HDAC inhibitors and uses thereof

Abstract

The present invention relates to HDAC inhibitors and uses thereof. Specifically, the present invention relates to a compound represented by general formula (1) and a method for preparing the same, as well as the use of the compound of general formula (1) and each of its isomers, each of its crystalline forms, pharmaceutically acceptable salts, hydrates, or solvates as HDAC inhibitors in the preparation of antitumor drugs.

Description

[Technical Field]

[0001] This application claims priority to Chinese Patent Application No. 2022115706657, filed on December 7, 2022, and Chinese Patent Application No. 2023106979240, filed on June 13, 2023, which are incorporated herein by reference in their entireties.

[0002] The present invention relates to the field of pharmaceutical chemistry, and in particular to a class of HDAC inhibitors, methods for their preparation, and the use of such compounds in the preparation of medicaments for treating or preventing cancer. [Background technology]

[0003] Histone deacetylases (HDACs) are a group of proteases that regulate gene expression and protein function by deacetylating histone and non-histone lysines. Currently, there are four major classes of HDACs, with 18 distinct subtypes. Class I includes four subtypes: HDAC1, HDAC2, HDAC3, and HDAC8. Class II includes six subtypes: HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10. Class IV includes only one subtype: HDAC11. While classes I, II, and IV are structurally homologous, class III includes a total of seven SIRT1-7 subtypes, which are structurally unrelated to the three aforementioned classes. HDAC inhibitors inhibit the proliferation and survival of various tumor cells in vitro. Several HDAC inhibitors, including chidamide, have been approved for clinical use as monotherapy or combination therapy for the treatment of relapsed or refractory peripheral T-cell lymphoma, multiple myeloma, large B-cell lymphoma, and breast cancer.

[0004] Recent studies have demonstrated that, in addition to their function in tumor cells, certain HDAC subtypes regulate tumor immunity. For example, inhibition of HDAC1 and HDAC2 upregulates NKG2D expression, enhancing the tumor-killing ability of NK cells (Molecules, 2021, 26(13):3952). Inhibition of HDAC3 upregulates CXCL10-mediated immune cell infiltration (Cancer Immunol Res, 2023, 11(5):657). Inhibition of HDAC6 downregulates inflammasome-mediated IL-1β release (Int J Mol Med, 2024, 53(1):1-14). HDAC10 regulates NK cell function by regulating CXCL10 expression (Proc Natl Acad Sci, 2021, 118(30):e2102718118). HDAC inhibitors exert antitumor effects by modulating the tumor immune microenvironment and suppressing angiogenesis. Inhibitors targeting various HDAC subtypes also exert differential regulatory effects on other important immune cells, such as T cells. Inhibitors targeting different HDAC subtypes can enhance the efficacy of tumor immunotherapies, such as immune checkpoint inhibitors and anti-PD-1 monoclonal antibodies, and overcome drug resistance to tumor immunotherapies. Therefore, developing drugs that selectively inhibit specific HDAC subtypes could effectively enhance the therapeutic efficacy of immune checkpoint inhibitors, which would be of great clinical value. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Molecules,2021,26(13):3952 [Non-patent document 2] Cancer Immunol Res,2023,11(5):657 [Non-patent document 3] Proc Natl Acad Sci,2021,118(30):e2102718118 Summary of the Invention [Problem to be solved by the invention]

[0006] (overview) The present disclosure unexpectedly discovered a group of unique HDAC inhibitors that possess selectivity for specific subtypes, excellent tumor-immune synergy, and metabolic stability. Furthermore, in in vivo studies, the compounds of the present disclosure have been observed to exhibit significant synergistic effects when combined with PD-1 inhibitors and VEGF inhibitors, resulting in tumor regression and even complete tumor disappearance, demonstrating potent antitumor effects. [Means for solving the problem]

[0007] The present disclosure provides a compound of general formula (1), or an isomer thereof, a crystalline form thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof. [ka] (In the general formula (1), X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13 , X 14 , and X 15 are each independently hydrogen or deuterium, and X1, X2, X3, X4, X5, X6, X7, X8, X9, and X 10 , X 11 , X 12 , X 13 , X 14 , or X 15 At least one of is selected from deuterium.

[0008] In another preferred embodiment, in general formula (1), X8 = X9.

[0009] In another specific embodiment of the present disclosure, the compound of the present disclosure has one of the following structures: [ka] TIFF2025540298000003.tif230168TIFF2025540298000004.tif130168

[0010] Another object of the present disclosure is to provide a pharmaceutical composition containing a pharmaceutically acceptable carrier, diluent and / or excipient and, as an active ingredient, the compound of general formula (1) of the present disclosure or an isomer thereof, a crystalline form thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof.

[0011] Yet another object of the present disclosure is to provide use of a compound of general formula (1) of the present disclosure, or an isomer thereof, a crystalline form thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof, or the above pharmaceutical composition, in the preparation of a medicament for treating, regulating or preventing a disease associated with HDAC, wherein said disease is preferably cancer, and said cancer is a hematological cancer or a solid cancer.

[0012] It is to be understood that both the foregoing general description and the following detailed description of the present disclosure are exemplary and explanatory and are intended to provide further explanation of the present disclosure as claimed.

[0013] (Compound synthesis) Methods for preparing the compounds of general formula (1) of the present disclosure are specifically described below, but these specific methods do not limit the present disclosure in any way.

[0014] The compounds of formula (1) above can be synthesized using standard synthetic techniques, well-known techniques, in combination with the methods described herein. Furthermore, the solvents, temperatures, and other reaction conditions described herein may vary. Starting materials for the synthesis of the compounds may be obtained synthetically or commercially available. The compounds described herein and other related compounds with different substituents are described in March, ADVANCED ORGANIC CHEMISTRY, 4 thEd., (Wiley 1992); Carey and Sundberg, ADVANCED ORGANIC CHEMISTRY, 4 th Ed., Vols. A and B (Plenum 2000, 2001), and Green and Wuts, Protective Groups in Organic Synthesis, 3 rd Ed., (Wiley 1999). General methods for preparing compounds can be modified by using appropriate reagents and conditions to introduce various groups into the molecular formulas described herein.

[0015] In one embodiment, the compounds described herein are prepared according to methods well known in the art. However, the conditions of the methods, such as reactants, solvents, bases, amounts of compounds used, reaction temperatures, and reaction times, are not limited to those described below. In addition, the compounds of the present disclosure can be easily prepared by any combination of various synthetic methods described herein or known in the art, and such combinations can be easily determined by those skilled in the art to which the present disclosure pertains. In one embodiment, the present disclosure further provides a method for preparing a compound of general formula (1), which is prepared using the following general reaction scheme 1.

[0016] General Reaction Scheme 1 [ka]

[0017] X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13 , X 14 , and X 15is as defined above. As shown in General Reaction Scheme 1, starting materials S1 and S2 are subjected to an amide condensation reaction to give compound A3, which is then deprotected under appropriate conditions to give compound A4, which is then subjected to a condensation reaction with S3 to give compound A5, which is then deprotected under acidic conditions to give target compound (1).

[0018] Further forms of the compound As used herein, the term "pharmaceutically acceptable" refers to a substance, such as a carrier or diluent, that does not cause a loss of biological activity or properties of a compound and is relatively non-toxic. For example, when administered to an individual, the substance does not cause undesired biological effects or adverse interactions with any of its components.

[0019] The term "pharmaceutically acceptable salt" refers to a form of a compound that does not significantly irritate the receiving organism or eliminate the biological activity and properties of the compound. In certain specific embodiments, the pharmaceutically acceptable salt is obtained by reacting a compound of the general formula with an acid or base, where the acid or base is selected from the group consisting of: Stahl and Wermuth, Handbook of Pharmaceutical Salts: properties, Selection, and Use, 1 st Ed., (Wiley, 2002), but are not limited to these.

