Benzimidazolone derivatives, processes for their preparation and antitumor use thereof

Abstract

The application belongs to the field of pharmaceutical chemistry, and particularly relates to a novel N-heterocyclic benzimidazolone derivative, a preparation method thereof and medical use. The benzimidazolone derivative has significant inhibitory effects on colorectal cancer, liver cancer, gastric cancer, pancreatic cancer, cervical cancer and ampullary cancer, has low cytotoxic side effects on normal intestinal epithelial cells and gastric mucosa cells, and has broad-spectrum solid tumor inhibitory activity. The anti-gastric cancer activity of the derivative 7a is superior to that of the clinical first-line drug 5-Fu, and the derivative 7a exhibits good in-vitro antitumor cytotoxicity on Caco-2 colorectal cancer cells, HepG-2 liver cancer cells, PANC-1 pancreatic cancer cells, Hela cervical cancer cells and DPC-X3 ampullary cancer cells, and can be used as a potential broad-spectrum antitumor candidate drug.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a class of N-heterocyclic systems with specific chemical structures possessing antitumor activity, namely N-alkylated benzimidazolone derivatives and their preparation methods and uses. Background Technology

[0002] A study published in *The Lancet* in September 2025 showed that there were approximately 18.5 million new cancer cases and 10.4 million cancer deaths globally in 2023. It is projected that by 2050, new cases will increase to 30.5 million and deaths to 18.6 million, representing increases of 60.7% and 74.5%, respectively. Faced with the increasingly heavy burden of cancer, traditional chemotherapy and radiotherapy, while effective, have significant toxic side effects and cause substantial damage to normal cells. New therapies are driving precision and diversification: monoclonal antibodies, antibody-drug conjugates (ADCs), cell therapy, and gene editing each have their own characteristics, but they generally face bottlenecks such as high cost, poor tissue penetration, off-target toxicity, low delivery efficiency, or poor efficacy in solid tumors. Against this backdrop, small molecule inhibitors exhibit irreplaceable core advantages: they can be administered orally, resulting in high patient compliance; their synthesis and production costs are relatively low, facilitating large-scale supply; their small molecular weight and strong penetrability allow them to act on multiple intracellular targets; and their rapid research and iteration speed allows for quick response to clinical drug resistance mutations. More importantly, small molecule drugs can precisely target tumor driver genes (such as EGFR, ALK, and BTK) and gradually overcome traditionally "difficult-to-drug" targets like KRAS through novel strategies such as covalent inhibition, allosteric regulation, and PROTAC degradation. Although small molecules also face challenges such as drug resistance and insufficient activity at some targets, their convenient administration methods, good tissue distribution, and mature production systems make them the cornerstone of combination therapy and long-term use. The continuous development of novel small molecule anti-tumor drugs is not only a key measure to meet the clinical needs of a broad range of patients and reduce medical costs, but also an important pillar for improving the accessibility and equity of global cancer prevention and control.

[0003] Benzimidazole is a bicyclic aromatic molecule formed by the fusion of a benzene ring and an imidazole ring at positions 4 and 5, with nitrogen atoms located at positions 1 and 3. Its derivatives have become important structural units in medicinal chemistry due to their wide range of pharmacological activities. Over the past few decades, the benzimazole core has attracted great interest from researchers and developed into an important research direction. Benzimidazole compounds possess a variety of pharmacological activities, including antiparasitic, antifungal, anticoagulant, anti-inflammatory, antihypertensive, anticonvulsant, antiulcer, antimalarial, antituberculosis, anti-HIV, antimicrobial, antiprotozoal, and antiviral effects. Notably, the NH group of benzimazole exhibits both weak basicity and strong acidity, a property that is helpful in the design of new drug molecules. Benzimidazole and its derivatives have also been reported to have analgesic effects. Representative drugs such as omeprazole (for treating peptic ulcers) and pimoben (for treating congestive heart failure) both contain this core. Since 1985, benzimidazole compounds have been identified as having anticancer activity, and some derivatives (such as Hoechst 33258 and Hoechst 33342) exert their effects by inhibiting topoisomerases. Modification of the benzimidazole moiety significantly affects its binding affinity to target proteins; alterations in electronic and steric effects can modulate its binding strength to enzymes. In recent years, numerous studies have revealed the chemical, structure-activity relationships, and biological activities of benzimidazole derivatives, inspiring researchers worldwide to develop sustainable, readily available, and highly effective new benzimidazole drugs. Summary of the Invention

[0004] Based on the above technical problems, the purpose of this invention is to propose a benzimidazole one derivative with broad-spectrum antitumor activity. This class of compounds exhibits good antitumor effects in vitro, especially against colorectal cancer and gastric cancer. Specifically, it includes the following:

[0005] In a first aspect, the present invention provides a benzimidazolone derivative or its tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, or cocrystal, wherein the structural formula of the benzimidazolone derivative is shown in formula (I) or formula (II) below:

[0006]

[0007] Equation (Ⅰ),

[0008]

[0009] Formula (II);

[0010] R1 is selected from H, halogen, C1~C6 alkyl, C1~C6 alkoxy, nitro, and hydroxyl.

[0011] R2 is selected from H, halogen, C1~C6 alkyl, C1~C6 alkoxy, nitro, hydroxyl, and C1~C2 haloalkyl;

[0012] R3 is selected from H, halogen, C1~C6 alkyl, C1~C6 alkoxy, nitro, hydroxyl, and C1~C2 haloalkyl;

[0013] R is selected from H or halogen.

[0014] Preferably, the benzimidazolone derivative is selected from compounds with any of the following structural formulas:

[0015] , , , , , , , , , , .

[0016] Preferably, the benzimidazolone derivative is selected from compounds with any of the following structural formulas:

[0017] , , , , , .

[0018] Preferably, the benzimidazolone derivative is selected from compounds with the following structural formulas:

[0019] .

