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

Abstract

To provide an inhibitor for V-ATPase activity, which can selectively and efficiently suppress the growth of the bacteria that cause diseases or the like.SOLUTION: The present invention provides an inhibitor for Na+-translocating V-ATPase activity including a compound represented by formula (1), where R1 represents a group selected from a hydroxy group, an alkoxy group, and a haloalkoxy group, each bonded to an adjacent phenyl group via oxygen, or a group selected from a dialkylamino group, a heterocyclic amine, and a carboxylic acid amide group, each bonded to an adjacent phenyl group via nitrogen, or represents bromine, iodine, or a straight-chain hydrocarbon group; R2 represents hydrogen or a haloalkoxy group; Z1 represents an aliphatic hydrocarbon group, an alicyclic hydrocarbon group, an aromatic hydrocarbon group, or a heterocyclic group, each optionally having any substituent).SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a V-ATPase activity inhibitor, an antibacterial agent, a medicine, an antibacterial method, and a screening method, and particularly relates to a V-ATPase activity inhibitor, an antibacterial agent, a medicine, an antibacterial method, and a screening method capable of efficiently inhibiting V-ATPase present in microorganisms causing diseases and the like.

Background Art

[0002] V-ATPase is a supramolecular complex present in the organelle membrane of eukaryotes and composed of a complex subunit structure. V-ATPase has a function as an ion-transporting molecular motor, and by the hydrolysis energy of ATP, specific subunits 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 grow under specific environments. For example, in Enterococcus, V-ATPase has a function of hydrolyzing ATP to transfer sodium ions (Na + ) outside the cell, thereby enabling Enterococcus to grow under high salt concentration and high pH conditions.

[0004] In addition, V-ATPase similar to that of Enterococcus exists in various pathogenic microorganisms and plays an important role in their growth under alkaline conditions. Therefore, compounds that inhibit the ion transport function of V-ATPase are useful as antibacterial agents against pathogenic bacteria causing diseases and are expected as new therapeutic agents.

[0005] Conventionally, various compounds are known as antibacterial agents. For example, Patent Document 1 describes that 2-substituted benzimidazoles having a specific structure are useful as antibacterial agents.

Prior Art Documents

Patent Documents

[0006] [Patent Document 1] U.S. Patent No. 5,942,532 (Claim 1, etc.) [Summary of the Invention] [Problems to be Solved by the Invention]

[0007] However, the antibacterial agent of Patent Document 1 exerts an antibacterial action as a histidine protein kinase inhibitor of microorganisms, which is different from those that inhibit V-ATPase.

[0008] An object of the present invention is to provide a V-ATPase activity inhibitor capable of selectively and efficiently suppressing the growth of bacteria causing diseases and the like, an antibacterial agent, a medicine, and an antibacterial method containing the same. Another object of the present invention is to provide a screening method for efficiently screening such a V-ATPase activity inhibitor. [Means for Solving the Problems]

[0009] The present inventors have conducted intensive studies to solve the above problems. As a result, they have found that a specific compound having an aromatic ring, an electron-donating group bonded thereto, and an arbitrary substituent having a double bond bonded to the aromatic ring exhibits an effect of inhibiting V-ATPase activity, and thus completed the present invention.

[0010] That is, the present invention is a Na + transport-type V-ATPase activity inhibitor, characterized by containing a compound represented by the following formula (1). [Chemical Formula] (Here, R1 is selected from a hydroxy group, an alkoxy group having 1 to 10 carbon atoms, and a haloalkoxy group having 1 to 3 carbon atoms, all of which are bonded to an adjacent phenyl group via oxygen, or a dialkylamino group having 1 to 10 carbon atoms in each alkyl group, a heterocyclic amine having 2 to 6 carbon atoms, and a carboxamide group which may have a substituent bonded to a carbon atom, all of which are bonded to an adjacent phenyl group via nitrogen, or represents bromine, iodine, or a linear hydrocarbon group having 2 to 5 carbon atoms.) R2 represents hydrogen or a haloalkoxy group having 1 to 3 carbon atoms. 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, all of which may have any substituent and has a structure containing a double bond selected from the following formulas (1-1) to (1-3) between it and an adjacent phenyl group. Here, * represents a bond to an adjacent phenyl group.)

Chemical formula

[0011] In this case, it is preferable that the compound represented by the formula (1) is a 2-phenylbenzimidazole derivative represented by the following formula (2).

Chemical formula

[0012] Further, in the above case, the V-ATPase activity inhibitor according to claim 1, wherein the compound represented by the formula (1) is a (1,2,4-oxadiazol-3-yl)phenyl derivative represented by the following formula (7).

Chemical formula

[0013] Also, in the above case, it is preferably characterized by binding to the inner membrane rotor ring (c-ring) of V-ATPase to inhibit its activity.

[0014] The compound of the 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-trimethylcyclohex-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.

[0015] The present invention is an antibacterial agent against bacteria having V-ATPase, and is characterized by containing the V-ATPase activity inhibitor described in any one of the above.

[0016] Furthermore, the present invention is a pharmaceutical characterized by containing the above antibacterial agent.

[0017] The present invention is a bacterial flora regulator that selectively reduces bacteria having V-ATPase from a bacterial flora containing bacteria having V-ATPase and bacteria not having V-ATPase, and is characterized by containing the V-ATPase activity inhibitor according to any one of claims 1 to 5.

[0018] Furthermore, the present invention is a medicament characterized by containing the above-mentioned bacterial flora regulator.

[0019] In addition, the present invention is an antibacterial method for suppressing the growth of bacteria using the above-mentioned antibacterial agent, comprising the step of administering the V-ATPase activity inhibitor to bacteria having V-ATPase, and the step of binding the V-ATPase activity inhibitor to the intramembrane rotor ring (c-ring) of the V-ATPase of the bacteria to inhibit the activity of the V-ATPase.

