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WO2025241650A1 - Kif18a抑制剂及其用途 - Google Patents

Kif18a抑制剂及其用途

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Publication number
WO2025241650A1
WO2025241650A1 PCT/CN2025/080391 CN2025080391W WO2025241650A1 WO 2025241650 A1 WO2025241650 A1 WO 2025241650A1 CN 2025080391 W CN2025080391 W CN 2025080391W WO 2025241650 A1 WO2025241650 A1 WO 2025241650A1
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WO
WIPO (PCT)
Prior art keywords
cancer
inhibitor
acid
mmol
compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/080391
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English (en)
French (fr)
Inventor
李兵
张科
宁文星
左小飞
黄琼琳
陈勤
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novostar Pharmaceuticals Ltd
Original Assignee
Novostar Pharmaceuticals Ltd
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Publication of WO2025241650A1 publication Critical patent/WO2025241650A1/zh
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • This invention relates to the pharmaceutical field, and in particular to deuterated small molecule compounds composed of benzamide structural units and fused heterobicyclic structural units, as well as methods and uses thereof for treating and/or preventing diseases.
  • Chromosomal instability refers to ongoing genomic alterations, including amplification or deletion of chromosome copy number or structure, ranging from point mutations to small-scale genomic changes and even changes in the number of entire chromosomes.
  • CIN is caused by persistent errors in chromosome segregation during mitosis and is a key characteristic of cancer cells, potentially playing a crucial role in tumorigenesis.
  • CIN is prevalent in various cancer types, particularly occurring with extremely high frequency in high-grade serous ovarian cancer (HGSOC), triple-negative breast cancer (TNBC), and colorectal cancer (CRC), and is closely associated with tumor metastasis, immune escape, and treatment resistance.
  • KIF18A a member of the kinin-8 family, is an enzyme that integrates microtubule motility and depolymerization activity. It reversibly binds to microtubules, influencing anchorid microtubule dynamics to control proper chromosome positioning and spindle tension, playing a key role in cell division. KIF18A is expressed at low levels in normal human tissues but is significantly overexpressed and functionally abnormal in various cancers, and KIF18A overexpression is associated with tumor grade, metastasis, and poor survival.
  • KIF18A could serve as a potential prognostic biomarker and a novel target for cancer therapy.
  • KIF18A inhibitors can selectively kill chromosomally unstable cancer cells, showing great promise as a potential tumor treatment strategy.
  • relatively few KIF18A inhibitors have been reported so far.
  • Patents such as US2022/0372018A1, US2022/0106293A1, CN115594664A, CN115772159A, CN115785068A, and WO2023/028564A1 disclose several aromatic heterocyclic compounds that can inhibit the enzymatic activity of KIF18A.
  • the present invention aims to provide a deuterated compound that can selectively inhibit KIF18A activity, which, compared with the corresponding non-deuterated small molecule compound, can provide comparable KIF18A inhibitory activity while offering additional advantages in terms of improved drug safety, solubility and liver microsomal stability, and improved pharmacokinetics.
  • a KIF18A inhibitor is provided, which is a compound of formula (I) or a pharmaceutically acceptable salt, solvate, ester, acid, metabolite or prodrug thereof.
  • X1 and X2 are each independently selected from N and CH;
  • R2a , R2b , R3a and R3b are each independently selected from hydrogen or deuterium, provided that at least one of R2a , R2b , R3a and R3b is deuterium;
  • n 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 not 0, R 4 is selected independently from halogens.
  • n and n are each independently 1.
  • q is 2.
  • R4 is fluorine, each of which is independent.
  • m is 1, q is 2, and R4 is substituted at the para position of the nitrogen atom on the nitrogen heterocycle.
  • X1 is CH.
  • X2 is CH.
  • both X1 and X2 are CH.
  • R2a , R2b , R3a , and R3b are deuterium. More preferably, R2a and R2b are deuterium. Even more preferably, R2a , R2b , R3a , and R3b are all deuterium.
  • R1 is hydrogen
  • the KIF18A inhibitor according to the present invention is preferably a compound selected from the following structures, or a pharmaceutically acceptable salt, solvate, ester, acid, metabolite or prodrug thereof:
  • a pharmaceutical composition comprising the compound of the invention, or a pharmaceutically acceptable salt, solvate, ester, acid, metabolite or prodrug thereof, and a pharmaceutically acceptable carrier or excipient, and optionally other therapeutic agents.
  • aspects of the invention relate to methods or uses of the compounds of the invention, or pharmaceutically acceptable salts, solvates, esters, acids, metabolites or prodrugs thereof, to selectively inhibit KIF18A activity, or the use of the compounds of the invention, or pharmaceutically acceptable salts, solvates, esters, acids, metabolites or prodrugs thereof, in the preparation of medicaments for selectively inhibiting KIF18A activity.
  • Further aspects of the invention relate to methods or uses of the compound of the invention, or a pharmaceutically acceptable salt, solvation, ester, acid, metabolite, or prodrug thereof, for treating or preventing diseases, disorders, or conditions regulated by or affected by KIF18A activity, or wherein KIF18A activity is involved; or the use of the compound of the invention, or a pharmaceutically acceptable salt, solvation, ester, acid, metabolite, or prodrug thereof, in the preparation of medicaments for treating or preventing diseases, disorders, or conditions regulated by or affected by KIF18A activity, or wherein KIF18A activity or overexpression is involved.
  • the disease, disorder, or condition regulated by or affected by KIF18A activity, or involving KIF18A activity or overexpression is cancer.
  • the disease, disorder, or symptom is selected from one or more of the following cancers with chromosomal instability: squamous cell carcinoma of the lung, adenocarcinoma of the lung, non-small cell lung cancer, small cell lung cancer, head and neck squamous cell carcinoma, breast cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, ovarian cancer, esophageal squamous cell 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.
  • cancers with chromosomal instability squamous cell carcinoma of the lung, adenocarcinoma of the lung, non-small cell lung
  • Figure 1 shows the inhibition curves of the proliferation of human embryonic kidney cells HEK293 by the compounds of the present invention and the control compounds.
  • this invention employs conventional methods within the scope of the art, such as mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology.
  • mass spectrometry NMR, HPLC, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology.
  • nomenclature and laboratory procedures and techniques related to analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein are known to those skilled in the art.
  • the foregoing techniques and steps can be practiced by conventional methods well-known in the art and described in various general and more specific documents, which are cited and discussed herein.
  • pharmaceutically acceptable salt in this article refers to a salt that retains the desired biological activity of the subject compound while exhibiting minimal undesirable toxicological effects.
  • pharmaceutically acceptable salts can be prepared in situ during the final isolation and purification of the compound, or by reacting the purified compound in its free acid or free base form with a suitable base or acid, respectively.
  • Solvate or “solvent compound” refers to a solvation compound containing a stoichiometric or non-stoichiometric solvent. Some compounds tend to trap solvent molecules in a fixed molar ratio in a crystalline solid state, thus forming a solvate compound. If the solvent is water, the formed solvate compound is a hydrate; if the solvent is an alcohol, the formed solvate compound is an alcohol. Hydrates are formed by the combination of one or more water molecules with a molecule of the substance, wherein the water retains its molecular state as H2O .
  • the “metabolites” of the compounds disclosed herein are derivatives of the compounds formed when the compounds are metabolized.
  • active metabolite refers to a biologically active derivative of the compound formed when the compound is metabolized.
  • the term “metabolized” refers to the sum of processes by which a particular substance is altered by an organism (including, but not limited to, hydrolysis and enzyme-catalyzed reactions, such as oxidation). Thus, enzymes can produce specific structures that are transformed into compounds.
  • cytochrome P450 catalyzes various oxidation and reduction reactions
  • glucosyl diphosphate transferases catalyze the conversion of activated glucuronic acid molecules to aromatic alcohols, aliphatic alcohols, carboxylic acids, amines, and free sulfhydryl groups.
  • Metabolites of the compounds disclosed herein can be identified by administering the compound to a host and analyzing tissue samples from that host, or by incubating the compound with hepatocytes in vitro and analyzing the resulting compound. Both methods are known in the art.
  • the metabolites of the compound are formed through an oxidation process and correspond to the corresponding hydroxyl-containing compounds.
  • the compound is metabolized into a drug-active metabolite.
  • modulation refers to direct or indirect interaction with a target to alter its activity, including, for example, enhancing, inhibiting, limiting, or prolonging the activity of a target.
  • prodrug or “prodrug precursor” refers to derivatives that may not be pharmacologically active, but in some cases can be administered orally or parenterally and subsequently metabolized in vivo to form the pharmacologically active compounds of the present invention.
  • prodrugs include esters, carbonates, hemiesters, phosphate esters, nitro esters, sulfate esters, sulfoxides, amides, carbamates, nitrogen-containing compounds, phosphoramides, glycosides, ethers, acetals, and ketoacetates, etc.
  • Effective amount refers to the amount of a drug or pharmaceutical preparation that will elicit a biological or medical response in an investigational tissue, system, animal, or human, such as that of an investigator or physician.
  • therapeutic effective amount refers to any amount that, compared to a corresponding subject who has not received that amount, results in a treatment, cure, prevention, or relief of disease, disorder, or side effects, or a reduction in the rate of disease or disorder progression. The term also includes amounts that effectively improve normal physiological function.
  • treatment refers to the relief of at least one symptom of a disease, disorder, or condition. This term includes administering medication to a subject and/or applying one or more of the compounds described herein to provide management or treatment of the condition.
  • treatment may, but does not necessarily, provide a cure; rather, it means that “treatment” can be a form of management of the condition.
  • treatment includes the partial or complete destruction of said harmful proliferating cells, but with minimal impact on normal cells.
  • the desired treatment mechanism for harmful, rapidly proliferating cells (including cancer cells) is apoptosis at the cellular level.
  • prevention includes the initiation of joint prevention or mitigation of the development of a clinically significant disease or the initiation of a preclinically significant disease stage in an individual at risk. This includes preventative treatment of individuals at risk of disease development.
  • subject or “patient” include organisms that may suffer from a condition or a condition associated with reduced or insufficient programmed cell death (apoptosis) or that may otherwise benefit from administration of the compounds of the present invention, such as humans and non-human animals.
  • Preferred humans include human patients who suffer from or are predisposed to suffer from the condition or related condition as described herein.
  • non-human animal includes vertebrates, such as mammals, such as non-human primates, sheep, cattle, dogs, cats, and rodents such as mice, as well as non-mammals such as chickens, amphibians, reptiles, etc.
  • the GI 50 used in this article refers to the drug concentration required to inhibit the growth of 50% of cells, that is, the drug concentration at which the growth of 50% of cells (such as cancer cells) is inhibited or controlled.
  • the IC 50 used in this article refers to the amount, concentration, or dose of a specific test compound that achieves 50% inhibition of the maximum effect in the analysis of the measured effect.
  • the EC 50 used in this article refers to the dose-dependent response of a assay compound, which is the maximum expression of 50% of the dose, concentration, or amount of the assay compound that induces, stimulates, or enhances a specific response.
  • This 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.
  • X1 and X2 are each independently selected from N and CH;
  • R2a , R2b , R3a and R3b are each independently selected from hydrogen or deuterium, provided that at least one of R2a , R2b , R3a and R3b is deuterium;
  • n 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 not 0, R 4 is selected independently from halogens.
  • n and n are each independently 1.
  • q is 2.
  • R4 is fluorine, each of which is independent.
  • m is 1, q is 2, and R4 is substituted at the para position of the nitrogen atom on the nitrogen heterocycle.
  • X1 is CH.
  • X2 is CH.
  • both X1 and X2 are CH.
  • R2a , R2b , R3a , and R3b are deuterium. More preferably, R2a and R2b are deuterium. Even more preferably, R2a , R2b , R3a , and R3b are all deuterium.
  • R1 is hydrogen
  • the present invention relates to the compounds shown in the table below, or pharmaceutically acceptable salts, solvates, esters, acids, metabolites or prodrugs thereof.
  • This article describes a novel KIF18A inhibitor.
  • Pharmaceutically acceptable salts, solvates, esters, acids, metabolites, and prodrugs of this compound are also described.
  • the compounds of the present invention can exist in a free form, such as a free base or free acid or zwitterion, or in the form of a salt.
  • the salt can be any salt, an organic or inorganic addition salt, particularly any physiologically acceptable organic or inorganic addition salt commonly used in pharmaceutical applications.
  • the preferred salts are those that are physiologically acceptable to the compounds of this invention.
  • this also includes salts that are not suitable for pharmaceutical applications but can be used, for example, to isolate or purify the compounds of this invention.
  • pharmaceutical acceptable salt refers to a relatively non-toxic inorganic or organic acid addition salt of the compounds of the present invention, see, for example, S.M. Berge et al., "Pharmaceutical Salts, J. Pharm. Sci. 1977, 66, 1-19".
  • Pharmaceutically acceptable salts of the compounds of this invention include acid addition salts of inorganic acids, carboxylic acids, and sulfonic acids, such as salts of the following acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid, nitric acid; or salts of organic acids, such as 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-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pyruvic acid, and pectin.
  • ester acids persulfate, 3-phenylpropionic acid, picric acid, tert-valeric acid, 2-hydroxyethanesulfonic acid, itaconic acid, aminosulfonic acid, trifluoromethanesulfonic acid, dodecyl sulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic 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, glucoheponic acid, glycerophosphate, aspartic acid, sul
  • Pharmaceutically acceptable salts of the compounds of the present invention also include salts of commonly used bases, such as and preferably alkali metal salts (e.g., sodium and potassium salts), alkaline earth metal salts (e.g., calcium and magnesium salts), and ammonium salts derived from ammonia or organic amines having 1 to 16 carbon atoms, said organic amines being, for example and preferably, ethylamine, diethylamine, triethylamine, ethyl diisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-methylmorpholine, arginine, lysine, ethylenediamine, N-methylpiperidine, N-methylglucosamine, dimethylglucosamine, ethylglucosamine, 1,6-hexanediamine, glucosamine, sarcosine, serine, tri
  • This invention includes all possible salts of the compounds of this invention, which may be a single salt or any mixture of said salts in any proportion.
  • solvent is used for the purposes of this invention to refer to those forms of the compounds of this invention that form complexes with solvent molecules through coordination in the solid or liquid state.
  • a hydrate is a specific form of solvate in which coordination with water occurs. Hydrates are preferably solvates within the scope of this invention.
  • the present invention also includes prodrugs of the compounds of the present invention.
  • prodrug includes compounds that may be biologically active or inert but are converted into (e.g., through metabolism or hydrolysis) the compounds of the present invention during their retention time in the body.
  • the present invention includes all possible crystalline forms or polymorphs of the compounds of the present invention, either as a single polymorph or as a mixture of more than one polymorph in any proportion.
  • the structural formula of the compound represents a specific isomer, but the invention includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbon atoms, stereoisomers, tautomers, etc.
  • the chiral compounds involved in this invention can have arbitrary configurations or mixtures of racemates.
  • the compounds used according to this invention contain more than one chiral center, they can exist in diastereomeric forms.
  • the diastereomeric isomers can be separated by methods known to those skilled in the art (e.g., chromatography or crystallization), while individual enantiomers can be separated as described above.
  • This invention includes the application of various diastereomeric compounds and mixtures thereof used according to this invention.
  • the compounds used in this invention can exist in different tautomeric forms or in different geometrical isomer forms, and this invention includes the application of various tautomeric and/or geometrical isomers of the compounds used according to this invention and mixtures thereof.
  • the compounds used in this invention can exist in zwitterionic forms. This invention includes the application of various zwitterionic forms of the compounds used according to this invention and mixtures thereof.
  • Screening and characterizing 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, and elemental analysis.
  • Various spectroscopic techniques used include, but are not limited to, Raman, FTIR, UVIS, and NMR (liquid and solid states).
  • Various microscopy techniques include, but are not limited to, IR microscopy and Raman microscopy.
  • the compounds of formula (I) of the present invention are capable of selectively inhibiting the enzymatic activity of KIF18A, and are therefore capable of being used to treat or prevent diseases, disorders or conditions that are regulated by or affected by KIF18A activity, or in which KIF18A activity or overexpression is involved.
  • diseases, disorders, or conditions regulated by or affected by KIF18A activity, or involving KIF18A activity or overexpression are cancers, particularly cancers with chromosomal instability, including but not limited to one or more of the following: squamous cell carcinoma of the lung, adenocarcinoma of the lung, non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma of the head and neck, breast cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, ovarian cancer, squamous cell carcinoma of the esophagus, 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, or malignant glioma.
  • cancers particularly cancer
  • the compounds of the present invention can act systemically and/or locally.
  • they can be administered in suitable manner, such as via oral, parenteral, pulmonary, nasal, sublingual, lingual, sublingual, rectal, dermal, transdermal, conjunctival, or ocular routes, or in the form of implants or stents.
  • the drug comprising the compound of the present invention can be administered to a patient by at least one of injection, oral administration, inhalation, rectal administration, and transdermal administration.
  • the KIF18A inhibitors and/or pharmaceutical compositions of the present invention are formulated into pharmaceutically acceptable dosage forms using conventional methods known to those skilled in the art.
  • the amount of a given drug depends on many factors, such as the specific dosing regimen, the type and severity of the disease or condition, and the unique characteristics of the patient or host requiring treatment (e.g., weight). However, depending on the specific surrounding circumstances, including, for example, the specific drug used, the route of administration, the condition being treated, and the patient or host being treated, the dosage can be conventionally determined by methods known in the art. Typically, for adult treatment, the dosage is typically in the range of 0.02-5000 mg/day, for example, about 1-1500 mg/day.