[0020] It should be understood that pharmaceutically acceptable salts include solvent addition forms or crystalline forms, particularly solvates or polymorphs. Solvates contain stoichiometric or non-stoichiometric amounts of solvent and are preferentially formed upon crystallization in pharmaceutically acceptable solvents such as water and ethanol. Hydrates are formed when the solvent is water, and alcoholates are formed when the solvent is ethanol. Solvates of compounds of general formula (1) are conveniently prepared or formed according to the methods described herein. For example, hydrates of compounds of general formula (1) are conveniently prepared by recrystallization in a water / organic solvent mixture, where the organic solvent used includes, but is not limited to, tetrahydrofuran, acetone, ethanol, or methanol. Furthermore, the compounds described herein can exist in either unsolvated or solvated forms. In general, solvated forms are considered equivalent to unsolvated forms for the purposes of the compounds and methods provided herein.

[0021] In other specific examples, the compound of general formula (1) is prepared in different forms, including, but not limited to, amorphous, pulverized, and nanoparticle forms. Furthermore, the compound of general formula (1) may be a polymorph, including crystalline forms. Polymorphs include different lattice arrangements of the same elements of a compound. Polymorphs generally have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardnesses, crystalline forms, optical properties, electrical properties, stability, and solubility. Various factors, such as the recrystallization solvent, crystallization rate, and storage temperature, may result in a single predominant crystalline form.

[0022] In another embodiment, the compounds of general formula (1) may have chiral centers and / or axial asymmetry and therefore may exist in the form of racemates, racemic mixtures, single enantiomers, diastereomeric compounds, single diastereomers, and cis-trans isomers. Each chiral center or axial asymmetry independently produces two optical isomers, and all possible optical isomers, diastereomeric mixtures, and pure or partially pure compounds are included within the scope of the present disclosure. The present disclosure is meant to include all such isomers of these compounds.

[0023] The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may contain tritium ( 3 H), iodine-125( 125 I) and C-14( 14

[0013] The compounds may be labeled with radioactive isotopes such as CI (C). As another example, deuterium can be used to replace a hydrogen atom to form a deuterated compound. The bond formed between deuterium and carbon is stronger than the bond formed between normal hydrogen and carbon. Compared to non-deuterated drugs, deuterated drugs generally have advantages such as reduced toxicity and side effects, improved drug stability, enhanced efficacy, and prolonged in vivo drug half-life. All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are within the scope of the present disclosure.

[0024] Unless otherwise specified, any atom of the compounds of the present disclosure refers to the isotope of the atom in the stable state of the compound.Unless otherwise specified, when a site in a molecular structure is selected as "H" or "hydrogen", it should be understood that the site has the natural abundance of hydrogen isotopes.Similarly, unless otherwise specified, when a site is selected as "D" or "deuterium", it should be understood that the site has a deuterium isotope abundance that is at least 3000 times the natural abundance (the natural abundance of deuterium isotopes is 0.015%).

[0025] More preferably, each deuterated site of a deuterated compound of the present disclosure has a deuterium atom abundance that is at least 3500 times its natural abundance (52.2% deuterium atom enrichment). More preferably, the deuterium atom abundance is at least 4500 times its natural abundance (67.5% deuterium atom enrichment). More preferably, the deuterium atom abundance is at least 5000 times its natural abundance (75% deuterium atom enrichment). More preferably, the deuterium atom abundance is at least 6000 times its natural abundance (90% deuterium atom enrichment). More preferably, the deuterium atom abundance is at least 6333 times its natural abundance (95% deuterium atom enrichment). More preferably, the deuterium atom abundance is at least 6466.7 times its natural abundance (97% deuterium atom enrichment). More preferably, the deuterium atom abundance is at least 6600 times its natural abundance (99% deuterium atom enrichment). More preferably, the deuterium atom abundance is at least 6633.3 times the natural abundance (deuterium atom enrichment 99.5%).

[0026] Terminology Unless otherwise indicated, the terms used herein, including those set forth in the specification and claims, are defined as follows: It should be noted that, in this specification and the appended claims, the singular forms "a" and "an" include the plural reference unless otherwise indicated. Conventional methods of mass spectrometry, nuclear magnetic resonance spectroscopy, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology are employed unless otherwise indicated. As used herein, "or" means "and / or" unless otherwise indicated.

[0027] The term "isomer" refers to any tautomer, stereoisomer, atropisomer, isotopic isomer, enantiomer, or diastereomer of a compound of the present disclosure. The compounds of the present disclosure may have one or more asymmetric centers or double bonds and therefore exist in the form of stereoisomers, e.g., double bond isomers (i.e., E / Z geometric isomers), or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers). Thus, the compounds of the present disclosure encompass all corresponding stereoisomers, i.e., stereoisomerically pure (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) forms, as well as mixtures of enantiomers and stereoisomers, e.g., racemates. Enantiomeric and stereoisomeric mixtures of the compounds of the present disclosure can be resolved into their constituent enantiomers or stereoisomers by well-known methods such as chiral gas chromatography and chiral high-performance liquid chromatography, as well as by crystallizing the compounds in the form of chiral salt complexes or in chiral solvents. Enantiomers and stereoisomers can also be obtained from stereomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

[0028] The term "isotopomers" refers to distinct molecules that differ only by isotopic composition, but are otherwise identical in structure.

[0029] Specific pharmaceutical and medical terms The term "acceptable" as used herein means that the formulation or active ingredient does not have an excessive and deleterious effect on the health of the general subject to be treated.

[0030] As used herein, the terms "treatment," "course of treatment," and "treatment" include alleviating, inhibiting, or ameliorating a disease symptom or condition, inhibiting the development of complications, improving or preventing underlying metabolic syndrome, inhibiting the development of a disease or condition (e.g., controlling the progression of a disease or condition), alleviating a disease or condition, regressing a disease or condition, and alleviating complications caused by a disease or condition, or preventing or treating symptoms caused by a disease or condition. As used herein, a compound or pharmaceutical composition, when administered, can ameliorate a disease, symptom, or condition, and in particular, can improve the severity, delay the onset, slow the progression, or shorten the duration of a disease. Fixed or temporary administration, or continuous or intermittent administration, can result from or be associated with administration.

[0031] "Active ingredient" refers to compounds of general formula (1) and pharmaceutically acceptable inorganic or organic salts of compounds of general formula (1). The compounds of the present disclosure may contain one or more asymmetric centers (chiral centers or axial asymmetry) and therefore may exist in the form of racemates, racemic mixtures, single enantiomers, diastereomeric compounds, and single diastereomers. The number of asymmetric centers that may be present depends on the nature of the various substituents on the molecule. Each such asymmetric center independently generates two optical isomers, and all possible optical isomers, diastereomeric mixtures, and pure or partially pure compounds are included within the scope of the present disclosure. The present disclosure is meant to include all such isomeric forms of these compounds.

[0032] As used herein, terms such as "compound," "composition," "drug," or "medicine or pharmaceutical agent" are used interchangeably and refer to any compound or composition that, when administered to an individual (human or animal), is capable of eliciting a desired pharmacological and / or physiological response through local and / or systemic action.

[0033] The terms "administered, administering, or administration" as used herein means direct administration of a compound or composition, or administration of a prodrug, derivative, analog, etc. of an active compound.

