WO2020059660A1 - Compound having enac activation potency - Google Patents
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- WO2020059660A1 WO2020059660A1 PCT/JP2019/036105 JP2019036105W WO2020059660A1 WO 2020059660 A1 WO2020059660 A1 WO 2020059660A1 JP 2019036105 W JP2019036105 W JP 2019036105W WO 2020059660 A1 WO2020059660 A1 WO 2020059660A1
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- A—HUMAN NECESSITIES
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
- A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/12—Antidiarrhoeals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- the present invention relates to an activator of epithelial Na + channel hENaC ⁇ , a therapeutic agent for diseases and the like 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).
- ENaC ⁇ is mainly expressed in the stomach, kidney and colon, and ENaC ⁇ is mainly expressed in nervous tissues such as cerebral cortex, cerebellum, hippocampus and testis (Non-patent Documents 2-4). In the tongue, ENaC ⁇ is expressed in the anterior part where the chorda tympani nerve projects, and is involved in low-concentration salty taste reception ( ⁇ 150 mM NaCl) (Non-Patent Document 5). Further, as one of the grounds for exhibiting the salty taste enhancing effect and the salty taste inhibiting effect, the phenomenon of activating or inhibiting humanENaC (hENaC) ⁇ is widely used (prior patent documents 1-5).
- Non-Patent Documents 6, 7 Non-Patent Documents 6, 7
- activators that activate hENaC ⁇ can be considered for the treatment of pseudoaldosteronism type 1 and neonatal respiratory distress syndrome.
- Compound S3969 is known as a hENaCh ⁇ activator (activator), and is expected as a therapeutic agent for the above-mentioned diseases (Non-Patent Document 6).
- the indole ring is considered to be one partial structure that activates hENaC, but there is no report on a partial structure capable of activating hENaC except for the indole ring.
- hENaC ⁇ activators not only alleviate the genetic diseases associated with hENaC ⁇ , but also It is expected to modify salty taste reception and greatly improve taste.
- the object of the present invention was to find a compound having an excellent hENaC ⁇ activating action.
- Another object of the present invention is to provide a therapeutic agent for a disease or the like in the pharmaceutical field containing the compound and a salty taste enhancer in the food field.
- the present inventors have conducted intensive studies and have found that 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [ ⁇ ] thiophene. It has been found that a compound having the same or similar structure as -2-carboxamide or a salt thereof has an activity of activating hENaC.
- the first invention of the present application is: “3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [ ⁇ ] thiophene represented by the following formula (1)
- Activator of hENaC ⁇ comprising a compound having the same or similar structure as -2-carboxamide or a salt thereof as an active ingredient
- the compound is a component derived from a food or a natural extract. That is, the second invention of the present application is: The hENaC ⁇ activator according to claim 1, wherein the compound is a component derived from a food or a natural extract.
- the compound of the present application or a salt thereof can also be used as a therapeutic drug for diseases and the like. That is, the third invention of the present application: “A therapeutic agent for a disease or the like containing the hENaC ⁇ activator according to claim 1 or 2”. It is.
- the compound of the present invention or a salt thereof can also be used as a salty taste enhancer. That is, the fourth invention of the present application “A salty taste enhancer containing the hENaC ⁇ activator according to claim 1 or 2”.
- an activator of hENaC ⁇ is provided. Further, the 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [ ⁇ ] thiophen-2-carboxamide of the present invention
- a therapeutic agent for a genetic disease caused by hENaC ⁇ is provided by containing an activator containing a compound having the same or similar structure as described above or a salt thereof as an active ingredient. Further, a salty taste enhancer is provided by activating hENaC ⁇ which is also a salty taste receptor.
- FIG. 4 is a schematic view illustrating a method of Example 1 and Example 2. This is the result of Example 2, and using 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N- using a membrane potential sensitive dye.
- 3 shows hENaC ⁇ activity by ethylbenzo [ ⁇ ] thiophene-2-carboxamide and compounds (three kinds) having structures similar thereto. This is the result of Example 2, and using 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N- using a membrane potential sensitive dye. 3 shows hENaC ⁇ activity by compounds (three kinds) having a structure similar to ethylbenzo [ ⁇ ] thiophene-2-carboxamide.
- hENaC ⁇ refers to a human gene human ENaC ⁇ (or SCNN1a), human ENaC ⁇ (or SCNN1b), which is encoded by human ENaC ⁇ (or SCNN1g), forms ⁇ trimers on a biological membrane, Refers to a membrane protein that exhibits the properties of a sodium channel.
- the activator is a compound that has an effect on the opening structure of the pores of the trimeric membrane protein composed of hENaC ⁇ and has the property of increasing the inflow efficiency of mainly sodium ions flowing through hENaC.
- the 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [ ⁇ ] thiophen-2-carboxamide is as follows: Having a structure.
- the compound has a benzothiophenone ring.
- the present inventors have found that as a result of screening a large number of compounds, the present compounds have the ability to activate hENaC ⁇ . Furthermore, a compound having a structure similar to the compound was searched, and a plurality of compounds having the ability to activate hENaC ⁇ were found. The invention further contemplates salts of these compounds.
- a compound having a similar structure to 2-carboxyamide indicates a compound showing a similarity of 60% or more in a similar search using ChemBioFinder Ultra 12.0 (manufactured by CambridgeSoft).
- ChemBioFinder Ultra 12.0 a database management software for chemical structure information, can be used.
- a compound library to be searched a compound library owned by a university, various research institutions, or the like can be used.
- ChemBioFinder it is possible to combine structural key features such as Unity 2D Fingerprint, Similog key, and MaCCS key, and to convert the structural information of a compound into a descriptor using a modified unique structural key.
- a Tanimoto similarity determination method having commutability was used (Non-Patent Document 8).
- Q (query) and T (target) are expressed by the following formulas using Tanimoto coefficients:
- Q (query) and T (target) shown here are respectively 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N -Ethylbenzo [ ⁇ ] thiophen-2-carboxamide and 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [ ⁇ ]
- Descriptors of compounds having a structure similar to thiophene-2-carboxamide are shown.
- Components derived from foods and natural extracts Compound (3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2) capable of activating hENaC ⁇ of the present invention -Oxoethyl) -N-ethylbenzo [ ⁇ ] thiophene-2-carboxamide) can be obtained as an ingredient derived from foods and natural products.
- the activator of hENaC ⁇ of the present invention can be used as a therapeutic agent for diseases and the like.
- pseudohypoaldosteronism type I a disease in which re-absorption of sodium ions in the kidney is impaired, and in the lung, diseases such as pulmonary edema and neonatal respiratory syndrome caused by increasing intracellular sodium ions. It can be used (Non-Patent Document 7).
- Salty taste enhancer The hENaC ⁇ activator of the present invention can be used as a salty taste enhancer. That is, the salty taste enhancer means to exert an action of strongly feeling the salty taste of salt.
