JP2010024203A - Gene expression enhancer and enhancement method of gene expression using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel compound useful as a gene expression enhancer, and to provide a gene expression enhancer comprising the compound as an active ingredient, and an enhancement method of gene expression using the same. <P>SOLUTION: The compound used as the active ingredient has a cholesteryl group, an alkylcarbonyl group, an oxyalkyl group or a thioalkyl group bound to either a hydroxyl group or a thiol group of a compound of formula (1), either directly or via a linker. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a compound useful as a gene expression enhancer. The present invention also relates to a gene expression enhancer comprising the compound as an active ingredient and a gene expression enhancing method using the same.

  Technology that introduces a desired gene into a desired cell and efficiently expresses it in the cell is a key technology in the life science research field, and gene therapy based on such gene transfer is used for cancer, nervous system diseases, etc. It is considered to be an effective treatment for various diseases.

In order to achieve gene therapy, a gene having specific genetic information must be introduced into a somatic cell. In that case, in chronic diseases, it is desired that the gene to be introduced is incorporated into an appropriate site in the nuclear gene and expressed constitutively, whereas in the treatment of cancer etc., transient expression may be appropriate. is there. In gene therapy, a “vector” that is a gene carrier is usually used for gene introduction. Gene transfer methods using such vectors are roughly classified into the following two types according to the type of vector.
(1) Virus method: A method of infecting cells to be transfected with a virus such as adenovirus or retrovirus as a vector,
(2) Non-viral method: A method in which a desired gene is introduced into a target cell using an artificially prepared vector without using a virus.

  Although the virus method of (1) has the advantage of high expression efficiency, there is a problem that the virus itself is pathogenic, and further, an immune response to the virus protein occurs. There is a drawback that the effect is reduced. There is also a problem that the size of DNA to be introduced is limited.

  Since both the cell membrane and the gene are negatively charged, it is difficult to efficiently introduce the gene into the cell as it is by electric repulsion. Another problem is that the introduced gene is cleaved by DNase before reaching the nucleus.

  Therefore, as a non-viral method of (2), before reaching the cell, for the purpose of preventing enzymatic cleavage, DNA is wrapped with a positively charged liposome and attached to the cell by electrostatic interaction, A method of facilitating incorporation, that is, a gene introduction method using a positively charged cationic lipid (cationic liposome) as a vector has been proposed (for example, Non-Patent Document 1). The gene transfer technique using such a non-viral method has the advantage that the vector can be used for multiple treatments because it has no pathogenicity or immunogenicity, and can be effectively used for transient gene expression. There is. In addition, there is no limitation on the size of DNA to be introduced, and there is an advantage that it can be formulated and can respond to a drug delivery system (DDS).

However, the non-viral method has a problem that gene transfer efficiency and expression rate are inferior to those of the viral method. A number of methods for increasing gene transfer efficiency have been studied in the past (for example, Patent Documents 1 to 3 and Non-Patent Documents 2 to 3). There are currently no technologies to improve the gene expression rate.
International Publication WO01 / 40254 JP 2001-2592 A JP 2006-254877 A Yotsuyanagi, T. et al., Japanese Clinical Practice, 56, 153-160 (1998) Miller, AD, The problem with selective liposome / micelle-based non-viral vector systems for gene therapy. Curr. Med. Chem. 10, 1195-1211 (2003). Pietersz, GA, Tang, CK, Apostolopoulos, V., Structure and design of polycationic carriers for gene delivery.Mini Rev. Med. Chem. 6, 1285-1298 (2006).

  It is an object of the present invention to provide a compound useful for improving the gene expression efficiency in cells by improving the low gene expression efficiency, which is a problem of the gene transfer technique using the non-viral method. To do. Another object of the present invention is to provide a gene expression enhancer comprising a compound having the above characteristics as an active ingredient, and a gene expression enhancing method using the compound or gene expression enhancer.

  The inventors of the present invention have made extensive studies in order to solve the above-mentioned problems. As a result, the following compound having a strong inhibitory activity against histone deacetylase (HDAC) (histone deacetylase inhibitor (HDACi)): )

A compound in which a cholesteryl group, an alkylcarbonyl group, an oxyalkyl group, or a thioalkyl group is bonded to any one of the hydroxyl group or thiol group, either directly or via a linker, has the effect of enhancing gene expression. I found out. Specifically, a complex (nanoplex) in which a lipid membrane structure prepared by mixing the above compound with a cationic phospholipid or cholesterol and a target gene (DNA) is complexed is expressed in a cell. It was confirmed that the expression of the gene in the cells was enhanced by introduction into the cells.

The present invention has been completed based on such knowledge and has the following configuration.
(I) Effective compound of gene expression enhancer (I-1) The following formula (1)

A compound in which a cholesteryl group, an alkylcarbonyl group, an oxyalkyl group, or a thioalkyl group is bonded directly or via a linker to either a hydroxyl group or a sulfhydryl group of the compound represented by formula (1).

  (I-2) The compound described in (I-1), wherein the compound (1) is a compound described in the general formula (2):

(In the formula, X is an oxygen atom or a sulfur atom; A and B are a hydrogen atom, a cholesteryl group, an alkylcarbonyl group having 3 to 20 carbon atoms, an oxyalkyl group having 3 to 20 carbon atoms, or 3 to 3 carbon atoms, respectively. 20 thioalkyl groups; l is an integer of 1 or 2; m is an integer of 0 or 1; Y is a group represented by the following formula (3) or (4);

[Wherein, W represents an oxygen atom, a nitrogen atom or a sulfur atom; “SZ-CO” represents a residue of an amino acid having a thiol group (a thiol group in which a hydrogen atom of a thiol group and a hydroxyl group of a carboxyl group are substituted) A thioalkylcarbonyl group; n is an integer of 0 or 1; p is an integer of 1 to 8]
Means. However, one of “— (Y) _m —A” or “— (Y) _m —B” is a hydrogen atom, and in that case, the other is not a hydrogen atom. ).

(I-3) The compound described in the general formula (1) is at least one selected from the group consisting of compounds represented by the following formula, and is described in (I-1) or (I-2) Compound:
(1) 2-[{4-[(hydroxyamino) carbonyl] benzyl} (2-naphthylmethyl) amino] ethyl laurate,
(2) 1-cholest-5-en-3-yl 4- {2-[{4-[(hydroxyamino) carbonyl] benzyl} (2-naphthyl
methyl) amino] ethyl} succinate,
(3) cholest-5-en-3-yl 4-{[(4-{[(2-hydroxyethyl) (2-naphthylmethyl) amino] methyl}
benzoyl) amino] oxy} -4-oxobutanoate,
(4) N- (dodecanoyloxy) -4-{[(2-hydroxyethyl) (2-naphthylmethyl) amino] methyl}
benzamide,
(5) 2- (dodecyldisulfanyl) ethyl 2-[{4-[(hydroxyamino) carbonyl] benzyl} (2-naphthyl
methyl) amino] ethyl carbonate, and
(6) cholest-5-en-3-yl (13R) -13-amino-2- {4-[(hydroxyamino) carbonyl] benzyl} -1- (2-
naphthyl) -6-oxo-5,7-dioxa-10,11-dithia-2-azatetradecan-14-oate
(7) 4-{[[3- (dodecyldisulfanyl) propyl] (2-naphthylmethyl) amino] methyl} -N-
hydroxybenzamide.

(II) A gene expression enhancer comprising a compound described in any one of the gene expression enhancer and gene introduction kit (II-1) (I-1) to (I-3).
(II-2) The gene expression enhancer according to (II-1), comprising a compound according to any one of (I-1) to (I-3) and a cationic lipid or cholesterol.
(II-3) The gene expression enhancer according to (II-2), which has a lipid membrane cyclic structure or a granular structure.

(II-4) A gene introduction kit comprising the gene expression enhancer according to any one of (II-1) to (II-3).
(II-5) The gene introduction kit according to (II-4), comprising the gene expression enhancer according to any one of (II-1) to (II-3) and a gene to be introduced.

(III) Gene-containing composition (III-1) Gene expression with enhanced expression, comprising the gene expression enhancer and gene according to any one of (II-1) to (II-3) Composition.

(IV) Method for enhancing expression of endogenous or exogenous gene (IV-1) Gene expression enhancer described in any of (II-1) to (II-3), or (III-1) A method of introducing a gene-containing composition into a cell in vitro to enhance the expression of an endogenous or exogenous gene.
(IV-2) The method according to (IV-1), wherein the cell is a tumor cell.

  As described above, the gene expression enhancer of the present invention is a cholesteryl group, an alkylcarbonyl group, directly or via a linker at any one of the hydroxyl group or thiol group of the histone deacetylase inhibitor (HDACi). , An oxyalkyl group, or a thioalkyl group. Such a gene expression enhancer can enhance gene expression regardless of whether it is endogenous or exogenous. Nanoparticles (nanoplexes) prepared by injecting the gene expression enhancer into nanoparticles containing positively charged cholesterol are easy to introduce into cells, so they are effective for efficient gene transfer into cells. Can be used.

(I) Effective compound of gene expression enhancer The gene expression enhancer of the present invention is represented by the following formula (1):

A compound in which a cholesteryl group, an alkylcarbonyl group, an oxyalkyl group, or a thioalkyl group is condensed or disulfide bonded directly or via a linker to any one of the hydroxyl group or thiol group of the compound represented by It is also characterized in that it is also referred to as “active compound”) as an active ingredient.

  Here, as the alkyl group of the alkylcarbonyl group, the oxyalkyl group, and the thioalkyl group, a linear or branched alkyl group having 3 to 20 carbon atoms can be exemplified. A linear alkyl group having 12 to 20 carbon atoms is preferred. Specific examples include a lauryl group, a myristyl group, a palmityl group, a stearyl group, and an aralkyl group, and a lauryl group is preferable.

  Here, the linker means a bridging group capable of bonding a cholesteryl group, an alkylcarbonyl group, an oxyalkyl group, or a thioalkyl group to the hydroxyl group or thiol group of the compound (1).

Although not limited, specific examples of the linker include a group represented by “— (Y) _m —”. In the formula, m represents an integer of 0 or 1, and Y represents a group represented by the following formula (3) or (4).

  In the formula, W means an oxygen atom, a nitrogen atom or a sulfur atom. Preferably, it is an oxygen atom.

  “S—Z—CO” is a residue of an amino acid having a thiol group or a thioalkylcarbonyl group, and examples of the amino acid having such a thiol group include cysteine and homocysteine. Moreover, as an alkyl group (Z) of a thioalkylcarbonyl group, C1-C6, Preferably C1-C3 alkyl groups, such as a methyl group, an ethyl group, or a butyl group, can be mentioned.

  Moreover, in said formula, p is an integer of 1-8, Preferably it is 1, n means the integer of 0 or 1.

  Preferred examples of the active compound of the present invention include compounds described in the following general formula (2).

(Where "- (Y) _m -" it is as for the aforementioned .A and B are each a hydrogen atom, cholesteryl group, fatty acid residue having 3 to 20 carbon atoms, aliphatic C3-20 An alcohol residue or an aliphatic thiol residue having 3 to 20 carbon atoms, provided that either “— (Y) _m —A” or “— (Y) _m —B” is a hydrogen atom. In that case, the other is not a hydrogen atom.)
In the above formula (2), “— (Y) _m —” means the above-described linker or single bond. That is, in “− (Y) _m −”, when m is 0, “− (Y) _m −” is a single bond.

In this compound (2), the effective compound of the present invention (hereinafter also referred to as “compound A”) in which “— (Y) _m —A” is a hydrogen atom is described in, for example, Production Example 3 or 4 described later. Thus, N-hydroxy-4-{[(2-hydroxyethyl) (2-naphthylmethyl) amino] methyl} benzamide (K-182) can be used as a starting compound to produce as follows.

  K-182 is described in Synthesis and cancer antiproliferative activity of new histone deacetylase inhibitors: hydrophilic hydroxamates and 2-aminobenzamide-containing derivatives, Y. Nagaoka, et al., European Journal of Medicinal Chemistry 41 (2006) 697-708. It can be manufactured by the method that is being used.

