WO2006075580A1

WO2006075580A1 - Antibacterial substances injuring bacterial cell membarane and methods of using the same

Info

Publication number: WO2006075580A1
Application number: PCT/JP2006/300156
Authority: WO
Inventors: Hatsuo Yamamura; Masao Kawai; Takashi Katsu
Original assignee: National University Corporation Nagoya Institute Of Technology; National University Corporation Okayama University
Priority date: 2005-01-13
Filing date: 2006-01-04
Publication date: 2006-07-20
Also published as: JPWO2006075580A1; JP5098015B2

Abstract

A monomer injuring bacterial cell membrane which is a compound having a carbon cycle structure or a carbon/oxygen heterocycle structure, carrying two or more amino-type functional groups attached to one side of the cycle plane and showing an affinity for phospholipids in the other side of the cycle plane and exhibits a bacteriostatic effect of injuring bacterial cell wall, i.e., injuring bacterial cell wall while allowing the bacterium to sustain its proliferating ability; an oligomer injuring bacterial cell membrane which consists of two or more units of the above monomer bonded to each other; an antimicrobial monomer injuring bacterial cell membrane showing an activity of injuring bacterial cell membrane and an antimicrobial activity which belongs to the monomer injuring bacterial cell membrane as described above and in which an amino-type functional group has an aromatic ring; an antimicrobial oligomer injuring bacterial cell membrane which consists of two or more units of the above monomer bonded to each other; and various antimicrobial methods using these substances. Thus, it becomes possible to provide substances injuring bacterial cell membrane and antimicrobial substances injuring bacterial cell membrane which can be easily produced and methods of effectively utilizing the same.

Description

Bacterial membrane damage · Antibacterial substances and their use

〔Technical field〕

The present invention relates to a microbial cell membrane damaging / antibacterial substance and a method for using the same. More specifically, the present invention relates to a monomeric substance (monomer) that exhibits bacteriostatic cell membrane damage, which causes damage to the cell membrane while retaining a certain growth ability in the bacteria, an oligomer thereof, and bacteria. Against

In addition, the present invention relates to a monomeric substance having both microbial cell membrane damage and strong antibacterial properties, an oligomer thereof, and use thereof. The present invention is expected to have medical applications, hygiene applications, industrial applications, and the like.

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[Background Technology]

Chemotherapeutic agents have been continuously developed since the discovery of penicillin. However, recently, as represented by the emergence of new pathogens and mesitillin-resistant Staphylococcus aureus (MRSA), resistant bacteria to existing chemotherapeutic agents have emerged, which is a major problem. Therefore, there is a strong demand for the development of antibacterial substances that are novel chemotherapeutic agents that are particularly resistant to drug resistance.

Conventionally, substances that exhibit antibacterial properties by membrane-operated mechanisms such as polymyxin B are known as antibacterial substances that do not easily cause drug resistance. However, these substances are natural products having a complex structure, and artificial mass production is not always easy. In addition, in order to develop more effective antibacterial properties by applying artificial structural modifications to these substances, a complicated synthesis process is required, and there is a cost problem.

On the other hand, some of the existing antibacterial substances or antibiotics are inherently excellent in antibacterial properties, but cannot exhibit antibacterial properties because they cannot pass through the cell membrane as a physical barrier. As represented by the acquisition of drug resistance, it is important to effectively exert the original antibacterial properties of antibacterial substances or antibiotics in order to effectively combat pathogenic bacteria diversified by passage. From such a point of view, It is desirable to provide substances that can be damaged, in particular, membrane-acting cell membrane-damaging substances that are difficult for pathogens to handle. That is, it should be reasonably estimated that the combined use of an antibacterial and non-permeable antibiotic such as erythromycin novopiocin and the above-mentioned cell membrane damaging substance is effective. Can do.

Furthermore, in relation to the cell membrane as a physical barrier, the degree of damage to the cell membrane is not destructive, and it is bacteriostatic to the extent that a certain growth ability remains in bacteria. In this case, it can be effectively used as a means for introducing an arbitrary target substance into the bacterium, which is completely different from the antibacterial action.

Reference 1: Japanese Patent Application Laid-Open No. Sho 5 2-1 3 8 5 80 describes penta- (6-amino-6-deoxy) -one cyclodextrin and hexa- (6-amino-6-deoxy) — β -It discloses that cyclodextrin has strong antibacterial activity.

Document 2: Japanese Patent Application Laid-Open No. 2000-300-2 is a film mainly composed of a polysaccharide derivative, and an amino group is attached to a carbon atom of an anhydrous monosaccharide unit constituting the polysaccharide. In addition, it discloses that an alkylamino group is bonded. In this case, it is disclosed that the amino group or the like imparts antibacterial properties to the membrane.

However, in the above-mentioned literature 1 and literature 2, there is no disclosure or suggestion about the following items (1) to (4).

(1) The possibility that the polysaccharide structure disclosed in the literature damages the cell membrane.

(2) The technical idea of distinguishing between bactericidal cell membrane damage and bacteriostatic cell membrane damage and investigating the effective use of these cell membrane damage.

(3) The technical idea of achieving both antibacterial properties and cell membrane damage by spatial localization of the amino groups in the sugar structure.

