allowed to cool to room temperature and concentrated to remove methanol. The residue was adjusted to pH = 6 with hydrochloric acid (6 M) and extracted with ethyl acetate (2 x 150 mL). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate and concentrated. The resulting crude product was purified by column chromatography (dichloromethane / methanol = 10 / 1; V / V) to give 7-chloro-5-hydroxy-[l,2,4]triazolo[l,5-c]pyrimidine (600 mg, yellow solid) in 50.4% yield.

[0165] LCMS: (ESI) [M+H] = 171.0. +

[0166] 1 H NMR: (400 MHz, DMSO-d6) δ 8.13 (s, 1H), 6.58 (s, 1H).

[0167] Step D: Preparation of 5,7-dichloro-[l,2,4]triazolo[l,5-c]pyrimidine

[0168] To a solution of 7-chloro-5-hydroxy-[l,2,4]triazolo[l,5-c]pyrimidine (1.20 g, 7.04 mmol, 1.0 eq.) in phosphorus oxychloride (15 mL) was added. The reaction was stirred at 120 °C for 4 h. The reaction was allowed to cool to room temperature and quenched slowly with warm water (50 mL), adjusted to pH = 7 with saturated sodium bicarbonate and extracted with ethyl acetate (3 x 100 mL). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated. The resulting crude product was purified by column chromatography (petroleum ether / ethyl acetate = 3 / 1; V / V) to give 5,7-dichloro-[l,2,4]triazolo[l,5-c]pyrimidine (440 mg, yellow solid) in 33.1% yield.

[0169] LCMS: (ESI) [M+H] = 188.9. +

[0170] 1 H NMR: (400 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.25 (s, 1H).

[0171] ​​Step E: Preparation of 7-chloro-5-(3,3-difluoroazetidin-l-yl)- [l,2,4]triazolo[l,5-c]pyrimidine

[0172] Difluoroazetidine hydrochloride (441 mg, 2.79 mmol, 1.2 eq.) was dissolved in isopropanol (10 mL), and 5,7-dichloro-[l,2,4]triazolo[l,5-c]pyrimidine (440 mg, 2.33 mmol, 1.0 eq.) and N,N-diisopropylethylamine (903 mg, 6.98 mmol, 3.0 eq.) were added. The reaction was heated at 90 °C for 3 h. The reaction was cooled to room temperature and quenched with water (80 mL) and extracted with ethyl acetate (2 x 80 mL). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated. The resulting crude product was purified by column chromatography (petroleum ether / ethyl acetate = 4 / 1; V / V) to give 7-chloro-5-(3,3-difluoroazetidin-l-yl)- [l,2,4]triazolo[l,5-c]pyrimidine (280 mg, yellow solid) in 76.8% yield.

[0173] LCMS: (ESI) [M+H] = 246.0. + = 246.0.

[0174] 1 H NMR: (400 MHz, DMSO-d6) δ 8.51 (s, 1H), 7.32 (s, 1H), 4.90 (t, J = 12.0 Hz, 4H).

[0175] Step F: Preparation of N-(5-(3,3-difluoroazetidin-l-yl)-[l,2,4]triazolo[l,5- c]pyrimidin-7-yl)-4-nitro-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0176] To a solution of 7-chloro-5-(3,3-difluoroazetidin-l-yl)-[l,2,4]triazolo[l,5- c]pyrimidine (230 mg, 0.93 mmol, 1.0 eq.) in dioxane (15 mL) was added 4-nitro-2-(6- azaspiro[2.5]octan-6-yl)benzamide (257 mg, 0.93 mmol, 1.0 eq.), 2-dicyclohexylphosphino-2',6'-diisopropoxy-l,l'-biphenyl (87 mg, 0.19 mmol, 0.2 eq.), chloro(2-dicyclohexylphosphino-2',6'-diisopropoxy-l,l'-biphenyl-2-yl)palladium(II) (72 mg, 0.09 mmol, 0.1 eq.) and cesium carbonate (915 mg, 2.81 mmol, 3.0 eq.) successively. The reaction mixture was heated at 120 °C for 4 h under nitrogen atmosphere. The reaction mixture was allowed to cool to room temperature and diluted with water (15 mL) and extracted with ethyl acetate (2 x 30 mL). The combined organic phase was dried over anhydrous sodium sulfate and concentrated. The crude product obtained was purified by column chromatography (petroleum ether / ethyl acetate = 3 / 1; V / V) to give N-(5-(3,3-difluoroazetidin-l-yl)-[l,2,4]triazolo[l,5-c]pyrimidin-7-yl)-4-nitro-2-(6- azaspiro[2.5]octan-6-yl)benzamide (500 mg, red solid) in 92.6% yield.

[0177] LCMS: (ESI) [M+H] = 485.0. +

[0178] 1 H NMR: (400 MHz, DMSO-d6) δ 13.39 (s, 1H), 8.42 (s, 1H), 8.31 (d, J = 8.4 Hz, 1H), 8.24 (d, J = 2.0 Hz, 1H), 8.14 (dd, J = 8.4, 2.0 Hz, 1H), 7.82 (s, 1H), 4.89 (t, J = 12.4 Hz, 4H), 3.13 (t, J = 5.2 Hz, 4H), 1.78 - 1.71 (m, 4H), 0.42 (s, 4H).

[0179] Step G: Preparation of 4-amino-N-(5-(3,3-difluoroazetidin-l-yl)-[l,2,4]triazolo[l,5- c]pyrimidin-7-yl)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0180] ​N-(5-(3,3-difluoroazetidin-l-yl)-[l,2,4]triazolo[l,5-c]pyrimidin-7-yl)-4-nitro-2-(6- azaspiro[2.5]oct-6-yl)benzamide (420 mg, 0.86 mmol, 1.0 eq.) was dissolved in acetic acid (6 mL), and zinc powder (396 mg, 6.10 mmol, 7.0 eq.) was added. The reaction was stirred at 25 °C for 2 h. The reaction was filtered, and the filtrate was concentrated. The concentrate was diluted with water (30 mL), and the residual acetic acid was neutralized with saturated sodium carbonate, and extracted with ethyl acetate (3 x 45 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated. The obtained crude product was separated and purified by column chromatography (petroleum ether / ethyl acetate = 3 / 2; V / V) to give 4-amino-N-(5-(3,3-difluoroazetidin-l-yl)-[l,2,4]triazolo[l,5-c]pyrimidin-7-yl)-2-(6- azaspiro[2.5]oct-6-yl)benzamide (340 mg, yellow solid) in a yield of 70.5%.

[0181] LCMS: (ESI) [M+H] = 455.0. +

[0182] 1 H NMR: (400 MHz, CDC13) δ 13.63 (s, 1H), 8.16-8.08 (m, 3H), 6.60-6.55 (m, 2H), 4.85 (t, J = 12.0 Hz, 4H), 3.09-3.00 (m, 4H), 1.82-1.68 (m, 4H), 0.43 (s, 4H).

[0183] Step H: Preparation of ethyl 2-(N-(4-((5-(3,3-difluoroazetidin-l-yl)-[l,2,4]triazolo[l,5- c]pyrimidin-7-yl)carbamoyl)-3-(6-azaspiro[2.5]oct-6-yl)phenyl)sulfamoyl)acetate (Intermediate C)

[0184] ​To a solution of 4-amino-N-(5-(3,3-difluoroazetidin-l-yl)-[l,2,4]triazolo[l,5- c]pyrimidin-7-yl)-2-(6-azaspiro[2.5]oct-6-yl)benzamide (200 mg, 0.44 mmol, 1.0 eq.) and triethylamine (222 mg, 2.20 mmol, 5.0 eq.) in tetrahydrofuran (5 mL) was added (chlorosulfonyl)acetic acid ethyl ester (205 mg, 1.10 mmol, 2.5 eq.) dropwise at 0 °C under nitrogen. The reaction was stirred at 0 °C for 2 h. The reaction was quenched by the addition of saturated aqueous ammonium chloride solution (30 mL) and extracted with ethyl acetate (3 x 30 mL). The combined organic phase was dried over anhydrous sodium sulfate and concentrated. The resulting crude product was purified by column chromatography (petroleum ether / ethyl acetate = 1 / 1; V / V) to give 2-(N-(4-((5-(3,3-difluoroazetidin-l-yl)-[l,2,4]triazolo[l,5-c]pyrimidin-7-yl)carbamoyl)-3-(6- azaspiro[2.5]oct-6-yl)phenyl)sulfamoyl)acetic acid ethyl ester (240 mg, yellow solid) in 90.2% yield.