Specifically, the compound A reacts with K-182 and “HO- (Y) _m -B” with a condensing agent in a solvent such as methylene chloride, ethyl acetate, or dimethylformamide, preferably methylene chloride. It can be produced by a condensation reaction using an accelerating additive. Examples of the condensing agent include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, and DCC is preferable. Examples of the reaction promoting additive include dimethylaminopyridine (DMAP), N-hydroxysuccinimide, hydroxybenzotriazole, and imidazole, with DMAP being preferred.

Specific examples of the compound (2) (compound A) obtained by the method include the following compounds:
・ Cholest-5-en-3-yl (13R) -13-amino-2- {4-[(hydroxyamino) carbonyl] benzyl} -1- (2-
naphthyl) -6-oxo-5,7-dioxa-10,11-dithia-2-azatetradecan-14-oate (Production Example 3)
・ N- (dodecanoyloxy) -4-{[(2-hydroxyethyl) (2-naphthylmethyl) amino] methyl}
benzamide (Production Example 4).

In the compound (2), the effective compound of the present invention (hereinafter also referred to as “compound B”) in which “— (Y) _m —B” is a hydrogen atom includes, for example, Production Examples 1, 2, and 5 described later. As shown, 4-{[(2-hydroxyethyl) (2-naphthylmethyl) amino] methyl} -N- (tetrahydro-2H-pyran-2-yloxy)
Using benzamide (compound 5) as a starting compound, it can be produced by the method of Method A or Method B shown in the following formula. Compound 5 can be produced by the method described in Reference Production Example 1 described later.

Specifically, in the method of Method A, compound B is prepared by first treating compound 5 with “HO- (Y) _m -A” in a solvent such as methylene chloride, ethyl acetate, dimethylformamide, preferably methylene chloride. The reaction product can be produced by conducting a condensation reaction using a condensing agent and a reaction promoting additive, and then hydrolyzing the reaction product with acetic acid in a mixed solution of tetrahydrofuran and water. Examples of the condensing agent include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, and DCC is preferable. Examples of the reaction promoting additive include dimethylaminopyridine (DMAP), N-hydroxysuccinimide, hydroxybenzotriazole, imidazole, and the like, preferably DMAP.

  In the method B, compound B is prepared by first treating compound 5 with triphosgene in the presence of DMAP, condensing the resulting acid chloride with the compound represented by (I), and then reacting HS-A. It can be produced by hydrolysis with acetic acid in a mixed solution of tetrahydrofuran and water.

Specific examples of the compound (2) (compound B) obtained by the method include the following compounds:
2-[{4-[(hydroxyamino) carbonyl] benzyl} (2-naphthylmethyl) amino] ethyl laurate (Production Example 1),
・ 1-cholest-5-en-3-yl 4- {2-[{4-[(hydroxyamino) carbonyl] benzyl} (2-naphthyl
methyl) amino] ethyl} succinate (Production Example 2),
2- (dodecyldisulfanyl) ethyl 2-[{4-[(hydroxyamino) carbonyl] benzyl} (2-naphthyl
methyl) amino] ethyl carbonate (Production Example 5).
・ Cholest-5-en-3-yl (13R) -13-amino-2- {4-[(hydroxyamino) carbonyl] benzyl} -1- (2-
naphthyl) -6-oxo-5,7-dioxa-10,11-dithia-2-azatetradecan-14-oate (Production Example 6).

Moreover, the active compound (compound B) of the present invention in which “— (Y) _m —B” is a hydrogen atom is compound 4 which is a production intermediate of compound 5 as described in Production Examples 6 and 7 described later. Can be also produced by the method of Method C or Method D shown below.

Specifically, the compound 4 is first reacted with the aldehyde represented by (II), preferably in the presence of sodium triacetoxyborohydride or sodium tetraborohydride, and then in a mixed solution of tetrahydrofuran and water. Then, it is hydrolyzed with acetic acid and subsequently treated with trifluoroacetic acid (TFA) in the presence of triethylsilane (Et ₃ SiH) to obtain a compound having a thiol group represented by (III). Next, in the method of Method C, the compound represented by (IV) is condensed with the obtained thiol compound to form a residue represented by -SA on the above compound, thereby producing Compound B. Can do. In the method D, the thiol compound obtained above is condensed with the compound represented by (V), and the resulting pyridyl disulfide intermediate is reacted with the compound “HA” to produce —SA Compound B can be produced by forming a residue represented by:
Specific examples of the compound (2) (compound B) obtained by the method include the following compounds:
・ 4-{[[3- (dodecyldisulfanyl) propyl] (2-naphthylmethyl) amino] methyl} -N-
hydroxybenzamide (Production Example 7).

(II) Gene expression enhancer and kit for gene introduction The gene expression enhancer of the present invention comprises the above-mentioned active compound as an active ingredient, and is not particularly limited as long as it improves the cell introduction efficiency of the gene. For this purpose, it is preferable to contain a cationic lipid or cholesterol in addition to the above compound.

  The cationic lipid is not particularly limited as long as it forms a complex with a nucleic acid, and includes a phospholipid, a cholesterol skeleton, a glutamic acid skeleton, a lipid having 1, 2, 3 or quaternary ammonium. Can be mentioned. Here, examples of the cationic lipid having a cholesterol skeleton include cholest-5-en-3-yl 2-[(2-hydroxyethyl) amino] ethylcarbamate (OH-Chol), [N- (N ′ , N′-dimethylaminoethane) -carbamoyl] cholesterol (DC-Chol); Examples of cationic lipids having a glutamic acid skeleton include N- (α-trimethylammonioacetyl) -didodecyl-D-glutamic acid (TMAG). Examples of quaternary ammonium salts include DOTMA (N- (1- (2,3-dioleoxy) propyl) -N, N, N-trimethylammonium bromide), DDAB (dimethyldioctacresyl ammonium bromide), DOSPA ( 2,3-dioreyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanea Minium trifluoroacetate), DOGS (dioleoyl-D-glutamate-N-2 (sperminecarboxamido) ethyl), DMRIE (1,2-dimyristoxypropyl-3-dimethyl-hydroxyethylammonium bromide), etc. Can do.

  These cationic lipids or cholesterols can be used alone or in combination of two or more.

  The proportion of these cationic lipids or cationic cholesterol in the gene expression enhancer of the present invention is 5 to 90%, preferably 40 to 90%, more preferably 50 to 90% in terms of molar fraction. be able to. Moreover, as a ratio of the active compound in the gene expression enhancer of the present invention, 0.5 to 30%, preferably 1 to 10%, more preferably 1 to 5% can be mentioned in terms of molar fraction.

  As the form of the gene expression enhancer of the present invention, the active compound of the present invention and lipid and / or cholesterol may be present in the form of a mixture, or the compound of the present invention and lipid and / or cholesterol may be present. They may be combined to form a lipid membrane structure or particle. A preferred example of such a lipid membrane structure is a lipid membrane cyclic structure including the active compound of the present invention.

  The existence form of the lipid membrane structure or particle and the production method thereof are not particularly limited. Examples of the existence form include a dry form, a form in which the lipid membrane structure or particle is dispersed in an aqueous solvent, Further, it can be in a frozen form and a dried form (including spray drying and freeze drying).

  The dried lipid mixture can be produced by dissolving lipid and / or cholesterol together with the active compound in an organic solvent such as chloroform, and then drying under reduced pressure or spray drying.

  Examples of the form in which the lipid membrane structure or particles are dispersed in an aqueous solvent include monolayer liposomes, multilamellar liposomes, O / W emulsions, W / O / W emulsions, spherical micelles (above, lipid membrane cyclic structures) ), String-like micelles, and irregular layered structures. The size of the lipid membrane structure in a dispersed state is not particularly limited. For example, among the lipid membrane cyclic structures, liposomes, emulsions, nanoparticles, etc. have a particle diameter of several tens to several nanometers. It is 100 nm, preferably 50 to 1000 nm, more preferably 90 to 200 nm, and the spherical micelle has a particle size of about 5 to 50 nm. In the case of a string-like micelle or an irregular layered structure, it can be considered that the thickness per layer is 5 nm to 10 nm and these form a layer.

  The composition of the aqueous solvent (dispersion medium) is not particularly limited, but water; sugar aqueous solutions such as glucose, lactose, and sucrose; polyhydric alcohol aqueous solutions such as glycerin and propylene glycol; phosphate buffer, citric acid Buffers, buffer solutions such as phosphate buffered physiological saline; physiological saline; medium for cell culture and the like. In order to increase the chemical stability of the lipid, the pH of the aqueous solvent may be set from weakly acidic to near neutral (pH 3 to 8), or dissolved oxygen may be removed by nitrogen bubbling. Furthermore, it is preferable to use an aqueous sugar solution when storing freeze-dried or spray-dried, and an aqueous sugar solution or aqueous polyhydric alcohol solution when storing frozen.

  The concentration of these aqueous solvents is not particularly limited. For example, in an aqueous sugar solution, 2 to 20% (W / V), preferably 5 to 10% (W / V) can be mentioned. Moreover, in a polyhydric alcohol aqueous solution, 1 to 5% (W / V), Preferably 2 to 2.5% (W / V) can be mentioned. Furthermore, in a buffer solution, 5-50 mM, Preferably 10-20 mM can be mentioned.

  The concentration of the lipid membrane structure or particles in the aqueous solvent is not particularly limited, but the concentration of the total amount of lipid or cholesterol used as the lipid membrane structure is 0.001 to 100 mM, preferably 0.01. -20 mM can be mentioned.

  In the form in which the lipid membrane structure or particles are dispersed in an aqueous solvent, the dried lipid mixture described above is added to the aqueous solvent, and further emulsified by an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like. Can be manufactured. In addition, methods well known as methods for producing liposomes, such as ultrasonic irradiation method, extrusion method, French press method, homogenization method, thin film method, reverse phase evaporation method, ethanol injection method, dehydration-rewatering It can also be manufactured using the sum method. In order to control the size of the lipid membrane structure, a method of performing extrusion (extrusion filtration) under high pressure using a membrane filter having a uniform pore diameter can be mentioned.

  In addition, examples of a method for further drying the lipid membrane structure or particles dispersed in the aqueous solvent include normal freeze drying and spray drying. As the aqueous solvent at this time, as described above, a sugar aqueous solution, preferably a sucrose aqueous solution or a lactose aqueous solution may be used. Here, once the lipid membrane structure or particles dispersed in an aqueous solvent are produced and further dried, the lipid membrane structure or particles can be stored for a long period of time, and the dried lipid membrane structure or particles can be stored. When an aqueous solution of the gene to be introduced is added to the product, the lipid mixture is efficiently hydrated, so that the gene itself can be efficiently retained in the lipid membrane structure or particle.

  By using the gene expression enhancer of the present invention in combination with a target gene to be introduced into a cell, the expression of the target gene in the cell can be enhanced. Therefore, the gene expression enhancer of the present invention can be used as one component of a gene introduction kit.

  Therefore, the present invention provides a gene introduction kit containing the above gene expression enhancer from another viewpoint. The gene introduction kit may include the gene expression enhancer and a target gene to be introduced into cells in separate packaging forms.

  In the present invention, the target gene may be any of oligonucleotide, DNA, and RNA, and in particular, a gene for introduction in vitro such as transformation, a gene that acts by expression in vivo, for example, gene therapy Genes for use in breeding industrial animals such as laboratory genes and laboratory animals and livestock are preferred. Examples of genes for gene therapy include oligonucleotides (antisense DNA, short double stranded RNA (siRNA)), genes encoding physiologically active substances such as enzymes and cytokines, and the like.

(III) Gene-containing composition In addition, the present invention provides a gene-containing composition comprising the above gene expression enhancer and a target gene to be introduced into cells.