[0020] In a second aspect, the present invention provides the use of the benzimidazole derivatives or their tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, and cocrystals described in the first aspect above in the preparation of antitumor drugs.

[0021] Preferably, the tumor includes colorectal cancer, liver cancer, stomach cancer, pancreatic cancer, cervical cancer, and ampullary cancer.

[0022] Preferably, the tumor is gastric cancer.

[0023] Thirdly, the present invention provides a pharmaceutical formulation comprising the benzimidazole derivative or its tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, eutectic, and pharmaceutically acceptable excipients described in the first aspect above.

[0024] Preferably, the dosage form of the pharmaceutical preparation includes any one of capsules, granules, tablets, sprays, oral liquids, suspensions, and injections.

[0025] Fourthly, the present invention provides a method for synthesizing the benzimidazolone derivative described in the first aspect above, the method comprising:

[0026] Operation 1: A mixture of o-phenylenediamine and ethyl 2-oxocyclohexanecarboxylate was dissolved in xylene and refluxed. The mixture was separated by column chromatography to obtain a white solid intermediate, 1-(cyclohexen-1-yl)-1,3-dihydro-2H-benzimidazol-2-one (III), as shown in synthetic route 1.

[0027]

[0028] Synthetic route 1;

[0029] Operation 2: A mixture of 1-(cyclohexen-1-yl)-1,3-dihydro-2H-benzimidazol-2-one (III) and K2CO3 was dissolved in N,N-dimethylformamide. Using benzyltriethylammonium chloride as a catalyst, the mixture was stirred for 20-30 min. Then, a 2-chloro-N-phenylacetamide derivative (IV) was added. After the reaction was completed at room temperature, the mixture was filtered, washed with ice water, and recrystallized from ethanol to obtain the product shown in formula (I), as shown in synthetic route 2.

[0030]

[0031] Synthesis Route 2;

[0032] Alternatively, in operation two: a mixture of 1-(cyclohexen-1-yl)-1,3-dihydro-2H-benzimidazol-2-one (III) and K2CO3 is dissolved in N,N-dimethylformamide, with benzyltriethylammonium chloride as a catalyst. After stirring for 20-30 min, N-(2-benzoylphenyl)-2-chloroacetamide (V) is added. The reaction is carried out with stirring at room temperature. After the reaction is complete, the organic layer is extracted with dichloromethane, dried over anhydrous sodium sulfate, and separated by column chromatography using a mixture of hexane / ethyl acetate as the eluent after rotary evaporation. Finally, recrystallization in ethanol yields the product shown in formula (II), as shown in synthetic route 3.

[0033]

[0034] Synthesis route 3.

[0035] The beneficial effects of this invention are as follows: This invention provides a class of benzimidazole derivatives, which exhibit significant broad-spectrum antitumor activity against colorectal cancer, liver cancer, gastric cancer, pancreatic cancer, cervical cancer, and ampullary cancer cells. Pharmacological and pharmacodynamic experiments have confirmed that the benzimidazole derivatives of this invention show excellent selectivity against HGC27 and AGS gastric cancer cells, effectively inhibiting the proliferation, migration, and invasion of gastric cancer cells, and inducing apoptosis in gastric cancer cells through the mitochondrial pathway. Their anti-gastric cancer efficacy is significantly superior to the commonly used clinical chemotherapy drug 5-fluorouracil (5-Fu), while exhibiting low cytotoxicity against normal gastric mucosal cells GES-1 and normal intestinal epithelial cells HIEC. Furthermore, these benzimidazole derivatives also show good in vitro antitumor effects against colon cancer cells Caco-2, pancreatic cancer cells PANC-1, cervical cancer cells HeLa, liver cancer cells HepG-2, and ampullary cancer cells DPC-X3, demonstrating a broad antitumor spectrum and potential as broad-spectrum antitumor candidate drugs with good clinical application value and industrialization prospects. Attached Figure Description

[0036] Figure 1 The structural formulas of benzimidazolone derivatives 5a~5i and 7a-b.

[0037] Figure 2 Synthetic routes for benzimidazolone derivatives 5a~5i.

[0038] Figure 3 Synthetic routes for benzimidazolone derivatives 7a-b.

[0039] Figure 4 MTT assay for the survival rate of benzimidazole derivative 7a in gastric cancer cell lines.

[0040] Figure 5 MTT assay for the survival rate of 5-Fu gastric cancer cell lines.

[0041] Figure 6 Benzimidazole derivative 7a inhibits clonogenicity in gastric cancer cells AGS and HGC27.

[0042] Figure 7 Effects of benzimidazole derivative 7a on the migration of gastric cancer cells AGS and HGC27.

[0043] Figure 8 Annexin V-FITC / PI apoptosis detection results of gastric cancer cells AGS and HGC27 under the treatment of benzimidazole derivative 7a.

[0044] Figure 9 TMRE staining assay was used to analyze the effect of benzimidazolone derivative 7a on the mitochondrial membrane potential of gastric cancer cells AGS and HGC27. Detailed Implementation

[0045] To facilitate understanding of the technical solution of this invention, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only for illustrating the invention and are not intended to limit the scope of protection of this invention. After studying the content of this invention, those skilled in the art can make various changes and improvements based on the concept of this invention. Such equivalent substitutions or modifications all fall within the scope of protection defined by the claims of this invention.

[0046] The human colorectal cancer cells Caco-2, liver cancer cells HepG-2, gastric cancer cells AGS, HGC27, SGC7901, MKN45 and MGC803, pancreatic cancer cells PANC-1, cervical cancer HeLa, ampullary cancer DPC-X3, normal gastric mucosal cells GES-1, and normal intestinal epithelial cells HIEC described in the following examples are all commercially available.

[0047] The novel benzimidazolone derivatives of this invention have the following structural formulas: Figure 1 As shown, the specific operations of the synthesis method include:

[0048] Operation 1: First, the mixture of o-phenylenediamine and ethyl 2-oxocyclohexanecarboxylate was dissolved in xylene and refluxed. The white solid intermediate product 1-(cyclohexen-1-yl)-1,3-dihydro-2H-benzimidazol-2-one (III) was obtained by column chromatography.

