JP7701713B2 - Viral infection inhibitor and method for inhibiting viral infection of cells - Google Patents

Description

The present invention relates to a viral infection inhibitor and a method for inhibiting viral infection in cells.

In cell cultures and samples collected from biological organs, etc., a plurality of cells may aggregate to form cell clusters. When measuring a sample containing such cell clusters using a flow cytometer, etc., a process for dispersing the cell clusters and/or a process for preventing the cells from re-aggregating after the dispersion process may be required.
Patent Document 1 describes a cell aggregation inhibitor containing a specific compound.

JP 2020-48446 A

The present invention aims to provide a viral infection inhibitor capable of suppressing viral infection of cells. Another objective of the present invention is to provide a method for suppressing viral infection of cells.

As a result of extensive research into achieving the above object, the inventors have discovered that the above object can be achieved by the following configuration.

[1] A viral infection inhibitor comprising a compound represented by formula 1 described below, which inhibits viral infection of cells by inhibiting viral attachment to cells.
[2] The virus infection inhibitor according to [1], wherein the hydrophilic polymer Y in the formula 1 described below is at least one selected from the group consisting of polyalkylene glycols and polyglycerols.
[3] The virus infection inhibitor according to [1] or [2], wherein the hydrophilic polymer has a weight average molecular weight of 4,400 or more.
[4] The viral infection inhibitor according to any one of [1] to [3], wherein the chain length of the polymer chain of Y in formula 1 described below is 4.9 nm or more.
[5] A method for inhibiting viral infection in cells, comprising adding the viral infection inhibitor according to any one of [1] to [4] to an object containing at least one selected from the group consisting of a tissue consisting of cells and an intercellular matrix, and cells, and a liquid medium.

The present invention provides a virus infection inhibitor that can suppress virus infection in cells. The present invention also provides a method for suppressing virus infection in cells.

FIG. 1 is a schematic diagram showing a specific compound immobilized on a cell. Scanning electron microscope images of Hek293T cells bound with PEG-CLS.

The present invention will be described in detail below.
The following description of the components may be based on a representative embodiment of the present invention, but the present invention is not limited to such an embodiment.
In this specification, a numerical range expressed using "to" means a range that includes the numerical values before and after "to" as the lower and upper limits.

In the description of groups (atomic groups) in this specification, the description without indicating whether substituted or unsubstituted includes both unsubstituted and substituted groups, as long as it does not impair the effects of the present invention. For example, "alkyl group" includes not only alkyl groups without a substituent (unsubstituted alkyl groups), but also alkyl groups with a substituent (substituted alkyl groups). This also applies to each compound.
In this specification, the term "(poly)oxyalkylene" refers to both or either of polyoxyalkylene and oxyalkylene.

[Virus infection inhibitor]
The viral infection inhibitor according to an embodiment of the present invention contains a compound represented by formula 1 described below (hereinafter, also referred to as the "specific compound").

In formula 1, n and m each independently represent an integer of 1 or more, X is at least one group selected from the group consisting of a monovalent group obtained by removing any hydrogen atom from at least one selected from the group consisting of sterols and sterol derivatives, a linear or branched alkyl group having 8 or more carbon atoms, a linear or branched alkenyl group having 8 or more carbon atoms, and a linear or branched alkynyl group having 8 or more carbon atoms, multiple Xs may be the same or different, and multiple Xs may be linked together to form a ring, Y is a polymer chain made of a hydrophilic polymer having an effective length of 4.5 nm or more measured from the fluorescence resonance transfer efficiency of the terminal in an aqueous solution, when n+m is 2, L is a single bond or a divalent group, and when n+m is 3 or more, L is an n+m-valent group.

The above-mentioned virus infection inhibitor can be used by adding it to tissues consisting of cells and intercellular matrix, and cells (hereinafter also referred to as "tissues, etc."). In particular, it is preferable to add it to tissues, etc., to which a liquid medium has been added. It is also preferable to disperse the virus infection inhibitor in a liquid medium in advance, and then add the tissues, etc. to the liquid medium.

In the specific compound, the portion represented by X in formula 1 (hereinafter also referred to as the "hydrophobic portion") has a high affinity for the cell membrane of a cell. In other words, the above-mentioned hydrophobic portion is easily adsorbed to the cell membrane and/or easily binds to the cell membrane. In other words, the specific compound is easily fixed to the cell membrane using the hydrophobic portion as an anchor.

The specific compound also has a moiety represented by Y in formula 1 (hereinafter also referred to as a "hydrophilic moiety").
Fig. 1 is a schematic diagram showing a specific compound immobilized on a cell. As shown in Fig. 1, when a specific compound having a hydrophobic portion 2 and a hydrophilic portion 3 is added to a mixture containing a liquid medium and a cell 1, the hydrophobic portion is immobilized on the cell membrane side, and the hydrophilic portion protrudes to the outside of the cell (liquid medium side).

When this hydrophilic portion is made of a hydrophilic polymer whose effective length, measured from the fluorescence resonance transfer efficiency of the terminal in an aqueous solution, is 4.5 nm or more, it is surprisingly effective in suppressing the attachment of virus 4 to the cell membrane. On the other hand, if it is less than 4.5 nm, a sufficient effect cannot be obtained.

The above chain length refers to a value obtained by measuring the efficiency of fluorescence resonance transfer between a pair of fluorescent dyes with fluorescent properties that allow fluorescence resonance transfer to occur, which are attached to both ends of a polymer in an aqueous solution, by changing the length of the polymer, and calculating the distance between the polymer ends from a theoretical formula for fluorescence resonance transfer efficiency.

The content of the specific compound in the viral infection inhibitor is not particularly limited, but in order to obtain a viral infection inhibitor having a more excellent effect of the present invention, it is generally preferred that the content be 0.01 to 99.9 mass% relative to the total mass of the viral infection inhibitor.
The viral infection inhibitor may contain one specific compound alone or two or more specific compounds. When the viral infection inhibitor contains two or more specific compounds, the total content thereof is preferably within the above numerical range.

In formula 1, n and m are each independently an integer of 1 or greater and are not particularly limited, but are each independently preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 or 2.

Among these, the specific compound is preferably a compound represented by formula 1A: (X) _n -L _1a -Y, in that a viral infection inhibitor having the more excellent effect of the present invention can be obtained. In formula 1A, n, X, and Y have the same meanings as the respective symbols in formula 1. Furthermore, L _1a is an n+1 valent group, and its form is the same as L in formula 1.

