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CN114436972B - Pabendazole derivative, and preparation method and application thereof - Google Patents

Pabendazole derivative, and preparation method and application thereof Download PDF

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CN114436972B
CN114436972B CN202210088377.1A CN202210088377A CN114436972B CN 114436972 B CN114436972 B CN 114436972B CN 202210088377 A CN202210088377 A CN 202210088377A CN 114436972 B CN114436972 B CN 114436972B
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compound
butyl
derivative
benzo
imidazol
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CN114436972A (en
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李敏勇
杜吕佩
梁栋
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Shandong University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/24—Benzimidazoles; Hydrogenated benzimidazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
    • C07D235/30—Nitrogen atoms not forming part of a nitro radical
    • C07D235/32—Benzimidazole-2-carbamic acids, unsubstituted or substituted; Esters thereof; Thio-analogues thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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Abstract

The invention relates to the field of medicines, in particular to a Pabendazole derivative, a preparation method and application thereof, wherein the structure of the Pabendazole derivative is shown as a formula X:wherein R is 1 Selected from alkoxy or alkylamino; r is R 2 Selected from fluorine, cyano, methyl, trifluoromethyl, n-butyl or n-pentyl; r is R 3 The panbendazole derivative prepared by the invention has good inhibition effect on proliferation of head and neck squamous cell carcinoma HNSCC cell lines in vitro, can inhibit tubulin polymerization, inhibit cell invasion, cell migration and block cell cycle; can obviously control the growth of HN6 xenograft tumor nude mice model tumor in vivo, has good treatment effect, and also proves that the curative effect is obvious through the detection of related tumor markers.

Description

Pabendazole derivative, and preparation method and application thereof
Technical Field
The invention relates to the field of medicines, in particular to a Pabendazole derivative, and a preparation method and application thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Head and neck squamous cell carcinoma (head and neck squamous cell carcinoma, HNSCC) is a malignancy in which the mouth, pharynx and larynx are covered by squamous epithelium, which, although originating from mucosal epithelial cells, has significant heterogeneity. Most patients require conservative treatment, except surgical excision. Cytotoxic-based cetuximab, cisplatin and 5-fluorouracil combination therapy is a common treatment regimen, and immunotherapy is a promising treatment method, and although some progress has been made in the treatment of HNSCC, the optimal treatment method is still unclear and development of new drugs is still necessary and urgent.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide the Pabendazole derivative, and the preparation method and the application thereof, and the Pabendazole derivative prepared in the invention has good inhibition effect on proliferation of head and neck squamous cell carcinoma HNSCC cell lines in vitro, can inhibit tubulin polymerization, inhibit cell invasion, cell migration and block cell cycle; can obviously control the growth of HN6 xenograft tumor nude mice model tumor in vivo, has good treatment effect, and also proves that the curative effect is obvious through the detection of related tumor markers.
In order to achieve the above object, the technical scheme of the present invention is as follows:
in a first aspect of the present invention, there is provided a derivative of pabendazole having the structure shown in formula X:
wherein R is 1 Selected from alkoxy or alkylamino; r is R 2 Selected from fluorine, cyano, methyl, trifluoromethyl, n-butyl or n-pentyl; r is R 3 Selected from hydrogen, methyl or fluorine.
In one or more embodiments, the panbendazole derivative has a structure represented by formula (I) or formula (II):
preferably, said R 1 Selected from alkoxy or alkylamino; r is R 2 Selected from fluorine, cyano, methyl, trifluoromethyl, n-butyl or n-pentyl; r is R 3 Selected from hydrogen, methyl or fluorine.
Specifically, the panbendazole 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;
6g of 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 ethyl 2- (3- (6-butyl-1H-benzo [ d ] imidazol-2-yl) ureido) methacrylate;
compound 6k (6-butyl-1H-benzo [ d ] imidazol-2-yl) carbamic acid isobutyl ester;
compound 6l benzyl (6-butyl-1H-benzo [ d ] imidazol-2-yl) carbamate;
2-chloroethyl (6-butyl-1H-benzo [ d ] imidazol-2-yl) carbamate compound 6 m;
compound 6n (6-butyl-1H-benzo [ d ] imidazol-2-yl) carbamic acid propyl ester;
6o (6-butyl-1H-benzo [ d ] imidazol-2-yl) carbamic acid hexyl ester;
compound 6p (6-butyl-1H-benzo [ d ] imidazol-2-yl) carbamic acid butyl ester;
compound 6q (6-butyl-1H-benzo [ d ] imidazol-2-yl) carbamic acid isopropyl ester;
compound 6r (6-butyl-1H-benzo [ d ] imidazol-2-yl) carbamic acid heptyl ester;
compound 6s (6-butyl-1H-benzo [ d ] imidazol-2-yl) carbamic acid cyclopentester;
3-chloropropyl (6-butyl-1H-benzo [ d ] imidazol-2-yl) carbamate;
compound 6u (6-butyl-1H-benzo [ d ] imidazol-2-yl) carbamic acid sec-butyl ester;
compound 6v (6-butyl-1H-benzo [ d ] imidazol-2-yl) allyl carbamate;
2-ethylhexyl (6-butyl-1H-benzo [ d ] imidazol-2-yl) carbamate compound 6 w;
the compound 6x is 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 (6-cyano-1H-benzo [ d ] imidazol-2-yl) carbamic acid isopropyl ester;
compound 6ac isopropyl (6- (trifluoromethyl) -1H-benzo [ d ] imidazol-2-yl) carbamate;
compound 6ad, (6-methyl-1H-benzo [ d ] imidazol-2-yl) carbamic acid isopropyl ester;
compound 6ae, (6-fluoro-1H-benzo [ d ] imidazol-2-yl) carbamic acid isopropyl ester;
compound 6af, (6-pentyl-1H-benzo [ d ] imidazol-2-yl) carbamic acid isopropyl ester;
compound 6ag (isopropyl 5, 6-difluoro-1H-benzo [ d ] imidazol-2-yl) carbamate;
compound 6ah, (5, 6-dimethyl-1H-benzo [ d ] imidazol-2-yl) carbamic acid isopropyl ester;
in a second aspect of the present invention, there is provided a process for the preparation of the panbendazole derivative of the first aspect, comprising the steps of:
(1) The amino group of the 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 a compound 2; subsequently, compound 2 is hydrolyzed by potassium hydroxide solution in methanol to compound 3, and the nitro group is then cleaved by H in methanol 2 Reduction of Pd to give Compound 4;
