CN116640081A

CN116640081A - Adamantane sulfonamide compound as well as preparation method and application thereof

Info

Publication number: CN116640081A
Application number: CN202310613837.2A
Authority: CN
Inventors: 胡湘南; 刘颖菊; 王灰飞; 闫丽萍; 刘海林; 吴齐红; 蒋雪; 张明; 刘科江; 张豪
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2016-09-12
Filing date: 2016-09-12
Publication date: 2023-08-25
Also published as: CN106432025A; CN106432025B

Abstract

The application relates to adamantane sulfonamide compounds, a preparation method and application thereof. The adamantane sulfonamide compound of the application has the following structureThe structure of the compound shown in the formula (I). The preparation method of the adamantane sulfonamide compound is simple. Animal experiments show that the adamantane sulfonamide compound can obviously inhibit proliferation of cultured macrophages and microglia and produce inflammatory factors; meanwhile, the composition has obvious protection effect on endotoxin-induced lung injury of mice, so that inflammatory reaction is reduced, and injury is reduced; the symptoms and joint injury of rat arthritis induced by the Freund's adjuvant are reduced, the weight of rat cotton bud granuloma is reduced, and the auricle swelling caused by the mouse xylene is reduced.

Description

Adamantane sulfonamide compound as well as preparation method and application thereof

The application relates to an adamantane sulfonamide compound with application number of 201610818842.7 and the application name of the adamantane sulfonamide compound and a divisional application of an application patent application of preparation and application of the adamantane sulfonamide compound.

Technical Field

The application relates to the fields of pharmaceutical chemistry and pharmacotherapeutics, in particular to adamantane sulfonamide compounds, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof, a preparation method and application.

Background

The inflammatory reaction is a basic pathological process generated by the body on the damage of tissues caused by various inflammatory stimuli, is also an important defense reaction of the body on the damage or infection, can help the body to remove harmful substances, localize the damage and start the healing process to repair the tissues, thereby playing a role in protection. However, excessive and persistent inflammatory reactions, which unbalance the anti-inflammatory and pro-inflammatory reactions, aggravate tissue cell damage and eventually may cause organ dysfunction. Inflammatory response is a common and multiple pathological process, and various diseases are closely related to inflammatory response, such as infectious diseases, autoimmune diseases, gout, neurodegenerative diseases, atherosclerosis, tumors and the like, and especially sepsis caused by infectious diseases, rheumatoid arthritis in autoimmune diseases, ankylosing spondylitis and the like are overactive or persistent of inflammatory response. Thus, anti-inflammatory drugs are also the second most general class of drugs in clinical treatment to be inferior to anti-infective drugs. Especially in the antirheumatic market, the evaluation pharma company reports that antirheumatic drugs are being marketed in the second largest therapeutic area, with global revenue reaching $411 billion in 2013 and still remaining strongly growing in the next 5 years. It is estimated that global non-steroidal anti-inflammatory drugs have been prescribed in 1 million in 1986, non-prescription drugs are more common and have a year-by-year trend of increasing, with celecoxib sold in 2013 at $27 million. While the usage rate of the steroid anti-inflammatory glucocorticoid in the hospital outpatient prescription in China is different, but is more than 12 percent; there are studies reporting that glucocorticoid application history for hospitalized patients in 2014, month 4 of a certain hospital accounts for 21.8% of the total discharge history of the month. However, both of these drugs have their respective adverse reactions. The steroid anti-inflammatory drug has complex actions on organisms, can cause the decline of the body defense function while generating strong anti-inflammatory action, induces or aggravates infection, and can cause the metabolism of human body substances and the metabolic disturbance of water and salt, induce aggravation of peptic ulcer, cause osteoporosis and the like after long-term use, even generate serious complications. The nonsteroidal anti-inflammatory drug also has adverse reactions such as gastrointestinal tract injury hemorrhage, liver and kidney function injury, increased incidence rate of cardiovascular adverse events and the like. Therefore, searching for new anti-inflammatory drugs with strong anti-inflammatory effect and small adverse reaction becomes a focus of study of domestic and foreign scholars. The recently proposed biological agents for treating inflammatory diseases such as rheumatoid arthritis, such as humira (adalimumab), enbrel (etanercept) and the like, become the most popular medicines worldwide, and obtain great profits while achieving remarkable clinical effects, such as 140 billions of dollars in the sales of humira 2015, but the medicines are expensive (humira sells 1840 mg in U.S. with the highest retail price of 7900 yuan per minute and is injected 1 per minute every 2 weeks) and are a heavy burden to medical insurance and patients. While the first oral JAK inhibitor in recent years, xeljanz (tofacitinib), which was proposed by pyroxene, was sold in U.S. at dollars 3.51 in 9 months of 2015, the drug was still expensive, and was in U.S. at dollars 3374/month (5 mg/tablet, 2 times daily).

