Application of conjugated ketene skeleton bromophenol compound in preparation of anti-gout drugs

Abstract

The invention discloses an application of a conjugated ketene skeleton bromophenol compound in preparation of an anti-gout drug, the conjugated ketene skeleton bromophenol compound comprises BP-50 and BP-47, and experiments prove that the conjugated ketene skeleton bromophenol compound has remarkable inhibitory activity on xanthine oxidase, and can be used for preparing anti-gout drugs. The invention further studies the inhibition effect of the conjugated ketene skeleton bromophenol compound on MSU-induced inflammation taking NLRP3 inflammasome as a core, both BP-50 and BP-47 can inhibit secretion of MSU-induced characteristic inflammatory factors IL-1beta in a concentration-dependent manner, BP-50 down-regulates expression of NLRP3 inflammasome components by inhibiting an MAPK / NF-kappa B signal channel, and BP-47 can inhibit MSU-induced inflammation taking NLRP3 inflammasome components by inhibiting MAPK / NF-kappa B signal channel. The activation of the NLRP3 inflammasome is inhibited by reducing the ROS content, so that the activity of the NLRP3 inflammasome is reduced. It is proved that the conjugated ketene skeleton bromophenol compound can be used for preparing the anti-gout medicine, has a remarkable technical effect and has a wide market application prospect.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to application of a conjugated enone skeleton bromophenol compound in the preparation of anti-gout drugs. Background Art

[0002] Uric acid is the end product of purine metabolism in the human body. Endogenous purine nucleotides and exogenous purine nucleotides ingested through food are converted into uric acid in the liver through the action of a series of enzymes, among which xanthine oxidase (XOD) is the most critical enzyme for uric acid production. 2 / 3 of the generated uric acid is excreted through the kidneys and 1 / 3 through the intestines. Many transporters (such as URAT1 and ABCG2) mediate the excretion of uric acid in the kidneys and intestines. Under physiological conditions, the production and excretion of uric acid are in balance, and uric acid exists in the blood circulation in the form of soluble urate. Excessive intake of exogenous purines or impaired uric acid excretion in the body can lead to increased blood uric acid concentration. When the serum urate concentration is >7 mg / dL, it is called hyperuricemia. At this time, uric acid is close to the solubility limit under physiological conditions, and monosodium urate (MSU) crystals may precipitate, which are deposited in joints and surrounding tissues, activate NLRP3 inflammasomes in macrophages, trigger innate immune responses, and lead to intermittent episodes of severe painful arthritis, namely gout, which seriously affects the quality of life of patients. In addition, more and more clinical practice and basic research have shown that hyperuricemia and gout are closely related to the occurrence and development of a variety of diseases (such as acute and chronic kidney disease, diabetes, hypertension, etc.).

[0003] The main treatment strategy for gout attacks is analgesia and suppression of joint inflammation. Colchicine, nonsteroidal anti-inflammatory drugs and corticosteroids are first-line treatment drugs. However, the adverse reactions of the drugs themselves and the contraindications of comorbidities (such as nonsteroidal anti-inflammatory drugs are contraindicated in patients with kidney disease and heart disease) make clinical treatment options very scarce. The long-term treatment strategy for gout is uric acid-lowering therapy. Existing uric acid-lowering drugs are mainly divided into the following three categories: (1) XOD inhibitors, which are the first-line drugs for uric acid-lowering therapy, mainly including allopurinol and febuxostat, but both have serious adverse reactions such as life-threatening hypersensitivity syndrome and cardiovascular events; (2) Drugs that promote uric acid excretion, which are usually used as alternatives to uric acid-lowering therapy, include URAT1 inhibitors probenecid, benzbromarone and rasinade. In addition to urolithiasis as a common side effect, this type of drug also has a variety of prescription restrictions. For example, benzbromarone has been withdrawn from the market in most European markets due to its hepatotoxicity; (3) Recombinant uricases, which are the last-line drugs for refractory gout, include rasburicase and pegloticase. However, the high immunogenicity of this type of drug can induce anti-uricase antibodies and infusion reactions, which poses challenges to its effectiveness and safety. Therefore, although a variety of drugs have been approved for the treatment of gout, some patients still lack appropriate options. It is crucial to develop new anti-gout drugs with significant efficacy and good safety.