(2) The 2-methyl-2-thioisourea sulfate and isocyanate, chloroformate or acyl chloride undergo substitution reaction under alkaline conditions to obtain 5a-5aa;
(3) The compounds 4 and 5a-5aa undergo cyclization under acidic conditions to produce the Pabendazole derivative (6 a-6 ah);
wherein the sequence of the steps (1) and (2) is not sequential;
reagents and conditions: aniline, acetic anhydride, concentrated sulfuric acid at 45-55 deg.c for 0.5-1.5 hr; nitric acid at 0 deg.c for 30-50 min. (II) KOH, 60-70 ℃ and 0.5-1.5 h. (III) H 2 Pd/C, room temperature, 23-25 h. (IV) isocyanate, chloroformate or acyl chloride, N, N-diisopropylethylamine, at room temperature for 46-50 h. (V) CH 3 COOH:H 2 O＝3:10，90～110℃，2.5～3.5h。
Specifically, the preparation method comprises the following steps:
(1) Acetic acid and a small amount of concentrated sulfuric acid are cooled to 0 ℃, and compound 1 is added; stirring at 45-55 ℃ for 0.5-1.5 h, cooling the mixture to room temperature, and then adding acetic anhydride; then cooling the mixture again to 0 ℃, adding concentrated nitric acid, and stirring for 30-50 min at 0 ℃; adding the reaction solution into ice water to obtain a compound 2;
dissolving the compound 2 in methanol, adding 40% KOH solution, and stirring for 0.5-1.5 h at 60-70 ℃; drying the reaction solution, filtering, and removing the solvent to obtain a compound 3;
compound 3, pd/C and methanol were mixed and reduced with hydrogen; stirring the reaction solution at room temperature for 23-25 h, and filtering Pd/C to obtain a compound 4;
(2) Dropwise adding isocyanate, chloroformate or acyl chloride into a mixture of 2-methyl-2-thiourea sulfate, DIPEA and acetonitrile; stirring for 46-50 h at room temperature, and removing the solvent; adding ethyl acetate and water, fully stirring, separating liquid, washing and drying an organic phase, and purifying a product to obtain a compound 5a-5aa;
(3) Stirring the compound 4, 5a-5aa and a solvent at 90-110 ℃ for 2.5-3.5 h, cooling the reaction solution to room temperature, and adjusting the PH to be alkaline; and adding dichloromethane, fully stirring, separating, drying an organic phase, filtering, concentrating and purifying to obtain the panbendazole derivative.
Preferably, the solvent in step (3) is an aqueous acetic acid solution, wherein V CH3COOH :V H2O ＝3:10；
Preferably, the isocyanate is selected from ethyl isocyanate, cyclohexyl isocyanate, isopropyl isocyanate, propyl isocyanate, hexyl isocyanate, phenethyl isocyanate, t-butyl isocyanate, cyclopentyl isocyanate, allyl isocyanate or isocyanatoethyl methacrylate;
preferably, the chloroformate is selected from isobutyl chloroformate, benzyl chloroformate, chloroethyl chloroformate, propyl chloroformate, hexyl chloroformate, butyl chloroformate, isopropyl chloroformate, heptyl chloroformate, cyclopentyl chloroformate, 2-ethylhexyl chloroformate, chloropropyl chloroformate, 1-methylpropyl chloroformate or allyl chloroformate;
preferably, the acid chloride is selected from the group consisting of 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-described Pabendazole derivative.
In a fourth aspect of the present invention, there is provided a pharmaceutical formulation comprising the above-described Pabendazole derivative or the above-described pharmaceutical composition, together with a pharmaceutically acceptable carrier and/or adjuvant. The preparation is selected from oral preparation and parenteral preparation, and can be tablet, pill, capsule or injection.
In a fifth aspect of the invention there is provided the use of a pabendazole derivative as described above in the manufacture 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 carcinoma and human pharyngeal squamous carcinoma.
In vitro activity research shows that the Pabendazole derivative disclosed by the invention has a good inhibition effect on proliferation of three HNSCC cell strains tested, and can inhibit tubulin polymerization, inhibit cell invasion, cell migration and block cell cycle.
In vivo activity research shows that the panbendazole derivative disclosed by the invention can obviously control the growth of HN6 xenograft tumor nude mice model tumor at the dosage of 80mg/kg, has good treatment effect, and also proves that the treatment effect is obvious through detection of related tumor markers.
The in-vitro activity shows that the panbendazole derivative disclosed by the invention is a potential molecule for treating the HNSCC of head and neck squamous cell carcinoma, and provides a certain foundation for the research and development of medicines.
As used herein, the term "pharmaceutical composition," which may also refer to "compositions," may be used to effect treatment or prevention of a disease or disorder described herein in a subject, particularly a mammal.
The pharmaceutical composition of the compounds of the present invention may be administered in any of the following ways: oral, spray inhalation, rectal, nasal, vaginal, topical, parenteral, such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal or intracranial injection or infusion, or by means of an explanted reservoir, with oral, intramuscular, intraperitoneal or intravenous modes of administration being preferred.
The compounds of the present invention or pharmaceutical compositions containing them may be administered in unit dosage form. The administration dosage form may be liquid dosage form or solid dosage form. The liquid dosage form can be true solution, colloid, microparticle, emulsion, and mixed rotation. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, clathrate, landfill, patch, liniment, etc.
The pharmaceutical compositions of the present invention may also contain conventional carriers, including but not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum proteins, buffer substances such as phosphates, glycerol, sorbitol, 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 carboxymethylcellulose, polyacrylates, beeswax, lanolin and the like. The carrier may be present in the pharmaceutical composition in an amount of from 1% to 98% by weight, typically about 80% by weight. For convenience, local anesthetics, preservatives, buffers, and the like may be directly dissolved in the carrier.
Oral tablets and capsules may contain excipients such as binding agents, for example syrup, acacia, sorbitol, tragacanth, or polyvinylpyrrolidone, fillers, for example lactose, sucrose, corn starch, calcium phosphate, sorbitol, glycine, lubricants, for example magnesium stearate, talc, polyethylene glycol, silica, disintegrants, for example potato starch, or acceptable wetting agents, for example sodium lauryl sulfate. The tablets may be coated by methods known in the pharmaceutical arts.