Disclosure of Invention

The application aims at adamantane sulfonamide compounds shown in the following formula and the preparation and anti-inflammatory effects, immunosuppressive effects and therapeutic applications in inflammatory and inflammation-related diseases, such as autoimmune diseases (rheumatism, rheumatoid arthritis, ankylosing spondylitis, lupus erythematosus, crohn's disease, psoriasis, juvenile idiopathic arthritis, uveitis and the like), infectious diseases (sepsis, septicemia and the like), neurodegenerative diseases (multiple sclerosis, senile dementia, parkinsonism and the like), gout, transplant immune rejection and atherosclerosis and tumor treatment.

In the above formula, A may be substituted at the 2,3,4 positions of the adamantane ring, A may be H, X (F, cl, br, I), OH, OR, NH ₂ ，NHR，NR ₂ ，CHO，COR，COOH，COOR，CONH ₂ ，CONHR，CONR ₂ Commonly used organic groups such as COOCOR; b can be H, R, ar and other common organic groups; c may be a common organic group such as R, ar, etc.

Further, A may be OH, NH ₂ ，COR，COOH，CONH ₂ Etc., B may be (CH) ₂ ) _n CONH ₂ ，(CH ₂ ) _n CONHR，(CH ₂ ) _n CONR ₂ And the like (n is 1,2,3,4, etc.), and C may specifically be Me, et, n-Pr, i-Pr, n-Bu, i-Bu, s-Bu, t-Bu, ph, bn, etc.

Further, A is more specifically OH, NH ₂ COOH, etc., B is more specifically CH ₂ CONH ₂ ，CH ₂ CONHR，CH ₂ CONR ₂ And the like, and C is more specifically Me, et, ph and the like.

A specific adamantane sulfonamide example CQMUH-011 can be described as, but is not limited to, wherein A is substituted at the 3-position of the adamantane ring, A is OH, B

Is thatC is->

The following provides a process for producing adamantane sulfonamide compounds.

Using amantadine hydrochloride as a starting material, and using mixed acid and potassium hydroxide as reagents to carry out hydroxylation on the 3-position of an amantadine ring to obtain amantadine; and reacting the adamantane amino alcohol with halogenated B to prepare the adamantane amino alcohol substituted by the amino B, and then continuously reacting with p-toluenesulfonyl chloride to prepare the target adamantane sulfonamide compound. And other corresponding alternative preparation methods which can be carried out by the expert knowledge of the person skilled in the art.

The study of the application finds that the adamantane sulfonamide compound can be used for inflammatory reaction, the action mechanism possibly involves a plurality of targets, and the study of the application shows that the adamantane sulfonamide compound can obviously inhibit proliferation of cultured macrophages and microglial cells and produce inflammatory factors; meanwhile, the composition has obvious protection effect on endotoxin-induced lung injury of mice, so that inflammatory reaction is reduced, and injury is reduced; the symptoms and joint injury of rat arthritis induced by the Freund's adjuvant are reduced, the weight of rat cotton bud granuloma is reduced, and the auricle swelling caused by the mouse xylene is reduced. Furthermore, toxicity tests showed that mice were not seen to die and organ damage at 40-fold dose of the effective dose. The above results demonstrate that the adamantane sulfonamide compounds we found have significant anti-inflammatory effects with low toxicity.

The adamantane sulfonamide compound has anti-inflammatory effect and immunosuppression effect, and can be applied to treatment of inflammatory and inflammation-related diseases, such as autoimmune diseases (rheumatism, rheumatoid arthritis, ankylosing spondylitis, lupus erythematosus, crohn disease, psoriasis, juvenile idiopathic arthritis, uveitis and the like), infectious diseases (sepsis, septicemia and the like), neurodegenerative diseases (multiple sclerosis, senile dementia, parkinsonism and the like), gout, transplant immune rejection and atherosclerosis and tumor treatment.

