CN112574172A

CN112574172A - Gallic acid hydrogen sulfide derivative, preparation method and medical application thereof

Info

Publication number: CN112574172A
Application number: CN202011474559.XA
Authority: CN
Inventors: 游鸿维
Original assignee: Hangzhou Haiminsi Medical Technology Co ltd
Current assignee: Hangzhou Haiminsi Medical Technology Co ltd
Priority date: 2020-12-14
Filing date: 2020-12-14
Publication date: 2021-03-30
Anticipated expiration: 2040-12-14
Also published as: CN112574172B

Abstract

The invention discloses a gallic acid hydrogen sulfide derivative, a preparation method and medical application thereof. The general formula of the gallic acid hydrogen sulfide derivative is A-Y-X, wherein A is gallic acid; y is-C (O) O-, -C (O) NH-, -C (O) OC (O) -, -C (O) NHCH₂C (O) -or is absent, when Y is absent, the compound is A-X; x is a moiety capable of releasing hydrogen sulfide either alone or in combination with A. Compared with gallic acid and the existing classical non-steroidal anti-inflammatory drugs, the gallic acid hydrogen sulfide derivative has equivalent or enhanced anti-inflammatory activity, weakened gastrointestinal tract and cardiovascular side effects, stronger anti-tumor activity and good cardiovascular and neuroprotective effects.

Description

Gallic acid hydrogen sulfide derivative, preparation method and medical application thereof

Technical Field

The invention relates to the field of medicines, in particular to a gallic acid hydrogen sulfide derivative, a preparation method thereof and a medicine.

Background

Inflammation is a defensive response of living tissue with a vascular system to an injury factor. It is the most common pathological process in human diseases and can occur in various tissues and organs, and common people include appendicitis, pneumonia, hepatitis, nephritis, encephalitis, enteritis, rheumatoid arthritis, osteoarthritis, traumatic infection and the like. In addition, inflammation is also closely related to cardiovascular diseases (hypertension, thrombosis, atrial fibrillation, coronary heart disease), respiratory diseases (acute/chronic pneumonia, Chronic Obstructive Pulmonary Disease (COPD)), tumors, and the like.

Moderate inflammatory responses may make the body effective against the attack of various pathogens, but if the body regulates itself in an unbalanced function, the inflammatory response is out of control (excessive inflammation), which may affect the normal physiological functions of the body, may involve various tissues or organs in the body, and if left to progress, may cause Systemic Inflammatory Response Syndrome (SIRS), until Multiple Organ Dysfunction Syndrome (MODS) and Multiple Organ Failure (MOF), or even death. At this time, appropriate measures should be taken to intervene in the inflammatory response and prevent and control the adverse effects of inflammation on the body. The administration of anti-inflammatory drugs is a very effective method.

Anti-inflammatory drugs can be broadly divided into two groups in terms of chemical structure: steroidal and non-steroidal anti-inflammatory drugs (NSAIDs). Steroidal anti-inflammatory drugs are not often the first choice due to their wide and complex effects, susceptibility to adverse reactions and complications, lack of specificity and specificity in treatment, and lack of specificity and pertinence. The non-steroidal anti-inflammatory drugs have the effects of relieving fever, easing pain, resisting inflammation and the like, can effectively relieve red, swollen, hot and painful symptoms of inflammation, are widely applied clinically, and become one of the most widely used drugs in the world.

Nonsteroidal anti-inflammatory drugs have been shown to be effective in a number of diseases, such as rheumatic diseases: can improve the clinical symptoms of patients and promote the recovery of the patients, and is widely applied to the patients with ankylosing spondylitis, osteoarthritis, reactive arthritis, gouty arthritis, psoriatic arthritis and other diseases; inflammatory diseases: the non-steroidal anti-inflammatory drug can be used for treating pain, swelling and fever caused by various cancerous diseases, systemic infection and patients with focal inflammation, can shorten the course of the patients and improve the conditions of the patients while treating the primary diseases of the patients, and has the most remarkable effects on diseases including urinary infection, respiratory infection, schistosome fever, cancerous fever, periodontitis, tonsillitis and the like; motor injury of soft tissue: the main effect of the non-steroidal anti-inflammatory drug for treating the patients is to improve the symptoms of stiffness, weakness, pain, swelling, limited movement and the like of the affected parts of the patients, and the non-steroidal anti-inflammatory drug is commonly used for the phenomena of laceration, contusion, sprain and strain caused by tendonitis, lumbar muscle strain, scapulohumeral periarthritis and neck and shoulder syndrome; pain: the symptoms mainly refer to dysmenorrhea of women, and the non-steroidal anti-inflammatory drug can not only effectively exert the effect of relieving pain, but also inhibit the synthesis of local prostaglandin; prevention of tumors: the non-steroidal anti-inflammatory drug can play a certain role in inhibiting the metastasis, development and occurrence of tumors, can effectively reduce the risk of tumor patients, and can reduce the risk coefficient to 40% by long-term administration. For this reason, nsaids have become one of the largest prescribed and non-prescribed drugs, and their use is only second to antibacterial drugs in our country according to statistics. However, with the increase of application and the continuous and deep research, people find that the drug has serious toxic and side effects after being taken for a long time.

