A nitroxynil derivative hydroxamic acid compound and a preparation method and application thereof

Abstract

The application belongs to the field of pharmaceutical chemistry, and discloses a chloramine-b-derived hydroxamic acid compound, a preparation method and application thereof, the chloramine-b-derived hydroxamic acid compound is a compound shown in formula (I) or a pharmaceutically acceptable salt, solvate or hydrate thereof; wherein, Linker is selected from C2~C10 alkylene or C2~C10 alkylene interrupted by one or more of NH, O, S, carbonyl. The compound takes chloramine-b as a mother nucleus, introduces a connecting arm containing a hydroxamic acid zinc binding group through a phenolic hydroxyl site, and forms a novel small molecule with double-channel regulation potential. Preferably, the compound N04 has HDAC1 / 6 inhibitory activity and STAT3 phosphorylation inhibition ability, and shows good anti-solid tumor activity.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to a class of STAT3 / HDAC dual-pathway inhibitors constructed with niclosamide as the parent core and the introduction of zinc hydroxamate binding groups, as well as their preparation methods, pharmaceutical compositions, and their use in the preparation of drugs for the prevention and / or treatment of tumors, especially solid tumors. Background Technology

[0002] The STAT3 signaling pathway and the histone deacetylase (HDAC) pathway play crucial roles in the occurrence, development, invasion, drug resistance, and immune escape of various solid tumors. Existing research indicates a mutually reinforcing regulatory loop between STAT3 and HDAC: on the one hand, STAT3 can upregulate the expression of specific HDAC isoforms and participate in the tumor pro-survival transcriptional network; on the other hand, HDAC activity can enhance tumor-related signal transduction through chromatin remodeling, non-histone deacetylation, and chaperone protein regulation.

[0003] Some HDAC inhibitors exhibit compensatory activation of the JAK / STAT3 axis in solid tumors, leading to limited efficacy or even acquired resistance. Therefore, single-target small molecules often struggle to achieve sustained and stable therapeutic effects in the complex tumor microenvironment.

[0004] Niclosamide has been shown to inhibit STAT3 phosphorylation and exhibit antitumor activity in various solid tumor models, but its poor water solubility and low oral bioavailability limit its further translational application. Vorinostat (SAHA) is a classic HDAC inhibitor; its hydroxamic acid group can efficiently bind zinc ions in the HDAC catalytic pocket, exhibiting a distinct HDAC inhibitory pharmacophore characteristic.

[0005] Therefore, introducing a SAHA-derived hydroxamic acid pharmacophore around the niclosamide skeleton has the potential to construct a dual-pathway inhibitor that simultaneously regulates STAT3 and HDAC, and is expected to yield lead compounds with both activity and drug-like properties for both solid tumors and non-solid tumors. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a niclosamide-derived hydroxamic acid compound, its preparation method and application, wherein the niclosamide-derived hydroxamic acid compound retains the ability of the niclosamide benzamide skeleton to regulate the STAT3 pathway, and endows it with HDAC inhibitory activity by introducing a zinc hydroxamic acid binding group.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] A chloronitrosamine-derived hydroxamic acid compound, which is a compound of formula (I) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0009]

[0010] The Linker is selected from C2-C10 alkylene groups or C2-C10 alkylene groups separated by one or more of NH, O, S, and carbonyl groups, preferably C2-C10 alkylene groups or C2-C10 alkylene groups separated by NH.

[0011] Preferably, the niclosamide-derived hydroxamic acid compound is compound NO1~NO6 or its pharmaceutically acceptable salt, solvate or hydrate;

[0012] The structural formulas of compounds N01~N06 are as follows:

[0013] , , .

[0014] The most preferred is compound NO4 or its pharmaceutically acceptable salt, solvate or hydrate.

[0015] Preferably, the pharmaceutically acceptable salt is an acid addition salt or an alkali metal salt.