(4) Antibacterial properties and cell membrane damage when an amino functional group containing an aromatic ring, particularly a benzylamino group, is introduced into the sugar structure.

[Disclosure of the Invention]

The present invention relates to a substance exhibiting cell membrane damage, in particular, a substance exhibiting bacteriostatic cell membrane damage that damages the cell membrane while allowing bacteria to proliferate, and its effectiveness. The purpose is to provide useful usage. In addition, an object of the present invention is to provide a substance that exhibits both excellent cell membrane damage and strong antibacterial properties, and an effective method for using the same.

The first invention to the 26th invention of the present application will be described below including the best embodiment and the functions and effects thereof.

(First invention)

The first invention of the present application has a carbocyclic structure or a carbon Z oxygen heterocyclic structure capable of multimerization, and is amino for two or more carbon atoms not participating in multimerization bonds on one side of the ring plane A bacteriostatic cell that binds a functional group and exhibits affinity for phospholipids on the other side of the ring plane, and damages the cell membrane while allowing the bacteria to grow. It is a cell membrane damaging monomer showing membrane damaging properties.

In the first invention, the “carbocyclic structure” is a ring structure formed only by carbon atoms and capable of multimerization, and does not participate in multimerization bonds. This refers to those capable of binding an amino functional group. In order to be able to bond an amino functional group, it is necessary to have a substituent or bondable group such as a hydroxyl group on one side of the ring plane. As a carbocyclic structure capable of bonding two or more amino functional groups, cyclohexanol or the like has 4 or more carbon atoms constituting the ring and does not participate in multimerization bonding. An aliphatic ring structure in which an atom has a hydroxyl group can be exemplified, but is not limited thereto. `` Carbon Z oxygen heterocycle structure '' is a ring structure formed by carbon atoms and oxygen atoms that can be multimerized, and is composed of two or more carbon atoms that do not participate in multimerization bonds. This refers to those capable of binding an amino functional group. Examples thereof include, but are not limited to, pentose or hexose sugar structures having a miacetal bond in the molecule. In addition, “showing phospholipid affinity” means that, on one side of the ring plane, hydrogen is mainly bonded to carbon constituting the ring structure. Say to show.

In general, it is known that a compound having a relatively high molecular weight and a large number of amino groups bound thereto exhibits bacteriostatic or antibacterial activity. Bacterial cell A of the first invention In Nomaichi, amino functional groups are intensively bonded to one side of the ring plane, so that it interacts with the negatively charged portion of the phospholipid of the cell membrane based on the brass charge of the amino functional group, It exhibits a certain degree of antibacterial action. At the same time, since no amino group is bonded to the other side of the ring plane, this one side shows lipophilicity, that is, phospholipid affinity.

The cell membrane damaging monomer of the first invention is similar to the cell membrane damaging oligomer, and is attacked on the cell membrane by analogy with the action and effect of the cell membrane damaging oligomer described below. It is thought that it exhibits certain antibacterial properties. In the compound of the ring structure, such an effect when the amino functional group is localized on one side surface is not suggested at all from various known literatures including the literature 1 and literature 2 described above. (Second invention)

In the second invention of the present application, the amino functional group according to the first invention is a primary, secondary or tertiary amino group.

There is no limitation on the type of amino functional group, and primary, secondary or tertiary amino groups can be used arbitrarily. As described later in the 16th invention, it is known that the antibacterial property is particularly enhanced when a specific type of amino functional group is introduced.

(Third invention)

In the third invention of the present application, the carbon-oxygen heterocyclic structure according to the first invention or the second invention is a sugar structure (monosaccharide) having a miacetal bond in the molecule.

In general, a sugar structure that has a ring structure formed by a miacetal bond into the molecule has a large number of hydroxyl groups bonded to the carbon atoms that constitute the ring structure, and these hydroxyl groups are attached to the ring plane on one side (6 The ring plane on the other OH group side is a lipophilic ring plane mainly composed of carbon-hydrogen bonds. Therefore, if an amino functional group is introduced at two or more positions of the above-mentioned hydroxyl bonding site, a bacterial cell typically having the characteristics of the first invention can be prepared easily. (Fourth invention)

In the fourth invention of the present application, the sugar structure according to the third invention is a bilanose structure or a furanose structure.

As the sugar structure (monosaccharide) having a miacetal bond in the molecule, a biranose structure or a furanose structure can be preferably exemplified.

(Fifth invention)

In the fifth invention of the present application, the bilanose structure according to the fourth invention is dalcose.

Examples of the bilanose structure, which is a sugar structure constituting the ring structure of the cell membrane damaging monomer, include glucose, fructose, galactose, and the like. Preferred examples include glucose.

(Sixth invention)

According to a sixth invention of the present application, two or more units of the cell membrane-damaging monomer according to any of the first to fifth inventions are bonded, and the ring planes to which the amino functional groups in each monomer are bonded have the same direction. It is a suitable compound, and is a cell membrane-damaging oligomer that exhibits bacteriostatic cell membrane damage that damages the cell membrane while allowing bacteria to proliferate.