  Specifically, the gene-containing composition includes a mixture containing the active compound of the present invention and a gene; a mixture comprising the active compound of the present invention and phospholipid and / or cholesterol (preferably a lipid membrane structure or A form in which a gene is mixed in a particle); a form in which a gene is held in a mixture (preferably a lipid membrane structure or particle) of the active compound of the present invention and phospholipid and / or cholesterol. it can. The term “retention” as used herein means that a gene is present in a part of a lipid membrane structure or a particle, for example, in the membrane, surface, inside, in a lipid layer, or on the surface of a lipid layer. To do.

  The existence form of the gene-containing composition and the method for producing the same are not particularly limited, as in the case of the lipid membrane structure or particle described above. For example, the existence form may be a mixed dry product form or an aqueous solvent. Examples include a dispersed form, a dried form, and a frozen form.

  For the mixed dried product of lipid membrane structure or particle and gene, for example, the lipid membrane structure or particle and gene to be used are once dissolved in an organic solvent such as chloroform and then dried under reduced pressure by an evaporator. Or by spray drying with a spray dryer. Examples of the form in which a lipid membrane structure or a mixture of particles and genes is dispersed in an aqueous solvent include multilamellar liposomes, single membrane liposomes, O / W emulsions, W / O / W emulsions, spherical micelles, and string-like shapes Examples include micelles and irregular layered structures.

  Specifically, a method is used in which an aqueous solvent is added to a lipid membrane structure or a mixed and dried product of particles and genes, and further emulsification is performed by an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like. be able to. When it is desired to control the size (particle diameter), it is possible to perform extrusion (extrusion exclusion) under a high pressure by using a membrane filter having a uniform pore diameter. In this method, the gene must first be dissolved in an organic solvent in order to make a dry mixture of the lipid membrane structure and the gene, but the interaction between the gene and the lipid membrane structure is maximized. There are merits that can be used. That is, even when the lipid membrane structure has a layered structure, the gene can enter even within the multi-layer. In general, when this production method is used, the retention rate of the gene in the lipid membrane structure is increased. Can be high.

  As another method, a lipid membrane structure or particle is once dissolved in an organic solvent, and then an aqueous solvent containing a gene is added to the dried product obtained by distilling off the organic solvent, followed by emulsification. When it is desired to control the size (particle diameter), it is possible to perform extrusion (extrusion exclusion) under a high pressure by using a membrane filter having a uniform pore diameter. This method can be applied to genes that are difficult to dissolve in organic solvents but are soluble in aqueous solvents. As an advantage, when the lipid membrane structure is a liposome, the gene can also be retained in the inner aqueous phase portion.

  Still another method is a method of adding an aqueous solvent further containing a gene to a lipid membrane structure or particle such as a liposome, emulsion (nanoparticle), micelle, or layered structure already dispersed in an aqueous solvent. Therefore, in this case, the gene is limited to a water-soluble gene. Since this method is a method in which a gene is added from the outside to an already formed lipid membrane structure or particle, in the case of a gene or a polymer, the gene enters the lipid membrane structure or particle. Instead, it takes a form of existence bound to the surface of the lipid membrane structure or particle. When liposomes are used as the lipid membrane structure, when this method is used, a sandwich structure (generally called a complex or a complex) in which a gene is sandwiched between liposome particles is taken. The merit of this method is that not only one kind of gene is produced by manufacturing and storing lipid membrane structures such as liposomes, emulsions (nanoparticles), micelles, and layered structures that have already been dispersed in an aqueous solvent. In addition, it can be applied to other genes in common. In addition, since an aqueous dispersion of a lipid membrane structure or particles alone is produced in advance, it is not necessary to consider the degradation of genes during emulsification, and the size (particle diameter) can be easily controlled. It can be said that production is relatively easy compared to the production method.

  Furthermore, as another production method, there can be mentioned a method in which a lipid membrane structure or particles dispersed in an aqueous solvent are once produced and then further dried, and then an aqueous solvent containing a gene is added to the dried product. . Therefore, in this case as well, it is limited to water-soluble genes as in the above method. In this method, in order to produce a lipid membrane structure dispersed in an aqueous solvent and then dried further, the lipid membrane structure exists in a solid state as a lipid membrane fragment at this stage. In order to make this lipid membrane fragment exist in a solid state, it is necessary to use an aqueous sugar solution, preferably an aqueous sucrose solution or an aqueous lactose solution as the aqueous solvent, as described above. Here, when an aqueous solvent containing a gene is added, the lipid membrane fragments that existed in the solid state immediately start to hydrate as water enters, and the lipid membrane structure can be reconstituted. At this time, a gene in which the gene is held in the lipid membrane structure can be produced. The advantage of this method is that once it is manufactured, it can be applied not only to one gene but also to other genes in common, and an aqueous dispersion of lipid membrane structure or particles alone is prepared in advance. For this reason, it is not necessary to consider the degradation of the gene during emulsification, and the size (particle diameter) can be easily controlled. Therefore, it can be mentioned that the production is relatively easy compared to the method described above. In addition, since freeze-drying or spray-drying is performed, it is easy to guarantee storage stability as a formulation, and the size (particle diameter) can be restored even if the dried formulation is reconstituted with a gene aqueous solution. Even in the case of a high molecular gene, it is easy to retain the gene inside the lipid membrane structure or particle.

  As a method for preparing a lipid membrane structure or a mixture of particles and genes in a form dispersed in an aqueous solvent, there are other methods well known as methods for producing liposomes, for example, an ultrasonic irradiation method and an extrusion method. , French press method, homogenization method, thin film method, reverse phase evaporation method, ethanol injection method, dehydration-rehydration method and the like. When it is desired to control the size (particle diameter), it is possible to perform extrusion (extrusion exclusion) under a high pressure by using a membrane filter having a uniform pore diameter.

  In addition, examples of the method for further drying the dispersion liquid in which the lipid membrane structure or the mixture of particles and gene is dispersed in an aqueous solvent include freeze drying and spray drying. As an aqueous solvent at this time, a sugar aqueous solution, preferably a sucrose aqueous solution or a lactose aqueous solution, may be used as in the case of the lipid membrane structure or the particulate matter alone. As a method for further freezing the dispersion in which the lipid membrane structure or the mixture of particles and gene is dispersed in an aqueous solvent, a normal freezing method can be mentioned. As the aqueous solvent at this time, the lipid membrane structure As in the case of the body alone, an aqueous sugar solution or an aqueous polyhydric alcohol solution may be used.

  The ratio of the lipid membrane structure or particle contained in the gene-containing composition of the present invention is preferably 1 to 500 nmol and more preferably 5 to 10 nmol with respect to 1 μg of gene.

  Using the gene-containing composition of the present invention, a gene can be efficiently introduced into a desired cell both in vitro and in vivo, and the introduced gene can be efficiently expressed in the cell. .

  In the present invention, examples of cells into which genes are introduced include in vitro cultured cells, cells extracted from living organisms, and cells present in living organisms. Tumor cells are preferred.

  As a method for introducing a gene into such cells, in the case of in vitro, a method of adding the gene-containing composition of the present invention to a suspension containing target cells, a medium containing the gene-containing composition of the present invention And a method of culturing the target cells. A composition in the form of a solution, a lyophilized product, or an aerosol is used for gene transfer into in vitro cultured cells.

  Moreover, in the case of in vivo, the method of administering the gene containing composition of this invention to a host (human, animal except a human) can be mentioned. The means for administration to the host may be oral administration or parenteral administration, but parenteral administration is preferred. The dosage form may be a conventionally known dosage form, and examples of the dosage form for oral administration include tablets, powders, granules, syrups and the like. Examples of the dosage form for parenteral administration include injections, eye drops, ointments, suppositories and the like. In order to introduce a gene into a cell, it is injected directly into a tissue, administered into a blood vessel such as a vein or a controlling artery of a target tissue, aerosolized and administered to a respiratory organ, encapsulated in a biodegradable capsule, etc. A method of embedding or encapsulating in a capsule degradable in the digestive tract and orally administering is used.

  When the gene-containing composition of the present invention is incorporated into a cell, the gene is efficiently expressed in the cell or tissue, and exhibits a physiologically active action or a pharmacological activity action based on the produced protein. Therefore, the gene-containing composition of the present invention is useful as a gene therapy agent (pharmaceutical composition) effective for the treatment and prevention of various diseases.

  Hereinafter, the effects of the present invention will be clarified by production examples and experimental examples. However, these are merely examples, and the present invention is not limited thereto.

Reference Production Example 1 4-{[(2-hydroxyethyl) (2-naphthylmethyl) amino] methyl} -N- (tetrahydro-2H-pyran-2-yloxy)
Production of benzamide (Compound 5) The title compound 5 was produced according to the following reaction steps.

(1) Synthesis of compound 2
Methyl (4-aminomethyl) benzoate hydrochloride ( Compound 1) (Sigma-Aldrich) (10.0 g, 44.8 mmol) , Et 3 N 6.92 ml (44.8 mmol), and 2-Naphthaldehyde 7.74 g dehydrated CHCl ₃ a (44.8 mmol) in this order And NaBH (OAc) ₃ (14.7 g, 44.8 mmol) was added thereto, followed by stirring for 4.5 hr at room temperature. The reaction was stopped by adding 220 ml of saturated aqueous NaHCO ₃ solution, and the mixture was stirred at room temperature for 2 hours. This solution was extracted with CHCl ₃ , the organic layer was dried over sodium sulfate, and then the CHCl ₃ phase was concentrated under reduced pressure to obtain a yellow solid residue. The residue was purified by silica gel column chromatography (developing solvent: ethyl acetate: toluene = 8: 1) to give methyl 4-{[(2-naphthylmethyl) amino] methyl} benzoate (compound 2) (11.0 g, yield 80 (Non-patent Document 4: Synthesis and cancer antiproliferative activity of new histone deacetylase inhibitors: hydrophilic hydroxamates and 2-aminobenzamide-containing derivatives, Y. Nagaoka, et al., European Journal of Medicinal Chemistry 41 (2006) 697 -708).

The ¹ H NMR results for Compound 2 are as follows:
¹ H NMR (CDCl ₃ ) δ: 3.90 (2H, s, NaphCH ₂ ), 3.91 (3H, s, CO ₂ Me), 3.97 (2H, s, NHCH ₂ Ph), 7.42-7.49 (5H, m), 7.75-7.83 (4H, m), 7.99-8.01 (2H, m).

(2) Synthesis of Compound 3 Compound 2 (18.7 g, 61.2 mmol) and 1M LiOH aqueous solution (244.8 ml, 244.8 mmol) were dissolved in THF (357 ml) and stirred at room temperature for 10 hours. Under ice-cooling, 30% HCl was added dropwise to the reaction solution to adjust the solution to pH 1, and crystals of compound 3 (20 g, yield 100%) were precipitated as hydrochloride. Melting point, ¹ H NMR, IR and HR-FAB-MS results for compound 3 are as follows:
mp. 227-229 ° C
¹ H-NMR (399.65 MHz, DMSO-d ₆ ) δ: 3.78 (2H, s), 3.85 (2H, s), 7.43-7.90 (11H, m). ¹³ C-NMR (100.4 MHz CDCl ₃ ) δ: 51.66, 52.14, 125.57, 126.05, 126.29, 126.78, 127.52, 127.56, 127.69, 128.13, 129.27, 129.48, 132.21, 132.94, 137.60, 167.36
IR (KBr) cm ^-1 : 2781, 2592, 1595, 1537, 1373, 1012, 817, 781, 752.
HR-FAB-MS m / z: 292.1336 (calcd for C ₁₉ H ₁₈ NO ₂ , 292.1338).

