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WO2025241650A1 - Inhibiteur de kif18a et son utilisation - Google Patents

Inhibiteur de kif18a et son utilisation

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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)
Chinese (zh)
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/fr
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

L'invention concerne un inhibiteur de KIF18A, qui est un composé représenté par la formule (I) ou un sel pharmaceutiquement acceptable, un solvate, un ester, un acide, un métabolite ou un promédicament de celui-ci. L'invention concerne également une utilisation de l'inhibiteur de KIF18A dans l'inhibition sélective de l'activité de KIF18A, ou pour le traitement ou la prévention de maladies, de troubles ou d'états qui sont régulés ou affectés par l'activité de KIF18A ou dans lesquels l'activité ou la surexpression de KIF18A est impliquée, en particulier le cancer, et plus particulièrement le cancer avec une instabilité chromosomique.
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WO2024067465A1 (fr) * 2022-09-30 2024-04-04 山东轩竹医药科技有限公司 Inhibiteur de kif18a

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WO2024067465A1 (fr) * 2022-09-30 2024-04-04 山东轩竹医药科技有限公司 Inhibiteur de kif18a

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