  • This required dosage can be conveniently expressed as a single dose, or concurrent (or over a short period of time) or fractions at appropriate intervals, such as two, three, four, or more doses per day.
  • the specific effective amount can be appropriately adjusted according to the patient's condition and in conjunction with the physician's diagnosis.
  • the actual dosage level and time course of the compounds of the present invention can be varied to obtain an amount of active ingredient that effectively achieves the therapeutic response desired by a particular patient and is non-toxic to the patient.
  • compositions comprising a compound of formula (I) of the invention, 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.
  • the compounds of the present invention can be administered in the form of a single pharmaceutical agent or in combination with one or more other therapeutic agents, wherein said combination does not cause unacceptable side effects.
  • the pharmaceutical composition includes administration of a single pharmaceutical dose formulation comprising the compound of the present invention and one or more other therapeutic agents, as well as administration of the compound of the present invention and various other therapeutic agents in their own separate pharmaceutical dose formulations.
  • the compound of formula (I) may be administered to a patient together with other therapeutic agents in the form of a single oral dose composition, such as a tablet or capsule, or each agent may be administered in a separate dose formulation.
  • the compounds of the present invention and one or more other therapeutic agents may be administered at substantially the same time (e.g., simultaneously) or at separately staggered times (e.g., sequentially).
  • the compounds of the present invention can be used in fixed combinations or alone with the following substances: other antitumor agents, such as alkylating agents, antimetabolites, plant-derived antitumor agents, hormone therapy agents, topoisomerase inhibitors, camptothecin derivatives, kinase inhibitors, targeted drugs, antibodies, interferons and/or bioresponse modifiers, antiangiogenic compounds and other antitumor drugs.
  • other antitumor agents such as alkylating agents, antimetabolites, plant-derived antitumor agents, hormone therapy agents, topoisomerase inhibitors, camptothecin derivatives, kinase inhibitors, targeted drugs, antibodies, interferons and/or bioresponse modifiers, antiangiogenic compounds and other antitumor drugs.
  • the compounds of the present invention can also be used in conjunction with radiotherapy and/or surgical intervention for cancer treatment.
  • the compounds of the present invention can be synthesized using standard synthetic techniques known to those skilled in the art, or by combining methods known in the art with those described herein. Furthermore, the solvents, temperatures, and other reaction conditions given herein can be varied according to the art. As further guidance, the following synthetic methods can also be utilized.
  • the reactions may be used sequentially to provide the compounds described herein; or they may be used to synthesize fragments subsequently added by the methods described herein and/or methods known in the art.
  • the starting materials used to synthesize the compounds described herein can be synthesized or are available from commercial sources.
  • the compounds described herein and other related compounds with different substituents can be synthesized using techniques and starting materials known to those skilled in the art.
  • General methods for preparing the compounds disclosed herein can be derived from reactions known in the art, and these reactions can be modified by reagents and conditions deemed appropriate by those skilled in the art to introduce various moieties provided herein.
  • reaction products can be separated and purified using conventional techniques, including but not limited to filtration, distillation, crystallization, and chromatography. These products can be characterized using conventional methods, including physical constants and spectral data.
  • the structure of the compound was determined by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS).
  • the solvents used for NMR determination were deuterated dimethyl sulfoxide (DMSO- d6 ), deuterated chloroform ( CDCl3 ), or deuterated methanol ( CD3OD ).
  • solution refers to an aqueous solution.
  • reaction temperature is room temperature, for example, 20°C to 30°C.
  • ACN Acetonitrile
  • DBU 1,8-diazabicyclo[5.4.0]undec-7-ene
  • DIPEA N,N-diisopropylethylamine
  • HATU 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate
  • LiHMDS Lithium bis(trimethylsilylamine);
  • mCPBA m-chloroperoxybenzoic acid
  • NMP N-methylpyrrolidone
  • Ruphos 2-Dicyclohexylphosphine-2',6'-diisopropoxy-1,1'-biphenyl;
  • Ruphos Pd G2 Chloro(2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II);
  • TEA Triethylamine
  • THF Tetrahydrofuran
  • reaction mixture was quenched with water (1.2 L) and extracted with ethyl acetate (4 x 550 mL). The combined organic phases were washed with saturated ammonium chloride aqueous solution (2 x 600 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated to approximately 250 mL in volume. A solid product precipitated out, and after filtration, 2-fluoro-4-nitrobenzamide (21.00 g, yellow solid) was obtained, with a yield of 87.7%.
  • Step B Preparation of 4-nitro-2-(6-azaspiro[2.5]oct-6-yl)benzamide (intermediate A)
  • 2-Fluoro-4-nitrobenzamide (18.41 g, 100.00 mmol, 1.0 eq.) was dissolved in N-methylpyrrolidone (200 mL), followed by the addition of N,N-diisopropylethylamine (38.78 g, 300.00 mmol, 3.0 eq.) and 6-aza-spiro[2.5]octane hydrochloride (16.24 g, 110.00 mmol, 1.1 eq.). The reaction mixture was reacted at 135 °C for 20 h. The reaction mixture was cooled to room temperature and diluted with water (1.2 L) and ethyl acetate (800 mL). A solid precipitated from the reaction mixture.
  • 5-Bromopyrazolo[1,5-a]pyridine (10.00 g, 50.75 mmol, 1.0 eq.) was dissolved in tetrahydrofuran (100 mL). Under nitrogen protection, the solution was cooled to -78 °C in a dry ice/ethanol bath, and bis(trimethylsilylaminolithium) (1 M, 55.8 mL, 55.8 mmol, 1.1 eq.) was added dropwise. The reaction solution was incubated at -78 °C for 0.5 h.
  • Step B Preparation of 5-bromo-7-(3,3-difluoroazacyclobutane-1-yl)pyrazolo[1,5-a]pyridine
  • Step C Preparation of N-(7-(3,3-difluoroazacyclobutane-1-yl)pyrazolo[1,5-a]pyridin-5-yl)-4-nitro-2-(6-azaspiro [2.5]octane-6-yl)benzamide
  • reaction mixture was incubated at 120 °C for 6 h under nitrogen protection.
  • the reaction mixture was cooled to room temperature and diluted with water (40 mL), then extracted with dichloromethane (2 x 80 mL). The combined organic phases were dried over anhydrous sodium sulfate and concentrated.
  • Step D Preparation of 4-amino-N-(7-(3,3-difluoroazacyclobutane-1-yl)pyrazolo[1,5-a]pyridin-5-yl)-2-(6- azaspiro[2,5]octane-6-yl)benzamide
  • N-(7-(3,3-difluoroazacyclobutan-1-yl)pyrazolo[1,5-a]pyridin-5-yl)-4-nitro-2-(6-azaspiro[2.5]octane-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 solution was reacted at 30 °C for 1 h. The reaction solution was diluted with dichloromethane (150 mL) and filtered. The filtrate was neutralized with acetic acid by adding saturated sodium carbonate.
  • Step E Preparation of 2-(N-(4-((7-(3,3-difluoroazacyclobutane-1-yl)pyrazolo[1,5-a]pyridin-5-yl) carbamoyl )-3-(6-azaspiro[2,5]octane-6-yl)phenyl)aminosulfonyl)ethyl acetate (intermediate B)
  • 4,6-Dichloro-dimethylthiopyrimidine (80.00 g, 410.13 mmol, 1.0 eq.) was dissolved in tetrahydrofuran (400 mL), cooled to 0 °C in an 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 slowly added. The reaction mixture was reacted at 25 °C for 3 h. The reaction mixture was concentrated to 100 mL, water (180 mL) was added, and the mixture was stirred at 25 °C for 15 min.
  • Step B Preparation of 7-chloro-5-(methylthio)-[1,2,4]triazolo[4,3-c]pyrimidine
  • Step C Preparation of 7-chloro-5-hydroxy-[1,2,4]triazolo[1,5-c]pyrimidine
  • Step D Preparation of 5,7-dichloro-[1,2,4]triazolo[1,5-c]pyrimidine
  • Step E Preparation of 7-chloro-5-(3,3-difluoroazacyclobutane-1-yl)-[1,2,4]triazolo[1,5-c]pyrimidine
  • Step F Preparation of N-(5-(3,3-difluoroazacyclobutane-1-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-4-nitro -2-(6-azaspiro[2.5]oct-6-yl)benzamide
  • reaction mixture was reacted at 120 °C for 4 h under nitrogen protection.
  • the reaction mixture was cooled to room temperature and diluted with water (15 mL), and extracted with ethyl acetate (2 x 30 mL). The combined organic phases were dried over anhydrous sodium sulfate and concentrated.
  • Step G Preparation of 4-amino-N-(5-(3,3-difluoroazacyclobutane-1-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7- yl)-2-(6-azaspiro[2.5]oct-6-yl)benzamide
  • N-(5-(3,3-difluoroazacyclobutane-1-yl)-[1,2,4]triazolo[1,5-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 mixture was reacted at 25 °C for 2 h. The reaction mixture was filtered, and the filtrate was concentrated. The concentrate was diluted with water (30 mL), and residual acetic acid was neutralized with saturated sodium carbonate.
  • Step H Preparation of 2-(N-(4-((5-(3,3-difluoroazacyclobutane-1-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)carbamoyl )-3-(6-azaspiro[2.5]oct-6-yl)phenyl)aminosulfonyl)ethyl acetate (intermediate C)
  • Step A Preparation of 4-(2,2-dideuterium-2-hydroxyethylsulfonamido)-N-(7-(3,3-difluoroazacyclobutane-1-yl)pyrazolo [1,5-a]pyridin-5-yl)-2-(6-azaspiro[2.5]oct-6-yl)benzamide
  • Lithium aluminum deuterated hydride (19 mg, 0.45 mmol, 3.0 eq.) was added to tetrahydrofuran (3 mL), and the mixture was cooled to 0 °C under nitrogen protection.
  • reaction mixture was reacted at 0 °C for 0.5 h.
  • the reaction mixture was quenched with dilute hydrochloric acid (1 M, 3 mL) and diluted with water (20 mL), then extracted with ethyl acetate (2 x 50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated.
  • Step A Preparation of dibenzyl(1,1-dideuterium-2-(N-(4-((7-(3,3-difluoroazacyclobutane-1-yl)pyrazolo[1,5-a]pyridin- 5-yl)carbamoyl)-3-(6-azaspiro[2,5]octane-6-yl)phenyl)aminosulfonyl)ethyl)phosphate
  • the mixture was cooled to 0 °C in an ice bath, and tetrazolium (15 mg, 0.21 mmol, 1.3 eq.) and dibenzyl N,N-diisopropylphosphonamide (66 mg, 0.19 mmol, 1.2 eq.) were added sequentially.
  • the reaction mixture was incubated at 0 °C for 70 min.
  • m-chloroperoxybenzoic acid 33 mg, 0.19 mmol, 1.2 eq. was added to the reaction mixture.
  • the reaction mixture was incubated at 0 °C for another 10 min.
  • reaction mixture was quenched with saturated sodium bicarbonate aqueous solution (50 mL) and extracted with ethyl acetate (2 x 45 mL). The organic phases were combined, washed with saturated ammonium chloride aqueous solution (80 mL), dried over anhydrous sodium sulfate, and concentrated.
  • Step B Preparation of trifluoroacetate of 1,1-dideuterium-2-(N-(4-((7-(3,3-difluoroazacyclobutane-1-yl)pyrazolo[1,5-a]pyridin-5-yl) carbamoyl )-3-(6-azaspiro[2.5]oct-6-yl)phenyl)aminosulfonyl)ethyl phosphate dihydrogen ester
  • Step A Preparation of N-(7-(3,3-difluoroazacyclobutane-1-yl)pyrazolo[1,5-a]pyridin-5-yl)-2-(6-azaspiro[2.5] oct-6-yl)-4-(1,1,2,2-tetradeuter-2-hydroxyethylsulfonamide)benzamide
  • reaction mixture was reacted at 30 °C for 48 h.
  • the reaction mixture 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.
  • Step A Preparation of dibenzyl(2-(N-(4-((7-(3,3-difluoroazacyclobutane-1-yl)pyrazolo[1,5-a]pyridin-5-yl)carbamoyl )-3-(6-azaspiro[2.5]oct-6-yl)phenyl)aminosulfonyl)1,1,2,2-tetradeuter-ethyl) phosphate
  • N-(7-(3,3-difluoroazacyclobutane-1-yl)pyrazolo[1,5-a]pyridin-5-yl)-2-(6-azaspiro[2.5]oct-6-yl)-4-(1,1,2,2-tetradeuter-2-hydroxyethylsulfonamido)benzamide 120 mg, 0.21 mmol, 1.0 eq. was dissolved in a mixture of dichloromethane (4 mL) and acetonitrile (4 mL).
  • the mixture was cooled to 0 °C in an ice bath, and tetrazolium (19 mg, 0.27 mmol, 1.3 eq.) and dibenzyl N,N-diisopropylphosphonamide (86 mg, 0.25 mmol, 1.2 eq.) were added sequentially.
  • the reaction mixture was incubated at 0 °C for 70 min.
  • m-chloroperoxybenzoic acid 43 mg, 0.25 mmol, 1.2 eq. was added to the reaction mixture.
  • the reaction mixture was incubated at 0 °C for another 10 min.
  • reaction mixture was quenched with saturated sodium bicarbonate aqueous solution (40 mL) and extracted with ethyl acetate (2 x 35 mL). The combined organic phases were washed with saturated ammonium chloride aqueous solution (80 mL), dried over anhydrous sodium sulfate, and concentrated.
  • Step B Preparation of 2-(N-(4-((7-(3,3-difluoroazacyclobut-1-yl)pyrazolo[1,5-a]pyridin-5-yl) carbamoyl )-3-(6-azaspiro[2,5]octane-6-yl)phenyl)aminosulfonyl)ethyl-1,1,2,2-tetradeuterium phosphate dihydrogen ester
  • Step A Preparation of N-(5-(3,3-difluoroazacyclobutane-1-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-4-(1,1,2,2 -tetradeuter-2-hydroxyethylsulfonamido)-2-(6-azaspiro[2.5]oct-6-yl)benzamide
  • reaction mixture was reacted at 10 °C for 48 h.
  • the reaction mixture 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.
  • Step A Preparation of dibenzyl(2-(N-(4-((5-(3,3-difluoroazacyclobutane-1-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7- yl)carbamoyl)-3-(6-azaspiro[2.5]oct-6-yl)phenyl)aminosulfonyl)1,1,2,2-tetradeuter-ethyl) phosphate
  • Tetrazazole (10 mg, 0.14 mmol, 1.3 eq.) and dibenzyl N,N-diisopropylphosphonamide (44 mg, 0.13 mmol, 1.2 eq.) were added sequentially.
  • the reaction mixture was reacted at 0 °C for 70 min.
  • Add m-chloroperoxybenzoic acid (85%, 26 mg, 0.13 mmol, 1.2 eq.) to the reaction solution.
  • m-chloroperoxybenzoic acid 85%, 26 mg, 0.13 mmol, 1.2 eq.
  • Step B Preparation of 2-(N-(4-((5-(3,3-difluoroazacyclobutane-1-yl)-[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)carbamoyl )-3-(6-azaspiro[2.5]oct-6-yl)phenyl)aminosulfonyl)ethyl-1,1,2,2-tetradeuterium-phosphate dihydrogen ester trifluoroacetate
  • Step A Preparation of 2-(N-(4-((7-(3,3-difluoroazacyclobutane-1-yl)pyrazolo[1,5-a]pyridin-5-yl) carbamoyl )-3-(6-azaspiro[2.5]oct-6-yl)phenyl)aminosulfonyl)1,1,2,2- tetradeuter -ethyl sulfate trifluoroacetate
  • N-(7-(3,3-difluoroazacyclobutane-1-yl)pyrazolo[1,5-a]pyridin-5-yl)-2-(6-azaspiro[2.5]oct-6-yl)-4-(1,1,2,2-tetradeuter-2-hydroxyethylsulfonamide)benzamide 50 mg, 0.09 mmol, 1.0 eq.
  • dichloromethane 2.5 mL
  • triethylamine 90 mg, 0.89 mmol, 10.0 eq.
  • chlorosulfonic acid 30 mg, 0.26 mmol, 2.9 eq.
  • reaction mixture 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.
  • Step A Preparation of N-(7-(3,3-difluoroazacyclobutane-1-yl)pyrazolo[1,5-a]pyridin-5-yl)-4-(2-hydroxyethylsulfonamido )-2-(6-azaspiro[2.5]octane-6-yl)benzamide
  • reaction solution was incubated at -78 °C for another 1 h.
  • the reaction solution was quenched with saturated ammonium chloride (80 mL) and extracted with ethyl acetate (2 ⁇ 70 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated.
  • Step B Preparation of 5-bromo-7-(4,4-difluoropiperidin-1-yl)pyrazolo[1,5-a]pyridine
  • Step C Preparation of N-(7-(4,4-difluoropiperidin-1-yl)pyrazolo[1,5-a]pyridin-5-yl)-4-nitro-2-(6-azaspiro[2.5] octane-6-yl)benzamide
  • reaction mixture was heated to 120 °C in a microwave oven for 2 hours under nitrogen protection.
  • the reaction mixture was cooled to room temperature and diluted with water (100 mL), then extracted with ethyl acetate (2 ⁇ 100 mL).
  • the combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated.
