Artemisinin analogue compound containing hydroxamic acid structure or medicinal salt thereof as well as preparation method and application of artemisinin analogue compound

Abstract

The invention provides an artemisinin analogue compound containing a hydroxamic acid structure or a medicinal salt thereof as well as a preparation method and application of the artemisinin analogue compound. The structural formula of the artemisinin analogue compound is shown as a formula I in the specification. The compound has a brand new skeleton structure and excellent anti-tumor activity, and can be used for preparing anti-tumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a class of artemisinin-like compounds containing isohydroxamic acid structures or their pharmaceutically acceptable salts, their preparation methods, and their application in the preparation of antitumor drugs. Background Technology

[0002] Artemisinin and its derivatives are sesquiterpene lactone compounds with unique chemical structures, derived from the plant *Artemisia annua* L. Since its discovery, it has become one of the most important and effective antimalarial drugs. Modern pharmacological studies have not only confirmed the significant antimalarial effects of artemisinin and its derivatives (ARTs), but have also found that these drugs exhibit a variety of pharmacological activities, including anti-inflammatory, antiviral, and immunomodulatory effects, with good clinical safety.

[0003] Cancer is a leading cause of death worldwide, posing a serious threat to human life. Artemisinin has demonstrated potent anti-cancer activity in various cancers, including breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and lung cancer. Artemisinin acts on cancer cells through multiple pathways, such as inducing apoptosis, and by generating reactive oxygen species, leading to ferroptosis and cell cycle arrest. Therefore, artemisinin can target multiple sites and affect multiple signaling pathways. Furthermore, artemisinin is known to have good tolerability and safety at low doses, reducing the risk of intolerable toxicity. Therefore, artemisinin holds great potential as an anti-cancer drug.

[0004] Histones are the main structural proteins of eukaryotic chromatin. Histone deacetylation is an epigenetic mechanism regulating gene expression, catalyzed by histone deacetylases (HDACs). Disorders of HDAC gene expression and mutations lead to abnormal transcription of key genes regulating important cancer pathways such as cell proliferation, cell cycle, and apoptosis. Given their role in cancer pathways, histone deacetylase inhibitors (HDACIs) are considered promising therapeutic drugs, and their development is a hot topic in the search for new anticancer drugs. Currently, six HDAC inhibitors—vorinostat (SAHA), romidepsin, belinostat, panobinostat, chidamide, and givinostat—have been approved for clinical use, but they still face challenges such as low selectivity and limited efficacy against solid tumors.

[0005] A literature report (Pharmaceuticals 2022, 15, 333) describes how researchers designed and synthesized a series of histone deacetylase inhibitors containing the artemisinin structural backbone. While these compounds exhibited some antitumor activity by combining the peroxy-bridged structure of artemisinin with the pharmacophore of HDAC inhibitors, in vitro activity evaluations showed that the inhibitory efficacy of some compounds was lower than that of the marketed drug vorinostat (SAHA). Summary of the Invention

[0006] To address the problems of the prior art, this invention provides a class of artemisinin analogs containing isohydroxamic acid structures or pharmaceutically acceptable salts thereof, as well as their preparation methods and applications. These compounds possess novel skeletal structures and excellent antitumor activity, and can be used to prepare antitumor drugs.

[0007] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a class of artemisinin analog compounds containing an isohydroxamic acid structure or pharmaceutically acceptable salts thereof, the structural formula of which is shown in Formula I:

[0008] Where X is selected from -CH2- and carbonyl groups; Y is selected from C atoms and N atoms.

[0009] In some preferred embodiments of the present invention, X is -CH2-.

[0010] In some preferred embodiments of the present invention, Y represents N atoms.

[0011] Preferably, the artemisinin-like compound containing an isohydroxamic acid structure is selected from one of the following compounds: .

[0012] Furthermore, the artemisinin-like compound containing an isohydroxamic acid structure is selected from one of the following compounds: .

