HDAC inhibitors and uses thereof

Abstract

This invention discloses a class of HDAC inhibitors and their uses. Specifically, it relates to a compound of general formula (1) and its preparation method, and the use of the compound of general formula (1) and its isomers, crystal forms, pharmaceutically acceptable salts, hydrates or solvates as HDAC inhibitors in the preparation of antitumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, and more specifically, relates to a class of HDAC inhibitors, their preparation methods, and the application of such compounds in the preparation of drugs for treating or preventing cancer. Background Technology

[0002] Histone deacetylases (HDACs) are a class of proteases whose acetylation and deacetylation of histones in chromatin are key steps in regulating gene expression. Abnormal gene expression is the molecular biological basis for tumors and some genetic and metabolic diseases. The degree of histone acetylation is coordinated and controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). When HDACs are overexpressed and recruited by transcription factors, it leads to abnormal repression of specific genes, resulting in tumors and other diseases.

[0003] HDAC is a large family of enzymes, currently known to comprise four main classes and 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 (of which 4, 5, 7, and 9 belong to IIa, and 6 and 10 belong to IIb); Class IV consists of only one subtype, HDAC11, which shares some homology with the first two classes; and Class III includes seven subtypes, SIRT1-7, which do not share structural homology with the first three classes.

[0004] Patent WO2007045844 discloses a series of compounds with good selectivity for HDAC1,2,3, including a compound codenamed CXD101 that is in clinical trials. Based on published clinical results, this series of compounds exhibits significantly fewer toxic side effects compared to marketed compounds such as vorinostat, romidesin, belistat, pabistamine, and chidamide.

[0005] Summary of the Invention

[0006] This invention provides a compound of general formula (1) or its isomers, crystal forms, pharmaceutically acceptable salts, hydrates or solvates:

[0007]

[0008] In general formula (1):

[0009] R 1 It is H, D or halogen;

[0010] R 2 It is H, D or halogen;

[0011] R3 It can be H, D, (C1-C4)alkyl, (C1-C4)alkoxy, (C1-C4)haloalkyl, (C2-C4)alkenyl, (C2-C4)ynyl, (C3-C6)cycloalkyl or (3-6)heterocyclic alkyl;

[0012] R 4 It can be H, D, (C1-C4)alkyl, (C1-C4)alkoxy, (C1-C4)haloalkyl, (C2-C4)alkenyl, (C2-C4)ynyl, (C3-C6)cycloalkyl or (3-6)heterocyclic alkyl;

[0013] X is either N or CH;

[0014] m and n are integers of 1 or 2.

[0015] In another preferred embodiment, in the compound of general formula (1), R 1 It can be H, D, F, Cl, or Br.

[0016] In another preferred embodiment, in the compound of general formula (1), R 2 It can be H or D.

[0017] In another preferred embodiment, in the compound of general formula (1), R 3 It can be methyl, trifluoromethyl, difluoromethyl, ethyl, cyclopropyl, or oxecyclobutyl.

[0018] In another preferred embodiment, in the compound of general formula (1), R 4 It can be methyl, ethyl, cyclopropyl or oxecyclobutyl.

[0019] In another specific embodiment of the present invention, the compound of general formula (1) has one of the following structures:

[0020]

[0021]

[0022]

[0023] Another object of the present invention is to provide a pharmaceutical composition comprising a pharmaceutically acceptable carrier, a diluent and / or an excipient, and a compound of general formula (1) of the present invention, or its isomers, crystal forms, pharmaceutically acceptable salts, hydrates or solvates as active ingredients.

[0024] Another object of the present invention is to provide the use of the compound of general formula (1) of the present invention, or any isomer, crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof, or the pharmaceutical composition thereof, in the preparation of a medicament for treating, regulating or preventing diseases associated with HDAC inhibitors. The disease is preferably cancer, specifically hematologic malignancies and solid tumors. Preferred are breast cancer, colon cancer, uterine cancer, pancreatic cancer, lung cancer, gastric cancer, hematologic malignancies, lymphoma, prostate cancer, liver cancer, cervical cancer, neuroblastoma, melanoma, or intracranial tumors.

[0025] It should be understood that the foregoing general description of the invention and the following detailed description are exemplary and illustrative, and are intended to provide further explanation of the claimed invention.

[0026] Compound Synthesis

[0027] The preparation methods of the compounds of the present invention are described in detail below, but these specific methods do not constitute any limitation on the present invention.

[0028] The compounds described above can be synthesized using standard synthetic techniques or known techniques combined with the methods described herein. Furthermore, the solvents, temperatures, and other reaction conditions mentioned herein can be varied. Starting materials used for the synthesis of the compounds can be obtained synthetically or from commercial sources, such as, but not limited to, Aldrich Chemical Co. (Milwaukee, Wis.) or Sigma Chemical Co. (St. Louis, Mo.). The compounds described herein and other related compounds with different substituents can be synthesized using known techniques and starting materials, including those discovered in March, ADVANCED ORGANIC CHEMISTRY 4. th Ed., (Wiley 1992); Carey and Sundberg, ADVANCED ORGANIC CHEMISTRY 4 th Ed., Vols.A and B (Plenum 2000, 2001), Green and Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 3 rd The method described in Ed. (Wiley 1999) can be used to prepare compounds by employing appropriate reagents and by introducing different groups into the molecular formulas provided herein.