  • Step D Preparation of 4-amino-N-(7-(4,4-difluoropiperidin-1-yl)pyrazolo[1,5-a]pyridin-5-yl)-2-(6-azaspiro[2,5] octane-6-yl)benzamide
  • N-(7-(4,4-difluoropiperidin-1-yl)pyrazolo[1,5-a]pyridin-5-yl)-4-nitro-2-(6-azaspiro[2.5]octane-6-yl)benzamide (290 mg, 0.57 mmol, 1.0 eq.) was dissolved in ethanol (10 mL), and stannous chloride (517 mg, 2.73 mmol, 4.8 eq.) was added. The reaction mixture was reacted at 70 °C for 2 h. The reaction mixture was cooled to room temperature and diluted with water (80 mL), and extracted with ethyl acetate (2 ⁇ 100 mL).
  • Step E Preparation of ethyl acetate 2-(N-(4-((7-(4,4-difluoropiperidin-1-yl)pyrazolo[1,5-a]pyridin-5-yl)carbamoyl)-3-(6- azaspiro[2,5]octane-6-yl)phenyl)aminosulfonyl)
  • Step F Preparation of N-(7-(4,4-difluoropiperidin-1-yl)pyrazolo[1,5-a]pyridin-5-yl)-4-(2- hydroxyethylsulfonamido )-2-(6-azaspiro[2.5]octane-6-yl)benzamide
  • KIF18A enzyme activity was tested using the ADP-Glo luminescence assay.
  • Human KIF18A (1-467) protein (Chempartner, CP20220309-N-His cleaved-BV) was incubated in 384-well plates with different concentrations of the test compound or DMSO in reaction buffer (15 mM Tris, pH 7.5, 10 mM MgCl2, 0.01% Pluronic F-68, 2% DMSO, 1 ⁇ M paclitaxel, 30 ⁇ g/mL tubulin) at room temperature for 15 min.
  • the substrate (Cytoskeleton, MT002) and ATP (Promega, V916B) were added to the wells of the 384-well plate, and the plates were incubated at 28°C for 60 min.
  • ADP-Glo reagent 1 (Promega, V9102) was then added, and the reaction was allowed to proceed for 120 min.
  • the RLU values were read using an EnVision 2104 Multilable Reader (PerkinElmer, 411177291). The above data were converted into inhibition percentages using the following formula.
  • min is the reading of the control well without enzyme
  • max is the reading of the control well with DMSO added as a control.
  • 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 an incubator at 37°C and 5% CO2 . Cells in the logarithmic growth phase were collected, and the single-cell suspension concentration was adjusted, then added to 96-well plates to achieve a cell density of 3000 cells/well. Cell-free culture medium was added to the blank control wells. After overnight culture, cells were treated with different concentrations of compounds, with DMSO (0.25%) as a solvent control.
  • DMSO 0.25%
  • the number of viable cells was determined using the Promega CellTiter-Glo luminescence assay kit (Promega-G7573). 75 ⁇ L of CellTiter-Glo working solution was added to each well, and the cells were shaken for 2 minutes in the dark until lysis. After incubation at room temperature for 10 minutes, the luminescence signal was detected on an EnVision microplate reader (PerkinElmer). The measured RLU data are converted into inhibition rate using the following formula:
  • compounds 2-7 exhibited strong inhibitory activity against the proliferation of ovarian cancer cells OVCAR3, colorectal cancer cells HT-29, and triple-negative breast cancer cells HCC1806.
  • HEK293 cells Human embryonic kidney cells (HEK293 cells, ATCC, CRL-157) were cultured in an incubator at 37°C and 5% CO2 .
  • the cytotoxicity assay for the compound on HEK293 cells was performed using the same method as the assay for tumor cell proliferation inhibition activity; please refer to the above-described assay method for details.
  • the experimental results are shown in Figure 1.
  • the control compound AMG650 (MCE, HY-132840) showed some cytotoxicity at a concentration of 10 ⁇ M, and almost completely inhibited cell growth at a concentration of 30 ⁇ M, demonstrating strong cytotoxicity.
  • compounds 4 and 5 showed only half the growth-inhibiting activity against HEK293 cells as AMG650, indicating relatively low cytotoxicity.
  • Liver microsomes (Biopredic International, final concentration 0.2 mg/mL) were compared with different concentrations of compounds (final concentrations of 0.01 ⁇ M, 0.04 ⁇ M, 0.12 ⁇ M, 0.37 ⁇ M, 1.11 ⁇ M, 3.33 ⁇ M, and 10 ⁇ M) including the control compound AMG650 and positive controls (CYP1A2: ⁇ -naphthylflavonoid; CYP2C9: sulfadiazine; CYP2C19: omeprazole; CYP3A4: ketoconazole; CYP2D6: quinidine). All compounds were purchased from Sigma-Aldrich.
  • CYP1A2 30 ⁇ M phenacetin
  • CYP2C9 10 ⁇ M diclofenac sodium
  • CYP2C19 35 ⁇ M S-metphenytoin
  • CYP3A4 5 ⁇ M midazolam and 80 ⁇ M testosterone
  • CYP2D6 5 ⁇ M dextromethorphan; all compounds were purchased from Sigma-Aldrich.
  • the mixture was incubated at 37°C for 10 min.
  • Coenzyme NADPH (Roche, final concentration 1 mM) was then added, and the mixture was incubated at 37°C for the specified times (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 the internal standard working solution to each incubation tube, vortexing to mix, and centrifuging at 3220g for 15 min. 50 ⁇ L of the supernatant was added to an equal volume of ultrapure water and vortexed to mix.
  • the amount of metabolites generated was detected by LC-MS/MS.
  • Raw chromatograms, peak area ratios, and other data were output using SCIEX Analyst software (Analyst 1.6.3). The measured data were converted to inhibition rate using the following formula:
  • Inhibition rate % (1 - amount of metabolites produced in the experimental or positive control group / amount of metabolites produced in the negative control group) ⁇ 100%
  • control compound AMG650 had a certain inhibitory effect on the CYP450 subtype CYP2C9, suggesting that the compound has a potential risk of drug-drug interactions; while compounds 4 and 5 of the present invention had no inhibitory effect on the CYP450 enzyme subtype, including CYP2C9, and were of high safety.
  • HEK293-hERG cells human embryonic kidney cells stably expressing hERG channel protein, Sophion Biosciences
  • DMEM medium fetal bovine serum
  • PWMbio penicillin-streptomycin
  • the temperature of the recording chamber was maintained at 20°C–25°C during current recording.
  • the solvent control (0.1% DMSO), compound 4 (final concentration 10 or 30 ⁇ M), comparative compound 10 (final concentration 10 or 30 ⁇ M), and positive control (cisapride, MCE, final concentration 0.1 ⁇ M) were administered using an 8-channel perfusion system.
  • a micromanipulator was used to move the output of the administration system to the selected cells in the cell chamber.
  • the drug flowing from the output terminal can immediately infiltrate the cells below.
  • Voltage clamp parameters were set using Clampex 10.6 software.
  • cells were clamped at a clamping potential of -80 mV for 100 ms, then hyperpolarized to -90 mV for 100 ms, then returned to -80 mV for 100 ms; depolarized to +40 mV for 500 ms to activate the hERG channel; then a 100 ms ramp stimulation 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. Currents were acquired using Clampex 10.6 software; the current signal input was filtered at 2 kHz and digitized at a frequency of 5 kHz. The peak tail current was measured using Clampfit 10.6 software for data acquisition and analysis. The mean of the peak tail current recorded during the last 10 administrations for each concentration was statistically analyzed. The mean of the peak tail current recorded during continuous perfusion of the solvent reference standard was taken as 100%, and the inhibition rate was used to calculate the IC50 . The calculation formula is as follows:
  • Inhibition rate % (1 - mean peak tail current after drug administration / mean peak tail current in solvent control) ⁇ 100%
  • the experimental results are shown in Table 4.
  • the non-deuterated comparative compound 10 exhibited strong inhibitory effects on hERG potassium channel currents in HEK293 cells at both 10 and 30 ⁇ M, showing a dose-dependent effect, suggesting that this compound may have cardiotoxicity.
  • the deuterated compound 4 of this invention did not significantly inhibit hERG potassium channel currents at either 10 or 30 ⁇ M.
  • Compounds 4, 5, and the control compound AMG650 were prepared into 10 mM stock solutions using DMSO. 8 ⁇ L of the stock solution was added to 792 ⁇ L of phosphate buffer (100 mM, pH 7.4), and the mixture was shaken at room temperature for 1 hour, followed by centrifugation at 12000 rpm for 10 min. The supernatant was transferred to new tubes and diluted 10-fold and 100-fold with 100 mM phosphate buffer, respectively. 5 ⁇ L of the undiluted or diluted sample was added to acetonitrile containing the internal standard working solution, and the compound concentrations were determined by LC-MS/MS.