[0004] Bromophenol is a secondary metabolite of marine organisms, mainly found in seaweed, and can also be found in other marine organisms such as sponges and sea squirts. In terms of structure, bromophenol usually contains one or more benzene rings, as well as different numbers of hydroxyl, bromine or other groups. In the past few decades, bromophenol has been shown to have a wide range of biological activities, such as anti-inflammatory, antibacterial, anti-tumor, antioxidant and anti-diabetic, and has attracted the attention of many medical researchers. At the same time, since the content of naturally occurring bromophenol is usually low, its development and utilization are limited. At present, more research is mainly focused on the design and synthesis of new bromophenol derivatives. Structural modification of natural marine bromophenols with novel skeletons and (or) significant biological activities through synthetic and semi-synthetic routes to optimize their pharmacokinetic and pharmacodynamic parameters has become an important strategy for new drug development. Therefore, taking bromophenol and its derivatives as research objects, screening for anti-inflammatory activity and uric acid-lowering activity is expected to obtain new anti-gout drugs with multiple pharmacodynamics. Summary of the invention

[0005] The purpose of the present invention is to provide an application of a conjugated enone skeleton bromophenol compound in the preparation of an anti-gout drug. The present invention proves through experiments that the conjugated enone skeleton bromophenol compound has significant inhibitory activity on XOD and can inhibit MSU crystal-induced inflammation centered on NLRP3 inflammasome.

[0006] In order to solve the above technical problems, the technical solution to be adopted in the present invention is: The invention provides application of a conjugated enone skeleton bromophenol compound in preparing an anti-gout medicine.

[0007] Further: the conjugated ketene skeleton bromophenol compound includes BP-50 and / or BP-47, the structural formula of BP-50 is shown in Formula I, and the structural formula of BP-47 is shown in Formula II:

[0008] Formula I

[0009] Formula II.

[0010] Furthermore: the BP-50 and BP-47 can inhibit xanthine oxidase.

[0011] Furthermore: the inhibition type of the compound BP-50 on xanthine oxidase is reversible mixed inhibition, and the inhibition type of the compound BP-47 on xanthine oxidase is reversible mixed inhibition.

[0012] Furthermore, both BP-50 and BP-47 can inhibit the secretion of IL-1β, a characteristic inflammatory factor induced by MSU, in a concentration-dependent manner, and BP-50 can inhibit the secretion of IL-6 and TNF-α in a concentration-dependent manner.

[0013] Furthermore: the BP-50 inhibits the activity of NLRP3 inflammasome by inhibiting the expression of NLRP3 inflammasome components.

[0014] Furthermore: The compound BP-50 downregulates the expression of NLRP3 inflammasome components by inhibiting the MAPK / NF-κB signaling pathway.

[0015] Furthermore: the compound BP-50 can inhibit the assembly of NLRP3 inflammasome components.

[0016] The present invention also provides the use of a conjugated enone skeleton bromophenol compound in the preparation of an NLRP3 inflammasome inhibitor, wherein the conjugated enone skeleton bromophenol compound includes the BP-50 and / or the BP-47.

[0017] The present invention also provides the use of a conjugated enone skeleton bromophenol compound in the preparation of a drug having a uric acid-lowering effect, wherein the conjugated enone skeleton bromophenol compound includes the BP-50 and / or the BP-47.

[0018] Compared with the prior art, the present invention has the advantages and beneficial effects of: the present invention provides the use of a conjugated enone skeleton bromophenol compound in the preparation of an anti-gout drug, wherein the conjugated enone skeleton bromophenol compound includes BP-50 and / or BP-47, and the IC of BP-50 to XOD is confirmed by examples. 50 The IC value of BP-47 for XOD is 3.06 μM. 50 The inhibition rate of BP-50 on XOD was 6.78 μM. Enzyme kinetics studies showed that the inhibition type of BP-50 on XOD was reversible mixed inhibition, and the inhibition type of BP-47 on XOD was reversible mixed inhibition.

[0019] The present invention also studies the inhibitory effect of the conjugated enone skeleton bromophenol compound on MSU-induced inflammation centered on the NLRP3 inflammasome. Both BP-50 and BP-47 can inhibit the secretion of the characteristic inflammatory factor IL-1β induced by MSU in a concentration-dependent manner. BP-50 downregulates the expression of NLRP3 inflammasome components by inhibiting the MAPK / NF-κB signaling pathway, and inhibits the activation of NLRP3 inflammasome by reducing the ROS content, which together lead to a decrease in the activity of NLRP3 inflammasome. It is proved that the conjugated enone skeleton bromophenol compound can be used to prepare anti-gout drugs, has significant technical effects, and has broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0021] Figure 1 IC of XOD for BP-50 and BP-47 50 value; Figure 2 The results of enzyme kinetic studies of XOD by BP-50 and BP-47 are shown respectively; Figure 3 The results of the effects of BP-50 and BP-47 on the secretion of inflammatory factors are shown in the figure; Figure 4 This is a graph showing the effect of BP-50 on the expression of NLRP3 inflammasome components; Figure 5 This is the result diagram of the effect of BP-50 on the expression of proteins related to the MAPK / NF-κB signaling pathway; Figure 6 This is the result diagram of the effect of BP-50 on ROS content. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with embodiments. The following embodiments are for explanation of the present invention but the present invention is not limited to the following embodiments.