[0185] LCMS: (ESI) [M+H] = 605.0. +

[0186] 1 H NMR: (400 MHz, DMSO-d6) δ 13.69 (s, 1H), 10.71 (s, 1H), 8.39 (s, 1H), 8.12 (d, J = 8.4 Hz, 1H), 7.80 (s, 1H), 7.32 (d, J = 2.0 Hz, 1H), 7.18 (dd, J = 8.4, 2.0 Hz, 1H), 4.89 (t, J = 12.0 Hz, 4H), 4.41 (s, 2H), 4.10 (q, J = 7.2 Hz, 2H), 3.02 - 2.98 (m, 4H), 1.84 - 1.75 (m, 4H), 1.16 (t, J = 7.2 Hz, 3H), 0.42 (s, 4H).

[0187] Example 2: Synthesis of compound 2

[0188] 4-(2,2-Dideutero-2-hydroxyethylsulfonamido)-N-(7-(3,3-difluoroazetidin-l-yl)pyrazolo[l,5- a]pyrimidin-5-yl)-2-(6-azaspiro[2.5]oct-6-yl)benzamide

[0189] ​Step A: Preparation of 4-(2,2-dideutero-2-hydroxyethylsulfonamido)-N-(7-(3,3- difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridine-5-yl)-2-(6-azaspiro[2.5]octan-6- yl)benzamide

[0190] Deuterated lithium aluminum hydride (19 mg, 0.45 mmol, 3.0 eq.) was added to tetrahydrofuran (3 ml) under nitrogen protection, and cooled to 0 °C. 2-(N-(4-((7-(3,3- difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridine-5-yl)carbamoyl)-3-(6-azaspiro[2.5]octan-6- yl)phenyl)sulfamoyl)acetic acid ethyl ester (90 mg, 0.15 mmol, 1.0 eq.) was added. The reaction solution was reacted at 0 °C for 0.5 h. The reaction solution was quenched by adding dilute hydrochloric acid (1 M, 3 ml) and diluted with water (20 ml), and extracted with ethyl acetate (2 x 50 ml). The organic phase was combined, dried over anhydrous sodium sulfate and concentrated. The obtained crude product was separated and purified by column chromatography (petroleum ether / ethyl acetate = 2 / 3; V / V) to obtain 4-(2,2-dideutero-2-hydroxyethylsulfonamido)-N-(7-(3,3- difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridine-5-yl)-2-(6-azaspiro[2.5]octan-6- yl)benzamide (44.57 mg, yellow solid), yield: 55.7%.

[0191] LCMS: (ESI) [M+H] + = 563.2.

[0192] 1 H NMR: (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 10.09 (s, 1H), 7.92 (d, J = 2.0 Hz, 1H), 7.78 (d, J = 8.8 Hz, 1H), 7.70 (d, J = 1.6 Hz, 1H), 7.15 (d, J = 2.0 Hz, 1H), 7.02 (dd, J = 8.4, 2.0 Hz, 1H), 6.47 (d, J = 2.0 Hz, 1H), 6.30 (d, J = 2.0 Hz, 1H), 4.92 (s, 1H), 4.66 (t, J = 12.0 Hz, 4H), 3.38-3.33 (m, 2H), 3.02-2.95 (m, 4H), 1.62-1.51 (m, 4H), 0.35 (s, 4H).

[0193] Example 3: Synthesis of compound 3

[0194] 1,1-Dideutero-2-(N-(4-((7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridin-5- yl)carbamoyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl phosphate dihydrogen trifluoroacetate salt

[0195] Step A: Preparation of dibenzyl (1,1-dideutero-2-(N-(4-((7-(3,3-difluoroazetidin-1- yl)pyrazolo[1,5-a]pyridin-5-yl)carbamoyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl) phosphate

[0196] To a solution of 4-(2,2-dideutero-2-hydroxyethylsulfonamido)-N-(7-(3,3-difluoroazetidin-1- yl)pyrazolo[1,5-a]pyridin-5-yl)-2-(6-azaspiro[2.5]octan-6-yl)benzamide (92 mg, 0.16 mmol, 1.0 eq.) in dichloromethane (4 mL) and acetonitrile (4 mL) was cooled to 0 °C in an ice bath. Tetrazole (15 mg, 0.21 mmol, 1.3 eq.) and dibenzyl N,N-diisopropyl phosphoramidite (66 mg, 0.19 mmol, 1.2 eq.) were added sequentially. The reaction was stirred at 0 °C for 70 min. m-CPBA (33 mg, 0.19 mmol, 1.2 eq.) was added to the reaction. The reaction was stirred at 0 °C for an additional 10 min. The reaction was quenched with saturated aqueous sodium bicarbonate (50 mL) and extracted with ethyl acetate (2 x 45 mL). The organic layers were combined, washed with saturated aqueous ammonium chloride (80 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 2 / 1; V / V) to give dibenzyl (1,1-dideutero-2-(N-(4-((7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridin-5- yl)carbamoyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl) phosphate (32 mg, yellow solid) in 23.8% yield.

[0197] LCMS: (ESI) [M+H] = 823.2. +

[0198] 1 ​H NMR: (400 MHz, DMSO-d6) δ 12.54 (s, 1H), 8.19 (d, J = 8.4 Hz, 1H), 8.07 (s, 1H), 7.87 (d, J = 2.4 Hz, 1H), 7.48 (d, J = 2.0 Hz, 1H), 7.40 - 7.32 (m, 10H), 7.30 (d, J = 2.0 Hz, 1H), 7.10 (dd, J = 8.4, 2.0 Hz, 1H), 6.40 (d, J = 2.4 Hz, 1H), 6.36 (d, J = 1.6 Hz, 1H), 5.11 - 5.01 (m, 4H), 4.66 (t, J = 12.0 Hz, 4H), 3.33 (s, 2H), 3.03 (t, J = 5.2 Hz, 4H), 1.59 - 1.56 (s, 4H), 0.42 (s, 4H).

[0199] Step B: Preparation of 1,1-dideuterio-2-(N-(4-((7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5- a]pyridin-5-yl)carbamoyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl dihydrogen phosphate trifluoroacetate salt

[0200] Dibenzyl (1,1-dideuterio-2-(N-(4-((7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5- a]pyridin-5-yl)carbamoyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl) phosphate (37 mg, 0.05 mmol, 1.0 eq.) was dissolved in methanol (4 mL) under nitrogen protection, and palladium on carbon (10%, 37 mg) was added. Vacuum was replaced with hydrogen for 3 times, and the reaction was stirred at 25 °C for 2 hours under hydrogen atmosphere. The reaction was filtered, and the filtrate was concentrated. The obtained crude product was separated and purified by preparative HPLC to obtain 1,1-dideuterio-2-(N-(4-((7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5- a]pyridin-5-yl)carbamoyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl dihydrogen phosphate trifluoroacetate salt (5.9 mg, yellow solid), yield: 17.3%.

[0201] LCMS: (ESI) [M+H] = 643.2. +

[0202] 1 ​H NMR: (400 MHz, DMSO-d6) δ 11.56 (s, 1H), 10.25 (s, 1H), 7.92 (d, J = 2.4 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 1.6 Hz, 1H), 7.15 (d, J = 2.0 Hz, 1H), 7.05 (dd, J = 8.4, 2.0 Hz, 1H), 6.48 (d, J = 2.4 Hz, 1H), 6.31 (d, J = 1.6 Hz, 1H), 4.66 (t, J = 12.4 Hz, 4H), 3.54 (s, 2H), 3.04 - 2.97 (m, 4H), 1.65 - 1.44 (m, 4H), 0.35 (s, 4H).

