Use of a compound for the preparation of a medicament for the removal of protein-bound uremic toxins from blood

Abstract

The application belongs to the technical field of biological medicine, and more particularly relates to application of a compound in preparation of a medicine for removing protein-bound uremic toxins in blood. The compound has a structural general formula shown in formula (I): wherein R1 is an aromatic heterocycle with a carbon number of 6-50, a benzenepropyl aromatic heterocycle with a carbon number of 6-50, or an aromatic hydrocarbon with a carbon number of 6-50; and R2 is a flavonoid group with a carbon number of 13-50, a polyphenol group, or a terpenoid group. Experiments show that the compound has a competitive replacement removal effect significantly better than that of the currently best replacement medicine, shikonin acid (LA), when the compound is used to remove protein-bound uremic toxins (PBUTs) such as IS and PCS by using a competitive replacement method, and the advantage is more obvious at a low concentration.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to the application of compounds in the preparation of drugs that remove protein-bound uremic toxins from the blood. Background Technology

[0002] Chronic kidney disease (CKD) is widely recognized as a major global public health problem. The global prevalence of CKD is approximately 14%, with about 49.02 to 70.83 million patients with end-stage renal disease (ESKD) requiring renal replacement therapy. In CKD patients, kidney disease or impaired function leads to the accumulation of uremic toxins in the body that would normally be removed through glomerular filtration or tubular secretion. These uremic toxins are broadly classified into three categories: (1) small toxins (<500 Da); (2) medium and large uremic toxins (>500 Da); and (3) protein-bound uremic toxins (PBUTs). Small, medium, and large uremic toxins can be effectively removed through hemodialysis. However, PBUTs, due to their binding to albumin, have a molecular weight that is too large to be removed through the dialysis membrane, eventually accumulating in the body. Albumin transports these PBUTs to various parts of the body, and then through PBUT transporters OAT or OCT to the kidneys, central nervous system, osteoblasts, vascular endothelial cells, etc., inducing uremia and its complications, including vascular damage, bone disease, central nervous system dysfunction, chronic ischemic heart disease, physiological oxidative stress, etc., ultimately leading to death.

[0003] PBUTs mainly include indophenol sulfate (IS), p-formyl sulfate (PCS), 3-carboxy-4-methyl-5-propyl-2-furan propionate (CMPF), and indole-3-acetic acid (IAA). IS, PCS, and IAA bind to site II of albumin, while CMPF binds to site I. Furthermore, IS and PCS are two of the most harmful toxins in end-stage renal disease, but due to their albumin binding, they are difficult to remove using traditional hemodialysis (HD) techniques.

[0004] Currently, the most effective methods for removing PBUTs mainly include displacement and adsorption methods. Displacement methods primarily involve injecting drugs such as ibuprofen, fatty acids, and shikonin (LA) into the bloodstream. These drugs bind to albumin I or II sites, thereby increasing the free form of PBUTs and promoting their removal. Adsorption methods mainly involve adding adsorbents such as activated charcoal, albumin, liposomes, and multi-site polycyclodextrin (PβCD) to the dialysate to adsorb free PBUTs, maintain the diffusion gradient of PBUTs, and promote their removal. In addition, other technologies for removing PBUTs, such as developing novel dialysis membranes, are also under development. Competitive displacement is an effective means of removing PBUTs, but current displacement agents still have drawbacks such as low efficiency and significant side effects. Therefore, developing novel displacement agents to improve the removal rate of PBUTs and prolong the lives of ESKD patients is extremely important. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide the application of a class of compounds in the preparation of drugs for removing protein-bound uremic toxins from the blood, so as to solve the technical problems of low efficiency and large side effects of the existing displacement method for removing PBUTs.

[0006] To achieve the above objectives, the present invention provides the use of the compound represented by formula (I) in the preparation of a medicament for clearing protein-bound uremic toxins from the blood.