[0029] On the one hand, the compounds described herein are prepared according to methods known in the art. However, the conditions of the method, such as reactants, solvents, bases, amounts of compounds used, reaction temperatures, and reaction times, are not limited to the explanations below. The compounds of the present invention can also be conveniently prepared by combining various synthetic methods described in this specification or known in the art, such combinations being readily performed by those skilled in the art. On the other hand, the present invention also provides a method for preparing the compounds described herein, wherein compounds of general formula (1) can be prepared using the following general reaction procedure 1 or 2: General reaction procedure 1

[0030]

[0031] Where R 1 R 2 R 3 R 4 As defined above, X, m, and n represent commonly used amine protecting groups (such as Cbz, Fmoc, trifluoroacetyl, Boc, etc.). As shown in the general reaction flow 1, compound A1 is hydrolyzed under alkaline conditions to obtain compound A2. A2 and the starting material A3 undergo a condensation reaction to obtain compound A4. A4 is deprotected under appropriate conditions to obtain compound A5. A5 and A6 undergo a reductive amination reaction and deprotection to obtain the target compound (1).

[0032] General reaction process 2

[0033]

[0034] Where R 1 R 2 R 3 R 4 X, m, and n are defined as described above. As shown in the general reaction flow 2, compound B1 and A6 undergo a reductive amination reaction to obtain compound B2. Compound B2 is hydrolyzed under alkaline conditions to obtain compound B3. Compound B3 and the starting material A3 undergo a condensation reaction to obtain compound B4. B4 is deprotected under appropriate conditions to obtain the target compound (1).

[0035] Further forms of the compound

[0036] "Pharmaceutical acceptable" here means that a substance, such as a carrier or diluent, will not destroy the biological activity or properties of a compound and is relatively non-toxic. For example, when given to an individual, a substance will not cause unwanted biological effects or interact with any of its components in a harmful manner.

[0037] The term "pharmaceutically acceptable salt" refers to a form of a compound that does not cause significant irritation to the administered organism and does not diminish the compound's biological activity and properties. In some specific aspects, pharmaceutically acceptable salts are obtained by reacting a compound of a general formula with an acid or base, wherein said 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).

[0038] It should be understood that references to pharmaceutically acceptable salts include solvent-added forms or crystalline forms, especially solvates or polymorphs. Solvates contain stoichiometric or non-stoichiometric solvents and are selectively formed during crystallization with pharmaceutically acceptable solvents such as water, ethanol, etc. A hydrate is formed when the solvent is water, or an alcohol is formed when the solvent is ethanol. Solvates of compounds of general formula (1) are readily prepared or formed according to the methods described herein. For example, hydrates of compounds of general formula (1) are readily prepared by recrystallization from a mixture of water and organic solvents, including but not limited to tetrahydrofuran, acetone, ethanol, or methanol. Furthermore, the compounds mentioned herein can exist in both solvated and non-solvated forms. In summary, for the purposes of the compounds and methods provided herein, the solvated form is considered equivalent to the non-solvated form.

[0039] In other specific embodiments, compounds of general formula (1) are prepared in various forms, including but not limited to amorphous, pulverized, and nano-particle forms. Furthermore, compounds of general formula (1) include crystalline forms and can also be polymorphic. Polymorphs comprise different lattice arrangements of the same elemental composition of the compound. Polymorphs typically have different X-ray diffraction spectra, infrared spectra, melting points, densities, hardness, crystal forms, optical and electrical properties, stability, and solubility. Different factors such as recrystallization solvents, crystallization rates, and storage temperatures may cause a single crystal form to dominate.

[0040] In another aspect, compounds of general formula (1) may possess a chiral center and / or axial chirality, and thus appear as racemates, racemic mixtures, single enantiomers, diastereomers, 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 diastereomer mixtures, as well as pure or partially pure compounds, are included within the scope of this invention. This invention means including all such isomeric forms of these compounds.

[0041] The compounds of this invention may contain atomic isotopes in non-natural proportions on one or more atoms constituting 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, deuterium can be used to replace hydrogen atoms to form deuterated compounds. The bond between deuterium and carbon is stronger than that between ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs generally have advantages such as reduced toxicity, increased drug stability, enhanced efficacy, and prolonged drug half-life in vivo. All isotopic variations of the compounds of this invention, regardless of radioactivity, are included within the scope of this invention.

[0042] Unless otherwise specified, any atom in the compounds described in this invention refers to its stable-state isotope. Unless otherwise specified, when a site on the molecular structure is chosen as "H" or "hydrogen", that site should be understood as having a natural abundance of hydrogen isotopes. Similarly, unless otherwise specified, when a site is chosen as "D" or "deuterium", that site should be understood as having a deuterium isotope abundance of at least 3000 times its natural abundance (the natural abundance of deuterium isotopes is 0.015%).

[0043] In this invention, "isotope enrichment factor" means the ratio of the isotope to its natural isotope.

[0044] More preferably, the deuterium abundance at each deuteration site of the deuterated compound in this invention is at least 3500 times its natural abundance (52.2% deuterium enrichment). More preferably, at least 4500 times (67.5% deuterium enrichment). More preferably, at least 5000 times (75% deuterium enrichment). More preferably, at least 6000 times (90% deuterium enrichment). More preferably, at least 6333 times (95% deuterium enrichment). More preferably, at least 6466.7 times (97% deuterium enrichment). More preferably, at least 6600 times (99% deuterium enrichment). More preferably, at least 6633.3 times (99.5% deuterium enrichment).

[0045] The term "isotope isomers" refers to different molecules that are identical in structure except for their isotopes.