The cell membrane damaging oligomer of the sixth invention is a cell membrane damaging monomer in which a plurality of the cell membrane damaging monomers are bonded by, for example, a hydroxyl group condensation reaction or the like, and the skeleton portion of the ring structure is glucose, fructose, galactose. Monosaccharide dimers (disaccharides), trimers (trisaccharides), monosaccharide multimers (oligosac power rides), dimers of hexanol, etc. Can be constructed.

According to the research of the present inventor, it was found that the cell membrane damaging oligomer exhibits a cell membrane damaging effect and a certain antibacterial effect. The mechanism is thought to be as follows.

In other words, the amino functional group bonded to the microbial cell II damaging oligomer is a positive ion. This interacts with the negatively charged part of the phospholipid in the bacterial cell membrane. At the same time, one side of the phospholipid affinity in the cell membrane damaging oligomer attacks the cell membrane of the bacteria and damages the cell membrane. Moreover, the damage to the cell membrane is not destructive (bactericidal), but is a partial (bacteriostatic) cell membrane injury that increases membrane permeability. Along with these cell membrane damaging effects, a certain antibacterial property is exhibited by one side surface of the cell membrane damaging oligomer to which an amino functional group is bonded. In a compound having a ring structure, such an effect in the case where an amino functional group is localized on one side of the compound is not suggested at all from various known documents including Document 1 and Document 2 described above.

It is presumed that the cell membrane damaging effect and certain antibacterial effect of the cell membrane damaging oligomer are relatively stronger than the cell membrane damaging monomer. However, in order to obtain an effective effect, in each monomer constituting the oligomer, it is preferable that the ring plane to which the amino functional group is bonded is directed in the same direction.

(Seventh invention)

In the seventh invention of the present application, the cell membrane-damaging oligomer according to the sixth invention is a cell membrane-damaging monomer in which 6 to 8 units are linked in a chain.

The number of monomers bonded to the cell membrane-damaging oligomer is not particularly limited, but more preferably 6 to 8 units of monomers linked in a chain.

(Eighth invention)

In the eighth invention of the present application, the chain-like conjugate of the cell membrane-damaging monomer according to the seventh invention is a cyclic linkage.

When the monomer constituting the cell membrane damaging oligomer forms a cyclic linkage, and the ring plane to which the amino functional group is bonded in each monomer faces the same direction, one ring plane side of the oligomer Since the concentration density of amino functional groups is high, the interaction with the negatively charged portion of the cell membrane phospholipid due to the positive charge of these amino functional groups is strengthened, and the antibacterial effect is relatively enhanced. Ori Due to the high lipophilicity (phospholipid affinity) of the other ring plane side of sesame, the cell membrane damage is relatively enhanced. Furthermore, when each monomer constituting the oligomer forms a cyclic chain bond, the structural condition that “the ring plane to which the amino functional group in each monomer is bonded is directed in the same direction” is stable. Maintained.

(9th invention)

In the ninth invention of the present application, the cyclic chain conjugate according to the eighth invention is a cyclodextrin which is a cyclic chain conjugate of glucose units.

As is well known, cyclodextrin is a chain conjugate (see Fig. 1) in which a number of D-dalcobilanose groups are cyclized in a crown shape by glycosidic linkages. Cyclodextrins consisting of 8 glucose units are particularly preferred.

In cyclodextrin, the higher the degree of substitution of the hydroxyl group on the ring plane on the 6-position OH group side of each glucose unit with an amino functional group, the stronger the antibacterial and cell membrane damage properties.

(Invention 10)

The tenth invention of the present application is selected from the cell membrane-damaging monomer according to any of the first to fifth inventions and the cell membrane-damaging oligomer according to any of the sixth to ninth inventions 1 This is an antibacterial cell membrane injury method that uses antibacterial membrane injury agents containing two or more species as active ingredients against Gram-positive bacteria to obtain an antibacterial effect.

The cell membrane of gram-negative bacteria such as E. coli consists of the outer membrane, cell wall, and inner membrane (cytoplasmic membrane) and is relatively less susceptible to antibacterial action. However, the cell membrane of gram-positive bacteria such as Staphylococcus aureus and MR SA Because it consists of cell walls and inner membrane and does not have outer membrane, it is relatively susceptible to cell membrane damage. It has been confirmed that the cell membrane damaging monomer and the cell membrane damaging oligomer according to the present invention result in an effective bactericidal effect on Gram-positive bacteria through the cell membrane damaging action. Yes.

In the cell membrane damaging agent of the tenth invention, the cell membrane damaging monomer as an active ingredient and And Z or fungus membrane-damaging oligomers can be used as they are, or can be used as salts or derivatives within the range where the effect is maintained and pharmaceutically acceptable. It may also contain bulking agents, preservatives, pH stabilizers and the like. Furthermore, the dosage form of the cell membrane injury agent is not limited. For example, it can be administered in a dosage form such as a powder, a tablet, a capsule, a liquid, an emulsion, or a suspension.

In addition, the active ingredient in the cell membrane damaging agent of the invention of the tenth invention exhibits antibacterial properties through a membrane-actuated mechanism, so that it is difficult to cause drug resistance and such as polymyxin B which is a known antibacterial substance of this kind. Since it is not a natural product having a complicated structure, artificial mass production is easy, and artificial structural modification for improving the effect is technically and cost-effective.