[0016] The present invention also provides a pharmaceutical composition comprising any one of the above-mentioned compounds or a pharmaceutically acceptable salt, solvate, hydrate thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

[0017] The pharmaceutical composition is an oral or injectable preparation, preferably a tablet, capsule, granule, lyophilized powder for injection, injection solution, suspension, or sustained-release preparation.

[0018] This invention also provides the use of the above-mentioned compounds or pharmaceutical compositions in the preparation of antitumor drugs;

[0019] The antitumor drug is used for the prevention and / or treatment of tumors;

[0020] The active ingredient contained in the drug is the aforementioned niclosamide-derived hydroxyoxime compound.

[0021] In a preferred embodiment, the antitumor drug is a drug for inhibiting STAT3 phosphorylation and / or inhibiting HDAC enzyme activity; the inhibition of HDAC enzyme activity is preferably the inhibition of HDAC1 and / or HDAC6;

[0022] It is preferably used to inhibit HDAC1 / HDAC6 and reduce the phosphorylation level of STAT3 Tyr705.

[0023] Furthermore, the antitumor drug is used to increase Ac-H3 levels, induce tumor cell apoptosis, inhibit tumor cell migration, inhibit colony formation, and / or inhibit the growth of 3D tumor spheres. This invention also provides a method for preparing the above-mentioned compound, which includes using niclosamide as a starting material, introducing a hydroxamic acid precursor linker arm through phenolic hydroxyl functionalization, and then constructing the target hydroxamic acid terminal group through hydroxylamine conversion or a protection / deprotection strategy.

[0024] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0025] This invention introduces the hydroxamic acid pharmacophore of SAHA into the niclosamide skeleton to construct a novel compound with dual STAT3 and HDAC pathway regulatory capabilities. This not only retains the intervention potential of the niclosamide core on tumor-related signaling pathways, but also enhances the inhibitory effect on HDAC targets through the hydroxamic acid group.

[0026] Among the series of compounds obtained in this invention, compound NO4 is preferred in terms of its IC50 concentration in MDA-MB-231 and HCT116 cells. 50 The concentrations were 1.49 μM and 1.40 μM, respectively; in the enzymatic evaluation of HDAC1, HDAC3 and HDAC6, the IC50 values ​​were 129.1 nM, 300.5 nM and 230.4 nM, respectively, showing superior dual-pathway inhibition potential.

[0027] The preferred compound NO4 also significantly reduced STAT3 phosphorylation levels, increased Ac-H3 levels, induced apoptosis, and inhibited migration and colony formation; SPR results showed that its binding dissociation constant KD with STAT3 was 5.816 × 10⁻⁶. -6 M.

[0028] The preferred compound NO4 exhibited a superior transmembrane transport tendency compared to niclosamide in the Caco-2 model and demonstrated measurable metabolic stability in the rat liver microsomal system (t). 1 / 2 At approximately 48.6 min, Clint (approximately 28.53 μL / min / mg microsomal protein) showed sustained inhibitory effects in the 3D tumor spheroid model. Attached Figure Description

[0029] Figure 1 This is a schematic diagram illustrating the design concept of the compound of this invention.

[0030] Figure 2 This is a schematic diagram of the synthetic routes for compounds NO1-NO3 of the present invention.

[0031] Figure 3 This is a schematic diagram of the synthetic routes for compounds NO4-NO6 of the present invention. Detailed Implementation

[0032] The present invention will be further illustrated by the following examples, but the scope of protection of the present invention is not limited thereto. Unless otherwise stated, the raw materials, reagents and solvents used are commercially available and can be used directly; the target compounds are all subjected to... 1 HNMR, 13 Identified by C NMR and HRMS, the final product had an HPLC purity greater than 98%.

[0033] In this specification, "pharmaceutically acceptable salt" includes, but is not limited to, salts that can be used in pharmaceutical manufacturing and are formed with inorganic acids, organic acids, alkali metals, alkaline earth metals, or organic amines; "solvent" and "hydrate" include associative compounds formed by the target compound with one or more solvents or water.

