Use of a compound as a urea channel inhibitor

Abstract

The present invention belongs to the technical field of drug research and development, and particularly relates to the use of a compound as a urea channel inhibitor. The present invention provides a compound of formula (I) that inhibits the urea channel UT-A2. This compound can significantly inhibit the rapid transport of urea by UT-A2, but has no effect on UT-A3 and UT-B. It is the first discovered small molecule UT-A2 inhibitor with excellent inhibitory activity and high selectivity, and has the potential to be developed into a new diuretic targeting UT-A2 for clinical treatment of malnutritional edema caused by low-protein diet, liver lesions, etc.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug research and development, and particularly relates to the application of a compound as a urea channel inhibitor. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the overall understanding of the present invention, and it is not necessarily to be regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Diuretics are commonly used drugs in clinical practice, mainly used for treating hypertension, heart failure, and various edematous diseases, etc. Their action targets are mainly in the kidneys, and they exert diuretic effects by increasing urine output. However, since traditional diuretics diurese by excreting sodium and water, they can cause side effects such as electrolyte imbalance. Scientists have been searching for new diuretic targets and developing new diuretics.

[0004] Urea transporters (UTs) are a class of membrane channel proteins that selectively permeate urea and play important roles in maintaining the intrarenal urea cycle, establishing the urea concentration gradient in the renal medullary tissue, and the urine concentration mechanism. Two UT subfamilies, UT-A and UT-B, have been found in mammals, which are encoded by genes Slc14a2 and Slc14a1, respectively. The human Slc14a2 gene generates four UT-A members through different promoters and alternative mRNA splicing, named UT-A1, -A2, -A3, and -A6, respectively. These four UTs, UT-A1, -A2, -A3, and UT-B, are responsible for the rapid urea cycle in the kidney and play important roles in the renal urea cycle, while UT-A6 is expressed in human colon tissue. In addition, rat UT-A4 is only found in the renal medulla, while mouse UT-A5 is only expressed in the testis. UT-A1 is the largest member of the UT family and consists of 920 amino acids. UT-A2 consists of 396 amino acids and is identical to the amino acids at positions 524 - 920 of UT-A1, while UT-A3 consists of the amino acids at positions 1 - 451 of UT-A1. UT-B has 61.6% sequence similarity with UT-A2. In the kidney, UT-A1 and UT-A3 are expressed in the principal cells of the inner medullary collecting duct (IMCD), and UT-A2 is expressed in the thin descending limb (TDL) of the Henle loop. UT-B is widely distributed throughout the human body and is mainly expressed in the descending vasa recta (DVR) of the kidney. The journal Kidney International once published a comment listing UT as a potential target for developing diuretics, suggesting that its inhibitors are very likely to be used as new diuretics for the long-term treatment of clinical diseases such as heart failure, edema, and hypertension, and new diuretics that do not cause electrolyte imbalance can be developed. However, the currently developed UT inhibitors generally have the disadvantage of poor selectivity and are prone to act on UT proteins outside the kidney, such as UT-B, thus causing side effects. Therefore, developing highly selective urea channel inhibitors that selectively act on UT-A proteins in the kidney is crucial for developing UT inhibitor-type diuretics. Summary of the Invention

[0005] The present invention provides the use of a compound as a urea channel inhibitor, and the structural formula of the compound is shown as formula (I):

[0006]

[0007] Furthermore, the use of a compound as a urea channel inhibitor, wherein the compound comprises a pharmaceutically acceptable salt, hydrate, solvate, or prodrug of formula (I).

[0008] Furthermore, the use of the compound of formula (I) as a urea channel-A2 inhibitor.

[0009] Furthermore, the compound of formula (I) is used in the preparation of diuretic drugs.

[0010] Furthermore, the compound of formula (I) is used in the preparation of drugs for treating hypoproteinemia.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0012] The present invention provides a compound of formula (I) that inhibits the urea channel UT-A2. This compound can significantly inhibit the rapid transport of urea by UT-A2, but has no effect on UT-A3 and UT-B. It is the first discovered small molecule UT-A2 inhibitor with excellent inhibitory activity and high selectivity, and has the potential to be developed into a new diuretic targeting UT-A2 for clinical treatment of malnutritional edema caused by low-protein diet, liver lesions, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings forming a part of this specification are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0014] Figure 1 : Flow chart of the urea inhibitor activity detection experiment; compared with the blank control group, after administration of an effective UT inhibitor, the process of rapid transport of urea molecules from inside to outside the cells is blocked, and the intracellular urea concentration increases. After lysing the cells, the intracellular urea is released into the lysis solution, and the cell debris and supernatant are separated by high-speed centrifugation. Finally, the method of hydrolyzing urea by urease reaction to develop color is used to detect the urea concentration in the supernatant, thereby reflecting the process of inhibitor molecules inhibiting urea transport.

[0015] Figure 2 : Inhibitory activity of the compound of formula (I) against human UT-A2; the compound of formula (I) can effectively block the rapid transmembrane transport process of urea molecules by human UT-A2, and the inhibitory effect is dose-dependent.

[0016] Figure 3 : Inhibitory activity of the compound of formula (I) against human UT-A3; the compound of formula (I) cannot effectively block the rapid transmembrane transport process of urea molecules by UT-A3.

[0017] Figure 4 : Inhibitory activity of the compound of formula (I) against human UT-B; the compound of formula (I) cannot effectively block the rapid transmembrane transport process of urea molecules by UT-B.

