WO2020177744A1

WO2020177744A1 - Salicylic acid berberine-type alkaloid quaternary ammonium compound and use thereof for preparing medicines

Info

Publication number: WO2020177744A1
Application number: PCT/CN2020/078049
Authority: WO
Inventors: 秦海林; 吴练秋; 李景; 张海婧; 邓安珺; 唐晓楠; 李想; 王与菲; 李志宏; 王文杰
Original assignee: 中国医学科学院药物研究所
Priority date: 2019-03-05
Filing date: 2020-03-05
Publication date: 2020-09-10
Also published as: CN111662281B; CN111662281A

Abstract

Disclosed in the present invention are a salicylic acid berberine-type alkaloid quaternary ammonium compound as shown in general formula (I), a preparation method therefor, solubility characteristics, a pharmaceutical composition and an application thereof in the preparation of medicinal products. The salicylic acid berberine-type alkaloid quaternary ammonium compound as shown in general formula (I) may be prepared by an organic synthesis method, and shows obvious solubility of alcohol-water mixed solvents. The compound may be conveniently prepared on a large scale, and has significant anti-ulcerative colitis activity, with non-toxic or low toxicity characteristics. The compound may be used to prepare products for preventing, relieving and/or treating ulcerative colitis.

Description

Salicylic acid berberine type alkaloid quaternary ammonium salt and use for preparing medicine

Technical field

The invention relates to a salicylic acid berberine alkaloid quaternary ammonium synthesized by using salicylic acid aromatic organic acids and various berberine alkaloid quaternary ammonium compounds whose acid radicals are not salicylic acid radicals as substrates The salt compound, the preparation method of the compound of the present invention, the pharmaceutical composition containing the compound of the present invention, and the pharmaceutical use of the compound of the present invention for anti-ulcerative colitis. The compound of the present invention has shown remarkable effectiveness in the pharmacodynamic experiment for the treatment of ulcerative colitis; the compound of the present invention has shown remarkable safety in the toxicology experiment; the compound of the present invention is excellent in a 60% ethanol-water mixed solvent The solubility. The above-mentioned properties of the compound of the present invention have obvious prominence. Therefore, the compound of the present invention has significant creativity and practical value in pharmaceutical applications, and can be made into various pharmaceutical dosage forms including ordinary tablet oral and gastric administration forms. It is used to treat ulcerative colitis. The specific structure of the compound of the present invention is based on 5-aminohydrochloride or 4-aminosalicylate or salicylic acid radicals such as salicylic acid radical as the acid radical balance anion unit, with 5,6-dihydrodibenzo[a, g] Quinazine-7-cationic quaternary ammonium salts are base-balanced cation units, and the two form salicylic acid berberine-type alkaloid quaternary ammonium salt compounds. The compound of the present invention can be used to prepare pharmaceutical products for preventing, relieving and/or treating ulcerative colitis, and belongs to the technical field of medicine.

Background technique

Ulcerative colitis (UC) is a chronic, non-specific, recurrent, intestinal mucosal inflammatory bowel disease, and also an autoimmune disease; its focus is mainly located in the sigmoid colon and rectum, and it can also extend to descending The colon or even the entire colon; based on these characteristics, they are called rectal ulcerative colitis, left colon ulcerative colitis, and total colonic ulcerative colitis. Ulcerative colitis lesions are mainly confined to the mucosa and submucosa. Pathological examination or microscopy shows that the basic structures of the intestinal mucosa and submucosa are destroyed, including the exfoliation of the intestinal mucosa with congestion, hemorrhage, and edema. In severe cases, it can be seen Inflammatory cells invade the muscle layer. Clinically, abdominal pain, bloody diarrhea with pus and mucus are the most common early symptoms. Other symptoms include paroxysmal colonic spasm pain, pale complexion, weight loss, tenesmus, vomiting, etc.; therefore, ulcerative colon Inflammation is mainly characterized by abdominal pain, diarrhea and mucus pus and blood in the stool. Patients with diarrhea less than 5 times a day are considered as mild patients, and severe patients with diarrhea more than 5 times a day, even up to 30 times per day, or water Diarrhea or bloody stools, severe abdominal pain, fever, and body temperature can exceed 38.5℃. For patients who do not heal for a long time, clinical symptoms such as anemia, nutritional disorders, and weakness may appear; even some patients have extraintestinal manifestations, such as erythema nodosa, chronic active hepatitis, and pericholangiitis.

Ulcerative colitis can also have some serious complications, including toxic colon dilation, intestinal perforation, hemorrhage, polyps, cancer, enteritis, arthritis, skin and mucosal lesions (multiple abscesses, localized abscesses, pustular gangrene , Erythema multiforme, etc.), a variety of eye diseases (iritis, iridocyclitis, uveitis, corneal ulcers, etc.).

What makes some patients feel entangled and painful is that ulcerative colitis has common symptoms such as prolonged onset, difficult to cure, and malignant transformation. Although it is chronic and low-malignant in some patient groups, it is acute and catastrophic in another group (about 15%); these patients have frequent bloody stools, high fever, abdominal pain, etc. Statistical investigations show that the incidence of ulcerative colitis has been increasing in recent years worldwide [Russel MG. Changes in the incidence of inflammatory bowel disease: what does it mean? .Eur J Inter Med,2000,11:191.;Jiang XI,et al.An analysis of 10218 ulcerative colitis cases in China.World J Gastroenterol,2002,1:158]. Therefore, ulcerative colitis is a recognized malignant disease that seriously affects the quality of life of patients and even threatens survival, and is listed as a difficult disease by the WHO.

Acute severe ulcerative colitis (Acute severe ulcerative colitis, ASUC) refers to bloody stool frequency ≥6 times/day, accompanied by pulse rate>90 beats/min or body temperature>37.8℃ or hemoglobin level<105g/l or red blood cell sedimentation rate >30mm/h. 15% to 25% of patients with ulcerative colitis will have an episode of ASUC. The first-line treatment for ASUC is intravenous corticosteroids, but if the patient does not respond after 3-5 days, cyclosporine or infliximab treatment is salvage therapy, and the patient’s symptoms and serum C- Reactive protein and albumin levels; when the response to these treatments is poor, the patient needs to undergo colon resection. At present, the colon resection rate of ASUC in different regions is 8.5%～48% [Hindryckx P, et al. Acute severe ulcerative colitis: from pathophysiology to clinical management. Nat Rev Gastro Hepat, 2016, 13: 654-664], average At 15% [Ungaro R, et al,.Ulcerative colitis.Lancet,2017,389:1756-1770]. Therefore, it is a serious life-threatening disease (when all drug treatments are ineffective, colorectal resection is needed in time to prevent serious complications). One of the most serious complications of ulcerative colitis is the occurrence of colorectal cancer. The prevalence of colorectal cancer in patients with ulcerative colitis is 3.7% [Thorsteinsdottir S,etal.Pathogenesis and biomarkers of carcinogenesis in ulcerative colitis.Nat Rev Gastro Hepat, 2011, 8:395-404].

The etiology and pathogenesis of ulcerative colitis have not been fully elucidated in academic circles. The views that have been put forward and have been being discussed include the belief that it is related to genetics, immunity, infection, environmental and spiritual factors [Baumgart DC, et al. Inflammatory bowel disease: cause and immunobiology. Lancet, 2007; 369:1627]. Among the existing anti-ulcerative colitis drugs, 5-aminosalicylic acid drugs such as sulfasalazine (Salazosulfapyridine, SASP) work by inhibiting NF-κB and scavenging free radicals. More recent studies have found that the dysfunction of the downstream key transcription factor X-box-binding protein 1 (xbp1) related to the uncontrollable endoplasmic reticulum stress response in intestinal epithelial cells is closely related to the pathogenesis of ulcerative colitis Patients with ulcerative colitis often have mutations in xbp1, which makes them more sensitive to the predisposing factors of ulcerative colitis. The lack or down-regulation of Xbp1 expression will promote the occurrence of ulcerative colitis and aggravate the development of ulcerative colitis; therefore, it is speculated that xbp1 may become a new drug target for the treatment of ulcerative colitis. In addition, mutations in multiple targets (signaling pathways) such as STAT3 lead to abnormal signal transmission of IL-23 and other factors, which are also considered to play an important role in the pathogenesis of ulcerative colitis [Anderson CA,et al.Meta-analysis identifies 29 additional ulcerative colitis risk loci, increasing the number of confirmed associations to 47.Nature genetics,2011,43:246;Rovedatti L,etal.Differential regulation of interleukin-17 and Interferon-gamma production, in inflammatory. Bowel Disease 2009,58:1629].

At present, there is no radical and specific treatment for ulcerative colitis, and there is a very lack of drugs with significant therapeutic effects on ulcerative colitis. In view of this situation, the clinical treatment strategy adopted is mainly palliative therapy similar to tumor treatment, that is, although most patients with ulcerative colitis cannot obtain curative treatment, they still need active treatment and care to control the symptoms. Attention is paid to psychological, social and spiritual issues; special emphasis is placed on various strategies such as symptom control, patient support, and improvement of quality of life, to maintain long-term relief and minimize drug-related adverse reactions; the purpose is to win the best for patients and their families. Probably the best quality of life. Various therapies that have been tried include nuclear factor κB (nuclear factor kappa-B, NF-κB) inhibition and scavenging mechanism of free radicals in the sense of conventional anti-inflammatory, and single-line treatment for tumor necrosis factor-alpha (TNFα). Cloned antibodies (anti-TNFα antibodies), tyrosine kinases (Janus kinases, JAK) inhibitors, etc., but the clinical benefits of current anti-ulcerative colitis drugs are very limited. In the absence of compounds with significant anti-ulcerative colitis activity, new dosage forms and biological preparations of traditional therapeutic drugs have become an active direction in the development of new anti-ulcerative colitis drugs, while microecological preparations and traditional Chinese medicine preparations are mainly used to assist Treatment and alternative therapies. Therefore, it is necessary to find new molecular entities with high efficiency and low toxicity against ulcerative colitis and to develop innovative drugs.

The present invention has discovered a class of compounds with significant anti-ulcerative colitis activity, safe use, and excellent physical and chemical properties. Using various salicylic acid and substituted salicylic acid type aromatic organic acid compounds and various berberine chloride type alkaloid quaternary ammonium salt compounds as substrates to prepare the compound of the present invention, it includes two steps: (1) Under neutral conditions, 8-acetone dihydroberberine-type alkaloid intermediates can be obtained through the nucleophilic addition of the enol ion of acetone to the berberine chloride-type alkaloid quaternary ammonium salt substrate; (2) Dissolve 5-aminosalicylic acid in DMSO or dissolve 4-aminosalicylic acid or salicylic acid in THF, add the intermediates already obtained in step (1) and stir to react until the product precipitates, thereby obtaining the present invention Compound salicylic acid berberine type alkaloid quaternary ammonium compound. The structure of the compound of the present invention has been confirmed by various structural confirmation methods and combined with synthetic routes (see the experimental example part). Further through extensive screening of pharmacological activity, evidence of the pharmacological characteristics of the compound of the present invention having significant anti-ulcerative colitis activity was obtained, and a comparative test showed that the anti-ulcerative colitis pharmacological strength of the compound of the present invention was significantly better than that of The pharmacological effects of the berberine chloride type alkaloid quaternary ammonium salt and the salicylic acid aromatic acid, which are the synthetic raw materials of the compound of the present invention, and the two are mixed in the principle of equal molar ratio. The cytotoxicity evaluation and the acute toxicity evaluation results of animal experiments show that the compound of the present invention does not show obvious toxic effects on normal cells (line), experimental cells and experimental animals, and belongs to a non-toxic or low-toxic molecular entity. In addition, the solubility test experiment proved that the compound of the present invention has excellent solubility in alcohol-water mixed solvents; it is precisely because the compound of the present invention has excellent physical and chemical properties of solubility in alcohol-water mixed solvents, it is a large-scale preparation in line with general pharmaceutical technology. Standardization of salicylic acid berberine alkaloid quaternary ammonium salt active compounds and the search for new pharmacological activities have laid the foundation for improving the strength of the drug. The test results of the stability of physical and chemical properties show that the compound of the present invention is stable in physical and chemical properties, and the structure of the compound of the present invention is extremely stable even if it is placed in a solution. Therefore, the compound of the present invention has significant application value in the preparation of anti-ulcerative colitis products, and can be used to prepare products for preventing, alleviating and/or treating ulcerative colitis.

Summary of the invention

The technical problem solved by the present invention is to provide a class of aromatic salicylic acid radicals as acid radical counter anions and berberine alkaloid quaternary ammonium cations as base counter cations by means of chemical synthesis. A compound with excellent solubility in alcohol-water mixed solvents and significant anti-ulcerative colitis activity and medicinal safety, that is, the salicylic acid berberine type alkaloid quaternary ammonium salt compound represented by general formula I.

In order to solve the above technical problems, the present invention provides the following technical solutions:

The first aspect of the present invention provides a salicylic acid berberine type alkaloid quaternary ammonium salt compound represented by general formula I as the compound of the present invention.

The second aspect of the present invention provides a method for preparing the salicylic acid berberine type alkaloid quaternary ammonium salt compound represented by general formula I.

The third aspect of the present invention provides a product composition of a salicylic acid berberine type alkaloid quaternary ammonium salt compound represented by general formula I; the product is selected from drugs.

The fourth aspect of the present invention provides the use of the salicylic acid berberine type alkaloid quaternary ammonium salt compound represented by general formula I in the prevention, alleviation and/or treatment of ulcerative colitis.

The chemical structural formula of the salicylic acid berberine type alkaloid quaternary ammonium salt compound represented by the general formula I provided in the first aspect of the present invention is shown in the following formula I:

In formula I, R is independently selected from H, NH ₂ , OH, halogen, C2-C4 alkanoyloxy, C2-C4 alkanoyloxy, C1-C4 alkyl or C1-C4 alkoxy, R is a single Substitution or multi-substitution, when R is mono-substituted, the substitution position is 3-position or 4-position or 5-position or 6-position; when R is poly-substituted, it is selected from NH ₂ , OH, halogen, C2-C4 alkamido group, C2-C4 Alkanoyloxy, C1-C4 alkyl, or C1-C4 alkoxy of any combination of 2- or 3- or 4-substituted; further, the halogen is selected from fluorine, chlorine, bromine, and iodine; The C2-C4 alkanoyl group is selected from acetamido, propionamido, butyramido, and isobutyramido; the C2-C4 alkanoyloxy group is selected from acetoxy, propionyloxy, butyryloxy , Isobutyryloxy; the C1-C4 alkyl group is selected from methyl, ethyl, propyl, isopropyl, butyl; the C1-C4 alkoxy group is selected from methoxy, ethoxy Group, propoxy, isopropoxy, butoxy;

R _{1 is} independently selected from H, C2-C4 alkanoyl or C1-C4 alkyl; further, said C2-C4 alkanoyl is selected from acetyl, propionyl, butyryl, isobutyryl; said C1 -C4 alkyl is selected from methyl, ethyl, propyl, isopropyl, butyl;

R ₂ and R ₃ are each independently selected from H, OH, C2-C4 alkanoyloxy, C1-C4 alkyl or C1-C4 alkoxy, or R ₂ and R _{3 are} connected to form a hydrocarbylene dioxy group; further The C2-C4 alkanoyloxy groups described in R ₂ and R ₃ are each independently selected from acetoxy, propionyloxy, butyryloxy, isobutyryloxy; R ₂ , R ₃ The C1-C4 alkyl groups are each independently selected from methyl, ethyl, propyl, isopropyl, and butyl; the C1-C4 alkoxy groups described in R ₂ and R ₃ are each independently selected from methoxy Group, ethoxy group, propoxy group, isopropoxy group, butoxy group; the alkylene dioxy group in R ₂ and R ₃ is independently selected from methylene dioxy group, ethylene dioxy group, Propylenedioxy, butylenedioxy

R ₉ , R ₁₀ , R ₁₁ , and R ₁₂ are each independently selected from H, OH, C1-C4 alkyl, C2-C4 alkanoyloxy or C1-C4 alkoxy, or R ₉ and R _{10 are} connected to form a sub Hydrocarbyl dioxy and R ₁₁ and R ₁₂ are each independently selected from H, OH, C1-C4 alkyl, C2-C4 alkanoyloxy, C1-C4 alkoxy, or R ₉ and R ₁₂ are each independently selected From H, OH, C1-C4 alkyl, C2-C4 alkanoyloxy, C1-C4 alkoxy and R ₁₀ and R _{11 are} connected to form a hydrocarbylene dioxy group, or R ₉ and R ₁₀ are each independently selected from H, OH, C1-C4 alkyl, C2-C4 alkanoyloxy, C1-C4 alkoxy and R ₁₁ and R _{12 are} connected to form a hydrocarbylene dioxy group; further, R ₉ , R ₁₀ , R ₁₁ , The C1-C4 alkyl groups in R ₁₂ are each independently selected from methyl, ethyl, propyl, isopropyl, and butyl; the C2-C4 groups in R ₉ , R ₁₀ , R ₁₁ , and R ₁₂ Alkanoyloxy groups are each independently selected from acetoxy, propionyloxy, butyryloxy, isobutyryloxy; the C1-C4 alkoxy groups described in R ₉ , R ₁₀ , R ₁₁ , and R ₁₂ Each is independently selected from methoxy, ethoxy, propoxy, isopropoxy, butoxy; the alkylene dioxy groups described in R ₉ , R ₁₀ , R ₁₁ , and R ₁₂ are each independently selected From methylene dioxy, ethylene dioxy, propylene dioxy, butylene dioxy.

The most preferred salicylic acid berberine alkaloid quaternary ammonium salt compound of the present invention is selected from compounds 1-21 in the following compound groups:

The second aspect of the present invention provides a preparation method of the salicylic acid berberine alkaloid quaternary ammonium salt compound of the present invention.

The salicylic acid berberine-type alkaloid quaternary ammonium salt compound can be synthesized by the general formula of the following synthetic route (route 1; see experimental examples for specific synthetic conditions):

Synthesis steps: (a) Various berberine alkaloid quaternary ammonium compounds whose acid radicals are not salicylic acid radicals react with acetone under alkaline conditions to obtain 8-acetonyl dihydroberberine intermediates; b) The 8-acetonyl dihydroberberine intermediates are subjected to quaternary ammonium salting in the presence of salicylic aromatic organic acids to obtain various compounds of the present invention.

Among them, the synthesis method of 5-aminosalicylic acid berberine-type alkaloid quaternary ammonium salt compound is as follows: Weigh various berberine-type alkaloid quaternary ammonium salt compounds whose acid roots are not 5-aminosalicylic acid roots into the reaction flask Add sodium hydroxide aqueous solution, then add acetone dropwise, and stir the reaction until the raw material reaction is complete. The reaction mixture was filtered with suction, and the filter cake was washed with water to neutrality to obtain a solid 8-acetone dihydroberberine type alkaloid. Weigh 5-aminosalicylic acid into the reaction flask, add DMSO, after completely dissolving, add 8-acetone dihydroberberine type alkaloid intermediate under stirring to react, until the raw material has reacted completely; add to the reaction mixture Add tetrahydrofuran to dilute, stir until there is no excessive precipitation, filter the reaction mixture, the filter cake is the compound of the present invention 5-aminosalicylic acid berberine type alkaloid quaternary ammonium salt compound.

The synthesis method of 4-aminosalicylic acid or salicylic acid berberine type alkaloid quaternary ammonium salt compound is as follows: Weigh 8-acetone dihydroberberine type alkaloid into a reaction flask, add tetrahydrofuran to make it completely dissolved Add 4-aminosalicylic acid or salicylic acid under stirring. After the addition, reflux the reaction to completion, let stand and cool to room temperature, and filter the reaction mixture. The filter cake is the compound 4-aminosalicylic acid of the present invention. Berberine type alkaloid quaternary ammonium compound or salicylic acid berberine type quaternary ammonium compound.

The third aspect of the present invention also relates to a pharmaceutical composition using the salicylic acid berberine alkaloid quaternary ammonium salt compound as the active ingredient of the first aspect of the present invention. These pharmaceutical compositions can be prepared according to their well-known methods. The compound of the present invention can be combined with one or more pharmaceutically acceptable solid or liquid excipients and/or adjuvants to prepare any dosage form suitable for human or animal use. The content of the compound of the present invention in its pharmaceutical composition is usually 0.1-99.9% (W/W).

The compound of the present invention or the pharmaceutical composition containing the compound of the present invention can be administered in a unit dosage form, and the route of administration can be mainly the digestive tract, such as oral administration, enteral administration, and the like. However, parenteral administration is also acceptable, such as intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eyes, lungs and respiratory tract, vagina, application to the skin, and so on. Among them, in the application of treating ulcerative colitis, its outstanding advantage is that it can be made into ordinary oral preparations such as ordinary tablets and ordinary capsules for direct oral administration without special treatment, and is very convenient to use. Various other routes and methods of administration, such as suppositories, can also be used.