<Hydrophobic site>
In formula 1, the hydrophobic moiety represented by X is at least one group selected from the group consisting of a monovalent group obtained by removing any hydrogen atom from at least one group selected from the group consisting of a sterol and a sterol derivative (hereinafter also referred to as "substituent A"), a linear or branched alkyl group having 8 or more carbon atoms, a linear or branched alkenyl group having 8 or more carbon atoms, and a linear or branched alkynyl group having 8 or more carbon atoms. In terms of obtaining a viral infection inhibitor having a more excellent effect of the present invention, at least one group selected from the group consisting of the substituent A, a linear or branched alkyl group having 8 or more carbon atoms, and a linear or branched alkenyl group having 8 or more carbon atoms is preferred, at least one group selected from the group consisting of the substituent A and a linear or branched alkyl group having 8 or more carbon atoms is more preferred, and the substituent A is even more preferred.

(Substituent A)
The substituent A is a monovalent group obtained by removing any hydrogen atom from at least one member selected from the group consisting of sterol and sterol derivatives.
In this specification, a sterol derivative means a compound having a steroid nucleus, a hydroxy group bonded to the carbon atom at position 3 of the steroid nucleus, and at least one of the hydrogen atoms bonded to the carbon atom of the steroid nucleus is substituted with any monovalent group. The monovalent group is not particularly limited, but examples thereof include the substituent W described below, and among these, hydrocarbon groups, hydroxy groups, halogen atoms, and the like are preferred.

The substituent A is not particularly limited, but is preferably a monovalent group obtained by removing any one hydrogen atom from the compound represented by the following formula 2, in order to obtain a viral infection inhibitor having a more excellent effect of the present invention.

In formula 2, rings A to D represent a saturated or unsaturated steroid nucleus, and in formula 2, R is a hydrogen atom or a monovalent group. The monovalent group is not particularly limited, but examples include the substituent W described below. Of these, in order to obtain a viral infection inhibitor having a more excellent effect of the present invention, a monovalent hydrocarbon group which may have a heteroatom is more preferred, and a linear, branched, or cyclic alkyl group is more preferred. The number of carbon atoms in the alkyl group is not particularly limited, but is preferably 1 to 20, more preferably 2 to 10, and even more preferably 3 to 8.
Furthermore, the hydrogen atoms bonded to the carbon atoms of the steroid nucleus may be substituted with monovalent groups, and examples of the monovalent groups include the substituent W described below.

More specifically, the compounds represented by formula 2 include phytosterols such as campesterol, campestanol, brassicasterol, 22-dehydrocampesterol, stigmasterol, stigmastanol, 22-dihydrospinasterol, 22-dehydrostigmastanol, 7-dehydrostigmasterol, sitosterol, tirucallol, euhol, fucosterol, isofucosterol, kojisterol, clionasterol, poriferasterol, clerosterol, 22-dehydroclerosterol, fungisterol, chondrilasterol, avenasterol, vernosterol, and polinastanol;
Animal sterols such as cholesterol, dihydrocholesterol, cholestanol, coprostanol, epicoprosterol, epicoprostanol, 22-dehydrocholesterol, desmosterol, 24-methylenecholesterol, lanosterol, 24,25-dihydrolanosterol, norlanosterol, spinasterol, dihydroagnosterol, agnosterol, lophenol, and lathosterol;
Fungal sterols such as dehydroergosterol, 22,23-dihydroergosterol, episterol, ascosterol, and fecosterol;
Examples include:

The bonding position between the compound represented by formula 2 and L in formula 1 is not particularly limited, but a hydroxy group bonded to the carbon atom at position 3 of the steroid nucleus is preferred. The bond between the hydroxy group and L can be easily formed, for example, by an ether bond, an ester bond, an amide bond, or the like.
Although there is no particular limitation, the specific compound in which Y is the substituent A is preferably, for example, a compound represented by the following formula (3).

In formula 3, rings A to D and R have the same meanings as the respective symbols in formula 2. In addition, in formula 3, Y and m have the same meanings as the respective symbols in formula 1.
In formula 3, L _b is a single bond or —C(O)—, L _c is a single bond or an m+1 valent group, and the form of the m+1 valent group of L _c is the same as that of L in formula 1.

Among these, the monovalent group obtained by removing any hydrogen atom from at least one selected from the group consisting of sterols and sterol derivatives is preferably a group represented by the following formula 3, in that it provides a viral infection inhibitor having a more excellent effect of the present invention.

In formula 4, R is a hydrogen atom or a monovalent group, and the monovalent group is not particularly limited, but examples include the substituent W described below. Of these, in order to obtain a viral infection inhibitor having a more excellent effect of the present invention, a monovalent hydrocarbon group which may have a heteroatom is more preferred, and a linear, branched, or cyclic alkyl group is more preferred. The number of carbon atoms in the alkyl group is not particularly limited, but is preferably 1 to 20, more preferably 2 to 10, and even more preferably 3 to 8.
Additionally, * indicates a bonding position.

When the hydrophobic moiety is a linear or branched alkyl group having 8 or more carbon atoms, the number of carbon atoms is not particularly limited, but is preferably 10 or more, more preferably 12 or more, even more preferably 13 or more, particularly preferably 14 or more, and most preferably 15 or more. The upper limit of the number of carbon atoms is not particularly limited, but is generally preferably 30 or less, more preferably 24 or less.
As the linear or branched alkyl group having 8 or more carbon atoms, a linear alkyl group having 8 or more carbon atoms is preferred.

When the hydrophobic moiety is a linear or branched alkenyl group having 8 or more carbon atoms, the number of carbon atoms is not particularly limited, but is preferably 10 or more, more preferably 12 or more, even more preferably 13 or more, particularly preferably 14 or more, and most preferably 15 or more. The upper limit of the number of carbon atoms is not particularly limited, but is generally preferably 30 or less, more preferably 24 or less.
As the linear or branched alkenyl group having 8 or more carbon atoms, a linear alkenyl group having 8 or more carbon atoms is preferred.

When the hydrophobic moiety is a linear or branched alkynyl group having 8 or more carbon atoms, the number of carbon atoms is not particularly limited, but is preferably 10 or more, more preferably 12 or more, even more preferably 13 or more, particularly preferably 14 or more, and most preferably 15 or more. The upper limit of the number of carbon atoms is not particularly limited, but is generally preferably 30 or less, more preferably 24 or less.
As the linear or branched alkynyl group having 8 or more carbon atoms, a linear alkynyl group having 8 or more carbon atoms is preferred.

<L>
In formula 1, L is a single bond or a divalent group when n+m (the sum of n and m) is 2. When n+m is 3 or more, L is an n+m-valent group.

