Organic arsenic compound DMMAV, preparation method thereof and application of DMMAV in preparation of medicine for treating skin inflammation

Abstract

The invention belongs to the field of medicine and chemistry and chemical engineering, and relates to an application of an organic arsenic compound DMMAV in preparation of a medicine for treating scytitis, the preparation method comprises the following steps: dissolving DMAV and anhydrous sodium sulfide in ultrapure water, reacting under an ice bath condition, slowly dropwise adding concentrated sulfuric acid, magnetically stirring under the protection of argon, and carrying out suction filtration to obtain a filtrate; after the reaction is finished, pre-cooled methanol is added for a crystallization reaction, HCl is added for adjusting the pH value, a water bath is used for promoting a dehydration reaction, chloroform extraction, drying and evaporation concentration are performed, methanol / water is added for cooling crystallization, and vacuum drying is performed to obtain the organic arsenic compound DMMAV. The DMMAV can improve MMP-9 and reduce TIMP-1 to promote reconstruction of respiratory tracts and lungs, participate in angiogenesis and the like to relieve inflammation, and can also relieve damage to human skin keratinocytes and relieve skin inflammation by reducing excessive secretion of inflammatory factors 8 caused by lipopolysaccharide.

Description

Technical Field

[0001] The present invention belongs to the fields of medicine, chemical engineering, and chemistry, and relates to the organic arsenic compound DMMTA V and its preparation method and application in the preparation of drugs for treating skin inflammation. Background Art

[0002] As the largest organ of the human body, the skin's inflammation repair process involves complex biological mechanisms, including multiple stages such as inflammation control, tissue regeneration, angiogenesis, and scar formation.

[0003] Currently, there are various treatment methods for skin inflammation, but there are still many challenges. Traditional drugs such as antibiotics and anti-inflammatory drugs can control infections and inflammation to a certain extent, but they often have problems such as limited efficacy and obvious side effects, making it difficult to meet clinical needs. Therefore, it is particularly important to develop new, highly efficient, and safe drugs for treating skin inflammation. Especially for deep inflammation or chronic refractory inflammation, the efficacy of existing drugs is even more inadequate. Some drugs for treating skin inflammation, such as hormonal drugs, although they have significant anti-inflammatory effects, long-term use can cause side effects such as skin atrophy and pigmentation, seriously affecting the quality of life of patients. Many existing drugs only target a certain link in the skin inflammation healing process, such as inhibiting infection and promoting cell proliferation, and lack comprehensive therapeutic effects. This single mechanism of action limits the efficacy and scope of application of the drugs.

[0004] However, existing drugs based on arsenic compounds still have many limitations. In terms of the scope of application, they have only achieved certain results in the field of tumor treatment, and there is less research and application in other disease fields such as skin inflammation treatment, unable to meet the clinical needs for treating multiple diseases. From the perspective of drug safety, although arsenic compounds themselves are highly toxic and may have adverse effects on normal cells during treatment, causing a series of side effects, these side effects limit the dosage and treatment cycle, affecting the treatment effect. However, if used properly, it will have a certain therapeutic effect on skin inflammation. At the same time, the research on the mechanism of action of current drugs is not deep enough, and there is insufficient understanding of aspects such as the drug's metabolism process in the body and its interaction with other biomolecules, which brings difficulties to the further optimization and rational use of the drugs. In addition, existing drug R & D and production technologies also need to be improved. For example, problems such as drug stability and bioavailability affect the clinical application and promotion of arsenic compound drugs. These problems not only limit the application of organic arsenic compounds in the treatment of a wider range of diseases but also leave a broad space for new drug development, urgently requiring scientific researchers to explore and innovate to develop more efficient, safe, and widely applicable arsenic compound drugs. Summary of the Invention

