As (III)-containing compound as well as preparation method and application thereof

Abstract

The invention discloses an As (III)-containing compound as well as a preparation method and application thereof. The chemical formula of the As (III)-containing compound disclosed by the invention is as shown in a formula (I): # imgabs0 #, wherein an R group is selected from one of C1-C10 alkyl groups or hydroxyl groups. Tests find that the As (III) compound can relieve and / or improve body weight change, disease activity index increase, colon shortening and intestinal tract pathological injury caused by inflammatory bowel diseases, has small toxic and side effects, and can provide a new strategy for developing medicines related to treatment of enteritis and intestinal cancer.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an As(III)-containing compound and a preparation method and application thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory disease, mainly including ulcerative colitis and Crohn's disease. Its etiology is complex and involves multiple factors such as genetics, environment, and immune response. The treatment of IBD mainly focuses on controlling inflammation and relieving symptoms, but there are problems such as individual differences, uncertainty in treatment response, and potential side effects. Current treatment drugs include aminosalicylic acid drugs, glucocorticoids, immunosuppressants, and anti-inflammatory factor monoclonal antibodies. Among them, the use of anti-tumor necrosis factor (TNF) antibody drugs is considered to be one of the most successful IBD treatment options to date. However, clinical studies have shown that it still has certain adverse effects during treatment. For example, after long-term use of anti-TNF therapy, some patients may develop drug resistance, resulting in a decrease in treatment efficacy.

[0003] Related studies have shown that NOD2 (Nucleotide-binding oligomerization domain 2) is an important cytoplasmic pattern recognition receptor that recognizes muramyl dipeptide (MDP) in bacteria. Overactive NOD2 signaling is associated with a variety of inflammatory diseases, such as early-onset sarcoidosis, Blau syndrome, inflammatory arthritis, allergic inflammation, and multiple sclerosis, including IBD. Therefore, targeted inhibition of NOD2 signaling has become a potential therapeutic strategy for some inflammatory diseases. NOD2 signaling depends on XIAP protein (X-linked Inhibitor of Apoptosis Protein). During NOD2 signaling, the BIR2 (Baculovirus IAP Repeat 2) domain of XIAP protein binds to the kinase domain of RIPK2, and ubiquitinates RIPK2 through the RING (Really Interesting New Gene) domain of XIAP protein. The binding and ubiquitination modification of RIPK2 by XIAP protein is a necessary step for NOD2 signaling. Therefore, targeting XIAP can inhibit the NOD2 signaling pathway, and XIAP inhibitors may become a new strategy for treating inflammatory diseases mediated by immune pathway disorders.

[0004] Based on this, the present invention aims to explore new IBD treatment strategies to provide patients with more options, thereby improving the treatment effects of related diseases and improving their quality of life. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides an As(III)-containing compound (hereinafter referred to as DMA-NAC), which can be used to treat inflammatory bowel disease.

[0006] The invention also provides a method for preparing the As(III)-containing compound.

[0007] The present invention also proposes the use of the above-mentioned As(III)-containing compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating inflammatory bowel disease or intestinal cancer.

[0008] The present invention also provides a drug for alleviating and / or treating inflammatory bowel disease.

[0009] In a first aspect of the present invention, there is provided an As(III)-containing compound, the chemical formula of which is shown in formula (I):

[0010]

[0011] In the formula, the R group is selected from one of a C1-C10 alkyl group or a hydroxyl group.

[0012] The As(III)-containing compound according to the embodiment of the present invention has at least the following beneficial effects:

[0013] The As(III) compound of the present invention can reduce the severity of ulcerative colitis induced by dextran sulfate sodium (DSS) through the NOD2 signaling pathway, can be used to alleviate and / or improve weight changes, increased disease activity index, colon shortening and intestinal pathological damage caused by inflammatory bowel disease, and has less toxic side effects than inorganic arsenic, and can provide a new strategy for the development of drugs related to the treatment of enteritis and intestinal cancer.

[0014] In some embodiments of the present invention, the R group is selected from one of C1 to C5 alkyl groups.

[0015] In some embodiments of the present invention, the R group is a methyl group, and the chemical structure of the As(III)-containing compound is shown in Formula I-1:

[0016]

[0017] The second aspect of the present invention provides a method for preparing the above-mentioned As(III)-containing compound, comprising the following steps:

[0018] In a solvent, acetylcysteine ​​is mixed with sodium cacodylate, reacted, and separated and purified to obtain the product.

