A benzodiazepine compound having HDAC6 inhibitory activity and a preparation method and application thereof

Abstract

The application provides a benzodiazepine compound and a preparation method and application thereof, a benzodiazepine compound structural general formula is shown as formula (I), or an isomer thereof, or a pharmaceutically acceptable salt, ester or prodrug thereof. The benzodiazepine compound disclosed in the application is novel in structure, can better inhibit HDAC6\HDAC1, shows higher anti-proliferation activity on various tumor cells, has low toxicity on normal cells, has small potential cardiotoxicity, has low acute toxicity on animals, and has development prospects as an efficient and low-toxicity antitumor therapeutic agent.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to benzodiazepine compounds with both HDAC6 and HDAC1 inhibitory activities, their preparation methods, and applications. Background Technology

[0002] Histone deacetylases (HDACs) and histone acetyltransferases (HATs) work together to regulate intracellular acetylation levels, thereby regulating gene expression. HDACs are key regulators of gene expression, and to date, 18 HDAC subtypes have been identified in mammals. Based on their homology with yeast proteins, they are divided into four classes: Class I (HDAC1, HDAC2, HDAC3, HDAC8) are usually located in the cell nucleus and are universally expressed in various cell lines and tissues; Class II is further divided into two subfamilies, IIa (HDAC4, HDAC5, HDAC7, HDAC9) and IIb (HDAC6, HDAC10), which share homology with yeast proteins and mainly shuttle between the cell nucleus and cytoplasm; Class IV (HDAC11), as its only member, exists in both the cell nucleus and cytoplasm, and its catalytic site is similar to that of Class I and II enzymes. These three classes represent Zn2+-dependent deacetylases. Class III (Sirt1–Sirt7) NAD+-dependent deacetylases require the activity and homologs of the yeast protein SiR2, which contains NAD+.

[0003] Currently, there are five marketed histone deacetylase inhibitors (HDACi): vorinostat, belinostat, panobinostat, romidepsin, and chidamide. The first three are broad-spectrum inhibitors, while the latter two selectively target class I subtypes. Vorinostat and romidepsin are used to treat cutaneous T-cell lymphoma (CTCL), belinostat and chidamide are used to treat relapsed and refractory peripheral T-cell lymphoma (PTCL), and panobinostat is used in combination with bortezomib and dexamethasone to treat multiple myeloma (MM).

[0004] Although the aforementioned HDAC inhibitors have achieved good clinical efficacy, broad-spectrum HDAC inhibitors generally have the following drawbacks:

[0005] (1) Strong toxic side effects, such as nausea, vomiting, and bone marrow suppression;

[0006] (2) Genotoxicity;

[0007] (3) Poor pharmacokinetic characteristics, low bioavailability, short half-life, etc.

[0008] The above-mentioned drawbacks not only cause inconvenience to cancer patients, but also hinder the application of broad-spectrum HDAC inhibitors in areas other than cancer treatment.

[0009] Currently, HDAC subtype selective inhibitors have become a research hotspot in this field, and HDAC6, due to its unique structure and function, has become a new hotspot in tumor treatment.

[0010] HDAC6 is a widely expressed cytoplasmic protein deacetylase whose main targets include α-tubulin and HSP90. Through post-translational modification of these substrates and other cytoplasmic targets, it is involved in several key cellular processes, including primary cell cilia, intracellular signaling, and DNA damage responses. Inhibition of HDAC6 leads to the restoration of cell cilia and attenuation of malignant phenotypes, consistent with its role in cellular regulation. Simultaneously, inhibition of HDAC6 has been shown to reduce the oncogenic Hedgehog signaling pathway (which controls cell fate, proliferation, and differentiation; aberrant activation of this pathway leads to tumorigenesis and development). HDAC6 is an important intracellular chaperone, interacting with signaling mediators or directly modulating HSP90 to influence intracellular signaling. In summary, these studies link HDAC6 to multiple oncogenic processes and highlight the potential of HDAC6 inhibitors to induce cellular and immune-mediated antitumor activity.

[0011] Currently, all HDAC6 inhibitors under clinical investigation are used for the treatment of tumors. Clinical studies have shown that their side effects are significantly improved compared to broad-spectrum HDAC inhibitors. There are currently no reports of dose-limiting toxicities, and they have potential applications in the treatment of tumors.

[0012] The literature (Oncologist, 2021.26(3):184-e366.) reports that ACY-1215 is a first-in-class HDAC6 inhibitor (IC50 = 5 nM) and is currently in phase II clinical trials. It has shown a high safety profile in patients with relapsed and refractory lymphoma, acting synergistically with bortezomib. In clinical studies of ACY-1215, the drug was well-tolerated, with no dose-limiting toxicities observed. The most common grade 1-2 toxicities (diarrhea, nausea, fatigue, cough, vomiting, and pain) were mild and easily managed. However, in the pharmacokinetic curve of ACY-1215, its serum concentration tended to stabilize at higher doses.

[0013]

[0014] The literature (Blood (2015) 126(23): 3040.) reports that ACY-241 is a second-generation selective HDAC6 inhibitor (IC). 50 =2.6 nM), used for the treatment of multiple myeloma (MM). Clinical studies have found that at the same dose, higher serum concentrations can be obtained than ACY-1215. There are no reports of any dose-limiting toxicities. It is used for the treatment of patients with advanced solid tumors and has potential therapeutic prospects for cancer treatment.

[0015]

[0016] The literature (Clin Cancer Res. 2021, 27(13)) reports that KA2507 is an effective selective HDAC6 inhibitor with an IC50 value of 2.5 nmol for HDAC6 inhibition, while its inhibition of other HDAC subtypes is poor. In preclinical models, KA2507 has shown antitumor efficacy and immunomodulatory effects. In a phase I clinical study, KA2507 showed selective target involvement in a subset of patients and prolonged disease stability, with good tolerability and no reports of dose-limit toxicity or other significant toxicities. These results indicate that KA2507 is a promising candidate drug worthy of further clinical investigation.

[0017]

[0018] HDAC6 is a potential target for cancer treatment, and the above studies suggest that selective HDAC6 inhibitors hold promise for development into novel, highly effective, and low-toxicity anti-tumor drugs. Literature (J.Am.Chem.Soc.2010,132,31,10842-10846) reports TubastainA as a promising HDAC6 inhibitor in development (IC50 = 15 nM), but its physicochemical properties are unsatisfactory and its drug-likeness is poor. Summary of the Invention

[0019] This invention provides a novel benzodiazepine compound that can effectively inhibit HDAC6 and HDAC1, exhibits high anti-proliferative activity against various tumor cells, has low toxicity to normal cells, low potential cardiotoxicity, and low acute toxicity in animals, and shows promising development potential as a highly effective and low-toxicity antitumor therapeutic agent.

