Organoarsenic compound DMMTA V Its preparation method and application in preparing medicine for treating skin inflammation

Abstract

The present invention belongs to the field of medicine and chemical industry, and relates to an organic arsenic compound DMMTA V The application of DMA in the preparation of drugs for treating skin inflammation comprises the following steps: V The mixture was dissolved in ultrapure water and anhydrous sodium sulfide, reacted in an ice bath, and concentrated sulfuric acid was slowly added dropwise. The mixture was magnetically stirred under argon protection. After the reaction, pre-cooled methanol was added for crystallization. HCl was added to adjust the pH. The mixture was dehydrated in a water bath. Chloroform was used for extraction. After drying and evaporation, methanol / water was added for cooling and crystallization. The organic arsenic compound DMMTA was obtained after vacuum drying. V .DMMTA V It can not only relieve inflammation by increasing MMP‑9 and reducing TIMP‑1 to promote the reconstruction of the respiratory tract and lungs and participate in angiogenesis, but also reduce the excessive secretion of inflammatory factor 8 caused by lipopolysaccharide, reduce damage to human skin keratinocytes, and relieve skin inflammation.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and chemical industry, and relates to an organic arsenic compound DMMTA V The invention also relates to a preparation method thereof and application thereof in preparing medicine for treating skin inflammation. Background Art

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

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

[0004] However, existing arsenic-based drugs still have numerous limitations. In terms of application, they have achieved limited success only in the field of tumor treatment. Research and application in other disease areas, such as skin inflammation, are limited, failing to meet the clinical demand for treating a wide range of diseases. Regarding drug safety, although arsenic compounds are inherently highly toxic and can adversely affect normal cells during treatment, triggering a series of side effects that limit dosage and treatment duration, compromising therapeutic efficacy, they can, if used properly, have a modest therapeutic effect on skin inflammation. Furthermore, current research on the drug's mechanism of action is insufficient, and understanding of its metabolism in the body and its interactions with other biomolecules is insufficient, hindering further optimization and rational use. Furthermore, existing drug development and production technologies need improvement. For example, issues such as drug stability and bioavailability hinder the clinical application and widespread adoption of arsenic-based drugs. These challenges not only limit the application of organoarsenic compounds in treating a wider range of diseases but also leave ample room for new drug development. Researchers urgently need to explore and innovate to develop more effective, safe, and broadly applicable arsenic-based drugs. Summary of the Invention

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

[0006] The technical solutions provided by the present invention are as follows:

[0007] Organoarsenic compound DMMTA V The preparation method comprises the following steps: dimethylarsonic acid (DMA V ) and anhydrous sodium sulfide are dissolved in ultrapure water, reacted in an ice bath, concentrated sulfuric acid is slowly added dropwise, and magnetic stirring is carried out under argon protection. After the reaction is completed, pre-cooled methanol is added for crystallization reaction. HCl is added to adjust the pH, and dehydration reaction is promoted in a water bath. Chloroform extraction is used, and after drying and evaporation concentration, methanol / water is added for cooling crystallization, and vacuum drying is performed to obtain the organic arsenic compound DMMTA. V .

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

[0009] Furthermore, the temperature of the water bath-promoted 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 , prepared by the above preparation method.

[0011] The present invention also provides the above-mentioned organoarsenic compound DMMTA V Application in the preparation of medicines for treating skin inflammation.

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

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

[0014] Furthermore, the drug is used to promote keratinocyte migration and accelerate wound re-epithelialization.

[0015] Furthermore, the drug is used to upregulate the expression of matrix metalloproteinase-9 (MMP-9).

[0016] Furthermore, the drug is used to downregulate interleukin-8 (IL-8) expression.