[0023] The invention provides application of a conjugated enone skeleton bromophenol compound as an anti-gout drug. The conjugated enone skeleton bromophenol compound comprises BP-50 and / or BP-47.

[0024] In the present invention, the chemical name of BP-50 is (1E, 4E)-1,5-bis(2-bromo-4,5-dihydroxyphenyl) penta-1,4-dien-3-one (in Chinese), (1E, 4E)-1, 5-bis(2-bromo-4,5-dihydroxyphenyl) penta-1, 4-dien-3-one (in English), and its synthesis method and structural information refer to "

[0061] Preparation Example 8: Preparation of 5b-8" of the patent "Conjugated enone skeleton bromophenol compound and preparation method and anti-tumor application" (patent application number CN202410165534.3), and the structural formula is shown in Formula I:

[0025] Formula I The chemical name of BP-47 is (1E, 4E)-1,5-bis(2-bromo-3-hydroxy-4-methoxyphenyl) penta-1,4-dien-3-one (Chinese), (1E, 4E)-1, 5-bis(2-bromo-3-hydroxy-4-methoxyphenyl) penta-1, 4-dien-3-one (English). Its synthesis method and structural information refer to "

[0048] Preparation Example 5: Preparation of 5b-5" of the patent "Conjugated enone skeleton bromophenol compound and preparation method and anti-tumor application" (patent application number CN202410165534.3). The structural formula is shown in Formula II:

[0026] Formula II.

[0027] In the examples of the present invention, by studying the biological activities and mechanisms of action of BP-50 and BP-47 on XOD and NLRP3 inflammasomes, it was demonstrated that the compounds have significant inhibitory activity on both XOD and NLRP3 inflammasomes.

[0028] The present invention also provides the use of BP-50 and / or BP-47 in preparing a medicament having a uric acid-lowering effect.

[0029] The present invention also provides the use of BP-50 and / or BP-47 in preparing a medicament for relieving acute attacks of gout.

[0030] The application of the conjugated enone skeleton bromophenol compound provided by the present invention as an anti-gout drug is further described in detail below in conjunction with the test examples, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Unless otherwise specified, the reagents and raw materials involved in the present invention can be obtained from commercial channels.

[0032] The experimental materials, reagents and instruments involved in the embodiments of the present invention include: Xanthine oxidase was purchased from Beijing Solebow Technology Co., Ltd.; Xanthine was purchased from Beijing Solebow Technology Co., Ltd.; 96-well cell culture plates were purchased from Zhejiang Biodi Biotechnology Co., Ltd.; Microplate reader, purchased from Molecular Devices, model: SpectraMax i3x; J774A.1 cells were provided by the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences; 1640 culture medium, purchased from Shanghai Yuanpei Biotechnology Co., Ltd.; Fetal bovine serum, purchased from PAN-Seratech; Cell culture dishes were purchased from Shuohua; 24-well cell culture plates were purchased from Zhejiang Zhongzai Medical Technology Co., Ltd.; Uric acid, purchased from Maclean; LPS was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.; IL-1β, IL-6, and TNF-α content detection kits were purchased from Shenzhen Dakoway Biotechnology Co., Ltd.

[0033] NLRP3, ASC, Caspase-1, p-p38, p-JNK, p-ERK1 / 2, p-p65, JNK, and ERK1 / 2 antibodies were purchased from Cell Signaling Technology.

[0034] p65 and p38 antibodies were applied to Santa Cruz Biotechnology.

[0035] GAPDH antibody was purchased from Hangzhou Huaan Biotechnology Co., Ltd.

[0036] ROS detection kit was purchased from Shanghai Biotech Biotechnology Co., Ltd.

