V-ATPase activity inhibitor, antibacterial agent, drug, antibacterial method and screening method

Abstract

The present invention provides a Na containing compound represented by formula (1) + Transportable V-ATPase activity inhibitor. #imgabs0#R1 represents: any one selected from hydroxyl, alkoxy and halogenated alkoxy groups bonded to the adjacent phenyl group via oxygen, or any one selected from dialkylamino, heterocyclic amine and carboxylic acid amide groups bonded to the adjacent phenyl group via nitrogen, or bromine, iodine, or a straight-chain hydrocarbon group. R2 represents hydrogen or halogenated alkoxy. Z1 is an aliphatic hydrocarbon group, an alicyclic hydrocarbon group, an aromatic hydrocarbon group or a heterocyclic group, and may have any substituent, and between Z1 and the adjacent phenyl group, there is a structure containing a double bond selected from the following formulas (1-1) to (1-3). * represents a bond to the adjacent phenyl group. #imgabs1#

Description

Technical Field

[0001] The present invention relates to a V-ATPase activity inhibitor, an antibacterial agent, a drug, an antibacterial method and a screening method, and in particular, to a V-ATPase activity inhibitor, an antibacterial agent, a drug, an antibacterial method and a screening method that can effectively inhibit V-ATPase present in microorganisms that cause diseases. Background Art

[0002] V-ATPase (V-type ATPase) exists in the membrane of eukaryotic organelles and is a supramolecular complex composed of a complex subunit structure. V-ATPase functions as a molecular motor for ion transport. The energy from the hydrolysis of ATP causes specific micro-subunits to rotate within the membrane while transporting ions between the inside and outside of the membrane. V-ATPase is also known to exist in prokaryotes (bacteria) and is sometimes referred to as A-ATPase.

[0003] V-ATPase is necessary for cells to reproduce under certain conditions. For example, in enterococci, V-ATPase has the function of hydrolyzing ATP and converting sodium ions (Na ＋ ) are transported outside the cell, so enterococci can also grow under high salt concentration and high pH conditions.

[0004] In addition, V-ATPase similar to that of enterococci exists in many pathogenic microorganisms and plays an important role in their growth under alkaline conditions. Therefore, compounds that inhibit the ion transport function of V-ATPase can be used as antibacterial agents for pathogenic bacteria that cause diseases and are highly expected as new therapeutic drugs.

[0005] Conventionally, various compounds are known as antimicrobial agents. For example, Patent Document 1 discloses that 2-substituted benzimidazole having a specific structure is used as an antimicrobial agent.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: U.S. Patent No. 5,942,532 (Claim 1, etc.) Summary of the invention

[0009] Technical problem to be solved by the invention

[0010] However, the antimicrobial agent of Patent Document 1 is an antimicrobial agent that exhibits antimicrobial action as a histidine protein kinase inhibitor of microorganisms, and is different from an antimicrobial agent that inhibits V-ATPase.

[0011] The object of the present invention is to provide a V-ATPase activity inhibitor that can selectively and effectively inhibit the growth of bacteria that cause diseases, etc., an antibacterial agent, a drug, and an antibacterial method containing the V-ATPase activity inhibitor. In addition, another object of the present invention is to provide a screening method for efficiently screening out such V-ATPase activity inhibitors.

[0012] Solutions to technical problems

[0013] The present inventors have conducted intensive research to solve the above problems and have found that a specific compound has an effect of inhibiting V-ATPase activity, thereby completing the present invention. The specific compound has an aromatic ring, an electron-donating group bonded to the aromatic ring, and an arbitrary substituent bonded to the aromatic ring and having a double bond.

[0014] That is, the present invention is a Na ＋ A transportable V-ATPase activity inhibitor, characterized in that it comprises a compound represented by formula (1):

[0015]

[0016] (Wherein, R1 represents: any one selected from hydroxyl, alkoxy having 1 to 10 carbon atoms and haloalkoxy having 1 to 3 carbon atoms, which is bonded to the adjacent phenyl group via oxygen, or any one selected from dialkylamino having 1 to 10 carbon atoms in each alkyl group, heterocyclic amine having 2 to 6 carbon atoms and carboxylic acid amide group which may have a substituent bonded to a carbon atom, which is bonded to the adjacent phenyl group via nitrogen, or bromine, iodine, or a straight-chain hydrocarbon group having 2 to 5 carbon atoms.

[0017] R2 represents hydrogen or a halogenated alkoxy group having 1 to 3 carbon atoms.

[0018] Z1 is an aliphatic hydrocarbon group having 5 to 20 carbon atoms, an alicyclic hydrocarbon group having 3 to 20 carbon atoms, an aromatic hydrocarbon group having 6 to 20 carbon atoms, or a heterocyclic group having 2 to 20 carbon atoms, any of which may have an arbitrary substituent, wherein the adjacent phenyl group has a structure containing a double bond selected from the following formulae (1-1) to (1-3). * represents a bond to the adjacent phenyl group.

[0019] .

[0020] In this case, the compound represented by the formula (1) is preferably a 2-phenylbenzimidazole derivative represented by the following formula (2):

[0021]

[0022] (wherein, R1 and R2 are as defined in formula (1), R3 and R4 represent: a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, or a halogen atom selected from fluorine, chlorine, bromine and iodine, and R3 and R4 may be the same or different).

[0023] In the above case, the V-ATPase activity inhibitor according to claim 1 is characterized in that the compound represented by the formula (1) is a (1,2,4-oxadiazol-3-yl)phenyl derivative represented by the following formula (7):

[0024]

[0025] (Among them, R7~R 14 represents: a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an amino group, an ether group, a sulfonyl group, a boron group, or a halogen atom selected from fluorine, chlorine, bromine and iodine, R7 to R 14 can be the same or different).

[0026] In the above case, it is preferred that the feature is that the V-ATPase activity is inhibited by binding to the intramembrane rotor ring (c-ring).

[0027] The compound of formula (1) is preferably (Z)-5-(2,4-bis(difluoromethoxy)benzylidene)-2-(cyclopentylamino)thiazol-4(5H)-one, (2E,4E,6E,8E)-N-(4-hydroxyphenyl)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexyl-1-en-1-yl)nona-2,4,6,8-tetraenamide, 5-(4-(nonyloxy)benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione, N,N-dimethyl-4-(6-methyl-1H-benzo[d]imidazol-2-yl)aniline, or N-(4-(5-((o-tolyloxy)methyl)-1,2,4-oxadiazol-3-yl)phenyl)thiophene-2-carboxamide.

[0028] The present invention is an antibacterial agent for bacteria having V-ATPase, characterized by comprising any one of the V-ATPase activity inhibitors described above.

[0029] Furthermore, the present invention is a medicine characterized by comprising the above-mentioned antibacterial agent.