[0049] Synthesis Route 1:

[0050] .

[0051] Operation 2: 1-(cyclohexen-1-yl)-1,3-dihydro-2H-benzimidazol-2-one (III), benzyltriethylamine chloride as a catalyst, and K₂CO₃ were dissolved in N,N-dimethylformamide and stirred for 20-30 min. Then, an appropriate amount of 2-chloro-N-phenylacetamide derivative (IV) was added. After the reaction was completed at room temperature, the mixture was filtered, washed with ice water, and recrystallized from ethanol to obtain the product with good yield.

[0052] Synthesis Route 2:

[0053] .

[0054] Operation 3: A mixture of 1-(cyclohexen-1-yl)-1,3-dihydro-2H-benzimidazol-2-one (III) and K₂CO₃ was dissolved in N,N-dimethylformamide. Using benzyltriethylammonium chloride as a catalyst, the mixture was stirred for 20–30 min, and then an appropriate amount of N-(2-benzoylphenyl)-2-chloroacetamide (V) was added. The reaction was carried out with stirring at room temperature. After the reaction, the organic layer was extracted with dichloromethane, dried over anhydrous sodium sulfate, and separated by column chromatography using a hexane / ethyl acetate mixture as eluent after rotary evaporation. Finally, it was recrystallized from ethanol.

[0055] Synthesis Route 3:

[0056] .

[0057] In the above synthetic route, R1 is selected from H, halogen, C1~C6 alkyl, C1~C6 alkoxy, nitro, hydroxyl; R2 is selected from H, halogen, C1~C6 alkyl, C1~C6 alkoxy, nitro, hydroxyl, C1~C2 haloalkyl; R3 is selected from H, halogen, C1~C6 alkyl, C1~C6 alkoxy, nitro, hydroxyl, C1~C2 haloalkyl; R is selected from H or halogen.

[0058] Example 1 Synthesis of benzimidazolone derivatives

[0059] (1) The synthetic routes of phenylimidazole derivatives 5a~5i are as follows Figure 2 As shown, the specific synthesis method is as follows:

[0060] In a 100 mL round-bottom flask, 1–2 mmol of 1-(cyclohexen-1-yl)-1,3-dihydro-2H-benzimidazol-2-one and a certain amount of benzyltriethylammonium salt were added, followed by 5–6 mmol of potassium carbonate. After mixing thoroughly, the mixture was dissolved in 15–30 mL of N,N-dimethylformamide and stirred for 20–30 min. Subsequently, an appropriate amount of 2-chloro-N-phenylacetamide derivative 4a–4i (2 mmol) was added. The reaction was stirred at room temperature for 4–5 hours. The resulting mixture was then poured into ice water, the precipitated solid was filtered, washed with ice water, and recrystallized from ethanol to obtain products 5a–5i in good yield.

[0061] (2) The synthetic routes of benzoimidazole derivatives 7a and 7b are as follows: Figure 3 As shown, the specific synthesis method is as follows:

[0062] In a 100 mL round-bottom flask, 1–2 mmol of 1-(cyclohexen-1-yl)-1,3-dihydro-2H-benzimidazol-2-one and a certain amount of benzyltriethylammonium salt were added, followed by 5–6 mmol of potassium carbonate. After mixing thoroughly, the mixture was dissolved in 15–30 mL of N,N-dimethylformamide and stirred for 20–30 min. Subsequently, an appropriate amount of N-(2-benzoylphenyl)-2-chloroacetamide 6a–6b derivatives (1–2 mmol) was added. The reaction was stirred at room temperature for 4–5 h. The reaction mixture was diluted with distilled water (30 mL), and the organic layer was extracted with dichloromethane (3 × 10 mL), dried over anhydrous sodium sulfate, and evaporated under reduced pressure. Using a hexane / ethyl acetate mixture as eluent, compounds 7a–7b were separated by column chromatography and subsequently recrystallized in ethanol.

[0063] The structural formula of the synthesized product in Example 1 above is as follows: Figure 1 As shown, the characterization results are as follows:

[0064] 2-(3-(Cyclohex-1-enyl)-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)-N-phenylacetamide (derivative 5a):

[0065] White solid, 80% yield. Mp: 204-206 °C (ethanol). 1 H NMR (300 MHz, CDCl3), d (ppm): 1.61–1.78 (m,4H,2CH2-cyclohexenyl), 2.17–2.28 (m,4H, 2CH2-cyclohexenyl), 4.59 (s, 2H, CH2), 5.85 – 5.88 (m, 1H, C=CH, H-cyclohexenyl), 6.94 – 7.07 (m, 4H, H-Ar), 7.13 –7.19 (m, 2H, H-Ar), 7.39 – 7.42 (d, J = 7.6Hz, 2H, H-Ar), 8.80 (s, 1H, NH). 1313C NMR (75 MHz, CDCl3). δ (ppm): 21.50, 22.52, 24.69, 26.78 (4C, 4CH2, C-cyclohexenyl), 46.19 (1C, CH2), 127.80 (1C, CH=C, C-cyclohexenyl), 108.52, 109.10, 120.12, 122.13, 124.84, 129.46 (8C, CH=C, C–Ar), 128.87 131.87, 131.32, 137.57 (4C, =C–), 153.54, 165.58 (2C, C=O).