[0019] The preparation method according to the embodiment of the present invention has at least the following beneficial effects: the preparation method of the present invention is simple, the raw materials are easily available, and the cost is low, and it can be used for industrial production.

[0020] In some embodiments of the invention, the solvent comprises water.

[0021] In some embodiments of the present invention, the separation and purification comprises separation by column chromatography.

[0022] The third aspect of the present invention provides use of the As(III)-containing compound or a pharmaceutically acceptable salt thereof as described in the first aspect in the preparation of a medicament for alleviating and / or treating inflammatory bowel disease.

[0023] The application of the embodiments of the present invention has at least the following beneficial effects:

[0024] (1) The As(III) compound of the present invention or a pharmaceutically acceptable salt thereof is used as an active substance to prepare a drug for treating inflammatory bowel disease or intestinal cancer, which can not only alleviate and / or improve weight changes, increased disease activity index, colon shortening and intestinal pathological damage caused by inflammatory bowel disease, but also has little toxic and side effects, and has good application prospects.

[0025] (2) The As(III) compound of the present invention chelates acetylcysteine, which can greatly reduce the damage to normal colon epithelial cells compared with conventional sodium arsenite, and has excellent therapeutic effect.

[0026] In some embodiments of the present invention, the drug for alleviating and / or treating inflammatory bowel disease has any of the following effects:

[0027] a1) Alleviate and / or improve weight changes caused by inflammatory bowel disease;

[0028] a2) Alleviate and / or improve the increase in disease activity index caused by inflammatory bowel disease;

[0029] a3) Relieve and / or improve colon shortening caused by inflammatory bowel disease;

[0030] a4) Alleviate and / or improve intestinal pathological damage caused by inflammatory bowel disease.

[0031] In some embodiments of the present invention, the pharmaceutically acceptable salt of the As(III)-containing compound includes any one of hydrochloride, nitrate, acetate, methanesulfonate, phosphate, citrate, fumarate, sulfate, succinate, tartrate, citrate, hydrobromide, hydroiodide, lactate, toluenesulfonate, cinnamate, salicylate, malonate, glutarate, and malate.

[0032] The fourth aspect of the present invention provides a drug for alleviating and / or treating inflammatory bowel disease, wherein the active substance of the drug comprises the As(III)-containing compound or a pharmaceutically acceptable salt thereof as described in the first aspect.

[0033] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0034] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a solvent, an excipient, a diluent, a binder, a disintegrant, a dispersant, a flavoring agent, a suspending agent, a surfactant, an isotonic agent, a thickener, an emulsifier, a preservative, a glidant, and a lubricant.

[0035] In some embodiments of the present invention, the dosage form of the drug is any one of tablets, capsules, granules, injections, powder injections, eye drops, liniments, suppositories, ointments, and aerosols.

[0036] In some embodiments of the invention, the excipient comprises water.

[0037] In some embodiments of the present invention, the filler comprises at least one of starch and sucrose.

[0038] In some embodiments of the present invention, the binder includes at least one of a cellulose derivative, alginate, gelatin, and polyvinyl pyrrolidone.

[0039] In some embodiments of the invention, the humectant comprises glycerin.

[0040] In some embodiments of the present invention, the disintegrant comprises at least one of agar, calcium carbonate, and sodium bicarbonate.

[0041] In some embodiments of the invention, the absorption enhancer comprises a quaternary ammonium compound.

[0042] In some embodiments of the invention, the surfactant comprises cetyl alcohol.

[0043] In some embodiments of the present invention, the adsorption carrier includes at least one of kaolin and bentonite.

[0044] In some embodiments of the present invention, the lubricant includes at least one of talc, calcium stearate, magnesium stearate, and polyethylene glycol.

[0045] In some embodiments of the present invention, the dosage form of the drug is any conventional dosage form in the art.

[0046] In some embodiments of the present invention, the dosage form of the drug is in the form of solid, semisolid or liquid, and can be an aqueous solution, non-aqueous solution or suspension.

[0047] In some embodiments of the present invention, the dosage form of the drug is tablets, capsules, soft capsules, granules, pills, oral liquids, dry suspensions, pellets, dry extracts, injections, and infusions.