[0203] Example 4: Synthesis of compound 4

[0204] N-(7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridin-5-yl)-2-(6-azaspiro[2.5]octan-6- yl)-4-(1,1,2,2-tetradeuterio-2-hydroxyethylsulfonamido)benzamide

[0205] Step A: Preparation of N-(7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridin-5-yl)-2-(6- azaspiro[2.5]octan-6-yl)-4-(1,1,2,2-tetradeuterio-2-hydroxyethylsulfonamido)benzamide

[0206] Ethyl 2-(N-(4-((7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridin-5-yl)carbamoyl)-3-(6- azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)acetate (70 mg, 0.12 mmol, 1.0 eq.) was dissolved in deuterated methanol-d4 (2 mL) and cooled to 0 °C in an ice bath. Sodium borodeuteride (141 mg, 3.37 mmol, 28.0 eq.) was added. The reaction was allowed to react at 30 °C for 48 h. The reaction was quenched with water (30 mL) and extracted with ethyl acetate (2 x 40 mL). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated. The obtained crude product was separated and purified by column chromatography (petroleum ether / ethyl acetate = 1 / 1; V / V) to give N-(7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridin-5-yl)-2-(6-azaspiro[2.5]octan-6-yl)-4-(1,1,2,2-tetradeuterio-2-hydroxyethylsulfonamido)benzamide (20.49 mg, yellow solid) with a yield of 30.2%.

[0207] LCMS: (ESI) [M+H]+ = 565.2.

[0208] 1 H NMR: (400 MHz, CDC13) δ 12.46 (s, 1H), 8.15 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 2.4 Hz, 1H), 7.41 (d, J = 2.0 Hz, 1H), 7.27 (d, J = 2.4 Hz, 1H), 6.99 (dd, J = 8.4, 2.0 Hz, 1H), 6.89 (s, 1H), 6.33 (d, J = 2.0 Hz, 1H), 6.29 (d, J = 2.0 Hz, 1H), 4.59 (t, J = 12.0 Hz, 4H), 3.02 (t, J = 5.2 Hz, 4H), 1.74 - 1.52 (m, 4H), 0.38 (s, 4H).

[0209] Example 5: Synthesis of compound 5

[0210] 2-(N-(4-((7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridin-5-yl)carbamoyl)-3-(6- azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl-1,1,2,2-tetradeuterophosphonic acid dihydrogen ester

[0211] Step A: Preparation of dibenzyl (2-(N-(4-((7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5- a]pyridin-5-yl)carbamoyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)1,1,2,2- tetra-deuterio-ethyl)phosphonate

[0212] N-(7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridin-5-yl)-2-(6-azaspiro[2.5]octan-6- yl)-4-(1,1,2,2-tetradeuterio-2-hydroxyethylsulfonamido)benzamide (120 mg, 0.21 mmol, 1.0 eq.) was dissolved in a mixture solvent of dichloromethane (4 mL) and acetonitrile (4 mL), and cooled to 0 °C in an ice bath. Tetrazole (19 mg, 0.27 mmol, 1.3 eq.) and dibenzyl N,N-diisopropylphosphoramidite (86 mg, 0.25 mmol, 1.2 eq.) were added successively. The reaction solution was reacted at 0 °C for 70 minutes. To the reaction solution was added m-chloroperoxybenzoic acid (43 mg, 0.25 mmol, 1.2 eq.). The reaction solution was continued to react at 0 °C for 10 minutes. The reaction solution was quenched by adding saturated aqueous sodium bicarbonate solution (40 mL), and extracted with ethyl acetate (2 x 35 mL). The combined organic phase was washed with saturated aqueous ammonium chloride solution (80 mL), dried over anhydrous sodium sulfate, and concentrated. The obtained crude product was separated and purified by column chromatography (petroleum ether / ethyl acetate = 4 / 1; V / V) to obtain dibenzyl (2-(N-(4-((7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridin-5-yl)carbamoyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)1,1,2,2-tetradeuterio-ethyl)phosphonate (150 mg, white solid), yield: 85.6%.

[0213] LCMS: (ESI) [M+H] = 825.2. +

[0214] 1 H NMR: (400 MHz, DMSO-d6) δ 11.36 (s, 1H), 10.14 (s, 1H), 7.69 (d, J = 2.0 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.47 (d, J = 1.6 Hz, 1H), 7.21 - 7.06 (m, 10H), 6.95 - 6.89 (m, 1H), 6.81 (dd, J = 8.4, 2.0 Hz, 1H), 6.25 (d, J = 2.4 Hz, 1H), 6.05 (d, J = 1.6 Hz, 1H), 4.81 - 4.69 (m, 4H), 4.43 (t, J = 12.0 Hz, 4H), 2.75 - 2.66 (m, 4H), 1.36 - 1.23 (m, 4H), 0.13 - 0.05 (m, 4H).

[0215] ​Step B: Preparation of 2-(N-(4-((7-(3,3-difluoroazetidin-l-yl)pyrazolo[l,5- a]pyridin-5-yl)carbamoyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl- 1,1,2,2-tetradeuteriophosphonate dihydrogen

[0216] Dibenzyl (2-(N-(4-((7-(3,3-difluoroazetidin-l-yl)pyrazolo[l,5-a]pyridin-5- yl)carbamoyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)l, l,2,2-tetradeuter o-ethyl)phosphonate (150 mg, 0.18 mmol, 1.0 eq.) was dissolved in methanol (2 mL) under nitrogen protection, palladium on carbon (10%, 80 mg) was added. Vacuum was replaced by hydrogen for 3 times, the reaction was stirred at 25 °C for 2 hours under hydrogen atmosphere. The reaction was filtered, the filtrate was concentrated. The obtained crude product was separated and purified by preparative HPLC to give 2-(N-(4-((7-(3,3-difluoroazetidin-l-yl)pyrazolo[l,5-a]pyridin-5-yl)carbamoyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl- 1,1,2,2-tetradeuteriophosphonate dihydrogen (4.09 mg, green solid), yield: 3.5%.

[0217] LCMS: (ESI) [M+H] = 645.2. +

[0218] 1 H NMR: (400 MHz, DMSO-d6) δ 11.51 (s, 1H), 7.74 (d, J = 2.0 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.53 (s, 1H), 7.09 (s, 1H), 7.00 - 6.92 (m, 2H), 6.29 (d, J = 2.0 Hz, 1H), 6.13 (s, 1H), 4.48 (t, J = 12.0 Hz, 4H), 2.86 - 2.79 (m, 4H), 1.42 - 1.32 (m, 4H), 0.16 (s, 4H).

[0219] Example 6: Synthesis of compound 6

[0220] N-(5-(3,3-difluoroazetidin-l-yl)-[l,2,4]triazolo[l,5-c]pyrimidin-7-yl)-4-(l,l,2,2- tetra-deuterio-2-hydroxyethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0221] ​Step A: Preparation of N-(5-(3,3-difluoroazetidin-l-yl)-[l,2,4]triazolo[l,5- c]pyrimidin-7-yl)-4-(l,l,2,2-tetradeuterio-2-hydroxyethylsulfonamido)-2-(6- azaspiro[2.5]oct-6-yl)benzamide

[0222] Ethyl 2-(N-(4-((5-(3,3-difluoroazetidin-l-yl)-[l,2,4]triazolo[l,5-c]pyrimidin-7- yl)carbamoyl)-3-(6-azaspiro[2.5]oct-6-yl)phenyl)sulfamoyl)acetate (200 mg, 0.33 mmol, 1.0 eq.) was dissolved in deuterated methanol-d4 (3 mL) and cooled to 0 °C in an ice bath. Sodium borodeuteride (554 mg, 13.23 mmol, 40.0 eq.) was added. The reaction was allowed to react at 10 °C for 48 h. The reaction was quenched with water (100 mL) and extracted with ethyl acetate (2 x 80 mL). The organic phases were combined, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, and concentrated. The resulting crude product was separated and purified by column chromatography (petroleum ether / ethyl acetate = 1 / 1; V / V) to give N-(5-(3,3-difluoroazetidin-l-yl)-[l,2,4]triazolo[l,5-c]pyrimidin-7-yl)-4-(l,l,2,2-tetradeuterio-2-hydroxyethylsulfonamido)-2-(6-azaspiro[2.5]oct-6-yl)benzamide (80 mg, white solid) with a yield of 42.7%.

