CN114436972A - Pabendazole derivative and preparation method and application thereof - Google Patents

Abstract

The invention relates to the field of medicines, in particular to a parbendazole derivative, a preparation method and an application thereof, wherein the structure of the parbendazole derivative is shown as a formula X:

wherein R is₁Selected from alkoxy or alkylamino; r is₂Selected from fluoro, cyano, methyl, trifluoromethyl, n-butyl or n-pentyl; r₃The parbendazole derivative prepared in the invention has good inhibition effect on the proliferation of a head and neck squamous cell carcinoma HNSCC cell line in vitro, and can inhibit tubulin polymerization, inhibit cell invasion, cell migration and block cell cycle; can obviously control the growth of HN6 xenograft tumor nude mouse model tumor in vivo, and has good curative effectAnd the detection of related tumor markers proves that the curative effect is obvious.

Description

Pabendazole derivatives and preparation method and application thereof

technical field

The invention relates to the field of medicine, in particular to a pabendazole derivative and a preparation method and application thereof.

Background technique

The disclosure of information in this Background section is only for enhancement of understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

Head and neck squamous cell carcinoma (HNSCC) is a malignant tumor of the oral cavity, pharynx and larynx covered by squamous epithelium. Although it originates from mucosal epithelial cells, it has obvious heterogeneity . In addition to surgical resection, most patients require conservative treatment. Cytotoxicity-based combination therapy of cetuximab, cisplatin, and 5-fluorouracil is a commonly used treatment regimen, and immunotherapy is also a promising treatment, although some progress has been made for the treatment of HNSCC, the best The treatment method for the disease is still unclear, and the development of new drugs is still necessary and urgent.

SUMMARY OF THE INVENTION

In view of the problems existing in the prior art, the purpose of the present invention is to provide a Pabendazole derivative and its preparation method and application. The proliferation of cell lines has a good inhibitory effect, which can inhibit tubulin polymerization, inhibit cell invasion, cell migration and block cell cycle; it can significantly control the growth of HN6 xenograft tumor in nude mouse model in vivo, which plays a good role in The therapeutic effect is also proved by the detection of related tumor markers.

In order to achieve the above object, the technical scheme of the present invention is as follows:

In the first aspect of the present invention, a kind of Pabendazole derivative is provided, and the structure of this derivative is shown in formula X:

Wherein, R ₁ is selected from alkoxy or alkylamino; R ₂ is selected from fluorine, cyano, methyl, trifluoromethyl, n-butyl or n-pentyl; R ₃ is selected from hydrogen, methyl or fluorine.

In one or more embodiments, the pabendazole derivative has a structure represented by formula (I) or formula (II):

Preferably, the R ₁ is selected from alkoxy or alkylamino; R ₂ is selected from fluorine, cyano, methyl, trifluoromethyl, n-butyl or n-pentyl; R ₃ is selected from hydrogen, methyl or fluorine.

Specifically, the Pabendazole derivative is selected from the following compounds:

Compound 6a: 1-(6-butyl-1H-benzo[d]imidazol-2-yl)-3-ethylurea;

Compound 6b: 1-(6-butyl-1H-benzo[d]imidazol-2-yl)-3-cyclohexylurea;

Compound 6c: 1-(6-butyl-1H-benzo[d]imidazol-2-yl)-3-isopropylurea;

Compound 6d: 1-(6-butyl-1H-benzo[d]imidazol-2-yl)-3-propylurea;

Compound 6e: 1-(6-butyl-1H-benzo[d]imidazol-2-yl)-3-hexylurea;

Compound 6f: 1-(6-butyl-1H-benzo[d]imidazol-2-yl)-3-phenethylurea;

Compound 6g: 1-(tert-butyl)-3-(6-butyl-1H-benzo[d]imidazol-2-yl)urea;

Compound 6h: 1-(6-butyl-1H-benzo[d]imidazol-2-yl)-3-cyclopentylurea;

Compound 6i: 1-allyl-3-(6-butyl-1H-benzo[d]imidazol-2-yl)urea;:

Compound 6j: 2-(3-(6-butyl-1H-benzo[d]imidazol-2-yl)ureido)ethyl methacrylate;

Compound 6k: isobutyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate;

Compound 61: benzyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate;

Compound 6m: 2-Chloroethyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate;

Compound 6n: propyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate;

Compound 6o: (6-butyl-1H-benzo[d]imidazol-2-yl)hexylcarbamate;

Compound 6p: butyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate;

Compound 6q: isopropyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate;

Compound 6r: (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate heptyl ester;

Compound 6s: (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate cyclopentyl ester;

Compound 6t: 3-chloropropyl(6-butyl-1H-benzo[d]imidazol-2-yl)carbamate;

Compound 6u: sec-butyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate;

Compound 6v: (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate allyl ester;

Compound 6w: 2-ethylhexyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate;

Compound 6x: N-(6-butyl-1H-benzo[d]imidazol-2-yl)furan-2-carboxamide;

Compound 6y: N-(6-butyl-1H-benzo[d]imidazol-2-yl)-2-phenylacetamide;

Compound 6z: N-(6-butyl-1H-benzo[d]imidazol-2-yl)tetrahydro-2H-pyran-4-carboxamide;

Compound 6aa: N-(6-butyl-1H-benzo[d]imidazol-2-yl)-2-(thiophen-2-yl)acetamide;

Compound 6ab: isopropyl (6-cyano-1H-benzo[d]imidazol-2-yl)carbamate;

Compound 6ac: isopropyl (6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)carbamate;

Compound 6ad: isopropyl (6-methyl-1H-benzo[d]imidazol-2-yl)carbamate;

Compound 6ae: isopropyl (6-fluoro-1H-benzo[d]imidazol-2-yl)carbamate;

Compound 6af: isopropyl (6-pentyl-1H-benzo[d]imidazol-2-yl)carbamate;

Compound 6ag: isopropyl (5,6-difluoro-1H-benzo[d]imidazol-2-yl)carbamate;

Compound 6ah: isopropyl (5,6-dimethyl-1H-benzo[d]imidazol-2-yl)carbamate;

In the second aspect of the present invention, a preparation method of the Pabendazole derivative described in the first aspect is provided, comprising the following steps:

(1) The amine group of compound 1 is acetylated in acetic anhydride under the catalysis of concentrated sulfuric acid, and then the ortho-position of the acetyl group is nitrated by acetic anhydride and nitric acid to obtain compound 2; subsequently, compound 2 is acetylated by potassium hydroxide in methanol The solution is hydrolyzed into compound 3, and then the nitro group is reduced by H ₂ /Pd in methanol to obtain compound 4;

(2) 2-methyl-2-thioisourea sulfate undergoes substitution reaction with isocyanate, chloroformate or acid chloride under alkaline conditions to obtain 5a-5aa;

(3) Compounds 4 and 5a-5aa undergo cyclization reaction under acidic conditions to generate Pabendazole derivatives (6a-6ah);

Wherein steps (1) and (2) are in no particular order;

Reagents and conditions: (I) aniline, acetic anhydride, concentrated sulfuric acid, 45～55℃, 0.5～1.5h; nitric acid, 0℃, 30～50min. (II) KOH, 60～70℃, 0.5～1.5h. (III) H ₂ , Pd/C, room temperature, 23～25h. (IV) Isocyanate, chloroformate or acid chloride, N,N-diisopropylethylamine, room temperature, 46～50h. (V) CH ₃ COOH:H ₂ O=3:10, 90～110°C, 2.5～3.5h.

Specifically, the preparation method is as follows:

(1) Cool acetic acid and a small amount of concentrated sulfuric acid to 0°C, add compound 1; after stirring at 45-55°C for 0.5-1.5 h, cool the mixture to room temperature, then add acetic anhydride; then, cool the mixture to 0°C again, add concentrated Nitric acid, stirring at 0°C for 30-50min; adding the reaction solution to ice water to obtain compound 2;

Compound 2 was dissolved in methanol, 40% KOH solution was added, and stirred at 60-70°C for 0.5-1.5 h; the reaction solution was dried, filtered, and the solvent was removed to obtain compound 3;

Compound 3, Pd/C and methanol were mixed and reduced with hydrogen; the reaction solution was stirred at room temperature for 23-25 h, and Pd/C was filtered to obtain compound 4;

(2) Add isocyanate, chloroformate or acid chloride dropwise to the mixture of 2-methyl-2-thiourea sulfate, DIPEA and acetonitrile; stir at room temperature for 46-50 h, remove the solvent; add ethyl acetate and water , After fully stirring, the liquid is separated, the organic phase is washed and dried, and the product is purified to obtain compounds 5a-5aa;

(3) The compound 4, 5a-5aa and the solvent were stirred at 90 to 110°C for 2.5 to 3.5 h, the reaction solution was cooled to room temperature, and the pH was adjusted to be alkaline; The phases are dried, filtered, concentrated and purified to obtain the Pabendazole derivative.

Preferably, the solvent described in the step (3) is an aqueous acetic acid solution, wherein V _{CH COOH} : V _{H O} =3:10;

Preferably, the isocyanate is selected from ethyl isocyanate, cyclohexyl isocyanate, isopropyl isocyanate, propyl isocyanate, hexyl isocyanate, phenethyl isocyanate, isocyanate tert-butyl, cyclopentyl isocyanate, allyl isocyanate or isocyanoethyl methacrylate;

Preferably, the chloroformate is selected from isobutyl chloroformate, benzyl chloroformate, ethyl chloroformate, propyl chloroformate, hexyl chloroformate, butyl chloroformate, isopropyl chloroformate, chlorine Heptyl formate, cyclopentyl chloroformate, 2-ethylhexyl chloroformate, chloropropyl chloroformate, 1-methylpropyl chloroformate or allyl chloroformate;

Preferably, the acid chloride is selected from furoyl chloride, phenylacetyl chloride, tetrahydropyran-4-carbonyl chloride or 2-thiopheneacetyl chloride.

In a third aspect of the present invention, there is provided a pharmaceutical composition comprising the above-mentioned pabendazole derivative.