  • compound 4 exhibits significantly higher solubility than the control compound AMG650 (greater than 25 times), while phosphate compound 5 further enhances solubility (more than 875 times that of AMG650). Due to their improved safety and solubility, compounds 4 and 5 can achieve higher drug exposure at higher doses in clinical practice, thereby improving the clinical efficacy of the drugs.
  • mice Eighteen male CD-1 mice (Shanghai Jihui Experimental Animal Breeding Co., Ltd.), aged 6-8 weeks and weighing 28-31g, were randomly divided into two groups (Group A and Group B), with nine mice in each group. The mice were fasted for 12 hours. Group A mice were administered the test compound solution by gavage at a dose of 10 mg/kg; Group B mice were administered the test compound solution by tail vein injection at a dose of 3 mg/kg. Blank blood samples were collected before administration. For Group A mice, approximately 110 ⁇ L of venous blood was collected at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after administration.
  • mice approximately 110 ⁇ L of venous blood was collected at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after administration. All samples were placed in test tubes containing EDTA-K2 anticoagulant (GREAGENT, G41456A), centrifuged, and the plasma was collected and stored at -70°C for testing.
  • GREAGENT EDTA-K2 anticoagulant
  • NCA non-compartmental model
  • AUC ⁇ sub>last ⁇ /sub> represents the area under the drug-time curve from the start of administration to the last sampling point;
  • CL(iv) represents the drug clearance rate after intravenous administration;
  • T ⁇ sub>1/2 ⁇ /sub> represents the plasma half-life of the drug, i.e., the time required for the plasma drug concentration to decrease by half;
  • C ⁇ sub> max ⁇ /sub> represents the maximum drug concentration reached in plasma after administration;
  • T ⁇ sub>max ⁇ /sub> represents the time required for the drug to reach its maximum concentration in plasma after administration;
  • F% represents the oral bioavailability of the drug.
  • the mixture was vortexed and centrifuged at 3200 g for 10 min at 4°C. 50 ⁇ L of the supernatant was taken, 100 ⁇ L of ultrapure water was added, vortexed to mix, and the remaining amount of the parent compound in the test sample was detected by LC-MS/MS.
  • the raw spectrum and peak area ratio were output using SCIEX's Analyst software (Analyst 1.6.3), and the half-life (T 1/2 ) was calculated using Microsoft Office Excel.
  • This invention provides a KIF18A inhibitor compound that can be used to selectively inhibit KIF18A activity, or to treat or prevent diseases, disorders, or conditions regulated by or affected by KIF18A activity, or involving KIF18A activity or overexpression. Therefore, it can be formulated into a corresponding pharmaceutical product suitable for industrial application.

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Abstract

提供一种KIF18A抑制剂，其为式(I)的化合物或其药学可接受的盐、溶剂化物、酯、酸、代谢物或前药。还提供KIF18A抑制剂在选择性地抑制KIF18A活性、或治疗或预防由KIF18A活性调节的或受其影响的或者其中涉及KIF18A活性或过表达的疾病、障碍或病症的用途，特别是癌症，更特别是具有染色体不稳定性的癌症。

Description

KIF18A抑制剂及其用途 技术领域
本发明涉及制药领域，特别是涉及由苯甲酰胺结构单元和稠合杂双环结构单元构成的氘代小分子化合物，以及其用于治疗和/或预防疾病的方法和用途。
背景技术
染色体不稳定性(CIN)是指正在进行的基因组改变，包括染色体拷贝数或结构的扩增或缺失，范围从点突变到小规模基因组变化甚至整条染色体数目变化。CIN是由有丝分裂过程中染色体分离持续出现错误导致的，是癌细胞的重要特征之一，并且可能是肿瘤形成的重要机制。CIN在多种类型的癌症中普遍存在，尤其以极高频率发生在高级别浆液性卵巢癌(HGSOC)、三阴性乳腺癌(TNBC)、结直肠癌(CRC)等肿瘤中，并与肿瘤转移、免疫逃逸和治疗耐药性等密切相关。
染色体正确分离的关键是由各种激酶和驱动蛋白协同作用调控有丝分裂纺锤体的形状和功能。KIF18A是驱动蛋白-8家族的成员，是一种整合微管运动和解聚活性的酶，能够可逆地与微管结合，影响着丝粒微管的动力学以控制正确的染色体定位和纺锤体张力，在细胞分裂方面发挥着关键作用。KIF18A在人正常组织中低表达，但在多种癌症中显著过表达和功能异常，并且KIF18A过表达与肿瘤分级、转移和低生存率相关。
功能分析表明，KIF18A在人乳腺癌细胞中过表达可导致细胞多核，而抑制KIF18A在体内和体外均可抑制癌细胞的生长。除了对肿瘤细胞有丝分裂的影响外，KIF18A功能的减弱还会抑制肿瘤细胞迁移并诱导细胞凋亡。因此，KIF18A可作为一种潜在的预后标志物和癌症治疗的新靶点。
KIF18A抑制剂可以选择性地杀伤染色体不稳定的癌细胞，作为潜在肿瘤治疗策略具有广阔前景。但目前报导的KIF18A抑制剂相对较少，专利US2022/0372018A1、US2022/0106293A1、CN115594664A、CN115772159A、CN115785068A、WO2023/028564A1等公开了几种芳杂环化合物，可以抑制KIF18A的酶活性。
尽管在该领域中已经取得了一些进步，本领域中仍然极其需要开发出高效、特异、安全的KIF18A小分子抑制剂，用于治疗具有染色体不稳定性的癌症。
发明内容
本发明旨在提供一种能够选择性地抑制KIF18A活性的氘代化合物，其与非氘代的相应小分子化合物相比，能够在提供相当的KIF18A抑制活性的同时，在提高药物安全性、溶解度和肝微粒体稳定性、改善药代动力学等方面提供额外的优势。
根据本发明的一个方面，提供KIF18A抑制剂，其为式(I)的化合物或其药学可接受的盐、溶剂化物、酯、酸、代谢物或前药，
其中，
X1和X2各自独立地选自N和CH；
R1选自氢、-P(＝O)(OH)2、和-S(＝O)2(OH)；
R2a、R2b、R3a和R3b各自独立地选自氢或氘，条件是R2a、R2b、R3a和R3b中的至少一个为氘；
m为选自1、2或3的整数；
n为选自1、2或3的整数；
q为选自0、1或2的整数，且当q不为0时，R4各自独立地选自卤素。
在另外的实施方式中，m和n各自独立地为1。
在其他优选的实施方式中，q为2。
在又一优选的实施方式中，R4各自独立地为氟。
优选地，m为1，q为2，且R4在氮杂环上的取代位置为氮原子的对位。
在另外的实施方式中，X1为CH。
在又一实施方式中，X2为CH。
优选地，X1和X2均为CH。
在其他优选的实施方式中，R2a、R2b、R3a和R3b中的至少两个为氘。更优选地，其中R2a和R2b为氘。进一步优选地，R2a、R2b、R3a和R3b均为氘。
在其他优选的实施方式中，R1为氢。
根据本发明的KIF18A抑制剂，优选为选自以下结构的化合物，或其药学可接受的盐、溶剂化物、酯、酸、代谢物或前药：
在本发明的另一个方面，提供一种药物组合物，其包括本发明的化合物、或其药学可接受的盐、溶剂化物、酯、酸、代谢物或前药，和药学上可接受的载体或赋形剂，以及任选的其它治疗剂。
本发明的其他方面还涉及本发明的化合物、或其药学可接受的盐、溶剂化物、酯、酸、代谢物或前药选择性地抑制KIF18A活性的方法或用途，或者本发明的化合物、或其药学可接受的盐、溶剂化物、酯、酸、代谢物或前药在制备用于选择性地抑制KIF18A活性的药物中的用途。
在本发明的进一步方面还涉及发明的化合物、或其药学可接受的盐、溶剂化物、酯、酸、代谢物或前药治疗或预防由KIF18A活性调节的或受其影响的或者其中涉及KIF18A活性的疾病、障碍或病症的方法或用途，或者本发明的化合物、或其药学可接受的盐、溶剂化物、酯、酸、代谢物或前药在制备用于治疗或预防由KIF18A活性调节的或受其影响的或者其中涉及KIF18A活性或过表达的疾病、障碍或病症的药物中的用途。
优选地，由KIF18A活性调节的或受其影响的或者其中涉及KIF18A活性或过表达的疾病、障碍或病症为癌症。
在更优选地方面，所述疾病、障碍或病症选自以下一种或多种具有染色体不稳定性的癌症：肺鳞癌、肺腺癌、非小细胞肺癌、小细胞肺癌、头颈鳞癌、乳腺癌、胰腺癌、胰腺导管腺癌、结直肠癌、黑色素瘤、卵巢癌、食管鳞癌、胃癌、肝癌、口腔癌、尿路上皮癌、前列腺癌、膀胱癌、肾细胞癌、胃肠间质瘤、子宫颈癌、子宫内膜癌、横纹肌肉瘤、纤维肉瘤、神经内分泌瘤、间皮瘤、脑癌、和恶性胶质瘤的癌症。
附图说明
图1显示本发明的化合物和对照化合物对人胚胎肾细胞HEK293的增殖抑制曲线。
具体实施方式
定义
除非另外定义，否则本文中使用的所有技术和科学术语具有本发明所属领域普通技术人员通常所理解的含义。在说明书中，除非上下文另有明确说明，单数形式也包含复数形式。本文提到的所有出版物、专利申请、专利或其它参考文献均通过引入本文作为参考。在存在抵触的情况下，以本说明书(包括定义在内)为准。此外，材料、方法和实施例都仅是说明性的，并不意在限制本发明的范围。
除非另有说明，本发明采用本领域技术范围内的质谱、NMR、HPLC、蛋白质化学、生物化学、重组DNA技术和药理学等常规方法。除非提供具体的定义，否则与本文描述的分析化学、合成有机化学、以及医学和药物化学等化学上相关的命名和实验室操作和技术，是本领域技术人员已知的。一般而言，前述技术和步骤可以通过本领域众所周知的和在各种一般文献和更具体文献中描述的常规方法来实施，这些文献在本说明书中被引用和讨论。
本文术语“药学上可接受的盐”指的是保留主题化合物的所需生物学活性且显示最小的不希望的毒理学效应的盐。这些药学上可接受的盐可以在化合物的最终分离和纯化过程中原位制备，或通过单独使纯化化合物的游离酸或游离碱形式分别与合适的碱或酸反应来制备。
“溶剂化物”或“溶剂合物”指的是含有化学计量或非化学计量溶剂的溶剂加成形式。一些化合物趋于以晶状固态捕集固定摩尔比例的溶剂分子，从而形成溶剂合物。若溶剂是水，则形成的溶剂合物是水合物；若溶剂是醇，则形成的溶剂合物是醇化物。水合物通过使一个或多个水分子与所述物质的一个分子结合而形成，其中所述水保持其分子状态为H2O。
本文公开的化合物的“代谢物”是当该化合物被代谢时形成的化合物的衍生物。术语“活性代谢物”是指当该化合物被代谢时形成的化合物的生物活性衍生物。本文使用的术语“被代谢”，是指特定物质被生物体改变的过程总和(包括但不限于水解反应和由酶催化的反应，例如氧化反应)。因此，酶可以产生特定的结构转变为化合物。例如，细胞色素P450催化各种氧化和还原反应，同时二磷酸葡萄糖甘酸基转移酶催化活化的葡萄糖醛酸分子至芳香醇、脂肪族醇、羧酸、胺和游离的巯基的转化。新陈代谢的进一步的信息可以从《The Pharmacological Basis of Therapeutics》，第九版，McGraw-Hill(1996)获得。本文公开的化合物的代谢物可以通过将化合物给予宿主并分析来自该宿主的组织样品、或通过将化合物与肝细胞在体外孵育并且分析所得化合物来鉴别。这两种方法都是本领域已知的。在一些实施方式中，化合物的代谢物是通过氧化过程形成并与相应的含羟基化合物对应。在一些实施方式中，化合物被代谢为药物活性代谢物。
本文使用的术语“调节”，是指直接或间接与靶标相互作用，以改变靶标的活性，仅仅举例来说，包括增强靶标的活性、抑制靶标的活性、限制靶标的活性或者延长靶标的活性。
术语“前药”或“前体药物”是指如下衍生物，其可能不具有药理学活性，但在某些情况下，可口服或肠胃外给予并在这之后在体内代谢以形成具有药理学活性的本发明化合物。前药的非限制性实例包括：酯、碳酸酯、半酯、磷酸酯、硝基酯、硫酸酯、亚砜、酰胺、氨基甲酸酯、含氮化合物、磷酰胺、糖苷、醚、乙缩醛和酮缩醇等。
“有效量”指，将引发例如研究者或医师在研的组织、系统、动物或人的生物学或医学反应的药物或药学制剂的量。此外，术语“治疗有效量”指与没有接受该量的相应对象相比，引起疾病、紊乱、或副作用改良的治疗、治愈、预防、或缓解、或者疾病或紊乱发展速率降低的任何量。该术语范围内还包括有效提高正常生理功能的量。
本文所用的术语“治疗”指缓解疾病、紊乱或病症的至少一种症状。该术语包括向对象给药和/或应用一种或多种本文所述化合物以提供病症的管理或治疗。用于本公开目的的“治疗”可以但不必须提供治愈；而是指，“治疗”可以是病症的管理形式。当本文所述化合物用于处理有害的增殖细胞(包括癌)时，“治疗”包括部分或完全破坏所述有害的增殖细胞，但对正常细胞的破坏影响最小。有害的快速增殖细胞(包括癌细胞)的所需处理机制在细胞水平上是凋亡。
本文所用的术语“预防”包括共同预防或减缓临床上显著疾病发展的开始或者预防或减缓风险个体中的临床前显著疾病阶段的开始。这包括预防性治疗有疾病发展风险的个人。
术语“受试者”或“患者”包括能患有病症或与降低的或不足的程序性细胞死亡(细胞凋亡)相关的病症的有机体或能以其他方式从本发明化合物的给药中获益的有机体，例如人类和非人类动物。优选的人类包括患有或倾向患有如本文所述的病症或相关状况的人类患者。术语“非人类动物”包括脊椎动物，例如哺乳动物，如非人类灵长类动物、羊、牛、狗、猫和啮齿动物如小鼠，以及非哺乳动物，如鸡、两栖动物、爬行动物等。
本文使用的GI50是指使50％细胞生长被抑制所需的药物浓度，即药物使50％细胞(如癌细胞)的生长得到抑制或控制时的药物浓度。
本文使用的IC50是指在测量效应的分析中获得最大效应的50％抑制的特定测试化合物的量、浓度或剂量。
本文使用的EC50是指测定化合物的剂量、浓度或量，其引起特定测定化合物诱导、刺激或加强的特定反应的50％的最大表达的剂量依赖反应。
本发明的KIF18A抑制剂
本发明涉及一种KIF18A抑制剂，其为式(I)的化合物或其药学可接受的盐、溶剂化物、酯、酸、代谢物或前药，
其中，
X1和X2各自独立地选自N和CH；
R1选自氢、-P(＝O)(OH)2、和-S(＝O)2(OH)；
R2a、R2b、R3a和R3b各自独立地选自氢或氘，条件是R2a、R2b、R3a和R3b中的至少一个为氘；
m为选自1、2或3的整数；
n为选自1、2或3的整数；
q为选自0、1或2的整数，且当q不为0时，R4各自独立地选自卤素。
在另外的实施方式中，m和n各自独立地为1。
在其他优选的实施方式中，q为2。
在又一优选的实施方式中，R4各自独立地为氟。
优选地，m为1，q为2，且R4在氮杂环上的取代位置为氮原子的对位。
在另外的实施方式中，X1为CH。
在又一实施方式中，X2为CH。
优选地，X1和X2均为CH。
在其他优选的实施方式中，R2a、R2b、R3a和R3b中的至少两个为氘。更优选地，其中R2a和R2b为氘。进一步优选地，R2a、R2b、R3a和R3b均为氘。
在其他优选的实施方式中，R1为氢。
进一步优选地，本发明涉及下表所示的化合物、或其药学可接受的盐、溶剂化物、酯、酸、代谢物或前药。
本文描述的是新型的KIF18A抑制剂。本文也描述了此化合物的药学可接受的盐、溶剂化物、酯、酸、代谢物和前药。
本发明的化合物可以游离形式存在，例如游离碱或游离酸或两性离子形式，或者可以盐的形式存在。所述盐可以为任何盐，可为有机或无机加成盐，特别是常用于药学的任何生理学上可接受的有机或无机加成盐。
对于本发明的目的而言为优选的盐是本发明化合物的生理学上可接受的盐。然而，也包括自身不适于药学应用但例如可用于分离或纯化本发明化合物的盐。
