JP2008266161A - Potassium channel-opening drug - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a BK channel-opening drug for opening a BK channel by selectively acting on a β-subunit. <P>SOLUTION: The potassium channel-opening drug contains a compound constituting the most stable three-dimensional structure in which an oligomethylene chain including an oxo-oxo anion part is present nearly on the same plane, and two oxygen atoms constituting the oxo-oxo anion part are present at the same side based on the oligomethylene chain, or a pharmacologically acceptable salt thereof as an active ingredient. The distance between oxygen atoms in the oxo-oxo anion part in the most stable three-dimensional structure is ≥0.3 nm and ≤1 nm. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to large conductance Ca ²⁺ activated K ⁺ channel ^openers .

The large conductance Ca ²⁺ activated K ⁺ (BK) channel is activated by an increase in intracellular Ca ²⁺ concentration or depolarization of the cell membrane (Non-patent Document 1). BK channels are widely expressed in highly excitable cells and play a negative feedback mechanism for regulating intracellular Ca ²⁺ concentration.

In the BK channel, an α subunit (BKα) that forms a channel hole and a β subunit (BKβ) that plays an auxiliary role are combined to form a tetramer (Non-patent Document 2). The α subunit has a characteristic region necessary for coupling with the β subunit (Non-patent Documents 3 and 4). The α subunit forming the channel pore was a K ⁺ channel (KCNMA1) belonging to the slo family first identified in Drosophila (Non-patent Document 5). BKα is encoded by only one kind of gene with some splice variants, and is ubiquitously expressed in a wide range of tissues except the heart (Non-patent Documents 6 and 7).

On the other hand, four types of β subunits have been identified, and each has a different tissue-specific expression distribution (Non-Patent Documents 8, 8, 9, and 11). β1 subunit and β4 subunit are mainly expressed in smooth muscle and nerve, respectively (Non-Patent Documents 12 to 14). When the β subunit is co-expressed with the α subunit, the biophysical and pharmacological properties (apparent Ca ²⁺ sensitivity, potential sensitivity, gating rate, drug sensitivity) of the BK channel consisting of the α subunit play a dramatic role. The BK channel function is diversified (Non-Patent Documents 12, 13, 15-18).

Here, α subunit (KCNMA1) knockout mice not only suffer from diseases related to smooth muscle dysfunction such as hypertension, hypersensitivity bladder, urinary incontinence, erectile dysfunction, but also cerebellar ataxia, hearing loss, and hyperaldosteronism (Non-Patent Documents 20 to 23). On the other hand, the smooth muscle-specific β1 subunit (KCNMB1) increases the apparent Ca ²⁺ / potential sensitivity of the BK channel, but in the knockout mouse, the coupling function between Ca ²⁺ spark and BK channel activation is reduced. And increase the tone of the blood vessels and increase the transient contraction of the bladder (Non-patent Documents 24 and 25). Therefore, BK channel openers that are selective for the β1 subunit may effectively treat diseases with smooth muscle hyperexcitation with fewer side effects. This is also supported by the decrease in the incidence of coronary vascular disease in postmenopausal women due to β-estradiol that selectively activates only the BK channel co-expressing the β1 subunit (Non-patent Documents 26 and 27). ).

At present, little is known about compounds that selectively act on BKβ, even though BKβ is a potential drug discovery target. Most of the developed BK channel openers act on the α subunit (Non-patent Documents 28 and 29), and the β subunit selective BK channel opener is β-estradiol.
Only Non-patent Document 26, Tamoxifen (Non-patent Document 30), Dehydrosoyasaponin-I (DHS-I) (Non-patent Documents 31 and 32) have been reported.

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  Thus, one object of the present invention is to provide a screening method for a BK channel opener that selectively acts on the β subunit to open the BK channel and a β subunit selective BK channel opener.

We have found that DiBAC ₄ (3) (bis (1,3-dibutylbarbituric acid) trimethine oxonol) and related oxonol derivatives, which are voltage sensitive fluorescent dyes, are openers of the BK channel. The present invention has been completed. DiBAC ₄ (3) and related oxonol derivatives are generally screened for _KATP channel agonists by FLIPR (Gonzalez et al., 1999; Whiteaker
et al., 2001) and evaluation of bacterial viability by flow cytometry (Jepras et al., 1997). Previously, we used a low concentration of DiBAC ₄ (3) for screening of BK channel openers (Yamada et al., 2001), and pimaric acid and related pimar compounds act on the α subunit to produce BK. Revealed to activate the channel (Imaizumi et al., 2002). At this time, since 50 nM DiBAC ₄ (3) was used as an indicator of potential, it could not be found that the BK channel exhibited an opening action. Subsequently, the inventors found that DiBAC ₄ (3) at a concentration of 1 μM or more cannot detect the opening action of BKαβ1 channel of pimaric acid or NS-1619, and DiBAC ₄ (3) strongly activates the BK channel. I got the knowledge that Further, based on the findings by reviewing the DiBAC ₄ (3) pharmacological effects such as such, DiBAC ₄ (3) or the like to obtain a knowledge that activate BK channels dependently β subunit. The present invention is provided based on these findings.

According to the present invention, a potassium channel opener represented by the following formula (1):
[Wherein, R ¹ and R ² may be the same or different, and may be an acyclic hydrocarbon group which may be substituted, a cyclic hydrocarbon group which may be substituted or a substituted group. Represents a heterocyclic group, and in the case of a cyclic hydrocarbon group and a heterocyclic group, it may be bonded to a carbon atom constituting an oligomethine chain to form a cyclic structure, and l represents 0, 1 or 2 . ]
The most stable three-dimensional structure in which the oligomethine chain including the oxo-oxoanion part is present on substantially the same plane, and two oxygen atoms constituting the oxo-oxoanion part are present on the same side with respect to the oligomethine chain. A potassium channel opener is provided that contains a constituent compound or a physiologically acceptable salt thereof as an active ingredient. The potassium channel opener may be β subunit selective. In the compound of the present invention, the distance between oxygen atoms of the oxo-oxoanion moiety in the most stable steric structure can be 0.3 nm or more and 1 nm or less.

The compound of the present invention can be represented by the following formula (2).
[Wherein, A ¹ and A ² may be the same or different and each represents an optionally substituted cyclic hydrocarbon group or an optionally substituted heterocyclic group. ]

The compound represented by the formula (2) can be represented by the following formula (3).
[Wherein, R ³ and R ⁴ may be the same or different and each represents an optionally substituted acyclic hydrocarbon group or an optionally substituted cyclic hydrocarbon group, and X ¹ and X ² may be the same or different and each represents a sulfur atom or an oxygen atom. ]

In the formula (3), X ¹ is an oxygen atom, l can be 1 or 2, and the distance between X ¹ can be 0.40 nm or more and 0.67 nm or less. Furthermore, the compound represented by Formula (3) may be any selected from the following Formula (4).

The compound represented by the formula (2) can be a compound represented by the following formula (5).
[Wherein R ⁵ may be the same or different and each may be a non-cyclic hydrocarbon group that may be substituted, a cyclic hydrocarbon group that may be substituted, or a heterocyclic ring that may be substituted; Represents a group. ]

The compound of this embodiment can be any one selected from the following formula (6).

According to the present invention, a method for screening a potassium channel opener, comprising:
It is represented by the following formula (1),
[Wherein, R ¹ and R ² may be the same or different, and may be an acyclic hydrocarbon group which may be substituted, a cyclic hydrocarbon group which may be substituted or a substituted group. Represents a heterocyclic group, and in the case of a cyclic hydrocarbon group and a heterocyclic group, it may be bonded to a carbon atom constituting an oligomethine chain to form a cyclic structure, and l represents 0, 1 or 2 . ]
The most stable three-dimensional structure in which the oligomethine chain including the oxo-oxoanion part is present on substantially the same plane, and two oxygen atoms constituting the oxo-oxoanion part are present on the same side with respect to the oligomethine chain. There is provided a screening method comprising an evaluation step of evaluating a potassium channel opening ratio of the test component using a constituent compound or a physiologically acceptable salt thereof as the test component.

In the method of the present invention, the evaluation step can be a step of evaluating a potassium channel opening ratio based on an action of a large conductance Ca ²⁺ activated K ⁺ (BK) channel on β1 subunit or β4 subunit. .

According to the present invention, there is provided a method for screening a potassium channel opener, comprising a site consisting of the amino acid sequence (LLFSFFWPTFLLTGGLLIIAMV) described in SEQ ID NO: 1 in the β1 subunit of a large conductance Ca ²⁺ activated K ⁺ (BK) channel; There is provided a screening method comprising the step of evaluating an interaction with a test component.

  The potassium channel opener of the present invention is represented by the above formula (1), the oligomethine chain containing the oxo-oxoanion portion of the above formula (1) is present on substantially the same plane, and the oxo-oxoanion portion is A compound constituting a most stable steric structure in which two constituting oxygen atoms are present on the same side of the oligomethine chain or a physiologically acceptable salt thereof can be contained as an active ingredient.

  Since the BK channel opening agent of the present invention promotes the opening of the BK channel, it can be used as a preventive or therapeutic agent for a disease related to the opening of the BK channel in addition to the reagent related to the opening of the BK channel. Further, since the BK channel opener of the present invention selectively acts on BKαβ1 and BKαβ4 by selectively acting on the β1 subunit and β4 subunit of the BK channel, the tissues in which these β subunits are expressed. Can be used as a prophylactic or therapeutic agent for diseases associated with BK channel openers in such tissues. Furthermore, the BK channel opener of the present invention can also be used as a seed compound and a lead compound useful for optimization as a preventive or therapeutic agent for a disease associated with a BK channel opener.

  Hereinafter, the BK channel opener which is an embodiment of the present invention and a screening method for the BK channel opener based on new knowledge about the BK channel opener will be described in detail.

(BK channel opener)
The BK channel opener of the present invention can contain a compound represented by the following formula (1) or a physiologically acceptable salt thereof as an active ingredient. In the compound represented by the formula (1) (hereinafter simply referred to as the present compound), the oligomethine chain containing an oxo-oxoanion part is present on substantially the same plane, and constitutes the oxo-oxoanion part. It is preferable to constitute the most stable steric structure in which two oxygen atoms are present on the same side of the oligomethine chain.

Here, the most stable three-dimensional structure can be determined by calculation using a density-functional theory (DFT) method. In determining the most stable solid structure by the DFT method, Spartan '04 Software for Windows version 1.0.3. (Wavefunction,
Inc., Irvine, CA, USA) (Windows is a trademark) can be used. The distance between oxygen and oxygen atoms can be measured from the most stable structure. Furthermore, UCSF Chimera software can be used for the display. In the compound represented by the formula (1), the distance between oxygen atoms of the oxo-oxoanion part in the most stable steric structure is preferably 0.3 nm or more and 1 nm or less.

The following formula (7) shows an electron transfer coupling state in the oxo-oxoanion moiety in the compound represented by formula (1). As shown in this formula, the compound represented by formula (1) is considered to transfer electrons between oxygen atoms via an oligomethine chain.

