A benzamide derivative and use thereof

Abstract

The present application provides a benzamide derivative and its use. The aforementioned benzamide derivative is shown as formula (I), which can be used as a histone deacetylase inhibitor (HDACi) to treat tumors or hyperproliferative diseases.

Description

[0001] This application is a divisional application filed on November 13, 2025, with application number CN202511669884.4 and invention title "A benzamide derivative and its use". Technical Field

[0002] This invention belongs to the field of pharmaceutical chemistry, specifically relating to a benzamide derivative and its uses. Background Technology

[0003] Histone deacetylases (HDACs) are enzymes that hydrolyze the ε-N-acetyl (O=C-CH3) residues on histone lysine residues. This causes DNA to wrap more tightly around histone, thus DNA expression levels are influenced by the levels of histone acetylation and deacetylation. Histone deacetylases (HDACs) play a crucial role in chromosome structural modification and gene expression regulation. Within the cell nucleus, histone acetylation and deacetylation are in dynamic equilibrium, regulated by both histone acetyltransferase (HAT) and HDACs. HAT transfers the acetyl group of acetyl-CoA to a specific lysine residue at the N-terminus of histone, while HDACs deacetylate histone, causing it to bind tightly to negatively charged DNA, resulting in dense, coiled chromatin and repressed gene transcription.

[0004] Currently, histone deacetylases (HDACs) have been identified as belonging to four major classes and 18 different subtypes. Class I includes four subtypes: HDAC1, HDAC2, HDAC3, and HDAC8; Class II includes six subtypes: HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10; Class IV has only one subtype: HDAC11; and Class III includes seven subtypes: SIRT1-7. Classes I, II, and IV are structurally homologous, while Class III does not share structural homology with them.

[0005] In cancer cells, overexpression of HDAC leads to enhanced deacetylation, which, by restoring the positive charge of histones, increases the attraction between DNA and histones, making loose nucleosomes very tight, which is detrimental to the expression of specific genes, including some tumor suppressor genes. Histone deacetylase inhibitors (HDACi) can regulate the expression and stability of apoptosis and differentiation-related proteins by increasing histone acetylation in specific regions of chromatin, thereby inducing apoptosis and differentiation, and have become a new class of anti-tumor drugs. HDACi not only have good therapeutic effects on various hematologic malignancies and solid tumors, but also have the characteristics of relatively high selectivity and low toxicity to tumor cells. Therefore, the development of new HDAC inhibitors with excellent activity has potential clinical value and can bring tangible benefits to patients. Summary of the Invention

[0006] The technical problem that the invention aims to solve:

[0007] Given the high application prospects and development value of histone deacetylase inhibitors (HDACi) in tumor treatment, this application provides a new compound that can serve as an HDACi, which has excellent inhibitory activity and anti-tumor effects, while also having better safety.

[0008] This application also provides a pharmaceutical composition comprising the above-described HDACi.

[0009] Solution for solving the problem:

[0010] To address the aforementioned problems, the inventors of this application conducted in-depth research and discovered that compounds with specific structures can achieve unexpected inhibitory activity against tumor cells, exhibit superior anti-tumor effects, and possess better safety, thus completing this invention.

[0011] This invention protects the following specific embodiments:

[0012] This invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0013] ;

[0014] Where X is selected from -CH- or -N-;

[0015] R1 and R5 are each independently selected from hydrogen, amino, fluorine, chlorine, bromine, iodine, and C. 1-6 alkyl;

[0016] R2, R3, and R4 are each independently selected from hydrogen, chlorine, bromine, iodine, and C. 1-6 alkyl;

[0017] And when X is selected from -N-, R1, R2, R3, R4, and R5 are not all hydrogen at the same time.

[0018] In one embodiment, in the above-mentioned compound or its pharmaceutically acceptable salt, R1 and R5 are each independently selected from hydrogen, amino, fluorine, chlorine, bromine, iodine, C 1-3 Alkyl, C 4-6 alkyl.

[0019] In a preferred embodiment, R1 and R5 are each independently selected from hydrogen, amino, fluorine, chlorine, and bromine.