[0048] It is understood that the drug of the present invention can be introduced into the body (such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue) by oral administration, injection, spraying, penetration, absorption, physical or chemical mediation; or introduced into the body after being mixed or wrapped with other substances. The dosage can be changed according to the patient's condition or weight, disease severity, dosage form, administration route, administration cycle, and can also be determined by a person skilled in the art. For example, the dosage of the As(III) compound can be 0.01 to 100 mg / kg, which can be completed once or several times a day, or can be administered at intervals of multiple days.

[0049] In addition, in the present invention, the term "inflammatory bowel disease" is used to refer to a class of chronic nonspecific inflammatory diseases involving the intestine, including but not limited to Crohn's disease and ulcerative colitis. The clinical symptoms of Crohn's disease may include but are not limited to abdominal pain, diarrhea, blood in the stool, abdominal mass, systemic fever, accompanied by aphthous stomatitis, perianal fistula, abscess, ulcer, anal fissure, perianal stenosis, perianal skin tag, perianal hemorrhoids, endoscopic segmental longitudinal ulcers, cobblestone sign and intestinal stenosis. The clinical symptoms of ulcerative colitis may include but are not limited to diarrhea, blood in the stool, fever, anemia, malnutrition, water and electrolyte imbalance, hypoproteinemia, bone and joint lesions, skin and mucosal lesions, erythema nodosum, gangrenosum pyoderma, etc., and endoscopic biopsy observed inflammatory exudation, erosion and ulceration, neutrophil infiltration, crypt deformation, mucus loss, Paneth cell metaplasia, etc. in tissue samples.

[0050] Other features and advantages of the present invention will be set forth in the description which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0052] Figure 1 is the ESI-MS spectrum of the compound DMA-NAC of the present invention;

[0053] Figure 2 This is a statistical chart showing the effect of the compound DMA-NAC of the present invention on the body weight of mice with inflammatory bowel disease;

[0054] Figure 3 The statistical graphs of the disease activity index test results of each group of mice in the present invention;

[0055] Figure 4 The figure is a graph showing the changes in colon length of each group of mice in the present invention;

[0056] Figure 5 The colon tissue pathological sections of each group of mice in the present invention are shown;

[0057] Figure 6 The following are the safety test results of the compound DMA-NAC of the present invention. DETAILED DESCRIPTION

[0058] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0059] The words "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0060] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0061] In the description of the present invention, the reference term "and / or" includes all and any combinations of one or more of the associated listed items.

[0062] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0063] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0064] Example 1: Preparation of DMA-NAC

[0065] This embodiment provides N-acetyl-S-(dimethylarsinoyl)-L-cysteine ​​(hereinafter referred to as DMA-NAC) and a preparation method thereof, wherein the structural formula of the compound DMA-NAC of this embodiment is shown in Formula I-1:

[0066]

[0067] It specifically includes the following steps:

[0068] Dissolve 1 mmol of acetylcysteine ​​in 50 mL of deionized water. Add 1 mmol of sodium cacodylate to the above solution. Place the mixed solution under reflux conditions, which usually means heating the solution to boiling for a period of time (5 hours in this case) to promote the reaction. After the reaction is completed, remove the solution from the heat source and let it cool naturally to room temperature. Load the cooled solution onto a silica gel column. In this case, acetonitrile and deionized water in a ratio of 2:3 are used as eluents to elute the target compound through the silica gel column. Most of the acetonitrile solvent is removed by a rotary evaporator to obtain the target product.

[0069] The target product obtained above was subjected to electrospray ionization mass spectrometry (ESI-MS) analysis. The ESI-MS spectrum is as follows: Figure 1 Its related parameter information is shown in Table 1.

[0070] Table 1: ESI-MS spectrum related parameters

[0071] Meas.m / z Ion Formula m / z z err[ppm] mSigma rdb e-Conf N-Rule 267.9985 C7H15AsNO3S 267.9983 1+ 0.6 2.8 5 even OK

[0072] Based on the above analysis, it can be seen that the target product is DMA-NAC.

[0073] Example 2: Effects of DMA-NAC on Body Weight and Disease Activity Index in Mice with Inflammatory Bowel Disease

[0074] In this example, a relevant mouse model was constructed to detect the application of the above DMA-NAC in the treatment of inflammatory bowel disease. The specific method includes the following steps:

[0075] (1) Construction of inflammatory bowel disease model mice:

[0076] 6-8 week old male C57BL / 6 mice were selected and continuously fed with 5% dextran sulfate sodium (DSS) in pure water for 7 days to obtain dextran sulfate sodium model mice (i.e., inflammatory bowel disease mouse model). The criteria for identifying successful modeling were:

[0077] A) Disease Activity Index Score (DAI score): This score is assessed based on body weight, stool consistency, and fecal occult blood. For details of the score, please refer to Table 2.