NSAIDs-associated adverse reactions include gastrointestinal tract injury, cardiovascular events, altered renal function, liver injury, and hematopoietic damage, among others. Among them, the more common adverse reactions are gastrointestinal damage (such as ulcer and ulcer bleeding directly associated with NSAIDs) and cardiovascular events (hypertension, pulmonary embolism), and serious patients may threaten the life safety of patients taking the medicine.

Gastrointestinal tract injury: the symptoms are the most common adverse reactions of nonsteroidal anti-inflammatory drugs, such as phenylbutazone, indometacin, sodium salicylate and aspirin, which can cause dyspepsia, gastric erosion, gastroduodenal ulcer bleeding and even perforation of a patient. It has been found that patients who have used NSAIDs have a 3-5 times higher incidence of peptic ulcers than patients who have not used NSAIDs.

Cardiovascular events: specific COX-2 selective inhibitors have been shown to increase the incidence of cardiovascular events (hypertension, cardiac arrest, pulmonary embolism, myocardial infarction and stroke) when clinically applied, due to: although it can reduce the occurrence of gastrointestinal tract injury, it cannot effectively prevent the production of LTs, but increases the synthesis of LTs. More importantly, PGI after COX-2 inhibition₂Reduction of PGI₂/TXA₂The balance between them is broken, TXA₂The effect is relatively enhanced, leading to thrombosis and cardiovascular risk events (coronary occlusion).

Hydrogen sulfide (hydrogen sulfide, H)₂S) is a colorless toxic gas with the odor of rotten eggs. For a long time, H₂S is considered to be an important pollutant in the air and water supply, and excessive inhalation can affect the normal functions of the lung, brain, kidney, and other organs. Later found to have H in the human body₂S exists and can be used as a neuroactive substance, and new knowledge is provided for the neuroactive substance. It has now been shown that H₂S is a third gas signal molecule following Nitric Oxide (NO) and carbon monoxide (CO), and plays an important physiological role in the body, related to H₂The study of S has become one of the hot spots in the field of biological research. One finds H successively₂S has important pathophysiological effects in cardiovascular, nervous, inflammatory reaction, respiration, digestion, blood, metabolism, inflammation and other multiple systems or disease processes: h₂S has universal effect on regulating the structure and function of the whole cardiovascular system, and research shows that the S can regulate the myocardial contraction function, directly relax the body and pulmonary circulation vessels and inhibit the vascular smoothnessMyocytes (VSMCs) proliferate and regulate their phenotypic transformation. Furthermore, H₂S also has the effects of resisting myocardial ischemia reperfusion injury, participating in inflammatory reaction, resisting oxidative stress and the like, and mainly participates in the pathophysiological processes of pulmonary hypertension, coronary heart disease, myocardial ischemia reperfusion injury, shock and the like; endogenous H₂S also has important regulation effect on nervous system function, has the effects of nerve regulation, inflammatory reaction inhibition, antioxidant stress and the like, and also participates in the pathophysiological process of nervous system diseases such as Alzheimer Disease (AD), Parkinson Disease (PD), cerebral ischemia, febrile convulsion, Down syndrome and the like; it can also relieve inflammatory reaction induced by lipopolysaccharide such as rat edema and vascular permeability increase, H₂S has the functions of resisting inflammation and oxidation injury in the early stage of inflammation occurrence, and can also reduce the gastric mucosa injury caused by the non-steroidal anti-inflammatory drugs by 60 to 70 percent.

Human endogenous H₂S is mainly generated through 3 paths, wherein the first 2 paths are enzyme-dependent regulation paths of pyridoxal 5-phosphate (PLP), namely L-cysteine (Cys) and homocysteine (Hcy) are taken as substrates and are catalyzed by cystathionine-beta-synthetase (CBS) and cystathionine-gamma-lyase (CSE); item 3 is a PLP-independent 3-thiopyruvate thiotransferase (3-MST) pathway, catalyzed by a sulfur-containing amino acid as a substrate. The expression of CBS, CSE and 3-MST is tissue specific, CBS is mainly present in brain and nerve tissue, CSE is mainly expressed in liver, kidney, vascular smooth muscle and cardiovascular system. Unlike CBS and CSE, 3-MST is H present in mitochondria, not in the cytoplasm₂S synthetase belongs to the family of sulfhydryl transferase, and is mainly present in brain, liver, kidney, heart, lung, thymus, testis and thoracic aorta.

In the early research on hydrogen sulfide, the biological effect of hydrogen sulfide was generally studied by using sodium hydrosulfide as a donor of hydrogen sulfide, and sodium hydrosulfide dissolved in water can release a large amount of hydrogen sulfide gas in a short time, but the characteristic gradually shows a definite disadvantage. In recent years, new hydrogen sulfide donors have been discovered which are complexes of sulfur-containing structures bound to precursors of different drugs, also referred to as derivatives of these drugs. This new donor slowly releases hydrogen sulfide in vivo, exhibiting many effects not achieved with sodium hydrosulfide.

Aspirin is a nonsteroidal anti-inflammatory drug, and its derivative S-aspirin introduces a sulfur-containing structure (ADT-OH) on the parent structure, can release hydrogen sulfide, and thus can be used as a hydrogen sulfide donor, which may have some functions of both parent aspirin and product hydrogen sulfide. In recent years, S-aspirin is found to have good effects on rat anti-inflammation, cardiovascular protection and neuroprotection.