The oral liquid can be made into water and oil suspension, solution, emulsion, syrup, or dry product, and can be supplemented with water or other suitable medium 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 gelatin, hydrogenated edible fats and oils, emulsifying agents such as lecithin, sorbitan monooleate, gum arabic; or a non-aqueous carrier (possibly containing edible oils) such as almond oil, fats and oils such as glycerin, ethylene glycol, or ethyl alcohol; preservatives, such as methyl or propyl parahydroxybenzoate, sorbic acid. Flavoring or coloring agents may be added as desired.
Suppositories may contain conventional suppository bases such as cocoa butter or other glycerides.
For parenteral administration, liquid dosage forms are typically made of the compound and a sterile carrier. The carrier is water. Depending on the carrier and drug concentration selected, the compound may be dissolved in either the carrier or in suspension, and when preparing an injectable solution, the compound is first dissolved in water, filtered and sterilized, and filled into sealed bottles or ampoules.
It must be appreciated that the optimal dosage and spacing of the compounds of formula X, formulas I-II is determined by the nature of the compound and external conditions such as the form, route of administration and the particular mammal being treated, and that such optimal dosage may be determined by conventional techniques. It must also be appreciated that the optimal course of treatment, i.e., the daily dosage of compound X at the same time over the nominal time period, can be determined by methods well known in the art.
The specific embodiment of the invention has the following beneficial effects:
the in vitro activity research of the panbendazole derivative disclosed by the invention shows that the panbendazole derivative has a good inhibition effect on proliferation of three HNSCC cell strains tested, can inhibit tubulin polymerization, inhibit cell invasion, cell migration and block cell cycle;
the in vivo activity research of the panbendazole derivative disclosed by the invention shows that the panbendazole derivative can obviously control the growth of HN6 xenograft tumor nude mice model tumor at the dosage of 80mg/kg, has good treatment effect, and also proves that the treatment effect is obvious through the detection of related tumor markers;
in vivo and in vitro activities show that the panbendazole derivative disclosed by the invention is a potential molecule for treating HNSCC, and provides a certain basis for the research and development of medicines for treating HNSCC.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
Fig. 1 is: immunostaining of tubulin in HN6 cells. With a compound (100 nmol)/L) treatment of HN6 cells for 24h, immunofluorescence assay showed microtubule structure. Red (Alexa)555 For labeling tubulin, blue (DAPI) for labeling nuclei; the scale bar is 20 mu m; a is HN6 cells treated with DMSO; b is HN6 cells treated with 6 q; c is HN6 cells treated with pabendazole; d is HN6 cells treated with 6 v.
Fig. 2 is: 6q and 6v are effective in inhibiting tumor cell migration and invasion. A is that after 24 hours of compound treatment, cells growing in a 6-hole plate are photographed at 0 hour and 24 hours respectively, and the scratch healing rate is calculated; scratches were made with 200 μl of the tip and photographed with an inverted fluorescence microscope; b is the healing rate of cell scratches after treatment of the Pabendazole, 6q and 6v; data are expressed as mean ± SEM (n=3, P <0.05, P < 0.001); c is staining of cells passing through the matrix with crystal violet after 24h treatment with or without the compound; d is the invasion rate of the Pabendazole, 6q and 6v to cells; data are expressed as mean ± SEM (n=3, × P < 0.001).
Fig. 3 is: HN6 cells were induced to apoptosis for 6q and 6v, blocking the cell cycle in G2/M phase. A is that cells are treated with the compound for 24 hours; the Annexin-FITC and PI are selected to dye the apoptotic cells, and a representative flow cytometry point diagram is displayed; b is the percentage of apoptotic cells in each group; data are expressed as mean ± SEM (n=3, P <0.05, P <0.01, P < 0.001); c is to treat cells with the compound for 24 hours, select PI dye liquor to dye DNA, and detect the DNA content by a flow cytometer; HN6 cells have a representative DNA distribution histogram; d is the cell cycle distribution and data are expressed as mean ± SEM (n=3).
Fig. 4 is: 6q and 6v have better therapeutic effect on xenograft tumor models. A-D is the tumor volume and weight of the mice; the transplanted tumor mice were given 80mg/kg, 6q,6v or solvent of patadine and gavaged 1 time daily. Tumor volume was measured and body weight was recorded; results are expressed in standard deviation (n=5); b is that the solid tumor is surgically resected; E-G is the level of CYFRA21-1, SCCAg and TPS in the serum of nude mice; blank group is normal tumor-free nude mice, and control group is untreated xenograft nude mice; results are expressed in standard deviation (n=5); * P <0.05, P <0.01.
Fig. 5 is: toxicity of the compounds to HFF-1 and Kunming mice; A-C cell viability of HFF-1 cells treated with a range of concentrations of Pabendazole, 6q and 6v for 72 h; detecting the cell viability by a CCK8 method; data are presented as average SEM, n=3. Survival curves for D and E patadine, 6q, 6v; m represents a male, F represents a female; the design and implementation of the experiments were carried out as specified by the Korbor and Limit method experiments in GB 15193.3-2014.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, 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 is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The invention is further illustrated and described below in connection with specific examples.
Example 1:1- (6-butyl-1H-benzo [ d ]]Imidazol-2-yl) -3-ethylurea (6 a)
Reagents and conditions: 4-n-butylaniline, acetic anhydride, concentrated sulfuric acid, 50 ℃ for 1h; nitric acid at 0 ℃ for 4min; (II) KOH,65℃for 1h; (III) H 2 Pd/C, room temperature, 24h; (IV) ethyl isocyanate, N, N-diisopropylethylamine, at room temperature for 48h; (V) CH 3 COOH:H 2 O＝3:10，100℃，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 250mL bottle, cooled to 0 ℃, and compound 1 was added dropwise. After stirring at 50 ℃ for 1h, the mixture was cooled to room temperature and acetic anhydride was added. After that, the mixture was cooled again to 0℃and concentrated nitric acid was slowly added thereto, followed by stirring at 0℃for 40 minutes. The reaction solution was added to a large amount of ice water, and the resulting yellow precipitate was filtered off and dried to give a crude product. The crude product was further purified by silica gel chromatography to give a yellow solid product. 1 H NMR(400MHz,DMSO)δ10.17(s,1H),7.74(d,J＝1.1Hz，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℃.
