Synthesis and application of water-soluble organic nanotube with uric acid resistance

Abstract

The invention discloses synthesis and application of a water-soluble organic nanotube with an anti-uric acid effect, and belongs to the field of organic synthesis. The water-soluble organic covalent nanotube is successfully prepared by taking hepta (6-O-tert-butyldimethylsilyl)-beta-cyclodextrin and 1, 3-bis (bromomethyl) benzene as initial raw materials through multi-step reaction and functional group modification. The synthesis method is simple in step, a convenient way is provided for constructing a cyclodextrin-based covalent organic nanotube skeleton, the obtained nanotube structure has a complete hydrophobic cavity and good water solubility and biological safety, the cylindrical cavity structure can be combined with sodium urate molecules in a physiological environment, and by inhibiting deposition of urate crystal tissue, the stability of the nanotube structure is improved. The inflammatory response of the sodium urate enriched part is reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of organic synthesis, and particularly relates to synthesis and application of water-soluble organic nanotubes with anti-uric acid effect. BACKGROUND

[0002] Hyperuricemia is a metabolic disease caused by disorder of purine metabolism, which leads to abnormal increase of uric acid level in blood. It has become the "fourth high" after hypertension, hyperglycemia and hyperlipidemia. Uric acid is the end product of purine metabolism in the human body. Its excessive accumulation in the body can form urate crystals and deposit in joints, soft tissues and kidneys, etc., causing gouty arthritis, and ultimately causing joint deformities such as "telescope hand" and "claw toe", and losing basic activity. In addition, hyperuricemia can form a vicious cycle with metabolic syndrome, induce hypertension, diabetes, accelerate atherosclerosis and other adverse effects, and form a network of multi-organ tissue failure of joints, kidneys, blood vessels and metabolism. Therefore, it has become an urgent task in the field of biological medicine to develop efficient anti-uric acid drugs and treatment methods. The currently used drugs such as allopurinol and febuxostat have different degrees of toxic side effects, and it is urgent to develop safe and effective anti-uric acid drugs.

[0003] As a new class of supramolecular macrocyclic host compounds, organic nanotubes have shown broad application potential in material separation and purification, catalytic conversion, biological medicine and other fields due to their diversified structures, easy functional modification, good biocompatibility and excellent host-guest recognition performance. In particular, in the direction of biological active small molecule antagonists, such compounds show unique advantages, and the successful application of succinylcholine sodium is a typical example. It has been widely used in clinical practice to reverse the effects of muscle relaxants. However, for the core factor of gout, uric acid, the development of supramolecular antagonists for it is still relatively weak. The few existing inhibitors reported often have the problem of high biological toxicity, which greatly limits their clinical transformation and application.

[0004] Therefore, on the basis of the prior art, it is of great value to develop and synthesize new organic nanotubes with high efficiency and low toxicity for efficient removal of high concentration of uric acid sodium in tissues to promote the treatment of gout, and it is also a technical problem to be solved in the field. The synthesis of water-soluble organic nanotubes with anti-uric acid effect develops a new therapy, which has very important theoretical significance and practical prospect in the fields of anti-inflammatory and anti-gout. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application aims to provide a water-soluble organic nanotube, and also provides a synthesis method and application thereof in resisting uric acid.The water-soluble organic nanotube is synthesized by using seven (6-O-tert-butyldimethylsilyl)-beta-cyclodextrin and 1,3-bis(bromomethyl)benzene as raw materials, and has the advantages of mild reaction conditions and easily available raw materials; in addition, the compound can selectively bind sodium urate, and provides a new idea for the treatment of diseases caused by high uric acid, and has potential medicinal value in the fields of anti-inflammation and anti-gout.

[0006] The water-soluble organic nanotube has a general structure as follows: , In the formula, R is selected from SC2H4SO3Na or SC3H4O2Na.

[0007] The present application also provides a preparation method of the water-soluble organic nanotube, which is prepared by using the following three-step reaction. .

[0008] Further, in the above scheme, the first step reaction: compound 4 and 1,3-bis(bromomethyl)benzene are used as raw materials, sodium hydride and 1,4-dioxane are used, and the reaction is carried out by heating to reflux to obtain compound 5.

[0009] Further, in the above scheme, the second step reaction: compound 5 is reacted in dichloromethane by refluxing in the presence of bromine and triphenylphosphine to obtain compound 6.

[0010] Further, in the above scheme, the third step reaction: compound 6 is respectively reacted with 2-sodium mercaptoethanesulfonate and 3-sodium mercaptopropionate in N-methylpyrrolidone and sodium hydroxide by heating to 60-90 DEG C to obtain water-soluble organic nanotube 7 or 8.

[0011] The present application also provides application of the water-soluble organic nanotube in preparing anti-uric acid drugs.