In the compound represented by the formula (1), R ¹ and R ² may be the same or different as long as the most stable steric structure is obtained, and may be an acyclic ring that may be substituted. It is represented by a hydrocarbon group, an optionally substituted cyclic hydrocarbon group, or an optionally substituted heterocyclic group.

Examples of the non-cyclic hydrocarbon group include straight chain, branched chain, or a combination thereof. Such hydrocarbon groups include alkyl groups, alkenyl groups, and alkynyl groups. Here, as the “alkyl”, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, 1-methylpropyl, n-hexyl, isohexyl, Examples thereof include C _1-6 alkyl such as 1,1-dimethylbutyl, 2,2-dimethylbutyl, and 3,3-dimethylbutyl.

Examples of “alkenyl” include vinyl, allyl, isopropenyl, 2-methylallyl, 1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl and 2-ethyl-1-butenyl. 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 2-hexenyl, 3 And C _2-6 alkenyl such as -hexenyl, 4-hexenyl, 5-hexenyl and the like.

Examples of “alkynyl” include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, C _2-6 alkynyl such as 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl and the like can be mentioned.

  Examples of the cyclic hydrocarbon group include a hydrocarbon group or a heterocyclic group in which a linear hydrocarbon group and / or a branched hydrocarbon group are combined in addition to the cyclic hydrocarbon group. Examples of such cyclic hydrocarbon groups include alicyclic hydrocarbon groups, aryl groups, aralkyl groups, and arylalkenyl groups. Here, examples of the “alicyclic hydrocarbon group” include saturated or unsaturated hydrocarbon groups such as a cycloalkyl group, a cycloalkenyl group, and a cycloalkadienyl group. Here, examples of the “cycloalkyl group” include C3-C9 cycloalkyl such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl and the like.

Examples of the “cycloalkenyl group” include 2-cyclopenten-1-yl, 3-cyclopenten-1-yl, 2-cyclohexen-1-yl, 3-cyclohexen-1-yl, 1-cyclobuten-1-yl, C _3-9 cycloalkenyl groups such as 1-cyclopenten-1-yl, 1-cyclohexen-1-yl, 1-cyclohepten-1-yl and the like can be mentioned.

Examples of the “cycloalkadienyl group” include C _4-6 such as 2,4-cyclopentadien-1-yl, 2,4-cyclohexadien-1-yl, 2,5-cyclohexadien-1-yl and the like. And cycloalkadienyl group.

Examples of the “aryl group” as an example of the cyclic hydrocarbon group include monocyclic or condensed polycyclic aromatic hydrocarbon groups, and examples thereof include C _6-14 such as phenyl, naphthyl, anthryl, phenanthryl, acenaphthylenyl and the like. An aryl group and the like are preferable, and phenyl and the like are particularly preferable.

Examples of the “aralkyl group” include phenyl-C _1-6 alkyl groups such as benzyl, phenethyl, 3-phenylpropyl and 4-phenylbutyl.

Examples of the “arylalkenyl group” include C _8-10 arylalkenyl such as cinnamyl, preferably phenyl-C _2-4 alkenyl and the like.

  Examples of the cyclic hydrocarbon group include the same or different 2 to 3 rings (preferably 2 or more kinds) selected from the rings constituting the alicyclic hydrocarbon group and the aromatic hydrocarbon group. Bicyclic or tricyclic hydrocarbon groups derived from the condensation of (ring).

  Examples of the optionally substituted heterocyclic group include an optionally substituted monocyclic heterocyclic group. The “monocyclic heterocyclic group” in the optionally substituted monocyclic heterocyclic group is selected from, for example, an oxygen atom, a sulfur atom, a nitrogen atom, etc. as an atom (ring atom) constituting the ring system. An aromatic heterocyclic group containing at least one (preferably one to four, more preferably one to two) heteroatoms of 1 to 3 (preferably 1 to 2) heteroatoms, saturated or unsaturated Non-aromatic heterocyclic group (aliphatic heterocyclic group) etc. are mentioned.

  Examples of the “aromatic heterocyclic group” include furyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,3, 4-oxadiazolyl, furazanyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,4-triazolyl, tetrazolyl, pyridyl, 5- to 6-membered aromatic monocyclic heterocyclic groups such as pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and the like, and, for example, benzofuranyl, isobenzofuranyl, benzo [b] thienyl, indolyl, isoindolyl, 1H-indazolyl, benzimidazolyl , Benzoxazolyl, 1,2-benzisooxy Zolyl, benzothiazolyl, benzopyranyl, 1,2-benzisothiazolyl, 1H-benzotriazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, naphthyridinyl, purinyl, buteridinyl, carbazolyl, α-carbolinyl, β-carbolinyl, γ-carbolinyl, acridinyl, phenoxazinyl, phenothiazinyl, phenazinyl, phenoxathiinyl, thianthenyl, phenathidinyl, phenatrolinyl, indolizinyl, pyrrolo [1,2-b] pyridazinyl, pyrazolo [1,5-a] pyridyl, imidazo [1, 2-a] pyridyl, imidazo [1,5-a] pyridyl, imidazo [1,2-b] pyridazinyl, imidazo [1,2-a] pyrimidinyl, 1,2,4-triazolo [4,3-a] Pil Le, 1,2,4-triazolo [4,3-b] 8-16 membered like pyridazinyl (preferably, 8-12 membered) an aromatic fused heterocyclic group.

  Examples of the “non-aromatic heterocyclic group” include 3 to 8 members (preferably 5 to 5) such as oxiranyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, tetrahydrofuryl, thiolanyl, piperidyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, piperazinyl and the like. 6-membered) saturated or unsaturated (preferably saturated) non-aromatic monocyclic heterocyclic group (aliphatic monocyclic heterocyclic group), 1,3-dihydroisoindolyl, etc. And heterocyclic groups in which 1 to 2 (preferably 1) group monocyclic heterocyclic groups are condensed with 1 to 2 (preferably 1) benzene rings.

  The “substituent” in the acyclic hydrocarbon group which may be substituted, the cyclic hydrocarbon group which may be substituted or the heterocyclic group which may be substituted is not particularly limited, but a halogen atom ( Fluorine, chlorine, bromine, iodine, etc., preferably chlorine, bromine, etc.), alkyl group, alkenyl group, alkynyl group, aryl group, cycloalkyl group, cycloalkenyl group, aralkyl group, arylalkenyl group, heterocyclic group, hydroxyl group, Amino group, thiol group, carbonyl group, carboxyl group, carbamoyl group, acyl group, cyano group and the like can be mentioned. These hydrocarbon groups or heterocyclic groups may have an oxo group or a thioxo group. The hydrocarbon group and the heterocyclic group can have such a substituent at a substitutable position.

Here, as the “alkyl”, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, 1-methylpropyl, n-hexyl, isohexyl, Examples thereof include C _1-6 alkyl such as 1,1-dimethylbutyl, 2,2-dimethylbutyl, and 3,3-dimethylbutyl.

Examples of “alkenyl” include vinyl, allyl, isopropenyl, 2-methylallyl, 1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl and 2-ethyl-1-butenyl. 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 2-hexenyl, 3 And C _2-6 alkenyl such as -hexenyl, 4-hexenyl, 5-hexenyl and the like.

Examples of “alkynyl” include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, C _2-6 alkynyl such as 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl and the like can be mentioned.

Examples of “aryl” include C _6-14 aryl such as phenyl, naphthyl, anthryl, phenanthryl, acenaphthylenyl and the like.

Examples of “cycloalkyl” include C _3-7 cycloalkyl such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.

Examples of “cycloalkenyl” include C _3-6 cycloalkenyl such as cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl and the like.

Examples of the “aralkyl group” include phenyl-C _1-6 alkyl groups such as benzyl, phenethyl, 3-phenylpropyl and 4-phenylbutyl.

Examples of the “arylalkenyl group” include C _8-10 arylalkenyl such as cinnamyl, preferably phenyl-C _2-4 alkenyl and the like.

  Examples of the “heterocyclic group” include the same groups as the heterocyclic group in the “optionally substituted heterocyclic group” already described.

The substituent in the acyclic hydrocarbon group which may be substituted, the cyclic hydrocarbon group which may be substituted, and the heterocyclic group which may be substituted is further a lower alkoxy group (eg, methoxy, ethoxy C _1-6 alkoxy such as propoxy), halogen atom (eg, fluorine, chlorine, bromine, iodine, etc.), lower alkyl (eg, C _1-6 alkyl such as methyl, ethyl, propyl etc.), lower alkenyl ( Examples, C _2-6 alkenyl such as vinyl and allyl), lower alkynyl (eg C _2-6 alkynyl such as ethynyl, propargyl, etc.), optionally substituted amino, optionally substituted hydroxyl, cyano It may be substituted with a group or the like.

The cyclic hydrocarbon group or heterocyclic group is preferably bonded to a part of the carbon atoms of the oligomethine chain to form a cyclic structure as represented by the following formula (2). A ¹ and A ² may be the same or different and each represents an optionally substituted cyclic hydrocarbon group or heterocyclic group. A ¹ and A ² can be an optionally substituted cyclic hydrocarbon group or an optionally substituted heterocyclic group which has already been described within a range compatible with the formula (2).

The compound represented by the formula (2) is preferably a compound represented by the following formula (3). In the formula (3), R ³ and R ⁴ may be the same or different and each represents a hydrogen atom, an optionally substituted acyclic hydrocarbon group or an optionally substituted cyclic hydrocarbon group. To express.

Examples of the acyclic hydrocarbon group which may be substituted as R ³ and R ⁴ include linear, branched or a combination thereof, and acyclic as R ¹ and R ² And hydrocarbon groups similar to the hydrocarbon group of. Preferably, it is an alkyl group, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, etc., more preferably methyl, ethyl, n-propyl. , Isopropyl, n-butyl, n-pentyl group. More preferably, they are an ethyl group, n-propyl group, and n-butyl group. In addition, these hydrocarbon groups can also have a substituent suitably.

Examples of the optionally substituted cyclic hydrocarbon group as R ³ and R ⁴ include the same hydrocarbon groups as the cyclic hydrocarbon groups as R ¹ and R ² . In addition, these hydrocarbon groups can also have a substituent suitably.

X ¹ and X ² may be the same or different and each represents a sulfur atom or an oxygen atom. That is, it constitutes a carbon atom of the ring skeleton and a thioxo group or oxo group. X ¹ is preferably an oxygen atom, it is preferred that two X ¹ are both oxygen atoms. When both of X ¹ are oxygen atoms, the compound of the present invention further comprises an oxo-oxo anion structure at X ¹ -X ¹ .

In the compound represented by the formula (3), l is preferably 1 or 2. This is because when l is 1 or 2, the oxygen atoms constituting the oxo-oxoanion structure can easily maintain an appropriate distance. For example, when l is 1 or 2, the X ¹ -X ¹ distance is in the range of 0.40 nm or more and 0.67 nm or less, and an excellent potassium channel opening action is easily exhibited.