(First invention)

The 11th invention of the present application provides any purpose for the cell membrane-damaging monomer according to any one of the 1st to 5th inventions or the cell membrane-damaging oligomer according to any of the 6th to 9th inventions. In a bacteriostatic cell membrane injury method in which a substance is bound and used against bacteria, and the target substance is introduced into the cell body by damaging the cell membrane while retaining the growth ability of the bacteria. is there.

The first invention can be conceived for the first time based on the novel knowledge of the present inventor that the cell membrane-damaging monomer or cell membrane-damaging oligomer exhibits bacteriostatic cell membrane toxicity. It is an invention that has become possible.

As described above, these cell membrane-damaging substances (cell membrane-damaging monomers / oligomers) damage the cell membrane while allowing the bacteria to survive. That is, the damage of the cell membrane is not destructive, but is a partial cell membrane damage that increases the membrane permeability. Therefore, when any target substance is bound to these cell membrane damaging substances, the cell membrane damaging substances can be used as vectors to easily realize DDS (drug delivery system) for the cells. it can. The type of target substance can be arbitrarily selected and is not limited. Examples of preferred target substances include genes, proteins (antibodies, enzymes, etc.), inorganic compounds, and organic compounds that contribute to bacterial transformation can do

(Invention 12)

The first and second inventions of the present application are selected from the cell membrane-damaging monomers according to any one of the first to fifth inventions and the cell membrane-damaging oligomers according to any of the sixth to ninth inventions 1 It is a cell membrane injury / antibacterial agent comprising as an active ingredient a species or two or more species and one or more of any antibacterial or antibiotics.

The 12th invention is an invention that can be conceived for the first time on the basis of the novel knowledge of the present inventor that a cell membrane-damaging monomer or cell membrane-damaging oligomer exhibits cell membrane toxicity. It is.

That is, these cell membrane damaging substances do not necessarily show destructive cell membrane damaging action by themselves, but damage the bacterial cell membrane and increase the membrane permeability. As a result, when any antibacterial substances or antibiotics are used in combination, the antibacterial effects of these substances are exhibited without hindrance.

Moreover, since the cell membrane damaging substances are membrane-operated, pathogens are difficult to cope with, and it is difficult to produce resistant bacteria against antibacterial substances or antibiotics used in combination with cell membrane damaging substances. is there.

In the cell membrane injury / antibacterial agent of the 12th invention, the cell membrane injury substance, the antibacterial agent or the antibiotic which is the active ingredient may be used as it is, or the salt is maintained within the range where the effect is maintained and pharmaceutically acceptable. Alternatively, it can be used as a derivative. In addition, a bulking agent, a preservative, or a pH stabilizer may be included. Furthermore, the dosage form of the cell membrane injury agent is not limited. For example, it can be administered in dosage forms such as powders, tablets, capsules, solutions, emulsions, suspensions and the like.

(Invention 13)

In the thirteenth invention of the present application, the antibacterial substance or antibiotic according to the first and second inventions is a cell membrane impermeable substance.

Bacterial membrane injury / antibacterial agent according to the above-mentioned first and second inventions, There are no restrictions on the type of antibacterial or antibiotic used in combination, but the combination with an antibacterial or antibiotic that is impermeable to the bacterial membrane is particularly effective.

(14th invention)

In the 14th invention of the present application, the antibacterial substance impermeable to the cell membrane according to the 13th invention, wherein the antibiotic is erythromycin or novopyocin.

Erythromycin Novopiocin has excellent antibacterial activity, but its cell membrane impermeability, so its usefulness is limited. Therefore, it is very preferable to use these in combination with the cell membrane damaging substance according to the present invention.

(15th invention)

The 15th invention of the present application is an antibacterial method for obtaining an antibacterial effect by using the antibacterial membrane damage / antibacterial agent according to any of the 12th invention to the 14th invention against a gram positive bacterium or a gram negative bacterium. is there.

The cell membrane damaging substance (cell membrane damaging monomer and cell membrane damaging oligomer) according to the present invention is not necessarily effective against Gram-negative bacteria in its antibacterial effect, Is effective against Gram-positive or Gram-negative bacteria. Therefore, an antibacterial agent that damages the cell membrane using this substance in combination with an antibacterial substance or an antibiotic can effectively exert an antibacterial action against either gram-positive bacteria or gram-negative bacteria.

In particular, for gram-negative bacteria with a strong cell membrane consisting of three layers, the membrane is damaged by a cell membrane-damaging substance (a cell membrane-damaging monomer or cell membrane-damaging oligomer). The antibacterial method according to the 15th invention is particularly effective because the effective role sharing that the antibacterial action of the antibacterial substances and antibiotics that have entered the cells is fully exerted under the increased permeability is achieved. is there.

(Invention 16)

The 16th invention of the present application relates to a carbocyclic structure or carbon / oxygen heterocyclic structure capable of multimerization. In addition, an amino functional group containing an aromatic ring is bonded to two or more carbon atoms that do not participate in multimerization bonding on one side of the ring plane, and phosphorus is bonded on the other side of the ring plane. It is a compound showing lipophilicity, and is a cell membrane damage / antibacterial monomer that has both cell membrane damage and strong antibacterial properties.