Step 2: synthesis of 4-butyl-2-nitroaniline (3)
Compound 2 was dissolved in methanol in a 250mL bottle; 40% KOH solution (7.4 mL,50.78 mmol) was added and the mixture was stirred at 65℃for 1h. Anhydrous Na for reaction solution 2 SO 4 After drying, the mixture was 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)δ7.74(s,1H)，7.30(s,2H),7.27(d,1H),6.95(d,J＝8.6Hz,1H),2.47(d,J＝7.7Hz,2H),1.54–1.44(m,2H),1.34–1.23(m,2H),0.89(t,J＝7.3Hz,3H).ESI-MS:m/z[M-H] - 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 250mL three-necked flask, compound 3, pd/C and methanol were added. The whole system was sealed, air was exchanged with nitrogen, and nitrogen was replaced with hydrogen. The reaction solution was stirred at room temperature for 24h, filtered at Pd/C and concentrated to give the product as an oil. 1 H NMR(400MHz,DMSO)δ6.39(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 (5 a)
Ethyl isocyanate was added dropwise to a mixture of 2-methyl-2-thiouronium sulphate, DIPEA and acetonitrile. Stirred at room temperature for 48h, then the solvent was removed under reduced pressure. Adding ethyl acetate and water, stirring thoroughly, separating, washing the organic phase with saturated sodium chloride solution for 2 times, and then with anhydrous Na 2 SO 4 The product was dried and purified by silica gel chromatography to give a white oil 5a. 1 H NMR(400MHz,CDCl 3 )δ12.59(s,1H),5.40(d,J＝76.0Hz,2H),3.27(dq,J＝13.8,7.0Hz,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,found 232.79.
Step 5: synthesis of 1- (6-butyl-1H-benzo [ d ] imidazol-2-yl) -3-ethylurea (6 a)
The compounds 4, 5 are combined with a solvent (CH 3 COOH:H 2 O=3:10) was stirred at 100 ℃ for 3h, the reaction solution was cooled to room temperature, and saturated NaHCO was used 3 The aqueous solution was adjusted to pH to alkaline. Then adding dichloromethane, fully stirring, separating liquid, and using anhydrous Na for the organic phase 2 SO 4 After drying, filtering and concentrating, the product is purified by silica gel chromatography to obtain a white solid product. White solid (48 mg,37.9% yield). 1 HNMR(400MHz,DMSO)δ11.31(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)δ154.69,148.73,135.05,121.50,35.57,34.53,34.39,22.20,15.77,14.30.ESI-HRMS:m/z[M+H] + calcd for C 14 H 21 N 4 O + 261.1715,found 267.1712.HPLC purity 99.1％,t R ＝10.558min,250mm×4.6mm,CH 3 OH:H 2 O＝75:25,0.8mL/min.Mp 253.6–254.9℃.
The following compounds were synthesized in the same manner as in example 1, using different starting materials.
Example 2:1- (6-butyl-1H-benzo [ d ]]Imidazol-2-yl) -3-cyclohexylurea (6 b)
White solid (36 mg,37.6% yield). 1 H NMR(400MHz,DMSO)δ11.31(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％,t R ＝6.551min,250mm×4.6mm,CH 3 OH:H 2 O＝75:25,0.8mL/min.Mp 119.9–120.6℃.
Example 3:1- (6-butyl-1H-benzo [ d ]]Imidazol-2-yl) -3-isopropylurea (6 c)
White solid (42 mg,41.9% yield). 1 H NMR(400MHz,DMSO)δ11.32(s,1H),9.55(s,1H),7.30(d,J＝32.2Hz,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 ＝9.114min,250mm×4.6mm,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 (6 d)
White solid (39 mg,46.7% yield). 1 H NMR(400MHz,DMSO)δ11.27(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＝12.0,6.1Hz,6H).ESI-HRMS:m/z[M+H] + calcd for C 15 H 23 N 4 O + 275.1872,found 275.1871.HPLC purity 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℃.
ExamplesAnd (c) the following steps: 1- (6-butyl-1H-benzo [ d ]]Imidazol-2-yl) -3-hexylurea (6 e)
White solid (30 mg,31.1% yield). 1 H NMR(400MHz,DMSO)δ11.26(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)δ154.74,148.73,135.07,121.51,39.61,35.57,34.38,31.41,29.97,26.48,22.53,22.19,14.36,14.29.ESI-HRMS:m/z[M+H] + calcd for C 18 H 29 N 4 O + 317.2341,found 317.2336.HPLC purity 96.7％,t R ＝7.152min,250mm×4.6mm,CH 3 OH:H 2 O＝75:25,0.8mL/min.Mp 202.5–203.9℃.
Example 6:1- (6-butyl-1H-benzo [ d ]]Imidazol-2-yl) -3-phenethylurea (6 f)
White solid (52 mg,37.0% yield). 1 H NMR(400MHz,DMSO)δ11.30(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＝14.0,6.9Hz,2H),2.60(t,J＝7.6Hz,2H),1.60–1.50(m,2H),1.37–1.25(m,2H),0.90(t,J＝7.3Hz,3H). 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] + calcd for C 20 H 25 N 4 O + 337.2028,found 337.2023.HPLC purity 98.9％,t R ＝11.688min,250mm×4.6mm,CH 3 OH:H 2 O＝75:25,0.8mL/min.Mp 180.2–181.7℃.
Example 7:1- (tert-butyl) -3- (6-butyl-1H-benzo [ d ]]Imidazol-2-yl) urea (6 g)
Colorless transparent oil (62 mg,35.3% yield). 1 H NMR(400MHz,DMSO)δ11.37(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)δ153.63,148.59,134.95,121.40,50.27,35.60,34.43,29.29,22.22,14.30.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 ＝11.820min,250mm×4.6mm,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 (6 h)
White solid (104 mg,55.0% yield). 1 H NMR(400MHz,DMSO)δ11.36(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 C NMR(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 ＝18.058min,250mm×4.6mm,CH 3 OH:H 2 O＝75:25,0.8mL/min.Mp 180.5–182.9℃.
Example 9: 1-allyl-3- (6-butyl-1H-benzo [ d ]]Imidazol-2-yl) urea (6 i)
White solid (50 mg,60.2% yield). 1 H NMR(400MHz,DMSO)δ11.32(s,1H),9.84(s,1H),7.61(s,1H),7.23(d,J＝8.0Hz,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＝10.3Hz,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 ＝15.687min,250mm×4.6mm,CH 3 OH:H 2 O＝80:20,0.8mL/min.Mp 223.4–225.6℃.