- 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 above compound alone, or may be provided in the form of a solid composition or a liquid composition. When provided as a composition, additives that can be used in the production of foods and beverages, such as excipients, pigments, and flavors, may be contained as needed, as long as the salty taste enhancing effect is not hindered.
- Example 1 3-Chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [ ⁇ ] thiophene Using Cultured Cells Results of hENaC ⁇ activity by 2-carboxyamide.
- Plasmid and expression protein experimental methods were performed according to JP-A-2017-217005. That is, in order to express human human ENaC ⁇ (hENaC ⁇ ) in HEK293T cells, a plasmid vector pEAK10 (EdgeBiosystem) having an EF1 ⁇ promoter was used.
- Table 1 shows the accession numbers indicating the gene sequences of hENaC ⁇ and the inserted restriction enzyme sites.
- Lipofectamine LTX Reagent with PLUS & Reagent was used as a gene transfer reagent.
- the transfected cells are seeded on a 96-well plate (black wall, CellBIND surface; CORNING), and in a CO 2 incubator (37 ° C., 5% CO 2 ) for 24 to 72 hours, 10 ⁇ M amiloride (SIGMA) and 10% FBS (GIBCO) Were cultured in DMEM (Dulbecco's modified Eagle's medium SIGMA) containing
- Cell-based assay using FlexStation 3 FlexStation 3 is a plate reader with a dispensing function. Using this, a ligand solution was administered to cells loaded with a membrane potential-sensitive dye, and the change over time in the fluorescence intensity was measured. The culture medium of the cells seeded in the 96-well plate was washed and replaced with an assay buffer solution before measurement.
- 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 CaCl 2 , 1.2 mM MgCl 2 , The pH was adjusted to 7.4 using 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 the measurement, a ligand solution prepared to a concentration twice as much as the buffer used for washing was administered to the cells, and a change in fluorescence intensity over time was measured for 120 seconds. The measurement results could be more stabilized by adding 2.5 mM probenecid to Membrane potential assay kit blue.
- FIG. 2 shows a schematic diagram of the measurement results and a method of evaluating the activation ability. From the time course of fluorescence obtained by FlexStation 3, the value of the fluorescence intensity due to the effect of ligand addition at 100 seconds after administration of the ligand solution and the fluorescence intensity of the vehicle (assay buffer solution adjusted for ligand) at 100 seconds The value of delta relative fluorescence units ( ⁇ RFU) was calculated from the difference, and this was defined as the response value of the cell.
- ⁇ RFU delta relative fluorescence units
- Delta relative fluorescence units which is the difference between the fluorescence intensity value due to the effect of addition of -N-ethylbenzo [ ⁇ ] thiophene-2-carboxamide and the fluorescence intensity at 100 seconds with the vehicle (assay buffer solution in which the ligand was adjusted).
- the large ( ⁇ RFU) value was obtained in a concentration-dependent manner, indicating that hENaC ⁇ has the activating ability.
- the EC 50 of 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [ ⁇ ] thiophen-2-carboxamide is , 95.0 ⁇ 21.0 ⁇ M, and the maximum effect concentration (E max ) was 928 ⁇ 67.8 in ⁇ RFU value.
- FIG. 1 shows the concentration of 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [ ⁇ ] thiophen-2-carboxamide. The response curve is shown.
- ChemBioFinder ⁇ Ultra ⁇ 12.0 ⁇ a database management software for chemical structure information.
- a compound library to be searched a compound library owned by the University of Tokyo Drug Discovery Organization was used. Definition of analogous compounds of 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [ ⁇ ] thiophen-2-carboxamide The method is described below.
- Non-Patent Document 8 a Tanimoto similarity determination method having commutability was used.
- Q (query) and T (target) are expressed by the following formulas using Tanimoto coefficients:
- Q (query) and T (target) shown here are respectively 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N -Ethylbenzo [ ⁇ ] thiophen-2-carboxamide and 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [ ⁇ ]
- Descriptors of compounds having a structure similar to thiophene-2-carboxamide are shown.
- FIG. 2 shows a schematic diagram of the evaluation method. This evaluation method is the same method as described in JP-A-2017-217005.
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Abstract
[Problem] The present invention addresses the problem of providing a novel hENaC αβγ activating agent. [Solution] Provided is a hENaC αβγ activating agent including, as an effective component, a compound or a salt thereof having a structure identical to or similar to 3-chloro-N-(2-((1,1-dioxidetetrahydrothiophene-3-yl)amino)-2-oxoethyl)-N-ethylbenzo[β]thiophene-2-carboxyamide, which is represented by formula (1). Utilization of this hENaC αβγ activating agent contributes to further development of drugs, the food industry, and the health sciences.
Description
本発明は、上皮性Na+チャンネルhENaC αβγの活性化剤、及び、これを含有する疾患等の治療剤並びに塩味増強剤に関する。
The present invention relates to an activator of epithelial Na + channel hENaC αβγ, a therapeutic agent for diseases and the like containing the same, and a salty taste enhancer.
上皮性ナトリウムチャンネル（epithelial sodium channel ENaC）は、ENaC / degenerinファミリーに属する非電位依存性のNaイオンチャネルである（非特許文献１）。
Epithelial sodium channel ENaC is a non-voltage-dependent Na ion channel belonging to the ENaC / degenerin family (Non-Patent Document 1).
ENaCを構成する各々のサブユニットは二回膜貫通領域を持ち、αβγもしくはδβγの三量体で機能する。ENaCαβγは、主に胃、腎臓、結腸に発現し、ENaCδβγは、主に大脳皮質や小脳、海馬などの神経組織や精巣で発現する（非特許文献2-4）。また、舌ではENaC αβγが、鼓索神経が投射する前方部に発現し、低濃度の塩味受容（＜150 mM NaCl）に関与する（非特許文献5）。また塩味増強効果及び、塩味阻害効果を示す根拠の１つとしてhumanENaC(hENaC) αβγを活性化または、阻害する現象が広く用いられている（先行特許文献1-5）。
Each subunit constituting ENaC has a double transmembrane domain and functions as an αβγ or δβγ trimer. ENaCαβγ is mainly expressed in the stomach, kidney and colon, and ENaCδβγ is mainly expressed in nervous tissues such as cerebral cortex, cerebellum, hippocampus and testis (Non-patent Documents 2-4). In the tongue, ENaC αβγ is expressed in the anterior part where the chorda tympani nerve projects, and is involved in low-concentration salty taste reception (<150 mM NaCl) (Non-Patent Document 5). Further, as one of the grounds for exhibiting the salty taste enhancing effect and the salty taste inhibiting effect, the phenomenon of activating or inhibiting humanENaC (hENaC) Δαβγ is widely used (prior patent documents 1-5).