[0034] While the numerical ranges and parameters defining the broad scope of the present disclosure are approximations, the relevant values ​​set forth in specific embodiments are set forth herein as precisely as possible. However, any numerical value inherently contains standard deviations that necessarily result from certain testing methods. Herein, the term "about" generally means that the actual numerical value is within a particular numerical value or range ±10%, 5%, 1%, or 0.5%. Alternatively, the term "about" indicates that the actual numerical value falls within an acceptable standard error of the mean, as would be understood by one of ordinary skill in the art. Except in experimental examples or unless otherwise indicated, all ranges, amounts, values, and percentages used herein (e.g., to describe amounts of substances, lengths of time, temperatures, operating conditions, proportions of amounts, etc.) are understood to be modified by the term "about." Thus, unless otherwise indicated, all numerical parameters set forth in this specification and the appended claims are approximations that may vary, if desired. At the very least, these numerical parameters should be construed as representing significant digits or as derived using conventional rounding rules.

[0035] Unless otherwise defined herein, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Furthermore, as used herein, singular nouns include their plural forms, unless contradictory to the context, and plural nouns as used herein also include their singular forms.

[0036] therapeutic use The present invention provides methods of treating diseases including, but not limited to, related conditions involving HDAC enzymes (e.g., cancer) using compounds of general formula (1) or pharmaceutical compositions of the invention.

[0037] In some embodiments, a method for treating cancer is provided, the method comprising administering to an individual in need thereof an effective amount of any of the aforementioned pharmaceutical compositions containing a compound of general formula (1). In some embodiments, the cancer is mediated by the relevant HDAC enzyme. In some embodiments, a compound of the present disclosure is used in combination with an immune checkpoint inhibitor. In some embodiments, a compound of the present disclosure is used in combination with a PD-1 or PD-L1 inhibitor. In some embodiments, a compound of the present disclosure is used in combination with a PD-1 antibody. In some embodiments, a compound of the present disclosure is used in combination with a PD-L1 antibody. In some embodiments, a compound of the present disclosure is used in combination with a VEGF / VEGFR inhibitor. In some embodiments, a compound of the present disclosure is used in combination with an immune checkpoint inhibitor and a VEGF / VEGFR inhibitor. In some embodiments, a compound of the present disclosure is used in combination with a PD-1 inhibitor and a VEGF / VEGFR inhibitor. In some embodiments, a compound of the present disclosure is used in combination with a PD-1 antibody and a VEGF / VEGFR inhibitor. PD-1 antibodies include, but are not limited to, nivolumab, pembrolizumab, toripalimab, sintilimab, camrelizumab, tislelizumab, penprimab, zimberelimab, selplulimab, pucotenlimab, pidilizumab, cemiplimab, spartalizumab, AMG404, RN888, mAbl5, MEDI-0680, BGB-108, spartalizumab, IBI-308, mDX-400, SHR-1210, PF-06801591, PDR-001, GB-226, and STI-1110, and biosimilars, biobetters, and bioequivalents of these inhibitors. PD-L1 antibodies include, but are not limited to, durvalumab, atezolizumab, embafolimab, sugemalimab, avelumab, avelumab, BMS-936559, AMP-714, ALN-PDL, TSR-042, KD-033, CA-170, STI-1014, KY-1003, and biosimilars, biobetters, and bioequivalents of these inhibitors.VEGF / VEGFR inhibitors include, but are not limited to, bevacizumab, ranibizumab, ramucirumab, sorafenib, axitinib, apatinib, sunitinib, regorafenib, vandetanib, pazopanib, lenvatinib, cabozantinib, ponatinib, aflibercept, and fruquintinib. In another embodiment, the cancer is breast cancer, colon cancer, uterine cancer, pancreatic cancer, lung cancer, gastric cancer, leukemia, lymphoma, prostate cancer, liver cancer, cervical cancer, neuroblastoma, melanoma, or intracranial tumor.

[0038] Administration route The compounds of the present disclosure and their pharmaceutically acceptable salts can be prepared into various formulations containing a safe and effective amount of the compounds of the present disclosure or their pharmaceutically acceptable salts and a pharmaceutically acceptable excipient or carrier, where "safe and effective amount" means an amount of the compound sufficient to significantly improve the condition without causing serious side effects. The safe and effective amount of the compound is determined according to the age, condition, course of treatment, and other specific conditions of the subject to be treated.

[0039] "Pharmaceutically acceptable excipient or carrier" refers to one or more compatible solid or liquid fillers or gel substances that must be suitable for human use and have sufficient purity and low toxicity. "Compatible," as used herein, means that the components of the composition are capable of being intermixed with the compounds of the present disclosure without significantly reducing the pharmaceutical efficacy of the compounds. Examples of pharmaceutically acceptable excipients or carriers include cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, or cellulose acetate), gelatin, talc, solid lubricants (e.g., stearic acid or magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, or olive oil), polyols (e.g., propylene glycol, glycerol, mannitol, or sorbitol), emulsifiers (e.g., Tween®), wetting agents (e.g., sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

[0040] When administering the compounds of the present disclosure, they can be administered orally, rectally, parenterally (intravenously, intramuscularly, or subcutaneously) or topically.

[0041] Solid dosage forms for oral administration include capsules, tablets, pills, pulvises, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier) such as sodium citrate or dicalcium phosphate, or the following ingredients: (a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders such as hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants such as glycerol; (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solution retardants such as paraffin; (f) absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as cetyl alcohol and glycerol monostearate; (h) adsorbents such as kaolin; and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, and sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, said dosage forms may further comprise buffering agents.

[0042] Solid dosage forms such as tablets, sugar-coated tablets, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials known in the art.These may contain opacifying agents, and the active compound or compounds in such compositions can be delayed in certain parts of the digestive tract.The examples of embedding components that can be used include polymeric materials and wax-based materials.If necessary, the active compound can be formed into microcapsules with one or more of the above-mentioned excipients.

[0043] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, etc. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0044] Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0045] In addition to the active compound, suspensions may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methylate, and agar-agar, or mixtures of these substances.

[0046] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0047] Dosage forms for topical administration of the compounds of the present disclosure include ointments, powders, patches, sprays, inhalants, etc. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier, and any preservatives, buffers, or propellants that may be required, as may be required.

[0048] The compounds of the present disclosure may be administered alone or in combination with other pharmaceutically acceptable compounds. When the pharmaceutical composition of the present invention is used, a safe and effective amount of the compound of the present disclosure is administered to the mammal (e.g., human) to be treated, where the administered amount is a pharmaceutically effective dose. For a 60 kg human, the daily dose is typically 1 to 2000 mg, preferably 50 to 1000 mg. Of course, the specific dose will take into account factors such as the route of administration and the patient's health condition, but these are well known to those skilled in the art.

[0049] The features described in this disclosure or the features described above in the embodiments can be arbitrarily combined. All features disclosed herein can be used in any composition, and various features disclosed herein can be replaced with any alternative features that serve the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the features disclosed herein are merely generic examples of equivalent or similar features.

[0050] (Detailed explanation) Various specific aspects, features and advantages of the above compounds, methods and pharmaceutical compositions will be described in detail below, so that the contents of the present disclosure will be more clearly understood. It should be understood that the following detailed description and examples describe specific examples for reference only. After reading the description of the present disclosure, those skilled in the art can make various changes or modifications to the present invention, and such equivalents also fall within the scope of the present application as defined herein.

[0051] In all examples, 1 H-NMR spectra were recorded on a Varian Mercury 400 nuclear magnetic resonance spectrometer, and chemical shifts are expressed in ppm. Unless otherwise specified, 200-300 mesh silica gel was used for separation, and the ratio of eluents was expressed by volume.