In a fourth aspect of the present invention, a pharmaceutical preparation is provided, which comprises the above-mentioned pabendazole derivative or the above-mentioned pharmaceutical composition, and a pharmaceutically acceptable carrier and/or adjuvant. The preparations are selected from oral preparations and parenteral preparations, and can be tablets, pills, capsules or injections.

In a fifth aspect of the present invention, there is provided the application of the above-mentioned pabendazole derivatives in the preparation of a medicament for the treatment of head and neck squamous cell carcinoma HNSCC;

In one or more embodiments, the head and neck squamous cell carcinoma includes human tongue squamous cell carcinoma and human pharyngeal squamous cell carcinoma.

In vitro activity studies show that the pabendazole derivatives disclosed in the present invention have a good inhibitory effect on the proliferation of the three tested HNSCC cell lines, which can inhibit tubulin polymerization, inhibit cell invasion, cell migration and block cells. cycle.

In vivo activity studies show that the pabendazole derivatives disclosed in the present invention can significantly control the growth of HN6 xenograft nude mouse model tumors at an oral dose of 80 mg/kg, and have a good therapeutic effect. The test also proved that its curative effect is obvious.

The in vitro and in vivo activities show that the pabendazole derivative disclosed in the present invention is a potential molecule for the treatment of head and neck squamous cell carcinoma HNSCC, which provides a certain basis for the research and development of drugs.

As used herein, the term "pharmaceutical composition", which may also refer to a "composition", can be used to effect the treatment or prevention of the diseases or conditions of the present invention in a subject, particularly a mammal.

Pharmaceutical compositions of the compounds of the present invention may be administered in any of the following ways: oral, inhalation spray, rectal, nasal, vaginal, topical, parenteral such as subcutaneous, intravenous, intramuscular , intraperitoneal, intrathecal, intraventricular, intrasternal or intracranial injection or infusion, or via an explanted reservoir, wherein oral, intramuscular, intraperitoneal or intravenous administration is preferred.

A compound of the present invention or a pharmaceutical composition containing it may be administered in unit dosage form. The dosage form for administration can be a liquid dosage form, a solid dosage form. Liquid dosage forms can be true solutions, colloids, particulate dosage forms, emulsion dosage forms, and swirl dosage forms. Other dosage forms such as tablets, capsules, dropping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, lyophilized powders, inclusions, embeddings, patches, wipes agent, etc.

The pharmaceutical compositions of the present invention may also contain commonly used carriers, and the pharmaceutically acceptable carriers herein include but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffers Substances such as phosphates, glycerol, sorbates, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts , Colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, beeswax, lanolin, etc. The content of the carrier in the pharmaceutical composition can be 1% by weight to 98% by weight, and usually accounts for about 80% by weight. For convenience, local anesthetics, preservatives, buffers, etc. can be dissolved directly in the vehicle.

Oral tablets and capsules may contain excipients such as binders, such as syrup, acacia, sorbitol, tragacanth, or polyvinylpyrrolidone, fillers such as lactose, sucrose, cornstarch, calcium phosphate, sorbitol, amino acids Acetic acid, lubricants such as magnesium stearate, talc, polyethylene glycols, silica, disintegrants such as potato starch, or acceptable emollients such as sodium lauryl sulfate. Tablets can be coated by methods known in pharmacy.

Oral liquids can be made into water and oil suspensions, solutions, emulsions, syrups, and can also be made into dry products, which can be supplemented with water or other suitable vehicles before use. Such liquid preparations may contain conventional additives such as suspending agents, sorbitol, cellulose methyl ether, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, hydrogenated edible Fats, emulsifiers such as lecithin, sorbitan monooleate, acacia; or non-aqueous carriers (may contain edible oils) such as almond oil, oils such as glycerol, glycol, or ethanol; preservatives, Such as methyl or propyl paraben, sorbic acid. Flavoring or coloring agents may be added if desired.

Suppositories may contain conventional suppository bases such as cocoa butter or other glycerides.

For parenteral administration, liquid dosage forms are usually prepared from the compound and a sterile carrier. The preferred carrier is water. According to the selected carrier and drug concentration, the compound can be dissolved in the carrier or can be made into a suspension solution. When preparing an injection solution, the compound is first dissolved in water, filtered and sterilized, and then put into a sealed bottle or ampule.

It must be recognized that the optimal dosage and interval for administration of the compounds of general formula X and general formulae I to II are determined by the nature of the compound and external conditions such as the form of administration, the route of administration, and the particular mammal being treated, and this Optimal dosages for administration can be determined using conventional techniques. It must also be recognized that the optimal course of treatment, ie, the daily dose of Compound X over a given period of time, can be determined by methods well known in the art.

The specific embodiment of the present invention has the following beneficial effects:

The in vitro activity study of the pabendazole derivatives disclosed in the present invention shows that it has a good inhibitory effect on the proliferation of the three tested HNSCC cell lines, and it can inhibit tubulin polymerization, inhibit cell invasion, cell migration and block the cell cycle;

The in vivo activity study of the pabendazole derivatives disclosed in the present invention shows that the oral dose of 80 mg/kg can obviously control the growth of HN6 xenograft tumor nude mouse model, and has a good therapeutic effect. The detection of markers also proved its obvious curative effect;

The in vitro and in vivo activities show that the pabendazole derivative disclosed in the present invention is a potential molecule for treating HNSCC, and provides a certain basis for the research and development of drugs for treating HNSCC.

Description of drawings

The accompanying drawings forming a part of the present invention are used to provide further understanding of the present invention, and the exemplary embodiments of the present invention and their descriptions are used to explain the present invention, and do not constitute an improper limitation of the present invention.

555)用于标记微管蛋白，蓝色(DAPI)用于标记细胞核；比例尺为20μm；A为用DMSO处理的HN6细胞；B为用6q处理的HN6细胞；C为用帕苯达唑处理的HN6细胞；D为用6v处理的HN6细胞。Figure 1: Immunostaining of tubulin in HN6 cells. HN6 cells were treated with compound (100nmol/L) for 24h, and the microtubule structure was shown by immunofluorescence assay. red (Alexa

555) is used to label tubulin, blue (DAPI) is used to label nuclei; scale bar is 20 μm; A is HN6 cells treated with DMSO; B is HN6 cells treated with 6q; C is treated with pabendazole HN6 cells; D is HN6 cells treated with 6v.

Figure 2: 6q and 6v can effectively inhibit the migration and invasion of tumor cells. A: After compound treatment for 24 h, the cells grown in 6-well plates were photographed at 0 h and 24 h, respectively, to calculate the scratch healing rate; 200 μL pipette tips were used to make scratches, and photos were taken with an inverted fluorescence microscope; B: Pabendazole, 6q , 6v treatment of cell scratch healing rate; data are expressed as mean±SEM (n=3, *P<0.05, ***P<0.001); C is the cells that passed through the matrix after 24h treatment with or without compound Crystal violet staining; D is the invasion rate of cells by pabendazole, 6q, 6v; data are expressed as mean±SEM (n=3, ***P<0.001).

Figure 3 shows: 6q and 6v induce HN6 cell apoptosis and arrest the cell cycle in the G2/M phase. A is the cells treated with compounds for 24 h; Annexin-FITC and PI were selected to stain apoptotic cells, and the representative flow cytometry dot plots were shown; B was the percentage of apoptotic cells in each group; the data were expressed as the mean ± SEM ( n=3, *P<0.05, **P<0.01, ***P<0.001); C is the treatment of cells with compounds for 24 hours, PI staining solution was selected to stain DNA, and DNA content was detected by flow cytometry; HN6 cells had Representative DNA distribution histograms; D is cell cycle distribution, data are presented as mean ± SEM (n=3).

Figure 4 shows: 6q and 6v have better therapeutic effects on xenograft tumor models. A-D are the tumor volume and weight of mice; the tumor-transplanted mice were given pabendazole 80 mg/kg, 6q, 6v or solvent, once a day by gavage. The tumor volume was measured and the body weight was recorded; the results were expressed as standard deviation (n=5); B is the solid tumor that was surgically removed; E-G are the serum levels of CYFRA21-1, SCCAg, and TPS in nude mice; the blank group was normal tumor-free nude mice, The control group was untreated xenograft nude mice; the results were expressed as standard deviation (n=5); *P<0.05, **P<0.01.

Figure 5: Toxicity of compounds to HFF-1 and Kunming mice; A-C cell viability of HFF-1 cells treated with a series of concentrations of pabendazole, 6q and 6v for 72h; cell viability was detected by CCK8 method; data are averaged SEM representation, n=3. D and E survival curves of Pabendazole, 6q, 6v; M means male, F means female; the design and implementation of the experiment were carried out in accordance with the provisions of Korbor method and limit method test in GB 15193.3-2014.

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It should be noted that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural as well, furthermore, it is to be understood that when the terms "comprising" and/or "including" are used in this specification, it indicates that There are features, steps, operations, devices, components and/or combinations thereof.

The present invention will be further explained and illustrated below in conjunction with specific embodiments.

Example 1 : 1-(6-Butyl-1H-benzo[d]imidazol-2-yl)-3-ethylurea (6a)

Reagents and conditions: (I) 4-n-butylaniline, acetic anhydride, concentrated sulfuric acid, 50℃, 1h; nitric acid, 0℃, 4min; (II) KOH, 65℃, 1h; (III) H ₂ , Pd/ C, room temperature, 24h; (IV) ethyl isocyanate, N,N-diisopropylethylamine, room temperature, 48h; (V) _CH3COOH : _H2O =3:10, 100°C, 3h.