医薬分野では、hENaC αβγの遺伝的変異による疾患として、高Na+血症や腎疾患、リルド症候群等が挙げられる。これらの治療としてhENaCαβγの阻害剤であるアミロライドなどの使用が挙げられる(非特許文献6、7)
In the field of medicine, diseases caused by genetic mutation of hENaC αβγ include hyperna + bloodemia, kidney disease, Lildo syndrome, and the like. These treatments include the use of hENaCαβγ inhibitors amiloride and the like (Non-Patent Documents 6, 7)
一方で偽性アルドステロン症1型や新生児呼吸窮迫症候群の治療には、hENaC αβγを活性化させる活性化剤が考えられる。化合物S3969は、hENaC αβγ活性化剤（アクチベーター）として知られており、上記の疾患の治療薬として期待されている(非特許文献6)。
On the other hand, activators that activate hENaC αβγ can be considered for the treatment of pseudoaldosteronism type 1 and neonatal respiratory distress syndrome. Compound S3969 is known as a hENaChαβγ activator (activator), and is expected as a therapeutic agent for the above-mentioned diseases (Non-Patent Document 6).
しかしながら、S3969を除いてhENaCαβγを活性化させる小分子の報告例はない。
However, there are no reports of small molecules that activate hENaCαβγ except for S3969.
S3969の構造からインドール環がhENaCを活性化させる１つの部分構造であると考えられているが、インドール環を除くhENaC活性化能を持つ部分構造に関する報告例はない。
From the structure of S3969, the indole ring is considered to be one partial structure that activates hENaC, but there is no report on a partial structure capable of activating hENaC except for the indole ring.
そこで、hENaC αβγを活性化させる小分子や部分構造を探索、開発することで、hENaC αβγ活性化剤（アクチベーター）が、hENaC αβγが関連する遺伝的疾患を緩和するばかりでなく、口腔内の塩味受容を修飾し、呈味性を大きく向上させることが期待されている。
Therefore, by searching for and developing small molecules and partial structures that activate hENaC αβγ, hENaC αβγ activators (activators) not only alleviate the genetic diseases associated with hENaC αβγ, but also It is expected to modify salty taste reception and greatly improve taste.
Therefore, by searching for and developing small molecules and partial structures that activate hENaC αβγ, hENaC αβγ activators (activators) not only alleviate the genetic diseases associated with hENaC αβγ, but also It is expected to modify salty taste reception and greatly improve taste.
本発明は、優れたhENaC αβγの活性化作用を有する化合物を見出すことを課題とした。また、当該化合物を含有し、医薬分野での疾患等の治療剤、また食品分野での塩味増強剤を提供する事を課題とした。
The object of the present invention was to find a compound having an excellent hENaCβαβγ activating action. Another object of the present invention is to provide a therapeutic agent for a disease or the like in the pharmaceutical field containing the compound and a salty taste enhancer in the food field.
本発明者は、鋭意研究の結果、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同一若しくは類似構造を有する化合物又はその塩にhENaCを活性化させる作用があることを見出した。
すなわち、本願第一の発明は、
“下記式（１）で表される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同一若しくは類似構造を有する化合物又はその塩を有効成分とするhENaC αβγの活性化剤 The present inventors have conducted intensive studies and have found that 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophene. It has been found that a compound having the same or similar structure as -2-carboxamide or a salt thereof has an activity of activating hENaC.
That is, the first invention of the present application is:
“3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophene represented by the following formula (1) Activator of hENaC αβγ comprising a compound having the same or similar structure as -2-carboxamide or a salt thereof as an active ingredient
すなわち、本願第一の発明は、
“下記式（１）で表される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同一若しくは類似構造を有する化合物又はその塩を有効成分とするhENaC αβγの活性化剤 The present inventors have conducted intensive studies and have found that 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophene. It has been found that a compound having the same or similar structure as -2-carboxamide or a salt thereof has an activity of activating hENaC.
That is, the first invention of the present application is:
“3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophene represented by the following formula (1) Activator of hENaC αβγ comprising a compound having the same or similar structure as -2-carboxamide or a salt thereof as an active ingredient
さらに、前記化合物が食品、天然抽出物由来の成分であることが好ましい。
すなわち、本願第二の発明は、
“前記化合物が食品、天然抽出物由来の成分である請求項１に記載のhENaC αβγ活性化剤。”、である。 Further, it is preferable that the compound is a component derived from a food or a natural extract.
That is, the second invention of the present application is:
The hENaC αβγ activator according to claim 1, wherein the compound is a component derived from a food or a natural extract.
すなわち、本願第二の発明は、
“前記化合物が食品、天然抽出物由来の成分である請求項１に記載のhENaC αβγ活性化剤。”、である。 Further, it is preferable that the compound is a component derived from a food or a natural extract.
That is, the second invention of the present application is:
The hENaC αβγ activator according to claim 1, wherein the compound is a component derived from a food or a natural extract.
次に、本願の化合物又はその塩は、疾患等の治療薬としても利用できる。
すなわち、本願第三の発明は、
“請求項１又は２のいずれかに記載のhENaC αβγ活性化剤を含有する疾患等の治療剤。”
である。 Next, the compound of the present application or a salt thereof can also be used as a therapeutic drug for diseases and the like.
That is, the third invention of the present application:
“A therapeutic agent for a disease or the like containing the hENaC αβγ activator according to claim 1 or 2”.
It is.
すなわち、本願第三の発明は、
“請求項１又は２のいずれかに記載のhENaC αβγ活性化剤を含有する疾患等の治療剤。”
である。 Next, the compound of the present application or a salt thereof can also be used as a therapeutic drug for diseases and the like.
That is, the third invention of the present application:
“A therapeutic agent for a disease or the like containing the hENaC αβγ activator according to claim 1 or 2”.
It is.
次に、本願の化合物又はその塩は、塩味増強剤としても利用できる。
すなわち、本願第四の発明は、
“請求項１又は２のいずれかに記載のhENaC αβγ活性化剤を含有する塩味増強剤。”、である。 Next, the compound of the present invention or a salt thereof can also be used as a salty taste enhancer.
That is, the fourth invention of the present application
“A salty taste enhancer containing the hENaC αβγ activator according to claim 1 or 2”.
すなわち、本願第四の発明は、
“請求項１又は２のいずれかに記載のhENaC αβγ活性化剤を含有する塩味増強剤。”、である。 Next, the compound of the present invention or a salt thereof can also be used as a salty taste enhancer.
That is, the fourth invention of the present application
“A salty taste enhancer containing the hENaC αβγ activator according to claim 1 or 2”.