[0013] A second aspect of the present invention provides a method for preparing artemisinin analog compounds containing an isohydroxamic acid structure or pharmaceutically acceptable salts thereof, the method for preparing the artemisinin analog compounds comprising the following steps: S1, Dihydroartemisinin (DHA) is reacted with boron trifluoride diethyl ether, the product is reacted with a bromine-containing carbon tetrachloride solution, and then reacted with triethylamine to obtain intermediate 1-2; Alternatively, DHA is reacted with boron trifluoride diethyl ether, the resulting product is reacted with a bromine-containing carbon tetrachloride solution, and then reacted with triethylamine to obtain intermediate 1-2; intermediate 1-2 is reacted with aminosulfonic acid and sodium chlorite to obtain intermediate 2-1. S2, react intermediate 1-2 with intermediate M1, AcOH and NaHB(OAc)3 to give intermediate M2 with X as -CH2-; or react intermediate 2-1 with intermediate M1, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) to give intermediate M2 with X as -C=O; S3, react intermediate M2 with LiOH to obtain intermediate M3; S4, intermediate M3 is reacted with tetrahydropyranylhydroxylamine (THPONH2), N-hydroxy-7-azabenzotriazole (HOAT) and EDCI, and the resulting product is reacted with benzoyl chloride to give an artemisinin-like compound containing an isohydroxamic acid structure; .

[0014] A third aspect of the present invention provides the use of a class of artemisinin analogs containing an isohydroxamic acid structure or pharmaceutically acceptable salts thereof in the preparation of antitumor drugs.

[0015] Preferably, the tumor is selected from malignant solid tumors such as lung cancer, colon cancer, and liver cancer, and hematologic tumors such as leukemia.

[0016] In a fourth aspect, the present invention provides the use of a class of artemisinin analogs containing an isohydroxamic acid structure or pharmaceutically acceptable salts thereof in the preparation of histone deacetylase inhibitors.

[0017] Compared with the prior art, the present invention has the following beneficial effects: Based on the structure of artemisinin analogs, this invention designs and synthesizes a series of novel artemisinin analog-HDAC inhibitor hybrid compounds containing isohydroxamic acid structures. Research results show that the compounds of this invention exhibit excellent antitumor activity, demonstrating superior inhibitory effects on the proliferation of lung cancer cells A549, colon cancer cells HCT116, liver cancer cells HepG2, and leukemia cells MV4-11 and HL60. Some compounds show superior antitumor activity compared to the marketed drug SAHA; for example, compounds 1 and 4 showed better antitumor activity against the IC50 of three tested solid tumor cell lines. 50 All concentrations were below 3 μM, exhibiting extremely excellent and broad-spectrum antitumor activity. Compound 1 showed the best antitumor activity, as evidenced by its high IC50 concentration against three solid tumor cell lines. 50 The IC50 values ​​were 0.84 μM, 0.93 μM, and 0.33 μM, respectively, for the two leukemia cell lines. 50The concentrations were 96 nM and 220 nM, respectively, exhibiting excellent antitumor activity, significantly superior to SAHA, and with an IC50 of 96 nM against HepG2 cells. 50 The value was reduced by half compared to SAHA, and the IC50 value for leukemia cells MV4-11 and HL60 was [missing information]. 50 The values ​​were reduced by 2- and 3-fold, respectively, compared to SAHA, providing important evidence for the development of next-generation antitumor drugs based on the artemisinin structure. Furthermore, compounds 1 and 4 showed superior inhibitory selectivity against 293T cells compared to SAHA, indicating that these compounds may have lower toxicity than SAHA. The compounds provided by this invention not only exhibit excellent antitumor activity but also show improved selectivity against different HDAC subtypes, demonstrating promising development prospects. In particular, the existing clinical safety data for artemisinin-based drugs provides a unique advantage for their translational application in the antitumor field.

[0018] The method for preparing artemisinin-like compounds provided by this invention has the advantages of fewer reaction steps, easy availability of raw materials, and short synthesis cycle. Detailed Implementation

[0019] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0020] Unless otherwise specified, the experimental methods described in the following examples are generally performed under standard conditions or as recommended by the manufacturer.