[0020] In a more preferred embodiment, R1 and R5 are each independently selected from hydrogen and amino groups.

[0021] In a more preferred embodiment, R1 and R5 are each independently selected from amino, fluorine, chlorine, and bromine.

[0022] In a further preferred embodiment, one of R1 and R5 is selected from amino and the other is selected from hydrogen.

[0023] In a further preferred embodiment, one of R1 and R5 is selected from amino and the other is selected from fluorine, chlorine, or bromine.

[0024] In a further preferred embodiment, R1 is selected from amino groups and R5 is selected from fluorine, chlorine, or bromine; or R1 is selected from fluorine, chlorine, or bromine and R5 is selected from amino groups.

[0025] In a further preferred embodiment, R1 is selected from hydrogen and R5 is selected from amino; or R1 is selected from amino and R5 is selected from hydrogen.

[0026] In one embodiment, among the above-mentioned compounds or their pharmaceutically acceptable salts, R2, R3, and R4 are each independently selected from hydrogen, chlorine, bromine, iodine, and C. 1-3 Alkyl, C4-6 alkyl.

[0027] In a preferred embodiment, R2, R3, and R4 are each independently selected from hydrogen, chlorine, bromine, methyl, ethyl, propyl, and isopropyl.

[0028] In a more preferred embodiment, R2, R3, and R4 are each independently selected from hydrogen, chlorine, and bromine.

[0029] In a further preferred embodiment, R2, R3, and R4 are all selected from hydrogen.

[0030] In one embodiment, the above-mentioned compound or a pharmaceutically acceptable salt thereof...

[0031] X is selected from -N-;

[0032] R1 and R5 are each independently selected from hydrogen, amino, fluorine, chlorine, and bromine;

[0033] R2, R3, and R4 are each independently selected from hydrogen, chlorine, and bromine;

[0034] Furthermore, R1, R2, R3, R4, and R5 are not all hydrogen at the same time.

[0035] In a preferred embodiment, R1 and R5 are each independently selected from hydrogen and amino groups.

[0036] In a preferred embodiment, R2, R3, and R4 are all selected from hydrogen.

[0037] In one embodiment, the above-mentioned compound or a pharmaceutically acceptable salt thereof...

[0038] X is selected from -CH;

[0039] R1 and R5 are each independently selected from hydrogen, amino, fluorine, chlorine, and bromine;

[0040] R2, R3, and R4 are each independently selected from hydrogen, chlorine, and bromine.

[0041] In a preferred embodiment, R1 and R5 are each independently selected from hydrogen and amino groups.

[0042] In one embodiment, the above-described compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the following compounds:

[0043] .

[0044] In one embodiment, the above-described compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the following compounds:

[0045] .

[0046] The compounds of this application can be prepared by various synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples of this application. The chemical reactions in the specific embodiments of this application are carried out in a suitable solvent, which must be suitable for the chemical changes of this application and the reagents and materials required therefor. In order to obtain the compounds of this application, it is sometimes necessary for those skilled in the art to modify or select the synthetic steps or reaction processes based on existing embodiments.

[0047] An important consideration in planning synthetic routes in this field is selecting suitable protecting groups for reactive functional groups (such as the amino groups in this application). The products obtained from each reaction step are obtained using separation techniques known in the art, including but not limited to extraction, filtration, distillation, crystallization, and chromatographic separation. The starting materials and chemical reagents required for the synthesis can be routinely synthesized according to literature (such as those provided by SciFinder) or purchased.

[0048] Pharmaceutical composition:

[0049] The present invention also provides a pharmaceutical composition comprising a compound represented by formula (I) above or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutical carrier and / or excipient and / or diluent.

[0050] Methods for preparing various pharmaceutical compositions containing a certain amount of active ingredient are known, or will be obvious to those skilled in the art according to the disclosure of the present invention. Methods for preparing the pharmaceutical compositions include incorporating appropriate pharmaceutical excipients, carriers, diluents, etc.

[0051] Medical uses:

[0052] Another aspect of the invention relates to the compound represented by formula (I) above or a pharmaceutically acceptable salt thereof, and the use of the above composition in the preparation of a medicament for treating and / or preventing diseases related to the biological activity of HDAC.