[0066] 2-(3-(Cyclohex-1-enyl)-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)-N-(p-tolyl)acetamide (Derivative 5b):

[0067] White solid, 80% yield. M.p: °C (ethanol). 1 1H NMR (300 MHz, CDCl3), δ(ppm): 1.59 – 1.64 (m, 2H, CH2 -cyclohexenyl), 1.69 – 1.77 (m, 2H, CH2 -cyclohexenyl), 2.15 – 2.27 (m, 4H, 2CH2 –cyclohexenyl, 3H, p-CH3-C6H4), 4.57(s, 2H, CH2), 5.83 – 5.85 (m, 1H, C=CH, H-cyclohexenyl), 6.92 – 7.02 (m, 6H, H-Ar), 7.27 (d, J = 8.4 Hz, 2H, H-Ar), 8.76 (s, 1H, NH). 1313C NMR (75 MHz, CDCl3). δ (ppm): 20.86 (1C, p-CH3-C6H4), 21.57, 22.53, 24.70, 26.78 (4C, 4CH2, C-cyclohexenyl), 45.88 (1C, CH2), 127.70 (1C, CH=C, C-cyclohexenyl), 108.54, 109.03, 120.20, 122.02, 129.33 (8C, CH=C, C–Ar), 129.55, 132.00, 134.03, 135.04 (5C, =C–), 153.48, 165.46 (2C, C=O).

[0068] 2-(3-(Cyclohex-1-enyl)-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)-N-(2,6-dimethylphenyl) acetamide (Derivative 5c):

[0069] White solid, 80% yield. M.p: °C (ethanol). 1 1H NMR (300 MHz, CDCl3), δ(ppm): 1.75 – 1.80 (m, 2H, CH2-cyclohexenyl), 1.95 – 1.91 (m, 2H, CH2-cyclohexenyl), 2.14 (s, 6H, 2CH3), 2.30 – 2.33 (m, 2H, CH2-cyclohexenyl), 2.39 – 2.41 (m, 2H, CH2-cyclohexenyl), 4.72 (s, 2H, CH2), 5.98 – 6.01 (m, 1H, C=CH, H-cyclohexenyl), 7.02 – 7.18 (m, 7H, H-Ar), 7.72 (s, 1H, NH). 1313C NMR (75 MHz, CDCl3). δ (ppm): 20.18 (2C, 2CH3), 21.56, 22.53, 24.70, 26.78 (4C, 4CH2, C-cyclohexenyl), 45.55 (1C, CH2), 127.77 (1C, CH=C, C-cyclohexenyl), 108.27, 109.13, 122.05, 127.50, 128.17 (7C, CH=C, C–Ar), 128.87, 129.65, 131.95, 133.07, 135.29 (6C, =C–), 153.35, 166.03 (2C, C=O).

[0070] 2-(3-(cyclohex-1-enyl)-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)-N-(4-methoxyphenyl) acetamide (Derivative 5d):

[0071] White solid, 80% yield. M.p: °C (ethanol). 1 1H NMR (300 MHz, CDCl3), δ(ppm): 1.73 – 1.78 (m, 2H, CH2-cyclohexenyl), 1.83 – 1.90 (m, 2H, CH2-cyclohexenyl), 2.28 – 2.31 (m, 2H, CH2-cyclohexenyl), 2.37 – 2.40 (m, 2H, CH2-cyclohexenyl), 3.77 (s, 3H, p-OCH3-C6H4), 4.66 (s, 2H, CH2), 5.97 – 5.99 (m, 1H, C=CH, H-cyclohexenyl), 6.80 – 6.84 (m, 2H, H-Ar), 7.05 – 7.18 (m, 4H, H-Ar), 7.39 – 7.43 (m, 2H, H-Ar), 8.56 (s, 1H, NH). 1313C NMR (75 MHz, CDCl3). δ(ppm): 21.56, 22.52, 24.70, 26.77 (4C, 4CH2, C-cyclohexenyl), 46.31 (1C, CH2), 127.77 (1C, CH=C, C-cyclohexenyl), 108.44, 109.10, 114.05, 121.94, 122.05, 122.16 (8C, CH=C, C–Ar), 129.09, 129.61, 130.51, 131.94 (5C, =C–), 153.52, 165.34 (2C, C=O).

[0072] 2-(3-(Cyclohex-1-enyl)-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)-N-(4-fluorophenyl) acetamide (Derivative 5e):

[0073] White solid, 80% yield. M.p: °C (ethanol). 1 1H NMR (300 MHz, CDCl3), δ(ppm): 1.61 – 1.66 (m, 2H, CH2-cyclohexenyl), 1.71 – 1.76 (m, 2H, CH2-cyclohexenyl), 2.16 – 2.19 (m, 2H, CH2-cyclohexenyl), 2.26 – 2.28 (m, 2H, CH2-cyclohexenyl), 4.60 (s, 2H, CH2), 5.85 – 5.87 (m, 1H, C=CH, H-cyclohexenyl), 6.81 – 6.87 (m, 1H, H-Ar), 6.95 – 7.08 (m, 6H, H-Ar), 7.34 – 7.38 (m, 1H, H-Ar), 8.95 (s, 1H, NH). 1313C NMR (75 MHz, CDCl3). δ (ppm): 21.53, 22.50, 24.69, 26.78 (4C, 4CH2, C-cyclohexenyl), 46.14 (1C, CH2), 127.84 (1C, CH=C, C-cyclohexenyl), 108.47, 109.17, 115.32, 115.62, 122.14, 122.20 (8C, CH=C, C–Ar), 129.54, 131.90, 133.59, 133.65, 153.56 (5C, =C–), 161.01, 165.55 (2C, C=O).

[0074] N-(4-chlorophenyl)-2-(3-(cyclohex-1-enyl)-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl) acetamide (Derivative 5f):

[0075] White solid, 80% yield. M.p: °C (ethanol). 1 1H NMR (300 MHz, CDCl3), δ(ppm): 1.60 – 1.65 (m, 2H, CH2-cyclohexenyl), 1.70 – 1.77 (m, 2H, CH2-cyclohexenyl), 2.15 – 2.18 (m, 2H, CH2-cyclohexenyl), 2.26 – 2.28 (m, 2H, CH2-cyclohexenyl), 4.61 (s, 2H, CH2), 5.84 – 5.86 (m, 1H, C=CH, H-cyclohexenyl), 6.95 – 7.09 (m, 6H, H-Ar), 7.31 – 7.35 (m, 2H, H-Ar), 9.09 (s, 1H, NH). 13CNMR (75 MHz, CDCl3). δ (ppm): 21.53, 22.51, 24.69, 26.80 (4C, 4CH2, C-cyclohexenyl), 45.95 (1C, CH2), 127.86 (1C, CH=C, C-cyclohexenyl), 108.49, 109.18, 121.20, 122.16, 122.20, 128.80 (8C, CH=C, C–Ar), 129.33, 129.09, 129.50, 131.92, 136.27 (5C, =C–), 153.56, 165.61 (2C, C=O).

