HDAC inhibitor and application thereof

Abstract

The invention relates to an HDAC inhibitor and application thereof. Specifically, the invention relates to a compound as shown in a general formula (1) and a preparation method thereof, and application of the compound as shown in the general formula (1) and isomers, crystal forms, pharmaceutically acceptable salts, hydrates or solvates of the compound as HDAC inhibitors in preparation of antitumor drugs.

Description

HDAC inhibitor and use thereof

[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. This application incorporates the entirety of the aforementioned Chinese patent applications. Technical Field

[0002] The present invention belongs to the field of medicinal chemistry, and more specifically, relates to a class of HDAC inhibitors, a preparation method thereof, and the use of such compounds in preparing drugs for treating or preventing cancer. Background Art

[0003] Histone deacetylases (HDACs) are a class of proteases that regulate gene expression and protein function by deacetyling histone and non-histone lysine residues. HDAC members are currently divided into four major classes, with 18 different subtypes. Class I includes four subtypes: HDAC1, 2, 3, and 8; Class II includes six subtypes: HDAC4, 5, 6, 7, 9, and 10; and Class IV has only one subtype: HDAC11. Classes I, II, and IV are structurally homologous; whereas Class III, which includes seven subtypes, SIRT1-7, shares no structural homology with the first three classes. HDAC inhibitors inhibit the growth and survival of various tumor cells in vitro. Several HDAC inhibitors, represented by Chidamide (cedamide), have been approved for clinical use as monotherapy or in combination for the treatment of relapsed or refractory peripheral T-cell lymphoma, multiple myeloma, large B-cell lymphoma, and breast cancer.

[0004] Recently, studies have shown that in addition to playing a role in tumor cells, specific HDAC subtypes have the function of regulating tumor immunity, such as inhibiting HDAC1 and 2 to upregulate NKG2D expression and enhance the ability of NK cells to kill tumors (Molecules, 2021, 26(13):3952); inhibiting HDAC3 to upregulate CXCL10-mediated immune cell infiltration (Cancer Immunol Res, 2023, 11(5):657); inhibiting HDAC6 to downregulate inflammasome-mediated IL1β 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 can also regulate the tumor immune microenvironment and anti-tumor effects by inhibiting angiogenesis. Different subtypes of HDAC inhibitors also have different regulatory effects on other important immune cells such as T cells. HDAC inhibitors targeting different subtypes can enhance the efficacy of tumor immunotherapy drugs, such as the immune checkpoint inhibitor anti-PD-1 monoclonal antibody, and overcome resistance to tumor immunotherapy drugs. Therefore, the development of drugs with selective inhibitory effects on specific HDAC subtypes can effectively enhance the therapeutic effect of immune checkpoint inhibitors and has great clinical value.

[0005] Summary of the Invention

[0006] The present invention unexpectedly discovered a unique class of HDAC inhibitors that are selective for specific subtypes, with superior tumor immune synergy and metabolic stability. In vivo experiments observed that the compounds of the present invention, when combined with PD-1 inhibitors and VEGF inhibitors, exhibited outstanding synergistic effects, causing tumor regression or even disappearance, demonstrating a powerful anti-tumor effect.

[0007] The present invention provides a compound represented by general formula (1) or its isomers, crystal forms, pharmaceutically acceptable salts, hydrates or solvates:

[0008] In the general formula (1):

[0009] X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 、X 11 、X 12 、X 13 、X 14 and X 15 are independently hydrogen or deuterium, and X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 、X11 、X 12 、X 13 、X 14 or X 15 At least one is selected from deuterium.

[0010] In another preferred technical solution, in the compound of the general formula (1), X8=X9.

[0011] In another embodiment of the present invention, the compound of formula (1) has one of the following structures:

[0012] Another object of the present invention is to provide a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent and / or excipient, and a compound of the general formula (1) of the present invention, or its isomers, crystal forms, pharmaceutically acceptable salts, hydrates or solvates as active ingredients.

[0013] Another object of the present invention is to provide the use of the compound represented by general formula (1) of the present invention, or its isomers, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, or the pharmaceutical composition thereof, for preparing a drug for treating, regulating or preventing diseases associated with HDAC inhibitors, wherein the disease is preferably cancer, and the cancer is a blood cancer or a solid tumor.

[0014] It is to be understood that both the foregoing general description and the following detailed description of the present invention are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.

[0015] Synthesis of compounds

[0016] The following specifically describes the preparation methods of the compound of general formula (1) of the present invention, but these specific methods do not constitute any limitation to the present invention.

[0017] The compounds of formula (1) described above can be synthesized using standard synthetic techniques or known techniques in combination with the methods described herein. In addition, the solvents, temperatures and other reaction conditions mentioned herein may vary. The starting materials used in the synthesis of the compounds can be synthesized or obtained from commercial sources. The compounds described herein and other related compounds having different substituents can be synthesized using known techniques and starting materials, including those found in March, ADVANCED ORGANIC CHEMISTRY 4 th Ed., (Wiley 1992); Carey and Sundberg, ADVANCED ORGANIC CHEMISTRY 4 thEd., Vols.A and B (Plenum 2000, 2001), Green and Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 3 rd Ed., (Wiley 1999). The general methods for the preparation of compounds can be modified by using appropriate reagents and conditions to introduce various groups into the formulae provided herein.

[0018] In one aspect, the compounds described herein are prepared according to methods known in the art. However, the conditions of the methods, such as reactants, solvents, bases, amounts of the compounds used, reaction temperatures, reaction times, etc., are not limited to the following explanations. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, and such combinations can be easily performed by those skilled in the art. In one aspect, the present invention also provides a method for preparing the compound of formula (1), wherein the compound of formula (1) can be prepared using the following general reaction scheme 1:

[0019] General reaction scheme 1

[0020] X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 、X 11 、X 12 、X 13 、X 14 and X 15 As defined above. As shown in general reaction scheme 1, raw materials S1 and S2 undergo amine acid condensation reaction to obtain compound A3, A3 is deprotected under appropriate conditions to obtain compound A4, A4 is further condensed with S3 to obtain compound A5, and A5 is deprotected under acidic conditions to obtain the target compound (1).

[0021] Further forms of compounds

[0022] "Pharmaceutically acceptable" as used herein refers to a substance, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compound and is relatively non-toxic, i.e., a substance that does not cause undesirable biological effects or interact in a deleterious manner with any of its components when administered to a subject.