[0046] the term

[0047] Unless otherwise specified, the terms used in this application, including the specification and claims, are defined as follows. It must be noted that in the specification and appended claims, unless otherwise clearly indicated, the singular form "a" includes the plural meaning. Unless otherwise specified, conventional methods such as mass spectrometry, nuclear magnetic resonance, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology are used. In this application, unless otherwise specified, "or" or "and" refers to "and / or".

[0048] Unless otherwise specified, "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight-chain and branched groups with 1 to 6 carbon atoms. Lower alkyl groups containing 1 to 4 carbon atoms are preferred, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, and tert-butyl. As used herein, "alkyl" includes unsubstituted and substituted alkyl groups, especially alkyl groups substituted with one or more halogens. Preferred alkyl groups are selected from CH3, CH3CH2, CF3, CHF2, CF3CH2, CF3(CH3)CH, etc. i Pr、 n Pr、 i Bu、 n Bu or t Bu.

[0049] Unless otherwise specified, "alkenyl" refers to an unsaturated aliphatic hydrocarbon group containing a carbon-carbon double bond, including straight-chain or branched groups with 1 to 14 carbon atoms. Lower alkenyl groups containing 1 to 4 carbon atoms are preferred, such as vinyl, 1-propenyl, 1-butenyl, or 2-methylpropenyl.

[0050] Unless otherwise specified, "alkynyl" refers to an unsaturated aliphatic hydrocarbon group containing a carbon-carbon triple bond, including straight-chain and branched groups with 1 to 14 carbon atoms. Lower alkynyl groups containing 1 to 4 carbon atoms are preferred, such as ethynyl, 1-propynyl or 1-butynyl.

[0051] Unless otherwise specified, "cycloalkyl" refers to a non-aromatic hydrocarbon ring system (monocyclic, bicyclic, or polycyclic). A partially unsaturated cycloalkyl group may be referred to as "cycloalkenyl" if the carbon ring contains at least one double bond, or as "cycloynyl" if the carbon ring contains at least one triple bond. Cycloalkyl groups may include monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) groups and spirocyclic groups. In some embodiments, the cycloalkyl group is monocyclic. In some embodiments, the cycloalkyl group is monocyclic or bicyclic. The cycloforming carbon atom of the cycloalkyl group may optionally be oxidized to form an oxo or thio group. Cycloalkyl groups also include cycloalkylene groups. In some embodiments, the cycloalkyl group contains 0, 1, or 2 double bonds. In some embodiments, the cycloalkyl group contains 1 or 2 double bonds (partially unsaturated cycloalkyl). In some embodiments, the cycloalkyl group may be fused with aryl, heteroaryl, cycloalkyl, and heterocyclic alkyl groups. In some embodiments, the cycloalkyl group may be fused with aryl, cycloalkyl, and heterocyclic alkyl groups. In some embodiments, the cycloalkyl group may be fused with aryl and heterocyclic alkyl groups. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cyclohepttrienyl, norcamphenyl, norpinel, norcarel, bicyclo[1.1.1]pentyl, bicyclo[2.1.1]hexyl, and so on.

[0052] Unless otherwise specified, "alkoxy" refers to an alkyl group bonded to the remainder of the molecule via an ether oxygen atom. Representative alkoxy groups are those having 1-6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, and tert-butoxy. As used herein, "alkoxy" includes unsubstituted and substituted alkoxy groups, particularly those substituted with one or more halogens. Preferred alkoxy groups are selected from OCH3, OCF3, CHF2O, CF3CH2O, etc. i- PrO, n- PrO, i- BuO、 n- BuO or t- BuO.

[0053] Unless otherwise specified, "heterocyclic alkyl" refers to a non-aromatic ring or ring system that may optionally contain one or more alkenyl groups as part of a ring structure, having at least one heteroatom ring member independently selected from boron, phosphorus, nitrogen, sulfur, oxygen, and phosphorus, preferably a saturated or partially unsaturated ring containing 1 to 4 heteroatoms selected from oxygen, sulfur, or nitrogen. If a heterocyclic alkyl contains at least one double bond, then a partially unsaturated heterocyclic alkyl may be referred to as a "heterocyclic alkenyl," or if a heterocyclic alkyl contains at least one triple bond, then a partially unsaturated heterocyclic alkyl may be referred to as a "heterocyclic ynyl." Heterocyclic alkyl can include monocyclic, bicyclic, spirocyclic, or polycyclic (e.g., having two fused or bridging rings) ring systems. In some embodiments, a heterocyclic alkyl is a monocyclic group having 1, 2, or 3 heteroatoms independently selected from nitrogen, sulfur, and oxygen. The cyclic carbon atom and heteroatom of a heterocyclic alkyl group may optionally be oxidized to form an oxo or thio group or other oxidized bond (e.g., C(O), S(O), C(S) or S(O)2, N-oxide, etc.), or the nitrogen atom may be quaternized. Heterocyclic alkyl groups may be linked via cyclic carbon atoms or cyclic heteroatoms. In some embodiments, the heterocyclic alkyl group contains 0 to 3 double bonds. In some embodiments, the heterocyclic alkyl group contains 0 to 2 double bonds. The definition of a heterocyclic alkyl group also includes portions of an aromatic ring (also called partially unsaturated heterocycles) having one or more aromatic rings fused with (i.e., sharing bonds with) the heterocyclic alkyl ring, such as benzo[a] derivatives of piperidine, morpholine, aziridine-heptadiene, or thiophene. Heterocyclic alkyl groups containing fused aromatic rings may be linked via any cyclic atom, including the cyclic atom of the fused aromatic ring. Examples of heterocyclic alkyl groups include, but are not limited to, azirrobutyl, azirroheptyl, dihydrobenzofuranyl, dihydrofuranyl, dihydropyranyl, N-morpholinyl, 3-oxa-9-azaspiro[5.5]undecyl, 1-oxa-8-azaspiro[4.5]decyl, piperidinyl, piperazinyl, oxoperazinyl, pyranyl, pyrrolidinyl, quininyl, tetrahydrofuranyl, tetrahydropyranyl, 1,2,3,4-tetrahydroquinolinyl, scopolamine, 4,5,6,7-tetrahydrothiazo[5,4-c]pyridinyl, and 4,5,6,7-tetrahydro-1H-imidazolium. Azo[4,5-c]pyridine, N-methylpiperidinyl, tetrahydroimidazolyl, pyrazolyl, butyrolactam, valproic acid, imidazolinone, hydantoin, dioxolane, phthalimide, pyrimidin-2,4(1H,3H)-diketoyl, 1,4-dioxane, morpholinyl, thiomorpholinyl, thiomorpholin-S-oxide, thiomorpholin-S,S-oxide, piperazine, pyranyl, pyridinone, 3-pyrrololinyl, thiaranyl, pyranone, tetrahydrothiophene, 2-azaspiro[3,3]heptyl, indololinyl,