[0020] In addition, the present invention... + A screening method for selecting a candidate compound of a transportable V-ATPase activity inhibitor from test compounds, comprising an untreated evaluation step of evaluating the survival state of bacteria having V-ATPase at a first pH and a second pH on the alkaline side of the first pH without contacting the test compound with the bacteria, a treated evaluation step of evaluating the survival state of the bacteria at the first pH and the second pH after contacting the test compound with the bacteria, and a candidate compound identification step of identifying a test compound showing a difference in survival state between the untreated evaluation step and the treated evaluation step as a candidate compound for the antibacterial agent of the bacteria.

[0021] In addition, the present invention is an antibacterial medicament containing a compound having V-ATPase inhibitory activity as an active ingredient.

[0022] In addition, the present invention is an antibacterial method comprising the step of administering a compound having V-ATPase inhibitory activity to bacteria having V-ATPase, and the step of binding the V-ATPase activity inhibitor to the V-ATPase of the bacteria to inhibit the activity of the V-ATPase.

Advantages of the Invention

[0023] According to the present invention, it becomes possible to provide a V-ATPase activity inhibitor capable of selectively and efficiently suppressing the growth of bacteria that cause diseases and the like, an antibacterial agent, a medicament, and an antibacterial method containing the same. Further, according to the present invention, it becomes possible to provide a screening method for efficiently screening such a V-ATPase activity inhibitor.

Brief Description of the Drawings

[0024]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Modes for Carrying Out the Invention

[0025] 1. V-ATPase Activity Inhibitor The V-ATPase activity inhibitor of the present invention is a transportable V-ATPase activity inhibitor and contains a compound represented by the following formula (1). + (Here, R1 is selected from a hydroxy group, an alkoxy group having 1 to 10 carbon atoms, and a haloalkoxy group having 1 to 3 carbon atoms, all of which are bonded to an adjacent phenyl group via oxygen, or a dialkylamino group having 1 to 10 carbon atoms in each alkyl group, a heterocyclic amine having 2 to 6 carbon atoms, and a carboxylic acid amide group which may have a substituent bonded to a carbon atom, all of which are bonded to an adjacent phenyl group via nitrogen, or represents bromine (Br), iodine (I), or a linear hydrocarbon group having 2 to 5 carbon atoms.)

Chemical formula

Chemical formula

[0026] In R1, the hydroxy group is a substituent represented by “-OH”. The alkoxy group having 1 to 10 carbon atoms is a substituent represented by “-O-R 1a ” (R 1a represents an alkyl group having 1 to 10 carbon atoms). Further, the haloalkoxy group having 1 to 3 carbon atoms in R1 is “-O-R 1b -X n1 ” (R 1b represents an alkyl group having 1 to 3 carbon atoms, and X represents a halogen atom selected from fluorine, chlorine, bromine, and iodine. n1 = 1 to 3 is shown). The dialkylamino group having 1 to 10 carbon atoms in each alkyl group is “-N(R 1c )(R1d )」(R 1c represents an alkyl group having 1 to 10 carbon atoms, and R 1d represents an alkyl group having 1 to 10 carbon atoms, and R 1c and R 1d may be the same or different. ) is a substituent represented by. The heterocyclic amine having 2 to 6 carbon atoms is "-N-(CH2) n2 -"(n2 = 2 to 6 is shown). ) is a substituent represented by. The 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". The straight-chain hydrocarbon group having 2 to 5 carbon atoms is "-(CH2) n3 -CH3"(n3 = 1 to 4 is shown). ) is a substituent represented by.

[0027] In R2, hydrogen is a substituent represented by "-H". The haloalkoxy group having 1 to 3 carbon atoms is "-O-R 2a -X n4 "(R 2a represents a hydrocarbon group having 1 to 3 carbon atoms, and X represents a halogen atom selected from fluorine, chlorine, bromine, and iodine. n4 = 1 to 3 is shown). ) is a substituent represented by. R2 may be located at the ortho position or the meta position with respect to Z1 in the phenyl group.

[0028] In Z1, the aliphatic hydrocarbon group having 5 to 20 carbon atoms is a substituent selected from alkanes, alkenes, alkynes, alkadienes, conjugated dienes, etc.

[0029] 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, eicosane, etc.

[0030] Examples of alkenes 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, 6-decene, and the like.

[0031] Examples of alkynes 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-decyne, 2-decyne, 3-decyne, 4-decyne, 5-decyne, 6-decyne, and the like.

[0032] Examples of alkadienes having 5 to 20 carbon atoms include 1,3-pentadiene, 2,4-heptadiene, 1,4-pentadiene, 1,7-octadiene, 2,5-octadiene, 2,6-octadiene, and the like.

[0033] Examples of conjugated alkenes 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, 2,4,6,8-decatetraene, and the like.

[0034] The alicyclic hydrocarbon group having 3 to 20 carbon atoms is a substituent selected from cycloalkanes, cycloalkenes, cycloalkynes, and the like. 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, cyclooctyne, and the like.

[0035] Examples of the aromatic hydrocarbon group having 6 to 20 carbon atoms include benzene, naphthalene, anthracene, and the like.

[0036] The heterocyclic group having 2 to 20 carbon atoms is a substituent having 2 to 20 carbon atoms and containing at least one element 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-azirine, 2H-azirine, oxylene, thiylene, azetidine, oxetane, thietane, azeto, azolidine, oxolane, thiolane, azole, oxole, thiol, azinane, oxane, thiane, pyridine, azepane, oxepane, thiepane, azepine, oxepine, 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, and the like.