[0076] N-(4-bromophenyl)-2-(3-(cyclohex-1-enyl)-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl) acetamide (Derivative 5g):

[0077] White solid, 80% yield. M.p: °C (ethanol). 1 H NMR (300 MHz, CDCl3), δ(ppm): 1.59 – 1.64 (m, 2H, CH2-cyclohexenyl), 1.69 – 1.76 (m, 2H, CH2-cyclohexenyl), 2.14 – 2.17 (m, 2H, CH2-cyclohexenyl), 2.25 – 2.27 (m, 2H, CH2-cyclohexenyl), 4.61 (s, 2H, CH2), 5.83 – 5.85 (m, 1H, C=CH, H-cyclohexenyl), 6.94 – 7.04 (m, 4H, H-Ar), 7.18 – 7.21 (m, 2H, H-Ar), 7.26 – 7.29 (m, 2H, H-Ar), 9.19 (s, 1H, NH). 1313C NMR (75 MHz, CDCl3). δ (ppm): 21.53, 22.52, 24.70, 26.81 (4C, 4CH2, C-cyclohexenyl), 45.80 (1C, CH2), 127.84 (1C, CH=C, C-cyclohexenyl), 108.49, 109.17, 116.94, 121.50, 122.15, 122.18, 129.25 (8C, CH=C, C–Ar), 129.48, 131.73, 131.94, 136.82 (5C, =C–), 153.54, 165.62 (2C, C=O).

[0078] 2-(3-(Cyclohex-1-enyl)-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)-N-(4-nitrophenyl) acetamide (Derivative 5h):

[0079] White solid, 80% yield. M.p: °C (ethanol). 1 1H NMR (300 MHz, CDCl3), δ(ppm): 1.65 – 1.71 (m, 2H, CH2-cyclohexenyl), 1.75 – 1.81 (m, 2H, CH2-cyclohexenyl), 2.21 – 2.24 (m, 2H, CH2-cyclohexenyl), 2.29 – 2.31 (m, 2H, CH2-cyclohexenyl), 4.66 (s, 2H, CH2), 5.91 – 5.93 (m, 1H, C=CH, H-cyclohexenyl), 7.00 – 7.11 (m, 4H, H-Ar), 7.58 – 7.63 (m, 2H, H-Ar), 8.04 – 8.07 (m, 2H, H-Ar), 9.62 (s, 1H, NH). 1313C NMR (75 MHz, CDCl3). δ (ppm): 21.51, 22.52, 24.68, 26.83 (4C, 4CH2, C-cyclohexenyl), 46.53 (1C, CH2), 127.96 (1C, CH=C, C-cyclohexenyl), 108.31, 109.35, 119.41, 122.29, 122.43, 124.76 (8C, CH=C, C–Ar), 129.06, 129.62, 131.95, 143.50 (5C, =C–), 153.82, 165.97 (2C, C=O).

[0080] 2-(3-(Cyclohex-1-enyl)-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)-N-(2-nitrophenyl) acetamide (Derivative 5i):

[0081] White solid, 80% yield. M.p: °C (ethanol). 1 1H NMR (300 MHz, CDCl3), δ(ppm): 1.61 – 1.69 (m, 2H, CH2-cyclohexenyl), 1.72 – 1.82 (m, 2H, CH2-cyclohexenyl), 2.17 – 2.20 (m, 2H, CH2-cyclohexenyl), 2.25 – 2.33 (m, 2H, CH2-cyclohexenyl), 4.64 (s, 2H, CH2), 5.87 – 6.00 (m, 1H, C=CH, H-cyclohexenyl), 6.86 – 7.07 (m, 5H, H-Ar), 7.50 (t, J = 7.5 Hz, 1H, H-Ar), 8.01 (d, J = 8.4Hz, 1H, H-Ar), 8.67 (d, J = 8.5 Hz, 1H, H-Ar), 10.66 (s, 1H, NH). 1313C NMR (75 MHz, CDCl3). δ (ppm): 21.61, 22.58, 24.75, 26.66 (4C, 4CH2, C-cyclohexenyl), 45.68 (1C, CH2), 127.92 (1C, CH=C, C-cyclohexenyl), 107.64, 109.29, 121.82, 121.91, 122.45, 123.81, 125.74, 135.96 (8C, CH=C, C–Ar), 128.56, 129.98, 132.01, 133.95, 136.42 (5C, =C–), 152.84, 166.48 (2C, C=O).

[0082] N-(2-benzoylphenyl)-2-(3-(cyclohex-1-enyl)-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl) acetamide (Derivative 7a):

[0083] White solid, 80% yield. M.p: °C (ethanol). 1 1H NMR (300 MHz, CDCl3), δ(ppm): 1.62, 1.71, 2.19 (3m, 8H, CH2-cyclohexenyl), 4.03 (m, 2H, CH2), 5.78 (m, 1H, CH- cyclohexenyl), 6.84 – 7.62 (m. CH-Ar), 12.47 (s, 1H, NH). 1313C NMR (75 MHz, CDCl3). δ (ppm): 21.17, 22.03, 24.05, 25.98 (4C, 4CH2, C-cyclohexenyl), 60.70 (1C, CH2), 127.16 (1C, CH=C, C-cyclohexenyl), 108.19, 108.68, 115.73, 118.94, 121.19, 121.29, 122.30, 122.73, 126.26, 127.87, 127.94, 128.08 (13C, CH=C, C–Ar), 128.19, 129.12, 131.61, 133.06, 138.76 (6C, =C–), 152.17, 158.28, 170.88 (3C, C=O).