[0023] The term "pharmaceutically acceptable salt" refers to a form of a compound that does not cause significant irritation to the organism to which it is administered and does not abrogate the biological activity and properties of the compound. In certain specific aspects, pharmaceutically acceptable salts are obtained by reacting a compound of the formula with an acid or base, wherein the acid or base includes, but is not limited to, those found in Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use 1. st Acids and Bases in Ed., (Wiley, 2002).

[0024] It should be understood that references to pharmaceutically acceptable salts include solvent-added forms or crystallized forms, particularly solvates or polymorphs. Solvates contain stoichiometric or non-stoichiometric amounts of solvent and are selectively formed during crystallization with pharmaceutically acceptable solvents such as water, ethanol, etc. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is ethanol. Solvates of compounds of formula (1) are conveniently prepared or formed according to the methods described herein. For example, hydrates of compounds of formula (1) are conveniently prepared by recrystallization from a mixed solvent of water / organic solvent, using organic solvents including, but not limited to, tetrahydrofuran, acetone, ethanol or methanol. In addition, the compounds mentioned herein can exist in unsolvated and solvated forms. In general, for the purposes of the compounds and methods provided herein, the solvated forms are considered to be equivalent to the unsolvated forms.

[0025] In other embodiments, the compound of formula (1) is prepared in different forms, including but not limited to, amorphous, crushed and nano-particle forms. In addition, the compound of formula (1) includes crystalline forms and can also be polymorphic. Polymorphs include different lattice arrangements of the same elemental composition of the compound. Polymorphs generally have different X-ray diffraction spectra, infrared spectra, melting points, density, hardness, crystal form, optical and electrical properties, stability and solubility. Different factors such as recrystallization solvent, crystallization rate and storage temperature may cause a single crystalline form to dominate.

[0026] In another aspect, compounds of formula (1) may have chiral centers and / or axial chirality and thus occur as racemates, racemic mixtures, single enantiomers, diastereomeric compounds and single diastereomers, and cis-trans isomers. Each chiral center or axial chirality will independently produce two optical isomers, and all possible optical isomers and diastereomeric mixtures as well as pure or partially purified compounds are included within the scope of the present invention. The present invention is intended to include all such isomeric forms of these compounds.

[0027] The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, the compounds may be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125 ( 125 I) and C-14( 14 C). For example, deuterated compounds can be formed by replacing hydrogen atoms with heavy hydrogen. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs generally have advantages such as reduced toxic side effects, increased drug stability, enhanced efficacy, and prolonged drug half-life in vivo. All isotopic variations of the compounds of the present invention, whether radioactive or not, are included within the scope of this invention.

[0028] Unless otherwise specified, any reference to an atom in the compounds of the present invention refers to its stable atomic isotope. Unless otherwise specified, when a position in a molecular structure is designated as "H" or "hydrogen," such position should be understood to have the natural abundance of the hydrogen isotope. Similarly, when a position is designated as "D" or "deuterium," such position should be understood to have a deuterium isotope abundance at least 3000 times its natural abundance (the natural abundance of the deuterium isotope is 0.015%).

[0029] More preferably, the deuterium atom abundance at each deuterated site of the deuterated compound of the present invention is at least 3500 times its natural abundance (52.2% deuterium atom enrichment). More preferably, it is at least 4500 times (67.5% deuterium atom enrichment). More preferably, it is at least 5000 times (75% deuterium atom enrichment). More preferably, it is at least 6000 times (90% deuterium atom enrichment). More preferably, it is at least 6333 times (95% deuterium atom enrichment). More preferably, it is at least 6466.7 times (97% deuterium atom enrichment). More preferably, it is at least 6600 times (99% deuterium atom enrichment). More preferably, it is at least 6633.3 times (99.5% deuterium atom enrichment).

[0030] the term

[0031] Unless otherwise indicated, the terms used in this application, including the specification and claims, are defined as follows. It should be noted that, in the specification and the appended claims, the singular forms "a," "an," and "an" include plural references unless the context clearly indicates otherwise. Conventional methods such as mass spectrometry, nuclear magnetic resonance, HPLC, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology are employed, unless otherwise indicated. Throughout this application, the use of "or" or "and" means "and / or," unless otherwise indicated.

[0032] The term "isomer" means any tautomer, stereoisomer, isotope, enantiomer or diastereomer of any compound of the present invention. The compounds of the present invention may have one or more chiral centers or double bonds and therefore exist in stereoisomeric form, for example, as double bond isomers (i.e., E / Z geometric isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers). Therefore, the compounds of the present invention encompass all corresponding stereoisomers, i.e., stereoisomerically pure (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) forms as well as enantiomers and stereoisomer mixtures, such as racemates. Enantiomeric and stereoisomeric mixtures of the compounds of the present invention can be separated into their component enantiomers or stereoisomers by well-known methods, such as chiral gas chromatography, chiral high performance liquid chromatography, and crystallization of the compounds as chiral salt complexes or crystallization of the compounds in chiral solvents. Enantiomers and stereoisomers can also be obtained from stereoisomerically pure or enantiomerically pure intermediates, reagents and catalysts by well-known asymmetric synthetic methods.

[0033] The term "isotopomers" refers to different molecules whose structures differ only in one isotope but are otherwise identical.

[0034] Specific pharmaceutical and medical terms

[0035] The term "acceptable," as used herein, means that a prescribed ingredient or active ingredient has no undue adverse effect on health and well-being for the general purpose of treatment.

[0036] The terms "treat," "treatment," or "therapy" as used herein include alleviating, inhibiting, or ameliorating the symptoms of a disease or condition; inhibiting the development of complications; ameliorating or preventing underlying metabolic syndrome; inhibiting the development of a disease or symptom, such as controlling the progression of a disease or condition; alleviating a disease or symptom; causing a regression of a disease or symptom; alleviating complications caused by a disease or symptom, or preventing or treating signs caused by a disease or symptom. As used herein, a compound or pharmaceutical composition, upon administration, can improve a disease, symptom, or condition, particularly by improving its severity, delaying its onset, slowing its progression, or reducing its duration. Whether the administration is fixed or temporary, continuous or intermittent, the circumstances attributable to or related to the administration can be explained.

[0037] "Active ingredient" refers to the compound of formula (1), as well as pharmaceutically acceptable inorganic or organic salts of the compound of formula (1). The compounds of the present invention may contain one or more asymmetric centers (chiral centers or axial chirality) and therefore appear in the form of racemates, racemic mixtures, single enantiomers, diastereomeric compounds and single diastereomers. The asymmetric centers that may exist depend on the properties of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers, and all possible optical isomers and diastereomeric mixtures as well as pure or partially pure compounds are included within the scope of the present invention. The present invention is meant to include all such isomeric forms of these compounds.