[0030] The present invention is a bacterial population regulator for selectively reducing bacteria having V-ATPase in a bacterial population including bacteria having V-ATPase and bacteria not having V-ATPase, characterized in that the agent comprises the V-ATPase activity inhibitor according to any one of claims 1 to 5.

[0031] Furthermore, the present invention is a medicine characterized by comprising the above-mentioned bacterial flora regulator.

[0032] In addition, the present invention is an antibacterial method, which is an antibacterial method for inhibiting the reproduction of bacteria using the above-mentioned antibacterial agent, and is characterized in that it includes: a step of adding the V-ATPase activity inhibitor to bacteria having V-ATPase; and a step of allowing the V-ATPase activity inhibitor to bind to the rotor ring (c-ring) within the membrane of the V-ATPase of the bacteria to inhibit the activity of the V-ATPase.

[0033] In addition, the present invention is a screening method for selecting Na from test compounds. ＋ A candidate compound for a transportable V-ATPase activity inhibitor, characterized in that it includes: an untreated evaluation step, for bacteria having V-ATPase, evaluating the survival state of the bacteria at a first pH and a second pH that is more alkaline than the first pH when the test compound does not contact the bacteria; a treated evaluation step, after the test compound is contacted with the bacteria, evaluating the survival state of the bacteria at the first pH and the second pH; a candidate compound determination step, comparing the survival states of the untreated evaluation step and the treated evaluation step, and determining the test compound that produces a difference as a candidate compound for an antibacterial agent for the bacteria.

[0034] In addition, the present invention is an antibacterial drug, the active ingredient of which is a compound having V-ATPase inhibitory activity.

[0035] In addition, the present invention is an antibacterial method, characterized in that it includes: the steps of administering a compound having V-ATPase inhibitory activity to bacteria having V-ATPase; and the steps of allowing the V-ATPase activity inhibitor to bind to the V-ATPase of the bacteria to inhibit the activity of the V-ATPase.

[0036] Effects of the Invention

[0037] According to the present invention, a V-ATPase activity inhibitor that can selectively and efficiently inhibit the growth of bacteria that cause diseases, etc., an antibacterial agent, a drug, and an antibacterial method containing the V-ATPase activity inhibitor can be provided. In addition, according to the present invention, a screening method for efficiently screening such a V-ATPase activity inhibitor can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1It is a schematic diagram showing the overall structure of V-ATPase and its expression under specific circumstances.

[0039] Figure 2 It is a graph showing the inhibition rate of V-ATPase activity of candidate compounds and the compound concentration.

[0040] Figure 3 This is a graph showing the results of a growth experiment on enterococci of candidate compounds.

[0041] Figure 4 This is a three-dimensional structural diagram showing a state where a V-ATPase activity inhibitor is bound to the c-ring of V-ATPase.

[0042] Figure 5 This is a diagram showing the results of X-ray crystal structure analysis of the compound of Example 4 and the c-ring of V-ATPase.

[0043] Figure 6 This is a graph showing the results of a pathogen growth inhibition test using the compound of Example 4.

[0044] Figure 7 This is a graph showing the results of a pathogen growth inhibition test using the compound of Example 4.

[0045] Figure 8 This is a graph showing the results of evaluating the effect of the compound of Example 4 on the intestinal environment.

[0046] Fig. 9 This is a graph showing the results of measuring the intestinal pH of mice fed with a high-protein diet and antibiotics.

[0047] Fig.10 This is a graph showing the results of measuring the number of VRE bacteria in VRE-infected mice to which the compound of Example 4 was administered. DETAILED DESCRIPTION

[0048] 1. V-ATPase activity inhibitor

[0049] The V-ATPase activity inhibitor of the present invention is Na ＋ The transportable V-ATPase activity inhibitor comprises a compound represented by the following formula (1):

[0050]

[0051] (wherein R1 represents: any one selected from hydroxyl, alkoxy having 1 to 10 carbon atoms and haloalkoxy having 1 to 3 carbon atoms, which is bonded to the adjacent phenyl group via oxygen; or any one selected from dialkylamino having 1 to 10 carbon atoms in each alkyl group, heterocyclic amine having 2 to 6 carbon atoms and carboxylic acid amide having a substituent bonded to a carbon atom, which is bonded to the adjacent phenyl group via nitrogen; or bromine (Br), iodine (I), or a straight-chain hydrocarbon group having 2 to 5 carbon atoms.

[0052] R2 represents hydrogen or a halogenated alkoxy group having 1 to 3 carbon atoms.

[0053] Z1 is: an aliphatic hydrocarbon group having 5 to 20 carbon atoms, an alicyclic hydrocarbon group having 3 to 20 carbon atoms, an aromatic hydrocarbon group having 6 to 20 carbon atoms, or a heterocyclic group having 2 to 20 carbon atoms, any of which may have any substituent, wherein the structure containing a double bond selected from the following formulae (1-1) to (1-3) is present between the adjacent phenyl group. * represents a bond to the adjacent phenyl group.

[0054] .

[0055] In R1, a hydroxyl group is a substituent represented by "-OH". An alkoxy group having 1 to 10 carbon atoms is represented by "-O-R 1a 」(R 1a represents an alkyl group having 1 to 10 carbon atoms). In addition, a halogenated alkoxy group having 1 to 3 carbon atoms in R1 is represented by "-O-R 1b -X n1 」(R 1b represents an alkyl group having 1 to 3 carbon atoms, X represents a halogen atom selected from fluorine, chlorine, bromine, and iodine. And n1 = 1 to 3. ) represents a substituent. The dialkylamino group having 1 to 10 carbon atoms in each alkyl group is represented by "-N(R 1c )(R 1d )」(R 1c represents an alkyl group having 1 to 10 carbon atoms, R 1d represents an alkyl group having 1 to 10 carbon atoms, R 1c With R 1d The heterocyclic amines having 2 to 6 carbon atoms are represented by "-N-(CH2) n2 A carboxylic acid amide group is a substituent represented by "-NH-C(=O)-". Bromine is a substituent represented by "-Br", and iodine is a substituent represented by "-I". A straight-chain hydrocarbon group with 2 to 5 carbon atoms is represented by "-(CH2) n3 -CH3" (n3 = 1 to 4).

[0056] In R2, hydrogen is a substituent represented by "-H". A halogenated alkoxy group having 1 to 3 carbon atoms is represented by "-O-R 2a -X n4 」(R 2a represents an alkyl group having 1 to 3 carbon atoms, X represents a halogen atom selected from fluorine, chlorine, bromine and iodine. And n4=1 to 3. ) represents a substituent. R2, in the phenyl group, may be located at the ortho position or the meta position of Z1.

[0057] In Z1, the aliphatic hydrocarbon group having 5 to 20 carbon atoms is a substituent selected from alkanes, alkenes, alkynes, dienes, conjugated dienes and the like.