[0053] Another aspect of the invention relates to a method of treating and / or preventing diseases associated with the biological activity of HDAC, comprising administering to an individual in need a therapeutically / preventively effective amount of a compound of formula (I) above or a pharmaceutically acceptable salt thereof, or a combination thereof.

[0054] Another aspect of the present invention relates to compounds of formula (I) above or pharmaceutically acceptable salts thereof, and compositions thereof, for the treatment and / or prevention of diseases associated with the biological activity of HDAC.

[0055] According to a preferred embodiment of the present invention, the disease associated with the biological activity of HDAC is selected from cancer and hyperproliferative diseases.

[0056] Technical terms of this invention:

[0057] In the following description, certain specific details are set forth to provide a thorough understanding of the various embodiments. However, those skilled in the art will understand that the invention can be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments. Unless the context otherwise requires, throughout the specification and claims, the word “comprising” and its variations, such as “including” and “containing,” should be interpreted in an open-ended sense, meaning “including but not limited to.” Furthermore, the headings provided herein are for convenience only and are not intended to define the scope or meaning of the claimed invention.

[0058] Throughout this specification, the phrase "an embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described with respect to that embodiment is included in at least one embodiment. Therefore, the phrases "in an embodiment" or "in an embodiment" appearing in different places throughout the specification do not necessarily all refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic may be combined in any suitable manner in one or more embodiments. Moreover, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural indicators unless the context clearly specifies otherwise. It should also be noted that, unless the context clearly specifies otherwise, the term "or" is generally used to include the meaning of "and / or."

[0059] The term "pharmaceutically acceptable salt" refers to the salt of the compounds of this invention, prepared by reacting a compound having specific substituents discovered in this invention with a relatively non-toxic acid or base. When the compounds of this invention contain relatively acidic functional groups, base addition salts can be obtained by contacting such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. When the compounds of this invention contain relatively basic functional groups, acid addition salts can be obtained by contacting such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent.

[0060] In this invention, the term "alkyl" used alone or in combination with other terms refers to a saturated hydrocarbon group that can be straight-chain or branched. The term "C 1-6 "Alkyl" refers to an alkyl group having 1 to 6 carbon atoms. An alkyl group formally corresponds to an alkane where a CH bond is replaced by a junction between the alkyl group and the rest of the compound. In some embodiments, the alkyl group contains 1-6 carbon atoms, 1-4 carbon atoms, 1-3 carbon atoms, or 1-2 carbon atoms. Examples of alkyl groups include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, primary butyl, tert-butyl, and isobutyl; higher homologues such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, and 1,2,2-trimethylpropyl.

[0061] In this invention, the term "halogen" used alone or in combination with other terms refers to F, Cl, Br, and I. In some embodiments, the term "halogenated" refers to a halogen atom selected from F, Cl, or Br.

[0062] In this invention, the term "therapeutic effective amount" used alone or in combination with other terms refers to the amount of a compound administered to mammals and / or subjects as a single dose or as part of a series of doses that effectively produces the desired therapeutic effect.

[0063] The beneficial effects of this invention are:

[0064] The compounds and pharmaceutical compositions provided by this invention exhibit unexpectedly strong inhibitory activity and high selectivity against tumor cells, excellent anti-tumor effects, and better safety, and can be used as drugs for the treatment of diseases related to this effect.

