JP7330828B2 - Compound having ENaC activating ability - Google Patents

Description

TECHNICAL FIELD The present invention relates to an activator of epithelial Na ⁺ channel hENaC αβγ, a therapeutic agent for diseases containing the same, and a salty taste enhancer.

Epithelial sodium channel ENaC is a non-voltage-dependent Na ion channel belonging to the ENaC/degenerin family (Non-Patent Document 1).

Each subunit of ENaC has two transmembrane domains and functions as a trimer of αβγ or δβγ. ENaCαβγ is mainly expressed in the stomach, kidney, and colon, and ENaCδβγ is mainly expressed in neural tissues such as the cerebral cortex, cerebellum, and hippocampus, and testis (Non-Patent Documents 2-4). In addition, in the tongue, ENaC αβγ is expressed in the anterior region to which the chorda tympani nerve projects, and is involved in low-concentration salty taste reception (<150 mM NaCl) (Non-Patent Document 5). In addition, the phenomenon of activating or inhibiting humanENaC (hENaC) αβγ is widely used as one of the grounds for the salty taste enhancing effect and the salty taste inhibiting effect (Prior Patent Documents 1 to 5).

In the medical field, diseases caused by genetic mutations of hENaC αβγ include hyperna ⁺ emia, renal disease, Lild syndrome, and the like. These therapies include the use of hENaCαβγ inhibitors such as amiloride (Non-Patent Documents 6 and 7).

On the other hand, activators that activate hENaC αβγ are considered for the treatment of pseudoaldosteronism type 1 and neonatal respiratory distress syndrome. Compound S3969 is known as a hENaC αβγ activator (activator) and is expected as a therapeutic drug for the above diseases (Non-Patent Document 6).

However, with the exception of S3969, there are no reports of small molecules that activate hENaCαβγ.

Based on the structure of S3969, the indole ring is considered to be one of the partial structures that activates hENaC, but there are no reports of partial structures other than the indole ring that have the ability to activate hENaC.

Therefore, by searching and developing small molecules and partial structures that activate hENaC αβγ, hENaC αβγ activators (activators) not only alleviate genetic diseases associated with hENaC αβγ, but also affect oral health. It is expected to modify salty taste reception and greatly improve taste.

JP 2017-217005 Patent No. 5674984 JP 2016-161281 Special table 2009-539377 Special table 2008-529987

Canessa, C. M., Horisberger, J. D., & Rossier, B. C. (1993). Epithelial sodium channel related to proteins involved in neurodegeneration. Nature, 361(6411), 467. Waldmann, R., Champigny, G., Bassilana, F., et al. (1995). Molecular cloning and functional expression of a novel amiloride-sensitive Na+ channel. Journal of Biological Chemistry, 270(46), 27411-27414. Yamamura, H., Ugawa, S., Ueda, T., et al. (2004). Protons activate the δ-subunit of the epithelial Na+ channel in humans. Journal of Biological Chemistry, 279(13), 12529-12534. Staruschenko, A., Adams, E., Booth, R. E., et al. (2005). Epithelial Na+ channel subunit stoichiometry. Biophysical journal, 88(6), 3966-3975. Chandrashekar, J., Kuhn, C., Oka, Y., et al. (2010). The cells and peripheral representation of sodium taste in mice. Nature, 464(7286), 297. Baker, E., Jeunemaitre, X., Portal, A. J., et al. (1998). Abnormalities of nasal potential difference measurement in Liddle's syndrome. The Journal of clinical investigation, 102(1), 10-14. Lu, M., Echeverri, F., Kalabat, D., Laita, B., Dahan, D. S., Smith, R. D., ... & Hwang, N. (2008). Small molecule activator of the human epithelial sodium channel. Journal of Biological Chemistry, 283(18), 11981-11994. Swamidass SJ, Baldi P.(2007) Mathematical correction for fingerprint similarity measures to improve chemical retrieval J. Chem. Inf. Model., 2007, 47 (3), pp 952-964

An object of the present invention is to find a compound having an excellent hENaC αβγ activating action. Another object of the present invention is to provide a therapeutic agent for diseases in the pharmaceutical field and a salty taste enhancer in the food field containing the compound.