术语“药学可接受的盐”是指本发明化合物的相对无毒的无机酸或有机酸加成盐，例如参见S.M.Berge等人，“Pharmaceutical Salts,J.Pharm.Sci.1977,66,1-19。
本发明化合物的药学可接受的盐包括无机酸、羧酸和磺酸的酸加成盐，例如以下酸的盐：盐酸、氢溴酸、氢碘酸、硫酸、焦硫酸、磷酸、硝酸；或与有机酸的盐，例如甲酸、乙酸、乙酰乙酸、丙酮酸、三氟乙酸、丙酸、丁酸、己酸、庚酸、十一烷酸、月桂酸、苯甲酸、水杨酸、2-(4-羟基苯甲酰基)-苯甲酸、樟脑酸、肉桂酸、环戊烷丙酸、二葡糖酸、3-羟基-2-萘甲酸、烟酸、扑酸、果胶酯酸、过硫酸、3-苯基丙酸、苦味酸、叔戊酸、2-羟基乙磺酸、衣康酸、氨基磺酸、三氟甲磺酸、十二烷基硫酸、乙磺酸、苯磺酸、对甲苯磺酸、甲磺酸、2-萘磺酸、萘二磺酸、樟脑磺酸、柠檬酸、酒石酸、硬脂酸、乳酸、草酸、丙二酸、琥珀酸、苹果酸、己二酸、海藻酸、马来酸、富马酸、D-葡糖酸、扁桃酸、抗坏血酸、葡庚糖酸、甘油磷酸、天冬氨酸、磺基水杨酸、半硫酸或硫氰酸的盐。
本发明化合物的药学可接受的盐还包括常用碱的盐，例如且优选碱金属盐(例如钠盐和钾盐)、碱土金属盐(例如钙盐和镁盐)、以及衍生自氨或具有1至16个碳原子的有机胺的铵盐，所述有机胺例如且优选为乙胺、二乙胺、三乙胺、乙基二异丙胺、单乙醇胺、二乙醇胺、三乙醇胺、二环己胺、二甲基氨基乙醇、普鲁卡因、二苄胺、N-甲基吗啉、精氨酸、赖氨酸、乙二胺、N-甲基哌啶、N-甲基葡糖胺、二甲基葡糖胺、乙基葡糖胺、1,6-己二胺、氨基葡萄糖、肌氨酸、丝氨醇、三(羟甲基)氨基甲烷、氨基丙二醇、Sovak碱和1-氨基-2,3,4-丁三醇。
本发明包括本发明化合物的所有可能的盐，其可为单一盐或所述盐以任意比例的任意混合物。
溶剂化物是为了本发明的目的对通过在固态或液态下的配位作用与溶剂分子形成络合物的本发明化合物的那些形式而使用的术语。水合物是其中与水发生配位的特殊溶剂合物形式。水合物优选作为本发明范围内的溶剂化物。
此外，本发明还包括本发明化合物的前体药物。术语“前药”包括自身可以是有生物学活性的或是惰性的但在其于体内的保留时间中被转化成(例如通过代谢或水解)本发明化合物的化合物。
此外，本发明包括本发明化合物的所有可能的结晶形式或多晶型物，其作为单独的多晶型物，或作为多于一种多晶型物以任意比例的混合物。
在本说明书中，在一些情况中为方便起见，化合物的结构式代表特定的异构体，但本发明包括所有异构体，例如几何异构体、基于不对称碳原子的光学异构体、立体异构体、互变异构体等。
本发明所涉及带有手性的化合物，其构型可以是任意构型或者混合的外消旋体。当根据本发明使用的化合物包含多于一种手性中心时，其可以非对映体形式存在。所述非对映体异构体化合物可以通过本领域技术人员已知的方法分离(例如，色谱或结晶)，而单独对映体可以如上所述分离。本发明包括根据本发明使用的多种非对映体化合物及其混合物的应用。本发明使用的化合物可以不同互变异构形式或以不同几何异构体形式存在，本发明包括根据本发明使用的化合物的各个互变异构体和/或几何异构体及其混合物的应用。本发明使用的化合物可以两性离子形式存在。本发明包括根据本发明使用的化合物的各个两性离子形式及其混合物的应用。
筛选和表征药学可接受的盐、多晶型和/或溶剂化物可以使用多种技术完成，所述技术包括但不限于热分析、X射线衍射、光谱、显微镜方法、元素分析。使用的各种光谱技术包括但不限于Raman、FTIR、UVIS和NMR(液体和固体状态)。各种显微镜技术包括但不限于IR显微镜检术和拉曼(Raman)显微镜检术。
本发明的药物用途
本发明的式(I)的化合物、或其药学可接受的盐、溶剂化物、酯、酸、代谢物或前药，能够选择性地抑制KIF18A的酶活性，因此能够用于治疗或预防由KIF18A活性调节的或受其影响的或者其中涉及KIF18A活性或过表达的疾病、障碍或病症。
在优选的方面，由KIF18A活性调节的或受其影响的或者其中涉及KIF18A活性或过表达的疾病、障碍或病症为癌症，特别是具有染色体不稳定性的癌症，包括但不限于以下一种或多种：肺鳞癌、肺腺癌、非小细胞肺癌、小细胞肺癌、头颈鳞癌、乳腺癌、胰腺癌、胰腺导管腺癌、结直肠癌、黑色素瘤、卵巢癌、食管鳞癌、胃癌、肝癌、口腔癌、尿路上皮癌、前列腺癌、膀胱癌、肾细胞癌、胃肠间质瘤、子宫颈癌、子宫内膜癌、横纹肌肉瘤、纤维肉瘤、神经内分泌瘤、间皮瘤、脑癌、或恶性胶质瘤的癌症。
本发明的化合物可全身和/或局部地发挥作用。为此，其可以以适当的方式给药，例如通过口服途径、肠胃外途径、肺途径、鼻途径、舌下途径、舌途径、含服途径、直肠途径、皮肤途径、经皮途径、结膜途径或耳途径，或以植入物或支架的形式。
优选地，在本发明的实施方式中，可以通过注射、口服、吸入、直肠和经皮施用中的至少一种将包含本发明化合物的药物施用给患者。
不论所选的给药途径如何，都通过本领域技术人员已知的常规方法将本发明的KIF18A抑制剂和/或本发明的药物组合物配制成药学上可接受的剂型。
在根据本发明对患者进行治疗时，给定药物的量取决于诸多因素，如具体的给药方案、疾病或病症类型及其严重性、需要治疗的受治疗者或宿主的独特性(例如体重)，但是，根据特定的周围情况，包括例如已采用的具体药物、给药途径、治疗的病症、以及治疗的受治疗者或宿主，施用剂量可由本领域已知的方法常规决定。通常，就成人治疗使用的剂量而言，施用剂量典型地在0.02-5000mg/天，例如约1-1500mg/天的范围。该所需剂量可以方便地被表现为一剂、或同时给药的(或在短时间内)或在适当的间隔的分剂量，例如每天二、三、四剂或更多分剂。本领域技术人员可以理解的是，尽管给出了上述剂量范围，但具体的有效量可根据患者的情况并结合医师诊断而适当调节。
本发明的化合物的实际给药剂量水平和时间进程可以变化，从而获得有效实现特定患者所期望的治疗响应并对所述患者无毒的活性成分的量。
药物组合物
本发明的另一方面涉及药物组合物，其包含本发明的式(I)的化合物、或其药学可接受的盐、溶剂化物、酯、酸、代谢物或前药，和药学上可接受的稀释剂、载体或赋形剂，以及任选的一种或多种其他治疗剂。
本发明的化合物可以以单独的药剂或与一种或多种其他治疗剂组合的形式给药，其中所述组合不引发不可接受的副作用。该药物组合物包括给药包含本发明的化合物和一种或多种其他治疗剂的单一药物剂量制剂、以及以其自身的单独的药物剂量制剂形式给药本发明的化合物和各种其他治疗剂。例如，可以向患者以例如片剂或胶囊的单一口服剂量组合物形式将式(I)的化合物和其他治疗剂一起给药，或者可以以单独的剂量制剂形式给药每一药剂。
当使用单独的剂量制剂时，本发明的化合物和一种或多种其他治疗剂可以在基本上相同的时间(例如同时)或在分别错开的时间(例如，相继地)给药。
特别地，本发明的化合物可以以与下列物质的固定组合或单独组合的形式使用：其他抗肿瘤剂，例如烷化剂、抗代谢剂、得自植物的抗肿瘤剂、激素治疗剂、拓扑异构酶抑制剂、喜树碱衍生物、激酶抑制剂、靶向药物、抗体、干扰素和/或生物反应修饰剂、抗血管生成化合物以及其他抗肿瘤药物。
本发明的化合物还可以连同放射疗法和/或手术介入来用于癌症治疗。
化合物的制备
使用本领域技术人员已知的标准合成技术或使用本领域已知的方法与本文描述的方法组合，可以合成本发明的化合物。另外，本文给出的溶剂、温度和其它反应条件可以根据本领域技术而改变。作为进一步的指导，也可以利用以下的合成方法。
所述反应可以按顺序使用，以提供本文描述的化合物；或它们可以用于合成片段，所述片段通过本文描述的方法和/或本领域已知的方法随后加入。
用于合成本文描述的化合物的起始原料可以被合成或可以从商业来源获得。本文描述的化合物和其它相关具有不同取代基的化合物可以使用本领域技术人员已知的技术和原料合成。制备本文公开的化合物的一般方法可以来自本领域已知的反应，并且该反应可以通过由本领域技术人员所认为适当的试剂和条件修改，以引入本文提供的分子中的各种部分。
如果需要，反应产物可以使用常规技术分离和纯化，包括但不限于过滤、蒸馏、结晶、色谱等方法。这些产物可以使用常规方法表征，包括物理常数和图谱数据。
制备式(I)的化合物的合成方案的非限制性实施例如下描述。
实施例
以下具体的非限制性实施例将被解释为仅仅是说明性的，并不以任何方式限制本发明。虽然无需进一步详细描述，但是可以相信本领域技术人员能基于本文的描述，完全利用本公开。
化合物的结构是通过核磁共振(NMR)或/和质谱(MS)来确定的。NMR的测定溶剂为氘代二甲基亚砜(DMSO-d6)、氘代氯仿(CDCl3)或氘代甲醇(CD3OD)。
实施例中如无特殊说明，溶液是指水溶液。
实施例中如无特殊说明，反应的温度为室温，例如20℃～30℃。
英文缩写：
ACN：乙腈；
Brettphos：2-(二环己基膦)-3,6-二甲氧基-2',4',6'-三异丙基-1,1'-联苯；
Brettphos Pd G3：甲烷磺酸(2-二环己基膦)-3,6-二甲氧基-2',4',6'-三异丙基-1,1'-联苯)(2'-氨基-1,1'-联苯-2-基)钯(II)；
DBU：1,8-二氮杂二环[5.4.0]十一碳-7-烯；
DCM：二氯甲烷；
DIPEA：N,N-二异丙基乙胺；
DMF：N,N-二甲基甲酰胺；
HATU：2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯；
LiHMDS：双三甲基硅基胺基锂；
mCPBA：间氯过氧苯甲酸；
NMP：N-甲基吡咯烷酮；
Ruphos：2-二环己基磷-2',6'-二异丙氧基-1,1'-联苯；
Ruphos Pd G2：氯(2-二环己基膦基-2',6'-二异丙氧基-1,1'-联苯基)(2-氨基-1,1'-联苯-2-基)钯(II)；
TEA：三乙胺；
THF：四氢呋喃。
实施例1：中间体的合成
1.1中间体A的合成
4-硝基-2-(6-氮杂螺[2.5]辛-6-基)苯甲酰胺
步骤A：2-氟-4-硝基苯甲酰胺的制备
将2-氟-4-硝基苯甲酸(24.06克，130.00毫摩尔，1.0eq.)溶于N,N-二甲基甲酰胺(250毫升)，依次加入N,N-二异丙基乙胺(50.41克，390.00毫摩尔，3.0eq.)，2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(54.37克，143.00毫摩尔，1.1eq.)及氯化铵(9.04克，169.00毫摩尔，1.3eq.)。反应液20℃反应3小时。反应液加水(1.2升)淬灭，乙酸乙酯(4 x 550毫升)萃取。合并有机相，饱和氯化铵水溶液(2 x 600毫升)洗涤。用无水硫酸钠干燥，过滤，并浓缩有机相至约250毫升体积剩余。有固体产物析出，过滤得到2-氟-4-硝基苯甲酰胺(21.00克，黄色固体)，收率：87.7％。
LCMS:(ESI)[M+H]+＝185.0。
1H NMR:(400MHz,DMSO-d6)δ8.20(dd,J＝10.0,2.0Hz,1H),8.15-8.11(m,1H),8.07(s,1H),7.93(s,1H),7.87(dd,J＝8.4,6.8Hz,1H)。
步骤B：4-硝基-2-(6-氮杂螺[2.5]辛-6-基)苯甲酰胺(中间体A)的制备
将2-氟-4-硝基苯甲酰胺(18.41克，100.00毫摩尔，1.0eq.)溶于N-甲基吡咯烷酮(200毫升)，依次加入N,N-二异丙基乙胺(38.78克，300.00毫摩尔，3.0eq.)及6-氮杂-螺[2.5]辛烷盐酸盐(16.24克，110.00毫摩尔，1.1eq.)。反应液于135℃反应20小时。反应液降至室温并加水(1.2升)及乙酸乙酯(800毫升)稀释。反应体系有固体析出，将混合体系过滤，得到析出的固体，并用乙酸乙酯(400毫升)洗涤，干燥得到4-硝基-2-(6-氮杂螺[2.5]辛-6-基)苯甲酰胺(24.60克，黄色固体)，收率：89.4％。
LCMS:(ESI)[M+H]+＝276.0。
1H 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)。
1.2中间体B的合成
2-(N-(4-((7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰基)-3-(6-氮 杂螺[2.5]辛烷-6-基)苯基)氨磺酰基)乙酸乙酯
步骤A：5-溴-7-氯吡唑并[1,5-a]吡啶的制备
将5-溴吡唑并[1,5-a]吡啶(10.00克，50.75毫摩尔，1.0eq.)溶于四氢呋喃(100毫升)，氮气保护下，于干冰/乙醇浴中降至-78℃，滴加双三甲基硅基胺基锂(1M，55.8毫升，55.8毫摩尔，1.1eq.)。反应液于-78℃反应0.5小时。向反应液中滴加六氯乙烷(13.22克，55.83毫摩尔，1.1eq.)的四氢呋喃(20毫升)溶液。反应液于-78℃继续反应1小时。反应液加入饱和氯化铵水溶液(120毫升)淬灭，乙酸乙酯(2 x 100毫升)萃取。合并有机相，无水硫酸钠干燥并浓缩。得到的粗产物通过柱层析(石油醚/乙酸乙酯＝20/1；V/V)分离纯化得到5-溴-7-氯吡唑并[1,5-a]吡啶(9.68克，淡黄色固体)，收率：82.4％。
LCMS:(ESI)[M+H]+＝231.0。
1H 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)。
步骤B：5-溴-7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶的制备
将5-溴-7-氯吡唑并[1,5-a]吡啶(2.88克，12.44毫摩尔，1.0eq.)溶于N-甲基吡咯烷酮(20毫升)，依次加入3,3-二氟氮杂环丁烷盐酸盐(4.83克，37.33毫摩尔，3.0eq.)及1,8-二氮杂二环[5.4.0]十一碳-7-烯(9.47克，62.21毫摩尔，5.0eq.)。反应液于135℃反应20小时。将反应液降至室温并加入饱和氯化铵水溶液(150毫升)稀释，乙酸乙酯(2 x 200毫升)萃取。合并有机相，饱和盐水(2 x 150毫升)洗涤，无水硫酸钠干燥并浓缩。得到的粗产物通过柱层析(石油醚/乙酸乙酯＝35/1；V/V)分离纯化得到5-溴-7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶(2.32克，淡黄色固体)，收率：64.7％。
LCMS:(ESI)[M+H]+＝288.0。
1H NMR:(400MHz,CDCl3)δ7.87(d,J＝2.0Hz,1H),7.23(d,J＝2.0Hz,1H),6.38(d,J＝2.4Hz,1H),5.80(d,J＝1.6Hz,1H),4.65(t,J＝12.0Hz,4H)。
步骤C：N-(7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)-4-硝基-2-(6-氮杂 螺[2.5]辛烷-6-基)苯甲酰胺的制备
将5-溴-7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶(800毫克，2.78毫摩尔，1.0eq.)溶于二氧六环(15毫升)，依次加入4-硝基-2-(6-氮杂螺[2.5]辛-6-基)苯甲酰胺(764毫克，2.78毫摩尔，1.0eq.)，2-(二环己基膦)-3,6-二甲氧基-2',4',6'-三异丙基-1,1'-联苯(89毫克，0.17毫摩尔，0.06eq.)，甲烷磺酸(2-二环己基膦)-3,6-二甲氧基-2',4',6'-三异丙基-1,1'-联苯)(2'-氨基-1,1'-联苯-2-基)钯(II)(76毫克，0.08毫摩尔，0.03eq.)以及碳酸铯(2.71克，8.33毫摩尔，3.0eq.)。氮气保护下，反应液于120℃反应6小时。反应液降至室温并加水(40毫升)稀释，二氯甲烷(2 x 80毫升)萃取。合并有机相，无水硫酸钠干燥并浓缩。得到的粗产物通过乙酸乙酯(40毫升)打浆并过滤，滤饼真空干燥得到N-(7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)-4-硝基-2-(6-氮杂螺[2.5]辛烷-6-基)苯甲酰胺(840毫克，黄色固体)，收率：62.7％。
LCMS:(ESI)[M+H]+＝483.2。
1H NMR:(400MHz,DMSO-d6)δ10.90(s,1H),7.95-7.88(m,3H),7.80(dd,J＝7.6,1.2Hz,1H),7.75(d,J＝1.6Hz,1H),6.51(d,J＝2.4Hz,1H),6.28(d,J＝2.0Hz,1H),4.66(t,J＝12.0Hz,4H),3.16-3.09(m,4H),1.51-1.40(m,4H),0.31(s,4H)。
步骤D：4-氨基-N-(7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)-2-(6-氮杂 螺[2.5]辛烷-6-基)苯甲酰胺的制备
将N-(7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)-4-硝基-2-(6-氮杂螺[2.5]辛烷-6-基)苯甲酰胺(1.06克，2.20毫摩尔，1.0eq.)溶于乙酸(10毫升)，加入锌粉(1.15克，17.60毫摩尔，8.0eq.)。反应液于30℃反应1小时。反应液加入二氯甲烷(150毫升)稀释并过滤，滤液加入饱和碳酸钠中和乙酸。有机相通过无水硫酸钠干燥并浓缩。得到的粗产物通过柱层析(石油醚/乙酸乙酯＝3/2；V/V)分离纯化得到4-氨基-N-(7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)-2-(6-氮杂螺[2.5]辛烷-6-基)苯甲酰胺(957毫克，黄色固体)收率：96.1％。
LCMS:(ESI)[M+H]+＝453.2。
1H NMR:(400MHz,DMSO-d6)δ12.48(s,1H),7.90(d,J＝2.0Hz,1H),7.75(d,J＝8.4Hz,1H),7.67(d,J＝1.6Hz,1H),6.58(d,J＝2.0Hz,1H),6.47-6.40(m,2H),6.22(d,J＝2.0Hz,1H),5.84(s,2H),4.66(t,J＝12.0Hz,4H),2.95(t,J＝5.2Hz,4H),1.74-1.48(m,4H),0.39(s,4H)。
步骤E：2-(N-(4-((7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰 基)-3-(6-氮杂螺[2.5]辛烷-6-基)苯基)氨磺酰基)乙酸乙酯(中间体B)的制备
将4-氨基-N-(7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)-2-(6-氮杂螺[2.5]辛烷-6-基)苯甲酰胺(905毫克，2.00毫摩尔，1.0eq.)与三乙胺(708毫克，7.00毫摩尔，3.5eq.)溶于四氢呋喃(22毫升)，氮气保护下，于冰浴中降至0℃，滴加(氯磺酰基)乙酸乙酯(933毫克，5.00毫摩尔，2.5eq.)的四氢呋喃(3毫升)溶液。反应液于0℃反应2小时。反应液加入饱和氯化铵水溶液(80毫升)淬灭，乙酸乙酯(2 x 80毫升)萃取。合并有机相，无水硫酸钠干燥并浓缩。得到的粗产物通过柱层析(石油醚/乙酸乙酯＝7/3；V/V)分离纯化得到2-(N-(4-((7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰基)-3-(6-氮杂螺[2.5]辛烷-6-基)苯基)氨磺酰基)乙酸乙酯(717毫克，黄色固体)，收率：59.5％。
LCMS:(ESI)[M+H]+＝603.2。
