Use of a benzimidazole compound in the preparation of an anti-leukemia drug

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a benzimidazole compound in preparation of an anti-leukemia drug. The compound can target inhibition of interaction between macrophage nuclear receptor coactivator 4 and ferritin heavy chain, inhibition of iron autophagy, thereby inhibition of proliferation of tumor cells, particularly significant inhibition effect on leukemia cells, and significant anti-leukemia effect, which provides more choices for medical research and clinical application.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to the application of a benzimidazole compound in the preparation of an anti-leukemia drug. Background Technology

[0002] Malignant tumors are the leading cause of death worldwide. In recent years, the overall incidence and mortality rates of cancer in my country have been on a continuous upward trend, having a serious impact on society. Statistics show that the incidence of malignant tumors increases by 3.9% and the mortality rate increases by 2.5% annually. Although the survival rate of patients with common types of malignant tumors has improved with social development and advancements in drug research, malignant tumors remain a major challenge to human health.

[0003] Hematologic malignancies are a large category of malignant tumors caused by the malignant proliferation of cells in the hematopoietic system. Leukemia, lymphoma, and multiple myeloma are common types of hematologic malignancies. The classification of hematologic malignancies is complex, and most are accompanied by genetic alterations, increasing the difficulty of diagnosis and treatment. Acute myeloid leukemia (AML) is an acquired, clonal hematologic malignancy caused by abnormal development of hematopoietic stem cells or progenitor cells. It is one of the most common and aggressive hematologic malignancies, and its incidence increases with age. Standard treatment for AML includes intensive induction chemotherapy followed by consolidation chemotherapy and allogeneic stem cell suppression, or both. However, in clinical practice, due to various reasons, patients with myeloid hematologic malignancies have low induction remission rates, high mortality rates, and poor prognoses. To provide more drugs for treating leukemia, researchers have developed numerous compounds with leukemic activity. For example, Chinese patent application CN111346082A discloses the application of a benzoic acid compound in the preparation of a drug for treating acute leukemia. This compound can be used as an AKR1C3 inhibitor to treat acute myeloid leukemia and T-cell acute lymphoblastic leukemia, exhibiting good inhibitory effects on AKR1C3 and possessing advantages such as low toxicity and good drug-likeness. However, the number of drugs available for clinical treatment of leukemia remains limited, and there is an urgent need to provide more anti-leukemia drugs to offer more options for medical research and clinical application. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the limitations and deficiencies of existing anti-leukemia drugs and to provide an application of benzimidazole compounds in the preparation of anti-leukemia drugs.

[0005] The above-mentioned objective of this invention is achieved through the following technical solution:

[0006] The use of a benzimidazole compound in the preparation of an anti-leukemia drug, wherein the benzimidazole compound has the structure of formula (I):

[0007]

[0008] Where R1 is R 11 It can be monosubstituted or polysubstituted, selected from hydrogen, halogen, C 1～6 Alkyl, C 1～6 Alkoxy, C 1～6 One or more of alkyl acyl, amino, and hydroxyl groups;

[0009] R2 is R 21 It may be mono- or poly-substituted, selected from one or more of hydrogen, halogen, unsubstituted or substituted piperazine, indole, and morpholine, wherein the substituent of the substituted piperazine is C. 1～6 Alkyl, halogen, C 1～6 alkoxy, hydroxy, or amino groups;

[0010] X is a C or N atom.

[0011] Iron is an essential element for cell proliferation and plays a crucial role in the survival and invasion of cancer cells. In the blood, iron ions bind to ferrotransferrin and are transported to ferrotransferrin receptors on the cell surface. Excess iron ions are stored in ferritin to avoid toxic side effects. Nuclear receptor coactivator 4 (NCOA4) is a newly discovered autophagy receptor that mediates ferritin autophagy. Ferritin heavy chains bind to NCOA4, enter lysosomes, and are degraded by lysosomal hydrolases, releasing the stored iron ions to meet the cell's needs for growth and reproduction. Knocking out NCOA4 inhibits ferritin degradation, reduces intracellular ferrous ion levels, and suppresses lipid peroxidation and ferroptosis. Overexpression of NCOA4 promotes ferritin degradation, increases intracellular ferrous ion levels, and increases cellular sensitivity to ferroptosis.