[0058] Examples of the alkane having 5 to 20 carbon atoms include pentane, hexane, heptane, octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, nonadecane, and eicosane.

[0059] Examples of the olefins having 5 to 20 carbon atoms include 1-pentene, 2-pentene, 1-hexene, 2-hexene, 3-hexene, 1-heptene, 2-heptene, 3-heptene, 4-heptene, 1-octene, 2-octene, 3-octene, 4-octene, 5-octene, 1-nonene, 2-nonene, 3-nonene, 4-nonene, 5-nonene, 1-decene, 2-decene, 3-decene, 4-decene, 5-decene and 6-decene.

[0060] Examples of the alkyne having 5 to 20 carbon atoms include 1-pentyne, 2-pentyne, 1-hexyne, 2-hexyne, 3-hexyne, 1-heptyne, 2-heptyne, 3-heptyne, 4-heptyne, 1-octyne, 2-octyne, 3-octyne, 4-octyne, 5-octyne, 1-nonyne, 2-nonyne, 3-nonyne, 4-nonyne, 5-nonyne, 1-decene, 2-decene, 3-decene, 4-decene, 5-decene, and 6-decene.

[0061] Examples of the diene having 5 to 20 carbon atoms include 1,3-pentadiene, 2,4-heptadiene, 1,4-pentadiene, 1,7-octadiene, 2,5-octadiene, and 2,6-octadiene.

[0062] Examples of the conjugated olefin having 5 to 20 carbon atoms include 1,3,5-heptatriene, 1,3,7-octatriene, 2,4,6-octatriene, 1,2,7,8-nonatetraene, 1,3,6,8-nonatetraene, 2,4,6,8-nonatetraene, 1,2,8,9-decatetraene, and 2,4,6,8-decatetraene.

[0063] Examples of the alicyclic hydrocarbon group having 3 to 20 carbon atoms include substituents selected from cycloalkanes, cycloalkenes, cycloalkynes, etc. Examples of the alicyclic hydrocarbon group having 3 to 20 carbon atoms include cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclopropene, cyclobutene, cyclopropene, cyclohexene, cycloheptene, cyclooctene, and cyclooctyne.

[0064] Examples of the aromatic hydrocarbon group having 6 to 20 carbon atoms include benzene, naphthalene, anthracene and the like.

[0065] The heterocyclic group having 2 to 20 carbon atoms is a substituent having 2 to 20 carbon atoms and containing one or more elements selected from nitrogen, oxygen and sulfur in the ring structure. Examples of the heterocyclic group having 2 to 20 carbon atoms include aziridine, oxirane, thiirane, 1H-aziridine, 2H-aziridine, oxirane, thiirene, azetidine, oxetane, thiirane, azetadiene, azolidine, oxolane, thiolane, azole, oxole, thiol, azinane, oxane, thiane, pyridine, azepane, azinane, oxane, thiane, pyridine, azepane, thiol ... zepane), oxepane, thiepane, azepine, oxepin, thiepine, imidazole, pyrazole, oxazole, thiazole, imidazoline, dioxane, morpholine, thiazine, triazole, tetrazole, dioxolane, pyridazine, pyrimidine, pyrazine, indole, isoindole, benzimidazole, purine, benzotriazole, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline, pteridine, chromene, isochromene, anthracene, acridine, xanthene, carbazole, oxadiazole, benzo-C-cinnoline (en), tetracene, etc.

[0066] As Z1, in addition to the above-mentioned substituents, one or more alkyl groups, alkenyl groups, alkynyl groups, aryl groups, oxy groups, halogen atoms, hydroxyl groups, nitro groups, sulfone groups, ether groups, thiol groups, ester groups, carbonate groups, carbonyl groups, amide groups, amino groups, azido groups, carbamate groups, cyano groups, hydroxyl groups, carboxyl groups, sulfonate groups, sultone groups, lactone rings, lactam rings, etc. may be bonded to these substituents.

[0067] Z1 preferably contains a ring structure in the molecule. The ring structure is preferably a group selected from the above-mentioned alicyclic hydrocarbon groups with 3 to 20 carbon atoms, aromatic hydrocarbon groups with 6 to 20 carbon atoms, and heterocyclic groups with 2 to 20 carbon atoms. In Z1, there may be only one ring structure or two or more ring structures. The ring structure more preferably contains a structure including a double bond of the above-mentioned formulas (1-1) to (1-3) in the molecule.

[0068] In particular, the compound represented by the above formula (1) is preferably a 2-phenylbenzimidazole derivative represented by the following formula (2):

[0069]

[0070] (Wherein, R1 and R2 are as defined in formula (1), R3 and R4 represent: a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, or a halogen atom selected from fluorine, chlorine, bromine and iodine, and R3 and R4 may be the same or different.).

[0071] Examples of the 2-phenylbenzimidazole derivatives of the formula (2) include compounds represented by the following formulas (V-161, V-161-01 to V-161-04, V-161-10):

[0072] .

[0073] Among them, formula V-161 (N,N-dimethyl-4-(6-methyl-1H-benzo[d]imidazol-2-yl)aniline) is preferred due to its high effect of inhibiting V-ATPase activity.

[0074] Examples of the compound represented by the formula (1) include (1,2,4-oxadiazol-3-yl)phenyl derivatives represented by the following formula (6):

[0075]

[0076] (wherein, R1 and R2 are as defined in formula (1). R6 represents an aromatic hydrocarbon group having 6 to 20 carbon atoms, which may be substituted by an alkyl group having 1 to 4 carbon atoms.)

[0077] In particular, the compound represented by formula (6) is preferably a (1,2,4-oxadiazol-3-yl)phenyl derivative represented by the following formula (7):

[0078]

[0079] (Among them, R7~R 14 represents: a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an amino group, an ether group, a sulfonyl group, a boron group, or a halogen atom selected from fluorine, chlorine, bromine and iodine, R7 to R 14 They can be the same or different from each other. ).

[0080] Specific examples of the compound represented by the above formula (7) include compounds represented by the following formulae (V-234) and (V-234-01) to (V-234-03):

[0081] .

[0082] Examples of the compound represented by the above formula (1) include compounds represented by the following formula (3):

[0083]

[0084] (Wherein, R1 and R2 are as defined in formula (1).)

[0085] In addition, examples of the compound represented by the above formula (1) include compounds represented by the following formula (4):

[0086]

[0087] (Wherein, R1 and R2 are as defined in formula (1).)

[0088] In addition, examples of the compound represented by the above formula (1) include compounds represented by the following formula (5):

[0089]

[0090] (wherein, R1 and R2 are as defined in formula (1). R5 represents an alicyclic hydrocarbon group having 3 to 20 carbon atoms.)

[0091] Examples of the compound of the above formula (1) include the following compounds:

[0092] .