[0054] Unless otherwise specified, "halogen" (or halogenated group) means fluorine, chlorine, bromine or iodine. The term "halogenated" (or "halogen substituted") appearing before the group name indicates that the group is partially or completely halogenated, that is, substituted by F, Cl, Br or I in any combination, preferably substituted by F or Cl.

[0055] "Optional" or "optionally" means that the event or condition described below may, but is not required, occur, and the description includes both the scenario in which the event or condition occurs and the scenario in which the event or condition does not occur.

[0056] Unless otherwise specified, the word “comprising”, or variations thereof such as “including” or “containing”, may be understood to mean including the stated element or integer, or a group of elements or integers, but does not exclude any other element or integer, or a group of elements or integers.

[0057] The term "membered ring" includes any ring structure. The term "membered" refers to the number of skeleton atoms that make up the ring. For example, cyclohexyl, pyridyl, pyranyl, and thioranyl are six-membered rings, while cyclopentyl, pyrroleyl, furanyl, and thiophenyl are five-membered rings.

[0058] The term "fragment" refers to a specific part or functional group of a molecule. Chemical fragments are generally considered to be chemical entities contained in or attached to a molecule.

[0059] Unless otherwise specified, use wedge-shaped solid line keys. and wedge-shaped dashed key The absolute configuration of the center of a solid is represented by a straight solid line key. and straight dashed key The relative configuration of the center of a solid is indicated by a wavy line. Indicates wedge-shaped solid line key or wedge-shaped dashed key Or use wavy lines Indicates a straight solid line key Or straight dashed key

[0060] Unless otherwise stated, use Indicates a single bond or a double bond.

[0061] Specific pharmaceutical and medical terminology

[0062] The term “acceptable,” as used herein, means that a prescription component or active ingredient does not have an excessively harmful effect on health for general therapeutic purposes.

[0063] The terms “treatment,” “treatment process,” or “therapy” as used herein include alleviating, suppressing, or improving symptoms or conditions of a disease; suppressing the development of complications; improving or preventing underlying metabolic syndrome; suppressing the development of a disease or symptom, such as controlling the progression of a disease or condition; reducing a disease or symptom; reducing a disease or symptom; reducing complications arising from a disease or symptom, or preventing or treating signs arising from a disease or symptom.

[0064] As used herein, a compound or pharmaceutical composition, when administered, can improve a disease, symptom, or condition, particularly by improving its severity, delaying its onset, slowing its progression, or reducing its duration. This may be attributable to or related to the administration, whether administered regularly or intermittently.

[0065] "Active ingredient" refers to the compounds of this invention, as well as pharmaceutically acceptable inorganic or organic salts of the compounds of this invention. The compounds of this invention may contain one or more asymmetric centers and thus appear as racemates, racemic mixtures, single enantiomers, diastereomers, and single diastereomers. The asymmetric centers that may exist depend on the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers, and all possible optical isomers and diastereomer mixtures, as well as pure or partially pure compounds, are included within the scope of this invention. This invention means including all such isomeric forms of these compounds.

[0066] The terms “compound,” “composition,” “agent,” or “medicine or medicament” may be used interchangeably here, and all refer to a compound or composition that, when applied to an individual (human or animal), can induce a desired pharmaceutical and / or physiological response through local and / or systemic action.

[0067] The term “administered, administering, or administration” here refers to the direct application of the compound or composition described herein, or the application of a prodrug, derivative, or analog of the active compound.

[0068] While the numerical ranges and parameters used to define the broader scope of this invention are approximate values, the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "approximately" may mean that the actual value falls within the acceptable standard error of the mean, as determined by those skilled in the art. Except for experimental examples, or unless explicitly stated otherwise, it is understood that all ranges, quantities, values, and percentages used herein (e.g., to describe material usage, duration, temperature, operating conditions, quantity ratios, and others similar) are modified with "approximately". Therefore, unless otherwise stated, the numerical parameters disclosed in this specification and the accompanying claims are approximate values ​​and are subject to change as needed. At a minimum, these numerical parameters should be understood as the indicated significant digits and values ​​obtained using general rounding.

[0069] Unless otherwise defined in this specification, scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art. Furthermore, unless conflicting with the context, singular nouns used herein include their plural forms, and vice versa.

[0070] Therapeutic uses

[0071] The present invention provides methods for treating diseases using compounds or pharmaceutical compositions of general formula (1) of the present invention, including but not limited to conditions involving HDAC enzymes (e.g., cancer).