  As a compound represented by Formula (3), Preferably, the compound represented by Formula (4) can be mentioned. In any of these compounds, the oligomethine chain containing two oxo-oxoanion parts is on the same plane, and two oxygen atoms constituting each oxo-oxoanion part are arranged on the same side of the oligomethine chain. ing. Among these, a high potassium channel opening action tends to be found by 4a to 4c which are compounds of l = 1.

Moreover, the compound represented by Formula (2) is preferably a compound represented by Formula (5). In the formula (5), R ^{5 s} may be the same or different and each may be an acyclic hydrocarbon group that may be substituted, a cyclic hydrocarbon group that may be substituted, or a substituent. Represents a heterocyclic group.

The acyclic hydrocarbon group in the acyclic hydrocarbon group which may be substituted in R ⁵ may be linear, branched or a combination thereof. Such hydrocarbon groups include linear or branched alkyl groups, alkenyl groups, and alkynyl groups. Here, as "alkyl", "alkenyl" and "alkynyl", an acyclic hydrocarbon group which may be substituted, a cyclic hydrocarbon group which may be substituted or a heterocyclic group which may be substituted Examples of the “substituent” in the above include “alkyl”, “alkenyl” and “alkynyl” for the alkyl, alkenyl and alkynyl groups already mentioned. Any of these alkyl groups, alkenyl groups, and alkynyl groups may be substituted. Preferably, it is an alkyl group.

  Examples of the cyclic hydrocarbon group in the cyclic hydrocarbon group that may be substituted include an aryl group, a cycloalkyl group, a cycloalkenyl group, an aralkyl group, and an arylalkenyl group. As used herein, “aryl”, “cycloalkyl”, “cycloalkenyl”, “aralkyl” and “arylalkenyl” include an acyclic hydrocarbon group which may be substituted, a cyclic hydrocarbon which may be substituted The aryl, cycloalkyl, cycloalkenyl, aralkyl and arylalkyl groups already mentioned as the “substituent” in the group or the optionally substituted heterocyclic group, “aryl”, “cycloalkyl”, “cyclo Examples include “alkenyl”, “aralkyl” and “arylalkenyl”. Any of these aryl groups, cycloalkyl groups, cycloalkenyl groups, aralkyl groups, and arylalkenyl groups may be substituted. Preferably, it is an aryl group.

  As one compound represented by Formula (5), the compound represented by Formula (6) can be mentioned. In these compounds, the oligomethine chain containing the oxo-oxoanion part is on the same plane, and two oxygen atoms constituting the oxo-oxoanion part are arranged on one side of the oligomethine chain.

  The compound represented by the above formula (1) can be produced by performing functional group transformation well known to those skilled in the art. Any of the compounds exemplified in Formula (4) and Formula (6) can be obtained commercially.

Furthermore, you may have 1 or 2 or more substituents. The type, position and number of substituents are not particularly limited, and when having two or more substituents, they may be the same or different.

  As an active ingredient of the present opener, in addition to the present compound, a physiologically acceptable salt thereof can be used depending on the type of substituent. The kind of the salt is not particularly limited as long as it is physiologically acceptable, and may be either an acid addition salt or a base addition salt. Examples of the acid addition salt include mineral acid salts such as hydrochloride, sulfate, and nitrate, and organic acid salts such as p-toluenesulfonate, methanesulfonate, oxalate, maleate, and tartrate. Examples of the base addition salt include metal salts such as sodium salt, potassium salt and calcium salt, and organic amine salts such as ammonium salt, triethylamine salt and ethylamine salt. A salt with an amino acid such as glycine may be used.

Moreover, as an active ingredient of the present opener, in addition to a free form compound or a physiologically acceptable salt thereof, any hydrate or solvate thereof can also be used. Although the kind of solvent which forms a solvate is not specifically limited, For example, acetone, ethanol, tetrahydrofuran, etc. can be mentioned. In addition, the present compound may have one or more asymmetric carbons depending on the type of substituent. Thus, stereoisomers such as optically active or diastereoisomers may exist based on the presence of such asymmetric carbon, but pure forms of stereoisomers, any mixture of stereoisomers, Racemates and the like can also be used as the active ingredient of this opener.

  The opener of the present invention can also contain one or more selected from the group consisting of the present compound, physiologically acceptable salts thereof, and hydrates and solvates thereof.

  This opener is used to prevent and / or protect various diseases and nerve cells that can be prevented and / or treated by relaxing smooth muscles, particularly in diseases involving BK channels in smooth muscle and central / peripheral nerves. It can be applied to various diseases that can be treated.

  The opener acts on BKαβ1 channels that exist in smooth muscle, which are components of various organs such as blood vessels, bladder, bronchi, and gastrointestinal tract, and BKαβ4 channels that exist in central and peripheral nerves. Increases the probability of opening calcium-dependent potassium channels (opening probability), leading to increased potassium permeability of the cell membrane. This results in hyperpolarizing smooth muscle cells and reduces the activity of voltage-dependent calcium channels. And smooth muscle is relaxed by suppressing calcium inflow from an extracellular fluid, and a nerve cell is protected from the damage by calcium overload.

  Therefore, the opener of the present invention is constantly contracted (hypertensive) in diseases such as hypertension including essential hypertension, tension bladder, peripheral circulation disorder, airway hypersensitivity, sensory nerve hypersensitivity, and central convulsions. ) Is useful as a medicament for the prevention and / or treatment of these diseases. It is also useful as a medicament for protecting central and peripheral nerve cells, particularly central nerve cells.

  As the present opener, the present compound or the like may be administered, but preferably, it can be administered as an oral or parenteral pharmaceutical composition that can be produced by methods well known to those skilled in the art. Examples of the pharmaceutical composition suitable for oral administration include tablets, capsules, powders, fine granules, granules, liquids, and syrups. The pharmaceutical composition suitable for parenteral administration includes Examples include injections, drops, suppositories, inhalants, eye drops, nasal drops, ear drops, ointments, creams, transdermal absorbents, transmucosal absorbents, patches, and the like. .

  Said pharmaceutical composition can be manufactured by adding one or more pharmaceutical additives. Examples of the additives for preparation include excipients, disintegrants or disintegration aids, binders, lubricants, coating agents, dyes, diluents, bases, solubilizers or solubilizers, isotonic agents, Examples thereof include a pH adjuster, a stabilizer, a propellant, and a pressure-sensitive adhesive, and these can be appropriately selected by those skilled in the art depending on the form of the pharmaceutical composition.

The dose of the potassium channel opener of the present invention is not particularly limited and can be appropriately selected according to the type of action and the intensity of action, and further, the weight and age of the patient, the type and symptoms of the disease, the administration route It can be appropriately increased or decreased depending on various factors that should normally be considered. Generally, in the case of oral administration, it can be used in the range of about 0.01 to 1,000 mg per adult day.

  The potassium channel opener of the present invention can further be used as a seed compound and lead compound for a more appropriate potassium channel opener.

(Screening method)
The screening method of the present invention may comprise an evaluation step of evaluating the potassium channel opening ratio of the test component using the present compound or a physiologically acceptable salt thereof as the test component. By selecting a compound having an excellent potassium channel opening ratio in such an evaluation step, a compound more suitable as a potassium channel opening agent can be obtained. The potassium channel opening ratio can be measured by preparing cells that express BKβ1 or β4 together with BKα, and conducting electrophysiological experiments such as whole cell patch clamp method and inside-out patch clamp method using this cell. . These methods are described in Imaizumi et al., 2002 (Imaizumi Y, Sakamoto K, Yamada A, Hotta A, Ohya S, Muraki K,
Uchiyama M and Ohwada T (2002) Mol Pharmacol 62: 836-846.). That is, the whole cell patch clamp method is a well-established and well-established method for examining what functions ion channels expressed on a cell membrane show as a sum (Hamil, OP. , Et al., Pflgers Archiv., 391, pp. 85-100, 1981). The inside-patch clamp method is a method in which a single channel opening and closing can be directly observed while artificially adjusting the environment inside the cell membrane by isolating a cell membrane fragment containing the channel. This method is a well-established method not only as a method for analyzing channel function but also for conducting quantitative efficacy tests of channel agonists (Hamill, OP, supra). In the experiment, the electrode resistance when filled with intracellular fluid is 2 to 4 MΩ during whole cell membrane current recording (hole cell method), 15 to 30 MΩ during single channel current recording (inside out method), macro During current recording (inside-out method), electrodes of 2 to 4 MΩ were used. A piece of glass with isolated and cultured cells fixed in a chamber placed on the stage of an inverted microscope was fixed, perfused with HEPES buffered saline, and currents were recorded by various potential fixation methods.

  In addition, the cell used for this screening method can use suitably the mouse | mouth, rat, rabbit, and the cell derived from higher primates including a human. As the cells, HEK cells (Human Embryo Kidney Cells) and the like can be used.

The evaluation step can be a step of evaluating the potassium channel opening ratio based on the action of the large conductance Ca ²⁺ activated K ⁺ (BK) channel on the β1 subunit or β4 subunit. Since the present compound can selectively act on β1 subunit and β4 subunit, the screening as a channel opener can be efficiently performed by evaluating the opening ratio of a BK channel comprising such subunit.

The screening method of the present invention is a screening method for a potassium channel opener, characterized by the amino acid sequence (LLFSFFWPTFLLTGGLLIIAMV) described in SEQ ID NO: 1 in the β1 subunit of the large conductance Ca ²⁺ activated K ⁺ (BK) channel. A step of evaluating an interaction between the site to be tested and the test component. Here, the interaction may be binding to a site characterized by the amino acid sequence, or may be a BK channel opening ratio including the site characterized by the amino acid sequence.

  According to the inventors, among the amino acid sequence set forth in SEQ ID NO: 1, the second, third, fifth, tenth to thirteenth, sixteenth, seventeenth and nineteenth positions The site (10 amino acid residues in total) is considered to be a site related to the change in the opening ratio of the BK channel containing the β1 subunit. Therefore, in particular, the interaction with these amino acids can be evaluated. In particular, by evaluating the binding of the β1 subunit to the structure (three-dimensional structure coordinates, etc.) characterized by these amino acid residues, the compound library can be searched by searching for compounds that can have binding. Can be built.

  Subsequently, potassium channel openers can be efficiently screened by evaluating the potassium channel open ratio using these compounds as test components. By constructing a compound library within the range of the compound represented by the formula (1), screening of potassium channel openers can be performed more efficiently.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited to a following example.

The experimental methods used in the following examples are described below.
(Isolation of cells)
Mouse bladder smooth muscle cells (mUBSMCs) were isolated using previously described methods (Morimura et al., 2006). That is, the bladder was removed from C57BL / 6 male mice (8-12 weeks old) and incubated for 11-13 minutes at 37 ° C. with 0.2% collagenase (Amano Enzyme, Nagoya, Japan) for mouse bladder smooth muscle. Cells were obtained.