The divalent group for L is not particularly limited, and examples thereof include -C(O)-, -C(O)O-, -OC(O)-, -O-, -S-, -NR ₂ - (R ₂ represents a hydrogen atom or a monovalent organic group), -O-P(O)(OH)-, -O-P(O)(O ^- M ⁺ )-, an alkylene group (preferably having 1 to 10 carbon atoms), a cycloalkylene group (preferably having 3 to 10 carbon atoms), an alkenylene group (preferably having 2 to 10 carbon atoms), and combinations thereof. M ⁺ is a counter ion, and examples thereof include Na ⁺ and NH ₄ ⁺ .

The trivalent or higher group for L is not particularly limited, but examples include groups represented by the following formulas (1a) to (1d).

In formula 1a, _L3 represents a trivalent group. _T3 represents a single bond or a divalent group, and three _T3 may be the same or different.
_L3 includes a trivalent hydrocarbon group (preferably having 1 to 10 carbon atoms. The hydrocarbon group may be an aromatic hydrocarbon group or an aliphatic hydrocarbon group) or a trivalent heterocyclic group (preferably a 5- to 7-membered heterocyclic group), and the hydrocarbon group may contain a heteroatom (for example, -O-). Specific examples of _L3 include a glycerin residue, a trimethylolpropane residue, a phloroglucinol residue, and a cyclohexanetriol residue.

In formula 1b, _L4 represents a tetravalent group. _T4 represents a single bond or a divalent group, and the four _T4s may be the same or different from each other.
Suitable forms of _L4 include a tetravalent hydrocarbon group (preferably having 1 to 10 carbon atoms. The hydrocarbon group may be an aromatic hydrocarbon group or an aliphatic hydrocarbon group) and a tetravalent heterocyclic group (preferably a 5- to 7-membered heterocyclic group), and the hydrocarbon group may contain a heteroatom (for example, -O-). Specific examples of _L4 include a pentaerythritol residue and a ditrimethylolpropane residue.

In formula 1c, _L5 represents a pentavalent group. _T5 represents a single bond or a divalent group, and the five _T5s may be the same or different from one another.
Suitable examples of _L5 include a pentavalent hydrocarbon group (preferably having 2 to 10 carbon atoms. The hydrocarbon group may be an aromatic hydrocarbon group or an aliphatic hydrocarbon group) or a pentavalent heterocyclic group (preferably a 5- to 7-membered heterocyclic group), and the hydrocarbon group may contain a heteroatom (for example, -O-). Specific examples of _L5 include an arabinitol residue, a phloroglucidol residue, and a cyclohexanepentanol residue.

In formula 1d, _L6 represents a hexavalent group. _T6 represents a single bond or a divalent group, and the six _T6 may be the same or different from each other.
Suitable examples of _L6 include a hexavalent hydrocarbon group (preferably having 2 to 10 carbon atoms. The hydrocarbon group may be an aromatic hydrocarbon group or an aliphatic hydrocarbon group) or a hexavalent heterocyclic group (preferably a 6- to 7-membered heterocyclic group), and the hydrocarbon group may contain a heteroatom (e.g., -O-). Specific examples of _L6 include a mannitol residue, a sorbitol residue, a dipentaerythritol residue, a hexahydroxybenzene residue, and a hexahydroxycyclohexane residue.

In formulae 1a to 1d, specific examples and preferred forms of the divalent groups represented by T ₃ to T ₆ may be the same as the divalent groups of L already explained.
When L is a group having a valence of 7 or more, a group obtained by combining the groups represented by formulae 1a to 1d can be used.

<Hydrophilic site>
In formula 1, Y is a hydrophilic moiety, and is a polymer chain made of a hydrophilic polymer, the effective length of which is measured from the fluorescence resonance transfer efficiency of the terminal in an aqueous solution and is 4.5 nm or more. In this specification, the term "polymer chain made of a hydrophilic polymer" means a partial structure in a specific compound obtained by binding a hydrophilic polymer to L, and the binding position of L in the hydrophilic polymer is not particularly limited, but may be, for example, the terminal of the hydrophilic polymer.

In this specification, a hydrophilic polymer means a polymer having a hydrophilic group.
The type of hydrophilic group is not particularly limited, and examples thereof include a polyoxyalkylene group (e.g., a polyoxyethylene group, a polyoxypropylene group, a polyoxyalkylene group in which an oxyethylene group and an oxypropylene group are blocked or randomly bonded), an amino group, a carboxy group, an alkali metal salt of a carboxy group, a hydroxy group, an alkoxy group, an amide group, a carbamoyl group, a sulfonamide group, a sulfamoyl group, a sulfonic acid group, and an alkali metal salt of a sulfonic acid group.

The structure of the main chain of the hydrophilic polymer is not particularly limited, and examples thereof include polyurethane, poly(meth)acrylic acid ester, polystyrene, polyester, polyamide, polyimide, polyether, and polyurea.
The term "poly(meth)acrylic acid ester" is a concept including both polyacrylic acid ester and polymethacrylic acid ester.

The hydrophilic polymer may also be a protein (glycoprotein). There are no particular limitations on the protein, so long as it is a molecule that does not have the function of inducing viral infection of cells by binding to a virus or activating molecules of the viral coat. In other words, a protein that can be used as a hydrophilic polymer is a protein that is different from a protein that induces viral binding and infection of cells. Alternatively, even if it is a protein molecule that induces viral infection when expressed from its gene in a cell, there is no limitation on the molecule, so long as it is added to a cell culture solution as an amphipathic molecule with a hydrophobic group added thereto and binds to the cell membrane.

It is generally believed that cell membrane-expressed molecules such as proteins are involved in the infection of cells by viruses. Examples of such involvement include specific cell surface proteins or other molecules that have affinity and bind to proteins expressed in the viral coat, i.e., receptors, and proteins that have the function of cleaving part of a protein expressed in the viral coat and transforming it into a form that can bind to proteins on the cell surface, i.e., proteases.

In either of the above cases, when a viral infection inhibitor according to an embodiment of the present invention is introduced, a structure is formed in which hydrophilic polymers having a specific chain length protrude three-dimensionally from the cell membrane surface, expanding the distance between the surfaces of nearby cells and the virus, decreasing the frequency with which molecules such as proteins on these cell membranes interact with molecules on the viral shell to function; in other words, it is presumed that a three-dimensional repulsive force is generated by the hydrophilic polymers protruding from the cell membrane surface.

Examples of hydrophilic polymers that may be used include synthetic polymers, proteins (including glycosylation), peptides, oligo-DNA, and glycochains (not including proteins).