[0037] Example 1: IC of compounds BP-50 and BP-47 for inhibiting XOD 50 Determination The reaction volume was set to 200 μL, 98 μL XOD working solution and 2 μL active compound (DMSO was used as solvent control) were added, the final concentration gradient of BP-50 was 10, 5, 2.5, 1.25, 0.625, 0.3125 μM, and the final concentration gradient of BP-47 was 20, 10, 5, 2.5, 1.25, 0.625 μM, and incubated at 37 °C for 15 min. 100 μL substrate xanthine solution was added and immediately placed in the microplate reader. The absorbance of the system at 295 nm was detected at 37 °C, and the detection was performed every 1 min for 10 min. According to the relative enzyme activity (%) = compound group △OD 295 nm ÷Control group△OD 295 nm ×100 was used for calculation and IC was calculated using Graphpadprism 5 software 50 Fitting.

[0038] The results are as follows Figure 1 As shown, the IC of compound BP-50 for XOD 50 The value is 3.06 μM, and the IC of compound BP-47 for XOD 50 The value is 6.78 μM.

[0039] Example 2: Enzyme kinetics study of compounds BP-50 and BP-47 on XOD Reversibility detection: Set the reaction volume to 200 μL, add 98 μL XOD working solution, 2 μL active compound (DMSO as solvent control), the final concentration gradient of active compound is 5, 2.5, 1.25 μM, for the same concentration of compound, set different XOD concentration gradients, incubate at 37 ℃ for 15 min, add 100 μL substrate xanthine solution, immediately put into the microplate reader, detect the absorbance of the system at 295 nm at 37 ℃, detect once every 1 min, and continue to detect for 10 min. According to the reaction rate = (T2 OD 295 nm -T1 OD 295 nm )÷△T was used for calculation, with XOD concentration as the horizontal axis and reaction rate as the vertical axis, and Graphpad prism 5 software was used to draw the relationship curve under the action of different concentrations of compounds.

[0040] Competitive assay: Set the reaction volume to 200 μL, add 98 μL XOD working solution, 2 μL active compound (DMSO as solvent control), the final concentration gradient of active compound is 5, 2.5, 1.25 μM, incubate at 37 °C for 15 min, add 100 μL substrate xanthine solution, set different xanthine concentration gradients for compounds of the same concentration, immediately put into the microplate reader, detect the absorbance of the system at 295 nm at 37 °C, detect once every 1 min, and continue to detect for 10 min. According to the reaction rate = (T2 OD 295 nm -T1 OD 295 nm )÷△T was used for calculation, with the reciprocal of xanthine concentration as the horizontal axis and the reciprocal of reaction rate as the vertical axis. Graphpad prism 5 software was used to draw the Lineweaver-Burk curve under the action of different concentrations of compounds.

[0041] The results are as follows Figure 2 As shown, the inhibition type of compound BP-50 on XOD is reversible mixed inhibition, and the inhibition type of compound BP-47 on XOD is reversible mixed inhibition.

[0042] Example 3: Effects of Compounds BP-50 and BP-47 on MSU Crystal-Induced Inflammation J774A.1 cells were inoculated into 24-well plates. When the cells reached 70% confluence, the model group and the compound treatment group were stimulated with LPS combined with MSU crystals. The compound treatment group was added with the corresponding active compounds, with the final concentration gradient of BP-50 being 1, 2, and 4 μM, and the final concentration gradient of BP-47 being 0.4, 0.8, and 1.6 μM. The cells were incubated for 24 h, and the supernatant was collected. The contents of IL-1β, IL-6, and TNF-α were detected by ELISA kits.

[0043] The results are as follows Figure 3 As shown in the results, both BP-50 and BP-47 can inhibit the secretion of IL-1β, a characteristic inflammatory factor induced by MSU, in a concentration-dependent manner. At the same time, BP-50 can inhibit the secretion of IL-6 and TNF-α in a concentration-dependent manner. The above results indicate that both BP-50 and BP-47 can inhibit the activation of NLRP3 inflammasome induced by MSU, thereby exerting an anti-inflammatory effect.

[0044] Example 4: Effect of compound BP-50 on the expression of NLRP3 inflammasome components J774A.1 cells were seeded into six-well plates. When the cells reached 70% confluence, the model group and the compound treatment group were stimulated with LPS combined with MSU crystals. The compound treatment group was added with BP-50 at a final concentration gradient of 1, 2, and 4 μM, respectively. The cells were incubated for 24 h, and proteins were extracted. Western Blot was used to detect the levels of NLRP3 inflammasome components NLRP3, ASC, and pro-caspase-1.

[0045] The results are as follows Figure 4 As shown in the data, compared with the control group, the model group could significantly upregulate the expression of NLRP3, a component of the NLRP3 inflammasome, and had no significant effect on the expression of ASC and pro-caspase-1. However, after co-treatment with BP-50, the expression of NLRP3 was significantly downregulated, indicating that BP-50 inhibits the activity of NLRP3 inflammasome by inhibiting the expression of NLRP3 inflammasome components.