(3) Synthesis of Compound 4 Hydrochloride (16.8 g, 51.2 mmol) of Compound 3 and Et ₃ N (7.13 ml, 51.2 mmol) were added to 180 ml of DMF, and NH ₂ —O-THP (5.7 g , 48.6 mmol), HOBt (6.92 g, 51.2 mmol), and WSCI (8.98 ml, 51.2 mmol) were added in this order, and the mixture was stirred for 5 hours. Thereafter, the reaction solution was concentrated under reduced pressure, the residue was dissolved in chloroform, and it was washed with a saturated sodium bicarbonate solution and saturated brine. The organic layer was purified by silica gel column chromatography (developing solvent: ethyl acetate) to obtain Compound 4 (15.3 g, yield 80.9%). The IR, ¹ H NMR, ¹³ C-NMR and HR-FAB-MS results for compound 4 are as follows:
IR (KBr) cm ⁻¹ : 3165, 2945, 2848, 1658, 1454, 1128, 904, 873.
¹ H-NMR (399.65 MHz, CDCl ₃ ) d: 1.58-1.92 (6H, m, CH ₂ x3 of THP), 3.63-3.69 (1H, m, -CH _a -O of THP), 3.88 (2H, s , N- CH ₂ -Ar), 3.95 (2H, s, Naph- CH ₂ -N), 4.02 (1H, ddd, J = 11.4, 9.2, 2.6 Hz, -CH _b -O of THP), 5.08 (1H , m, O- CH -O of THP), 7.42-7.83 (11H, m).
¹³ C-NMR (100.4 MHz CDCl ₃ ) d: 18.70, 24.99, 28.08, 52.57, 53.14, 62. 72., 102.72, 125.61, 126.03, 126.47, 126.53, 127.30, 127.63, 127.65, 128.13, 128.29, 130.58, 132.67 , 133.37, 137.37, 144.65.
HR-FAB-MS m / z : 391.2025 (calcd for C 24 H 27 N 2 O 3, 391.2022).

(4) Production of compound 5
4-{[(2-naphthylmethyl) amino] methyl} -N- (tetrahydro-2H-pyran-2-yloxy) benzamide (compound 4) (1.0 g, 0.94 mmol), Et ₃ N 0.71 ml (5.12 mmol), Then, 2-bromoethanol (0.36 ml, 5.12 mmol) was sequentially dissolved in 20 ml of CH ₃ CN, and this mixed solution was stirred at 60 ° C. for 6 hours. The reaction mixture was concentrated under reduced pressure, and the resulting mixture was fractionated by silica gel column chromatography (developing solvent: ethyl acetate / hexane = 3/2), and the fraction containing the title compound 5 was extracted with chloroform and hexane. Reprecipitation gave compound 5 (650 mg, yield 58.5%). The results of ¹ H-NMR, ¹³ C-NMR, IR and HR-FAB-MS of the compound 5 are as follows.
¹ H-NMR (399.65 MHz, CDCl ₃ ) d: 1.62-1.86 (6H, m, CH ₂ x 3 of THP), 2.71 (2H, t, J = 5.6 Hz, N- CH ₂ -CH ₂ ), 3.62 (2H, t, J = 5.6 Hz, CH ₂ - CH ₂ -OH), 3.64-3.68 (1H, m, -CH _a -O of THP), 3.69 (2H, s, N- CH ₂ -Ar), 3.78 (2H, s, Naph- CH ₂ -N), 4.00 (1H, ddd, J = 11.2, 9.2, 2.8 Hz, -CH _b -O of THP) 5.07 (1H, t, J = 2.8 Hz, O- CH -O of THP), 7.38-7.84 (11H, m, Ar- H ), 8.81 (1H, s, CO- NH -O ).
¹³ C-NMR (100.40 MHz, CDCl ₃ ) d: 18.61, 24.99, 28.03, 54.98, 57.97, 58.54, 58.71, 62.63, 102.65, 125.79, 126.14, 126.83, 127.37, 127.59, 127.67, 127.73, 128.29, 129.09, 130.97 , 132.79, 133. 24, 135.91, 143.33.
IR (neat) cm ^-1 3221, 2936, 1719, 1653, 1275, 1204, 1051, 905.
HR-FAB-MS m / z : 435.2289 (calcd for C 26 H 31 N 2 O 4, 435.2284).

Production Example 1 Production of 2-[{4-[(hydroxyamino) carbonyl] benzyl} (2-naphthylmethyl) amino] ethyl laurate (hereinafter, also referred to as “compound DD-K-182”) Compound DD- K182 was manufactured.

(1) Preparation of 2-[(2-naphthylmethyl) (4-{[(tetrahydro-2H-pyran-2-yloxy) amino] carbonyl} benzyl) amino] ethyl laurate (Compound 6) 4-{[(2-hydroxyethyl) (2-naphthylmethyl) amino] methyl} -N- (tetrahydro-2H-pyran-2-yloxy)
To a solution of benzamide (compound 5) (26.2 mg, 60.3 μmol) in CH ₂ Cl ₂ (2.0 ml), Lauric acid (12.1 mg, 60.3 μmol), dicyclohexylcarbodiimide (DCC) (12.4 mg, 60.3 μmol), dimethylaminopyridine (DMAP) (7.37 mg, 60.3 μmol) was added, and the mixture was stirred at room temperature for 5 hours. After removing the precipitate from the reaction solution by filtration, the filtrate was concentrated under reduced pressure, and the resulting concentrated mixture was purified by silica gel thin layer chromatography (SiO ₂ TLC) (developing solvent: EtOAc / hexane = 1/4). This gave the target compound 6 (22.4 mg, 36.3 μmol, yield 60.2%) as a colorless oil. The results of ¹ H-NMR, ¹³ C-NMR, IR and HR-FAB-MS of the compound 6 are as follows.
¹ H-NMR (CDCl ₃ , 399.65 MHz) d: 0.87 (t, 3H, J = 7.2Hz, CH ₃ CH ₂ ), 1.25-1.28 (m, 16H), 1.43-1.58 (m, 6H), 1.84- 1.92 (m, 2H), 2.28 (t, 2H, J = 7.6Hz, COCH ₂ ), 2.77 (t, 2H, J = 5.6Hz, NCH ₂ CH ₂ ), 3.72 (s, 2H, NCH ₂ Ph), 3.78 (s, 2H, NCH ₂ ), 4.20 (t, 2H, J = 6.0Hz, OCH ₂ CH ₂ N), 5.08 (s, 1H, OCH ₂ O), 7.43-7.52 (m, 5H, Ar-H ), 7.70-7.83 (m, 6H, Ar-H).
¹³ C-NMR (100.40 MHz CDCl ₃ ) d: 14.09, 18.66, 22.66, 24.91, 25.02, 25.60, 28.06, 29.18 29.28, 29.31, 29.46, 29.59, 31.86, 33.91, 34.34, 51.91, 58.31, 58.89, 62.05, 62.69 , 102.71, 125.62, 126.00, 126.92, 127.20, 127.30, 127.61, 127.65, 128.01, 128.88, 130.75, 132.80, 133.27, 136.56, 144.06, 173.74.
^{_{IR (CHCl 3) cm -1:}} 3010, 2927, 1728, 1683, 1456, 1112, 777, 669.
HR-FAB-MS m / z: 617.3960 (calcd for C ₃₈ H ₅₃ N ₂ O ₅ , 617.3876).

(2) Production of 2-[{4-[(hydroxyamino) carbonyl] benzyl} (2-naphthylmethyl) amino] ethyl laurate (Compound DD-K-182) Compound 6 obtained above (16.5 mg, 26.8 μmol) A mixed solution of acetic acid (2.0 ml), THF (1.0 ml), and water (0.5 ml) was added to the mixture, and the mixture was heated to reflux at 60 ° C. for 10 hours. After the reaction solution was concentrated under reduced pressure, the obtained concentrated mixture was purified by preparative SiO ₂ TLC (developing solvent: EtOAc / hexane = 1/2) to obtain the desired brown oily compound DD-K-182 (11.5 mg, 21.6 μmol, yield 80.6%). The results of ¹ H-NMR, ¹³ C-NMR, IR and HR-FAB-MS of the compound DD-K-182 are as follows.

¹ H-NMR (CDCl ₃ , 399.65 MHz) d: 0.874 (t, 3H, J = 6.8 Hz), 1.03-1.25 (m, 16H), 1.57-1.70 (m, 2H), 1.90-1.93 (d, 1H ), 2.27 (t, 2H, J = 7.6 Hz), 2.76 (t, 2H, J = 5.6 Hz), 3.70 (s, 2H), 3.78 (s, 2H), 4.18 (t, 2H, J = 5.6 Hz) ), 7.42-7.50 (m, 5H), 7.68-7.82 (m, 6H).
¹³ C-NMR (100.40 MHz CDCl ₃ ) d: 14.09, 22.66, 24.90, 24.93, 25.58, 29.18, 29.28, 29.32, 29.46, 29.60, 31.89, 33.87, 34.36, 52.04, 58.36, 59.00, 62.08, 125.66, 126.03, 126.87, 126.92, 127.34, 127.62, 127.66, 128.04, 129.01, 129.37, 132.83, 133.30, 136.54, 144.41, 173.78.
IR (CHCl ₃ ) cm ^-1 : 3405, 3210, 2927, 2854, 1728, 1612, 1454, 763.
HR-FAB-MS m / z : 533.3373 (calcd for C 33 H 45 N 2 O 4, 533.3301).

Production Example 2 1-cholest-5-en-3-yl 4- {2-[{4-[(hydroxyamino) carbonyl] benzyl} (2-naphthylmethyl) amino] ethyl} succinate (hereinafter referred to as “compound CM-K- according to the manufacturer following reaction steps also referred) and 182 ', to prepare the compound CM-K182.

(1) 1-cholest-5-en-3-yl 4- {2-[(2-naphthylmethyl) (4-{[(tetrahydro-2H-pyran-2-yloxy) amino] carbonyl} benzyl) amino] ethyl } Production of succinate (Compound 7) To a solution of Compound 5 (48.6 mg, 111.6 μmol) prepared in Reference Production Example 1 in CH ₂ Cl ₂ (2.0 ml), Cholesteryl succinate (54.4 mg, 111.6 μmol), DCC (23.0 mg) , 111.6 μmol) and DMAP (13.6 mg, 111.6 μmol) were added, and the mixture was stirred at room temperature for 4 hours. After the precipitate was filtered off from the reaction solution, the filtrate was concentrated under reduced pressure, and the resulting mixture was purified by preparative SiO ₂ TLC (developing solvent: EtOAc / hexane = 1/2), and the above-mentioned colorless oil was purified. Compound 7 (66.3 mg, 73.4 μmol, yield 65.8%) was obtained. The results of ¹ H-NMR, ¹³ C-NMR, IR and HR-FAB-MS of the compound 7 are as follows.
¹ H-NMR (CDCl ₃ , 399.65 MHz) d: 0.67-2.04 (m, 48H), 2.29 (d, 2H, J = 8.0 Hz, CCH ₂ CH), 2.57 (m, 2H, 2OCH ₂ CH ₂ O) , 2.76 (t, 2H, J = 5.6 Hz, NCH ₂ CH ₂ ), 3.69 (s, 2H, NCH ₂ Ph), 3.78 (s, 2H, NCH ₂ ), 4.20 (t, 2H, J = 5.6 Hz, OCH ₂ CH ₂ N), 4.62 (m, 1H, OCHCH ₂ ), 5.08 (s, 1H, OCH ₂ O), 5.35 (d, 1H, CCHCH ₂ ), 7.43-7.53 (m, 5H, Ar-H) , 7.70-7.82 (m, 6H, Ar-H).
¹³ C-NMR (100.40 MHz CDCl ₃ ) d: 11.83, 18.63, 18.70, 19.27, 21.00, 22.54, 22.80, 23.81, 24.26, 24.91, 25.04, 25.60, 27.71, 27.99, 28.07, 28.21, 29.41, 31.83, 31.88, 33.91, 35.76, 36.17, 36.55, 36.92, 38.04, 39.50, 39.71, 42.29, 49.99, 51.96, 56.12, 56.67, 58.31, 59.01, 62.44, 62.61, 74.43, 102.63, 122.71, 125.63, 126.02, 126.94, 127.24, 127.34 127.62, 127.66, 128.06, 128.87, 130.82, 132.81, 133.27, 136.52, 144.01, 171.74, 172.17.
IR (CHCl ₃ ) cm ^-1 : 3008, 2947, 2854, 1728, 1683, 1456, 1363, 1112, 761.
HR-FAB-MS m / z: 903.5882 (calcd for C ₅₇ H ₇₈ N ₂ O ₇ : 903.5809).