[0034] Unless otherwise specified, “inhibition of STAT3” as described herein includes reducing the phosphorylation level of STAT3 at the Tyr705 site; “inhibition of HDAC” includes inhibiting the enzyme activity of one or more HDAC isoforms, preferably HDAC1 and / or HDAC6.

[0035] Example 1: Preparation of compound NO1

[0036] Under nitrogen protection, 5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide (1.00 g, 3.06 mmol) and oxetane (0.23 mL, 3.67 mmol) were dissolved in anhydrous THF (10 mL), followed by the addition of KOtBu (1.03 g, 9.17 mmol), and the reaction was stirred at 50 °C for 16 h. After the reaction was completed, the mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The residue was separated by preparative reversed-phase HPLC using a C18 column with a mobile phase of MeCN / H2O (20–80%) containing 0.01% NH4HCO3, yielding a pale yellow solid intermediate 1 (yield 46%).

[0037] Step B. Synthesis of N01

[0038] Under nitrogen protection, intermediate 1 (200 mg, 0.48 mmol), NH₂OH·HCl (67.3 mg, 0.97 mmol), and Et₃N (0.20 mL, 1.45 mmol) were dissolved in anhydrous DMF (10 mL), followed by the addition of HATU (239.3 mg, 0.63 mmol). The reaction mixture was stirred at 25 °C for 16 h. The crude product was separated by preparative reversed-phase HPLC using a C18 column with a mobile phase of MeCN / H₂O (20–80%) containing 0.01% NH₄HCO₃, yielding a pale yellow solid NO₁ (20% yield).

[0039] The characterization data are as follows:

[0040] HPLC Purity: 99.1%; ¹H NMR (400 MHz, DMSO-d6): δ 10.55 (s, 2H), 8.82 (s, 1H), 8.62-8.52 (m, 1H), 8.41-8.31 (m, 1H), 8.28-8.20 (m, 1H), 7.93 (d, J =2.8 Hz, 1H), 7.71-7.58 (m, 1H), 7.41-7.35 (m, 1H), 4.57 (t, J = 6.3 Hz, 2H),2.61 (t, J = 6.3 Hz, 2H); ¹³C NMR (101 MHz, DMSO-d6): δ 166.42, 162.48,155.54, HRMS (ESI): m / z calcd forC 16 H 13 Cl2N3O6 [M+H] + 414.0249, found 414.0218.

[0041] Example 2: Preparation of compounds NO2 and NO3

[0042] Step A. Synthesis of intermediates 2 and 3

[0043] Under nitrogen protection, 5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide (500 mg, 1.52 mmol) was dissolved in 1,4-dioxane (10 mL), followed by the addition of ethyl 5-bromopentanoate (for NO2) or ethyl 7-bromoheptanoate (for NO3, 2.0 equivalent), KI (126.8 mg, 0.76 mmol), TBAB (123.1 mg, 0.38 mmol), and K2CO3 (633.7 mg, 4.58 mmol). The reaction mixture was stirred at 85 °C for 16 h. After cooling to room temperature, it was diluted with H2O (20 mL) and extracted with EtOAc (30 mL × 3). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography (petroleum ether / EtOAc, 1:1) to give a white solid intermediate 2 or 3.

[0044] Step B. Synthesis of NO2 and NO3

[0045] Intermediate 2 or 3 (1.0 equivalent) was dissolved in NH2OH·HCl (2.0 equivalent) in THF / MeOH (1:1, 2 mL), followed by the addition of NaOH aqueous solution (3.0 equivalent). The reaction system was stirred at 25 °C for 16 h. After removing the solvent under reduced pressure, the residue was separated by preparative HPLC using a C18 column with MeCN / H2O (20–95%) containing 0.1% NH4HCO3 as the mobile phase, yielding white solid NO2 (from intermediate 2, yield 18%) or NO3 (from intermediate 3, yield 21%).