[0223] LCMS: (ESI) [M+H] = 567.2. + = 567.2.

[0224] 1 H NMR: (400 MHz, DMSO-d6) δ 13.72 (s, 1H), 10.28 (s, 1H), 8.39 (s, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.81 (s, 1H), 7.32 (d, J = 2.0 Hz, 1H), 7.17 (dd, J = 8.4, 2.0 Hz, 1H), 4.88 (t, J = 12.0 Hz, 4H), 3.00 (t, J = 4.8 Hz, 4H), 1.92 - 1.40 (m, 4H), 0.43 (s, 4H).

[0225] Example 7: Synthesis of compound 7

[0226] 2-(N-(4-((5-(3,3-difluoroazetidin-1-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)carbamoyl)-3-(6-azaspiro[2.5]oct-6-yl)phenyl)sulfamoyl)ethyl-1,1,2,2-tetra-deuterio-phosphoric acid dihydrogen ester trifluoroacetic acid salt

[0227] Step A: Preparation of dibenzyl (2-(N-(4-((5-(3,3-difluoroazetidin-1-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)carbamoyl)-3-(6-azaspiro[2.5]oct-6-yl)phenyl)sulfamoyl)1,1,2,2-tetra-deuterio-ethyl)phosphonate

[0228] N-(5-(3,3-difluoroazetidin-1-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-4-(1,1,2,2-tetra-deuterio-2-hydroxyethylsulfonamido)-2-(6-azaspiro[2.5]oct-6-yl)benzamide (60 mg, 0.11 mmol, 1.0 eq.) was dissolved in a mixture solvent of dichloromethane (2 mL) and acetonitrile (2 mL), cooled to 0 °C under nitrogen protection, and then added with tetrazole (10 mg, 0.14 mmol, 1.3 eq.) and dibenzyl N,N-diisopropyl phosphoramidite (44 mg, 0.13 mmol, 1.2 eq.) successively. The reaction solution was reacted at 0 °C for 70 min. M-chloroperbenzoic acid (85%, 26 mg, 0.13 mmol, 1.2 eq.) was added to the reaction solution. The reaction solution was continuously reacted at 0 °C for 10 min. The reaction solution was quenched by adding saturated aqueous sodium bicarbonate solution (50 mL), and extracted with ethyl acetate (2 x 45 mL). The combined organic phase was washed with saturated aqueous ammonium chloride solution (80 mL), dried over anhydrous sodium sulfate, and concentrated. The obtained crude product was separated and purified by column chromatography (petroleum ether / ethyl acetate = 2 / 1; V / V) to obtain dibenzyl (2-(N-(4-((5-(3,3-difluoroazetidin-1-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)carbamoyl)-3-(6-azaspiro[2.5]oct-6-yl)phenyl)sulfamoyl)1,1,2,2-tetra-deuterio-ethyl)phosphonate (41 mg, yellow solid) with a yield of 46.8%.

[0229] LCMS: (ESI) [M+H] + = 827.2.

[0230] 1H NMR: (400 MHz, DMSO-d6) δ 13.67 (s, 1H), 10.60 (s, 1H), 8.45 (d, J = 1.2 Hz, 1H), 8.15 (d, J = 8.8 Hz, 1H), 7.86 (s, 1H), 7.45 - 7.34 (m, 10H), 7.33 (d, J = 2.0 Hz, 1H), 7.23 (dd, J = 8.8, 2.0 Hz, 1H), 5.07 - 4.99 (m, 4H), 4.93 (t, J = 12.4 Hz, 4H), 2.97 (t, J = 4.8 Hz, 4H), 1.95 - 1.60 (m, 4H), 0.42 (s, 4H).

[0231] Step B: Preparation of 2-(N-(4-((5-(3,3-difluoroazetidin-1-yl)-[1,2,4]triazolo[1,5- c]pyrimidin-7-yl)carbamoyl)-3-(6-azaspiro[2.5]oct-6-yl)phenyl)sulfamoyl)ethyl-1,1,2,2- tetra-deuterio-phosphoric acid dihydrogen ester trifluoroacetic acid salt

[0232] Dibenzyl (2-(N-(4-((5-(3,3-difluoroazetidin-1-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7- yl)carbamoyl)-3-(6-azaspiro[2.5]oct-6-yl)phenyl)sulfamoyl)1,1,2,2-tetra-deuterio- ethyl)phosphonate (35 mg, 0.04 mmol, 1.0 eq.) was dissolved in tetrahydrofuran (2 mL) under nitrogen protection, palladium on carbon (10%, 35 mg) was added. The reaction was stirred at 20 °C for 2 hours under hydrogen atmosphere. The reaction was filtered, the filtrate was concentrated. The obtained crude product was separated and purified by prep-HPLC (trifluoroacetic acid) to give 2-(N-(4-((5-(3,3-difluoroazetidin-1-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7- yl)carbamoyl)-3-(6-azaspiro[2.5]oct-6-yl)phenyl)sulfamoyl)ethyl-1,1,2,2-tetra-deuterio- phosphoric acid dihydrogen ester trifluoroacetic acid salt (6.62 mg, white solid), yield: 21.8%.

[0233] LCMS: (ESI) [M+H] = 647.2. +

[0234] 1 ​H NMR: (400 MHz, DMSO-d6) δ 13.73 (s, 1H), 10.43 (s, 1H), 8.40 (s, 1H), 8.11 (d, J = 8.8 Hz, 1H), 7.81 (s, 1H), 7.31 (d, J = 2.0 Hz, 1H), 7.19 (dd, J = 8.8, 2.0 Hz, 1H), 4.88 (t, J = 12.4 Hz, 4H), 3.01 (t, J = 4.8 Hz, 4H), 2.07 - 1.47 (m, 4H), 0.43 (s, 4H).

[0235] Example 8: Synthesis of compound 8

[0236] 2-(N-(4-((7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridin-5-yl)carbamoyl)-3-(6- azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)-1,1,2,2-tetradeuterio-ethyl sulfate trifluoroacetate

[0237] Step A: Preparation of 2-(N-(4-((7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridin-5- yl)carbamoyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)-1,1,2,2-tetradeuterio- ethyl sulfate trifluoroacetate

[0238] N-(7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridin-5-yl)-2-(6-azaspiro[2.5]octan-6- yl)-4-(1,1,2,2-tetradeuterio-2-hydroxyethylsulfonamido)benzamide (50 mg, 0.09 mmol, 1.0 eq.) was dissolved in dichloromethane (2.5 mL) and cooled to -30 °C under nitrogen protection. Triethylamine (90 mg, 0.89 mmol, 10.0 eq.) and chlorosulfonic acid (30 mg, 0.26 mmol, 2.9 eq.) were added successively. The reaction solution was reacted at 25 °C for 18 hours. The reaction was quenched with water (5 mL) and extracted with dichloromethane (3 x 20 mL). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated. The obtained crude product was separated and purified by preparative HPLC (trifluoroacetic acid) to obtain 2-(N-(4-((7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridin-5-yl)carbamoyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)-1,1,2,2-tetradeuterio-ethyl sulfate trifluoroacetate (1.74 mg, yellow solid), yield: 2.6%.