As a result of intensive research, the present inventors have found that 3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β]thiophene It was found that a compound having a structure identical with or similar to that of -2-carboxamide or a salt thereof has an activating effect on hENaC.
That is, the first invention of the present application is
"3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β]thiophene represented by the following formula (1) -hENaC αβγ activator containing a compound having the same or similar structure as 2-carboxamide or a salt thereof as an active ingredient

。”である。

. ”.

Furthermore, it is preferable that the compound is a component derived from foods or natural extracts.
That is, the second invention of the present application is
"The hENaC αβγ activator according to claim 1, wherein said compound is a component derived from foodstuffs, natural extracts."

Next, the compounds or salts thereof of the present application can also be used as therapeutic agents for diseases and the like.
That is, the third invention of the present application is
“A therapeutic agent for diseases, etc., containing the hENaC αβγ activator according to claim 1 or 2.”
is.

Next, the compound of the present application or a salt thereof can also be used as a salty taste enhancer.
That is, the fourth invention of the present application is
"A salty taste enhancer containing the hENaC αβγ activator according to claim 1 or 2."

The present invention provides activators of hENaC αβγ. Also, 3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β]thiophene-2-carboxamide of the present invention A therapeutic agent for genetic diseases thought to be caused by hENaC αβγ is provided by containing an activating agent containing a compound having the same or similar structure as or a salt thereof as an active ingredient. Further provided is a salty taste enhancer by activating hENaC αβγ, which is also a salty taste receptor.

3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β]thiophene-2-carboxamide and 3-chloro A structural analogy to -N-(2-((1,1-dioxydotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β]thiophene-2-carboxamide gives hENaCαβγ. Structural information on activation is useful as a research and development tool, and can contribute to the further development of medicine, the food industry, and health science.

3-Chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β] using membrane potential-sensitive dye in Example 1 Concentration-response curves of the hENaC αβγ activating effect of thiophene-2-carboxamide are shown. 1 is a schematic diagram showing the methods of Examples 1 and 2. FIG. Results of Example 2, 3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N- hENaC αβγ activity by ethylbenzo[β]thiophene-2-carboxamide and compounds (three kinds) having a similar structure. Results of Example 2, 3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N- hENaC αβγ activity by compounds (3 types) having a similar structure to ethylbenzo[β]thiophene-2-carboxamide is shown.

1. Activator of hENaC αβγ
The hENaC αβγ referred to in the present invention is encoded by the human genes human ENaCα (or SCNN1a), human ENaCβ (or SCNN1b), human ENaCγ (or SCNN1g), forms αβγ trimers on biological membranes, A membrane protein that exhibits the properties of a sodium channel.
The activator is a compound that affects the pore opening structure of the trimeric membrane protein composed of hENaC αβγ and increases the influx efficiency of mainly sodium ions that flow through hENaC. say.

2. Compounds having hENaC αβγ activating ability of the invention - A compound having the same or similar structure as ethylbenzo[β]thiophene-2-carboxamide, or a salt thereof.
This 3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β]thiophene-2-carboxamide is have a structure.

当該化合物は、ベンゾチオフェノン環を有することを特徴とする。本発明者は、多くの化合物のスクリーニングの結果、本化合物にhENaC αβγの活性化能を有することを見出した。
さらに、当該化合物に類似する構造を有する化合物を検索し、複数のhENaC αβγの活性化能を有する化合物を見出した。さらに、本発明はこれらの化合物の塩も意図する。

The compound is characterized by having a benzothiophenone ring. As a result of screening many compounds, the present inventor found that the present compound has the ability to activate hENaC αβγ.
Furthermore, by searching for compounds having a structure similar to that of the compound, multiple compounds having the ability to activate hENaC αβγ were found. Additionally, the present invention contemplates salts of these compounds.

3. Compounds having a similar structure "3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β]thiophene" referred to in the present invention A compound having a similar structure to -2-carboxamide means a compound showing a similarity of 60% or more in a similarity search using ChemBioFinder Ultra 12.0 (manufactured by CambridgeSoft).
ChemBioFinder Ultra 12.0, which is database management software for chemical structure information, can be used for sampling of compounds with a similarity of 60% or more. As the compound library to be searched, compound libraries owned by universities, various research institutes, etc. can be used.