[0093] The names of these compounds are as follows.

[0094] ・V-6: (Z)-5-(2,4-bis(difluoromethoxy)benzylidene)-2-(cyclopentylamino)thiazol-4(5H)-one

[0095] ・V-84: (2E,4E,6E,8E)-N-(4-hydroxyphenyl)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexyl-1-en-1-yl)nona-2,4,6,8-tetraenamide

[0096] ・V-130: 5-(4-(nonyloxy)benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione

[0097] ・V-161: N,N-dimethyl-4-(6-methyl-1H-benzo[d]imidazol-2-yl)aniline

[0098] ・V-234: N-(4-(5-((o-tolyloxy)methyl)-1,2,4-oxadiazol-3-yl)phenyl)thiophene-2-carboxamide

[0099] Among them, V-6, V-161, and V-234 are particularly preferred from the viewpoint of having little non-specific binding other than binding to V-ATPase.

[0100] 2. Principle of V-ATPase activity inhibition

[0101] Next, V-ATPase will be described. Figure 1 Schematic diagram of V-ATPase of Enterococcus hirae (E. hirae), an intestinal streptococcus, showing Na ＋ Transport V-ATPase. Figure 1 As shown in (a), V-ATPase is a complex composed of multiple subunits, with a hydrophilic catalyst head (V1 part) and a sodium ion (Na ＋ ) The membrane-intrinsic portion of the transport (V o The V1 part is the ATP-driven motor part. Under the action of the A3B3 subunit, ATP is hydrolyzed, causing the V1 part to rotate. o The V-ATPase part uses rotational motion to transport ions. It contains a membrane-internal rotor ring (c-ring) composed of a decamer of c subunits. The c-ring has ion binding sites and is involved in the transport of ions from inside to outside the membrane. V-ATPase hydrolyzes ATP through the V1 part and uses the energy to make V o Part of the c-ring rotates, thereby expelling the sodium ions in the membrane to the outside.

[0102] Figure 1 (b) Figure 1 (c) Schematic diagram showing the state in which the pH of the external environment causes changes in the activity of V-ATPase in E. hirae. Figure 1 As shown in (b), near neutral pH (pH ≤ 7.0), V-ATPase is not expressed. Figure 1 As shown in (c), at alkaline pH (pH > 7.0) and high salt concentration, V-ATPase is expressed and the sodium ions in the membrane are expelled to the outside of the membrane. ＋ It can be kept low and can grow in alkaline and high salt concentration environments.

[0103] The V-ATPase activity inhibitor of the present invention has the function of binding to V-ATPase and inhibiting its activity. In particular, the V-ATPase activity inhibitor of the present invention has the function of inhibiting the transport of ions by binding to the c-ring of V-ATPase and inhibiting its rotation. The activity inhibition principle of the V-ATPase activity inhibitor is described below.

[0104] The V-ATPase activity inhibitor of the present invention is a compound represented by formula (1). As shown in the following formula, the compound comprises a phenyl group and an arbitrary substituent, and has an electron donating group at the para position of the phenyl group relative to the position where the arbitrary substituent is bonded.

[0105]

[0106] In addition, for the V-ATPase activity inhibitors exemplified above, figures illustrating the structures thereof are shown. As shown in these figures, any substituent Z1 and the phenyl group are connected via a double bond-containing structure of formula (1-1) to (1-3). In addition, Z1 has a ring structure.

[0107]

[0108] The phenyl group having an electron-donating group, together with an arbitrary substituent, binds to a specific site on the periphery of the c-ring of V-ATPase and is sandwiched between the c-ring and the a-subunit, thereby inhibiting the rotation of the c-ring. The phenyl group and the arbitrary substituent are hydrophobic and bind to the c-ring through hydrophobic interactions with amino acid residues having hydrophobic side chains on the periphery of the c-ring. In addition, it is believed that the electron density of the π electrons of the benzene ring increases due to the electron-donating group bound to the para position of the phenyl group, so that the benzene ring is activated, which contributes to the binding affinity of the V-ATPase activity inhibitor to the c-ring. For example, in the compound "V-161" described in the Examples described below, it interacts with the amino acid residues around the 52nd phenylalanine on the c-ring to ensure the binding ability of the inhibitor.

[0109] It is speculated that the hydrogen atoms provided by the NH group or the like of the arbitrary substituent and / or electron-donating group become donors, and the N atom, O atom, and S atom become acceptors, forming multiple hydrogen bonds with the acceptors and donors of the c-ring. Therefore, the arbitrary substituent and the electron-donating group contribute to improving the binding affinity between V-ATPase and the c-ring. The arbitrary substituent can be a relatively arbitrary structure as long as the molecular weight is appropriately large and it is hydrophobic. The substituent preferably has a ring structure. By having a hydrophobic ring structure and increasing the volume of the arbitrary substituent, the rotation inhibition effect of the c-ring of the V-ATPase activity inhibitor can be improved.

[0110] First, in order to bind to the c-ring of V-ATPase, a structure including a phenyl group and a double bond in a specific direction of the phenyl group is the minimum necessary basic structure. In particular, in the case where the double bond is adjacent to the phenyl group as in formula (1-2) and formula (1-3), it is believed that the double bond and the phenyl group resonate to fix the structure, and the π electrons of the resonance play an important role in the binding of V-ATPase to the c-ring.

[0111] It should be noted that the structure containing a double bond may be contained in a linker connecting a phenyl group and a ring structure in any substituent (e.g., V-84, etc.), or in a ring structure when there is no linker between the phenyl group and the ring structure (e.g., V-161). Due to such a structure, the V-ATPase activity inhibitor binds to the c-ring of V-ATPase and inhibits its rotation, thereby exerting an effect of inhibiting V-ATPase activity.

[0112] 3. Antimicrobial agents, antimicrobial methods, and drugs

[0113] The V-ATPase activity inhibitor of the present invention can be used as an antibacterial agent for microorganisms expressing V-ATPase because it inhibits the activity of V-ATPase as described above. The list of microorganisms expressing V-ATPase is shown in the following table. These microorganisms can be inhibited from having V-ATPase activity by the V-ATPase activity inhibitor of the present invention, and their reproduction can be expected to be inhibited. In addition, the related disease name indicates a disease that can be expected to be treated or prevented by inhibiting the reproduction of microorganisms.