[0072] In some embodiments, a method for treating cancer is provided, comprising administering to an individual in need an effective amount of any of the aforementioned pharmaceutical compositions comprising a compound of general 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 immune checkpoint inhibitors; in some embodiments, the compounds of the present invention are used in combination with PD-1 or PD-L1 inhibitors; in some embodiments, the compounds of the present invention are used in combination with PD-1 antibodies; in some embodiments, the compounds of the present invention are used in combination with PD-L1 antibodies; in some embodiments, the compounds of the present invention are used in combination with VEGF / VEGFR inhibitors; in some embodiments, the compounds of the present invention are used in combination with immune checkpoint inhibitors and VEGF / VEGFR inhibitors; in some embodiments, the compounds of the present invention are used in combination with PD-1 inhibitors and VEGF / VEGFR inhibitors; in some embodiments, the compounds of the present invention are used in combination with PD-1 antibodies and VEGF / VEGFR inhibitors; wherein the PD-1 antibodies include, but are not limited to, nivolumab, pembrolizumab, toripalimab, sintilimab, camrelizumab, tislelizumab, penaprilimab, cepalimumab, slulimab, pectilimab, cimiprimab, spartazumab, and AMG404. RN888, mAbl5, MEDI-0680, BGB-108, spartazumab, 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, envorimab, sugemalimab, velumab, avilmab, and 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, hematological malignancies, lymphoma, prostate cancer, liver cancer, cervical cancer, neuroblastoma, melanoma, or intracranial tumors.

[0073] route of administration

[0074] The compounds of this invention and their pharmaceutically acceptable salts can be formulated into various preparations, comprising, within a safe and effective range, the compounds of this invention or their pharmaceutically acceptable salts and pharmacologically acceptable excipients or carriers. "Safe and effective range" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects. The safe and effective range of the compound is determined based on the age, condition, and duration of treatment of the patient.

[0075] "Pharmaceutically acceptable excipients or carriers" refers to one or more compatible solid or liquid fillers or gelling substances that 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 and with the compounds of the present invention without significantly reducing the efficacy of the compounds. 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, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0076] When applying the compounds of this invention, they can be administered orally, rectally, parenterally (intravenously, intramuscularly, or subcutaneously), or topically.

[0077] 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 components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in the dosage forms of capsules, tablets, and pills.

[0078] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0079] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., 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.

[0080] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0081] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0082] 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.

[0083] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0084] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable compounds. When using the pharmaceutical composition, a safe and effective amount of the compound of this invention is applied to the mammal (such as a human) requiring treatment, wherein the dose administered is a pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 50–100 mg. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health condition, which are within the scope of a skilled physician's expertise.

[0085] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features. Detailed Implementation

[0086] The following description will elaborate on the specific aspects, characteristics, and advantages of the aforementioned compounds, methods, and pharmaceutical compositions, making the content of this invention readily apparent. It should be understood that the detailed descriptions and examples described below are specific embodiments and are for reference only. After reading this description, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by this application.

[0087] In all embodiments, 1 H-NMR was recorded using a Vian Mercury 400 NMR spectrometer, and chemical shifts are expressed as δ (ppm). Unless otherwise specified, the silica gel used for separation was 200-300 mesh, and all eluent ratios were by volume.

[0088] The following abbreviations are used in this invention: AcOH represents glacial acetic acid; Boc2O represents ditert-butyl dicarbonate; CDCl3 represents deuterated chloroform; D2 represents deuterium; 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; Flash represents rapid medium-pressure preparative chromatography; h represents hours; H2 represents hydrogen; HATU represents 2-(7-diethylaminophenyl)-2-ethylhexylene. (N,N,N',N'-Tetramethylurea hexafluorophosphate); HCl represents hydrochloric acid; 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 minutes; mL represents milliliters; MS represents mass spectrometry; NaBH(OAc)3 represents sodium triacetoxyborohydride; NMR represents nuclear magnetic resonance; Pd / C represents palladium on carbon; TFA represents trifluoroacetic acid; THF represents tetrahydrofuran; Zn represents zinc powder.

[0089] Preparation Example 1: Synthesis of (2-aminophenyl-4-d) tert-butyl carbamate (S1-1)

[0090]

[0091] Synthesis of S1-1a:

[0092] 4-Bromo-2-nitroaniline (2.17 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 for 20 h under argon protection. After the reaction was completed as detected by LC-MS, water (50 mL) and DCM (50 mL) were added to the mixture, which was stirred, separated, and the organic phase was washed with 1N HCl (50 mL), saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to give a yellow-brown solid product (3.5 g, yield: >100%).

[0093] ESI-MS m / z: 317.0 [M+H] + .

[0094] Synthesis of S2-1:

[0095] Compound S1-1b (3.5 g, crude product, 10.0 mmol), 10% Pd / C (100 mg), and sodium acetate (500 mg) were added to MeOH (30 mL). The system was purged with deuterium three times, and then stirred at room temperature for 20 h using a deuterium gas bag. After the reaction was completed, the mixture was filtered by LC-MS, and the filtrate was concentrated under reduced pressure to a small amount. The residue was added to EA (50 mL) and saturated sodium bicarbonate solution (50 mL), stirred, and separated. The organic phase was washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by column chromatography to give a brown solid product (970 mg, 46.4%).

[0096] ESI-MS m / z: 210.1 [M+H] + .

[0097] Using different raw materials, the target intermediates S1-2 to S1-9 in Table 1 were obtained by the synthesis method of intermediate S1-1.