[0007]

[0008] Wherein, R1 is an aromatic heterocycle with 6-50 carbon atoms, a phenylpropanoid aromatic heterocycle with 6-50 carbon atoms, or an aromatic hydrocarbon with 6-50 carbon atoms; wherein the aromatic heterocycle is pyrroleyl, furanyl, thiopheneyl, pyridinyl, pyridazinyl, pyrazinyl, pyranoneyl, imidazolyl, pyrazolyl, thiazolyl, or oxazolyl, etc.; the phenylpropanoid aromatic heterocycle is indolyl, benzofuranyl, benzothiopheneyl, or quinolinyl, etc.; the aromatic hydrocarbon is phenyl, naphthyl, anthraceneyl, or phenanthrene, etc.; R2 is a flavonoid group, polyphenol group, or terpene group with 13-50 carbon atoms, etc.

[0009] Preferably, R1 is an aromatic heterocycle, phenylpropionic aromatic heterocycle or aromatic hydrocarbon with 6-30 carbon atoms, more preferably an aromatic heterocycle or aromatic hydrocarbon with 6-20 carbon atoms; R2 is a flavonoid group or terpene group with 13-30 carbon atoms, more preferably a flavonoid group or terpene group with 13-20 carbon atoms.

[0010] Preferably, the uremic toxin is one or more of indophenol sulfate, p-formyl sulfate, 3-carboxy-4-methyl-5-propyl-2-furan propionate, and indole-3-acetic acid.

[0011] Preferably, when applied, the drug is in the form of an injection.

[0012] According to another aspect of the invention, a dialysate for removing uremic toxins from blood via dialysis displacement is provided, comprising the compound shown in formula (I).

[0013]

[0014] Wherein, R1 is an aromatic heterocycle with 6-50 carbon atoms, a phenylpropanoid aromatic heterocycle with 6-50 carbon atoms, or an aromatic hydrocarbon with 6-50 carbon atoms; wherein the aromatic heterocycle is pyrroleyl, furanyl, thiopheneyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyranoneyl, imidazolyl, pyrazolyl, thiazolyl, or oxazolyl, etc., the phenylpropanoid aromatic heterocycle is indolyl, benzofuranyl, benzothiopheneyl, or quinolinyl, etc., and the aromatic hydrocarbon is phenyl, naphthyl, anthraceneyl, or phenanthrene, etc.; R2 is a flavonoid group, polyphenolic group, or terpene group with 13-50 carbon atoms, etc.

[0015] Preferably, the effective concentration of the compound represented by formula (I) in the dialysate is 100-500 μM, more preferably 150-400 μM.

[0016] Preferably, the dialysate is an injectable dialysate.

[0017] Preferably, it also includes a dialysis perfusion solution, wherein the dialysis perfusion solution is a compound sodium chloride solution.

[0018] In summary, compared with the prior art, the above-described technical solutions conceived by this invention have the following advantages:

[0019] Beneficial effects:

[0020] This invention provides the use of a class of compounds in the preparation of drugs for clearing protein-bound uremic toxins from the blood, and dialysates containing these compounds. Experiments have shown that these compounds exhibit significantly better competitive displacement removal effects than the currently best displacement agent, shikonin (LA), especially at low concentrations, in the competitive displacement removal of protein-bound uremic toxins such as IS and PCS, thus effectively treating cardiovascular and cerebrovascular diseases and uremia caused by protein-bound uremic toxins. Attached Figure Description

[0021] Figure 1 The results of the competitive substitution efficiency test for the three compounds YW1, YW2 and YW3 in Example 1 are shown, with a compound concentration of 200 μmol / L.

[0022] Figure 2 The results of the competitive substitution efficiency test for the three compounds YW1, YW2 and YW3 in Example 1 are shown, with a compound concentration of 400 μmol / L. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0024] This invention proposes using the compound represented by formula (I) to clear protein-bound uremic toxins from the blood, thereby treating a series of diseases caused by the accumulation of protein-bound uremic toxins in the blood. The general structural formula of this compound is as follows:

[0025]