[0114]

Table 1

[0115]

[0116] "Bacteria" in the table indicates the name of the microorganism, and "Related disease name" indicates an example of a disease related to the microorganism. In addition, "V-ATPase ring identification (%)" in the table indicates the percentage of identity between the c-ring of the V-ATPase of the microorganism and the c-ring of the V-ATPase of enterococci. The inventors, for enterococcal V-ATPase, confirmed in the examples described below that the V-ATPase activity inhibitor binds to the c-ring of the V-ATPase and shows the growth inhibitory effect on enterococci. Therefore, microorganisms having V-ATPase that have a high identity with the c-ring of the V-ATPase of enterococci can be expected to exert the growth inhibitory effect of the microorganism through the V-ATPase activity inhibitor of the present invention in the same way as enterococci. On the other hand, although the V-ATPase activity inhibitor interacts with the outer peripheral surface of the c-ring as described above, the inventors found that the amino acid sequence on the outer peripheral surface of the c-ring of V-ATPase is highly conserved in a variety of microorganisms. Therefore, it is considered that V-ATPase activity inhibitors are not necessarily ineffective even for microorganisms with low similarity to the c-ring of V-ATPase of enterococci, and V-ATPase activity inhibitors may also have a growth-inhibiting effect on microorganisms.

[0117] Since the V-ATPase activity inhibitor of the present invention is a novel antibacterial agent, it is expected to be effective against drug-resistant bacteria that are resistant to existing antibacterial agents. For example, it is useful in the case of enterococci (VRE) such as E. faecalis (Enterococcus faecalis) and E. faecium (Enterococcus faecium) that are resistant to vancomycin and the like.

[0118] The V-ATPase activity inhibitor of the present invention can be used as a bacterial population regulator for selectively reducing bacteria having V-ATPase in a bacterial population including bacteria having V-ATPase and bacteria not having V-ATPase.

[0119] For example, Clostridium perfringens and the like are pathogenic bacteria called bad bacteria that corrupt proteins in the intestine and produce harmful amines and carcinogenic substances. These microorganisms associated with the deterioration of the intestinal environment usually have V-ATPase and thrive in an alkaline environment. On the other hand, Bifidobacterium (Bifidobacterium genus) and lactic acid bacteria (Lactobacillus genus) are called probiotics, which ferment dietary fiber in the intestine and produce short-chain fatty acids such as butyric acid and acetic acid that are important for maintaining host health, and can inhibit the growth of bad bacteria and diseases related to them. These microorganisms that have the effect of improving the intestinal environment usually do not have V-ATPase. Therefore, the V-ATPase activity inhibitor of the present invention does not have an effect on the above-mentioned probiotics but only has an effect on bad bacteria, and thus selectively inhibits the growth of only bad bacteria with V-ATPase, so the intestine can be improved to an environment where probiotics can easily thrive.

[0120] It should be noted that VRE infection, enteritis caused by Clostridium difficile, etc. are usually caused by the disturbance of intestinal bacterial flora caused by the use of antibiotics. Abnormalities in the immune system and intestinal bacterial flora in the intestine may induce autoimmune diseases. The deterioration of the intestinal environment is related to intestinal cancer and liver cancer, and lifestyle diseases such as obesity and diabetes are also closely related to intestinal bacterial flora. Therefore, the V-ATPase activity inhibitor of the present invention can be used as a drug to regulate the balance of good bacteria and bad bacteria in the intestinal bacterial flora and prevent and treat various diseases.

[0121] The V-ATPase activity inhibitor (antibacterial agent, bacterial flora regulator) of the present invention can be used as an active ingredient in drugs, pesticides and other agents, and is particularly preferably used for pharmaceutical purposes. Examples of the types of drugs include tablets, capsules, pills, powders, granules, fine particles, gels, liquids and the like.

[0122] The concentration of the V-ATPase activity inhibitor contained in the drug can be appropriately determined, and can be, for example, in the range of 1 μM to 100 mM, preferably in the range of 100 μM to 10 mM. The V-ATPase activity inhibitor can be used by dissolving in a solvent such as water, or can also be used in a powdered state.

[0123] The above-mentioned drugs, in addition to the V-ATPase activity inhibitor (antibacterial agent, bacterial flora regulator) of the present invention, may contain additives such as solvents, excipients, binders, disintegrants, lubricants, stabilizers, and suspending agents within the range that does not impair the effects of the present invention. Examples of solvents for preparations include water, ethanol, glycerol, and the like. Examples of excipients include lactose, sucrose, glucose, mannitol, sorbitol, corn starch, potato starch, α-starch, dextrin, carboxymethyl starch, crystalline cellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, gum arabic, dextran, pullulan, silicates, calcium phosphate, calcium carbonate, and calcium sulfate. Examples of silicates include light silicic anhydride, synthetic aluminum silicate, and magnesium aluminosilicate. Examples of binders include gelatin, polyvinyl pyrrolidone, and polyethylene glycol. Examples of disintegrants include crosslinked carboxymethyl cellulose sodium, carboxymethyl starch sodium, and crosslinked polyvinyl pyrrolidone. Examples of lubricants include talc, stearic acid, calcium stearate, magnesium stearate, colloidal silicon dioxide, propolis, pearl wax, spermaceti, boric acid, ethylene glycol, fumaric acid, adipic acid, sodium benzoate, sodium sulfate, leucine, sodium lauryl sulfate, magnesium lauryl sulfate, silicic anhydride, silicic acid hydrate, etc. Examples of stabilizers include methyl paraben, propyl paraben, chlorobutanol, benzyl alcohol, phenylethyl alcohol, benzalkonium chloride, phenol, cresol, thimerosal, acetic anhydride, sorbic acid, etc. Examples of suspending agents include polysorbate 80, sodium carboxymethylcellulose, etc.

[0124] 4. Screening method

[0125] Next, Na was selected from the test compounds. ＋ The screening method of the present invention comprises the following steps: (a) a primary screening step and (b) a secondary screening step.

[0126] (a) Primary screening step

[0127] In a primary screening step, only compounds showing ATPase inhibitory activity are selected from a large number of compounds. First, test compounds having V-ATPase inhibitory activity are selected from a compound library. As a compound library, a compound database stored in a university or the like can be used.

[0128] Next, for each selected compound, V-ATPase is reacted with the compound and the ATPase activity is measured to evaluate whether the compound inhibits ATPase. ATPase activity can be measured using the molybdenum blue method, in which the absorbance of inorganic phosphate produced by the hydrolysis of ATP is measured. The reaction conditions can be the conditions used to measure ATPase activity. As a buffer used in the reaction, Tris-HCl buffer can be used. The reaction time is usually 10 to 60 minutes, but it can be appropriately adjusted according to the temperature, etc. The reaction of V-ATPase is stopped by adding a surfactant such as SDS, and after adding a color developing solution (ferrous sulfate, etc.) for color development, the absorbance at 650nm is measured to calculate the concentration of inorganic phosphate.

[0129] The inhibition rate of the V-ATPase reaction can be calculated from the calculated inorganic phosphate concentration. That is, a graph is prepared with the inhibition rate on the vertical axis and the concentration of the compound on the horizontal axis, and the concentration at which the inhibition rate is 50% (IC 50 ). To reach IC 50 The lower the concentration of the compound, the higher the V-ATPase inhibitory effect is obtained with a small amount, and thus it becomes a candidate compound for V-ATPase activity inhibitor. In the examples described below, the IC 50 Compounds with concentrations less than 10 μM (hereinafter referred to as “IC 50 <10μM". ) were used as candidate compounds.