Oral administration or other routes of administration can also adopt other dosage forms, including various liquid dosage forms, solid dosage forms or semi-solid dosage forms prepared by new technologies. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including O/W type, W/O type and double emulsion), suspensions, injections (including water injection, powder injection and infusion), eye drops Tablets, nasal drops, lotions and liniments; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules ( Including hard capsules, soft capsules, enteric-coated capsules), granules, powders, pellets, dripping pills, suppositories, films, patches, air (powder) sprays, sprays, etc.; semi-solid dosage forms can be ointments, Cream type, gel, paste, etc.

The compounds of the present invention can be made into ordinary preparations, and can also be made into sustained-release preparations, controlled-release preparations, targeted preparations and various particle delivery systems.

In order to prepare the compound of the present invention into tablets, various excipients known in the related art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, and glidants. The diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent can be water, ethanol, Isopropanol, etc.; the binder can be starch syrup, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia syrup, gelatin syrup, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl Methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrant can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked Polyvinylpyrrolidone, croscarmellose sodium, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium lauryl sulfonate, etc.; lubricant and auxiliary The flow agent can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol and the like.

The tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multilayer tablets.

In order to make the dosing unit into a capsule, the compound of the present invention can be mixed with a diluent, glidant, and the mixture can be directly placed in a hard or soft capsule; or the compound of the present invention can be mixed with a diluent, a binder, The disintegrant is made into granules or pellets, and then placed in hard or soft capsules. The various diluents, binders, wetting agents, disintegrants, and glidants used to prepare the compound tablets of the present invention can also be used to prepare the compound capsules of the present invention.

In order to prepare the compound of the present invention into injections, water, ethanol, isopropanol, propylene glycol or their mixtures can be used as solvents and appropriate solubilizers, cosolvents, pH regulators, and osmotic pressure regulators commonly used in the pharmaceutical field can be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH regulator can be phosphate, acetate, hydrochloride, sodium hydroxide, etc.; osmotic pressure adjustment The agent can be sodium chloride, mannitol, glucose, phosphate, acetate and the like. For the preparation of freeze-dried powder injections, mannitol, glucose, etc. can also be added as proppants.

In addition, if necessary, coloring agents, preservatives, perfumes, flavoring agents or other additives can also be added to the pharmaceutical preparations.

In order to achieve medicinal purposes and enhance the therapeutic effect, the drug (compound) or pharmaceutical composition of the present invention can be administered and used by any known administration method and application mode.

The administration (application) or dosage (use) of the compound pharmaceutical composition of the present invention depends on the severity of the prevention or treatment of ulcerative colitis, the individual condition of the patient or animal, the administration (application) route and dosage form, etc. Changes in scope. Generally speaking, the appropriate daily dose range of the compound of the present invention is 0.001-500 mg/kg body weight, preferably 0.1-100 mg/kg body weight, more preferably 1-60 mg/kg body weight, and most preferably 2-40 mg/kg body weight. The above-mentioned dosage can be administered in one dosage unit or divided into several dosage units, depending on the doctor's clinical experience, the progress of treatment, and the administration (use) plan including the use of other treatment (application) means.

The compound or product composition of the present invention can be taken alone or combined with other therapeutic drugs or symptomatic drugs. When the compound of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

The fourth aspect of the present invention provides the use of the salicylic acid berberine-type alkaloid quaternary ammonium salt compound represented by general formula I in the preparation of products for the prevention, relief and/or treatment of ulcerative colitis. Wherein, the product includes medicine. See the experimental example of the present invention for the remarkable pharmacodynamic effect of the compound of the present invention in treating ulcerative colitis.

Beneficial technical effect

Experimental studies on pharmacodynamics show that the compound of the present invention has significant pharmacological activities against ulcerative colitis, including pharmacological activities against acute ulcerative colitis and chronic ulcerative colitis. The evaluation results of in vitro cytotoxicity and acute toxicity experiments in animals show that the compounds of the present invention are non-toxic or low-toxic compounds. In terms of physical and chemical properties, the solubility test experiment proved that the compound of the present invention has significantly improved solubility in alcohol-water mixed solvents compared with the corresponding berberine chloride alkaloid quaternary ammonium salts and 5-aminosalicylic acid aromatic acid compounds Therefore, it has significant practical value for the large-scale preparation of active molecular entities of salicylic acid berberine alkaloid quaternary ammonium salt that meets the general technical specifications of pharmaceuticals, and for finding new pharmacological activities and improving the strength of the drug; stability test results It shows that the compound of the present invention is not only stable in physical and chemical properties in the solid state, but its structure is extremely stable even when placed in a solution. Of course, according to the theory of organic chemistry, the compound of the present invention exists in the form of ion pairs or ion clusters in a solution, and has a specific arrangement rather than a mixture. Therefore, the compound of the present invention has significant medicinal effectiveness, safety and quality controllability, and its application prospects in the pharmaceutical field are very significant. In particular, the compound of the present invention has significant application value in the preparation of anti-ulcerative colitis products, and can be used to prepare products for preventing, alleviating and/or treating ulcerative colitis.

In the animal experiment for evaluating the anti-acute ulcerative colitis activity in an animal model of acute C57BL/6J mouse ulcerative colitis induced by Dextran Sulfate Sodium (DSS), the weight change percentage of the model animal after treatment , Percentage of colonic contracture, the effect on the disease activity index (DAI) score and the inhibition rate of the disease activity index (DAI) score and the inhibition rate of the disease activity index and the colon tissue in the acute C57BL/6J mouse ulcerative colitis animal model induced by dextran sodium sulfate Pathological examinations are used as inspection indicators to evaluate curative effects, and the results of each evaluation indicator all show that the compound of the present invention has significant anti-acute ulcerative colitis activity, and has a very prominent therapeutic effect in vivo. The compound of the present invention can significantly improve the symptoms of weight loss, loose stools, blood in the stool, colon contracture and other symptoms of ulcerative colitis model animals, and is significantly better than the current clinically commonly used anti-ulcerative colitis treatment drug sulfasalazine, and better than other related compounds. At a dosage of 100 mg/kg, the compound of the present invention 5-aminosalicylic acid berberine quaternary ammonium salt (1), 5-aminosalicylic acid isocberine quaternary ammonium salt (2), 5-aminosalicylic acid The therapeutic effects of palmatine quaternary ammonium salt (3) and 5-aminosalicylic acid berberine quaternary ammonium salt (4) against acute ulcerative colitis are significantly higher than the positive control drug sulfasalazine given 500 mg/kg. The therapeutic effect of the drug dosage, and parallel experiments show that the anti-ulcerative colitis of the compound of the present invention is also significantly better than the berberine-type alkaloid quaternary ammonium salt (including the quaternary berberine chloride Ammonium salt, quaternary ammonium salt of isoxarine chloride, quaternary ammonium salt of palmatine chloride and quaternary berberine chloride) and 5-aminosalicylic acid monomer compounds; especially 5-aminosalicylic acid in In the same batch of experiments, the percentage of body weight change of model animals, the percentage of colon contracture, the dextran sodium sulfate-induced acute C57BL/6J mouse ulcerative colitis animal model mouse disease activity composite index score and disease activity composite index inhibition rate In terms of the influence, the data are only -15.09%, 39.78% (colon length 4.80±0.28cm), 2.97±0.11 and 7.76%, which are significantly lower than the data of all the detected compounds of the present invention (see experimental examples for specific data). In addition, with 5-aminosalicylic acid berberine quaternary ammonium salt (1) and 5-aminosalicylic acid palmatine quaternary ammonium salt (3) as representative compounds, the amount of the compound of the present invention against acute ulcerative colitis The effect relationship was investigated, and further comparative tests were carried out; the results showed that 5-aminosalicylic acid berberine quaternary ammonium salt (1) and 5-aminosalicylic acid palmatine quaternary ammonium salt (3) are not only anti-ulcerative The efficacy of colitis is strong, and the dose-effect relationship is significant, and it is further confirmed that its anti-acute ulcerative colitis pharmacodynamic effect is significantly stronger than berberine chloride quaternary ammonium salt and dihydroberberine chloride and stronger than palmatine chloride Quaternary ammonium salt; for example, in the experiment, 5-aminosalicylic acid berberine quaternary ammonium salt (1) in the high, medium and low dose groups were administered at 200mg/kg, 100mg/kg and 50mg/kg, and Comparison of animal weight values at the beginning of the experiment. After the experiment, the percentage of animal weight changes (increase to + or decrease to -) were +2.73％ ^## , +2.52％ ^## and -2.51％ ^## ( ^## p<0.01, and Compared with the model group), the weight change percentage of animals treated in parallel in the model group was -18.44%** (**p<0.01, compared with the normal control group); compared with the colon length of the normal control group after the experiment The contracture percentages were 7.57% ^## , 16.26% ^## and 22.18% ^# ( ^# p<0.05, ^## p<0.01, compared with the model group), while the model group animals’ colonic contracture percentage was 36.96%**(* *p<0.01, compared with the normal control group); the comprehensive disease activity index scores are 0.19±0.12 ^## , 0.28±0.19 ^## and 0.80±0.19 ^## , and the corresponding comprehensive disease activity index inhibition rate values are respectively 93.85 %, 90.93% and 74.11% ( ^## p<0.01, compared with the model group) (see experimental examples for specific data); pathological examination of colon tissue shows that the compound of the present invention can significantly reduce colonic tissue ulcer formation, and intestinal epithelial cells after treatment The arrangement is complete and almost restored to normal physiological state. And the conclusion that the dose-effect relationship is significant and that the effective dose of the compound of the present invention is less than 50 mg/kg is clear. The above treatment data of the compound of the present invention is compared with the corresponding data when the dose of the positive control drug sulfasalazine in the parallel experiment is 500 mg/kg, including the percentage change (decrease) of animal weight in the sulfasalazine administration group- 13.47% (no significant difference compared with the model group), animal colon contracture percentage 32.23% (no significant difference compared with the model group), disease activity comprehensive index score 2.12±0.28 ^# and disease activity comprehensive index inhibition rate experimental data 31.39 % And the results of colonic histopathological examination, the curative effect is very significant. Compared with the parallel experiments of berberine chloride quaternary ammonium salt, palmatine chloride quaternary ammonium salt, berberine chloride quaternary ammonium salt and dihydro berberine quaternary ammonium salt, it also has outstanding advantages (see experimental examples for specific data) ).

In an animal experiment for evaluating anti-chronic ulcerative colitis activity in an animal model of chronic BALB/c mouse ulcerative colitis induced by oxazolone (OXZ), the percentage of body weight change and the percentage of colon contracture in the model animal after treatment As well as the effect on the disease activity comprehensive index score and the disease activity comprehensive index inhibition rate of ulcerative colitis model mice, the evaluation indexes are used to evaluate the efficacy. The results of each evaluation index indicate that the compound of the present invention has significant anti-chronic ulcerative colitis activity , The therapeutic effect in the body is very prominent. The compound of the invention can significantly improve the symptoms of chronic ulcerative colitis model animals such as weight loss, loose stools, blood in the stool, colonic contracture, etc., and is significantly better than the current clinically commonly used anti-ulcerative colitis treatment drug sulfasalazine. In the specific experiment, 5-aminosalicylic acid berberine quaternary ammonium salt (1) was used as a representative compound to investigate the effect of the compound of the present invention against oxazolone-induced chronic ulcerative colitis in BALB/c mice and the dose-effect relationship. The results show that 5-aminosalicylic acid berberine quaternary ammonium salt (1) not only has a strong anti-chronic ulcerative colitis, but also has a significant dose-effect relationship. 5-aminosalicylic acid berberine quaternary ammonium salt (1) When the doses of the high, medium and low dose groups are 200mg/kg, 100mg/kg and 50mg/kg respectively, compared with the initial animal weight value of the experiment, the experiment ends The percentages of post-animal body weight change were -11.00% ^# , -16.50% and -20.91% ( ^# p<0.05, compared with the model group), while the percentage change of animal weight in the model group treated in parallel was -27.70%**(** p<0.01, compared with the normal control group); compared with the colon length of the normal control group after the experiment, the percentage of colon contracture is 7.25% ^## , 15.53% ^## and 17.18% ^## ( ^## p<0.01 , Compared with the model group), and the percentage of colonic contracture in the model group was as high as 28.57%** (**p<0.01, compared with the normal control group); the disease activity comprehensive index inhibition rate value reached 69.64% ^## ,46.43 % ^## and 32.14% ( ^## p<0.01, compared with the model group). The above treatment data of the compound of the present invention is compared with the corresponding data when the dose of the positive control drug sulfasalazine in the parallel experiment is 500 mg/kg, including the percentage change (decrease) of animal weight in the sulfasalazine administration group- 22.82% (no significant difference compared with the model group), animal colon contracture percentage 24.64% (no significant difference compared with the model group), and disease activity comprehensive index inhibition rate value experimental data 14.29% (no significant difference compared with the model group) ) Comparison, the effect is very significant. Although some treatment data, such as the percentage of body weight change after treatment in the middle and low dose groups of the compound of the present invention and the inhibition rate of disease activity composite index after low dose treatment, there is no significant difference compared with the model group, but the experimental dose The effect of gradient is also very obvious, and the dose-effect relationship is very clear.

In animal experiments using dextran sodium sulfate to induce chronic C57BL/6J mouse ulcerative colitis animal model to evaluate anti-chronic ulcerative colitis activity, the compound of the present invention has a significant therapeutic effect on chronic ulcerative colitis model animals The effect is significantly better than that of sulfasalazine, a commonly used clinical treatment for anti-ulcerative colitis. When the doses of the high, medium and low dose groups were 200mg/kg, 100mg/kg and 50mg/kg, the disease activity comprehensive index inhibition rate values were 94.44% ^## 、58.33% ^# and 38.89% ^# ( ^# p <0.05, ^## p<0.01, compared with the model group), compared with the experimental data of 50.00% of the inhibitory rate of the disease activity composite index when the dose of the positive control drug sulfasalazine in the parallel experiment is 500mg/kg, Especially in the high-dose and middle-dose administration groups, the curative effect is very significant, and the dose-effect relationship is clear.

In view of the fact that 5-aminosalicylic acid is not suitable for direct oral administration in the treatment of ulcerative colitis due to its physical and chemical properties, 5-aminosalicylic acid is used as the acid radical to balance the anion unit, and the berberine type organism Alkaline quaternary ammonium cations are base-balanced cation units, and the two form salicylic acid berberine-type alkaloid quaternary ammonium salt compounds, but they show exceptional antiulcerative colitis activity in direct oral administration experiments. Therefore, The effective mechanism of the compound of the present invention in the treatment of ulcerative colitis remains to be further studied. According to the compound of the present invention, 5-aminosalicylic acid, berberine chloride quaternary ammonium salt, palmatine chloride quaternary ammonium salt, berberine chloride quaternary ammonium salt, and berberine chloride quaternary ammonium salt, and berberine chloride are formulated according to the principle of equal A mixture of quaternary ammonium salt and 5-aminosalicylic acid, a mixture of palmatine chloride quaternary ammonium salt and 5-aminosalicylic acid, and a mixture of berberine chloride quaternary ammonium salt and 5-aminosalicylic acid The effect comparison test also shows that the compound of the present invention is significantly different from the mixture of berberine chloride type quaternary ammonium salt and 5-aminosalicylic acid prepared simply by the principle of equal moles. The compound of the present invention is based on 5-aminohydrochloride (or 4-aminosalicylate or salicylate) as the acid-balanced anion unit, and 5,6-dihydrodibenzo[a,g]quinazine-7- The cationic quaternary ammonium structure is a base-balanced cationic unit. The two match through a specific intermolecular attraction to form a salicylic acid berberine alkaloid quaternary ammonium salt compound. Each structural unit is arranged regularly in the substance. The cloth, the structure is stable, and the mixture of berberine-type alkaloid quaternary ammonium salt and salicylic acid formulated on the principle of simply equaling the number of moles has essential differences; the compound of the present invention has significant anti-ulcerative colitis efficacy. Better than 5-aminosalicylic acid, berberine chloride type quaternary ammonium salt substrate and a mixture of berberine chloride type quaternary ammonium salt substrate and 5-aminosalicylic acid prepared on the principle of equal moles (specific For the data, see pharmacodynamic experiment examples 1, 4, 6 and 7).

In addition, according to the research and correlation of the present invention, the pharmacological effects of the compounds of the present invention related to anti-ulcerative colitis are also significantly better than those of tartrate, citrate, oxalate, maleate, malate, fumarate and Other organic acid radicals such as benzenesulfonate are acid radical counter-anion, and other organic acid berberine-type alkaloid quaternary ammonium salt compounds are prepared with berberine-type alkaloid quaternary ammonium cation as base counter-cation. First, parallel animal pharmacodynamic experiments proved that the anti-ulcerative colitis of the compound of the present invention is significantly better than dihydroberberine, and dihydroberberine is better than berberine chloride quaternary ammonium salt (see the drug for specific data Efficacy experiment example). Secondly, molecular-level pharmacological experiments confirmed that the xbp1 promoter transcriptional activation effect of dihydroberberine was significantly better than that of organic tartrate, citrate, oxalate, maleate, malate, fumarate and benzenesulfonate. Acid root is the acid root balancing anion, and the organic acid berberine alkaloid quaternary ammonium salt compound prepared with the berberine alkaloid quaternary ammonium cation as the base balancing cation has the transcriptional activation effect of the xbp1 promoter of these other types of organic acids The berberine-type alkaloid quaternary ammonium salt compound has a positive or ineffective activation factor for xbp1 promoter transcription within the range of 1.02-1.31, while the activation factor of dihydroberberine in parallel experiments is 1.64, which has obvious activation Effect (see pharmacodynamic experiment example for specific data). The above pharmacological experiment results are consistent with published literature data [Zhang ZH, et al. Versatile methods for synthesizing organic acid salts of quaternary berberine-type alkaloids as anti-ulcerative colitis agents. J. Asian Nat.Prod.Res., 2016 ,18,576-586; Zhang ZH,et al. Synthesis and structure-activity relationships of quaternary coptisine derivatives as potential anti-ulcerative Colitis agents. J.Med.Chem.,2015,58,7557-7571]. Therefore, the experimental results of the present invention confirm the outstanding anti-ulcerative colitis effect of the compound of the present invention, which is superior to other related compounds.

In addition to the significant pharmacological activity of the compound of the present invention compared with related compounds, based on the research on the specificity of the pharmacological action, another outstanding feature of the compound of the present invention is that it also has the advantages of non-toxicity or low toxicity. In the test of toxicity (cell viability) of

compounds

1, 2, 3 and 4 by culturing normal human embryonic kidney 293T epithelial cells in vitro, the inhibition rates of

compounds

1, 2, 3 and 4 on normal cell growth were 12.35, respectively. %, 14.94%, 3.96% and 14.68%. In the acute toxicity experiment in animals with Kunming mice (weight range of 18-22g), the compound of the present invention 5-aminosalicylic acid berberine quaternary ammonium salt (1), 5-aminosalicylic acid isoberberine When both the quaternary ammonium salt (2) and the 5-aminosalicylic acid palmatine quaternary ammonium salt (3) were administered at a dose of 5.0 g/kg, the animals were not dead and were in good general condition. The 5-aminosalicylic acid berberine quaternary ammonium salt (4) has an LD ₅₀ value of 3.0 g/kg. Therefore, the compounds of the present invention are all non-toxic or low-toxic specific anti-ulcerative colitis compounds.

Compared with the corresponding berberine chloride-type alkaloid quaternary ammonium salt substrates and 5-aminosalicylic acid, the solubility of the compound of the present invention in a mixed solvent of ethanol and water is significantly improved; at an ambient temperature of 25℃±2℃ To determine the solubility, the amount of 60% ethanol-water mixed solvent needed to dissolve each gram of the compound of the present invention is 5-aminosalicylic acid berberine quaternary ammonium salt (1) 300ml, 5-aminosalicylic acid isocberine quaternary ammonium salt (2) 200ml, 5-aminosalicylic acid palmatine quaternary ammonium salt (3) 400ml, 5-aminosalicylic acid berberine quaternary ammonium salt (4) 350ml. In parallel determinations, it takes 60% ethanol water mixed solvent to dissolve each gram of berberine-type alkaloid quaternary ammonium cation substrates of the present invention. The amounts are 1200ml and 680ml respectively, and the amount of 60% ethanol-water mixed solvent required to dissolve each gram of 5-aminosalicylic acid is 800ml, which is significantly larger than the amount required for compound 1-4. Under reflux conditions, the alcohol-water mixed solvent of the compound of the present invention has higher solubility, and the amount of 60% ethanol-water mixed solvent required to dissolve the compound of the present invention is 5-aminosalicylic acid berberine quaternary ammonium salt (1) 38ml, 5-aminosalicylic acid isoberberine quaternary ammonium salt (2) 26ml, 5-aminosalicylic acid palmatine quaternary ammonium salt (3) 47ml, 5-aminosalicylic acid berberine quaternary ammonium salt (4) ) 73ml; these physical and chemical properties are very suitable for large-scale preparation of compounds.