[0052] The following abbreviations are used in this disclosure: ACN is acetonitrile; AcOH is glacial acetic acid; AIBN is azobisisobutyronitrile; BocO is di-tert-butyl dicarbonate; CDCl is deuterated chloroform; (COCl) is oxalyl chloride; D is deuterium gas; DO is heavy water; DBU is 1,8-diazabicyclo[5.0]undec-7-ene; DCM is dichloromethane; dioxane is 1,4-dioxane; DIPEA is diisopropylethylamine; DMSO is dimethyl sulfoxide; DMAP is 4-dimethylaminopyridine; DMF is N,N-dimethylformamide; EA is ethyl acetate; EtOH is ethanol; EDCI is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; FA is formic acid; Flash is flash preparative medium pressure liquid chromatography; h is hour; H is hydrogen; HOBt is 1-hydroxybenzotriazoline. KCO is anhydrous potassium carbonate; KOH is potassium hydroxide; LC-MS is liquid chromatography-mass spectrometry; LiOH is lithium hydroxide; LiOH.H0 is lithium hydroxide monohydrate; MeOH is anhydrous methanol; MeOD is monodeuterated methanol; min is minute; mL is milliliter; MS is mass spectrometry; NaBD is sodium deuterated borohydride; NaOAc is anhydrous sodium acetate; n-BuLi is n-butylaluminum; NaBH(OAc) is sodium triacetoxyborohydride; NBS is N-bromosuccinimide; NHCl is ammonium chloride; NMR is nuclear magnetic resonance; Pd / C is palladium on carbon; PE is petroleum ether; PPTS is pyridinium 4-toluenesulfonate; TFA is trifluoroacetic acid; TFAA is trifluoroacetic anhydride; THF is tetrahydrofuran; Zn is zinc powder.

[0053] Preparation Example 1: Synthesis of 4-((2,2,2-trifluoroacetamido)methyl)benzoic acid-2-d (S1-1) [ka]

[0054] Synthesis of S1-1a: A 100 mL single-neck flask was charged with methyl 2-bromo-4-(aminomethyl)benzoate (2.0 g, 8.2 mmol), sodium acetate (1 g), 10% Pd / C (200 mg), and MeOH (40 mL). The system was purged with deuterium gas three times and then stirred at room temperature for 6 hours under a deuterium atmosphere supplied via a balloon. After LC-MS confirmed the completion of the reaction, the mixture was filtered, and the filtrate was concentrated under reduced pressure until a small volume remained. EA (100 mL) and saturated sodium bicarbonate solution (50 mL) were added to the residue, and the mixture was stirred and subjected to liquid separation. The organic phase was washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to give a colorless oily product (1.82 g, >100%). ESI-MS m / z: 167.1 [M+H] + .

[0055] Synthesis of S1-1b: To a 100 mL single-neck flask, the above compound S1-1a (1.82 g, crude, 8.2 mmol), THF (20 mL), MeOH (10 mL), and NaOH (1.6 g, 40.0 mmol) were added. The mixture was stirred at room temperature for 5 hours. After completion of the reaction was confirmed by LC-MS, the mixture was stirred at room temperature for 5 hours. The mixture was concentrated, and the residue was purified by flash filtration and lyophilized to give a white solid product (840 mg, 67.4%). ESI-MS m / z: 153.0 [M+H] + .

[0056] Synthesis of S1-1: The above compound S1-1b (840 mg, 5.52 mmol), DCM (84 mL), and DIPEA (1.78 g, 13.8 mmol) were added to a 250 mL one-neck flask, and a solution of TFAA (1.74 g, 8.28 mmol) in DCM (10 mL) was added dropwise under an ice bath. After the addition was complete, the mixture was stirred at room temperature for 6 hours. After the completion of the reaction was confirmed by LC-MS, 2N HCl solution (30 mL) was added to the mixture. The mixture was stirred for 20 minutes and then subjected to liquid separation. The organic phase was then washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was slurried in EA (5 mL) / PE (30 mL) at room temperature for 30 minutes, filtered, and dried to give an off-white solid product (1.21 g, 88.4%). ESI-MS m / z: 249.0 [M+H] + .

[0057] Following the synthesis method of intermediate S1-1, target intermediates S1-2 to S1-6 were obtained using different starting materials.

[0058] [Table 1]

[0059] Preparation Example 2: Synthesis of 4-((2,2,2-trifluoroacetamido-d)methyl-d2)benzoic acid (S1-7) [ka]

[0060] Synthesis of S1-7a: A 500 mL single-neck flask was charged with 4-cyanobenzoic acid (2.94 g, 20.0 mmol), 4 M HCl / MeOH (5 mL, 20.0 mmol), and 10% Pd / C (300 mg) in MeOH (100 mL). The system was purged with deuterium gas three times and then stirred at room temperature for 20 h under a deuterium atmosphere supplied via a balloon. After LC-MS showed the reaction was complete, the mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure to give a white solid product (4.1 g, >100%). ESI-MS m / z: 156.1 [M+H] + .

[0061] Synthesis of S1-7: The above compound S1-1b (4.1 g, 20.0 mmol), DCM (160 mL), and DIPEA (12.9 g, 0.1 mol) were added to a 500 mL single-neck flask, and a solution of TFAA (6.3 g, 30 mmol) in DCM (20 mL) was added dropwise under an ice bath. After the addition was complete, the mixture was stirred at room temperature for 6 hours. After the completion of the reaction was confirmed by LC-MS, 2N hydrochloric acid solution (30 mL) was added to the mixture. The mixture was stirred for 20 minutes and then subjected to liquid separation. The organic phase was then washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was slurried in EA (20 mL) / PE (100 mL) at room temperature for 30 minutes, filtered, and dried to give an off-white solid product (2.65 g, 53%). ESI-MS m / z: 251.0 [M+H] + .

[0062] Preparation Example 3: Synthesis of tert-butyl (2-amino-5-fluorophenyl-4-d)carbamate (S2-1) [ka]

[0063] Synthesis of S2-1a: 5-Fluoro-4-bromo-2-nitroaniline (2.35 g, 10.0 mmol) was dissolved in DCM (30 mL) and DMAP (610 mg, 5.0 mmol), DIPEA (2.58 g, 20.0 mmol), and BocO (2.62 g, 12.0 mmol) were added. The mixture was heated to reflux under an argon atmosphere for 20 hours. After LC-MS confirmed the reaction was complete, water (50 mL) and DCM (50 mL) were added to the mixture. The resulting mixture was stirred and allowed to separate. The organic phase was then washed with 1 N HCl (50 mL), saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to give a tan solid product (3.7 g, >100% yield). ESI-MS m / z: 335.0 [M+H] + .

[0064] Synthesis of S2-1b: The crude product S2-1a was added to THF (50 mL) and zinc powder (6.5 g, 0.1 mol) was added. A solution of AcOH (6 g, 0.1 mol) in THF (10 mL) was slowly added dropwise under ice bath. After the addition was completed, the mixture was stirred at room temperature for 4 hours. After the completion of the reaction was confirmed by LC-MS, the mixture was filtered. The filter cake was washed with THF, and the filtrate was concentrated. The residue was purified by column chromatography to give a brown solid product (1.52 g, 49.8%). ESI-MS m / z: 305.0 [M+H]+.

[0065] Synthesis of S2-1: The aforementioned compound S2-1b (1.43 g, 4.69 mmol), 10% Pd / C (100 mg), and sodium acetate (500 mg) were added to MeOH (30 mL). The system was purged with deuterium gas three times and then stirred at room temperature for 20 hours under a deuterium atmosphere supplied via a balloon. After LC-MS confirmed the completion of the reaction, the mixture was filtered, and the filtrate was concentrated under reduced pressure until a small amount remained. EA (50 mL) and saturated sodium bicarbonate solution (50 mL) were added to the residue, and the mixture was stirred and subjected to liquid separation. The organic phase was then washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to give a brown solid product (970 mg, 91.1%). ESI-MS m / z: 228.1 [M+H] + .