[0084] N-(2-Benzoyl-4-chlorophenyl)-2-(3-(cyclohex-1-enyl)-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)acetamide (Derivative 7b):

[0085] White solid, 83% yield. M.p: °C (ethanol). 1 1H NMR (300 MHz, CDCl3), δ(ppm): 1.62, 1.71, 2.19 (3m, 8H, CH2-cyclohexenyl), 4.03 (m, 2H, CH2), 5.78(m, 1H, CH- cyclohexenyl), 6.84 – 7.62 (m. CH-Ar), 12.47 (s, 1H, NH). 13C NMR(75 MHz, CDCl3). d (ppm): 21.17, 22.03, 24.05, 25.98 (4C, 4CH2, C-cyclohexenyl), 60.70 (1C, CH2), 127.16 (1C, CH=C, C-cyclohexenyl), 108.19,108.68, 115.73, 118.94, 121.19, 121.29, 122.30, 122.73, 126.26, 127.87,127.94, 128.08 (13C, CH=C, C–Ar), 128.19, 129.12, 131.61, 133.06, 138.76 (6C,=C–), 152.17, 158.28, 170.88 (3C, C=O).

[0086] Example 2: Inhibitory effect of benzodiazepine derivatives on different tumor cells

[0087] Different concentrations of benzimidazolone derivatives 5a-5i and 7a-7b prepared in Example 1 were used to treat colorectal cancer cells Caco-2, liver cancer cells HepG-2, gastric cancer cells AGS, pancreatic cancer cells PANC-1, cervical cancer cells HeLa, ampullary cancer cells DPC-X3, and normal intestinal epithelial cells HIEC for 48 h. After the culture was completed, 10 μL of 5 mg / mL MTT working solution was added to each cell well, and the cells were incubated in a constant temperature incubator at 37°C and 5% CO2 for 4 h in the dark. After the incubation was completed, the original culture medium and MTT solution were carefully discarded, and 100 μL of DMSO solution was added. The cells were shaken at 120 r / min for 15 min on a shaker to fully dissolve the formazan crystals generated in the cells. The absorbance of each well was then measured at 490 nm using a microplate reader. Based on the results, the inhibition rate of each derivative on the proliferation of different cell types was calculated. Finally, the whiteboard inhibition rate (IC50) of each compound was calculated using SPSS 29.0.2.0 software. 50 ).

[0088] The cytotoxic IC50 values ​​of benzimidazole derivatives 5a-5i and 7a-7b against colorectal cancer cells Caco-2, liver cancer cells HepG-2, gastric cancer cells AGS, pancreatic cancer cells PANC-1, cervical cancer cells HeLa, ampullary cancer cells DPC-X3, and normal intestinal epithelial cells HIEC 50 The test results are shown in Table 1 below. Among them, the benzimidazolone derivative 7a described in this application exhibited potent cytotoxicity against human gastric cancer cells AGS, with a half-maximal inhibitory concentration (IC50) of [missing value]. 50The concentration was 3.77 ± 0.37 μM. This compound also exhibited significant antitumor activity against colorectal cancer cells Caco-2, liver cancer cells HepG-2, pancreatic cancer cells PANC-1, and cervical cancer cells HeLa, with corresponding IC50 values ​​of 3.77 ± 0.37 μM. 50 The effective concentrations were 4.44±0.61 μM, 7.81±0.50 μM, 7.75±0.37 μM, and 5.49±0.24 μM, respectively; the IC50 values ​​for human ampullary cancer cells DPC-X3 were... 50 The IC50 value for HIEC in normal human intestinal epithelial cells was 35.62 ± 2.22 μM. 50 The value was 27.62 ± 2.16 μM. Except for derivative 7a, the other benzimidazolone derivatives also exhibited varying degrees of cytotoxicity against the aforementioned tumor cell types.

[0089] Table 1. IC50 of benzimidazole derivatives against tumor cells 50 Detection results (μM)

[0090]

[0091] Example 3: Inhibitory effect of benzimidazole derivatives on other gastric cancer cells

[0092] 1. MTT assay for the inhibitory effects of benzimidazole derivatives and 5-Fu on other gastric cancer cells.

[0093] Five gastric cancer cell lines (AGS, HGC27, SGC7901, MKN45, and MGC803) and normal human gastric mucosal cells (GES-1) were extracted from liquid nitrogen and routinely resuscitated and passaged. When the second-generation cells reached 80% confluence, they were removed and digested with trypsin containing phenol red and EDTA. The cells were then resuspended in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. After hemocytometer counting, the cell concentration was adjusted to 5000–6000 cells / 100 μL, and 100 μL was seeded per well in 96-well plates. The plates were incubated at 37°C with 5% CO2 for 24 h. The original medium was then discarded, and 100 μL of medium containing different concentrations of benzimidazolone derivatives 7a and 5-Fu was added to each well, and the plates were cultured for another 48 h. Subsequently, 10 μL of 5 mg / mL MTT solution was added to each well, and the mixture was incubated for 4 h. The liquid in the wells was then aspirated, and 100 μL of DMSO was added. The mixture was shaken at 120 r / min for 15 min until the formazan was completely dissolved. The absorbance was measured at 490 nm using a microplate reader, the cell proliferation inhibition rate was calculated, and the IC50 was determined using SPSS software. 50 value.