本発明により、hENaC αβγの活性化剤が提供される。また、本発明の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同一若しくは類似構造を有する化合物又はその塩を有効成分とする活性化剤を含有することで、hENaC αβγが原因と考えられる遺伝子疾患の治療剤が提供される。さらに、塩味受容体でもあるhENaC αβγ活性化による塩味増強剤が提供される。
According to the present invention, an activator of hENaCΔαβγ is provided. Further, the 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2-carboxamide of the present invention A therapeutic agent for a genetic disease caused by hENaCΔαβγ is provided by containing an activator containing a compound having the same or similar structure as described above or a salt thereof as an active ingredient. Further, a salty taste enhancer is provided by activating hENaC αβγ which is also a salty taste receptor.
また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-カルボキシアミド及び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した構造がhENaC αβγを活性化するという構造情報は、研究開発のツールとして有用であり、医薬、食品産業及び健康科学のさらなる発展に貢献することができる。
3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2-carboxamide and 3-chloro A structure similar to -N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2-carboxyamide gives hENaC αβγ The structural information of activation is useful as a research and development tool and can contribute to the further development of the pharmaceutical, food industry and health sciences.
１．hENaC αβγの活性化剤
本発明にいう、hENaC αβγとは、ヒトの遺伝子human ENaCα（あるいはSCNN1a）, human ENaCβ（あるいはSCNN1b）human ENaCγ（あるいはSCNN1g）にコードされ生体膜上で、αβγの三量体を形成し、上皮性ナトリウムチャネルの性質を示す膜タンパク質のことをいう。
また、その活性化剤とは、hENaC αβγで構成される三量体膜タンパク質の孔の開口構造に影響を与え、hENaCを介して流入する主にナトリウムイオンの流入効率を上げる性質をもつ化合物をいう。 1. Activator of hENaC αβγ
In the present invention, hENaC αβγ refers to a human gene human ENaCα (or SCNN1a), human ENaCβ (or SCNN1b), which is encoded by human ENaCγ (or SCNN1g), forms αβγ trimers on a biological membrane, Refers to a membrane protein that exhibits the properties of a sodium channel.
The activator is a compound that has an effect on the opening structure of the pores of the trimeric membrane protein composed of hENaC αβγ and has the property of increasing the inflow efficiency of mainly sodium ions flowing through hENaC. Say.
本発明にいう、hENaC αβγとは、ヒトの遺伝子human ENaCα（あるいはSCNN1a）, human ENaCβ（あるいはSCNN1b）human ENaCγ（あるいはSCNN1g）にコードされ生体膜上で、αβγの三量体を形成し、上皮性ナトリウムチャネルの性質を示す膜タンパク質のことをいう。
また、その活性化剤とは、hENaC αβγで構成される三量体膜タンパク質の孔の開口構造に影響を与え、hENaCを介して流入する主にナトリウムイオンの流入効率を上げる性質をもつ化合物をいう。 1. Activator of hENaC αβγ
In the present invention, hENaC αβγ refers to a human gene human ENaCα (or SCNN1a), human ENaCβ (or SCNN1b), which is encoded by human ENaCγ (or SCNN1g), forms αβγ trimers on a biological membrane, Refers to a membrane protein that exhibits the properties of a sodium channel.
The activator is a compound that has an effect on the opening structure of the pores of the trimeric membrane protein composed of hENaC αβγ and has the property of increasing the inflow efficiency of mainly sodium ions flowing through hENaC. Say.
２．本発明のhENaC αβγ活性化能を有する化合物
本発明においては、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同一若しくは類似構造を有する化合物又はその塩、を対象とする。
本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-カルボキシアミドは、以下の構造を有する。 2. HENaC αβγ activating compound of the present invention In the present invention, 3-chloro-N- (2-((1,1- dioxidetetrahydrothiophen -3-yl) amino) -2- Oxoethyl) -N-ethylbenzo [β] thiophene-2-carboxamide or a salt thereof having the same or similar structure.
The 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2-carboxamide is as follows: Having a structure.
本発明においては、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同一若しくは類似構造を有する化合物又はその塩、を対象とする。
本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-カルボキシアミドは、以下の構造を有する。 2. HENaC αβγ activating compound of the present invention In the present invention, 3-chloro-N- (2-((1,1- dioxidetetrahydrothiophen -3-yl) amino) -2- Oxoethyl) -N-ethylbenzo [β] thiophene-2-carboxamide or a salt thereof having the same or similar structure.
The 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2-carboxamide is as follows: Having 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 has a benzothiophenone ring. The present inventors have found that as a result of screening a large number of compounds, the present compounds have the ability to activate hENaC αβγ.
Furthermore, a compound having a structure similar to the compound was searched, and a plurality of compounds having the ability to activate hENaC αβγ were found. The invention further contemplates salts of these compounds.
３．類似構造を有する化合物
本発明にいう“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有する化合物とは、ChemBioFinder Ultra 12.0 (CambridgeSoft製)による類似検索で60%以上の類似度を示すものを指すものとする。
60%以上の類似度を持つ化合物のサンプリングに関して、化学構造情報のデータベース管理ソフトであるChemBioFinder Ultra 12.0 を用いることができる。検索対象とする化合物ライブラリーは、大学、種々の研究機関等が所有する化合物ライブラリーを用いることができる。 3. Compound having a similar structure "3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophene according to the present invention A compound having a similar structure to 2-carboxyamide indicates a compound showing a similarity of 60% or more in a similar search using ChemBioFinder Ultra 12.0 (manufactured by CambridgeSoft).
For sampling of compounds having a similarity of 60% or more, ChemBioFinder Ultra 12.0, a database management software for chemical structure information, can be used. As a compound library to be searched, a compound library owned by a university, various research institutions, or the like can be used.
本発明にいう“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有する化合物とは、ChemBioFinder Ultra 12.0 (CambridgeSoft製)による類似検索で60%以上の類似度を示すものを指すものとする。
60%以上の類似度を持つ化合物のサンプリングに関して、化学構造情報のデータベース管理ソフトであるChemBioFinder Ultra 12.0 を用いることができる。検索対象とする化合物ライブラリーは、大学、種々の研究機関等が所有する化合物ライブラリーを用いることができる。 3. Compound having a similar structure "3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophene according to the present invention A compound having a similar structure to 2-carboxyamide indicates a compound showing a similarity of 60% or more in a similar search using ChemBioFinder Ultra 12.0 (manufactured by CambridgeSoft).
For sampling of compounds having a similarity of 60% or more, ChemBioFinder Ultra 12.0, a database management software for chemical structure information, can be used. As a compound library to be searched, a compound library owned by a university, various research institutions, or the like can be used.