Example 10:2- (3- (6-butyl-1H-benzo [ d ])]Imidazol-2-yl) ureido) ethyl methacrylate (6 j)
White solid (135 mg,64.4% yield). 1 H NMR(400MHz,DMSO)δ11.18(s,1H),10.18(dd,J＝221.4,53.7Hz,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＝5.3Hz,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 ＝8.850min,250mm×4.6mm,CH 3 OH:H 2 O＝75:25,0.8mL/min.Mp 221.4–223.9℃.
Example 11: (6-butyl-1H-benzo [ d ]]Isobutyl imidazol-2-yl) carbamate (6 k)
White solid (51 mg,51.8% yield). 1 H NMR(400MHz,DMSO)δ11.57(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)δ135.61,121.97,71.33,35.58,34.39,28.04,22.22,19.35,14.29.ESI-HRMS:m/z[M+H] + calcd for C 16 H 24 N 3 O 2 + 290.1869,found 190.1863.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: (6-butyl-1H-benzo [ d ]]Imidazol-2-yl) carbamic acid benzyl ester (6 l)
White solid (50 mg, 50.8%). 1 H NMR(400MHz,DMSO)δ11.77(s,2H),7.49–7.34(m,5H),7.26(d,J＝8.1Hz,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). 13 C NMR(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 ＝15.432min,250mm×4.6mm,CH 3 OH:H 2 O＝75:25,0.8mL/min.Mp 199.2–201.7℃.
Example 13: 2-chloroethyl (6-butyl-1H-benzo [ d ]]Imidazol-2-yl) carbamate (6 m)
White solid (41 mg,44.6% yield). 1 H NMR(400MHz,DMSO)δ11.88(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 ＝8.219min,250mm×4.6mm,CH 3 OH:H 2 O＝75:25,0.8mL/min.Mp 199.2–201.7℃.Mp 182.8–184.7℃.
Example 14: (6-butyl-1H-benzo [ d ]]Imidazol-2-yl) carbamic acid propyl ester (6 n)
White solid (38 mg,45.8% yield). Delta.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=39.7 Hz, 4H), 1.31 (s, 2H), 0.91 (d, J=6.5 Hz, 6H). 13C NMR (101 MHz, DMSO) delta.121.96, 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,found 276.1705.HPLC purity 99.3%, tR=9.068 min,250mm×4.6mm, CH3OH: H=80:20, 0.8mL/min, 0.204.206-8.206.8 ℃.
Example 15: (6-butyl-1H-benzo [ d ]]Imidazol-2-yl) carbamic acid hexyl ester (6 o)
White solid (56 mg,60.0% yield). 1 H NMR(400MHz,DMSO)δ11.50(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,found 318.2173.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: (6-butyl-1H-benzo [ d ]]Imidazol-2-yl) carbamic acid butyl ester (6 p)
White solid (53 mg,50.1% yield). 1 H NMR(400MHz,DMSO)δ11.58(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 ＝10.557min,250mm×4.6mm,CH 3 OH:H 2 O＝80:20,0.8mL/min.Mp 211.7–213.2℃
Example 17: (6-butyl-1H-benzo [ d ]]Isopropyl imidazol-2-yl) carbamate (6 q)
White solid (49 mg,58.5% yield). DELTA.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 (101 MHz, DMSO) delta 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: (6-butyl-1H-benzo [ d ]]Imidazol-2-yl) carbamic acid heptyl ester (6 r)
White solid (91 mg,75.2% yield). 1 H NMR(400MHz,DMSO)δ11.45(s,2H),7.26(d,J＝7.5Hz,1H),7.18(s,1H),6.89(d,J＝8.3Hz,1H),4.14(s,2H),2.62(s,2H),1.60(d,J＝32.7Hz,4H),1.28(s,10H),0.88(s,6H). 13 C 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℃.
Example 19: cyclopentyl (6-butyl-1H-benzo [ d ]]Imidazol-2-yl) carbamate (6 s)
White solid (45 mg,49.0% yield). 1 H NMR(400MHz,DMSO)δ11.56(s,2H),7.30–7.26(m,1H),7.20(s,1H),6.89(d,J＝8.1Hz,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,found 302.1860.HPLC purity 99.6％,t R ＝11.135min,250mm×4.6mm,CH 3 OH:H 2 O＝80:20,0.8mL/min.Mp 171.8–174.8℃.
Example 20: 3-chloropropyl (6-butyl-1H-benzo [ d ]]Imidazol-2-yl) carbamate (6 t)
White solid (40 mg,42.4% yield). 1 H NMR(400MHz,DMSO)δ11.58(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)δ155.01,151.30,148.54,147.31,136.80,123.02,113.40,112.97,62.82,42.23,35.47,34.24,31.84,22.17,14.27.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％,t R ＝8.599min,250mm×4.6mm,CH 3 OH:H 2 O＝80:20,0.8mL/min.Mp 184.2–185.9℃.
Example 21: (6-butyl-1H-benzo [ d ]]Imidazol-2-yl) carbamic acid sec-butyl ester (6 u)
White solid (46 mg,52.2% yield). 1 H NMR(400MHz,CDCl 3 )δ10.68(s,1H),7.56(s,1H),7.25(s,1H),7.02(d,J＝7.9Hz,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 ＝10.562min,250mm×4.6mm,CH 3 OH:H 2 O＝80:20,0.8mL/min.
Example 22: (6-butyl-1H-benzo [ d ]]Imidazol-2-yl) carbamic acid allyl ester (6 v)
White solid (36 mg,43.3% yield). 1 H NMR(400MHz,DMSO)δ11.58(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)δ135.69,133.61,122.05,118.14,65.79,35.54,34.34,22.20,14.29.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％,t R ＝8.042min,250mm×4.6mm,CH 3 OH:H 2 O＝80:20,0.8mL/min.Mp 211.2–212.9℃.