Specific examples of hydrophilic polymers include polyvinylpyrrolidone, polyalkylene glycols, polyglycerin, poly(meth)acrylic acid esters, cellulose derivatives (e.g., carboxymethylcellulose, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, and hydroxyethylcellulose), starch derivatives (pullulan), polyvinyl alcohol, ethyl vinyl acetate, Eudragit, gelatin, polyacrylic acid, sodium polyacrylate, polyisobutylene maleic anhydride copolymer, alginic acid, sodium alginate, carrageenan, gum arabic, tragacanth, gum karaya, and polyvinyl methacrylate.

As the hydrophilic polymer, at least one selected from the group consisting of polyalkylene glycols and polyglycerins is preferred, in that a viral infection inhibitor having the effects of the present invention can be obtained.
Polyalkylene glycols and polyglycerins are presumed to have weak interactions with most biomolecules and weak interactions with viruses, and therefore are presumed to be more effective at suppressing infection regardless of the type of virus.

The hydrophilic moiety represented by Y in formula 1 is a partial structure (polymer chain) in a specific compound formed by binding the hydrophilic polymer to L, and the hydrophilic polymer has an effective length (chain length) of 5.0 nm or more as measured from the fluorescence resonance transfer efficiency of its terminal in an aqueous solution.
The chain length is not particularly limited as long as it is 4.5 nm or more; however, in terms of obtaining a viral infection inhibitor having a more excellent effect of the present invention, the chain length is preferably 4.9 nm or more, more preferably 7.0 nm or more, even more preferably 7.3 nm or more, particularly preferably 10 nm or more, and most preferably 10.9 nm or more.

On the other hand, although there is no particular upper limit to the chain length, a chain length of 20.0 nm or less is preferable in that the hydrophilicity of the viral infection inhibitor can be easily adjusted within an appropriate range, and the viral infection inhibitor can be more easily attached to the cell membrane of the cell to be inhibited.

The above chain length is measured by binding a fluorescent dye with fluorescent properties that can cause fluorescence resonance transfer to both ends of the hydrophilic polymer to be measured, dispersing the resulting sample in an aqueous solution, measuring the efficiency of fluorescence resonance transfer, and calculating the distance between the polymer ends from a theoretical formula for the efficiency of fluorescence resonance transfer. This method makes it possible to measure the effective length of the polymer chain.

For example, when the hydrophilic polymer is a polyalkylene glycol, one end is biotinylated and the other end is maleimide-modified, the biotin end is bound to fluorescent (DyLight649)-labeled streptavidin bound to a biotin-immobilized plastic plate, and the maleimide end is covalently bound to a cysteine group of another fluorescently labeled protein (e.g., IEkMCC labeled with Dylight549) to prepare a sample. When this sample is dispersed in water and the fluorescence intensity is measured, the fluorescence spectrum of DyLight549 and the absorption spectrum of DyLight649 overlap, so that fluorescence resonance transfer (FRET) occurs according to the distance between the two dyes, and the distance between the polymer ends can be calculated.

The above measurement method is described in PLoS One. 2014 Nov 10; 9(11): e112292, the contents of which are incorporated herein by reference.
The method for adjusting the chain length of the hydrophilic polymer is not particularly limited, but can be adjusted by the primary structure and molecular weight of the hydrophilic polymer, and such methods are known to those skilled in the art.

The molecular weight of the hydrophilic polymer is not particularly limited, but in order to obtain a viral infection inhibitor having a more excellent effect of the present invention, it is preferably 4,400 or more, more preferably 5,000 or more, even more preferably 9,400 or more, particularly preferably 10,000 or more, and most preferably 18,000 or more.
The upper limit of the molecular weight is not particularly limited, but in general, it is preferably 200,000 or less, and more preferably 100,000 or less.
In this specification, the molecular weight means a weight average molecular weight measured by gel permeation chromatography.

The specific compound is not particularly limited, but examples thereof include compounds represented by the following formulas.

In each formula, R represents a hydrogen atom or a monovalent group, _R1 represents a polymer chain of Y, p represents an integer of 2 or more, q represents an integer of 0 to 2, and M represents a counter ion (for example, Na ⁺ and _NH4 ⁺ ).
The specific compound can be synthesized by a known method, or a commercially available product manufactured by Sigma, Nanocs, Biochempeg, or Avanti Polar Lipids can also be used.

[Other ingredients]
The virus infection inhibitor according to the embodiment of the present invention may contain other components besides the specific compound within the range in which the effects of the present invention are exhibited. The other components are not particularly limited, but include a buffering agent, a chelating agent, and the like.

<Buffering Agent>
The viral infection inhibitor according to the embodiment of the present invention may contain a buffering agent.
The content of the buffering agent in the viral infection inhibitor is not particularly limited, but in order to obtain a viral infection inhibitor having the effects of the present invention that are more excellent, it is generally preferable that the content be 0.001 to 99% by mass relative to the total mass of the viral infection inhibitor. The viral infection inhibitor may contain one type of buffering agent alone, or may contain two or more types. When the viral infection inhibitor contains two or more types of buffering agents, it is preferable that the total content is within the above numerical range.

Buffering agents include, but are not limited to, sodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium acetate, and epsilon-aminocaproic acid.

<Chelating Agent>
The viral infection inhibitor according to the embodiment of the present invention may contain a chelating agent. The content of the chelating agent in the viral infection inhibitor is not particularly limited, but in order to obtain a viral infection inhibitor having a more excellent effect of the present invention, it is generally preferable that the content is 0.001 to 99 mass% relative to the total mass of the viral infection inhibitor. The viral infection inhibitor may contain one type of chelating agent alone, or may contain two or more types. When the viral infection inhibitor contains two or more types of chelating agents, the total content thereof is preferably within the above numerical range.

The chelating agent is not particularly limited, but examples thereof include hydroxy acid-based agents such as citric acid, malic acid, lactic acid, tartaric acid, gluconic acid, ascorbic acid, etidronic acid, and dihydroxyethylglycine (DHEG);
Aminocarboxylic acids such as ethylenediaminetetraacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, hydroxyethylethylenediaminetriacetic acid, tetrasodium glutamate diacetate, glycol ether diaminetetraacetic acid, triethylenetetraminehexaacetic acid, hydroxyethyliminodiacetic acid, ethylenediamine-N,N'-disuccinic acid, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid;
Ether carboxylic acids such as carboxymethyltartronic acid (CMT) and carboxymethyloxysuccinic acid (CMOS);
etc.

[Uses of viral infection inhibitors]
The viral infection inhibitor according to an embodiment of the present invention can be used by dispersing the viral infection inhibitor in a liquid medium and adding it to tissues, etc., for the purpose of inhibiting attachment of viruses to cells. It can also be added to tissues, etc., to which a liquid medium has been added. The viral infection inhibitor may also be dispersed in advance in a liquid medium, to which tissues, etc. are added. By using the viral infection inhibitor according to an embodiment of the present invention, attachment of viruses to the surface of each cell can be inhibited.