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-カルボキシアミドの類似化合物の定義方法を下記に示す。即ち、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-カルボキシアミドの構造情報をChemBioFinder Ultra 12.0を用いて数値化された記述子に変換し、類似度を計算することができる。ChemBioFinder では、Unity 2D Fingerprint、Similog key、MaCCS keyのといった代表的な構造キー特徴を組み合わせ、改変した独自の構造キーを用いて化合物の構造情報を記述子への変換が行う事が可能である。
化合物の構造情報を類似性評価尺度については、可換性を持つTanimoto類似性判定法を用いた（非特許文献８）。Tanimoto類似判定法は、Tanimoto係数を用いてQ（クエリ）、T（ターゲット）が以下の数式： Definition of analogous compounds of 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2-carboxamide The method is described below. That is, structural information of 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2-carboxamide Can be converted into a numerical descriptor using ChemBioFinder Ultra 12.0, and the similarity can be calculated. In ChemBioFinder, it is possible to combine structural key features such as Unity 2D Fingerprint, Similog key, and MaCCS key, and to convert the structural information of a compound into a descriptor using a modified unique structural key.
As for the similarity evaluation scale of the structural information of the compound, a Tanimoto similarity determination method having commutability was used (Non-Patent Document 8). In the Tanimoto similarity determination method, Q (query) and T (target) are expressed by the following formulas using Tanimoto coefficients:
化合物の構造情報を類似性評価尺度については、可換性を持つTanimoto類似性判定法を用いた（非特許文献８）。Tanimoto類似判定法は、Tanimoto係数を用いてQ（クエリ）、T（ターゲット）が以下の数式： Definition of analogous compounds of 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2-carboxamide The method is described below. That is, structural information of 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2-carboxamide Can be converted into a numerical descriptor using ChemBioFinder Ultra 12.0, and the similarity can be calculated. In ChemBioFinder, it is possible to combine structural key features such as Unity 2D Fingerprint, Similog key, and MaCCS key, and to convert the structural information of a compound into a descriptor using a modified unique structural key.
As for the similarity evaluation scale of the structural information of the compound, a Tanimoto similarity determination method having commutability was used (Non-Patent Document 8). In the Tanimoto similarity determination method, Q (query) and T (target) are expressed by the following formulas using Tanimoto coefficients:
３．食品、天然抽出物由来の成分
本発明のhENaC αβγの活性化能を有する化合物（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同一又は類似構造を持つ化合物）は、食品、天然物由来成分として得ることができる。 3. Components derived from foods and natural extracts Compound (3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2) capable of activating hENaC αβγ of the present invention -Oxoethyl) -N-ethylbenzo [β] thiophene-2-carboxamide) can be obtained as an ingredient derived from foods and natural products.
本発明のhENaC αβγの活性化能を有する化合物（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同一又は類似構造を持つ化合物）は、食品、天然物由来成分として得ることができる。 3. Components derived from foods and natural extracts Compound (3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2) capable of activating hENaC αβγ of the present invention -Oxoethyl) -N-ethylbenzo [β] thiophene-2-carboxamide) can be obtained as an ingredient derived from foods and natural products.
４．疾患等の治療剤
本発明のhENaC αβγの活性化剤は、疾患等の治療剤として利用することができる。具体的には、腎臓におけるナトリウムイオンの再吸収が損なわれてしまう疾病である偽性低アルドステロン症I型、肺では、細胞内ナトリウムイオンを増加させることによる肺水腫、新生児呼吸症候群等の疾患に利用することができる（非特許文献７）。 4. Therapeutic agent for diseases and the like The activator of hENaCαβγ of the present invention can be used as a therapeutic agent for diseases and the like. Specifically, pseudohypoaldosteronism type I, a disease in which re-absorption of sodium ions in the kidney is impaired, and in the lung, diseases such as pulmonary edema and neonatal respiratory syndrome caused by increasing intracellular sodium ions. It can be used (Non-Patent Document 7).
本発明のhENaC αβγの活性化剤は、疾患等の治療剤として利用することができる。具体的には、腎臓におけるナトリウムイオンの再吸収が損なわれてしまう疾病である偽性低アルドステロン症I型、肺では、細胞内ナトリウムイオンを増加させることによる肺水腫、新生児呼吸症候群等の疾患に利用することができる（非特許文献７）。 4. Therapeutic agent for diseases and the like The activator of hENaCαβγ of the present invention can be used as a therapeutic agent for diseases and the like. Specifically, pseudohypoaldosteronism type I, a disease in which re-absorption of sodium ions in the kidney is impaired, and in the lung, diseases such as pulmonary edema and neonatal respiratory syndrome caused by increasing intracellular sodium ions. It can be used (Non-Patent Document 7).
５．塩味増強剤
本発明のhENaC αβγの活性化剤は塩味増強剤として利用できる。すなわち、塩味増強剤とは、食塩の塩味を強く感じさせる作用を発揮することをいう。当該塩味増強剤は、飲食品等に幅広く利用することができる。
本発明の塩味増強剤は、上記化合物のみの形態で提供されるものであっても、固形組成
物または液体組成物の形態により提供されるものであってもよい。組成物として提供され
る場合は、塩味増強作用を妨げない範囲において、必要に応じて、賦形剤、色素、香料等
の飲食品の製造に使用可能な添加剤を含有してもよい。
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 to exert an action of strongly feeling the salty taste of salt. 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 above compound alone, or may be provided in the form of a solid composition or a liquid composition. When provided as a composition, additives that can be used in the production of foods and beverages, such as excipients, pigments, and flavors, may be contained as needed, as long as the salty taste enhancing effect is not hindered.
本発明のhENaC αβγの活性化剤は塩味増強剤として利用できる。すなわち、塩味増強剤とは、食塩の塩味を強く感じさせる作用を発揮することをいう。当該塩味増強剤は、飲食品等に幅広く利用することができる。
本発明の塩味増強剤は、上記化合物のみの形態で提供されるものであっても、固形組成
物または液体組成物の形態により提供されるものであってもよい。組成物として提供され
る場合は、塩味増強作用を妨げない範囲において、必要に応じて、賦形剤、色素、香料等
の飲食品の製造に使用可能な添加剤を含有してもよい。
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 to exert an action of strongly feeling the salty taste of salt. 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 above compound alone, or may be provided in the form of a solid composition or a liquid composition. When provided as a composition, additives that can be used in the production of foods and beverages, such as excipients, pigments, and flavors, may be contained as needed, as long as the salty taste enhancing effect is not hindered.
以下、実施例により本発明をより詳細に説明する。これらの実施例は、本発明を限定するものではない。
Hereinafter, the present invention will be described in more detail with reference to examples. These examples do not limit the invention.
［実施例１］培養細胞を用いた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よるhENaC αβγ活性の結果。
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よるhENaC αβγの活性化能を見るために、hENaC αβγを発現するHEK293T細胞を用いたセルベースアッセイ法で濃度依存曲線を作成した（図１）。
Example 1 3-Chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophene Using Cultured Cells Results of hENaC αβγ activity by 2-carboxyamide.
Activity of hENaC αβγ by 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2-carboxyamide In order to check the chemopotency, a concentration-dependent curve was prepared by a cell-based assay using HEK293T cells expressing hENaC αβγ (FIG. 1).
