Dimethyl arsonic acid glutamine compound as well as preparation method and application thereof

Abstract

The invention belongs to the field of medicine and chemistry and chemical engineering, and relates to a dimethyl arsonic acid glutamine compound and a preparation method and application thereof.The preparation method comprises the following steps that dimethyl arsonic acid and glutathione protected by nitrogen are dissolved in ultrapure water, after argon is introduced and stirred, heating reaction is conducted, and after reaction liquid is cooled, the dimethyl arsonic acid glutamine compound is obtained; and adding precooled methanol for crystallization reaction to obtain primary crystals, dissolving the primary crystals in methanol / water for recrystallization, and drying under reduced pressure to obtain the dimethyl arsonic acid glutamine compound. The dimethyl arsonic acid glutamine compound prepared by the invention can improve MMP-9 and reduce TIMP-1 to promote the reconstruction of respiratory tracts and lungs, participate in angiogenesis and the like to relieve inflammation. Meanwhile, DMAIII can also significantly reduce the excessive secretion of IL-8 increased due to LPS stimulation so as to reduce the damage of LPS to HaCaT cells, thereby achieving the effect of relieving skin inflammation.

Description

Technical Field

[0001] The present invention belongs to the fields of medicine, chemistry and chemical engineering, and relates to dimethylarsinic acid glutamine complex and its preparation method and application. Background Art

[0002] In the field of treating skin inflammation, the research and development of highly effective and low-toxic anti-inflammatory drugs has always been the focus of scientific research and clinical attention. Dimethylarsinic acid glutamine complex (DMA III -SG), as a metabolite of arsenic in mammals, although it has strong toxicity, its unique biochemical properties bring new possibilities for the development of new drugs for treating skin inflammation, and its anti-inflammatory potential shown in organisms has attracted much attention.

[0003] At present, drugs developed based on arsenic compounds in the market have achieved certain results in the field of tumor treatment. Taking arsenic compound drugs used to treat tumor-related diseases as an example, it mainly exerts its efficacy by targeting and inducing the production of reactive oxygen species (ROS) in mitochondria. This process depends on the activation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. After activation, mitochondrial function is damaged, and then a series of cellular reactions are triggered. Specifically, the increase in ROS will directly affect the mitochondrial membrane potential, leading to the release of cytochrome C, activating apoptotic bodies, and finally initiating the apoptosis program of tumor cells, achieving G2 / M cell cycle arrest. At the same time, this drug also has anti-angiogenic activity, further enhancing its application value in tumor treatment.

[0004] However, existing drugs based on arsenic compounds still have many limitations. In terms of the application scope, they only have certain effects in the field of tumor treatment, and there is less research and application in other disease fields such as skin inflammation treatment, which cannot meet the clinical needs for treating various diseases. From the perspective of drug safety, although arsenic compounds themselves are highly toxic and may have adverse effects on normal cells during treatment, triggering a series of side effects, these side effects limit the dosage and treatment cycle of the drug, 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 metabolic process of drugs in the body and the interaction with other biomolecules, which brings difficulties for the further optimization and rational use of drugs. In addition, the existing drug R & D and production technologies also need to be improved. For example, problems such as the stability and bioavailability of drugs affect the clinical application and promotion of arsenic compound drugs. These problems not only limit the application of dimethylarsinic acid glutamine complex in the treatment of a wider range of diseases, but also leave a broad space for the development of new drugs. Therefore, it is urgent for scientific research personnel to develop more efficient, safe and widely applicable arsenic compound drugs. Summary of the Invention

[0005] The present invention aims to provide a dimethylarsinic acid glutamine complex, its preparation method and application. By optimizing the synthesis route of the dimethylarsinic acid glutamine complex DMA III -SG, its toxicity is reduced while its anti-inflammatory effect is retained or enhanced, bringing new breakthroughs to the field of skin inflammation treatment.