In addition, when used at an appropriate concentration and chain length range, the virus infection inhibitor according to the embodiment of the present invention can function as a cell dispersant for another purpose (JP 2020-048446 A). In other words, it can expand the distance between cells that adhere to and interact with each other.
Therefore, by adding a viral infection inhibitor according to an embodiment of the present invention, it is possible to suppress attachment of the virus to cells in the external solution, and to suppress intercellular infection of the virus budding from an already virally infected cell to adjacent/adherent cells.

In particular, when the hydrophilic polymer is polyalkylene glycol, it is more advantageous in that it is non-toxic and the chain length is easy to control. In the viral infection inhibitor according to the embodiment of the present invention, it is presumed that the polymer chain consisting of the hydrophilic polymer is not introduced into the cell. On the other hand, the polymer chain is fixed onto the cell membrane by the hydrophobic portion that is easily bonded and peeled off, and the hydrophilic polymer is held in the solution (in the cell suspension containing the cells) when necessary, and can be quickly removed by washing the solution when it is no longer needed.

(Substituent W)
Examples of the substituent W include a halogen atom, an alkyl group (e.g., methyl, ethyl, propyl, isopropyl, tert-butyl, pentyl, hexyl, octyl, dodecyl, tridecyl, tetradecyl, and pentadecyl), a cycloalkyl group (e.g., cyclopentyl and cyclohexyl), an alkenyl group (e.g., vinyl and allyl), an alkynyl group (e.g., ethynyl and propargyl), an aromatic hydrocarbon ring group (also called an aromatic carbon ring, aryl, etc., e.g., phenyl, p-chlorophenyl, mesityl, tolyl, xylyl, naphthyl, anthryl, azulenyl, acenaphthenyl, fluorenyl, phenanthryl, indenyl, pyrenyl, and biphenylyl), and an aromatic heterocyclic group (a 5- or 6-membered aromatic heterocyclic group, and a 5- or 6-membered aromatic heterocyclic group is also preferred). The heteroatom is preferably a sulfur atom, a nitrogen atom, an oxygen atom, a silicon atom, a boron atom, or a selenium atom, and examples thereof include pyridyl, pyrimidinyl, furyl, pyrrolyl, imidazolyl, benzimidazolyl, pyrazolyl, pyrazinyl, triazolyl (e.g., 1,2,4-triazol-1-yl and 1,2,3-triazol-1-yl), oxazolyl, benzoxazolyl, thiazolyl, isoxazolyl, isothiazolyl, furazanyl, thienyl, quinolyl, benzofuryl, dibenzofuryl, benzothienyl, dibenzothienyl, indolyl, carbazolyl, carbolinyl, diazacarbazolyl (representing a group in which one of the carbon atoms constituting the carboline ring of the carbolinyl group is replaced with a nitrogen atom), quinoxalinyl, pyridazinyl, triazinyl, quinazolinyl, phthalazinyl, borole, azaborine, etc.), heterocyclic groups (non-aromatic heterocyclic groups, which may be saturated or unsaturated, preferably 5- or 6-membered, and the heteroatoms constituting the ring are preferably sulfur, nitrogen, oxygen, silicon, or selenium atoms, for example, pyrrolidyl, imidazolidyl, morpholyl, oxazolidyl, etc.), alkoxy groups (for example, methoxy, ethoxy, propyloxy, pentyloxy, hexyloxy, octyloxy, dodecyloxy, etc.), cycloalkoxy groups (for example, cyclopentyloxy, cyclohexyloxy, etc.), aryloxy groups (for example, phenoxy, naphthyloxy, etc.), alkylthio groups (for example, methylthio, ethylthio, propylthio, pentylthio, hexylthio, octylthio, dodecylthio ... alkylthio groups (e.g., cyclopentylthio, cyclohexylthio, etc.), arylthio groups (e.g., phenylthio, naphthylthio, etc.), alkoxycarbonyl groups (e.g., methyloxycarbonyl, ethyloxycarbonyl, butyloxycarbonyl, octyloxycarbonyl, dodecyloxycarbonyl, etc.), aryloxycarbonyl groups (e.g., phenyloxycarbonyl, naphthyloxycarbonyl, etc.), sulfamoyl groups (e.g., aminosulfonyl, methylaminosulfonyl, dimethylaminosulfonyl, butylaminosulfonyl, hexylaminosulfonyl, cyclohexylaminosulfonyl, octylaminosulfonyl, dodecylaminosulfonyl, phenylaminosulfonyl, naphthylaminosulfonyl, 2-pyridylaminosulfonyl, etc.),

Acyl groups (e.g., acetyl, ethylcarbonyl, propylcarbonyl, pentylcarbonyl, cyclohexylcarbonyl, octylcarbonyl, 2-ethylhexylcarbonyl, dodecylcarbonyl, acryloyl, methacryloyl, phenylcarbonyl, naphthylcarbonyl, pyridylcarbonyl, etc.), acyloxy groups (e.g., acetyloxy, ethylcarbonyloxy, butylcarbonyloxy, octylcarbonyloxy, dodecylcarbonyloxy, phenylcarbonyloxy, etc.), amido groups (e.g., methylcarbonylamino, ethylcarbonylamino, dimethylcarbonylamino, propylcarbonylamino, pentyl carbonylamino, cyclohexylcarbonylamino, 2-ethylhexylcarbonylamino, octylcarbonylamino, dodecylcarbonylamino, phenylcarbonylamino, naphthylcarbonylamino, etc.), carbamoyl groups (for example, aminocarbonyl, methylaminocarbonyl, dimethylaminocarbonyl, propylaminocarbonyl, pentylaminocarbonyl, cyclohexylaminocarbonyl, octylaminocarbonyl, 2-ethylhexylaminocarbonyl, dodecylaminocarbonyl, phenylaminocarbonyl, naphthylaminocarbonyl, 2-pyridylaminocarbonyl, etc.), ureido groups (for example, methylurethane, ureido, ethylureido, pentylureido, cyclohexylureido, octylureido, dodecylureido, phenylureido, naphthylureido, 2-pyridylaminoureido, etc.), sulfinyl groups (e.g., methylsulfinyl, ethylsulfinyl, butylsulfinyl, cyclohexylsulfinyl, 2-ethylhexylsulfinyl, dodecylsulfinyl, phenylsulfinyl, naphthylsulfinyl, 2-pyridylsulfinyl, etc.), alkylsulfonyl groups (e.g., methylsulfonyl, ethylsulfonyl, butylsulfonyl, cyclohexylsulfonyl, 2-ethylhexylsulfonyl, dodecylsulfonyl, etc.) , arylsulfonyl groups or heteroarylsulfonyl groups (e.g., phenylsulfonyl, naphthylsulfonyl, 2-pyridylsulfonyl, etc.), amino groups (including amino groups, alkylamino groups, alkenylamino groups, arylamino groups, and heterocyclic amino groups, e.g., amino, ethylamino, dimethylamino, butylamino, cyclopentylamino, 2-ethylhexylamino, dodecylamino, anilino, naphthylamino, 2-pyridylamino, etc.), cyano groups, nitro groups, hydroxy groups, mercapto groups, silyl groups (e.g., trimethylsilyl, triisopropylsilyl, triphenylsilyl, phenyldiethylsilyl, etc.), and the like.
Each of these groups may further have a substituent, and the substituent may be the above-mentioned substituent. For example, an aralkyl group in which an aryl group is substituted on an alkyl group, a hydroxyalkyl group in which a hydroxy group is substituted on an alkyl group, etc. may be mentioned. In addition, when the substituent W further has a plurality of substituents, the plurality of substituents may be bonded to each other to form a ring.