[0094] The results were verified by an in vitro MTT cell proliferation inhibition experiment as follows: Figure 4 and Figure 5 As shown, the benzimidazole derivative 7a described in this application exhibits significant inhibitory activity against the proliferation of HGC27, AGS, MKN45, MGC803, and SGC7901 gastric cancer cell lines. Furthermore, its overall in vitro cytotoxicity is superior to that of the first-line clinical chemotherapy drug 5-fluorouracil (5-Fu), and it also shows low cytotoxicity against normal gastric mucosal cells GES-1. Its overall efficacy demonstrates the advantages of high efficiency and low toxicity, selectively inhibiting the proliferation of gastric cancer cells while also exhibiting broad-spectrum inhibitory effects against various tumor cell lines.

[0095] 2. Cell cloning experiments

[0096] Human gastric cancer HGC27 and AGS cells were cultured in medium-sized cell culture dishes. When the cell confluence reached 80%, the cells were processed according to the aforementioned cell digestion method to prepare a homogeneous single-cell suspension. The prepared cell suspension was seeded into 24-well culture plates at a cell density of 500 cells / well, with a final culture medium volume of 500 μL per well. The culture plates were incubated at 37°C in a 5% CO2 incubator for approximately 24 h. After incubation, the original culture medium in the wells was discarded, and fresh culture medium containing different concentrations of benzimidazolone derivative 7a was added. The cells were incubated for 7–10 days. During the experiment, the corresponding concentration of drug-containing culture medium was replaced every 48 h to ensure a stable drug concentration. After the cell clones in the wells grew to a suitable observation density, the old culture medium was discarded, and the cells were washed twice with 1×PBS buffer. 300–400 μL of 4% paraformaldehyde solution was added to each well to fix the cells for 40 min. After fixation, the fixative was aspirated, and the wells were washed again with 1×PBS buffer. Subsequently, 0.1% crystal violet staining solution was added to stain the cell clones for 20 min. After staining, the staining solution was recovered, and the cells were washed twice with 1×PBS buffer. After air drying at room temperature, the morphology and number of cell clones in each group were photographed and recorded.

[0097] Experimental results are as follows Figure 6 As shown, the benzimidazole derivative 7a of the present invention can significantly inhibit the clonal formation ability of gastric cancer cells and effectively inhibit the proliferation and clonal growth of gastric cancer cells.

[0098] 3. Cell scratch assay

[0099] Before the experiment, horizontal lines were evenly drawn on the back of 6-well plates using a marker, with a spacing of 0.5–1 cm, ensuring at least 5 horizontal lines in each well for subsequent scratch localization. Human gastric cancer HGC27 and AGS cells in logarithmic growth phase were selected. The original culture medium in the wells was discarded, and the cells were washed twice with 1×PBS buffer. After trypsin digestion, a single-cell suspension was prepared and seeded into the pretreated 6-well plates. The final volume of culture medium in each well was 2 mL. The seeding amount was adjusted to ensure 100% confluence after overnight culture, followed by routine culture at 37℃ and 5% CO2 for 24 h. After the cells had covered the bottom of the plate, a 10 μL pipette tip was used to make scratches perpendicular to the marking lines on the back of the plate at a uniform speed, ensuring the pipette tip remained vertical and untilted throughout the process to guarantee uniform scratch width in all groups. After scratching, the cells were washed three times with PBS buffer to thoroughly remove detached cell debris, and the horizontal marking lines on the back of the plate were wiped off. Pure serum-free culture medium and serum-free culture medium containing different concentrations of benzimidazolone derivative 7a were added to each well. After standing, samples were taken and photographed. Finally, ImageJ software was used to quantify and analyze the scratch images of each group.

[0100] Experimental results are as follows Figure 7 As shown, the benzimidazole derivative 7a of the present invention can significantly inhibit the migration ability of human gastric cancer HGC27 and AGS cells, and has excellent anti-tumor migration activity.

[0101] 4. Flow cytometry Annexin V-FITC / PI apoptosis detection

[0102] AGS and HGC27 cells in logarithmic growth phase were seeded at 150,000 cells per well in 6-well cell culture plates and incubated at 37°C with 5% CO2 for 24 h. Tumor cells in the experimental groups were then treated with different concentration gradients of benzimidazolone derivatives for 48 h. After treatment, the supernatant was collected, and the cells were washed twice with 1×PBS, digested with trypsin without EDTA, and collected from each well. The cells were centrifuged at 1800 r / min for 5 min, the supernatant was discarded, and the cells were resuspended in PBS. The cells were centrifuged again, and the cell pellet was collected. A certain amount of 1×Binding Buffer was added to resuspend the cells. The cell suspension was transferred to flow cytometry tubes, and 5 μL of Annexin V-FITC staining solution was added to each tube. The tubes were incubated at room temperature in the dark for 5 min, followed by the addition of 5 μL LPI and 400 μL of 1×PBS. The mixture was then immediately flow-cytometry analyzed.

[0103] The results are as follows Figure 8 As shown, the benzimidazolone derivative 7a of the present invention can induce apoptosis in gastric cancer cells AGS and HGC27 cells in a concentration-dependent manner.

[0104] 5. TMRE staining experiment

[0105] Human gastric cancer AGS and HGC27 cells were used at a rate of 3×10⁻⁶. 5 Cells were seeded at a density of [number] cells / well in 6-well cell culture plates and cultured at 37°C with 5% CO2 for 24 h to allow for stable adherence and growth. Once the cells were stable, different concentrations of benzimidazolone derivative 7a were added for intervention, with treatment lasting 48 h. After drug incubation, the culture medium in the wells was discarded, and the cells were gently washed twice with pre-warmed sterile PBS buffer to remove residual culture medium and drug impurities. Then, an appropriate concentration of TMRE staining working solution was added to each well, and the cells were incubated at 37°C for 30 min under complete darkness to ensure sufficient probe binding to mitochondria. After staining, the staining solution was aspirated, and the cells were washed again with pre-warmed PBS to remove unbound free dye and reduce background fluorescence interference. Finally, inverted fluorescence microscopy was used to observe and acquire fluorescence images of each group of cells. The effect of derivative 7a on the mitochondrial membrane potential level of gastric cancer cells was analyzed by fluorescence intensity changes to evaluate its mechanism of action in inducing tumor cell apoptosis.