[0037] In addition to the above substituents, Z1 may be one in which one or more of an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an oxo group, a halogen atom, a hydroxy group, a nitro group, a sulfo group, an ether group, a thiol group, an ester group, a carbonate group, a carbonyl group, an amide group, an amino group, an azide group, a carbamate group, a cyano group, a hydroxy group, a carboxyl group, a sulfonic acid ester group, a sultone group, a lactone ring, a lactam ring, etc. are bonded to these substituents.

[0038] Z1 preferably contains a ring structure in the molecule. As the ring structure, a group selected from the above alicyclic hydrocarbon groups having 3 to 20 carbon atoms, aromatic hydrocarbon groups having 6 to 20 carbon atoms, and heterocyclic groups having 2 to 20 carbon atoms is preferred. In Z1, there may be only one ring structure or two or more ring structures may be included. The ring structure more preferably contains a structure containing a double bond of the above formulas (1-1) to (1-3) in the molecule.

[0039] In particular, as the compound represented by the above formula (1), a 2-phenylbenzimidazole derivative represented by the following formula (2) is preferred.

Chemical formula

[0040] Examples of the 2-phenylbenzimidazole derivative of the above formula (2) include compounds represented by the following formulas (V-161, V-161-01 to V-161-04, V-161-10).

Chemical formula

[0041] Among these, formula V-161 (N,N-dimethyl-4-(6-methyl-1H-benzo[d]imidazol-2-yl)aniline) is preferred because of its high effect of inhibiting V-ATPase activity.

[0042] Examples of the compound represented by the above formula (1) include (1,2,4-oxadiazol-3-yl)phenyl derivatives represented by the following formula (6).

Chemical formula

[0043] In particular, it is preferable that the compound represented by formula (6) is a (1,2,4-oxadiazol-3-yl)phenyl derivative represented by the following formula (7).

Chemical formula

[0044] Specific examples of the compound represented by the following formula (7) include the compounds represented by the following formulas (V-234), (V-234-01) to (V-234-03).

Chemical formula

[0045] Examples of the compound represented by the above formula (1) include the compound represented by the following formula (3).

Chemical formula

[0046] In addition, examples of the compound represented by the above formula (1) include the compound represented by the following formula (4).

Chemical formula

[0047] In addition, examples of the compound represented by the above formula (1) include the compound represented by the following formula (5).

Chemical formula

[0048] Examples of the compound of the above formula (1) include the compounds shown below.

Chemical formula

[0049] These compound names are as follows. ·V-6: (Z)-5-(2,4-bis(difluoromethoxy)benzylidene)-2-(cyclopentylamino)thiazol-4(5H)-one ·V-84: (2E,4E,6E,8E)-N-(4-hydroxyphenyl)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraenamide ·V-130: 5-(4-(nonyloxy)benzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione ·V-161: N,N-dimethyl-4-(6-methyl-1H-benzo[d]imidazol-2-yl)aniline ·V-234: N-(4-(5-((o-tolyloxy)methyl)-1,2,4-oxadiazol-3-yl)phenyl)thiophene-2-carboxamide

[0050] Among these, V-6, V-161, and V-234 are particularly preferred in that they have less non-specific binding to proteins other than V-ATPase.

[0051] 2. V-ATPase Activity Inhibition Mechanism Next, the V-ATPase will be described. FIG. 1 is a schematic diagram of the V-ATPase of Enterococcus hirae (E. hirae), which is an intestinal streptococcus, and shows the + transport-type V-ATPase. As shown in (a) of this figure, V-ATPase is a complex composed of multiple subunits, and has a hydrophilic catalytic head portion (V1 portion) and a membrane-intrinsic portion (V + ) responsible for sodium ion (Na o ) transport. The V1 portion is an ATP-driven motor portion, and ATP is hydrolyzed by the A3B3 subunits to rotate the V1 portion. The V o portion uses the rotational movement to transport ions and includes an intramembrane rotor ring (c ring) composed of a decamer of c subunits. The c ring has an ion binding site and is involved in the transport of ions across the membrane. V-ATPase hydrolyzes ATP by the V1 portion, and by using the energy to rotate the c ring of the V o portion, it discharges sodium ions in the membrane to the outside of the membrane.

[0052] (b) and (c) of FIG. 1 are schematic diagrams showing how the activity of V-ATPase changes depending on the pH of the external environment in E. hirae. As shown in (b) of this figure, near neutral pH (pH ≦ 7.0), V-ATPase is not expressed. On the other hand, as shown in (c) of this figure, under alkaline pH (pH > 7.0) or high salt concentration, V-ATPase is expressed and discharges sodium ions in the membrane to the outside of the membrane. As a result, E. hirae keeps the intracellular Na + low and can grow even in an environment of alkali or high salt concentration.

[0053] The V-ATPase activity inhibitor of the present invention has an action of binding to V-ATPase and inhibiting its activity. In particular, the V-ATPase activity inhibitor of the present invention has a function of inhibiting ion transport by binding to the c ring of V-ATPase and inhibiting its rotation. Hereinafter, the activity inhibition mechanism by the V-ATPase activity inhibitor will be described.

[0054] The V-ATPase activity inhibitor of the present invention is a compound represented by the formula (1). As shown in the following formula, it has a phenyl group and an arbitrary substituent, and the phenyl group has an electron-donating group at the para position with respect to the bonding position with the arbitrary substituent.

Chemical formula

[0055] In addition, for the V-ATPase activity inhibitors exemplified above, figures explaining the structures are shown. As shown in these figures, any substituent Z1 is linked to the phenyl group via a structure containing a double bond of the formula (1-1) to (1-3). Also, all Z1 have a ring structure.