[0065] Patent CN1284772C discloses a class of benzamide compounds, specifically synthesizing two compounds. Experimental results show that these compounds exhibit good anti-proliferative activity as histone deacetylase inhibitors (HDACi). However, the applicant has discovered new compounds with completely unexpected technical effects. Cell proliferation inhibition assays using this invention show that compound 1 exhibits excellent inhibitory activity against human leukemia T lymphocytes. Compared to Chidamide in patent CN1284772C, the inhibitory activity of compound 1 is more than 10 times that of Chidamide. Furthermore, the 293T IC50 / Jurkat IC50 ratio of compound 1 is more than twice that of Chidamide, indicating that compound 1 has less impact on normal cells while killing human leukemia T lymphocytes, exhibiting higher selectivity and safety. In summary, compared to Chidamide, one of the best-performing similar HDACi compounds in the prior art, the compounds of this invention show more than a 10-fold increase in inhibitory activity against tumor cells, as well as higher selectivity and safety. This is an unexpected discovery and an unforeseen improvement in technical effect. Furthermore, the applicant also discovered that compound 1 has unexpected advantages over chidamide in various aspects, including HDAC enzyme activity inhibition, proliferation inhibition of various cell types such as HL60 / K562 / MV-4-11 / HepG-2, IV clearance rate in mice, and tumor inhibition rate in a mouse colon cancer cell line MC38 xenograft model.

[0066] Patent CN118772048A discloses an HDAC inhibitor, specifically synthesizing 91 compounds. However, the applicant discovered that, compared to compound 4 in CN118772048A, which has better activity, pharmacokinetics, and efficacy, compound 1 of this invention has unexpected advantages in various indicators, such as IV clearance rate in mice and survival rate in the MC38 colon cancer cell line xenograft model.

[0067] In summary, the compounds of this invention, compared with Chidamide in the prior art CN1284772C and the superior molecular compound 4 in CN118772048A, have a number of unexpected advantages. Attached Figure Description

[0068] Figure 1 The drug efficacy curve is shown in the mouse colon cancer cell line MC38 xenograft model.

[0069] Figure 2 Survival curves of animals in each group after drug administration. Detailed Implementation Plan

[0070] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Various other modifications, substitutions, or alterations can be made based on ordinary technical knowledge and common practice in the art, without departing from the basic technical concept of this invention described above.

[0071] The present invention will be described in detail below with reference to examples, but this does not imply any adverse limitation on the invention. The compounds of the present invention can be prepared by various synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the invention, and these should also be considered within the scope of protection of the present invention.

[0072] Example 1: Preparation of Compound 1

[0073]

[0074] Step 1: Compound 1b (406 mg, 1.95 mmol), O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (808 mg, 2.13 mmol), and triethylamine (538 mg, 5.31 mmol) were added to a solution of compound 1a (500 mg, 1.77 mmol) in N,N-dimethylformamide (10 mL). After addition, the mixture was reacted at 25 °C for 1.5 h. After the reaction was complete, saturated brine (30 mL) was added, and the mixture was extracted with ethyl acetate (20 mL*2). The organic phase was washed with saturated brine (20 mL*3), dried over anhydrous sodium sulfate, and concentrated to obtain 1c (500 mg). ESI-MS (m / z): 473.1 [M+H]+.

[0075] Step 2: Add 1,4-dioxane hydrochloric acid solution (2 M, 10 mL) to compound 1c (500 mg, 1.06 mmol), and react at 25°C for 1 hour. After the reaction is complete, concentrate the reaction solution and perform reversed-phase column chromatography (acetonitrile: 0.1% ammonium bicarbonate aqueous solution = 0~45%) to obtain compound 1 (200 mg), ESI-MS (m / z): 373.1 [M+H]+.

[0076] 1 H NMR (400 MHz, DMSO-d6)δ9.65 (s, 1H), 8.92–8.72 (m, 2H), 8.58 (d, J= 4.7 Hz, 1H), 8.03–7.98 (m, 3H), 7.55 (d, J = 15.9 Hz, 1H), 7.51-7.37 (m,3H), 7.19 (d, J = 7.8 Hz, 1H), 6.99 (t, J = 7.6 Hz, 1H), 6.90 – 6.75 (m, 2H), 6.62 (t, J = 7.6 Hz, 1H), 4.90 (s, 2H), 4.52 (d, J = 6.0 Hz, 2H).

[0077] Example 2: Inhibition of proliferation of HEK293T and human acute T-lymphoblastic leukemia cell line JurkatE6.1 by the compounds of the present invention.