Still another object of the present application is to provide a pharmaceutical composition comprising an effective amount of the above adamantane sulfonamide compound, and various dosage forms and compositions thereof, each of which is formulated with a pharmaceutically acceptable carrier or excipient.

Drawings

FIG. 1 is a graph showing the effect of CQMUH-011 on LPS-stimulated microglial proliferation (IC with CQMUH-011 action for 48h ₅₀ The values are:

1.305×10-8mol/L ^-1 )；

FIG. 2 is a graph showing the effect of various concentrations of CQMUH-011 on LPS-stimulated macrophage RAW264.7 proliferation;

FIG. 3 is a graph showing the effect of various concentrations of CQMUH-011 on LPS-stimulated macrophage RAW264.7 secretion of TNF- α and IL-1β;

FIG. 4 is a graph showing the effect of CQMUH011 on pathological changes in LPS-induced lung tissue (HE staining, ×200 times) (A: normal group, B: LPS group, C: DXM+LPS group, D: CQMUH-011+LPS group);

FIG. 5 is a graph showing the effect of CQMUH-011 on wet/dry specific gravity of LPS-induced lung injury in mice;

FIG. 6 is a graph showing the effect of CQMUH-011 on inflammatory factors TNF- α and IL-6 in serum of LPS-induced lung injury mice;

FIG. 7 is a graph showing the effect of CQMUH-011 on MDA content and MPO activity in lung tissue of mice with LPS-induced lung injury;

FIG. 8 is a graph showing the effect of CQMUH011 on the pathological changes of ankle joint tissue in Freund's adjuvant-induced arthritis rats (. Times.40) (A: normal group; B: AAModel group; C: DXM+Model group; D: CQMUH-011+Model group);

FIG. 9 is a graph of the effect of CQMUH-011 on the extent of perifoot swelling in Freund's adjuvant arthritic rats;

FIG. 10 is a graph of the effect of CQMUH-011 on the degree of perifoot swelling in Freund's adjuvant arthritic rats (14D) (A: normal group; B: AAModel group; C: DXM+Model group; D: CQMUH-011+Model group);

FIG. 11 is a graph showing the effect of CQMUH-011 on inflammatory factors TNF- α and IL-6 in serum of Freund's adjuvant arthritic rats;

FIG. 12 is a graph showing the effect of CQMUH-011 on MDA content and MPO activity in serum of Freund's adjuvant arthritic rats;

FIG. 13 is a graph of the effect of CQMUH-011 on dry weight of granuloma of cotton ball in rats;

FIG. 14 is a graph showing the effect of CQMUH-011 on NO and MDA and MPO in serum of cotton bud granulomatous rats;

FIG. 15 is a graph showing the effect of CQMUH-011 on xylene induced ear swelling in mice.

Detailed Description

The application will be further understood by the following specific examples, which are not to be construed as limiting the scope of the application. Extension extensions available to the expert knowledge of the present application to those skilled in the art given the above-described premise of the present application should be within the scope of the claimed application.

Example 1

Synthesis of adamantane sulfonamide Compound CQMUH-011:

38ml of 98% concentrated sulfuric acid and 4ml of 65% concentrated nitric acid are mixed and cooled in an ice bath, 3.75 g of amantadine hydrochloride is added to the mixed acid in portions, and the mixed acid is stirred in the ice bath for reaction for 3 hours; 60 g of crushed ice is added and stirred for 0.5 hour; adding potassium hydroxide solid, regulating the pH to 12-13, and stirring in ice bath for reaction for 2 hours; suction filtering, regulating pH of the filtrate to 8-9 with concentrated hydrochloric acid, and concentrating to dryness; adding 80ml of absolute ethyl alcohol, refluxing for 1 hour, cooling, filtering, and concentrating the filtrate to dryness; 10ml of the mixture (acetone: ethyl acetate=3:1) was added and refluxed for 1 hour, and the mixture was left under ice bath to give 3 g of 3-aminoadamantanol as a solid with a melting point of more than 256 ℃.