Description of the drawings

Figure 1. Positive drugs sulfasalazine, berberine chloride quaternary ammonium salt, dihydroberberine, and different doses of the compound 1 administration group of the present invention on weight loss in mice with acute ulcerative colitis induced by sodium dextran sulfate Improvement effect (**p<0.01, compared with the normal control group; ^## p<0.01, compared with the model group).

Figure 2. The positive drug sulfasalazine, berberine chloride quaternary ammonium salt, dihydroberberine, compound 1 of the present invention, high, medium, and low dose administration groups in the acute ulcerative colitis model induced by sodium dextran sulfate The percentage of mouse colonic contracture during the treatment of mice (Note: ^** p<0.01, compared with the normal control group; ^# p<0.05, ^## p<0.01, compared with the model group).

Figure 3. The positive drug sulfasalazine, berberine chloride quaternary ammonium salt, dihydroberberine, compound 1 of the present invention in high, medium and low dose administration groups in the acute ulcerative colitis model induced by sodium dextran sulfate Mouse Disease Activity Comprehensive Index (DAI) score during treatment of mice (Note: ^** p<0.01, compared with normal control group; ^# p<0.05, ^## p<0.01, compared with model group).

Figure 4. The therapeutic effect of compound 1 of the present invention on pathological damage of colon tissue in mice with acute ulcerative colitis induced by sodium dextran sulfate (HE, 40 times).

Figure 5. The improvement effect of the positive drug sulfasalazine and different doses of compound 1 of the present invention on the weight loss of oxazolone-induced chronic ulcerative colitis model mice (**p<0.01, compared with the normal control group; ^# p<0.05, compared with the model group).

Figure 6. The improving effect of compound 1 of the present invention on oxazolone-induced colonic contracture in mice with chronic ulcerative colitis.

Figure 7. The composite index score of disease activity of mice in the treatment of oxazolone-induced chronic ulcerative colitis model mice in the low, medium and high dose administration groups of positive drug sulfasalazine and compound 1 of the present invention (Note: ^** p<0.01, compared with the normal control group; ^## p<0.01, compared with the model group).

Figure 8. Experimental results of the activation effect of dihydroberberine and 10 organic acid berberine-type alkaloid quaternary ammonium compounds on the xbp1 promoter.

detailed description

The specific embodiments of the invention do not limit the invention in any way.

The preparation process and structure identification data of the active compound of the present invention, wherein the compound number corresponds to the specific compound number in the content of the present invention.

1. Preparation experiment example of the compound of the present invention

Experimental example (1): Synthesis and structure identification data of compound 1 of the present invention

Weigh the quaternary ammonium salt of berberine chloride (2.00g, 5.62mmol) into the reaction flask, add 5N aqueous sodium hydroxide solution (12ml), and then add acetone (4ml, 54.27mmol) dropwise, stir at room temperature and react for 4h. complete. The reaction mixture was suction filtered, and the filter cake was washed with water to neutrality to obtain 1.99 g of 8-acetone dihydroberberine as a pale yellow solid with a yield of 93.87%.

Weigh 5-aminosalicylic acid (43mg, 0.28mmol) into the reaction flask, add DMSO (3ml), sonicate to completely dissolve, add 8-acetone dihydroberberine (100mg, 0.26mmol) under stirring at room temperature, After the addition, the reaction was carried out at 25°C for 5.5 hours until the reaction of the raw materials was complete; add tetrahydrofuran (6ml) to the reaction mixture to dilute, stir until no excessive precipitation precipitated, filter the reaction mixture, and wash the filter cake with tetrahydrofuran three times to obtain Compound 1 is a yellow solid 76 mg, with a yield of 60.61%. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ: 9.94 (s, 1H, ArH), 8.95 (s, 1H, ArH), 8.04 (d, J = 8.4 Hz, 1H, ArH), 7.82 (d, J = 8.4Hz, 1H, ArH), 7.79 (s, 1H, ArH), 7.08 (s, 1H, ArH), 6.98 (d, J = 2.8 Hz, 1H, ArH), 6.53 (s, 2H, OCH ₂ O), 6.42 (dd, J = 8.4, 2.8 Hz, 1H, ArH), 6.32 (d, J = 8.4 Hz, 1H, ArH), 6.17 (s, 2H, OCH ₂ O), 4.87 (t, J = 6.4 Hz, 2H, N CH ₂ CH ₂ ), 3.20 (t, J=6.4 Hz, 2H, NCH ₂ CH ₂ ). ¹³ C-NMR (150MHz, DMSO-d ₆ ) δ: 171.3, 154.0, 149.8, 147.7, 147.1, 144.6, 143.8, 137.8, 136.8, 132.3, 130.6, 121.7, 121.1, 121.03, 120.97, 120.5, 118.3, 116.0, 115.1,111.6,108.4,105.3,104.5,102.1,55.1,26.3.(+)HRESI-MS(m/z):320.09158[MC ₇ H ₆ NO ₃ ] ⁺ (calcd for C ₁₉ H ₁₄ NO ₄ ,320.09173) ; (-)HRESI-MS(m/z): 152.03560[MC ₁₉ H ₁₄ NO ₄ ] ^- (calcd for C ₇ H ₆ NO ₃ , 152.0353).

Experimental example (2): Synthesis and structure identification data of compound 2 of the present invention

Weigh out the quaternary ammonium salt of isoflavine chloride (3.84g, 10.80mmol) in the reaction flask, add 5N sodium hydroxide aqueous solution (100ml), and then add acetone (10ml, 0.14mol) dropwise, heat and stir at 50°C for 4h , The raw material reaction is complete. The reaction mixture was suction filtered, and the filter cake was washed with water to neutrality. A pale yellow solid crude product was obtained, and then recrystallized with a mixed solvent of acetone and water (acetone: water = 3:1, v/v) to obtain 2.26 g of yellow granular crystals of 8-acetone dihydroisoberberine, yield 55.52%.

Weigh 5-aminosalicylic acid (43mg, 0.28mmol) into the reaction flask, add DMSO (3ml), sonicate to completely dissolve, add 8-acetone dihydroisoberberine (100mg, 0.26mmol) under stirring at room temperature After the addition, the reaction was carried out at 25°C for 5.5 hours until the reaction of the raw materials was complete; the reaction mixture was diluted by adding tetrahydrofuran (6ml), stirred until no excessive precipitation was precipitated, the reaction solution was filtered, and the filter cake was washed with tetrahydrofuran three times to obtain Compound 2 is a yellow solid of 68 mg, with a yield of 54.09%. ¹ H-NMR(400MHz,DMSO-d ₆ )δ: 9.55(s,1H,ArH), 8.73(s,1H,ArH), 7.73(s,1H,ArH), 7.71(s,1H,ArH), 7.51(s,1H,ArH),7.08(s,1H,ArH),6.99(d,J=2.8Hz,1H,ArH),6.43(dd,J=8.0,2.8Hz,1H,ArH),6.41( s, 2H, OC H ₂ O), 6.32 (d, J = 8.0 Hz, 1H, ArH), 6.17 (s, 2H, OC H ₂ O), 4.75 (t, J = 6.4 Hz, 2H, NC H ₂ CH ₂ ), 3.18 (t, J=6.4 Hz, 2H, NCH ₂ C H ₂ ). ¹³ C-NMR (150MHz, DMSO-d ₆ ) δ: 171.9, 156.4, 154.5, 151.3, 150.4, 148.1, 146.3, 139.2,139.1,138.4,131.3,124.0,121.6,120.7,119.4,118.7,116.4,115.6,109.0,105.9,104.4,104.2,103.1,102.6,54.9,26.8. (+)HRESI-MS(m/z): 320.09418[MC ₇ H ₆ NO ₃ ] ⁺ (calcd for C ₁₉ H ₁₄ NO ₄ ,320.09173); (-)HRESI-MS(m/z):152.03355[MC ₁₉ H ₁₄ NO ₄ ] ^- (calcd for C ₇ H ₆ NO ₃ ,152.03532).

Experimental example (3): Synthesis and structure identification data of compound 3 of the present invention

Weigh palmatine chloride quaternary ammonium salt (4.00g, 10.30mmol) in the reaction flask, add 5N sodium hydroxide aqueous solution (24ml), then add acetone (9.16ml, 123.20mmol) dropwise, stir at room temperature for 4h, The raw material reaction is complete. The reaction mixture was suction filtered, and the filter cake was washed with water to neutrality, to obtain 3.36 g of 8-acetone dihydropalmatine as a pale yellow solid, with a yield of 79.71%.

Weigh 5-aminosalicylic acid (40mg, 0.26mmol) into the reaction flask, add DMSO (3ml), sonicate to completely dissolve, add 8-acetone dihydropalmatine (100mg, 0.24mmol) under stirring at room temperature After the addition, the reaction was carried out at 25°C for 5.5 hours until the reaction of the raw materials was complete; the reaction mixture was diluted by adding tetrahydrofuran (6ml), stirred until no excessive precipitation was precipitated, the reaction solution was filtered, and the filter cake was washed with tetrahydrofuran three times to obtain Compound 3 is a yellow solid of 80 mg, with a yield of 64.77%. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ: 9.89 (s, 1H, ArH), 9.01 (s, 1H, ArH), 8.21 (d, J = 9.2 Hz, 1H, ArH), 8.02 (d, J = 9.2Hz, 1H, ArH), 7.71 (s, 1H, ArH), 7.10 (s, 1H, ArH), 6.98 (d, J = 2.8 Hz, 1H, ArH), 6.43 (dd, J = 8.4, 2.8Hz,1H,ArH), 6.32(d,J=8.4Hz,1H,ArH), 4.95(t,J=6.4Hz,2H,NC H ₂ CH ₂ ), 4.10(s, 3H, OCH ₃ ), 4.07 (s, 3H, OCH ₃ ), 3.94 (s, 3H, OCH ₃ ), 3.88 (s, 3H, OCH ₃ ), 3.23 (t, J = 6.4 Hz, 2H, NCH ₂ C H ₂ ). ¹³ C NMR (150MHz, DMSO-d ₆ ) δ: 171.8, 154.5, 152.0, 150.7, 149.2, 145.9, 144.1, 138.3, 138.2, 133.5, 129.1, 127.3, 123.8, 121.8, 121.5, 120.3, 119.4, 118.8, 116.4, 115.6 ,111.8,109.2,62.4,57.5,56.6,56.3,55.8,26.5.(+)HRESI-MS(m/z):352.15759[MC ₇ H ₆ NO ₃ ] ⁺ (calcd for C ₂₁ H ₂₂ NO ₄ ,352.15433 ); (-)HRESI-MS(m/z):152.03357[MC ₂₁ H ₂₂ NO ₄ ] ^- (calcd for C ₇ H ₆ NO ₃ ,152.03532).

Experimental example (4): Synthesis and structure identification data of compound 4 of the present invention

Weigh berberine chloride quaternary ammonium salt (2.00g, 5.38mmol) into the reaction flask, add 5N sodium hydroxide aqueous solution (12ml), then add acetone (4ml, 53.80mmol) dropwise, stir at room temperature for 4h, raw materials The reaction is complete. The reaction mixture was filtered with suction, and the filter cake was washed with water until it was neutral to obtain 1.72 g of 8-acetone dihydroberberine as a pale yellow solid with a yield of 81.33%.

Weigh 5-aminosalicylic acid (42mg, 0.26mmol) into the reaction flask, add DMSO (3ml), sonicate to completely dissolve, add 8-acetone dihydroberberine (100mg, 0.24mmol) under stirring at room temperature After the addition, the reaction was carried out at 25°C for 5.5 hours until the reaction of the raw materials was complete; the reaction mixture was diluted by adding tetrahydrofuran (6ml), stirred until no excessive precipitation was precipitated, the reaction solution was filtered, and the filter cake was washed with tetrahydrofuran three times to obtain Compound 4 is a yellow solid of 69 mg, with a yield of 55.18%. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ: 9.89 (s, 1H, ArH), 8.93 (s, 1H, ArH), 8.20 (d, J = 9.2 Hz, 1H, ArH), 8.00 (d, J = 9.2Hz, 1H, ArH), 7.80 (s, 1H, ArH), 7.09 (s, 1H, ArH), 6.98 (d, J = 2.8 Hz, 1H, ArH), 6.43 (dd, J = 8.4, 2.8Hz,1H,ArH),6.32(d,J=8.4Hz,1H,ArH),6.17(s,2H,OC H ₂ O), 4.93(t,J=6.0Hz,2H,NC H ₂ CH ₂ ), 4.10 (s, 3H, OCH ₃ ), 4.07 (s, 3H, OCH ₃ ), 3.20 (t, J = 6.0 Hz, 2H, NCH ₂ C H ₂ ). ¹³ C NMR (150MHz, DMSO-d ₆ )δ: 171.7, 154.5, 150.9, 150.3, 148.2, 145.9, 144.1, 138.3, 138.0, 133.4, 131.2, 127.2, 124.0, 121.9, 121.6, 120.9, 120.7, 118.7, 116.4, 115.6, 108.9, 105.9, 102.6, 62.4 ,57.5,55.7,26.8.(+)HRESI-MS(m/z):336.12604[MC ₇ H ₆ NO ₃ ] ⁺ (calcd for C ₂₀ H ₁₈ NO ₄ ,336.12303);(-)HRESI-MS(m /z):152.03360[MC ₂₀ H ₁₈ NO ₄ ] ^- (calcd for C ₇ H ₆ NO ₃ ,152.03532).

Experimental example (5): Synthesis and structure identification data of compound 5 of the present invention

Weigh 2,3-methylenedioxy-10,11-ethylenedioxy-5,6-dihydrodibenzo[a,g]quinazine quaternary ammonium chloride (500mg, 1.35mmol) in Into the reaction flask, 5N aqueous sodium hydroxide solution (3ml) was added, and then acetone (1ml, 13.5mmol) was added dropwise. The reaction was stirred at room temperature for 4h, and the reaction of the raw materials was complete. The reaction mixture was filtered with suction, and the filter cake was washed with water to neutrality to obtain a light yellow solid. The product was directly used in the next step without purification. Weigh 5-aminosalicylic acid (177mg, 1.14mmol) into the reaction flask, add DMSO (4ml), sonicate until it is completely dissolved, add the above unpurified product under stirring at room temperature, and proceed the reaction at 25°C after addition 5.5h, until the reaction of the raw materials is complete; add tetrahydrofuran (8ml) to the reaction mixture to dilute, stir until no excessive precipitation is precipitated, filter the reaction solution, and wash the filter cake with tetrahydrofuran three times to obtain compound 5 as yellow solid 366mg, yield 55.71% . ¹ H-NMR(400MHz,DMSO-d ₆ )δ: 9.61(s,1H,ArH), 8.72(s,1H,ArH), 7.84(s,1H,ArH), 7.73(s,1H,ArH), 7.58(s,1H,ArH),7.09(s,1H,ArH), 6.99(d,J=2.8Hz,1H,ArH), 6.46(dd,J=8.0,2.8Hz,1H,ArH), 6.34( d,J=8.0Hz,1H,ArH),6.17(s,2H,OCH ₂ O),4.76(t,J=6.4Hz,2H,NC H ₂ CH ₂ ),4.57,4.50(2×m,2 ×2H,OC H ₂ C H ₂ O), 3.19(t,J=6.4Hz,2H,NCH ₂ C H ₂ ). ¹³ C-NMR(150MHz,DMSO-d ₆ )δ: 171.4, 154.0, 152.9, 149.7, 147.6, 147.2, 147.1, 137.9, 137.5, 135.3, 130.5, 121.7, 121.1, 120.5, 118.3, 118.2, 115.9, 115.1, 114.0, 111.4, 108.4, 105.3, 102.1, 65.1, 64.2, 54.6, 26.5. (+ )HRESI-MS(m/z):334.10739[MC ₇ H ₆ NO ₃ ] ⁺ (calcd for C ₂₀ H ₁₆ NO ₄ ,334.10738).

Experimental example (6): Synthesis and structure identification data of compound 6 of the present invention

Weigh 2,3-methylenedioxy-9,10,11-trimethoxy-5,6-dihydrodibenzo[a,g]quinazine quaternary ammonium chloride (500mg, 1.25mmol) in Into the reaction flask, 5N aqueous sodium hydroxide solution (3ml) was added, and then acetone (0.92ul, 12.5mmol) was added dropwise. The reaction was stirred at room temperature for 4h, and the reaction of the raw materials was complete. The reaction mixture was filtered with suction, and the filter cake was washed with water to neutrality to obtain a light yellow solid. The product was directly used in the next step without purification. Weigh 5-aminosalicylic acid (165mg, 1.06mmol) into the reaction flask, add DMSO (4ml), sonicate to completely dissolve, add the above unpurified product under stirring at room temperature, and proceed the reaction at 25℃ after adding 5.5h, until the reaction of the raw materials is complete; add tetrahydrofuran (8ml) to the reaction mixture to dilute, stir until no excessive precipitation is precipitated, the reaction mixture is suction filtered, the filter cake is washed with tetrahydrofuran three times to obtain compound 6 yellow solid 380mg, yield 58.91%. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ: 9.64 (s, 1H, ArH), 8.74 (s, 1H, ArH), 7.75 (s, 1H, ArH), 7.40 (s, 1H, ArH), 7.08(s,1H,ArH), 6.98(d,J=2.8Hz,1H,ArH), 6.43(dd,J=8.0,2.8Hz,1H,ArH), 6.32(d,J=8.0Hz,1H, ArH), 6.17(s,2H,OC H ₂ O), 4.84(t,J=6.4Hz,2H,NC H ₂ CH ₂ ), 4.15(s,3H,OCH ₃ ),4.09(s,3H,OCH ₃ ), 3.94 (s, 3H, OCH ₃ ), 3.18 (t, J = 6.4 Hz, 2H, NCH ₂ C H ₂ ). ¹³ C-NMR (150MHz, DMSO-d ₆ ) δ: 171.3, 162.0, 154.0 ,150.0,148.8,147.7,143.8,142.0,139.2,137.9,136.8,131.1,121.1,120.3,118.2,118.1,117.1,115.9,115.1,108.5,105.5,102.1,101.7,62.3,61.4,57.0,54.3,26.4 .(+)HRESI-MS(m/z):366.13364[MC ₇ H ₆ NO ₃ ] ⁺ (calcd for C ₂₁ H ₂₀ NO ₅ ,366.13360).

Experimental example (7): Synthesis and structure identification data of compound 7 of the present invention

Weigh 2,3-methylenedioxy-9,10-dimethyl-5,6-dihydrodibenzo[a,g]quinazine quaternary ammonium chloride (500mg, 1.47mmol) in the reaction flask Add 5N sodium hydroxide aqueous solution (3ml), then add acetone (1.08ml, 14.7mmol) dropwise, stir and react at room temperature for 4h, the reaction of the raw materials is complete. The reaction mixture was filtered with suction, and the filter cake was washed with water to neutrality to obtain a light yellow solid. The product was directly used in the next step without purification. Weigh 5-aminosalicylic acid (195mg, 1.26mmol) into the reaction flask, add DMSO (4ml), sonicate until it is completely dissolved, add the above unpurified product under stirring at room temperature, and proceed the reaction at 25°C after addition 5.5h, until the reaction of the raw materials is complete; add tetrahydrofuran (8ml) to the reaction mixture to dilute, stir until no excessive precipitation precipitates, filter the reaction mixture, and wash the filter cake with tetrahydrofuran three times to obtain compound 7 as yellow solid 412mg, yield 61.40 %. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ: 9.98 (s, 1H, ArH), 8.89 (s, 1H, ArH), 7.98 (d, J = 8.4 Hz, 1H, ArH), 7.93 (d, J = 8.4Hz, 1H, ArH), 7.81 (s, 1H, ArH), 7.07 (s, 1H, ArH), 6.94 (d, J = 2.8 Hz, 1H, ArH), 6.39 (dd, J = 8.4, 2.8Hz, 1H, ArH), 6.28 (d, J = 8.4 Hz, 1H, ArH), 6.14 (s, 2H, OCH ₂ O), 4.90 (t, J = 6.0 Hz, 2H, NC H ₂ CH ₂ ) , 3.19 (t, J = 6.0 Hz, 2H, NCH ₂ C H ₂ ), 2.69 (s, 3H, ArCH ₃ ), 2.51 (s, 3H, ArCH ₃ ). ¹³ C-NMR (150MHz, DMSO-d ₆ )δ: 171.8, 154.5, 150.5, 148.2, 147.9, 140.0, 139.1, 138.7, 138.3, 138.0, 136.1, 131.4, 125.6, 125.1, 121.5, 120.9, 120.7, 118.7, 116.4, 115.6, 108.9, 106.1, 102.6, 55.5 ,26.8,20.6,14.5.(+)HRESI-MS(m/z):304.13327[MC ₇ H ₆ NO ₃ ] ⁺ (calcd for C ₂₀ H ₁₈ NO ₂ ,304.13321).