[0012] Further, in the above scheme, the anti-uric acid is achieved by combining sodium urate with the water-soluble organic nanotube molecules to realize the inhibitory effect on tissue inflammation. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 It is an isothermal titration calorimetry test result graph of compound 7; Figure 2 It is a fitting graph of the isothermal titration calorimetry test result of compound 7; Figure 3 It is a cytotoxicity experiment test result graph of compound 7. DETAILED DESCRIPTION

[0014] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Example 1

[0015] , Under nitrogen protection, 1.24 g of sodium hydride (NaH, 60% mineral oil dispersion) was washed twice with 80 mL of n-hexane. 5.0 g of dried hepta(6-O-tert-butyldimethylsilyl)-β-cyclodextrin dissolved in 200 mL of 1,4-dioxane was added to the system, and the mixture was refluxed at 100 °C for 1 h. Subsequently, 3.38 g of 1,3-bis(bromomethyl)benzene dissolved in 40 mL of 1,4-dioxane was slowly added to the reaction system, and the mixture was refluxed again for 12 h. After the reaction was complete, methanol was added to quench the reaction, and the solvent was removed under vacuum. The residue was dissolved in 300 mL of chloroform and washed three times with saturated brine. After drying with anhydrous sodium sulfate and concentrating under reduced pressure, the residue was purified by silica gel column chromatography using n-hexane / ethyl acetate (30 / 1-10 / 1) as eluent to give compound 5 in 20% yield. 1 H NMR (400 MHz, CDCl3) δ 7.72 (s, 7H), 7.29-7.25 (m, 7H), 7.11 (m, 14H), 5.23 (d, J = 12.0 Hz, 14H), 4.88 (s, 14H), 4.80 (d, J = 3.6 Hz, 14H), 4.20-4.11(m, 28H), 3.97-3.94 (m, 14H), 3.66-3.57 (m, 28H), 3.41 -3.34 (m, 28H), 0.85(s, 126H), 0.01 (d, J = 8.0, 84H). HRMS: calcd for C 224 H 378 O 70 Si 14 Na₂[M+2Na] 2+ :2315.1323, found 2315.1321. Example 2

[0016] , Under nitrogen protection, 450 mg of triphenylphosphine (PPh3) was added to 10 mL of ultra-dry dichloromethane solution. Then, 88 μL of bromine (Br2) was added dropwise in an ice bath, stirring until a white precipitate appeared. 200 mg of compound 5 was dissolved in 5 mL of ultra-dry dichloromethane and added to the reaction system. The mixture was heated to reflux for 1.5 h. After the reaction was complete, the solvent was removed under vacuum, and the product was purified by silica gel column chromatography using hexane / dichloromethane (1 / 3) as the eluent to give the target product 6 in 30% yield. 1 H NMR (400 MHz, CDCl3) δ 7.67 (s, 7H), 7.30 (t, J = 8.0 Hz, 7H), 7.12 (d, J = 8.0 Hz, 14H), 5.23(d, J = 12.0 Hz, 14H), 4.94 (d, J = 4.0 Hz, 14H), 4.87(s, 14H), 4.18-4.13 (m,28H), 3.91-3.87 (m, 28H), 3.72 -3.67(m, 14H), 3.49-3.46 (m, 14H), 3.28-3.23(m, 14H). HRMS: calcd for C 140 H 168 Na2O 56 Br 14 [M+2Na] 2+ : 1955.9278, found1955.9282. Example 3

[0017] , Under nitrogen protection, 100 mg of compound 6 was dissolved in 3 mL of anhydrous N-methylpyrrolidone (NMP). After dissolution, 127 mg of sodium 2-mercaptoethanesulfonate and 83 mg of sodium hydroxide were added sequentially, and the mixture was heated to 70 °C and stirred for 24 h. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was washed three times with 30 mL of acetone. The precipitate was collected by filtration and dried under vacuum at 80 °C to give compound 7, with a yield of 33%. 1 H NMR (600 MHz, D2O) δ 7.63 (s, 7H), 7.38 (t, J = 6.0 Hz, 7H), 7.27 (d, J = 6.0 Hz, 14H), 5.12 (s, 14H), 4.95 (d, J= 12.0 Hz, 7H), 4.37(d, J = 12.0 Hz, 7H), 4.21 (t, J = 12.0 Hz, 14H), 4.10 (s, 14H), 3.58-3.52(m,28H), 3.23-3.12 (m, 56H), 3.02-2.93(m, 56H). HRMS: calcd for C 168 H 224 Na 16 O 84 S 14 [M+2Na] 2+ : 2526.6624, found 2526.6629. Example 4