[0017] Beneficial effects

[0018] During the production process of this product, dimethylarsonic acid was recrystallized twice to further improve 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 It can not only play an anti-cancer role by inducing cell cycle arrest, cell apoptosis, DNA damage, protein dysfunction, lipid peroxidation, reducing superoxide dismutase (SOD) activity and free radical production, but also can relieve inflammation by increasing MMP-9 and reducing TIMP-1, promoting the reconstruction of the respiratory tract and lungs, participating in angiogenesis, etc. At the same time, DMMTA V It can also significantly reduce the excessive secretion of IL-8 caused by LPS stimulation, thereby reducing the damage of LPS to HaCaT cells and thus alleviating skin inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 DMMTA V Structural diagram;

[0021] Figure 2 DMMTA V Preparation process;

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

[0023] Figure 4 Different concentrations of DMMTA V Effects on HaCaT cell survival;

[0024] Figure 5 Different concentrations of DMMTA V The changes of MMP-9 expression in the LPS-induced HaCaT injury model were treated;

[0025] Figure 6 Different concentrations of DMMTA V The changes of IL-8 expression in HaCaT injury model induced by LPS were analyzed. DETAILED DESCRIPTION

[0026] Example 1

[0027] Organoarsenic compound dimethyl arsenic acid DMMTA VThe preparation method comprises the following steps (such as Figure 2 shown):

[0028] Weigh DMA V 5.00 g (36.2 mmol) of methyl paraben (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) were dissolved in 150 mL of ultrapure water. The reaction temperature was controlled at 0-5°C in an ice bath. 3.5 mL (63.2 mmol) of 98% concentrated sulfuric acid was slowly added dropwise through a constant pressure dropping funnel. The mixture was magnetically stirred (800 rpm) under argon protection for 12 h. 5°C cold methanol was added to crystallize upon cooling, and 6 mol / L methyl paraben was added dropwise. Adjust the pH to 1.2-1.5 with HCl, and promote the dehydration reaction in a 35°C water bath for 1 hour. Extract with chloroform three times (3×50 mL). Combine the organic phases, backwash with saturated NaCl solution, add anhydrous CaCl2 and dry for 2 hours. After evaporation and concentration to 1 / 3 of the original volume, add methanol / water (volume ratio 3:1) at 60°C (close to the boiling point of methanol, but avoid violent boiling). After dissolution, slowly cool to room temperature for recrystallization. After 4°C overnight, dry in a vacuum desiccator (20 mmHg, 40°C) for 4 hours to obtain white crystalline DMMTA. V The obtained powder was verified by high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The mass spectrum of HPLC-ICP-MS analysis is shown in the figure below. Figure 3 As shown, DMMTA can be clearly observed in the mass spectrum V The purity of the eluted peak was >99% after analysis by mass spectrometry software.

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

[0030]

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

[0032] Comparative Example 1

[0033] According to the preparation method of Example 1, only one crystallization was performed until the DMMTA was crystallized by adding cold methanol. V purity.

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

[0035]

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

[0037] DMMTA prepared in Comparative Example 1 V The purity is only 94%. Since Comparative Example 1 does not have the subsequent second step of recrystallization process, the purity of the product of Example 1 is better than that of Comparative Example 1, indicating that using methanol for two recrystallizations can achieve better technical effects.

[0038] Comparative Example 2

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

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

[0041]

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

[0043] DMMTA prepared in Comparative Example 2 V The purity is only 95%. Since Comparative Example 2 did not extract with chloroform three times (3×50 mL) after the first recrystallization and the organic phases were combined, the purity of the product of Example 1 was better than that of Comparative Example 2, indicating that better technical effects can be achieved by extracting and recrystallizing with chloroform three times.

[0044] Comparative Example 3

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

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

[0047]

