Dimethylarsinic acid-glutamine complex and its preparation method and application

Abstract

The present invention belongs to the fields of medicine and chemical engineering, and relates to a dimethylarsinic acid glutamine complex, a preparation method and application thereof. The preparation method comprises the following steps: dissolving dimethylarsinic acid and glutathione protected by nitrogen in ultrapure water, stirring with argon, heating for reaction, cooling the reaction solution, adding pre-cooled methanol for crystallization reaction to obtain primary crystals, dissolving the primary crystals in methanol / water for recrystallization, and drying under reduced pressure to obtain a dimethylarsinic acid glutamine complex. The dimethylarsinic acid glutamine complex prepared by the present invention can promote the reconstruction of the respiratory tract and lungs, participate in angiogenesis, etc. to relieve inflammation by increasing MMP-9 and reducing TIMP-1. At the same time, DMA III 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.

Description

Technical Field

[0001] The invention belongs to the fields of medicine and chemistry, and relates to a dimethylarsinic acid-glutamine complex and a preparation method and application thereof. Background Art

[0002] In the field of skin inflammation treatment, the development of highly effective and low-toxic anti-inflammatory drugs has always been the focus of scientific research and clinical attention. III -SG), as a metabolite of arsenic in mammals, is highly toxic, but its unique biochemical properties have brought new possibilities for the development of new drugs for the treatment of skin inflammation, and its anti-inflammatory potential in vivo has attracted much attention.

[0003] Currently, arsenic-based drugs have achieved some success in cancer treatment. For example, arsenic compounds used to treat cancer-related diseases primarily exert their efficacy by inducing the production of reactive oxygen species (ROS) within mitochondria. This process relies on the activation of the NADPH oxidase complex, which impairs mitochondrial function and triggers a series of cellular responses. Specifically, increased ROS directly affects mitochondrial membrane potential, leading to the release of cytochrome c and activation of apoptotic bodies, ultimately initiating the apoptotic process in tumor cells and achieving G2 / M cell cycle arrest. Furthermore, these drugs exhibit anti-angiogenic activity, further enhancing their value in cancer treatment.

[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 needs for treating a wide range of conditions. Regarding drug safety, while 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, when used appropriately, have a modest therapeutic effect on skin inflammation. Furthermore, current research into 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 dimethylarsinic acid-glutamine complexes in the treatment of a wider range of diseases but also leave ample room for new drug development. Therefore, researchers urgently need to develop more effective, safe, and broadly applicable arsenic-based drugs. Summary of the Invention

[0005] The present invention aims to provide a dimethylarsinic acid glutamine complex and its preparation method and application. The present invention optimizes the dimethylarsinic acid glutamine complex DMA III -SG's synthetic pathway reduces its toxicity while retaining or enhancing its anti-inflammatory effect, bringing new breakthroughs in the field of skin inflammation treatment.

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

[0007] A method for preparing a dimethylarsinic acid-glutamine complex comprises the following steps: V ) and glutathione (GSH) protected by nitrogen were dissolved in ultrapure water, stirred with argon, and heated to react. After the reaction solution was cooled, pre-cooled methanol was added for crystallization to obtain primary crystals. The primary crystals were dissolved in methanol / water for recrystallization and dried under reduced pressure to obtain dimethylarsinic acid glutamine complex DMA. III -SG.

[0008] Furthermore, the temperature of the methanol after precooling is -22~-18°C.

[0009] Furthermore, the temperature of the heating reaction is 42-48°C, the reaction time is 1-3 hours, and the temperature of the reaction liquid during cooling is 3-5°C.

[0010] The present invention also provides a dimethylarsinic acid-glutamine complex prepared by the above-mentioned preparation method.

[0011] The present invention also provides use of the dimethylarsinic acid-glutamine complex in preparing a drug 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), promoting extracellular matrix (ECM) remodeling and cell migration.

[0016] Furthermore, the drug is used to downregulate interleukin-8 (IL-8) expression and inhibit activation of nuclear factor κB (NF-κB) or mitogen-activated protein kinase (MAPK) inflammatory signaling pathways.