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よるhENaC αβγの活性化能を見るために、hENaC αβγを発現するHEK293T細胞を用いたセルベースアッセイ法で濃度依存曲線を作成した（図１）。
Example 1 3-Chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophene Using Cultured Cells Results of hENaC αβγ activity by 2-carboxyamide.
Activity of hENaC αβγ by 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2-carboxyamide In order to check the chemopotency, a concentration-dependent curve was prepared by a cell-based assay using HEK293T cells expressing hENaC αβγ (FIG. 1).
＜方法＞
プラスミド及び発現タンパク質
実験手法は、特開2017-217005に準じて行った。即ちヒトhumanENaC αβγ（hENaC αβγ）をHEK293T細胞に発現させるために、EF1αプロモーターを持つプラスミドベクター pEAK10（EdgeBiosystem社）を用いた。pEAK10 のマルチクローニングサイト間にhENaC α、hENaC β、hENaC γのcDNA領域を挿入した発現コンストラクトをα：β：γ＝1：1：1の割合でミックスし、1 μg/35mm dishの比率で遺伝子導入を行った。 hENaC αβγの各遺伝子配列を示すaccession numberと挿入した制限酵素サイトを表1に示す。遺伝子導入試薬にはLipofectamine LTX Reagent with PLUS＆ Reagent（Thermo Fisher社）を用いた。 <Method>
Plasmid and expression protein experimental methods were performed according to JP-A-2017-217005. That is, in order to express human human ENaCαβγ (hENaCαβγ) in HEK293T cells, a plasmid vector pEAK10 (EdgeBiosystem) having an EF1α promoter was used. The expression construct in which the cDNA regions of hENaCα, hENaCβ, and hENaCγ are inserted between the multiple cloning sites of pEAK10 is mixed at a ratio of α: β: γ = 1: 1: 1, and the gene is mixed at a ratio of 1 μg / 35 mm dish. Introduced. Table 1 shows the accession numbers indicating the gene sequences of hENaC αβγ and the inserted restriction enzyme sites. Lipofectamine LTX Reagent with PLUS & Reagent (Thermo Fisher) was used as a gene transfer reagent.
プラスミド及び発現タンパク質
実験手法は、特開2017-217005に準じて行った。即ちヒトhumanENaC αβγ（hENaC αβγ）をHEK293T細胞に発現させるために、EF1αプロモーターを持つプラスミドベクター pEAK10（EdgeBiosystem社）を用いた。pEAK10 のマルチクローニングサイト間にhENaC α、hENaC β、hENaC γのcDNA領域を挿入した発現コンストラクトをα：β：γ＝1：1：1の割合でミックスし、1 μg/35mm dishの比率で遺伝子導入を行った。 hENaC αβγの各遺伝子配列を示すaccession numberと挿入した制限酵素サイトを表1に示す。遺伝子導入試薬にはLipofectamine LTX Reagent with PLUS＆ Reagent（Thermo Fisher社）を用いた。 <Method>
Plasmid and expression protein experimental methods were performed according to JP-A-2017-217005. That is, in order to express human human ENaCαβγ (hENaCαβγ) in HEK293T cells, a plasmid vector pEAK10 (EdgeBiosystem) having an EF1α promoter was used. The expression construct in which the cDNA regions of hENaCα, hENaCβ, and hENaCγ are inserted between the multiple cloning sites of pEAK10 is mixed at a ratio of α: β: γ = 1: 1: 1, and the gene is mixed at a ratio of 1 μg / 35 mm dish. Introduced. Table 1 shows the accession numbers indicating the gene sequences of hENaC αβγ and the inserted restriction enzyme sites. Lipofectamine LTX Reagent with PLUS & Reagent (Thermo Fisher) was used as a gene transfer reagent.
遺伝子導入した細胞を96ウェルプレート(black wall, CellBIND surface; CORNING)に播き、24から72時間CO2インキュベータ内(37℃, 5％ CO2)、10μMアミロライド（SIGMA）と10 ％FBS(GIBCO)を含むDMEM(ダルベッコ改変イーグル培地 SIGMA)で培養した。
The transfected cells are seeded on a 96-well plate (black wall, CellBIND surface; CORNING), and in a CO 2 incubator (37 ° C., 5% CO 2 ) for 24 to 72 hours, 10 μM amiloride (SIGMA) and 10% FBS (GIBCO) Were cultured in DMEM (Dulbecco's modified Eagle's medium SIGMA) containing
FlexStation 3を用いたセルベースアッセイ
FlexStation 3(Molecular Devices)は分注機能付きのプレートリーダーである。これを用いて膜電位感受性色素を負荷した細胞にリガンド溶液を投与し、その蛍光強度の経時的変化を測定した。96ウェルプレートに播種した細胞の培養液は、測定前にアッセイバッファー液で洗浄し、置換した。アッセイバッファー液の組成は、130 mM NaCl、10 mM Dグルコース、10 mM HEPES（4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid）、5 mM KCl、2 mM CaCl2、1.2 mM MgCl2であり、NaOHを用いてpH 7.4に調整した。 Cell-based assay using FlexStation 3 FlexStation 3 (Molecular Devices) is a plate reader with a dispensing function. Using this, a ligand solution was administered to cells loaded with a membrane potential-sensitive dye, and the change over time in the fluorescence intensity was measured. The culture medium of the cells seeded in the 96-well plate was washed and replaced with an assay buffer solution 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 CaCl 2 , 1.2 mM MgCl 2 , The pH was adjusted to 7.4 using NaOH.
FlexStation 3(Molecular Devices)は分注機能付きのプレートリーダーである。これを用いて膜電位感受性色素を負荷した細胞にリガンド溶液を投与し、その蛍光強度の経時的変化を測定した。96ウェルプレートに播種した細胞の培養液は、測定前にアッセイバッファー液で洗浄し、置換した。アッセイバッファー液の組成は、130 mM NaCl、10 mM Dグルコース、10 mM HEPES（4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid）、5 mM KCl、2 mM CaCl2、1.2 mM MgCl2であり、NaOHを用いてpH 7.4に調整した。 Cell-based assay using FlexStation 3 FlexStation 3 (Molecular Devices) is a plate reader with a dispensing function. Using this, a ligand solution was administered to cells loaded with a membrane potential-sensitive dye, and the change over time in the fluorescence intensity was measured. The culture medium of the cells seeded in the 96-well plate was washed and replaced with an assay buffer solution 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 CaCl 2 , 1.2 mM MgCl 2 , The pH was adjusted to 7.4 using NaOH.