[0012] This invention discovered that one of the main causes of chemotherapy resistance in AML is the existence of a group of quiescent leukemia stem cells (QLSCs) within leukemia stem cells (LSCs). QLSCs possess the ability to self-renew and differentiate into various leukemia cells. They can remain in a quiescent state after chemotherapy to avoid responding to anticancer drugs and survive, thus re-proliferating malignantly upon disease relapse. This state of QLSCs relies on ferritin autophagy to provide iron ions for survival. Inhibiting ferritin autophagy can kill QLSCs, thereby achieving a therapeutic effect on drug-resistant AML. Given the high expression of NCOA4 in acute myeloid leukemia stem cells, NCOA4 inhibitors can selectively kill drug-resistant LSCs without harming normal hematopoietic stem cells, making them a lead compound for combating drug-resistant acute myeloid leukemia stem cells.

[0013] The benzimidazole compounds provided by this invention can target and inhibit the interaction between macrophage nuclear receptor coactivator 4 and ferritin heavy chain, inhibit ferrophagy, thereby inhibiting the proliferation of tumor cells and achieving a significant therapeutic effect on tumors, especially leukemia.

[0014] Furthermore, the R 11 It is hydrogen or halogen; R 21 The substituted piperazine has a C substituent. 1～3 Alkyl group; X is a C or N atom.

[0015] Furthermore, the R 11 It is fluorine; R 21 The substituted piperazine has a methyl group as its substituent and X is a C atom.

[0016] Preferably, the benzimidazole compound has the following structure:

[0017]

[0018] Furthermore, the benzimidazole compounds may also be pharmaceutically acceptable salts, solvates, or chiral isomers thereof.

[0019] Furthermore, the pharmaceutically acceptable salt is obtained by reacting benzimidazole compounds with inorganic or organic acids.

[0020] Furthermore, the inorganic acid is hydrochloric acid, phosphoric acid, sulfuric acid, or nitric acid.

[0021] Furthermore, the organic acid is citric acid, acetic acid, oxalic acid, isonicotinic acid, lactic acid, salicylic acid, acidic citric acid, tartaric acid, oleic acid, tannic acid, pantothenic acid, tartaric acid, ascorbic acid, succinic acid, maleic acid, gentic acid, fumaric acid, gluconic acid, glucuronic acid, glycolic acid, formic acid, benzoic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or dihydroxynaphthyl acid.

[0022] Furthermore, the leukemia described is acute myeloid leukemia.

[0023] Furthermore, the drug is an oral, injectable, topical, or inhaled formulation.

[0024] Preferably, the oral medication is a capsule, tablet, pill, granule, etc.; the inhalant is a spray.

[0025] The present invention has the following beneficial effects:

[0026] This invention provides a benzimidazole compound that can target and inhibit the interaction between macrophage nuclear receptor coactivator 4 and ferritin heavy chain, inhibiting ferrophagy and thus inhibiting the proliferation of tumor cells. In particular, it has a significant inhibitory effect on leukemia cells and has a significant anti-leukemia effect, providing more options for medical research and clinical application. Attached Figure Description

[0027] Figure 1 This is a statistical graph showing the effect of compound 7 on the iron ion concentration in AML cells in Experiment Example 1.

[0028] Figure 2 This is a statistical graph showing the effect of compound 7 on AML cell clone formation in Experiment Example 1.

[0029] Figure 3 This is a statistical graph showing the effect of compound 7 on tumor stem cells in AML transplanted mice in Experiment Example 2. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0031] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0032] Example 1: Preparation of benzimidazole compound 7

[0033] The synthetic route for the benzimidazole compound 7 is as follows:

[0034]

[0035] Specifically, the following steps are included:

[0036] Preparation of S1 and intermediate 2:

[0037] 2-Nitro-5-chloroaniline (997.6 mg, 5.8 mmol), N-methylpiperazine (6.9 mmol), and potassium carbonate (1.28 g, 9.3 mmol) were added sequentially to a pressure-resistant tube, followed by the addition of dried N,N-dimethylformamide (10 mL). The mixture was heated in an oil bath to 110 °C and magnetically stirred for 16 hours. After the reaction was completed, an appropriate amount of water was added to the reaction system, and the mixture was extracted with ethyl acetate (3 × 150 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The solid obtained by rotary evaporation was purified by column chromatography (dichloromethane:ethyl acetate = 2:1) to obtain a yellow solid, which was compound 1 (0.97 g, 71%).