Definition of analogues of 3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β]thiophene-2-carboxamide The method is shown below. Namely, structural information of 3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β]thiophene-2-carboxamide can be converted to numerical descriptors using ChemBioFinder Ultra 12.0 and the similarity can be calculated. In ChemBioFinder, it is possible to combine representative structural key features such as Unity 2D Fingerprint, Similog key, MacCCS key, and convert structural information of compounds into descriptors using unique modified structural keys.
As a similarity evaluation scale for structural information of compounds, the Tanimoto similarity determination method having commutativity was used (Non-Patent Document 8). In the Tanimoto similarity judgment method, Q (query) and T (target) are expressed by the following formula using the Tanimoto coefficient:

を満たすものを60%以上の類似度と定義できる。ここで示さるQ（クエリ）、T（ターゲット）は、それぞれ3-クロロ-Ｎ-(2-((1,1-ジオキシドテトラハイドロチオフェン-3-イル)アミノ)-2-オキソエチル)-Ｎ-エチルベンゾ[β]チオフェン-2-カルボキシアミドと3-クロロ-Ｎ-(2-((1,1-ジオキシドテトラハイドロチオフェン-3-イル)アミノ)-2-オキソエチル)-Ｎ-エチルベンゾ[β]チオフェン-2-カルボキシアミドに類似構造を持つ化合物の記述子を示す。

can be defined as a similarity of 60% or more. Q (query) and T (target) shown here are 3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N -ethylbenzo[β]thiophene-2-carboxamide and 3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β ] provides descriptors for compounds with similar structures to thiophene-2-carboxamide.

3. Ingredients derived from foods and natural extracts The compound (3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2 having the ability to activate hENaC αβγ of the present invention) -oxoethyl)-N-ethylbenzo[β]thiophene-2-carboxamide) can be obtained as a component derived from foods and natural products.

4. Therapeutic Agent for Diseases The hENaC αβγ activator of the present invention can be used as a therapeutic agent for diseases. Specifically, it is effective for pseudohypoaldosteronism type I, a disease in which reabsorption of sodium ions in the kidney is impaired, and for diseases such as pulmonary edema and neonatal respiratory syndrome caused by increased intracellular sodium ions in the lungs. can be used (Non-Patent Document 7).

5. Salty Taste Enhancer The hENaC αβγ activator of the present invention can be used as a salty taste enhancer. That is, the salty taste enhancer means that it exerts the action of making the salty taste of salt stronger. The salty taste enhancer can be widely used for foods and drinks.
The salty taste enhancer of the present invention may be provided in the form of the compound alone, or may be provided in the form of a solid composition or a liquid composition. When provided as a composition, it may contain additives that can be used in the production of food and drink, such as excipients, pigments, and flavorings, as necessary, as long as they do not interfere with the salty taste-enhancing action.

The present invention will be described in more detail below with reference to examples. These examples do not limit the invention.

[Example 1] 3-Chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β]thiophene using cultured cells Results of hENaC αβγ activity with -2-carboxamide.
Activation of hENaC αβγ by 3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β]thiophene-2-carboxamide In order to examine the potency, a concentration-dependent curve was constructed by a cell-based assay using HEK293T cells expressing hENaC αβγ (Fig. 1).

<Method>
Plasmids and expressed proteins
The experimental method was performed according to JP-A-2017-217005. Specifically, a plasmid vector pEAK10 (EdgeBiosystem) having an EF1α promoter was used to express human ENaC αβγ (hENaC αβγ) in HEK293T cells. Expression constructs with cDNA regions of hENaC α, hENaC β, and hENaC γ inserted between multiple cloning sites of pEAK10 were mixed at a ratio of α:β:γ = 1:1:1, and the gene was added at a ratio of 1 µg/35 mm dish. introduced. Table 1 shows the accession number indicating each gene sequence of hENaC αβγ and the inserted restriction enzyme site. Lipofectamine LTX Reagent with PLUS & Reagent (Thermo Fisher) was used as a gene introduction reagent.