[0005] The present invention aims to provide the organic arsenic compound dimethylmonothioarsinic acid (DMMTAV ), its preparation method and application in the preparation of drugs for treating skin inflammation. DMMTA V As a pentavalent arsenic compound, it has strong cytotoxicity. It can relieve inflammatory effects by increasing matrix metalloproteinase-9 (MMP-9) and decreasing tissue inhibitor of metalloproteinase-1 (TIMP-1) to promote the reconstruction of the respiratory tract and lungs and participate in angiogenesis, etc. It can also reduce the excessive secretion of inflammatory interleukin-8 (IL-8) caused by lipopolysaccharide (LPS), reduce the damage to human skin keratinocytes (HaCaT), and relieve skin inflammation.

[0006] The technical solution provided by the present invention is as follows:

[0007] Organic arsenic compound DMMTA V The preparation method of, comprising the following steps: Dissolve dimethylarsinic acid (DMA V ) and anhydrous sodium sulfide in ultrapure water, react under ice bath conditions, slowly add concentrated sulfuric acid dropwise, stir magnetically under argon protection, after the reaction is completed, put it into pre-cooled methanol for crystallization reaction, add HCl to adjust the pH, promote dehydration reaction by water bath, extract with chloroform, dry and evaporate and concentrate, then add methanol / water for cooling crystallization, and obtain organic arsenic compound DMMTA after vacuum drying V .

[0008] Further, add HCl to adjust the pH to 1.2 - 1.5.

[0009] Further, the temperature of the water bath for promoting dehydration reaction is 32 - 38 °C, and the reaction time is 40 - 80 min.

[0010] The present invention also provides an organic arsenic compound DMMTA V , which is prepared by the above preparation method.

[0011] The present invention also provides the above organic arsenic compound DMMTA V The application in the preparation of drugs for treating skin inflammation.

[0012] Further, the drug concentration is 10 - 20 μM.

[0013] Further, the drug is used to promote skin repair.

[0014] Further, the drug is used to promote the migration of keratinocytes and accelerate wound re-epithelialization.

[0015] Further, the drug is used to up-regulate the expression of matrix metalloproteinase-9 (MMP-9).

[0016] Further, the drug is used to down-regulate the expression of interleukin-8 (IL-8).

[0017] Beneficial effects

[0018] During the production process of this product, dimethylarsinic acid was recrystallized twice, further improving the purity of the product. The purity was strictly analyzed using advanced mass spectrometry analysis software (Agilent), and the purity exceeded 99%. This result fully demonstrates the extremely high stability and reliability of the product.

[0019] DMMTA V DMMTA can exert its anti-cancer effect by inducing cell cycle arrest, apoptosis, DNA damage, protein dysfunction, lipid peroxidation, reducing the activity of superoxide dismutase (SOD) and free radical production, etc., to cause cell damage; it can also alleviate the inflammatory effect by increasing MMP-9 and decreasing TIMP-1 to promote the reconstruction of the respiratory tract and lungs and participate in angiogenesis. At the same time, DMMTA V can also significantly reduce the excessive secretion of IL-8 elevated by LPS stimulation to reduce the damage of LPS to HaCaT cells, thereby achieving the effect of alleviating skin inflammation. Brief description of the drawings

[0020] Figure 1 is the structural diagram of DMMTA V ;

[0021] Figure 2 is the preparation process of DMMTA V ;

[0022] Figure 3 is the HPLC-ICP-MS analysis mass spectrum of DMMTA V ;

[0023] Figure 4 is the effect of different concentrations of DMMTA V on the survival rate of HaCaT cells;

[0024] Figure 5 is the change in the expression of MMP-9 in the HaCaT injury model induced by LPS treated with different concentrations of DMMTA V ;

[0025] Figure 6 is the change in the expression of IL-8 in the HaCaT injury model induced by LPS treated with different concentrations of DMMTA V ; Detailed implementation manners

[0026] Example 1

[0027] The organoarsenic compound dimethylmonothioarsinic acid DMMTA VPreparation method, including the following steps (as Figure 2 shown):