[0021] The chemical structural formulas of the compounds prepared in the following examples are as follows: 1 Detailed H-NMR and HRMS data are shown in Table 1, where numbers 1 to 4 correspond to compounds 1 to 4 prepared in Examples 1 to 4, respectively.

[0022] Table 1. Chemical structural formulas of compounds 1-4 1 H-NMR and HRMS data

[0023] Example 1 Synthesis of Compound 1

[0024] Reaction 1: DHA, i.e., intermediate 1-1 (850 mg, 3.0 mmol), was dissolved in 10 mL of diethyl ether. 1 mL of boron trifluoride diethyl ether was added dropwise at 0 °C, and the reaction was allowed to proceed for 4 h. The mixture was washed with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane (DCM), and the organic phase was evaporated under reduced pressure to dryness. The mixture was purified by column chromatography to obtain a white solid. 400 mg of the obtained white solid was added to 20 mL of carbon tetrachloride. At 0 °C, 2 mL of a bromine-containing carbon tetrachloride solution (bromine water:carbon tetrachloride = 1:20, v / v) was added dropwise. After stirring for 30 min, 5 mL of pure water was added, and the mixture was stirred at room temperature for 1 h. When a yellow solid was observed to precipitate, the solvent was evaporated under reduced pressure to obtain the yellow solid. The yellow solid (300 mg) was dissolved in dichloromethane, and triethylamine (300 μL) was added. The mixture was reacted overnight at room temperature. After the reaction was completed, the solvent was evaporated to dryness, and the mixture was purified by column chromatography (volume ratio of petroleum ether: ethyl acetate = 10: 1) to obtain a pure white solid, which was intermediate 1-2, with a yield of 53%.

[0025]

[0026] Reaction 2: Intermediate 1-2 (120 mg, 0.42 mmol), intermediate 1-3 (39.2 mg, 0.28 mmol), AcOH (77 μL, 1.34 mmol), and NaHB(OAc)3 (212 mg, 1.0 mmol) were added to dichloroethane and stirred overnight at room temperature. After the reaction was complete, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 12:1, v / v) to give intermediate 1-4 as a white solid in 72% yield.

[0027]

[0028] Reaction 3: Intermediates 1-4 (45 mg, 0.11 mmol) and LiOH (11.6 mg, 0.28 mmol) were added to a mixed solvent system of tetrahydrofuran and water (volume ratio of tetrahydrofuran:water = 1:2) and stirred at room temperature for 2 h. After the reaction was completed, the solvent was evaporated under reduced pressure, and 2 mL of dichloromethane was added to redissolve the solvent. 3-4 drops of HOAc were added to the solution for acidification, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (volume ratio of dichloromethane:methanol = 8:1) to obtain intermediates 1-5, a white solid with a yield of 92%.

[0029]

[0030] Reaction 4: Intermediates 1-5 (32 mg, 0.08 mmol), THPONH2 (28.8 mg, 0.23 mmol), HOAT (15.8 mg, 0.12 mmol), and EDCI (22.3 mg, 0.12 mmol) were added to dichloromethane and stirred overnight at room temperature. After the reaction was complete, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 30:1, v / v). The obtained compound was added to 10 μL of benzoyl chloride in ethanol and stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 35:1) to give compound 1, a grayish-white solid, with a yield of 64%.

[0031] Example 2 Synthesis of Compound 2

[0032] Reaction 1: Intermediate 1-2 (245 mg, 0.87 mmol) was dissolved in 10 mL of dioxane, and aminosulfonic acid (340 mg, 3.55 mmol) and water (3.5 mL) were added. The mixture was stirred at 0 °C for 20 h. Sodium chlorite (320 mg, 3.55 mmol) and 2.5 mL of water were added, and the mixture was stirred at room temperature for 30 min. The reaction solution was evaporated to dryness, dissolved in dichloromethane, filtered, and the filtrate was evaporated to dryness. The solution was purified by column chromatography (dichloromethane:methanol = 100:1, v / v) to obtain a white solid, which was intermediate 2-1, with a yield of 43%.