Step 1: Synthesis of N-(4-butyl-2-nitrophenyl)acetamide (2)

Acetic acid and a small amount of concentrated sulfuric acid were added to a 250 mL bottle, cooled to 0 °C, and compound 1 was added dropwise. After stirring at 50 °C for 1 h, the mixture was cooled to room temperature, and acetic anhydride was added. After that, the mixture was cooled to 0°C again, concentrated nitric acid was slowly added, and the mixture was stirred at 0°C for 40 min. The reaction solution was added to a large amount of ice water, and the produced yellow precipitate was filtered out and dried to obtain a crude product. The crude product was further purified by silica gel chromatography to give the product as a yellow solid. ¹ H NMR (400MHz, DMSO) δ 10.17 (s, 1H), 7.74 (d, J=1.1 Hz, 1H), 7.51 (d, 2H), 2.67–2.60 (m, 2H), 2.05 (s, 3H) ), 1.60–1.52 (m, 2H), 1.35–1.25 (m, 2H), 0.90 (t, J=7.3Hz, 3H). ESI-MS: m/z[M+H] ⁺ calcd for C ₁₂ H ₁₇ N ₂ O ₃ ⁺ 237.12, found 237.49. Mp 72.8–73.6°C.

Step 2: Synthesis of 4-butyl-2-nitroaniline (3)

In a 250 mL bottle, compound 2 was dissolved in methanol; 40% KOH solution (7.4 mL, 50.78 mmol) was added, and the mixture was stirred at 65 °C for 1 h. The reaction solution was dried over anhydrous Na ₂ SO ₄ and filtered, and the solvent was removed under reduced pressure. Purification by silica gel chromatography gave the product as a yellow oil. ¹ H NMR (400MHz, DMSO) δ 7.74 (s, 1H), 7.30 (s, 2H), 7.27 (d, 1H), 6.95 (d, J=8.6Hz, 1H), 2.47 (d, J=7.7 Hz, 2H), 1.54–1.44 (m, 2H), 1.34–1.23 (m, 2H), 0.89 (t, J=7.3Hz, 3H). ESI-MS: m/z[MH] ^- calcd for C ₁₀ H ₁₃ N ₂ O ₂ ^- 193.10, found 193.23.

Step 3: Synthesis of 4-butylbenzene-1,2-diamine (4)

In a 250 mL three-necked flask, compound 3, Pd/C and methanol were added. The entire system was sealed and replaced with nitrogen for air and hydrogen for nitrogen. The reaction solution was stirred at room temperature for 24 h, filtered through Pd/C, and concentrated to obtain an oily product. ¹ H NMR (400MHz, DMSO) δ 6.39 (d, J=7.7Hz, 1H), 6.33 (d, J=1.8Hz, 1H), 6.19 (dd, J=7.7, 1.8Hz, 1H), 4.26 ( s, 4H), 2.32 (t, J=7.6Hz, 2H), 1.48–1.40 (m, 2H), 1.31–1.22 (m, 2H), 0.87 (t, J=7.3Hz, 3H). ESI-MS :m/z[M+H] ⁺ calcd for C ₁₀ H ₁₇ N ₂ ⁺ 165.14, found 165.14.

Step 4: Synthesis of 1,3-bis(ethylaminocarbonyl)-2-methyl-2-thioisourea (5a)

To the mixture of 2-methyl-2-thiourea sulfate, DIPEA and acetonitrile, ethyl isocyanate was added dropwise. Stir at room temperature for 48 h, then remove the solvent under reduced pressure. Ethyl acetate and water were added, the mixture was fully stirred, and the layers were separated. The organic phase was washed twice with saturated sodium chloride solution, and then dried with anhydrous Na ₂ SO ₄ . The product was purified by silica gel chromatography to obtain a white oily substance 5a. ¹ H NMR (400 MHz, CDCl ₃ ) δ 12.59 (s, 1H), 5.40 (d, J=76.0 Hz, 2H), 3.27 (dq, J=13.8, 7.0 Hz, 4H), 2.31 (s, 3H) ,1.16(dt,J=15.4,7.6Hz,6H).ESI-MS: m/z[M+H] ⁺ calcd for C ₈ H ₁₇ N ₈ O ₂ S ⁺ 233.11, found232.79.

Step 5: Synthesis of 1-(6-butyl-1H-benzo[d]imidazol-2-yl)-3-ethylurea (6a)

Compounds 4, 5 and solvent (CH ₃ COOH:H ₂ O=3:10) were stirred at 100° C. for 3 h, the reaction solution was cooled to room temperature, and the pH was adjusted to basic with saturated aqueous NaHCO ₃ solution. Then, dichloromethane was added, and the mixture was sufficiently stirred to separate the layers. The organic phase was dried with anhydrous Na ₂ SO ₄ , filtered, concentrated, and purified by silica gel chromatography to obtain the product as a white solid product. White solid (48mg, 37.9% yield). ¹ HNMR (400MHz, DMSO) δ 11.31(s, 1H), 9.80(s, 1H), 7.37(s, 1H), 7.23(d, J=8.0Hz, 1H ),7.16(s,1H),6.85(d,J＝8.0Hz,1H),3.21(dt,J＝14.0,7.1Hz,2H),2.59(dd,J＝16.8,9.1Hz,2H),1.56 (dt, J＝15.1, 7.5Hz, 2H), 1.30 (dq, J＝14.6, 7.3Hz, 2H), 1.11 (t, J＝7.2Hz, 3H), 0.89 (t, J＝7.3Hz, 3H) . ¹³ C NMR (101MHz, DMSO) δ 154.69, 148.73, 135.05, 121.50, 35.57, 34.53, 34.39, 22.20, 15.77, 14.30. ESI-HRMS: m/z[M+H] ⁺ calcd for C ₁₄ H ₂₁ N ₄ O ⁺ 261.1715, found 267.1712. HPLC purity 99.1%, _tR =10.558min, 250mm×4.6mm, CH3OH: _{H2O =} 75:25, 0.8mL/min.Mp 253.6-254.9°C.

According to the same method as in Example 1, using different starting materials, the following compounds were synthesized.

Example 2 : 1-(6-Butyl-1H-benzo[d]imidazol-2-yl)-3-cyclohexylurea (6b)

White solid (36mg, 37.6% yield). ¹ H NMR (400MHz, DMSO) δ 11.31(s, 1H), 9.66(s, 1H), 7.50(s, 1H), 7.23(d, J=8.0Hz, 1H), 7.16(s, 1H), 6.85(d, J=8.0Hz, 1H), 3.63-3.54(m, 1H), 2.60(t, J=7.6Hz, 2H), 1.89-1.81(m, 2H) ), 1.72–1.64 (m, 2H), 1.60–1.51 (m, 3H), 1.37–1.21 (m, 7H), 0.89 (t, J=7.3Hz, 3H). ¹³ C NMR (101MHz, DMSO)δ153 .88,148.70,135.08,121.52,48.29,35.57,34.38,33.12,25.66,24.64,22.20,14.29.ESI-HRMS: m/z[M+H] ⁺ calcd for C ₁₈ H ₂₇ N ₄ O ⁺ 315.2185, found 315.2178 .HPLC purity 97.0%, _tR =6.551min, 250mm×4.6mm, CH3OH: _{H2O =} 75:25, 0.8mL/min.Mp 119.9-120.6°C.

Example 3 : 1-(6-butyl-1H-benzo[d]imidazol-2-yl)-3-isopropylurea (6c)

White solid (42 mg, 41.9% yield). ¹ H NMR (400 MHz, DMSO) δ 11.32 (s, 1H), 9.55 (s, 1H), 7.30 (d, J=32.2 Hz, 1H), 7.22 (d, J=7.8Hz, 1H), 7.15 (s, 1H), 6.84 (d, J=7.8Hz, 1H), 3.90–3.78 (m, 1H), 2.65–2.57 (m, 2H), 1.55 (dt, J ＝15.1,7.5Hz,2H),1.30(dd,J＝14.7,7.4Hz,2H),1.15(d,J＝6.5Hz,6H),0.89(t,J＝7.3Hz,3H).ESI-HRMS : m/z [M+H] ⁺ calcd for C ₁₅ H ₂₃ N ₄ O ⁺ 275.1872, found 275.1871. HPLC purity 95.8%, t _R = 9.114 min, 250 mm×4.6 mm, CH ₃ OH:H ₂ O=75: 25,0.8mL/min.Mp 144.5–147.4℃.

Example 4 : 1-(6-Butyl-1H-benzo[d]imidazol-2-yl)-3-propylurea (6d)

White solid (39mg, 46.7% yield). ¹ H NMR (400MHz, DMSO) δ 11.27(s, 1H), 9.78(s, 1H), 7.48(s, 1H), 7.23(d, J=8.0Hz, 1H), 7.15(s, 1H), 6.84(d, J=8.0Hz, 1H), 3.15(dd, J=12.8, 6.5Hz, 2H), 2.60(t, J=7.5Hz, 2H), 1.56( dd, J＝14.8, 7.4Hz, 2H), 1.48 (dd, J＝14.3, 7.2Hz, 2H), 1.30 (dd, J＝14.7, 7.4Hz, 2H), 1.18 (t, J＝11.8Hz, 2H) ), 0.90 (dt, J=12.0, 6.1 Hz, 6H). ESI-HRMS: m/z[M+H] ⁺ calcd for C ₁₅ H ₂₃ N ₄ O ⁺ 275.1872, found 275.1871. HPLCpurity 99.6%, t _R =9.261min,250mm×4.6mm,CH ₃ OH:H ₂ O=75:25,0.8mL/min.Mp 171.8–172.9℃.

Example ·: 1-(6-butyl-1H-benzo[d]imidazol-2-yl)-3-hexylurea (6e)

White solid (30mg, 31.1% yield). ¹ H NMR (400MHz, DMSO) δ 11.26(s, 1H), 9.71(s, 1H), 7.43(s, 1H), 7.23(d, J=8.0Hz, 1H), 7.15(s, 1H), 6.84(d, J=8.1Hz, 1H), 3.18(dd, J=12.8, 6.6Hz, 2H), 2.60(t, J=7.6Hz, 2H), 1.61– 1.52 (m, 2H), 1.51–1.44 (m, 2H), 1.35–1.26 (m, 8H), 0.88 (q, J=7.1Hz, 6H). ¹³ C NMR (101MHz, DMSO) δ 154.74, 148.73, 135.07 ,121.51,39.61,35.57,34.38,31.41,29.97,26.48,22.53,22.19,14.36,14.29.ESI-HRMS: m/z[M+H] ⁺ calcd for C ₁₈ H ₂₉ N ₄ O ⁺ 317.2341, found 317.2336 .HPLC purity 96.7%, _tR =7.152min, 250mm×4.6mm, CH3OH: _{H2O =} 75:25, 0.8mL/min.Mp 202.5-203.9°C.