[0066] Following the synthetic method for intermediate S2-1, the target intermediates S2-2 and S2-3 were obtained using different starting materials.

[0067] [Table 2]

[0068] Preparation Example 4: Synthesis of (E)-3-(pyridin-3-yl-4-d)acrylic acid (S3-1) [ka]

[0069] Synthesis of S3-1a 4-Bromopyridine-3-carbaldehyde (1.86 g, 10.0 mmol), 10% Pd / C (200 mg), and sodium acetate (1 g) were added to MeOH (30 mL). The system was purged with deuterium gas three times and then stirred at room temperature for 20 hours under a deuterium atmosphere supplied via a balloon. After LC-MS confirmed the completion of the reaction, the mixture was filtered, and the filtrate was concentrated under reduced pressure until a small amount remained. EA (50 mL) and saturated sodium bicarbonate solution (50 mL) were added to the residue, and the mixture was stirred and subjected to liquid separation. The organic phase was then washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to give a white solid product (1.1 g, >100%). ESI-MS m / z: 109.1 [M+H] + .

[0070] Synthesis of S3-1 To a 100 mL single-neck flask, the above compound S3-1a (1.1 g, 10.0 mmol), malonic acid (2.08 g, 20 mmol), piperidine (2.55 g, 30 mmol), and pyridine (10 mL) were added. The mixture was purged with argon, heated to reflux, and stirred for 20 hours. After LC-MS showed the reaction was complete, the mixture was concentrated, and the residue was purified by flash filtration and lyophilized to give an off-white solid product (853 mg, 56.9%). ESI-MS m / z: 151.0 [M+H] + .

[0071] Following the synthesis method of intermediate S3-1, the target intermediates S3-2 to S3-7 were obtained using different starting materials.

[0072] [Table 3]

[0073] Preparation Example 5: Synthesis of (E)-3-(pyridin-3-yl)-2,3-d2 acrylic acid (S3-8) [ka]

[0074] Synthesis of S3-8a: Methyl 3-(pyridin-3-yl)propiolate (1.61 g, 10.0 mmol) and 10% Pd / C (200 mg) were added to MeOH (30 mL). The system was purged with deuterium gas three times and then stirred at room temperature for 20 hours under a deuterium atmosphere supplied via a balloon. After LC-MS showed the reaction was complete, the mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure to give the anhydrous oily product (1.8 g, >100%). ESI-MS m / z: 170.1 [M+H] + .

[0075] Synthesis of S3-8b: To a 100 mL single-neck flask, the above compound S3-8a (1.8 g, 10.0 mmol), AIBN (164 mg, 1.0 mmol), and acetonitrile (40 mL) were added, followed by the addition of NBS (1.96 g, 11.0 mmol) in portions. The mixture was heated at reflux for 6 hours. After LC-MS showed the reaction was complete, the mixture was cooled and concentrated, and the residue was used directly in the next step.

[0076] Synthesis of S3-8c: To a 100 mL single-neck flask, the above compound S3-8b (5.2 g, crude product, 10.0 mmol), DBU (3.04 g, 20.0 mmol), and acetonitrile (40 mL) were added, and the mixture was heated to 50 °C and reacted for 4 h. After LC-MS confirmed the completion of the reaction, the mixture was cooled and concentrated, and the residue was purified by Flash to give a colorless oily product (930 mg, 56.4%). ESI-MS m / z: 166.1 [M+H] + .

[0077] Synthesis of S3-8: To a 100 mL single-neck flask, the above compound S3-8c (930 mg, 5.64 mmol), THF (5 mL), MeOH (5 mL), and LiOH.HO (474 ​​mg, 11.3 mmol) were added. The mixture was stirred at room temperature for 5 hours. After completion of the reaction was confirmed by LC-MS, the mixture was concentrated, and the residue was purified by flash filtration and lyophilized to give a white solid product (716 mg, 83.5%). ESI-MS m / z: 152.0 [M+H] + .

[0078] Following the synthesis method of intermediate S3-8, the target intermediates S3-9 to S3-15 were obtained using different starting materials.

[0079] [Table 4]

[0080] Preparation Example 6: Synthesis of (E)-3-(pyridin-3-yl)-3-dacrylic acid (S3-16) [ka]

[0081] Synthesis of S3-16a: A 100 mL single-neck flask was charged with 3-oxo-3-(pyridin-3-yl)propionic acid (2 g, 12.12 mmol), THF (30 mL), and MeOD (2 g), and NaBD4 (509 mg, 12.12 mmol) was added in portions at room temperature. After the addition was complete, the mixture was stirred at room temperature for 2 h. After LC-MS showed the reaction was complete, the mixture was quenched by the dropwise addition of DO (5 mL). The mixture was concentrated, and the residue was purified by Flash HPLC to give the product (1.66 g, 81%) as a colorless solid. ESI-MS m / z: 170.0 [M+H] + .

[0082] Synthesis of S3-16: The above compound S3-16a (1.66 g, 9.82 mmol), toluene (20 mL), PPTS (250 mg, 1.0 mmol), and 4A molecular sieves (5 g) were added to a 100 mL single-neck flask, and the mixture was heated to 100 °C and stirred for 16 h. After completion of the reaction was confirmed by LC-MS, the mixture was filtered. The filter cake was washed with THF, and the filtrate was concentrated to dryness. The residue was purified by flash filtration to give a white solid product (1.03 g, 69.9%). ESI-MS m / z: 151.0 [M+H] + .

[0083] Following the synthesis of intermediate S3-16, but using different starting materials, the desired intermediate S3-17 was obtained. [Table 5]

[0084] Example 1: Synthesis of (E)-N-(2-amino-4-fluorophenyl-5-d)-4-((3-(pyridin-3-yl)acrylamido)methyl)benzamide (Compound 1) [ka]

[0085] Step 1: Synthesis of compound 1-1: A 100 mL single-neck flask was charged with 4-((2,2,2-trifluoroacetamido)methyl)benzoic acid (S1-0, 446 mg, 1.8 mmol), DMF (20 mg), and DCM (10 mL), and a solution of (COCl)2 (343 mg, 2.7 mmol) in DCM (2 mL) was added dropwise at room temperature under an argon atmosphere. After the addition was complete, the mixture was stirred at room temperature for 2 hours. After completion of the reaction was confirmed by LC-MS, the mixture was concentrated to dryness under pressure. Subsequently, DCM (10 mL) was added to the residue, and then solutions of DIPEA (700 mg, 5.43 mmol) and S2-1 (410 mg, 1.81 mmol) in DCM (5 mL) were added dropwise at room temperature. After the addition was complete, the mixture was stirred at room temperature for 1 hour. After LC-MS confirmed the completion of the reaction, DCM (30 mL) and water (30 mL) were added to the mixture. The mixture was stirred and then subjected to liquid separation. The organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was slurried in EA (2 mL) / PE (8 mL) at room temperature, filtered, and dried to give a light brown solid product (581 mg, 70.8%). ESI-MS m / z: 457.1 [M+H] + .