[Method for inhibiting viral infection]
A method for inhibiting viral infection in a cell according to an embodiment of the present invention is a method for inhibiting viral infection in a cell, which comprises adding the above-mentioned viral infection inhibitor to an object containing at least one selected from the group consisting of tissue consisting of cells and intercellular matrix, and cells, and a liquid medium.
There are no particular limitations on the tissues to which the viral infection suppression method of the embodiment of the present invention can be applied, and the tissues may be tissues collected from biological organs (such as the bronchi, nasal cavity, and pharynx), cultured cells or tissues, or the biological organ itself.

The liquid medium is not particularly limited, but for example, water, a water-soluble organic solvent, and a mixed solvent of an aqueous solution and a water-soluble organic solvent can be used. Of these, water or a mixed solvent of water and a water-soluble organic solvent is preferred.

The method of adding the viral infection inhibitor to the target object is not particularly limited, but examples include a method of adding a predetermined amount of the viral infection inhibitor to the target object (which may be stirred if necessary), and a method of mixing the viral infection inhibitor with a liquid medium in advance and adding the resulting mixture to the target object.
The amount of the viral infection inhibitor to be added is not particularly limited, but generally, it is preferably 0.1 nM to 10 mM in the target substance.

The present invention will be described in more detail below with reference to the following examples. The materials, amounts used, ratios, processing contents, processing procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be interpreted as being limited by the following examples.

(Preparation of specific compounds)
All of the specific compounds were purchased from Biochempeg. The specific compounds are compounds represented by the following formulas, and the specific compounds used and their chain lengths are shown in Table 1. In the following, the specific compounds are collectively referred to as PEG-CLS, and are referred to as "PEG-2000CLS" or the like according to the weight average molecular weight of the polymer chain (PEG chain).

In addition, "chain length" in Table 1 means a value obtained by a method in which the efficiency of fluorescence resonance transfer between a pair of fluorescent dyes having fluorescent properties that can cause fluorescence resonance transfer, which are bound to both ends of a polymer in an aqueous solution, is measured by changing the length of the polymer, and the distance between the polymer ends is calculated from a theoretical formula for fluorescence resonance transfer efficiency. In the case of PEG-CLS, the "chain length" calculated by (chain length) = 0.035 × (weight average molecular weight of PEG chain) ^0.5797 is rounded off to one decimal place to obtain a number to one decimal place.

(Virus preparation)
Lentivirus pCAG-HIVgp and pCMV-VSV-G-RSV-Rev (VSV-G recombined with HCV E1E2) were obtained from RIKEN BioResource Research Center (RIKEN BRC), and pLV-eGFP (or pLenti CMV Puro LUC) was obtained from Addgene. These plasmid vectors were transfected into HEK293T cells cultured in DMEM (Dulbecco's Modified Eagle Medium) using polyethyleneimine, and lentiviruses were collected from the medium solution 48 hours later.
The recovered lentivirus (or recombinant thereof) solution was filtered to remove impurities using a 0.22 μm filter, concentrated by coprecipitation with PEG 8000 or ultracentrifugation as necessary, and stored at −80° C. in a deep freezer.

Adeno-associated virus AAV-GFP, pAAV2/2, and pAdDeltaF6 were obtained from Addgene. These plasmid vectors were transfected into HEK293T cells cultured in DMEM medium using polyethyleneimine, and adeno-associated viruses were collected from the medium solution and cell lysate 48 hours later.

Adenovirus Ax1-CA-gfp was obtained from RIKEN BRC. This was amplified by infecting HEK293T cells cultured in DMEM medium, and was recovered from the medium solution and cell lysate 48 hours later.
The adeno-associated virus and adenovirus crude extracts were stored at -80°C as they were, or, if necessary, after purification and concentration by density gradient centrifugation using cesium chloride.

Of these prepared viruses, lentivirus (having the G protein of VSV (vesicular stomatitis virus) in its outer shell) was used to infect Hek293T cells and Jurkat cells.
The lentivirus expressing the E1-E2 molecule of HCV (hepatitis C virus) instead of VSV-G in the envelope of the lentivirus was infected into Hek293T cells transfected with the human Occludin gene.
Adenovirus was used to infect Hek293T cells. Adeno-associated virus was used to infect Hek293T cells.

(Infection experiment method)
Suspension cells or detached adherent cells: Suspension cells such as Jurkat cells and adherent cells such as Hek293T cells suspended in the presence of PBS (phosphate-buffered saline) containing EDTA (ethylenediaminetetraacetic acid); cells that adherent cells such as Hek293T cells were suspended by a short enzyme treatment with trypsin; and cells that were detached in the presence of PBS containing EDTA from adherent cells such as Hek293T cells that had been cultured on a microplate for one day, then the necessary genes were introduced by transfection with a plasmid vector and cultured for another day to reach a confluence of up to 80%. These were each prepared and suspended at a concentration of 1.5 x ¹⁰ cells/mL.

In the presence of 5% _CO2 , PEG-CLS was added to the cell suspension at a final concentration of 0.01 to 25 μM, and the mixture was incubated at 37°C for 30 minutes with gentle rotation to allow PEG-CLS to bind to the cell membrane. After 30 minutes of incubation, a molecular binding density close to saturation was obtained.

After incubation, each virus was added to the cell suspension at an arbitrary concentration. The virus was diluted appropriately based on the previously measured titer of the virus stock so that the percentage of infected cells in the control sample (sample not containing PEG-CLS) was 20% or less, ensuring a sufficient dynamic range.