3 g of 3-aminoadamantanol, 0.25 g of potassium iodide and 10g of potassium carbonate are added to 30ml of tetrahydrofuran, the temperature is raised to 40 ℃ with stirring, and a solution of 3 g of N-chloroacetyl-2-cyanotetrahydropyrrole (i.e. chloro B) dissolved in 30ml of tetrahydrofuran is slowly added dropwise at the temperature for about 1.5 hours. After the dripping is finished, the temperature is kept at 40 ℃ for reaction for 1 hour, and then the temperature is raised to reflux for reaction for 2 hours. At the end of the reflux, the hot filtrate was filtered, the filter cake was washed with a small amount of tetrahydrofuran, the filtrates were combined and concentrated to an oil. Adding a proper amount of butanone into the oily matter for dissolution, standing for crystallization, filtering and drying to obtain 4 g of amantadine amino alcohol solid substituted by amino B, wherein the melting point is 147-149 ℃. The purity of the product is 98.1% by HPLC normalization, and the structural characteristics such as IR, NMR and the like meet the requirements.

Into a reaction flask, 1 g of the above-mentioned amino-B-substituted adamantanol, an appropriate amount of p-toluenesulfonyl chloride, an appropriate amount of pyridine and 20ml of tetrahydrofuran were added, stirred and slowly warmed to reflux, and reacted for 20 hours. After the completion of the reaction, 20ml of a 5% sodium carbonate solution was added to the reaction mixture, followed by addition of 40ml of ethyl acetate, and the mixture was poured into a separating funnel, and an organic layer was collected. The aqueous layer was extracted twice with 50ml of ethyl acetate. The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and after drying, the organic solvent was removed by rotary evaporation to give a crude white solid. Purifying by column chromatography, and recrystallizing with acetone to obtain adamantane sulfonamide compound CQMUH-011 solid with melting point of 192-195 deg.C. The purity of the product is 95.1% by HPLC normalization, and the structural characteristics such as IR, NMR and the like meet the requirements.

Example 2

In vitro cell experiment of adamantane sulfonamide CQMUH-011

CQMUH-011 with different concentrations is added into the cultured primary microglia and macrophage RAW264.7, the influence of the CQMUH-011 on the proliferation and inflammatory factor production of the cultured microglia and macrophages after LPS stimulation is observed, and the result shows that the CQMUH-011 has obvious inhibition effect on the proliferation and inflammatory factor production of the microglia and macrophages caused by LPS stimulation

(see FIG. 1. FIG. 2. FIG. 3. Table 1)

TABLE 1 influence of CQMUH-011 on microglial TNF-alpha and IL-1 beta production

Example 3

In vivo anti-inflammatory experiment of adamantane sulfonamide CQMUH-011

1. Protection of lipopolysaccharide-induced lung injury in mice by CQMUH-011

The purpose is as follows: the inhibitory effect of CQMUH-011 on sepsis was evaluated.

Dosing regimen and experimental procedure: balb/c male mice were weighed and numbered. The animals were randomly divided into a normal group, a Lipopolysaccharide (LPS) group, a Dexamethasone (DXM) +LPS group and a CQMUH-011+LPS group, each group comprising 10 mice. LPS group mice were intraperitoneally injected with LPS (20 mg/kg), normal group mice were intraperitoneally injected with an equal dose of physiological saline, and each group of mice was administered physiological saline, dexamethasone solution (1.5 mg/kg), and novel CQMUH-011 solution (457 μg/kg) (all solutions contained 4%1, 2-propanediol) 2 hours before modeling. Half of mice selected from each group were collected from eyeballs and bled 6 hours after modeling, serum was isolated (3000 r,4 ℃ C., centrifuged for 10 min), and TNF- α and IL-6 values were measured according to the kit procedure; the remaining mice were bled 12 hours later, and a portion of their right lung was excised and fixed with 4% paraformaldehyde, the remaining right lung was examined for wet dry weight of the lung, a portion of the left lung was homogenized with cold saline for Malondialdehyde (MDA) and Myeloperoxidase (MPO) detection, and the remaining left lung was stored at-80℃for use.

1. Tissue observation: 4% paraformaldehyde fixed specimens were embedded in paraffin, sectioned, and observed for morphological changes under an optical microscope after HE staining.