[0038] Compound 1 (5.0 mmol) was dissolved in methanol (30 mL), hydrazine hydrate (5 mL) and a catalytic amount of nickel were added, and the mixture was magnetically stirred at 60 °C for 2 hours. After the reaction was completed, the mixture was cooled to room temperature, filtered, and the filtrate was concentrated to give intermediate 2 (black solid, 90-95%).

[0039] Preparation of S2 and Compound 7:

[0040] 5-Fluoro-1-indanone (3.99 g, 26.6 mmol) was dissolved in a mixture of 40 mL dichloromethane and 40 mL methanesulfonic acid. After cooling to 0 °C, sodium azide (3.46 g, 53.2 mmol) was slowly added to the reaction flask. The mixture was stirred overnight at room temperature. After the reaction was completed, 20% sodium hydroxide solution was slowly added under ice bath to adjust the pH to neutral. The mixture was then extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The solution was purified by column chromatography (petroleum ether: ethyl acetate = 1:2) to give a white solid, which was intermediate 3 (2.67 g, 61%).

[0041] Intermediate 3 (1.65 g, 10 mmol) was dissolved in dry tetrahydrofuran (5 mL), and a tetrahydrofuran suspension of 1 mol / L lithium aluminum hydride (20 mL) was slowly added at 0 °C. The mixture was refluxed for 4 hours. After the reaction was completed, the reaction was quenched with 30% sodium hydroxide solution in an ice bath. The mixture was filtered through diatomaceous earth and washed with methanol. The filtrate was concentrated under reduced pressure and then purified by column chromatography (ethyl acetate:methanol = 10:1) to give a white oily substance, compound 4 (0.82 g, 51%).

[0042] Compound 4 (30 mmol), ethyl 3-iodobenzoate (5.52 g, 20 mmol), potassium carbonate (8.34 g, 60 mmol), cuprous iodide (0.8 g, 4 mmol), and L-proline (0.92 g, 8 mmol) were dissolved in dimethyl sulfoxide (25 mL). The mixture was heated in an oil bath to 80 °C under nitrogen protection and magnetically stirred for 24 hours. After the reaction was complete, an appropriate amount of ice-water mixture was added to the reaction system, followed by extraction with ethyl acetate (3 × 150 mL). The organic layer was concentrated under reduced pressure, and the residue was subjected to column chromatography. Compound 7a (5.65 g, 63%) was obtained by chromatography (petroleum ether: ethyl acetate = 5:1). Intermediate 5 (10 mmol) was added to 50 mL of tetrahydrofuran, followed by the addition of lithium hydroxide (1.19 g, 50 mmol) dissolved in ethanol:water (5:1, 5 mL). The reaction was carried out at 90 °C for 3 hours. After the reaction was completed, the reaction solution was evaporated to dryness under vacuum, dissolved in a small amount of ice water, and then the pH was slowly adjusted to neutral with dilute hydrochloric acid under ice bath until a solid precipitated. The precipitated solid was filtered and dried to obtain compound 6.

[0043] Compound 6 (1 mmol) was dissolved in 5 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (0.21 mL, 1.2 mmol) and O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (385 mg, 1.2 mmol) were added under ice bath conditions. After stirring for 30 minutes, compound 2a (1.1 mmol) was added. The reaction was carried out at room temperature for 6 hours, and then the reaction was quenched with ice water. The mixture was extracted with ethyl acetate, and the organic layer was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure. The solid obtained by evaporation was dissolved in 10 mL of glacial acetic acid and refluxed overnight. After the reaction was completed, the mixture was cooled to room temperature, and the pH was adjusted to neutral with saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate, and the organic layer was dried over reduced pressure and evaporated to dryness under reduced pressure. The mixture was purified by column chromatography to obtain the corresponding compound 7 (yellow solid, 48%).