[0033]

[0034] Reaction 2: Intermediate 2-1 (100 mg, 0.34 mmol), intermediate 1-3 (36.7 mg, 0.28 mmol), EDCI (96.4 mg, 0.5 mmol), and DMAP (4-dimethylaminopyridine, 6.8 mg, 0.06 mmol) were added to dichloromethane and stirred at room temperature for 6 h. After the reaction was complete, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 30:1, v / v) to give a pale yellow solid as intermediate 2-2, with a yield of 49%.

[0035]

[0036] Reaction 3: Intermediate 2-2 (45 mg, 0.11 mmol) and LiOH (11.6 mg, 0.28 mmol) were added to a mixed solvent system of tetrahydrofuran and water (volume ratio of tetrahydrofuran:H2O = 1:2) and stirred at room temperature for 2 h. After the reaction was completed, the solvent was evaporated under reduced pressure, and 2 mL of dichloromethane was added to redissolve the solvent. 3-4 drops of HOAc were added to the solution for acidification, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (volume ratio of dichloromethane:methanol = 8:1) to obtain intermediate 2-3, a pale yellow solid, with a yield of 88%.

[0037]

[0038] Reaction 4: Intermediates 2-3 (52 mg, 0.08 mmol), THPONH2 (28.8 mg, 0.23 mmol), HOAT (15.8 mg, 0.12 mmol), and EDCI (22.3 mg, 0.12 mmol) were added to dichloromethane and stirred overnight at room temperature. After the reaction was complete, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 30:1, v / v). The obtained compound was added to 10 μL of benzoyl chloride in ethanol and stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 35:1, v / v) to give compound 2, a pale yellow solid, with a yield of 51%.

[0039] Example 3 Synthesis of Compound 3

[0040] Referring to Example 1, intermediates 1-3 in reaction 2 were replaced with intermediate 3-1 to obtain intermediate 3-2; intermediates 1-4 in reaction 3 were replaced with intermediate 3-2 to obtain intermediate 3-3; intermediates 1-5 in reaction 4 were replaced with intermediate 3-3, and the reaction yielded compound 3, a yellow solid (36 mg, yield 31%).

[0041] Example 4 Synthesis of Compound 4

[0042] Referring to Example 1, intermediates 1-3 in reaction 2 were replaced with intermediate 4-1 to obtain intermediate 4-2; intermediates 1-4 in reaction 3 were replaced with intermediate 4-2 to obtain intermediate 4-3; intermediates 1-5 in reaction 4 were replaced with intermediate 4-3, and the reaction yielded compound 4, a brown solid (23.8 mg, yield 33%).

[0043] Example 5 Antitumor activity and toxicity tests of the compounds of this invention.

[0044] In the tumor cell proliferation inhibition experiment and in vitro toxicity assay of the compound of this invention, adherent cells such as lung cancer cells A549, colon cancer cells HCT116, and liver cancer cells HepG2 were tested using the CCK8 assay, while leukemia cells MV4-11 and HL60 were tested using the resazurin assay. Before the experiment, the control drug and the compound of this invention were prepared into a 10 mM stock solution using DMSO and stored at -20 °C. Cells in the exponential growth phase (lung cancer cells A549, colon cancer cells HCT116, and liver cancer cells HepG2) were prepared into a high-density suspension, and 100 μL of a 1×10⁻⁶ density solution was added to different wells of 96-well plates according to their different cell division and growth rates. 5 Cells were cultured at a concentration of 10 cells / mL. After 24 h of cell culture, the supernatant was aspirated from the wells. 200 μL of culture medium was added to each well in the blank control group, and 200 μL of drug-containing culture medium was added to each well in the experimental group. 200 μL of PBS was added to each well in the outermost wells of the 96-well plate. After incubation for 72 h, the supernatant was aspirated, and 100 μL of CCK8 diluent was added to each well. After incubation for 30 min, the plate was shaken for 20 s using a microplate reader, and the absorbance (OD) was measured at 450 nm to calculate the half-maximal inhibitory concentration (IC50). 50 .