2. W/D value determination of lung: the remaining right lung of the mice was taken, surface fluid was gently blotted with absorbent paper and weighed with a ten-thousandth balance and each sample reading was recorded. Then wrapping with tinfoil paper, drying in a drying oven at 60 ℃ for 72 hours, taking out, weighing again, and recording. The ratio of change in dry-wet weight of lung tissue was calculated as an index for evaluating the degree of pulmonary edema.

3. Detecting inflammatory factors: the isolated sera were assayed for TNF- α and IL-6 levels using a corresponding enzyme-linked immunosorbent assay (ELISA). The specific operation method is as follows:

(1) Taking out the kit from the refrigerator, balancing at room temperature for 20min, taking out the required strips from the aluminum foil bag, and placing the rest strips back into the self-sealing bag for preservation at 4 ℃.

(2) Adding 50 mu L of standard substances with different concentrations into the standard substance holes; sample 10. Mu.L and sample diluent 40. Mu.L were added to the sample wells.

(3) Standard wells and sample wells were filled with 100 μl of HRP-labeled antibody, the reaction wells were blocked, and incubated for 60min at 37 ℃.

(4) The liquid was discarded and the plate was washed 5 times.

(5) Add 50. Mu.L of each of substrates A and B and incubate at 37℃for 15min in the dark.

(6) The OD of each well was measured at a wavelength of 450nm by adding 50. Mu.L of the stop solution for 15min.

The corresponding inflammatory factor concentration can be obtained from the OD value and the standard curve.

MDA content determination: centrifuging 10% tissue homogenate to obtain supernatant, measuring by referring to the corresponding detection kit instruction, and calculating according to the kit calculation method. The specific operation is as follows:

(1) The lung tissue homogenate protein content was determined, according to the instructions, as follows:

the supernatant was diluted 1% with physiological saline at 1:9 to give a 1% homogenate to be tested.

TABLE 2 Lung tissue protein determination procedure

Calculation formula of lung tissue homogenate supernatant protein content: protein content (μg/ml) = (determination tube OD value-blank tube OD value) ×standard concentration (563 μg/ml) ×dilution before sample testing/(standard tube OD value-blank tube OD value)

(2) The detection of MDA content in lung tissue homogenates is performed according to the specification of an MDA detection kit, and comprises the following steps: configuration of working solution I: reagent one: reagent two-use liquid: reagent three application liquid=a: 3:1, formulated as needed, and used the day after formulation. Configuration of working solution I: reagent one: reagent two-use liquid: reagent three application liquid=a: 3:1, formulated as needed, and used the day after formulation.

TABLE 3 MDA determination procedure for lung tissue

	Blank pipe	Standard tube	Measuring tube	Control tube
					10nmol/ml standard (ml)	a
Absolute ethyl alcohol (ml)	a
					Measuring sample (ml)		a	a
Working fluid I (ml)	4.0ml	4.0ml	4.0ml
					Working solution II (ml)			4.0ml

Mixing by a vortex mixer, carrying out water bath at 95 ℃ for 40 minutes, taking out, cooling by running water, centrifuging for 10 minutes at 3500-4000 rpm, taking supernatant, and measuring absorbance value of each tube at 532 nm.

Lung tissue homogenate supernatant MDA content calculation formula:

MDA content (nmol/mgprot) = (measurement tube OD value-control tube OD value)/(standard tube OD value-standard blank tube OD value) ×Standard concentration (10 nmol/ml)/sample protein content (mg prot/ml)

Mpo activity assay: taking 10% of non-centrifuged lung tissue homogenate, adding the reagent II according to the ratio of 1:1, and uniformly mixing to obtain 5% of lung tissue homogenate. Fully and uniformly mixing 5% of lung tissue homogenate and a third reagent according to the proportion of 9:1, carrying out water bath for 15min in a 37 ℃ water bath, taking out, and operating according to instructions, wherein the steps are as follows:

TABLE 4 operation procedure for MPO determination in lung tissue

MPO activity (U/g tissue) = (assay tube OD value-control tube OD value)/11.3 x sample size (g)

Experimental results:

the CQMUH-011 can obviously relieve the lung injury of mice caused by LPS, reduce the wet/dry specific gravity of the lung, reduce the inflammatory factors TNF-alpha and IL-6 in the serum of the mice and reduce the MDA content and the MPO activity in the lung tissues of the mice (see figure 4, figure 5, figure 6 and figure 7), and the novel medicine CQMUH-O11 has a certain protection effect on acute lung injury of mice caused by LPS. 2. Protection of Freund's complete adjuvant induced arthritis in rats by CQMUH-011: the protective effect of CQMUH-011 on immune inflammation was evaluated.