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

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

[0050] Comparative Example 4

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

[0052] Dissolve 5.24 g of DMA in a 50 mL centrifuge tube. V Dissolve 14.41 g of sodium sulfide nonahydrate (Na2S·9H2O) in 50 mL of deionized, nitrogen-purged water in a 250 mL flask. Slowly add 3.3 mL of concentrated sulfuric acid (96%) to 40 mL of deionized, nitrogen-purged water in a 50 mL centrifuge tube. V The solution was added to a 250 mL flask (containing 50 mL of Na2S solution). DMA was flushed with 10 mL of nitrogen-purged water. V Centrifuge tube, and add the rinsing liquid to the flask. Seal the flask with a three-hole rubber stopper, and connect the three holes respectively: nitrogen inlet glass tube, outlet glass tube, and sulfuric acid solution injection glass tube. Immediately after sealing, introduce nitrogen, control the gas pressure so that the airflow is gentle and covers the liquid surface to avoid splashing of the solution. Connect the sulfuric acid injection tube through a syringe, and slowly add 40mL H2SO4 solution from step 3 to the flask step by step. Pause after adding each 4-5mL of sulfuric acid, observe the color change of the reaction solution, and keep it in a white turbid state. Ensure that the reaction solution is allowed to stand for 1 hour. Pour the collected ether layer back into the same separatory funnel and add about 100mL of deionized water purged with nitrogen. Oscillate for 5-10 minutes, discard the nitrogen-purged deionized water and a small amount of ether layer. Collect the remaining ether layer into a glass culture dish. Transfer the glass culture dish to a nitrogen atmosphere glove box to prevent material conversion and dry until a white precipitate forms in the glass culture dish.

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

[0054] Example 2

[0055] To further verify the high concentration of DMMTA VTo investigate the effect of cytotoxicity on cell viability, a CCK8 assay was performed. Normal human immortalized keratinocytes (HaCaT cells) were prepared into single-cell suspensions and seeded at a density of 10,000 cells / well in 96-well plates. The cells were incubated in a cell culture incubator until the cell confluence reached 80%. The culture medium was discarded, and DMMTA was added at various 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). V Incubate cells with the toxin solution for 24 hours. Add 10 μL of CCK8 working solution to each well and incubate in a cell culture incubator for 4 hours. Measure the absorbance of each well at 450 nm using a microplate reader. Calculate cell viability as follows: (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 follows Figure 4 As shown, with DMMTA V With the increase of the dose, the survival rate of human immortalized keratinocytes (HaCaT cells) showed a trend of increasing first and then decreasing. The cell survival rate was the highest at 1.0 μM, and then the survival rate gradually decreased with the increase of the dose (P<0.05). The results showed that at high concentrations, DMMTA V Significantly inhibits cell proliferation.

[0057] Example 3

[0058] Human immortalized keratinocytes (HaCaT cells) were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C, 5% CO2, and humidified incubator. When the cells reached 80% confluency, the medium was removed, the cells were washed twice with phosphate-buffered saline (PBS), and then digested with 0.25% trypsin (containing ethylenediaminetetraacetic acid (EDTA)) and centrifuged. Logarithmic phase human immortalized keratinocytes (HaCaT cells) were maintained at 1×10 4 Cells were seeded evenly in a 96-well plate at a density of 1 cell / well, with 100 μL of cell suspension per well. The cells were incubated in an incubator for 24 hours. The supernatant was removed, and LPS solutions at concentrations of 25, 50, 100, 200, 400, 800, and 1000 μg / mL were added, with the experiment repeated five times. After an additional 24-hour culture, cell viability was assessed using the CCK-8 assay, and MMP-9 content was measured using a kit. Taking both cell viability and MMP-9 content into account, the final modeling condition was established, with cells stimulated with 200 μg / mL LPS solution for 24 hours.

[0059] DMMTA prepared by the method of Example 1V Prepare different concentrations (5.0 μM, 10 μM, 15 μM and 20 μM) of DMMTA with deionized water. V After 48 h of treatment with LPS-induced human immortalized keratinocyte (HaCaT) injury model, the expression of MMP-9 protein in cells was detected by WB method. Figure 5 As shown, within this dose range, DMMTA V The MMP-9 protein level in the treatment group was significantly higher than that in the control group. V The level at the concentration of 10.0 μM was higher than that in other groups (P<0.05).

[0060] MMP-9 can promote keratinocyte migration and accelerate wound re-epithelialization by degrading extracellular matrix (ECM). V Leading to high expression of MMP-9, which may in turn cause DMA III It may promote inflammation repair by inhibiting the nuclear translocation of AP-1 or NF-κB.