Diclofenac itself is an anti-inflammatory drug, but has side effects of damaging gastrointestinal mucosa, S-diclofenac is a compound which connects a sulfur-containing structure (ADT-OH) capable of releasing hydrogen sulfide with diclofenac through an aliphatic bond, and the S-diclofenac can protect the gastrointestinal tract and has strong anti-inflammatory effect, has unique protective effect in vascular occlusive diseases and relieves ischemia/reperfusion injury, and shows that the S-diclofenac can become a new drug with smaller side effects while exerting anti-inflammatory and cardiovascular protection of the S-diclofenac, but the S-diclofenac has yet to be further clinically researched.

As is well known, naproxen has the functions of resisting inflammation, relieving fever and easing pain, and is mainly applied to treating rheumatoid arthritis. ATB-346 is a complex linking a sulfur-containing structure to naproxen, and in recent years in carrageenan-induced synovitis in rats, effective doses of ATB-346 and naproxen were found to reduce edema and pain scores, with tactile allodynia similarly inhibited by both drugs, and with similar anti-inflammatory effects. However, in rats with arthritis, ATB-346 showed a faster anti-inflammatory effect than naproxen. In terms of toxic side effects, ATB-346 does not damage the gastrointestinal tract and may even promote healing of pre-existing gastric ulcers, while naproxen increases gastrointestinal damage. Although ATB-346 has no obvious difference in anti-inflammatory effect from naproxen, it can shorten the time for exerting the drug effect and relieve the pain of rheumatoid arthritis patients in a short time, and most importantly, compared with naproxen, ATB-346 does not damage gastrointestinal tracts and has a certain protective effect. Therefore, ATB-346 may replace naproxen, a traditional anti-inflammatory agent, in the treatment of rheumatoid arthritis.

Due to the discovery of the above-mentioned peculiar biological effects of hydrogen sulfide, there is a new understanding and appreciation of many life phenomena, and there is a growing awareness that hydrogen sulfide can be used in the treatment of diseases.

Gallic Acid (GA), also known as gallic acid, with chemical name of 3,4, 5-trihydroxybenzoic acid, is one of the important components of Chinese medicinal gallnut, and is also present in medicinal plants such as radix et rhizoma Rhei Palmati, Corni fructus, cortex moutan, and fructus Punicae Granati. The gallic acid is a natural polyphenol structure with simple structure, has various biological activities, and has the functions of anti-inflammation, antioxidation, liver protection, anti-tumor and the like which gradually become research hotspots along with the deep research on the pharmacological activities of the gallic acid and the derivatives thereof. But the gallic acid has poor lipid solubility and poor bioavailability, so that the activity of the gallic acid is difficult to exert. When the carboxyl of the gallic acid reacts with the alcohol or the phenolic hydroxyl to form an esterification product, the lipid solubility is improved, and the biological activity of the gallic acid can be obviously improved. At present, no compound for linking gallic acid to a sulfur-containing structure has been reported.

Disclosure of Invention

The invention provides a gallic acid hydrogen sulfide derivative, which comprises gallic acid and a hydrogen sulfide donor, wherein the gallic acid hydrogen sulfide derivative has equivalent or enhanced anti-inflammatory activity, has reduced side effects on gastrointestinal tract and cardiovascular system, and shows stronger anti-tumor activity and good cardiovascular and neuroprotective effects compared with gallic acid and the existing classical non-steroidal anti-inflammatory drugs.

The technical scheme provided by the invention for solving the technical problem is as follows:

a compound having the following general formula, an isomer thereof or a pharmaceutically acceptable salt thereof,

A-Y-X

wherein the content of the first and second substances,

a is gallic acid;

y is-C (O) O-, -C (O) NH-, -C (O) OC (O) -, -C (O) NHCH₂C (O) -or is absent, when Y is absent, the compound is A-X;

x is a moiety capable of releasing hydrogen sulfide either alone or in combination with A.

After a great deal of research and experiments, the inventor finds that after the gallic acid is grafted with a hydrogen sulfide releasing group, on one hand, the lipid solubility of the gallic acid is increased, on the other hand, the derivative can release the hydrogen sulfide, the biological activity of the derivative is enhanced or basically equivalent to that of the existing clinical drugs, the derivative with lower side effect is formed, and simultaneously, the compound is endowed with new potential biological activity or functions such as cardiovascular and nervous system protection, metabolic regulation function and the like.

A is

X is

The compound is

The invention provides a preparation method of the compound, the isomer or the pharmaceutically acceptable salt thereof, which comprises the following steps: carrying out nucleophilic substitution reaction on ADT-OH and TAGC in the presence of an acid-binding agent, and purifying to obtain TAGT; partially hydrolyzing TAGT to obtain GT;

the ADT-OH and the TAGC are subjected to nucleophilic substitution reaction in the presence of an acid-binding agent, and the method specifically comprises the following steps: dissolving TAGC in a solvent, then sequentially adding an acid-binding agent and ADT-OH for nucleophilic substitution reaction, and after the nucleophilic substitution reaction is finished, purifying the reaction solution to obtain TAGT.

The molar ratio of ADT-OH, TAGC and the acid-binding agent is 1: 0.9-1.1: 0.9 to 1.1.

The solvent is dichloromethane.

The acid-binding agent is triethylamine.

The reaction temperature of the nucleophilic substitution reaction is 15-30 ℃, and the reaction time is 2-4 h.

The GT is obtained after partial hydrolysis of the TAGT, and specifically comprises the following steps: dissolving TAGT in organic solvent, adding hydrazine hydrate for hydrolysis, and purifying to obtain GT.