[0020] In order to achieve the above-mentioned objectives, the first aspect of the present invention provides a benzodiazepine compound with the general structural formula shown in formula (I), or an isomer thereof, or a pharmaceutically acceptable salt, ester or prodrug thereof;

[0021]

[0022] in,

[0023] R1 and R2 are independently selected from hydrogen, deuterium, hydroxyl, halogen, alkyl, alkoxy, cycloalkyl, benzyl, heterocycloalkyl, aryl, heteroaryl, cyano, haloalkyl, acyl, sulfonyl, or aminoalkyl, each of which may optionally be substituted.

[0024] Preferably, the alkyl group is an alkyl group containing 1 to 4 carbon atoms, which may optionally be substituted with 0 to 3 halogens;

[0025] Preferably, the cycloalkyl group is a cycloalkyl group containing 3 to 6 carbon atoms, and may optionally be replaced by 0 to 3 halogens;

[0026] Preferably, the heterocyclic alkyl group is selected from pyrroleyl, morpholinyl, piperidinyl, piperazine ring, tetrahydroquinolinyl, tetrahydrotriazolylpyrazineyl, diazacycloheptyl or piperazineyl, which may optionally be substituted;

[0027] Preferably, the aryl or heteroaryl group is selected from phenyl, naphthyl, anthracene, pyridyl, pyrimidinyl, pyrazinyl, indolyl, imidazolyl, benzoxazolyl, benzofuranyl, benzothiophene, benzothiazolyl, triazolyl, isoxazolyl, quinolinyl, pyrroleyl, pyrazolyl or 5,6,7,8-tetrahydroisoquinoline; it may optionally be substituted.

[0028] Preferably, the acyl group is selected from acetyl, propionyl, isobutyryl, or aryl acyl, and may optionally be substituted;

[0029] Preferably, the sulfonyl group is selected from methanesulfonyl or arylsulfonyl, which may optionally be substituted;

[0030] Preferably, the aminoalkyl group is selected from dimethylaminoalkyl, methylaminoalkyl, piperazinealkyl, or piperidinylalkyl, and may optionally be substituted;

[0031] Preferably, the halogen is selected from fluorine, chlorine, and bromine;

[0032] Preferably, the alkoxy group is selected from alkoxy groups containing 1 to 4 carbon atoms.

[0033] Preferably, pharmaceutically acceptable salts of the compound of formula (I) include anionic salts formed by reacting the compound of formula (I) with hydrochloric acid, hydrobromic acid, sulfuric acid, acetic acid, trifluoroacetic acid, citric acid, tartaric acid, maleic acid, fumaric acid, methanesulfonic acid, malic acid, p-toluenesulfonic acid or oxalic acid; or cationic salts formed by reacting the compound of formula (I) with sodium ion solution or potassium ion solution.

[0034] Preferably, R1 and R2 are independently selected from hydrogen, methyl, F, Cl, Br or methoxy.

[0035] In some specific embodiments of the present invention, benzodiazepines include the following compounds or isomers thereof, or pharmaceutically acceptable salts, esters or prodrugs thereof:

[0036] Table 1

[0037]

[0038]

[0039]

[0040] A second aspect of the present invention provides a method for preparing the benzodiazepine compounds described above, comprising the following steps:

[0041] S1, under nitrogen protection, compound (II) reacts with methyl 4-bromomethylbenzoate to give compound (III);

[0042]

[0043] R1 and R2 are as shown in the above technical solution;

[0044] S2, the compound of formula (III) reacts with an alkaline hydroxylamine solution to give the compound of formula (I);

[0045]

[0046] Preferably, the reaction temperature in step S1 is 60–100°C, and the reaction time is 1.5–4 h.

[0047] Preferably, the preparation method of compound (II) includes any of the following methods:

[0048] Method 1 includes the following steps:

[0049] A1, Compound (IV) reacts with acrylic acid under acidic conditions to give compound (II);

[0050]

[0051]

[0052] Method 2 includes the following steps:

[0053] B1, Compound (V) reacts with acrylic acid upon heating to give compound (VI);

[0054]

[0055] B2, Compound (VI) reacts with hydrogen under Pt / C catalysis to produce compound (VII), or, Compound (VI) reacts with concentrated hydrochloric acid under Zn catalysis to produce compound (VII).

[0056]

[0057] B3, Compound (VII) reacts with concentrated hydrochloric acid under heating conditions to give compound (II);

[0058]

[0059] Preferably, in step A1, the compound of formula (V), acrylic acid, and concentrated hydrochloric acid are mixed and reacted at 60–85°C for 10–16 h;

[0060] Preferably, in step B1, the reaction temperature is 120–145°C and the reaction time is 6–10 h;

[0061] Preferably, in step B2, compound (VII) and Pt / C are dissolved in an organic solvent, and hydrogen gas is introduced for 1.5 to 3 hours to react and generate compound (VIII).

[0062] Preferably, in step B2, after the compound of formula (Ⅶ), concentrated hydrochloric acid, and organic solvent are mixed, zinc powder is slowly added until the reaction is terminated;

[0063] Preferably, in step B3, the reaction temperature is 60–80°C and the reaction time is 3–6 hours.

[0064] A third aspect of the present invention provides an intermediate compound for preparing the benzodiazepine compounds described in the foregoing technical solutions, comprising a compound of formula (II) or an isomer thereof, a pharmaceutically acceptable salt, an ester, or a prodrug:

[0065]

[0066] And / or, compounds of formula (III) or their isomers, pharmaceutically acceptable salts, esters, or prodrugs:

[0067]

[0068] And / or, compounds of formula (IV) or their isomers, pharmaceutically acceptable salts, esters, or prodrugs:

[0069]

[0070] And / or, compounds of formula (V) or their isomers, pharmaceutically acceptable salts, esters, or prodrugs:

[0071]

[0072] And / or, compounds of formula (VI) or their isomers, pharmaceutically acceptable salts, esters, or prodrugs:

[0073]

[0074] And / or, compounds of formula (VII) or their isomers, pharmaceutically acceptable salts, esters, or prodrugs:

[0075]

[0076] R1 and R2 are as shown in the aforementioned technical solution.

[0077] The fourth aspect of the present invention provides the use of the benzodiazepine compounds described in the foregoing technical solutions, the benzodiazepine compounds obtained by the preparation methods described in the foregoing technical solutions, or the intermediate compounds described in the foregoing technical solutions in the preparation of histone deacetylase inhibitors or antitumor drugs.

[0078] Preferably, the histone deacetylase inhibitor is an HDAC6 and / or HDAC1 inhibitor.

[0079] Preferably, the antitumor drugs include drugs for treating breast cancer, colon cancer, liver cancer, multiple myeloma, sarcoma, lung cancer, prostate cancer, rectal cancer, kidney cancer, pancreatic cancer, leukemia, neuroblastoma, glioma, head cancer, neck cancer, thyroid cancer, ovarian cancer, vulvar cancer, cervical cancer, endometrial cancer, testicular cancer, bladder cancer, esophageal cancer, gastric cancer, nasopharyngeal cancer, buccal cancer, oral cancer, gastrointestinal stromal tumor, and skin cancer; more preferably, they include drugs for treating myeloma, neuroblastoma, and chronic myeloid leukemia.