[0046] The characterization data of compound NO2 are as follows:

[0047] HPLC Purity: 99.8%; ¹H NMR (400 MHz, DMSO-d6): δ 10.39 (s, 2H), 8.67 (d,J = 9.2 Hz, 1H), 8.67 (s, 1H), 8.38 (d, J = 2.5 Hz, 1H), 8.25 (dd, J = 9.2,2.5 Hz, 1H), 7.93 (d, J = 2.8 Hz, 1H), 7.63 (dd, J = 8.9, 2.7 Hz, 1H), 7.35(d, J = 9.0 Hz, 1H), 4.36 (t, J = 6.7 Hz, 2H), 2.01 (t, J = 7.3 Hz, 2H),1.89-1.75 (m, 2H), 1.71-1.53 ​​(m, 2H); ¹³C NMR (101 MHz, DMSO-d6): δ 169.16,162.61, 155.71, 143.28, 141.16, 134.25, 131.12, 125.63, 125.17, 124.11,123.35, 122.54, 121.96, 116.32, 70.19, 32.18, 28.25, 21.97; HRMS (ESI): m / zcalcd for C 18 H 17 Cl2N3O6 [M+H] + 442.0562, found 442.0572.

[0048] The characterization data of compound NO3 are as follows:

[0049] HPLC Purity: 98.4%; ¹H NMR (400 MHz, DMSO-d6): δ 10.36 (s, 2H), 8.67 (d, J = 9.2 Hz, 1H), 8.67 (s, 1H), 8.39 (d, J = 2.6 Hz, 1H), 8.26 (dd, J =9.2, 2.6 Hz, 1H), 7.96 (d, J = 2.8 Hz, 1H), 7.63 (dd, J = 8.9, 2.8 Hz, 1H), 7.35 (d, J = 9.0 Hz, 1H), 4.34 (t, J = 6.8 Hz, 2H), 2.23-1.71 (m, 4H), 1.52-1.25 (m, 6H). ¹³C NMR (101 MHz, DMSO-d6): δ 169.46, 162.66, 155.80, 143.28,141.17, 134.26, 131.11, 125.61, 125.18, 124.12, 123.28, 122.56, 121.98,116.34, 70.58, 32.62, 28.74, 28.66, 25.48. HRMS (ESI): m / z calcd forC 20 H 21 Cl2N3O6 [M+H] + 470.0875, found 470.0880.

[0050] Example 3: Preparation of compounds NO4-NO6

[0051] Step A. Synthesis of Intermediate 4

[0052] Under nitrogen protection, 5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide (981.4 mg, 3.00 mmol) and N-(tert-butyloxycarbonyl)ethanolamine (532.0 mg, 3.30 mmol) were dissolved in anhydrous toluene (30 mL), followed by the addition of (cyanomethylene)tributylphosphonane (CMBP, 796.5 mg, 3.30 mmol), and the reaction was stirred at 100 °C for 16 h. After the reaction was completed, the mixture was cooled to room temperature and diluted with EtOAc (50 mL). The solution was washed successively with saturated NaHCO3 solution (20 mL), H2O (20 mL), and saturated brine (20 mL). The organic phase was dried over anhydrous MgSO4, filtered, concentrated, and then separated by silica gel column chromatography (petroleum ether / EtOAc, 3:1 to 1:1) to give a white solid intermediate 4 (yield 47%).

[0053] Step B. Synthesis of Intermediate 5

[0054] Intermediate 4 (470.3 mg, 1.00 mmol) was dissolved in anhydrous CH2Cl2 (6 mL) and cooled to 0 °C. After adding TFA (3 mL), the mixture was stirred at room temperature for 2 h. Volatile components were removed under reduced pressure, and the residue was dissolved in H2O (10 mL). The pH was carefully adjusted to 8-9 with saturated NaHCO3 solution under ice bath conditions. The aqueous phase was extracted again with CH2Cl2 (15 mL × 3). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous MgSO4, filtered, and concentrated. The residue was separated by silica gel column chromatography (CH2Cl2 / MeOH, 20:1 to 10:1) to give a pale yellow solid intermediate 5 (yield 39%).