[0098] Table 1. Intermediates S1-2 to S1-9

[0099]

[0100]

[0101] Example 1: Synthesis of N-(2-aminophenyl-5-d)-4-(1-((1,3-dimethyl-1H-pyrazol-4-yl)methyl)piperidin-4-yl)benzamide (compound 1)

[0102]

[0103] Step 1: Synthesis of compounds 1-2:

[0104] Compound 1-1 (3.53 g, 10.0 mmol), THF (50 ml), MeOH (20 ml), water (10 ml), and lithium hydroxide monohydrate (2.1 g, 50.0 mmol) were added to a 500 ml single-necked flask. The mixture was stirred at room temperature for 20 h. After the reaction was completed as detected by LC-MS, the mixture was concentrated to about one-third of its original volume, and then the pH was adjusted to 3–4 with 2N HCl. The mixture was extracted twice with EA (50 ml * 2), and the combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to give a colorless, viscous solid product (3.1 g, yield: 91.4%).

[0105] ESI-MS m / z: 340.2 [M+H] + .

[0106] Step 2: Synthesis of compounds 1-3:

[0107] Compounds 1-2 (3.0 g, 8.84 mmol), DIPEA (1.72 g, 13.3 mmol), S1-1 (1.85 g, 8.85 mmol), HATU (5.05 g, 13.3 mmol), and DMF (50 ml) were added to a 250 ml single-necked flask. After purging the mixture with argon, the mixture was stirred at room temperature for 20 h. After the reaction was complete as detected by LC-MS, the mixture was quenched with water (100 ml), then extracted twice with EA (100 ml * 2). The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to give a white solid product (3.52 g, yield: 75.0%).

[0108] ESI-MS m / z: 531.3 [M+H] + .

[0109] Step 3: Synthesis of compounds 1-4:

[0110] Compounds 1-3 (3.52 g, 6.63 mmol), MeOH (100 ml), and 10% Pd / C (350 mg) were added to a 250 ml single-necked flask. After the mixture was purged with hydrogen three times, it was stirred at room temperature and atmospheric pressure for 20 h. After the reaction was completed, the mixture was filtered through diatomaceous earth and the filtrate was concentrated to dryness to give a white solid product (2.72 g, yield: 100%).

[0111] ESI-MS m / z: 397.2 [M+H] + .

[0112] Step 4: Synthesis of Compound 1:

[0113] Compounds 1-4 (100 mg, 0.252 mmol), DCM (5 ml), AcOH (30 mg, 0.5 mmol), and 1,3-dimethyl-1H-pyrazole-4-carboxaldehyde (34 mg, 0.274 mmol) were added to a 50 ml single-necked flask. The mixture was stirred at room temperature for 30 min, and then NaBH(OAc)3 (106 mg, 0.5 mmol) was added. The mixture was stirred at room temperature for 20 h. After the reaction was completed by LC-MS, DCM (10 ml) and water (10 ml) were added to the system, the mixture was stirred, separated, and the aqueous phase was extracted again with DCM (10 ml). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product.

[0114] The crude product was dissolved in DCM (5 ml), followed by TFA (1 ml), and the mixture was stirred at room temperature for 2 hours. After the reaction was completed by LC-MS, the mixture was concentrated, the residue was flash purified, and lyophilized to obtain a white solid product (46 mg, yield: 45.1%).

[0115] ESI-MS m / z: 405.2 [M+H] + .

[0116] 1 H NMR (400MHz, DMSO-d6) δ9.57(s,1H),7.90(d,J=7.8Hz,2H),7.47(s,1H),7.35(d,J=8.0Hz,2H),7.14(s,1H),6.94(dd,J=8.0,1.6Hz,1H) ,6.79–6.71(m,1H),4.86(s,2H),3.72(s,3H),3.30(s,2H),2.94(m,2H),2.58(m,1H),2.12(s,3H),2.07–1.89(m,2H),1.88–1.50(m,4H).

[0117] Example 2 Synthesis of N-(2-amino-4-fluorophenyl)-4-(1-((1,3-dimethyl-1H-pyrazol-4-yl)methyl)piperidin-4-yl)benzamide (compound 7)

[0118]

[0119]

[0120] Step 1: Synthesis of compound 7-2:

[0121] Compound 7-1 (4.38 g, 20.0 mmol), DCM (100 ml), AcOH (2.4 g, 40.0 mmol), and 1,3-dimethyl-1H-pyrazole-4-carboxaldehyde (2.73 g, 22.0 mmol) were added to a 250 ml single-necked flask. The mixture was stirred at room temperature for 30 min, followed by the addition of NaBH(OAc)3 (8.48 g, 40.0 mmol). The mixture was stirred at room temperature for 20 h. After the reaction was complete as detected by LC-MS, DCM (100 ml) and water (100 ml) were added, the mixture was stirred, separated, and the aqueous phase was extracted again with DCM (100 ml). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give a colorless oily product (5.38 g, yield: 82.0%).

[0122] ESI-MS m / z: 328.2 [M+H] + .

[0123] Step 2: Synthesis of compound 7-3:

[0124] Compound 7-2 (5.38 g, 16.42 mmol), THF (100 ml), MeOH (50 ml), water (20 ml), and lithium hydroxide monohydrate (3.45 g, 82.1 mmol) were added to a 500 ml single-necked flask. The mixture was stirred at room temperature for 20 h. After the reaction was completed as detected by LC-MS, the mixture was concentrated to about one-third of its original volume, and then the pH was adjusted to 4–5 with 2N HCl. The mixture was extracted twice with EA (100 ml * 2), and the combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to give a colorless, viscous solid product (3.65 g, yield: 70.9%).