[0028] Weigh 5.00 g (36.2 mmol) of DMA V (Wako Pure Chemical Industries, Ltd., Osaka, Japan, molecular weight 138.0) and 4.52 g (57.9 mmol) of anhydrous sodium sulfide (Na2S, molecular weight 78.0), dissolve them in 150 mL of ultrapure water, control the reaction temperature at 0 - 5 °C in an ice bath, slowly add 3.5 mL (63.2 mmol) of 98% concentrated sulfuric acid dropwise through a constant pressure dropping funnel, stir magnetically (800 rpm) for 12 hours under argon protection, add cold methanol at 5 °C to cause crystallization when it cools, slowly add 6 mol / L HCl dropwise to adjust the pH to 1.2 - 1.5, promote the dehydration reaction in a 35 °C water bath for 1 h, extract three times with chloroform (3 × 50 mL), combine the organic phases, wash with saturated NaCl solution, dry with anhydrous CaCl2 for 2 h, evaporate and concentrate to 1 / 3 of the original volume, then add it to methanol / water (volume ratio 3:1) at 60 °C (close to the boiling point of methanol, but avoid violent boiling), wait for it to dissolve and then slowly cool to room temperature for recrystallization, after overnight at 4 °C, dry in a vacuum dryer (20 mmHg, 40 °C) for 4 h to obtain white crystals of DMMTA V . Use the analytical method of high performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS) to verify the obtained powder. The HPLC-ICP-MS analysis mass spectrum is as Figure 3 shown. The elution peak of DMMTA V can be clearly observed in the mass spectrum. Use mass spectrometry analysis software to analyze its purity > 99%.

[0029] Table 1 Mass and purity of DMMTA prepared by the method of Example 1 V

[0030]

[0031] Note: The number of times in the table is 5 repeated experiments.

[0032] Comparative Example 1

[0033] According to the preparation method of Example 1 until the cold methanol is added and crystallization occurs when it cools, only perform crystallization once, and verify the purity of the obtained DMMTA V .

[0034] Table 2 Mass and purity of DMMTA prepared by the method of Comparative Example 1 V

[0035]

[0036] Note: The number of times in the table is 5 repeated experiments.

[0037] DMMTA prepared in Comparative Example 1 V The purity was only 94%. Since there was no subsequent second-step recrystallization process in Comparative Example 1, the product purity of Example 1 was better than that of Comparative Example 1, indicating that better technical effects could be obtained by recrystallizing twice with methanol.

[0038] Comparative Example 2

[0039] Remove the step of extracting three times with chloroform (3×50 mL) and combining the organic phases in Example 1, and verify the purity of the obtained DMMTA V purity.

[0040] Table 3 Quality and purity of DMMTA prepared by the method of Comparative Example 2 V purity

[0041]

[0042] Note: The number of times in the table is 5 repeated experiments.

[0043] DMMTA prepared in Comparative Example 2 V The purity was only 95%. Since in Comparative Example 2, there was no extraction three times with chloroform (3×50 mL) and combination of the organic phases after the first recrystallization, the product purity of Example 1 was better than that of Comparative Example 2, indicating that better technical effects could be obtained by extracting three times with chloroform and then recrystallizing.

[0044] Comparative Example 3

[0045] According to the preparation method of Example 1, but finally dried under standard atmospheric pressure, and verify the purity of the obtained DMMTA V purity.

[0046] Table 4 Quality and purity of DMMTA prepared by the method of Comparative Example 3 V purity

[0047]

[0048] Note: The number of times in the table is 5 repeated experiments.

[0049] DMMTA prepared in Comparative Example 3 V The purity was only 96%. Since Example 1 focused on the crystallization process in a vacuum environment, while Comparative Example 3 did not have such a process, the product purity of Example 1 was better than that of Comparative Example 3, indicating that better technical effects could be obtained by drying and crystallizing under reduced pressure in an adsorption dryer.