Example 23: 2-ethylhexyl (6-butyl-1H-benzo [ d ]]Imidazol-2-yl) carbamate (6 w)
Colorless oil (39 mg,38.9% yield). 1 H NMR(400MHz,DMSO)δ11.58(d,J＝32.0Hz,2H),7.28(dd,J＝8.0,3.1Hz,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)δ155.72,148.22,135.58,121.97,113.54,67.59,38.97,35.58,34.39,30.20,28.85,23.61,22.92,22.22,14.38,14.29,11.29.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 (6 x)
White solid (39 mg,45.2% yield). 1 H NMR(400MHz,DMSO)δ12.10(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＝7.3Hz,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 (6 y)
White solid (45 mg,48.1% yield). 1 H NMR(400MHz,DMSO)δ11.99(s,2H),7.38–7.25(m,7H),6.92(d,J＝11.5Hz,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)δ170.76,146.84,135.73,129.67,128.84,127.23,42.78,35.60,34.38,22.22,14.30.ESI-HRMS:m/z[M+H] + calcd for C 19 H 22 N 3 O + 308.1763,found 315.2178.HPLC purity 98.3％,t R ＝9.064min,250mm×4.6mm,CH 3 OH:H 2 O＝80:20,0.8mL/min.Mp 171.0–173.0℃.
Example 26: n- (6-butyl-1H-benzo [ d ]]Imidazol-2-yl) tetrahydro-2H-pyran-4-carboxamide (6 z)
White solid (48 mg,52.3% yield). 1 H NMR(400MHz,DMSO)δ11.92(s,1H),11.41(s,1H),7.31(d,J＝8.1Hz,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℃.
Example 27: n- (6-butyl-1H-benzo [ d ]]Imidazol-2-yl) -2- (thiophen-2-yl) acetamide (6 aa)
Brown oil (23 mg,22.9% yield). 1 H NMR(400MHz,CDCl 3 )δ7.44(d,J＝8.2Hz,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),1.39–1.32(m,2H),0.92(t,J＝7.3Hz,3H). 13 C NMR(101MHz,CDCl 3 )δ151.88,137.82,137.75,127.40,126.94,125.32,123.55,35.81,34.24,30.04,22.31,13.97.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＝80:20,0.8mL/min.
Example 28: (6-cyano-1H-benzo [ d ]]Isopropyl imidazol-2-yl) carbamate (6 ab)
Gray solid (60 mg,65.4% yield). 1 H NMR(400MHz,DMSO)δ12.26(s,1H),11.59(s,1H),7.84(s,1H),7.56(d,J＝8.2Hz,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 ＝4.666min,250mm×4.6mm,CH 3 OH:H 2 O＝80:20,0.8mL/min.Mp>300℃.
Example 29: (6- (trifluoromethyl) -1H-benzo [ d ]]Isopropyl imidazol-2-yl) carbamate (6 ac)
Gray solid (56 mg,68.7% yield). 1 H NMR(400MHz,DMSO)δ11.96(s,2H),7.76(s,1H),7.60(d,J＝8.3Hz,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)δ153.91,149.68,127.05,124.36,118.13,69.87,22.19.ESI-HRMS:m/z[M+H] + calcd for C 12 H 13 F 3 N 3 O 2 + 288.0960,found 288.0952.HPLC purity 99.8％,t R ＝7.905min,250mm×4.6mm,CH 3 OH:H 2 O＝75:25,0.8mL/min.Mp 202.2–204.9℃.
Example 30: (6-methyl-)1H-benzo [ d ]]Isopropyl imidazol-2-yl) carbamate (6 ad)
White solid (33 mg,34.6% yield). 1 H NMR(400MHz,DMSO)δ11.67(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)δ154.69,147.92,130.28,122.54,69.23,22.32,21.72.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 ＝6.709min,250mm×4.6mm,CH 3 OH:H 2 O＝75:25,0.8mL/min.Mp 195.0–197.0℃.
Example 31: (6-fluoro-1H-benzo [ d ]]Isopropyl imidazol-2-yl) carbamate (6 ae)
White solid (39 mg,41.5% yield). 1 H NMR(400MHz,DMSO)δ11.70(s,2H),7.39(dd,J＝8.6,5.0Hz,1H),7.20(dd,J＝9.6,2.2Hz,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 ＝6.020min,250mm×4.6mm,CH 3 OH:H 2 O＝75:25,0.8mL/min.Mp>300℃.
Example 32: (6-pentyl-1H-benzo [ d ]]Isopropyl imidazol-2-yl) carbamate (6 af)
White solid (61 mg,755.2% yield). 1 H NMR(400MHz,DMSO)δ7.27(dd,J＝8.0,3.7Hz,1H),7.19(s,1H),6.89(d,J＝8.1Hz,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). 13 C 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 ＝17.844min,250mm×4.6mm,CH 3 OH:H 2 O＝75:25,0.8mL/min.Mp 128.0–129.0℃.
Example 33: (5, 6-difluoro-1H-benzo [ d ]]Isopropyl imidazol-2-yl) carbamate (6 ag)
Pale yellow solid (51 mg,51.6% yield). 1 H NMR(400MHz,DMSO)δ11.98(s,1H),11.37(s,1H),7.39(t,J＝9.2Hz,2H),5.06–4.93(m,1H),1.31(d,J＝6.2Hz,6H). 13 C NMR(101MHz,DMSO)δ153.83,148.83,69.72,22.21.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 ＝6.511min,250mm×4.6mm,CH 3 OH:H 2 O＝75:25,0.8mL/min.Mp>300℃.
Example 34: (5, 6-dimethyl-1H-benzo [ d ]]Isopropyl imidazol-2-yl) carbamate (6 ah)
White solid (42 mg,47.4% yield). 1 H NMR(400MHz,DMSO)δ11.52(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(101MHz,DMSO)δ155.02,147.56,129.30,114.38,69.04,22.35,20.33.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℃.
Example 35: compound inhibition CAL-27, HN6 and Fadu cell proliferation activity assay
This example was used to determine the inhibitory activity of the panbendazole derivatives of the present invention on proliferation of HNSCC cell lines CAL-27, hn6 and Fadu cells. The test protocol is as follows: cells were seeded at a concentration of 5000 cells/well in 96-well transparent plates at 100 μl per well. At 5% CO 2 Culturing at 37deg.C for 12 hr. Then, a series of compound solutions (100. Mu.L) prepared in serum-free medium were added to the wells, the culture was continued for 72 hours, the cell viability was evaluated by CCK8 method, absorbance at 450nm was recorded by CLARIOstar microplate reader (BMG Co.), and finally IC50 value was calculated by GraphPad prism 7.00. IC of the test compounds of the invention for inhibiting tumor cell proliferation 50 The values are given in the following table.
TABLE 1 Pabendazole derivatives inhibit HN6 cell proliferation Activity and inhibit tubulin polymerization Activity
TABLE 2 inhibition of HN6, CAL-27, fadu cell proliferation Activity by Pabendazole derivatives
Conclusion: in-vitro cell proliferation of the compound shows that the activity of the compounds 6q and 6v is superior to that of the panbendazole, and a thought is provided for designing novel medicines.