(Transfection and cell culture)
HEK293 cells are 10% fetal bovine serum (Cell Culture Technologies,
Buhnrain / Zurich, Switzerland), 100 units / ml penicillin (Wako Pure Chemical Industries, Osaka,
Japan), MEM medium (Invitrogen) supplemented with 100 μg / ml streptomycin (Meiji Seika, Tokyo, Japan)
Corp., Carlsbad, CA, USA) at 37 ° C., 5% CO ₂ . HEK293 cells (HEK-rBKα, HEK-rSK2, HEK-mSK4, HEK-rKv1.1, HEK-rKv4.3) in which rBKα, rSK2, mSK4, rKv1.1, and rKv4.3 were expressed in a constant manner were previously used as rBKα, It was prepared by transfection of pcDNA3.1 (+) (Invitrogen) incorporating rSK2, mSK4, rKv1.1, rKv4.3 (Yamada et al., 2001; Imaizumi et al.,
2002; Hatano et al., 2004; Sakamoto et al., 2006). Similarly, HEK293 cells (HEK-rBKαβ1, HEK-rBKαβ4) in which rBKα and rBKβ1 or rBKβ4 were constantly expressed were also prepared previously. HEK293 cells (HEK-rBKαβ2) in which rBKα and rBKβ2 were transiently expressed were prepared by simultaneously transfecting pcDNA3.1 (+) (Invitrogen) incorporating rBKα and pTracer-CMV2 (Invitrogen) incorporating rBKβ2. did. Transfected cells were determined by the presence or absence of GFP signal derived from pTracer-CMV2 24-96 hours after transfection. The transfected cells were cultured on small pieces of glass in a culture dish and used for electrophysiological experiments.

(Electrophysiology)
Whole cell patch clamp method and inside-out patch clamp method are CEZ-2200, CEZ-2300, CEZ-2400 (Nihon
Kohden, Tokyo, Japan), EPC-7 (List Electronik, Darmstadt,
(Germany) was used as previously reported (Imaizumi et al., 2002). Electrophysiological recording and data acquisition / analysis in whole cells were performed using two programs (Data-Acquisition, Cell-Soft) developed at the University of Calgary. Single channel current analysis was performed using PATV 7.0C developed at Strathclyde University. For the whole cell patch clamp method, an electrode resistance of 2-5 MΩ when the solution in the electrode was filled was used, and for the inside-out patch clamp method, a 10-15 MΩ one was used.

(solution)
(Record of total cell currents of isolated mouse bladder smooth muscle isolated cells and transfected HEK293 cells)
Standard HEPES buffer [(mM): 137
NaCl, 5.9 KCl, 2.2 CaCl ₂ , 1.2 MgCl ₂ , 10 HEPES, 14 D-glucose
(Adjusted to pH 7.4 with NaOH)] was used as the perfusate.
(Record of total cell current of mouse bladder smooth muscle isolated cells)
300 nM Ca ²⁺ electrode solution [(mM): 140 KCl, 4.5 CaCl ₂ , 3
MgCl ₂ , 10 EGTA, 10 HEPES, 2 ATP (adjusted to pH 7.2 with KOH)] and 100
Standard HEPES buffer containing μM Cd ²⁺ was used.
(Recording total cell current of HEK-rBKα, HEK-rBKαβ1, HEK-rBKαβ4)
300 nM Ca ²⁺ electrode solution [(mM): 140 KCl, 3.3 CaCl ₂ , 1
MgCl ₂ , 5 EGTA, 10 HEPES, 2 ATPNa ₂ (pH with KOH
Prepared in 7.2)].
(Record of whole cell current of HEK-rBKαβ2)
1 μM Ca ²⁺ electrode solution [(mM): 140 KCl, 4.3 CaCl ₂ , 4 MgCl ₂ , 5 EGTA, 10
HEPES, 2 ATPNa ₂ (prepared to pH 7.2 with KOH)] was used.
(Recording of total cell current of HEK-rSK2 and HEK-mSK4)
1 μM Ca ²⁺ solution in electrode [(mM): 138 K-aspartate, 8.6 CaCl ₂ , 2 MgCl ₂ , 10
EGTA, 10 HEPES, 1 ATPK ₂ (prepared to pH 7.2 with KOH)] was used. The inter-liquid potential was corrected by -14 mV by measurement.
(Recording of total cell current of HEK-rKv1.1 and HEK-rKv4.3)
No nominal Ca ²⁺ (~ 10
nM) Electrode solution [(mM): 138 KCl, 0.58 CaCl ₂ , 2 MgCl ₂ ,
10 EGTA, 10 HEPES, 1 ATPK ₂ (prepared to pH 7.2 with KOH)].
(Recording single channel current under inside-out patch clamp)
HEPES buffer solution for both perfusate and electrode solution [(mM) 140 KCl,
2.2 CaCl ₂ , 1.2 MgCl ₂ , 5 EGTA, 10 HEPES, 14 D-glucose (pCa
7.0, adjusted to pH 7.2 with NaOH)].
Free Ca ²⁺ concentration was calculated using (WEBMAXC program (http://www.stanford.edu/ ^~ cpatton / webmaxcS.htm).

(Used compounds)
The compounds used this time are as follows. Shown with the source.
DiBAC ₄ (3): bis (1,3-dibutylbarbituric acid) trimethine oxonol (Dojindo (Kumamoto, Japan)
DiBAC ₄ (5): bis (1,3-dibutylbarbituric acid) pentamethine oxonol, DiSBAC (3): bis (1,3-diethylthiobarbituric acid) trimethine oxonol (Molecular
Probes, Inc. )
DiSBAC ₂ (5): bis (1,3-diethylthiobarbituric acid) pentamethine oxonol (AnaSpec, Inc.)
DiSBAC ₂ (1): bis (1,3-diethylthiobarbituric acid methine oxonol (Specs, Inc.)
BA: Barbituric acid (Wako Pure Chemical Industries, Ltd.)
DiSBAC ₄ (3): bis (1,3-dibutylthiobarbituric acid) trimethine oxonol, DiSBAC ₀ (5): bis (barbituric acid) pentamethine oxonol, Oxonol 595: bis (3-cyano-1- Ethyl-4-methyl-2,6-dioxo-1,2,5,6-tetrahydropyridine) trimethine oxonol, Oxonol V: bis (3-phenyl-5-oxoisoxazol-4-yl) pentamethine oxo Nord, DMBA: 1,3-dimethylbarbituric acid, DETBA: 1,3-diethyl l-2-thiobarbituric acid, penitremA (Sigma Aldrich)
Oxonol VI: bis (3-propyl-5-oxoisoxazol-4-yl) pentamethine oxonol (Fluka (Buchs, Switzerland)
DiSBAC ₁₀ (3): bis (1,3-didecylthiobarbituric acid) trimethine oxonol (Cayman (Ann
Arbor, MI, U. S. A. ))

DiBAC ₄ (3), DiBAC ₄ (5), DiSBAC ₂ (3), DiSBAC ₄ (3), DiSBAC ₀ (5), DiSBAC ₂ (1), Oxonol 595, Oxonol V, Oxonol VI, penitremA in DMSO at a concentration of 10 mM. After dissolution, BA, DMBA, and DETBA were dissolved in DMSO at a concentration of 100 mM and stored at -20 ° C. In order to dissolve the drug in the perfusate, the compound which is difficult to dissolve was sonicated for about 1 minute and used in the experiment.

(Optimization of three-dimensional structure of compound and calculation of pKa and intramolecular distance)
The most stable structure of an oxonol compound is Spartan '04 software for Windows version 1.0.3 in the form of a monovalent anion, except for DiSBAC ₀ (5), which is a tautomer in structure and exists as a divalent anion. . (Wavefunction,
Inc., Irvine, CA, USA) and determined by calculation using the density-functional theory (DFT) method and displayed by UCSF Chimera software. The distance between oxygen atoms in the molecule was measured from its most stable structure. The pKa value that determines the degree of ionization of molecules was calculated using Solaris V4.76 software (Advanced Chemistry Development, Inc., Toronto, Ontario, Canada).

(statistics)
The experimental results are expressed as mean ± standard error. For testing between two groups or between multiple groups, after performing Ftest or one-way analysis of variance, Student's t-test, Scheffe '
Evaluated by test. * And ** in the figure mean that the risk factors are statistically significant at 5% and 1%, respectively.

In this example, it was confirmed whether DiBAC ₄ (3) activates BK current in isolated mouse bladder smooth muscle cells. First, we examined the effect of DiBAC ₄ (3) on the outward current induced when depolarizing stimulation was applied to isolated mouse bladder smooth muscle cells using the patch clamp method. Measurement of whole-cell BK current elicited when depolarizing stimulation is applied to mouse bladder smooth muscle cells is 100 μM Cd ² in standard HEPES buffer to block voltage-dependent Ca ²⁺ channels (VDCC) ⁺ Was added, and pCa of the solution in the electrode was fixed at 6.5 with Ca ²⁺ -EGTA buffer.

As shown in FIG. 1A, the outward current (black circle in the figure) elicited when depolarizing from holding potential -60 mV to +40 mV for 150 milliseconds is significantly increased by administration of 300 nM DiBAC ₄ (3). Furthermore, it was suppressed by administration of peninitrem A, which is a selective BK channel inhibitor. Also 60
A current density-voltage relationship was obtained from the outward current (white circle in the figure) induced when depolarized in a 10 mV step in the range of mV to +60 mV for 150 milliseconds (FIG. 1B). As shown in FIG. 1B, the peak outward current density when depolarized to +60 mV is 16.9 ± 0.4 pA / pF before drug administration, 28.8 at 300 nM DiBAC ₄ (3) administration.
± 1.3 pA / pF (p <0.01 compared to pre-drug administration), 6.6 after peninitrem A administration
± 1.6 pA / pF (p <0.01 compared to DiBAC ₄ (3) administration, p <0.05 compared to drug administration) (n = 4 respectively). These results indicate that DiBAC ₄ (3) has a strong activating effect on the BK channel current of mouse bladder smooth muscle cells composed of α and β1 subunits.

In this example, the presence or absence of DiBAC ₄ (3) BK channel opening action was evaluated. HEK293 cells (HEK-rBKα) in which only rat BK channel α subunit (rBKα) is constantly expressed, α subunit and rat BK channel β1 subunit, β2 subunit, β4 subunit (rBKβ1, rBKβ2, rBKβ4) The heterologous expression system using HEK293 cells (HEK-rBKαβ1, HEK-rBKαβ2, HEK-rBKαβ4), which either or both of these are co-expressed steadily or transiently, exerts the effect of DiBAC ₄ (3) on the BK channel. investigated.