In the 16th invention, the meanings of “carbocyclic structure”, “carbon Z oxygen heterocyclic structure” and “showing phospholipid affinity” are the same as in the first invention.

According to the research of the present inventor, among the above-mentioned cell membrane-damaging monomers, those having two or more amino functional groups containing an aromatic ring exhibit strong antibacterial properties along with the above-mentioned cell membrane membrane-damaging properties. It has been found. A compound having a carbocyclic structure or a carbon-oxygen heterocyclic structure and having two or more amino functional groups containing an aromatic ring bonded to one side of the ring plane has not been known as an antibacterial substance. The 16th invention provides a novel and powerful cell membrane damage / antibacterial substance.

Cell membrane damage according to the 16th invention · The reason why the antibacterial monomer exhibits strong antibacterial properties is under investigation and has not been elucidated, but the aromatic ring in the amino functional group brings hydrophobicity to the functional group. As a result of a strong attack on the hydrophobic part of the cell membrane phospholipid, it is considered that the cell membrane is severely damaged and exhibits strong antibacterial properties.

(Invention 17)

In the seventeenth invention of the present application, the amino functional group containing an aromatic ring according to the sixteenth invention is a benzylamino group.

In the above 16th invention, the kind of an amino functional group containing an aromatic ring is not limited, but a benzylamino group is particularly preferred. The benzylamino group includes a benzylamino group derivative in which any functional group is bonded to an aromatic ring as long as the effect of the invention is not inhibited.

(Invention 18)

The 18th invention of the present application is an amino official who contains two or more units of the cell membrane damage / antibacterial monomer according to the 16th invention or the 17th invention and contains an aromatic ring in each monomer. It is a compound in which the ring plane to which the functional group is bonded is oriented in the same direction, and is a cell membrane injury / antibacterial oligomer that has both cell membrane damage and strong antibacterial properties.

The cell membrane injury / antibacterial oligomer according to the eighteenth aspect of the invention is a combination of a plurality of the cell membrane injury / antibacterial monomers by, for example, a hydroxyl group condensation reaction. Dimers (disaccharides), trimers (trisaccharides), monosaccharides (oligosaccharides), or cyclohexanols of monosaccharides consisting of one or more of glucose, fructose, galactose, etc. It may be composed of a dimer or the like. .

Bacterial membrane damage and antibacterial effects of bacterial membrane damage oligomers are even stronger than those of cell membrane damage and antibacterial monomers. However, in order to obtain an effective effect, in each monomer constituting the oligomer, it is preferable that ring planes to which an amino functional group containing an aromatic ring is bonded face the same direction.

(Invention 19)

In the nineteenth invention of the present application, the bacterial membrane injury / antibacterial oligomer according to the eighteenth invention is a combination of 6-8 units of bacterial membrane injury / antibacterial monomers linked in a chain.

Bacterial membrane damage · The number of monomer bonds in the antibacterial oligomer is not particularly limited, but it is preferably one in which about 6 to 8 units of monomers are linked in a chain.

(20th invention)

In the 20th invention of the present application, the cell membrane injury / antibacterial oligomer according to the 19th invention is a cyclic chain conjugate of the cell membrane injury / antibacterial monomer.

Bacterial membrane damage · Monomers constituting antibacterial oligomers form a cyclic linkage, and the ring plane in which each monomer is bonded to an amino functional group containing an aromatic ring faces the same direction. And the antibacterial effect is particularly enhanced due to the high concentration density of the amino functional group containing an aromatic ring on one ring plane side of the oligomer. At the same time, the lipophilicity (phospholipid affinity on the other ring plane side of the oligomer) Cell) due to its high Membrane damage is particularly enhanced. Furthermore, when each monomer constituting the oligomer forms a cyclic linkage, “the ring planes to which amino functional groups containing aromatic rings in each monomer are bonded face the same direction.” The structural condition is maintained stably.

(Second invention)

In the 21st invention of the present application, the cyclic chain conjugate in the membrane damage / antibacterial oligomer according to the 20th invention is a cyclodextrin.

The cyclodextrin is as described above, but as the cell membrane damage / antibacterial oligomer, those in which the cyclic chain conjugate is cyclodextrin are particularly preferable.

Substituting most or all of the hydroxyl groups on the 6-position OH group side of each glucose constituting cyclodextrin with the above benzylamino group exhibits excellent cell membrane damage and antibacterial properties, and against gram-positive bacteria. Of course, it is effective, but it may show effective antibacterial properties against gram-negative bacteria.

(Second invention)

The 22nd invention of the present application is the fungus body according to the 16th invention or the 17th invention)] injuries ■ antibacterial monomers and cell membrane damage / antibacterial according to any of the 18th invention to the 21st invention A cell membrane injury / antibacterial agent comprising one or more selected from sex oligomers as an active ingredient.