Transfected cells were plated on a 96-well plate (black wall, CellBIND surface; CORNING) and placed in a CO ₂ incubator (37°C, 5% CO ₂ ) for 24 to 72 hours with 10 µM amiloride (SIGMA) and 10% FBS (GIBCO). was cultured in DMEM (Dulbecco's Modified Eagle's Medium SIGMA) containing

Cell-based assays using FlexStation 3
FlexStation 3 (Molecular Devices) is a plate reader with dispensing function. Using this, a ligand solution was administered to cells loaded with a membrane potential-sensitive dye, and changes in fluorescence intensity over time were measured. The culture medium of cells seeded in 96-well plates was washed and replaced with assay buffer before measurement. The composition of the assay buffer solution is 130 mM NaCl, 10 mM D-glucose, 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 5 mM KCl, 2 _mM CaCl2, 1.2 mM _MgCl2 , Adjusted to pH 7.4 with NaOH.

The assay was performed at 27° C., and the fluorescence (560 nm) of Membrane potential assay kit blue (R8042 Molecular Devices) when excited at 530 nm was measured once every 2 seconds. Twenty seconds after the start of measurement, a ligand solution prepared with the same buffer as that used for washing was administered to the cells to double the concentration, and changes in fluorescence intensity over time were measured for 120 seconds. Adding 2.5 mM probenecid to the membrane potential assay kit blue stabilized the measurement results.

FIG. 2 shows a schematic diagram of the measurement results and a method for evaluating the activation ability. From the change in fluorescence over time obtained by FlexStation 3, the fluorescence intensity due to the ligand addition effect at 100 seconds after administration of the ligand solution and the fluorescence intensity of the vehicle (ligand-adjusted assay buffer solution) at 100 seconds. A delta relative fluorescence units (ΔRFU) value was calculated from the difference of , and defined as the cell response value.

The final concentration of the ligand solution was 10 nM, 50 nM, 100 nM, 1 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 75 μM, 100 μM, 300 μM, 500 μM, 1 mM (n = 6 each). .

─ Results ─
As shown in "sample" in FIG. For thiophene-2-carboxamide, 3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl )-N-Ethylbenzo[β]thiophene-2-carboxamide addition effect, delta relative fluorescence units, which is the difference in fluorescence intensity between vehicle (ligand-adjusted assay buffer) and fluorescence intensity at 100 seconds. Since the (ΔRFU) value was large in a dose-dependent manner, it was shown that hENaC αβγ has an activating ability.
and the EC ₅₀ of 3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β]thiophene-2-carboxamide is , 95.0±21.0 μM, and the maximum effective concentration (E _max ) was 928±67.8 in ΔRFU values.

Figure 1 shows the concentration of 3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β]thiophene-2-carboxamide Response curves are shown.

[Example 2] 3-Chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β using a membrane potential-sensitive dye hENaC αβγ activity by compounds with structural similarity to ]thiophene-2-carboxamide

<Method>
Structurally similar to 3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β]thiophene-2-carboxamide A compound with a similarity of 60% or higher in a similarity search using ChemBioFinder Ultra 12.0 (manufactured by CambridgeSoft).

ChemBioFinder Ultra 12.0, which is database management software for chemical structure information, was used for sampling of compounds with a similarity of 60% or more. A compound library owned by the University of Tokyo Drug Discovery Organization was used as the compound library to be searched. Definition of analogues of 3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β]thiophene-2-carboxamide The method is shown below. Namely, structural information of 3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β]thiophene-2-carboxamide were converted to numerical descriptors using ChemBioFinder Ultra 12.0 and the similarity was calculated. In ChemBioFinder, it is possible to combine representative structural key features such as Unity 2D Fingerprint, Similog key, MacCCS key, and convert structural information of compounds into descriptors using unique modified structural keys.