[0050] Comparative Example 4

[0051] A method for preparing DMMTA VThe method includes the following steps:

[0052] Dissolve 5.24 g of DMA in a 50 mL centrifuge tube V In 40 mL of deionized and nitrogen-purged (for at least 30 minutes) water. In a 250 mL flask, dissolve 14.41 g of sodium sulfide nonahydrate (Na2S·9H2O) in 50 mL of deionized and nitrogen-purged water. In a 50 mL centrifuge tube, slowly add 3.3 mL of concentrated sulfuric acid (96%) to 40 mL of deionized and nitrogen-purged water. Add the prepared 40 mL DMA V solution to the 250 mL flask (containing 50 mL of Na2S solution). Rinse the DMA V centrifuge tube with 10 mL of nitrogen-purged water and add the rinsing liquid to the flask. Seal the flask with a three-hole rubber stopper. The three holes are respectively connected to: a nitrogen inlet glass tube, an outlet glass tube, and a sulfuric acid solution injection glass tube. Immediately after sealing, introduce nitrogen, control the gas pressure to make the gas flow gently cover the liquid surface to avoid splashing of the solution. Connect a syringe to the sulfuric acid injection tube and slowly add the 40 mL H2SO4 solution in step 3 to the flask in portions. Pause after adding 4 - 5 mL of sulfuric acid each time and observe the color change of the reaction solution, which should maintain a white turbid state. Ensure that the reaction solution stands for 1 hour. Pour the collected ether layer back into the same separatory funnel, add about 100 mL of nitrogen-purged deionized water. Oscillate for 5 - 10 minutes and discard the nitrogen-purged deionized water and a small amount of the ether layer. Collect the remaining ether layer in a glass petri dish. Transfer the glass petri dish to a nitrogen atmosphere glove box to prevent material transformation and dry it until a white precipitate forms in the glass petri dish.

[0053] The DMMTA prepared in Comparative Example 4 V has a purity of only 92%. Since Example 1 focuses on low temperature and long reaction time, while Comparative Example 4 optimizes the reaction process by adding acid step by step, strict nitrogen protection, and real-time monitoring, the product purity of Example 1 is better than that of Comparative Example 4, indicating that better technical effects can be obtained by using low temperature and long reaction time.

[0054] Example 2

[0055] To further verify high-concentration DMMTA VTo investigate the effect on cell viability, a CCK8 assay was performed. Normal human immortalized keratinocytes (HaCaT cells) were prepared into single-cell suspensions and seeded in 96-well plates at a cell density of 10,000 cells / well. The plates were then incubated in a cell culture incubator until the cell confluence reached 80%. The culture medium was discarded, and DMMTA at different concentrations (0.0 μM, 0.05 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1.0 μM, 2.0 μM, 5.0 μM, 10.0 μM, 20.0 μM, 50.0 μM, and 100.0 μM) was added. V The poisoned solution was incubated for 24 h. Then, 10 μL of CCK8 working solution was added to each well, and the plates were further incubated in the cell culture incubator for 4 h. The absorbance of each well was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The cell viability was calculated as follows: Cell viability = (A value of the experimental group - A value of the blank group) × 100% / (A value of the control group - A value of the blank group).

[0056] The results are as Figure 4 shown. As the dose of DMMTA V increased, the viability of human immortalized keratinocytes (HaCaT cells) first increased and then decreased. The cell viability was highest at 1.0 μM, and then gradually decreased as the dose of the poisoned solution increased (P < 0.05). The results indicate that at high concentrations, DMMTA V significantly inhibited cell proliferation.