[0078] HEK293T cell treatment: HEK293T cells were cultured in complete DMEM (Hyclone, SH30243.01) / 10% FBS (Hyclone, SH30406.05) medium (inside) to evaluate the inhibitory effect of the compound on cell proliferation. One day before drug administration, 1500 HEK293T cells were added to each well of a 96-well cell culture plate (Quanpin, QB-315-096-C) and incubated overnight (16-18 h) at 37°C in a 5% CO2 incubator. On the second day, a stepwise dilution method was used to prepare compounds at two-fold final concentrations: First, compounds (compound 1 and chidamide) were serially diluted 3-fold with DMSO (starting at 10000 μM, for a total of 10 concentration points); second, the compounds prepared in step one with DMSO in the first step were diluted 50-fold with complete culture medium DMEM / 10% FBS to obtain compounds 1 and chidamide at an initial concentration of 200 μM (each concentration point containing 2% DMSO). Equal volumes of the compounds prepared at two-fold final concentrations with complete culture medium were added to 96-well cell culture plates (50 μl / well, each concentration point containing 1% DMSO), with two replicates per concentration. The 96-well plates were returned to the incubator and incubated for 3 days at 37 ℃ and 5% CO2, marked as Day 0.

[0079] Jurkat E6.1 cells were treated: Jurkat E6.1 cells (Nanjing Kebai, CBP60942) were cultured in complete medium RPMI 1640 (Hyclone, SH30255.FS) / 10% FBS (Hyclone, SH30406.05) to evaluate the inhibitory effect of the compounds on cell proliferation. 3000 Jurkat E6.1 cells were added to each well of a 96-well cell culture plate (Quanpin, QB-315-096-C) and incubated at 37 ℃ with 5% CO2 for further culture. On the day of plating, the drugs were added using a stepwise dilution method to prepare compounds at two-fold final concentrations: First, compounds (compound 1 and chidamide) were serially diluted 3-fold with DMSO (Sigma, D5879) (compound 1 starting at 2000 μM, chidamide starting at 4000 μM, for a total of 10 concentration points); Second, the compounds prepared in step one with DMSO were diluted 50-fold with complete culture medium RPMI 1640 / 10% FBS to obtain compound 1 at an initial concentration of 40 μM and chidamide at an initial concentration of 80 μM (DMSO content at each concentration point was 2%). Equal volumes of the compounds prepared at two-fold final concentrations with complete culture medium were added to 96-well cell culture plates (50 μl / well, DMSO content at each concentration point was 1%), with two replicates per concentration. The 96-well plates were returned to the incubator and incubated for 3 days at 37 ℃ and 5% CO2, marked as Day 0.

[0080] Three days later (Day 3), the 96-well culture plate was removed, examined under a microscope, and equilibrated at room temperature for 30 min. An equal volume (100 μL) of CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7572) was added to each well. The plate was incubated at room temperature in the dark on a shaker for 2 min to lyse the luminescent signal, followed by 10 min of static incubation at room temperature to stabilize the luminescent signal. Luminescence readings were obtained using Tecan Spark and recorded. Data analysis was performed using GraphPad Prism 10 software, employing the equation "log (inhibitor) vs. normalized response -- variable slope" (formula Y=Bottom + (Top-Bottom) / (1+10^((LogIC 50 Data analysis was performed using -X)*HillSlope))) to obtain the IC50 of the compound. 50Values ​​(Table 1). Where Y represents cell viability, X represents compound concentration, Top refers to maximum cell viability (cell viability at the lowest compound concentration), Bottom refers to baseline response (cell viability at the highest compound concentration), and Hill Slope refers to IC50. 50 The slope of the curve, IC 50 The concentration of the compound at half-inhibition.

[0081]

[0082] The results above show that compound 1 exhibits excellent inhibitory activity against human leukemia T lymphocytes. Compared to chidamide, the inhibitory activity of compound 1 is more than 10 times that of chidamide, which is unexpected. Furthermore, the 293T IC50 / Jurkat IC50 ratio shows that the ratio of compound 1 is more than twice that of chidamide, indicating that compound 1 has less impact on normal cells while killing human leukemia T lymphocytes, exhibiting higher selectivity and safety, which is also unexpected.

[0083] In summary, the compounds of this invention exhibit excellent inhibitory activity against tumor cells, with an inhibitory activity more than 10 times higher than that of Chidamide, and also have higher safety.