The 22nd invention provides a novel antibacterial agent for cell membrane injury that has both excellent cell membrane injury and strong antibacterial properties. In this antibacterial agent / antibacterial agent, the active ingredient cell membrane damage, antibacterial monomer and / or cell membrane injury, antibacterial oligomer can be used as they are, and the effect is maintained and pharmaceutically acceptable. To the extent possible, it can also be used as a salt or a derivative. In addition, it may contain a bulking agent, a preservative, or a pH stabilizer. Furthermore, the dosage form of the cell membrane damaging agent is not limited, and for example, it can be administered in dosage forms such as powders, tablets, capsules, liquids, emulsions, suspensions and the like. In addition, the active ingredient in the cell membrane damaging agent of the second aspect of the invention exhibits antibacterial properties through a membrane-actuated mechanism, and thus is difficult to cause drug resistance. Since it is not a natural product having such a complicated structure, artificial mass production is easy, and artificial structural modification for improving the effect is technically and cost-effective.

(Second invention)

The second invention of the present application is the fungus membrane injury / antibacterial monomer according to the 16th invention or the 17th invention, and the fungus membrane injury according to any of the 18th invention to the 21st invention. A cell membrane injury / antibacterial agent comprising one or more selected from sesame and one or more of any antibacterial or antibiotics as active ingredients.

The 23rd invention is based on the novel knowledge of the present inventor that cell membrane injury, antibacterial monomer or cell membrane injury, and antibacterial oligomer exhibit excellent cell membrane damage as well as strong antibacterial properties. This is the first invention that can be conceived.

In other words, these bacterial membrane damages ■ antibacterial substances (bacterial membrane injury, antibacterial monomer and Z or fungal body)] huge injury / antibacterial oligomer) itself show excellent antibacterial action and bacteria The membrane permeability is increased by damaging the cell membrane. As a result, when any antibacterial substance or antibiotic is used in combination, these antibacterial effects also work effectively, so that both antibacterial effects are exerted synergistically, and an extremely strong antibacterial action can be expected as a whole.

Cell membrane damage · Antibacterial substances are membrane-acting, so pathogens are difficult to cope with. Bacterial membrane injury · Bacteria resistant to antibiotics or antibiotics used in combination with antibacterial substances There is an advantage that it is difficult to do.

In the antibacterial agent of the second aspect of the invention, the antimicrobial agent may be used as it is, or the antibacterial substance, antibacterial substance or antibiotic may be used as it is, and the effect is maintained and pharmaceutically acceptable. It can also be used as a salt or derivative in the range. It may also contain bulking agents, preservatives, pH stabilizers and the like. Furthermore, the dosage form of the cell membrane damaging agent is not limited. For example, powders, tablets, capsules, liquids, emulsions, suspensions, etc. It can be administered in dosage form. (24th invention)

In the 24th invention of the present application, the antibacterial substance or the antibiotic according to the 23rd invention is a cell membrane impermeable substance.

In the above-mentioned cell membrane injury / antibacterial agent according to the second aspect of the invention, the type of antibacterial substance or antibiotic used in combination with the cell membrane injury / antibacterial agent is not limited, but the cell membrane membrane has non-permeability antibacterial properties Combination with substances or antibiotics is particularly effective.

(25th invention)

In the 25th invention of the present application, the antibacterial substance or antibiotic that is impermeable to cell membranes according to the 24th invention is erythromycin or novobiocin.

Erythromycin Novopiocin has excellent antibacterial activity, but its cell membrane impermeability, so its usefulness is limited. Therefore, it is extremely preferable to use these in combination with the cell membrane injury / antibacterial substance according to the present invention.

(26th invention)

The 26th invention of the present application is an antibacterial method for obtaining an antibacterial effect by using an antibacterial agent against Gram-positive bacteria or Gram-negative bacteria according to any one of the 22nd to 25th inventions. is there.

The cell membrane injury / antibacterial substance (cell membrane injury / antibacterial monomer and cell membrane injury 'antibacterial oligomer) according to the present invention is not only effective against gram positive bacteria but also gram negative In some cases, antibacterial action is also effective against bacteria.

In addition to this bacterial membrane damage 'antibacterial substance', in combination with any antibacterial substance or antibiotic effective against gram positive bacteria and / or gram negative bacteria, A more advantageous antibacterial effect can be obtained.

[Brief description of the drawings] Fig. 1 shows a simplified structure of cyclodextrin. FIG. 2 is a list showing the effects of the embodiment. FIG. 3 is a list showing the effects of the embodiment.

〔Example〕

(Example 1)

This example relates to the synthesis of octakis (6-benzenoreamino) 1γ-cyclodextrin.

Okutakisu (6- o-tosyl) Single γ- cyclodextrin (150 mg, 5. 93 XI 0 one ⁵ mol) vacuum distilled Benjiruamin the (24 ml) was added, and stirred between 4:00 5 at 9 0 ° C . Thereafter, the reaction solution was distilled off under reduced pressure, and water (1 ml) was added to the resulting residue. After neutralizing this with lmol / 1 hydrochloric acid aqueous solution, the resulting precipitate was filtered off and the solution obtained by gel filtration chromatography was used to obtain white octakis (6-benzylamino) γ-cyclodextrin. A solid (11.8 mg) was obtained. This structure was determined by a 300 MHz proton-nuclear magnetic resonance spectrum.