Dosing regimen and experimental method: male SD rats weighing 180+ -20 g were selected and randomly grouped into a normal group, an AA model group, a dexamethasone+AA group and a CQMUH-011 (300 μg/kg) +AA group, and the right hind foot plantar skin of each rat except the normal group was injected with 0.1ml FCA to cause inflammation. 2 hours prior to inflammation, each group was given the corresponding drug, normal and AA model groups were given normal saline, dexamethasone+AA group was given dexamethasone solution, and CQMUH-011+AA group was given CQMUH-011 solution (all containing 4%1, 2-propanediol). The swelling degree and weight change of the right hind foot of each group of rats were measured 18 hours after inflammatory; starting administration after one week of inflammation, and observing the swelling degree and weight change of the right hind feet of each group of rats for 7 days continuously; the drug was administered again 21 days after the inflammatory process, and the change in the swelling degree and weight of the right hind foot was observed for each group of rats for 7 consecutive days. After 28 days of inflammation, blood is obtained from the operation of taking out eyeballs from the rat to obtain serum for detecting inflammatory factors, and the right hind ankle joint of the rat is taken out and fixed in 10% paraformaldehyde.

1. Ankle joint pathological section observation: samples fixed with 10% paraformaldehyde were decalcified with 8% nitric acid, embedded in paraffin, sectioned, and HE stained for morphological changes under an optical microscope.

2. Rat right hind foot swelling degree determination: the circumference of the same portion of the right hindfoot of each rat was measured with a tape for 18h, 7d, 14d, 21d, and 28d before and after the inflammation.

3. TNF- α and IL-6 assays in rat serum: the detection is carried out by using corresponding ELISA kits respectively, and the specific operation steps are as follows

(1) Preparation: and (5) placing the kit at room temperature, and placing the redundant ELISA strips back to the refrigerator. Standard substance solution preparation: 1mL of the standard diluent was added to each bottle of standard, and after capping, the mixture was allowed to stand at room temperature for about 10 minutes while the mixture was repeatedly inverted to aid dissolution, at a concentration of 200pg/mL (stock). After the sample was diluted to 100pg/mL (standard curve highest concentration), 7 EP tubes for diluting the standard were prepared, and 500. Mu.L of standard diluent was added to each EP tube, and the dilution was sequentially performed at a ratio of 100pg/mL, 50pg/mL, 25pg/mL, 12.5pg/mL, 6.25pg/mL, 3.12pg/mL, 1.56pg/mL, and standard diluent (0 pg/mL) as blank wells. And (5) standby.

(2) Sample detection:

1) Sample adding: and respectively arranging a standard hole, a sample hole to be tested and a blank hole. A standard well 7 was set and 100. Mu.l of standards of different concentrations (as indicated above) were added sequentially. 100 mu L of standard diluent is added to the blank hole, 100 mu L of sample to be detected is added to the Yu Kongjia, the ELISA plate is added with a coating film, and the incubation is carried out for 2 hours at 37 ℃.

2) Discard the liquid, spin-dry, and do not need washing.

3) 100 mu L of the working solution of the detection solution A (prepared before use) is added to each well, and the ELISA plate is covered with a film and incubated for 1 hour at 37 ℃.

4) Removing liquid in the holes, washing with 350 mu L of washing liquid in each hole, soaking for 1-2 minutes, spin-drying the liquid in the ELISA plate, and repeatedly washing for 3 times by beating the ELISA plate with water-absorbing paper. And the liquid in the holes is completely dried after the last washing.

5) 100 mu L of working solution (prepared before use) of detection solution B is added to each well, and an ELISA plate is coated with a film and incubated for 30 minutes at 37 ℃.