Example 36: in vitro tubulin polymerization experiments
This example was used to determine the inhibitory activity of the inventive panbendazole derivatives on tubulin polymerization in vitro. TestThe scheme is as follows: the test was performed using a tubulin polymerization kit according to the manufacturer's protocol. Tubulin, compound (20 μm) and GTP were mixed into the wells at 37 ℃. As microtubules polymerize, reporter groups are added to the microtubules, and fluorescence gradually increases. Fluorescent signals were recorded with a BMG CLARIOstar microplate reader every minute over 1 hour, and IC of the compounds inhibiting microtubule polymerization was calculated at the tubulin polymerization rate 50 . The test results are shown in Table 1.
Conclusion: the activity of a plurality of compounds such as 6n, 6q, 6u, 6v, 6z, 11e, 11g and the like for independently inhibiting the polymerization of the microtubulin is better than that of the panbendazole, and a thinking is provided for designing novel compounds.
Example 37: determination of the influence of the derivatives 6q,6v on the cellular tubulin structure
This example was used to determine the effect of derivatives 6q,6v on cellular tubulin structure. The test protocol is as follows: HN6 cells were seeded in confocal laser dishes (20000 cells/dish) and 100nmol/L of compound solution was added 12h later. After 24h incubation, the medium was discarded, 4% paraformaldehyde was added and the mixture was fixed at room temperature for 15min. The cells were blocked in blocking solution (5% normal goat serum and 0.3% Triton X-100 PBS) for 1h after 3 washes in PBS. After discarding the blocking solution, alpha-tubulin antibodies were added and the cells were incubated overnight at 4 ℃. PBS was used for 3 washes, and Anti-Rabbit IgG (H+L), F (ab') 2Fragment (Alexa)555 Conjugate), incubated for 1h at room temperature in the dark. Nuclei were stained with DAPI (10. Mu.g/mL). And finally, acquiring an image by using a high-speed confocal platform.
Conclusion: from FIG. 1, it can be seen that the DMSO-treated cells (FIG. 1A) are in good morphology and that tubulin forms a regular network structure; whereas 100nmol/L of Pabendazole (FIG. 1C), 6q (FIG. 1B) or 6v (FIG. 1D) treated cells lost the regular network of tubulin, with most of the tubulin accumulating around the nucleus.
Example 38: determination of the influence of the derivatives 6q,6v on cell invasion and cell migration
The implementation isExamples are for determining the effect of derivatives 6q,6v on cell invasion and cell migration. The test protocol is as follows: cell migration assay: HN6 cells were seeded in 6-well plates at 1X 10 per well 6 Individual cells. After 12h incubation, 200. Mu.L of the tip created scratches. The wells were washed 3 times with serum-free medium and the scratches were photographed with an inverted fluorescence microscope (IX 73+DP80, objective: 10X). Then, a compound solution prepared by 0.1. Mu. Mol/L serum-free medium was added thereto, and after culturing for 24 hours, the scratch healing was photographed again. The percent scratch healing was calculated. Cell invasion assay: after 20-fold dilution of the basal membrane matrix (Corning, cat# 356234) with serum-free medium, 100. Mu.L of cell culture insert (24 well plate, pore size 8.0 μm, corning, cat# 353097) was added to the upper chamber. Then, the cell was left to stand at 37℃for 5 hours, and then the liquid was sucked out. The chamber was washed 1 time with serum-free medium and 100. Mu.L of the compound solution prepared in the serum-free medium was added. HN6 cells (100. Mu.L, 10) 5 The cells/well) was added to the upper cell culture dish and 600 μl of serum-containing medium was added to the lower cell. After incubation for 24h, the chamber was removed from the 24-well plate and the upper cells were gently rubbed. Washed once with PBS and fixed with 4% paraformaldehyde solution for 10min at room temperature. After washing 2 times with PBS, the cells were stained with a 0.1% crystal violet solution for 10min, washed, inverted, air-dried and photographed. Finally, the crystal violet was dissolved in 33% acetic acid, and the absorbance was measured at 570 nm.
Conclusion: scratch healing rates were tested after 24h treatment with patadine, 6q or 6 v. As can be seen from fig. 2, the scratch healing rate was significantly reduced in the panbendazole group (12.8%), the 6q group (6.27%) and the 6v group (8.1%) compared to the control group (44.0%). Furthermore, 6q inhibited cell migration more significantly than patadine. In the cell invasion experiment, the compound also significantly inhibits the invasion process of cells crossing the matrix, and the concentration is 200nmol/L. When the concentration was increased to 500nmol/L, only a few cells in the Pabendazole group could penetrate the matrix, while few cells in the 6q and 6q groups could successfully invade the lower layer. In conclusion, 6q and 6v can effectively inhibit migration and invasion of tumor cells.
Example 39: determination of the influence of the derivatives 6q,6v on apoptosis and cell cycle
This example was used to determine the effect of derivatives 6q,6v on apoptosis and cell cycle. The test protocol is as follows: apoptosis assay: HN6 cells were seeded in 6-well plates with 30 ten thousand cells per well. After 12h, cells were treated with 0.1. Mu. Mol/L or 1. Mu. Mol/L compound solution for 24h and collected, washed 1 time with PBS and 1 time with binding buffer. Then, 100. Mu.L of binding buffer was added to suspend the cells, 5. Mu.L of Annexin FITC and 5. Mu.L of PI staining solution were added, and incubated at room temperature for 15min in the dark. Staining results were analyzed by flow cytometry (CytoFLEX S). Cell cycle assay: HN6 cells were seeded in 6-well plates with 30 ten thousand cells per well. After 12h, cells were treated with 0.1. Mu. Mol/L compound solution in serum-free medium for 24h, collected, washed with PBS, and fixed overnight with 70% ethanol. PBS was washed 1 time, resuspended in 100. Mu.L RNase A solution, and stained in a water bath at 37℃for 30 min. Add 400. Mu.L PI staining solution, incubate at 4deg.C for 30min, and analyze staining results by flow cytometry (CytoFLEX S).