(Action on HEK-rBKαβ1)
Total cell current was measured with pCa 6.5 electrode solution and standard perfusate. The current density-voltage relationship is shown in FIG. 2A. The outward current elicited when HEK-rBKαβ1 is depolarized from a holding potential of -60 mV to 150 mV in 10 mV steps is significantly increased at a potential higher than -40 mV by administration of 300 nM DiBAC ₄ (3), Furthermore, it was completely suppressed by administration of 1 μM penitrem A. The peak outward current density when depolarized to +60 mV is 142.8 ± 14.6 pA / pF, 300 before drug administration.
nM DiBAC ₄ (3) administration 219.1 ± 35.8 pA / pF (p <0.01 compared to pre-drug administration), penitem A administration 2.4 ± 2.0 pA / pF
(P <0.01 compared to the time before drug administration and DiBAC ₄ (3) administration) (n = 3 respectively). As shown in FIG. 2B, the outward current when HEK-rBKαβ1 was depolarized to +20 mV was activated in a concentration-dependent manner by cumulative administration of 10 nM to 1 μM DiBAC ₄ (3). Moreover, the outward current was almost completely suppressed by administration of 1 μM penitrem A. Further, in FIG. 2C, the concentration of DiBAC ₄ (3) in HEK-rBKαβ1 - response relationships, normalized by DiBAC ₄ (3) Peak outward current before drug administration the peak outward current during administration, relative Shown as an increase rate. As shown in FIG. 2C, the relative increase rate when depolarized to +20 mV is 10
nM DiBAC ₄ (3) (1.440 ± 0.242; p <0.05 compared to before drug administration) and higher concentrations of DiBAC ₄ (3) and higher. In addition, BK current activated by administration of low concentration DiBAC ₄ (3) (less than 100 nM) under whole cell patch clamp was reversibly recovered by washout, but high concentration DiBAC ₄ (3)
The BK current activated by administration (300 nM or more) was not completely recovered by washing out (not shown).

Further, the single channel current at +20 mV of HEK-rBKαβ1 was measured using the inside-out patch clamp method under the condition that the inside and outside of the cell were 140 mM K ⁺ . The pCa of the perfusate and the solution in the electrode was fixed at 7.0. DiBAC ₄ (3) a P _o upon administration normalized by P _o before drug administration, indicated as a relative open probability (P _o). The results show that the relative P _o of rBKαβ1 channel is 100 as shown in FIGS. 3A and 3B.
It increased in a concentration-dependent manner by the cumulative administration of nM to 3 μM DiBAC ₄ (3). DiBAC ₄ (3) increase of the relative P _o of rBKαβ1 channel by administration Unlike effect on whole cell rBKαβ1 current, wash
Recovered quickly and completely with out. DiBAC ₄ (3) Concentration - shown in Figure 3C a relation relative P _o.

Also, as shown in FIG. 3D, the single channel current and the single channel conductance at +50 mV were 12.37 ± 0.07 pA, 260.2 pS, 300 before drug administration.
nM DiBAC ₄ (3) administration 12.76 ± 0.24 pA, 263.4
pS (p> 0.05 compared to before drug administration, n = 5 respectively). P _o is 300
It increased at all potentials measured by administration of nM DiBAC ₄ (3), and the probability of opening-voltage curve was well fitted to the Boltzmann equation shown below.
P _o = 1/1 [1 + exp {V ₁ / ₂ -V _m ) / S}]
V _1/2 , V _m , and S represent 50% activation potential, membrane potential, and slope factor, respectively.

FIG. 3E shows V _1/2 and S. As shown in FIG. 3E, V _1/2 and S are 96.3 ± 2.7 mV before drug administration, 11.4 ± 2.2 mV, 300 nMDiBAC ₄ (3) 74.8 ± 8.4 mV (drug administration) P <0.01) vs. 17.3 ± 4.3 mV (p> 0.05 vs. before drug administration) (n = 5 for each). From the above, it was found that the opening probability-voltage curve moved to the low potential side by about 30 mV by administration of 300 nMDiBAC ₄ (3).

(Action on HEK-rBKα)
Similarly, the whole cell current and single channel current of HEK-rBKα were measured under the same conditions as HEK-rBKαβ1. Under inside-out patch clamp, P ₀ at the time of DiBAC ₄ (3) administration is normalized by P ₀ before drug administration, and is shown in FIG. 4A as relative opening probability (P ₀ ). As shown in FIGS. 4A and 4B, the relative P ₀ of the rBKα channel did not change significantly with cumulative administration of 10 nM to 3 μM DiBAC ₄ (3). FIG. 4C shows the DiBAC ₄ (3) concentration-relative P ₀ relationship. As shown in FIG. 4C, the relative P ₀ of rBKα proportional to the concentration of DiBAC ₄ (3) did not increase. As is clear from the current density-voltage relationship shown in FIG. 4D, the outward current of HEK-rBKα is 300 nM under the whole cell patch clamp.
DiBAC ₄ (3) administration showed no significant change. Furthermore, the outward current was suppressed by administration of 1 μM penitrem A. Peak outward current density when depolarized to +60 mV is 152.8 ± 70.2 pA / pF, 300 prior to drug administration
nM DiBAC ₄ (3) administration 145.5 ± 67.2 pA / pF (p> 0.05 compared to pre-drug administration), penitrem A administration 6.8 ± 3.3 pA / pF
(P <0.01 compared to the time before drug administration and DiBAC ₄ (3) administration) (n = 3 respectively).

(Selectivity for subtype of BK channel β subunit of DiBAC ₄ (3))
Next, the selectivity of DiBAC ₄ (3) to the subtype of the BK channel β subunit was examined using HEK-rBKαβ2 and HEK-rBKαβ4 in addition to HEK-rBKαβ1.

(Action on HEK-rBKαβ2)
In order to observe the fully inactivated BK current in HEK-rBKαβ2 in the +20 mV to +80 mV range, rBKαβ2 total cell current was measured with pCa 6.0 in-electrode solution and standard perfusate (Wallner
et al., 1999). FIG. 5A shows the concentration-response relationship of DiBAC ₄ (3) in HEK-rBKαβ2. As shown in Fig. 5A, the outward current induced when HEK-rBKαβ2 is depolarized from the holding potential of -80 mV in a 20 mV step for 1000 milliseconds has a fast deactivation on the higher voltage side than +20 mV. Indicated. Unlike the HEK-rBKα or HEK-rBKαβ1, outward current showing inactivation of HEK-rBKαβ2 is DiBAC ₄ (3) without being influenced by the administration, 3 μM DiBAC ₄ (3) is significantly suppressed by administration It was. Furthermore, it was completely suppressed by administration of 1 μM penitrem A. The peak outward current density when depolarized to +80 mV is 199.1 ± 35.6 pA / pF before drug administration, 300
nM DiBAC ₄ (3) 135.0 ± 26.0 pA / pF (p> 0.05 compared to pre-drug), penitrem A 7.8 ± 2.3 pA / pF
(P <0.01 compared to the time before drug administration and DiBAC ₄ (3) administration) (n = 3 respectively). As shown in FIG. 5B, +80
The relative increase in mV is 0.878 ± 0.090 (p> 0.05) at 1 μM DiBAC ₄ (3) administration, 3
It was 0.627 ± 0.069 (p <0.01) at the time of μM DiBAC ₄ (3) administration (n = 5 for each).

(Action on HEK-rBKαβ4)
The total cell current of HEK-rBKαβ4 was measured under the same conditions as HEK-rBKα and HEK-rBKαβ1. As shown in FIG. 6B, the outward current of HEK-rBKαβ4 is markedly increased on the higher potential side than −20 mV by administration of 300 nM DiBAC ₄ (3).
Peak outward current density when depolarized to +60 mV completely suppressed by μM penitrem A administration was 127.6 ± 17.9 pA / pF (n = 6) before drug administration, 300 nM DiBAC BAC ₄ (3) administration 230.5 ± 36.1 pA / pF (p <0.01 vs n before drug administration, n = 6), penitrem A administration 9.4
± 2.3 pA / pF (p <0.01, n = 4 before drug administration and DiBAC BAC ₄ (3) administration).

Moreover, the outward current of HEK-rBKαβ4 when depolarized to +20 mV increased in a concentration-dependent manner by cumulative administration of 10 nM to 3 μM DiBAC BAC ₄ (3). The outward current was completely suppressed by administration of 1 μM penitrem A (FIG. 6B). The concentration-response relationship of DiBAC BAC ₄ (3) in HEK-rBKαβ4 is shown in FIG. 6C superimposed with the concentration-response relationship of HEK-rBKαβ1 shown in FIG. 2C. Of note is the DiBAC of rBKαβ4 current at concentrations from 10 nM to 3 μM.
The activation by BAC ₄ (3) is almost the same as the activation of rBKαβ1 current. +20
The relative increase in rBKαβ4 current when depolarized to mV is 1.322 ± 0.096 for 10 nM DiBAC BAC ₄ (3) (p <0.05 vs. pre-drug administration).
There was no significant difference from the relative increase rate by nM DiBAC BAC ₄ (3) (1.440 ± 0.242; p> 0.05 for rBKαβ1 current).

(Selectivity for other K ⁺ channels of DiBAC ₄ (3))
Next, DiBAC BAC ₄ (3) selectivity for other K ⁺ channels such as small conductance Ca ²⁺ activated K ⁺ channels (rSK2, mSK4) and voltage-dependent K ⁺ channels (rKv1.1, rKv4.3) Therefore, HEK293 cells (HEK-rSK2, HEK-mSK4, HEK-rKv1.1, HEK-rKv4.3) in which these channels are constantly expressed were prepared.

(Action on HEK-rSK2 and HEK-mSK4)
HEK-rSK2 and whole-cell currents HEK-mSK4 is Cl ^- and aspartate ^-
Using a solution in the electrode with a pCa fixed at 6.0 and a standard perfusion solution, measurement was performed by depolarizing from −160 mV to +20 mV by a ramp pulse for 250 milliseconds.

As shown in FIG. 7A, the total cell current of HEK-rSK2 is 10 μM DiBAC.
10 nM which is slightly reduced by BAC ₄ (3) administration and is a selective SK1-3 channel inhibitor
It was suppressed by administration of UCL 1684. As shown in FIG. 7B, the total cell current of HEK-mSK4 is 10 μM DiBAC.
It was not affected by administration of BAC ₄ (3) and was suppressed by administration of 1 μM clotrimazole, a selective SK4 channel inhibitor.

(Action on HEK-rKv1.1 and HEK-rKv4.3)
The total cell current of HEK-rKv1.1 and HEK-rKv4.3 is depolarized from the holding potential of -80 mV to +20 mV for 1000 milliseconds using the electrode solution with pCa fixed at 8.0 and the standard perfusate. And measured. As shown in FIG. 7C, the total cell current of HEK-rKv1.1 was not affected by administration of 10 μM DiBAC ₄ (3), and was suppressed by administration of 100 nM margatoxin, a Kv1.x channel inhibitor. In addition, as shown in FIG. 7D, the total cell current of HEK-rKv4.3 was not affected by the administration of 10 μM DiBAC BAC ₄ (3).

The effects of 10 μM DiBAC BAC ₄ (3) on these channels are summarized in FIG. 7E. The relative rate of increase in HEK-rSK2 and HEK-mSK4 is calculated by Cl ^- reversal DiBAC BAC ₄ (3) at -50 mV which is the potential amount of current dose normalized by the amount of current prior to drug administration Asked. The relative increase rate in HEK-rKv1.1 and HEK-rKv4.3 is the peak outward current during DiBAC BAC ₄ (3) administration when depolarized to +20 mV. It was obtained by standardizing with the direction current.