Example 6 : 1-(6-Butyl-1H-benzo[d]imidazol-2-yl)-3-phenethylurea (6f)

White solid (52 mg, 37.0% yield). ¹ H NMR (400 MHz, DMSO) δ 11.30 (s, 1H), 9.73 (s, 1H), 7.47 (s, 1H), 7.33–7.26 (m, 4H), 7.24–7.20(m, 2H), 7.14(s, 1H), 6.84(d, J=8.1Hz, 1H), 3.43(q, J=13.2, 6.8Hz, 2H), 2.80(t, J=14.0, ¹³ C NMR (101MHz, DMSO) δ139.81, 139.51, 135.10, 129.20, 129.12, 128.86, 126.69, 126.64, 121.52, 41.27, 36.13, 35.56, 34.37, 22.19, 14.30. ESI-HRMS: m/z[M+H] ⁺ calc for C ₂₀ H ₂₅ N ₄ O ⁺ 337.2028, found337.2023. HPLC purity 98.9%, t _R = 11.688 min, 250 mm×4.6 mm, CH ₃ OH:H ₂ O=75:25, 0.8 mL/min. Mp 180.2 –181.7℃.

Example 7 : 1-(tert-butyl)-3-(6-butyl-1H-benzo[d]imidazol-2-yl)urea (6g)

Colorless transparent oil (62mg, 35.3% yield). ¹ H NMR (400MHz, DMSO) δ 11.37(s, 1H), 9.46(s, 1H), 7.23(d, J=8.0Hz, 2H), 7.15 (s, 1H), 6.84(d, J=8.0Hz, 1H), 2.60(t, J=7.6Hz, 2H), 1.59–1.51(m, 2H), 1.35(s, 9H), 1.28(d, J=4.6Hz, 2H), 0.89 (t, J=7.3Hz, 3H). ¹³ C NMR (101MHz, DMSO) δ 153.63, 148.59, 134.95, 121.40, 50.27, 35.60, 34.43, 29.29, 22.22, 14.30.ESI- HRMS: m/z [M+H] ⁺ calcd for C ₁₆ H ₂₅ N ₄ O ⁺ 289.2089, found 289.2022. HPLC purity 98.2%, t _R = 11.820 min, 250 mm×4.6 mm, CH ₃ OH:H ₂ O =75:25,0.8mL/min.Mp 84.4–86.9℃.

Example 8 : 1-(6-Butyl-1H-benzo[d]imidazol-2-yl)-3-cyclopentylurea (6h)

White solid (104mg, 55.0% yield). ¹ H NMR (400MHz, DMSO) δ 11.36(s, 1H), 9.60(s, 1H), 7.46(s, 1H), 7.23(d, J=8.0Hz, 1H), 7.15(s, 1H), 6.85(d, J=8.0Hz, 1H), 4.07–3.98 (m, 1H), 2.61 (t, J=7.0Hz, 2H), 1.92–1.85 (m, 2H) ),1.70–1.65(m,2H),1.60–1.53(m,4H),1.43(dd,J=12.2,6.2Hz,2H),1.31(dd,J=14.8,7.4Hz,2H),0.90( t, J＝7.3Hz, 3H). ¹³ CNMR(101MHz, DMSO)δ154.26,148.68,135.09,121.52,51.52,35.57,34.39,33.23,23.65,23.60,22.21,14.30.ESI-HRMS:m/z[M +H] ⁺ calcd for C ₁₇ H ₂₅ N ₄ O ⁺ 301.2028, found 301.2022. HPLC purity 95.6%, t _R = 18.058 min, 250 mm×4.6 mm, CH ₃ OH:H ₂ O=75:25, 0.8 mL /min.Mp 180.5–182.9℃.

Example 9 : 1-Allyl-3-(6-butyl-1H-benzo[d]imidazol-2-yl)urea (6i)

White solid (50 mg, 60.2% yield). ¹ H NMR (400 MHz, DMSO) δ 11.32 (s, 1H), 9.84 (s, 1H), 7.61 (s, 1H), 7.23 (d, J=8.0 Hz, 1H), 7.16(s, 1H), 6.85(d, J=8.0Hz, 1H), 5.97–5.85(m, 1H), 5.20(d, J=17.2Hz, 1H), 5.11(d, J=10.3 Hz, 1H), 3.91–3.81 (m, 2H), 2.63–2.58 (m, 2H), 1.63–1.51 (m, 2H), 1.37–1.25 (m, 2H), 0.89 (t, J=7.3Hz, 3H). ¹³ C NMR (101MHz, DMSO) δ154.71, 148.66, 136.19, 135.42, 135.15, 121.59, 115.45, 41.97, 35.56, 34.38, 22.20, 14.30. ESI-HRMS: m/z[M+H] ⁺ calcd for C ₁₅ H ₂₁ N ₄ O ⁺ 273.1715, found 273.1710. HPLC purity 99.7%, t _R = 15.687 min, 250 mm x 4.6 mm, CH ₃ OH:H ₂ O = 80:20, 0.8 mL/min. Mp 223.4 - 225.6℃.

Example 10 : Ethyl 2-(3-(6-butyl-1H-benzo[d]imidazol-2-yl)ureido)methacrylate (6j)

White solid (135 mg, 64.4% yield). ¹ H NMR (400 MHz, DMSO) δ 11.18 (s, 1H), 10.18 (dd, J=221.4, 53.7 Hz, 1H), 7.77 (s, 1H), 7.23 ( d, J=8.0Hz, 1H), 7.15(s, 1H), 6.86(d, J=8.1Hz, 1H), 6.12(s, 1H), 5.71(s, 1H), 4.20(t, J=5.3 Hz, 2H), 3.51(q, J=5.4Hz, 2H), 2.61(t, J=7.6Hz, 2H), 1.90(s, 3H), 1.61–1.51(m, 2H), 1.35–1.25(m , 2H), 0.89(t, J=7.3Hz, 3H). ¹³ C NMR(101MHz, DMSO)δ166.94,154.95,148.64,136.29,135.18,126.43,121.63,64.07,38.68,35.55,34.36,22.18,18.46, 14.29. ESI-HRMS: m/z [M+H] ⁺ calcd for C ₁₈ H ₂₅ N ₄ O ₃ ⁺ 345.1927, found 345.1919. HPLC purity 99.6%, t _R = 8.850 min, 250 mm×4.6 mm, CH ₃ OH: H ₂ O = 75:25, 0.8 mL/min. Mp 221.4–223.9°C.

Example 11 : Isobutyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate (6k)

White solid (51mg, 51.8% yield). ¹ H NMR (400MHz, DMSO) δ 11.57(s, 2H), 7.28(d, J=7.7Hz, 1H), 7.20(s, 1H), 6.89(d, J=8.0Hz, 1H), 3.92 (t, J=8.3Hz, 2H), 2.61 (t, J=7.5Hz, 2H), 2.01–1.90 (m, 1H), 1.59–1.51 (m, 2H), 1.35–1.26 (m, 2H), 0.93 (d, J=6.7Hz, 6H), 0.89 (t, J=7.3Hz, 3H). ¹³ C NMR (101MHz, DMSO) δ 135.61, 121.97, 71.33, 35.58, 34.39 , 28.04, 22.22, 19.35, 14.29. ESI-HRMS: m/z [M+H] ⁺ calcd for C ₁₆ H ₂₄ N ₃ O ₂ ⁺ 290.1869, found190.1863. HPLC purity 97.2%, t _R =16.446min, 250mm×4.6mm, CH ₃ OH:H ₂ O=75:25, 0.8mL/min.Mp 135.9–137.4℃.

Example 12 : Benzyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate (6l)

White solid (50 mg, 50.8%). ¹ H NMR (400 MHz, DMSO) δ 11.77 (s, 2H), 7.49–7.34 (m, 5H), 7.26 (d, J=8.1 Hz, 1H), 7.19 (s ,1H),6.89(d,J=8.0Hz,1H),5.24(s,2H),2.60(t,J=7.5Hz,2H),1.59–1.50(m,2H),1.29(dt,J= 14.5,7.3Hz,2H),0.89(t,J=7.3Hz,3H). 13CNMR( ^101MHz ,DMSO)δ155.89,148.61,137.00,135.78,128.93,128.47,122.14,113.46,113.04,66.87,35.54,34.35 , 22.20, 14.29. ESI-HRMS: m/z [M+H] ⁺ calcd for C ₁₉ H ₂₁ N ₃ O ₂ ⁺ 324.1712, found 324.1704. HPLC purity 99.8%, t _R = 15.432 min, 250 mm × 4.6 mm, CH ₃ OH:H ₂ O=75:25, 0.8 mL/min. Mp 199.2–201.7°C.

Example 13 : 2-Chloroethyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate (6m)

White solid (41mg, 44.6% yield). ¹ H NMR (400MHz, DMSO) δ 11.88(s, 1H), 7.35(d, J=8.1Hz, 1H), 7.27(s, 1H), 6.93(d, J＝8.1Hz, 1H), 4.57(t, J＝8.0Hz, 2H), 4.20(t, J＝8.0Hz, 2H), 2.63(t, J＝7.6Hz, 2H), 1.62–1.53(m, 2H), 1.37–1.26 (m, 2H), 0.90 (t, J=7.3Hz, 3H). ¹³ C NMR (101MHz, DMSO) δ155.05, 145.48, 135.79, 122.28, 117.20, 111.37, 63.97, 44.38, 35.59, 34.35, 22.20, 14.29. ESI-HRMS: m/z [M+H] ⁺ calcd for C ₁₄ H ₁₉ ClN ₃ O ₂ ⁺ 296.1166, found 315.2178. HPLC purity 99.8%, t _R = 8.219 min, 250 mm×4.6 mm, CH ₃ OH:H ₂ O=75:25, 0.8 mL/min. Mp 199.2–201.7°C. Mp 182.8–184.7°C.