The organic solvent is tetrahydrofuran.

The molar ratio of TAGT to hydrazine hydrate is 1: 3 to 3.2.

The reaction temperature of the hydrolysis reaction is 15-30 ℃, and the reaction time is 20-40 min.

The invention provides a pharmaceutical composition, which is prepared from the compound, the isomer or the pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier or auxiliary material.

The invention also provides application of the compound, the isomer thereof or the pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparing medicaments for preventing or treating tumors.

The invention also provides application of the compound, the isomer thereof or the pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparing a pain treatment drug or an antipyretic, analgesic and anti-inflammatory drug.

The invention also provides application of the compound, the isomer thereof or the pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparing medicaments for treating cardiovascular and cerebrovascular diseases.

The invention also provides application of the compound, the isomer thereof or the pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparing a medicament for treating nervous system diseases.

The compounds of the invention have relatively high pharmacological activity and good safety and tolerance, and can be used for treating and preventing various diseases, such as various disease states in which oxidative stress is involved, respiratory diseases, cardiovascular diseases, cancer, diffuse scleroderma, systemic sclerosis, glioblastomas, viral hepatitis, drug-induced hepatitis, arteriolar fibrosis, arteriosclerosis, acute and chronic glomerulonephritis, interstitial nephritis, pyelonephritis, renal arteriosclerosis, renal insufficiency, digestive tract ulcer, gastric cancer, Kaschin's disease and keshan disease. Also, the compounds may be used in a variety of diseases significantly associated with inflammation, such as, but not limited to, cancer, asthma, cerebral stroke, myocardial infarction, myocardial ischemia, coronary sclerosis, angina, hypertension, rheumatic heart disease, myocarditis, keshan disease, pericarditis, atherosclerosis.

More related, the compounds of the invention may be used, but are not limited to, in the treatment of inflammation, or in the treatment of diseases associated with inflammation, for example, as antipyretic, analgesic and anti-inflammatory agents. For example, the compounds of the present invention may be used to treat arthritis, including, but not limited to, rheumatoid arthritis, osteoarthritis, rheumatoid arthritis, gouty arthritis, lupus erythematosus syndrome. Inflammation related to the skin, psoriasis, eczema, burns, dermatitis. It can also be used for treating gastrointestinal disorders such as inflammatory bowel disease, Crohn's disease, gastritis, irritable bowel syndrome and ulcerative colitis. The compounds of the invention may be used in the treatment of asthma, bronchitis, bursitis, tenosynovitis. For the prevention or treatment of cancer, such as colorectal cancer. Such compounds may also be used to treat pain, including, but not limited to, headache, toothache, maxillofacial pain, neck pain, shoulder, upper limb pain, chest pain, abdominal pain, low back pain, pelvic pain, lower limb pain, anal, perineal pain, surgical pain, muscle pain, cancer pain. Can also be used for inflammation in certain diseases including, but not limited to, vascular disease, migraine, arteritis nodosa, thyroiditis, aplastic anemia, scleropathy, rheumatic fever, diabetes, myasthenia gravis, gingivitis, nephritis, allergies, swelling after injury, myocardial ischemia, etc.; it can also be used for treating pulmonary inflammation, acute lung injury, acute respiratory distress syndrome, infectious (such as bacterial, viral, mycoplasma, fungal, and parasitic) pneumonia, physicochemical (such as metabolic, inhalational, and lipidic) pneumonia, allergic (such as allergic and rheumatic) pneumonia, chronic obstructive pulmonary disease, and endotoxin shock. Inflammation, spasmodic anal pain and rectal fissures associated with hemorrhoids; gallbladder and/or biliary tract disorders, for example, cholangitis, sclerosing cholangitis, primary biliary cirrhosis and cholecystitis, intestinal carbuncles. Can also be used for treating central nervous system diseases, such as Alzheimer disease (senile dementia), Parkinson disease, atherosclerosis and central nervous system injury caused by apoplexy, ischemia, and trauma. In addition to human disease treatment, it can be used in other mammals, including pigs, cattle, horses, sheep, cats, dogs, rats.

The compounds of the invention may be used to prevent or treat a variety of diseases, particularly inflammation of the GI tract, including, but not limited to, oral inflammation such as mucositis, infectious diseases (e.g., viral, bacterial, fungal diseases) and crohn's disease, inflammation of the esophagus such as esophagitis, diseases resulting from chemical injury (e.g., ingestion of lye), gastroesophageal reflux disease, bile acid reflux, barrett's esophagus, esophageal stricture, inflammation such as gastritis (e.g., helicobacter pylori, acid, digestive diseases, and atrophic gastritis), celiac disease, peptic ulcer, precancerous lesions of the stomach, non-ulcerative dyspepsia, salmonella enteritis, shigella infection, yersinia disease, cryptosporidiosis, microsporidian infection and viral infection, radiation-induced colitis, colitis in an immunocompromised host (e.g., ceitis), precancerous lesions of the colon (e.g., dysplasia, inflammation of the bowel and polyps of the colon), proctitis.