[0044] Compound 7 was structurally characterized, and the characterization data are as follows:

[0045] 1 H NMR (400MHz, CD3OD) δ7.65(t,J=1.8Hz,1H),7.42(d,J=8.8Hz,1H),7.37(m,1H),7.31(t,J=7.9Hz,1H),7.15(dd,J=9.4,5.6Hz,1H),7.07 -7.03(m,2H),6.98(dd,J=8.8,2.2Hz,1H),6.87-6.80(m,2H),4.39(s,2H),3.57(t,J=6.0Hz,2H),2.94(m,6H),2.85(s,4H),2.48(s,3H).

[0046] 13 C NMR (100MHz, CD3OD) δ162.8(d,J=242.8Hz),153.7,152.3,149.1,138.4(d,J=7.7Hz),131.7,131.4(d,J=2.6Hz),130.9(2C),130.6,129.3(d ,J=8.1Hz),117.9,117.7,116.7,115.6(d,J=21.1Hz),114.1(2C),114 .0(d,J=21.8Hz),102.7,55.7(2C),51.0,50.9,47.3,45.1,43.5,30.0.

[0047] HRMS(ESI):calcd for C 27 H 28 N5F[M+H] + 442.2402, found 442.2412.

[0048] Example 2 Injection

[0049] Compound 7 prepared in Example 1 was dissolved in a small amount of DMSO, and then mixed with water for injection according to standard procedures. After fine filtration, it was filled, sealed, and sterilized to prepare an injection solution.

[0050] Example 3 Tablets

[0051] Compound 7 prepared in Example 1 was granulated and compressed into tablets with cyclodextrin at a weight ratio of 6:1 to obtain tablets.

[0052] Example 4 Capsules

[0053] Compound 7 prepared in Example 1 was mixed with microcrystalline cellulose in a weight ratio of 6:1 to form capsules.

[0054] Inhibitory effect of compound 7 on acute leukemia cells in Experiment Example 1

[0055] 1. Experimental Methods

[0056] Cell Source: The original cells used in this study were derived from anonymized acute myeloid leukemia (AML) patients. These cells came from the Swiss HIMIP sample bank (BB-0033-0060), which had been formally registered with the Ministry of Higher Education and Research (DC 2008-307, collection number 1). Prior to obtaining the samples, an ethics committee approval and a signed transfer agreement (AC 2008-129) were obtained. To comply with ethical guidelines, all participants provided written informed consent authorizing the use of their samples for research purposes.

[0057] Cell culture: Cells were cultured at 37°C in a 5% CO2 incubator. Frozen samples were obtained from patients diagnosed with AML or normal CD34+ hematopoietic cells, thawed as quickly as possible in a 37°C water bath, and immediately placed in 20% fetal bovine serum-modified IMDM medium with 10 μg / mL DNASE I added. Next, the cells were centrifuged at 1500 rpm for 5 minutes and then resuspended in complete medium at a concentration of 2-5 × 10⁻⁵. 6 Cells / mL. Thawed cells were then cultured in complete medium: IMDM medium supplemented with 10% fetal bovine serum (FBS) and BIT (bovine serum albumin 4 g / L, insulin 5 μg / mL, transferrin 60 μg / mL, all from Sigma-Aldrich). To promote and maintain cell growth, specific cytokines and components were added: 50 ng / mL FLT3 ligand, 10 ng / mL IL-6, 50 ng / mL stem cell factor (SCF), 25 ng / mL thrombopoietin (TPO), 10 ng / mL IL-3, and 10 ng / mL granulocyte colony-forming factor (G-CSF). Additionally, the medium contained 50 μM β-mercaptoethanol (Sigma-Aldrich).