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

[0007] A preparation method of a dimethylarsinic acid glutamine complex, comprising the following steps: Dissolve dimethylarsinic acid (DMA V ) and glutathione (GSH) protected by nitrogen in ultrapure water. After stirring with argon, heat for reaction. After the reaction solution is cooled, add pre-cooled methanol for crystallization reaction to obtain primary crystals. Dissolve the primary crystals in methanol / water for recrystallization, and obtain the dimethylarsinic acid glutamine complex DMA III -SG after drying under reduced pressure.

[0008] Further, the temperature of the pre-cooled methanol is -22~-18°C.

[0009] Further, the temperature of the heating reaction is 42~48°C, the reaction time is 1~3h, and the temperature of the reaction solution cooling is 3~5°C.

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

[0011] The present invention also provides the application of the above dimethylarsinic acid glutamine complex 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), promote the remodeling of the extracellular matrix (ECM) and cell migration.

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

[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] DMA III It can play an anti-cancer role by inducing cell cycle arrest, apoptosis, DNA damage, protein dysfunction, lipid peroxidation, reducing the activity of superoxide dismutase (SOD) and the generation of free radicals, etc., thereby causing cell damage; it can also relieve inflammatory effects by increasing MMP-9 and reducing tissue inhibitor of metalloproteinase-1 (TIMP-1) to promote the reconstruction of the respiratory tract and lungs and participate in angiogenesis, etc. At the same time, DMA III can also significantly reduce the excessive secretion of IL-8 elevated by lipopolysaccharide (LPS) stimulation, reduce the damage of LPS to human immortalized skin keratinocytes (HaCaT) cells, and thus achieve the effect of relieving skin inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the structural diagram of DMA III ;

[0021] Figure 2 is the preparation process of DMA III ;

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

[0023] Figure 4 is the effect of different concentrations of DMA III 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 DMA III ;

[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 DMA III ;

[0026] Figure 7 is the effect of the venom solution prepared with different purities of DMA III on the survival rate of cells. DETAILED DESCRIPTION OF THE INVENTION

[0027] Example 1

[0028] Preparation method of dimethylarsinic acid glutamine complex for treating skin inflammation, comprising the following steps (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 glutathione GSH 18.4 g (molecular weight 307.33, 60.0 mmol) protected by nitrogen, dissolve them in 120 mL of ultrapure water according to a molar ratio of 1:3, stir for 30 min by passing argon gas, heat to 45 °C and react for 2 h. After the reaction solution is cooled to 4 °C, add precooled methanol (-20 °C) for crystallization. Take 5.0 g of the primary crystal and dissolve it in 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, and dry it under reduced pressure (380 mmHg, 40 °C) in an adsorption dryer for 24 h to obtain a white solid powder of dimethylarsinic acid glutamine complex DMA III -SG. The obtained powder was verified by the analytical method of high performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The HPLC-ICP-MS analysis mass spectrum is as Figure 3 shown. In the mass spectrum, a broad-tailed outflow peak of DMA III can be clearly observed. The purity was analyzed by mass spectrometry software and found to be >99% (Note: The finally obtained white solid powder is DMA III -SG. When DMA III -SG is dissolved in water, it will rapidly hydrolyze and finally form dimethylarsinic acid DMA III ).

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

[0031]

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

[0033] Comparative Example 1

[0034] According to the preparation method of Example 1, until adding precooled methanol (-20 °C) for crystallization, only one crystallization was carried out, and the purity of the obtained DMA III -SG was verified.

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

[0036]

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

[0038] The DMA prepared in Comparative Example 1 III has a purity of only 90%. Since there is no subsequent second-step recrystallization process in Comparative Example 1, the product purity of Example 1 is superior to that of Comparative Example 1, indicating that recrystallization with methanol twice can achieve better technical effects.