(2) 1-cholest-5-en-3-yl 4- {2-[{4-[(hydroxyamino) carbonyl] benzyl} (2-naphthylmethyl) amino] ethyl} succinate (compound CM-K-182) Production To compound 7 (33.0 mg, 36.6 μmol) synthesized above, a mixed solution of acetic acid (2.0 ml), THF (1.0 ml) and water (0.5 ml) was added and heated to reflux at 60 ° C. for 7 hours. The reaction mixture was concentrated under reduced pressure, and the obtained mixture was purified by preparative SiO ₂ TLC (developing solvent: EtOAc / hexane = 2/3) to give the title brown oily compound DM-K-182 (18.4 mg, 22.5). μmol, yield 61.5%). The results of ¹ H-NMR, ¹³ C-NMR, IR and HR-FAB-MS of the compound DM-K-182 are as follows.
¹ H-NMR (CDCl ₃ , 399.65 MHz) d: 0.67-1.98 (m, 41H), 2.30 (m, 2H, CCH ₂ CH), 2.56 (m, 4H, 2OCH ₂ CH ₂ O), 2.74 (m, 2H, NCH ₂ CH ₂ ), 3.68 (s, 2H, NCH ₂ ), 3.80 (s, 2H, NCH ₂ Ph), 4.19 (t, 2H, J = 4.8Hz, OCH ₂ CH ₂ N), 4.61 (m , 1H, OCHCH ₂ ), 5.34 (s, 1H, CCHCH ₂ ), 7.44-7.54 (m, 5H) 7.69-7.80 (m, 6H).
¹³ C-NMR (100.40 MHz CDCl ₃ ) d: 11.85, 18.71, 19.28, 21.02, 22.55, 22.80, 23.83, 24.27, 27.72, 28.00, 28.21, 29.18, 29.41, 29.68, 31.85, 31.88, 35.78, 36.19, 36.56, 36.91, 38.03, 39.52, 39.72, 42.32, 49.98, 52.22, 56.15, 56.67, 58.37, 59.26, 62.42, 74.64, 122.77, 125.68, 126.05, 126.94, 127.39, 127.63, 127.67, 128.09, 128.97, 132.84, 133.300, 136.50, 139.50, 144.36, 171.99, 172.15.
IR (CHCl ₃ ) cm ^-1 : 3421, 2935, 2854, 1728, 1612, 1465, 1365, 1164.
HR-FAB-MS m / z : 819.5304 (calcd for C 52 H 71 N 2 O 6, 819.5234).

Production Example 3 cholest-5-en-3-yl 4-{[(4-{[(2-hydroxyethyl) (2-naphthylmethyl) amino] methyl} benzoyl) amino] oxy} -4-oxobutanoate (hereinafter referred to as “compound Production of Compound K-182-CM According to the following reaction process, Compound K-182-CM was produced. In addition, K-182 N-hydroxy-4-{[(2-hydroxyethyl) (2-naphthylmethyl) amino] methyl} benzamide (K-182) used as a synthetic starting compound is described in Non-Patent Document 4 described above. It was synthesized by the method.

Add CHholesteryl succinate (29.5 mg, 60.5 μmol), DCC (12.5 mg, 60.5 μmol) and DMAP (7.4 mg, 60.5 μmol) to a CH ₂ Cl ₂ (2.0 ml) solution of K-182 (21.2 mg, 60.5 μmol). The mixture was further stirred at room temperature for 6 hours. After removing the precipitate from the reaction solution by filtration, the filtrate was concentrated under reduced pressure, and the resulting mixture was purified by preparative SiO ₂ TLC (EtOAc / hexane = 4/5) to give the title colorless oily compound K- 182-CM (10.5 mg, 12.8 μmol, yield 21.2%) was obtained. The results of ¹ H-NMR, ¹³ C-NMR, IR and HR-FAB-MS of the compound K-182-CM are as follows.
¹ H-NMR (CDCl ₃ , 400 MHz) d: 2.32 (d, 2H, J = 7.6 Hz, CCH2CH), 2.71 (m, 4H, 2OCH2CH2O), 2.87 (t, 2H, J = 6.8Hz, NCH2CH2), 3.62 (t, 2H, J = 5.6 Hz, HOCH2CH2), 3.70 (s, 2H, NCH2Ph), 3.78 (s, 2H, NCH2), 4.63 (m, 1H, OCHCH2), 5.36 (m, 1H, CCHCH2), 7.41-7.48 (m, 5H, Ar-H), 7.70-7.83 (m, 6H, Ar-H).
¹³ C-NMR (100.40 MHz CDCl ₃ ) d: 11.85, 18.70, 19.28, 21.02, 22.54, 22.80, 23.83, 24.27, 27.05, 27.69, 28.00, 28.21, 29.14, 31.84, 31.89, 35.78, 36.18, 36.58, 36.94, 38.00, 39.52, 39.73, 42.32, 50.01, 55.17, 56.16, 56.69, 58.07, 58.65, 58.82, 74.79, 122.77, 125.84, 126.19, 126.87, 127.64, 127.68, 127.71, 127.79, 128.36, 129.29, 129.61, 132.86, 133.30, 135.87, 139.49, 144.29, 171.06.
IR (CHCl ₃ ) cm ^-1 : 3320, 2949, 2868, 1759, 1724, 1466, 1364, 1105, 667.
HR-FAB-MS m / z : 819.5320 (calcd for C 52 H 71 N 2 O 6, 819.5234).

Production Example 4 N- (dodecanoyloxy) -4 - { [(2-hydroxyethyl) (2-naphthylmethyl) amino] methyl} benzamide ( hereinafter, referred to as "compound K-182-DD" and also) according to the manufacturer following reaction steps, compounds K-182-DD was produced.

In a CH ₂ Cl ₂ (4.0 ml) solution of K-182 (33.2 mg, 94.6 μmol), Lauric acid (19.0 mg, 94.6 μmol), DCC (19.5 mg, 94.6 μmol), and DMAP (11.56 mg, 94.6 μmol) And stirred at room temperature for 5 hours. After the precipitate was filtered off from the reaction solution, the filtrate was concentrated under reduced pressure, and the resulting mixture was purified by preparative SiO ₂ TLC (EtOAc / hexane = 1/1) to give the title compound K-182 as a colorless oil. -DD (12.23 mg, 22.96 μmol, 24.3% yield) was obtained. The results of ¹ H-NMR, ¹³ C-NMR, IR and HR-FAB-MS of the compound K-182-DD are as follows.
¹ H-NMR (CDCl ₃ , 400 MHz) d: 0.88 (t, 3H, J = 7.2 Hz, CH ₃ CH ₂ ), 2.55 (t, 2H, J = 7.6 Hz, COCH ₂ ), 2.71 (t, 2H, J = 5.2Hz, NCH ₂ CH ₂ ), 3.62 (t, 2H, J = 5.2 Hz, HOCH ₂ CH ₂ ), 3.70 (s, 2H, NCH ₂ Ph), 3.78 (s, 2H, NCH ₂ ), 7.41 -7.52 (m, 5H, Ar-H), 7.71-7.84 (m, 6H, Ar-H).
¹³ C-NMR (100.40 MHz CDCl ₃ ) d: 14.10, 22.66, 24.70, 24.90, 25.59, 28.99, 29.14, 29.31, 29.37, 29.56, 31.78, 31.89, 33.90, 55.11, 58.03, 58.61, 58.77, 125.85, 126.19, 126.86, 127.63, 127.67, 127.71, 127.80, 128.37, 129.29, 129.78, 132.84, 133.29, 135.86, 144.22, 172.20.
IR (CHCl ₃ ) cm ^-1 : 3341, 3010, 2927, 2854, 1780, 1697, 14111, 1141, 773.
HR-FAB-MS m / z : 533.3370 (calcd for C 33 H 45 N 2 O 4, 533.3301).

Production Example 5 Production of 2- (dodecyldisulfanyl) ethyl 2-[{4-[(hydroxyamino) carbonyl] benzyl} (2-naphthylmethyl) amino] ethyl carbonate (hereinafter also referred to as “compound DDTS-K-182”) According to the process, compound DDTS-K-182 was prepared.

(1) 2-[(2-naphthylmethyl) (4-{[(tetrahydro-2H-pyran-2-yloxy) amino] carbonyl} benzyl) amino] ethyl 2- (2-pyridinyldisulfanyl) ethyl carbonate (Compound 8) Production Reference Compound 5 (100 mg, 0.230 mmol) and DMAP (167 mg, 1.38 mmol) produced in Preparation Example 1 were dissolved in dehydrated CH ₂ Cl ₂ (2.0 ml), and triphosgene (23.9 mg, 0.0805 mmol) was dissolved in this solution. ) And stirred for 15 minutes at room temperature. 2- (2-pyridinyldisulfanyl) ethanol (Jones, Lisa R. et al.,: Journal of the American Chemical Society (2006), 128 (20), 6526-6527) (43.0 mg, 0.230 mmol) was added to this reaction solution. The mixture was further stirred for 7 hours at room temperature. The reaction solution was diluted with CHCl ₃ (20 ml), and this solution was washed with water and saturated brine. The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to obtain an oily residue. This was purified by preparative SiO ₂ TLC (developing solvent: ethyl acetate / hexane = 3/2) to give the title compound 8 (55.0 mg, yield 36.9%) as a colorless oil. ^{1 H-NMR, 13 C-} NMR, the results of IR and HR-FAB-MS of the compound 8 is as follows.

¹ H-NMR (399.65 MHz, CDCl ₃ ) δ: 1.60-1.86 (6H, m, CH ₂ x 3 of THP), 2.79 (2H, t, J = 6Hz, CH ₂ - CH ₂ -SS), 3.05 ( 2H, t, J = 3.2 Hz , N- CH 2 -CH 2), 3.64 (1H, m, - CH a -O of THP), 3.72 (2H, s, N- CH ₂ -Ar), 3.80 (2H, s, Naph- CH ₂ -N), 3.99 (1H, ddd, J = 11.2, 9.2, 2.8 Hz, -CH _b -O of THP), 4.23 (2H, t, J = 3.2Hz, CH ₂ - CH ₂ -OCOO), 4.35 (2H, t, J = 6.0 Hz, OCOO- CH ₂ -CH ₂ ), 5.08 (1H, m, O- CH -O), 7.07-7.81 (15H, m, Ar-H), 8.45 (1H, m, Pyr- H ).
¹³ C-NMR (100.40 MHz CDCl ₃ ) d: 18.65, 24.99, 28.07, 37.00, 51.79, 58.33, 58.86, 62.61, 65.34, 65.84, 76.68, 77.00, 77.31, 102.60, 119.88, 120.88, 125.63, 125.99, 126.87, 127.24, 127.32, 127.62, 128.04, 128.82 132.78, 133.25, 136.32, 137.05, 143.73, 149.68, 154.76, 159.47.
IR (neat) cm ^-1 : 3400, 3027, 3010, 2950, 2824, 1743, 1684, 703.
HR-FAB-MS m / z: 648.2205 (calcd for C ₃₄ H ₃₈ N ₃ O ₆ S ₂ [M + H ⁺ ], 648.2202).

(2) 2- (dodecyldisulfanyl) ethyl 2-[(2-naphthylmethyl) (4-{[(tetrahydro-2H-pyran-2-yloxy) amino] carbonyl} benzyl) amino] ethyl carbonate (Compound 9)
Compound 8 (55 mg, 0.085 mmol) obtained above was dissolved in DMSO (1.5 ml), dodecanethiol (0.02 ml, 0.085 mmol) was added, and the mixture was stirred at room temperature for 2.5 hours. After diluting the reaction solution in CHCl ₃ (10 ml), the solution was washed with water and saturated brine. The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to obtain an oily residue. This was purified by preparative SiO ₂ TLC (developing solvent: ethyl acetate / hexane = 3/2) to give the title compound 9 (59.0 mg, yield 93.9%) as a colorless oil. The results of ¹ H-NMR, ¹³ C-NMR, IR and HR-FAB-MS of the compound 9 are as follows.