[0130] (b) Secondary screening step

[0131] The secondary screening step is a step to confirm whether the compound screened in the primary screening step can inhibit the growth of microorganisms. The secondary screening step includes at least (b-1) an untreated evaluation step, (b-2) a treated evaluation step, and (b-3) a candidate compound identification step.

[0132] (b-1) Unprocessed evaluation steps

[0133] In the untreated evaluation step, for bacteria having V-ATPase, the survival state of the bacteria is evaluated at a first pH and a second pH that is more alkaline than the first pH, in a state where the compound does not contact the bacteria. V-ATPase is usually expressed in microorganisms that reproduce in an alkaline environment. Therefore, in this step, the microorganisms are grown in two environments, a specific pH (first pH) and a more alkaline pH (second pH), and their growth conditions are observed. In the case of enterococci, it is preferred that the first pH is appropriately selected within the range of pH ≤ 7.0, and the second pH is appropriately selected within the range of pH > 7.0. For example, the first pH is pH 7.0, the second pH is pH 8.0, etc.

[0134] The culture conditions (culture medium, culture temperature) of the microorganism in the untreated evaluation step can be appropriately determined according to the type of microorganism. For example, when the microorganism is enterococcus, it is preferably cultured in a liquid culture medium containing tryptone and yeast extract, and glucose and the like can be added as needed. A culture medium with different pH values ​​is used at the first pH and the second pH. In addition, the culture temperature of enterococcus is preferably 30 to 37°C.

[0135] The growth state of microorganisms can be evaluated by observing the number of microorganisms. The number of microorganisms can be observed by measuring the absorbance of a culture medium in which the microorganisms are cultured, or by counting the number of microorganisms by culturing the microorganisms in a dish or the like. In the case of enterococci, the absorbance at a wavelength of 600 nm (OD 600 ), which can assess fertility status.

[0136] (b-2) Processed evaluation steps:

[0137] Next, after the test compound is brought into contact with the bacteria, the survival state of the bacteria is evaluated at a specific pH (first pH) and an alkaline pH (second pH) in the same manner as in (b-1). The culture of the microorganisms and the evaluation of the growth state are performed using the same conditions and methods as in (b-1).

[0138] (b-3) Candidate compound specific steps:

[0139] Next, the survival state of the microorganisms in the (a) untreated evaluation step and the (b) treated evaluation step are compared, and the test compound that shows a difference is identified as a candidate compound for an antibacterial agent for bacteria. If the (b) treated suppresses the growth of the microorganism more than the (a) untreated at a pH more alkaline than the specific pH, the test compound is judged to have the possibility of inhibiting V-ATPase activity and is selected as a candidate compound for a V-ATPase activity inhibitor. On the other hand, if the growth is also suppressed at a specific pH, or if the treated case (a) grows more when compared with the untreated case (b) at a pH more alkaline than the specific pH, or if the growth of the two cases is roughly the same, the test compound can be judged to have no effect on V-ATPase activity and is excluded from the candidate compound for a V-ATPase activity inhibitor.

[0140] Example

[0141] Hereinafter, the present invention will be specifically described based on examples, but these examples do not limit the content of the present invention in any way. In addition, in the following examples, "%" represents a mass basis (mass percentage) unless otherwise specified.

[0142] (1) Preparation of compounds and V-ATPase

[0143] (a) Obtaining compounds

[0144] Compounds that are candidates for ATPase activity inhibitors were obtained from the compound library of the Institute for Pharmaceutical Research, University of Tokyo. There were approximately 70,000 compounds obtained, and all of them were screened once by measuring ATPase activity as described below.

[0145] (b) Regulation of V-ATPase

[0146] V-ATPase was prepared according to the method described in the following paper.

[0147] Paper 1: Takeshi Murata, et. al, “Purification and Reconstitution of Na ＋ -translocatingVacuolar ATPase from Enterococcus hirae", J. Biol. Chem. 272, 24885-24890 (1997)

[0148] Paper 2: Takeshi Murata, et. al, "Torque Generation of Enterococcus hiraeV-ATPase", J. Biol. Chem. 289, 31212-31223 (2014)

[0149] (2) ATPase activity measurement (primary screening)

[0150] ATPase activity was measured by the molybdenum blue method, in which inorganic phosphate produced by the hydrolysis of ATP is quantified by absorbance measurement. The measurement was performed using a 96-well plate, and 0.05% DDM (n-dodecyl-β-D-maltoside) and 10μg / mL V-ATPase (purified from enterococci or recombinant Escherichia coli) and 20μM of the compound were added to the buffer (100mM Tris-HCl pH8.5, 100mM NaCl, 5mM MgSO4, 10% glycerol). ATP was added to 5mM, the total volume was 100μL, and the reaction was started while stirring with a stirrer for 30 minutes. ATPase activity varies greatly depending on the reaction temperature, so the reaction time was adjusted within the range of 10 minutes to 60 minutes depending on the temperature on the day of the experiment. The reaction was stopped by adding 50 μL of 20% SDS to denature V-ATPase, and 75 μL of a color developing solution (5% ferrous sulfate, 1.6% ammonium molybdate, 1M sulfuric acid) was added to develop color. The absorbance at 650 nm was immediately measured using a plate reader. The activity of V-ATPase was measured by determining the concentration of inorganic phosphate using a standard curve prepared by reacting four KH2PO4 aqueous solutions of 0.2 mM, 0.5 mM, 1.0 mM, and 1.5 mM with MQ (ultrapure water). Here, for compounds that inhibited activity by more than 50%, the concentrations were diluted in stages to 10 μM, 5 μM, 2 μM, 1 μM, 500 nM, 200 nM, 100 nM, and 50 nM, and the measurement was performed in the same manner as above.

[0151] (3) Primary screening results

[0152] Based on the results of the above ATPase activity measurement, the activity without adding the compound was recorded as 100, and the inhibition rate was calculated based on the decrease in activity compared with 100. That is, a graph was prepared with the inhibition rate on the vertical axis and the concentration of the compound on the horizontal axis ( Figure 2). According to the graph, calculate the concentration of the compound that produces a 50% inhibition rate (IC 50 ). As a result, IC 50 Below 10 μM (IC 50 There are 280 compounds with IC 50 Less than 100nM (IC 50 There are 12 compounds with IC 50 280 compounds with a concentration of <10 μM were tested for their effects on human HeLa cells. As a result, 24 compounds showed toxicity, so the remaining 256 compounds were selected as candidate inhibitors. From these 256 compounds, 24 compounds were selected based on their high activity and diversity. The structural formulas of these 24 compounds are shown below.