[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] DMA III 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, causing cell damage; it can also relieve inflammation by increasing MMP-9 and reducing tissue inhibitor of metalloproteinase-1 (TIMP-1) to promote the reconstruction of the respiratory tract and lungs, participate in angiogenesis, etc. At the same time, DMA III It can also significantly reduce the excessive secretion of IL-8 caused by lipopolysaccharide (LPS) stimulation, thereby reducing the damage of LPS to human immortalized skin keratinocytes (HaCaT) cells, thereby alleviating skin inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 DMA III Structural diagram;

[0021] Figure 2 DMA III Preparation process;

[0022] Figure 3 DMA III HPLC-ICP-MS analysis mass spectrum;

[0023] Figure 4 For different concentrations of DMA III Effects on HaCaT cell survival;

[0024] Figure 5 For different concentrations of DMA III The changes of MMP-9 expression in the LPS-induced HaCaT injury model were treated;

[0025] Figure 6 For different concentrations of DMA III The changes of IL-8 expression in the HaCaT injury model induced by LPS were treated;

[0026] Figure 7 DMA of different purity III Effect of the prepared poison solution on cell survival rate. DETAILED DESCRIPTION

[0027] Example 1

[0028] The preparation method of dimethylarsinic acid glutamine complex for treating skin inflammation comprises the following steps (such as Figure 2 shown):

[0029] Weigh DMA V (Wako Pure Chemical Industries, Ltd., Osaka, Japan, molecular weight 138.0, 2.76 g, 20.0 mmol) and 18.4 g of nitrogen-protected glutathione (GSH) (molecular weight 307.33, 60.0 mmol) were dissolved in 120 mL of ultrapure water at a molar ratio of 1:3. After stirring under argon for 30 minutes, the mixture was heated to 45°C for 2 hours. After cooling the reaction solution to 4°C, pre-cooled methanol (-20°C) was added for crystallization. 5.0 g of the initial crystals were dissolved in methanol / water (3:1 volume ratio) at 60°C (close to the boiling point of methanol, but avoiding violent boiling). After dissolution, the mixture was slowly cooled to room temperature for recrystallization and dried under reduced pressure in an adsorption dryer (380 mmHg, 40°C) for 24 hours to obtain dimethylarsinic acid-glutamine complex DMA as a white solid powder. III -SG. 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 FIG. Figure 3 As shown, DMA can be clearly observed in the mass spectrum III The broad tail elution peak was analyzed by mass spectrometry software and its purity was >99% (Note: the white solid powder obtained was DMA III -SG, and when DMA III -SG dissolves in water and quickly hydrolyzes to form dimethylarsinic acid (DMA) III ).

[0030] Table 1 DMA prepared by the method of Example 1 III -SG quality and purity

[0031]

[0032] Note: The number of times in the table refers to 5 repeated experiments.

[0033] Comparative Example 1

[0034] According to the preparation method of Example 1 until the addition of pre-cooled methanol (-20 ° C) for crystallization, only one crystallization was performed to verify the obtained DMA III - Purity of SG.

[0035] Table 2 DMA prepared by the method of Comparative Example 1 III -SG quality and purity

[0036]

[0037] Note: The number of times in the table refers to 5 repeated experiments.

[0038] DMA prepared in Comparative Example 1 III The purity is only 90%. 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.

[0039] Comparative Example 2

[0040] According to the preparation method of Example 1, the DMA was finally dried under standard atmospheric pressure to verify that III - Purity of SG.

[0041] Table 3 DMA prepared by the method of Comparative Example 2 III -SG quality and purity

[0042]

[0043] Note: The number of times in the table refers to 5 repeated experiments.

[0044] DMA prepared in Comparative Example 2 III The purity is only 97%. In Example 1, the product is dried under reduced pressure after recrystallization, while in Comparative Example 2, it is dried under standard atmospheric pressure. Finally, the purity of the product of Example 1 is better than that of Comparative Example 2, indicating that better technical effects can be achieved by drying under reduced pressure after recrystallization.

[0045] Example 2

[0046] To further verify the high concentration of DMA III To determine whether apoptosis or autophagy is induced, a CCK8 assay was performed. Normal human immortalized keratinocytes (HaCaT cells) were prepared into a single-cell suspension and seeded at a density of 10,000 cells / well in a 96-well plate. The cells were incubated in a cell culture incubator until the cell confluence reached 80%. The culture medium was discarded, and DMA 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) was added. III 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).

[0047] The results are as follows Figure 4 As shown, with DMAIII With increasing doses of the drug, the survival rate of human immortalized keratinocytes (HaCaT cells) showed a trend of first increasing and then decreasing. The highest survival rate was achieved at 0.1 μM, and then the survival rate gradually decreased with increasing doses (P<0.05).

[0048] The results showed that at high concentrations, DMA III The effect of inhibiting cell proliferation is greater than its ability to promote skin repair, which makes DMA III The best concentration for treating skin inflammation is 15μM.

[0049] Example 3

[0050] 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.

[0051] The DMA prepared by the method of Example 1 III DMA was prepared with deionized water at different concentrations (5.0 μM, 10 μM, 15 μM, and 20 μM). III 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, DMA III The MMP-9 protein level in the treatment group was III The level at the concentration of 15.0 μM was higher than that in other groups (P<0.05).