¹ H-NMR (399.65 MHz, CDCl ₃ ) δ: 0.88 (3H, t, J = 7.2 Hz, (CH ₂ ) ₉ - CH ₃ ) 1.25 (18H, m, CH _2- ( CH ₂ ) ₉ -CH ₃ ), 1.35 (2H, t, J = 7.2Hz, CH ₂ - CH _2- (CH2) ₉ ), 1.62-1.86 (6H, m, CH ₂ x 3 of THP) δ: 2.69 (2H, t, J = 7.2Hz, SS- CH ₂ -CH ₂ ), 2.81 (2H, t, J = 6.0Hz, N- CH ₂ -CH ₂ ), 2.90 (2H, t, J = 6.8Hz, CH ₂ - CH ₂ -SS ), 3.64 (1H, m, CH ₂ - CH _a -O), 3.73 (2H, s, N- CH ₂ -Ar), 3.80 (2H, s, Naph- CH ₂ -N), 4.00 (1H, ddd , J = 11.2, 9.2, 2.8 Hz, CH ₂ - CH _b -O), 4.25 (2H, t, J = 6.0Hz, CH ₂ - CH ₂ -OCOO), 4.34 (2H, t, J = 6.8Hz, OCOO- CH ₂ -CH ₂ ), 5.07 (1H, m, O- CH -O), 7.45-7.93 (11H, m, Ar-H).
¹³ C-NMR (100.40 MHz CDCl ₃ ) δ: 14.053, 18.680, 22.631, 24.994, 28,057. 28.460, 29.094, 29.160, 29.283, 29.440, 29.530, 29.580, 31.860, 36.816, 39.088, 39.187, 41.180, 51.824, 58.345, 58.905, 60.345, 62.700, 65.812, 65.869, 76.679, 77.000, 77.313, 102.719, 125.614, 125.976, 126.857, 127.219, 127.318, 127.615, 128.035, 128.578, 128.866, 129.714, 130.776, 132.801, 133.270, 136.341, 143.825, 4.8
IR (neat) cm ^-1 : 3400, 3030, 2927, 2855, 1744, 1683.
HR-FAB-MS m / z: 738.3816 (calcd for C ₄₁ H ₅₉ N ₂ O ₆ S ₂ [M + H ⁺ ], 739.3815).

(3) Preparation of 2- (dodecyldisulfanyl) ethyl 2-[{4-[(hydroxyamino) carbonyl] benzyl} (2-naphthylmethyl) amino] ethyl carbonate (Compound DDTS-K-182) Compound 9 obtained above ( Acetic acid (0.80 ml) was added to a solution of 29.0 mg, 39.0 μmol) in THF / H ₂ O (2/1) (0.60 ml), and the mixture was stirred at 60 ° C. for 6 hours. Thereafter, the reaction solution was concentrated under reduced pressure to obtain an oily residue. This was purified by preparative SiO ₂ TLC (developing solvent: ethyl acetate / hexane = 1/2) to give the title compound DDTS-K-182 (19.0 mg, yield 76.0%) as a colorless oil. . The results of ¹ H-NMR, ¹³ C-NMR, IR and HR-FAB-MS of the compound DDTS-K-182 are as follows.
^{1 H-NMR (399.65 MHz,} CDCl 3) d: 0.88 (3H, t, J = 7.2 Hz, (CH 2) 9 - CH 3), 1.25 (20H, m, CH 2 - (CH 2) 10 -CH ₃ ), 2.69 (2H, t, J = 7.2 Hz, SS- CH ₂ -CH ₂ ), 2.81 (2H, t, J = 5.2 Hz, N- CH ₂ -CH ₂ ), 2.90 (2H, t, J = 5.6 Hz, CH ₂ - CH ₂ -SS), 3.73 (2H, s, N- CH ₂ -Ar), 3.80 (2H, s, Naph- CH ₂ -N), 4.24 (2H, t, J = 5.2 Hz, CH ₂ - CH ₂ -OCOO), 4.34 (2H, t, J = 5.6Hz, OCOO- CH ₂ -CH ₂ ), 7.47-7.81 (11H, m, Ar-H).
¹³ C-NMR (100.40 MHz CDCl ₃ ) 14.061, 14.135, 20.985, 22.639, 28.468, 29.102, 29.176, 29.300, 29.456, 29.547, 29.588, 29.604, 29.654, 31.877, 36.816, 39.195, 51.907, 58.386, 59.028, 60.411, 65.828, 65.902, 125.663, 126.017, 126.874, 127.368, 127.631, 128.067, 128.989, 132.818, 133.279, 136.317, 144.047, 154.906.
IR (neat) cm ^-1 : 3300, 3026, 2928, 2855, 1730, 1653, 705.
HR-FAB-MS m / z : 654.3243 (calcd for C 36 H 51 N 2 O 5 S 2 [M + H +], 655.3239).

Production Example 6 cholest-5-en-3-yl (13R) -13-amino-2- {4-[(hydroxyamino) carbonyl] benzyl} -1- (2-naphthyl) -6-oxo-5,7- Production of dioxa-10,11-dithia-2-azatetradecan-14-oate (hereinafter also referred to as “compound CCS-K-182”) Compound CCS-K-182 was produced according to the following reaction steps.

(1) cholest-5-en-3-yl (2R) -2-{[(9H-fluoren-9-ylmethoxy) carbonyl] amino} -3-
Production of {[(4-methoxyphenyl) (diphenyl) methyl] sulfanyl} propanoate (Compound 10)
Fmoc-Cys (Mmt) -OH (500 mg, 0.812 mmol), cholesterol (314 mg, 0.812 mmol), DMAP (99.2 mg, 0.812 mmol), and DCC (178 mg, 0.812 mmol) in CH ₂ Cl ₂ (ml ) And the solution was stirred at room temperature for 3 hours. After the reaction solution was filtered to remove DCU, the filtrate was diluted with chloroform (ml), and this solution was washed with water and saturated brine. The organic layer was collected, dried and filtered with Na ₂ SO ₄ , and concentrated under reduced pressure to give an oily residue. This was purified by silica gel column chromatography (developing solvent: ethyl acetate / hexane = 1/5) to give the title compound 10 (581 mg, yield 72.9%) as a colorless oil. The results of ¹ H-NMR, ¹³ C-NMR, IR and HR-FAB-MS of the compound 10 are as follows.
¹ H-NMR (399.65 MHz, CDCl ₃ ) d: 0.68-1.86 (43H, m, chol), 2.04 (1H, m, S- CH _a -CH), 2.30 (2H, m, CH- CH ₂ -C =), 2.59-2.65 (1H, m, S- CH _b -CH), 3.76 (3H, s, O- CH ₃ ), 4.24 (1H, m, CH ₂ - CH- (Ar) ₂ ), 4.35 ( 2H, m, COO- CH ₂ -of Fmoc), 4.63 (1H, m, NH- CH -COO), 5.32 (1H, m, CH ₂ - CH = C), 6.80-7.77 (22H, m, Ar- H).
¹³ C-NMR (100.40 MHz CDCl ₃ ) 11.846, 14.102, 18.704, 19.305, 21.018, 22.549, 22.639, 22.804, 23.817, 24.270, 27.637, 27.999, 28.213, 31.835, 31.893, 34.322, 35.779, 36.174, 36.553, 36.866, 37.837, 39.508, 39.706, 42.307, 47.091, 49.972, 53.051, 55.051, 56.130, 56.665, 66.437, 67.170, 75.658, 113.249, 119.950, 122.980, 125.145, 126.791, 127.080, 127.697, 127.969, 129.393, 129.418, 130.727 139.313, 141.264, 143.775, 143.907, 144.599, 144.640, 155.573, 158.207, 169.890.
IR (neat) cm ^-1 : 3428, 3028, 2951, 2855, 1728, 1669, 700.
HR-FAB-MS m / z: 1006.5414 (calcd for C ₆₅ H ₇₇ NNaO ₅ S [M + Na ⁺ ], 1006.5420).

(2) Preparation of cholest-5-en-3-yl (2R) -2-amino-3-{[(4-methoxyphenyl) (diphenyl) methyl] sulfanyl} propanoate (Compound 11) Compound 10 obtained above Was dissolved in 20% piperidine / CH ₂ Cl ₂ and the solution was stirred at room temperature for 20 minutes. The reaction mixture was diluted with chloroform (ml), and this solution was washed with water and saturated brine. The organic layer was collected and the solution was filtered dried over Na ₂ SO ₄ to give a residue and concentrated under reduced pressure an oily. This was purified by silica gel column chromatography (developing solvent: ethyl acetate / hexane = 1/5) to obtain the desired colorless oily compound 11 (101 mg, yield 39.4%). The results of ¹ H-NMR, ¹³ C-NMR, IR and HR-FAB-MS of the compound 11 are as follows.
¹ H NMR (399.65 MHz, CDCL) d: 0.68-1.83 (43H, m, chol), 1.99 (1H, m, S- CH _a -CH), 2.25 (2H, m, CH- CH ₂ -C =) , 2.49 (1H, m, S- CH _b -CH), 3.20 (1H, m, O- CH- (CH ₂ ) ₂ ), 3.78 (3H, s, O- CH ₃ ), 4.52-4.60 (1H, m, NH- CH -COO), 5.36-5.36 (1H, m, CH ₂ - CH = C), 6.80-7.43 (14H, m , Ar-H).
¹³ C NMR (100.40 MHz CDCL ₃ ) 11.846, 14.185, 18.712, 19.305, 21.026, 22.541, 22.796, 23.825, 24.270, 27.645, 27.999, 28.205, 31.860, 31.893, 35.779, 36.182, 36.561, 36.907, 37.195, 37.928, 39.517 , 39.722, 42.316, 50.005, 54.121, 55.200, 56.147, 56.682, 60.353, 66.372, 74.785, 113.183, 122.807, 126.660, 127.895, 129.508, 130.801, 136.662, 139.445, 144.912, 144.936, 158.158, 173.232.
IR (neat) cm ^-1 : 3400, 3050, 2951, 2855, 1718, 1650, 701.
HR-FAB-MS m / z: 784.4734 (calcd for C ₅₀ H ₆₇ NNaO ₃ S, 784.4739).

(3) cholest-5-en-3-yl (13R) -13-amino-1- (2-naphthyl) -6-oxo-2- (4-{[(tetrahydro-2H-pyran-2- yloxy) amino] carbonyl} benzyl) -5,7- dioxa-10,11-dithia-2-azatetradecan-14-oate ( compound 12) compound 11 (35. 1 mg, a 0.0462 mmol), TFA (16 μl ) , Triethylsilane (TESH) (20 μl), and CH ₂ Cl ₂ (2.0 ml) were dissolved, and the solution was stirred under an ice-cooled argon atmosphere for 4 hours. Thereafter, the reaction solution was concentrated under reduced pressure to obtain an oily residue. This residue was dissolved in DMSO (0.5 ml), compound 5 (30 mg, 0.069 mmol) was added to this solution, and the mixture was stirred at room temperature for 2 hours. The reaction solution was dissolved in chloroform (20 ml), and this solution was washed with water and saturated brine. The organic layer was dried over Na ₂ SO ₄ and filtered, and then concentrated under reduced pressure to obtain an oily residue. This was purified by preparative SiO ₂ TLC (developing solvent: ethyl acetate / hexane = 3/2) to give the title compound 12 (30.0 mg, yield 71.0%) as a colorless oil. The results of ¹ H-NMR, ¹³ C-NMR, IR and HR-FAB-MS of the compound 12 are as follows.