The physician or patient can use the compounds of the present invention in dosages adjusted to suit the particular situation or disease state in question, provided that the dosage is within the effective and safe limits. As will be readily appreciated by those skilled in the art. Generally, the daily dosage of these compounds is about 1-5000mg for an adult, and may be administered once or more than once, although it is necessary to individually adjust the dosage to achieve the optimal therapeutic effect depending on the disease treatment needs and the weight or disease state of the patient. However, the most suitable dosage level should be in the range of 0.1-500mg/kg, preferably 5-100mg/kg, but will vary depending on the body weight of the patient to be treated, the condition and their sensitivity to the drug, as well as the type of pharmaceutical formulation selected and the time and interval at which it is to be administered. In some cases, dosage levels below the lower limit or higher than the upper limit of the above range may be used so long as they do not produce any side effects which affect the treatment.

The compounds of the present invention may be formulated into various pharmaceutical dosage forms, the nature of which depends on the route of administration, and these pharmaceutical compositions may be prepared by conventional methods using compatible, pharmaceutically acceptable excipients or carriers. The composition can be in the form of tablet, capsule, granule, spray, transdermal patch, lozenge, syrup, powder, gel, suppository, etc., and can be prepared into temporary solution, injectable preparation, rectal, ocular, vaginal preparation, etc., preferably by oral and rectal administration.

When used for oral administration, various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate and glycine and various disintegrants such as starch, sodium alginate and certain complex silicates, and particulate binders such as polyvinylpyrrolidone, sucrose, gelatin and acacia may be used. Additionally, lubricants such as magnesium stearate, sodium lauryl sulfate and talc may be used for tableting, solid compositions of similar type may also be used with fillers in the capsule, preferred materials relating thereto also include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions are intended for oral use, the active ingredient may be combined with sweetening or flavoring agents, coloring matter and emulsifying and/or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin and various combinations thereof.

The dosage form may be designed for slow release, controlled release, immediate release, delayed release, or targeted delayed release. The definitions of these terms are well known to those skilled in the art.

For parenteral use, solutions of the active compound in sesame or peanut oil or in aqueous propylene glycol may be employed, the aqueous solution should be suitably buffered (preferably to a pH greater than 8) if necessary, the liquid diluent being isotonic first, the aqueous solution being suitable for intravenous purposes. The preparation of all of these solutions under sterile conditions is accomplished by standard pharmaceutical techniques well known to those of ordinary skill in the art.

The invention has the beneficial effects that:

the compound of the present invention has excellent anti-inflammatory effect and no obvious gastrointestinal tract damage is found. And the compound in the invention can release a proper amount of hydrogen sulfide, so that the occurrence of cardiovascular events can be reduced. Therefore, the compound of the invention can possibly avoid gastrointestinal tract injury and/or adverse cardiovascular events or other toxic and side effects caused by non-steroidal anti-inflammatory drugs in clinical application, can possibly replace the existing clinical application, and has very wide application prospect.

Drawings

FIG. 1 shows the effect of gallic acid hydrogen sulfide derivatives on ear swelling in mice caused by xylene.

FIG. 2 is a graph showing the effect of gallic acid hydrogen sulfide derivatives on croton oil induced ear swelling in mice.

FIG. 3 shows the effect of gallic acid hydrogen sulfide derivatives on tumor cell growth.

In FIGS. 1 to 3, GA is gallic acid, DI is diclofenac, and GT is a hydrogen sulfide derivative of gallic acid.

FIG. 4 shows the effect of a single administration of a gallic acid hydrogen sulfide derivative on the gastrointestinal tract of rats.

FIG. 5 shows the effect of continuous administration of gallic acid hydrogen sulfide derivatives on the gastrointestinal tract of rats.

In FIGS. 4 to 5, CTR is a vehicle control group, GA is gallic acid, DI is diclofenac, and GT is a gallic acid hydrogen sulfide derivative.

Detailed Description

The present invention is described in detail below by way of examples, but is not meant to be limited to any of the disadvantages of the present invention. Having described the invention in detail and having disclosed specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Example 1.

1. Synthesis of 5- (4-hydroxyphenyl) -3H-1, 2-dithiocyclopent-4-ene-3-thione (ADT-OH)

(1) Synthesis of 5- (4-methoxyphenyl) -3H-1, 2-dithiole-3-thione (ADT-OCH3)

Anethole (3.17g,0.021mol), sulfur (4.70g,0.146mol) and DMF30mL were added to the flask and stirred under reflux for 6 h. Pouring the mixture into cold water after the reaction is finished, separating out a large amount of precipitates, filtering, and recrystallizing by ethanol-water to obtain red crystals with the yield of 78 percent and the mp: 110-.

(2) Synthesis of 5- (4-hydroxyphenyl) -3H-1, 2-dithiole-3-thione (ADT-OH)

ADT-OCH3(20.6g,0.1mol) and pyridine hydrochloride (69.0g,0.6mol) were heated under reflux for 1h, cooled to become a solid, triturated, washed several times with water, filtered, and the filter cake was recrystallized from acetone-water in 98.0% yield, mp:191 and 192 ℃.

2. Triacetyl gallic acid (TAGA)

To a 150ml round bottom flask were added in the order 17g (0.1mol) of Gallic Acid (GA), 0.51g of p-toluenesulfonic acid (3% by mass of GA), 30ml of acetic acid, a stirrer, and 49.2ml (0.52mol) of acetic anhydride slowly with stirring in a thermostatically heated electromagnetic stirring oil bath at 90 ℃. The reaction solution is kept at 90 ℃ for 1 to 2 hours and is slightly yellow. Sampling and spotting to check the experiment progress by using thin layer chromatography. The developing solvent is EtOAc (volume ratio) PE (6: 4), and one drop of acetic acid is added. The lamella plates show an impurity with a polarity slightly less than the target product. And after the reaction is finished, cooling, crystallizing in ice bath, filtering, washing a filter cake with water, transferring the filter cake to a surface dish, and drying under an infrared lamp to obtain white powder, namely triacetyl gallic acid. Sampling and detecting by thin layer chromatography without the above impurities.