[0038] The terms "compound," "composition," "agent," or "medicine or medicament" are used interchangeably herein and refer to a compound or composition that, when administered to a subject (human or animal), induces a desired pharmaceutical and / or physiological response through local and / or systemic action.

[0039] The term "administered," "administering," or "administration" as used herein refers to the direct administration of the compound or composition, or the administration of a prodrug, derivative, or analog of the active compound.

[0040] Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate, the numerical values ​​of the specific examples are presented herein as precisely as possible. However, any numerical value inherently and inevitably contains standard deviations resulting from individual testing methods. As used herein, "about" generally refers to the actual value being within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "about" means that the actual value falls within an acceptable standard error of the mean, as determined by one skilled in the art. Except in the experimental examples, or unless otherwise expressly indicated, all ranges, amounts, values, and percentages used herein (e.g., to describe material amounts, time periods, temperatures, operating conditions, quantitative ratios, and the like) are to be understood as modified by the word "about." Therefore, unless otherwise indicated, the numerical parameters disclosed in this specification and the appended claims are approximate and may be modified as needed. At a minimum, these numerical parameters should be understood to include the number of significant digits indicated and to include normal rounding.

[0041] Unless otherwise defined in this specification, the scientific and technical terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, unless otherwise defined in this specification, singular terms used in this specification include the plural form of the term, and plural terms also include the singular form of the term, unless otherwise defined in the context.

[0042] Therapeutic uses

[0043] The present invention provides methods for treating diseases using the compounds of formula (1) or pharmaceutical compositions of the present invention, including but not limited to conditions involving HDAC enzymes (eg, cancer).

[0044] In some embodiments, a method for treating cancer is provided, comprising administering to a subject in need thereof an effective amount of any of the aforementioned pharmaceutical compositions comprising a compound of formula (1). In some embodiments, the cancer is mediated by HDAC enzymes. In some embodiments, the compounds of the present invention are used in combination with an immune checkpoint inhibitor; in some embodiments, the compounds of the present invention are used in combination with a PD-1 or PD-L1 inhibitor; in some embodiments, the compounds of the present invention are used in combination with a PD-1 antibody; in some embodiments, the compounds of the present invention are used in combination with a PD-L1 antibody; in some embodiments, the compounds of the present invention are used in combination with a VEGF / VEGFR inhibitor; in some embodiments, the compounds of the present invention are used in combination with an immune checkpoint inhibitor and a VEGF / VEGFR inhibitor; in some embodiments, the compounds of the present invention are used in combination with a PD-1 inhibitor and a VEGF / VEGFR inhibitor; in some embodiments, the compounds of the present invention are used in combination with a PD-1 antibody and a VEGF / VEGFR inhibitor; wherein the PD-1 antibody includes but is not limited to nivolumab, pembrolizumab, toripalimab, sintilimab, camrelizumab, tislelizumab, penampalimab, sepalimab, slulizumab, putalimab, pidilizumab, cemiprilizumab, spartalizumab, AMG404 , RN888, mAb15, MEDI-0680, BGB-108, spartalizumab, IBI-308, mDX-400, SHR-1210, PF-06801591, PDR-001, GB-226 and STI-1110, as well as biosimilars, bioenhancers and bioequivalents of these inhibitors; wherein the PD-L1 antibodies include but are not limited to durvalumab, atezolizumab, envomalimab, sugemalimab, velumab, evolozumab, BMS-93 6559, AMP-714, ALN-PDL, TSR-042, KD-033, CA-170, STI-1014 and KY-1003, as well as biosimilars, bioenhancers and bioequivalents of these inhibitors; wherein the 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 other embodiments, the tumor range includes but is not limited to breast cancer, colon cancer, uterine cancer, pancreatic cancer, lung cancer, gastric cancer, blood cancer, lymphoma, prostate cancer, liver cancer, cervical cancer, neuroblastoma, melanoma or intracranial tumor.

[0045] Route of administration

[0046] The compounds of the present invention and their pharmaceutically acceptable salts can be formulated into various formulations containing a safe and effective amount of the compounds of the present invention or their pharmaceutically acceptable salts and a pharmacologically acceptable excipient or carrier. "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 will be determined based on the patient's age, condition, and duration of treatment, among other factors.

[0047] "Pharmaceutically acceptable excipients or carriers" refer to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmacologically acceptable excipients or carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0048] The compounds of the present invention may be administered orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), or topically.

[0049] Solid dosage forms for oral administration include capsules, tablets, pills, powders, 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 with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0050] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compounds in such compositions can be delayed in a certain portion of the digestive tract. Examples of useful encapsulating components are polymeric substances and waxes. If desired, the active compound can also be microencapsulated with one or more of the above-mentioned excipients.

[0051] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, or mixtures thereof.

[0052] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0053] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0054] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous 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.

[0055] Dosage forms for topical administration of the compounds of this invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0056] The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds. When using a pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage. For a 60 kg human, the daily dosage is generally 1 to 2000 mg, preferably 20 to 1000 mg. Of course, the specific dosage will also take into account factors such as the route of administration and the patient's health status, all of which are within the skill of a skilled physician.

[0057] The features described above, or in the embodiments, may be combined in any combination. All features disclosed in this specification may be used in any combination, and each feature disclosed in this specification may be replaced by any alternative feature that serves the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the features disclosed are merely general examples of equivalent or similar features. DETAILED DESCRIPTION

[0058] The following description will elaborate on various specific aspects, characteristics, and advantages of the above-mentioned compounds, methods, and pharmaceutical compositions so that the present invention will be readily apparent. It should be understood that the following detailed description and examples describe specific embodiments and are provided for reference only. After reading the present description, those skilled in the art may make various changes or modifications to the present invention, and such equivalents are within the scope of the present invention.

[0059] In all embodiments, 1 H-NMR was recorded on a Varian Mercury 400 nuclear magnetic resonance instrument, and chemical shifts are expressed in δ (ppm). Silica gel used for separation was 200-300 mesh unless otherwise specified, and the eluent ratios were by volume.