6) Discarding the liquid, spin-drying, washing the plate for 5 times, and the method is the same as that in the step 4.

7) Each well was filled with 90. Mu.L of the primer solution, the ELISA plate was covered with a film, and developed in a dark place at 37℃for 15-25 minutes.

8) The reaction was terminated by adding 50. Mu.L of the termination solution to each well, and the optical density of each well was measured at a wavelength of 450nm using an ELISA reader.

4. Determination of MDA content in rat serum: the procedure is as for the first part according to the kit of parts.

5. Determination of MPO viability in rat serum: according to the steps of the kit, the specific operation is as follows

And adding the second reagent into the rat serum according to the ratio of 1:1, and fully and uniformly mixing. Adding the solution and the reagent III according to the ratio of 9:1 after uniformly mixing, fully uniformly mixing, carrying out water bath in a 37 ℃ water bath for 15min, taking out, and operating according to instructions, wherein the steps are as follows:

TABLE 5 operation procedure for determining MPO Activity in rat serum

MPO activity (U/L) = (measurement tube OD value-control tube OD value)/11.3 x sample size (L)

Experimental results: CQMUH-011 has protective effect on the pathological injury of rat arthritis ankle joint caused by Freund's adjuvant, reduces the perifoot swelling degree of the arthritis rat, reduces the inflammatory factors (TNF-alpha and IL-6) and MDA level and MPO activity in serum (see figure 8, figure 9, figure 10 and figure 11), and has inhibitory (treatment) effect on the immune arthritis.

3. Inhibition effect of CQMUH-011 on rat cotton granuloma: evaluation of the inhibition of subacute inflammation by CQMUH-011

Dosing regimen and experimental method: male SD rats weighing 150+ -10 g were selected, weighed and numbered, and randomly grouped into normal, model, dexamethasone, and CQMUH-011 (300 μg/kg) groups, respectively. Except for the normal group, each group of rats was cut with a small opening at each of the left and right parts under anesthesia, the subcutaneous tissue was expanded with vascular forceps, and a sterilized cotton ball (weight 10.+ -. 1 mg) was buried in each side, followed by suturing. 2 hours prior to surgery, i.e. normal and model groups with normal saline, dexamethasone with dexamethasone solution, CQMUH-011 with the new drug solution (all drugs contain 4%1, 2-propanediol); the animals were then sacrificed on day 7 and day 8 for one daily dosing, whole blood was obtained, serum was isolated (3000 r,4 ℃, 10min centrifugation) and cotton balls were removed to remove adipose tissue for use.

1. Rat cotton ball granuloma weight determination: wrapping the obtained cotton ball with tinfoil paper, drying in a 60 ℃ oven for 48 hours, taking out the dried cotton ball, weighing, and subtracting the weight of the cotton ball, namely the granuloma weight, and expressing the granuloma weight as mg/100g body weight.

2. Determination of NO in rat serum: according to the steps of the kit, the specific operation is as follows

(1) The kit was removed and Griess Reagent I and Griess Reagent II were allowed to return to room temperature.

(2) Standard preparation with different concentrations: the standard was diluted with physiological saline to 9 concentrations of 0, 1,2, 5, 10, 20, 40, 60, 100 μm, respectively.

(3) Standard and test samples were added to 96-well plates at 50 μl/well.

(4) At 50 μl/well, room temperature Griess Reagent I was added to each well.

(5) At 50. Mu.l/well, room temperature Griess Reagent II was added to each well.

(6) The absorbance was measured at 540nm wavelength.

The NO concentration can be obtained from the absorbance and standard curve.

3. Determination of MDA content in rat serum: the procedure is as for the first part according to the kit of parts.

4. Determination of MPO viability in rat serum: the procedure is as for the second part according to the kit procedure.

Experimental results: CQMUH-011 can obviously reduce the dry weight of cotton ball granuloma of rats, reduce the levels of NO and MDA in serum of the granuloma rats and the activity of MPO (see figure 12 and figure 13), and the novel medicine CQMUH-011 has a certain inhibition effect on subacute inflammation.