Experimental example (8): Synthesis and structure identification data of compound 8 of the present invention

Weigh 2,3-methylenedioxy-10,11-dimethoxy-5,6-dihydrodibenzo[a,g]quinazine quaternary ammonium chloride (500mg, 1.34mmol) in the reaction Into the bottle, 5N aqueous sodium hydroxide solution (3ml) was added, and then acetone (1ml, 13.5mmol) was added dropwise. The reaction was stirred at room temperature for 4h, and the reaction of the raw materials was complete. The reaction mixture was filtered with suction, and the filter cake was washed with water to neutrality to obtain a light yellow solid. The product was directly used in the next step without purification. Weigh 5-aminosalicylic acid (165mg, 1.06mmol) into the reaction flask, add DMSO (4ml), sonicate to completely dissolve, add the above unpurified product under stirring at room temperature, and proceed the reaction at 25℃ after adding 5.5h, to complete the reaction of the raw materials; add tetrahydrofuran (8ml) to the reaction mixture to dilute, stir until no excessive precipitation precipitates, filter the reaction mixture, and wash the filter cake with tetrahydrofuran three times to obtain compound 8 as a yellow solid 350mg, the yield is 53.35 %. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ: 9.53 (s, 1H, ArH), 8.76 (s, 1H, ArH), 7.74 (s, 1H, ArH), 7.72 (s, 1H, ArH), 7.57(s,1H,ArH),7.10(s,1H,ArH),6.99(d,J=2.8Hz,1H,ArH),6.44(dd,J=8.0,2.8Hz,1H,ArH),6.33( d,J=8.0Hz,1H,ArH),6.18(s,2H,OCH ₂ O),4.78(t,J=6.4Hz,2H,NC H ₂ CH ₂ ),4.07(s,3H,OCH ₃ ) ,4.00(s,3H,OCH ₃ ), 3.20(t,J=6.4Hz,2H,NCH ₂ C H ₂ ). ¹³ C-NMR(150MHz,DMSO-d ₆ )δ: 171.8, 157.9, 154.5, 152.8 ,150.3,148.1,146.0,138.7,138.3,137.1,131.2,122.6,121.5,121.0,118.8,118.7,116.4,115.6,109.0,107.1,105.8(×2C),102.5,57.1,56.8,55.0,26.9. +)HRESI-MS(m/z):336.12308[MC ₇ H ₆ NO ₃ ] ⁺ (calcd for C ₂₀ H ₁₈ NO ₄ ,336.12303).

Experimental example (9): Synthesis and structure identification data of compound 9 of the present invention

Weigh 2,3-methylenedioxy-10,11,12-trimethoxy-5,6-dihydrodibenzo[a,g]quinazine quaternary ammonium chloride (500mg, 1.24mmol) in Into the reaction flask, 5N aqueous sodium hydroxide solution (3ml) was added, and then acetone (0.92ml, 12.4mmol) was added dropwise. The reaction was stirred at room temperature for 4h, and the reaction of the raw materials was complete. The reaction mixture was filtered with suction, and the filter cake was washed with water to neutrality to obtain a light yellow solid. The product was directly used in the next step without purification. Weigh 5-aminosalicylic acid (165mg, 1.06mmol) into the reaction flask, add DMSO (4ml), sonicate to completely dissolve, add the above unpurified product under stirring at room temperature, and proceed the reaction at 25℃ after adding 5.5h, to complete the reaction of the raw materials; add tetrahydrofuran (8ml) to the reaction mixture to dilute, stir until no excessive precipitation precipitates, filter the reaction mixture, and wash the filter cake three times with tetrahydrofuran to obtain compound 9 as a yellow solid 395mg, the yield is 61.24 %. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ: 9.68 (s, 1H, ArH), 8.63 (s, 1H, ArH), 7.99 (s, 1H, ArH), 7.63 (s, 1H, ArH), 7.10(s,1H,ArH), 6.98(d,J=2.8Hz,1H,ArH), 6.44(dd,J=8.0,2.8Hz,1H,ArH), 6.32(d,J=8.0Hz,1H, ArH), 6.18(s,2H,OCH ₂ O), 4.82(t,J=6.4Hz,2H,NC H ₂ CH ₂ ),4.08(s,3H,OCH ₃ ),4.06(s,3H,OCH ₃ ), 4.03 (s, 3H, OCH ₃ ), 3.20 (t, J = 6.4 Hz, 2H, NCH ₂ C H ₂ ). ¹³ C-NMR (150MHz, DMSO-d ₆ ) δ: 171.8, 156.4, 154.5, 150.3, 149.2, 148.2, 147.5, 146.3, 138.6, 138.3, 131.3, 131.2, 123.2, 121.5, 121.0, 118.7, 116.4, 115.6, 114.4, 108.8, 106.6, 104.2, 102.5, 62.8, 61.8, 57.2, 55.5, 26.9. (+)HRESI-MS(m/z): 366.13367[MC ₇ H ₆ NO ₃ ] ⁺ (calcd for C ₂₁ H ₂₀ NO ₅ ,366.13360).

Experimental example (10): synthesis and structure identification data of compound 10 of the present invention

Weigh 2,3-dihydro-5-formylbenzo[b][1,4]dioxene (382mg, 2.26mmol) into the reaction flask, add CH ₃ OH (5ml) and pepper in turn Ethylamine (267μl, 1.88mmol), reflux for 3h. The temperature of the reaction mixture was lowered to room temperature, then NaBH ₄ (85 mg, 2.26 mmol) was added in batches, and the reaction was refluxed for 1 h. Water (30 mL) was added to the reaction system and extracted with chloroform; the organic phase was extracted and washed with a saturated aqueous sodium chloride solution, dried with anhydrous MgSO ₄ , filtered, and the solvent was evaporated under reduced pressure. The residue was added with THF (10 ml) and stirred After it was completely dissolved, 2N HCl (0.2ml) was added dropwise, and the reaction was stirred at room temperature until no excessive precipitation was precipitated. The reaction solution was filtered under reduced pressure to obtain N-(2,3-dihydrobenzo[b][1,4 ]Dioxen-5-ylmethyl)-2-(2,3-dihydrobenzo[b][1,4]dioxen-6-yl)ethylamine hydrochloride White solid 520mg, yield 79.15%.

Weigh anhydrous CuSO ₄ (685mg, 4.30mmol) into the reaction flask, add formic acid (12ml), heat and dehydrate in an oil bath at 50℃ for 30min, add N-(2,3-dihydrobenzo[b][1 ,4]Dioxen-5-ylmethyl)-2-(2,3-dihydrobenzo[b][1,4]dioxen-6-yl)ethylamine salt Salt (519mg, 1.43mmol), glyoxal (0.72ml, 5.67mmol), sodium chloride (841mg, 14.41mmol), warm up to 100°C and react for 4h, filter it while it is hot to obtain a filter cake; Transfer to a beaker and add water (20ml) to ultrasonic treatment for 15min, then heat to 80°C for 15min, place to cool, filter, and wash the filter cake with water. The obtained filter cake was put into 0.17L NaHCO ₃ solution with a concentration of 0.5mol/l, stirred and placed in a water bath at 80℃ for 2h, filtered while hot, added 9ml concentrated hydrochloric acid to the filtrate, cooled to room temperature, crystals were precipitated, filtered and dried 137 mg of 2,3-methylenedioxy-9,10-ethylenedioxy-5,6-dihydrodibenzo[a,g]quinazine quaternary ammonium chloride was obtained with a yield of 24.90%.

Weigh 2,3-methylenedioxy-9,10-ethylenedioxy-5,6-dihydrodibenzo[a,g]quinazine quaternary ammonium chloride (500mg, 1.28mmol) in Into the reaction flask, 5N aqueous sodium hydroxide solution (3ml) was added, and then acetone (1ml, 12.8mmol) was added dropwise. The reaction was stirred at room temperature for 4h, and the reaction of the raw materials was complete. The reaction mixture was filtered with suction, and the filter cake was washed with water to neutrality to obtain a light yellow solid. The product was directly used in the next step without purification. Weigh 5-aminosalicylic acid (154mg, 0.99mmol) into the reaction flask, add DMSO (4ml), sonicate to completely dissolve, add the above unpurified product under stirring at room temperature, and proceed the reaction at 25°C after the addition 5.5h, until the raw material reaction is complete; add tetrahydrofuran (8ml) to the reaction mixture to dilute, stir until there is no excessive precipitation, filter the reaction mixture, and wash the filter cake with tetrahydrofuran three times to obtain compound 10 as yellow solid 380mg, yield 57.84 %. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ: 9.82 (s, 1H, ArH), 8.91 (s, 1H, ArH), 7.85 (d, J = 8.4 Hz, 1H, ArH), 7.80 (s, 1H,ArH),7.73(d,J=8.4Hz,1H,ArH),7.08(s,1H,ArH),6.98(d,J=2.8Hz,1H,ArH),6.44(dd,J=8.4, 2.8Hz, 1H, ArH), 6.32 (d, J = 8.4 Hz, 1H, ArH), 6.17 (s, 2H, OCH ₂ O), 4.91 (t, J = 6.4 Hz, 2H, NC H ₂ CH ₂ ) ,4.61,4.52(2×m,2×2H,OC H ₂ C H ₂ O), 3.19(t,J=6.4Hz,2H,NCH ₂ C H ₂ ). ¹³ C-NMR(150MHz,DMSO-d ₆ )δ: 171.7, 154.5, 150.4, 148.2, 143.9, 142.4, 139.2, 138.4, 138.3, 134.2, 131.3, 130.3, 121.5, 120.9, 120.8, 120.6, 118.7, 118.4, 116.4, 115.6, 108.9, 105.9, 102.6, 65.5,65.1,55.4,26.8.(+)HRESI-MS(m/z):334.10739[MC ₇ H ₆ NO ₃ ] ⁺ (calcd for C ₂₀ H ₁₆ NO ₄ ,334.10738).

Experimental example (11): Synthesis and structure identification data of compound 11 of the present invention

Weigh dopamine hydrochloride (500mg, 2.58mmol) into the reaction flask, add THF (6.7ml), saturated aqueous sodium bicarbonate solution (4ml), (BOC) ₂ O (691mg, 3.10mmol) in turn, stir at room temperature for 24 hours and then add Water (30ml), extracted three times with ethyl acetate, combined the ethyl acetate extracts, washed the ethyl acetate solution with saturated sodium chloride aqueous solution; the organic phase was dried over anhydrous MgSO ₄ and filtered, and the solvent was evaporated under reduced pressure. The residue Purified by silica gel column chromatography (petroleum ether/ethyl acetate=5:1, v/v), 573 mg of white solid was obtained with a yield of 85.80%. Weigh this white solid (600mg, 2.37mmol) in the reaction flask, add DMF (10ml), cesium carbonate (3.86g, 11.84mmol), 1,2-dibromoethane (0.8ml, 9.48mmol) in order, and add DMF (10ml), cesium carbonate (3.86g, 11.84mmol), 1,2-dibromoethane (0.8ml, 9.48mmol) in order to 110 The reaction was stirred at ℃ for 5h; the reaction mixture was concentrated under reduced pressure and then added with water (30ml), extracted three times with ethyl acetate, combined the ethyl acetate extracts, the organic phase was extracted and washed with saturated sodium chloride aqueous solution and then dried with anhydrous MgSO ₄ , Filter, and evaporate the solvent under reduced pressure to obtain 525 mg of pure white solid with a yield of 79.40%. Weigh this white solid (525mg, 1.88mmol) into a reaction flask, add CH ₂ Cl ₂ (5ml), place in an ice-water bath, add TFA (4.64ml, 18.8mmol) dropwise to the reaction solution, stir and react for 1h After the reaction mixture was concentrated under reduced pressure, 2N NaOH (30ml) was added, extracted three times with chloroform, the chloroform extracts were combined, the organic phase was extracted and washed with saturated aqueous sodium chloride solution, dried with anhydrous MgSO ₄ , filtered, and evaporated under reduced pressure From the solvent, 515 mg of 2-(2,3-dihydrobenzo[b][1,4]dioxe-6-yl)ethylamine was obtained as a pure white solid, with a yield of 93.47%.

Weigh piperonal (339mg, 2.26mmol) into the reaction flask, and add CH ₃ OH (5ml) and 2-(2,3-dihydrobenzo[b][1,4]dioxene- 6-yl)ethylamine (551mg, 1.88mmol), reflux for 3h. When the temperature of the reaction mixture dropped to room temperature, NaBH4 (85mg, 2.26mmol) was added in batches, and the reaction was refluxed for 1h; then water (30ml) was added to the reaction system and extracted with chloroform; the organic phase was extracted and washed with saturated sodium chloride aqueous solution Then it was dried with anhydrous MgSO ₄ , filtered, and the solvent was evaporated under reduced pressure. After adding THF (10ml) to the residue until it was completely dissolved, 2N HCl (0.2ml) was added dropwise, and the reaction was stirred at room temperature until no excessive precipitation was precipitated. The reaction solution was filtered under suction to obtain N-(benzo[d][1,3]dioxolane-5-ylmethyl)-2-(2,3-dihydrobenzo[b][1,4] Dioxan-6-yl)ethylamine hydrochloride is a white solid of 460 mg, with a yield of 70.09%.

Weigh anhydrous CuSO ₄ (685mg, 4.30mmol) into the reaction flask, add formic acid (12ml), heat and dehydrate in an oil bath at 50℃ for 30min, add N-(benzo[d][1,3]dioxolane Cyclo-5-ylmethyl)-2-(2,3-dihydrobenzo[b][1,4]dioxen-6-yl)ethylamine hydrochloride (500mg, 1.43mmol) , Glyoxal (0.72ml, 5.67mmol), Sodium chloride (841mg, 14.41mmol), warm up to 100℃ and react for 4h, stop heating, filter it while it is hot, to obtain a filter cake; transfer the filter cake to a beaker and add Water (20ml) was ultrasonically treated for 15min, then heated to 80℃ for 15min, left to cool, and filtered; the filter cake was washed with water, and the resulting filter cake was placed in 0.17l NaHCO ₃ solution with a concentration of 0.5mol/l, stirred and placed at 80°C Incubate in a water bath at ℃ for 2h, filter while it is hot, add 9ml of concentrated hydrochloric acid to the filtrate, cool to room temperature, precipitate crystals, filter and dry to obtain 10,11-methylenedioxy-2,3-ethylenedioxy-5,6- Dihydrodibenzo[a,g]quinazine quaternary ammonium salt 832mg, the yield is 84.35%.

Weigh 10,11-methylenedioxy-2,3-ethylenedioxy-5,6-dihydrodibenzo[a,g]quinazine quaternary ammonium chloride (500mg, 1.28mmol) in Into the reaction flask, 5N aqueous sodium hydroxide solution (3ml) was added, and then acetone (1ml, 12.8mmol) was added dropwise. The reaction was stirred at room temperature for 4h, and the reaction of the raw materials was complete. The reaction mixture was filtered with suction, and the filter cake was washed with water to neutrality to obtain a light yellow solid. The product was directly used in the next step without purification. Weigh 5-aminosalicylic acid (167mg, 1.07mmol) into the reaction flask, add DMSO (4ml), sonicate to completely dissolve, add the above unpurified product under stirring at room temperature, and proceed the reaction at 25℃ after the addition 5.5h, until the raw material reaction is complete; add tetrahydrofuran (8ml) to the reaction mixture to dilute, stir until there is no excessive precipitation, filter the reaction mixture, and wash the filter cake with tetrahydrofuran three times to obtain compound 11 as yellow solid 400mg, yield 60.88 %. ¹ H-NMR(400MHz,DMSO-d ₆ )δ: 9.50(s,1H,ArH), 8.76(s,1H,ArH), 7.70(s,2H,2×ArH), 7.54(s,1H,ArH) ),7.00(s,1H,ArH),6.99(d,J=2.8Hz,1H,ArH),6.45(dd,J=8.0,2.8Hz,1H,ArH),6.42(s,2H,OC H ₂ O), 6.33(d,J=8.0Hz,1H,ArH), 4.75(t,J=6.4Hz,2H,NC H ₂ CH ₂ ), 4.35(m,4H,OC H ₂ C H ₂ O), 3.16(t,J=6.4Hz,2H,NCH ₂ C H ₂ ). ¹³ C-NMR(150MHz, DMSO-d ₆ )δ: 171.8, 156.4, 154.5, 151.3, 146.8, 146.3, 143.8, 139.2, 138.9, 138.3,129.2,124.0,121.5,120.2,119.3,118.7,117.1,116.4,115.6,114.8,104.4,104.2,103.1,65.1,64.6,55.1,26.2.(+)HRESI-MS(m/z):334.10733[ MC ₇ H ₆ NO ₃ ] ⁺ (calcd for C ₂₀ H ₁₆ NO ₄ ,334.10738).

Experimental example (12): Synthesis and structure identification data of compound 12 of the present invention

Weigh 2,3-dihydro-6-formylbenzo[b][1,4]dioxane (382mg, 2.26mmol) in the reaction flask, add CH ₃ OH (5ml) and 2 -(2,3-Dihydrobenzo[b][1,4]dioxan-6-yl)ethylamine (551mg, 1.88mmol), reflux for 3h. The temperature of the reaction mixture was lowered to room temperature, then NaBH ₄ (85 mg, 2.26 mmol) was added in batches, and the reaction was refluxed for 1 h. Then water (30mL) was added to the reaction system, extracted with chloroform; the organic phase was extracted and washed with saturated sodium chloride aqueous solution, dried with anhydrous MgSO ₄ , filtered, and the solvent was evaporated under reduced pressure. The residue was added with THF (10ml) to After it was completely dissolved, 2N HCl (0.2ml) was added dropwise, and the reaction was stirred at room temperature until no excessive precipitation was precipitated. The reaction solution was filtered under reduced pressure to obtain N-(2,3-dihydrobenzo[b][1,4] Dioxan-6-ylmethyl)-2-(2,3-dihydrobenzo[b][1,4]dioxen-6-yl)ethylamine hydrochloride white Solid 485mg, yield 71.11%.

Weigh anhydrous CuSO ₄ (685mg, 4.30mmol) into the reaction flask, add formic acid (12ml), heat and dehydrate in an oil bath at 50℃ for 30min, then add the above N-(2,3-dihydrobenzo[b] [1,4]dioxe-6-ylmethyl)-2-(2,3-dihydrobenzo[b][1,4]dioxe-6-yl)ethyl Amine hydrochloride white solid (519mg, 1.43mmol), glyoxal (0.72ml, 5.67mmol), sodium chloride (841mg, 14.41mmol), warm up to 100°C and react for 4h, filter it while it is hot to obtain a filter cake ; Transfer the resulting filter cake to a beaker and add water (20ml) for ultrasonic treatment for 15min, then heat to 80°C for 15min, place to cool, filter, and wash the filter cake with water. The obtained filter cake was put into 0.17l NaHCO ₃ solution with a concentration of 0.5mol/l and stirred and placed in a water bath at 80°C for 2h, filtered while hot, and 9ml concentrated hydrochloric acid was added to the filtrate, cooled to room temperature, crystals precipitated, filtered and dried 331 mg of 2,3:10,11-bisethylenedioxy-5,6-dihydrodibenzo[a,g]quinazine quaternary ammonium chloride was obtained, with a yield of 60.00%.

Weigh 2,3:10,11-bisethylenedioxy-5,6-dihydrodibenzo[a,g]quinazine quaternary ammonium chloride (510mg, 1.35mmol) into the reaction flask and add 5N aqueous sodium hydroxide solution (3ml), then acetone (1ml, 13.5mmol) was added dropwise, the reaction was stirred at room temperature for 4h, and the reaction of the raw materials was complete. The reaction mixture was filtered with suction, and the filter cake was washed with water to neutrality to obtain a light yellow solid. The product was directly used in the next step without purification. Weigh 5-aminosalicylic acid (205mg, 1.30mmol) into the reaction flask, add DMSO (5ml), sonicate until it is completely dissolved, add the above unpurified product under stirring at room temperature, and proceed the reaction at 25°C after addition 5.5h, to complete the reaction of the raw materials; add tetrahydrofuran (10ml) to the reaction mixture to dilute, stir until no excessive precipitation is precipitated, filter the reaction mixture, and wash the filter cake with tetrahydrofuran three times to obtain compound 12 as a yellow solid 400mg, with a yield of 60.15 %. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ: 9.60 (s, 1H, ArH), 8.74 (s, 1H, ArH), 7.83 (s, 1H, ArH), 7.69 (s, 1H, ArH), 7.60(s,1H,ArH),6.99 (s,1H,ArH),6.98(d,J=2.8Hz,1H,ArH),6.44(dd,J=8.0,2.8Hz,1H,ArH),6.33( d,J=8.0Hz,1H,ArH),4.75(t,J=6.4Hz,2H,NC H ₂ CH ₂ ),4.56,4.49(2×m,2×2H,OC H ₂ C H ₂ O) , 4.34(m,4H,OC H ₂ C H ₂ O), 3.16(t, J=6.4Hz, 2H, NCH ₂ C H ₂ ). ¹³ C-NMR(150MHz, DMSO-d ₆ )δ: 171.8, 154.5,153.4,147.7,147.6,146.5,143.8,138.3,137.8,135.8,129.0,122.2,121.5,120.5,118.7,118.6,117.1,116.4,115.6,114.7,114.4,111.9,65.6,65.1,64.7,64.6, 55.3,26.3.(+)HRESI-MS(m/z):348.12280[MC ₇ H ₆ NO ₃ ] ⁺ (calcd for C ₂₁ H ₁₈ NO ₄ ,348.12303).