[0057] Example 3

[0058] Human immortalized keratinocytes (HaCaT cells) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibiotics in an incubator at 37°C and 5% CO2 with saturated humidity. When the cell confluence reached 80%, the culture medium was removed, and the cells were washed twice with phosphate-buffered saline (PBS). Then, 0.25% trypsin (containing ethylenediaminetetraacetic acid (EDTA)) was added for digestion and centrifugation. Logarithmic-phase human immortalized keratinocytes (HaCaT cells) were evenly seeded in 96-well plates at a density of 1×10 4 cells / well, with 100 μL of cell suspension in each well. The plates were incubated in the incubator for 24 h. The supernatant was removed, and LPS solutions at concentrations of 25, 50, 100, 200, 400, 800, and 1000 μg / mL were added respectively. The experiment was repeated 5 times. After culturing for another 24 h, the cell viability was detected by the CCK-8 method, and the MMP-9 content was detected using a kit. Considering both the cell viability and the MMP-9 content, 200 μg / mL LPS solution was finally used to stimulate cell culture for 24 h as the modeling condition.

[0059] The DMMTA prepared by the method of Example 1V Prepare DMMTA with different concentrations (5.0 μM, 10 μM, 15 μM, and 20 μM) using deionized water V After treating the LPS-induced human immortalized keratinocyte (HaCaT cell) injury model with DMMTA at different concentrations and 1 μg / L of recombinant human epidermal growth factor (rhEGF) (positive control) for 48 h respectively, detect the expression of MMP-9 protein in cells by WB method. The results are as Figure 5 shown. In this dose range, the level of MMP-9 protein in the DMMTA V treatment group is higher than that of other groups when the concentration of DMMTA V is 10.0 μM (P < 0.05).

[0060] MMP-9 can promote keratinocyte migration and accelerate wound re-epithelialization by degrading the extracellular matrix (ECM). Example 3 shows that DMMTA V results in high expression of MMP-9, and then may promote inflammatory repair by DMA III may inhibit the nuclear translocation of AP-1 or NF-κB, thereby promoting inflammatory repair.

[0061] Combined with Figure 4 the dose-effect curve of cell viability:

[0062] Low concentration (0 - 5 μM): The drug concentration is insufficient to fully induce high expression of MMP-9 and activate the downstream signaling pathway or bind to the target, resulting in limited pro-repair effect.

[0063] Optimal concentration (10 μM): Achieve the best binding efficiency between the drug and the target, maximize the activation of MMP-9 expression, and at the same time do not cause cytotoxicity.

[0064] High concentration (15 - 20 μM): May trigger a cellular stress response leading to a decrease in cell viability, or inhibit MMP-9 expression through a negative feedback mechanism.

[0065] The results of Example 3 suggest that DMMTA V significantly upregulates the expression of MMP-9, indicating that it can directly participate in inflammatory repair by promoting ECM remodeling and cell migration. And the MMP-9 level of 10.0 μM DMMTA V is comparable to that of rhEGF (a clinically used pro-repair factor), which suggests its potential for substitution or synergistic application.

[0066] Table 5 Changes in MMP-9 expression in the LPS-induced HaCaT injury model treated with different concentrations of DMMTA V

[0067] ​

[0068] Note: The number of times in the table is 9 repeated experiments.

[0069] Example 4

[0070] Human immortalized keratinocytes (HaCaT cells) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibiotics, and placed in an incubator at 37 °C and 5% CO2 saturated humidity. When the cells were fused to 80%, the medium was removed, washed twice with PBS, and digested and centrifuged with 0.25% trypsin (containing EDTA). The human immortalized keratinocytes (HaCaT cells) in the logarithmic phase were maintained at a density of 1×10 4 cells / well and evenly inoculated into a 96-well plate, with 100 μL of cell suspension in each well, and incubated in the incubator for 24 h. The supernatant was removed, and LPS solutions with concentrations of 25, 50, 100, 200, 400, 800, and 1000 μg / mL were added respectively, and the experiment was repeated 5 times. After culturing for another 24 h, the cell viability was detected by the CCK-8 method, and the IL-8 content was detected using a kit. Considering the cell viability and IL-8 content comprehensively, finally, the cells were stimulated with 200 μg / mL LPS solution for 24 h as the modeling condition.