[0055] Step C. Synthesis of intermediates 6–8

[0056] Under nitrogen protection, the corresponding ω-bromoalkyl acid (1.0 equivalent) and N-methylmorpholine (1.1 equivalent) were dissolved in anhydrous THF (30 mL) and cooled to -10 °C. Isobutyl chloroformate (1.3 equivalent) and Trt-ONH2 (1.0 equivalent) were then added dropwise. The reaction mixture was heated to room temperature and stirred overnight. The reaction solution was diluted with H2O (20 mL) and extracted with EtOAc (30 mL × 3). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography to give Trt-protected ω-bromoalkyl hydroxyoxime intermediates 6–8 as white solids, in yields of 38–46%.

[0057] Step D. Synthesis of intermediates 9–11

[0058] Intermediate 5 (1.0 equivalent) and DIPEA (3.0 equivalent) were dissolved in anhydrous DMF (30 mL). The corresponding Trt protecting intermediates (6, 7, or 8; 1.0 equivalent) were added at room temperature, and the reaction mixture was stirred at 50 °C for 16 h. After routine post-treatment (including EtOAc extraction, washing with saturated brine, drying, and concentration), the residue was purified by silica gel column chromatography to obtain white solid Trt protecting intermediates 9-11, in yields of 29–35%.

[0059] Step E. Synthesis of NO4-NO6

[0060] Intermediates 9, 10, or 11 (1.0 equivalent) were dissolved in CH₂Cl₂ (3 mL), and 4 M HCl / 1,4-dioxane solution (1 mL) was added at room temperature. The reaction system was stirred for 2 h, concentrated under reduced pressure, and purified by preparative HPLC to obtain white solids NO₄, NO₅, or NO₆ in yield of 22-26%.

[0061] The characterization data of compound NO4 are as follows:

[0062] Yield: 26%; HPLC purity: 99.2%; ¹H NMR (400 MHz, DMSO-d6): δ 10.38 (s, 1H), 10.37 (s, 1H), 8.72 (s, 2H), 8.60 (d, J = 9.2 Hz, 1H), 8.47 (d, J = 2.6 Hz, 1H), 8.32 (dd, J = 9.2, 2.6 Hz, 1H), 7.93 (d, J = 2.8 Hz, 1H), 7.72 (dd, J = 8.9, 2.8 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H), 4.59 (s, 2H), 3.44 (s, 2H), 2.99 (s, 2H). 2H), 1.95 (t, J = 6.8 Hz, 2H), 1.66-1.41 (m, 4H). ¹³C NMR (101 MHz, DMSO-d6): δ 169.02, 163.26, 154.73, 143.81, 141.26, 133.76, HRMS (ESI): m / z calcd for C 20 H 22 Cl2N4O6 [M+H] + 485.0989, found485.1018.

[0063] The characterization data of compound N05 are as follows:

[0064] Yield: 25%; HPLC purity: 98.5%; ¹H NMR (400 MHz, DMSO-d6): δ 10.49–10.26 (m, 2H), 9.07–8.82 (m, 2H), 8.60 (d, J = 9.2 Hz, 1H), 8.47–8.41 (m, 1H), 8.33–8.26 (m, 1H), 7.91 (s, 1H), 7.74–7.65 (m, 1H), 7.39 (d, J = 9.0 Hz, 1H), 4.61 (s, 2H), 3.46 (s, 2H), 2.98 (s, 2H), 1.91 (t, J = 7.3 Hz, 2H). 1.60-1.38(m, 4H), 1.31-1.13 (m, 4H); ¹³C NMR (101 MHz, DMSO-d6): δ 169.46, 163.16,154.72, 143.68, 141.29, 133.75, 130.98, 126.07, 125.28, 124.61, 124.24,123.95, 123.05, 116.11, 65.75, 47.52, 45.87, 32.52, 28.48, 26.07, 25.75,25.28. m / z calcd for C 22 H 26 C 12 N4O6 [M+H] + 513.1302, found 513.1306.