Experimental example (13): Synthesis and structure identification data of compound 13 of the present invention

Weigh berberine chloride quaternary ammonium salt (5g, 13.45mmol) into the reaction flask, add dry CH ₂ Cl ₂ (200ml), at -40℃, add dropwise containing BBr ₃ (6.83ml, 73.55 After the addition of CH ₂ Cl ₂ (50 ml), the mixture was gradually heated to room temperature, and the reaction was stirred for 6 h. Then CH ₃ OH (2ml) was added dropwise to quench the excess BBr ₃ , and filtered under reduced pressure to obtain 2,3,9,10-tetrahydroxy-5,6-dihydrodibenzo[a,g]quinazine chloride The yellow solid of quaternary ammonium salt is 4.10g, the yield is 91.93%.

Weigh 2,3,9,10-tetrahydroxy-5,6-dihydrodibenzo[a,g]quinazine quaternary ammonium chloride yellow solid (1g, 3.01mmol) into the reaction flask, and add DMF sequentially (60ml), cesium fluoride (4.58g, 30.01mmol), 1,2-dibromoethane (1.04ml, 12.06mmol), stirred at 110°C for 24h. The reaction mixture was cooled to room temperature and suction filtered. The filtrate was evaporated to remove the solvent under reduced pressure. 1% dilute aqueous hydrochloric acid (30ml) was added to the residue, and the mixture was extracted with a mixed solvent of chloroform/methanol=10:1 (v/v). The phase was extracted and washed with saturated aqueous sodium chloride solution, dried with anhydrous MgSO ₄ and filtered; the filtrate was evaporated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (chloroform/methanol=20:1, v/v) 2,3:9,10-bisethylenedioxy-5,6-dihydrodibenzo[a,g]quinazine quaternary ammonium salt is a yellow solid 180mg, the yield is 15.58%.

Weigh 2,3:9,10-bisethylenedioxy-5,6-dihydrodibenzo[a,g]quinazine quaternary ammonium chloride (510mg, 1.35mmol) into the reaction flask and add 5N aqueous sodium hydroxide solution (3ml), then acetone (1ml, 13.5mmol) was added dropwise, the reaction was stirred at room temperature for 4h, and the reaction of the raw materials was complete. The reaction mixture was filtered with suction, and the filter cake was washed with water to neutrality to obtain a light yellow solid. The product was directly used in the next step without purification. Weigh 5-aminosalicylic acid (205mg, 1.30mmol) into the reaction flask, add DMSO (5ml), sonicate until it is completely dissolved, add the above unpurified product under stirring at room temperature, and proceed the reaction at 25°C after addition 5.5h, until the raw material reaction is complete; add tetrahydrofuran (10ml) to the reaction mixture to dilute, stir until there is no excessive precipitation, filter the reaction mixture, and wash the filter cake three times with tetrahydrofuran to obtain compound 13 as yellow solid 375mg, yield 56.39 %. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ: 9.81 (s, 1H, ArH), 8.93 (s, 1H, ArH), 7.85 (d, J = 9.2 Hz, 1H, ArH), 7.76 (s, 1H,ArH),7.75(d,J=9.2Hz,1H,ArH),7.00(s,1H,ArH),6.99(d,J=2.8Hz,1H,ArH),6.45(dd,J=8.4, 2.8Hz, 1H, ArH), 6.34 (d, J = 8.4 Hz, 1H, ArH), 4.90 (t, J = 6.4 Hz, 2H, NC H ₂ CH ₂ ), 4.61, 4.52 (2×m, 2× 2H, OC H ₂ C H ₂ O), 4.35 (m, 4H, OC H ₂ C H ₂ O), 3.16 (t, J = 6.4 Hz, 2H, NCH ₂ C H ₂ ). ¹³ C-NMR (150MHz ,DMSO-d ₆ )δ: 171.3,154.0,146.2,143.5,143.4,141.9,138.7,137.8(×2C),133.7,129.8,128.8,120.6,120.3,120.2,119.9,118.6,118.0,116.6,115.9, 115.2,114.4,65.0,64.64,64.61,64.1,55.2,25.7.ESI-MS(m/z):348.12[M] ⁺ .

Experimental example (14): Synthesis and structure identification data of compound 14 of the present invention

The synthesis method of 8-acetone dihydroberberine is the same as compound 1 of the present invention. Weigh 8-acetone dihydroberberine (100mg, 0.26mmol) into the reaction flask, add tetrahydrofuran (5ml), sonicate to completely dissolve, add 4-aminosalicylic acid (43mg, 0.28mmol) under stirring at room temperature, After the addition, the reaction was refluxed for 2.5 hours. The raw materials were completely reacted. The reaction mixture was filtered off by suction and the filter cake was washed with tetrahydrofuran three times to obtain 79 mg of compound 14 as a yellow solid with a yield of 63.00%. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ: 9.94 (s, 1H, ArH), 8.95 (s, 1H, ArH), 8.04 (d, J = 8.4 Hz, 1H, ArH), 7.82 (d, J = 8.4Hz, 1H, ArH), 7.79 (s, 1H, ArH), 7.29 (d, J = 8.2 Hz, 1H, ArH), 7.08 (s, 1H, ArH), 6.53 (s, 2H, OCH ₂ O), 6.17 (s, 2H, OCH ₂ O), 5.82 (br d, J = 8.2 Hz, 1H, ArH), 5.77 (br s, 1H, ArH), 4.88 (t, J = 6.4 Hz, 2H, NC H ₂ CH ₂ ), 3.20 (t, J=6.4Hz, 2H, NCH ₂ C H ₂ ).

Experimental example (15): Synthesis and structure identification data of compound 15 of the present invention

The synthesis method of 8-acetone dihydroisoberine is the same as compound 2 of the present invention. Weigh 8-acetone dihydroisoberberine (100mg, 0.26mmol) in the reaction flask, add tetrahydrofuran (5ml), sonicate to completely dissolve, add 4-aminosalicylic acid (43mg, 0.28mmol) under stirring at room temperature After the addition, the reaction was refluxed for 2.5 hours. The raw materials were completely reacted. The reaction solution was filtered and the filter cake was washed with tetrahydrofuran three times to obtain 118 mg of compound 15 as a yellow solid with a yield of 94.10%. ¹ H-NMR(500MHz,DMSO-d ₆ )δ: 9.53(s,1H,ArH), 8.74(s,1H,ArH), 7.74(s,1H,ArH), 7.71(s,1H,ArH), 7.52(s,1H,ArH),7.30(d,J=8.2Hz,1H,ArH),7.09(s,1H,ArH),6.41(s,2H,OCH ₂ O),6.17(s,2H,OCH ₂ O), 5.83 (br d, J = 8.2 Hz, 1H, ArH), 5.79 (br s, 1H, ArH), 4.74 (br, 2H, NC H ₂ CH ₂ ), 3.19 (br, 2H, NCH ₂ C H ₂ ).

Experimental example (16): Synthesis and structure identification data of compound 16 of the present invention

The synthesis method of 8-acetone dihydropalmatine is the same as that of compound 3 of the present invention. Weigh 8-acetone dihydropalmatine (100mg, 0.24mmol) into the reaction flask, add tetrahydrofuran (5ml), sonicate to completely dissolve, add 4-aminosalicylic acid (40mg, 0.26mmol) under stirring at room temperature After the addition, the reaction was refluxed for 2.5 hours. The raw materials were completely reacted. After standing and cooling to room temperature, the reaction mixture was suction filtered and the filter cake was washed with tetrahydrofuran three times to obtain 95 mg of compound 16 as a yellow solid with a yield of 77.24%. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ: 9.89 (s, 1H, ArH), 9.02 (s, 1H, ArH), 8.21 (d, J = 9.2 Hz, 1H, ArH), 8.02 (d, J = 9.2Hz, 1H, ArH), 7.71 (s, 1H, ArH), 7.27 (dd, J = 8.0, 2.0 Hz, 1H, ArH), 7.09 (s, 1H, ArH), 5.76 (dt, J = 8.0,2.0Hz,1H,ArH),5.74(t,J=2.0Hz,1H,ArH), 4.95(t,J=6.4Hz,2H,NC H ₂ CH ₂ ), 4.10(s, 3H, OCH ₃ ), 4.07 (s, 3H, OCH ₃ ), 3.93 (s, 3H, OCH ₃ ), 3.87 (s, 3H, OCH ₃ ), 3.22 (t, J = 6.4 Hz, 2H, NCH ₂ C H ₂ ). ¹³ C-NMR(150MHz, DMSO-d ₆ )δ: 171.9, 164.6, 151.7, 151.5, 150.2, 148.7, 145.5, 143.6, 137.7, 133.1, 130.7, 128.6, 126.8, 123.4, 121.3, 119.9, 118.9, 111.3, 109.9, 108.7, 103.1, 99.6, 61.9, 57.0, 56.1, 55.9, 55.4, 26.0.

Experimental example (17): Synthesis and structure identification data of compound 17 of the present invention

The synthesis method of 8-acetone dihydroberberine is the same as compound 4 of the present invention. Weigh 8-acetone dihydroberberine (100mg, 0.24mmol) into the reaction flask, add tetrahydrofuran (5ml), sonicate until completely dissolved, add 4-aminosalicylic acid (42mg, 0.26mmol) under stirring at room temperature After the addition, the reaction was refluxed for 2.5 hours. The raw materials were completely reacted. The reaction mixture was filtered off by suction and the filter cake was washed with tetrahydrofuran three times to obtain 93 mg of compound 17 as a yellow solid with a yield of 74.37%. ¹ H-NMR (500MHz, DMSO-d ₆ ) δ: 9.89 (s, 1H, ArH), 8.93 (s, 1H, ArH), 8.19 (d, J = 9.2 Hz, 1H, ArH), 7.99 (d, J = 9.2Hz, 1H, ArH), 7.79 (s, 1H, ArH), 7.28 (d, J = 8.2 Hz, 1H, ArH), 7.08 (s, 1H, ArH), 6.17 (s, 2H, OCH ₂ O), 5.80 (br d, J = 8.2 Hz, 1H, ArH), 5.76 (br s, 1H, ArH), 4.93 (br, 2H, NC H ₂ CH ₂ ), 4.09 (s, 3H, OCH ₃ ) , 4.07 (s, 3H, OCH ₃ ), 3.20 (br, 2H, NCH ₂ C H ₂ ).

Experimental example (18): Synthesis and structure identification data of compound 18 of the present invention

Weigh 8-acetone dihydroberberine (100mg, 0.26mmol) into the reaction flask, add tetrahydrofuran (5ml), sonicate to completely dissolve, add salicylic acid (39mg, 0.28mmol) under stirring at room temperature, after the addition is complete The reaction was refluxed for 2.5 hours, and then allowed to stand to cool to room temperature, the reaction mixture was filtered, and the filter cake was washed three times with tetrahydrofuran to obtain 76 mg of compound 18 as a yellow solid with a yield of 62.81%. ¹ H-NMR(400MHz, DMSO-d ₆ )δ: 9.94(s,1H,ArH), 8.95(s,1H,ArH), 8.04(d,J=8.8Hz,1H,ArH), 7.82(d, J = 8.8Hz, 1H, ArH), 7.79 (s, 1H, ArH), 7.62 (dd, J = 8.0, 2.0 Hz, 1H, ArH), 7.09 (s, 1H, ArH), 7.08 (m, 1H, ArH), 6.56 (m, 1H, ArH), 6.53 (m, 1H, ArH), 6.53 (s, 2H, OC H ₂ O), 6.17 (s, 2H, OC H ₂ O), 4.88 (t, J _{= 6.4Hz, 2H, NC H 2} CH 2), 3.20 (t, J = 6.4Hz, 2H, NCH 2 C H 2).

Experimental example (19): Synthesis and structure identification data of compound 19 of the present invention

Weigh 8-acetone dihydroisoberberine (100mg, 0.26mmol) into the reaction flask, add tetrahydrofuran (5ml), sonicate to completely dissolve, add salicylic acid (39mg, 0.28mmol) under stirring at room temperature, add After completion, the reaction was refluxed for 2.5 hours, and then allowed to stand to cool to room temperature, the reaction mixture was filtered, and the filter cake was washed three times with tetrahydrofuran to obtain 89 mg of compound 19 as a yellow solid with a yield of 73.55%. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ: 9.50 (s, 1H, ArH), 8.74 (s, 1H, ArH), 7.75 (s, 1H, ArH), 7.71 (s, 1H, ArH), 7.62(dd,J=8.0,2.0Hz,1H,ArH),7.52(s,1H,ArH),7.09(s,1H,ArH),7.08(ddd,J=9.0,7.0,2.0Hz,1H,ArH) ), 6.56(ddd,J=9.0,8.0,2.0Hz,1H,ArH), 6.53(dd,J=7.0,2.0Hz,1H,ArH), 6.42(s,2H,OC H ₂ O), 6.18( s, 2H, OC H ₂ O), 4.75 (t, J = 6.4Hz, 2H, NC H ₂ CH ₂ ), 3.18 (t, J = 6.4Hz, 2H, NCH ₂ C H ₂ ).

Experimental example (20): Synthesis and structure identification data of compound 20 of the present invention

Weigh 8-acetone dihydropalmatine (100mg, 0.24mmol) in the reaction flask, add tetrahydrofuran (5ml), sonicate until completely dissolved, add salicylic acid (36mg, 0.26mmol) at room temperature while stirring, add After completion, the reaction was refluxed for 2.5 hours, and then allowed to stand to cool to room temperature, the reaction mixture was filtered, and the filter cake was washed three times with tetrahydrofuran to obtain 95 mg of compound 20 as a yellow solid with a yield of 77.24%. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ: 9.89 (s, 1H, ArH), 9.02 (s, 1H, ArH), 8.21 (d, J = 9.2 Hz, 1H, ArH), 8.02 (d, J = 9.2Hz, 1H, ArH), 7.71 (s, 1H, ArH), 7.62 (dd, J = 8.0, 2.0 Hz, 1H, ArH), 7.09 (s, 1H, ArH), 7.08 (ddd, J = 9.0,7.0,2.0Hz,1H,ArH),6.56(ddd,J=9.0,8.0,2.0Hz,1H,ArH), 6.53(dd,J=7.0,2.0Hz,1H,ArH), 4.95(t, J=6.4Hz, 2H, NC H ₂ CH ₂ ), 4.10 (s, 3H, ArOCH ₃ ), 4.07 (s, 3H, ArOCH ₃ ), 3.93 (s, 3H, ArOCH ₃ ), 3.87 (s, 3H, ArOCH ₃ ), 3.22 (t, J=6.4 Hz, 2H, NCH ₂ C H ₂ ).

Experimental example (21): Synthesis and structure identification data of compound 21 of the present invention

Weigh 8-acetonyldihydroberberine (100mg, 0.24mmol) into the reaction flask, add tetrahydrofuran (5ml), sonicate to completely dissolve, add salicylic acid (37mg, 0.26mmol) under stirring at room temperature, add After the completion, the reaction was refluxed for 2.5 hours, and then allowed to stand to cool to room temperature, the reaction mixture was filtered, and the filter cake was washed with tetrahydrofuran three times to obtain 83 mg of compound 21 as a yellow solid with a yield of 69.17%. ¹ H-NMR (500MHz, DMSO-d ₆ ) δ: 9.89 (s, 1H, ArH), 8.93 (s, 1H, ArH), 8.20 (d, J = 9.2 Hz, 1H, ArH), 7.99 (d, J = 9.2Hz, 1H, ArH), 7.80 (s, 1H, ArH), 7.62 (dd, J = 8.0, 2.0 Hz, 1H, ArH), 7.09 (s, 1H, ArH), 7.08 (ddd, J = 9.0,7.0,2.0Hz,1H,ArH), 6.56(ddd,J=9.0,8.0,2.0Hz,1H,ArH), 6.53(dd,J=7.0,2.0Hz,1H,ArH), 6.17(s, 2H,OC H ₂ O), 4.93 (t, J = 6.4Hz, 2H, NC H ₂ CH ₂ ), 4.09 (s, 3H, ArOCH ₃ ), 4.07 (s, 3H, ArOCH ₃ ), 3.22 (t, J=6.4 Hz, 2H, NCH ₂ C H ₂ ).

2. Experimental example of solubility detection of the compound of the present invention

Weigh each compound of the present invention and place it in a certain amount of 60% ethanol-water mixed solvent at 25°C±2°C, shake vigorously for 30 seconds every 5 minutes, and observe the dissolution within 30 minutes, if there is no visible When solute particles are considered completely dissolved.

Experimental results: The solubility was measured at a temperature of 25°C±2°C. The amount of 60% ethanol-water mixed solvent required to dissolve the compound of the present invention was 5-aminosalicylic acid berberine quaternary ammonium salt (1) 300ml, 5 -Aminosalicylic acid isoberberine quaternary ammonium salt (2) 200ml, 5-aminosalicylic acid palmatine quaternary ammonium salt (3) 400ml, 5-aminosalicylic acid berberine quaternary ammonium salt (4) 350ml; In parallel determinations, it takes 60 per gram of berberine-type alkaloid quaternary ammonium salt substrates to dissolve berberine-type alkaloid quaternary ammonium salt and isberberine chloride quaternary ammonium salt and 5-aminosalicylic acid. The amount of% ethanol water mixed solvent is 1200ml, 680ml and 800ml respectively. Measured under reflux conditions, the amount of 60% ethanol-water mixed solvent required to dissolve each gram of the compound of the present invention is 5-aminosalicylic acid berberine quaternary ammonium salt (1) 38ml, 5-aminosalicylic acid isocericine quaternary Ammonium salt (2) 26ml, 5-aminosalicylic acid palmatine quaternary ammonium salt (3) 47ml, 5-aminosalicylic acid berberine quaternary ammonium salt (4) 73ml.

3. Experimental example of the efficacy and toxicology evaluation of the compound of the present invention

Experimental example 1: The anti-ulcerative colitis activity (therapeutic effect) of the

compounds

1, 2, 3, and 4 of the present invention on the acute C57BL/6J mouse ulcerative colitis model induced by sodium dextran sulfate and the study with berberine chloride Examples of comparative study on the curative effect of quaternary ammonium salt, isoflavine chloride quaternary ammonium salt, palmatine chloride quaternary ammonium salt, berberine chloride quaternary ammonium salt and 5-aminosalicylic acid

(1) Materials and methods

1) Animals: C57BL/6J mice, male, with a weight range of 20-22 g.

2) Grouping: This experiment is divided into normal control group, dextran sodium sulfate model group, positive drug sulfasalazine group,

compound

1, 2, 3, 4 administration group of the present invention, 5-aminosalicylic acid administration Group, berberine chloride quaternary ammonium salt administration group, isoflavine chloride quaternary ammonium salt administration group, palmatine chloride quaternary ammonium salt administration group and berberine chloride quaternary ammonium salt administration group. Randomly group, 6 mice in each group.

3) Dosage and frequency of administration: The dose of the positive drug sulfasalazine administration group is 500 mg/kg; the

compounds

1, 2, 3 and 4 of the present invention, 5-aminosalicylic acid, berberine chloride quaternary ammonium salt The dosage of the quaternary ammonium salt of isberberine chloride, the quaternary ammonium salt of palmatine chloride, and the quaternary berberine chloride group were all 100mg/kg; once a day for 7 days.

4) Experimental method: C57BL/6J mice were adaptively reared in an SPF animal room (experimental animal license number: SYXK (京)2014-0023) for one week, and then randomly grouped according to the experimental design. Mice in the model group and each compound administration group were given dextran sodium sulfate (MP, CA9011-18-1, US) every day to model ulcerative colitis model established in the laboratory [Zhang ZH, et al. Synthesis] and structure-activity relationships of quaternary coptisine derivatives as potential anti-ulcerative Colitis agents.J.Med.Chem.,2015,58,7557-7571]. The normal control group and the model group were given 0.5% sodium carboxymethylcellulose aqueous solution by gavage once a day. Sulfasalazine group, each compound of the present invention, 5-aminosalicylic acid, quaternary ammonium salt of berberine chloride, quaternary ammonium salt of berberine chloride, quaternary ammonium salt of palmatine chloride and berberine chloride The ammonium salt administration group was administered intragastrically according to the experimental design plan, once a day. Sulfasalazine, each compound of the present invention and comparative compounds were all prepared with 0.5% sodium carboxymethyl cellulose aqueous solution according to the dosage of the experimental plan.