[0046] Example 5: Effect of compound BP-50 on MAPK / NF-κB signaling pathway J774A.1 cells were seeded into six-well plates. When the cells reached 70% confluence, the model group and the compound treatment group were stimulated with LPS combined with MSU crystals. The compound treatment group was added with BP-50 at a final concentration gradient of 1, 2, and 4 μM, respectively. The cells were incubated for 24 h, and proteins were extracted. Western Blot was used to detect the levels of p38 / p-p38, ERK / p-ERK, JNK / p-JNK, and p65 / p-p65, proteins related to the MAPK / NF-κB signaling pathway.

[0047] like Figure 5 As shown in the figure, compared with the control group, the model group can significantly upregulate the phosphorylation level of proteins related to the MAPK / NF-κB signaling pathway, indicating that the MAPK / NF-κB signaling pathway is significantly activated, and after the intervention of compound BP-50, the phosphorylation levels of p38 MAPK, JNK MAPK and p65 are significantly downregulated, indicating that the pathway is inhibited. Given that NF-κB activation is the main signal mediating the expression of NLRP3 inflammasome components, the above results can indicate that compound BP-50 downregulates the expression of NLRP3 inflammasome components by inhibiting the MAPK / NF-κB signaling pathway.

[0048] Example 6: Effect of compound BP-50 on ROS J774A.1 cells were inoculated into 96-well plates. When the cells reached 70% confluence, the model group and the compound treatment group were stimulated with LPS combined with MSU crystals. The compound treatment group was added with BP-50 at a final concentration gradient of 1, 2, and 4 μM, respectively, and incubated for 24 hours. After the incubation, the culture medium was discarded, 100 μL DCFH-DA was added to each well, and the incubation continued for 20 min. The cells were washed three times with 1640 culture medium and immediately placed in a microplate reader to detect the fluorescence value (excitation light 488 nm, emission light 525 nm).

[0049] like Figure 6 As shown in the figure, compared with the control group, the ROS level in the model group was significantly increased, while after the intervention of compound BP-50, the ROS level was significantly downregulated. Given that ROS is an important factor in activating the NLRP3 inflammasome, the above results show that compound BP-50 can not only inhibit the NLRP3 inflammasome by downregulating the expression of NLRP3 inflammasome components, but also inhibit its assembly process, further inhibiting its activity.

[0050] In addition, it should be noted that the shapes and names of the parts and components of the specific embodiments described in this specification may be different. Any equivalent or simple changes made based on the structure, features and principles described in the patent concept of the present invention are included in the protection scope of the patent of the present invention. The technicians in the technical field of the present invention can make various modifications or supplements to the specific embodiments described or replace them in a similar manner, as long as they do not deviate from the structure of the present invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

Claims

1. Application of conjugated enone skeleton bromophenol compounds in the preparation of anti-gout drugs.

2. The use according to claim 1, characterized in that: The conjugated ketene skeleton bromophenol compound includes BP-50 and / or BP-47. The structural formula of BP-50 is shown in Formula I, and the structural formula of BP-47 is shown in Formula II: ， Formula I, ， Formula II.

3. The use according to claim 2, characterized in that: The BP-50 and BP-47 can inhibit xanthine oxidase.

4. The use according to claim 3, characterized in that: The inhibition type of the compound BP-50 on xanthine oxidase is reversible mixed inhibition, and the inhibition type of the compound BP-47 on xanthine oxidase is reversible mixed inhibition.

5. The use according to claim 2, characterized in that: Both BP-50 and BP-47 can inhibit the secretion of IL-1β, a characteristic inflammatory factor induced by MSU, in a concentration-dependent manner, and BP-50 can inhibit the secretion of IL-6 and TNF-α in a concentration-dependent manner.

6. The use according to claim 2, characterized in that: The BP-50 inhibits the activity of NLRP3 inflammasome by inhibiting the expression of NLRP3 inflammasome components.

7. The use according to claim 6, characterized in that: The compound BP-50 downregulates the expression of NLRP3 inflammasome components by inhibiting the MAPK / NF-κB signaling pathway.

8. The use according to claim 1, characterized in that: The compound BP-50 can inhibit the assembly of NLRP3 inflammasome components.

9. Application of conjugated enone skeleton bromophenol compounds in the preparation of NLRP3 inflammasome inhibitors.

10. Application of conjugated enone skeleton bromophenol compounds in the preparation of drugs with uric acid lowering effect.

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