[0086] Step 2: Synthesis of Compound 1-2: To a 100 mL single-neck flask, the above compound 1-1 (581 mg, 1.27 mmol), MeOH (10 mL), and anhydrous potassium carbonate (351 mg, 2.54 mmol) were added. The mixture was purged with argon, heated to 60 °C, and stirred for 6 hours. After LC-MS confirmed the completion of the reaction, the mixture was concentrated. DCM (30 mL) and saturated aqueous sodium chloride solution (20 mL) were added to the resulting residue. The mixture was stirred and subjected to liquid separation. The organic phase was then washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to give an off-white solid product (486 mg, 106%). ESI-MS m / z: 361.1 [M+H] + .

[0087] Step 3: Synthesis of compounds 1-3: A 100 mL single-neck flask was charged with (E)-3-(pyridin-3-yl)acrylic acid (S3-0, 42 mg, 0.278 mmol), DIPEA (72 mg, 0.556 mmol), HOBt (56 mg, 0.417 mmol), EDCI (80 mg, 0.417 mmol), and DMF (5 mL). The mixture was stirred at room temperature for 15 minutes, and then compound 1-2 (106 mg, 0.278 mmol) was added. The resulting mixture was stirred for an additional 16 hours. After LC-MS showed the reaction was complete, the mixture was purified by flash filtration to give the yellowish solid product (118 mg, 86.4%). ESI-MS m / z: 492.1 [M+H] + .

[0088] Step 4: Synthesis of Compound 1: Compound 1-3 (118 mg, 0.24 mmol), DCM (5 mL), and TFA (0.5 mL) were added to a 100 mL single-neck flask, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction was confirmed by LC-MS, the mixture was concentrated, and the residue was purified by preparative liquid chromatography to give a white solid product (60 mg, 64%). ESI-MS m / z: 392.1 [M+H] + .

[0089] Example 2: Synthesis of (E)-N-(2-amino-4-fluorophenyl)-4-((3-(pyridin-3-yl)acrylamido)methyl)-3-dbenzamide (Compound 5) [ka]

[0090] Step 1: Synthesis of compound 5-1: A 100 mL single-neck flask was charged with 4-((2,2,2-trifluoroacetamido)methyl)-3-d-benzoic acid (S1-2, 600 mg, 2.42 mmol), DMF (30 mg), and DCM (10 mL). A solution of (COCl)2 (461 mg, 3.63 mmol) in DCM (2 mL) was added dropwise at room temperature under an argon atmosphere. After the addition was complete, the mixture was stirred at room temperature for 2 hours. After completion of the reaction was confirmed by LC-MS, the mixture was concentrated to dryness under pressure. Subsequently, DCM (10 mL) was added to the residue, followed by the dropwise addition of DIPEA (1.03 g, 8.0 mmol) and tert-butyl (2-amino-5-fluorophenyl)carbamate (547 mg, 2.42 mmol) solutions in DCM (5 mL) at room temperature. After the addition was complete, the mixture was stirred at room temperature for 1 hour. After LC-MS confirmed the completion of the reaction, DCM (30 mL) and water (30 mL) were added to the mixture. The mixture was stirred and then subjected to liquid separation. The organic phase was washed twice with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was slurried in EA (2 mL) / PE (8 mL) at room temperature, filtered, and dried to give a light brown solid product (853 mg, 77.3%). ESI-MS m / z: 457.1 [M+H] + .

[0091] Step 2: Synthesis of compound 5-2: The above compound 5-1 (853 mg, 1.87 mmol), MeOH (10 mL), and anhydrous potassium carbonate (516 mg, 3.74 mmol) were added to a 100 mL single-neck flask. The mixture was purged with argon, heated to 60 °C, and stirred for 6 h. After LC-MS confirmed the completion of the reaction, the mixture was concentrated. DCM (30 mL) and saturated sodium chloride solution (20 mL) were added to the resulting residue. The mixture was stirred and subjected to liquid separation. The organic phase was then washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to give an off-white solid product (660 mg, 98.0%). ESI-MS m / z: 361.1 [M+H] + .

[0092] Step 3: Synthesis of compound 5-3: A 100 mL single-neck flask was charged with (E)-3-(pyridin-3-yl)acrylic acid (S3-0, 63 mg, 0.417 mmol), DIPEA (108 mg, 0.837 mmol), HOBt (84 mg, 0.622 mmol), EDCI (120 mg, 0.626 mmol), and DMF (10 mL). The mixture was stirred at room temperature for 15 min, and then the above compound 5-2 (160 mg, 0.417 mmol) was added. The resulting mixture was stirred for an additional 16 h. After LC-MS confirmed the completion of the reaction, the mixture was purified by flash filtration to give the yellowish solid product (140 mg, 68.3%). ESI-MS m / z: 492.1 [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ 9.77 (s, 1H), 8.84 - 8.72 (m, 3H), 8.56 (dd, J = 4.7, 1.7 Hz, 1H), 8.01 (dt, J = 8.0, 2.0 Hz, 1H), 7.97 - 7.90 (m, 2H), 7.53 (dd, J = 15.0, 5.4 Hz, 2H), 7.46 (dt, J = 8.4, 2.1 Hz, 3H), 6.97 (td, J = 8.4, 3.0 Hz, 1H), 6.83 (d, J = 15.9 Hz, 1H), 4.51 (d, J = 6.0 Hz, 2H), 1.45 (s, 9H).

[0093] Step 4: Synthesis of compound 5: Compound 5-3 (140 mg, 0.285 mmol), DCM (5 mL), and TFA (0.5 mL) were added to a 100 mL single-neck flask, and the mixture was stirred at room temperature for 2 h. After completion of the reaction was confirmed by LC-M, the mixture was concentrated, and the residue was purified by preparative liquid chromatography to give a white solid product (82 mg, 73.5%). ESI-MS m / z: 392.1 [M+H] + .

[0094] By following similar procedures to the synthesis of Compound 1 and Compound 5, other target compounds in Table 6 can be obtained using different intermediates as starting materials.

[0095] [Table 6] TIFF2025540298000020.tif234168TIFF2025540298000021.tif238168TIFF2025540298000022.tif129168

[0096] Nuclear magnetic resonance data for some of the compounds of the present disclosure is shown in Table 7 below.

[0097] [Table 7] TIFF2025540298000024.tif153168

[0098] Biological Example 1: Assay of proliferation inhibition of Jurkat and 293T cells by compounds of the present disclosure Jurkat or 293T cells were seeded in a 96-well plate at 3000 cells / well. After overnight adherent culture, compounds serially diluted in DMSO were added. After 72 hours, intracellular ATP levels were measured using CTG. The IC values ​​for compound-induced cell growth inhibition were calculated. 50 was calculated in comparison with the DMSO group. The results are shown in Table 8 below.

[0099] [Table 8]

[0100] As can be seen from the data in the table above, the compounds of the present disclosure selectively kill Jurkat cells, and show significant differences in cytotoxicity between human embryonic kidney cells 293T and tumor cells Jurkat.Compared to chidamide, compound 1 and compound 5 show stronger cytotoxicity to tumor cells Jurkat and weaker cytotoxicity to 293T cells.

[0101] Biological Example 2: Measurement of Intracellular Acetyllysine / H3K 27 Acetyllysine Levels with Compounds of the Disclosure HeLa cells were seeded in 96-well plates at 20,000 cells / well. After overnight adherent culture, serially diluted compounds were added. After 24 hours of treatment, intracellular acetyl-lysine and H3K27 acetyl-lysine levels were quantified by ELISA. The results are shown in Table 9 below.