3. Preparation of triacetylgalloyl chloride (TAGC)

A150 ml round bottom flask was charged with 16.28g (55mmol) of TAGA, 70ml of dichloromethane, stirred with a stirrer and dissolved, and 4ml (165mmol) of thionyl chloride, 0.2ml (1.2% by mass of TAGA) of DMF were added. The reaction was refluxed at 60 ℃ for 3 hours. After the reaction is finished, the temperature is raised to 90 ℃, dichloromethane and thionyl chloride are distilled out, yellowish white solid is obtained, and nitrogen is filled for protection. The pure product can be obtained by recrystallization of carbon tetrachloride.

4. Preparation of triacetylgallate (TAGT)

15.73g (50mmol) of triacetylgalloyl chloride (TAGC) and 60ml of dichloromethane are added into a 150ml round-bottom flask, a stirrer is placed into the flask, the materials are stirred and dissolved, 7.12ml (50mmol) of triethylamine is added, white smoke appears on the solution, the solution gradually disappears, the solution gradually turns yellow and brown, 11.3g (50mmol) of ADT-OH is added, the solution turns black, the mixture is stirred for 3 hours at room temperature, a little heat is generated in the reaction, and the temperature can be reduced by proper water bath. Sampling, spotting by using thin layer chromatography to check the progress of the reaction, and developing by using volume ratio of EtOAc: PE-4: 6, the polarity of the product is slightly larger than ADT-OH. After the reaction, the triethylamine salt generated in the reaction is removed by washing with 50ml of water twice, and then washed with 50ml of saturated sodium bicarbonate aqueous solution for three times, so as to remove the triacetyl benzoyl chloride (TAGC) which is not completely reacted and the triacetyl gallic acid (TAGA) which is not removed in the previous step, and finally washed with saturated salt water, dried by anhydrous sodium sulfate, filtered, and the solvent is evaporated by rotary evaporation to obtain a brownish black solid. Silica gel column chromatography purification was performed using EtOAc to PE 2:8 as eluent to give a brown solid powder.

5. Preparation of gallic acid hydrogen sulfide derivative (GT)

25.2g (50mmol) of the crude triacetylgallate (TAGT) obtained in the above step and 60ml of tetrahydrofuran were added to a 150ml round bottom flask, stirred and dissolved. 8.8ml (150mmol) of hydrazine hydrate is added and stirred for 30min at room temperature, and the temperature can be reduced in a proper water bath due to a small amount of exothermic reaction so as to prevent side reaction caused by temperature rise. Sampling, carrying out thin layer chromatography, developing by using a developing agent in a volume ratio of EtOAc to PE of 5:5, and adding a drop of acetic acid. After completion of the reaction, 8.6ml (150mmol) of acetic acid was added. The reaction was removed from the reaction flask, 100ml of water was added, extracted twice with 50ml of EtOAc, and the organic phases were combined and washed with saturated aqueous sodium chloride solution. Adding anhydrous Na₂SO₄Drying, filtering, and rotary evaporating to remove solvent to obtain black solid crude product with slight ammonia odor. Purifying by silica gel column chromatography, eluting with EtOAc (volume ratio) and PE (proportion of PE to PE) 3:7, and evaporating the solvent to dryness to obtain reddish brownA colored solid powder was the final product.

ESI-HRMS m/z 378.9757(M+H⁺)。

IR：3392cm^-1，1689cm^-1，1604cm^-1，1517cm^-1，1487cm^-1，1261cm^-1，1095cm^-1，1020cm^-1，802cm^-1。

NMR：9.47ppm(2H)，9.24ppm(1H)，8.01ppm(2H)，7.86ppm(1H)，7.43ppm(2H)，7.13ppm(2H)。

Example 2: pharmacological experiments and results:

anti-inflammatory and anti-tumor effects

1. Xylene induced ear swelling test in mice

Kunming mouse, purchased from Shanghai laboratory animal center of Chinese academy of sciences, weighing 18-22g, male. Temperature is 20-24 deg.C, humidity is 65-70%, standard fodder is fed, padding and food are replaced in time every day, and squirrel cage is kept clean.

Test compounds were formulated as suspensions in 0.5% CMC-Na solution, fasted for 12h before administration, and allowed to drink freely. Mice were gavaged (125.6. mu. mol/kg) with a dose volume of 0.2mL/10g body weight. After 1h of administration, the right auricle of the mouse was evenly coated on both sides with 20. mu.L of xylene by a microsyringe to cause inflammation, and the left auricle was used as a control. After 1h of inflammation, the mice were sacrificed by taking off the cervical vertebrae, taking off both ears along the base line of the auricle, and taking off one ear at the same position by a puncher (diameter 7mm) and weighing by an electronic balance. The swelling degree is obtained by subtracting the weight of the control side ear piece from the weight of the inflammation-causing ear piece. The swelling inhibition rate was calculated according to the following formula, and the degree of swelling of the control group and the administration group was statistically treated: the swelling inhibition ratio (%) was (average degree of swelling in model group-average degree of swelling in administration group)/average degree of swelling in model group × 100%.