アッセイは27 ℃で行い、530 nmで励起した時のMembrane potential assay kit blue(R8042 Molecular Devices社)の蛍光(560 nm)を2秒に1度測定した。測定開始から20秒後に2倍の濃度に洗浄と同じバッファーで調製したリガンド溶液を細胞に投与し、120秒間にわたり経時的な蛍光強度変化を測定した。Membrane potential assay kit blue に2.5mM プロベネシドを添加することでより測定結果が安定させることができた。
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 the measurement, a ligand solution prepared to a concentration twice as much as the buffer used for washing was administered to the cells, and a change in fluorescence intensity over time was measured for 120 seconds. The measurement results could be more stabilized by adding 2.5 mM probenecid to Membrane potential assay kit blue.
測定結果の模式図と活性化能の評価方法を図２に示す。 FlexStation 3によって得られた蛍光の経時的変化から、リガンド溶液投与後、100秒時点でのリガンド添加効果による蛍光強度の値と100秒時点でのビークル（リガンドを調整したアッセイバッファー液）の蛍光強度の差分から delta relative fluorescence units(ΔRFU)値を算出してこれを細胞の応答値と定義した。
FIG. 2 shows a schematic diagram of the measurement results and a method of evaluating the activation ability. From the time course of fluorescence obtained by FlexStation 3, the value of the fluorescence intensity due to the effect of ligand addition at 100 seconds after administration of the ligand solution and the fluorescence intensity of the vehicle (assay buffer solution adjusted for ligand) at 100 seconds The value of delta relative fluorescence units (ΔRFU) was calculated from the difference, and this was defined as the response value of the cell.
FIG. 2 shows a schematic diagram of the measurement results and a method of evaluating the activation ability. From the time course of fluorescence obtained by FlexStation 3, the value of the fluorescence intensity due to the effect of ligand addition at 100 seconds after administration of the ligand solution and the fluorescence intensity of the vehicle (assay buffer solution adjusted for ligand) at 100 seconds The value of delta relative fluorescence units (ΔRFU) was calculated from the difference, and this was defined as the response value of the cell.
リガンド溶液の濃度は、終濃度が10nM、50nM、100nM、1μM、5μM、10μM、20μM、30μM、40μM、50μM、75μM、100μM、300μM、500μM、1mMの12点（各n=6）を測定した。
The concentration of the ligand solution was measured at 12 points (n = 6) at final concentrations of 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, and 1 mM. .
The concentration of the ligand solution was measured at 12 points (n = 6) at final concentrations of 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, and 1 mM. .
─結果─
図２における“sample”に示すように、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は、各種濃度を添加後、100秒時点で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-カルボキシアミドの添加効果による蛍光強度の値と100秒時点でのビークル（リガンドを調整したアッセイバッファー液）との蛍光強度の差分である delta relative fluorescence units(ΔRFU)値が濃度依存的に大きな値を得られたことから、hENaC αβγ に活性化能が有ることが示された。
また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-カルボキシアミドのEC50は、95.0±21.0 μMであり、最大効果濃度（Emax）は、ΔRFU値で928±67.8であった。
─Result─
As shown in “sample” in FIG. 2, 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] After addition of various concentrations, 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl was added at 100 seconds to thiophene-2-carboxamide. ) Delta relative fluorescence units, which is the difference between the fluorescence intensity value due to the effect of addition of -N-ethylbenzo [β] thiophene-2-carboxamide and the fluorescence intensity at 100 seconds with the vehicle (assay buffer solution in which the ligand was adjusted). The large (ΔRFU) value was obtained in a concentration-dependent manner, indicating that hENaC αβγ has the activating ability.
The EC 50 of 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2-carboxamide is , 95.0 ± 21.0 μM, and the maximum effect concentration (E max ) was 928 ± 67.8 in ΔRFU value.
図２における“sample”に示すように、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は、各種濃度を添加後、100秒時点で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-カルボキシアミドの添加効果による蛍光強度の値と100秒時点でのビークル（リガンドを調整したアッセイバッファー液）との蛍光強度の差分である delta relative fluorescence units(ΔRFU)値が濃度依存的に大きな値を得られたことから、hENaC αβγ に活性化能が有ることが示された。
また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-カルボキシアミドのEC50は、95.0±21.0 μMであり、最大効果濃度（Emax）は、ΔRFU値で928±67.8であった。
─Result─
As shown in “sample” in FIG. 2, 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] After addition of various concentrations, 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl was added at 100 seconds to thiophene-2-carboxamide. ) Delta relative fluorescence units, which is the difference between the fluorescence intensity value due to the effect of addition of -N-ethylbenzo [β] thiophene-2-carboxamide and the fluorescence intensity at 100 seconds with the vehicle (assay buffer solution in which the ligand was adjusted). The large (ΔRFU) value was obtained in a concentration-dependent manner, indicating that hENaC αβγ has the activating ability.
The EC 50 of 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2-carboxamide is , 95.0 ± 21.0 μM, and the maximum effect concentration (E max ) was 928 ± 67.8 in ΔRFU value.
図１に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-カルボキシアミドの濃度応答曲線を示す。
FIG. 1 shows the concentration of 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2-carboxamide. The response curve is shown.
FIG. 1 shows the concentration of 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2-carboxamide. The response curve is shown.
[実施例２] 膜電位感受性色素を用いた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する構造をもつ化合物によるhENaC αβγ活性
[Example 2] {3-Chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β using a membrane potential sensitive dye ] Activity of hENaC αβγ by compounds having a structure similar to thiophene-2-carboxamide
＜方法＞
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もつとする化合物は、ChemBioFinder Ultra 12.0 (CambridgeSoft製)による類似検索で60%以上の類似度を示すものを指す。 <Method>
Has a similar structure to 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2-carboxamide Indicates a compound showing a similarity of 60% or more in a similar search using ChemBioFinder Ultra 12.0 (manufactured by CambridgeSoft).
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もつとする化合物は、ChemBioFinder Ultra 12.0 (CambridgeSoft製)による類似検索で60%以上の類似度を示すものを指す。 <Method>
Has a similar structure to 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2-carboxamide Indicates a compound showing a similarity of 60% or more in a similar search using ChemBioFinder Ultra 12.0 (manufactured by CambridgeSoft).
60%以上の類似度を持つ化合物のサンプリングに関して、化学構造情報のデータベース管理ソフトであるChemBioFinder Ultra 12.0 を用いた。検索対象とした化合物ライブラリーは、東京大学創薬機構が所有する化合物ライブラリーを用いた。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-カルボキシアミドの類似化合物の定義方法を下記に示す。即ち、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-カルボキシアミドの構造情報をChemBioFinder Ultra 12.0を用いて数値化された記述子に変換し、類似度を計算した。ChemBioFinder では、Unity 2D Fingerprint、Similog key、MaCCS keyのといった代表的な構造キー特徴を組み合わせ、改変した独自の構造キーを用いて化合物の構造情報を記述子への変換が行う事が可能である。
For sampling of compounds with similarity of {60% or more, ChemBioFinder {Ultra} 12.0}, a database management software for chemical structure information, was used. As a compound library to be searched, a compound library owned by the University of Tokyo Drug Discovery Organization was used. Definition of analogous compounds of 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2-carboxamide The method is described below. That is, structural information of 3-chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2-carboxamide Was converted to a numerical descriptor using ChemBioFinder Ultra 12.0, and the similarity was calculated. In ChemBioFinder II, it is possible to combine structural key features such as Unity 2D Fingerprint, Similog key, and MaCCS key, and convert the structural information of a compound into a descriptor using a modified unique structure key.