[0102] [Table 9]

[0103] Biological Example 3. Assay of Inhibitory Activity of Compounds of the Present Disclosure Against HDAC Enzymes The effect of compound 5 of the present disclosure on the activity of HDAC enzymes was measured using a fluorometric assay. Serially diluted DMSO sample solutions were added to reaction wells, enzyme was added to a 384-well plate, and reaction buffer was added to control wells. The plate was incubated at room temperature for 15 minutes. The reaction was initiated after adding fluorescent substrate solution. Fluorescence intensity measurements (excitation: 355 nM, emission: 460 nM) were recorded every minute for 60 minutes using a Paradigm detector, and slope values ​​were calculated. Inhibition (%) = (maximum value - sample value) / (maximum value - minimum value) × 100%. IC 50 Values ​​were obtained by fitting the curve using the following formula: Y = Bottom + (Top - Bottom) / (1 + (IC 50 / X)^HillSlope), where Y represents the inhibition rate and X represents the concentration of the compound. The results are shown in Table 10 below.

[0104] [Table 10]

[0105] Results: Compound 5 of the present disclosure exhibited relatively good inhibitory activity against the four subtypes HDAC1, HDAC2, HDAC3, and HDAC10. Its activity against HDAC1 was slightly stronger (1.2-fold) than chidamide, its activity against HDAC2 was slightly weaker (0.83-fold) than chidamide, and its activity against HDAC10 was 2.5-fold higher than chidamide. The IC values ​​of compound 5 and chidamide for inhibitory activity against HDAC6 were 50 Both values ​​exceeded 30 mM.

[0106] Biological Example 4: NK Cell-Mediated Tumor Cell Killing by Compounds of the Present Disclosure GFP-expressing OVCAR3 or NK92 cells were seeded at 4000 cells / well in a 96-well plate. After overnight adherent culture, serially diluted compounds were added and incubated for an additional 72 hours. The two cell types were mixed and cultured for an additional 1-4 hours, after which the NK92 cells were removed by washing with PBS, and the adherent GFP-positive tumor cells were counted. The EC of compounds promoting NK92 cell-mediated OVCAR3 cell death was calculated. 50 was calculated in comparison with the DMSO group. The results are shown in Table 11 below.

[0107] [Table 11]

[0108] The results showed that Compounds 1 and 5 of the present disclosure had significantly superior NK cell-mediated tumor cell killing activity than the control drug, chidamide.

[0109] Biological Example 5: Assay of Stability of Disclosed Compounds in Liver Microsomes After incubating 1 μM of the compound with 0.5 mg / mL human or mouse liver microsomes and an NADPH-regenerating system at 37°C for different periods of time, the remaining amount of the compound was analyzed using LC-MS-MS. 1 / 2 The results are shown in Table 12 below.

[0110] [Table 12]

[0111] As can be seen from the data in the table above, the stability of several compounds of the present disclosure in human liver microsomes was significantly superior to that of chidamide.

[0112] Biological Example 6: Assays to identify metabolites of compounds of the present disclosure in liver microsomes and hepatocytes of various species 10 μM compounds were incubated with 1.0 mg / mL liver microsomes or hepatocytes (five species: mouse, rat, dog, monkey, and human) and an NADPH-regenerating system at 37°C for 120 min. The reaction was stopped by adding stop solution, and the incubated samples were removed from the water bath. After stopping the reaction, the samples were vortexed and centrifuged at 3260 × g for 15 min at 4°C. After centrifugation, all supernatants were transferred to a 96-well plate and either used directly for LC-MS analysis or diluted or concentrated and reconstituted before injection. An LC-UV-HRMSn (n = 1-2) analytical method was established, and data acquisition was performed using SCIEX OS software. Parent drug and metabolites could be analyzed by mass spectrometry using different scan modes (MS / MS or IDA) and a full UV wavelength scan (λ = 190-500 nm). Potential metabolites were identified by comparing the UV and mass spectra of the incubation samples with those of blank samples from the same species. Potential metabolite structures were deduced by comparative analysis of collision-induced dissociation (CID) fragments of the test compound and its metabolites. Metabolites and their relative abundances were tabulated for interspecies comparison.

[0113] The experimental results showed that some compounds of the present disclosure, especially compound 5, exhibit simple metabolic pathways and good stability in liver microsomes of all species.

[0114] TIFF2025540298000030.tif65168

[0115] TIFF2025540298000031.tif74168

[0116] TIFF2025540298000032.tif72168

[0117] TIFF2025540298000033.tif74168

[0118] TIFF2025540298000034.tif95168

[0119] Biological Example 7: Pharmacokinetic studies of compounds of the present disclosure in mice Female CD-1 mice aged 7–10 weeks were administered 1 mg / kg intravenously and 10 mg / kg orally, respectively. Mice were fasted for at least 12 hours before administration, given food 4 hours after administration, and allowed free access to water throughout the experiment. On the day of the experiment, animals in the intravenous administration group received a single injection of the corresponding compound via the tail vein at a dose of 10 mL / kg, while animals in the oral administration group received a single intragastric injection of the corresponding compound at a dose of 10 mL / kg. Animals were weighed before administration, and the dose was calculated based on their body weight. Sample collection time points were 0.083, 0.167, 0.5, 1, 2, 4, 8, and 24 h. Approximately 200 μL of whole blood was collected from the retro-orbital venous plexus at each time point and used to prepare plasma for concentration measurement by high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). Plasma concentrations were processed using a non-compartmental model in Winnolin pharmacokinetic software, and pharmacokinetic parameters were calculated using the log-linear trapezoidal method. The results are shown in Table 13 below.

[0120] [Table 13]

[0121] As can be seen from the data in the table above, some compounds of the present disclosure showed significant differences in pharmacokinetics (PK) in mice, indicating that deuterium substitution at different positions significantly affected the PK of the compounds. Among them, the bioavailability of compound 5 in mice was superior to that of chidamide.

[0122] Biological Example 8: Pharmacokinetic Study of Compounds of the Present Disclosure in Rats Healthy female rats aged 6 to 8 weeks were administered intravenously and orally at doses of 1 mg / kg and 10 mg / kg. The rats were fasted overnight before administration. On the day of the experiment, animals in the intravenous administration group received a single injection of the corresponding compound at 10 mL / kg via the tail vein, while animals in the oral administration group received a single injection of the corresponding compound at 10 mL / kg via intragastric injection. Sample collection time points were 0.083, 0.167, 0.5, 1, 2, 4, 8, and 24 hours. Approximately 200 μL of whole blood was collected from the neck at each time point and used to prepare plasma for concentration measurement by high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). Plasma concentrations were processed using a non-compartmental model in Winnolin pharmacokinetic software, and pharmacokinetic parameters were calculated using the log-linear trapezoidal method. The results are shown in Table 14 below.

[0123] [Table 14]

[0124] Biological Example 9: In vivo efficacy studies of compounds of the present disclosure in the MC-38 model Female C57BL6N mice (6 weeks old, 18–22 g) were provided by Vital River Laboratory Animal Technology Co., Ltd. (China) and were used after one week of quarantine and acclimation. All animals were housed in a room at 23 ± 2°C and 50 ± 5% relative humidity, with artificial lighting from 08:00 to 20:00 daily and air changes 13–18 times per hour. Mouse colon carcinoma MC38 cells were cultured in 1640 medium containing 10% fetal bovine serum in a conventional manner at 37°C / 5% CO2 incubator. After subculture, cells were harvested when they reached the desired number. 2 × 10 6 MC38 cells were injected subcutaneously into the right flank of C57BL6N mice to form tumors. 3 After the animals reached maturity, they were randomly assigned to groups and administered the treatment. Tumor volumes were measured using calipers on days 3, 7, 10, 14, 17, and 21 after administration. The tumor growth inhibitory ability of the compounds was evaluated using the tumor growth inhibition ratio (TGI) = 1 - (tumor volume of the treatment group on day 28 - tumor volume of the treatment group on day 1) / (tumor volume of the control group on day 28 - tumor volume of the control group on day 1). Compound toxicity was evaluated based on the body weight and condition of the mice.