[0239] LCMS: (ESI) [M+H]+ = 645.4 (free).

[0240] 1 H NMR: (400 MHz, DMSO-d6) δ 11.46 (s, 1H), 10.18 (s, 1H), 7.92 (d, J = 2.0 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 2.0 Hz, 1H), 7.19-7.15 (m, 1H), 7.05 (dd, J = 8.4, 2.0 Hz, 1H), 6.48 (d, J = 2.4 Hz, 1H), 6.34 (d, J = 1.6 Hz, 1H), 4.66 (t, J = 12.4 Hz, 4H), 3.07-3.00 (m, 4H), 1.58-1.49 (m, 4H), 0.35 (s, 4H).

[0241] Comparative Example 1: Synthesis of Comparative Compound 9

[0242] N-(7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridin-5-yl)-4-(2-hydroxyethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0243] Step A: Preparation of N-(7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridin-5-yl)-4-(2-hydroxyethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0244] Ethyl 2-(N-(4-((7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridin-5-yl)carbamoyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)acetate (700 mg, 1.16 mmol, 1.0 eq.) was dissolved in tetrahydrofuran (10 mL), and sodium borohydride (132 mg, 3.49 mmol, 3.0 eq.) was added. The reaction solution was reacted at 25 °C for 4 hours. The reaction solution was quenched with water (30 mL) and extracted with ethyl acetate (3 x 40 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated. The obtained crude product was separated and purified by reverse phase column chromatography (water / acetonitrile = 3 / 2; V / V) to obtain N-(7-(3,3-difluoroazetidin-1-yl)pyrazolo[1,5-a]pyridin-5-yl)-4-(2-hydroxyethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide (411 mg, yellowish solid) with a yield of 63.1%.

[0245] LCMS: (ESI) [M+H] = 231.0. + = 561.0.

[0246] 1 H NMR: (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 10.10 (s, 1H), 7.92 (d, J = 2.4 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 1.6 Hz, 1H), 7.16 (d, J = 1.6 Hz, 1H), 7.03 (dd, J = 8.4, 2.0 Hz, 1H), 6.48 (d, J = 2.4 Hz, 1H), 6.31 (d, J = 1.6 Hz, 1H), 4.96 (t, J = 5.4 Hz, 1H), 4.66 (t, J = 12.0 Hz, 4H), 3.82 - 3.73 (m, 2H), 3.37 - 3.34 (m, 2H), 3.03 - 2.94 (m, 4H), 1.61 - 1.51 (m, 4H), 0.36 (s, 4H).

[0247] Comparative Example 2: Synthesis of Comparative Compound 10

[0248] N-(7-(4,4-difluoropiperidin-l-yl)pyrazolo[l,5-a]pyridin-5-yl)-4-(2- hydroxyethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0249] Step A: Preparation of 5-bromo-7-chloropyrazolo[l,5-a]pyridine

[0250] Dissolve 5-bromopyrazolo[l,5-a]pyridine (1.18 g, 6.00 mmol, 1.0 eq.) in tetrahydrofuran (15 mL) and cool to -78 °C in a dry ice / ethanol bath under nitrogen. Add lithium bis(trimethylsilyl)amide (1 M, 7.2 mL, 7.20 mmol, 1.2 eq.) dropwise. Continue to stir the reaction at -78 °C for 0.5 h. Add a solution of hexachloroethane (1.70 g, 7.20 mmol, 1.2 eq.) in tetrahydrofuran (4 mL) dropwise. Continue to stir the reaction at -78 °C for 1 h. Quench the reaction by adding saturated ammonium chloride (80 mL) and extract with ethyl acetate (2 x 70 mL). Dry the combined organic extracts over anhydrous sodium sulfate and concentrate. Purify the resulting crude product by column chromatography (petroleum ether / ethyl acetate = 20 / 1; V / V) to give 5-bromo-7-chloropyrazolo[l,5-a]pyridine (1.20 g, yellow solid) in 86.3% yield.

[0251] LCMS: (ESI) [M+H] = 231.0. + = 561.0.

[0252] 1 H NMR: (400 MHz, CDC13) δ 8.06 (d, J = 2.0 Hz, 1H), 7.72 (d, J = 2.0 Hz, 1H), 7.03 (d, J = 1.6 Hz, 1H), 6.61 (d, J = 2.0 Hz, 1H).

[0253] Step B: Preparation of 5-bromo-7-(4,4-difluoropiperidin-l-yl)pyrazolo[l,5- a]pyridine

[0254] Dissolve 5-bromo-7-chloropyrazolo[l,5-a]pyridine (926 mg, 4.00 mmol, 1.0 eq.) in N-methylpyrrolidine (8 mL), add 4,4-difluoropyridine (1.45 g, 12.00 mmol, 3.0 eq.). The reaction solution is reacted at 135 °C for 20 hours. The reaction solution is reduced to room temperature and diluted with saturated aqueous ammonium chloride solution (50 mL), extracted with ethyl acetate (2 x 70 mL). The combined organic phase is washed with saturated brine (2 x 70 mL), dried over anhydrous sodium sulfate and concentrated. The obtained crude product is separated and purified by column chromatography (petroleum ether / ethyl acetate = 20 / 1; V / V) to obtain 5-bromo-7-(4,4-difluoropiperidin-l-yl)pyrazolo[l,5-a]pyridine (864 mg, yellow solid), yield: 68.6%.

[0255] LCMS: (ESI) [M+H] + = 318.0.

[0256] 1 H NMR: (400 MHz, CDC13) δ 8.06 (d, J = 2.0 Hz, 1H), 7.72 (d, J = 2.0 Hz, 1H), 7.03 (d, J = 1.6 Hz, 1H), 6.61 (d, J = 2.0 Hz, 1H).

[0257] Step C: Preparation of N-(7-(4,4-difluoropiperidin-l-yl)pyrazolo[l,5-a]pyridin-5-yl)-4- nitro-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0258] To a solution of 5-bromo-7-(4,4-difluoropiperidin-l-yl)pyrazolo[l,5- a]pyridine (300 mg, 0.95 mmol, 1.0 eq.) in dioxane (10 mL) was added 4-nitro-2-(6- azaspiro[2.5]octan-6-yl)benzamide (314 mg, 1.14 mmol, 1.2 eq.), 2-(dicyclohexylphosphino)-3,6-dimethoxy-2',4',6'-triisopropyl-l,r-biphenyl (102 mg, 0.19 mmol, 0.2 eq.), methane sulfonic acid (2-dicyclohexylphosphino)-3,6-dimethoxy-2',4',6'-triisopropyl-l,r-biphenyl (2'-amino-l,r-biphenyl-2-yl)palladium(II) (86 mg, 0.10 mmol, 0.1 eq.) and cesium carbonate (928 mg, 2.85 mmol, 3.0 eq.) successively. The reaction mixture was heated to 120 °C for 2 h under microwave irradiation. The reaction mixture was allowed to cool to room temperature and diluted with water (100 mL) and extracted with ethyl acetate (2 x 100 mL). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate and concentrated. The crude product obtained was purified by column chromatography (ethyl acetate / petroleum ether = 3 / 7; V / V) to give N-(7-(4,4-difluoropiperidin-l-yl)pyrazolo[l,5-a]pyridin-5-yl)-4-nitro-2-(6- azaspiro[2.5]octan-6-yl)benzamide (450 mg, yellow solid) in 94.7% yield.