The cells were infected with the virus for 2 hours at 37°C with gentle rotation in the presence of 5% _CO2 . After 2 hours, the mixture was either left as is or washed by suspending the cells in PBS and centrifuging the cells multiple times to remove the virus and free PEG-CLS from the culture medium, and then transferred to a cell culture plate for culture for 2 days, after which viral infection in the cells was quantified by gene expression (GFP fluorescence or activity of the expressed luciferase enzyme).

Adherent cells: adherent cells such as Hek293T cells cultured on a 24-well microplate for one day; adherent cells such as Hek293T cells cultured on a 24-well microplate for one day, into which a necessary gene has been introduced by transfection of a plasmid vector and further cultured for one day to reach a confluence of up to 80%; the volume of the medium for these cell suspensions was reduced to 200 μL, and PEG-CLS was added at a final concentration of 0.01 to 25 μM, followed by incubation for 30 minutes at 37° C. in the presence of 5% _CO2 , after which a virus appropriately diluted based on the titer of the stock solution measured in advance was added so that the percentage of infected cells was up to 20%, and infection was carried out for 3 hours at 37° C. in the presence of 5% _CO2 .
The medium containing the virus and PEG-CLS was then removed, 500 μL of fresh medium was added, and the cells were cultured for 2 days, after which viral infection in the cells was quantified by gene expression (GFP fluorescence or activity of the expressed luciferase enzyme).

(Measuring method)
- Flow cytometry After the incubation period, cells infected with a virus containing a GFP gene were collected from the culture plate and suspended in a PBS solution, and the infection was measured by measuring the expression level of the GFP protein using flow cytometry. The average intensity of GFP fluorescence was measured as the infection level, or the fluorescence value of uninfected cells was set as a threshold, and cells showing a fluorescence value equal to or higher than this were considered to be GFP-expressing cells, i.e., infected cells, and the proportion of these cells was measured as the infection level.

(Luciferase Assay)
The adherent cells infected with a virus containing a luciferase gene were washed in the culture plate wells with PBS while still in the adherent state to remove the medium solution, and then Passive Lysis Buffer (Promega Dual-Luciferase Reporter Assay System) containing a surfactant and the like was added, followed by incubation at room temperature for 15 minutes or more with shaking.

The floating cells infected with a virus containing the luciferase gene were collected from the culture plate, the medium was removed by centrifugation, and the cells were washed with PBS. After that, a passive lysis buffer (Promega Dual-Luciferase Reporter Assay System) containing a surfactant was added to each cell, and the cells were suspended in the solution and incubated at room temperature with shaking for 15 minutes or more.

Then, each lysis solution was collected and the cells were removed by centrifugation, after which up to 20 μL of the solution was mixed with 100 μL of LAR II solution (Promega Dual-Luciferase Reporter Assay System) containing luciferin and ATP, and the amount of luciferase expression in the cell solution was quantified by measuring the enzyme reaction with a luminometer.

Microscopic observation The deformation of cells into which PEG-CLS was introduced was observed using a scanning electron microscope (SEM). 1.5 x ¹⁰⁶ cells/mL Hek293T cells were incubated at 37°C for 1 hour with a sample containing no PEG-CLS, 25 μM PEG-2000CLS, or PEG-20000CLS, and then fixed in the following manner and observed with a SEM.

Fixation method: The cells were centrifuged to remove the PEG-CLS solution, then suspended in PBS containing 2.5% glutaraldehyde and left at 4°C for one day for pre-fixation. The glutaraldehyde was then removed and the cells were resuspended in PBS. The suspension was dripped onto a holder and aspirated to allow the cells to adhere to the membrane. The adhered cells were fixed for one hour with a post-fixative (1% osmium tetrachloride - 0.1M phosphate buffer, 1:1). The cells were then dehydrated for 10 minutes each in 50%, 70%, 80%, 90%, 95%, 100%, 100%, and 100% ethanol. The dehydrated samples were immersed in 3-methylbutyl acetate for approximately 10 minutes and then dried in a critical point drying apparatus. The dried samples were vapor-deposited with osmium (5 nm) using an osmium plasma coater.

- Fluorescence Microscopy Observation The deformation of the cells into which PEG-CLS was introduced was observed using a fluorescence microscope.
In RPMI medium containing 1 μg/mL calcein-AM, 1.5 x ¹⁰⁶ cells/mL Jurkat cells were incubated with a sample containing no PEG-CLS, a sample containing 25 μM PEG-2000CLS, and a sample containing PEG-20000CLS at 37°C for 1 hour, and then the cells were photographed using a fluorescence microscope.The cells were then washed with RPMI medium three times, for 15 minutes, 15 minutes, and 2 hours, to reduce the concentration of PEG-CLS in the liquid or bound to the cells, and the cells were photographed again using the fluorescence microscope.
Fluorescence microscopy images were analyzed using ImageJ (software, NIH), and the roundness of each cell was calculated as 4×area/(π×major-axis ² ).

As a result, it was found that the roundness of cells into which PEG-2000CLS and PEG-20000CLS were introduced increased. It was also found that the cells expanded and became spherical. This result was consistent with the SEM observations. It was also found that PEG-CLS was removed by washing, restoring the cell state.

(result)
FIG. 2 is an SEM image of Hek293T cells bound with PEG-CLS. The state of cell expansion found from the fluorescence microscopy photograph analysis was observed at high magnification. From the results of FIG. 2, regardless of the introduction of PEG-CLS, no structure that would interfere with viral adhesion could be observed on the cell surface. In other words, it was suggested that the inhibition of viral infection is achieved by a structure (molecule) smaller than the resolution of the electron microscope SEM. Furthermore, no structural difference was found from the SEM image for PEG-2000CLS and PEG-20000CLS, which have different chain lengths. In FIG. 2, "2KPEG-CLS" represents "PEG-2000CLS" and 20KPEG-CLS represents "PEG-20000CLS".

Table 2 shows the results of adding PEG-CLS to Hek293T cells at a concentration of 25 μmol/L, infecting them with lentivirus for 2 hours, removing the virus and PEG-CLS, and then culturing the cells for 48 hours, after which the proportion of infected cells (GFP-expressing cells) was measured by FACS, and the results were normalized to the proportion of expressing cells in control cells without added PEG-CLS.
A value below 1.00 indicates that the virus infection has been suppressed.

Table 3 shows the results of adding PEG-CLS to Hek293T cells at a concentration of 25 μmol/L, infecting them with adenovirus for 2 hours, removing the virus and PEG-CLS, and then culturing the cells for 48 hours, after which the proportion of infected cells (GFP-expressing cells) was measured by FACS, and the results were normalized to the proportion of expressing cells in control cells without PEG-CLS added.
A value below 1.00 indicates that the virus infection has been suppressed.