After modeling, the drug was administered continuously for 7 days, until the model group animals showed obvious symptoms of ulcerative colitis such as malaise, reduced activity, weight loss, loose stools, blood in the stool, etc., the experiment was terminated in due course, the animals in each group were killed, and the ulcerative colon was detected. All related evaluation indicators of inflammation (see the experimental results section), comprehensively evaluate the anti-ulcerative colitis pharmacodynamic activity of each group of experiments.

(2) Experimental results

The

compounds

1, 2, 3, and 4 of the present invention have significant therapeutic effects in vivo on the acute C57BL/6J mouse ulcerative colitis model induced by sodium dextran sulfate, which is better than other comparative compounds; the results of each evaluation index are as follows.

1) The

compounds

1, 2, 3 and 4 of the present invention can effectively reduce the weight loss of acute C57BL/6J mouse ulcerative colitis model animal induced by sodium dextran sulfate (see Table 1).

Table 1. Improving effects of the

compound

1, 2, 3, 4 administration groups of the present invention on the weight loss of mice with acute ulcerative colitis induced by sodium dextran sulfate

Note: ^** p<0.01, compared with the normal control group; ^# p<0.05, ^## p<0.01, compared with the model group. Positive drug: sulfasalazine; 5-ASA: 5-aminosalicylic acid; comparison 1: berberine chloride quaternary ammonium salt; comparison 2: isberberine chloride quaternary ammonium salt; comparison 3: palmatine chloride Quaternary ammonium salt; comparison 4: berberine chloride quaternary ammonium salt.

It can be seen from Table 1 that compared with the initial value of animal weight in each group, after the end of the experiment, the weight of the normal control group increased by +9.24% (increase to +, decreased to -), while the model group animal weight decreased by -11.99% ^** ( ^** p<0.01, compared with the normal control group), in line with the change trend of the ulcerative colitis model, indicating that the model was successful. However, the animal weight change rate of the

compound

1, 2, 3, and 4 administration groups of the present invention at a dosage of 100 mg/kg was -0.87% ^## ( ^## p<0.01, compared with the model group), -5.48 % ^# ( ^# p<0.05, compared with the model group), -4.42% ^# ( ^# p<0.05, compared with the model group) and -6.54% ^# ( ^# p<0.05, compared with the model group). Therefore, the

compounds

1, 2, 3, and 4 of the present invention can slow down or significantly slow down the weight loss of model animals, which is statistically significantly different from the model group. It is particularly worth pointing out that the improvement effect of the

compound

1, 2, 3, and 4 administration group of the present invention is significantly better than that of the 5-aminosalicylic acid administration group in parallel experiments (the animal body weight change rate is -15.09, compared with the model group There is no significant difference), and they are also superior to the corresponding berberine chloride alkaloid quaternary ammonium salt.

2) The

compound

1, 2, 3 and 4 of the present invention improve the effect of dextran sodium sulfate on the colonic contracture induced by acute C57BL/6J mouse ulcerative colitis model animals (see Table 2)

Table 2 shows the colon length value and colon contracture percentage of each group of animals after the experiment. The results showed that compared with the normal control group, the mouse colon in the model group was significantly shorter, with a length of 4.87±0.15 cm, and the colon contracture ratio reached 38.91% ^** ( ^** p<0.01, compared with the normal control group). At the dose of 100 mg/kg used in the experiment, the mice in the

compound

1, 2, 3, and 4 administration groups of the present invention had significantly increased colon lengths compared with the model mice; the colon length of the mice in the compound 1 group was 6.08±0.12cm, the colonic contracture ratio was 23.64% ^## ( ^## p<0.01, compared with the model group), the colon length of mice in the compound 2 group was 5.90±0.13cm, and the colonic contracture ratio was 25.94% ^## ( ^{# #} p<0.01, compared with the model group), the length of the mouse colon in the compound 3 group was 5.97±0.14cm, and the colon contracture ratio was 25.11% ^## ( ^## p<0.01, compared with the model group), the compound 4 group The length of the mouse colon was 5.80±0.27cm, and the colon contracture ratio was 27.20% ^# ( ^# p<0.05, compared with the model group). The colonic contracture ratio of the positive drug sulfasalazine 500 mg/kg dose group was 37.45% (compared to the model group). Therefore, the

compounds

1, 2, 3 and 4 of the present invention have a very significant improvement effect on dextran sodium sulfate-induced acute C57BL/6J mouse ulcerative colitis model animal colon contracture; and the improvement effect is significantly better than the 5 in parallel experiments. -Aminosalicylic acid administration group and berberine chloride type alkaloid quaternary ammonium salt administration group.

Table 2. The improving effect of the

compound

1, 2, 3 and 4 administration groups of the present invention on the colonic contracture induced by dextran sodium sulfate in acute C57BL/6J mouse ulcerative colitis model animals

3) The effects of

compounds

1, 2, 3 and 4 of the present invention on dextran sodium sulfate-induced acute C57BL/6J mouse ulcerative colitis model animal disease activity comprehensive index score and disease activity comprehensive index inhibition rate (see Table 3)

The comprehensive disease activity index score evaluates the effect of active compound treatment from indicators such as the percentage of animal weight loss, stool characteristics and blood in the stool that are closely related to the clinical symptoms of ulcerative colitis; the lower the comprehensive disease activity index score, the greater the inhibition rate of the comprehensive disease activity index , Indicating that the model animals are closer to the normal physiological state of the animals after treatment. Through the investigation of the effects of

compounds

1, 2, 3 and 4 of the present invention on the dextran sodium sulfate-induced acute C57BL/6J mouse ulcerative colitis model animal disease activity comprehensive index score and disease activity comprehensive index inhibition rate, the results show that, The

compounds

1, 2, 3, and 4 of the present invention have significant anti-ulcerative colitis activity at a dosage of 100 mg/kg; and their pharmacological effects are significantly better than the 5-aminosalicylic acid administration group and the parallel experiment The corresponding berberine chloride type alkaloid quaternary ammonium salt administration group.

Table 3 The effects of the

compound

1, 2, 3 and 4 administration groups of the present invention on the dextran sodium sulfate-induced acute C57BL/6J mouse ulcerative colitis model animal disease activity comprehensive index score and disease activity comprehensive index inhibition rate

Experimental example 2: The therapeutic effect of compound 1 of the present invention on the acute C57BL/6J mouse ulcerative colitis model animal induced by sodium dextran sulfate, the dose-effect relationship and the comparative study with related compounds

(1) Materials and methods

1) Animals: C57BL/6J mice, male, with a weight range of 20-22 g.

2) Grouping: This experiment is divided into normal control group, dextran sodium sulfate model group, sulfasalazine positive drug group, compound 1 high-dose, middle-dose and low-dose administration groups of the present invention; for further comparison, this The experiment also set up the quaternary ammonium salt group of berberine chloride and the group of dihydroberberine. Randomly group, 7 mice in each group.

3) Dosage and frequency of administration: The dosage of the sulfasalazine administration group is 500 mg/kg; the dosage of the berberine chloride quaternary ammonium salt and the dihydroberberine administration group are both 100 mg/kg; the compound of the present invention 1. The dosages of the high-dose, medium-dose and low-dose administration groups were 200, 100 and 50 mg/kg respectively; once a day, the administration was administered for 7 days.

4) Experimental method: C57BL/6J mice were adaptively reared in an SPF animal room (experimental animal license number: SYXK (京)2014-0023) for one week, and then randomly grouped according to the experimental design. Mice in the model group and each compound administration group were given dextran sodium sulfate (MP, CA9011-18-1, US) every day to model ulcerative colitis model established in the laboratory [Zhang ZH, et al. Synthesis] and structure-activity relationships of quaternary coptisine derivatives as potential anti-ulcerative Colitis agents.J.Med.Chem.,2015,58,7557-7571]. The normal control group and the model group were given 0.5% sodium carboxymethylcellulose aqueous solution by gavage once a day. The sulfasalazine administration group, the berberine chloride quaternary ammonium salt administration group, the dihydroberberine administration group, and the compound 1 high-dose, middle-dose and low-dose administration groups of the present invention were administered intragastrically according to the experimental protocol. Once a day. The positive drug, berberine chloride quaternary ammonium salt, dihydroberberine and compound 1 of the present invention were all prepared with 0.5% sodium carboxymethyl cellulose aqueous solution according to the experimental protocol.

(2) Experimental results

1) The compound 1 of the present invention can effectively reduce the weight loss of acute C57BL/6J mouse ulcerative colitis model animals induced by sodium dextran sulfate (see Table 4 and Figure 1), and the effect intensity is significantly better than the positive drug sulfasalazine , Coptisine Chloride Quaternary Ammonium Salt and Dihydro Coptisine. The dose-effect relationship is clear.

Table 4. The improvement of each administration group of positive drugs sulfasalazine, berberine chloride quaternary ammonium salt, dihydroberberine, compound 1 of the present invention on the weight loss of mice with acute ulcerative colitis induced by sodium dextran sulfate effect

Note: ^** p<0.01, compared with the normal control group; ^## p<0.01, compared with the model group. Positive drug: Sulfasalazine; Comparison 1: Coptisine Chloride Quaternary Ammonium Salt; Comparison 2: Dihydroberberine.

It can be seen from Table 4 and Figure 1 that compared with the normal control group, the body weight of the mice in the model group was significantly reduced (a decrease of -18.44%), and the difference was statistically significant, indicating that the model of ulcerative colitis was successful. The high-dose (200mg/kg), medium-dose (100mg/kg) and low-dose (50mg/kg) administration groups of compound 1 of the present invention can effectively alleviate the acute ulcerative colitis model mice induced by dextran sodium sulfate. Weight loss, especially in the high-dose and middle-dose administration groups, even showed animal weight gain, which was statistically significant and the dose-effect relationship was significant. However, the positive drug sulfasalazine at a dosage of 500 mg/kg has an improvement effect on the weight loss of mice even not as good as the compound 1 low-dose administration group of the present invention. The quaternary ammonium salt of berberine chloride and dihydroberberine at the same dosage as the compound 1 of the present invention in reducing the effect of dextran sodium sulfate in inducing the weight loss of acute C57BL/6J mouse ulcerative colitis model animals It is obviously not as effective as the parallel experiment of the compound of the present invention.

2) The effects of positive drugs sulfasalazine, berberine chloride quaternary ammonium salt, dihydroberberine, compound 1 of the present invention on the colon contracture of mice with acute ulcerative colitis induced by sodium dextran sulfate are shown in Table 5 and Figure 2.

Table 5. Positive drug sulfasalazine, berberine chloride quaternary ammonium salt, dihydroberberine, compound 1 of the present invention, each administration group improves colon contracture in mice with acute ulcerative colitis induced by sodium dextran sulfate effect

Note: ^** p<0.01, compared with the normal control group; ^# p<0.05, ^## p<0.01, compared with the model group. Positive drug: Sulfasalazine; Comparison 1: Coptisine Chloride Quaternary Ammonium Salt; Comparison 2: Dihydroberberine.

It can be seen from Table 5 and Figure 2 that in the acute C57BL/6J mouse ulcerative colitis model induced by sodium dextran sulfate, compared with the normal control group, the mouse colon in the model group was significantly shorter and the contracture ratio was as high as 36.96%. , The difference is statistically significant. The high-dose, middle-dose and low-dose administration groups of the compound 1 of the present invention can effectively and significantly improve the colonic contracture of model animals, with statistically significant differences and a significant dose-effect relationship. However, the positive drug sulfasalazine at a dosage of 500 mg/kg has an effect on improving colonic contracture in mice even significantly less than the compound 1 low-dose group of the present invention. The improvement effect of berberine chloride quaternary ammonium salt and dihydroberberine at the same dosage as the compound 1 of the present invention on the colonic contracture induced by sodium dextran sulfate in acute C57BL/6J mouse ulcerative colitis model mice It is obviously not as effective as the parallel experiment of the compound of the present invention.

3) The positive drug sulfasalazine, berberine chloride quaternary ammonium salt, dihydroberberine, compound 1 of the present invention, high-dose, medium-dose, and low-dose, can induce acute ulcerative colitis in mice with dextran sodium sulfate See Table 6 and Figure 3 for the influence of the comprehensive activity index score and the inhibition rate of the comprehensive disease activity index.

Table 6. The comprehensive index of disease activity of each administration group of positive drugs sulfasalazine, berberine chloride quaternary ammonium salt, dihydroberberine, and compound 1 of the present invention on dextran sodium sulfate-induced acute ulcerative colitis model mice The improvement effect of scoring and disease activity comprehensive index inhibition rate

Note: ^** p<0.01, compared with the normal control group; ^# p<0.05, ^## p<0.01, compared with the model group. Positive drug: Sulfasalazine;

Comparison 1: Coptisine chloride quaternary ammonium salt; Comparison 2: Dihydro berberine.

From Table 6 and Figure 3, it can be seen that compared with the normal control group, the animal disease activity comprehensive index score of the model group increased significantly, and the statistical difference was very significant, indicating that the model was successful. Compared with the model group, the animal disease activity comprehensive index score of the positive drug group was significantly lower, and there was a significant difference compared with the model group, indicating that the positive drug was effective. The high-dose, medium-dose and low-dose administration groups of the compound 1 of the present invention can significantly reduce the comprehensive index score of the disease activity of experimental animals, the difference is very significant in statistics and the dose-effect relationship is clear. The numerical value shows that the curative effect of compound 1 is significantly better than that of positive drugs. The berberine chloride quaternary ammonium salt and dihydroberberine at the same dosage as the compound 1 of the present invention are used to induce acute C57BL/6J mouse ulcerative colitis model animal disease activity comprehensive index scores and The effect of improving the inhibition rate of the comprehensive index of disease activity is significantly lower than that of the parallel experiment of the compound of the present invention.

(Note: The comprehensive disease activity index score is evaluated from the animal's weight loss, stool characteristics, blood in the stool and other indicators. The lower the disease activity comprehensive index score, the closer the animal is to the normal physiological state, see Table 7.)

Table 7. Scoring criteria for the comprehensive index of disease activity

Note: ^a normal stool: formed stool; ^b loose stool: mushy, semi-formed stool that does not adhere to the anus; ^c loose stool: watery stool.

4) The high-dose, medium-dose and low-dose administration groups of compound 1 of the present invention have a significant improvement effect on the pathological damage of colon tissue in mice with acute ulcerative colitis induced by sodium dextran sulfate (Figure 4).

Compared with the normal control group, the basic structure of the colonic mucosa in the dextran sodium sulfate model group was destroyed, the polarity of epithelial cells disappeared, the inflammation involved the mucosa and the submucosal lamina propria, and crypts were destroyed; a large number of inflammatory cells infiltrated in the inflammatory lesions; The lower edema is obvious; a large number of red blood cells and exudates can be seen in the intestinal cavity. The positive drug sulfasalazine group only slightly improved the colon tissue damage of the acute ulcerative colitis model mice induced by sodium dextran sulfate, which showed symptoms of ulcerative colitis (destruction of the basic structure of the intestinal mucosa, disorder and loss of the arrangement of epithelial cells) Polarity phenomenon) No significant improvement (although there is improvement), there is mild edema and inflammatory cell infiltration in the submucosa and muscle layer. After administration of the compound 1 of the present invention in the low, medium, and high-dose administration groups, colonic mucosa shedding and structural damage were significantly improved, and submucosal edema was significantly reduced; inflammatory cell infiltration at inflammatory lesions was reduced; some epithelial cells in the high-dose administration group The polarity has even recovered. The above results indicate that the compound 1 of the present invention can significantly improve the pathological damage of colon tissue in mice with acute ulcerative colitis induced by sodium dextran sulfate, and present a significant dose-effect relationship.

Experimental example 3: The therapeutic effect of compound 1 of the present invention on oxazolone-induced chronic BALB/c mouse ulcerative colitis model animal and the research implementation example of the dose-effect relationship

(1) Materials and methods

1) Animal: BALB/c mouse, male, with a weight range of 20-22 g.

2) Grouping: This experiment is divided into normal control group, oxazolone model group, positive drug sulfasalazine group, low-dose administration group of compound 1 of the present invention, medium-dose administration group of compound 1 of the present invention, compound 1 of the present invention High-dose administration group. Randomly group, 6 mice in each group.

3) Dosage and frequency of administration: the dosage of the positive drug sulfasalazine administration group is 500 mg/kg, and the dosage of the compound 1 of the present invention is 50, 100 and 200 mg for the low-dose, middle-dose and high-dose administration groups, respectively /kg; once a day for 6 days.

4) Experimental method: BALB/c mice were adaptively reared in an SPF animal room (experimental animal license number: SYXK (京)2014-0023) for one week, and then randomly grouped according to the experimental design. Mice in the model group, positive drug and compound 1 administration group of the present invention were modeled with oxazolone (Sigma, E0753, 15646-46-5, USA) according to the literature oxazolone-induced animal ulcerative colitis model [Heller F,et al. Oxazolone Colitis,a Th2 colitis model resembling ulcerative colitis, is mediated by IL-13-proding NK-T cells.Immunity,2002,17,629-638]. The normal control group and the model group were given 0.5% sodium carboxymethylcellulose aqueous solution by gavage once a day. The sulfasalazine administration group, the compound 1 low-dose, middle-dose and high-dose administration groups of the present invention were administered intragastrically according to the experimental protocol, once a day. Both the positive drug and the compound 1 of the present invention were prepared with 0.5% sodium carboxymethyl cellulose aqueous solution according to the experimental protocol.

After modeling, the drug was continuously administered for 6 days, the experiment was terminated at an appropriate time, the animals in each group were sacrificed, and the relevant evaluation indexes of ulcerative colitis were tested (see the experimental results section), and the anti-chronic ulcerative colitis efficacy of each group experiment was comprehensively evaluated Learning effect.

(2) Experimental results

1) The compound 1 of the present invention can effectively reduce the weight loss of oxazolone-induced chronic BALB/c mouse ulcerative colitis model animals (see Table 8 and Figure 5), and the effect intensity is significantly better than the positive drug sulfasalazine. The effective relationship is clear.

Table 8. The improvement effect of each administration group of the positive drug sulfasalazine and the compound 1 of the present invention on the weight loss of oxazolone-induced chronic ulcerative colitis model mice

Note: ^** p<0.01, compared with the normal control group; ^# p<0.05, compared with the model group. Positive drug: Sulfasalazine.

Note to Table 8 and Figure 5: Compared with the normal control group, the weight of the mice in the model group was significantly reduced (decreased by -26.70%), and the difference was statistically significant, indicating that the model of ulcerative colitis was successful. The positive drug sulfasalazine group, the low-dose group and the middle-dose group of compound 1 of the present invention are all effective in alleviating the weight loss of oxazolone-induced chronic ulcerative colitis mice, but they do not show statistical differences; the present invention The compound 1 high-dose group has a significant improvement effect on the weight loss of oxazolone-induced chronic ulcerative colitis model mice, and the difference is statistically significant. Although the low-dose group and the middle-dose group of the compound 1 of the present invention did not show statistical differences in effectively alleviating the weight loss of oxazolone-induced chronic ulcerative colitis model mice, the trend of the compound 1 of the present invention to alleviate the weight loss effect Obviously, and has a good dose-effect relationship.

2) The effects of the positive drug sulfasalazine and compound 1 of the present invention on the colon contracture of mice with chronic ulcerative colitis induced by oxazolone are shown in Table 9 and Figure 6.

Table 9. The improvement effect of each administration group of the positive drug sulfasalazine and the compound 1 of the present invention on the colon contracture of mice with chronic ulcerative colitis induced by oxazolone

Note: ^** p<0.01, compared with the normal control group; ^## p<0.01, compared with the model group. Positive drug: Sulfasalazine.

Table 9 and Figure 6 Note: In the oxazolone-induced chronic BALB/c mouse ulcerative colitis animal model, compared with the normal control group (colon length is 9.66 cm), the model group mouse colon contracture is significantly shorter ( 6.90cm), the contracture percentage is as high as 28.57%, which is statistically significant. The compound 1 low-dose group, middle-dose group and high-dose group of the present invention can effectively improve the colonic contracture of model animals, significantly reduce the percentage of colonic contracture, and have statistically significant differences, and have a good dose-effect relationship. The positive drug sulfasalazine failed to effectively improve colonic contracture, and there was no statistically significant difference.