1H NMR:(400MHz,DMSO-d6)δ11.55(s,1H),10.53(s,1H),7.92(d,J＝2.4Hz,1H),7.79(d,J＝8.4Hz,1H),7.71(d,J＝2.0Hz,1H),7.16(d,J＝2.0Hz,1H),7.04(dd,J＝8.8,2.0Hz,1H),6.48(d,J＝2.4Hz,1H),6.31(d,J＝1.6Hz,1H),4.66(t,J＝12.4Hz,4H),4.34(s,2H),4.11(q,J＝7.2Hz,2H),3.02-2.96(m,4H),1.59-1.51(m,4H),1.18(t,J＝7.2Hz,3H),0.35(s,4H)。
1.3中间体C的合成
2-(N-(4-((5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶-7-基)氨基甲酰 基)-3-(6-氮杂螺[2.5]辛-6-基)苯基)氨磺酰基)乙酸乙酯
步骤A：4-氯-6-肼基-2-(甲硫基)嘧啶的制备
将4,6-二氯-二甲硫基嘧啶(80.00克，410.13毫摩尔，1.0eq.)溶于四氢呋喃(400毫升)，冰浴下冷却至0℃，缓慢加入溶于四氢呋喃(240毫升)及乙醇(80毫升)的水合肼(80％，20.51克，328.10毫摩尔，0.8eq.)。反应液于25℃反应3小时。反应液浓缩至100毫升体积，加水(180毫升)并于25℃搅拌15分钟。过滤收集析出固体，并用石油醚/乙酸乙酯(19/1，V/V；60毫升)浆化，过滤并将产品通过真空干燥得到4-氯-6-肼基-2-(甲硫基)嘧啶(50.00克，白色固体)，收率：64.0％。
LCMS:(ESI)[M+H]+＝191.0。
1H NMR:(400MHz,DMSO-d6)δ8.85(s,1H),6.51(s,1H),4.60(s,2H),2.42(s,3H)。
步骤B：7-氯-5-(甲硫基)-[1,2,4]三唑并[4,3-c]嘧啶的制备
将4-氯-6-肼基-2-(甲硫基)嘧啶(33.00克，173.09毫摩尔，1.0eq.)加入甲酸(90毫升)中。反应液于105℃反应18小时。反应液降至室温并浓缩。得到的粗产物通过柱层析(石油醚/乙酸乙酯＝1/1；V/V)分离纯化得到7-氯-5-(甲硫基)-[1,2,4]三唑并[4,3-c]嘧啶(3.70克，黄色固体)，收率：10.7％。
LCMS:(ESI)[M+H]+＝201.0。
1H NMR:(400MHz,DMSO-d6)δ9.50(d,J＝0.8Hz,1H),7.81(d,J＝0.8Hz,1H),2.77(s,3H)。
步骤C：7-氯-5-羟基-[1,2,4]三唑并[1,5-c]嘧啶的制备
将7-氯-5-(甲硫基)-[1,2,4]三唑并[4,3-c]嘧啶(3.70克，18.4毫摩尔，1.0eq.)溶于甲醇(600毫升)，加入氢氧化钾水溶液(2M，50毫升)。反应液于85℃反应3小时。反应液降至室温并浓缩除去甲醇。残留物用盐酸(6M)调节至pH＝6，乙酸乙酯(2 x 150毫升)萃取。合并有机相，饱和盐水(100毫升)洗涤，无水硫酸钠干燥并浓缩。得到的粗产物通过柱层析(二氯甲烷/甲醇＝10/1；V/V)分离纯化得到7-氯-5-羟基-[1,2,4]三唑并[1,5-c]嘧啶(600毫克，黄色固体)，收率：50.4％。
LCMS:(ESI)[M+H]+＝171.0。
1H NMR:(400MHz,DMSO-d6)δ8.13(s,1H),6.58(s,1H)。
步骤D：5,7-二氯-[1,2,4]三唑并[1,5-c]嘧啶的制备
将7-氯-5-羟基-[1,2,4]三唑并[1,5-c]嘧啶(1.20克，7.04毫摩尔，1.0eq.)加入三氯氧磷(15毫升)中。反应液于120℃反应4小时。反应液降至室温并缓慢加入温水(50毫升)中淬灭，用饱和碳酸氢钠调节至pH＝7，乙酸乙酯(3 x 100毫升)萃取。合并有机相，无水硫酸钠干燥并浓缩。得到的粗产物通过柱层析(石油醚/乙酸乙酯＝3/1；V/V)分离纯化得到5,7-二氯-[1,2,4]三唑并[1,5-c]嘧啶(440毫克，黄色固体)，收率：33.1％。
LCMS:(ESI)[M+H]+＝188.9。
1H NMR:(400MHz,DMSO-d6)δ8.79(s,1H),8.25(s,1H)。
步骤E：7-氯-5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶的制备
将5,7-二氯-[1,2,4]三唑并[1,5-c]嘧啶(440毫克，2.33毫摩尔，1.0eq.)及N,N-二异丙基乙胺(903毫克，6.98毫摩尔，3.0eq.)溶于异丙醇(10毫升)，加入3,3-二氟氮杂环丁烷盐酸盐(441毫克，2.79毫摩尔，1.2eq.)。反应液于90℃反应3小时。反应液冷却至室温并加水(80毫升)淬灭，乙酸乙酯(2 x 80毫升)萃取。合并有机相，无水硫酸钠干燥并浓缩。得到的粗产物通过柱层析(石油醚/乙酸乙酯＝4/1；V/V)分离纯化得到7-氯-5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶(280毫克，黄色固体)，收率：76.8％。
LCMS:(ESI)[M+H]+＝246.0。
1H NMR:(400MHz,DMSO-d6)δ8.51(s,1H),7.32(s,1H),4.90(t,J＝12.0Hz,4H)。
步骤F：N-(5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶-7-基)-4-硝基 -2-(6-氮杂螺[2.5]辛-6-基)苯甲酰胺的制备
将7-氯-5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶(230毫克，0.93毫摩尔，1.0eq.)溶于二氧六环(15毫升)，依次加入4-硝基-2-(6-氮杂螺[2.5]辛烷-6-基)苯甲酰胺(257毫克，0.93毫摩尔，1.0eq.)，2-二环己基磷-2',6'-二异丙氧基-1,1'-联苯(87毫克，0.19毫摩尔，0.2eq.)，氯(2-二环己基膦基-2',6'-二异丙氧基-1,1'-联苯基)(2-氨基-1,1'-联苯-2-基)钯(II)(72毫克，0.09毫摩尔，0.1eq.)以及碳酸铯(915毫克，2.81毫摩尔，3.0eq.)。氮气保护下，反应液于120℃反应4小时。反应液降至室温并加水(15毫升)稀释，乙酸乙酯(2 x 30毫升)萃取。合并有机相，无水硫酸钠干燥并浓缩。得到的粗产物通过柱层析(石油醚/乙酸乙酯＝3/1；V/V)分离纯化得到N-(5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶-7-基)-4-硝基-2-(6-氮杂螺[2.5]辛-6-基)苯甲酰胺(500毫克，红色固体)，收率：92.6％。
LCMS:(ESI)[M+H]+＝485.0。
1H NMR:(400MHz,DMSO-d6)δ13.39(s,1H),8.42(s,1H),8.31(d,J＝8.4Hz,1H),8.24(d,J＝2.0Hz,1H),8.14(dd,J＝8.4,2.0Hz,1H),7.82(s,1H),4.89(t,J＝12.4Hz,4H),3.13(t,J＝5.2Hz,4H),1.78-1.71(m,4H),0.42(s,4H)。
步骤G：4-氨基-N-(5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶-7- 基)-2-(6-氮杂螺[2.5]辛-6-基)苯甲酰胺的制备
将N-(5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶-7-基)-4-硝基-2-(6-氮杂螺[2.5]辛-6-基)苯甲酰胺(420毫克，0.86毫摩尔，1.0eq.)溶于乙酸(6毫升)，加入锌粉(396毫克，6.10毫摩尔，7.0eq.)。反应液于25℃反应2小时。反应液过滤，滤液浓缩。浓缩液加水(30毫升)稀释，加入饱和碳酸钠中和残留乙酸，乙酸乙酯(3 x 45毫升)萃取。合并有机相，无水硫酸钠干燥并浓缩。得到的粗产物通过柱层析(石油醚/乙酸乙酯＝3/2；V/V)分离纯化得到4-氨基-N-(5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶-7-基)-2-(6-氮杂螺[2.5]辛-6-基)苯甲酰胺(340毫克，黄色固体)，收率：70.5％。
LCMS:(ESI)[M+H]+＝455.0。
1H NMR:(400MHz,CDCl3)δ13.63(s,1H),8.16-8.08(m,3H),6.60-6.55(m,2H),4.85(t,J＝12.0Hz,4H),3.09-3.00(m,4H),1.82-1.68(m,4H),0.43(s,4H)。
步骤H：2-(N-(4-((5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶-7-基)氨基 甲酰基)-3-(6-氮杂螺[2.5]辛-6-基)苯基)氨磺酰基)乙酸乙酯(中间体C)的制备
将4-氨基-N-(5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶-7-基)-2-(6-氮杂螺[2.5]辛-6-基)苯甲酰胺(200毫克，0.44毫摩尔，1.0eq.)及三乙胺(222毫克，2.20毫摩尔，5.0eq.)溶于四氢呋喃(5毫升)，氮气保护下，于冰浴中降至0℃，滴加(氯磺酰基)乙酸乙酯(205毫克，1.10毫摩尔，2.5eq.)溶液。反应液于0℃反应2小时。反应液加入饱和氯化铵水溶液(30毫升)淬灭，乙酸乙酯(3 x 30毫升)萃取。合并有机相，无水硫酸钠干燥并浓缩。得到的粗产物通过柱层析(石油醚/乙酸乙酯＝1/1；V/V)分离纯化得到2-(N-(4-((5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶-7-基)氨基甲酰基)-3-(6-氮杂螺[2.5]辛-6-基)苯基)氨磺酰基)乙酸乙酯(240毫克，黄色固体)，收率：90.2％。
LCMS:(ESI)[M+H]+＝605.0。
1H NMR:(400MHz,DMSO-d6)δ13.69(s,1H),10.71(s,1H),8.39(s,1H),8.12(d,J＝8.4Hz,1H),7.80(s,1H),7.32(d,J＝2.0Hz,1H),7.18(dd,J＝8.4,2.0Hz,1H),4.89(t,J＝12.0Hz,4H),4.41(s,2H),4.10(q,J＝7.2Hz,2H),3.02-2.98(m,4H),1.84-1.75(m,4H),1.16(t,J＝7.2Hz,3H),0.42(s,4H)。
实施例2：化合物2的合成
4-(2,2-二氘-2-羟乙基磺酰胺基)-N-(7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶 -5-基)-2-(6-氮杂螺[2.5]辛-6-基)苯甲酰胺
步骤A：4-(2,2-二氘-2-羟乙基磺酰胺基)-N-(7-(3,3-二氟氮杂环丁烷-1-基)吡唑并 [1,5-a]吡啶-5-基)-2-(6-氮杂螺[2.5]辛-6-基)苯甲酰胺的制备
将氘代氢化锂铝(19毫克，0.45毫摩尔，3.0eq.)加入四氢呋喃(3毫升)，氮气保护下，冷却至0℃，加入2-(N-(4-((7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰基)-3-(6-氮杂螺[2.5]辛烷-6-基)苯基)氨磺酰基)乙酸乙酯(90毫克，0.15毫摩尔，1.0eq.)。反应液于0℃反应0.5小时。反应液加入稀盐酸(1M，3毫升)淬灭并加水(20毫升)稀释，乙酸乙酯(2 x 50毫升)萃取。合并有机相，无水硫酸钠干燥并浓缩。得到的粗产物通过柱层析(石油醚/乙酸乙酯＝2/3；V/V)分离纯化得到4-(2,2-二氘-2-羟乙基磺酰胺基)-N-(7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)-2-(6-氮杂螺[2.5]辛-6-基)苯甲酰胺(44.57毫克，黄色固体)，收率：55.7％。
LCMS:(ESI)[M+H]+＝563.2。
1H NMR:(400MHz,DMSO-d6)δ11.58(s,1H),10.09(s,1H),7.92(d,J＝2.0Hz,1H),7.78(d,J＝8.8Hz,1H),7.70(d,J＝1.6Hz,1H),7.15(d,J＝2.0Hz,1H),7.02(dd,J＝8.4,2.0Hz,1H),6.47(d,J＝2.0Hz,1H),6.30(d,J＝2.0Hz,1H),4.92(s,1H),4.66(t,J＝12.0Hz,4H),3.38-3.33(m,2H),3.02-2.95(m,4H),1.62-1.51(m,4H),0.35(s,4H)。
实施例3：化合物3的合成
1,1-二氘-2-(N-(4-((7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰 基)-3-(6-氮杂螺[2.5]辛-6-基)苯基)氨磺酰基)乙基磷酸二氢酯三氟乙酸盐
步骤A：二苄基(1,1-二氘-2-(N-(4-((7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶 -5-基)氨基甲酰基)-3-(6-氮杂螺[2.5]辛烷-6-基)苯基)氨磺酰基)乙基)磷酸酯的制备
将4-(2,2-二氘-2-羟乙基磺酰胺基)-N-(7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)-2-(6-氮杂螺[2.5]辛-6-基)苯甲酰胺(92毫克，0.16毫摩尔，1.0eq.)溶于二氯甲烷(4毫升)及乙腈(4毫升)的混合溶剂，冰浴下降温至0℃，依次加入四氮唑(15毫克，0.21毫摩尔，1.3eq.)及二苄基N,N-二异丙基亚磷酰胺(66毫克，0.19毫摩尔，1.2eq.)。反应液于0℃反应70分钟。向反应液中加入间氯过氧苯甲酸(33毫克，0.19毫摩尔，1.2eq.)。反应液于0℃继续反应10分钟。反应液加入饱和碳酸氢钠水溶液(50毫升)淬灭，乙酸乙酯(2 x 45毫升)萃取。合并有机相，饱和氯化铵水溶液(80毫升)洗涤，无水硫酸钠干燥并浓缩。得到的粗产品通过柱层析(石油醚/乙酸乙酯＝2/1；V/V)分离纯化得到二苄基(1,1-二氘-2-(N-(4-((7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰基)-3-(6-氮杂螺[2.5]辛烷-6-基)苯基)氨磺酰基)乙基)磷酸酯(32毫克，黄色固体)，收率：23.8％。
LCMS:(ESI)[M+H]+＝823.2。
1H NMR:(400MHz,DMSO-d6)δ12.54(s,1H),8.19(d,J＝8.4Hz,1H),8.07(s,1H),7.87(d,J＝2.4Hz,1H),7.48(d,J＝2.0Hz,1H),7.40-7.32(m,10H),7.30(d,J＝2.0Hz,1H),7.10(dd,J＝8.4,2.0Hz,1H),6.40(d,J＝2.4Hz,1H),6.36(d,J＝1.6Hz,1H),5.11-5.01(m,4H),4.66(t,J＝12.0Hz,4H),3.33(s,2H),3.03(t,J＝5.2Hz,4H),1.59-1.56(s,4H),0.42(s,4H)。
步骤B：1,1-二氘-2-(N-(4-((7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)氨 基甲酰基)-3-(6-氮杂螺[2.5]辛-6-基)苯基)氨磺酰基)乙基磷酸二氢酯三氟乙酸盐的 制备
将二苄基(1,1-二氘-2-(N-(4-((7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰基)-3-(6-氮杂螺[2.5]辛烷-6-基)苯基)氨磺酰基)乙基)磷酸酯(37毫克，0.05毫摩尔，1.0eq.)溶于甲醇(4毫升)，氮气保护下，加入钯碳(10％，37毫克)。抽真空置换氢气3次，氢气氛围下，反应液于25℃反应2小时。反应液过滤，滤液浓缩。得到的粗产品通过制备型HPLC分离纯化得到1,1-二氘-2-(N-(4-((7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰基)-3-(6-氮杂螺[2.5]辛-6-基)苯基)氨磺酰基)乙基磷酸二氢酯三氟乙酸盐(5.9毫克，黄色固体)，收率：17.3％。
LCMS:(ESI)[M+H]+＝643.2。
1H NMR:(400MHz,DMSO-d6)δ11.56(s,1H),10.25(s,1H),7.92(d,J＝2.4Hz,1H),7.79(d,J＝8.4Hz,1H),7.71(d,J＝1.6Hz,1H),7.15(d,J＝2.0Hz,1H),7.05(dd,J＝8.4,2.0Hz,1H),6.48(d,J＝2.4Hz,1H),6.31(d,J＝1.6Hz,1H),4.66(t,J＝12.4Hz,4H),3.54(s,2H),3.04-2.97(m,4H),1.65-1.44(m,4H),0.35(s,4H)。
实施例4：化合物4的合成
N-(7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)-2-(6-氮杂螺[2.5]辛-6- 基)-4-(1,1,2,2-四氘-2-羟乙基磺酰胺基)苯甲酰胺
步骤A：N-(7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)-2-(6-氮杂螺[2.5] 辛-6-基)-4-(1,1,2,2-四氘-2-羟乙基磺酰胺基)苯甲酰胺的制备
将2-(N-(4-((7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰基)-3-(6-氮杂螺[2.5]辛烷-6-基)苯基)氨磺酰基)乙酸乙酯(70毫克，0.12毫摩尔，1.0eq.)溶于氘代甲醇-d4(2毫升)，于冰浴下冷却至0℃，加入硼氘化钠(141毫克，3.37毫摩尔，28.0eq.)。反应液于30℃反应48小时。反应液加水(30毫升)淬灭，乙酸乙酯(2 x 40毫升)萃取。合并有机相，无水硫酸钠干燥并浓缩。得到的粗产物通过柱层析(石油醚/乙酸乙酯＝1/1；V/V)分离纯化得到N-(7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)-2-(6-氮杂螺[2.5]辛-6-基)-4-(1,1,2,2-四氘-2-羟乙基磺酰胺基)苯甲酰胺(20.49毫克，黄色固体)，收率：30.2％。
LCMS:(ESI)[M+H]+＝565.2。
1H NMR:(400MHz,CDCl3)δ12.46(s,1H),8.15(d,J＝8.4Hz,1H),7.80(d,J＝2.4Hz,1H),7.41(d,J＝2.0Hz,1H),7.27(d,J＝2.4Hz,1H),6.99(dd,J＝8.4,2.0Hz,1H),6.89(s,1H),6.33(d,J＝2.0Hz,1H),6.29(d,J＝2.0Hz,1H),4.59(t,J＝12.0Hz,4H),3.02(t,J＝5.2Hz,4H),1.74-1.52(m,4H),0.38(s,4H)。
实施例5：化合物5的合成