[0259] LCMS: (ESI) [M+H] = 511.2. +

[0260] Step D: Preparation of 4-amino-N-(7-(4,4-difluoropiperidin-l-yl)pyrazolo[l,5- a]pyridin-5-yl)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0261] ​N-(7-(4,4-difluoropiperidin-l-yl)pyrazolo[l,5-a]pyridin-5-yl)-4-nitro-2-(6- azaspiro[2.5]octan-6-yl)benzamide (290 mg, 0.57 mmol, 1.0 eq.) was dissolved in ethanol (10 mL), stannous chloride (517 mg, 2.73 mmol, 4.8 eq.) was added. The reaction was heated at 70 °C for 2 h. The reaction was cooled to room temperature and diluted with water (80 mL) and extracted with ethyl acetate (2 x 100 mL). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate and concentrated. The resulting crude product was purified by column chromatography (ethyl acetate / petroleum ether = 1 / 1; V / V) to give 4-amino-N-(7-(4,4-difluoropiperidin-l-yl)pyrazolo[l,5-a]pyridin-5-yl)-2-(6- azaspiro[2.5]octan-6-yl)benzamide (120 mg, yellow oil) in 44.0% yield.

[0262] LCMS: (ESI) [M+H] = 481.2. +

[0263] 1 H NMR: (400 MHz, CDC13) δ 12.78 (s, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.86 (d, J = 2.4 Hz, 1H), 7.72 (d, J = 2.0 Hz, 1H), 6.64 (d, J = 1.6 Hz, 1H), 6.53-6.48 (m, 2H), 6.38 (d, J = 2.0 Hz, 1H), 4.04 (s, 2H), 3.54-3.49 (m, 4H), 2.99 (t, J = 4.8 Hz, 4H), 2.26-2.18 (m, 4H), 1.58-1.48 (s, 4H), 0.37 (s, 4H).

[0264] Step E: Preparation of 2-(N-(4-((7-(4,4-difluoropiperidin-l-yl)pyrazolo[l,5-a]pyridin-5- yl)carbamoyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)ethyl acetate

[0265] ​To a solution of 4-amino-N-(7-(4,4-difluoropiperidin-l-yl)pyrazolo[l,5- a]pyridin-5-yl)-2-(6-azaspiro[2.5]octan-6-yl)benzamide (120 mg, 0.25 mmol, 1.0 eq.) and triethylamine (76 mg, 0.75 mmol, 3.0 eq.) in tetrahydrofuran (8 mL) was added (chlorosulfonyl)acetic acid ethyl ester (94 mg, 0.50 mmol, 2.0 eq.) dropwise at 0 °C under nitrogen. The reaction was stirred at 18 °C for 2 h. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (2 x 60 mL). The organic layers were combined, dried over anhydrous sodium sulfate and concentrated. This resulted in 2-(N-(4-((7-(4,4-difluoropiperidin-l-yl)pyrazolo[l,5-a]pyridin-5-yl)carbamoyl)-3-(6- azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)acetic acid ethyl ester (120 mg, crude, yellow oil).

[0266] LCMS: (ESI) [M+H] + = 631.2.

[0267] 1 H NMR: (400 MHz, CDC13) δ 12.56 (s, 1H), 8.19 (d, J = 8.8 Hz, 1H), 7.89 (d, J = 2.0 Hz, 1H), 7.73 (d, J = 2.0 Hz, 1H), 7.33 (s, 1H), 7.30 (d, J = 2.0 Hz, 1H), 7.08 (dd, J = 8.8, 2.0 Hz, 1H), 6.59 (d, J = 2.0 Hz, 1H), 6.41 (d, J = 2.4 Hz, 1H), 4.20 (q, J = 7.2 Hz, 2H), 3.93 (s, 2H), 3.54 - 3.49 (m, 4H), 3.03 (t, J = 5.2 Hz, 4H), 2.26 - 2.17 (m, 4H), 1.69 - 1.56 (m, 4H), 1.24 (t, J = 7.2 Hz, 3H), 0.38 (s, 4H).

[0268] Step F: Preparation of N-(7-(4,4-difluoropiperidin-l-yl)pyrazolo[l,5-a]pyridin-5-yl)-4- (2-hydroxyethylsulfonamido)-2-(6-azaspiro[2.5]octan-6-yl)benzamide

[0269] Ethyl 2-(N-(4-((7-(4,4-difluoropiperidin-l-yl)pyrazolo[l,5-a]pyridin-5- yl)carbamoyl)-3-(6-azaspiro[2.5]octan-6-yl)phenyl)sulfamoyl)acetate (60 mg, 0.09 mmol, 1.0 eq.) was dissolved in tetrahydrofuran (5 mL), sodium borohydride (29 mg, 0.76 mmol, 8.0 eq.) was added. The reaction was stirred at 18 °C for 18 h. The reaction was quenched with water (30 mL) and extracted with ethyl acetate (2 x 50 mL). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated. The resulting crude product was separated and purified by preparative HPLC to give N-(7-(4,4-difluoropiperidin-l-yl)pyrazolo[l,5-a]pyridin-5-yl)-4-(2- hydroxyethylsulfamoyl)-2-(6-azaspiro[2.5]octan-6-yl)benzamide (24.07 mg, yellowish solid), yield: 43.0%.

[0270] LCMS: (ESI) [M+H] = 589.2. +

[0271] 1 H NMR: (400 MHz, CDC13) δ 12.72 (s, 1H), 8.23 (d, J = 8.4 Hz, 1H), 7.98 (d, J = 2.4 Hz, 1H), 7.82 (d, J = 1.2 Hz, 1H), 7.38 (d, J = 1.6 Hz, 1H), 7.23 (s, 1H), 7.08 (dd, J = 8.4, 1.6 Hz, 1H), 6.66 (d, J = 1.6 Hz, 1H), 6.50 (d, J = 2.4 Hz, 1H), 4.22 - 4.11 (m, 2H), 3.64 - 3.57 (m, 4H), 3.40 - 3.31 (m, 2H), 3.11 (t, J = 4.8 Hz, 4H), 2.78 (s, 1H), 2.38 - 2.26 (m, 4H), 1.82 - 1.68 (m, 4H), 0.47 (s, 4H).

[0272] Example 9

[0273] Biological Assay

[0274] Determination of the inhibitory activity of the compounds on KIF18A enzyme

[0275] ​The ADP-Glo Luminescence Assay was used to test the inhibition of KIF18A enzyme activity by the compounds. Human KIF18A (1-467) protein (Chempartner, CP20220309-N-His cleaved-BV) was incubated with different concentrations of test compounds or DMSO in reaction buffer (15 mM Tris, pH 7.5, 10 mM MgCl2, 0.01% Pluronic F-68, 2% DMSO, 1 mM taxol, 30 pg / mL tubulin) in a 384-well plate for 15 minutes at room temperature. Substrate (Cytoskeleton, MT002) and ATP (Promega, V916B) were added to the 384-well plate reaction wells, and incubated at 28°C for 60 minutes. ADP-Glo Reagent 1 (Promega, V9102) was added, and the reaction was allowed to proceed for 120 minutes. After 60 minutes at room temperature following the addition of ADP-Glo Reagent 2, the RLU values were read using an EnVision 2104 Multilable Reader (PerkinElmer, 411177291). The above data was converted to percent inhibition using the following formula.

[0276] where "min" is the reading of the control well with no enzyme added; "max" is the reading of the control well with DMSO added as a control.

[0277] The data was imported into MS Excel and curve fitting was performed using XLFit excel add-in version 5.4.0.8, with the following formula:

[0278] where "Bottom" represents the minimum value of the curve; "Top" represents the maximum value of the curve; "IC 50 " represents the concentration at which the maximum effect is half of the maximum; and "HillSlope" represents the slope of the curve.

[0279] The results of the experiments are shown in Table 1.

[0280] Table 1. Determination of KIF18A enzyme inhibition activity (IC 50 , nM) of compounds

[0281] The results show that deuterated compounds 2, 4 and 6 have good inhibitory activity against KIF18A protein.