Chemical formula

[0056] The phenyl group having an electron-donating group binds to a specific site on the outer periphery of the c-ring of V-ATPase together with an arbitrary substituent and is sandwiched between the c-ring and the a-subunit, thereby playing a role of inhibiting the rotation of the c-ring. The phenyl group and an arbitrary substituent are hydrophobic and bind to the c-ring by hydrophobic interaction with amino acid residues having hydrophobic side chains on the outer periphery of the c-ring. Also, it is considered that the activation of the benzene ring due to the increase in the electron density of the π electrons of the benzene ring by the electron-donating group bonded to the para position of the phenyl group 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 below, it interacts with the amino acid residues around the 52nd phenylalanine of the c-ring, ensuring the binding ability of the inhibitor.

[0057] A hydrogen atom supplied from an arbitrary substituent and / or an NH group of an electron-donating group serves as a donor, and an N atom, an O atom, or an S atom serves as an acceptor. It is presumed that a plurality of hydrogen bonds are formed between the acceptor or donor of the c-ring. Thereby, the arbitrary substituent and the electron-donating group contribute to the improvement of the binding affinity with the c-ring of V-ATPase. The arbitrary substituent may have a relatively arbitrary structure as long as its molecular weight is moderately large and it is hydrophobic. This substituent preferably has a ring structure. By having a hydrophobic ring structure and increasing the bulkiness of the arbitrary substituent, the inhibitory effect of the V-ATPase activity inhibitor on the rotation of the c-ring becomes higher.

[0058] First, in order to bind to the c-ring of V-ATPase, a phenyl group and a structure containing a double bond existing in a specific direction therefrom are the minimum necessary basic structures. In particular, when a double bond such as in formula (1-2) or formula (1-3) is adjacent to the phenyl group, the structure is fixed by the resonance between the double bond and the phenyl group, and it is considered that the resonating π electrons play an important role in binding to the c-ring of V-ATPase.

[0059] Note that the structure containing a double bond may be included in the linker that connects the phenyl group and the ring structure among the arbitrary substituents (for example, V-84, etc.), or may be included in the ring structure when there is no linker between the phenyl group and the ring structure (for example, V-161). By having such a structure, the V-ATPase activity inhibitor binds to the c-ring of V-ATPase and suppresses its rotation, thereby exerting an effect of inhibiting V-ATPase activity.

[0060] 3. Antibacterial agent, antibacterial method, medicine Since the V-ATPase activity inhibitor of the present invention inhibits the activity of V-ATPase as described above, it is useful as an antibacterial agent for microorganisms expressing V-ATPase. A list of microorganisms expressing V-ATPase is shown in the following table. It is expected that the activity of V-ATPase of these microorganisms will be inhibited by the V-ATPase activity inhibitor of the present invention, and their growth will be suppressed. Also, the related disease names indicate diseases that are expected to be treated and prevented by the growth inhibition of the microorganisms.

Table 1

[0061] In the table, "Bacteria" indicates the name of the microorganism, and "Related disease name" indicates examples of diseases related to that microorganism. Also, "V-ATPase ring identity (%)" in the table indicates the percentage of homology between the c-ring of the V-ATPase of that microorganism and the c-ring of the V-ATPase of Enterococcus. The inventors have confirmed in the examples described below that the V-ATPase activity inhibitor binds to the c-ring of V-ATPase and exhibits an effect of inhibiting the growth of Enterococcus. Therefore, for microorganisms having a V-ATPase with high homology to the c-ring of Enterococcus V-ATPase, it is expected that the V-ATPase activity inhibitor of the present invention will exhibit an effect of inhibiting the growth of the microorganisms in the same manner as Enterococcus. On the other hand, as described above, the V-ATPase activity inhibitor interacts with the outer peripheral surface of the c-ring. As the inventors have confirmed, the amino acid sequence is relatively highly conserved in many microorganisms on the outer peripheral surface of the c-ring of V-ATPase. Therefore, even for microorganisms with low homology to the c-ring of Enterococcus V-ATPase, it is not necessarily the case that the effect of the V-ATPase activity inhibitor is absent, and it is considered that the V-ATPase activity inhibitor may exhibit an effect of inhibiting the growth of the microorganisms.

[0062] 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 conventional antibacterial agents. For example, it is useful against E. faecalis, E. faecium, etc. of vancomycin-resistant enterococci (VRE).

[0063] The V-ATPase activity inhibitor of the present invention is useful as a microbiota regulator that selectively reduces bacteria having V-ATPase from a microbiota containing bacteria having V-ATPase and bacteria not having V-ATPase.

[0064] For example, Clostridium perfringens, etc. are so-called bad bacteria, and are pathogenic bacteria that cause protein corruption in the intestine to produce harmful amines and carcinogenic substances. Many of these microorganisms that lead to the deterioration of the intestinal environment have V-ATPase and grow predominantly under alkaline conditions. On the other hand, Bifidobacterium spp., Lactobacillus spp., etc. are so-called good bacteria, and ferment dietary fiber in the intestine to produce short-chain fatty acids important for maintaining the health of the host, such as butyric acid and acetic acid, and suppress the growth of bad bacteria and related diseases. Many of these microorganisms that help improve the intestinal environment do not have V-ATPase. Therefore, the V-ATPase activity inhibitor of the present invention selectively inhibits the growth of only the bad bacteria having V-ATPase by exerting an effect only on the bad bacteria and not on the above-mentioned good bacteria, thereby improving the intestine into an environment in which good bacteria can easily grow.

[0065] Note that VRE infections, enteritis caused by Clostridium difficile, etc. are often caused by disturbances in the intestinal flora due to the administration of antibiotics. Abnormalities in the immune system and intestinal flora in the intestinal tract may induce autoimmune diseases. Deterioration of the intestinal environment is said to lead to colorectal cancer and liver cancer, and the intestinal flora is closely related to lifestyle diseases such as obesity and diabetes. Therefore, the V-ATPase activity inhibitor of the present invention is useful as a medicine for preventing and treating various diseases by adjusting the balance between beneficial bacteria and harmful bacteria in the intestinal flora of intestinal bacteria.