[0060] The present invention uses the following abbreviations: ACN represents acetonitrile; AcOH represents glacial acetic acid; AIBN represents azobisisobutyronitrile; Boc2O represents di-tert-butyl dicarbonate; CDCl3 represents deuterated chloroform; (COCl)2 represents oxalyl chloride; D2 represents deuterium gas; D2O represents heavy water; DBU represents 1,8-diazabicyclo[5.4.0]undec-7-ene; DCM represents dichloromethane; Dioxane represents 1,4-dioxane; DIPEA represents diisopropylethylamine; DMSO represents dimethyl sulfoxide; DMAP represents 4-dimethylaminopyridine; DMF represents N,N-dimethylformamide; EA represents ethyl acetate; EtOH represents ethanol; EDCI represents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; FA represents formic acid; Flash represents fast medium pressure preparative chromatography; h represents hours; H2 represents hydrogen ; HOBt represents 1-hydroxybenzotriazole; K2CO3 represents anhydrous potassium carbonate; KOH represents potassium hydroxide; LC-MS represents liquid chromatography-mass spectrometry; LiOH represents lithium hydroxide; LiOH.H2O represents lithium hydroxide monohydrate; MeOH represents anhydrous methanol; MeOD represents deuterated methanol; min represents minute; mL represents milliliter; MS represents mass spectrometry; NaBD4 represents sodium tetradeuterated borohydride; NaOAc represents anhydrous sodium acetate; n-BuLi represents n-butylaluminum; NaBH(OAc)3 represents sodium triacetoxyborohydride; NBS represents N-bromosuccinimide; NH4Cl represents ammonium chloride; NMR represents nuclear magnetic resonance; Pd / C represents palladium on carbon; PE represents petroleum ether; PPTS represents 4-methylbenzenesulfonate pyridine; TFA represents trifluoroacetic acid; TFAA represents trifluoroacetic anhydride; THF represents tetrahydrofuran; Zn represents zinc powder.

[0061] Preparation Example 1 Synthesis of 4-((2,2,2-trifluoroacetylamino)methyl)benzoic acid-2-d acid (S1-1)

[0062] Synthesis of S1-1a:

[0063] To a 100 mL single-necked flask was added 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 deuterium-purged three times, then connected to a deuterium bag and stirred at room temperature for 6 h. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated under reduced pressure to a small amount. The residue was added with EA (100 mL) and saturated sodium bicarbonate solution (50 mL), stirred, and the layers separated. The organic phase was washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to give the product as a colorless oil (1.82 g, >100%).

[0064] ESI-MS m / z:167.1[M+H] + .

[0065] Synthesis of S1-1b:

[0066] To a 100 mL single-necked flask was added compound S1-1a (1.82 g, crude product, 8.2 mmol), THF (20 mL), MeOH (10 mL), and NaOH (1.6 g, 40.0 mmol). The mixture was stirred at room temperature for 5 h. After completion of the reaction as determined by LC-MS, the mixture was concentrated, the residue flash purified, and lyophilized to afford the product as a white solid (840 mg, 67.4%).

[0067] ESI-MS m / z:153.0[M+H] + .

[0068] Synthesis of S1-1:

[0069] 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 single-necked 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 reaction was stirred at room temperature for 6 h. After the reaction was completed by LC-MS detection, 2N HCl solution (30 mL) was added to the mixture, stirred for 20 min, and then the liquid was separated. The organic phase was washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was slurried with EA (5 mL) / PE (30 mL) at room temperature for 30 min, filtered, and dried to give an off-white solid product (1.21 g, 88.4%).

[0070] ESI-MS m / z:249.0[M+H] + .

[0071] Target intermediates S1-2 to S1-6 were obtained by using different raw materials and adopting the synthesis method of intermediate S1-1.

[0072] Table 1. Structural formulas of intermediates S1-2 to S1-6

[0073] Preparation Example 2 Synthesis of 4-((2,2,2-trifluoroacetylamino-d)methyl-d2)benzoic acid (S1-7)

[0074] Synthesis of S1-7a:

[0075] To a 500 mL single-necked flask, 4-cyanobenzoic acid (2.94 g, 20.0 mmol), 4 M HCl / MeOH (5 mL, 20.0 mmol), and 10% Pd / C (300 mg) were added to MeOH (100 mL). The system was deuterium-purged three times, then connected to a deuterium bag and stirred at room temperature for 20 h. After completion of the reaction as determined by LC-MS, the mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure to afford the product as a white solid (4.1 g, >100%).

[0076] ESI-MS m / z:156.1[M+H] + .

[0077] Synthesis of S1-7:

[0078] 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-necked 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 reaction was stirred at room temperature for 6 h. After the reaction was completed by LC-MS detection, 2N HCl solution (30 mL) was added to the mixture, stirred for 20 min, and then the liquid was separated. The organic phase was washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was slurried with EA (20 mL) / PE (100 mL) at room temperature for 30 min, filtered, and dried to give an off-white solid product (2.65 g, 53%).

[0079] ESI-MS m / z:251.0[M+H] + .

[0080] Preparation Example 3 Synthesis of tert-butyl (2-amino-5-fluorophenyl-4-d)carbamate (S2-1)

[0081] Synthesis of S2-1a:

[0082] 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 Boc2O (2.62 g, 12.0 mmol) were added. The mixture was heated to reflux under argon for 20 h. After completion of the reaction as determined by LC-MS, water (50 mL) and DCM (50 mL) were added to the mixture, stirred, and separated. The organic phase was washed with 1N HCl (50 mL), washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to afford the product as a yellow-brown solid (3.7 g, yield: >100%).

[0083] ESI-MS m / z:335.0[M+H] + .

[0084] Synthesis of S2-1b:

[0085] The crude product S2-1a was added to THF (50 mL), followed by zinc powder (6.5 g, 0.1 mol). A solution of AcOH (6 g, 0.1 mol) in THF (10 mL) was slowly added dropwise under an ice bath. The mixture was stirred at room temperature for 4 h. After completion of the reaction, the mixture was filtered, the filter cake was rinsed with THF, the filtrate was concentrated, and the residue was purified by column chromatography to afford the product as a brown solid (1.52 g, 49.8%).

[0086] ESI-MS m / z:305.0[M+H] + .

[0087] Synthesis of S2-1:

[0088] 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 deuterium-purged three times, then connected to a deuterium bag and stirred at room temperature for 20 h. After LC-MS analysis, the mixture was filtered, and the filtrate was concentrated under reduced pressure to a small amount. The residue was added with EA (50 mL) and saturated sodium bicarbonate solution (50 mL), stirred, and the layers separated. The organic phase was washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to obtain the product as a brown solid (970 mg, 91.1%).

[0089] ESI-MS m / z:228.1[M+H] + .

[0090] The target intermediates S2-2 and S2-3 were obtained by using different raw materials and the synthesis method of intermediate S2-1.