As a similarity evaluation scale for structural information of compounds, the Tanimoto similarity determination method having commutativity was used (Non-Patent Document 8). In the Tanimoto similarity judgment method, Q (query) and T (target) are expressed by the following formula using the Tanimoto coefficient:

を満たすものを60%以上の類似度と定義した。ここで示さるQ（クエリ）、T（ターゲット）は、それぞれ3-クロロ-Ｎ-(2-((1,1-ジオキシドテトラハイドロチオフェン-3-イル)アミノ)-2-オキソエチル)-Ｎ-エチルベンゾ[β]チオフェン-2-カルボキシアミドと3-クロロ-Ｎ-(2-((1,1-ジオキシドテトラハイドロチオフェン-3-イル)アミノ)-2-オキソエチル)-Ｎ-エチルベンゾ[β]チオフェン-2-カルボキシアミドに類似構造を持つ化合物の記述子を示す。

was defined as a similarity of 60% or higher. Q (query) and T (target) shown here are 3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N -ethylbenzo[β]thiophene-2-carboxamide and 3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β ] provides descriptors for compounds with similar structures to thiophene-2-carboxamide.

The compounds selected in this way are shown below.
(((1,1-dioxybenzo[beta]thiophen-2-yl)methoxy)carbonyl)-L-asparagine,

1-(4-(3-((benzo[beta]thiophen-2-ylmethyl)(methyl)amino)-2-ethylbenzo[1,2-alpha]pyridine-6-carbonyl)piperazin-1-yl)ethane- 1-on

N-(2-(tert-butylamino)-2-oxyoethyl)-3-chloro-N-ethylbenzo[beta]thiophene-2-carboxamide

N-(2-((1,1-Dioxydotetrahydrothiophen-3-yl)amino)-2-oxyethyl)-N-ethyl-4-fluorobenzo[beta]thiophene-2-carboxamide

2-benzyl-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxyethyl)-N-ethylbenzamide

1-(4-chlorophenyl)-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxyoethyl)-N-ethylcyclobutane-1-carboxamide

プラスミド及び発現タンパク質
実験方法及び材料は、

Plasmids and expressed proteins Experimental methods and materials were

Methods and materials similar to those described in [0023] were used.

<Evaluation method>
From the change in fluorescence over time obtained by the aforementioned FlexStation 3, the difference between the fluorescence intensity value at 100 seconds after administration of the ligand solution and the value at the start of measurement was calculated, and this was taken as the cell response value. Defined. If necessary, the response values of all cells were converted as relative response values when the response value of 1 μM S3969 cells measured as a control was defined as 1. An evaluation score of 0.18 or higher was regarded as active. A schematic diagram of the evaluation method is shown in FIG. This evaluation method is the same method as described in JP-A-2017-217005.

─ Results ─
3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β]thiophene-2- shown in FIGS. Carboxamides and six compounds with similar structures were shown to have the ability to activate hENaCαβγ.

Claims (4)

3-chloro-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β]thiophene- represented by the following formula (1) 2-carboxamide,
(((1,1-dioxybenzo[beta]thiophen-2-yl)methoxy)carbonyl)-L-asparagine represented by the following formula (2),
1-(4-(3-((benzo[beta]thiophen-2-ylmethyl)(methyl)amino)-2-ethylbenzo[1,2-alpha]pyridine-6-carbonyl represented by the following formula (3) )piperazin-1-yl)ethan-1-one,

N-(2-(tert-butylamino)-2-oxyoethyl)-3-chloro-N-ethylbenzo[beta]thiophene-2-carboxamide represented by the following formula (4),
N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxyethyl)-N-ethyl-4-fluorobenzo[beta]thiophene represented by the following formula (5) -2-carboxamide,
2-benzyl-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxyethyl)-N-ethylbenzamide represented by the following formula (6),
as well as,
1-(4-chlorophenyl)-N-(2-((1,1-dioxidotetrahydrothiophen-3-yl)amino)-2-oxyoethyl)-N-ethylcyclobutane represented by the following formula (7) -1-carboxamide,
A hENaC αβγ activator containing, as an active ingredient, one or more compounds selected from the group consisting of or a salt thereof. The hENaC αβγ activator according to claim 1, wherein said compound is a component derived from food or natural extract. A therapeutic agent for diseases, etc., comprising the hENaC αβγ activator according to claim 1 or 2. A salty taste enhancer containing the hENaC αβγ activator according to claim 1 or 2.

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