[0039] Comparative Example 2

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

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

[0042]

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

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

[0045] Example 2

[0046] To further verify whether high-concentration DMA III causes cell apoptosis or autophagy, a CCK8 assay was performed. Normal immortalized keratinocytes (HaCaT cells) were prepared into a single-cell suspension and seeded in a 96-well plate at a cell density of 10,000 cells / well, and incubated in a cell culture incubator until the cell confluence reached 80%. The culture medium was discarded, and 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) of DMA III were added as the poisoned solution and cultured for 24 h. 10 μL of CCK8 working solution was added to each well, and then incubated in a cell culture incubator for 4 h. The absorbance value of each well was measured by an enzyme-labeled instrument at 450 nm. The cell survival rate was calculated according to the formula: cell survival rate = (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 Figure 4 shown. As the DMAIII With the increase of the dosing amount, the survival rate of human immortalized keratinocytes (HaCaT cells) showed a trend of first increasing and then decreasing. The cell survival rate was the highest at 0.1 μM, and then gradually decreased with the increase of the dosing amount (P<0.05).

[0048] The results showed that at high concentrations, DMA III had a greater inhibitory effect on cell proliferation than its ability to promote skin repair, so that DMA III was optimal for treating skin inflammation at 15 μM.

[0049] Example 3

[0050] 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 cell confluence reached 80%, the medium was removed, washed twice with phosphate buffered saline (PBS), and digested and centrifuged with 0.25% trypsin (containing ethylenediaminetetraacetic acid (EDTA)). The human immortalized keratinocytes (HaCaT cells) in the logarithmic phase were evenly inoculated into 96-well plates at a density of 1×10 4 cells / well, with 100 μL of cell suspension in each well, and incubated in an 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 survival rate was detected by the CCK-8 method, and the MMP-9 content was detected using a kit. Considering both the cell survival rate and the MMP-9 content, finally, the cells were stimulated with 200 μg / mL LPS solution for 24 h as the modeling condition.

[0051] The DMA prepared by the method of Example 1 III was prepared with deionized water into different concentrations (5.0 μM, 10 μM, 15 μM, and 20 μM) of DMA III and 1 μg / L of recombinant human epidermal growth factor (rhEGF) (positive control) were used to treat the LPS-induced damage model of human immortalized keratinocytes (HaCaT cells) for 48 h, and then the expression of MMP-9 protein in the cells was detected by the WB method. The results were as Figure 5 shown. In this dose range, the MMP-9 protein level in the DMA III treatment group was higher than that of other groups when the DMA III concentration was 15.0 μM (P<0.05).

[0052] MMP-9 can promote keratinocyte migration and accelerate wound re-epithelialization by degrading the extracellular matrix (ECM). Example 3 showed that DMAIII It may promote inflammatory repair by inhibiting the nuclear translocation of AP-1 or NF-κB.

[0053] Combined with Figure 4 Regarding cell viability:

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

[0055] Optimal concentration (15 μM): Achieves the best binding efficiency between the drug and the target, maximally activates the expression of MMP-9, and does not cause cytotoxicity at the same time.

[0056] High concentration (20 μM): May trigger a cellular stress response or inhibit the expression of MMP-9 through a negative feedback mechanism, resulting in a decrease in cell viability.

[0057] The results of Example 3 suggest that DMA III 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 15.0 μM DMA III is comparable to that of rhEGF (a clinically used pro-repair factor), suggesting its potential for alternative or synergistic applications.

[0058] Table 4 Changes in the expression of MMP-9 in the LPS-induced HaCaT injury model treated with different concentrations of DMA III Note: The number of times in the table is 9 repeated experiments.

[0059]

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

[0061] Example 4

[0062] 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 reached 80% confluence, the medium was removed, washed twice with PBS, and digested and centrifuged with 0.25% trypsin (containing EDTA). The HaCaT cells in the logarithmic phase were maintained at 1×10 4Cells were evenly inoculated at a density of [number] cells / well into a 96-well plate, with 100 μL of cell suspension in each well, and incubated in an 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 IL-8 content was detected using a kit. Considering both the cell viability and the IL-8 content, finally, cells were stimulated with 200 μg / mL LPS solution for 24 h as the modeling condition.