[0078] Table 2

[0079]

[0080]

[0081] B) Histological change score: evaluated by observing the histological changes of DSS ulcerative colitis.

[0082] C) Colon length: In the acute colitis model, a reduction in colon length could be detected on day 8.

[0083] If common clinical enteritis-related symptoms appear, such as weight loss, loose stools, diarrhea, bloody stools or fecal occult blood, ulcers, etc., it can be considered that the DSS drug is effective and the model is successful.

[0084] The mice in the control group were fed with purified water for seven days.

[0085] (2) Drug administration:

[0086] The inflammatory bowel disease model mice and control group mice constructed above were divided into three groups: control group (CTR), model group (DSS) and drug administration group (DSS+DMA-NAC), among which:

[0087] ① Control group: control group mice, 6-8 weeks old, male, fed with purified water for seven days;

[0088] ②Model group: inflammatory bowel disease model mice, 6-8 weeks old, male;

[0089] ③Administration group: inflammatory bowel disease model mice, 6-8 weeks old, male.

[0090] The control group and model group mice were intraperitoneally injected with 0.5 mL of normal saline, and the drug group mice were intraperitoneally injected with 50 mg / kg of the compound DMA-NAC. The drug was administered once a day, and the weight changes, stool characteristics and blood in the stool of the mice were observed and recorded every day during the drug administration process, and the scores were calculated to calculate the disease activity index (DAI). There were 5 replicates in each group, and the values ​​were expressed as mean ± SEM, ***p < 0.001, **p < 0.01, *p < 0.05.

[0091] The scoring rules and calculation methods are as follows:

[0092] DAI = (weight score + diarrhea score + occult blood score) / 3.

[0093] In the formula, weight scoring: weight increase or no change is recorded as 0 points, weight loss of 1-5% is recorded as 1 point, weight loss of 6-10% is recorded as 2 points, weight loss of 11-15% is recorded as 3 points, and weight loss of more than 15% is recorded as 4 points;

[0094] Diarrhea score: normal stool was scored as 0 points, soft stool was scored as 2 points, and sticky stool or diarrhea was scored as 4 points;

[0095] Occult blood score: 0 points for negative occult blood, 2 points for positive occult blood, and 4 points for visible bleeding.

[0096] (3) Experimental results:

[0097] The weight changes of mice in each group are shown in Figure 2 As shown, the weight change of mice in the control group increased, while the weight change of mice in the model group showed a significant downward trend on the second day after injection, and continued to decrease over time. The weight change of the group that received intraperitoneal injection of the compound DMA-NAC increased compared with the model group.

[0098] The disease activity index test results of each group of mice are as follows Figure 3 As shown, after a period of drug treatment, the DAI score of the drug-treated group was significantly lower than that of the model group (DDS), indicating that the compound DMA-NAC has a therapeutic effect on inflammatory bowel disease and is expected to be used in the preparation of drugs for the prevention or treatment of inflammatory bowel disease.

[0099] Example 3: Effect of DMA-NAC on Colon Length in Mice with Inflammatory Bowel Disease

[0100] This example detects the effect of the above compound DMA-NAC on the colon length of ulcerative colitis in mice modeled with dextran sodium sulfate (DSS), and the specific method is as follows:

[0101] Inflammatory bowel disease mice were constructed with reference to the above Example 2, and then divided into three groups: a control group (CTR), a model group (DSS), and a drug administration group (DSS+DMA-NAC), and the same drug administration was performed. On the eighth day of drug administration, the mice were killed by cervical dislocation, colon tissue was taken, its length was measured, and statistical analysis was performed. There were 5 replicates in each group, and the values ​​were expressed as mean ± SEM, ***p<0.001, **p<0.01, *p<0.05.

[0102] Test results such as Figure 4As shown, compared with the model group, the colon length of the drug-treated group was significantly longer than that of the model group, indicating that the compound DMA-NAC can alleviate the shortening of the colon caused by inflammatory bowel disease and can be used as an effective therapeutic drug for inflammatory bowel disease.