[0080] A fifth aspect of the present invention provides a pharmaceutical composition comprising at least one active ingredient and one or more pharmaceutically acceptable excipients; said active ingredient comprising benzodiazepines as described in the foregoing technical solutions or benzodiazepines obtained by the preparation methods described in the foregoing technical solutions.

[0081] Preferably, the pharmaceutically acceptable excipients include one or more of the following: diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, flavorings, and sweeteners.

[0082] The pharmaceutical composition of this invention can be formulated into various forms such as tablets, powders, granules, capsules, oral liquids, and injectable drugs. All of these dosage forms can be prepared using conventional methods in the pharmaceutical field. The active component in the pharmaceutical composition of this invention can also be combined with other effective ingredients that have therapeutic effects or enhance therapeutic effects, reduce toxic side effects, or prolong metabolic time to form a pharmaceutical composition.

[0083] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0084] This invention uses Tubastatin A as a lead compound and modifies its cap region to obtain benzodiazepine compounds as shown in formula (I). These compounds have strong HDAC6 / HDAC1 inhibitory activity, low toxicity, and high efficacy, and are candidate drugs with certain therapeutic effects on tumors and good drug-like properties.

[0085] 1) The benzodiazepine compounds of the present invention have strong inhibitory activity against both HDAC6 and HDAC1. Most of the compounds have better inhibitory activity against HDAC6 than Rocilinostat (ACY-1215), an HDAC6 inhibitor in clinical Phase I / II trials. Most of the benzodiazepine compounds of the present invention have better inhibitory activity against HDAC1 than Rocilinostat.

[0086] 2) The benzodiazepine compounds described in this invention have good inhibitory effects on multiple tumor cell lines, including human myeloma cells (NCI-H929), human neuroblastoma cells (SH-SY5Y), and human chronic myeloid leukemia cells (K562), while exhibiting weak inhibitory effects on normal cells, thus demonstrating good cell selective inhibitory activity.

[0087] 3) The benzodiazepine compounds described in this invention have weak inhibitory activity against hERG and low potential cardiotoxicity; they are well tolerated by rats after a single oral gavage and have low acute toxicity. Detailed Implementation

[0088] In this invention, the term "isomer" includes, but is not limited to, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, tautomers, mixtures of racemic mixtures and diastereomers, and their pharmaceutically acceptable salts. Unless otherwise stated, when an isomer component is not specifically specified, all possible isomers are included.

[0089] In this invention, "pharmaceutically acceptable salt" refers to a compound modified by forming an acidic or basic salt of the benzothiadiazine compound described in this invention, including but not limited to salts of inorganic acids selected from, for example, hydrochlorides, phosphates, hydrogen phosphates, hydrobromic acids, sulfates, sulfites, and nitrates; and salts of organic salts selected from, for example, malates, maleates, fumarates, tartrates, succinates, citrates, lactates, methanesulfonates, p-toluenesulfonates, 2-hydroxyethylsulfonates, benzoates, salicylates, stearates, alkanoates such as acetates, and salts of HOOC-(CH2)n-COOH, where n can be any integer from 0 to 4. If the compound is obtained as an acid addition salt, the free base can be obtained by alkalizing a solution of the acidic salt. Conversely, if the product is a free base, the addition salt (e.g., a pharmaceutically acceptable addition salt) can be prepared by dissolving the free base in a suitable organic solvent and treating the solution with acid, consistent with the conventional process for preparing acid addition salts from basic compounds. Those skilled in the art will understand the various synthetic methods that can be used to prepare non-toxic, pharmaceutically acceptable addition salts without excessive experimentation. Similarly, "pharmaceutically acceptable ester" refers to an ester derivative formed by the formation of the small molecule inhibitors of the present invention, and "pharmaceuticalally acceptable prodrugs" include precursor compounds having the ability to form the small molecule inhibitors of the present invention in vitro and in vivo.

[0090] In this invention, the term "aromatic ring" or "aryl" refers to a monocyclic or fused polycyclic group with 5-12 carbon atoms, possessing a fully conjugated π-electron system. Non-limiting examples of aromatic rings include benzene rings, biphenyl rings, naphthyl rings, and anthracene rings. Aromatic rings can be unsubstituted or substituted. Substituents in aromatic rings can be selected from halogens, nitro groups, amino groups, C1-C6 alkyl groups, C1-C6 alkoxy groups, halo-C1-C6 alkyl groups, halo-C1-C6 alkoxy groups, C3-C6 cycloalkyl groups, and halo-C3-C6 cycloalkyl groups.

[0091] In this invention, "heteroaryl" refers to an unsaturated carbon ring with 5-12 ring atoms, wherein one or more carbon atoms are replaced by heteroatoms such as oxygen, nitrogen, sulfur, etc. The heteroaryl ring can be monocyclic or bicyclic, i.e., formed by the fusion of two rings. Specific heterocyclic aryl groups can be: pyrrole, pyrazolyl, imidazolyl, furanyl, thiophene, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrazinyl, pyrrole, morpholinyl, piperidinyl or piperazinyl, thiophene, benzothiophene, pyrazolyl, benzopyrazolyl, indolyl, dioxopentyl, benzo[1,3]dioxopentyl, oxazolyl, benzooxazolyl, furanyl, benzofuranyl, thiazolyl or benzothiazolyl, etc. Heterocyclic aryl groups can be unsubstituted or substituted. The substituents of the heterocyclic aryl group can be selected from halogen, nitro, amino, C1-C6 alkyl, C1-C6 alkoxy, halo-C1-C6 alkyl, halo-C1-C6 alkoxy, C3-C6 cycloalkyl, and halo-C3-C6 cycloalkyl.

[0092] In this invention, "alkoxy" refers to an -O-alkyl group, wherein the alkyl group is as defined above. Examples of "alkoxy" as used in this invention include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, and tert-butoxy, and the alkoxy group may be unsubstituted or substituted.

[0093] In this invention, "halogen" or "halogenated" means fluorine, chlorine, bromine or iodine.

[0094] In this invention, the pharmaceutically acceptable excipients include, but are not limited to, conventional pharmaceutical diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc., and flavoring agents, sweeteners, etc., may be added if necessary. The pharmaceutical compositions of this invention can be formulated into various forms such as tablets, powders, granules, capsules, oral liquids, and injectable drugs, and all of the above dosage forms can be prepared according to conventional pharmaceutical methods.

[0095] In some specific embodiments of the present invention, the compounds of the present invention can be prepared using the following synthetic routes:

[0096] Route 1:

[0097] Route 2:

[0098] Route 3:

[0099]

[0100] Compound of formula (I) can be obtained by using any of the above synthetic routes. Compound of formula (I) can be further reacted with inorganic acid or organic acid in a solvent, and the salt of the corresponding compound of formula (I) structure is precipitated by cooling.