[0052] MMP-9 can promote keratinocyte migration and accelerate wound re-epithelialization by degrading the extracellular matrix (ECM).III It may promote inflammation repair by inhibiting the nuclear translocation of AP-1 or NF-κB.

[0053] Combine Figure 4 From the perspective of cell survival rate:

[0054] Low concentration (0-10μM): The drug concentration is insufficient to fully activate downstream signaling pathways or bind to targets, resulting in limited pro-repair effects.

[0055] Optimal concentration (15 μM): Achieve the best binding efficiency between the drug and the target, maximize the activation of MMP-9 expression, and do not cause cytotoxicity.

[0056] High concentration (20 μM): May trigger cell stress response or inhibit MMP-9 expression through negative feedback mechanism, leading to decreased cell survival rate.

[0057] Example 3 results suggest that DMA III Significantly upregulated MMP-9 expression, indicating that it can directly participate in inflammation repair by promoting ECM remodeling and cell migration. III 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.

[0058] Table 4 Different concentrations of DMA III Changes in MMP-9 expression in LPS-induced HaCaT injury model

[0059]

[0060] Note: The number of times in the table refers to 9 repeated experiments.

[0061] Example 4

[0062] 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. HaCaT cells in the logarithmic phase were maintained at 1×10 4Cells 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.

[0063] The DMA prepared by the method of Example 1 III DMA was prepared with deionized water at different concentrations (5.0 μM, 10 μM, 15 μM, and 20 μM). III After the LPS-induced injury model of human immortalized keratinocytes (HaCaT cells) was treated with 1 μg / L of rhEGF (positive control) for 48 h, the expression of IL-8 protein, a marker of inflammation, was detected by WB. Figure 6 As shown, within this dose range, DMA III The IL-8 protein level in the treatment group was significantly decreased at 15.0 μM (P<0.05).

[0064] IL-8 is a key factor in inflammatory response, and its reduced level indicates DMA III It can effectively inhibit the activation of inflammatory signaling pathways such as NF-κB or MAPK.

[0065] Combine Figure 4 From the perspective of cell survival rate:

[0066] Low concentration (0-10μM): The drug concentration is insufficient to fully inhibit the protein level of the inflammatory factor IL-8, resulting in limited pro-repair effect.

[0067] Optimal concentration (15 μM): achieves the best binding efficiency between the drug and the target, significantly inhibits IL-8 expression, and does not cause cytotoxicity.

[0068] High concentration (20 μM): May trigger cell stress response, or activate IL-8 expression through negative feedback mechanism, and even lead to decreased cell survival rate.

[0069] Example 4 Results suggest that DMA III 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. III 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.

[0070] Table 5 Different concentrations of DMA III Changes in IL-8 expression in the LPS-induced HaCaT injury model

[0071]

[0072] Note: The number of times in the table refers to 9 repeated experiments.

[0073] Example 5

[0074] To further verify that the optimized synthesis pathway reduced toxicity and was more suitable for use as a skin inflammation drug, a CCK8 assay was performed. Normal HaCaT cells were prepared into a single-cell suspension and seeded at a density of 10,000 cells per well in a 96-well plate. The cells were then incubated in a cell culture incubator until the cell confluence reached 80%. The culture medium was discarded, and DMA (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 at different concentrations (90% and 99%). III 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).

[0075] The results are as follows Figure 7 As shown, using DMA with a purity of 99% III The cell survival rate was significantly higher when incubated with the prepared dye solution than when incubated with 90% DMA. III The possible reason for the configuration of the poison solution is that at the same concentration, the DMA with higher purity is used. III The smaller the amount, the less toxicity to cells.

[0076] The results show that the dimethylarsinic acid-glutamine complex of the present invention has reduced toxicity after optimizing the synthesis route, making it more suitable as a drug for skin inflammation.

Claims

1. The use of dimethylarsinic acid in the preparation of a drug for treating skin inflammation, characterized in that: The concentration of dimethylarsinic acid is 15.0 μM. The preparation method of dimethylarsinic acid comprises the following steps: dissolving dimethylarsinic acid and nitrogen-protected glutathione in ultrapure water, stirring under argon for 30 minutes, heating to 45°C for reaction for 2 hours, cooling the reaction solution to 4°C, adding pre-cooled -20°C methanol for crystallization to obtain primary crystals, dissolving the primary crystals in methanol / water for recrystallization, and drying under reduced pressure to obtain a dimethylarsinic acid-glutamine complex, and hydrolyzing the dimethylarsinic acid-glutamine complex to obtain dimethylarsinic acid.

2. The use according to claim 1, characterized in that The drug is used for up-regulating the expression of matrix metalloproteinase-9.

3. The use according to claim 1, characterized in that The drug is used to downregulate interleukin-8 expression.

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