[0091] Table 2. Structural formulas of intermediates S2-2 to S2-3

[0092] Preparation Example 4 Synthesis of (E)-3-(pyridin-3-yl-4-d)acrylic acid (S3-1)

[0093] Synthesis of S3-1a:

[0094] 4-Bromopyridine-3-carboxaldehyde (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 deuterium-purged three times, then connected to a deuterium bag and stirred at room temperature for 20 hours. After LC-MS analysis, the mixture was filtered, and the filtrate was concentrated under reduced pressure to a small amount. The residue was added with EA (50 mL) and saturated sodium bicarbonate solution (50 mL), stirred, and the layers separated. The organic phase was washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to obtain the product as a white solid (1.1 g, >100%).

[0095] ESI-MS m / z:109.1[M+H] + .

[0096] Synthesis of S3-1:

[0097] To a 100 mL single-necked flask was added 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). After argon replacement, the mixture was heated to reflux with stirring for 20 h. After completion of the reaction as determined by LC-MS, the mixture was concentrated, the residue flash purified, and lyophilized to afford the product as an off-white solid (853 mg, 56.9%).

[0098] ESI-MS m / z:151.0[M+H] + .

[0099] The target intermediates S3-2 to S3-7 were obtained by using different raw materials and the synthesis method of intermediate S3-1.

[0100] Table 3. Structural formulas of intermediates S3-2 to S3-7

[0101] Preparation Example 5 Synthesis of (E)-3-(pyridin-3-yl)acrylic acid-2,3-d2 acid (S3-8)

[0102] Synthesis of S3-8a:

[0103] 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 deuterium-purged three times, then connected to a deuterium bag and stirred at room temperature for 20 hours. After completion of the reaction as determined by LC-MS, the mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure to afford the product as an anhydrous oil (1.8 g, >100%).

[0104] ESI-MS m / z:170.1[M+H]+ .

[0105] Synthesis of S3-8b:

[0106] To a 100 mL single-necked flask, compound S3-8a (1.8 g, 10.0 mmol), AIBN (164 mg, 1.0 mmol), and acetonitrile (40 mL) were added, followed by portionwise addition of NBS (1.96 g, 11.0 mmol). The mixture was heated to reflux and reacted for 6 h. Upon completion of the reaction as determined by LC-MS, the mixture was cooled, concentrated, and the residue was directly used in the next step.

[0107] Synthesis of S3-8c:

[0108] To a 100 mL single-necked flask was added compound S3-8b (5.2 g, crude product, 10.0 mmol), DBU (3.04 g, 20.0 mmol), and acetonitrile (40 mL). The mixture was then heated to 50°C for 4 h. After completion of the reaction as determined by LC-MS, the mixture was cooled, concentrated, and the residue was flash purified to afford the product as a colorless oil (930 mg, 56.4%).

[0109] ESI-MS m / z:166.1[M+H] + .

[0110] Synthesis of S3-8:

[0111] To a 100 mL single-necked vial was added compound S3-8c (930 mg, 5.64 mmol), THF (5 mL), MeOH (5 mL), and LiOH.H2O (474 ​​mg, 11.3 mmol). The mixture was stirred at room temperature for 5 h. After completion of the reaction, as determined by LC-MS, the mixture was concentrated, the residue flash purified, and lyophilized to afford the product as a white solid (716 mg, 83.5%).

[0112] ESI-MS m / z:152.0[M+H] + .

[0113] The target intermediates S3-9 to S3-15 were obtained by using different raw materials and the synthetic method of intermediate S3-8.

[0114] Table 4. Structural formulas of intermediates S3-9 to S3-15

[0115] Preparation Example 6 Synthesis of (E)-3-(pyridin-3-yl)acrylic acid-3-d acid (S3-16)

[0116] Synthesis of S3-16a:

[0117] To a 100 mL single-necked flask, 3-oxo-3-(pyridin-3-yl)propanoic acid (2 g, 12.12 mmol), THF (30 mL), and MeOD (2 g) were added. NaBD4 (509 mg, 12.12 mmol) was then added portionwise at room temperature. After addition, the mixture was stirred at room temperature for 2 h. After completion of the reaction as determined by LC-MS, the mixture was quenched by dropwise addition of D2O (5 mL), concentrated, and the residual amount purified by Flash chromatography to afford the product as a colorless solid (1.66 g, 81%).

[0118] ESI-MS m / z:170.0[M+H] + .

[0119] Synthesis of S3-16:

[0120] To a 100 mL single-necked flask, 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. The mixture was heated to 100°C and stirred for 16 h. After completion of the reaction as determined by LC-MS, the mixture was filtered, the filter cake rinsed with THF, and the filtrate concentrated to dryness. The residue was flash purified to afford the product as a white solid (1.03 g, 69.9%).

[0121] ESI-MS m / z:151.0[M+H] + .

[0122] The target intermediate S3-17 was obtained by using different raw materials and the synthetic method of intermediate S3-16.

[0123] Table 5 Structural formula of intermediate S3-17

[0124] Example 1 Synthesis of (E)-N-(2-amino-4-fluorophenyl-5-d)-4-((3-(pyridin-3-yl)acrylamido)methyl)benzamide (Compound 1)

[0125] Step 1: Synthesis of compound 1-1:

[0126] To a 100 mL single-necked flask, 4-((2,2,2-trifluoroacetylamino)methyl)benzoic acid (S1-0, 446 mg, 1.8 mmol), DMF (20 mg), and DCM (10 mL) were added. Under argon, a solution of (COCl)2 (343 mg, 2.7 mmol) in DCM (2 mL) was added dropwise at room temperature. After completion of the addition, the mixture was stirred at room temperature for 2 h. After completion of the reaction as determined by LC-MS, the mixture was concentrated to dryness under pressure. The residue was further added to DCM (10 mL), followed by a solution of DIPEA (700 mg, 5.43 mmol) and S2-1 (410 mg, 1.81 mmol) in DCM (5 mL) at room temperature. After completion of the addition, the mixture was stirred at room temperature for 1 h. After the reaction was complete as determined by LC-MS, the mixture was added with DCM (30 mL) and water (30 mL), stirred, and separated. 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 further slurried with EA (2 mL) / PE (8 mL) at room temperature, filtered, and dried to give the product as a light brown solid (581 mg, 70.8%).

[0127] ESI-MS m / z:457.1[M+H] + .

[0128] Step 2: Synthesis of compound 1-2:

[0129] To a 100 mL single-necked flask, 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 and heated to 60°C with stirring for 6 h. After completion of the reaction as determined by LC-MS, the mixture was concentrated. The resulting residue was added with DCM (30 mL) and saturated sodium chloride solution (20 mL), stirred, and separated. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to afford an off-white solid product (486 mg, 106%).