[0026] Wherein, R1 is an aromatic heterocycle, phenylpropanoid aromatic heterocycle, or aromatic hydrocarbon with 6-50 carbon atoms, wherein the aromatic heterocycle includes, but is not limited to, pyrroleyl, furanyl, thiopheneyl, pyridinyl, pyrazinyl, pyrimidinyl, pyrazinyl, pyranoneyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, etc., and the phenylpropanoid aromatic heterocycle includes, but is not limited to, indolyl, benzofuranyl, benzothiopheneyl, quinolinyl, etc., and the aromatic hydrocarbon includes, but is not limited to, phenyl, naphthyl, anthraceneyl, phenanthrene, etc. R2 is a flavonoid group, a polyphenol group, or a terpene group with 13-50 carbon atoms. The flavonoid group is a group derived from flavonoid compounds. Flavonoids refer to a general term for a series of compounds in which two benzene rings are connected by three carbon atoms, that is, a class of compounds with a C6-C3-C6 structure.

[0027] In a preferred embodiment, R1 is an aromatic heterocycle, phenylpropionic aromatic heterocycle or aromatic hydrocarbon with 6-30 carbon atoms, more preferably an aromatic heterocycle or aromatic hydrocarbon with 6-20 carbon atoms; R2 is a flavonoid group or terpene group with 13-20 carbon atoms, more preferably a flavonoid group or terpene group with 13-20 carbon atoms.

[0028] In the preferred embodiment, R1 is Phenyl group whose benzene ring is substituted with methyl group at any position Benzyl group with methyl substituted at any position Phenylethyl group with methyl substitution at any position R2 is

[0029]

[0030] Where * represents the connection site of the substituent on the molecule of formula (i).

[0031] In some embodiments of the present invention, the molecules represented by formulas (ii), (iii), and (iv) have been shown to have the effect of clearing protein-bound uremic toxins from the blood:

[0032]

[0033]

[0034] The present invention also provides a dialysate comprising the compound shown in formula (I) and a dialysate for removing protein-bound uremic toxins from the blood using a dialysis displacement method.

[0035] In some embodiments, the effective concentration of the compound represented by formula (I) in the dialysate is 100-500 μM, preferably 150-400 μM, and more preferably 200-400 μM. The dialysate is a dialysate for injection.

[0036] In some embodiments, the dialysis solution also includes dialysis perfusion solution (compound sodium chloride solution). The dialysis perfusion solution used in the embodiments of the present invention was provided by Anhui Shuanghe Pharmaceutical Co., Ltd.

[0037] The molecules shown in equations (ii), (iii), and (iv) have ZINC database numbers ZINC000008791789, ZINC000012297018, and ZINC000012296493, respectively. These molecules can be commercially available using their ZINC numbers (e.g., purchased from InterBioScreen Co., Ltd. (Russia)) or synthesized in-house. Typical synthetic methods for these molecules are given below.

[0038] Taking the compound shown in formula (iv) as an example, its preparation method includes the following steps:

[0039] Step 1: Under stirring, anhydrous potassium carbonate (2073.2 mg) was added to an acetone (100 mL) solution of apigenin (2524.4 mg) and stirred at 60 °C for 30 min. Ethyl 2-bromoacetate (2724 μL) and potassium iodide (166.0 mg) catalyst were slowly added dropwise using a constant pressure dropping funnel, and the mixture was stirred and refluxed at 60 °C. After the reaction was completed by TLC, the mixture was cooled, filtered, and the residue was washed with a small amount of acetone. The organic solvent was removed from the filtrate using a rotary evaporator. The filtrate was then purified by silica gel column chromatography (dichloromethane / acetone = 50:1) to obtain compound 1.

[0040] Step 2: Compound 1 (2075 mg), 1 mol / L potassium hydroxide (12.5 mL), and 50 mL of methanol were added sequentially to a 100 mL three-necked flask and stirred under reflux at 60 °C. After the reaction was completed as detected by TLC, the reaction solution was filtered. 0.5 mol / L sulfuric acid was added dropwise to the filtrate. A pale yellow solid gradually precipitated as the acid was added. The pH of the filtrate was adjusted to 2-3, and the solution was placed in ice water for 2 hours. After complete crystallization, the solution was filtered. The filter cake was washed three times each with 3.8% hydrochloric acid solution, saturated sodium chloride solution, and distilled water, and then dried under vacuum to obtain compound 2.