[0066] The V-ATPase activity inhibitor (antibacterial agent, flora regulator) of the present invention is useful as an active ingredient in drugs such as pharmaceuticals and agricultural chemicals, and in particular, pharmaceutical use is preferred. Examples of the types of pharmaceuticals include tablets, capsules, pills, powders, granules, fine granules, jelly agents, liquid agents, and the like.

[0067] The concentration of the V-ATPase activity inhibitor contained in the medicine can be determined appropriately, but for example, it is in the range of 1 μM to 100 mM, and preferably in the range of 100 μM to 10 mM. The V-ATPase activity inhibitor can be used by dissolving it in a solvent such as water, or can also be used in a powder state.

[0068] In addition to the above-mentioned medicament which is a V-ATPase activity inhibitor (antibacterial agent, bacterial flora regulator) of the present invention, additives such as solvents, excipients, binders, disintegrants, lubricants, stabilizers, suspending agents, etc. may be included within the range that does not impair the effects of the present invention. Examples of solvents for preparations include water, ethanol, glycerin, etc. Examples of excipients include lactose, sucrose, glucose, mannitol, sorbitol, corn starch, potato starch, α-starch, dextrin, carboxymethyl starch, crystalline cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, gum arabic, dextran, pullulan, silicate salts, calcium phosphate, calcium carbonate, calcium sulfate, etc. Examples of silicate salts include light anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminometasilicate, etc. Examples of binders include gelatin, polyvinylpyrrolidone, macrogol, etc. Examples of disintegrants include croscarmellose sodium, sodium carboxymethyl starch, crosslinked polyvinylpyrrolidone, etc. Examples of lubricants include talc, stearic acid, calcium stearate, magnesium stearate, colloidal silica, beeswax, beeswax, gaylussite, boric acid, glycol, fumaric acid, adipic acid, sodium benzoate, sodium sulfate, leucine, sodium lauryl sulfate, magnesium lauryl sulfate, anhydrous silicic acid, hydrated silicic acid, 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 carboxymethyl cellulose, etc.

[0069] 4. Screening method Next, Na +A screening method for selecting candidate compounds of a transportable V-ATPase activity inhibitor from test compounds will be described. The screening method of the present invention comprises the following (a) primary screening step and (b) secondary screening step.

[0070] (a) Primary screening step In the primary screening step, only those 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 or the like. As the compound library, a compound database held by a university or the like can be used.

[0071] Next, for each of the selected compounds, V-ATPase and the compound are reacted, and the ATPase activity is measured to evaluate whether the compound inhibits ATPase. The ATPase activity can be measured by the molybdenum blue method of measuring the absorbance of inorganic phosphate generated by the hydrolysis of ATP. As the reaction conditions, conditions for measuring the ATPase activity can be adopted. As the buffer solution used in the reaction, Tris-HCl buffer can be used. The reaction time is usually 10 to 60 minutes, but can be appropriately adjusted depending on the temperature and the like. The reaction of V-ATPase is stopped by adding a surfactant such as SDS, and after adding a coloring solution (such as ferrous sulfate) to develop color, the concentration of inorganic phosphate can be calculated by measuring the absorbance at 650 nm.

[0072] From the concentration of inorganic phosphate calculated above, the inhibition rate of the V-ATPase reaction can be calculated. That is, a graph is created with the inhibition rate on the vertical axis and the concentration of the compound on the horizontal axis, and the concentration (IC 50 ) at which the inhibition rate becomes 50% is determined. The lower the concentration of the compound at which IC 50 is obtained, the higher the V-ATPase inhibitory effect at a small amount, so it becomes a candidate compound for a V-ATPase activity inhibitor. In the examples described later, IC 50A compound with an IC50 of less than 10 μM (hereinafter expressed as "IC50 < 10 μM") is used as a candidate compound selected in the primary screening step.

[0073] (b) Secondary screening step The secondary screening step is a step of confirming whether the growth of microorganisms is inhibited for the compounds narrowed down in the primary screening step. The secondary screening step has at least (b-1) an untreated evaluation step, (b-2) a treated evaluation step, and (b-3) a candidate compound identification step.

[0074] (b-1) Untreated evaluation step 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 on the alkaline side of the first pH without contacting the test compound with the bacteria. V-ATPase is often expressed in microorganisms that grow in an alkaline environment. Therefore, in this step, the microorganisms are grown in two environments of a specific pH (the first pH) and a pH on the alkaline side thereof (the second pH), and their growth status is confirmed. When targeting Enterococcus, it is preferable to appropriately select the first pH within the range of pH ≦ 7.0 and the second pH within the range of pH > 7.0. For example, the first pH is pH 7.0 and the second pH is pH 8.0.

[0075] The culture conditions (culture medium, culture temperature) of the microorganisms in the untreated evaluation step can be appropriately determined according to the type of microorganisms. For example, when the microorganism is Enterococcus, it is preferably cultured in a liquid medium containing tryptone and yeast extract, and glucose or the like may be added as necessary. Culture media with different pH values are used at the first pH and the second pH. Also, the culture temperature of Enterococcus is preferably 30 to 37°C.

[0076] The growth state of microorganisms can be evaluated by checking the number of microorganisms. The number of microorganisms can be confirmed by methods such as measuring the absorbance of the medium in which the microorganisms are cultured, or culturing the microorganisms on a plate or the like and counting the number of microorganisms. In the case of enterococci, the growth state can be evaluated by measuring the absorbance (OD 600 ) at a wavelength of 600 nm.