Example 14 : Propyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate (6n)

White solid (38mg, 45.8% yield). 1H NMR (400MHz, DMSO) δ 11.48(s, 2H), 7.26(s, 1H), 7.18(s, 1H), 6.90(s, 1H), 4.11(s ,2H),2.61(s,2H),1.61(d,J=39.7Hz,4H),1.31(s,2H),0.91(d,J=6.5Hz,6H).13C NMR(101MHz,DMSO)δ121 .96, 66.91, 35.57, 34.38, 22.31, 22.21, 14.30, 10.70. ESI-HRMS: m/z[M+H]+calcd for C15H22N3O2+276.1712, found276.1705.HPLC purity 99.3%, tR=9.068min, 250mm×4.6mm, CH3OH:H2O＝80:20,0.8mL/min.Mp 204.5-206.8℃.

Example 15 : Hexyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate (6o)

White solid (56mg, 60.0% yield). ¹ H NMR (400MHz, DMSO) δ 11.50(s, 2H), 7.26(d, J=8.1Hz, 1H), 7.18(s, 1H), 6.89(d, J=8.1Hz, 1H), 4.14 (t, J=6.6Hz, 2H), 2.65–2.58 (m, 2H), 1.65 (dd, J=13.7, 6.9Hz, 2H), 1.55 (dd, J=15.1 , 7.7Hz, 2H), 1.38–1.26 (m, 8H), 0.93–0.86 (m, 6H). ¹³ C NMR (101MHz, DMSO) δ135.49, 121.90, 65.39, 35.57, 34.38, 31.37, 28.90, 25.44, 22.49 , 22.21, 14.35, 14.30. ESI-HRMS: m/z[M+H] ⁺ calcd for C ₁₈ H ₂₈ N ₃ O ₂ ⁺ 318.2182, found318.2173. HPLC purity 99.4%, t _R =12.993min, 250mm× 4.6mm, CH ₃ OH:H ₂ O=75:25, 0.8mL/min.Mp 201.2–203.5℃.

Example 16 : Butyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate (6p)

White solid (53mg, 50.1% yield). ¹ H NMR (400MHz, DMSO) δ 11.58(s, 2H), 7.28(d, J=7.8Hz, 1H), 7.20(s, 1H), 6.89(d, J=7.7Hz, 1H), 4.15 (t, J=6.1Hz, 2H), 2.61 (t, J=6.8Hz, 2H), 1.67–1.51 (m, 4H), 1.43–1.27 (m, 4H), 0.96–0.85(m, 6H). ¹³ C NMR(101MHz, DMSO)δ148.23,135.55,121.94,113.61,65.15,35.58,34.39,31.00,22.22,19.03,14.30,14.08.ESI-HRMS:m/z[M +H] ⁺ calcd for C ₁₆ H ₂₄ N ₃ O ₂ ⁺ 290.1869, found 290.1860. HPLC purity 99.3%, t _R = 10.557 min, 250 mm×4.6 mm, CH ₃ OH:H ₂ O=80:20, 0.8 mL/min.Mp 211.7–213.2℃

Example 17 : Isopropyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate (6q)

White solid (49 mg, 58.5% yield). 1H NMR (400 MHz, DMSO) δ 11.65(s, 2H), 7.30(d, 1H), 7.23(s, 1H), 6.91(d, 1H), 5.09–4.90 (m, 1H), 2.62 (t, 2H), 1.67–1.49 (m, 2H), 1.43–1.20 (m, 8H), 0.90 (t, 3H). 13C NMR (101MHz, DMSO) δ 154.72, 147.98, 135.56 , 121.94, 113.54, 69.21, 35.60, 34.44, 22.32, 22.24, 14.29. ESI-HRMS: m/z[M+H]+calcd for C15H22N3O2+276.1712, found 276.1706.HPLC purity 99.6%, t _R =8.792min, 250mm ×4.6mm, CH ₃ OH:H ₂ O=80:20, 0.8mL/min.Mp 122.4–125.1℃.

Example 18 : Heptyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate (6r)

White solid (91mg, 75.2% yield). ¹ H NMR (400MHz, DMSO) δ 11.45(s, 2H), 7.26(d, J=7.5Hz, 1H), 7.18(s, 1H), 6.89(d, ^13C NMR(101MHz,DMSO)δ135.56,121.97,65.43,35.56,34.36,31.66,28.92,28.80,25.73,22.48,22.20,14.40,14.29.ESI-HRMS:m/z[M+H] ⁺ calcd for C ₁₉ H ₃₀ N ₃ O ₂ ⁺ 332.2338, found 332.2328. HPLC purity 97.8%, t _R =12.155min, 250mm×4.6mm, CH ₃ OH:H ₂ O=85:15, 0.8mL/min.Mp 194.1-195.2°C.

Example 19 : Cyclopentyl (6-butyl-lH-benzo[d]imidazol-2-yl)carbamate (6s)

White solid (45 mg, 49.0% yield). ¹ H NMR (400 MHz, DMSO) δ 11.56 (s, 2H), 7.30–7.26 (m, 1H), 7.20 (s, 1H), 6.89 (d, J=8.1 Hz,1H),5.16(t,J＝5.7Hz,1H),2.62(t,J＝7.6Hz,2H),1.92(dd,J＝12.5,6.5Hz,2H),1.76–1.66(m,4H) ), 1.63–1.52 (m, 4H), 1.36–1.27 (m, 2H). ¹³ C NMR (101MHz, DMSO) δ155.12, 148.05, 135.54, 121.92, 78.24, 35.59, 34.42, 32.74, 23.79, 22.24, 14.30. ESI-HRMS: m/z[M+H] ⁺ calcd for C ₁₇ H ₂₄ N ₃ O ₂ ⁺ 302.1869, found302.1860. HPLC purity 99.6%, t _R = 11.135 min, 250 mm×4.6 mm, CH ₃ OH: H ₂ O = 80:20, 0.8 mL/min. Mp 171.8–174.8°C.

Example 20 : 3-Chloropropyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate (6t)

White solid (40mg, 42.4% yield). ¹ H NMR (400MHz, DMSO) δ 11.58(s, 2H), 7.27(d, J=8.1Hz, 1H), 7.19(s, 1H), 6.91(d, J＝8.1Hz, 1H), 4.26(t, J＝6.2Hz, 2H), 3.75(t, J＝6.4Hz, 2H), 2.62(t, J＝7.6Hz, 2H), 2.16–2.05(m, 2H), 1.61–1.51 (m, 2H), 1.35–1.25 (m, 2H), 0.90 (t, J=7.3Hz, 3H). ¹³ C NMR (101MHz, DMSO) δ 155.01, 151.30, 148.54, 147.31, 136.80 , 123.02, 113.40, 112.97, 62.82, 42.23, 35.47, 34.24, 31.84, 22.17, 14.27. ESI-HRMS: m/z[M+H] ⁺ calcd for C ₁₆ H ₂₁ ClN ₃ O ₂ ⁺ 310.1322, found 310.1314. HPLC purity 99.6%, _tR =8.599min, 250mm×4.6mm, CH3OH: _{H2O =} 80:20, 0.8mL/min.Mp 184.2-185.9°C.

Example 21 : sec-butyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate (6u)

White solid (46 mg, 52.2% yield). ¹ H NMR (400 MHz, CDCl ₃ ) δ 10.68 (s, 1H), 7.56 (s, 1H), 7.25 (s, 1H), 7.02 (d, J=7.9 Hz ,1H),5.08–4.98(m,1H),2.71(t,J=7.7Hz,2H),1.95–1.83(m,1H),1.80–1.69(m,1H),1.69–1.60(m,2H ),1.44(d,J＝6.3Hz,3H),1.38(dt,J＝12.9,6.5Hz,2H),1.02(t,J＝7.4Hz,3H),0.94(t,J＝7.3Hz,3H) ).ESI-HRMS: m/z[M+H] ⁺ calcd for C ₁₆ H ₂₄ N ₃ O ₂ ⁺ 290.1869, found 290.1862. HPLC purity 99.4%, t _R = 10.562 min, 250 mm×4.6 mm, CH ₃ OH :H ₂ O=80:20,0.8mL/min.

Example 22 : Allyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate (6v)

White solid (36mg, 43.3% yield). ¹ H NMR (400MHz, DMSO) δ 11.58(s, 2H), 7.26(d, J=7.9Hz, 1H), 7.18(s, 1H), 6.90(d, J=7.8Hz, 1H), 6.05–5.94 (m, 1H), 5.38 (d, J=17.2Hz, 1H), 5.25 (d, J=10.2Hz, 1H), 4.67 (d, J=4.2Hz, 2H), 2.62(t, J＝7.2Hz, 2H), 1.60–1.50(m, 2H), 1.31(q, J＝14.2, 7.0Hz, 2H), 0.90(t, J＝7.1Hz, 3H). ¹³ C NMR (101MHz, DMSO) δ 135.69, 133.61, 122.05, 118.14, 65.79, 35.54, 34.34, 22.20, 14.29. ESI-HRMS: m/z[M+H] ⁺ calcd for C ₁₅ H ₂₀ N ₃ O ₂ ⁺ 274.1556 , found 274.1550.HPLC purity 98.8%, _tR =8.042min, 250mm×4.6mm, CH3OH: _{H2O =} 80:20, 0.8mL/min.Mp 211.2-212.9℃.

Example 23 : 2-Ethylhexyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate (6w)

Colorless oil (39 mg, 38.9% yield). ¹ H NMR (400 MHz, DMSO) δ 11.58 (d, J=32.0 Hz, 2H), 7.28 (dd, J=8.0, 3.1 Hz, 1H), 7.20 ( d, J=2.6Hz, 1H), 6.90 (d, J=8.1Hz, 1H), 4.07 (dd, J=11.0, 7.8Hz, 2H), 2.62 (t, J=7.6Hz, 2H), 1.64– 1.52 (m, 3H), 1.38–1.26 (m, 10H), 0.91–0.86 (m, 9H). ¹³ C NMR (101MHz, DMSO) δ 155.72, 148.22, 135.58, 121.97, 113.54, 67.59, 38.97, 35.58, 34.39 , 30.20, 28.85, 23.61, 22.92, 22.22, 14.38, 14.29, 11.29. ESI-HRMS: m/z [M+H] ⁺ calcd for C ₂₀ H ₃₂ N ₃ O ₂ ⁺ 346.2495, found 346.2488. HPLC purity 97.1% , t _R =8.608min,250mm×4.6mm,CH ₃ OH:H ₂ O =90:10,0.8mL/min.Mp 61.3-63.4℃.