(Example 2)

In this example, octakis (6-amino) 1γ-cyclodextrin, heptakis (6-amino) 1 | 3-cyclodextrin, hexakis (6-amino) 1 α-cyclodextrin, octakis (6-benzylamino) 1 γ-cyclodextrin, heptakis (6-benzylamino) 1 / 3-cyclodextrin, and hexakis (6-benzylamino) 1 a-cyclodextrin.

Escherichia coli K12 and W3110 were cultured in a minimal salt medium containing 1% polypeptone at 37 ° C under aerobic conditions. Staphylococcus aureus 209P strain, 0 - ^5% meat extract, 1.5% Po Ripeputon were cultured in liquids medium containing 0.5% sodium chloride and 0.5% dipotassium hydrogen phosphate. The bacteria were diluted with physiological saline and used for measurement. The MIC was measured by a method based on micro liquid dilution. At this time, the drug was prepared in a 2-fold dilution series with purified water. A thing was used. Bacteria diluted to 1 × 10 ⁴ (“C” means the number of bacteria) were incubated in Mueller-Hinton medium (Difco) at 37 ° C. for 20 hours. The MIC was determined at the lowest drug concentration at which the bacteria could not grow.

As a result, the minimum growth inhibitory concentration (MIC) for E. coli (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacteria) of the former three was 128 g / ml or more. On the other hand, the minimum growth inhibitory concentrations of the latter three are 64, 64, 32 g / ml for Escherichia coli (Gram-negative bacteria) and 4, 4, 8 for Staphylococcus aureus (Gram-positive bacteria), respectively. The antibacterial activity was remarkable against gram positive bacteria at g / ml.

(Example 3)

This example shows octakis (6-amino) 1 γ-cyclodextrin, heptakis (6-amino) 1] 3-cyclodextrin, and hexakis (6-amino) _ _α -cyclodextrin for bacterial cell membrane permeability. This is related to the increase effect.

For Escherichia coli, K12 and W3110 strains were used, and for Staphylococcus aureus, 209 strains were used. Escherichia coli K12 and W3110 were cultured in a minimal salt medium containing 1% polypeptone at 37 ° C under aerobic conditions. S. aureus strain 209P was cultured in a liquid medium containing 0.5 ° / ₀ meat extract, 1.5% polybeptone, 0.5% sodium chloride, and 0.5% dibasic hydrogen phosphate. Bacteria collected in the logarithmic growth phase were washed twice with a buffer solution (lOOmM choline chloride, 50 mM M0PS-TRIS (pH 7.2)) and then suspended in the same buffer solution to a protein concentration of 10 mg / ml. . Escherichia coli and Staphylococcus aureus should be buffered (100 mM choline chloride, 10 m TRIS-lactic acid, 50 mM MOPS-TRIS (pH 7.2)) so that the final protein concentrations are 0.5 and 0.2 mg / ml, respectively. ).

Each of the above compounds was administered to bacteria, and the efflux of potassium ions from the bacteria was examined using an ion selective electrode. The table shown in Fig. 2 shows the results of potassium ion efflux rates (%) for E. coli and S. aureus.

The observed outflow of potassium ions causes these compounds to invade the outer membrane of the bacteria, and further attacks the cytoplasmic membrane (inner membrane), causing turbulence, destabilization, and destruction of the inner membrane. Thus, it can be seen that intracellular potassium ions are released to the outside of the cell. In particular, it was concluded that the activity of benzylamineated derivatives against Staphylococcus aureus was prominent and thus showed antibacterial activity.

(Example 4)

In this example, octakis (6-amino) 1 γ-cyclodextrin, heptakis (6-amino) 1 | 3-cyclodextrin, and hexakis (6-amino) —α-cyclodextrin and other antibiotics It is related with the effect in combination with.

From the results of Example 3, it is clear that these compounds damage the cytoplasmic membrane of Escherichia coli and cause the release of force lithium ions. Therefore, we investigated the combined effects of novobiocin and erythromycin, which are antibiotics that cannot exhibit their original antibacterial properties due to their low outer membrane permeability. The results are shown in the table shown in Fig. 3.

The minimum growth inhibition concentrations (MIC) for E. coli with novobiocin and erythromycin are 128 / ^ 1 and 64 g / nil, respectively, and their effects are weak. On the other hand, Ml C of the above antibiotics under the combined administration of the cyclodextrin derivative was remarkably reduced and showed a remarkable antibacterial activity. Based on the above results, these cyclodextrin derivatives have the effect of damaging the bacterial outer membrane and significantly increasing its permeability, resulting in the original antibacterial activity of non-transmembrane antibiotics. Their usefulness in combination with antibiotics has been revealed.

[Industrial application fields]

According to the present invention, a compound that is easy to produce, and has a cell membrane damage property, particularly a bacteriostatic cell membrane damage property that damages the cell membrane while remaining proliferative ability in bacteria. Provided are substances that exhibit both excellent cell membrane damage and strong antibacterial properties, and effective use thereof.