Effect of gallic acid hydrogen sulfide derivative (GT) on xylene-induced ear swelling in mice: the anti-inflammatory activity of GT was evaluated in a xylene-induced mouse ear swelling model, and the results are shown in FIG. 1, (GA: gallic acid, DI: diclofenac, GT: hydrogen sulfide derivative of gallic acid, the same applies below); from fig. 1, it can be seen that the tested compound showed a better ear swelling inhibition effect, which exceeds twenty percentage points (P <0.01) of diclofenac and gallic acid, indicating that the compound has a stronger anti-inflammatory activity.

Experiment of ear swelling caused by 2 croton oil

Preparing croton oil into a mixed solution according to the following proportion: ethanol: diethyl ether: water ═ 2:20:73:5(v/v), used as an inflammatory agent. Healthy adult ICR mice are selected and purchased from the animal experiment center of Nantong university, the weight of the mice is 18-22g, and the animals are divided into a solvent group, a positive control group and a tested drug group. Standard feed is used for feeding, padding and rat food are replaced in time every day, and the cage is kept clean.

Test compounds were formulated as suspensions in 0.5% CMC-Na solution, fasted for 12h before administration, and allowed to drink freely. Mice were gavaged (125.6. mu. mol/kg) with a dose volume of 0.2mL/10g body weight. After 0.5h of administration, both sides of the right auricle of the mouse were evenly coated with 0.1mL of croton oil inflammation-causing agent by a microsyringe, and the left auricle was used as a control. After 4h of inflammation, the mice were sacrificed by taking off the cervical vertebrae, taking off both ears along the auricle base line, and taking off one ear at the same position by a puncher and weighing by an electronic balance. The swelling degree is obtained by subtracting the weight of the control side ear piece from the weight of the inflammation-causing ear piece. The swelling inhibition rate was calculated according to the following formula, and the degree of swelling of the control group and the test drug group was statistically treated: the swelling inhibition ratio (%) was (average degree of swelling in model group-average degree of swelling in administration group)/average degree of swelling in model group × 100%.

Effect of gallic acid hydrogen sulfide derivative (GT) on croton oil induced ear swelling in mice: the anti-inflammatory activity of GT was evaluated in a croton oil-induced mouse ear swelling model, and the results are shown in fig. 2.

From fig. 2, it can be seen that the test compound showed a better ear swelling inhibition effect, both exceeding over a dozen percentage points (P <0.01) of diclofenac and gallic acid, indicating that the compound has an excellent anti-inflammatory activity.

3 antitumor action

Material

MCF-7 and HT-29 cell strains (Shanghai cell bank of Chinese academy of sciences) are recovered and subcultured. RPMI1640 (or DMEM) medium, tetramethyl azo blue (MTT, Sigma), trypsin (guino bio-pharmaceutical technology, ltd), fetal bovine serum (hangzhou siji qing), dimethyl sulfoxide (DMSO, guangzhou chemical reagent factory), SDS, 96-well plate.

Sample processing

The test compound is dissolved in DMSO to prepare a high-concentration solution, and then the solution is diluted to the required concentration by using a culture solution for later use. The culture solution is used for preparing DMSO, and the DMSO is prepared just before use.

Cell culture

The tumor cell line was inoculated in RPMI1640 (or DMEM) medium containing 10% inactivated fetal bovine serum in 5% CO₂Culturing in an incubator at 37 ℃ under a fully humidified condition, and replacing the culture solution 2-3 times per week.

Test method for inhibiting tumor cell proliferation in vitro

Taking a bottle of cells with good logarithmic phase state, adding 0.25% trypsin for digestion to make adherent cells fall off, and preparing into a solution containing 5 × 10 cells per ml⁶～6×10⁶A suspension of individual cells. Seeding the cell suspension on a 96-well plate, and placing in constant temperature CO₂Culturing in an incubator for 24 h. After changing the medium, test compounds (compounds dissolved in DMSO and diluted in culture medium at concentrations of 10, 20, 40, 80, 160. mu.M, and partial compounds at concentrations of 5, 10, 20, 40, 80. mu.M) were added and incubated for 48 hours. MTT was added to a 96-well plate at 20. mu.L per well and reacted in an incubator for 4 hours. SDS was then added, and the absorbance of each well was measured at a wavelength of 570nm using an enzyme-linked immunoassay instrument, and the cell inhibition rate was calculated.

Inhibition (%) - (control well mean OD value-experimental well mean OD value)/control empty mean OD value × 100%.

Gallic acid hydrogen sulfide derivative (GT) for treating tumor cells HT-29, MCF-7 and Hep G₂The effect of proliferation is shown in FIG. 3. The half inhibitory concentration of the compound on the tumor cells is shown in the figure<50 mu M, which is obviously stronger than that of diclofenac and gallic acid, indicating that the gallic acid is connected into H₂The anti-tumor activity is enhanced after S donor.