Example 24 : N-(6-Butyl-1H-benzo[d]imidazol-2-yl)furan-2-carboxamide (6x)

White solid (39mg, 45.2% yield). ¹ H NMR (400MHz, DMSO) δ 12.10(s, 2H), 7.89(s, 1H), 7.37(s, 1H), 7.32(d, J=7.6Hz, 1H), 7.23(s, 1H), 6.97(d, J=7.6Hz, 1H), 6.66(s, 1H), 2.67–2.59(m, 2H), 1.61–1.51(m, 2H), 1.36–1.27 (m, 2H), 0.90 (t, J=7.3 Hz, 3H). ESI-HRMS: m/z [M+H] ⁺ calcd for C ₁₆ H ₁₈ N ₃ O ₂ ⁺ 284.1399, found 284.1393. HPLC purity 98.2 %, t _R =7.137min,250mm×4.6mm,CH ₃ OH:H ₂ O =80:20,0.8mL/min.Mp 149.2-152.3℃.

Example 25 : N-(6-Butyl-1H-benzo[d]imidazol-2-yl)-2-phenylacetamide (6y)

White solid (45 mg, 48.1% yield). ¹ H NMR (400 MHz, DMSO) δ 11.99 (s, 2H), 7.38–7.25 (m, 7H), 6.92 (d, J=11.5 Hz, 1H), 3.72 ( s, 2H), 2.63 (t, J=15.1, 7.6Hz, 2H), 1.61–1.51 (m, 2H), 1.34–1.27 (m, 2H), 0.89 (t, J=7.3Hz, 3H). ¹³ C NMR (101MHz, DMSO) δ 170.76, 146.84, 135.73, 129.67, 128.84, 127.23, 42.78, 35.60, 34.38, 22.22, 14.30. ESI-HRMS: m/z[M+H] ⁺ calcd for C ₁₉ H ₂₂ N ₃ O ⁺ 308.1763, found 315.2178. HPLC purity 98.3%, _tR =9.064min, 250mm×4.6mm, CH3OH: _{H2O =} 80:20, 0.8mL/min.Mp 171.0-173.0°C.

Example 26 : N-(6-Butyl-1H-benzo[d]imidazol-2-yl)tetrahydro-2H-pyran-4-carboxamide (6z)

White solid (48 mg, 52.3% yield). ¹ H NMR (400 MHz, DMSO) δ 11.92 (s, 1H), 11.41 (s, 1H), 7.31 (d, J=8.1 Hz, 1H), 7.23 (s, 1H), 6.91(d, J=8.1Hz, 1H), 3.91(d, J=11.3Hz, 2H), 3.35(dd, J=8.5, 6.1Hz, 2H), 2.79–2.72(m, 1H), 2.62(t,J=7.6Hz,2H),1.78-1.66(m,4H),1.59-1.53(m,2H),1.35-1.28(m,2H),0.90(t,J=7.3Hz,3H) . ¹³ C NMR (101MHz, DMSO) δ174.60, 147.01, 135.57, 122.10, 66.71, 41.19, 35.61, 34.39, 29.02, 22.23, 14.30. ESI-HRMS: m/z[M+H] ⁺ calcd for C ₁₇ H ₂₄ N ₃ O ₂ ⁺ 302.1869, found 302.1861. HPLC purity 99.0%, t _R =7.284min, 250mm×4.6mm, CH ₃ OH:H ₂ O=80:20, 0.8mL/min.Mp 158.0-159.2°C.

Example 27 : N-(6-Butyl-1H-benzo[d]imidazol-2-yl)-2-(thiophen-2-yl)acetamide (6aa)

Brown oil (23 mg, 22.9% yield). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.44 (d, J=8.2 Hz, 1H), 7.31 (s, 1H), 7.25-7.21 (m, 1H), 7.06(d,J＝8.2Hz,1H),6.98(d,J＝3.3Hz,2H),4.48(s,2H),2.70(t,J＝7.7Hz,2H),1.65–1.58(m,2H) _The ^_ 30.04, 22.31, 13.97. ESI-HRMS: m/z [M+H] ⁺ calcd for C ₁₇ H ₂₀ N ₃ OS ⁺ 314.1327, found 315.2178. HPLC purity 98.7%, t _R =8.935min, 250mm×4.6mm , CH ₃ OH:H ₂ O=80:20, 0.8 mL/min.

Example 28 : Isopropyl (6-cyano-1H-benzo[d]imidazol-2-yl)carbamate (6ab)

Gray solid (60 mg, 65.4% yield). ¹ H NMR (400 MHz, DMSO) δ 12.26 (s, 1H), 11.59 (s, 1H), 7.84 (s, 1H), 7.56 (d, J=8.2 Hz, 1H), 7.48(d, J=8.2Hz, 1H), 5.08–4.96(m, 1H), 1.32(d, J=6.3Hz, 6H). ¹³ C NMR (101MHz, DMSO) δ153.81, 150.04, 125.29, 120.73, 69.98, 22.20. ESI-HRMS: m/z [M+H] ⁺ calcd for C ₁₂ H ₁₃ N ₄ O ₂ ⁺ 245.1039, found 245.1031. HPLC purity 99.7%, t _R = 4.666 min, 250 mm×4.6 mm, CH ₃ OH:H ₂ O=80:20, 0.8mL/min. Mp>300℃.

Example 29 : Isopropyl (6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)carbamate (6ac)

Gray solid (56 mg, 68.7% yield). ¹ H NMR (400 MHz, DMSO) δ 11.96 (s, 2H), 7.76 (s, 1H), 7.60 (d, J=8.3 Hz, 1H), 7.41 (d, J=8.3Hz, 1H), 5.09–4.97 (m, 1H), 1.34 (s, 3H), 1.32 (s, 3H). ¹³ C NMR (101MHz, DMSO) δ 153.91, 149.68, 127.05, 124.36, 118.13, 69.87 , 22.19. ESI-HRMS: m/z[M+H] ⁺ calcd for C ₁₂ H ₁₃ F ₃ N ₃ O ₂ ⁺ 288.0960, found 288.0952. HPLCpurity 99.8%, t _R =7.905min, 250mm×4.6mm, CH ₃ OH:H ₂ O=75:25, 0.8 mL/min. Mp 202.2–204.9°C.

Example 30 : Isopropyl (6-methyl-1H-benzo[d]imidazol-2-yl)carbamate (6ad)

White solid (33mg, 34.6% yield). ¹ H NMR (400MHz, DMSO) δ 11.67(s, 2H), 7.29(d, J=8.0Hz, 1H), 7.23(s, 1H), 6.89(d, J=8.1Hz, 1H), 5.06–4.92 (m, 1H), 2.36 (s, 3H), 1.31 (d, J=6.2Hz, 6H). ¹³ C NMR (101MHz, DMSO) δ 154.69, 147.92, 130.28, 122.54, 69.23, 22.32, 21.72. ESI-HRMS: m/z [M+H] ⁺ calcd for C ₁₂ H ₁₆ N ₃ O ₂ ⁺ 234.1243, found 234.1236. HPLC purity 99.9%, t _R = 6.709 min, 250 mm ×4.6mm, CH ₃ OH:H ₂ O=75:25, 0.8mL/min.Mp 195.0–197.0℃.

Example 31 : Isopropyl (6-fluoro-1H-benzo[d]imidazol-2-yl)carbamate (6ae)

White solid (39 mg, 41.5% yield). ¹ H NMR (400 MHz, DMSO) δ 11.70 (s, 2H), 7.39 (dd, J=8.6, 5.0 Hz, 1H), 7.20 (dd, J=9.6, 2.2 Hz,1H),6.94–6.87(m,1H),5.06–4.94(m,1H),1.31(d,J=6.3Hz,6H).ESI-HRMS: m/z[M+H] ⁺ calcd for C ₁₁ H ₁₃ FN ₃ O ₂ ⁺ 238.0992, found 238.0985. HPLC purity 99.9%, t _R = 6.020 min, 250 mm×4.6 mm, CH ₃ OH:H ₂ O=75:25, 0.8 mL/min. Mp> 300℃.

Example 32 : Isopropyl (6-pentyl-1H-benzo[d]imidazol-2-yl)carbamate (6af)

White solid (61 mg, 755.2% yield). ¹ H NMR (400 MHz, DMSO) δ 7.27 (dd, J=8.0, 3.7 Hz, 1H), 7.19 (s, 1H), 6.89 (d, J=8.1 Hz, 1H), 5.03–4.91 (m, 1H), 2.61 (t, J=7.6Hz, 2H), 1.63–1.51 (m, 2H), 1.37–1.22 (m, 10H), 0.86 (t, J=6.7Hz) ,3H) ^.13C NMR(101MHz,DMSO)δ154.79,147.96,135.56,121.92,69.16,35.87,31.87,31.38,22.46,22.32,14.40.ESI-HRMS:m/z[M+H] ⁺ calcd for C ₁₆ H ₂₄ N ₃ O ₂ ⁺ 290.1869, found 290.1864. HPLC purity 99.8%, t _R = 17.844 min, 250 mm x 4.6 mm, CH ₃ OH:H ₂ O = 75:25, 0.8 mL/min. Mp 128.0-129.0 °C.