4. Protection of mouse otoxylene inflammation by CQMUH-011: evaluation of the inhibitory Effect of CQMUH-011 on acute inflammation

Dosing regimen and experimental method: about 25g of male mice are taken and randomly grouped into a normal group, a model group, a dexamethasone group and a CQMUH-011 (457 mu g/kg) group, 10 mice in each group are respectively dripped with 0.03-0.05 ml of dimethylbenzene in the right ear of the mouse except the normal group, and the left ear is not treated. Each group was given the corresponding drug 2 hours before the inflammatory, i.e., normal and model groups were given physiological saline, dexamethasone was given dexamethasone solution, and CQMUH-011 group was given the new drug solution. After 2 hours of inflammation, the mice were sacrificed, two ears were cut off along the auditory canal base line, round ear pieces were punched on the same parts of the left and right ears with a punch having a diameter of 5mm, and the difference between the weights of the left and right ear pieces was weighed to obtain the swelling degree.

Experimental results: CQMUH-011 can significantly reduce ear swelling in mice caused by xylene, resulting in reduced ear swelling (see FIG. 14). The novel medicine CQMUH-011 has a certain inhibiting effect on acute inflammation.

5. Acute toxicity test

CQMUH-011 was given to mice at a therapeutic dose (457. Mu.g/kg) 10 times (457. Mu.g/kg), 20 times (9142. Mu.g/kg), and 40 times (18284. Mu.g/kg) as much as the therapeutic dose, and the mice were intraperitoneally injected, and after 2 weeks of observation, only 40 times of the administration had sleepiness within 2 hours after the administration, and then recovered to normal. All mice did not die within 2 weeks, were psychoactive, and were pathologically dissected without abnormalities in various organs.

Claims

1. An adamantane sulfonamide compound represented by the following formula, which is characterized in that,

wherein A is substituted at the 2,3 OR 4 position of the adamantane ring, A is H, F, cl, br, I, OH, OR, NH ₂ ，NHR，NR ₂ ，CHO，COR，COOH，COOR，CONH ₂ ，CONHR，CONR ₂ Or COOCOR; b is H, R or Ar; c is R or Ar.

2. A compound according to claim 1, wherein: the A is OH, NH ₂ ，COR，COOH，CONH ₂ The method comprises the steps of carrying out a first treatment on the surface of the B is (CH) ₂ ) _n CONH ₂ ，(CH ₂ ) _n CONHR，(CH ₂ ) _n CONR ₂ The method comprises the steps of carrying out a first treatment on the surface of the n is 1,2,3 or 4; c is Me, et, n-Pr, i-Pr, n-Bu, i-Bu, s-Bu, t-Bu, ph or Bn.

3. A compound according to claim 1, wherein: a is OH, NH ₂ Or COOH; b is CH ₂ CONH ₂ ，CH ₂ CONHR or CH ₂ CONR ₂ C is Me, et or Ph.

4. A compound according to claim 1, wherein: b is

5. A compound according to claim 1, wherein: c is

6. Use of an adamantane sulfonamide compound of any one of claims 1-5 in the manufacture of an anti-inflammatory medicament; the application in preparing medicines for preventing or treating immunosuppression; application in preparing medicines for preventing or treating autoimmune diseases; the application in preparing medicines for preventing or treating infectious diseases; application in preparing medicines for preventing or treating neurodegenerative diseases; application in preparing medicine for preventing or treating gout; application in preparing medicines for preventing or treating transplant rejection; application in preparing medicines for preventing or treating tumor.

7. The use according to claim 6, wherein the autoimmune disease is selected from the group consisting of rheumatism, rheumatoid arthritis, ankylosing spondylitis, lupus erythematosus, crohn's disease, psoriasis, juvenile idiopathic arthritis, or uveitis; the infectious disease is selected from sepsis or sepsis; the neurodegenerative disease is selected from multiple sclerosis, senile dementia or parkinson's disease.

8. A pharmaceutical composition comprising an adamantane sulfonamide compound of any one of claims 1-5, and a pharmaceutically acceptable carrier.

9. The process for producing adamantane sulfonamide compound according to any one of claims 1 to 5, wherein adamantanol is reacted with halogeno B to produce an amino B-substituted adamantanol, which is then reacted with p-toluenesulfonyl chloride to produce the objective adamantane sulfonamide compound.

10. The method of claim 9, wherein: the halogenated B is N-chloroacetyl-2-cyano tetrahydropyrrole.