[0041] Step 3: Compound 2 (396.0 mg), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) (766.8 mg), 1-hydroxybenzotriazole (HOBt) (540.6 mg), and solvent N,N-dimethylformamide (DMF) (20 mL) were added to a 50 mL three-necked flask under ice bath conditions. The mixture was stirred for 1 h. Then, 4 mL of DMF solution containing phenylalanine methyl ester hydrochloride (430 mg) was added. Using a constant pressure dropping funnel, 2 mL of DMF solution containing the acid-binding agent N,N'-diisopropylethylamine (DIPEA) (700 μL) and 4-dimethylaminopyrrole (DMAP) (146.4 mg) was added dropwise. The mixture was reacted under ice bath conditions for 30 min and then gradually raised to room temperature for 12 h. After the reaction was completed by TLC detection, the reaction solution was put into a beaker containing 50 mL of ice water. The beaker was placed in the ice water and allowed to stand for 2 hours. After filtration, the filter cake was washed three times each with saturated sodium chloride solution and distilled water. The mixture was then separated and purified using a silica gel column (dichloromethane / acetone = 50:1) to obtain a pale yellow solid compound 3.

[0042] Step 4: Add 12.0 mg (0.25 mmol) of salicylic acid methyl ester compound 3 and 50 mL of anhydrous ethanol sequentially to a 100 mL single-necked round-bottom flask. Adjust the pH of the reaction solution to 10–11 with 0.1 mol·L⁻¹ potassium hydroxide (KOH) solution. Stir at room temperature for about 3 hours, and monitor the complete hydrolysis of the starting material by TLC. Filter the reaction solution, and adjust the pH of the filtrate to 2–3 with 0.5 mol·L⁻¹ sulfuric acid. A pale yellow solid gradually precipitates as acid is added. Place the precipitate in ice water and let it stand for 2 hours. Filter the solution, wash the filter cake three times with distilled water, and dry it under vacuum to obtain a pale yellow solid, which is the compound shown in formula (IV).

[0043] The protein-bound uremic toxins (PBUTs) described in this invention are one or more of indophenol sulfate (IS), p-formyl sulfate (PCS), benzamide acetic acid, 3-carboxy-4-methyl-5-propyl-2-furan propionate (CMPF), and indole-3-acetic acid (IAA). When applied, the drug is in the form of an injection. IS, PCS, and IAA bind to site II of albumin, while CMPF binds to site I of albumin. Furthermore, IS and PCS are the two most harmful toxins in advanced uremia, but due to their binding to albumin, they are difficult to remove using traditional hemodialysis (HD) techniques. Current technologies mainly involve injecting ibuprofen, salvianolic acid, fatty acids, and shikonin (LA) into the bloodstream, utilizing their binding to sites I or II of albumin to increase the free form of PBUTs and promote their removal, with LA showing the best competitive displacement efficiency. To test the effectiveness of the compounds provided in this invention in removing PBUTs from blood using a competitive displacement method, the following embodiments of this invention provide three compounds of formula (II), formula (III) and formula (IV) as dialysate drug molecules, and conduct in vitro hemodialysis experiments to test their competitive displacement effect and compare it with LA.

[0044] Example 1

[0045] Accurately weigh IS and PCS powders, dissolve them separately in ultrapure water to prepare stock solutions with a concentration of 1 mg / ml, and then dilute them sequentially to a final concentration of 50 μg / ml for subsequent HPLC detection of response values. Weigh the corresponding amounts of LA standard (positive control) and candidate molecules (the three compounds of formulas (II), (III), and (IV) above, represented by their ZINC numbers ZINC000008791789, ZINC000012297018, and ZINC000012296493 respectively) and dissolve them quantitatively in 2 ml of ultrapure water to prepare stock solutions with a concentration of 4 mmol / L, and then dilute them to 1 ml of 2 mmol / L stock solution for later use.