[0063] The DMA prepared by the method of Example 1 III DMA at different concentrations (5.0 μM, 10 μM, 15 μM, and 20 μM) was prepared with deionized water III and 1 μg / L rhEGF (positive control) were used to treat the LPS-induced injury model of human immortalized keratinocyte cell line (HaCaT cells) for 48 h, and the protein expression of the intracellular inflammatory marker IL-8 was detected by WB method. The results are as Figure 6 shown. In this dose range, the level of IL-8 protein in the DMA III treatment group was significantly decreased at 15.0 μM (P < 0.05).

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

[0065] Combined with Figure 4 the cell viability:

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

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

[0069] The results of Example 4 suggest that DMA III significantly downregulates IL-8 expression, confirming that it can regulate the skin inflammatory microenvironment, meeting the requirements of skin inflammation drugs to have both anti-inflammatory and repair-promoting functions. And the IL-8 level of 10.0 μM DMA III is comparable to that of rhEGF (a clinically used repair-promoting factor), indicating its potential for substitution or synergistic application.

[0070] Table 5 Different Concentrations of DMA III Changes in the Expression of IL-8 in the LPS-Induced HaCaT Injury Model Treated with Different Concentrations

[0071]

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

[0073] Example 5

[0074] To further verify that the toxicity is reduced after optimizing the synthesis route and it is more conducive to being used as a skin inflammation drug, a CCK8 assay was performed. Normal HaCaT cells were prepared into a single-cell suspension and seeded in a 96-well plate at a cell density of 10,000 cells / well, and incubated in a cell culture incubator until the cell confluence reached 80%. The culture medium was discarded, and different concentrations of 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) prepared with different concentrations (90% and 99%) were added respectively. III The poisoned liquid was cultured for 24 h. 10 μL of CCK8 working solution was added to each well, and then incubated in a cell culture incubator for 4 h. The absorbance value of each well was measured at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. The cell survival rate was calculated according to the formula: cell survival rate = (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 Figure 7 shown. When incubated with the poisoned liquid prepared with 99% pure DMA III the cell survival rate was significantly higher than that with the poisoned liquid prepared with 90% DMA III The possible reason is that at the same concentration, less DMA was used with higher purity, resulting in less toxic effect on cells. III

[0076] The results indicate that the toxicity of the dimethylarsinic acid glutamine complex described in the present invention is reduced after optimizing the synthesis route, and it is more conducive to being used as a skin inflammation drug.

Claims

1. A method for preparing a dimethylarsinic acid glutamine complex, characterized in that, It includes the following steps: Dissolve dimethylarsinic acid and glutathione protected by nitrogen in ultrapure water. After stirring by passing argon gas, heat for reaction. After the reaction solution is cooled, add pre-cooled methanol for crystallization reaction to obtain primary crystals. Dissolve the primary crystals in methanol / water for recrystallization, and obtain the dimethylarsinous acid glutamine complex after drying under reduced pressure.

2. The preparation method of the dimethylarsinic acid glutamine complex according to claim 1, characterized in that, The temperature of the pre-cooled methanol is -22~-18°C.

3. The preparation method of the dimethylarsinic acid glutamine complex according to claim 1, characterized in that, The temperature of the heating reaction is 42~48°C, the reaction time is 1~3 h, and the temperature for cooling the reaction solution is 3~5°C.

4. A dimethylarsinic acid glutamine complex, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the dimethylarsinous acid glutamine complex according to claim 4 in the preparation of a drug for treating skin inflammation.

6. The application according to claim 5, characterized in that The drug concentration is 10~20 μM.

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

8. The application according to claim 5, wherein 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, promoting extracellular matrix remodeling and cell migration.

10. The application according to claim 5, characterized in that, The drug is used for down-regulating the expression of interleukin-8 and inhibiting the activation of the nuclear factor κB or mitogen-activated protein kinase inflammatory signaling pathway.

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