化合物の構造情報を類似性評価尺度については、可換性を持つTanimoto類似性判定法を用いた（非特許文献８）。Tanimoto類似判定法は、Tanimoto係数を用いてQ（クエリ）、T（ターゲット）が以下の数式：
As for the similarity evaluation scale of the structural information of the compound, a Tanimoto similarity determination method having commutability was used (Non-Patent Document 8). In the Tanimoto similarity determination method, Q (query) and T (target) are expressed by the following formulas using Tanimoto coefficients:
このようにして選択した化合物を以下に示す。
(((1,1-ジオキシベンゾ[ベータ]チオフェン-2-イル)メトキシ)カルボニル)-Ｌ-アスパラギン、 The compounds thus selected are shown below.
(((1,1-dioxybenzo [beta] thiophen-2-yl) methoxy) carbonyl) -L-asparagine,
(((1,1-ジオキシベンゾ[ベータ]チオフェン-2-イル)メトキシ)カルボニル)-Ｌ-アスパラギン、 The compounds thus selected are shown below.
(((1,1-dioxybenzo [beta] thiophen-2-yl) methoxy) carbonyl) -L-asparagine,
1-(4-(3-((ベンゾ[ベータ]チオフェン-2-イルメチル)(メチル)アミノ)-2-エチルベンゾ[1,2-アルファ]ピリジン-6-カルボニル)ピペラジン-1-イル)エタン-1-オン
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-(ターシャリーブチルアミノ)-2-オキシオエチル)-3-クロロ-N-エチルベンゾ[ベータ]チオフェン-2-カルボキシアミド
N- (2- (tert-butylamino) -2-oxyoethyl) -3-chloro-N-ethylbenzo [beta] thiophen-2-carboxamide
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-オキシエチル)-N-エチル-4-フルオロベンゾ[ベータ]チオフェン-2-カルボキシアミド
N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxyethyl) -N-ethyl-4-fluorobenzo [beta] thiophen-2-carboxamide
2-ベンジル-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-オキシエチル)-N-エチルベンザミド
2-benzyl-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxyethyl) -N-ethylbenzamide
1-(4-クロロフェニル)-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-オキシオエチル)-N-エチルシクロブタン-1-カルボキシアミド
1- (4-chlorophenyl) -N- (2-((1,1-dioxidetetrahydrothiophen-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
から[００２３]に記載した方法と同様の方法及び材料を用いた。
To [0023], using the same method and materials.
To [0023], using the same method and materials.
＜評価方法＞
前述のFlexStation 3によって得られた蛍光の経時的変化から、リガンド溶液投与後後、100秒時点での蛍光強度の値と計測開始時点での値の差分を算出してこれを細胞の応答値と定義した。また必要な場合は、対照として計測した1μMのS3969の細胞の応答値を1としたときの相対応答値として、すべての細胞の応答値を換算した。評価スコア0.18以上を活性ありとした。評価方法の模式図を図２に示す。尚、本評価方法は、特開2017-217005と同様に記載されているものと同様の方法である。 <Evaluation method>
From the time-dependent change in fluorescence obtained by the above-mentioned FlexStation 3, the difference between the value of the fluorescence intensity at 100 seconds after administration of the ligand solution and the value at the start of measurement was calculated, and this was used as the response value of the cell. 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 set to 1. An evaluation score of 0.18 or more was determined to be active. FIG. 2 shows a schematic diagram of the evaluation method. This evaluation method is the same method as described in JP-A-2017-217005.
前述のFlexStation 3によって得られた蛍光の経時的変化から、リガンド溶液投与後後、100秒時点での蛍光強度の値と計測開始時点での値の差分を算出してこれを細胞の応答値と定義した。また必要な場合は、対照として計測した1μMのS3969の細胞の応答値を1としたときの相対応答値として、すべての細胞の応答値を換算した。評価スコア0.18以上を活性ありとした。評価方法の模式図を図２に示す。尚、本評価方法は、特開2017-217005と同様に記載されているものと同様の方法である。 <Evaluation method>
From the time-dependent change in fluorescence obtained by the above-mentioned FlexStation 3, the difference between the value of the fluorescence intensity at 100 seconds after administration of the ligand solution and the value at the start of measurement was calculated, and this was used as the response value of the cell. 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 set to 1. An evaluation score of 0.18 or more was determined to be active. FIG. 2 shows a schematic diagram of the evaluation method. This evaluation method is the same method as described in JP-A-2017-217005.
─結果─
図３及び図４に示す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その類似構造を持つ6化合物には、hENaCαβγに対して活性化能が示された。 ─Result─
3-Chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2- shown in FIGS. Carboxamide and six compounds having a similar structure were able to activate hENaCαβγ.
図３及び図４に示す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その類似構造を持つ6化合物には、hENaCαβγに対して活性化能が示された。 ─Result─
3-Chloro-N- (2-((1,1-dioxidetetrahydrothiophen-3-yl) amino) -2-oxoethyl) -N-ethylbenzo [β] thiophen-2- shown in FIGS. Carboxamide and six compounds having a similar structure were able to activate hENaCαβγ.
Claims (4)
- 下記式（１）で表される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同一若しくは類似構造を有する化合物又はその塩を有効成分とするhENaC αβγ活性化剤。
- 前記化合物が食品、天然抽出物由来の成分である請求項１に記載のhENaC αβγ活性化剤。
The hENaC αβγ activator according to claim 1, wherein the compound is a component derived from a food or a natural extract.
- 請求項１又は２のいずれかに記載のhENaC αβγ活性化剤を含有する疾患等の治療剤。
A therapeutic agent for a disease or the like containing the hENaC αβγ activator according to claim 1.
- 請求項１又は２のいずれかに記載のhENaC αβγ活性化剤を含有する塩味増強剤。 塩 A salty taste enhancer comprising the hENaC αβγ activator according to claim 1 or 2.
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| JP2018176045 | 2018-09-20 | ||
| JP2018-176045 | 2018-09-20 | ||
| JP2019-165439 | 2019-09-11 | ||
| JP2019165439A JP7330828B2 (en) | 2018-09-20 | 2019-09-11 | Compound having ENaC activating ability |
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| CN112898286A (en) * | 2021-01-28 | 2021-06-04 | 中国药科大学 | Benzothiophene compound or pharmaceutically acceptable salt and isomer thereof, and preparation method, pharmaceutical composition and application thereof |
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