Claims

1. An amino functional group is bonded to two or more carbon atoms that have a carbocyclic structure or carbon-oxygen heterocyclic structure capable of multimerization and that do not participate in multimerization bonding on one side of the ring plane On the other side of the ring plane, it is a compound that exhibits affinity for phospholipids, and is a bacteriostatic cell that damages the cell membrane while allowing the bacterium to retain its growth ability)! Bacterial membrane-damaging monomer that exhibits enormous damage.

Contract

2. The cell membrane-damaging monomer according to claim 1, wherein the amino functional group is a primary, secondary or tertiary amino group.

of

3. The cell membrane-damaging monomer according to claim 1 or 2, wherein the carbon-oxygen heterocyclic structure is a sugar structure having a miacetal bond in the molecule.

4. The cell membrane-damaging monomer according to claim 3, wherein the sugar structure is a pyranose structure or a furanose structure.

5. The cell membrane-damaging monomer according to claim 4, wherein the bilanose structure is glucose.

6. A compound in which two or more units of the cell membrane-damaging monomer according to any one of claims 1 to 5 are bonded, and ring planes to which an amino functional group is bonded in each monomer are directed in the same direction. A cell membrane-damaged oligomer that exhibits bacteriostatic cell membrane damage, which causes damage to the cell membrane while allowing bacteria to proliferate.

7. The cell membrane damaging oligomer according to claim 6, wherein the cell membrane damaging monomer is 6-8 units linked in a chain.

8. The cell membrane-damaging oligomer according to claim 7, which is a cyclic chain-bound product of the cell membrane-damaging monomer.

9. The cell membrane-damaging oligomer according to claim 8, wherein the cyclic chain conjugate is cyclodextrin.

10. A cell membrane damaging monomer according to any one of claims 1 to 5 and a cell membrane damaging oligomer according to any one of claims 6 to 9, Using a cell membrane injury agent containing two or more active ingredients against Gram-positive bacteria, An antibacterial cell membrane injury method for obtaining an antibacterial effect.

1 1. Any desired substance may be added to the cell membrane damaging monomer according to any one of claims 1 to 5 or the cell membrane damaging oligomer according to any one of claims 6 to 9. A bacteriostatic cell membrane injury method in which the target substance is introduced into the microbial cell by damaging the cell membrane while remaining microbial growth ability, using the same against bacteria.

1 2. A cell membrane-damaging monomer according to any one of claims 1 to 5 and a cell membrane-damaging oligomer according to any one of claims 6 to 9, A cell membrane injury / antibacterial agent comprising two or more and one or more of any antibacterial substance or antibiotic as active ingredients.

13. The cell membrane injury / antibacterial agent according to claim 12, wherein the antibacterial substance or antibiotic is a cell membrane impermeable substance.

14. The cell membrane injury / antibacterial agent according to claim 13, wherein the cell membrane impermeable antibacterial substance or antibiotic is erythromycin or novobiocin.

15. An antibacterial method for obtaining an antibacterial effect by using the antibacterial agent against gram-positive bacteria or gram-negative bacteria according to any one of claims 1 to 14.

16 6. Having a carbocyclic structure or a carbon-oxygen heterocyclic structure capable of multimerization, and an aromatic ring for two or more carbon atoms that do not participate in multimerization bonds on one side of the ring plane. Is a compound that binds an amino functional group containing phosphatase and exhibits phospholipid affinity on the other side of the ring plane. Antibacterial monomer.

17. The cell membrane damage / antibacterial monomer according to claim 16, wherein the amino functional group containing an aromatic ring is a benzylamino group.

1 8. Claims 16 or 17 Cell membrane damage · Antibacterial monomers are bonded in 2 units or more, and the ring planes where the amino functional groups containing aromatic rings in each monomer are bonded are the same. It is a compound that is facing the direction, and is a cell membrane injury / antibacterial oligomer that has both cell membrane damage and strong antibacterial properties.

1 9. Cell membrane damage · Antibacterial monomer linked 6-8 units in a chain The cell membrane injury / antibacterial oligomer according to claim 18, which is

20. The cell membrane injury / antibacterial oligomer according to claim 19, wherein the cell membrane injury is a cyclic linkage of antibacterial monomers.

21. The rod membrane damage / antibacterial oligomer according to claim 20, wherein the cyclic chain conjugate is cyclodextrin.

2 2. Claims 16 or 17 Bacterial Membrane Damage ■ Antibacterial Monomer and Claims 18 to 21 Bacterial Membrane Damage ■ Antibacterial Oligomer Bacterial membrane injury / antibacterial agent comprising one or more selected from as active ingredients.

2 3. Claims 16 or 17 Bacterial membrane injury / antibacterial monomer according to claim 18 and Claims 18 to 21 Bacterial membrane injury / antibacterial oligomer A cell membrane injury / antibacterial agent comprising as an active ingredient one or more selected from the group consisting of one or more of any antibacterial substance or antibiotic.

24. The cell membrane injury / antibacterial agent according to claim 23, wherein the antibacterial substance or antibiotic is impermeable to the cell membrane.

25. The cell membrane injury / antibacterial agent according to claim 24, wherein the cell membrane impermeable antibacterial substance or antibiotic is erythromycin or novopyocin.

26. An antibacterial method for obtaining an antibacterial effect by using the antibacterial agent against gram-positive or gram-negative bacteria according to any one of claims 2 to 25.