[0065] The characterization data of compound N06 are as follows:

[0066] Yield: 22%; HPLC purity: 99.5%; ¹H NMR (400 MHz, DMSO-d6): δ 10.62–10.21 (m, 2H), 8.92 (s, 2H), 8.77–8.49 (m, 2H), 8.45 (s, 1H), 8.34–8.24 (m, 1H), 7.91 (d, J = 2.6 Hz, 1H), 7.76–7.64 (m, 1H), 7.39 (d, J = 9.0 Hz, 1H), 4.70–4.53 (m, 2H), 3.45 (s, 2H), 2.97 (s, 2H), 1.91 (t, J = 7.3 Hz, 2H). 1.61-1.38(m, 4H), 1.29-1.13 (m, 6H); ¹³C NMR (101 MHz, DMSO-d6): δ 169.51, 163.25,154.72, 143.74, 141.30, 133.72, 130.94, 126.06, 125.31, 124.70, 124.39,123.98, 123.19, 116.13, 65.76, 47.54, 45.90, 32.63, 28.80, 28.64, 26.21,25.86, 25.39; HRMS (ESI): m / z calcd for C 23 H 28 Cl2N4O6 [M+H] + 527.1459, found527.1453.

[0067] Table 1 Representative compounds and linker types

[0068]

[0069] Example 4: Evaluation of in vitro antitumor activity

[0070] The inhibitory activity of each compound against six solid tumor cell lines was evaluated using the CCK-8 assay. The IC50 of compound N04 was preferred in MDA-MB-231 and HCT116 cells. 50 The values ​​were 1.49 μM and 1.40 μM, respectively.

[0071] The CCK-8 assay process is as follows: Cells (MDA-MB-231, MCF-7, HCT116, SW620, SGC-7901, and BGC-823) are introduced at a density of 5 × 10⁶ cells per well. 3Cells were seeded at a density of 1,000 cells in 96-well plates and allowed to adhere overnight.

[0072] The test compound was dissolved in DMSO and further diluted with culture medium to the specified concentration (0.01–10 µM). After 48 hours of treatment, 10 µL of CCK-8 solution was added to each well, and incubation was continued at 37°C for 2 hours. Absorbance was measured at 450 nm using a BioTek microplate reader. Cell viability is expressed as a percentage relative to the solvent control.

[0073] Niclosamide and SAHA were used as positive controls. IC50 was determined using nonlinear regression analysis with GraphPad Prism 9.5. 50 Values. All experiments were performed in triplicate, and each experiment was repeated independently at least three times.

[0074] Example 5: HDAC Enzymology and Cellular Mechanism Experiment

[0075] The HDAC inhibition activity was evaluated using a fluorescence substrate method. The IC50 values ​​of NO4 for HDAC1, HDAC3, and HDAC6 were determined. 50 The concentrations were 129.1 nM, 300.5 nM, and 230.4 nM, respectively. Western blot results showed that NO4 increased Ac-H3 levels and decreased p-STAT3 levels.

[0076] The procedure for in vitro HDAC enzyme activity assay is as follows:

[0077] The assay was performed in a black 96-well plate with a final volume of 80 µL, containing HDAC assay buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM MgCl2), recombinant HDAC enzyme (final concentration 0.1 µg / µL), acetylated H4K16 peptide (100 µM), and the test compound.