[0130] ESI-MS m / z:361.1[M+H] + .

[0131] Step 3: Synthesis of compound 1-3:

[0132] To a 100 mL single-necked flask was added (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 min before the addition of compound 1-2 (106 mg, 0.278 mmol). The reaction was stirred for 16 h. After completion of the reaction by LC-MS, the mixture was flash purified to afford the product as a slightly yellow solid (118 mg, 86.4%).

[0133] ESI-MS m / z:492.1[M+H] + .

[0134] Step 4: Synthesis of compound 1:

[0135] To a 100 mL single-necked vial was added compound 1-3 (118 mg, 0.24 mmol), DCM (5 mL), and TFA (0.5 mL). The mixture was stirred at room temperature for 2 h. After completion of the reaction as determined by LC-MS, the mixture was concentrated and the residue purified by liquid phase preparative purification to afford the product as a white solid (60 mg, 64%).

[0136] ESI-MS m / z:392.1[M+H] + .

[0137] Example 2 Synthesis of (E)-N-(2-amino-4-fluorophenyl)-4-((3-(pyridin-3-yl)acrylamido)methyl)-3-d-benzamide (Compound 5)

[0138] Step 1: Synthesis of compound 5-1:

[0139] To a 100 mL single-necked flask, 4-((2,2,2-trifluoroacetylamino)methyl)-3-d-benzoic acid (S1-2, 600 mg, 2.42 mmol), DMF (30 mg), and DCM (10 mL) were added dropwise. Under argon, a solution of (COCl)2 (461 mg, 3.63 mmol) in DCM (2 mL) was added dropwise at room temperature. After completion of the reaction, the mixture was stirred at room temperature for 2 h. After completion of the reaction, as determined by LC-MS, the mixture was concentrated to dryness under pressure. The residue was further added to DCM (10 mL), followed by a solution of DIPEA (1.03 g, 8.0 mmol) and tert-butyl (2-amino-5-fluorophenyl)carbamate (547 mg, 2.42 mmol) in DCM (5 mL) at room temperature. After completion of the addition, the mixture was stirred at room temperature for 1 h. After the reaction was complete as determined by LC-MS, the mixture was added with DCM (30 mL) and water (30 mL), stirred, and separated. 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 further slurried with EA (2 mL) / PE (8 mL) at room temperature, filtered, and dried to give the product as a light brown solid (853 mg, 77.3%).

[0140] ESI-MS m / z:457.1[M+H] + .

[0141] Step 2: Synthesis of compound 5-2:

[0142] To a 100 mL single-necked flask, compound 5-1 (853 mg, 1.87 mmol), MeOH (10 mL), and anhydrous potassium carbonate (516 mg, 3.74 mmol) were added. After argon replacement, the mixture was heated to 60°C and stirred for 6 h. After completion of the reaction as determined by LC-MS, the mixture was concentrated. The resulting residue was added with DCM (30 mL) and saturated sodium chloride solution (20 mL), stirred, and separated. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to afford an off-white solid product (660 mg, 98.0%).

[0143] ESI-MS m / z:361.1[M+H] + .

[0144] Step 3: Synthesis of compound 5-3:

[0145] To a 100 mL single-necked flask was added (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 before the addition of compound 5-2 (160 mg, 0.417 mmol). The reaction was stirred for 16 h. After completion of the reaction by LC-MS, the mixture was flash purified to afford the product as a slightly yellow solid (140 mg, 68.3%).

[0146] ESI-MS m / z:492.1[M+H] + .

[0147] 1 H NMR (600MHz, DMSO-d6) δ9.77(s,1H),8.84–8.72(m,3H),8.56(dd,J=4.7,1.7Hz,1H),8.01(dt,J=8.0,2.0Hz,1H),7.97–7.90(m,2H),7.53(d d,J=15.0,5.4Hz,2H),7.46(dt,J=8.4,2.1Hz,3H),6.97(td,J=8.4,3.0Hz,1H),6.83(d,J=15.9Hz,1H),4.51(d,J=6.0Hz,2H),1.45(s,9H).

[0148] Step 4: Synthesis of compound 5:

[0149] To a 100 mL single-necked vial was added compound 5-3 (140 mg, 0.285 mmol), DCM (5 mL), and TFA (0.5 mL). The mixture was stirred at room temperature for 2 h. After completion of the reaction, as determined by LC-MS, the mixture was concentrated and the residue purified by liquid phase preparative purification to afford the product as a white solid (82 mg, 73.5%).

[0150] ESI-MS m / z:392.1[M+H] + .

[0151] Similar to the synthesis of compound 1 and compound 5, other target compounds in Table 6 can be obtained by using different intermediates as raw materials.

[0152] Table 6

[0153] The NMR data of some compounds in this patent are listed in Table 7 below:

[0154] Table 7

[0155] Biological Example 1 Inhibitory test of Jurkat and 293T cell proliferation by the compounds of the present invention

[0156] 3000 Jurkat or 293T cells were seeded in a 96-well plate. After overnight attachment, serial dilutions of the compound were added to the plate. After 72 hours, intracellular ATP levels were measured using CTG. The IC value of the compound for inhibiting cell proliferation was calculated compared to that of DMSO. 50 The results are shown in Table 8 below.

[0157] Table 8 Inhibitory activity of the compounds of the present invention on Jurkat / 293T cell proliferation (IC 50 ,μM)

[0158] As shown in the above table, the compounds of the present invention selectively kill Jurkat cells, with significant differences in their killing activity against human renal fibroblasts 293T and Jurkat tumor cells. Compounds 1 and 5 are more potent than Chidamide against Jurkat tumor cells, but less potent than Chidamide against 293T cells.

[0159] Biological Example 2 Determination of the Effect of Compounds of the Present Invention on Intracellular Acetyl Lysine / H3K 27Acetyl Lysine Levels

[0160] 20,000 HeLa cells were seeded per well in a 96-well plate and allowed to adhere overnight. A gradient of compound dilutions was then added. After 24 hours of treatment, intracellular acetyl lysine and H3K 27 acetyl lysine levels were measured by ELISA. The results are shown in Table 9 below.