[0101] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. Experimental methods in the following embodiments without specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. All raw materials without specified synthesis methods were purchased from manufacturers such as Exploration Platform, Aladdin, and Sigma-Aldrich, and were of analytical grade.

[0102] Example 1: Preparation of 4-((4-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine-1-yl)methyl)-N-hydroxybenzamide (T-1)

[0103] The preparation was carried out according to route 1, and the synthetic route diagram is as follows:

[0104]

[0105] Synthesis of 1,3,4,5-tetrahydro-2H-benzo[b][1,4]diazepine-2-one (1-2)

[0106] In a 500 ml three-necked flask, 1,2-phenylenediamine (4 g, 36.99 mmol, 1 eq), acrylic acid (4 g, 1.5 eq), 10 ml concentrated hydrochloric acid, and 50 ml water were added sequentially. The mixture was stirred at 70 °C for 12 h. After cooling to room temperature, the pH was adjusted to 7-8 with sodium bicarbonate. The mixture was extracted with 100 ml ethyl acetate, and the organic phase was separated and concentrated to dryness. Column chromatography was used to separate the solid, yielding 3.2 g of a white solid (53.3% yield). 1H NMR(600MHz,DMSO-d6)δ9.40(s,1H),6.87(dd,J=7.9,1.5Hz,1H),6.82(ddd,J=8.5,7.2,1.5Hz,1H),6.75(dd,J=8.0,1.5Hz,1H),6 .61(td,J=7.5,1.5Hz,1H),5.81–5.68(m,1H),3.43(dtd,J=5.7,4.0,1.8Hz,2H),2.50–2.46(m,2H).ESI-MS(+)m / z=163.3[M+H]+.

[0107] Synthesis of methyl 4-((4-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine-1-yl)methyl)benzoate (1-3)

[0108] Intermediate 1-2 (1 g, 6.17 mmol, 1 eq) was added to a 1 L three-necked flask and dissolved in 40 mL of DMF. n-BuOK (0.6919 g, 1 eq) was added, and the mixture was stirred at room temperature for 30 min under N2 protection. Methyl 4-bromomethylbenzoate (1.41 g, 1 eq) was added, and the mixture was stirred at 80 °C for 3 h. After cooling to room temperature, 100 mL of water and 150 mL of ethyl acetate were added for extraction. The organic phase was dried, and column chromatography was used to separate the solid, yielding 1.394 g of a white solid (73% yield). 1H NMR (600MHz, DMSO-d6) δ7.90–7.74(m,2H),7.47–7.32(m,2H),7.19(dd,J=8.1,1.4Hz,1H),6.99(ddd,J=8.4,7.1,1.4Hz,1H),6.95(dd,J=7.9,1.7Hz,1H ),6.89–6.77(m,1H),5.25(t,J=3.0Hz,1H),5.11(s,2H),3.81(s,3H),3.64( td,J=6.6,2.9Hz,2H),2.47(t,J=6.6Hz,2H).ESI-MS(+)m / z=310.23[M+H]+.

[0109] Synthesis of 4-((4-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine-1-yl)methyl)-N-hydroxybenzamide (1);

[0110] 20 g of hydroxylamine hydrochloride was dissolved in 200 ml of methanol, and 19 g of potassium hydroxide (85% purity) was dissolved in 200 ml of methanol. The solutions were slowly mixed under ice-water bath cooling and stirred at room temperature for 2 hours. The filtrate was filtered to obtain a hydroxylamine methanol solution. Intermediate 1-3 (1 g, 3.22 mmol) and 50 ml of the hydroxylamine methanol solution were added to a 100 ml three-necked flask, and the reaction was allowed to proceed overnight at room temperature. The pH was adjusted to neutral with acetic acid, and the solution was concentrated. Ethyl acetate was added, and the hydroxylamine was washed away with water. The organic phase was dried and concentrated to dryness. 10 ml of ethanol was added, and the mixture was stirred until a white solid of 0.4 g was obtained, with a yield of 40%. 1H NMR (400MHz, DMSO-d6) δ11.16(s,1H),9.06(s,1H),7.65(d,J=8.1Hz,2H),7.33(d,J=8.0Hz,2H),7.29–7.22(m,1H),7.08–6.95(m,2H),6.88(td, J=7.5,6.9,1.9Hz,1H),5.30(q,J=5.8,4.4Hz,1H),5.11(s,2H),3.67(td,J=6.6,2.7Hz,2H),2.50(t,J=6.6Hz,2H).ESI-MS(+)m / z=311.2[M+H]+.

[0111] Example 2: Preparation of 4-((7,8-dimethyl-4-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine-1-yl)methyl)-N-hydroxybenzamide (T-2)

[0112]

[0113] T-2 was prepared according to general method 1. ¹H NMR (400 MHz, DMSO-d6) δ 11.09 (s, ¹H), 8.97 (s, ¹H), 7.60 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H), 7.02 (s, ¹H), 6.73 (s, ¹H), 5.05 (s, 2H), 4.98 (d, J = 2.8 Hz, 1H), 3.58 (td, J = 6.6, 2.8 Hz, 2H), 2.41 (t, J = 6.6 Hz, 2H), 2.06 (d, J = 3.2 Hz, 6H). ESI-MS (+) m / z = 340.00 [M+H]+.

[0114] Example 3: Preparation of 4-((8-methyl-4-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine-1-yl)methyl)-N-hydroxybenzamide (T-3)

[0115] The preparation was carried out according to general synthetic method 2, and the specific synthetic route is as follows:

[0116]

[0117] Synthesis of 3-((5-methyl-2-nitrophenyl)amino)propionic acid (3-2)

[0118] 5-Methyl-2-nitroaniline (10 g, 65.29 mmol, 1 eq) and acrylic acid (7.06 g, 1.5 eq) were added to a 250 mL three-necked flask and stirred at 135 °C for 8 h. After cooling to room temperature, 15 mL of dichloromethane was added and stirred at room temperature for 2 h. The mixture was filtered to obtain a yellow solid. The crude product weighed 7.3 g after drying, with a yield of 49.9%.

[0119] Synthesis of 3-((2-amino-5-methylphenyl)amino)propionic acid (3-3)

[0120] Add 7.3 g (32.56 mmol) of 3-2, 1 g of Pt / C, and 50 ml of ethanol to a 250 ml pressure vessel. Purge with 10 psi of hydrogen gas, seal the vessel, and react at room temperature for 2 h. The reaction is complete at half-second. Filter the Pt / C, concentrate the filtrate to dryness under reduced pressure to obtain 4.79 g of black solid, which is directly added to the next step without purification.