[0071] The DMMTA prepared by the method of Example 1 V was prepared with deionized water at different concentrations (5.0 μM, 10 μM, 15 μM, and 20 μM) of DMMTA V and 1 μg / L of rhEGF (positive control) were used to treat the LPS-induced human immortalized keratinocyte (HaCaT cell) injury model for 48 h, and the protein expression of the intracellular inflammatory marker IL-8 was detected by WB method. The results were as Figure 6 shown. In this dose range, the IL-8 protein level in the DMMTA V treatment group was significantly decreased at 10.0 μM (P<0.05).

[0072] IL-8 is a key factor in the inflammatory response, and the decrease in its level indicates that DMMTA V can effectively inhibit the activation of inflammatory signaling pathways such as NF-κB or MAPK.

[0073] Combined Figure 4 with the dose-effect curve of cell viability:

[0074] Low concentration (0-5 μM): The drug concentration is insufficient to fully inhibit the protein level of the inflammatory factor IL-8, resulting in limited promotion of repair.

[0075] Optimal concentration (10 μM): Achieves the best binding efficiency between the drug and the target, significantly inhibits IL-8 expression, and does not cause cytotoxicity at the same time.

[0076] High concentration (15 - 20 μM): May trigger a cellular stress response, or activate IL-8 expression through a negative feedback mechanism, and even lead to a decrease in cell viability.

[0077] The results of Example 4 suggest that DMMTA V significantly downregulates IL-8 expression, confirming its ability to regulate the skin inflammatory microenvironment, meeting the requirements for skin inflammation drugs to have both anti-inflammatory and pro-repair functions. Moreover, the IL-8 level of 10.0 μM DMMTA V is comparable to that of rhEGF (a clinically used pro-repair factor), indicating its potential for substitution or synergistic application.

[0078] Table 6 Changes in IL-8 expression in the LPS-induced HaCaT injury model treated with different concentrations of DMMTA V

[0079]

[0080] Note: The number of times in the table is 9 repeated experiments.

Claims

1. Preparation method of organic arsenic compound DMMTA V characterized in that It includes the following steps: Dissolve DMA V and anhydrous sodium sulfide in ultrapure water, react under ice bath conditions, slowly add concentrated sulfuric acid dropwise, stir magnetically under argon protection. After the reaction is completed, add pre-cooled methanol for crystallization reaction. After adjusting the pH with HCl, promote the dehydration reaction in a water bath, extract with chloroform, dry and evaporate to concentrate, then add methanol / water for cooling crystallization, and obtain the organoarsenic compound DMMTA after vacuum drying V .

2. The preparation method of the organoarsenic compound DMMTA according to claim 1 V , characterized in that Add HCl to adjust the pH to 1.2 - 1.

5.

3. The preparation method of the organic arsenic compound DMMTA according to claim 1 V , characterized in that The temperature of the water bath for promoting the dehydration reaction is 32 - 38 °C, and the reaction time is 40 - 80 min.

4. An organic arsenic compound DMMTA V , characterized in that It is prepared by the preparation method according to any one of claims 1 - 3.

5. Use of the organoarsenic compound DMMTA according to claim 4 V in the preparation of a medicament for treating skin inflammation.

6. The application according to claim 5, wherein The drug concentration is 10 - 20 μM.

7. The application according to claim 5, characterized in that, The drug is used for promoting skin repair.

8. The application according to claim 5, characterized in that, The drug is used for promoting the migration of keratinocytes and accelerating wound re - epithelialization.

9. The application according to claim 5, characterized in that, The drug is used for up - regulating the expression of matrix metalloproteinase - 9.

10. The application according to claim 5, characterized in that, The drug is used for down - regulating the expression of interleukin - 8.

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