[0084] Example 3: Inhibitory activity test of the compound of the present invention against HDAC enzyme.

[0085] Dissolve the compound to 10 mM with DMSO, then serially dilute the compound with DMSO. The initial concentration was 30000 nM, with 3-fold serial dilutions for a total of 10 concentration gradients. Add 50 μL / well to a 384-well plate, with 3 replicates per well and blank wells. Add 2.5 μL of HDAC (including HDAC1, HDAC2, HDAC3, and HDAC10) sequentially as per the manufacturer's instructions and incubate at room temperature for 15 minutes; add 2.5 μL of HDAC substrate and incubate at 37°C for 40 / 180 minutes; then add 5 μL of Developer mixture and incubate at room temperature for 10 minutes. Detect the fluorescence signal using a microplate reader (Em: 340 nM; Ex: 450 nm).

[0086] Using wells without compounds as negative controls (Y) and wells without enzymes as positive controls (Z), the inhibition rate of each compound against the corresponding kinase was calculated based on the fluorescence signal (X) of each test well, using the following formula: Inhibition rate (%) = 100% - (XZ) / (YZ) × 100%. The half-maximal inhibitory concentration (IC50) of each compound against the corresponding HDAC enzyme was then calculated using GraphPad. 50 ).

[0087] The enzymatic results in Table 2 show that compound 1 of the present invention has good inhibitory activity against the four isoforms of HDAC1, 2, 3, and 10; compared with Chidamide, compound 1 has 4 times stronger activity against HDAC1 enzyme and 2 times stronger activity against HDAC10 enzyme.

[0088]

[0089] Example 4: Inhibition test of the proliferation of the compound of the present invention on various cell types

[0090] 90 μL of cell suspension was aspirated using a pipette and added to a 96-well plate (Corning, catalog number 3599). The plate was incubated at 37°C in a 5% CO2 incubator for 24 h. On the second day, the compound was prepared as a 10 mM stock solution using DMSO (Sigma, catalog number D2650-100 mL), diluted with culture medium to the desired concentration gradient, and then added to each well. Chidamide and compound 1 of this invention were diluted in HL60 / K562 / MV-4-11 / HepG-2 / BT549 / LO2 cells using a 20 μM starting dilution, followed by 2x dilutions, for a total of 9 concentration points. In Daudi / HCT116 / SW480 / LN-18 cells, the dilution protocol was 30 μM starting dilution, followed by 2x dilutions, for a total of 9 concentration points. Compound 1 of this invention was diluted in 20 μM starting dilutions, followed by 3x dilutions, for a total of 9 concentration points. Two replicates were set up for each concentration. After the drug was added, the cell culture plate was incubated in a 37°C, 5% CO2 incubator for 3 days.

[0091] On day 5, 20 μL of MTS (Promega, catalog number G3581) was added to each well of HL60 / K562 / MV-4-11 / HepG-2 / BT549 / LO2 / Daudi / HCT116 / SW480 / LN-18 cells and incubated at 37°C in a 5% CO2 incubator for 2 h. After 2 h, the template was detected at a wavelength of 490 nm using a microplate reader.

[0092] The inhibition rate was calculated using the following formula, and the IC was calculated using the log(inhibitor) vs. response - variable slope analysis method in GraphPad Prism 6.0 software. 50 For those using MTS detection, the OD value was used for calculation, while for those using Celltiter-Glo detection, the luminescence value was used for calculation. The test results are shown in Table 3.

[0093]

[0094]

[0095] Test results show that compound 1 of the present invention has a 5-15 times stronger inhibitory effect on the proliferation of the above-mentioned cells such as HL60 / K562 / MV-4-11 / HepG-2 than Chidamide.

[0096] Example 5: Metabolic kinetics of the compounds of the present invention in CD-1 mice

[0097] Experimental materials: The experimental animals were healthy adult male CD-1 mice (provided by Sichuan Vitonlihua Laboratory Animal Technology Co., Ltd.).