[0077] (b-2) Treated evaluation step: Next, after bringing the test compound into contact with the bacteria, the survival state of the bacteria is evaluated at a specific pH (first pH) and an alkaline-side pH (second pH) in the same manner as in (b-1) above. The culturing and evaluation of the growth state of the microorganisms are carried out under the same conditions and by the same method as in (b-1).

[0078] (b-3) Candidate compound identification step: Next, the survival states of the microorganisms in the (a) untreated evaluation step and the (b) treated evaluation step are compared, and a test compound showing a difference is identified as a candidate compound for the antibacterial agent of the bacteria. If the growth of the microorganisms is more suppressed in (b) the treated state than in (a) the untreated state at a pH on the alkaline side of the specific pH, it is judged that the test compound may have inhibited the V-ATPase activity, and it becomes a candidate compound for a V-ATPase activity inhibitor. On the other hand, if the growth is suppressed even at the specific pH, or if the growth is more in the (b) treated state than in (a) the untreated state, or the growth is almost the same in both at a pH on the alkaline side of the specific pH, it is judged that the test compound has no effect on the V-ATPase activity, and it is excluded from the candidate compounds for the V-ATPase activity inhibitor.

Example

[0079] Hereinafter, the present invention will be specifically described based on examples, but these do not limit the content of the present invention. In the following examples, the “%” indication is based on mass (mass percent) unless otherwise specified.

[0080] (1) Preparation of compound and V-ATPase (a) Obtaining the compound Compounds that are candidates for ATPase activity inhibitors were obtained from the compound library of the Institute of Innovative Research, The University of Tokyo. Approximately 70,000 compounds were obtained, and primary screening was performed for all compounds by measuring ATPase activity, which will be described later. (b) Preparation of V-ATPase V-ATPase was prepared by the method described in the following papers. Paper 1: Takeshi Murata, et.al, “Purification and Reconstitution of Na + -translocating Vacuolar ATPase from Enterococcus hirae”, J. Biol. Chem. 272, 24885-24890 (1997) Paper 2: Takeshi Murata, et.al, “Torque Generation of Enterococcus hirae V-ATPase”, J. Biol. Chem. 289, 31212-31223 (2014)

[0081] (2) Measurement of ATPase activity (primary screening) The ATPase activity was measured using the molybdenum blue method, which quantifies the inorganic phosphate generated by the hydrolysis of ATP through absorbance measurement. The measurement was performed in a 96-well plate. 0.05% DDM (n-dodecyl-β-D-maltoside), 10 μg / mL of V-ATPase (purified from Enterococcus or recombinant Escherichia coli), and 20 μM of the compound were added to Buffer (100 mM Tris-HCl pH 8.5, 100 mM NaCl, 5 mM MgSO4, 10% glycerol). ATP was added to a concentration of 5 mM, and the reaction was initiated with stirring using a stirrer while adjusting the total volume to 100 μL and setting the reaction time to 30 minutes. Since the ATPase activity varies significantly depending on the reaction temperature, the reaction time was adjusted within the range of 10 minutes to 60 minutes according to the ambient temperature on the day of the experiment. The reaction was stopped by denaturing V-ATPase by adding 50 μL of 20% SDS, and color development was achieved by adding 75 μL of the color-developing solution (5% ferrous sulfate, 1.6% ammonium molybdate, 1 M sulfuric acid). Immediately, the absorbance at 650 nm was measured using a plate reader. The activities of V-ATPase were measured by reacting four types of KH2PO4 aqueous solutions at 0.2 mM, 0.5 mM, 1.0 mM, 1.5 mM and MQ (ultrapure water) in the same experimental system and determining the concentration of the generated inorganic phosphate using the prepared calibration curve. Therefore, for the compounds that inhibited the activity by 50% or more, the concentrations were serially diluted to 10 μM, 5 μM, 2 μM, 1 μM, 500 nM, 200 nM, 100 nM, and 50 nM, and the same measurements as above were performed.

[0082] (3) Primary screening results From the results of the above ATPase activity measurement, with the activity under the condition of not adding the compound set as 100, the inhibition rate was calculated from the decrease in activity compared to it. A graph was created with the inhibition rate on the vertical axis and the concentration of the compound on the horizontal axis (Figure 2). From the graph, the concentration of the compound (IC 50 ) at which the inhibition rate became 50% was determined. As a result, there were 280 types of compounds with IC 50 less than 10 μM (IC 50 < 10 μM), and compounds with IC 50 less than 100 nM (IC50 There were 12 types of compounds with an IC<100 nM). 50 Regarding 280 types of compounds with <10 μM, the effects on human-derived HeLa cells were verified. As a result, 24 types showed toxicity, so 256 types excluding them were used as candidate inhibitors. From these 256 types, 24 types of compounds were selected considering the compounds with the highest activity and diversity. The structural formulas of these 24 types of compounds are shown below.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0083] The following secondary screening was performed on the above 24 types of compounds in the growth experiment of Enterococcus. (4) Growth experiment of Enterococcus (secondary screening) For the culture of Enterococcus hirae, a buffer was added based on 2% tryptone and 1% yeast extract, and a medium with the concentration of Na + adjusted was used. When adding glucose, it was prepared as a 50% solution and added to 1% when using the medium. A total of 200 μL of culture was carried out using a 96-well plate, and OD 600 was measured using a plate reader every hour. Media with pH 7.0 and pH 8.5 were used to observe the influence of pH on growth. In the culture of bacteria, the pH often fluctuates greatly due to the decomposition of carbohydrates necessary for growth, and in this experiment, it is extremely important to maintain a constant pH, so the pH was kept constant by adding a buffer suitable for each pH. Since V-ATPase is expressed only under alkaline conditions, the growth inhibition observed by the addition of inhibitors depends greatly on pH. In addition, the inhibitors do not affect the growth of bacteria without V-ATPase.