[0103] Example 4: Effects of DMA-NAC on Colon Tissue of Mice with Inflammatory Bowel Disease

[0104] This example detects the effect of DMA-NAC on colon tissue of mice with inflammatory bowel disease, and specifically includes the following steps:

[0105] Inflammatory bowel disease mice were constructed with reference to the above Example 2, and then divided into three groups: a control group (CTR), a model group (DSS), and a drug-treated group (DSS+DMA-NAC), and the same drug-treated treatment was performed. On the eighth day of drug-treated treatment, the mice were killed by cervical dislocation, colon tissue was taken, and the middle section of the colon tissue was selected, with a tissue length of about 1 cm, immersed in formalin solution, fixed overnight, embedded in paraffin, sliced, and HE stained for observation.

[0106] The results are as follows Figure 5 As shown, compared with the control group, the intestinal mucosa of the model group was destroyed, the tissue was edematous and congested, and the glandular structure was damaged; further, compared with the model group, the intestinal mucosal layer of the drug-treated group was intact in morphology, indicating that the compound DMA-NAC has a significant therapeutic effect on mucosal damage caused by enteritis.

[0107] Example 5: Safety testing of DMA-NAC

[0108] This example detects the toxicity of the above compound DMA-NAC to human colon epithelial cells, and the specific method is as follows:

[0109] The FHC human colon epithelial cell line (0.2×10 5 / mL, 100μL) were inoculated in a 96-well plate (three replicate wells were set for each treatment group), and each well was cultured with 90μL of DMEM suspension. Then the drugs (DMA-NAC, NAC, DMA, NaAsO2) were diluted, and the drugs of the specified concentrations were added to each cell well, and an equal amount of DMSO was used as a control, and the 96-well plate was returned to the cell culture incubator for 24h.

[0110] The MTT cell proliferation and cytotoxicity detection kit (purchased from Beyotime) was used to determine the drug inhibitory effect. The ELISA reader selected an emission wavelength of 570 nm to detect the empty absorbance OD value of each sample.

[0111] The cell survival rate was calculated as follows: cell survival rate = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%. GraphPad Prism 8.0.1 was used to generate the dose response curve.

[0112] Test results such as Figure 6 As shown, it is shown that compared with inorganic arsenic, the compound DMA-NAC of the present invention has better safety than conventional inorganic arsenic.

[0113] In summary, the present invention provides an As(III)-containing compound and a preparation method and application thereof. The As(III)-containing compound of the present invention has excellent therapeutic effects on inflammatory bowel disease, and relatively low toxic and side effects, and has good application prospects.

[0114] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. An As(III)-containing compound, characterized in that: The chemical formula is shown in formula (I): In the formula, the R group is selected from one of a C1-C10 alkyl group or a hydroxyl group.

2. The As(III)-containing compound according to claim 1, characterized in that The R group is methyl.

3. A method for preparing an As(III)-containing compound as claimed in claim 2, characterized in that: The following steps are involved: In a solvent, acetylcysteine ​​is mixed with sodium cacodylate, reacted, and separated and purified to obtain the product.

4. The method for preparing the As(III)-containing compound according to claim 3, characterized in that: The solvent includes water.

5. The method for preparing the As(III)-containing compound according to claim 3, characterized in that: The separation and purification includes separation by column chromatography.

6. Use of the As(III)-containing compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2 in the preparation of a medicament for alleviating and / or treating inflammatory bowel disease.

7. The use according to claim 6, characterized in that: The drug for alleviating and / or treating inflammatory bowel disease has any of the following effects: a1) Alleviate and / or improve weight changes caused by inflammatory bowel disease; a2) Alleviate and / or improve the increase in disease activity index caused by inflammatory bowel disease; a3) Relieve and / or improve colon shortening caused by inflammatory bowel disease; a4) Alleviate and / or improve intestinal pathological damage caused by inflammatory bowel disease.

8. The use according to claim 6 or 7, characterized in that: The pharmaceutically acceptable salt of the As(III)-containing compound includes any one of hydrochloride, nitrate, acetate, methanesulfonate, phosphate, citrate, fumarate, sulfate, succinate, tartrate, citrate, hydrobromide, hydroiodide, lactate, toluenesulfonate, cinnamate, salicylate, malonate, glutarate and malate.

9. A drug for alleviating and / or treating inflammatory bowel disease, characterized in that: The active substance of the medicine comprises the As(III)-containing compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof.

10. The drug according to claim 9, characterized in that The drug further comprises pharmaceutically acceptable excipients.

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