[0282] Determination of the inhibitory activity of compounds on tumor cell proliferation

[0283] Ovarian cancer cells OVCAR3 (ATCC, HTB-161), colorectal cancer cells HT-29 (ATCC, HTB-38) and triple-negative breast cancer cells HCC1806 (ATCC, CRL-2335) were cultured in a 37 °C, 5% CO2 incubator. The logarithmic growth phase cells were collected, the single cell suspension concentration was adjusted, and the 96-well plate was added to make the cell density 3000 cells per well. The blank control well was added with culture solution without cells. After overnight culture, the cells were treated with different concentrations of compounds, and DMSO (0.25%) was set as the solvent control. After 6 days of continuous culture, the number of living cells was determined by Promega CellTiter-Glo Luminescent Cell Viability Assay Kit (Promege-G7573). 75 μL of CellTiter-Glo working solution was added to each well, and the cells were lysed by shaking in the dark for 2 minutes. After 10 minutes of room temperature standing, the luminescent signal was detected on the EnVision multifunctional enzyme labeler (PerkinElmer). The determination data RLU was converted to the inhibition rate by the following formula:

[0284] Then the Graphpad Prism software was used to draw the inhibition curve and calculate the IC 50 .

[0285] The experimental results are shown in Table 2.

[0286] Table 2. Determination of the proliferation inhibition activity of compounds on OVCAR3, HT-29 and HCC1806 tumor cells (IC 50 , nM)

[0287] As shown in Table 2, compounds 2-7 have strong proliferation inhibition activity on ovarian cancer cells OVCAR3, colorectal cancer cells HT-29 and triple-negative breast cancer cells HCC1806.

[0288] Cytotoxicity determination of compounds on normal cells

[0289] Human embryonic kidney cells HEK293 cells (ATCC, CRL-157) were cultured in a 37 °C, 5% CO2 incubator. The cytotoxicity test method of the compound on HEK293 cells was consistent with the test method of the tumor cell proliferation inhibition activity, which is described above.

[0290] The experimental results are shown in Figure 1. The control compound AMG650 (MCE, HY-132840) showed certain cytotoxicity at a concentration of 10 μΜ and almost completely inhibited the growth of cells at a concentration of 30 μΜ, showing strong cytotoxicity. However, the growth inhibition activity of compounds 4 and 5 on HEK293 cells was only half that of AMG650, and the cytotoxicity was relatively low.

[0291] Test of inhibitory activity of compounds on CYP450 enzymes

[0292] After mixing liver microsomes (Biopredic International, final concentration 0.2 mg / mL) with different concentrations of compounds (final concentrations 0.01 μΜ, 0.04 μΜ, 0.12 μΜ, 0.37 μΜ, 1.11 μΜ, 3.33 μΜ and 10 μΜ), control compound AMG650, positive controls (CYP1A2: α-naphthoflavone; CYP2C9: sulfaphenazole; CYP2C19: omeprazole; CYP3A4: ketoconazole; CYP2D6: quinidine; all of the above compounds were purchased from Sigma-Aldrich) or negative control (1% DMSO), the respective CYP isozyme indicator substrates (CYP1A2: 30 μΜ phenacetin; CYP2C9: 10 μΜ diclofenac sodium; CYP2C19: 35 μΜ S-mephenytoin; CYP3A4: 5 μΜ midazolam and 80 μΜ testosterone; CYP2D6: 5 μΜ dextromethorphan; all of the above compounds were purchased from Sigma-Aldrich) were added, and incubation was carried out at 37°C for 10 min. Coenzyme NADPH (Roche, final concentration 1 mM) was added, and incubation was carried out at 37°C for a specified time (CYP 3A4: 5 min; CYP 1A2, CYP 2C9 and CYP 2D6: 10 min; CYP 2C19: 45 min). The reaction was terminated by adding acetonitrile containing internal standard working solution to each incubation tube, vortexing and centrifuging at 3220 g for 15 min. After vortexing, 50 μL of supernatant was added to an equal amount of ultrapure water, and the amount of metabolite produced was detected by LC-MS / MS method. The Analyst software (Analyst 1.6.3) of SCIEX was used to output raw spectra, peak area ratio and other data. The determination data were converted to inhibition rate by the following formula:

[0293] Inhibition rate % = (1 - amount of metabolite produced in the experimental or positive control group / amount of metabolite produced in the negative control group) x 100%

[0294] Then the Graphpad Prism software was used to draw the inhibition curve and calculate IC 50 .

[0295] The experimental results are shown in Table 3.

[0296] Table 3. Test of the inhibitory activity of compounds (IC 50 , μM)

[0297] The experimental results show that the control compound AMG650 has a certain inhibitory effect on CYP450 subtype CYP2C9, suggesting that the compound has a potential risk of drug-drug interaction; and the compounds 4 and 5 of the application have no inhibitory effect on CYP450 enzyme subtypes, including CYP2C9, and are higher in safety.

[0298] Test of the inhibitory activity of compounds on hERG potassium channel current

[0299] HEK293-hERG cells (human embryonic kidney cells stably expressing the hERG channel protein, Sophion Biosciences) were cultured in DMEM medium (Cytiva) in a 37°C cell culture incubator with 5% CO2. The cell culture medium was supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin-streptomycin mix (Solarbio). After digestion with 0.25% trypsin (Gibco) and addition of cell culture medium, a single cell suspension was obtained and added to recording chambers in an inverted microscope, and the temperature of the recording chamber was controlled at 20-25°C during the current recording process. The vehicle control (0.1% DMSO), compound 4 (final concentration 10 or 30 mM), comparative example compound 10 (final concentration 10 or 30 mM) and positive control (cisapride, MCE, final concentration 0.1 mM) were administered using an 8-channel perfusion system. When administering, the output end of the administration system was moved to the selected cells in the cell chamber using a micro-manipulator. When the three-way valve was opened, the drug flowing out of the output end could immediately infiltrate the cells below it. Voltage clamp parameter settings were performed using Clampex 10.6 software. In the whole-cell mode, the cells were clamped at a clamping potential of -80 mV for 100 ms, first hyperpolarized to -90 mV for 100 ms, then returned to -80 mV for 100 ms; then depolarized to +40 mV for 500 ms to activate the hERG channel; then a 100 ms ramp stimulus was applied to repolarize to -80 mV (1.2 V / s) to induce the characteristic tail current of the hERG channel; finally, -80 mV was maintained for 3000 ms. The stimulation frequency was 0.2 Hz (stimulation time was 5 s, start to start). Membrane currents were recorded using a patch-clamp amplifier and a digital-to-analog converter. The current signal was input through a 2 kHz filter and digitized at a frequency of 5 kHz using Clampex 10.6 software. The tail current peak was measured using Clampfit 10.6 software and used for data acquisition and analysis. The data obtained were statistically analyzed as the mean of the tail current peak of the last 10 recordings at each concentration. The mean of the tail current peak recorded by continuous perfusion of the vehicle control was taken as 100%, and the inhibition rate was used for IC 50 calculation, the formula is as follows:

[0300] Inhibition rate % = (1 - mean of tail current peak after administration / mean of tail current peak of vehicle control) x 100%

[0301] The experimental results are shown in Table 4. The non-deuterated comparative compound 10 has strong inhibitory effect on the hERG potassium channel current of HEK293 cells at 10 and 30 μM, and shows dose dependence, suggesting that the compound can have cardiotoxicity. The deuterated compound 4 of the present application has no significant inhibitory effect on the hERG potassium channel current at 10 μM and 30 μM. The above results suggest that the cardiotoxicity risk of the compound of the present application is lower than that of the non-deuterated comparative compound.

[0302] Table 4. Inhibition rate (%) of compounds on hERG potassium channel current (average value ± standard deviation)

[0303] Solubility test of compounds

[0304] The compound 4, 5 and the control compound AMG650 were prepared into 10 mM stock solution with DMSO, 8 μL of the stock solution was added into 792 μL of phosphate buffer (100 mM, pH 7.4), and after shaking at room temperature for 1 hr, centrifugation was performed at 12000 rpm for 10 min. The supernatant was transferred to a new tube, and diluted 10 times and 100 times with 100 mM phosphate buffer respectively, 5 μL of the undiluted or diluted sample was added into acetonitrile containing internal standard working solution, and the compound concentration was detected by LC-MS / MS method.