[0058] Intracellular Fe 2+ Scale assay: Cells were seeded in 96-well laser confocal microscopy plates, with 5000 cells per well containing 100 μL of culture medium. After overnight culture, the old medium was discarded, and the cells were treated with medium containing 0.5 μM compound 7 for 6 hours. A probe working solution containing 1 μM FerroOrange and 1 μg / mL Hoechst 33342 was prepared using serum-free medium and added to the 96-well plates. The cells were incubated at 37°C for 30 min, followed by washing with 1×PBS. Cell analysis was performed using an FV3000 laser confocal microscope or a Cellmics ArrayScan Vti high-content system.

[0059] L-CFU method for detecting colony formation: AML cells were used at a concentration of 1×10⁻⁶ 5 L-CFU (cell colonies) were seeded at 10% concentration in H4230 medium supplemented with: IMDM, 50 ng / mL FLT3 ligand, 10 ng / mL IL-6, 50 ng / mL stem cell factor (SCF), 25 ng / mL thrombopoietin (TPO), 10 ng / mL IL-3, and 10 ng / mL granulocyte colony-forming factor (G-CSF). On day 7, L-CFU (>10 cell colonies) were counted using an inverted microscope.

[0060] 2. Experimental Results

[0061] After incubation with cells, compound 7 significantly reduced the concentration of ferrous ions in AML cells. Figure 1 Further L-CFU analysis showed that compound 7 was able to reduce AML cell colony formation in a dose-dependent manner (1 μM and 5 μM). Figure 2 ).

[0062] The above results indicate that compound 7 can bind to NCOA4, inhibit ferrophagy, thereby eliminating AML stem cells and inhibiting AML proliferation.

[0063] Inhibitory effect of compound 7 on AML transplanted mice in Experiment Example 2

[0064] 1. Experimental Methods

[0065] To establish a humanized mouse model for studying AML, 6-8 week old adult NOD / scidgamma-null (NSG) mice were used. Mice were treated with busulfan (Busilvex, Sigma-Aldrich) via intraperitoneal injection. The day after busulfan treatment, live primary human AML cells obtained from patients were injected into the tail vein of the mice, with cell numbers ranging from 0.5 to 2 × 10⁻⁶ cells. 6 To assess the engraftment of human AML cells into the bone marrow hematopoietic environment of mice, mice were monitored for different time periods, typically between 10 and 16 weeks. At the designated endpoint, mice were euthanized, and the presence of surviving human CD45+CD33+ cells in the bone marrow was quantitatively detected using flow cytometry. Twelve weeks after AML modeling in mice, mice were treated with placebo and compound 7 for five consecutive days, and the number of stem cells in AML cells was measured in the mice.

[0066] 2. Experimental Results:

[0067] The number of CD34+CD38- cells is a marker of tumor stem cell production and is often used to evaluate the generation of tumor stem cells. Results are as follows... Figure 3 As shown, the experimental results indicate that compound 7 treatment significantly reduced CD34+CD38- cells compared to the placebo group.

[0068] The above results indicate that compound 7 can inhibit the proliferation of AML tumor stem cells in vivo, thereby inhibiting AML.

[0069] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of a benzimidazole compound in the preparation of an anti-leukemia drug, characterized in that, The benzimidazole compounds have the following structures: ； The leukemia mentioned is acute myeloid leukemia.

2. The application according to claim 1, characterized in that, The benzimidazole compounds can also be their pharmaceutically acceptable salts.

3. The application according to claim 2, characterized in that, The pharmaceutically acceptable salt is obtained by reacting benzimidazole compounds with inorganic or organic acids.

4. The application according to claim 3, characterized in that, The inorganic acid is hydrochloric acid, phosphoric acid, sulfuric acid, or nitric acid.

5. The application according to claim 3, characterized in that, The organic acids are citric acid, acetic acid, oxalic acid, isonicotinic acid, lactic acid, salicylic acid, acidic citric acid, tartaric acid, oleic acid, tannic acid, pantothenic acid, ascorbic acid, succinic acid, maleic acid, gentian acid, fumaric acid, gluconic acid, glucuronic acid, succinic acid, formic acid, benzoic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or dihydroxynaphthalic acid.

6. The application according to claim 2, characterized in that, The drug is an oral, injectable, topical, or inhaled preparation.

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