[0061] Combine Figure 4 From the dose-effect curve of cell survival:

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

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

[0064] High concentrations (15-20 μM): May trigger cell stress responses, leading to decreased cell survival, or inhibit MMP-9 expression through a negative feedback mechanism.

[0065] Example 3 The results suggest that DMMTA V Significantly upregulated MMP-9 expression, indicating that it can directly participate in inflammation repair by promoting ECM remodeling and cell migration. V The MMP-9 level in the cells was comparable to that of rhEGF (a clinically used pro-repair factor), suggesting that it has the potential for alternative or synergistic applications.

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

[0067]

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

[0069] Example 4

[0070] Human immortalized keratinocytes (HaCaT cells) were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C, 5% CO2, and saturated humidity incubator. When the cells reached 80% confluency, the medium was removed, the cells were washed twice with PBS, and then digested with 0.25% trypsin (containing EDTA) and centrifuged. Human immortalized keratinocytes (HaCaT cells) in the logarithmic phase were maintained at 1×10 4 Cells were seeded evenly in a 96-well plate at a density of 1 cell / well, with 100 μL of cell suspension per well. The cells were incubated in an incubator for 24 hours. The supernatant was removed, and LPS solutions at concentrations of 25, 50, 100, 200, 400, 800, and 1000 μg / mL were added, with the experiment repeated five times. After an additional 24-hour culture, cell viability was assessed using the CCK-8 assay, and interleukin (IL)-8 levels were measured using a kit. Taking both cell viability and IL-8 levels into account, the final modeling condition was established, with cells stimulated with 200 μg / mL LPS solution for 24 hours.

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

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

[0073] Combine Figure 4 From the dose-effect curve of cell survival:

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

[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.

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

[0077] Example 4 The results suggest that DMMTA V Significantly down-regulating IL-8 expression confirms that it can regulate the skin inflammatory microenvironment, which meets the requirement that skin inflammation drugs must have both anti-inflammatory and pro-repair functions. V The IL-8 level of the drug was comparable to that of rhEGF (a clinically used pro-repair factor), suggesting that it has the potential for alternative or synergistic applications.

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

[0079]

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

Claims

1. Organic arsenic compound DMMTA V The application of the preparation method in preparing a drug for increasing the level of MMP-9 is characterized in that: The organic arsenic compound DMMTA V The concentration of the organoarsenic compound DMMTA is 10.0 μM. V The preparation method comprises the following steps: V The mixture was dissolved in ultrapure water and anhydrous sodium sulfide, reacted in an ice bath, and concentrated sulfuric acid was slowly added dropwise. The mixture was magnetically stirred under argon protection. After the reaction, pre-cooled methanol was added for crystallization. HCl was added to adjust the pH. The mixture was dehydrated in a water bath. Chloroform was used for extraction. After drying and evaporation, methanol / water was added for cooling and crystallization. The organic arsenic compound DMMTA was obtained after vacuum drying. V .

2. Organic arsenic compound DMMTA V The application of the preparation method in preparing a drug for reducing IL-8 protein level is characterized in that: The organic arsenic compound DMMTA V The concentration of the organoarsenic compound DMMTA is 10.0 μM. V The preparation method comprises the following steps: V The mixture was dissolved in ultrapure water and anhydrous sodium sulfide, reacted in an ice bath, and concentrated sulfuric acid was slowly added dropwise. The mixture was magnetically stirred under argon protection. After the reaction, pre-cooled methanol was added for crystallization. HCl was added to adjust the pH. The mixture was dehydrated in a water bath. Chloroform was used for extraction. After drying and evaporation, methanol / water was added for cooling and crystallization. The organic arsenic compound DMMTA was obtained after vacuum drying. V .

3. The use according to claim 1 or 2, characterized in that HCl was added to adjust the pH to 1.2-1.

5.

4. The use according to claim 1 or 2, characterized in that The temperature of the water bath dehydration reaction is 32~38℃, and the reaction time is 40~80min.

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