[0046] Take 160 μl of fresh rat plasma, add 10 μl of IS stock solution (1 mg / ml) and 10 μl of PCS stock solution (1 mg / ml), mix well, and incubate at 37 °C for 30 min. Then add 20 μl of ultrapure water as a blank control, 20 μl of LA (4 mmol / L) as a positive control, and 20 μl of candidate molecule (4 mmol / L), mix well, and incubate at 37 °C for 1 h. The total sample volume is 200 μl, the final concentration of IS and PCS is 50 μg / ml, and the final concentration of LA / candidate molecule is 400 μmol / L. (PS: The method for 200 μmol / L is similar; add 20 μl of ultrapure water / LA (2 mmol / L) / candidate molecule (2 mmol / L) and mix well.)

[0047] The microdialysis system consisted of a microdialysis syringe pump (CMA402, Stockholm, Sweden) and a dialysis probe (CMAMicrodialysis AB, Torshamnsgatan 30A, 16440Kista, Sweden). The dialysis membrane mounted on the probe was 4 mm long, 0.5 mm in outer diameter, and had a molecular weight cutoff of 20,000 Da. The system was pre-started with PBS buffer (pH 7.4) for 15 min and equilibrated with a blank control sample for 60 min. During the actual dialysis, the dialysate from the blank control group, positive control group (treated with LA), and sample group (treated with candidate molecule samples) was collected every 1 h for HPLC detection of IS and PCS content. The dialysate flow rate was set to 2 μl / min, and the sample temperature was maintained at 37 °C throughout the dialysis process using a PCMT thermal shaker.

[0048] HPLC system for detecting the concentration of target substances in dialysate:

[0049] Prior to formal testing, HPLC methodology was evaluated, including standard curves and precision. The concentrations of IS and PCS in each dialysate group were then measured.

[0050] Chromatographic analysis was performed using an Alltima high-performance liquid chromatography system (HPLC, Waters Corp., MA, USA). A C18 column (250 mm × 4.6 mm, 5 μm, GRACE) was used. The column and autosampler temperatures were maintained at 35 °C and 4 °C, respectively. The mobile phase consisted of (A) ammonium formate (200 mM, pH 4.5) and (B) acetonitrile. Elution conditions were optimized as follows: 25% B (0–8 min). The IS elution time was approximately 6.2 min, and the PCS elution time was approximately 5.6 min. Fluorescence detection of IS and PCS was performed at specific excitation / emission wavelengths (IS: 280 / 375 nm; PCS: 260 / 300 nm), with an injection volume of 5 μl.

[0051] Dialysis efficiency (%) of IS and PCS = (Response value of IS or PCS in dialysate / Response value of IS or PCS in standard) × 100%

[0052] The final three compounds are of formula (ii), formula (iii), and formula (iv). Figure 1 and Figure 2 The effects of competitive displacement dialysis IS and PCS (represented by YW1, YW2, and YW3 respectively) are as follows: Figure 1 , Figure 2 As shown, Figure 1 The concentrations of the three compounds were 200 μmol / L. Figure 2The concentration of the three compounds was 400 μmol / L. As can be seen from the figure, the three compounds in Example 1 exhibited better competitive displacement efficiency for both IS and PCS uremic toxins than LA, especially at lower concentrations of 200 μmol / L, where the advantage was more pronounced.

[0053] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of the compound represented by formula (I) in the preparation of drugs for treating uremia. Formula (1) in, R1 is , , R2 is or , where * represents the linking site of the substituent on the molecule of formula (i).

2. The application as described in claim 1, characterized in that, When applied, the drug is in the form of an injection.

3. A dialysate for treating uremia using dialysis replacement therapy, characterized in that, Including the compounds shown in formula (a), Formula (1) Where R1 is , , R2 is or , where * represents the linking site of the substituent on the molecule of formula (i).

4. The dialysis solution as described in claim 3, characterized in that, The concentration of the compound represented by formula (I) in the dialysate is 100-500 μM.

5. The dialysis solution as described in claim 3, characterized in that, The concentration of the compound represented by formula (I) in the dialysate is 150-400 μM.

6. The dialysis solution as described in claim 3, characterized in that, The dialysate is an injectable dialysate.

7. The dialysis solution as described in claim 3, characterized in that, It also includes dialysis perfusion fluid, which is a compound sodium chloride solution.

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