Application of compound as urea channel inhibitor

Abstract

The invention belongs to the technical field of medicine research and development, and particularly relates to application of a compound as a urea channel inhibitor. The invention provides a compound for inhibiting a urea channel UT-A2 as shown in formula (I), the compound can significantly inhibit rapid transfer of UT-A2 to urea, is ineffective to UT-A3 and UT-B, is a UT-A2 inhibitor small molecule which is found for the first time and has excellent inhibitory activity and high selectivity, has potential to be developed into a novel diuretic drug targeting UT-A2, and has broad application prospects. The traditional Chinese medicine composition is used for clinically treating malnutrition edema caused by low-protein diet, hepatic lesion and the like.

Description

Technical Field

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

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Diuretics are commonly used drugs in clinical practice, mainly used to treat hypertension, heart failure and various edema diseases. Their main target is the kidney, and they exert a diuretic effect by increasing urine volume. However, since traditional diuretics diuresis by excreting sodium and water, they can cause side effects such as electrolyte imbalance. Scientists have been looking for new diuretic drug targets and developing new diuretics.

[0004] Urea transporters (UTs) are a class of membrane channel proteins that selectively permeate urea and play an important role in maintaining the intrarenal urea cycle, establishing the urea concentration gradient in the renal medulla tissue, and the urine concentration mechanism. Two UT subfamilies, UT-A and UT-B, are found in mammals, encoded by the genes Slc14a2 and Slc14a1, respectively. The human Slc14a2 gene produces four UT-A members, named UT-A1, -A2, -A3, and -A6, through different promoters and alternative mRNA splicing. UT-A1, -A2, -A3, and UT-B, the four UTs are responsible for the rapid urea cycle in the kidney and play an important role in the urea cycle in the kidney, 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, consisting of 920 amino acids. UT-A2 is composed of 396 amino acids, which are identical to the amino acids 524-920 of UT-A1, while UT-A3 is composed of the amino acids 1-451 of UT-A1. UT-B has a 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 loop of Henle. UT-B is widely distributed throughout the human body, and in the kidney it is mainly expressed in the straight segment of the descending renal vascular segment (DVR). Kidney International magazine once published a commentary listing UT as a potential target for the development of diuretics, and proposed 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 that new diuretics that do not cause electrolyte imbalance can be developed. However, the UT inhibitors currently developed generally have the disadvantage of poor selectivity and are prone to act on UT proteins outside the kidney, such as UT-B, thereby causing side effects. Therefore, the development of highly selective urea channel inhibitors that selectively act on the UT-A protein in the kidney is crucial in the development of UT inhibitor-type diuretics. Summary of the invention

[0005] The present invention provides a compound for use as a urea channel inhibitor, wherein the compound has a structural formula as shown in formula (I):

[0006]

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

[0008] Furthermore, the compound of formula (I) is used 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 a drug for treating hypoproteinemia.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The present invention provides a compound of formula (I) for inhibiting urea channel UT-A2. The compound can significantly inhibit the rapid transport of urea by UT-A2, but is ineffective against UT-A3 and UT-B. It is a small molecule UT-A2 inhibitor with excellent inhibitory activity and high selectivity that has been discovered for the first time. It has the potential to be developed into a new diuretic targeting UT-A2, and is used for the clinical treatment of malnutrition edema caused by low-protein diet, liver lesions, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0014] Figure 1 :Flow chart of the urea inhibitor activity detection experiment; Compared with the blank control group, after the administration of effective UT inhibitors, the process of rapid transport of urea molecules from the inside to the outside of the cells was blocked, and the intracellular urea concentration increased. After the cells were lysed, the intracellular urea was released into the lysate, and the cell fragments and the supernatant were separated by high-speed centrifugation. Finally, the urease reaction was used to hydrolyze urea and develop color to detect the urea concentration in the supernatant, thereby reflecting the process of the inhibitor molecules inhibiting urea transport.

[0015] Figure 2 : Compound (I) has inhibitory activity against human UT-A2; Compound (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 : Compound (I) has an inhibitory activity against human UT-A3; Compound (I) cannot effectively block the rapid transmembrane transport process of urea molecules by UT-A3.

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

[0018] Figure 5:Effects of compound formula (I) on the osmotic pressure and urea concentration of mouse renal medulla; UT-A2 is mainly distributed in the thin descending limb (TDL) of the loop of Henle in the renal nephron, mediating the transmembrane transport process of urea in the inner medulla. After compound formula (I) selectively blocked the urea transport of UT-A2, the urea concentration and osmotic pressure of the mouse renal medulla were significantly higher than those of the control group. The significance analysis was performed using the Student's t-test, ** indicates P is less than or equal to 0.01; * indicates P is less than or equal to 0.05.