2-(N-(4-((7-(3,3-二氟氮杂环丁-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰基)-3-(6-氮杂 螺[2.5]辛烷-6-基)苯基)氨基磺酰基)乙基-1,1,2,2-四氘磷酸二氢酯
步骤A：二苄基(2-(N-(4-((7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)氨基 甲酰基)-3-(6-氮杂螺[2.5]辛-6-基)苯基)氨磺酰基)1,1,2,2-四氘-乙基)磷酸酯的制备
将N-(7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)-2-(6-氮杂螺[2.5]辛-6-基)-4-(1,1,2,2-四氘-2-羟乙基磺酰胺基)苯甲酰胺(120毫克，0.21毫摩尔，1.0eq.)溶于二氯甲烷(4毫升)及乙腈(4毫升)的混合溶剂，冰浴下降温至0℃，依次加入四氮唑(19毫克，0.27毫摩尔，1.3eq.)及二苄基N,N-二异丙基亚磷酰胺(86毫克，0.25毫摩尔，1.2eq.)。反应液于0℃反应70分钟。向反应液中加入间氯过氧苯甲酸(43毫克，0.25毫摩尔，1.2eq.)。反应液于0℃继续反应10分钟。反应液加入饱和碳酸氢钠水溶液(40毫升)淬灭，乙酸乙酯(2 x 35毫升)萃取。合并有机相，饱和氯化铵水溶液(80毫升)洗涤，无水硫酸钠干燥并浓缩。得到的粗产品通过柱层析(石油醚/乙酸乙酯＝4/1；V/V)分离纯化得到二苄基(2-(N-(4-((7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰基)-3-(6-氮杂螺[2.5]辛-6-基)苯基)氨磺酰基)1,1,2,2-四氘-乙基)磷酸酯(150毫克，白色固体)，收率：85.6％。
LCMS:(ESI)[M+H]+＝825.2。
1H NMR:(400MHz,DMSO-d6)δ11.36(s,1H),10.14(s,1H),7.69(d,J＝2.0Hz,1H),7.56(d,J＝8.4Hz,1H),7.47(d,J＝1.6Hz,1H),7.21-7.06(m,10H),6.95-6.89(m,1H),6.81(dd,J＝8.4,2.0Hz,1H),6.25(d,J＝2.4Hz,1H),6.05(d,J＝1.6Hz,1H),4.81-4.69(m,4H),4.43(t,J＝12.0Hz,4H),2.75-2.66(m,4H),1.36-1.23(m,4H),0.13-0.05(m,4H)。
步骤B：2-(N-(4-((7-(3,3-二氟氮杂环丁-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰 基)-3-(6-氮杂螺[2.5]辛烷-6-基)苯基)氨基磺酰基)乙基-1,1,2,2-四氘磷酸二氢酯的制 备
将二苄基(2-(N-(4-((7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰基)-3-(6-氮杂螺[2.5]辛-6-基)苯基)氨磺酰基)1,1,2,2-四氘-乙基)磷酸酯(150毫克，0.18毫摩尔，1.0eq.)溶于甲醇(2毫升)，氮气保护下，加入钯碳(10％，80毫克)。抽真空置换氢气3次，氢气氛围下，反应液于25℃反应2小时。反应液过滤，滤液浓缩。得到的粗产品通过制备型HPLC分离纯化得到2-(N-(4-((7-(3,3-二氟氮杂环丁-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰基)-3-(6-氮杂螺[2.5]辛烷-6-基)苯基)氨基磺酰基)乙基-1,1,2,2-四氘磷酸二氢酯(4.09毫克，绿色固体)，收率：3.5％。
LCMS:(ESI)[M+H]+＝645.2。
1H NMR:(400MHz,DMSO-d6)δ11.51(s,1H),7.74(d,J＝2.0Hz,1H),7.58(d,J＝8.4Hz,1H),7.53(s,1H),7.09(s,1H),7.00-6.92(m,2H),6.29(d,J＝2.0Hz,1H),6.13(s,1H),4.48(t,J＝12.0Hz,4H),2.86-2.79(m,4H),1.42-1.32(m,4H),0.16(s,4H)。
实施例6：化合物6的合成
N-(5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶-7-基)-4-(1,1,2,2-四氘-2- 羟乙基磺酰胺基)-2-(6-氮杂螺[2.5]辛-6-基)苯甲酰胺
步骤A：N-(5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶-7-基)-4-(1,1,2,2- 四氘-2-羟乙基磺酰胺基)-2-(6-氮杂螺[2.5]辛-6-基)苯甲酰胺的制备
将2-(N-(4-((5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶-7-基)氨基甲酰基)-3-(6-氮杂螺[2.5]辛-6-基)苯基)氨磺酰基)乙酸乙酯(200毫克，0.33毫摩尔，1.0eq.)溶于氘代甲醇-d4(3毫升)，于冰浴下冷却至0℃，加入硼氘化钠(554毫克，13.23毫摩尔，40.0eq.)。反应液于10℃反应48小时。反应液加水(100毫升)淬灭，乙酸乙酯(2 x 80毫升)萃取。合并有机相，饱和盐水(200毫升)洗涤，无水硫酸钠干燥并浓缩。得到的粗产物通过柱层析(石油醚/乙酸乙酯＝1/1；V/V)分离纯化得到N-(5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶-7-基)-4-(1,1,2,2-四氘-2-羟乙基磺酰胺基)-2-(6-氮杂螺[2.5]辛-6-基)苯甲酰胺(80毫克，白色固体)，收率：42.7％。
LCMS:(ESI)[M+H]+＝567.2。
1H NMR:(400MHz,DMSO-d6)δ13.72(s,1H),10.28(s,1H),8.39(s,1H),8.11(d,J＝8.4Hz,1H),7.81(s,1H),7.32(d,J＝2.0Hz,1H),7.17(dd,J＝8.4,2.0Hz,1H),4.88(t,J＝12.0Hz,4H),3.00(t,J＝4.8Hz,4H),1.92-1.40(m,4H),0.43(s,4H)。
实施例7：化合物7的合成
2-(N-(4-((5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶-7-基)氨基甲酰 基)-3-(6-氮杂螺[2.5]辛-6-基)苯基)氨磺酰基)乙基-1,1,2,2-四氘-磷酸二氢酯三氟乙 酸盐
步骤A：二苄基(2-(N-(4-((5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶-7- 基)氨基甲酰基)-3-(6-氮杂螺[2.5]辛-6-基)苯基)氨磺酰基)1,1,2,2-四氘-乙基)磷酸酯 的制备
将N-(5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶-7-基)-4-(1,1,2,2-四氘-2-羟乙基磺酰胺基)-2-(6-氮杂螺[2.5]辛-6-基)苯甲酰胺(60毫克，0.11毫摩尔，1.0eq.)溶于二氯甲烷(2毫升)及乙腈(2毫升)的混合溶剂，氮气保护下，冰浴下降温至0℃，依次加入四氮唑(10毫克，0.14毫摩尔，1.3eq.)及二苄基N,N-二异丙基亚磷酰胺(44毫克，0.13毫摩尔，1.2eq.)。反应液于0℃反应70分钟。向反应液中加入间氯过氧苯甲酸(85％，26毫克，0.13毫摩尔，1.2eq.)。反应液于0℃继续反应10分钟。反应液加入饱和碳酸氢钠水溶液(50毫升)淬灭，乙酸乙酯(2 x 45毫升)萃取。合并有机相，饱和氯化铵水溶液(80毫升)洗涤，无水硫酸钠干燥并浓缩。得到的粗产品通过柱层析(石油醚/乙酸乙酯＝2/1；V/V)分离纯化得到二苄基(2-(N-(4-((5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶-7-基)氨基甲酰基)-3-(6-氮杂螺[2.5]辛-6-基)苯基)氨磺酰基)1,1,2,2-四氘-乙基)磷酸酯(41毫克，黄色固体)，收率：46.8％。
LCMS:(ESI)[M+H]+＝827.2。
1H NMR:(400MHz,DMSO-d6)δ13.67(s,1H),10.60(s,1H),8.45(d,J＝1.2Hz,1H),8.15(d,J＝8.8Hz,1H),7.86(s,1H),7.45-7.34(m,10H),7.33(d,J＝2.0Hz,1H),7.23(dd,J＝8.8,2.0Hz,1H),5.07-4.99(m,4H),4.93(t,J＝12.4Hz,4H),2.97(t,J＝4.8Hz,4H),1.95-1.60(m,4H),0.42(s,4H)。
步骤B：2-(N-(4-((5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶-7-基)氨基 甲酰基)-3-(6-氮杂螺[2.5]辛-6-基)苯基)氨磺酰基)乙基-1,1,2,2-四氘-磷酸二氢酯三 氟乙酸盐的制备
将二苄基(2-(N-(4-((5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶-7-基)氨基甲酰基)-3-(6-氮杂螺[2.5]辛-6-基)苯基)氨磺酰基)1,1,2,2-四氘-乙基)磷酸酯(35毫克，0.04毫摩尔，1.0eq.)溶于四氢呋喃(2毫升)，氮气保护下，加入钯碳(10％，35毫克)。氢气氛围下，反应液于20℃反应2小时。反应液过滤，滤液浓缩。得到的粗产品通过prep-HPLC(三氟乙酸)分离纯化得到2-(N-(4-((5-(3,3-二氟氮杂环丁烷-1-基)-[1,2,4]三唑并[1,5-c]嘧啶-7-基)氨基甲酰基)-3-(6-氮杂螺[2.5]辛-6-基)苯基)氨磺酰基)乙基-1,1,2,2-四氘-磷酸二氢酯三氟乙酸盐(6.62毫克，白色固体)，收率：21.8％。
LCMS:(ESI)[M+H]+＝647.2。
1H NMR:(400MHz,DMSO-d6)δ13.73(s,1H),10.43(s,1H),8.40(s,1H),8.11(d,J＝8.8Hz,1H),7.81(s,1H),7.31(d,J＝2.0Hz,1H),7.19(dd,J＝8.8,2.0Hz,1H),4.88(t,J＝12.4Hz,4H),3.01(t,J＝4.8Hz,4H),2.07-1.47(m,4H),0.43(s,4H)。
实施例8：化合物8的合成
2-(N-(4-((7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰基)-3-(6-氮 杂螺[2.5]辛-6-基)苯基)氨磺酰基)1,1,2,2-四氘-乙基硫酸酯三氟乙酸盐
步骤A：2-(N-(4-((7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰 基)-3-(6-氮杂螺[2.5]辛-6-基)苯基)氨磺酰基)1,1,2,2-四氘-乙基硫酸酯三氟乙酸盐的 制备
将N-(7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)-2-(6-氮杂螺[2.5]辛-6-基)-4-(1,1,2,2-四氘-2-羟乙基磺酰胺基)苯甲酰胺(50毫克，0.09毫摩尔，1.0eq.)溶于二氯甲烷(2.5毫升)，氮气保护下，降温至-30℃，依次加入三乙胺(90毫克，0.89毫摩尔，10.0eq.)及氯磺酸(30毫克，0.26毫摩尔，2.9eq.)。反应液于25℃反应18小时。反应加水(5毫升)淬灭，二氯甲烷(3 x 20毫升)萃取。合并有机相，无水硫酸钠干燥并浓缩。得到的粗产品通过制备型HPLC(三氟乙酸)分离纯化得到2-(N-(4-((7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰基)-3-(6-氮杂螺[2.5]辛-6-基)苯基)氨磺酰基)1,1,2,2-四氘-乙基硫酸酯三氟乙酸盐(1.74毫克，淡黄色固体)，收率：2.6％。
LCMS:(ESI)[M+H]+＝645.4(free)。
1H NMR:(400MHz,DMSO-d6)δ11.46(s,1H),10.18(s,1H),7.92(d,J＝2.0Hz,1H),7.76(d,J＝8.4Hz,1H),7.71(d,J＝2.0Hz,1H),7.19-7.15(m,1H),7.05(dd,J＝8.4,2.0Hz,1H),6.48(d,J＝2.4Hz,1H),6.34(d,J＝1.6Hz,1H),4.66(t,J＝12.4Hz,4H),3.07-3.00(m,4H),1.58-1.49(m,4H),0.35(s,4H)。
比较例1：比较例化合物9的合成
N-(7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)-4-(2-羟乙基磺酰胺 基)-2-(6-氮杂螺[2.5]辛烷-6-基)苯甲酰胺
步骤A：N-(7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)-4-(2-羟乙基磺酰 胺基)-2-(6-氮杂螺[2.5]辛烷-6-基)苯甲酰胺的制备
将2-(N-(4-((7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰基)-3-(6-氮杂螺[2.5]辛烷-6-基)苯基)氨磺酰基)乙酸乙酯(700毫克，1.16毫摩尔，1.0eq.)溶于四氢呋喃(10毫升)，加入硼氢化钠(132毫克，3.49毫摩尔，3.0eq.)。反应液于25℃反应4小时。反应液加水(30毫升)淬灭，乙酸乙酯(3 x 40毫升)萃取。合并有机相，无水硫酸钠干燥并浓缩。得到的粗产物通过反相柱层析(水/乙腈＝3/2；V/V)分离纯化得到N-(7-(3,3-二氟氮杂环丁烷-1-基)吡唑并[1,5-a]吡啶-5-基)-4-(2-羟乙基磺酰胺基)-2-(6-氮杂螺[2.5]辛烷-6-基)苯甲酰胺(411毫克，淡黄色固体)，收率：63.1％。
LCMS:(ESI)[M+H]+＝561.0。
1H NMR:(400MHz,DMSO-d6)δ11.58(s,1H),10.10(s,1H),7.92(d,J＝2.4Hz,1H),7.79(d,J＝8.4Hz,1H),7.71(d,J＝1.6Hz,1H),7.16(d,J＝1.6Hz,1H),7.03(dd,J＝8.4,2.0Hz,1H),6.48(d,J＝2.4Hz,1H),6.31(d,J＝1.6Hz,1H),4.96(t,J＝5.4Hz,1H),4.66(t,J＝12.0Hz,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)。
比较例2：比较例化合物10的合成
N-(7-(4,4-二氟哌啶-1-基)吡唑并[1,5-a]吡啶-5-基)-4-(2-羟乙基磺酰胺基)-2-(6-氮杂 螺[2.5]辛烷-6-基)苯甲酰胺
步骤A：5-溴-7-氯吡唑并[1,5-a]吡啶的制备
将5-溴吡唑并[1,5-a]吡啶(1.18克，6.00毫摩尔，1.0eq.)溶于四氢呋喃(15毫升)，氮气保护下，于干冰/乙醇浴中降至-78℃，滴加双三甲基硅基胺基锂(1M，7.2毫升，7.20毫摩尔，1.2eq.)。反应液于-78℃反应0.5小时。向反应液中滴加六氯乙烷(1.70克，7.20毫摩尔，1.2eq.)的四氢呋喃(4毫升)溶液。反应液于-78℃继续反应1小时。反应液加入饱和氯化铵(80毫升)淬灭，用乙酸乙酯(2×70毫升)萃取。合并有机相，无水硫酸钠干燥并浓缩。得到的粗产物通过柱层析(石油醚/乙酸乙酯＝20/1；V/V)分离纯化得到5-溴-7-氯吡唑并[1,5-a]吡啶(1.20克，淡黄色固体)，收率：86.3％。
LCMS:(ESI)[M+H]+＝231.0。
1H NMR:(400MHz,CDCl3)δ8.06(d,J＝2.0Hz,1H),7.72(d,J＝2.0Hz,1H),7.03(d,J＝1.6Hz,1H),6.61(d,J＝2.0Hz,1H)。
步骤B：5-溴-7-(4,4-二氟哌啶-1-基)吡唑并[1,5-a]吡啶的制备
将5-溴-7-氯吡唑并[1,5-a]吡啶(926毫克，4.00毫摩尔，1.0eq.)溶于N-甲基吡咯烷酮(8毫升)，加入4,4-二氟吡啶(1.45克，12.00毫摩尔，3.0eq.)。反应液于135℃反应20小时。反应液降至室温并加入饱和氯化铵水溶液(50毫升)稀释，用乙酸乙酯(2×70毫升)萃取。合并有机相，饱和食盐水(2×70毫升)洗涤，无水硫酸钠干燥并浓缩。得到的粗产物通过柱层析(石油醚/乙酸乙酯＝20/1；V/V)分离纯化得到5-溴-7-(4,4-二氟哌啶-1-基)吡唑并[1,5-a]吡啶(864毫克，黄色固体)，收率：68.6％。
LCMS:(ESI)[M+H]+＝318.0。
1H NMR:(400MHz,CDCl3)δ7.97(d,J＝2.0Hz,1H),7.45(d,J＝2.0Hz,1H),6.49(d,J＝2.0Hz,1H),6.29(d,J＝1.6Hz,1H),3.61-3.50(m,4H),2.36-2.22(m,4H)。
步骤C：N-(7-(4,4-二氟哌啶-1-基)吡唑并[1,5-a]吡啶-5-基)-4-硝基-2-(6-氮杂螺[2.5] 辛烷-6-基)苯甲酰胺的制备
将5-溴-7-(4,4-二氟哌啶-1-基)吡唑并[1,5-a]吡啶(300毫克，0.95毫摩尔，1.0eq.)溶于二氧六环(10毫升)，依次加入4-硝基-2-(6-氮杂螺[2.5]辛-6-基)苯甲酰胺(314毫克，1.14毫摩尔，1.2eq.)，2-(二环己基膦)-3,6-二甲氧基-2',4',6'-三异丙基-1,1'-联苯(102毫克，0.19毫摩尔，0.2eq.)，甲烷磺酸(2-二环己基膦)-3,6-二甲氧基-2',4',6'-三异丙基-1,1'-联苯)(2'-氨基-1,1'-联苯-2-基)钯(II)(86毫克，0.10毫摩尔，0.1eq.)以及碳酸铯(928毫克，2.85毫摩尔，3.0eq.)。氮气保护下，反应液于微波下加热至120℃反应2小时。反应液降至室温并加水(100毫升)稀释，用乙酸乙酯(2×100毫升)萃取。合并有机相，饱和食盐水(100毫升)洗涤，无水硫酸钠干燥并浓缩。得到的粗产物通过柱层析(乙酸乙酯/石油醚＝3/7；V/V)分离纯化得到N-(7-(4,4-二氟哌啶-1-基)吡唑并[1,5-a]吡啶-5-基)-4-硝基-2-(6-氮杂螺[2.5]辛烷-6-基)苯甲酰胺(450毫克，黄色固体)，收率：94.7％。
LCMS:(ESI)[M+H]+＝511.2。
步骤D：4-氨基-N-(7-(4,4-二氟哌啶-1-基)吡唑并[1,5-a]吡啶-5-基)-2-(6-氮杂螺[2.5] 辛烷-6-基)苯甲酰胺的制备
将N-(7-(4,4-二氟哌啶-1-基)吡唑并[1,5-a]吡啶-5-基)-4-硝基-2-(6-氮杂螺[2.5]辛烷-6-基)苯甲酰胺(290毫克，0.57毫摩尔，1.0eq.)溶于乙醇(10毫升)，加入氯化亚锡(517毫克，2.73毫摩尔，4.8eq.)。反应液于70℃反应2小时。反应液降至室温并加水(80毫升)稀释，用乙酸乙酯(2×100毫升)萃取。合并有机相，饱和食盐水(100毫升)洗涤，无水硫酸钠干燥并浓缩。得到的粗产物通过柱层析(乙酸乙酯/石油醚＝1/1；V/V)分离纯化得到4-氨基-N-(7-(4,4-二氟哌啶-1-基)吡唑并[1,5-a]吡啶-5-基)-2-(6-氮杂螺[2.5]辛烷-6-基)苯甲酰胺(120毫克，黄色油状物)，收率：44.0％。