¹ H-NMR (399.65 MHz, CDCl ₃ ) d: 0.67-2.04 (52H, m), 2.31-2.33 (m, 2H, SS- CH ₂ -CH,), 2.81 (2H, t, J = 5.6 Hz, N- CH ₂ -CH ₂ ), 2.96 (2H, m, CH ₂ - CH ₂ -SS), 3.09-3.14 (1H, m, O- CH- (CH ₂ ) ₂ ), 3.64 (2H, s, N -CH ₂ -Ar), 3.72 (1H, m, CH ₂ - CH _a -O), 3.80 (2H, s, Naph- CH ₂ -N), 4.00 (1H, ddd, J = 11.2, 9.2, 2.8 Hz , CH ₂ - CH _b -O), 4.25 (2H, t, J = 5.6 Hz, CH ₂ - CH ₂ -OCOO), 4.36 (2H, t, J = 6.4 Hz, OCOO- CH ₂ -CH ₂ ), 4.64-4.67 (1H, m, NH- CH -COO), 5.07 (1H, m, O- CH -O), 5.30-5.36 (1H, m, CH ₂ - CH = C), 7.43-7.81 (11H, m, Ar-H).
¹³ C-NMR (100.40 MHz CDCl ₃ ) 11.838, 18.696, 18.811, 19.281, 21.009, 22.541, 22.796, 23.809, 24.253, 25.035, 27.670, 27.991, 28.155, 28.197, 29.678, 31.819, 31.868, 35.762, 36.166, 36.545, 36.890. 37.993, 39.500, 39.698, 42.291, 49.980, 51.890, 53.817, 56.122, 56.657, 58.353, 58.946, 52.815, 65.614, 65.878, 75.279, 102.801, 122.955, 125.655, 126.017, 126.898, 127.252, 127.359, 127.656, 8.0 128.874, 133.287, 136.341, 143.792, 154.898.
IR (neat) cm ^-1 : 3400, 3030, 2935, 2855, 1734, 1684, 699.
HR-FAB-MS m / z: 1026.5697 (calcd for C ₅₉ H ₈₄ N ₃ O ₈ S ₂ [M + H ⁺ ], 1026.5700).

(4) cholest-5-en-3-yl (13R) -13-amino-2- {4-[(hydroxyamino) carbonyl] benzyl} -1- (2-naphthyl) -6-oxo-5,7- dioxa-10,11-dithia-2- azatetradecan-14-oate (CCS-K-182) for the preparation of compound 12 (20.0 mg, 0.0195 mmol) in acetic acid (0.8 ml), THF (0.4 ml), H 2 O ( 0.2 ml) was added, and the mixture was stirred at 60 ° C. for 2.5 hours. Thereafter, the reaction solution was concentrated under reduced pressure to obtain an oily residue. This was purified by preparative SiO ₂ TLC (developing solvent: ethyl acetate) to give the title compound CCS-K-182 (16 mg, yield 87.0%) as a colorless oil. The results of ¹ H-NMR, ¹³ C-NMR, IR and HR-FAB-MS of the compound CCS-K-182 are as follows.
¹ H-NMR (399.65 MHz, CDCl ₃ ) d: 0.81-2.01 (47H, m), 2.31 (m, 2H, SS- CH ₂ -CH,), 2.77 (2H, t, J = 5.6 Hz, N- _{CH 2 -CH 2), 2.94 (} 2H, m, CH 2 - CH 2 -SS), 3.13 (1H, m, O- CH - (CH 2) 2), 3.64 (2H, s, N- CH 2 - Ar), 3.80 (2H, s, Naph- CH ₂ -N), 4.23 (2H, t, J = 5.6 Hz, CH ₂ - CH ₂ -OCOO) 4.33 (2H, t, J = 6.4 Hz, OCOO- CH ₂ -CH ₂ ), 4.65 (1H, m, NH- CH -COO), 5.34 (1H, m, CH ₂ - CH = C), 7.43-7.81 (11H, m, Ar-H).
¹³ C-NMR (100.40 MHz CDCl ₃ ) 11.863, 14.127, 18.729, 19.297, 21.026, 22.566, 22.698, 22.821, 23.850, 24.278, 27.670, 28.024, 28.222, 29.366, 29.704, 31.828, 31.893, 35.796, 36.199, 36.553, 36.891, 36.981, 37.986, 39.525, 39.715, 42.316, 49.981, 52.195, 56.147, 56.666, 58.337, 59.259, 65.648, 65.763, 70.530, 75.576, 123.021, 125.705, 126.067, 126.948, 127.426, 127.681, 128.150, 128.924, 132.8 133.312, 136.350, 139.240, 144.031, 154.906.
IR (neat) cm ^-1 : 3400, 3019, 2930, 2855, 1740, 1650, 706.
HR-FAB-MS m / z: 942.5115 (calcd for C ₅₄ H ₇₆ N ₃ O ₇ S ₂ [M + H ⁺ ], 942.5125).

Production Example 7 4-{[[3- (dodecyldisulfanyl) propyl] (2-naphthylmethyl) amino] methyl} -N-
Hydroxybenzamide (hereinafter, also referred to as "compound 15") according to the manufacturer following reaction steps, to produce a compound 15.

(1) 4-({(2-naphthylmethyl) [3- (tritylsulfanyl) propyl] amino} methyl) -N-
Preparation of (tetrahydro-2H-pyran-2-yloxy) benzamide (Compound 13) Compound 4 (100 mg, 0.25 mmol), 3- (tritylsulfanyl) propanal (86.8 mg, 0.25 mmol), Et ₃ N (42 ml, 0.30 mmol) was dissolved in chloroform (2.0 ml), NaBH (OAc) ₃ (53 mg, 0.5 mmol) was added to the solution, and the mixture was stirred at room temperature for 2 hours. Thereafter, 2 ml of a saturated aqueous NaHCO ₃ solution was added to the reaction solution, followed by stirring at room temperature for 1 hour. This chloroform was added 5 ml, and the organic layer was washed with saturated brine and saturated aqueous NaHCO _3, dried over Na ₂ SO _4, and evaporated. The obtained residue was purified by SiO ₂ column chromatography (EtOAc / toluene = 1/6) to obtain the desired colorless oily compound 13 (100 mg, 56% yield). The results of ¹ H-NMR and ¹³ C-NMR of the compound 13 are as follows.
^{1 H-NMR (399.65 MHz,} CDCl 3) d: 1.47-1.84 (6H, m, CH 2 x 3 of THP), 1.53-1.58 (2H, m, N-CH 2 - CH 2 -), 2.14 (2H , t, J = 7.5 Hz, S-CH _2- ), 2.33 (2H, t, J = 6.8 Hz, N- CH ₂ -CH ₂ ), 3.45 (2H, s, N- CH ₂ -Ar), 3.53 (2H, s, Naph- CH ₂ -N), 3.54-3.64 (1H, m, -CH _a -O of THP), 3.93-4.02 (1H, m, -CH _b -O of THP) 5.05 (1H, s, O- CH -O of THP), 7.15-7.78 (26H, m, Ar- H ), 9.15 (1H, s, CO- NH -O ).
¹³ C-NMR (100.40 MHz, CDCl ₃ ) d: 18.61, 24.90, 26.15, 28.00, 29.88, 52.69, 57.75, 58.26, 62.60, 66.37, 77.21, 102.65, 125.19, 125.44, 125.83, 126.45, 126.96, 127.05, 127.20 , 127.53, 127.73, 127.79, 127.94, 128.12, 128.75, 128.92, 129.14, 129.20, 129.46, 129.73, 130.40, 132.61, 133. 14, 136.70, 144.15, 144.85, 166.78.

(2) Production of N-hydroxy-4-({(2-naphthylmethyl) [3- (tritylsulfanyl) propyl] amino} methyl) benzamide (Compound 14) Compound 13 (100 mg, 142 mmol) obtained above , Acetic acid (1.0 ml), THF (0.5 ml), and water (0.5 ml) were added, and the mixture was heated to reflux at 60 ° C. for 15 hours. After the reaction solution was concentrated under reduced pressure, the obtained concentrated mixture was purified by preparative SiO ₂ TLC (developing solvent: EtOAc / hexane = 1/1) to obtain the desired brown oily compound 14 (56.9 mg, 91.4 mmol, yield). 80.6%) was obtained. The results of ¹ H-NMR and ¹³ C-NMR of the compound 14 are as follows.
^{1 H-NMR (399.65 MHz,} CDCl 3) d: 1.52-1.64 (2H, m, N-CH 2 - CH 2 -), 2.10-2.20 (2H, m, S-CH 2 -), 2.33 -2.42 ( 2H, m, N- CH ₂ -CH ₂ ), 3.47 (2H, s, N- CH ₂ -Ar), 3.56 (2H, s, Naph- CH ₂ -N), 7.10-7.90 (26H, m, Ar -H ).
¹³ C-NMR (100.40 MHz, CDCl ₃ ) d: 26.15, 29.93, 52.74, 57.83, 58.37, 66.43, 77.20, 125.51, 125.91, 126.51, 126.79, 127.03, 127.29, 127.59, 127.79, 127.85, 129.00, 129.52, 132.66 , 133. 18, 136.70, 144.20, 144.90, 165.30.

(3) 4-{[[3- (dodecyldisulfanyl) propyl] (2-naphthylmethyl) amino] methyl} -N-
Preparation of hydroxybenzamide (Compound 15) Compound 14 (45 mg, 72 mmol) was dissolved in CH ₂ Cl ₂ (0.14 ml), and CF ₃ CO ₂ H (134 ml) and Et ₃ SiH (12 ml) were added to this solution. The mixture was further stirred at room temperature for 1 hour. After concentrating this solution under reduced pressure, the concentrated residue was dissolved in CH ₂ Cl ₂ (0.5 ml), 2- (dodecyldisulfanyl) pyridine (27 mg, 86 mmol) was added to this solution, and the mixture was stirred at room temperature for 3 hours. After concentration of the reaction solution under reduced pressure, the obtained concentrated mixture was purified by preparative SiO ₂ TLC (developing solvent: EtOAc / hexane = 1/1) to obtain the desired brown oily compound 15 (30 mg, 71% yield). Got. The results of ¹ H-NMR of the compound 15 are as follows.
¹ H-NMR (399.65 MHz, CDCl ₃ ) d: 0.87 (3H, t, J = 6.8 Hz), 1.21-1.45 (20H, m), 1.54-1.62 (2H, m), 2.33-2.51 (6H, m ), 3.46 (2H, s, N- CH 2 -Ar), 3.54 (2H, s, Naph- CH 2 -N), 7.39-7.90 (11H, m, Ar- H).

Experimental Example 1 Preparation of nanoparticles for gene transfer
5 mol% surfactant (Tween 80, obtained from NOF Co. Ltd) and positively charged cholesterol (cholest-5-en-3-yl 2-[(2-hydroxyethyl) amino] ethylcarbamate, hereinafter “OH” -Chol ") was prepared (hereinafter referred to as" NP "). To this, a modified ethanol injection method (Non-patent Document 5: Y. Hattori, et al., Folate-linked nanoparticles formed with DNA complexes in sodium chloride solution enhance transfection efficiency, J. Biomed. Nanotech. 1 (2005) 176-184 ) Compound DD-K-182, CM-K-182, K-182-CM, K-182-DD, DDTS-K-182, or CCS- K-182 was injected at a final concentration of 10 mol% to prepare gene expression enhancing nanoparticles (hereinafter referred to as “NP for gene expression enhancement”).

  Specifically, for the preparation of NP, 9 ml of water heated to 60 ° C. was first added to an eggplant flask in which 10 mg OH-Chol and 1.3 mg Tween 80 were dissolved in 1 ml ethanol solution. Thereafter, ethanol was distilled off by an evaporator, and water was added to adjust to 10 ml. Thereafter, NP was prepared by reducing the particle size by ultrasonic treatment. In addition, NP for gene expression was first produced in Production Examples 1 to 5 in an eggplant flask in which 10 mg OH-Chol and 1.3 mg Tween 80 were dissolved in 1 ml ethanol solution so as to be 10 mol% with respect to the amount of NP lipid. DD-K182, CM-K182, K182-CM, K182-DD, or DDTS-K182 was added, respectively, and the rest were prepared by the same operation as the preparation of NP.