[0098] Administration and Sample Collection: In mice, CD-1 male mice were administered a single intravenous dose (1 mg / kg, solvent: 5% DMSO + 5% solvent + 90% saline) (DMSO: AR 500 mL, Chengdu Kelong Chemical Co., Ltd.; Solutol: 42966-1 KG, BASF; Saline: Sichuan Meida Kangjiale Pharmaceutical Co., Ltd., 100 mL 0.9 g). Whole blood (50 μL) from the fundus venous plexus was collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h post-administration. Plasma was collected by centrifugation at 4000 rpm for 6 min.

[0099] Sample analysis: Take 10 μL of mouse plasma sample, add 110 μL of acetonitrile solution containing internal standard to precipitate protein, vortex for 10 min, then centrifuge at 4000 rpm for 10 min, take 80 μL of supernatant into 96-well plate, add 80 μL of pure water to dilute, vortex for 10 min, and finally centrifuge at 4000 rpm for 5 min.

[0100] The drug concentration in the plasma of mice at different time points after intravenous administration of the compound of the example was determined by LC-MS / MS, and the relevant pharmacokinetic parameters were calculated to study the pharmacokinetic behavior of the compound in mice and evaluate its pharmacokinetic characteristics.

[0101]

[0102] Comparative Example 1 is compound 4 described in patent CN118772048A, which has the following structure and was prepared according to the method in patent CN118772048A.

[0103]

[0104] The results in Table 4 show that the scavenging rate of compound 1 of the present invention is higher than that of comparative example 1 and Chidamide, and the high scavenging rate can reduce the toxicity risk.

[0105] Example 6: In vivo efficacy experiment of the compound of the present invention in a mouse colon cancer cell line MC38 xenograft model

[0106] MC38 cells (Suzhou Beina Chuanglian Biotechnology Co., Ltd., BNCC353445) in logarithmic growth phase were digested and collected. After counting, the cell density was adjusted to 5 × 10⁶ cells using RPMI 1640 medium (Gibco). 6 Cells / mL. Twenty C57BL / 6N mice (20 ± 2 g mice, Chengdu Vital River Laboratory Animal Technology Co., Ltd.) were subcutaneously inoculated with 100 μL of MC38 cell suspension. On day 13 after inoculation, the average tumor volume reached 115 mm. 3 Animals were randomly divided into 4 groups (N = 5). The compound was administered by gavage once daily at a concentration of 15 mg / kg in a suspension prepared with solvent (0.2% CMC-Na / 0.1% Tween 80) at a concentration of 1.5 mg / mL.

[0107] After animals were inoculated with MC38 cells, tumor volume was measured 2-3 times per week, with the tumor volume calculated as length × width. 2 Calculate using ×0.5.

[0108] The tumor inhibition rate (TGI) (tumor volume) for each treatment group is calculated using the following formula: TGI TV =1-((V Tt -V T0 ) / (V Ct -V C0 ))×100%. Where V Tt V represents the tumor volume at each measurement in the treatment group. T0 Tumor volume was measured when grouping the treatment group; V Ct The group represents the tumor volume at each measurement, V. C0 The tumor volume was measured when the control group was grouped.

[0109] Survival observations were conducted on each group of animals during the experiment, and survival curves were plotted.

[0110] Data were statistically analyzed using GraphPad Prism 9.0 software, and are expressed as Mean ± SEM (standard error). One-way ANOVA was used to compare each drug-treated group with the control group. Homogeneity of variance was determined using the Brown-Forsythe method, and heterogeneity of variance was determined using Dunnett's T3 method.

[0111] The results are shown in Table 5 and Appendix. Figure 1 Appendix Figure 2 As shown.

[0112]

[0113] Comparative Example 1 is the same as Comparative Example 1 in Example 5.

[0114] Experimental Results and Analysis: In the mouse colon cancer cell line MC38 CDX, after 12 days of administration, compound 1 of the present invention achieved unexpected technical effects in tumor inhibition compared with Chidamide (P < 0.0001), and the TGI was nearly doubled; compared with Comparative Example 1, compound 1 of the present invention also had an unexpected advantage in survival rate, with a survival rate of 100%, while the survival rate of Comparative Example 1 was 20%.