Example 33 : Isopropyl (5,6-difluoro-1H-benzo[d]imidazol-2-yl)carbamate (6ag)

Pale yellow solid (51 mg, 51.6% yield). ¹ H NMR (400 MHz, DMSO) δ 11.98 (s, 1H), 11.37 (s, 1H), 7.39 (t, J=9.2 Hz, 2H), 5.06–4.93 (m, 1H), 1.31 (d, J=6.2Hz, 6H). ¹³ C NMR (101MHz, DMSO) δ 153.83, 148.83, 69.72, 22.21. ESI-HRMS: m/z[M+H] ⁺ calcd for C ₁₁ H ₁₂ F ₂ N ₃ O ₂ ⁺ 256.0898, found 256.0893. HPLC purity 99.7%, t _R = 6.511 min, 250 mm×4.6 mm, CH ₃ OH:H ₂ O=75:25, 0.8 mL/min. Mp> 300℃.

Example 34 : Isopropyl (5,6-dimethyl-1H-benzo[d]imidazol-2-yl)carbamate (6ah)

White solid (42 mg, 47.4% yield). ¹ H NMR (400 MHz, DMSO) δ 11.52 (s, 2H), 7.18 (s, 2H), 5.04–4.90 (m, 1H), 2.25 (s, 6H), 1.30(d, J=6.1Hz, 6H). ¹³ C NMR (101 MHz, DMSO) δ 155.02, 147.56, 129.30, 114.38, 69.04, 22.35, 20.33. ESI-HRMS: m/z[M+H] ⁺ calcd for C ₁₃ H ₁₈ N ₃ O ₂ ⁺ 248.1399, found 248.1393. HPLC purity 99.6%, t _R =7.806min, 250mm×4.6mm, CH ₃ OH:H ₂ O=75:25, 0.8mL/min.Mp>300°C.

Example 35 : Compound Inhibition of CAL-27, HN6 and Fadu Cell Proliferation Activity Assay

This example is used to determine the inhibitory activity of the pabendazole derivatives of the present invention on the proliferation of HNSCC cell lines CAL-27, HN6 and Fadu cells. The experimental protocol was as follows: cells were seeded in a 96-well transparent plate at a concentration of 5000 cells/well, 100 μL per well. Incubate at 37°C for 12h with 5% CO ₂ . Then, a series of compound solutions (100 μL) prepared in serum-free medium were added to the wells, and the culture was continued for 72 h. The CCK8 method was used to evaluate the cell viability, and the CLARIOstar microplate reader of BMG Company was used to record the absorbance value at 450 nm. IC50 values were calculated with GraphPad prism 7.00. The IC ₅₀ values of the test compounds of the present invention for inhibiting tumor cell proliferation are shown in the following table.

Table 1 Pabendazole derivatives inhibit HN6 cell proliferation activity and inhibit tubulin polymerization activity

Table 2 Pabendazole derivatives inhibit the proliferation activity of HN6, CAL-27, Fadu cells

Conclusion: The in vitro cell proliferation of the compounds in the present invention shows that compounds 6q and 6v are superior to pabendazole in activity, which provides ideas for designing new drugs.

Example 36 : In vitro tubulin polymerization assay

This example is used to determine the inhibitory activity of the pabendazole derivatives of the present invention on tubulin polymerization in vitro. The experimental protocol was as follows: The assay was performed using a tubulin polymerization kit according to the manufacturer's protocol. Tubulin, compound (20 μM) and GTP were mixed into wells at 37°C. As the microtubules polymerize, reporter groups are added to the microtubules, and the fluorescence gradually increases. The fluorescence signal was recorded with BMG CLARIOstar microplate reader every minute within 1 h, and the IC ₅₀ of the compound inhibiting microtubule polymerization was calculated by the tubulin polymerization rate. The test results are shown in Table 1.

Conclusion: 6n, 6q, 6u, 6v, 6z, 11e, 11g and many other compounds have better inhibitory activity on tubulin polymerization than pabendazole alone, which provides ideas for designing new compounds.

Example 37 : Determination of the effect of derivatives 6q, 6v on the structure of cellular tubulin

555Conjugate)，室温避光孵育1h。用DAPI(10μg/mL)对细胞核进行染色。最后，利用高速共聚焦平台采集图像。This example is used to determine the effect of derivatives 6q, 6v on the structure of cellular tubulin. The experimental protocol was as follows: HN6 cells were seeded in a laser confocal culture dish (20,000 cells/culture dish), and 100 nmol/L compound solution was added after 12 h. After culturing for 24 h, the medium was discarded, 4% paraformaldehyde was added, and the cells were fixed at room temperature for 15 min. After washing 3 times with PBS, cells were blocked in blocking solution (5% normal goat serum and 0.3% Triton X-100 PBS) for 1 h. After discarding the blocking solution, α-tubulin antibody was added and the cells were incubated overnight at 4°C. Wash the dishes 3 times with PBS, add Anti-Rabbit IgG(H+L), F(ab')2Fragment(Alexa

555Conjugate), incubated at room temperature for 1 h in the dark. Nuclei were stained with DAPI (10 μg/mL). Finally, images are acquired using a high-speed confocal platform.

Conclusion: It can be seen from Figure 1 that the cells treated with DMSO (Figure 1A) have good morphology, and tubulin forms a regular network structure; Following 6v (Fig. 1D) treatment, cells lost the regular tubulin network and most tubulin aggregated around the nucleus.

Example 38 : Determination of the effect of derivatives 6q, 6v on cell invasion and cell migration

This example was used to determine the effects of derivatives 6q, 6v on cell invasion and cell migration. The experimental protocol was as follows: Cell migration assay: HN6 cells were seeded in 6-well plates at 1×10 ⁶ cells per well. After 12 h of incubation, a 200 μL pipette tip created a scratch. The wells were washed three times with serum-free medium, and the scratches were photographed with an inverted fluorescence microscope (IX73+DP80, objective lens: 10×). Then, the compound solution prepared in 0.1 μmol/L serum-free medium was added, and the wound healing was recorded again after 24 h of culture. Calculate the percentage of scratch healing. Cell Invasion Assay: Basement membrane matrix (Corning, cat#356234) was diluted 20-fold with serum-free medium, and 100 μL was added to the upper chamber cell culture insert (24-well plate, 8.0 μm pore size, Corning, cat#353097). Then, the chamber was placed at 37°C for 5 h, and the liquid was then aspirated. The chamber was washed once with serum-free medium, and 100 μL of compound solutions prepared in serum-free medium were added. HN6 cells (100 μL, 10 ⁵ cells/well) were added to the upper chamber cell culture dish, and 600 μL serum-containing medium was added to the lower chamber. After culturing for 24 h, the chamber was removed from the 24-well plate, and the cells in the upper layer were gently wiped. Wash once with PBS and fix with 4% paraformaldehyde solution for 10 min at room temperature. After washing twice with PBS, stained with 0.1% crystal violet solution for 10 min, washed, inverted, air-dried and photographed. Finally, the crystal violet was dissolved in 33% acetic acid, and the absorbance was measured at a wavelength of 570 nm.

Conclusions: After 24 h of treatment with pabendazole, 6q or 6v, the rate of scratch healing was tested. As can be seen from Figure 2, compared with the control group (44.0%), the scratch healing rate of the pabendazole group (12.8%), 6q group (6.27%) and 6v group (8.1%) was significantly reduced. Furthermore, 6q inhibited cell migration more significantly than pabendazole. In the cell invasion assay, the compound also significantly inhibited the process of cell invasion across the matrix at a concentration of 200 nmol/L. When the concentration was increased to 500nmol/L, only a few cells in the pabendazole group could penetrate the matrix, while almost no cells in the 6q and 6q groups could successfully invade the lower layer. Taken together, 6q and 6v can effectively inhibit tumor cell migration and invasion.

Example 39 : Determination of the effect of derivatives 6q, 6v on apoptosis and cell cycle

This example is used to determine the effects of derivatives 6q, 6v on apoptosis and cell cycle. The experimental protocol is as follows: Apoptosis assay: HN6 cells were seeded in 6-well plates with 300,000 cells per well. After 12 h, cells were treated with 0.1 μmol/L or 1 μmol/L compound solution for 24 h and collected, washed once with PBS and once with binding buffer. Then 100 μL of binding buffer was added to suspend the cells, 5 μL of Annexin FITC and 5 μL of PI staining solution were added, and the cells were incubated at room temperature for 15 min in the dark. Staining results were analyzed by flow cytometry (CytoFLEXS). Cell cycle assay: HN6 cells were seeded in 6-well plates with 300,000 cells per well. After 12 h, cells were treated with compound solution prepared in 0.1 μmol/L serum-free medium for 24 h, cells were collected, washed with PBS, and fixed with 70% ethanol overnight. Washed once with PBS, resuspended in 100 μL of RNase A solution, and stained in a water bath at 37 °C for 30 min. Add 400 μL of PI staining solution, incubate in the dark at 4°C for 30 min, and analyze the staining results by flow cytometry (CytoFLEX S).

Conclusion: As can be seen from Figure 3, compared with the control group, both 0.1 μmol/L and 1 μmol/L compounds can effectively induce cell apoptosis after 24 h of treatment. As the concentration increased, the proportion of apoptotic cells also increased. Given the important role of tubulin in cell mitosis, the effects of compounds on the cell cycle were also evaluated. Apparently, either 0.1 μmol/L 6q or 6v could successfully arrest the cell cycle in G2/M phase. In conclusion, 6q and 6v can effectively induce HN6 cell apoptosis and arrest the cell cycle in G2/M phase, thereby inhibiting the pathological process of tumor.

Example 40 : Determination of therapeutic effect of derivatives 6q, 6v on xenograft tumor model nude mice

This example is used to determine the therapeutic effect of derivatives 6q, 6v on xenograft tumor model nude mice. The experimental protocol was as follows: 4-week-old male nude mice, after 5 days of adaptive feeding, were subcutaneously injected with 1 million HN6 tumor cells to grow to an appropriate size. Mice were randomly divided into 4 groups with 5 mice in each group. Pabendazole, 6q, 6v powder and 0.5% sodium carboxymethyl cellulose solution were prepared into a suspension, which was administered by gavage once a day for 2 consecutive weeks. The tumor size was measured with an electronic caliper, and the content of SCCAg, TPS and CYFRA21-1 in serum was measured by ELISA. All procedures were performed in accordance with the Laboratory Animal Care and Use Guidelines of the Laboratory Animal Research Institute.