[0018] , Under nitrogen protection, 100 mg of compound 6 was dissolved in 3 mL of anhydrous N-methylpyrrolidone (NMP). After dissolution, 100 mg of sodium 3-mercaptopropionate and 83 mg of sodium hydroxide were added sequentially, and the mixture was heated to 70 °C and stirred for 24 h. After the reaction was completed, the mixture was diluted with 30 mL of water, and hydrochloric acid was added to pH=2. The precipitate was collected by filtration and dried under vacuum at 80 °C to give compound 8, with a yield of 30%. 1 H NMR (600 MHz, D2O) δ 7.59 (s, 7H), 7.35 (t, J = 6.0 Hz, 7H), 7.23(d, J = 6.0 Hz, 14H), 5.06 (d, J = 3.6 Hz, 14H), 4.91 (d, J = 6.0 Hz, 14H), 4.33(d, J = 12.0 Hz, 14H), 4.16 (t, J = 12.0 Hz, 14H), 4.00 (s, 14H), 3.54-3.52 (m,14H), 3.45 (t, J = 6.0 Hz, 14H), 3.12 (d, J = 12.0 Hz, 14H), 2.98-2.95 (m, 14H), 2.81-2.79 (m, 42H), 2.49-2.45 (m, 28H).HRMS: calcd for C 182 H 224 Na16 O 84 S 14 [M+ 2Na] 2+ : 2296.8854, found 2296.8862. Example 5

[0019] Isothermal titration calorimetry for detecting binding interactions: Taking compound 7 as an example, in the ITC experiment, 200 μM sodium urate solution was injected into the sample cell of the ITC instrument, and 4 μM of compound 7 solution was loaded into the titration needle. The experimental temperature was set to 25 °C, the stirring speed to 750 rpm, and the titration volume to be 2 μL each time, for a total of 19 titrations, with an interval of 180 s between titrations. The instrument was started to begin titration, and it automatically recorded the heat change generated during each titration. After titration, the raw data was processed using ITC analysis software, and a binding isotherm was obtained by fitting a suitable binding model, thereby obtaining thermodynamic parameters such as binding constant, stoichiometry, and binding enthalpy change. Figure 1 and 2 As shown, compound 7 exhibits a specific binding interaction with uric acid analogues. The binding process is exothermic and spontaneous, with moderate affinity, consistent with the single binding site model.

[0020] . Example 6

[0021] Cell culture: Mouse macrophages RAW264.7 were cultured in DMEM high glucose medium containing 10% (v / v) FBS, 100 U / mL penicillin and streptomycin, and were placed in an incubator at 37°C, saturated humidity, and containing 5% (v / v) CO2. Example 7

[0022] CCK8 assay for cell viability: Taking compound 7 as an example, RAW264.7 cells were seeded at a density of 5000 / well in 96-well plates. After treatment with different drug concentrations for 24 h, 10 μL of CCK8 was added to each well, and the cells were incubated in an incubator for 2 h. The absorbance was then measured at 450 nm using a microplate reader. Figure 3 As shown, cell viability was not significantly affected with increasing concentrations of compounds 7 and 8, demonstrating that they have almost no toxicity to cells at a concentration of 40 µM. Example 8

[0023] RT-qPCR: Taking compound 7 as an example, target gene-specific primers were designed using Primer Premier 5.0 software (Table 1) and synthesized by Sangon Biotech (Shanghai) Co., Ltd. Total RNA was isolated from macrophages using the RNA-Quick purification kit (ESScience, China). Using the obtained RNA as a template, cDNA was obtained by reverse transcription according to the instructions of the Hiscript III first-strand cDNA synthesis kit (Vazyme, China). RT-qPCR was performed using Taq Pro UniversalSYBR qPCR Master Mix (Vazyme, China). Each sample was replicated in triplicate, with GAPDH as an internal control. 2^ (-△△CT) The corresponding CT values ​​were calculated using a method, and the experiment was repeated three times. Experimental verification showed that the addition of compounds 7 and 8 significantly reduced the expression level of anti-inflammatory factors by more than 20%, indicating that the molecules achieved an inhibitory effect on tissue inflammation by binding to sodium urate.

[0024] Table 1 lists the specific primers for designing the target gene. .

[0025] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Water-soluble organic nanotubes, characterized in that, Its general structural formula is: ， Wherein: R is selected from SC2H4SO3Na or SC3H4O2Na.

2. The method for synthesizing water-soluble organic nanotubes as described in claim 1, characterized in that, Includes the following steps: 。 3. The method for synthesizing water-soluble organic nanotubes according to claim 2, characterized in that: The first step involves reacting compound 4 and 1,3-bis(bromomethyl)benzene as raw materials in sodium hydride and 1,4-dioxane at reflux to obtain compound 5.

4. The method for synthesizing water-soluble organic nanotubes according to claim 2, characterized in that: The second step involves reacting compound 5 and bromine in dichloromethane under reflux in the presence of triphenylphosphine to obtain compound 6.

5. The method for synthesizing water-soluble organic nanotubes according to claim 2, characterized in that: The third step involves reacting compound 6 with sodium 2-mercaptoethanesulfonate and sodium 3-mercaptopropionate in N-methylpyrrolidone and sodium hydroxide at 60-90°C to obtain water-soluble organic nanotubes 7 or 8.

6. The application of the water-soluble organic nanotubes as described in claim 1 in the preparation of antiuric acid drugs.

7. The application of the water-soluble organic nanotubes according to claim 6 in the preparation of anti-uric acid drugs, wherein the anti-uric acid effect is achieved by the water-soluble organic nanotube molecules binding sodium urate to achieve the inhibitory effect on tissue inflammation.

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