[0125] The groups are as follows: 1) Vehicle control group, 2) PD-1 group, 3) BDO group, 4) Chidamide group, 5) Compound 1 group, 6) Compound 5 group, 7) PD-1 + BDO group, 8) Compound 1 and PD-1 combination group, 9) Compound 1 and BDO combination group, 10) Compound 5 and PD-1 combination group, 11) Compound 5 and BDO combination group, 12) Compound 1, PD-1 and BDO combination group, 13) Compound 5, PD-1 and BDO combination group, 14) Chidamide, PD-1 and BDO combination group (6 mice per group). The results are shown in Table 15 below.

[0126] [Table 15]

[0127] NOTE: BDO stands for anti-VEGF antibody, PD-1 stands for anti-PD-1 antibody, IV stands for intravenous administration, IP stands for intraperitoneal administration, PO stands for oral administration, QD stands for once-daily administration, QW stands for once-weekly administration, PR stands for partial response (tumor volume reduction of more than 30% compared to the initial volume at the completion of the administration cycle), and SD stands for stable disease (tumor volume reduction or growth does not exceed 30% compared to the initial volume at the completion of the administration cycle).

[0128] As can be seen from the results of the in vivo experiments above, the compounds of the present disclosure, when administered in combination with PD-1 and BDO, showed relatively good inhibitory effects on the MC-38 in vivo tumor model. Among them, compound 5 + PD-1 + BDO (Group 13) was able to induce tumor regression in 50% (3 / 6) of mice, and its effect was significantly superior to that of chidamide + PD-1 + BDO (Group 14, 1 / 6).

[0129] Biological Example 10: In vivo efficacy studies of compounds of the present disclosure in the CT-26 model Female BALB / c mice (6 weeks old, 18–22 g) were provided by Vital River Laboratory Animal Technology Co., Ltd. (China) and were used after one week of quarantine and acclimation. All animals were housed in a room at 23 ± 2°C and 50 ± 5% relative humidity, with artificial lighting from 08:00 to 20:00 daily and air exchanged 13–18 times per hour. Mouse colon carcinoma CT-26 cells were cultured in 1640 medium containing 10% fetal bovine serum in a conventional manner in an incubator at 37°C and 5% CO2. After subculture, cells were harvested when they reached the desired number. 2 × 10 5 CT-26 cells were subcutaneously injected into the right flank of BALB / c mice to form tumors. 3After the animals reached maturity, they were randomly assigned to groups and administered the compound. Tumor volumes were measured with calipers on days 3, 7, 10, 14, 17, and 21 after administration. The tumor growth inhibitory ability of the compound was evaluated using the tumor growth inhibition ratio (TGI) = 1 - (tumor volume on day 28 in the treatment group - tumor volume on day 1 in the treatment group) / (tumor volume on day 28 in the control group - tumor volume on day 1 in the control group). Compound toxicity was evaluated based on the body weight and condition of the mice.

[0130] The groups are as follows: 1) Vehicle control group, 2) PD-1 group, 3) BDO group, 4) Chidamide group, 5) Compound 1 group, 6) Compound 5 group, 7) PD-1 + BDO group, 8) Compound 1 and PD-1 combination group, 9) Compound 1 and BDO combination group, 10) Compound 5 and PD-1 combination group, 11) Compound 5 and BDO combination group, 12) Compound 1, PD-1 and BDO combination group, 13) Compound 5, PD-1 and BDO combination group, 14) Chidamide, PD-1 and BDO combination group (6 mice per group). The results are shown in Table 16 below.

[0131] [Table 16]

[0132] NOTE: BDO stands for anti-VEGF antibody, PD-1 stands for anti-PD-1 antibody, IV stands for intravenous administration, IP stands for intraperitoneal administration, PO stands for oral administration, QD stands for once-daily administration, QW stands for once-weekly administration, PR stands for partial response (tumor volume reduction of more than 30% compared to the initial volume at the completion of the administration cycle), SD stands for stable disease (tumor volume reduction or growth does not exceed 30% compared to the initial volume at the completion of the administration cycle), and CR stands for complete tumor regression.

[0133] As can be seen from the results of the in vivo experiments above, the compounds of the present disclosure, when administered in combination with PD-1 and BDO, showed relatively good inhibitory effects on the CT-26 in vivo tumor model. Among them, compound 5 + PD-1 + BDO (Group 13) was able to induce tumor regression or slow tumor growth in mice (5 / 6), and its effect was significantly better than that of chidamide + PD-1 + BDO (Group 14, 2 / 6).

[0134] Although specific embodiments of the present disclosure have been described above, it will be understood by those skilled in the art that these embodiments are merely examples and that many changes or modifications can be made to these embodiments without departing from the principles and spirit of the present disclosure. Therefore, the scope of protection of the present disclosure is defined by the appended claims.

Claims

1. A compound of general formula (1), or an isomer thereof, a crystalline form thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof. 【Chemistry 1】 (In general formula (1), X １ , X ２ , X ３ , X ４ , X ５ , X ６ , X ７ , X ８ , X ９ , X １０ , X １１ , X １２ , X １３ , X １４ , and X １５ are each independently hydrogen or deuterium, and X １ , X ２ , X ３ , X ４ , X ５ , X ６ , X ７ , X ８ , X ９ , X １０ , X １１ , X １２ , X １３ , X １４ , or X １５ At least one of is selected from deuterium.

2. In general formula (1), X ８ =X ９ 2. The compound according to claim 1, wherein:

3. The compound has the following structure: 【Chemistry 2】 【change】 【change】 3. The compound according to claim 1 or 2, or an isomer thereof, a crystalline form thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof, having one of the following formula:

4. The compound has the following structure: 【Transformation 3】 3. The compound according to claim 1 or 2, or an isomer thereof, a crystalline form thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof, having one of the following formula:

5. The compound has the following structure: 【Chemistry 4】 3. The compound according to claim 1 or 2, having the formula:

6. The compound has the following structure: 【Transformation 5】 3. The compound according to claim 1 or 2, having the formula:

7. A pharmaceutical composition comprising a pharmaceutically acceptable excipient or carrier and, as an active ingredient, a compound according to any one of claims 1 to 6, or an isomer thereof, a crystalline form thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof.

8. A pharmaceutical composition comprising a pharmaceutically acceptable excipient or carrier, and as active ingredients, a therapeutically effective amount of a compound according to any one of claims 1 to 6 or an isomer thereof, a crystalline form thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof, and a therapeutically effective amount of an immune checkpoint inhibitor.

9. A pharmaceutical composition comprising a pharmaceutically acceptable excipient or carrier, and as active ingredients, a therapeutically effective amount of the compound according to any one of claims 1 to 6 or an isomer thereof, a crystalline form thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, a therapeutically effective amount of an immune checkpoint inhibitor, and a therapeutically effective amount of a VEGFR inhibitor.

10. Use of a compound according to any one of claims 1 to 6, or an isomer thereof, a crystalline form thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof, or a pharmaceutical composition according to claims 7 to 9, in the preparation of a medicament for treating, regulating and / or preventing an HDAC inhibitor-associated disease.

11. The use according to claim 10, wherein the disease is cancer, and the cancer is a blood cancer or a solid cancer.

12. 12. The use of claim 11, wherein the cancer comprises breast cancer, colon cancer, uterine cancer, pancreatic cancer, lung cancer, gastric cancer, leukemia, lymphoma, prostate cancer, liver cancer, cervical cancer, neuroblastoma, melanoma, or intracranial tumor.

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