[0121] Synthesis of 7-methyl-1,3,4,5-tetrahydro-2H-benzo[b][1,4]diazepine-2-one (3-4)

[0122] Add 4.79 g of crude intermediate 3-3, 10 ml of concentrated hydrochloric acid, and 30 ml of purified water to a 100 ml three-necked flask, and heat to 70 °C and reflux for 4 h. Cool to room temperature, slowly add sodium bicarbonate to neutralize, extract with ethyl acetate, dry the organic layer with anhydrous sodium sulfate, filter, concentrate the filtrate to dryness under reduced pressure, and purify by column chromatography to give 1.23 g of white solid. The two-step yield is 21.43%. 1H NMR (600MHz, DMSO-d6) δ9.36 (s, 1H), 6.80 (d, J = 8.0 Hz, 1H), 6.60 (s, 1H), 6.56–6.46 (m, 1H), 5.62 (t, J = 3. 8Hz, 1H), 3.47 (dt, J = 6.8, 3.6Hz, 2H), 2.51 (t, J = 5.6Hz, 2H), 2.18 (s, 3H). ESI-MS (+) m / z = 177.00 [M + H] +.

[0123] Synthesis of methyl 4-((8-methyl-4-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine-1-yl)methyl)benzoate (3-5)

[0124] Intermediate 3-4 (1 g, 5.67 mmol, 1 eq) was added to a 250 mL three-necked flask and dissolved in 40 mL DMF. n-BuOK (0.64 g, 1 eq) was added, and the mixture was stirred at room temperature for 30 min under N2 protection. Methyl 4-bromomethylbenzoate (1.3 g, 1 eq) was added, and the mixture was stirred at 80 °C for 3 h. After cooling to room temperature, 100 mL of water and 150 mL of ethyl acetate were added for extraction. The organic phase was dried, and column chromatography was used to separate the solid, yielding 1.13 g of a white solid (61.4% yield). 1H NMR (600MHz, DMSO-d6) δ7.90–7.76(m,2H),7.38(d,J=8.1Hz,2H),7.08(d,J=8.1Hz,1H),6.76(d,J=2.1Hz,1H),6.65(dd,J=8.2,2.0Hz,1H),5. 16(d,J=3.1Hz,1H),5.09(s,2H),3.81(s,3H),3.62(td,J=6.6,2.9Hz,2H),2.46(t,J=6.5Hz,2H),2.16(s,3H)..ESI-MS(+)m / z=310.23[M+H]+.

[0125] Synthesis of 4-((8-methyl-4-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine-1-yl)methyl)-N-hydroxybenzamide (3)

[0126] In a 100 mL three-necked flask, intermediate 3-5 (1 g, 3.08 mmol) and 50 mL of hydroxylamine methanol solution were added, and the mixture was reacted overnight at room temperature. The pH was adjusted to neutral with acetic acid, and the mixture was concentrated. Ethyl acetate was added, and the hydroxylamine was washed away with water. The organic phase was dried and concentrated to dryness. 10 mL of ethanol was added, and the mixture was recrystallized. The solution was filtered to give 0.4 g of white solid, with a yield of 40%.

[0127] 1H NMR(400MHz,DMSO-d6)δ11.11(s,1H),9.04(s,1H),7.65–7.54(m,2H),7.28 (d,J=8.1Hz,2H),7.09(d,J=8.2Hz,1H),6.74(d,J=2.0Hz,1H),6.65(dd,J=8 .1,2.0Hz,1H),5.15(q,J=8.1,5.5Hz,1H),5.04(s,2H),3.61(td,J=6.7,2. 7Hz, 2H), 2.44 (t, J = 6.5Hz, 2H), 2.15 (s, 3H). ESI-MS (+) m / z = 326.01 [M + H] +.

[0128] Example 4: Preparation of 4-(9-methyl-4-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine-1-yl)methyl)-N-hydroxybenzamide (T-4)

[0129]

[0130] T-4 was prepared according to general method 2. ¹H NMR (400 MHz, DMSO-d6) δ 11.12 (s, ¹H), 9.00 (s, ¹H), 7.66–7.54 (m, 2H), 7.37–7.28 (m, 2H), 7.09 (dd, J = 8.1, 1.5 Hz, ¹H), 6.99–6.90 (m, ¹H), 6.80 (t, J = 7.7 Hz, 1H), 5.05 (s, 2H), 4.57 (d, J = 2.5 Hz, 1H), 3.67 (td, J = 6.9, 2.1 Hz, 2H), 2.41 (t, J = 6.8 Hz, 2H), 2.23 (s, 3H). ESI-MS (+) m / z = 326 [M+H]+.

[0131] Example 5: Preparation of 4-((7-fluoro-4-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine-1-yl)methyl)-N-hydroxybenzamide (T-5)

[0132] The preparation was carried out according to general synthetic method 3, and the specific synthetic route is as follows:

[0133]

[0134] Synthesis of 3-((4-fluoro-2-nitrophenyl)amino)propionic acid (5-2)

[0135] 4-fluoro-2-nitroaniline (10 g, 64.05 mmol, 1 eq) and acrylic acid (6.92 g, 1.5 eq) were added to a 250 mL three-necked flask, and the mixture was stirred at 135 °C for 8 h. After cooling to room temperature, 15 mL of dichloromethane was added and stirred at room temperature for 2 h. The mixture was filtered to obtain a yellow solid, and the crude product weighed 8.1 g after drying, with a yield of 55.4%.

[0136] Synthesis of 3-((4-fluoro-2-aminophenyl)amino)propionic acid (5-3)

[0137] Add 8.1 g (35.50 mmol) of 5-2, 10 ml of concd HCl, and 50 ml of ethanol to a 250 ml three-necked flask. Slowly add zinc powder while stirring. When the reaction is confirmed to be complete by TLC, stop adding zinc powder. Cool to room temperature and concentrate the ethanol in the reaction solution under reduced pressure. Continue to the next step without purification.

[0138] Synthesis of 8-fluoro-1,3,4,5-tetrahydro-2H-benzo[b][1,4]diazepine-2-one (5-4)

[0139] Add 5-3g of the crude product obtained in the previous step, 10ml of concentrated hydrochloric acid, and 30ml of purified water to a 100ml three-necked flask, and heat to 70℃ and reflux for 4h. Cool to room temperature, slowly add sodium bicarbonate to neutralize, extract with ethyl acetate, dry the organic layer with anhydrous sodium sulfate, filter, concentrate the filtrate to dryness under reduced pressure, and purify by column chromatography to give 2.1g of white solid. The two-step yield is 32.8%. ¹H NMR (400MHz, DMSO-d6) δ 9.52 (s, 1H), 6.92–6.59 (m, 3H), 5.55 (t, J=3.7Hz, 1H), 3.40 (td, J=5.9, 3.7Hz, 2H), 2.47 (s, 2H). ESI-MS (+) m / z=181.00 [M+H]+.