Second, gastrointestinal tract injury experiment

1. Effect of a Single administration of test Compounds on the gastrointestinal tract of rats

Healthy male SD rats were purchased from Shanghaisi Lett experimental animal center, and randomly grouped by weight (average 100g), and fasted for 24h before the experiment without water deprivation. Feeding with standard feed, timely replacing padding and mouse food every day, and keeping the mouse cage clean. In the experiment, a solvent control group (CTR), a diclofenac group (DI), a gallic acid Group (GA) and a gallic acid hydrogen sulfide derivative Group (GT) are respectively arranged. The vehicle control group is administered by intragastric administration of the vehicle to each animal; the diclofenac sodium solution is infused into each animal of the diclofenac control group according to the dosage of 20 mg/kg; test drug groups (GA, GT), animals of each group were gavaged at 62.8. mu. mol/kg, and animals of each group were sacrificed 6 hours after administration, and the effects of the drugs on the gastrointestinal tract of rats were observed, compared, and the correlation index was calculated as follows. The stomach was cut open along the greater curvature side of the stomach and flattened, and the gastric Ulcer Index (UI) was calculated according to Guth's criteria: the length of the ulcer surface is 1 minute when the length is less than 1mm, 2 minutes when the length is 1-2 mm, 3 minutes when the length is 2-3 mm, 4 minutes when the length is 3-4 mm, and the ulcer surface is divided into a plurality of sections when the length is more than 4mm, wherein each section is calculated according to the method. Score x 2 for ulcer width >1mm, dot bleeding point calculated per 0.5 point, and cumulative additive score for each rat is the total gastric ulcer index for that rat. And analyzing whether the gastric ulcer indexes have significant difference among groups by adopting a t test. The results of the experiment are shown in FIG. 4.

The results in fig. 4 show that the index of gastric ulcer in GA and GT groups is very small and significantly different (P <0.01) compared to diclofenac group. Meanwhile, after the dissection, the stomach of the rat with the diclofenac acid group has color change of different degrees, mainly presents yellow-white or pale-white, while the rat with the GA and GT groups does not observe the change with naked eyes, and other abnormal changes are not obviously detected.

2 Effect of continuous administration of test Compounds on gastrointestinal tract of rats

Healthy male SD rats were purchased from Shanghaisi Lett experimental animal center, and randomly grouped by weight (average 100g), and fasted for 24h before the experiment without water deprivation. Feeding with standard feed, timely replacing padding and mouse food every day, and keeping the mouse cage clean. In the experiment, a solvent control group (CTR), a diclofenac group (DI), a gallic acid Group (GA) and a gallic acid hydrogen sulfide derivative Group (GT) are respectively arranged. The vehicle control group is administered by intragastric administration of the vehicle to each animal; the diclofenac sodium solution is infused into each animal of the diclofenac control group according to the dosage of 40 mu mol/kg; test drug groups (GA, GT), each group of animals was gavaged at 40. mu. mol/kg once daily for 5 consecutive days. Food and water were not controlled during administration. On day 5, animals were sacrificed 6h after each group was dosed, the effect of the drug on the gastrointestinal tract of rats was observed, compared and the correlation index was calculated as described above. And analyzing whether the gastric ulcer indexes have significant difference among groups by adopting a t test. The results of the experiment are shown in FIG. 5.

The results in fig. 5 show that the index of gastric ulcer in GA and GT groups is very small and significantly different (P <0.01) compared to diclofenac group. Meanwhile, in the experimental process, rats in the diclofenac group have the symptoms that the abdomen is swollen, and yellow or even brown ascites flows out of the cut abdominal cavity. The mucosa covering the abdominal cavity becomes hard, the gastric mucosa has little elasticity and erosion, and some rats can not eat food in the experimental process. The two experiments show that no obvious linear gastrointestinal hemorrhagic injury is found in the test object no matter single administration or continuous administration, and the stomach injury caused by the diclofenac in the test process is very obvious, which indicates that the gastrointestinal safety of the test object is very good.

Claims

1. A compound having the following general formula, an isomer thereof or a pharmaceutically acceptable salt thereof,

A-Y-X

wherein the content of the first and second substances,

a is gallic acid;

2. The compound, its isomers or pharmaceutically acceptable salts thereof according to claim 1, wherein a is

3. The compound, its isomers or pharmaceutically acceptable salts thereof according to claim 1, wherein X is

4. The compound, isomer, or pharmaceutically acceptable salt according to claim 1, wherein the compound is represented by formula GT:

5. the process for preparing a compound, an isomer thereof, or a pharmaceutically acceptable salt thereof according to claim 4, comprising the steps of: carrying out nucleophilic substitution reaction on ADT-OH and TAGC in the presence of an acid-binding agent, and purifying to obtain TAGT; partially hydrolyzing TAGT to obtain GT;

6. a pharmaceutical composition comprising a therapeutically effective amount of a compound of any one of claims 1 to 4, an isomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier or adjuvant.

7. Use of the compound, isomer or pharmaceutically acceptable salt thereof according to any of claims 1 to 4 or the pharmaceutical composition according to claim 6 for preparing a medicament for preventing or treating tumor.

8. Use of the compound, isomer or pharmaceutically acceptable salt thereof according to any one of claims 1-4 or the pharmaceutical composition according to claim 6 in preparation of a medicament for treating pain or antipyretic, analgesic and anti-inflammatory drugs.

9. Use of a compound according to any one of claims 1 to 4, an isomer thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 6 for the preparation of a medicament for the treatment of cardiovascular and cerebrovascular diseases.

10. Use of a compound according to any one of claims 1 to 4, an isomer thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 6 for the manufacture of a medicament for the treatment of neurological diseases.