[0045] In the in vitro proliferation inhibition experiment of two cell lines, human promyelocytic leukemia (HL-60) and human myeloid monocytic leukemia (MV4-11), the resazurin indicator method was used. Cells in the exponential growth phase were prepared into a high-density suspension, and 100 μL of a 1×10⁻⁶ m³ density solution was added to a dedicated black 96-well plate. 5 Cells were cultured at a concentration of 100 μL / mL. After 24 h of cell culture, 100 μL of culture medium was added to each well in the blank control group, and 100 μL of drug-containing culture medium (drug concentration started from 100 μM and diluted 3-fold) was added to each well in the experimental group. 200 μL of PBS was added to each of the outermost wells of the 96-well plate. Cells were incubated for 72 h. Then, 20 μL of resveratrol dilution was added, and the cells were incubated for another 4 h. After shaking for 20 s in a microplate reader, the fluorescence OD value was measured at 450 nm, and the half-maximal inhibitory concentration (IC50) was calculated. 50 .

[0046] Table 2. In vitro inhibitory activity (IC50) of compounds against solid tumor cells and hematological malignancies. 50 (μM)

[0047] Table 2 shows that the series of compounds designed and synthesized in this invention exhibited significant inhibitory effects on proliferation in in vitro antitumor activity screening, especially showing excellent inhibitory effects on the proliferation of three solid tumor cell lines: lung cancer A549, colon cancer HCT116, and liver cancer HepG2 (IC50, 110, 120, 130, 140, 150, 160, 160, 160, 160, 160, 170, 18 ... 50 All values ​​were less than 20 μM. Compound 1 exhibited the best antitumor activity, superior to the clinically used histone deacetylase inhibitor SAHA. Compound 1 showed an IC50 value against lung cancer cells A549, colon cancer cells HCT116, and liver cancer cells HepG2. 50 The concentrations reached 0.84±0.11 μM, 0.93±0.06 μM, and 0.33±0.10 μM, respectively; the IC50 of compound 4 was... 50 The specific values ​​were 1.16±0.16 μM, 0.76±0.12 μM, and 2.92±0.12 μM, respectively, for all ICs. 50 The values ​​were all below 3 μM. Furthermore, compounds 1 and 4 showed superior in vitro antitumor activity against leukemia cells MV4-11 and HL60 compared to SAHA and DHA, especially compound 1, which exhibited a significantly higher IC50 value against MV4-11 and HL60 leukemia cells. 50 The optimal activity was achieved at 96 nM and 220 nM, respectively.

[0048] The IC50 values ​​of the compounds listed in Table 2 were used to assess the effects on three solid tumor cell lines: lung cancer A549, colon cancer HCT116, and liver cancer HepG2, as well as leukemia cells MV4-11 and HL60. 50 The assay results screened out highly active antitumor compounds 1 and 4, and their cytotoxicity against human embryonic kidney endothelial cells (293T) was determined. The results are shown in Table 3. Compounds 1 and 4 showed in vitro inhibitory activity against human embryonic kidney endothelial cells (293T) above 10 μM, with selectivity superior to the marketed drug SAHA.

[0049] Table 3. Cytotoxicity of highly active compounds to 293T cells (IC50) 50 (μM)

[0050] Example 6 In vitro inhibitory activity assay of highly active compounds against HADC enzyme.

[0051] The enzymes, substrates, drugs, and developing agents required for the experiment were prepared using working solutions at the necessary concentrations. The experiment included a positive control group, a blank control group, an experimental group, and a background group. 25 μL of drug solution and 50 μL of enzyme solution were added sequentially to the experimental and positive control groups; 25 μL of working solution and 50 μL of enzyme solution were added to the blank control group wells; 75 μL of working solution was added to the background group wells, omitting both drug and enzyme solutions. After sample addition, the 96-well plate was placed in a microplate reader and shaken for 30 seconds to ensure thorough mixing, then incubated at 37 ℃ for 10 min. 25 μL of substrate solution was added to each well, mixed, and incubated at 37 ℃ for 60 min. Finally, 10 μL of developing agent was added to each well, mixed, and incubated at 37 ℃ for 5 min. The fluorescence OD value was then detected using a microplate reader at 380 / 500 nm wavelengths.