[0125] ESI-MS m / z: 314.2 [M+H] + .

[0126] Step 3: Synthesis of compound 7-4:

[0127] Compound 7-3 (313 mg, 1.0 mmol), DIPEA (194 mg, 1.5 mmol), tert-butyl (2-amino-5-fluorophenyl)carbamate (226 mg, 1.0 mmol), HATU (570 mg, 1.5 mmol), and DMF (10 ml) were added to a 100 ml single-necked flask. After purging the mixture with argon, the mixture was stirred at room temperature for 20 h. After the reaction was completed by LC-MS, the mixture was quenched with water (20 ml), then extracted twice with EA (20 ml * 2). The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to give a white solid product (306 mg, yield: 58.5%).

[0128] ESI-MS m / z: 522.3 [M+H] + .

[0129] Step 4: Synthesis of Compound 7:

[0130] Compound 7-4 (306 mg, 0.585 mmol), DCM (10 ml), and TFA (1 ml) were added to a 100 ml single-necked flask and stirred at room temperature for 2 h. After the reaction was completed as detected by LC-MS, the mixture was concentrated, the residue was flash purified, and lyophilized to give a white solid product (175 mg, yield: 71%).

[0131] ESI-MS m / z: 422.2 [M+H] + .

[0132] 1 H NMR(400MHz,Chloroform-d)δ7.82(d,J=7.9Hz,2H),7.66(s,1H),7.33(d,J=8.0Hz,2H),7.20–7.11(m,1H),6.52(ddt,J=11.1,8.2,4.0Hz,2H),4 .00(s,2H),3.81(s,3H),3.40(s,2H),3.05(d,J=11.0Hz,2H),2.62–2.5 2(m,1H),2.24(s,3H),2.12–2.01(m,2H),1.81(dt,J=13.7,10.6Hz,4H).

[0133] Similar to the synthesis of compounds 1 and 7, using different intermediates as raw materials, target compounds 2-6 and 8-49 in Table 2 can be obtained.

[0134] Table 2

[0135]

[0136]

[0137]

[0138] The NMR data for some of the compounds in this patent are listed in Table 3 below:

[0139] Table 3

[0140]

[0141] Biological Example 1: Assay on the inhibitory activity of the compounds of the present invention against HDAC1,2,3 enzymes

[0142] The inhibitory activities of the compounds of this invention against HDAC1, HDAC2, and HDAC3 were determined using the Fluorometric method. HDAC1, HDAC2, and HDAC3 were obtained by purification or by purchasing reagents directly.

[0143] Specific method: Add serially diluted DMSO sample solution to the reaction wells, add enzyme to the 384-well plate, and add reaction buffer to the control wells. Incubate at room temperature for 15 min, then add fluorescent substrate solution to start the reaction. Use a Paradigm multi-label microplate reader to detect the fluorescence intensity readings per minute over 60 min (excitation: 355 nM, emission: 460 nM), and calculate the slope value. Inhibition rate (%) = (maximum value - sample value) / (maximum value - minimum value) × 100%. Use Y = Bottom + (Top - Bottom) / (1 + (IC) 50 The formula / X)^HillSlope) is fitted to a curve to obtain IC. 50 Value, Y is the inhibition rate, and X is the compound concentration.

[0144] Table 4. Inhibitory activity of the compounds of this invention against HDAC1, 2, and 3 enzymes (IC50). 50 ,nM)

[0145] compound HDAC1 HDAC2 HDAC3 7 240 356 473

[0146] Biological Example 2: Inhibition of Jurkat and 293T cell proliferation by the compounds of the present invention.

[0147] 3000 Jurkat or 293T cells per well were seeded in 96-well plates. After overnight adhesion, serially diluted compounds were added. Intracellular ATP levels were measured using CTG after 72 hours. The IC50 of the compounds was calculated compared to DMSO to inhibit cell proliferation. 50 .

[0148] Table 5. Inhibitory activity of the compounds of the present invention against the proliferation of Jukat / 293T cells (IC50) 50 ,uM)

[0149] compound Jukat cells 293T cells compound Jukat cells 293T cells 1 0.83 4.92 7 3.2 >10 2 1.80 2.26 9 0.93 3.93

[0150] Biological Example 3: Determination of Intracellular Acetyl Lysine / H3K27 Acetyl Lysine Levels by Compounds of the Present Invention

[0151] 20,000 HeLa cells per well were seeded in 96-well plates and allowed to adhere overnight. Then, serially diluted compounds were added and the cells were treated for 24 hours. The levels of Acetyl lysine and H3K27Acetyl lysine in the cells were detected by ELISA.

[0152] Table 6. Effects of the compounds of this invention on Acetyl lysine and H3K27Acetyl lysine levels.

[0153]

[0154] Biological Example 4: Metabolic kinetics of the compounds of the present invention in mice.

[0155] Female CD-1 mice aged 7 to 10 weeks were selected, and the intravenous and oral doses were 2 mg / kg and 10 mg / kg, respectively. Mice were fasted for at least 12 hours before administration, and were given food 4 hours after administration. They had free access to water throughout the experiment.