The relative rate of increase with 10 μM DiBAC BAC ₄ (3) was 0.754 ± 0.052 for HEK-rSK2 (p <0.05 compared to pre-drug administration) and 1.051 ± 0.057 for HEK-mSK4.
(p> 0.05), HEK-rKv1.1 is 1.010 ± 0.013 (p> 0.05), HEK-rKv4.3 is 0.942
± 0.055 (p> 0.05) (n = 4 for each). From these results, DiBAC
BAC ₄ (3) were found to be selective for BK channels than Ca ²⁺ activated K ⁺ channels and voltage-gated K ⁺ channels was examined in this example.

(Structure-activity relationship between DiBAC BAC ₄ (3) and related oxonol compounds as BK channel openers)
DiBAC ₄ (3) has a unique structure in which two 1,3-dialkylbarbituric acids are conjugated and linked by an oligomethine chain, and therefore determines the molecular structure required for activating the BK channel of DiBAC BAC ₄ (3) Therefore, the structure-efficacy relationship for BK channel activation of 16 kinds of DiBAC ₄ (3) and its similar compounds was elucidated. FIG. 8A shows the names and structures of DiBAC ₄ (3) and its similar compounds.

HEK-rBKαβ1 holding potential -60 mV to +20
FIG. 8 shows the effects of 100 nM and 1 μM DiSBAC ₂ (5) and 1 and 10 μM DiSBAC ₂ (1) on the total amount of outward current induced when depolarized to mV. As shown in FIG. 8B, rBKαβ1 current was clearly activated by administration of 1 μM DiSBAC ₂ (5) and slightly activated by administration of 10 μM DiSBAC ₂ (1). Similar experiments were performed to determine the activity of the illustrated compounds (not shown). The peak outward BK current at the time of administration of similar compounds is normalized by the peak outward BK current before drug administration, and is shown in FIG. 8C as a relative increase rate.

As shown in FIG. 8C, the total cell current of HEK-rBKαβ1 is 100 nM.
DiSBAC ₄ (3) administration increased about 2-fold (2.415 ± 0.390, n = 5). This is similar to the degree of activation by administration of 100 nM DiBAC ₄ (3) (2.262 ± 0.096, n = 7; p> 0.05 compared to DiSBAC ₄ (3)), and DiBAC C = O binding It was found that substitution of DiSBAC with C = S bond did not affect BK channel activation. Furthermore, the total cell current of HEK-rBKαβ1 is 100 nM
DiSBAC ₂ (3) administration increased about 2-fold (2.044 ± 0.32, n = 4; p> 0.05 for DiBAC ₄ (3) and DiSBAC ₄ (3)).

Similarly, as shown in FIG. 8C, the total cell current of HEK-rBKαβ1 is 1, 10
No effect was shown by administration of μM DiSBAC ₀ (5) (1.017
± 0.011, n = 5; p> 0.05 vs. 1.003 ± 0.006, n before drug administration
= 5; p> 0.05 compared to before drug administration). The same applies to the inside-out patch clamp, and the NP _{o of} HEK-rBKαβ1 is 1.
μM DiSBAC ₀ (5) was not affected by the administration but, 10 μM DiSBAC ₀ (5) was significantly reduced in the administration. _{1 μM, 10 μM DiSBAC 0 (} 5) relative increase rate 1.180 ± of NP _o by administration 0.217 (n = 6; p> 0.05 for the previous drug administration), 0.422 ± 0.146 (n = 4; drug administration P <0.05). Therefore, it appears that the efficacy is significantly reduced when the alkyl side chain is gone. These results indicate that the alkyl side chain at positions 1 and 3 of barbituric acid is effective for BK channel opening characteristics, but the length of the carbon atom is greatly related to the efficacy of both ethyl (C2) and butyl (C4) groups. Did not seem to.

Furthermore, as shown in FIG. 8C, hypnotic drugs (hexobarbital, barbital), hydantoin-type antiepileptic drugs (phenytoin), barbituric acid / thiobarbituric acid (BA, DMBA, DETBA) used in clinical practice are -rBKαβ1 had no effect on the total cell current (all 10 μM) (data not shown for hexobarbital, barbital and phenytoin). The relative increase in BK current with administration of 10 μM BA, TMBA, and TETBA was 0.996 ± 0.039 (n = 5; p> 0.05 compared to pre-drug), 0.938 ± 0.028 (n = 4; pre-drug) P> 0.05) and 1.125 ± 0.096 (n = 4; p> 0.05 before drug administration). From these results, it was found that two Barbitur rings conjugated with oligomethine chains are effective for BK channel opening characteristics of DiBAC ₄ (3).

In addition, the effects of DiBAC ₄ (3) and DiSBAC ₂ (3) on the trimethine chain were compared with those of DiBAC ₄ (5) and DiSBAC ₂ (5) obtained by extending the trimethine chain to a pentamethine chain. In addition, we examined the effect of DiSBAC ₂ (1) in which the trimethine chain was shortened to the monomethine chain. Interestingly, the relative rate of increase by administration of 100 nM and 1 μM DiBAC ₄ (5) was 1.257 ± 0.025 (n = 5; p <0.01 before drug administration, 1.918 ± 0.287 (n = 5; p <0.05 vs drug administration), relative to 100 nM, 1 μM DiSBAC ₂ (5) administration The rate of increase was 1.293 ± 0.033 (n = 4; p <0.01 compared to pre-drug administration) and 2.107 ± 0.053 (n = 4; p <0.01 compared to pre-drug administration). BK channels were not activated by 1 μM DiSBAC ₂ (1) administration (1.044 ± 0.050, n = 4; p> 0.05 compared to pre-drug administration), but were activated by 10 μM DiSBAC ₂ (1) administration ( 1.252 ± 0.063, n = 4; p <0.05 compared to before drug administration (FIG. 8C). Therefore, it was found that in the oxonol compound having 1,3-alkyl barbituric acid, the trimethine chain is a preferred length of the oligomethine chain. From these results, it can be seen that the length of the conjugated oligomethine chain is an important factor determining the efficacy as a BK channel opener.

In addition, Oxonol 595, Oxonol V, Oxonol were used to elucidate the number and localization of oxo moieties required for a molecule with efficacy.
The action of VI was examined. Oxonol 595 has a structure similar to DiBAC ₄ (3), although the oxo moiety at the 4-position of the pyrimidine ring has disappeared. Oxonol V and Oxonol VI have a 5-isoxazolone ring. Oxonol 595 showed no activity (at 1 μM administration: 1.002 ± 0.025,
n = 4; p> 0.05 before drug administration, 10 μM administration: 1.071 ± 0.078, n = 5; p> 0.05 before drug administration). In contrast, Oxonol V, Oxonol
Both VI act as BK channel openers, but Oxonol V was about 10 times more potent than Oxonol VI (FIG. 8D). Oxonol compounds with acidic pKa form oxoanions almost completely at pH 7.4. Nevertheless, Oxonol 595 did not show efficacy as a BK channel opener even at a concentration of 10 μM.

(Structure of DiBAC ₄ (3) required for BK channel activation based on stereo analysis)
In this example,
The three-dimensional structure for BK channel activation and the most effective length of the oligomethine chain between the oxo-oxoanion moieties in the molecule were investigated. In the three-dimensional structure analysis, the most stable three-dimensional structure of the oxonol compound was calculated using the DFT method using Spartan software. FIG. 9A shows a top view and a side view of the most stable steric structure of ten oxonol compounds used in Example 6. FIG. Table 1 also shows the relative increase rate of the total cell current of HEK-rBKαβ1 and the distance between oxo-oxoanion moieties on both sides of the oligomethine chain.

As is apparent from the side view of FIG. 9A, in the active compound, the negatively charged oxygen atom indicated by the arrow and its oligomethine chain are present on one plane in the most stable structure. That is, two Barbitur rings / Thiobarbitur rings linked by an oligomethine chain in the DiBAC / DiSBAC compound also exist on one plane. The same applies to oxonol V in which the two rings are 5-isoxazolone rings. This is thought to be necessary for the negatively charged oxo group and the oligomethine chain to effectively transfer electrons. Twist was observed in the two thiobarbitur rings in the molecular image of DiSBAC ₂ (1), which showed little activity. In addition, a slight twist in the oligomethine chain was observed in oxonol VI, which was significantly less active than oxonol V (FIG. 9A).

  The active DiBAC / DiSBAC compound and the oxo-oxoanion part of oxonol V / VI are located on the same side of the oxonol chain, whereas the oxo-oxoanion part of oxonol 595 is located on the opposite side of the oligomethine chain. Position one by one. In addition, the oxo-oxoanion part of oxonol 595 is located in opposite directions with respect to the conjugated oligomethine chain.

In addition, as shown in Table 1, the distance between two oxygen atoms in the two oxo-oxoanion moieties located on both sides of the oligomethine chain in the molecule of each compound was calculated to be 4 angstroms and 7 angstroms.
It was Angstrom. In the DiBAC / DiSBAC compound, the two oxygen atoms present in the oxo-oxoanion part with a short distance (4 angstroms) conjugated with the trimethine chain give high activity due to the most efficient transmission of conjugated π electrons. It's thought to be.

  From the above, as shown in FIG. 9B, it is possible to adopt a most stable structure in which at least one oxo-oxoanion portion conjugated with an oligomethine chain is located on the same side of the oligomethine chain and these have a substantially planar structure. It was found to be effective in acting as a β-unit selective BK channel opener.

(Molecular mechanism of β subunit selective BK channel activation)
(Identification of DiBAC4 (3) sensitive region of β subunit)
Between the BK channel β1 subunit and β2 subunit, there is about 41% homology at the amino acid level, and the highest homology among BK channel β subunits. Nonetheless, the BK current co-expressed with the α1 subunit is sensitive to DiBAC ₄ (3), whereas the BK current co-expressed with the β2 subunit is DiBAC ₄ (3). (See Examples 3 and 4). Therefore, we aimed to elucidate the mechanism of BK channel activation via β subunit by oxonol compounds by creating chimeric mutants of β1 subunit and β2 subunit.

Unlike the β1 subunit, the BK channel β2 subunit exhibits inactivation characteristics because an inactivated ball exists in the N-terminal region. Therefore, the functional analysis of β-subunit mutants, .beta.2 subunit mutants exhibiting no deactivation characteristics deficient the inactivation ball (Beta2IR subunit; IR: i nactivation
r emoved) was made and used in the experiment. Administration of 300 nM DiBAC ₄ (3) with a macro BK current of V _0.5 observed under an inside-out patch clamp with the intracellular K ⁺ concentration fixed at 140 mM and the intracellular Ca ²⁺ concentration fixed at 3 μM The amount of change (ΔV _0.5 ) due to was used as an index of DiBAC ₄ (3) sensitivity. FIG. 10 shows the effect of DiBAC ₄ (3) on the BKα current, BKαβ1 current, and BKαβ2IR current.