[0153]

[0154]

[0155]

[0156]

[0157] For the above 24 compounds, secondary screening was performed through the growth test of enterococci described below.

[0158] (4) Growth test of enterococci (secondary screening)

[0159] Enterococcus hirae was cultured using a buffer solution based on 2% tryptone and 1% yeast extract and adjusted for Na ＋ When glucose is added, add a 50% glucose solution, and when using culture medium, add 1% glucose. Use a 96-well plate to culture a total of 200 μL, and measure the OD every 1 hour using a plate reader 600 . Using culture media at pH 7.0 and pH 8.5, the effect of pH on growth was observed. In bacterial culture, the decomposition of sugars necessary for growth usually causes a large change in pH. In this experiment, it is very important to maintain a constant pH, so the pH was kept constant by adding buffers suitable for various pH values. Since V-ATPase is only expressed under alkaline conditions, the growth inhibition observed by adding inhibitors depends largely on pH. In addition, the inhibitors have no effect on the growth of bacteria that do not have V-ATPase.

[0160] (5) Secondary screening results

[0161] In the table below, the IC values ​​of the above 24 compounds are recorded. 50 Etc. It should be noted that the "+" in the "Specificity" column in the table below indicates that there is no growth inhibition at pH 7.0 and there is growth inhibition at pH 8.5, "-" indicates that there is no growth inhibition in either case, and "na" indicates that there is growth inhibition in either case. Specifically, for "+", a case where the growth inhibition rate at pH 7.0 is 20% or less is judged as no growth inhibition. In addition, for "-", a case where the growth inhibition rate at either pH 7.0 or pH 8.5 is 20% or less is judged as no growth inhibition. In addition, for "na", a case where the growth inhibition rate at pH 7.0 is 60% or more is judged as growth inhibition. In addition, for compounds for which no clear inhibition was observed, the growth inhibition rate is also recorded as a reference.

[0162]

Table 2

[0163]

[0164] In addition, a graph showing the reproductive results of the 14 species with a "+" "specificity" is shown ( Figure 3 ). The horizontal axis represents the candidate compound, and the vertical axis represents the number of bacteria measured by absorbance (%). The left end of the chart is a control group, the left side of each compound represents the results under the culture condition of pH 7.0, and the right side of the chart represents the results under the culture condition of pH 8.5. As shown in the chart, it can be seen that any candidate compound inhibits the growth of enterococci more at pH 8.5 than at pH 7.0.

[0165] Among the above 14 compounds, the one with the highest affinity for V-ATPase (IC 50 <=400), high growth inhibition effect (growth inhibition rate (%) >=70%) and no non-specific inhibition, are specifically compounds that show high inhibition efficiency of V-ATPase activity inhibitors. Specifically, they are Example 1 (V-6), Example 4 (V-161), and Example 5 (V-234). In particular, according to IC 50 The results of the inhibition of growth and fertility showed that the compound of Example 4 (V-161) (2-phenylbenzimidazole derivative) was the most useful candidate compound. 50 <=400) and had a high growth inhibition effect (growth inhibition rate (%) >=70%), but two compounds (Example 2 (V-84) and Example 3 (V-130)) that had non-specific inhibition were also identified as candidate compounds.

[0166] (6) Eucrystallization and X-ray crystal structure analysis

[0167] The compound of Example 4 (V-161) and the c-ring of V-ATPase were co-crystallized and subjected to X-ray crystal structure analysis. Using the methods of the above-mentioned Papers 1 and 2, the V-ATPase from Enterococcus was expressed in recombinant Escherichia coli, purified into a complex, and then the c-ring was isolated. The c-ring was adjusted to 2 mg / mL, and V-161 was added to 500 μM. Crystallization was carried out at 23°C under the conditions of 100 mM Tris-HCl pH 8.0, 220 mM sodium nitrate, and 32% PEG400. For the obtained crystals, an X-ray diffraction experiment was carried out using the beamline of the PhotonFactory (PF), which is a synchrotron radiation device. The diffraction image was analyzed at 2.3 The structure was determined at a resolution of Figure 4 as well as Figure 5 Shown in.

[0168] For the detailed method of X-ray crystal structure analysis, reference can be made to the following paper related to X-ray crystal structure analysis of the c-ring of Enterococcus V-ATPase.

[0169] Paper 3: Takeshi Murata, et. al, “Structure of the Rotor of the V-Type Na ＋ -ATPase from Enterococcus hirae" Science 308, 654-659 (2005)

[0170] like Figure 4 As shown in (a), the ribbon represents the helix of the c-ring, the surrounding sticks represent lipid molecules and surfactant molecules, and the particles in the center represent sodium ions. Figure 4 (b) is the enlarged display Figure 4 (a) is a diagram of the area enclosed by a box. Figure 5 The electron density map shows the overall structure of the c-ring on the left, and the structures of the binding positions of V-161 are gradually magnified in the center and on the right. From these structures, it can be seen that V-161 interacts with the surrounding amino acid residues including the 52nd phenylalanine of the c-ring and binds to its outer surface.

[0171] (7) Compounds of 2-phenylbenzimidazole derivatives

[0172] Other 2-phenylbenzimidazole derivatives having the same skeleton as V-161 were synthesized and developed. The following 10 compounds are (V-161-01) to (V-161-10). The structural formulas of the compounds are shown below.

[0173]

[0174] The inhibitory effect of ATPase activity was evaluated for these compounds using the same method as in "(2) Measurement of ATPase activity". The results are shown in the table below. It should be noted that Reference Example 4-02 (V-161-06) had no specificity in activity due to pH (high activity at both pH 7.0 and pH 8.5), and Reference Example 4-05 (V-161-09) had no activity observed.

[0175]

Table 3

[0176]

[0177] Among these results, the one with the highest affinity for V-ATPase (IC 50 The five compounds of Example 4-01 (V-161-01) to Example 4-04 (V-161-04) and Example 4-05 (V-161-10) which had high inhibitory effects (inhibition rate (%) >= 70%) and no non-specific binding were specifically identified as V-ATPase activity inhibitors with high inhibitory effects. On the other hand, Reference Example 4-04 (V-161-08) and Reference Example 4-05 (V-161-09) did not show any inhibitory effects on ATPase activity.

[0178] (8) Confirmation of antibacterial effect against pathogenic bacteria

[0179] For pathogenic bacteria having a V-ATPase similar to that of Enterococcus hirae, confirm whether V161 shows growth inhibition in the same manner as in the above "(4) Growth test of Enterococci". As pathogenic bacteria, vancomycin-resistant enterococci (VRE) Enterococcus faecalis, Enterococcus faecium, and Clostridium difficile, a multidrug-resistant bacterium associated with deterioration of the intestinal environment, were prepared. VRE was cultured using culture media at pH 7.0, pH 7.5, and pH 8.0, and C. difficile was cultured using culture media at pH 6.5, pH 7.0, and pH 7.5, and OD was measured. 600 Confirm the difference in bacterial count. Figure 6 , Figure 7 Shown in.