[0140] Synthesis of methyl 4-((7-fluoro-4-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine-1-yl)methyl)benzoate (5-5)

[0141] Intermediate 5-4 (1 g, 5.55 mmol, 1 eq) was added to a 250 mL three-necked flask and dissolved in 40 mL DMF. n-BuOK (0.623 g, 1 eq) was added, and the mixture was stirred at room temperature for 30 min under N2 protection. Methyl 4-bromomethylbenzoate (1.27 g, 1 eq) was added, and the mixture was stirred at 80 °C for 3 h. After cooling to room temperature, 100 mL of water and 150 mL of ethyl acetate were added for extraction. The organic phase was dried, and column chromatography was used to separate the solid, yielding 0.94 g of a white solid (51.6% yield). 1H NMR (600MHz, DMSO-d6) δ7.88–7.76(m,2H),7.38(d,J=8.2Hz,2H),7.17(dd,J=10.4,2.9Hz,1H),6.97(dd,J=8.7,5.9Hz,1H),6.87(td,J=8.4 ,2.9Hz,1H),5.19–5.16(m,1H),5.14(s,2H),3.81(s,3H),3.61(td,J=6.7,2.7Hz,2H),2.47(t,J=6.7Hz,2H).ESI-MS(+)m / z=329.10[M+H]+.

[0142] Synthesis of 4-((7-fluoro-4-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine-1-yl)methyl)-N-hydroxybenzamide (5)

[0143] In a 100 mL three-necked flask, intermediate 5-5 (1 g, 3.05 mmol) and 50 mL of hydroxylamine methanol solution were added, and the reaction was carried out overnight at room temperature. The pH was adjusted to neutral with acetic acid, concentrated, and then ethyl acetate was added. The hydroxylamine was washed away with water, the organic phase was dried, concentrated to dryness, and recrystallized with 10 mL of ethanol. The mixture was filtered to give 0.437 g of white solid, with a yield of 43.7%. 1H NMR (600MHz, DMSO-d6) δ11.12(s,1H),9.00(s,1H),7.61(d,J=7.5Hz,2H),7.30(d,J=7.4Hz,2H),7.18(d,J=9.7Hz,1H ),7.03–6.94(m,1H),6.87(s,1H),5.17(s,1H),5.11(s,2H),3.61(s,2H),2.47(s,2H).ESI-MS(+)m / z=330.6[M+H]+.

[0144] Example 6: Preparation of 4-((7-chloro-4-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine-1-yl)methyl)-N-hydroxybenzamide (T-6)

[0145]

[0146] T-6 was prepared according to general method 3. ¹H NMR (400 MHz, DMSO-d6) δ 11.12 (s, ¹H), 9.02 (s, ¹H), 7.70–7.57 (m, 2H), 7.31 (d, J = 2.3 Hz, ¹H), 7.30–7.25 (m, 2H), 7.03 (dd, J = 8.5, 2.3 Hz, 1H), 6.94 (d, J = 8.5 Hz, 1H), 5.43 (q, J = 3.3 Hz, 1H), 5.09 (s, 2H), 3.63 (td, J = 6.5, 2.8 Hz, 2H), 2.48 (d, J = 6.2 Hz, 2H). ESI-MS (+) m / z = 345.9 [M+H]+

[0147] Example 7: Preparation of 4-((7-methyl-4-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine-1-yl)methyl)-N-hydroxybenzamide (T-7)

[0148]

[0149] T-7 was prepared according to general method 2. ¹H NMR (600 MHz, DMSO-d6) δ 11.13 (s, ¹H), 9.01 (s, ¹H), 7.62 (d, J = 7.8 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.07 (s, ¹H), 6.87 (d, J = 8.0 Hz, 1H), 6.82 (d, J = 7.9 Hz, 1H), 5.07 (s, 3H), 3.60 (t, J = 6.7 Hz, 2H), 2.44 (t, J = 6.7 Hz, 2H), 2.16 (s, 3H). ESI-MS (+) m / z = 326 [M+H]+.

[0150] Example 8: Preparation of 4-((8-fluoro-4-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine-1-yl)methyl)-N-hydroxybenzamide (T-8)

[0151]

[0152] T-8 was prepared according to general method 3. ¹H NMR (600 MHz, DMSO-d6) δ 11.13 (s, ¹H), 9.00 (s, ¹H), 7.62 (d, J = 8.2 Hz, 2H), 7.27 (d, J = 8.2 Hz, 2H), 7.23 (d, J = 2.9 Hz, 1H), 6.73 (dd, J = 10.3, 2.9 Hz, 1H), 6.63 (td, J = 8.5, 2.9 Hz, 1H), 5.56 (s, ¹H), 5.05 (s, 2H), 3.68–3.62 (m, 2H), 2.49 (d, J = 6.5 Hz, 2H). ESI-MS (+) m / z = 330 [M+H]+.

[0153] Example 9: Preparation of 4-((7-bromo-4-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine-1-yl)methyl)-N-hydroxybenzamide (T-9)

[0154]

[0155] T-9 was prepared according to general method 3. ¹H NMR (400 MHz, DMSO-d6) δ 11.05 (s, ¹H), 9.17 (s, ¹H), 7.62 (d, J = 7.9 Hz, 2H), 7.47–7.38 (m, ¹H), 7.27 (d, J = 7.9 Hz, 2H), 7.14 (dd, J = 8.3, 2.3 Hz, 1H), 6.89 (d, J = 8.5 Hz, 1H), 5.46 (s, ¹H), 5.09 (s, 2H), 3.63 (q, J = 5.7 Hz, 2H), 2.48 (d, J = 7.0 Hz, 2H). ESI-MS (+) m / z = 391.9 [M+H]+.

[0156] Example 10: Preparation of 4-((6-methyl-4-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine-1-yl)methyl)-N-hydroxybenzamide (T-10)

[0157]

[0158] T-10 was prepared according to general method 2. ¹H NMR (600 MHz, DMSO-d⁶) δ 11.07 (s, ¹H), 9.05 (s, ¹H), 7.57 (d, J = 7.9 Hz, 2H), 7.37 (d, J = 7.9 Hz, 2H), 6.98 (t, J = 7.6 Hz, 1H), 6.82 (t, J = 7.0 Hz, 2H), 5.33 (d, J = 15.8 Hz, 1H), 5.06 (d, J = 4.9 Hz, 1H), 4 .28(d,J=15.9Hz,1H),3.54(ddd,J=14.0,10.0,4.9Hz,1H),3.45(dt,J=10.9,5.8Hz,1H),2.53 (dd,J=13.1,7.1Hz,1H),2.28(s,3H),2.15(dd,J=12.7,4.7Hz,1H).ESI-MS(+)m / z=326[M+H]+.