[0084] (5) Secondary screening results

[0085] The IC of the above 24 compounds is shown in the table below. 50 In the "Specificity" column of the table below, "+" indicates that there was no growth inhibition at pH 7.0 and at pH 8.5, "-" indicates that there was no growth inhibition at any of the conditions, and "na" indicates that there was growth inhibition at any of the conditions. Specifically, for "+", compounds with a growth inhibition rate of 20% or less at pH 7.0 were judged to have no growth inhibition. For "-", compounds with a growth inhibition rate of 20% or less at both pH 7.0 and pH 8.5 were judged to have no growth inhibition. For "na", compounds with a growth inhibition rate of 60% or more at pH 7.0 were judged to have growth inhibition. For reference, the growth inhibition rate is also listed for compounds for which no clear inhibition was observed. [Table 2]

[0086] Additionally, a graph of the growth results for 14 of these compounds with a "+" for "specificity" is shown (Figure 3). The horizontal axis shows the candidate compound, and the vertical axis shows the number of bacteria (%) measured by absorbance. The leftmost graph is the control, while the graphs to the left of each compound show the results under culture conditions of pH 7.0, and the graphs to the right show the results under pH 8.5. As shown in this graph, it can be seen that for all of the candidate compounds, the growth of enterococci is inhibited at pH 8.5 compared to pH 7.0.

[0087] Among the 14 compounds listed above, the compounds with the highest affinity for V-ATPase (IC 50 Three compounds with high inhibitory activity (growth inhibition rate (%) >= 70%) and no non-specific inhibition were identified as compounds exhibiting V-ATPase activity inhibitors with high inhibitory efficiency. Specifically, they are Example 1 (V-6), Example 4 (V-161), and Example 5 (V-234). In particular, the compound in Example 4 (V-161) (a 2-phenylbenzimidazole derivative) exhibited high inhibitory activity (IC50 Based on the results of growth inhibition, it is considered to be the most useful candidate compound. In addition, it has a high affinity for V-ATPase (IC 50 <=400), and has a high growth inhibition effect (growth inhibition rate (%) >= 70%). However, two compounds with non-specific inhibition (Example 2 (V-84), Example 3 (V-130)) were also identified as candidate compounds.

[0088] (6) Co-crystallization and X-ray crystal structure analysis 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. The V-ATPase derived from Enterococcus was expressed in recombinant Escherichia coli by the methods of the above-mentioned Papers 1 and 2, purified into a complex, and then the c-ring was isolated. The c-ring was prepared at 2 mg / mL, and V-161 was added to a concentration of 500 μM. Crystallization was carried out at 23 °C under the conditions of 100 mM Tris-HCl pH 8.0, 220 mM sodium citrate, and 32% PEG400. The obtained crystals were subjected to X-ray diffraction experiments at the beamline of the Photon Factory (PF), a synchrotron radiation facility. Analysis was performed from the diffraction image, and the structure was determined at a resolution of 2.3 Å. The results are shown in FIGS. 4 and 5. Regarding the detailed method of X-ray crystal structure analysis, the following paper on the X-ray crystal structure analysis of the c-ring of Enterococcus V-ATPase can be referred to. Paper 3: Takeshi Murata, et.al, “Structure of the Rotor of the V-Type Na + -ATPase from Enterococcus hirae” Science 308, 654-659 (2005)

[0089] As shown in Fig. 4(a), the ribbon-like shape represents the helix of the c-ring, the surrounding stick-like shapes represent lipid molecules and surfactant molecules, and the central granule represents sodium ions. Fig. 4(b) is an enlarged view of the area surrounded by the square in Fig. 4(a). Fig. 5 is an electron density map, with the left side showing the overall structure of the c-ring and the central and right sides showing the structures of the binding positions of V-161 successively enlarged. From these structures, it was found that V-161 binds to the outer peripheral surface by interacting with the amino acid residues around the 52nd phenylalanine of the c-ring.

[0090] (7) Compound development of 2-phenylbenzimidazole derivatives For other 2-phenylbenzimidazole derivatives having the same skeleton as V-161, synthetic development was carried out. The compounds are 10 types of the following (V-161-01) to (V-161-10). The structural formulas of the compounds are shown below.

[0091] [Chemical formula]

[0092] For these compounds, the inhibitory effect on ATPase activity was evaluated by the same method as in "(2) Measurement of ATPase activity". The results are shown in the following table. Note that in Reference Example 4-02 (V-161-06), there is no specificity of activity depending on pH (high activity at both pH 7.0 and pH 8.5), and in Reference Example 4-05 (V-161-09), no activity was observed. [Table 3]

[0093] Among these results, the affinity with V-ATPase is high (IC 50Examples 4-01 (V-161-01) to 4-04 (V-161-04) and Example 4-05 (V-161-10), which have a growth inhibitory effect <= 400), a high growth inhibitory effect (growth inhibition rate (%) >= 70%), and no non-specific binding, were specified as V-ATPase activity inhibitors with high inhibitory efficiency. On the other hand, for Reference Example 4-04 (V-161-08) and Reference Example 4-05 (V-161-09), no inhibitory effect on ATPase activity was observed.