[0078] For initial screening, compounds N01-N06 and SAHA were tested at a final concentration of 10 µM, with DMSO as a solvent control. After incubation at 37°C for 30–60 minutes, the reaction was terminated by adding the chromogenic solution, followed by incubation at room temperature for 10–15 minutes. Fluorescence intensity was measured using a microplate reader at excitation wavelengths of 360 nm and emission wavelengths of 460 nm. Enzyme activity was normalized to the solvent control, and inhibition rate was calculated accordingly.

[0079] For compounds showing >50% inhibition at 10 µM, dose-response studies were conducted against HDAC1, HDAC3, and HDAC6 to determine the IC50. 50The values ​​were calculated using nonlinear regression analysis with GraphPad Prism 9.5. Compounds were serially diluted six times to a concentration starting at 10,000 nM and determined under identical conditions. 50 Values. All measurements were repeated in at least three independent experiments.

[0080] Example 6: Functional Experiments and ADME-Related Evaluation

[0081] Compound NO4 exhibited significant functional antitumor activity in clonogenic, transwell migration, and Annexin V / PI apoptosis assays; SPR analysis revealed a KD of 5.816 × 10⁻⁶ for NO4 and STAT3. -6 M. Caco-2 results suggest its transmembrane transport tendency is superior to niclosamide; in rat liver microsomes, compound NO4's t 1 / 2 The time to reach the target was approximately 48.6 min, and Clint was approximately 28.53 μL / min / mg microsomal protein. In the HCT116 3D tumor sphere model, NO4 also showed a sustained inhibitory effect.

[0082] The clone formation experiment process is as follows:

[0083] Cells were seeded into six-well plates at a density of 500 cells per well and allowed to adhere overnight. The cells were then treated with compounds NO4 (1 µM), niclosamide (1 µM), SAHA (1 µM), niclosamide plus SAHA (1 µM each), or DMSO (solvent control). The culture medium containing the appropriate compound was changed every 3 days.

[0084] After 10 days of incubation, colonies were fixed with 4% paraformaldehyde for 15 minutes and stained with 0.1% crystal violet for 10 minutes. Colonies with a diameter greater than 50 µm were manually counted under a microscope. Each treatment was tested in triplicate in at least three independent experiments.

[0085] The Transwell migration experiment process is as follows:

[0086] Cell migration was assessed using a Transwell assay in HCT116 cells. In short, 5 × 10⁶ cells were used... 4 Cells suspended in serum-free DMEM were seeded into the upper chamber of a 24-well Transwell chamber (8 µm pore size, Corning, USA), while the lower chamber was filled with pure DMEM containing 10% FBS as a chemical inducer.

[0087] After incubation at 37°C and 5% CO2 for 48 hours, unmigrated cells on the upper surface of the membrane were wiped off with a cotton swab. Cells that migrated to the lower surface were fixed with 4% paraformaldehyde for 15 minutes and stained with 0.1% crystal violet for 10 minutes. Representative images were taken under a microscope at 20x magnification. Migrating cells were counted in five randomly selected fields of view for each well. Treatment groups included DMSO, niclosamide (1 µM), SAHA (1 µM), niclosamide plus SAHA (1 µM each), and NO4 (1 µM). Data are presented as mean ± standard deviation from three independent experiments.

[0088] The apoptosis experiment procedure is as follows:

[0089] The apoptosis induced by compound N04 in HCT116 cells was analyzed using Annexin V-FITC / PI double staining combined with flow cytometry. Cells were treated with N04 at 1 or 2 µM for 48 hours. As a comparison, cells were also treated with niclosamide (1 µM), SAHA (1 µM), or niclosamide plus SAHA (1 µM each). DMSO was used as a solvent control. After treatment, cells were collected, washed twice with cold PBS, and analyzed by 1×10⁻⁶ cells. 6 Resuspend the cells at a density of 100 cells / mL in binding buffer. Incubate 100 µL of the cell suspension with 5 µL Annexin V-FITC and 5 µL PI at room temperature in the dark for 15 minutes. Then, add 400 µL of binding buffer, and analyze the samples using a BD FACSCanto II flow cytometer (BD Biosciences, USA). Data were processed using FlowJo software (version X). Total apoptosis rate was calculated from the Annexin V-positive cell population.