[0161] Table 9 Effects of the compounds of the present invention on the levels of acetyl lysine and H3K 27

[0162] Biological Example 3 Inhibitory Activity Test of the Compounds of the Present Invention on HDAC Enzymes

[0163] Fluorometric method was used to determine the effect of compound 5 of the present invention on HDAC enzyme activity. A gradient dilution of DMSO sample solution was added to the reaction wells, enzyme was added to the 384-well plate, reaction buffer was added to the control wells, and the mixture was incubated at room temperature for 15 minutes. The reaction was started after the fluorescent substrate solution was added. The fluorescence intensity readings per minute within 60 minutes were measured using a Paradigm detector (excitation light: 355 nM, emission light: 460 nM), and the slope value was calculated. Inhibition rate (%) = (maximum value - sample value) / (maximum value - minimum value) × 100%. Y = Bottom + (Top-Bottom) / (1 + (IC 50 / X)^HillSlope) formula is fitted into a curve to obtain IC 50 The results are shown in Table 10 below.

[0164] Table 10 Inhibitory activity of the compounds of the present invention on HDAC1, 2, 3, 10 enzymes (IC 50 ,nM)

[0165] The results showed that compound 5 of the present invention had good inhibitory activity against four subtypes of HDAC1, 2, 3, and 10. Its activity against HDAC1 was slightly stronger than that of chidamide (1.2 times), its activity against HDAC2 was slightly weaker than that of chidamide (0.83 times), and its activity against HDAC10 was 2.5 times that of chidamide. The activity against HDAC6 was higher than 30 mM.

[0166] Biological Example 4 Effect of the Compounds of the Present Invention on NK Cell-Mediated Tumor Cell Killing

[0167] 4000 GFP-expressing OVCAR 3 cells or NK92 cells were seeded in a 96-well plate. After overnight adherence, serial dilutions of the compound were added and incubated for 72 hours. After 1-4 hours of mixed culture, the NK92 cells were washed with PBS to remove the adherent GFP-positive tumor cells. The EC of the compound that promoted NK92 cell-mediated OVCAR 3 cell death was calculated compared to DMSO. 50 The results are shown in Table 11 below.

[0168] Table 11 Effect of the compounds of the present invention on NK cell-mediated tumor cell killing

[0169] The results showed that the compounds 1 and 5 of the present invention had significantly better NK cell-mediated tumor cell killing effects than the control drug Chidamide.

[0170] Biological Example 5 Liver microsomal stability test of the compound of the present invention

[0171] After incubation of 1 μM compound with 0.5 mg / ml human or mouse liver microsomes and NADPH regeneration system at 37°C for different time periods, the residual amount of compound was analyzed by LC-MS-MS and T was calculated. 1 / 2 The results are shown in Table 12 below.

[0172] Table 12 Evaluation results of the stability of the compounds of the present invention in human and mouse liver microsomes

[0173] It can be seen from the data in the above table that the stability of some compounds of the present invention in human liver microsomes is significantly better than that of Chidamide.

[0174] Biological Example 6 Identification of Metabolites of the Compounds of the Invention in Various Liver Microsomes and Hepatocytes

[0175] 10 μM compound was incubated with 1.0 mg / ml liver microsomes or hepatocytes (5 species: mouse, rat, dog, monkey and human) and NADPH regeneration system at 37 degrees for 120 minutes, then the stop solution was added to terminate the reaction and the incubated samples were removed from the water bath. After the reaction was terminated, the sample was shaken and centrifuged at 4°C and 3260×g for 15 minutes. After centrifugation, all supernatants were transferred to a 96-well plate and used directly for LC-MS analysis or diluted or concentrated for reconstitution and injection. An LC-UV-HRMSn (n=1-2) analysis method was established on LC-UV-HRMSn, and SCIEX OS software was used for data acquisition. Different mass spectrometry scanning modes (MS / MS or IDA) and UV full wavelength (λ=190-500nm) scanning were performed on the parent drug and metabolites. The blank sample and the incubated sample of the same species were compared, and the UV image and mass spectrum were compared to identify possible metabolites. By comparing the CID (collision-induced fragmentation) fragments of the test sample and its metabolites, the possible structures of the metabolites can be inferred. The metabolites and their relative abundances are listed in a table for comparison between different species.

[0176] The experimental results show that some of the compounds of the present invention, especially compound 5, have simple metabolic pathways and have good stability in various liver microsomes.

[0177] LC-MS analysis conditions:

[0178] Elution gradient:

[0179] LC-MS / MS analysis conditions:

[0180] Elution gradient:

[0181] Mass spectrometry parameters:

[0182] Biological Example 7 Metabolic Kinetics Experiment of the Compounds of the Present Invention in Mice

[0183] Female CD-1 mice aged 7 to 10 weeks were administered intravenously and orally at doses of 1 mg / kg and 10 mg / kg, respectively. Mice were fasted for at least 12 hours before dosing and resumed feeding 4 hours after dosing. Water was available ad libitum throughout the experiment.

[0184] On the day of the experiment, the animals in the intravenous group were given a single injection of the corresponding compound via the tail vein at a volume of 10 mL / kg; the animals in the oral group were given a single injection of the corresponding compound via gavage at a volume of 10 mL / kg. The animals were weighed before administration, and the administration volume was calculated based on their body weight. Sample collection times were: 0.083, 0.167, 0.5, 1, 2, 4, 8, and 24 h. Approximately 200 μL of whole blood was collected from the orbital venous plexus at each time point for plasma preparation for concentration determination by high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). Plasma concentrations were processed using a non-compartmental model using Winnolin pharmacokinetic software, and pharmacokinetic parameters were calculated using the linear logarithmic trapezoidal method. The results are shown in Table 13 below.

[0185] Table 13 In vivo pharmacokinetic evaluation results of the compounds of the present invention

[0186] As can be seen from the data in the table above, some compounds of the present invention have significant differences in PK in mice, indicating that deuterium substitution at different positions has a significant effect on the PK of the compounds. Among them, compound 5 has better bioavailability in mice than chidamide.

[0187] Biological Example 8 Metabolic Kinetics Experiment of the Compounds of the Present Invention in Rats

[0188] Healthy 6-8 week old female rats were selected and administered 1 mg / kg and 10 mg / kg intravenously and orally. The rats were fasted overnight before administration.

[0189] On the day of the experiment, the intravenous group of animals was given a single injection of the corresponding compound via the tail vein at a volume of 10 mL / kg; the oral group of animals was given a single injection of the corresponding compound via gavage at a volume of 10 mL / kg. Sample collection times were: 0.083, 0.167, 0.5, 1, 2, 4, 8, and 24 h. At each time point, approximately 200 μL of whole blood was collected from the neck to prepare plasma for concentration determination by high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). Plasma concentrations were processed using a non-compartmental model using Winnolin pharmacokinetic software, and pharmacokinetic parameters were calculated using the linear logarithmic trapezoidal method. The results are shown in Table 14 below.