[0106] The results are as follows Figure 9 As shown, the blank group of gastric cancer AGS and HGC27 cells exhibited high fluorescence intensity and uniform fluorescence distribution, maintained a high mitochondrial membrane potential, and showed stable overall cell growth. After treatment with different concentrations of benzimidazole derivative 7a, the red fluorescence intensity of gastric cancer cells in both groups decreased in a concentration-dependent manner. With increasing concentration, the fluorescence quenching phenomenon became more pronounced. These results indicate that benzimidazole derivative 7a can effectively reduce the mitochondrial membrane potential of gastric cancer cells, disrupt mitochondrial structural stability, and thus induce apoptosis in gastric cancer cells, exhibiting a significant dose-dependent characteristic.

[0107] In summary, the benzimidazole derivatives described in this application exhibit activity against colorectal cancer, liver cancer, gastric cancer, pancreatic cancer, and cervical cancer, with low toxicity to normal intestinal epithelial cells and gastric mucosal cells. These benzimidazole derivatives possess excellent broad-spectrum antitumor activity and can serve as potential broad-spectrum antitumor drug candidates. Specifically, derivative 7a demonstrates good selectivity against gastric cancer cells, effectively inhibiting their proliferation and migration, and significantly inducing apoptosis. Its antitumor efficacy against gastric cancer is superior to that of the commonly used chemotherapy drug 5-FU, and it possesses the combined advantages of high efficiency and low toxicity, showing promising application prospects in the fields of targeted therapy for gastric cancer and the development of broad-spectrum antitumor drugs.

[0108] The specific embodiments listed in this invention are only used to illustrate the technical solutions of this invention in detail, so that those skilled in the art can fully understand them, and are not intended to limit the scope of protection of this invention. Without departing from the core technical concept and innovative essence of this invention, those skilled in the art can reasonably optimize, adjust, or equivalently replace the specific implementation parameters, experimental conditions, and equipment selection of this invention based on existing known technologies. Any modifications, substitutions, or improvements made based on the technical essence of this invention fall within the protection scope defined by the claims of this invention.

Claims

1. A benzimidazolone derivative or its tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, or cocrystal, characterized in that, The structural formula of the benzimidazolone derivative is shown in formula (I) or formula (II) below: Equation (Ⅰ), Formula (II); R1 is selected from H, halogen, C1~C6 alkyl, C1~C6 alkoxy, nitro, and hydroxyl. R2 is selected from H, halogen, C1~C6 alkyl, C1~C6 alkoxy, nitro, hydroxyl, and C1~C2 haloalkyl; R3 is selected from H, halogen, C1~C6 alkyl, C1~C6 alkoxy, nitro, hydroxyl, and C1~C2 haloalkyl; R is selected from H or halogen.

2. The benbenzimidazolone derivative or its tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, or cocrystals as described in claim 1, characterized in that, The benzimidazole derivative is selected from any of the compounds with the following structural formulas: 、 、 、 、 、 、 、 、 、 、 。 3. The benzimidazolone derivative or its tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, or cocrystal as described in claim 2, characterized in that, The benzimidazole derivative is selected from any of the compounds with the following structural formulas: 、 、 、 、 、 。 4. The benzimidazolone derivative or its tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, or cocrystal as described in claim 3, characterized in that, The benzimidazole derivative is selected from compounds with the following structural formulas: 。 5. The use of the benzimidazolone derivatives or their tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, or eutectics as described in any one of claims 1-4 in the preparation of antitumor drugs.

6. The application as described in claim 5, characterized in that, The tumors include colorectal cancer, liver cancer, stomach cancer, pancreatic cancer, cervical cancer, and ampullary cancer.

7. The application as described in claim 6, characterized in that, The tumor is stomach cancer.

8. A pharmaceutical preparation, characterized in that, The pharmaceutical formulation comprises any of the benzimidazolone derivatives or their tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, eutectics, and pharmaceutically acceptable excipients as described in any one of claims 1-4.

9. The pharmaceutical preparation according to claim 8, characterized in that, The dosage form of the pharmaceutical preparation includes any one of capsules, granules, tablets, sprays, oral liquids, suspensions, and injections.

10. The method for synthesizing the benzimidazolone derivative as described in claim 1, characterized in that, The method includes: Operation 1: A mixture of o-phenylenediamine and ethyl 2-oxocyclohexanecarboxylate was dissolved in xylene and refluxed. The mixture was separated by column chromatography to obtain a white solid intermediate, 1-(cyclohexen-1-yl)-1,3-dihydro-2H-benzimidazol-2-one (III), as shown in synthetic route 1. Synthetic route 1; Operation 2: A mixture of 1-(cyclohexen-1-yl)-1,3-dihydro-2H-benzimidazol-2-one (III) and K2CO3 was dissolved in N,N-dimethylformamide. Using benzyltriethylammonium chloride as a catalyst, the mixture was stirred for 20-30 min. Then, a 2-chloro-N-phenylacetamide derivative (IV) was added. After the reaction was completed at room temperature, the mixture was filtered, washed with ice water, and recrystallized from ethanol to obtain the product shown in formula (I), as shown in synthetic route 2. Synthesis Route 2; Alternatively, in operation two: a mixture of 1-(cyclohexen-1-yl)-1,3-dihydro-2H-benzimidazol-2-one (III) and K2CO3 is dissolved in N,N-dimethylformamide, with benzyltriethylammonium chloride as a catalyst. After stirring for 20-30 min, N-(2-benzoylphenyl)-2-chloroacetamide (V) is added. The reaction is carried out with stirring at room temperature. After the reaction is complete, the organic layer is extracted with dichloromethane, dried over anhydrous sodium sulfate, and separated by column chromatography using a mixture of hexane / ethyl acetate as the eluent after rotary evaporation. Finally, recrystallization in ethanol yields the product shown in formula (II), as shown in synthetic route 3. Synthesis route 3.

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