It can be seen from Figure 4 that 6q and 6v have better therapeutic effects on the xenograft tumor model. Conclusion: It can be seen from Figure 4 that the compound can effectively control the growth of tumor volume in nude mice, and there is no significant decrease in body weight, showing its good efficacy and biocompatibility at therapeutic doses. The serum levels of all three tumor markers were significantly increased in nude mice transplanted with tumors. After two weeks of treatment, tumor growth was effectively controlled, and serum levels of three tumor markers were significantly reduced.

Example 41 : Acute toxicity of derivatives 6q, 6v to SPF Kunming mice and determination of toxicity to human skin fibroblasts (HFF-1)

This example is used to determine the acute toxicity of derivatives 6q, 6v to SPF Kunming mice and the toxicity to human skin fibroblasts (HFF-1). After adaptive feeding, mice were randomly divided into two groups, male and female, with 10 mice in each group. Compounds were prepared as a suspension with 0.5% sodium carboxymethyl cellulose solution, and each 0.4 mL was administered by gavage. Fasting for 6h before gavage and free drinking water; fasting for 2h after gavage. Mice were observed and their body weight and survival were recorded. The procedure for the determination of the toxicity of derivatives 6q, 6v on human skin fibroblasts (HFF-1) is shown in Example 35.

Conclusion: As can be seen from Figure 5, the mice given 1g/kg pabendazole or 6v had no adverse reactions, and were in good condition without death. Mice given 150 mg/kg or 250 mg/kg 6q did not show any adverse effects and remained active until the end of the experiment without dying. Of the mice dosed with 630 mg/kg 6q, one female and one male survived. Mice given 1 g/kg 6q developed symptoms of weakness, lethargy and hypothermia after 3 hours, and mice given 630 mg/kg 6q developed the above after 12 hours. After calculation, the LD ₅₀ values of pabendazole and 6v were both greater than 1 g/kg, and the LD ₅₀ value of 6q was 341 mg/kg. The LD ₅₀ of 6q was much higher than its effective therapeutic dose, and the mice showed no abnormality at the therapeutic dose.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. a Pabendazole derivative, is characterized in that, the structure of described Pabendazole derivative is as shown in formula X:

Wherein, R ₁ is selected from alkoxy or alkylamino; R ₂ is selected from fluorine, cyano, methyl, trifluoromethyl, n-butyl or n-pentyl; R ₃ is selected from hydrogen, methyl or fluorine. 2. Pabendazole derivative as claimed in claim 1, is characterized in that, described Pabendazole derivative has the structure shown in formula (I) or formula (II):

Preferably, the R ₁ is selected from alkoxy or alkylamino; R ₂ is selected from fluorine, cyano, methyl, trifluoromethyl, n-butyl or n-pentyl; R ₃ is selected from hydrogen, methyl or fluorine. 3. Pabendazole derivative as claimed in claim 1, is characterized in that, described Pabendazole derivative is selected from following compound: Compound 6a: 1-(6-butyl-1H-benzo[d]imidazol-2-yl)-3-ethylurea; Compound 6b: 1-(6-butyl-1H-benzo[d]imidazol-2-yl)-3-cyclohexylurea; Compound 6c: 1-(6-butyl-1H-benzo[d]imidazol-2-yl)-3-isopropylurea; Compound 6d: 1-(6-butyl-1H-benzo[d]imidazol-2-yl)-3-propylurea; Compound 6e: 1-(6-butyl-1H-benzo[d]imidazol-2-yl)-3-hexylurea; Compound 6f: 1-(6-butyl-1H-benzo[d]imidazol-2-yl)-3-phenethylurea; Compound 6g: 1-(tert-butyl)-3-(6-butyl-1H-benzo[d]imidazol-2-yl)urea; Compound 6h: 1-(6-butyl-1H-benzo[d]imidazol-2-yl)-3-cyclopentylurea; Compound 6i: 1-allyl-3-(6-butyl-1H-benzo[d]imidazol-2-yl)urea;: Compound 6j: 2-(3-(6-butyl-1H-benzo[d]imidazol-2-yl)ureido)ethyl methacrylate; Compound 6k: isobutyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate; Compound 61: benzyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate; Compound 6m: 2-Chloroethyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate; Compound 6n: propyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate; Compound 6o: (6-butyl-1H-benzo[d]imidazol-2-yl)hexylcarbamate; Compound 6p: butyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate; Compound 6q: isopropyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate; Compound 6r: (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate heptyl ester; Compound 6s: (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate cyclopentyl ester; Compound 6t: 3-chloropropyl(6-butyl-1H-benzo[d]imidazol-2-yl)carbamate; Compound 6u: sec-butyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate; Compound 6v: (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate allyl ester; Compound 6w: 2-ethylhexyl (6-butyl-1H-benzo[d]imidazol-2-yl)carbamate; Compound 6x: N-(6-butyl-1H-benzo[d]imidazol-2-yl)furan-2-carboxamide; Compound 6y: N-(6-butyl-1H-benzo[d]imidazol-2-yl)-2-phenylacetamide; Compound 6z: N-(6-butyl-1H-benzo[d]imidazol-2-yl)tetrahydro-2H-pyran-4-carboxamide; Compound 6aa: N-(6-butyl-1H-benzo[d]imidazol-2-yl)-2-(thiophen-2-yl)acetamide; Compound 6ab: isopropyl (6-cyano-1H-benzo[d]imidazol-2-yl)carbamate; Compound 6ac: isopropyl (6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)carbamate; Compound 6ad: isopropyl (6-methyl-1H-benzo[d]imidazol-2-yl)carbamate; Compound 6ae: isopropyl (6-fluoro-1H-benzo[d]imidazol-2-yl)carbamate; Compound 6af: isopropyl (6-pentyl-1H-benzo[d]imidazol-2-yl)carbamate; Compound 6ag: isopropyl (5,6-difluoro-1H-benzo[d]imidazol-2-yl)carbamate; Compound 6ah: (5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)carbamate isopropyl ester. 4. the preparation method of the described Pabendazole derivative of any claim in claim 1-3, is characterized in that, comprises the steps: (1) The amine group of compound 1 is acetylated by acetic anhydride under the catalysis of concentrated sulfuric acid, and then the ortho-position of the acetyl group is nitrated by acetic anhydride and nitric acid to obtain compound 2; then, compound 2 is acetylated by potassium hydroxide in methanol The solution is hydrolyzed into compound 3, and then the nitro group is reduced by H ₂ /Pd in methanol to obtain compound 4; (2) 2-methyl-2-thioisourea sulfate undergoes substitution reaction with isocyanate, chloroformate or acid chloride under alkaline conditions to obtain 5a-5aa; (3) Compounds 4 and 5a-5aa undergo cyclization reaction under acidic conditions to generate Pabendazole derivatives (6a-6ah); Wherein steps (1) and (2) are in no particular order;

5. preparation method as claimed in claim 4, is characterized in that, described preparation method is as follows: (1) Cool acetic acid and a small amount of concentrated sulfuric acid to 0°C, add compound 1; after stirring at 45-55°C for 0.5-1.5 h, cool the mixture to room temperature, then add acetic anhydride; then, cool the mixture to 0°C again, add concentrated Nitric acid, stirring at 0°C for 30-50min; adding the reaction solution to ice water to obtain compound 2; Compound 2 was dissolved in methanol, 35-45% KOH solution was added, and stirred at 60-70 °C for 0.5-1.5 h; the reaction solution was dried and filtered, and the solvent was removed to obtain compound 3; Compound 3, Pd/C and methanol were mixed and reduced with hydrogen; the reaction solution was stirred at room temperature for 23-25 h, and Pd/C was filtered to obtain compound 4; (2) Add isocyanate, chloroformate or acid chloride dropwise to the mixture of 2-methyl-2-thiourea sulfate, DIPEA and acetonitrile; stir at room temperature for 46-50 h, remove the solvent; add ethyl acetate and water , After fully stirring, the liquid is separated, the organic phase is washed and dried, and the product is purified to obtain compounds 5a-5aa; (3) The compound 4, 5a-5aa and the solvent were stirred at 90 to 110°C for 2.5 to 3.5 h, the reaction solution was cooled to room temperature, and the pH was adjusted to be alkaline; The phases are dried, filtered, concentrated and purified to obtain the Pabendazole derivative. 6. preparation method as claimed in claim 5 is characterized in that, the solvent described in step (3) is acetic acid aqueous solution, wherein V _{CH COOH} : V _{H O} =3:10. 7. preparation method as claimed in claim 5 is characterized in that, described isocyanate is selected from ethyl isocyanate, cyclohexyl isocyanate, isopropyl isocyanate, propyl isocyanate, isocyanate Hexyl, phenethyl isocyanate, tert-butyl isocyanate, cyclopentyl isocyanate, allyl isocyanate or isocyanoethyl methacrylate; The chloroformate is selected from isobutyl chloroformate, benzyl chloroformate, ethyl chloroformate, propyl chloroformate, hexyl chloroformate, butyl chloroformate, isopropyl chloroformate, heptyl chloroformate , cyclopentyl chloroformate, 2-ethylhexyl chloroformate, chloropropyl chloroformate, 1-methylpropyl chloroformate or allyl chloroformate; The acid chloride is selected from furoyl chloride, phenylacetyl chloride, tetrahydropyran-4-carbonyl chloride or 2-thiopheneacetyl chloride. 8. A pharmaceutical composition, characterized in that, the pharmaceutical composition comprises the Pabendazole derivative according to any one of claims 1-3. 9. A pharmaceutical preparation, characterized in that it comprises the pabendazole derivative of any one of claims 1-3 or the pharmaceutical composition of claim 8, and a pharmaceutically acceptable carrier and/or adjuvant. 10. The application of any one of the described Pabendazole derivatives of claims 1-3 in the preparation of a medicament for the treatment of head and neck squamous cell carcinoma HNSCC; Preferably, the head and neck squamous cell carcinoma includes human tongue squamous cell carcinoma and human pharyngeal squamous cell carcinoma.

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