[0180] Figure 6The results for vancomycin-resistant enterococci (VRE) are shown on the left, with data for E.faecalis and data for E.faecium on the right. The data for pH 7.0, pH 7.5, and pH 8.0 are shown from top to bottom. The figure shows that the growth of any pathogen is inhibited depending on pH, and V-161 has a specific growth inhibition activity under alkaline conditions that inhibits the activity of V-ATPase. It can also be confirmed that E.faecium is more strongly affected by V-161 than E.faecalis. Figure 7 This is the data of pathogenic Clostridium bacteria. From top to bottom, the data are pH 6.5, pH 7.0, and pH 7.5. This figure gives the following insights: C. difficile growth is also inhibited under neutral and acidic conditions, the pH range of V-ATPase expression in pathogenic Clostridium bacteria is wide, and V-161 also functions under neutral and acidic conditions.

[0181] (9) Confirmation of the conditions affecting the intestinal environment

[0182] Mice (C57BL / 6, ♀, 6 weeks old, weighing about 15g) were used to investigate the effect of V-161 on the intestinal environment. SoybeanPC and sodium bicarbonate (メイロン) were used to suspend V-161 and make it 20mM. 0.5ml of the suspension was orally administered to 4 mice once a day. After 5 days, the mice were dissected and the feces and intestinal contents were collected to measure the intestinal pH and sodium ion concentration. The results showed that Figure 8 The upper half of the graph shows intestinal pH, and the lower half shows sodium ion concentration.

[0183] The results show that the small intestine is the most suitable place for V-ATPase expression and for V-161 to work. In addition, even when V-161 was fed in large quantities, no toxicity was observed in mice.

[0184] (10) Confirmation of the intestinal environment of mice (high-protein diet and antibiotic administration)

[0185] Under a high-protein diet, the so-called bad bacteria usually increase and the intestinal environment becomes disturbed. The administration of antibiotics usually causes a significant decrease in intestinal bacteria and is associated with the fixation and infection of pathogens. Therefore, verification was performed under these two models.

[0186] First, three mice were fed a high-protein diet (65% protein) and a normal diet (25% protein) respectively. In addition, a model was created in which mice were fed antibiotics (1 g / L ampicillin) through drinking water and a model was created in which mice were fed normal drinking water. All mice were dissected after one week, and the intestinal contents were collected and the pH was measured. The results were as follows: Fig. 9 The upper part of the figure shows the results of mice fed with a high-protein diet, and the lower part shows the results of mice fed with antibiotics.

[0187] In all models, it was observed that the pH slightly increased in the small intestine and significantly increased in the cecum and large intestine, and the normally acidic intestinal environment became alkaline. Based on these results, it can be inferred that in an aggravated intestinal environment, the pH tends to increase, and V-ATPase inhibitors are more likely to work than in normal situations.

[0188] (11) Experiments on mice infected with VRE

[0189] Mice treated with antibiotics were infected with E. faecium of VRE and fed with V-161. The experimental conditions, such as the amount of antibiotics and V-161 fed, were the same as those in "(9) Confirmation of the conditions of the intestinal environment" and "(10) Confirmation of the intestinal environment of mice (high-protein diet and antibiotic feeding)". Fig.10 The upper half of the figure shows the results for the small intestine, and the lower half shows the results for the cecum, large intestine, and feces. The vertical axis of the figure shows the number of live VRE bacteria.

[0190] As can be seen from the figure, the VRE in the small intestine was significantly reduced (97% reduction) when V-161 was added compared to the control group. In addition, the VRE in the cecum, large intestine, and feces was significantly reduced (about 50% reduction) when V-161 was added compared to the control group. In other words, it can be seen that V-161 has an inhibitory effect on the proliferation of VRE in the mouse intestine.

[0191] (12) (1,2,4-oxadiazol-3-yl)phenyl derivatives

[0192] Other (1,2,4-oxadiazol-3-yl)phenyl derivatives having the same skeleton as V-234 were synthesized and developed. The following three compounds are (V-234-01) to (V-161-03). The structural formulas of the compounds including V-234 are shown below.

[0193]

[0194] The inhibitory effect of these compounds on ATPase activity was evaluated by the same method as in "(2) Measurement of ATPase activity". The results are shown in the following table.

[0195]

Table 4

[0196]

[0197] Any of the compounds of Examples 5-01 to 5-03 has a high affinity for V-ATPase (IC 50 <=400), high fertility inhibition effect (fertility inhibition rate (%) >=70%), and no non-specific binding.

Claims

1. A V-ATPase activity inhibitor for Na from bacteria ＋ The transport V-ATPase is inhibited, characterized in that The present invention includes 2-phenylbenzimidazole derivatives represented by the following formula (2): wherein R1 represents: any one selected from hydroxyl, alkoxy with 1 to 10 carbon atoms, and haloalkoxy with 1 to 3 carbon atoms, which is bonded to the adjacent phenyl group via oxygen, or any one selected from dialkylamino with 1 to 10 carbon atoms of each alkyl group and heterocyclic amine with 2 to 6 carbon atoms, which is bonded to the adjacent phenyl group via nitrogen, or bromine, iodine, or a straight-chain hydrocarbon group with 2 to 5 carbon atoms; R2 represents hydrogen; R3 and R4 represent a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, or a halogen atom selected from fluorine, chlorine, bromine and iodine. R3 and R4 may be the same as or different from each other.

2. The V-ATPase activity inhibitor according to claim 1, characterized in that It binds to the intramembrane rotor ring (c-ring) of V-ATPase and inhibits its activity.

3. The V-ATPase activity inhibitor according to claim 1, characterized in that The compound represented by formula (2) is a 2-phenylbenzimidazole derivative represented by the following formula (V-161-01), formula (V-161-02), formula (V-161-03), (V-161-04) or formula (V-161-10): 。 4. An antibacterial agent for bacteria having V-ATPase, characterized in that: Contains the V-ATPase activity inhibitor according to any one of claims 1 to 3.

5. A drug, characterized in that Contains the antibacterial agent as claimed in claim 4.

6. A bacterial population regulator for selectively reducing bacteria having V-ATPase in a bacterial population comprising bacteria having V-ATPase and bacteria not having V-ATPase, characterized in that: The method comprises the V-ATPase activity inhibitor according to any one of claims 1 to 3.

7. A drug, characterized in that Contains the bacterial flora regulator as described in claim 6.

8. Use of the antibacterial agent according to claim 4 in the manufacture of a drug for treating an infection caused by bacteria having V-ATPase.

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