Conclusion: as can be seen from FIG. 3, the compounds of 0.1. Mu. Mol/L and 1. Mu. Mol/L were effective in inducing apoptosis after 24 hours of treatment of cells, compared with the control group. As the concentration increases, the proportion of apoptotic cells increases. Given the important role of tubulin in cell mitosis, the effect of compounds on cell cycle was also evaluated. Obviously, 0.1. Mu. Mol/L6 q or 6v was successful in blocking the cell cycle in the G2/M phase. In conclusion, 6q and 6v are effective in inducing HN6 apoptosis and blocking the cell cycle in G2/M phase, thereby inhibiting the pathological process of tumor.
Example 40: determination of therapeutic effect of derivative 6q,6v on xenograft tumor model nude mice
This example was used to determine the therapeutic effect of derivatives 6q,6v on xenograft tumor model nude mice. The test protocol is as follows: after 4-week-old male nude mice are adaptively fed for 5 days, 100 ten thousand HN6 tumor cells are subcutaneously injected to grow to an appropriate size. Mice were randomly divided into 4 groups of 5 mice each. Pabendazole, 6q,6v powder and 0.5% sodium carboxymethyl cellulose solution were formulated into suspensions, which were intragastric 1 time a day for 2 weeks. Tumor size was measured with electronic calipers and the content of SCCAg, TPS and CYFRA21-1 in serum was measured by ELISA. All procedures were performed according to the guidelines for laboratory animal care and use of the laboratory animal research institution.
From fig. 4, it can be seen that 6q and 6v have better therapeutic effect on xenograft tumor models. Conclusion: as can be seen from fig. 4, the compound is effective in controlling tumor volume growth in nude mice without significant weight loss, showing good therapeutic effect and biocompatibility at therapeutic doses. The levels of three tumor markers in the serum of nude mice transplanted with tumors are all significantly increased. After two weeks of treatment, tumor growth was effectively controlled, and serum levels of all three tumor markers were significantly reduced.
Example 41: determination of acute toxicity of derivative 6q,6v to SPF Kunming mice and toxicity to human skin fibroblast (HFF-1)
This example was used to determine the toxicity of derivatives 6q,6v to SPF Kunming mice acutely and to human skin fibroblasts (HFF-1). After adaptive feeding, mice were randomly divided into two groups, male and female, 10 each. The compound was formulated as a suspension with 0.5% sodium carboxymethyl cellulose solution, each time administered as 0.4mL lavage. Fasted for 6 hours before stomach irrigation, and free drinking water is carried out; fasted for 2 hours after gastric lavage. Mice were observed and their body weight and survival were recorded. Toxicity of the derivatives 6q,6v to human skin fibroblasts (HFF-1) was determined as described in example 35.
Conclusion: as can be seen from FIG. 5, the mice given 1g/kg of Pabendazole or 6v had no adverse reaction, and were good in condition and free from death. Mice given 150mg/kg or 250mg/kg 6q did not show any adverse reactions, remained active until the end of the experiment, and did not die. Of the mice given 630mg/kg 6q, one female and one male survived. Mice given 1g/kg 6q developed symptoms of weakness, somnolence and hypothermia after 3 hours, and mice given 630mg/kg 6q developed the above after 12 hours. Calculated, pabendazole and 6v LD 50 LD with values greater than 1g/kg and 6q 50 The value was 341mg/kg. LD of 6q 50 Far above its effective therapeutic dose, and at therapeutic doses no abnormalities were seen in the mice.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (4)

1. Use of a derivative of pabendazole for the preparation of a medicament for the treatment of head and neck squamous cell carcinoma HNSCC, characterized in that the derivative of pabendazole is selected from the group consisting of:
compound 6q (6-butyl-1H-benzo [ d ] imidazol-2-yl) carbamic acid isopropyl ester;
compound 6v (6-butyl-1H-benzo [ d ] imidazol-2-yl) allyl carbamate;
the squamous cell carcinoma of the head and neck is selected from the group consisting of human tongue squamous carcinoma and human pharyngeal squamous carcinoma.
2. The use according to claim 1, wherein the process for the preparation of the panbendazole derivative comprises the steps of:
(1) The amino group of the 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 a compound 2; subsequently, compound 2 is hydrolyzed by potassium hydroxide solution in methanol to compound 3, and the nitro group is then cleaved by H in methanol 2 Reduction of Pd to give Compound 4;
(2) The 2-methyl-2-thioisourea sulfate and chloroformate undergo substitution reaction under alkaline conditions to obtain 5a-5aa, wherein the chloroformate is selected from isopropyl chloroformate or allyl chloroformate;
(3) The compounds 4 and 5a-5aa undergo cyclization under acidic conditions to produce the Pabendazole derivative 6q or 6v;
wherein the sequence of the steps (1) and (2) is not sequential;
the method comprises the steps of carrying out a first treatment on the surface of the Wherein R is 2 Selected from n-butyl; r is R 3 Selected from hydrogen.
3. The use according to claim 2, wherein the preparation method is as follows:
(1) Cooling acetic acid and a small amount of concentrated sulfuric acid to 0 o C, adding the compound 1; 45-55 o C, stirring for 0.5-1.5 h, cooling the mixture to room temperature, and then adding acetic anhydride; thereafter, the mixture is cooled again to 0 o C, adding concentrated nitric acid, at 0 o C, stirring for 30-50 min; adding the reaction solution into ice water to obtain a compound 2;
dissolving the compound 2 in methanol, adding 35-45% KOH solution, and performing treatment on the mixture at 60-70% o Stirring for 0.5-1.5 h under the condition of C; drying the reaction solution, filtering, and removing the solvent to obtain a compound 3;
compound 3, pd/C and methanol were mixed and reduced with hydrogen; stirring the reaction solution at room temperature for 23-25 h, and filtering Pd/C to obtain a compound 4;
(2) Dropwise adding chloroformate into a mixture of 2-methyl-2-thioisourea sulfate, DIPEA and acetonitrile; stirring for 46-50 h at room temperature, and removing the solvent; adding ethyl acetate and water, fully stirring, separating liquid, washing and drying an organic phase, and purifying a product to obtain a compound 5a-5aa;
(3) Mixing the compound 4, 5a-5aa and a solvent in a range of 90 to 110 o Stirring for 2.5-3.5 h under the condition of C, cooling the reaction solution to room temperature, and regulating the PH to be alkaline; and adding dichloromethane, fully stirring, separating, drying an organic phase, filtering, concentrating and purifying to obtain the panbendazole derivative.
4. The use according to claim 3, wherein the solvent in step (3) is an aqueous acetic acid solution, wherein V CH3COOH : V H2O =3:10。
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