As shown in FIG. 10, V _0.5 of BKα current and BKα + β2IR current observed under the inside-out patch clamp was not significantly changed by 300 nM DiBAC ₄ (3) administration, but BKαβ1 current V _0.5 moved to the low potential side by about 30 mV by administration of 300 nM DiBAC ₄ (3). This is the same results as the effects of DiBAC ₄ (3) for the previous HEK-rBKαβ2 whole cell currents, also be deficient inactivated ball DiBAC ₄ (3) did not show sensitivity to BKα + β2IR current . In contrast, the slope factor increased significantly in all measured BK currents. This is considered to be an effect on the BKα subunit independent of the presence or absence of the β subunit of DiBAC ₄ (3). From the above results, it was confirmed that BKαβ1 current was sensitive to 300 nM DiBAC ₄ (3), but BKα current and BKαβ2IR current were not sensitive.

Next, a chimeric mutant (β1NCβ2) in which the intracellular NC-terminal region of β1 subunit was replaced with the intracellular NC-terminal region of β2IR subunit, and the extracellular loop of β2IR subunit was replaced with the extracellular loop of β1 subunit Chimera mutants (β2Lβ1) and chimeric mutants (β2TMsβ1) in which the two transmembrane regions of β2IR subunit are replaced with two transmembrane regions of β1 subunit were prepared, respectively, and β1 required for DiBAC ₄ (3) sensitivity. We examined the area of subunits. FIG. 11 shows the effect of DiBAC ₄ (3) on the BKαβ2Lβ1 current, BKαβ1NCβ2 current, and BKαβ2TMsβ1 current.

As shown in FIG. 11, V _0.5 of the BKαβ1NCβ2 current and BKαβ2TMsβ1 current moved to the lower potential side by administration of 300 nM DiBAC ₄ (3), whereas V _0.5 of the BKαβ2Lβ1 current was 300 nM DiBAC ₄ (3). There was no significant change with administration. From this, it was speculated that the DiBAC ₄ (3) binding region exists in the two transmembrane regions of the β1 subunit.

In addition, chimeric mutants (β1TM1β2, β2TM1β1), β1, and β2IR subunit Cs in which the N-terminal transmembrane region (TM1) of β1 and β2IR subunits are replaced with the N-terminal transmembrane region of β2IR and β1 subunits, respectively. Chimeric mutants (β1TM2β2, β2TM2β1) were prepared by replacing the terminal transmembrane region (TM2) with the C2 terminal transmembrane region of β2IR and β1 subunits, respectively, and DiBAC ₄ (3) sensitivity was examined. FIG. 12 shows the effect of DiBAC ₄ (3) on the BKαβ1-LFF / FHL current, BKαβ1-FLLT / CMMA current, and BKαβ1-LLI / VAV current.

As shown in FIG. 12, V _0.5 of the BKαβ1TM1β2 current was shifted to the low potential side by administration of 300 nM DiBAC ₄ (3) (not shown), whereas V _0.5 of BKαβ1TM2β2 current and BKαβ2TM1β1 current was 300 nM DiBAC. ₄ (3) No significant change was observed by administration. Interestingly, V _0.5 of the BKαβ2TM2β1 current shifted to the low potential side by administration of 300 nM DiBAC ₄ (3). These results suggest that the C1-terminal transmembrane region of β1 subunit regulates BK channel activity as a drug-sensitive region of DiBAC ₄ (3).

(Identification of DiBAC ₄ (3) sensitive site in β subunit C-terminal transmembrane site)
From the analysis using β1 and β2IR subunit chimeric mutants, we compared the amino acid sequences of the C-terminal transmembrane region of rat β1 subunit and rat β2 subunit used in the experiment. As shown in FIG. 13, 12 amino acids out of 23 amino acids predicted as the transmembrane region were conserved, and there were 11 amino acids presumed to be DiBAC ₄ (3) sensitive sites. In addition, isoleucine (I) at the 156th position (1st C-terminal transmembrane region) of rat β1 subunit is valine (V) in mice, alanine (A) in humans and rabbits, serine (S) in cattle, and dogs. So it differs from threonine (T) by species (not shown). Since the whole cell BK current of mouse bladder smooth muscle was markedly activated by administration of 300 nM DiBAC ₄ (3) (see Example 2), the 156th isoleucine (I) of the β1 subunit was sensitive to DiBAC ₄ (3). Can be excluded from the site. Therefore, at the present time, 10 amino acids different in β1 subunit and β2 subunit were estimated as DiBAC ₄ (3) sensitive sites.

Next, the C-terminal transmembrane region of β1 subunit, which is presumed to be DiBAC ₄ (3), is divided into an N-terminal part, a central part, and a C-terminal part. Point mutants (β1LFF / FHL, β1FLLT / CMMA, β1LLI / VAV) substituted at the N-terminal part, the central part and the C-terminal part were prepared, and DiBAC ₄ (3) sensitivity was examined. FIG. 13 also shows the effect of DiBAC ₄ (3) on the BKαβ1-LFF / FHL current, BKαβ1-FLLT / CMMA current, and BKαβ1-LLI / VAV current.

As shown in FIG. 13, V _0.5 of BKαβ1LFF / FHL, BKαβ1FLLT / CMMA, and BKαβ1LLI / VAV currents all moved to the low potential side by administration of 300 nM DiBAC ₄ (3). From the above, .beta.1 although C-terminal transmembrane region of the subunits is necessary to BK channel activation by DiBAC ₄ (3), C-terminal membrane only partial mutation of transmembrane domain DiBAC ₄ (3) sensitivity It was shown not to disappear.

(Effects of DiBAC ₄ (3) on rat mesenteric artery)
The pharmacological action of DiBAC ₄ (3) was examined using rat mesenteric artery. A cannula was inserted into the first branch vessel of the mesenteric artery isolated from the Wister / ST rat, and Kreb's solution was refluxed using a peristaltic pump at a rate of 10 ml / min. The measurement was performed after the reflux pressure was stabilized. As shown in FIG. 14, the reflux pressure was significantly increased from 42.96 ± 1.21 mmHg to 218.16 ± 19.92 mmHg by administration of 3 μM phenylephrine. When 100 nM DiBAC ₄ (3) was pre-administered for 10 minutes and 3 μM phenylephrine was administered in the same manner, the reflux pressure increased from 40.14 ± 0.73 mmHg to 74.86 ± 8.81 mmHg, but before and after 100 nM DiBAC ₄ (3) administration The increase in reflux pressure by 3 μM phenylephrine was 175.20 ± 20.52 mmHg and 34.72 ± 8.12 mmHg, respectively, and the effect was remarkably attenuated. These results indicate that DiBAC ₄ (3) has extremely strong muscle relaxation.