[0156] On the day of the experiment, animals in the intravenous group were administered the corresponding compound via a single tail vein injection at a volume of 10 mL / kg; animals in the oral group were administered the corresponding compound via a single gavage injection at a volume of 10 mL / kg. Animal weight was measured before administration, and the volume of administration was calculated based on body weight. Samples were collected at 0.083, 0.167, 0.5, 1, 2, 4, 8, and 24 hours. At each time point, approximately 200 μL of whole blood and plasma were collected via the orbital venous plexus for concentration determination using high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). The non-compartmental model of Winnolin pharmacokinetic software was used to process plasma concentrations, and the linear logarithmic trapezoidal method was used to calculate pharmacokinetic parameters.

[0157] Table 7. In vivo pharmacokinetic evaluation results of the compounds.

[0158]

[0159] As shown in the table above, the compound 7Vdss in this invention has a high bioavailability of 100% and excellent ADME properties.

[0160] Biological Example 5: In vivo efficacy experiment of the compound of the present invention

[0161] Female C57BL6N mice (6 weeks old, 18-22g) were provided by Vital River Pharmaceuticals, China, and used after one week of quarantine and acclimatization. All animals were housed in a room at 23±2℃ and 50±5% relative humidity, with artificial lighting from 08:00 to 20:00 daily and ventilation at 13-18 times per hour. They had free access to standard laboratory diet and water.

[0162] Mouse colon cancer MC38 cells were routinely cultured in a 37°C, 5% CO2 incubator using 1640 containing 10% fetal bovine serum. After passage, cells were harvested when the desired cell count was reached. 2 × 10⁻⁶ cells were subcutaneously injected into the right side of C57BL6N mice. 6One MC38 tumor formed, and the tumor grew to 100mm. 3 After administration, animals were randomly divided into a solvent control group, a test compound monotherapy group, a test compound + PD-1 combination group, and a PD-1 monotherapy group, and then drug administration began. Tumor volume was measured using calipers on days 3, 7, 10, 14, 17, 21, 24, and 28 post-administration. The tumor growth inhibition capacity of the compound was evaluated using the formula: Tumor Growth Inhibition Rate (TGI) = 1 - (Tumor volume on day 28 in the treated group - Tumor volume on day 1 in the treated group) / (Tumor volume administered to the control group on day 28 - Tumor volume on day 1 in the control group). The toxicity of the compound was evaluated based on the mice's body weight and condition.

[0163] The groups are as follows:

[0164] 1) Solvent control group; 2) PD-1 group; 3) BDO group; 4) Compound 7 group; 5) PD1+BDO group; 6) Compound 7 and PD-1 and BDO combination group, 6 mice in each group. The results are shown in Table 7 below.

[0165] Table 8. In vivo efficacy of some compounds of the present invention in the MC-38 model.

[0166]

[0167]

[0168] Notes: 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 remission (tumor volume shrinks by more than 30% relative to initial volume after completing a dosing cycle); SD indicates stable disease (tumor volume shrinks or increases by no more than 30% relative to initial volume after completing a dosing cycle).

[0169] The in vivo experimental results above show that compound 7 of the present invention, in combination with PD-1 and BDO, has a good inhibitory effect on the MC-38 in vivo tumor model.

[0170] Biological Example 6: The killing of tumor cells mediated by PBMCs by the compounds of the present invention

[0171] 4000 OVCAR 3 cells / well were seeded in 96-well plates and allowed to adhere overnight. After incubation, serially diluted compounds were added, and the cells were incubated for another 72 hours. OVCAR 3 cells were stained with Calcein AM, then PBMCs were added, and the cells were cultured for another 1-4 hours. After washing with PBS, tumor cells were counted. The EC50-90% cell death response rate of the compound was calculated compared to DMSO. 50 .

[0172] Biological Example 7: Determination of PD-L1 expression in cells by the compounds of the present invention

[0173] 3000 MD-MBA 231 cells per well were seeded in 96-well plates. After overnight adhesion, serially diluted compounds were added, and after 72 hours of treatment, PD-L1 expression on the cell surface was detected by ELISA.

[0174] Biological Example 8: Liver microsomal stability test of the compounds of the present invention

[0175] After incubating 1 μM of the compound with 500 mg / ml liver microsomes from humans, monkeys, beagle dogs, rats, or mice, as well as an NADPH regeneration system at 37°C for different times, the remaining amount of the compound was analyzed by LC-MS-MS, and T was calculated. 1 / 2 .

[0176] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can 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 of formula (1) or a pharmaceutically acceptable salt thereof: ； In general formula (1): R 1 R is halogen; R 2 is H or D; R 3 R is (C1-C4)alkyl; R 4 R is (C1-C4)alkyl; X is either N or CH; m and n are both 1.

2. The compound of claim 1 or pharmaceutically acceptable salt thereof, wherein in the general formula (1), R 1 is F.

3. The compound or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein R in the general formula (1) is methyl or ethyl. 3 is methyl or ethyl.

4. The compound or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein R in the general formula (1) is methyl or ethyl. 4 is methyl or ethyl.

5. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound has one of the following structures: 。 6. A pharmaceutical composition, characterized by, It contains a pharmaceutically acceptable excipient or carrier, and a compound or a pharmaceutically acceptable salt thereof as any one of claims 1-5 as the active ingredient.

7. Use of a compound as described in any one of claims 1-5, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in claim 6, in the preparation of a medicament for treating, modulating, and / or preventing diseases associated with HDAC inhibitors.

8. The use as claimed in claim 7, wherein the disease is cancer, and the cancer is a hematologic malignancy or a solid tumor.

9. The use as described in claim 8, wherein the cancer includes breast cancer, colon cancer, uterine cancer, pancreatic cancer, lung cancer, stomach cancer, leukemia, lymphoma, prostate cancer, liver cancer, cervical cancer, neuroblastoma, melanoma, or intracranial tumor.

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