3) The effects of the positive drug sulfasalazine and the compound 1 of the present invention at low, medium and high doses on the disease activity index score and disease activity index inhibition rate of oxazolone-induced chronic ulcerative colitis model mice are shown in the table 10 and Figure 7.

Table 10. The improvement effect of each administration group of the positive drug sulfasalazine and the compound 1 of the present invention on the disease activity index score and the disease activity index inhibition rate of oxazolone-induced chronic ulcerative colitis model mice

Note: ^** p<0.01, compared with the normal control group; ^## p<0.01, compared with the model group; positive drug: sulfasalazine.

Note to Table 10 and Figure 7: Compared with the normal control group, the disease activity comprehensive index score of the model group increased significantly, and the statistical difference was very significant, indicating that the model was successful. Compared with the model group, the positive drug sulfasalazine and the compound 1 low-dose group of the present invention showed no statistical difference in the effect of improving the comprehensive index score of the disease activity in mice. The medium-dose group and the high-dose group of the compound 1 of the present invention can significantly Reduce the comprehensive index score of laboratory animal disease activity, statistically significant differences. Although the compound 1 low-dose group of the present invention did not show a statistical difference in the effective alleviation of oxazolone-induced chronic ulcerative colitis model mice disease activity comprehensive index score, the trend of the remission effect of the compound 1 of the present invention is obvious.

Experimental Example 4: Comparison of the effect (therapeutic effect) of a mixture of berberine chloride quaternary ammonium salt and 5-aminosalicylic acid prepared by the principle of equal mole number of compound 1 of the present invention against acute ulcerative colitis induced by sodium dextran sulfate Experimental implementation examples

(1) Materials and methods

1) Animals: C57BL/6J mice, male, with a weight range of 20-22 g.

2) Grouping: This experiment is divided into normal control group, dextran sodium sulfate model group, positive drug sulfasalazine group, compound 1 (100mg) administration group of the present invention, and berberine chloride prepared on the principle of equal moles The quaternary ammonium salt (75mg) and 5-aminosalicylic acid (32mg) mixture (c1+A) administration group. Randomly group, 5 mice in each group.

3) Dosage and frequency of administration: the administration dose of the positive drug sulfasalazine administration group is 500 mg/kg, the administration dose of the compound 1 administration group of the present invention is 100 mg/kg, and the number of moles is equal. The dosage of the mixture administration group of alkali quaternary ammonium salt and 5-aminosalicylic acid was 107 mg/kg; once a day, the administration was administered for 7 days.

4) Experimental method: C57BL/6J mice were adaptively reared in an SPF animal room (experimental animal license number: SYXK (京)2014-0023) for one week, and then randomly grouped according to the experimental design. Mice in the model group and each compound administration group were given dextran sodium sulfate (MP, CA9011-18-1, US) every day to model ulcerative colitis model established in the laboratory [Zhang ZH, et al. Synthesis] and structure-activity relationships of quaternary coptisine derivatives as potential anti-ulcerative Colitis agents.J.Med.Chem.,2015,58,7557-7571]. The normal control group and the model group were given 0.5% sodium carboxymethylcellulose aqueous solution by gavage once a day. The sulfasalazine administration group, the compound 1 administration group of the present invention, and the mixture of berberine chloride quaternary ammonium salt and 5-aminosalicylic acid (c1+A) administration group prepared by the principle of equal moles were designed according to the experiment The regimen was administered by gavage, once a day, all were prepared with 0.5% sodium carboxymethyl cellulose aqueous solution according to the experimental regimen.

After modeling, the drug was administered continuously for 7 days, until the model group animals showed obvious symptoms of ulcerative colitis such as malaise, reduced activity, weight loss, loose stools, blood in the stool, etc., the experiment was terminated in due course, the animals in each group were killed, and the ulcerative colon was detected. All relevant evaluation indicators of inflammation (see the experimental results section), comprehensively evaluate the pharmacodynamic effects of anti-ulcerative colitis in each group of experiments.

(2) Experimental results

The compound 1 of the present invention has a significant therapeutic effect in vivo on the acute C57BL/6J mouse ulcerative colitis animal model induced by dextran sodium sulfate, which is significantly better than the quaternary ammonium chloride and berberine chloride formulated on the principle of equal moles Each evaluation index of 5-aminosalicylic acid mixture (c1+A) administration group.

1) The compound 1 of the present invention effectively reduces the weight loss of dextran sodium sulfate-induced acute C57BL/6J mouse ulcerative colitis model animal, which is significantly better than the berberine chloride quaternary ammonium salt formulated on the principle of equal moles and 5- The aminosalicylic acid mixture (c1+A) administration group (see Table 11).

Table 11. Compound 1 administration group of the present invention and a mixture of berberine chloride quaternary ammonium salt and 5-aminosalicylic acid (c1+A) administration group prepared on the principle of equal moles induce acute acute treatment of sodium dextran sulfate Comparison of improving effects of weight loss in mice with ulcerative colitis

Note: ^** p<0.01, compared with the normal control group; ^## p<0.01, compared with the model group. Positive drug: sulfasalazine; c1+A: a mixture of berberine chloride quaternary ammonium salt and 5-aminosalicylic acid prepared on the principle of equal moles.

It can be seen from Table 11 that compared with the initial value of animal weight in each group, after the end of the experiment, the weight of the animals in the normal control group increased by +9.99%, while the weight of the animals in the model group decreased by -11.96% ^** ( ^** p<0.01, compared with normal Compared with the control group), it is in line with the change trend of ulcerative colitis model, indicating that the model is successful. The compound 1 administration group of the present invention has an animal body weight change rate of -2.47% at a dose of 100 mg/kg ^## ( ^## p<0.01, compared with the model group), which is significantly better than the principle of equal number of moles. The corresponding value of the berberine chloride quaternary ammonium salt and 5-aminosalicylic acid mixture administration group is -13.11% (compared with the model group, there is no significant difference).

2) The compound 1 of the present invention has a significantly better effect of improving colonic contracture in the acute C57BL/6J mouse ulcerative colitis model animal induced by dextran sodium sulfate than the berberine chloride quaternary ammonium salt and 5- A mixture of aminosalicylic acid (see Table 12).

Table 12 shows the colon length value and colon contracture percentage of each group of animals after the experiment. The results showed that compared with the normal control group’s colon length value of 8.08±0.22cm, the model group’s colon was significantly shorter to 5.58±0.12cm, and the colon contracture ratio reached 30.94% ( ^** p<0.01, compared with the normal control group ). At the dose of 100 mg/kg used in the experiment, the mice in the compound 1 administration group of the present invention had significantly increased colon length compared with the model group mice; the colon length of the mice in the compound 1 group was 7.12±0.12 cm, and the colon contracture The ratio is 11.88% ^## ( ^## p<0.01, compared with the model group). The colonic contracture ratio of the group administered with the mixture of berberine chloride quaternary ammonium salt and 5-aminosalicylic acid prepared on the principle of equal moles reached 30.20% (no significant difference compared with the model group). Therefore, the compound 1 of the present invention has a very significant improvement effect on the colonic contracture induced by dextran sodium sulfate in the acute C57BL/6J mouse ulcerative colitis model animal, which is significantly better than the quaternary ammonium chloride of berberine prepared by the principle of equal mole number And 5-aminosalicylic acid mixture.

Table 12. The compound 1 administration group of the present invention and the mixture administration group of berberine chloride quaternary ammonium salt and 5-aminosalicylic acid prepared on the principle of equal moles induced acute C57BL/6J mice with sodium dextran sulfate Comparison of improving effects of ulcerative colitis model animals with colonic contracture

3) The compound 1 of the present invention and the mixture of berberine chloride quaternary ammonium salt and 5-aminosalicylic acid (c1+A) prepared by the principle of equal moles can induce acute ulceration of C57BL/6J mice by sodium dextran sulfate Comparison of the effect of colitis model animal disease activity comprehensive index score and disease activity comprehensive index inhibition rate (see Table 13)

The mixture of berberine chloride quaternary ammonium salt and 5-aminosalicylic acid prepared by the principle of the compound 1 of the present invention and the molar number is equal to dextran sodium sulfate inducing acute disease activity of C57BL/6J mouse ulcerative colitis model animal The investigation of the influence of the comprehensive index score and the inhibition rate of the disease activity comprehensive index showed that the compound 1 of the present invention has a significant anti-ulcerative colitis effect at a dosage of 100 mg/kg, which is significantly better than the number of moles in the parallel experiment A mixture of berberine chloride quaternary ammonium salt and 5-aminosalicylic acid prepared by the principle of equality. It can be seen from Table 13 that compared with the normal control group, the animal disease activity comprehensive index score of the model group was significantly increased, which was 3.00±0.42**, which was statistically significant, indicating that the model was successful. Compared with the model group, the 100 mg/kg administration group of compound 1 of the present invention can significantly reduce the comprehensive index score of experimental animal disease activity (score of 0.80±0.37 ^## ), and the difference is statistically significant. A mixture of berberine chloride quaternary ammonium salt and 5-aminosalicylic acid prepared according to the principle of equal mole number on dextran sodium sulfate-induced acute C57BL/6J mouse ulcerative colitis model animal disease activity comprehensive index score and disease activity The improvement effect values of the comprehensive index inhibition rate were 2.87±0.33 and 4.44%, respectively, and there was no statistically significant difference, which was significantly lower than the compound of the present invention.

Table 13 The compound 1 administration group of the present invention and the molar number are equal to the mixture administration group of berberine chloride quaternary ammonium salt and 5-aminosalicylic acid induced acute ulcer in C57BL/6J mice with sodium dextran sulfate Of disease activity index score and disease activity index inhibition rate of animal model of colitis

Experimental example 5: The therapeutic effect of compound 1 of the present invention on the chronic C57BL/6J mouse ulcerative colitis model animal induced by sodium dextran sulfate and the implementation example of the dose-effect relationship

(1) Materials and methods

1) Animal: C57BL/6J mouse, male, with a weight range of 18-20g.

2) Grouping: This experiment is divided into normal control group, dextran sodium sulfate model group, positive drug sulfasalazine group, low-dose administration group of compound 1 of the present invention, medium-dose administration group of compound 1 of the present invention, and the present invention Compound 1 high-dose administration group. Randomly group, 6 mice in each group.

3) Dosage and frequency of administration: The dosage of the positive drug sulfasalazine administration group is 500 mg/kg, and the dosage of compound 1 of the present invention is 50, 100 and 200 mg for the low-dose, middle-dose and high-dose administration groups, respectively /kg; once a day for 40 days.

4) Experimental method: C57BL/6J mice were adaptively reared in an SPF animal room (experimental animal license number: SYXK (京)2014-0023) for one week, and then randomly grouped according to the experimental design. The mice in the model group, the positive drug group and each administration group of compound 1 of the present invention were given 2% dextran sodium sulfate (MP, CA9011-18-1, US) on day 1-6 and normal drinking on day 7-20 Water: 2% dextran sodium sulfate is given on 21-26 days, normal drinking water is given on 27-40 days, and 2% dextran sodium sulfate is given on 41-46 days. The normal control group and the model group were given 0.5% sodium carboxymethyl cellulose aqueous solution by gavage, once a day after the start of administration. The sulfasalazine administration group, the compound 1 low-dose, middle-dose and high-dose administration groups of the present invention were administered intragastrically from day 7 according to the experimental protocol, once a day. The experiment was terminated on the 46th day for a total of 40 days of administration. Both the positive drug and the compound 1 of the present invention were prepared with 0.5% sodium carboxymethyl cellulose aqueous solution according to the experimental protocol.

After the completion of the administration, each group of animals was sacrificed, and related evaluation indexes were detected (see the experimental results section), and the anti-ulcerative colitis efficacy of the compound of the present invention was comprehensively evaluated by the mouse disease activity comprehensive index score and the disease activity comprehensive index inhibition rate Learning effect.

(2) Experimental results

The effects of the positive drug sulfasalazine and the compound 1 of the present invention at low dose, medium dose and high dose on dextran sodium sulfate-induced chronic ulcerative colitis model mice disease activity comprehensive index score and disease activity comprehensive index inhibition rate are shown in the table 14.

Table 14. Positive drug sulfasalazine and compound 1 of the present invention improve disease activity index score and disease activity index inhibition rate of chronic ulcerative colitis model mice induced by sodium dextran sulfate

Note: ^** p<0.01, compared with the normal control group; ^# p<0.05, ^## p<0.01, compared with the model group; positive drug: sulfasalazine.

Table 14 Note: In an animal experiment for evaluating anti-chronic ulcerative colitis activity in an animal model of chronic ulcerative colitis in C57BL/6J mice induced by sodium dextran sulfate, the compound of the present invention has an effect on chronic ulcerative colitis model animals. It has a significant therapeutic effect and is significantly better than the current first-line treatment drug sulfasalazine for anti-ulcerative colitis. When the doses of the high, medium and low dose groups were 200mg/kg, 100mg/kg and 50mg/kg, the disease activity comprehensive index inhibition rate values were 94.44% ^## 、58.33% ^# and 38.89% ^# ( ^# p <0.05, ^## p<0.01, compared with the model group), compared with the positive control drug sulfasalazine in the parallel experiment, when the dose of sulfasalazine is 500 mg/kg, the comprehensive index of disease activity inhibition rate value is 50.00% (with Compared with the model group, there is no significant difference), the curative effect is very significant, and the dose-effect relationship is clear.

Experimental Example 6: Compound 3 of the present invention, single-dose palmatine chloride, single-dose palmatine chloride, single-dose 5-aminosalicylic acid, and palmatine chloride quaternary ammonium salt and Example of comparative experiment on pharmacological effects of 5-aminosalicylic acid combined administration of dextran sodium sulfate-induced acute ulcerative colitis in mice

(1) Materials and methods

1) Animals: C57BL/6J mice, male, with a weight range of 20-22 g.

2) Grouping: This experiment is divided into normal control group, dextran sodium sulfate model group, positive drug dexamethasone single-dose group, low-dose single-dose group of compound 3 of the present invention, and single-dose medium dose of compound 3 of the present invention Group, compound 3 high-dose single-administration group of the present invention, palmatine chloride single-administration group, 5-aminosalicylic acid single-administration group, and palmatine chloride formulated on the principle of equal moles Ammonium salt (549mg) and 5-aminosalicylic acid (221mg) homogeneous mixture (c3+A) combined administration group. Randomly group, 6 mice in each group.

3) Dosage and frequency of administration: the dosage of the positive drug dexamethasone in single administration group is 0.5 mg/kg, and the dosage of compound 3 of the present invention in the low, middle and high dose single administration groups is 50 mg/kg, respectively , 100mg/kg and 200mg/kg, the dosage of palmatine chloride quaternary ammonium salt single-administration group and 5-aminosalicylic acid single-administration group are both 100mg/kg, and the chlorinated chlorination is prepared based on the principle of equal number of moles The dosage of the combined administration group of the mixture of palmatine quaternary ammonium salt and 5-aminosalicylic acid was 100 mg/kg; once a day, the administration was administered for 7 days.

4) Experimental method: C57BL/6J mice were adaptively reared in an SPF animal room (experimental animal license number: SYXK (京)2014-0023) for one week, and then randomly grouped according to the experimental design. Mice in the model group and each compound administration group were given dextran sodium sulfate (MP, CA9011-18-1, US) every day to model ulcerative colitis model established in the laboratory [Zhang ZH, et al. Synthesis] and structure-activity relationships of quaternary coptisine derivatives as potential anti-ulcerative Colitis agents.J.Med.Chem.,2015,58,7557-7571]. The normal control group and the model group were given 0.5% sodium carboxymethylcellulose aqueous solution by gavage once a day. Positive drug dexamethasone single-administration group, compound 3 of the present invention low-dose single-administration group, medium-dose single-administration group and high-dose single-administration group, palmatine chloride single-administration group, 5- The aminosalicylic acid single administration group and the palmatine quaternary ammonium chloride and 5-aminosalicylic acid homogenous mixture (c3+A) prepared by the principle of equal moles were administered by gavage according to the experimental design plan , Once a day, are prepared with 0.5% sodium carboxymethyl cellulose aqueous solution according to the experimental protocol.

(2) Experimental results

The compound 3 of the present invention shows a significant therapeutic effect in vivo on the acute C57BL/6J mouse ulcerative colitis animal model induced by dextran sodium sulfate, which is significantly better than palmatine chloride quaternary ammonium salt and 5-aminosalicylic acid And a homogeneous mixture of palmatine chloride quaternary ammonium salt and 5-aminosalicylic acid (c3+A) prepared on the principle of equal moles.

1) The compound 3 of the present invention effectively reduces the pharmacological effects of dextran sodium sulfate-induced acute C57BL/6J mouse ulcerative colitis model animal weight loss significantly better than palmatine chloride quaternary ammonium salt, 5-aminosalicylic acid and A homogeneous mixture of palmatine chloride quaternary ammonium salt and 5-aminosalicylic acid (c3+A) prepared according to the principle of equal mole number (see Table 15).

Table 15. Compound 3 of the present invention, single administration of palmatine chloride quaternary ammonium salt, single administration of 5-aminosalicylic acid, and the formula of palmatine chloride quaternary ammonium salt and 5 -Aminosalicylic acid homogenous mixture (c3+A) combined administration to improve the pharmacological effects of dextran sodium sulfate-induced acute ulcerative colitis model mice

Note: ^** p<0.01, compared with the normal control group; ^# p<0.05, ^## p<0.01, compared with the model group. Positive drug: dexamethasone; comparison 1: single administration of palmatine chloride quaternary ammonium salt; c3+A: a mixture of palmatine chloride quaternary ammonium chloride and 5-aminosalicylic acid prepared on the principle of equal moles Combined administration.

It can be seen from Table 15 that compared with the initial value of animal weight in each group, after the end of the experiment, the weight of the normal control group increased by +2.96%, while the weight of the model group decreased by -21.70% ^** ( ^** p<0.01, compared with normal Compared with the control group), it is in line with the change trend of ulcerative colitis model, indicating that the model is successful. The compound 3 single-administered group of the present invention under the doses of 50mg/kg, 100mg/kg and 200mg/kg, the animal body weight change rate was -16.38%, -2.75% ^# and -5.70% ^## ( ^# p<0.05 , ^## p<0.01, compared with the model group), the dose-effect relationship is clear. Among them, at the same dose of 100mg/kg, the compound 3 single-administration group of the present invention effectively reduces the pharmacological effect of the weight loss of model animals significantly better than the single-administration palmatine chloride quaternary ammonium salt group and 5-aminosalicylic acid In the single administration group and the combined administration group of palmatine quaternary ammonium chloride and 5-aminosalicylic acid homogeneous mixture (c3+A) prepared on the principle of equal moles, the corresponding values of the latter three are -19.80% respectively , -14.68% and -23.94% (there are no significant differences compared with the model group). Single administration of the positive drug dexamethasone showed significant pharmacological effects in the experiment.

2) The compound 3 of the present invention is significantly better than palmatine quaternary ammonium chloride, 5-aminosalicylic acid and massage in improving the effect of dextran sodium sulfate-induced acute C57BL/6J mouse ulcerative colitis model animal colon contracture A homogeneous mixture of palmatine chloride quaternary ammonium salt and 5-aminosalicylic acid (c3+A) prepared by the principle of equal number of mols (see Table 16).

Table 16 shows the colon length value and colon contracture percentage of each group of animals after the experiment. The results showed that compared with the normal control group’s colon length value of 7.32±0.29cm, the model group’s colon was significantly shorter to 4.48±0.76cm, and the colon contracture ratio reached 38.75% ^** ( ^** p<0.01, compared with the normal control group compared to). Compared with the mice in the model group, the length of the colon of the mice in the low-dose, medium-dose and high-dose single administration group of compound 3 of the present invention was significantly increased, which were 5.60±0.40cm, 6.03±0.76cm and 7.03±0.80cm, respectively. The contracture ratios were 23.50% ^# , 17.58% ^## and 3.92% ^## ( ^# p<0.05, ^## p<0.01, compared with the model group). The palmatine chloride quaternary ammonium salt single-administration group, the 5-aminosalicylic acid single-administration group, and the homogeneous mixture of palmatine chloride quaternary ammonium chloride and 5-aminosalicylic acid prepared on the principle of equal moles ( c3+A) The colonic contracture rate of the combined administration group at a dose of 100 mg/kg reached 32.38%, 25.77% and 33.61% respectively (compared with the model group, there is no significant difference). Therefore, the compound 3 of the present invention has a very significant improvement effect on dextran sodium sulfate-induced acute C57BL/6J mouse ulcerative colitis model animal colon contracture, and the dose-effect relationship is prominent. In the experiment, the same dosage is used. Inventive compound 3 is significantly better than palmatine quaternary ammonium chloride, 5-aminosalicylic acid and equal moles in improving colonic contracture induced by dextran sodium sulfate in acute C57BL/6J mouse ulcerative colitis model animals A homogeneous mixture of palmatine chloride quaternary ammonium salt and 5-aminosalicylic acid (c3+A) prepared in principle.