LCMS:(ESI)[M+H]+＝481.2。
1H NMR:(400MHz,CDCl3)δ12.78(s,1H),8.05(d,J＝8.0Hz,1H),7.86(d,J＝2.4Hz,1H),7.72(d,J＝2.0Hz,1H),6.64(d,J＝1.6Hz,1H),6.53-6.48(m,2H),6.38(d,J＝2.0Hz,1H),4.04(s,2H),3.54-3.49(m,4H),2.99(t,J＝4.8Hz,4H),2.26-2.18(m,4H),1.58-1.48(s,4H),0.37(s,4H)。
步骤E：2-(N-(4-((7-(4,4-二氟哌啶-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰基)-3-(6- 氮杂螺[2.5]辛烷-6-基)苯基)氨磺酰基)乙酸乙酯的制备
将4-氨基-N-(7-(4,4-二氟哌啶-1-基)吡唑并[1,5-a]吡啶-5-基)-2-(6-氮杂螺[2.5]辛烷-6-基)苯甲酰胺(120毫克，0.25毫摩尔，1.0eq.)与三乙胺(76毫克，0.75毫摩尔，3.0eq.)溶于四氢呋喃(8毫升)，氮气保护下，于冰浴中降至0℃，滴加(氯磺酰基)乙酸乙酯(94毫克，0.50毫摩尔，2.0eq.)。反应液于18℃反应2小时。反应液加水(50毫升)淬灭，用乙酸乙酯(2×60毫升)萃取。合并有机相，无水硫酸钠干燥并浓缩。得到2-(N-(4-((7-(4,4-二氟哌啶-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰基)-3-(6-氮杂螺[2.5]辛烷-6-基)苯基)氨磺酰基)乙酸乙酯(120毫克，粗品，黄色油状物)。
LCMS:(ESI)[M+H]+＝631.2。
1H NMR:(400MHz,CDCl3)δ12.56(s,1H),8.19(d,J＝8.8Hz,1H),7.89(d,J＝2.0Hz,1H),7.73(d,J＝2.0Hz,1H),7.33(s,1H),7.30(d,J＝2.0Hz,1H),7.08(dd,J＝8.8,2.0Hz,1H),6.59(d,J＝2.0Hz,1H),6.41(d,J＝2.4Hz,1H),4.20(q,J＝7.2Hz,2H),3.93(s,2H),3.54-3.49(m,4H),3.03(t,J＝5.2Hz,4H),2.26-2.17(m,4H),1.69-1.56(m,4H),1.24(t,J＝7.2Hz,3H),0.38(s,4H)。
步骤F：N-(7-(4,4-二氟哌啶-1-基)吡唑并[1,5-a]吡啶-5-基)-4-(2-羟乙基磺酰胺 基)-2-(6-氮杂螺[2.5]辛烷-6-基)苯甲酰胺的制备
将2-(N-(4-((7-(4,4-二氟哌啶-1-基)吡唑并[1,5-a]吡啶-5-基)氨基甲酰基)-3-(6-氮杂螺[2.5]辛烷-6-基)苯基)氨磺酰基)乙酸乙酯(60毫克，0.09毫摩尔，1.0eq.)溶于四氢呋喃(5毫升)，加入硼氢化钠(29毫克，0.76毫摩尔，8.0eq.)。反应液于18℃反应18小时。反应液加水(30毫升)淬灭，用乙酸乙酯(2×50毫升)萃取。合并有机相，无水硫酸钠干燥并浓缩。得到的粗产物通过制备型HPLC分离纯化得到N-(7-(4,4-二氟哌啶-1-基)吡唑并[1,5-a]吡啶-5-基)-4-(2-羟乙基磺酰胺基)-2-(6-氮杂螺[2.5]辛烷-6-基)苯甲酰胺(24.07毫克，淡黄色固体)，收率：43.0％。
LCMS:(ESI)[M+H]+＝589.2。
1H NMR:(400MHz,CDCl3)δ12.72(s,1H),8.23(d,J＝8.4Hz,1H),7.98(d,J＝2.4Hz,1H),7.82(d,J＝1.2Hz,1H),7.38(d,J＝1.6Hz,1H),7.23(s,1H),7.08(dd,J＝8.4,1.6Hz,1H),6.66(d,J＝1.6Hz,1H),6.50(d,J＝2.4Hz,1H),4.22-4.11(m,2H),3.64-3.57(m,4H),3.40-3.31(m,2H),3.11(t,J＝4.8Hz,4H),2.78(s,1H),2.38-2.26(m,4H),1.82-1.68(m,4H),0.47(s,4H)。
实施例9
生物学检测
化合物对KIF18A酶抑制活性的测定
使用ADP-Glo发光法测试化合物对KIF18A酶活性的抑制。在384孔板中将人KIF18A(1-467)蛋白(Chempartner，CP20220309-N-His cleaved-BV)在反应缓冲液(15mM Tris，pH 7.5，10mM MgCl2，0.01％Pluronic F-68，2％DMSO，1μM紫杉醇，30μg/mL微管蛋白)中与不同浓度的待测化合物或DMSO在室温孵育15分钟。将底物(Cytoskeleton，MT002)和ATP(Promega，V916B)加入384孔板反应孔中，28℃孵育60分钟。再加入ADP-Glo试剂1(Promega，V9102)，反应120分钟。加入ADP-Glo试剂2室温放置60分钟后，使用EnVision2104 Multilable Reader(PerkinElmer，411177291)读取RLU值。将上述数据通过以下公式转换为抑制百分率。
其中，“min”为不加酶进行反应的对照样孔读数；“max”为加入DMSO作为对照时的对照孔读数。
将数据导入MS Excel并使用XLFit excel add-in version 5.4.0.8进行曲线拟合，拟合公式：
其中“Bottom”代表曲线最小值；“Top”代表曲线最大值；“IC50”代表最大效应浓度的一半；“HillSlope”代表曲线的斜率。
实验结果如表1所示。
表1.化合物对KIF18A酶抑制活性的测定(IC50，nM)
结果表明，氘代化合物2、4和6对KIF18A蛋白都有较好的抑制活性。
化合物对肿瘤细胞增殖抑制活性的测定
将卵巢癌细胞OVCAR3(ATCC，HTB-161)、结直肠癌细胞HT-29(ATCC，HTB-38)和三阴乳腺癌细胞HCC1806(ATCC，CRL-2335)在37℃、5％CO2的培养箱中进行培养。收集对数生长期细胞，调整单细胞悬液浓度，加入96孔板中，使细胞密度为3000个/孔。空白对照孔中加入不含细胞的培养液。培养过夜后，用不同浓度的化合物处理细胞，同时设置DMSO(0.25％)的溶媒对照。继续培养6天后，采用Promega CellTiter-Glo发光法细胞活性检测试剂盒(Promege-G7573)测定活细胞数量。每孔加入75μL CellTiter-Glo工作液，避光震荡2分钟至细胞裂解，室温放置10分钟后在EnVision多功能酶标仪(PerkinElmer)上检测发光信号。将测定数据RLU通过下述公式转换为抑制率：
然后用Graphpad Prism软件作抑制曲线图和计算IC50。
实验结果如表2所示。
表2.化合物对OVCAR3、HT-29和HCC1806肿瘤细胞增殖抑制活性的测定(IC50，nM)
如表2所示，化合物2-7对卵巢癌细胞OVCAR3、结直肠癌细胞HT-29和三阴乳腺癌细胞HCC1806都有较强的抑制增殖活性。
化合物对正常细胞的细胞毒性测定
将人胚胎肾细胞HEK293细胞(ATCC，CRL-157)在37℃、5％CO2的培养箱中进行培养。化合物对HEK293细胞的细胞毒性测试方法与肿瘤细胞增殖抑制活性的测试方法一致，具体参见上述测试方法。
实验结果如图1所示，对照化合物AMG650(MCE，HY-132840)在10μM浓度下显示出一定的细胞毒性，在30μM浓度下几乎完全抑制细胞的生长，显示出很强的细胞毒性。但是化合物4和5对HEK293细胞的生长抑制活性仅为AMG650的一半，细胞毒性相对较低。
化合物对CYP450酶抑制活性的测试
将肝微粒体(Biopredic International，终浓度0.2mg/mL)与不同浓度的化合物(终浓度0.01μM、0.04μM、0.12μM、0.37μM、1.11μM、3.33μM和10μM)对照化合物AMG650、阳性对照(CYP1A2：α-萘黄酮；CYP2C9：磺胺苯吡唑；CYP2C19：奥美拉唑；CYP3A4：酮康唑；CYP2D6：奎尼丁；上述化合物均购自Sigma-Aldrich公司)或阴性对照(1％DMSO)混合后，分别加入各CYP亚酶的指针底物(CYP1A2：30μM非那西汀；CYP2C9：10μM双氯芬酸钠；CYP2C19：35μM S-美芬妥英；CYP3A4：5μM咪达唑仑和80μM睾酮；CYP2D6：5μM右美沙芬；上述化合物均购自Sigma-Aldrich公司)，37℃孵育10min。加入辅酶NADPH(Roche，终浓度1mM)，37℃孵育指定的时间(CYP 3A4：5min；CYP 1A2、CYP 2C9和CYP 2D6：10min；CYP 2C19：45min)。各孵育管中加入含内标工作液的乙腈终止反应，涡旋混匀，3220g离心15min。取50μL上清液加入等量超纯水后涡旋混匀，用LC-MS/MS方法检测代谢产物生成量。使用SCIEX公司的Analyst软件(Analyst 1.6.3)输出原始图谱、峰面积比等数据。将测定数据通过下述公式转换为抑制率：
抑制率％＝(1-实验或阳性对照组中代谢产物生成量/阴性对照组中代谢产物生成量)×100％
然后用Graphpad Prism软件作抑制曲线图和计算IC50。
实验结果如表3所示。
表3.化合物对CYP450酶抑制活性的测试(IC50，μM)
实验结果表明，对照化合物AMG650对CYP450亚型CYP2C9有一定抑制作用，提示该化合物有药物-药物相互作用的潜在风险；而本发明的化合物4和5对CYP450酶亚型，包括CYP2C9，均没有抑制作用，安全性较高。
化合物对hERG钾通道电流抑制活性的测试
HEK293-hERG细胞(稳定表达hERG通道蛋白的人胚肾细胞，Sophion Biosciences)在含有5％CO2的37℃细胞培养箱中的DMEM培养液里(Cytiva)培养。细胞培养液中添加10％的胎牛血清(Sigma-Aldrich)及1％的青链霉素混合液(Solarbio)。用0.25％胰蛋白酶(Gibco)消化后加入细胞培养液，得到单个细胞悬液，加入到倒置显微镜的记录槽中，电流记录过程中将记录槽温度控制在20℃-25℃。溶媒对照品(0.1％DMSO)、化合物4(终浓度10或30μM)、比较例化合物10(终浓度10或30μM)及阳性对照(西沙比利，MCE，终浓度0.1μM)用8通道灌流系统完成给药。给药时用微操纵器将给药系统输出端移至细胞槽中的选定的细胞周围。打开三通开关时，输出端流出的药物可以立即将其下方的细胞浸润。使用Clampex 10.6软件进行电压钳参数设置。在全细胞模式下，将细胞钳制在钳制电位-80mV持续100ms，首先超极化至-90mV持续100ms，之后回到-80mV持续100ms；再去极至+40mV持续500ms以启动hERG通道；然后施以100ms的斜坡刺激复极至-80mV(1.2V/s)，诱发hERG通道特征性的尾电流；最后维持-80mV持续3000ms。刺激频率为0.2Hz(刺激时间为5s，start to start)。使用膜片钳放大器和数模转换器记录膜电流。使用Clampex 10.6软件采集电流，电流信号输入经过2kHz的滤波，以5kHz的频率进行数字化处理。使用Clampfit 10.6软件测量尾电流峰值并用于数据获取及分析。统计分析所取数据为每个浓度给药最后10次记录尾电流峰值的均值。以溶媒对照品持续灌流记录的尾电流峰值的均值作为100％，抑制率用于IC50的计算，其计算公式如下：
抑制率％＝(1-给药后尾电流峰值的均值/溶媒对照尾电流峰值的均值)×100％
实验结果如表4所示，非氘代比较例化合物10在10和30μM下对HEK293细胞hERG钾通道电流均有较强的抑制作用，并呈现剂量依赖性，提示该化合物可能具有心脏毒性。而本发明的氘代化合物4在10μM和30μM下对hERG钾通道电流均没有显著的抑制作用。以上结果提示本发明的化合物的心脏毒性风险低于非氘代的比较例化合物。
表4.化合物对hERG钾通道电流的抑制率(％，平均值±标准差)
化合物的溶解度测试
将化合物4、5和对照化合物AMG650用DMSO配制成10mM储存液，取8μL储存液加入792μL磷酸盐缓冲液(100mM，pH7.4)中，室温振摇1hr后，12000rpm离心10min。将上清转移至新管，用100mM磷酸盐缓冲液分别稀释10倍和100倍，取5μL未稀释或稀释样品加入含内标工作液的乙腈中，用LC-MS/MS方法检测化合物浓度。
实验结果如表5所示。
表5.化合物的溶解度(μM)
如表5所示，化合物4的溶解度远高于对照化合物AMG650(大于25倍)，磷酸酯化合物5则进一步提高了溶解度(是AMG650的875倍以上)。化合物4和5由于安全性和溶解度的提高，在临床上可在较高的剂量下获得较高的药物暴露量，从而提升药物的临床疗效。
化合物的药代动力学测试
针对每种待测化合物各取CD-1小鼠(上海吉辉实验动物饲养有限公司)18只，雄性，6-8周龄，体重28-31g，随机分为2组(A组和B组)，每组9只，禁食12小时。A组动物按10mg/kg剂量灌胃给予待测化合物溶液；B组动物按3mg/kg剂量尾静脉注射给予待测化合物溶液。给药前取空白血，A组动物分别于给药后0.25h、0.5h、1h、2h、4h、8h、24h取静脉血约110μL，B组动物分别于给药后0.083h、0.25h、0.5h、1h、2h、4h、8h、24h取静脉血约110μL，均置于加有EDTA-K2抗凝(GREAGENT，G41456A)的试管中，离心后，取血浆，-70℃保存供测试。
使用代谢动力学数据分析软件WinNonlin(version 8.0.0.3176)对全血浓度数据进行分析。利用非房室模型法(NCA)计算T1/2、Cmax、Tmax、AUC、CL和F％等。其中AUClast表示从给药时间开始到最后一个采样点的时间段内的药时曲线下面积，即血药浓度-时间曲线下面积；CL(iv)表示静脉给药的药物清除率；T1/2表示药物的血浆半衰期，即指血浆药物浓度下降一半所需时间；Cmax表示给药后药物在血浆中所达到的最大药物浓度；Tmax表示给药后药物在血浆中达到最大药物浓度所需时间；F％表示药物的口服生物利用度。
实验结果如表6所示，化合物4在小鼠体内表现出优异的药代动力学性质：尾静脉注射3mg/kg后，化合物4的血浆暴露量较高(AUClast为6737ng/mL*hr)，清除率较低(CL(iv)为0.433L/hr/kg)，因而血浆半衰期较长(T1/2为1.57hr)；口服10mg/kg的剂量，体内暴露量可达到21156ng/mL*hr，最大血药浓度为4517ng/mL，生物利用度高。
表6.化合物4的药代动力学测试
由于磷酸酯化合物5可在体内代谢为化合物4，因此化合物5的药代动力学测试中同时测定化合物5和化合物4的药代动力学参数。实验结果如表7所示，化合物5在体内可迅速完全地转化为化合物4。
表7.化合物5的药代动力学测试
氘代化合物与非氘代比较例化合物9和10的肝微粒体稳定性测试
化合物4、比较例化合物9、比较例化合物10(终浓度1μM)或阳性对照酮色林(MCE，终浓度1μM)分别与各种属肝微粒体(Biopredic International，终浓度0.5mg/mL)在37℃预孵育10min。然后，每管中加入37℃水浴预热5min的NADPH溶液(Roche，终浓度1mM)，轻摇混匀，置于37℃恒温振荡水浴锅(50rpm)中开始计时。孵育0、5、15、30、45min后，加入400μL含内标工作液(Sigma-Aldrich)的乙腈终止反应。涡旋混匀，于4℃、3200g离心10min。取上清液50μL，加入100μL超纯水，涡旋混匀，用LC-MS/MS检测供试品的母体剩余量。使用SCIEX公司的Analyst软件(Analyst 1.6.3)输出原始图谱、峰面积比等数据，利用Microsoft Office Excel计算半衰期(T1/2)。
实验结果如表8所示，相较于非氘代比较例化合物9和10，氘代化合物4在啮齿类(大鼠、小鼠)和非啮齿类(猴、人)肝微粒体中的稳定性均有不同程度的提升，提示其较好的药代动力学性质。
表8.化合物的肝微粒体稳定性测试(T1/2，min)
工业应用性
本发明提供一种KIF18A抑制剂化合物，其可以用于选择性地抑制KIF18A活性、或治疗或预防由KIF18A活性调节的或受其影响的或者其中涉及KIF18A活性或过表达的疾病、障碍或病症。因而，可将其制成相应的药物，适于工业应用。
尽管本文对本发明作了详细说明，但本发明不限于此，本技术领域的技术人员可以根据本发明的原理进行修改，因此，凡按照本发明的原理进行的各种修改都应当理解为落入本发明的保护范围。

Claims (14)

  1. 一种抑制剂，其为式(I)的化合物或其药学可接受的盐、溶剂化物、酯、酸、代谢物或前药，
    其中，
    X1和X2各自独立地选自N和CH；
    R1选自氢、-P(＝O)(OH)2、和-S(＝O)2(OH)；
    R2a、R2b、R3a和R3b各自独立地选自氢或氘，条件是R2a、R2b、R3a和R3b中的至少一个为氘；
    m为选自1、2或3的整数；
    n为选自1、2或3的整数；
    q为选自0、1或2的整数，且当q不为0时，R4各自独立地选自卤素。
  2. 如权利要求1所述的抑制剂，其中m和n各自独立地为1。
  3. 如权利要求1或2所述的抑制剂，其中q为2，和/或R4各自独立地为氟。
  4. 如权利要求1-3中任一项所述的抑制剂，其中m为1，q为2，且R4在氮杂环上的取代位置为氮原子的对位。
  5. 如权利要求1-4中任一项所述的抑制剂，其中X1为CH，和/或X2为CH。
  6. 如权利要求1-5中任一项所述的抑制剂，其中R2a、R2b、R3a和R3b中的至少两个为氘。
  7. 如权利要求6所述的抑制剂，其中R2a和R2b为氘。
  8. 如权利要求6所述的抑制剂，其中R2a、R2b、R3a和R3b均为氘。
  9. 如权利要求1-8中任一项所述的抑制剂，其中R1为氢。
  10. 如权利要求1中所述的抑制剂，其为选自以下的式(I)的化合物或其药学可接受的盐、溶剂化物、酯、酸、代谢物或前药：
  11. 一种药物组合物，其包括根据权利要求1-10中任一项所述的抑制剂，和药学上可接受的载体或赋形剂，以及任选的其它治疗剂。
  12. 如权利要求1-10中任一项所述的抑制剂，用于治疗或预防由KIF18A活性调节的或受其影响的或者其中涉及KIF18A活性或过表达的疾病、障碍或病症的用途。
  13. 如权利要求12所述的抑制剂的用途，其中所述疾病、障碍或病症为癌症。
  14. 如权利要求12或13所述的抑制剂的用途，其中所述疾病、障碍或病症选自以下一种或多种具有染色体不稳定性的癌症：肺鳞癌、肺腺癌、非小细胞肺癌、小细胞肺癌、头颈鳞癌、乳腺癌、胰腺癌、胰腺导管腺癌、结直肠癌、黑色素瘤、卵巢癌、食管鳞癌、胃癌、肝癌、口腔癌、尿路上皮癌、前列腺癌、膀胱癌、肾细胞癌、胃肠间质瘤、子宫颈癌、子宫内膜癌、横纹肌肉瘤、纤维肉瘤、神经内分泌瘤、间皮瘤、脑癌、和恶性胶质瘤的癌症。
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