[0159] Example 11: Preparation of 4-((8-methoxy-4-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine-1-yl)methyl)-N-hydroxybenzamide (T-11)

[0160]

[0161] T-11 was prepared according to general method 2. ¹H NMR (400 MHz, DMSO) δ 11.10 (s, ¹H), 8.96 (s, ¹H), 7.61 (d, J = 8.2 Hz, 2H), 7.29 (d, J = 8.2 Hz, 2H), 7.12 (d, J = 8.8 Hz, 1H), 6.51 (d, J = 2.8 Hz, 1H), 6.41 (dd, J = 8.8, 2.8 Hz, 1H), 5.27 (s, ¹H), 5.02 (s, 2H), 3.65 (s, 3H), 3.61 (d, J = 2.2 Hz, 2H), 2.45 (t, J = 6.4 Hz, 2H). ESI-MS (+) m / z = 343.4 [M+H]+.

[0162] Example 12: Preparation of 4-((7,8-difluoro-4-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine-1-yl)methyl)-N-hydroxybenzamide (T-12)

[0163]

[0164] T-12 was prepared according to general method 1. ¹H NMR (400 MHz, DMSO-d6) δ 11.10 (s, ¹H), 8.97 (s, ¹H), 7.69–7.55 (m, 2H), 7.43 (dd, J = 12.3, 8.3 Hz, ¹H), 7.34–7.21 (m, 2H), 6.93 (dd, J = 12.0, 8.3 Hz, 1H), 5.37 (d, J = 2.9 Hz, 1H), 5.08 (s, 2H), 3.61 (td, J = 6.6, 2.8 Hz, 2H), 2.47 (d, J = 6.6 Hz, 2H). ESI-MS (+) m / z = 348.40 [M+H]+.

[0165] Example 13: Preparation of 4-((7,8-dichloro-4-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine-1-yl)methyl)-N-hydroxybenzamide (T-13)

[0166]

[0167] T-13 was prepared according to general method 1. ¹H NMR (400 MHz, DMSO-d⁶) δ 11.11 (s, ¹H), 8.98 (s, ¹H), 7.68–7.57 (m, 2H), 7.51 (s, ¹H), 7.25 (d, J = 8.1 Hz, 2H), 7.13 (s, ¹H), 5.68 (t, J = 3.1 Hz, 1H), 5.10 (s, 2H), 3.64 (td, J = 6.4, 3.0 Hz, 2H), 2.53 (d, J = 6.3 Hz, 2H). ESI-MS (+) m / z = 378.90 [M+H]⁺.

[0168] Example 14 Compound's HDAC Inhibition Activity

[0169] The in vitro inhibitory activity of the compounds against histone deacetylases was determined according to the manufacturer's instructions for use with the HDAC6 and HDAC1 inhibitor screening kits (HDAC6: BPS; HDAC1: Active Motif). The clinically investigated HDAC6 inhibitor Rocilinostat (ACY-1215) was used as a positive control.

[0170] The experimental results are shown in Table 2:

[0171] Table 2

[0172]

[0173] Experimental results show that the compounds of this invention exhibit strong inhibitory activity against HDAC6 (IC50 < 9 nM) and also show strong inhibitory activity against HDAC1. Except for compound T10, the inhibitory activity of the compounds against HDAC1 is superior to that of the positive control Rocilinostat (ACY-1215).

[0174] Example 15: In vitro anti-proliferative activity test against tumor cells

[0175] The antiproliferative activity of some compounds of this invention against NCI-H929 (human myeloma cells), SH-SY5Y (human neuroblastoma cells), K562 (human chronic myeloid leukemia cells), and MRC-5 normal human embryonic lung fibroblasts was determined using the CCK-8 assay, with Rocilinostat (ACY-1215) selected as a control. Specific results are shown in the table (units: Inh%in 20μM and Inh%in 2μM):

[0176] Experimental steps

[0177] (1) Cell Culture

[0178] Collect cells in the logarithmic growth phase, count them, resuspend them in complete culture medium, adjust the cell concentration to a suitable level, and seed them into 96-well plates, with 100 μl of cell suspension in each well. Incubate the cells at 37°C, 100% relative humidity, and 5% CO2 for 12–24 hours.

[0179] (2) Filtering

[0180] Collect cells in the logarithmic growth phase, count them, resuspend them in complete culture medium, adjust the cell concentration to an appropriate level (determined based on cell density optimization assay results), and seed them in 96-well plates, adding 100 μl of cell suspension to each well. Incubate the cells at 37°C, 100% relative humidity, and 5% CO2 for 24 hours.

[0181] After the cells had fully adhered, the test compounds were diluted to 1 mM and 10 mM with DMSO. 1 μl was added to each well to achieve final concentrations of 10 μM and 100 μM. The final DMSO concentration was 1%.

[0182] The cells were incubated at 37°C, 100% relative humidity, and 5% CO2 for 48 hours.

[0183] Add 10 μl of CCK-8 solution and incubate at 37°C for 4 hours.

[0184] After gentle shaking, the absorbance at 450 nm was measured on a SpectraMax M5 Microplate Reader. The absorbance at 650 nm was used as a reference to calculate the suppression rate.

[0185] (3) Data processing

[0186] The inhibition rate of drug on tumor cell growth is calculated using the following formula: Tumor cell growth inhibition rate % = [(Ac-As) / (Ac-Ab)] × 100%

[0187] As: OA of the sample (cells + CCK-8 + test compound)

[0188] Ac: Negative control OA (cells + CCK-8 + DMSO)

[0189] Ab: Positive control OA (culture medium + CCK-8 + DMSO)

[0190] Table 3 Results of cell antiproliferative activity tests

[0191]

[0192] As shown in the table above, compared with the positive control Rocilinostat, the compounds of this invention exhibited good in vitro anti-tumor cell proliferation activity against various tumor cell types, and showed high inhibitory activity against human myeloma cells (NCI-H929), human neuroblastoma cells (SH-SY5Y), and human chronic myeloid leukemia cells (K562). In particular, at the tested concentrations, some compounds showed significantly better anti-proliferative activity against SH-SY5Y than the positive control.

[0193] Meanwhile, compared with Rocilinostat, the compound of the present invention has weaker inhibitory activity against MRC-5 human normal embryonic lung fibroblasts and has lower toxicity, revealing that the compound of the present invention has better selectivity in inhibiting the proliferation of tumor cells and normal cells, suggesting that it may have lower toxicity when used as an anti-tumor drug.

[0194] Example 16: Experiment on the effect of compounds on hERG potassium channels

[0195] The potential cardiotoxic side effects of some compounds of this invention were preliminarily investigated in vitro using the hERG potassium channel inhibition assay. The experimental procedure is as follows:

[0196] 1) Cell preparation

[0197] CHO-hERG cells were cultured in 175cm² culture flasks. When the cell density reached 60-80%, the culture medium was removed, the cells were washed once with 7mL PBS, and then 3mL Detachin was added for digestion.

[0198] After complete digestion, add 7 mL of culture medium to neutralize, then centrifuge, remove the supernatant, and add 5 mL of culture medium to resuspend the cells to ensure a cell density of 2–5 × 10⁶ / mL.