Table 16. Compound 3 of the present invention, single-drug administration of palmatine chloride, single-drug administration of palmatine chloride, single-drug administration of 5-aminosalicylic acid, and palmatine quaternary ammonium chloride formulated on the principle of equal moles Comparison of the effect of combined administration of salt and 5-aminosalicylic acid homogeneous mixture (c3+A) on the improvement of colonic contracture in mice with acute ulcerative colitis induced by sodium dextran sulfate

Note: ^** p<0.01, compared with the normal control group; ^# p<0.05, ^## p<0.01, compared with the model group. Positive drug: dexamethasone; comparison 1: palmatine chloride quaternary ammonium salt; c3+A: a mixture of palmatine chloride quaternary ammonium salt and 5-aminosalicylic acid prepared on the principle of equal moles.

3) The compound 3 administration group of the present invention, the palmatine chloride quaternary ammonium salt administration group, the 5-aminosalicylic acid administration group, and the palmatine chloride quaternary ammonium salt and 5-aminosalicylic acid administered on the principle of equal moles Comparison of the effect of the administration group of aminosalicylic acid homogeneous mixture (c3+A) on the comprehensive disease activity index score and the inhibition rate of the disease activity comprehensive index in mice with acute ulcerative colitis induced by sodium dextran sulfate (see Table 17)

The compound 3 of the present invention, palmatine chloride quaternary ammonium salt, 5-aminosalicylic acid, and the principle of equal molar number of palmatine chloride and 5-amino water prepared by using the same batch of whole animal experiments The effect of the homogeneous mixture of salicylic acid (c3+A) on the dextran sodium sulfate-induced acute C57BL/6J mouse ulcerative colitis model animal disease activity comprehensive index score and disease activity comprehensive index inhibition rate, the results show that the compound 3 of the present invention Not only has significant anti-ulcerative colitis activity and a clear dose-effect relationship, it is significantly better than the palmatine chloride quaternary ammonium salt administration group and 5-aminosalicylic acid administration in parallel experiments at the same dosage Group and the administration group of homogeneous mixture of palmatine chloride quaternary ammonium salt and 5-aminosalicylic acid (c3+A) prepared on the principle of equal moles. It can be seen from Table 17 that compared with the normal control group, the animal disease activity comprehensive index score of the model group was significantly increased, which was 3.50±0.51**. The statistical difference was very significant, indicating that the model was successful. Compared with the model group, the hormone-positive drug dexamethasone can significantly reduce the comprehensive disease activity index score of experimental animals (score 1.61±0.44 ^## ) at a dose of 0.5 mg/kg, which is statistically compared with the model group The difference is very significant, which fully proves the reliability of the overall animal experiment. The low-dose (50mg/kg), medium-dose (100mg/kg) and high-dose (200mg/kg) administration groups of Compound 3 of the present invention significantly reduced the comprehensive index scores of the disease activity of experimental animals to 2.56±0.40 ^## , 2.06± 0.98 ^## and 1.33±1.05 ^## , both have statistically significant differences compared with the model group. The palmatine chloride quaternary ammonium salt administration group, the 5-aminosalicylic acid administration group and the homogeneous mixture of palmatine chloride quaternary ammonium chloride and 5-aminosalicylic acid prepared on the principle of equal massage (c3+A ) In the administration group, at a dosage of 100 mg/kg, the values of the improvement effect of the comprehensive disease activity index score and the inhibition rate of the comprehensive disease activity index inhibition rate of the acute C57BL/6J mouse ulcerative colitis model induced by dextran sodium sulfate are respectively 3.00±0.92, 2.61±0.74 ^# and 2.78±1.50, 5-aminosalicylic acid administration groups have statistically significant differences, but they are significantly lower than the compound 3 of the present invention; the other two administration groups are not statistically significant The sex difference is significantly lower than that of compound 3 of the present invention.

Table 17. Compound 3 of the present invention, palmatine chloride quaternary ammonium salt, 5-aminosalicylic acid, and palmatine chloride quaternary ammonium salt and 5-amino water prepared on the principle of equal moles The effect of combined administration of homogeneous mixture of salicylic acid (c3+A) on dextran sodium sulfate-induced acute C57BL/6J mouse ulcerative colitis model animal disease activity composite index score and disease activity composite index inhibition rate

Experimental Example 7: The compound 4 of the present invention is single-administered with berberine chloride quaternary ammonium salt, 5-aminosalicylic acid is single-administered, and berberine chloride quaternary ammonium salt and The pharmacological effect of the homogeneous mixture of 5-aminosalicylic acid combined with dextran sodium sulfate-induced acute ulcerative colitis in mice is comparative experimental example

(1) Materials and methods

1) Animals: C57BL/6J mice, male, with a weight range of 20-22 g.

2) Grouping: This experiment is divided into the normal control group, the dextran sodium sulfate model group, the positive drug dexamethasone single administration group, the low-dose, middle-dose and high-dose single administration group of compound 4 of the present invention, The berberine quaternary ammonium salt single administration group, the 5-aminosalicylic acid single administration group and the berberine chloride quaternary ammonium salt (553mg) and 5-aminosalicylic acid (231mg) formulated on the principle of equal moles are uniform Mixture (c4+A) combined administration group. Randomly group, 6 mice in each group.

3) Dosage and frequency of administration: the dosage of the positive drug dexamethasone single administration group is 0.5 mg/kg, and the dosage of compound 4 of the present invention is 25 mg/kg for the low, middle and high dose single administration groups, respectively , 50mg/kg and 100mg/kg, the dosage of berberine chloride quaternary ammonium salt single-administration group and 5-aminosalicylic acid single-administration group is 100mg/kg, and the number of moles is equal. The dosage of the combined administration group of the uniform mixture of berberine quaternary ammonium salt and 5-aminosalicylic acid was 100 mg/kg; once a day, the administration was administered for 7 days.

4) Experimental method: C57BL/6J mice were adaptively reared in an SPF animal room (experimental animal license number: SYXK (京)2014-0023) for one week, and then randomly grouped according to the experimental design. Mice in the model group and each compound administration group were given dextran sodium sulfate (MP, CA9011-18-1, US) every day to model ulcerative colitis model established in the laboratory [Zhang ZH, et al. Synthesis] and structure-activity relationships of quaternary coptisine derivatives as potential anti-ulcerative Colitis agents.J.Med.Chem.,2015,58,7557-7571]. The normal control group and the model group were given 0.5% sodium carboxymethylcellulose aqueous solution by gavage once a day. Positive drug dexamethasone single administration group, compound 4 of the present invention low-dose, middle-dose and high-dose single administration group, berberine chloride quaternary ammonium salt single administration group, 5-aminosalicylic acid single administration group And the combined administration group of berberine chloride quaternary ammonium salt and 5-aminosalicylic acid homogenous mixture (c4+A) prepared on the principle of equal moles was administered by gavage according to the experimental design plan, once a day, both at 0.5 % Sodium carboxymethyl cellulose aqueous solution was prepared according to the experimental dosage.

After modeling, the drug was continuously administered for 7 days, until the model group animals showed obvious symptoms of ulcerative colitis such as malaise, reduced activity, weight loss, loose stools, blood in the stool, etc., the experiment was terminated in due course, the animals in each group were killed, and the relevant evaluation indicators were tested. (See the experimental results part), comprehensively evaluate the anti-ulcerative colitis pharmacodynamic effect of the compound 4 of the present invention through the mouse disease activity comprehensive index score and the disease activity comprehensive index inhibition rate.

(2) Experimental results

The compound 4 of the present invention has a significant therapeutic effect in vivo on the acute C57BL/6J mouse ulcerative colitis animal model induced by dextran sodium sulfate, and its pharmacological effect is significantly better than berberine chloride quaternary ammonium salt and 5-amino water A homogeneous mixture of berberine chloride quaternary ammonium salt and 5-aminosalicylic acid (c4+A) prepared on the principle that salicylic acid and moles are equal.

The compound of the present invention 4 single administration group, berberine chloride quaternary ammonium salt single administration group, 5-aminosalicylic acid single administration group, and berberine chloride quaternary ammonium salt and 5 -Aminosalicylic acid homogenous mixture (c4+A) combined administration group on the dextran sodium sulfate-induced acute ulcerative colitis model mice disease activity comprehensive index score and disease activity comprehensive index inhibition rate, see Table 18 .

By using the same batch of whole animal experiments to investigate the compound 4 of the present invention, single-dose, single-dose berberine chloride quaternary ammonium salt, single-dose 5-aminosalicylic acid, and chlorinated berberis prepared on the principle of equal moles Combined administration of alkali quaternary ammonium salt and 5-aminosalicylic acid homogeneous mixture (c4+A) to dextran sodium sulfate-induced acute C57BL/6J mouse ulcerative colitis model animal disease activity comprehensive index score and disease activity comprehensive index The results show that the compound 4 of the present invention not only has a significant anti-ulcerative colitis pharmacological effect, the dose-effect relationship is clear, and it is significantly better than the parallel experimental berberine quaternary ammonium chloride at the same dosage. Salt, 5-aminosalicylic acid and a homogeneous mixture of berberine chloride quaternary ammonium salt and 5-aminosalicylic acid (c4+A) prepared on the principle of equal moles. It can be seen from Table 18 that compared with the normal control group, the animal disease activity comprehensive index score of the model group was significantly increased, which was 3.00±0.67**, which was statistically significant, indicating that the model was successful. Compared with the model group, the hormone-positive drug dexamethasone can significantly reduce the comprehensive disease activity index score of experimental animals at a dose of 0.5 mg/kg, with a score of 1.50±0.84 ^## , which is statistically very significant, which proves The reliability of overall animal experiments. The low-dose (25mg/kg) single-administered compound 4 of the present invention has no obvious pharmacological effect in reducing the comprehensive index score of experimental animal disease activity, but both the middle-dose (50mg/kg) and high-dose (100mg/kg) single-administered groups Significantly reduce the comprehensive index scores of laboratory animal disease activity to 1.78±0.89 ^# and 1.56±0.66 ^## , both of which are statistically significantly different from the model group. Berberine chloride quaternary ammonium salt for single administration group, 5-aminosalicylic acid single administration group, and a homogeneous mixture of berberine chloride quaternary ammonium salt and 5-aminosalicylic acid prepared on the principle of equal moles ( c4+A) The combined administration group improves the dextran sodium sulfate-induced acute C57BL/6J mouse ulcerative colitis model animal disease activity comprehensive index score and disease activity comprehensive index inhibition rate at a dose of 100 mg/kg The effect values were 2.72±0.98, 2.11±0.54 ^# and 2.17±0.69. The pharmacological effects of the 5-aminosalicylic acid single-administered group were statistically significantly different ( ^# p<0.05, compared with the model group) , But obviously inferior to the compound 4 of the present invention ( ^## p<0.01, compared with the model group); the other two administration groups have no statistically significant difference, which is significantly inferior to the compound 4 of the present invention.

Table 18. The compound of the present invention 4 single administration, single administration of berberine chloride quaternary ammonium salt, 5-aminosalicylic acid single administration and the principle of equal molar number of berberine chloride and 5 -Aminosalicylic acid homogenous mixture (c4+A) combined administration on dextran sodium sulfate-induced acute C57BL/6J mouse ulcerative colitis model animal disease activity composite index score and disease activity composite index inhibition rate

Note: ^** p<0.01, compared with the normal control group; ^# p<0.05, ^## p<0.01, compared with the model group. Positive drug: dexamethasone; comparison 1: berberine chloride quaternary ammonium salt; c4+A: a mixture of berberine chloride quaternary ammonium salt and 5-aminosalicylic acid prepared on the principle of equal moles.

Experimental example 8: Experimental example of the transcriptional activation effect of related compounds on pGL3-pxbp1

(1) Experimental method: Inoculate the IEC-6 cells in the vigorous growth stage in a 48-well plate, the number of cells per well is 5×10 ⁴ , so that the cells are evenly dispersed in the well, and placed at 37°C, 5% CO ₂ Culture in humidified cell incubator. After the cells are confluent to 70%-80%, the cells are transfected with the corresponding plasmids (0.6μg/well), 4h later, 1×10 ^-5 mol/L of related compounds are added and incubated with the transfected cells (n=3 ). After co-cultivation for 36h-48h, the samples were collected, and the experimental samples were tested for luciferase activity using the dual luciferase reporter gene detection kit (Promega, USA).

(2) Results: The plasmid-free cells were used as the control group 1, and the pGL-xbp1 non-medicated cells were used as the control group 2. The statistical analysis showed that the detected 10 organic acid berberine-type organisms Alkaline quaternary ammonium compounds have a certain activation effect on the xbp1 promoter, and the activation factor is in the range of 1.02-1.31 times, while the parallel experiment of dihydroberberine has a stronger activation effect on the xbp1 promoter, the activation factor is 1.64 times.

The experimental results are shown in Figure 8.

Experimental Example 9: Acute toxicity test results of

compounds

1, 2, 3, 4 of the present invention in animals

Take Kunming mice (weight range of 18-22g) into groups, each group of 10, male and female, a total of 8 dose groups, according to the Bliss method from the highest dose (5g/kg) in descending proportions to set each The dose of the administration group (1:0.8). Mice were given intragastric administration. The animals were fasted and watered the night before the administration. The mice were given a normal diet 4h after administration. After a single dose, continuous observation of the animals 14 days mental state, weight, diet, behavior, secretions, excretions, and the reaction poisoning death and _IC50 values _are calculated index LD. The results of the acute toxicity test in animals of the

compounds

1, 2, 3 and 4 of the present invention are as follows:

Compounds

1, 2, and 3 have no animal death and abnormal changes in physiological indicators at a dose of 5.0 g/kg. The LD ₅₀ value is 3.0 g/kg. Therefore, the compounds of the present invention are all non-toxic or low-toxic specific anti-ulcerative colitis compounds.

Experimental Example 10: Detection of the cytotoxicity (cell survival rate) of the compound of the present invention on normal human embryonic kidney epithelial cells 293T

(1) Experimental method: normal 293T human embryonic kidney epithelial cells grown in vitro to 90% confluence were digested with 0.25% trypsin/0.1% EDTA and seeded on a 96-well cell culture plate, the number of cells per well was 2×10 ³ . On the next day of culture, the original medium was removed, and a working solution containing 1×10 ^-5 mol/L of each compound of the present invention was added to each well to continue the culture. The toxicity (survival rate) of each compound of the present invention to 293T cells was detected by the MTT method at 0h, 24h and 72h after the 293T cells were co-cultured with the compounds of the present invention. Calculate the 293T cell survival rate of the test product according to the following formula:

(2) Results: Within the time range determined by the experiment, 1×10 ^-5 mol/L compounds 1, 2, 3, and 4 of the present invention have no significant cytotoxicity to normal human embryonic kidney epithelial cells 293T cells, and the inhibition rates are respectively 12.35%, 14.94%, 3.96% and 14.68%. Compared with the normal control group, there was no statistically significant difference.

Claims

Salicylic acid berberine type alkaloid quaternary ammonium salt compound represented by general formula I:

R is independently selected from H, NH 2 , OH, halogen, C2-C4 alkamido, C2-C4 alkanoyloxy, C1-C4 alkyl or C1-C4 alkoxy, R is mono- or poly-substituted, When R is mono-substituted, the substitution position is 3-position or 4-position or 5-position or 6-position, and when R is multiple substitution, it is selected from 2 substitution, 3 substitution or 4 substitution;

R 1 is independently selected from H, C1-C4 alkyl or C2-C4 alkanoyl;

R 2 and R 3 are each independently selected from H, OH, C1-C4 alkyl, C2-C4 alkanoyloxy or C1-C4 alkoxy, or R 2 and R 3 are connected to form a hydrocarbylene dioxy group;

R 9 , R 10 , R 11 , and R 12 are each independently selected from H, OH, C1-C4 alkyl, C2-C4 alkanoyloxy or C1-C4 alkoxy, or R 9 and R 10 are connected to form a sub Hydrocarbyl dioxy and R 11 and R 12 are each independently selected from H, OH, C1-C4 alkyl, C2-C4 alkanoyloxy, C1-C4 alkoxy, or R 9 and R 12 are each independently selected From H, OH, C1-C4 alkyl, C2-C4 alkanoyloxy, C1-C4 alkoxy and R 10 and R 11 are connected to form a hydrocarbylene dioxy group, or R 9 and R 10 are each independently selected from H, OH, C1-C4 alkyl, C2-C4 alkanoyloxy, C1-C4 alkoxy and R 11 and R 12 are connected to form an alkylenedioxy group.
The salicylic acid berberine alkaloid quaternary ammonium salt compound according to claim 1, wherein the halogen is selected from fluorine, chlorine, bromine, and iodine.
The salicylic acid berberine-type alkaloid quaternary ammonium salt compound according to claim 1, wherein the C2-C4 alkyl amide group is selected from the group consisting of acetamide, propionamide, butyramide, and isobutyramide base.
The salicylic acid berberine-type alkaloid quaternary ammonium salt compound according to claim 1, wherein the C2-C4 described in R, R 2 , R 3 , R 9 , R 10 , R 11 , and R 12 The alkanoyloxy groups are each independently selected from acetoxy, propionyloxy, butyryloxy, and isobutyryloxy.
The salicylic acid berberine alkaloid quaternary ammonium salt compound according to claim 1, wherein the C2-C4 alkanoyl group is selected from the group consisting of acetyl, propionyl, butyryl, and isobutyryl.
The salicylic acid berberine-type alkaloid quaternary ammonium salt compound according to claim 1, wherein R, R 1 , R 2 , R 3 , R 9 , R 10 , R 11 , R 12 are described in The C1-C4 alkyl groups are each independently selected from methyl, ethyl, propyl, isopropyl, and butyl.
The salicylic acid berberine type alkaloid quaternary ammonium salt compound according to claim 1, wherein the C1-C4 in R, R 2 , R 3 , R 9 , R 10 , R 11 , and R 12 The alkoxy groups are each independently selected from methoxy, ethoxy, propoxy, isopropoxy, and butoxy.
The salicylic acid berberine-type alkaloid quaternary ammonium salt compound according to claim 1, wherein said R 2 and R 3 are connected to form a hydrocarbylene dioxy group or R 9 and R 10 are connected to form a hydrocarbylene dioxy group. The oxy group or R 10 and R 11 are connected to form a hydrocarbylene dioxy group or R 11 and R 12 are connected to form a hydrocarbylene dioxy group, each independently selected from methylene dioxy, ethylene dioxy, and propylene dioxy , Butylenedioxy.
The salicylic acid berberine alkaloid quaternary ammonium salt compound according to claim 1, wherein the compound is selected from compounds 1-21 in the following compound group:
A method for preparing the salicylic acid berberine-type alkaloid quaternary ammonium salt compound according to claim 1, wherein the method is as follows: weighing 8-acetone dihydroberberine-type alkaloid for reaction In the bottle, add tetrahydrofuran to dissolve completely and then add salicylic acid under stirring. After the addition, reflux to complete the reaction. Let stand and cool to room temperature. The reaction mixture is suction filtered. The filter cake is the salicylic acid berberine type organism. Alkali quaternary ammonium compound.
A method for preparing 5-aminosalicylic acid berberine type alkaloid quaternary ammonium salt compound is characterized in that the adopted method for preparing 5-aminosalicylic acid berberine type alkaloid quaternary ammonium salt compound is as follows Description: Weigh various berberine-type alkaloid quaternary ammonium compounds whose acid root is not 5-aminosalicylate into the reaction flask, add sodium hydroxide aqueous solution, then add acetone dropwise, and stir to react until the raw material has reacted completely; The reaction mixture was suction filtered, and the filter cake was washed with water to neutrality to obtain a solid 8-acetone dihydroberberine type alkaloid; weigh 5-aminosalicylic acid into the reaction flask, add DMSO, and dissolve it completely Under stirring, add 8-acetone dihydroberberine alkaloid compound to react until the raw material reaction is complete; add tetrahydrofuran to the reaction mixture to dilute, stir until no excessive precipitation precipitates, filter the reaction mixture, and use tetrahydrofuran for the filter cake After washing three times, a 5-aminosalicylic acid berberine-type alkaloid quaternary ammonium salt compound is obtained; the 5-aminosalicylic acid berberine-type alkaloid quaternary ammonium salt compound is one in which R is the 5-position in claim 1 Amino monosubstituted.
A pharmaceutical composition, characterized in that it contains an effective dose of the salicylic acid berberine alkaloid quaternary ammonium salt compound of any one of claims 1-9 and a pharmaceutically acceptable carrier or excipient.
The use of the salicylic acid berberine alkaloid quaternary ammonium salt compound of any one of claims 1-9 or the pharmaceutical composition of claim 12 in the preparation of a medicine for preventing, relieving and/or treating ulcerative colitis.