The results in Tables 2 and 3 show that PEG-CLS with a PEG chain length of 4.5 nm or more has an excellent effect of inhibiting viral infection. On the other hand, PEG-CLS with a chain length of less than 4.5 nm does not have an effect of inhibiting viral infection.

Table 4 shows the results of infecting Jurkat cells with lentivirus for 48 hours in the presence of various concentrations of PEG-20000CLS, measuring the proportion of infected cells (GFP-expressing cells) by FACS, and normalizing the proportion of expressing cells in control cells without added PEG-CLS.
The results in Table 4 show that PEG-CLS has an inhibitory effect on lentivirus infection.

Table 5 shows the results of infecting Hek293T cells with adeno-associated virus in the presence of various concentrations of PEG-20000CLS for 2 hours, then removing the virus and PEG-CLS and culturing the cells for 48 hours, after which the proportion of infected cells (GFP-expressing cells) was measured by FACS, and the results were normalized to the proportion of expressing cells in control cells without added PEG-CLS.
The results in Table 5 show that PEG-CLS has an inhibitory effect on infection with adeno-associated virus.

(Method of experiment to measure amount of lentivirus cellular uptake)
The following cells were prepared: suspended cells such as Jurkat cells, and adherent cells such as Hek293T cells in the presence of PBS (phosphate-buffered saline) containing EDTA (ethylenediaminetetraacetic acid); suspended adherent cells such as Hek293T cells by a short enzyme treatment with trypsin; and adherent cells such as Hek293T cells cultured on a microplate for one day, then transfected with a plasmid vector to introduce a required gene and cultured for another day until the cells reached 80% confluence, and detached in the presence of PBS containing EDTA. These were then suspended at a concentration of 1.5 x ¹⁰ cells/mL.

In the presence of 5% _CO2 , PEG-CLS was added to the cell suspension at a final concentration of 0.01 to 25 μM, and the mixture was incubated at 37°C for 30 minutes with gentle rotation to allow PEG-CLS to bind to the cell membrane. After 30 minutes of incubation, a molecular binding density close to saturation was obtained.

After incubation, fluorescently labeled lentivirus was added to the cell solution by premixing it with DID (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine Perchlorate).

The cells were infected with the virus for 2 hours at 37°C with gentle rotation in the presence of 5% _CO2 . After 2 hours, the mixture was left as is, or the mixture was washed by suspending the cells in PBS and centrifuging the cells multiple times to remove the virus and free PEG-CLS from the culture medium, and the amount of virus that had bound to and been taken up by the infected cells was evaluated by measuring the fluorescence intensity of DID by flow cytometry.

Table 6 shows the results of adding PEG-20000CLS to Hek293T cells at a concentration of 0.1 to 25 μmol/L, infecting them with lentivirus fluorescently labeled with DID for 2 hours, and then measuring the percentage of cells that had taken up the virus (cells displaying DID fluorescence) by FACS immediately after removing the virus and PEG-CLS, and normalizing the results to the expression cells in control cells without added PEG-CLS.
A value below 1.00 indicates that the virus infection has been suppressed.

This virus infection inhibitor can be added to tissues, etc., for the purpose of inhibiting the attachment of viruses to cells. By using this virus infection inhibitor, it is possible to inhibit the attachment of viruses to the surface of each cell.

1. Cell 2. Hydrophobic region 3. Hydrophilic region 4. Virus

Claims (8)

A virus infection inhibitor comprising a compound represented by the following formula 1:
In formula 1, n and m each independently represent an integer of 1 or more.
X is a monovalent group obtained by removing the hydrogen atom of the hydroxy group bonded to the carbon atom at position 3 of the steroid nucleus in the compound represented by formula 2, and a plurality of X may be the same or different .
Y is a polymer chain made of a hydrophilic polymer, the effective chain length of which is 4.5 nm or more as measured from the fluorescence resonance transfer efficiency of the terminal in an aqueous solution, the hydrophilic polymer is a polyalkylene glycol, and a plurality of Y's may be the same or different,
When n+m is 2, L is a single bond or a divalent group;
A viral infection inhibitor, wherein when n+m is 3 or more, L is an n+m-valent group.


In formula 2, rings A to D represent a saturated or unsaturated steroid nucleus.
R is a hydrogen atom or a linear, branched, or cyclic alkyl group having 3 to 8 carbon atoms;
The hydrogen atoms bonded to the carbon atoms of the steroid nucleus may be substituted with a monovalent substituent selected from the group consisting of a hydrocarbon group, a hydroxyl group, and a halogen atom. 2. The virus infection inhibitor according to claim 1, wherein in formula 1, n and m each represent 1, and L represents a single bond or a divalent group. In formula 1, L is -C(O)-, -C(O)O-, -OC(O)-, -O-, -S-, or -NR ₂ - (R ₂ represents a hydrogen atom or a monovalent organic group), -O-P(O)(OH)-, -O-P(O)(O ^- M ⁺ )-(M ⁺ is Na ⁺ or NH ₄ ⁺ (representing (i) above), an alkylene group having 1 to 10 carbon atoms, a cycloalkylene group having 3 to 10 carbon atoms, an alkenylene group having 2 to 10 carbon atoms, and a divalent group selected from the group consisting of these groups. The virus infection inhibitor according to any one of claims 1 to 3, wherein X in formula 1 is a monovalent group represented by the following formula 4:




In formula 4,
R is a hydrogen atom or a linear, branched, or cyclic alkyl group having 3 to 8 carbon atoms;
The hydrogen atoms bonded to the carbon atoms of the steroid nucleus may be substituted with a monovalent substituent selected from the group consisting of a hydrocarbon group, a hydroxyl group, and a halogen atom. The viral infection inhibitor according to any one of claims 1 to 4, wherein the compound represented by formula 1 is one selected from the group consisting of compounds represented by the following formulas (i) to (iii):
In formulas (i) to (iii), R represents a hydrogen atom or a monovalent group, and p and n each represent an integer of 2 or more. The virus infection inhibitor according to any one of claims 1 to 5 , wherein the hydrophilic polymer has a weight average molecular weight of 4,400 or more. The virus infection inhibitor according to any one of claims 1 to 6 , wherein the chain length is 4.9 nm or more. A method for inhibiting viral infection in cells, comprising adding a viral infection inhibitor according to any one of claims 1 to 7 to a subject (excluding humans) containing at least one selected from the group consisting of tissues consisting of cells and intercellular matrix, and cells, and a liquid medium.