[0019] Figure 6 : Effect of compound formula (I) on urine osmotic pressure and urea concentration in mice. Since compound formula (I) selectively blocks the ability of UT-A2 to transport urea, under low-protein diet conditions, the urea concentration and osmotic pressure of mouse urine showed a significant decrease compared with the control group, suggesting that compound formula (I) can be used as a diuretic under low-protein conditions. Student's t-test was used for significance analysis, and **** indicates that P is less than or equal to 0.0001. DETAILED DESCRIPTION

[0020] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0021] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit 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 "comprising" and / or "including" are used in this specification, it indicates 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 embodiments and comparative examples.

[0023] Example 1: Selective Inhibition of Compounds on UT-A2

[0024] Experimental method: The experimental process of urea inhibitor activity detection is as follows ( Figure 1), 293F cells were transfected with UT-A2 protein. After 2 days, urea with a final concentration of 50mM was added to the cell fluid. After shaking on a shaker for 20min, the urea concentration inside and outside the cells was the same. 1ml of cell fluid was added to each centrifuge tube, centrifuged for 8s, and the supernatant was discarded. Then, 1ml of HBSS containing a concentration gradient of compound formula (I) was used for washing for 50s. At this time, a urea concentration difference was formed inside and outside the cells. If the ligand has an antagonistic effect, less urea will flow out through transmembrane transport, and the urea concentration in the cell will increase. After the cells were centrifuged at low speed for 6s, the supernatant was sucked clean and the cell pellet was retained. The cells were ruptured with 1ml of single distilled water, and the cell fragments and supernatant were separated by high-speed centrifugation. The urea concentration in the supernatant was detected by a urease detection kit, thereby reflecting the effect of small molecules blocking urea transport.

[0025] Experimental results: Through the urea inhibitor activity detection experiment, it is proved that compound (I) only selectively inhibits UT-A2 protein, and its IC 50 349±30nM, ineffective against UT-A3 and UT-B ( Figure 2-Figure 4 ). As far as the inventors know, this is the only known small molecule inhibitor that is highly selective for UT-A2.

[0026] Example 2: Compounds inhibit urea transport in mouse kidney medulla

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

[0028] (1) Male C57BL / 6J mice were placed in metabolic cages for 2 days to adapt, and mice with abnormal conditions were removed. During the adaptation period, sufficient water and food were provided. Urine was collected every 24 hours using metabolic cages.

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

[0030] (3) The experimental mice were placed on a low-protein diet and deprived of water after 7 days, but were still provided with sufficient food. After 30 hours of water deprivation, the control group was given 0.1 ml / 10 g of solvent, and the drug-treated group was given 0.1 ml / 10 g of compound (I) (concentration: 8 mg / ml).

[0031] (4) After 36 hours of water deprivation, gently massage the bladder and abdomen to expel urine and collect urine.

[0032] (5) The mice were then dissected and the inner medullary tissue was collected and placed in an EP tube. The tissue was weighed using a balance and 10 times the weight of double distilled water was added. The tissue was homogenized using a homogenizer and centrifuged at 12,000 rpm for 15 min. The supernatant was collected.

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

[0034] Experimental results: Compound (I) showed an inhibitory effect on UT-A2 in mice, and its physiological performance was similar to that of UT-A2 knockout mice. UT-A2 mainly mediates the urea transport process in the renal medulla. Compound (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, the urine urea concentration and urine osmotic pressure of mice under low-protein diet conditions are significantly lower than those of the control group ( Figure 6 ). The above results show that under low-protein diet conditions, compound formula (I) can effectively block UT-A2-mediated urea transmembrane transport, thereby significantly reducing the urea concentration and osmotic pressure in the renal medulla and urine, indicating that it has a diuretic effect. Therefore, formula (I) can be developed into a diuretic that targets and blocks UT-A2, and is used to treat malnutrition edema (also known as hypoproteinemia) caused by factors such as long-term malnutrition, liver disease, 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 equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of a compound as a urea channel inhibitor, characterized in that: The structural formula of the compound is shown in formula (I):

2. Use of a compound as claimed in claim 1 as a urea channel inhibitor, wherein the compound comprises a pharmaceutically acceptable salt, hydrate, solvate or prodrug of formula (I).

3. A use as claimed in claim 1, characterized in that: Use of the compound of formula (I) as a urea channel-A2 inhibitor.

4. A use of the compound of formula (I) as claimed in claim 1 in the preparation of diuretic drugs.

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

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