[0115] Survival rate is a core endpoint in in vivo pharmacodynamics. A high survival rate indicates good drug safety, effective inhibition of cancer cell proliferation, invasion, or metastasis, and prolongation of survival time in tumor-bearing mice. Therefore, compound 1 of this invention exhibits excellent tumor-suppressive activity while demonstrating even better safety, achieving unexpected technical benefits.

[0116] Therefore, based on the experimental data described in Examples 1-6, it is evident that, compared to Chidamide, Compound 1 of the present invention achieved unexpected technical effects in terms of enzymology, cell activity and selectivity, clearance rate, and tumor suppression effect; compared to Comparative Example 1, Compound 1 of the present invention achieved unexpected technical effects in terms of clearance rate and survival rate. In summary, Compound 1 of the present invention achieved unexpected technical effects in both efficacy and safety.

[0117] 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 compound of formula (I) or a pharmaceutically acceptable salt thereof: ； in, X is selected from -CH- or -N-; R1 and R5 are each independently selected from hydrogen, amino, fluorine, chlorine, bromine, iodine, and C. 1-6 alkyl; R2, R3, and R4 are each independently selected from hydrogen, chlorine, bromine, iodine, and C. 1-6 alkyl; And when X is selected from -N-, R1, R2, R3, R4, and R5 are not all hydrogen at the same time.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: R1 and R5 are each independently selected from hydrogen, amino, fluorine, chlorine, bromine, iodine, and C. 1-3 Alkyl, C 4-6 alkyl; Preferably, R1 and R5 are each independently selected from hydrogen, amino, fluorine, chlorine, and bromine; More preferably, R1 and R5 are each independently selected from hydrogen and amino groups; More preferably, R1 and R5 are each independently selected from amino, fluorine, chlorine, and bromine; Preferably, one of R1 and R5 is selected from amino and the other is selected from hydrogen; Preferably, one of R1 and R5 is selected from amino, and the other is selected from fluorine, chlorine, or bromine; More preferably, R1 is selected from amino groups and R5 is selected from fluorine, chlorine, and bromine; or R1 is selected from fluorine, chlorine, and bromine and R5 is selected from amino groups. More preferably, R1 is selected from hydrogen and R5 is selected from amino; or R1 is selected from amino and R5 is selected from hydrogen.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: R2, R3, and R4 are each independently selected from hydrogen, chlorine, bromine, iodine, and C. 1-3 Alkyl, C4-6 alkyl; Preferably, R2, R3, and R4 are each independently selected from hydrogen, chlorine, bromine, methyl, ethyl, propyl, and isopropyl. More preferably, R2, R3, and R4 are each independently selected from hydrogen, chlorine, and bromine; Preferably, R2, R3, and R4 are all selected from hydrogen.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: X is selected from -N-; R1 and R5 are each independently selected from hydrogen, amino, fluorine, chlorine, and bromine; preferably, R1 and R5 are each independently selected from hydrogen and amino. R2, R3, and R4 are each independently selected from hydrogen, chlorine, and bromine; preferably, R2, R3, and R4 are all selected from hydrogen. Furthermore, R1, R2, R3, R4, and R5 are not all hydrogen at the same time.

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The compound is selected from the following compounds: ； ； ； ； 。 6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The compound is selected from the following compounds: 。 7. A pharmaceutical composition comprising the compound of any one of claims 1-6 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable auxiliary component.

8. Use of the compound of any one of claims 1-6 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 7 in the preparation of a medicament for the treatment, regulation and / or prevention of biological activity related to HDAC; Preferably, the disease is a tumor or a disease of excessive proliferation.

9. A method for treating, modulating, and / or preventing diseases related to the biological activity of HDAC, comprising administering to a patient in need a therapeutically effective amount of the compound of any one of claims 1-6 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 7; Preferably, the disease is a tumor or a disease of excessive proliferation.

10. The compound of any one of claims 1-6 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 7, for the treatment, regulation, and / or prevention of diseases related to the biological activity of HDAC; Preferably, the disease is a tumor or a disease of excessive proliferation.

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