[0018] Figure 5: Effects of Compound (I) on the osmotic pressure and urea concentration in the renal medulla of mice; UT-A2 is mainly distributed in the thin descending limb (TDL) of the Henle loop of the renal nephron, mediating the transmembrane transport process of urea in the inner medulla. After selectively blocking the urea transport of UT-A2 with Compound (I), the urea concentration and osmotic pressure in the renal medulla of mice were significantly higher than those in the control group. Student's t-test was used for significance analysis, ** indicates P ≤ 0.01; * indicates P ≤ 0.05.

[0019] Figure 6 : Effects of Compound (I) on the osmotic pressure and urea concentration in the urine of mice. Since Compound (I) selectively blocked the ability of UT-A2 to transport urea, under the condition of a low-protein diet, the urea concentration and osmotic pressure in the urine of mice showed a significant decrease compared with the control group, suggesting that Compound (I) can be used as a diuretic under low-protein conditions. Student's t-test was used for significance analysis, **** indicates P ≤ 0.0001. Detailed implementation manners

[0020] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0021] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0022] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific examples and comparative examples.

[0023] Example 1 Selective inhibitory effect of the compound on UT-A2

[0024] Experimental method: The experimental procedure for detecting the activity of urea inhibitors is as follows ( Figure 1) After transfecting 293F cells with UT-A2 protein, 2 days later, urea with a final concentration of 50 mM was added to the cell culture medium. After shaking on a shaker for 20 min, the urea concentrations inside and outside the cells were the same. 1 ml of cell culture medium was added to each centrifuge tube, and after centrifuging for 8 s, the supernatant was discarded. Then, the cells were washed with 1 ml of HBSS containing a concentration gradient of compound (I) for 50 s. At this time, a urea concentration difference was formed inside and outside the cells. If the ligand had an antagonistic effect, less urea would flow out through transmembrane transport, and the urea concentration inside the cells would increase. After centrifuging the cells at low speed for 6 s, the supernatant was aspirated completely, and the cell pellet was retained. The cells were lysed with 1 ml of distilled water, and the cell debris and supernatant were separated by high-speed centrifugation. The urea concentration in the supernatant was detected using a urease detection kit to reflect the effect of small molecules blocking urea transport.

[0025] Experimental results: Through the urea inhibitor activity detection experiment, it was proved that compound (I) only selectively inhibited UT-A2 protein, and its IC 50 was 349 ± 30 nM and was ineffective against UT-A3 and UT-B ( Figures 2 - 4 ). To the inventors' knowledge, this is the only known small molecule inhibitor with high-efficiency and selective action on UT-A2 so far.

[0026] Example 2. Inhibition of urea transport in the renal medulla of mice by the compound

[0027] Experimental method: The in vivo experimental verification process for mice is as follows.

[0028] (1) Male C57BL / 6J mice were placed in a metabolic cage for 2 days to adapt. Abnormal mice were excluded, and sufficient water and food were provided during the adaptation period. Urine was collected every 24 hours using the metabolic cage.

[0029] (2) The experimental mice were paired and grouped according to the urine volume during the adaptation period into a drug administration group and a control group.

[0030] (3) The experimental mice were fed a low-protein diet. After 7 days, water was withheld, but sufficient food was still provided. When water was withheld for 30 h, the control group was given 0.1 ml / 10 g of the solvent, and the drug administration group was given 0.1 ml / 10 g of compound (I) (concentration: 8 mg / ml).

[0031] (4) When water was withheld for 36 h, the bladder and abdomen were gently massaged to excrete urine, and the urine was collected.

[0032] (5) Subsequently, the mice were dissected, and the inner medulla tissue was placed in an EP tube. The tissue was weighed using a balance, and 10 times the weight of double-distilled water was added. Homogenization was performed using a homogenizer, and centrifugation was carried out at 12,000 rpm for 15 min to obtain the supernatant.

[0033] (6) The urea concentration of each sample was measured using a urea kit; the osmotic pressure of each sample was measured using a freezing point osmometer.

[0034] Experimental results: Compound of formula (I) showed an inhibitory effect on UT-A2 in mice, and its physiological manifestations were similar to those of UT-A2 knockout mice. UT-A2 mainly mediates the urea transport process in the renal medulla. Compound of formula (I) acts on UT-A2 without affecting the normal functions of UT-A1, A3 and -B, blocking the urea transport in the medulla, thereby significantly reducing the osmotic pressure and urea concentration in the renal medulla ( Figure 5 ). Since formula (I) blocks the urea transport process of UT-A2, under the condition of a low-protein diet, the urine urea concentration and urine osmotic pressure of the mice were significantly lower than those of the control group ( Figure 6 ). The above results indicate that under the condition of a low-protein diet, the compound of formula (I) can effectively block the urea transmembrane transport mediated by UT-A2, thereby significantly reducing the urea concentration and osmotic pressure in the renal medulla and urine, suggesting its diuretic effect. Therefore, formula (I) can be developed into a diuretic targeting the blockade of UT-A2 for the treatment of malnutrition edema (also known as hypoproteinemia) caused by factors such as long-term malnutrition, liver diseases, and short-term massive blood loss.

[0035] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these changes and modifications.

Claims

1. Use of a compound in the preparation of a urea channel inhibitor drug, characterized in that, The structural formula of the said compound is shown as formula (I): , the urea channel inhibitor drug is a diuretic drug and a drug for treating hypoproteinemia.

2. An application as claimed in claim 1, wherein, The inhibition of the urea channel is the inhibition of the urea channel-A2.

3. Use of a compound of formula (I) as claimed in claim 1 in the preparation of a diuretic drug.

4. Use of a compound of formula (I) as claimed in claim 1 in the preparation of a drug for treating hypoproteinemia.

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