[0094] (8) Confirmation of antibacterial action against pathogenic bacteria For pathogenic bacteria having a V-ATPase similar to Enterococcus hirae, it was confirmed whether V161 shows growth inhibition in the same manner as the above-mentioned "(4) Growth experiment of Enterococcus". As pathogenic bacteria, Enterococcus faecalis and Enterococcus faecium of vancomycin-resistant Enterococcus (VRE) and Clostridium difficile with multi-drug resistance leading to deterioration of the intestinal environment were prepared. VRE was cultured using media at pH 7.0, pH 7.5, and pH 8.0, and C. difficile was cultured using media at pH 6.5, pH 7.0, and pH 7.5, and OD 600 was measured to confirm the difference in the number of bacteria. The results are shown in Figures 6 and 7.

[0095] Figure 6 shows the results of vancomycin-resistant enterococci (VRE). The data on the left side is for E. faecalis, and the data on the right side is for E. faecium. In order from the top of this figure, the data are for pH 7.0, pH 7.5, and pH 8.0. From this figure, it was found that the growth of any of the pathogenic bacteria was inhibited in a pH-dependent manner, and V-161 had a specific growth inhibitory activity under alkaline conditions due to the inhibition of V-ATPase activity. Also, it was confirmed that E. faecium was more strongly affected by V-161 than E. faecalis. Figure 7 shows the data of pathogenic Clostridium bacteria. In order from the top of this figure, the data are for pH 6.5, pH 7.0, and pH 7.5. From this figure, it was suggested that C. difficile growth was inhibited even under neutral and acidic conditions, and the pH range in which V-ATPase is expressed in pathogenic Clostridium bacteria is wide, and V-161 acts even under neutral and acidic conditions.

[0096] (9) Confirmation of the action conditions of the intestinal environment The effect of V-161 on the intestinal environment was examined using mice (C57BL / 6, ♀, 6 weeks old, body weight about 15 g). V-161 was suspended in soybean PC and melon to a concentration of 20 mM. 0.5 ml of the suspension was orally administered to each of 4 mice once a day, and after 5 days, the mice were dissected to collect feces and intestinal tract contents, and the intestinal pH and sodium ion concentration were measured. The results are shown in Figure 8. The upper part of the figure shows the intestinal pH, and the lower part shows the sodium ion concentration.

[0097] From these results, it was found that especially in the small intestine, it is a pH environment suitable for the expression of V-ATPase and the action of V-161. Also, no toxicity was observed in the mice despite the administration of a large amount of V-161.

[0098] (10) Confirmation of the mouse intestinal environment (high-protein diet·antibiotic administration) In a high-protein diet, so-called bad bacteria often increase and the intestinal environment is often disrupted. Since the administration of antibiotics often causes a significant decrease in intestinal bacteria and leads to the colonization and infection of pathogenic bacteria, verification was carried out in these two models. First, models were prepared for every three mice each, one model fed with a high-protein diet (65% protein) and the other model fed with a normal diet (25% protein). Also, models were prepared for mice administered with an antibiotic (1 g / L ampicillin) in drinking water and mice administered with normal drinking water. After one week, all of them were dissected, intestinal contents were collected, and pH was measured. The results are shown in Fig. 9. The upper part of the figure shows the results for the mice fed with the high-protein diet, and the lower part shows the results for the mice administered with the antibiotic.

[0099] In all models, it was confirmed that the pH increased slightly in the small intestine, and increased significantly in the cecum and large intestine, and the normally acidic intestinal environment changed to alkaline. From these results, it is inferred that in the deteriorated intestinal environment, the pH tends to be high, and the V-ATPase inhibitor is more likely to act than in the normal environment.

[0100] (11) Test in VRE-infected mice Mice treated with antibiotics were infected with E. faecium of VRE, and V-161 was administered. The experimental conditions such as the dosage of the antibiotic and V-161 were the same as those in “(9) Confirmation of the action conditions of the intestinal environment” and “(10) Confirmation of the mouse intestinal environment (high-protein diet and antibiotic administration)”. The results are shown in Fig. 10. The upper part of this figure shows the results for the small intestine, and the lower part shows the results for the cecum, large intestine, and feces. Also, the vertical axis of this figure shows the viable count of VRE.

[0101] From this figure, VRE in the small intestine decreased significantly (97% decrease) when V-161 was added compared with the control. Also, VRE in the cecum, large intestine, and feces all decreased significantly (about 50% decrease) when V-161 was added compared with the control. That is, it was found that V-161 shows an effect of suppressing the growth of VRE in the mouse intestine.

[0102] (12) (1,2,4-oxadiazol-3-yl)phenyl derivative Synthetic development was carried out on other (1,2,4-oxadiazol-3-yl)phenyl derivatives having a skeleton common to V-234. The compounds are three types of the following (V-234-01) to (V-161-03). The structural formulas of the compounds containing V-234 are shown below.

[0103] [Chemical formula]

[0104] For these compounds, the inhibitory effect on ATPase activity was evaluated by the same method as in "(2) Measurement of ATPase activity". The results are shown in the table below. [Table 4]

[0105] The compounds of Examples 5-01 to 5-03 all had high affinity for V-ATPase (IC 50 <= 400), high growth inhibitory effect (growth inhibition rate (%) >= 70%), and no non-specific binding.

Claims

1. A V-ATPase activity inhibitor derived from bacteria, which comprises a compound represented by the following formula (1). ＋ Having sodium transportability. 【Chemical 1】

2. An antibacterial agent against bacteria having V-ATPase, characterized by containing the V-ATPase activity inhibitor according to Claim 1.

3. A bacterial flora regulator that selectively reduces bacteria having V-ATPase from a bacterial flora containing bacteria having V-ATPase and bacteria not having V-ATPase, characterized by containing the V-ATPase activity inhibitor according to Claim 1.

4. A medicament for the prevention or treatment of a disease associated with bacteria having V-ATPase, characterized by containing the antibacterial agent according to Claim 2 or the bacterial flora regulator according to Claim 3.

5. The medicament according to Claim 4, wherein the disease is an infectious disease or enteritis.

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