[0190] Table 14 In vivo pharmacokinetic evaluation results of the compounds

[0191] Biological Example 9 In vivo efficacy experiment of the compound of the present invention in MC-38 model

[0192] Female C57BL6N mice (6 weeks, 18-22 g) were provided by Weitong Lihua Co., Ltd., China, and used after one week of quarantine and acclimatization. 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 13-18 air changes per hour.

[0193] Mouse colon cancer MC38 cells were routinely cultured in 1640 medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. After passage, the cells were collected when the cells reached the required number. 2×10 6 MC38 cells were used to form tumors, and the tumors were grown to 100 mm. 3 After approximately 14 days, the animals were randomly divided into groups and dosing began. Tumor volumes were measured with calipers on days 3, 7, 10, 14, 17, and 21 after dosing. The ability of the compounds to inhibit tumor growth was evaluated using the formula: tumor growth inhibition (TGI) = 1 - (tumor volume of the dosing group on day 28 - tumor volume of the dosing group on day 1) / (tumor volume of the control group on day 28 - tumor volume of the control group on day 1). The toxicity of the compounds was assessed based on the weight and condition of the mice.

[0194] The groups are as follows:

[0195] 1) Solvent 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, with 6 mice in each group. The results are shown in Table 15 below.

[0196] Table 15 In vivo efficacy of some compounds of the present invention in MC-38 model

[0197] Note: BDO denotes anti-VEGF antibody; PD-1 denotes anti-PD-1 antibody; IV denotes intravenous administration; IP denotes intraperitoneal administration; PO denotes oral administration; QD denotes once-daily administration; QW denotes once-weekly administration; PR denotes partial response (after completion of the dosing cycle, the tumor volume decreases by more than 30% relative to the initial volume); SD denotes stable disease (after completion of the dosing cycle, the tumor volume decreases or increases by no more than 30% relative to the initial volume).

[0198] The in vivo results demonstrate that the compounds of the present invention, combined with PD-1 and BDO, exhibit significant inhibitory activity against the MC-38 tumor model. Compound 5 combined with PD-1 and BDO (Group 13) resulted in tumor regression in 50% (3 / 6) of the mice, significantly outperforming Chidamide combined with PD-1 and BDO (Group 14, 1 / 6).

[0199] Biological Example 10 In vivo efficacy experiment of the compound of the present invention in CT-26 model

[0200] Female BALB / c mice (6 weeks, 18-22 g) were provided by Weitong Lihua Co., Ltd., China, and used after one week of quarantine and acclimatization. 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 13-18 air changes per hour.

[0201] Mouse colon cancer CT-26 cells were routinely cultured in 1640 medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. After passage, the cells were collected when the cells reached the required number. 2×10 5 CT-26 cells were used to form tumors, and the tumors were grown to 100 mm. 3After approximately 14 days, the animals were randomly divided into groups and dosing began. Tumor volumes were measured with calipers on days 3, 7, 10, 14, 17, and 21 after dosing. The ability of the compounds to inhibit tumor growth was evaluated using the formula: tumor growth inhibition (TGI) = 1 - (tumor volume of the dosing group on day 28 - tumor volume of the dosing group on day 1) / (tumor volume of the control group on day 28 - tumor volume of the control group on day 1). The toxicity of the compounds was assessed based on the weight and condition of the mice.

[0202] The groups are as follows:

[0203] 1) Solvent 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, with 6 mice in each group. The results are shown in Table 16 below.

[0204] Table 16 In vivo efficacy of some compounds of the present invention in the CT-26 model

[0205] Note: BDO indicates anti-VEGF antibody; PD-1 indicates anti-PD-1 antibody; IV indicates intravenous administration; IP indicates intraperitoneal administration; PO indicates oral administration; QD indicates once-daily administration; QW indicates once-weekly administration; PR indicates partial response (tumor volume decreases by more than 30% relative to the initial volume after the administration cycle); SD indicates stable disease (tumor volume decreases or increases by no more than 30% relative to the initial volume after the administration cycle). CR indicates complete tumor regression;

[0206] The in vivo experimental results above demonstrate that the compounds of the present invention, combined with PD-1 and BDO, exhibited significant inhibitory effects against the CT-26 tumor model. Compound 5 + PD-1 + BDO (Group 13) was able to induce tumor regression or slow tumor growth in 5 / 6 mice, demonstrating significantly superior efficacy to Chidamide + PD-1 + BDO (Group 14, 2 / 6).

[0207] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A compound represented by the general formula (1) or its isomers, crystal forms, pharmaceutically acceptable salts, hydrates or solvates: 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 independently hydrogen or deuterium, and X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 、X 11 、X 12 、X 13 、X 14 or X 15 At least one is selected from deuterium.

2. The compound according to claim 1 or its isomers, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein in the general formula (1), X8 = X9.

3. The compound according to claim 1 or 2, or any isomer, crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof, wherein the compound has one of the following structures:

4. The compound according to claim 1 or 2, or any isomer, crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof, wherein the compound has one of the following structures:

5. The compound according to claim 1 or 2, or any isomer, crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof, wherein the compound has the following structure:

6. The compound according to claim 1 or 2, or any isomer, crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof, wherein the compound has the following structure:

7. A pharmaceutical composition, characterized in that It contains a pharmaceutically acceptable excipient or carrier, and the compound according to any one of claims 1 to 6 or its isomers, crystal forms, pharmaceutically acceptable salts, hydrates or solvates as active ingredients.

8. A pharmaceutical composition, characterized in that It contains a pharmaceutically acceptable excipient or carrier, a therapeutically effective amount of the compound according to any one of claims 1 to 6 or its respective isomers, respective crystal forms, pharmaceutically acceptable salts, hydrates or solvates, and a therapeutically effective amount of an immune checkpoint inhibitor as active ingredients.

9. A pharmaceutical composition, characterized in that It contains a pharmaceutically acceptable excipient or carrier, and a therapeutically effective amount of the compound according to any one of claims 1 to 6 or its respective isomers, respective crystal forms, pharmaceutically acceptable salts, hydrates or solvates, and a therapeutically effective amount of an immune checkpoint inhibitor and a therapeutically effective amount of a VEGFR inhibitor as active ingredients.

10. Use of the compound according to any one of claims 1 to 6 or its isomers, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, or the pharmaceutical composition according to claims 7 to 9 in the preparation of a drug for treating, regulating and / or preventing diseases associated with HDAC inhibitors.

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

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