[0090] Table 2. Representative activity data of compound NO4

[0091]

[0092] In summary, this invention provides a class of niclosamide-derived hydroxamic acid compounds that achieve dual regulation of the STAT3 and HDAC pathways by introducing hydroxamic acid linkers of different lengths and topologies onto the niclosamide core.

[0093] The compounds, especially the preferred compound NO4, possess good anti-tumor cell activity, HDAC1 / 6 inhibitory activity, and STAT3 regulatory ability. They also exhibit sustained functional anti-tumor effects in migration, apoptosis, colony formation, and 3D tumor sphere experiments, thus serving as candidate scaffolds for developing novel anti-solid tumor drugs.

Claims

1. A chloronitrosamine-derived hydroxyoxime acid compound, characterized in that, The compound of formula (I) or a pharmaceutically acceptable salt, solvate or hydrate thereof; ； Linker is selected from C2-C10 alkylene groups or C2-C10 alkylene groups separated by one or more of NH, O, S, and carbonyl groups.

2. The chloronitrosamine-derived hydroxyoxime acid compound according to claim 1, characterized in that, Compounds NO1-NO6 or their pharmaceutically acceptable salts, solvates or hydrates; The structural formulas of compounds N01~N06 are as follows: 、 、 。 3. The chloronitrosamine-derived hydroxyoxime acid compound according to claim 1 or 2, characterized in that, The pharmaceutically acceptable salt is an acid addition salt or an alkali metal salt.

4. A pharmaceutical composition, characterized in that, Includes the niclosamide-derived hydroxamic acid compounds according to any one of claims 1-3, and one or more pharmaceutically acceptable carriers; The carrier is a diluent, excipient, stabilizer, disintegrant, adhesive, lubricant, cosolvent, or sustained-release excipient.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition is an oral or injectable formulation.

6. Use of a niclosamide-derived hydroxyoxime compound as described in any one of claims 1-3 or a pharmaceutical composition as described in claim 4 or 5 in the preparation of an antitumor drug; The antitumor drug is used for the prevention and / or treatment of tumors; The active ingredient contained in the drug is a chloronitrosamine-derived hydroxyoxime compound as described in any one of claims 1-3.

7. The use according to claim 6, characterized in that, The tumor is a solid tumor; The solid tumors include one or more of the following: breast cancer, colorectal cancer, stomach cancer, lung cancer, liver cancer, pancreatic cancer, ovarian cancer, prostate cancer, glioma, and melanoma.

8. The use according to claim 6 or 7, characterized in that, The antitumor drug is a drug used to inhibit STAT3 phosphorylation and / or inhibit HDAC enzyme activity; The inhibition of HDAC enzyme activity is the inhibition of HDAC1 and / or HDAC6.

9. The use according to claim 6 or 7, characterized in that, The antitumor drug is used to increase Ac-H3 levels, induce tumor cell apoptosis, inhibit tumor cell migration, inhibit clonogenicity, and / or inhibit the growth of 3D tumor spheres.

10. A method for preparing the chloronitrosamine-derived hydroxyoxime acid compound according to any one of claims 1-3, characterized in that, Includes the following steps: Starting with 5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide, a linker precursor was introduced via O-alkylation, followed by the construction of a hydroxyxamic acid terminal group through hydroxylamine conversion or protection / deprotection reaction to obtain the chloronitrosamine-derived hydroxyxamic acid compound. The O-alkylation is introduced into the linker precursor using a haloester, haloacid, Boc-protected amino alcohol derivative, or Trt-protected hydroxyoxime acid halide. The step of constructing the terminal group of the hydroxamic acid involves reacting hydroxylamine or its salt with an ester precursor, or coupling a Trt-protected hydroxamic acid precursor followed by acidic deprotection.

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