[0052] Table 4. Enzyme inhibitory activities of compound 1 and SAHA against different isoforms of HDACs (IC50) 50 , nM)

[0053] The in vitro half-maximal inhibitory concentrations (IC50) of compound 1 and the positive control drug SAHA against several different isotypes of HDAC enzymes are shown in Table 4. The IC50 of compound 1 against HDAC6 is also shown. 50 Its value is 24 nM, significantly higher than its inhibitory activity against several other HDAC enzymes, making it a selective inhibitor of HDAC6. Furthermore, its inhibitory activity against HDAC8 is similar to that of SAHA (IC). 50 = 1574±484 nM) is 14 times higher than that of SAHA. In addition, compound 1, like SAHA, has no inhibitory effect on SIRT2.

[0054] In summary, the compounds prepared in Examples 1 to 4 of this invention all possess excellent and broad-spectrum antitumor activity, and have the potential and value for in-depth research and development.

Claims

1. A class of artemisinin analogues or their pharmaceutically acceptable salts containing an isohydroxamic acid structure, characterized in that, The structural formula of the artemisinin-like compound is shown in Formula I: Where X is selected from -CH2- and carbonyl groups; Y is selected from C atoms and N atoms.

2. The artemisinin analogue compound containing an isohydroxamic acid structure or its pharmaceutically acceptable salt according to claim 1, characterized in that, X is -CH2-.

3. The artemisinin analogue compound containing an isohydroxamic acid structure or its pharmaceutically acceptable salt according to claim 1, characterized in that, Y represents an N atom.

4. The artemisinin analogue compound containing an isohydroxamic acid structure or its pharmaceutically acceptable salt according to claim 1, characterized in that, The artemisinin-like compound is one of the following compounds: 。 5. The artemisinin analogue compound containing an isohydroxamic acid structure or its pharmaceutically acceptable salt according to claim 4, characterized in that, The artemisinin-like compound is one of the following compounds: 。 6. The method for preparing artemisinin analogs containing an isohydroxamic acid structure or pharmaceutically acceptable salts thereof according to any one of claims 1 to 5, characterized in that, The method for preparing the artemisinin analogue includes the following steps: S1, DHA is reacted with boron trifluoride diethyl ether, the product is reacted with a bromine-containing carbon tetrachloride solution, and then reacted with triethylamine to obtain intermediate 1-2; Alternatively, DHA is reacted with boron trifluoride diethyl ether, the resulting product is reacted with a bromine-containing carbon tetrachloride solution, and then reacted with triethylamine to obtain intermediate 1-2; intermediate 1-2 is reacted with aminosulfonic acid and sodium chlorite to obtain intermediate 2-1. S2, react intermediate 1-2 with intermediate M1, AcOH and NaHB(OAc)3 to obtain intermediate M2 with X as -CH2-; or react intermediate 2-1 with intermediate M1, EDCI and DMAP to obtain intermediate M2 with X as carbonyl. S3, react intermediate M2 with LiOH to obtain intermediate M3; S4, intermediate M3 is reacted with THPONH2, HOAT and EDCI, and the resulting product is reacted with benzoyl chloride to give an artemisinin-like compound containing an isohydroxamic acid structure; 。 7. The use of the artemisinin analogue compound containing the isohydroxamic acid structure as described in any one of claims 1 to 5, or its pharmaceutically acceptable salt, in the preparation of antitumor drugs.

8. The application according to claim 7, characterized in that, The tumors were selected from lung cancer, colon cancer, liver cancer, and leukemia.

9. The use of the artemisinin analogue containing the isohydroxamic acid structure as described in any one of claims 1 to 5, or its pharmaceutically acceptable salt, in the preparation of histone deacetylase inhibitors.

Images