FIG. 7 is a graph showing the effect of DiBAC ₄ (3) on the total cell BK current of mouse bladder smooth muscle cells. A shows the total cell BK current before administration of the drug and 1 μM penitrem A when 300 nM DiBAC ₄ (3) is administered. B shows the effect of DiBAC ₄ (3) on the total cell BK current of mouse bladder smooth muscle cells, and shows the current before administration, during administration, and at 1 μM penitrem A administration. Indicates the voltage relationship. Is a diagram showing the effect of DiBAC ₄ (3) to the total cell current of HEK-rBKαβ1, A is prior to drug administration, and at 1 μM penitrem A administered at a current - shows the voltage relationship, B is HEK It is a diagram showing the effect of DiBAC ₄ (3) on the total cellular current of rBKαβ1, and at the time when 10 μM to 1 μM DiBAC ₄ (3) was depolarized while cumulatively administered and 1 μM penitrem A was added. It is the figure which plotted the amount of peak outward currents. Current plots at different concentrations are shown as superimposed insets. C is a graph showing the effect of DiBAC ₄ (3) on the total cell current of HEK-rBKαβ1, and shows the concentration-response relationship of DiBAC ₄ (3) with respect to the total cell current of HEK-rBKαβ1. Is a diagram showing the effect of DiBAC ₄ (3) for a single channel current of HEK-rBKαβ1, A is, DiBAC ₄ (3) is open probability upon administration (P _o) were normalized by P _o before drug administration, Indicated as relative _Po . C, O1, O2, and O3 indicate the closed and open states of the BK channel, respectively. B is a diagram showing an original current diagram and a current amount histogram after washing out before administration of the drug obtained from A, at the time of 300 nM DiBAC ₄ (3) administration, at the time of 3 μM DiBAC ₄ (3) administration. The head of the arrow indicates the closed state of the BK channel. C is a graph showing the concentration-response relationship of DiBAC ₄ (3) with respect to the relative opening probability of the rBKαβ1 channel obtained from the experiment of A. FIG. D is a graph showing rBKαβ1 single channel current measured before and after administration of 300 nMDiBAC ₄ (3) at a potential of 0 mV to +50 mV. Single channel conductance was obtained by linearly fitting each data. E shows the relative P _o -voltage relationship of the rBKαβ1 single channel current. The data was fitted by Boltzmann equation. Is a diagram showing the effect of DiBAC ₄ (3) for a single channel current of HEK-rBKα, A is, DiBAC ₄ (3) is open probability upon administration (P _o) were normalized by P _o before drug administration, Shown as relative _Po . C, O1, and O2 indicate the closed and open states of the BK channel, respectively. B shows a current map and a current amount histogram after washout before administration of the drug obtained from A, at the time of 300 nM DiBAC ₄ (3) administration, at the time of 3 μM DiBAC ₄ (3) administration. The head of the arrow indicates the closed state of the BK channel, and C indicates the concentration-response relationship of DiBAC ₄ (3) with respect to the relative P _o of the rBKα channel obtained from the experiment of A. It was also plotted again to compare the relative P _o of rBKαβ1 channel shown in FIG. 3C. The inset shows the concentration-response relationship of DiBAC ₄ (3) to the relative P _o of the rBKα channel expanded. D is a diagram showing a current-voltage relationship before drug administration, at the time of 300 nM DiBAC ₄ (3) administration, and at the time of 1 μM penitrem A administration. It is a figure which shows the effect | action of DiBAC ₄ (3) with respect to the whole cell electric current of HEK-rBKαβ2, and A shows the current-voltage relationship at the time of 3 μM DiBAC ₄ (3) administration and 1 μM penitrem A administration before drug administration. FIG. 4B shows the concentration-response relationship of DiBAC ₄ (3) with respect to the total cell current of HEK-rBKαβ2. The peak outward current at the time of DiBAC ₄ (3) administration is normalized by the peak outward current before drug administration and is shown as a relative increase rate. ** indicates a significant difference with a 1% risk rate compared to before drug administration. It is a figure which shows the effect of DiBAC ₄ (3) on the whole cell current of HEK-rBKαβ4. A shows the current-voltage relationship at the time of 300 nM DiBAC ₄ (3) administration and 1 μM penitrem A administration before drug administration. B is a graph in which the peak outward current amount is plotted against the time when 10 nM to 3 μM DiBAC ₄ (3) is polarized while cumulatively administered and 1 μM penitrem A is added. Current plots at different concentrations are shown as superimposed insets. C is a graph in which the peak outward current at the time of DiBAC ₄ (3) administration is normalized by the peak outward current before drug administration and is shown as a relative increase rate. The concentration-response relationship of DiBAC ₄ (3) of the total cell current of HEK-rBKαβ1 shown in FIG. 2C was plotted again for comparison. Is a diagram showing the effect of DiBAC ₄ (3) with respect to K ⁺ channels other than BK channels were expressed HEK293 cells, A is shows the effect on small-conductance Ca ²⁺ activated K ⁺ channel, type 2 (rSK2) , B shows the action on the same type 4 (mSK4), C shows the action on the voltage-dependent K ⁺ channel rKv1.1, D shows the action on the same rKv4.3, and E shows A to D These experimental results are collectively shown, and show the relative increase rate of rSK2 channel, mSK4 channel, rKv1.1 channel and rKv4.3 channel by administration of 10 μM DiBAC ₄ (3). * Indicates that there is a significant difference at a risk rate of 5% compared to before drug administration. It is a figure which shows the structure activity relationship of DiBAC / DiSBAC and its related compound, A shows the structure of a similar compound containing DiBAC ₄ (3) and barbituric acid and its related compound, B is 100 nM, 1 μM ₂ shows the time course of BK current when DiSBAC ₂ (5) was administered and when 1 μM and 10 μM DiSBAC ₂ (1) was administered. C is the time when iBAC ₄ (3) and its similar structural compounds were administered. It is the figure which normalized the peak outward current by the peak outward current before drug administration, and showed it as a relative increase rate. * And ** indicate significant differences at 5% and 1% risk rates, respectively, before drug administration. D is the relative increase in oxonol V, oxonol VI and oxonol 595 concentrations and rBKαβ1 current. The relationship with the rate is shown. The dotted lines indicated by C1, C3, and C5 are plotted again based on the C results. It is a figure which shows the most stable three-dimensional structure of an oxonol compound, and the most stable three-dimensional structure of the oxonol compound was calculated using the Spartan software and using the density-functional theory (DFT) method. A shows its top and side views with UCSF Chimera software. The hydrogen atom, carbon atom, nitrogen atom, oxygen atom, and sulfur atom in the oxonol compound are shown in white, gray, dark gray, black, and light gray, respectively. Two sets of oxo-oxoanion moieties are indicated by sky blue and pink arrows, respectively. B shows the three-dimensional structure required as a BK channel opener. It is a diagram showing the action of DiBAC ₄ (3) on BKα current, BKαβ1 current, and BKαβ2IR current. Inside out and outside conditions are fixed at 140 mM K ⁺ concentration inside and outside and Ca ²⁺ concentration at 3 μM in the intracellular solution. Conductance-potential relationship when 300 nM DiBAC ₄ (3) is administered to macro BK current observed under patch clamp. V _0.5 and slope factor were calculated by fitting the obtained data by the Boltzmann equation. The number of cases is as follows (BKα current; n = 3, BKαβ1 current; n = 7, BKαβ2IR current; n = 7). D, E: V _0.5 and slope factor obtained with AC were summarized (**; p <0.01, *; p <0.05 vs. control). F: Changes in V _0.5 before and after administration of 300 nM DiBAC ₄ (3) were summarized (##; p <0.01 vs. α + β1, $$; p <0.01 vs. α + β2IR). BKαβ2Lβ1 current, BKarufabeta1NCbeta2 current is a diagram showing the effect of DiBAC ₄ (3) for BKαβ2TMsβ1 current, A through C is a K ⁺ concentration of intracellular 140 mM, Ca ²⁺ concentration of intracellular fluid fixed to 3 [mu] M Conductance-potential relationship when 300 nM DiBAC ₄ (3) was administered to the macro BK current observed under an inside-out patch clamp under the above conditions. V _0.5 and slope factor were calculated by fitting the obtained data by the Boltzmann equation. The number of cases is as follows (BKαβ2Lβ1 current; n = 4, BKαβ1NCβ2 current; n = 5, BKαβ2TMsβ1 current; n = 5). D, E: V _0.5 and slope factor obtained with AC were summarized (**; p <0.01, *; p <0.05 vs. control). F: Changes in V _0.5 before and after administration of 300 nM DiBAC ₄ (3) were summarized (##; p <0.01 vs. α + β1, $$; p <0.01 vs. α + β2IR). BKαβ2TM1β1 current, BKarufabeta2TM2beta1 current is a diagram showing the effect of DiBAC ₄ (3) for BKαβ1TM2β2 current, A through C is a K ⁺ concentration of intracellular 140 mM, Ca ²⁺ concentration of intracellular fluid fixed to 3 [mu] M Conductance-potential relationship when 300 nM DiBAC ₄ (3) was administered to the macro BK current observed under an inside-out patch clamp under the above conditions. V _0.5 and slope factor were calculated by fitting the obtained data by the Boltzmann equation. The number of cases is as follows (BKαβ2TM1β1 current; n = 4, BKαβ2TM2β1 current; n = 5, BKαβ1TM2β2 current; n = 4). D, E: V _0.5 and slope factor obtained with AC were summarized (**; p <0.01, *; p <0.05 vs. control). F: Changes in V _0.5 before and after administration of 300 nM DiBAC ₄ (3) were summarized (##; p <0.01 vs. α + β1, $$; p <0.01 vs. α + β2IR). It is a figure which shows the effect | action of DiBAC ₄ (3) with respect to BKαβ1-LFF / FHL current, BKαβ1-FLLT / CMMA current, and BKαβ1-LLI / VAV current, and A is the C terminal of rat BK channel β1 subunit and β2 subunit. Comparison of amino acid sequences of the transmembrane region on the side. The different amino acids in the β1 subunit and β2 subunit are inverted, and B to D are inside-out patches under the condition that the intracellular K ⁺ concentration is 140 mM and the Ca ²⁺ concentration in the intracellular fluid is fixed at 3 μM. Conductance-potential relationship when 300 nM DiBAC ₄ (3) is administered to the macro BK current observed under the clamp. V _0.5 and slope factor were calculated by fitting the obtained data by the Boltzmann equation. The number of cases is as follows (BKαβ1-LFF / FHL current; n = 5, BKαβ1-FLLT / CMMA current; n = 4, BKαβ1-LLI / VAV current; n = 4). E and F summarized V _0.5 and slope factor obtained from B to D (**; p <0.01 vs. control). G: Changes in V _0.5 before and after administration of 300 nM DiBAC ₄ (3) were summarized ($$; p <0.01 vs. α + β2IR). FIG. 2 is a graph showing the action of DiBAC ₄ (3) on rat mesenteric artery, and A shows the time course of reflux pressure with administration of 3 μM phenylephrine in the presence or absence of 100 nM DiBAC ₄ (3). B is a graph summarizing the results obtained from A. The black and white columns indicate the reflux pressure before and after administration of 3 μM phenylephrine (n = 4, **; p <0.01, *; p <0.05 vs. 3 μM phenylephrine before reflux). C shows a comparison of the amount of change in reflux pressure due to administration of 3 μM phenylephrine in the presence or absence of DiBAC ₄ (3). (N = 4, *; p <0.05 vs. change in reflux pressure by administration of 3 μM phenylephrine in the absence of DiBAC4 (3)).

Claims (13)

A potassium channel opener,
It is represented by the following formula (1),
[Wherein, R ¹ and R ² may be the same or different, and may be an acyclic hydrocarbon group which may be substituted, a cyclic hydrocarbon group which may be substituted or a substituted group. Represents a heterocyclic group, and in the case of a cyclic hydrocarbon group and a heterocyclic group, it may be bonded to a carbon atom constituting an oligomethine chain to form a cyclic structure, and l represents 0, 1 or 2 . ]
The most stable three-dimensional structure in which the oligomethine chain including the oxo-oxoanion part is present on substantially the same plane, and two oxygen atoms constituting the oxo-oxoanion part are present on the same side with respect to the oligomethine chain. A potassium channel opener comprising a constituent compound or a physiologically acceptable salt thereof as an active ingredient. The potassium channel opener according to claim 1, wherein the compound is represented by the following formula (2).
[Wherein, A ¹ and A ² may be the same or different and each represents an optionally substituted cyclic hydrocarbon group or an optionally substituted heterocyclic group. ]   The potassium channel opener according to claim 2, wherein a distance between oxygen atoms of the oxo-oxoanion moiety in the most stable steric structure is 0.3 nm or more and 1 nm or less. The said compound is a potassium channel opener in any one of Claims 1-3 represented by following formula (3).
[Wherein, R ³ and R ⁴ may be the same or different and each represents an optionally substituted acyclic hydrocarbon group or an optionally substituted cyclic hydrocarbon group, and X ¹ and X ² may be the same or different and each represents a sulfur atom or an oxygen atom. ] 5. The potassium channel opener according to claim 4, wherein X ¹ is an oxygen atom, and 1 is 1 or 2. 6. Wherein X ¹ between the distance is less than 0.40 nm 0.67 nm, potassium channel openers according to claim 5. The potassium channel opener according to any one of claims 4 to 6, wherein the compound is any one selected from the following formula (4).
The said compound is a potassium channel opener in any one of Claims 1-3 represented by following formula (5).
[Wherein R ⁵ may be the same or different and each may be a non-cyclic hydrocarbon group that may be substituted, a cyclic hydrocarbon group that may be substituted, or a heterocyclic ring that may be substituted; Represents a group. ] The potassium channel opener according to claim 8, wherein the compound is any one selected from the following formula (6).
  The potassium channel opener according to any one of claims 1 to 9, which is β subunit selective. A method for screening potassium channel openers, comprising:
It is represented by the following formula (1),
[Wherein, R ¹ and R ² may be the same or different, and may be an acyclic hydrocarbon group which may be substituted, a cyclic hydrocarbon group which may be substituted or a substituted group. Represents a heterocyclic group, and in the case of a cyclic hydrocarbon group and a heterocyclic group, it may be bonded to a carbon atom constituting an oligomethine chain to form a cyclic structure, and l represents 0, 1 or 2 . ]
The most stable three-dimensional structure in which the oligomethine chain including the oxo-oxoanion part is present on substantially the same plane, and two oxygen atoms constituting the oxo-oxoanion part are present on the same side with respect to the oligomethine chain. A screening method comprising an evaluation step of evaluating a potassium channel opening ratio of the test component using a constituent compound or a physiologically acceptable salt thereof as the test component. The screening method according to claim 11, wherein the evaluation step is a step of evaluating a potassium channel opening ratio based on an action of a large conductance Ca ²⁺ activated K ⁺ (BK) channel on a β1 subunit or a β4 subunit. . A screening method for a potassium channel opener, wherein a site identified by the amino acid sequence (LLFSFFWPTFLLTGGLLIIAMV) described in SEQ ID NO: 1 in the β1 subunit of a large conductance Ca ²⁺ -activated K ⁺ (BK) channel and a test component A screening method comprising a step of evaluating an interaction.