[0305] The experimental results are shown in Table 5.

[0306] Table 5. Solubility of compounds (μM)

[0307] As shown in Table 5, the solubility of compound 4 is much higher than that of the control compound AMG650 (more than 25 times), and the phosphate ester compound 5 further improves the solubility (more than 875 times of AMG650). Due to the improvement of safety and solubility, the compounds 4 and 5 can obtain higher drug exposure at higher dose in clinic, thereby improving the clinical efficacy of the drug.

[0308] Pharmacokinetic test of compounds

[0309] CD-1 mice (Shanghai Jihui Experimental Animal Breeding Co., Ltd.) were used for each test compound, male, 6-8 weeks old, weighing 28-31 g, and randomly divided into two groups (group A and group B), 9 in each group, fasting for 12 hours. The animals in group A were given test compound solution by gavage at a dose of 10 mg / kg; the animals in group B were given test compound solution by tail vein injection at a dose of 3 mg / kg. Before administration, the blank blood was taken, and the animals in group A were taken about 110 μL of venous blood at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after administration, and the animals in group B were taken about 110 μL of venous blood at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after administration, which were placed in test tubes added with EDTA-K2 anticoagulant (GREAGENT, G41456A). After centrifugation, the plasma was taken and stored at -70°C for testing.

[0310] The whole blood concentration data were analyzed using the metabolic kinetics data analysis software WinNonlin (version 8.0.0.3176). The non-compartment model method (NCA) was used to calculate T 1 / 2 , C max , T max , AUC, CL and F% and the like. Among them, AUC last represents the area under the concentration-time curve in the time period from the administration time to the last sampling point, i.e. the area under the concentration-time curve; CL(iv) represents the drug clearance rate of intravenous administration; T 1 / 2 represents the plasma half-life of the drug, i.e. the time required for the plasma drug concentration to decrease by half; C max represents the maximum drug concentration reached in the plasma after administration; T max represents the time required for the drug to reach the maximum drug concentration in the plasma; F% represents the oral bioavailability of the drug.

[0311] The experimental results are shown in Table 6. Compound 4 showed excellent pharmacokinetic properties in mice: after tail vein injection of 3 mg / kg, the plasma exposure of compound 4 was high (AUC last was 6737 ng / mL*hr), the clearance rate was low (CL(iv) was 0.433 L / hr / kg), and thus the plasma half-life was long (T 1 / 2 was 1.57 hr); the in vivo exposure could reach 21156 ng / mL*hr, the maximum plasma concentration was 4517 ng / mL, and the bioavailability was high.

[0312] Table 6. Pharmacokinetic test of compound 4

[0313] Since the phosphate compound 5 can be metabolized to compound 4 in vivo, the pharmacokinetic parameters of both compound 5 and compound 4 were determined in the pharmacokinetic test of compound 5. The experimental results are shown in Table 7, and compound 5 can be rapidly and completely converted to compound 4 in vivo.

[0314] Table 7. Pharmacokinetic test of compound 5

[0315] Hepatic microsomal stability test of deuterated compound compared with non-deuterated comparative compounds 9 and 10

[0316] Compound 4, comparative compound 9, comparative compound 10 (final concentration 1 μM) or positive control ketanserin (MCE, final concentration 1 μM) were pre-incubated with various species of liver microsomes (Biopredic International, final concentration 0.5 mg / mL) at 37 °C for 10 min, respectively. Then, NADPH solution (Roche, final concentration 1 mM) preheated at 37 °C for 5 min was added to each tube, mixed gently, and placed in a 37 °C constant temperature shaking water bath (50 rpm) to start timing. After incubation for 0, 5, 15, 30 and 45 min, the reaction was terminated by adding 400 μL of acetonitrile containing internal standard working solution (Sigma-Aldrich). After vortex mixing, the supernatant was centrifuged at 3200 g for 10 min at 4 °C. 50 μL of the supernatant was mixed with 100 μL of ultrapure water, vortexed, and the residual amount of the parent compound in the test sample was detected by LC-MS / MS. The raw spectrum and peak area ratio data were output using the Analyst software (Analyst 1.6.3) of SCIEX, and the half-life (T 1 / 2 ) was calculated using Microsoft Office Excel.

[0317] The experimental results are shown in Table 8. Compared with non-deuterated comparative compounds 9 and 10, the stability of deuterated compound 4 in rodent (rat, mouse) and non-rodent (monkey, human) liver microsomes was improved to varying degrees, indicating its good pharmacokinetic properties.

[0318] Table 8. Hepatic microsomal stability test (T 1 / 2 , min) of compounds

[0319] Industrial applicability

[0320] The present application provides a KIF18A inhibitor compound, which can be used to selectively inhibit KIF18A activity, or treat or prevent diseases, disorders or conditions modulated by or affected by KIF18A activity or in which KIF18A activity or overexpression is involved. Thus, it can be made into a corresponding medicine, suitable for industrial application.

[0321] While the application has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes can be made therein without departing from the spirit and scope of the application. Accordingly, all such modifications are intended to be included within the scope of the application.

Claims

1. An inhibitor which is a compound of formula (I) or a pharmaceutically acceptable salt, solvate, ester, acid, metabolite or prodrug thereof, ###00001### (I) wherein, X1and X2are each independently selected from N and CH; R 1 selected from hydrogen, -P(=0)(OH)2, and -S(=0)2(OH); R 2a , R 2b , R 3a , and R 3b are each independently selected from hydrogen or deuterium, provided that at least one of R 2a , R 2b , R 3a , and R 3b is deuterium; m is an integer selected from 1, 2, or 3; n is an integer selected from 1, 2, or 3; q is an integer selected from 0, 1, or 2, and when q is other than 0, R 4 each is independently selected from halogen.

2. The inhibitor of claim 1, wherein m and n are each independently 1.

3. The inhibitor of claim 1 or 2, wherein q is 2, and / or R 4 each independently is fluoro.

4. The inhibitor of any one of claims 1-3, wherein m is 1, q is 2, and R 4 The substituent position on the nitrogen heterocycle is the para position to the nitrogen atom.

5. The inhibitor of any one of claims 1-4, wherein X1is CH, and / or X2is CH.

6. The inhibitor of any one of claims 1-5, wherein at least two of R 2a , R 2b , R 3a , and R 3b are deuterium.

7. The inhibitor of claim 6, wherein R 2a and R 2b are deuterium.

8. The inhibitor of claim 6, wherein R 2a , R 2b , R 3a , and R 3b are each deuterium.

9. The inhibitor of any one of claims 1-8, wherein R 1 is hydrogen.

10. The inhibitor of claim 1, wherein it is a compound selected from the group consisting of formula (I) or a pharmaceutically acceptable salt, solvate, ester, acid, metabolite or prodrug thereof:

11. A pharmaceutical composition comprising the inhibitor of any one of claims 1-10, and a pharmaceutically acceptable carrier or excipient, and optionally other therapeutic agents.

12. The inhibitor of any one of claims 1-10 for use in the treatment or prevention of a disease, disorder, or condition modulated by or affected by KIF18A activity, or in which KIF18A activity or overexpression is implicated.

13. The inhibitor for use of claim 12, wherein the disease, disorder, or condition is a cancer.

14. The inhibitor for use of claim 12 or 13, wherein the disease, disorder, or condition is selected from one or more of the following cancers with chromosomal instability: lung squamous carcinoma, lung adenocarcinoma, non-small cell lung cancer, small cell lung cancer, head and neck squamous carcinoma, breast cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, ovarian cancer, esophageal squamous carcinoma, gastric cancer, liver cancer, oral cancer, urothelial carcinoma, prostate cancer, bladder cancer, renal cell carcinoma, gastrointestinal stromal tumor, cervical cancer, endometrial cancer, rhabdomyosarcoma, fibrosarcoma, neuroendocrine tumor, mesothelioma, brain cancer, and malignant glioma.

Images