[0199] 2) Electrophysiological recording process

[0200] The entire process of single-cell high-impedance sealing and whole-cell pattern formation was automated by the Qpatch instrument. After obtaining the whole-cell recording pattern, the cells were clamped at -80 mV. Before applying a 5-second +20 mV depolarization stimulus, a 50-millisecond -50 mV pre-voltage was applied, followed by repolarization to -50 mV for 5 seconds, and then back to -80 mV. This voltage stimulus was applied every 15 seconds. After recording for 2 minutes, extracellular fluid was applied for another 2 minutes of recording, and then the drug administration process began. The compound concentration started from the lowest test concentration, and each test concentration was administered for 2 minutes. After all concentrations were administered, 10 μM Cisapride, a positive control compound, was administered. At least 3 cells (n≥3) were tested for each concentration.

[0201] 3) Compound preparation

[0202] The stock solution of the compound was diluted with extracellular fluid. 2 μL of the stock solution was added to 998 μL of extracellular fluid, and then the solution was serially diluted 5-fold in extracellular fluid containing 0.2% DMSO to obtain the final concentration to be tested. Experimental data were analyzed using XLFit software.

[0203] The experimental results are shown in Table 4 below:

[0204] Table 4

[0205]

[0206] hERG experimental results showed that compounds T1–T6, T9, and T10 all exhibited inhibitory activity against hERG potassium ion channels greater than 20 μM, suggesting that the compounds of this invention have low potential cardiotoxicity.

[0207] Example 17 Acute Toxicity Test

[0208] Using the methods reported in "Modern Pharmacological Experimental Methods" edited by Zhang Juntian, preliminary screening was conducted. The LD50 of compounds T-9 and T-12 in mice after a single oral gavage was statistically analyzed using the Bliss method. 50 The concentrations were 1700 mg / kg and 1500 mg / kg, respectively. The test results indicate that T-9 and T-12 have a low risk of acute toxicity.

[0209] Example 18 Pharmaceutical Composition 1

[0210] The compound T-9 prepared in Example 9 was mixed with a filler, a disintegrant, and a lubricant, granulated, and tableted to obtain a pharmaceutical composition 1 with compound T-9 as the active ingredient.

[0211] Example 19 Pharmaceutical Composition 2

[0212] The compound T-12 prepared in Example 12 was mixed with a solvent and a stabilizer, filtered, and packaged to obtain pharmaceutical composition 2 with compound T-12 as the active ingredient.

[0213] Example 20 Pharmaceutical Composition 3

[0214] Compound T-2 prepared in Example 2 and compound T-12 prepared in Example 12 were mixed with filler, disintegrant, and lubricant, granulated, and tableted to obtain pharmaceutical composition 3 with compounds T-2 and T-12 as active ingredients.

[0215] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A benzodiazepine compound having the general structural formula shown in formula (I), or a pharmaceutically acceptable salt thereof; （Ⅰ） in, R1 and R2 are independently selected from hydrogen, halogens, alkyl groups containing 1 to 4 carbon atoms, or alkoxy groups containing 1 to 4 carbon atoms.

2. The benzodiazepine compound according to claim 1, characterized in that, The halogens mentioned are selected from fluorine, chlorine, and bromine.

3. The benzodiazepine compound according to claim 1, characterized in that, Pharmaceutically acceptable salts of compounds of formula (I) include anionic salts formed by the reaction of compounds of formula (I) with hydrochloric acid, hydrobromic acid, sulfuric acid, acetic acid, trifluoroacetic acid, citric acid, tartaric acid, maleic acid, fumaric acid, methanesulfonic acid, malic acid, p-toluenesulfonic acid or oxalic acid; or cationic salts formed by the reaction of compounds of formula (I) with sodium ion solution or potassium ion solution.

4. The benzodiazepine compound according to claim 1 or 2, characterized in that, R1 and R2 are independently selected from hydrogen, methyl, F, Cl, Br or methoxy.

5. The benzodiazepine compound according to claim 4, characterized in that, Including the following compounds or their pharmaceutically acceptable salts: 。 6. A method for preparing a benzodiazepine compound according to any one of claims 1-5, characterized in that, Includes the following steps: S1, under nitrogen protection, compound (II) reacts with methyl 4-bromomethylbenzoate to give compound (III); Wherein, R1 and R2 are as described in any one of claims 1-5; S2, the compound of formula (III) reacts with an alkaline hydroxylamine solution to give the compound of formula (I); 。 7. The preparation method according to claim 6, characterized in that, The reaction temperature in step S1 is 60~100℃, and the reaction time is 1.5~4h.

8. The preparation method according to claim 6, characterized in that, The preparation method of compound (II) includes any of the following methods: Method 1 includes the following steps: A1, Compound (IV) reacts with acrylic acid under acidic conditions to give compound (II); Method 2 includes the following steps: B1, Compound (V) reacts with acrylic acid upon heating to give compound (VI); B2, Compound (VI) reacts with hydrogen under Pt / C catalysis to produce compound (VII), or, Compound (VI) reacts with concentrated hydrochloric acid under Zn catalysis to produce compound (VII). B3, Compound (VII) reacts with concentrated hydrochloric acid under heating conditions to give compound (II); 。 9. The preparation method according to claim 8, characterized in that, In step A1, the compound of formula (Ⅳ), acrylic acid and concentrated hydrochloric acid are mixed and reacted at 60~85℃ for 10~16h; And / or, in step B1, the reaction temperature is 120~145℃ and the reaction time is 6~10h; And / or, in step B2, compound (VI) and Pt / C are dissolved in an organic solvent and hydrogen is introduced for 1.5 to 3 hours to react and generate compound (VII); And / or, in step B3, the reaction temperature is 60~80℃ and the reaction time is 3~6h.

10. The preparation method according to claim 8, characterized in that, In step B2, after mixing the compound of formula (VI), concentrated hydrochloric acid, and organic solvent, zinc powder is slowly added until the reaction is terminated.

11. The use of the benzodiazepine compound according to any one of claims 1-5 or the benzodiazepine compound obtained by the preparation method according to any one of claims 6-10 in the preparation of histone deacetylase inhibitors or antitumor drugs, wherein the histone deacetylase inhibitor is an HDAC6 and / or HDAC1 inhibitor, and the antitumor drug is a drug for multiple myeloma, neuroblastoma, or leukemia.

12. A pharmaceutical composition comprising at least one active ingredient and one or more pharmaceutically acceptable excipients; said active ingredient comprising a benzodiazepine compound as described in any one of claims 1-5 or a benzodiazepine compound prepared by any one of claims 6-10.

13. The pharmaceutical composition according to claim 12, characterized in that, Pharmaceutically acceptable excipients include one or more of the following: diluents, fillers, binders, humectants, disintegrants, surfactants, adsorbents, lubricants, flavorings, and sweeteners.

14. The pharmaceutical composition according to claim 12, characterized in that, Pharmaceutically acceptable excipients include excipients.

15. The pharmaceutical composition according to claim 12, characterized in that, Pharmaceutically acceptable excipients include absorption enhancers.

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