  NP thus prepared (control example) and NPs for enhancing gene expression (Example 1 (NP-DD-K182), Example 2 (NP-CM-K182), Example 3 (NP-K182-CM)) The compositions of Example 4 (NP-K182-DD) and Example 5 (NP-DDTS-K182)) are shown in Table 1.

  Positively charged cholesterol (OH-Chol) was synthesized and used according to the method described in Non-Patent Document 5 above.

  Next, for the prepared NP (Control Example 1) and each NP for gene expression enhancement (Examples 1 to 5), the particle size distribution was measured using a dynamic light scattering particle size distribution analyzer (ELS-Z2, manufactured by Otsuka Electronics Co., Ltd.). ). Table 2 shows the average particle size (nm) of the prepared NP and each NP for gene expression enhancement.

  From these results, it was confirmed that all of the obtained NPs for gene expression enhancement had an average particle diameter in the range of 100 to 200 nm.

Experimental Example 2 Preparation of Complex (Nanoplex) of DNA and Nanoparticle for Gene Introduction (1) Preparation of Plasmid DNA Plasmid pCMV-Gluc encoding a secreted luciferase gene controlled by cytomegalovirus promoter is New England Purchased from Biolabs (MA, USA). Moreover, pGL3-basic (Promega) was used as a control plasmid that does not express the gene. Each plasmid is It was purified using a maxiprep column (Qiagen, Hilden, Germany) to obtain plasmid DNA for gene transfer.

(2) Preparation of Nanoplex Preparation of NP or complex of each gene expression enhancing NP and plasmid DNA (nanoplex) was carried out by preparing NP prepared in Experimental Example 1 or each gene expression enhancing NP (Examples 1 to 5). ) And plasmid DNA were mixed. Specifically, 9.5 μl of the above NP or each NP for gene expression enhancement was added to 50 μl of 50 mM NaCl aqueous solution containing 2 μg of plasmid DNA, gently stirred, and then stirred at room temperature (25 ± 5 ° C.) at 10 ° C. Desired nanoplexes (Control Example 2, Examples 6 to 10) were prepared by standing for a minute.

Experimental example 3
Each nanoplex prepared in Experimental Example 2 was evaluated for gene expression and cytotoxicity using human prostate cancer cells (PC-3 cells).
(1) PC-3 cell culture
PC-3 cells were obtained from Tohoku University Medical Research Center for Aging Medicine. The cells were cultured in RPMI-1640 medium supplemented with 10% non-immobilized fetal bovine serum (FBS) (Gibco, Inc.) and kanamycin (100 μg / ml) at 37 ° C and 5% CO ₂ . Performed under conditions. In gene expression and cytotoxicity assays, PC-3 cells were seeded in 96-well plates and used in experiments.

(2) Measurement of gene expression (luciferase assay)
Each nanoplex was prepared by adding 9.5 μl of the NP or each gene expression enhancing NP to 50 μl of 50 mM NaCl aqueous solution containing 2 μg of pCMV-Gluc. Each nanoplex was diluted with 1 ml medium supplemented with 10% FBS so that the DNA concentration in the medium was 2 μg / ml (K182 concentration was 2 μM), and the human prostate cancer cells (PCs) cultured in a 96-well plate were used. -3 cells) was added at 100 μl. Thereafter, the cells were cultured under the conditions described in (1) above, and 10 μL of the medium was collected 48 hours after the addition of the nanoplex, and the activity of luciferase secreted into the medium was measured.

  The luciferase activity (counts per second (cps) / μL) was measured using a Gaussia Luciferase Assay Kit (New England BioLabs, Inc., MA, USA). For control, luciferase activity was measured in the same manner as described above for cells transfected with NP of Control Example 1 instead of Nanoplex. As Comparative Examples 1 and 2, NP was diluted with media containing 2 μM K182 and 10 μM K182, respectively, and luciferase activity was measured in the same manner as described above.

  Example 6-9 Nanoplex (Example 6: NP-DD-K182, Example 9: NP-K182-DD), Control Example (NP), and Comparative Examples 1 and 2 (2 μM or 10 μM K182) Luciferase FIG. 1 shows the activity, and FIG. 2 shows the luciferase activities of the nanoplex (NP-DDTS-K182) of Example 10 and the control example (NP). In addition, each result is shown by relative% when the luciferase activity of the control example (NP) is 100%.

  As shown in FIG. 1, it was found that the nanoplexes (NP-DD-K182, NP-K182-DD) of Examples 6 and 9 had increased luciferase activity and enhanced luciferase gene expression. Moreover, as shown in FIG. 2, it was found that the nanoplex (NP-DDTS-K182) of Example 10 also increased the luciferase activity and enhanced the expression of the luciferase gene.

(3) Evaluation of cytotoxicity Nanoplex of Examples 6 to 10 (Example 6: NP-DD-K182, Example 7: NP-CM-K182, Example 8: NP-K182-CM, Example 9: NP-K182-DD), Control (NP), and Comparative Examples 1 and 2 (2 μM or 10 μM K182) were diluted with 1 ml RPMI-1640 medium to 2 μg pCMV-Gluc / mL, and the culture supernatant was removed. 100 μl was added to human prostate cancer cells (PC-3 cells) cultured in a 96-well plate. This was cultured under the conditions described in (1) above, and cell viability was measured at 48 hours using Cell Counting Kit-8 (Dojindo Laboratories). Cell viability was calculated as the ratio (%) of living cells to cells not administered with nanoplex.

  The results are shown in FIG. As shown in FIG. 3, each of the gene expression enhancing NPs has higher cytotoxicity than NP, and thus it has been found that K182 is released in the cells and exhibits an anti-cancer activity together with an effect of increasing gene expression.

Experimental Example 4
For each nanoplex prepared in Experimental Example 2, the endogenous gene expression increasing effect was evaluated using human prostate cancer cells (PC-3 cells).

(1) PC-3 cell culture
A stable expression strain of PC-3 cells that constantly express Gluc was prepared by geneticin (G418) at 48 hours after the introduction of pCMV-Gluc into PC-3 cells using Lipofectamine 2000 (Invitrogen). After culturing in RPMI-1640 medium containing 800 μM for 2 weeks, the cells transfected with the chromosome were cloned by limiting dilution. A PC-3 cell stable strain that efficiently secretes Gluc into the cell supernatant was selected from the obtained cells and used as a Gluc expression stable strain PC-3 cell (PC-3-Luc). PC-3-Luc cells were cultured using RPMI-1640 medium supplemented with 10% non-immobilized fetal bovine serum (FBS) (Gibco, Inc.) and kanamycin (100 μg / ml) at 37 ° C, 5% The test was performed under the condition of CO ₂ . In the gene expression and cytotoxicity assays, each cell was seeded in a 96-well plate and used for experiments.

(2) Measurement of endogenous gene expression (luciferase assay)
The nanoplex was prepared by adding 9.5 μl of the above-mentioned NP or each gene expression enhancing NP to 50 μl of 50 mM NaCl aqueous solution containing 2 μg of pGL3-basic. Each nanoplex (Examples 7 to 10) was diluted with 1 ml of medium supplemented with 10% FBS so that the DNA concentration in the medium was 2 μg / ml (K182 concentration was 2 μM), and cultured in a 96-well plate. 100 μl was added to PC-3-Luc cells. Thereafter, the cells were cultured under the conditions described in (1) above, and 10 μl of the medium was collected 48 hours after the addition of the nanoplex, and the activity of luciferase secreted into the medium was measured. For control, luciferase activity was measured in the same manner as above for cells transfected with NP of the control example instead of Nanoplex.

  The luciferase activities of the nanoplexes of Examples 7 to 9 (Example 7: NP-CM-K182, Example 8: NP-K182-CM, Example 9: NP-K182-DD) and the control example (NP) FIG. 4 shows the luciferase activity of the nanoplex of Example 10 (NP-DDTS-K182) and the control example (NP). In addition, each result is shown by relative% when the luciferase activity of the control example (NP) is 100%.

  As shown in FIGS. 4 and 5, it was found that the luciferase activity was increased and the expression of the luciferase gene was enhanced for the nanoplexes of the present invention of Examples 7-10. This shows that endogenous gene expression is increased by nanoplex administration.

  In addition, K182 was dissolved in a cell culture medium and then added to a Gluc stable expression strain, and the effect on Gluc gene expression was observed. Specifically, K182 was added to the cells at 1 μM, 2 μM, and 10 μM, and the luciferase activity secreted into the medium after 48 hours was measured. FIG. 6 shows the value of the increased gene expression rate (%) compared to the medium without K182. As shown in FIG. 6, it was found that when a medium containing K-182 was added, the luciferase activity was increased and the expression of the luciferase gene was increased. This shows that K182 increases endogenous gene expression by histone acetylase inhibitory action.

Regarding the nanoplex of the present invention (complex of NP and gene for enhancing gene expression) (Example 6: NP-DD-K182, Example 9: NP-K182-DD), luciferase gene expression enhancement in human prostate cancer cells The result of investigating the effect is shown (Experimental Example 3). Nanoplex was prepared using pCMV-Gluc and administered to cells. The culture supernatant was collected 24 hours after administration, and luciferase activity was measured. About the nanoplex of this invention (complex of NP for gene expression enhancement and a gene) (Example 10: NP-DDTS-K182), the result of having investigated the luciferase gene expression enhancement effect in a human prostate cancer cell is shown (experimental example) 3). Nanoplex of the present invention (complex of gene expression enhancing NP and gene) (Example 6: NP-DD-K182, Example 7: NP-CM-K182, Example 8: NP-K182-CM, Implementation) (Example 9: NP-K182-DD) shows the results of examining cytotoxicity against human prostate cancer cells (Experimental Example 3). Nanoplex was prepared using pCMV-Gluc and administered to cells. Cell viability was measured by WST-8 assay 48 hours after administration. About the nanoplex (complex of NP for gene expression enhancement, and a gene) of this invention, the result of having investigated the gene expression with respect to a human prostate cancer cell is shown (experimental example 4). This is the result when pGL3-basic is introduced by NP-K182-DD, NP-CM-K182, or NP-K182-DD for Gluc stable expression strain, and endogenous gene expression is increased by nanoplex administration. Indicates that The result of having investigated the gene expression with respect to the human prostate cancer cell about the nanoplex (complex of NP for gene expression enhancement and a gene) (Example 10: NP-DDTS-K182) of the present invention is shown (Experimental Example 4). It is a result when pGL3-basic is introduced by NP-DDTS-K182 to the Gluc stable expression strain, and shows that endogenous gene expression is increased by nanoplex administration. The results of observing the effect on expression of Gluc gene after adding K182 in cell culture medium and then adding to Gluc stable expression strain are shown (Experimental Example 4). K182 was added to the cells at 1, 2, and 10 μM, and the luciferase activity secreted into the medium was measured 48 hours later. The value of gene rise expression rate (%) compared with the culture medium without K182 is shown.

Claims (11)

The following formula (1)

A compound in which a cholesteryl group, an alkylcarbonyl group, an oxyalkyl group, or a thioalkyl group is bonded directly or via a linker to either a hydroxyl group or a sulfhydryl group of the compound represented by formula (1). The compound according to claim 1, wherein the compound (1) is a compound described in the general formula (2):

(In the formula, X is an oxygen atom or a sulfur atom; A and B are a hydrogen atom, a cholesteryl group, an alkylcarbonyl group having 3 to 20 carbon atoms, an oxyalkyl group having 3 to 20 carbon atoms, or 3 to 3 carbon atoms, respectively. 20 thioalkyl groups; l is an integer of 1 or 2; m is an integer of 0 or 1; Y is a group represented by the following formula (3) or (4);

[Wherein, W represents an oxygen atom, a nitrogen atom or a sulfur atom; “S—Z—CO” represents a residue of an amino acid having a thiol group or a thioalkylcarbonyl group; n represents an integer of 0 or 1; Means an integer of 1 to 8]
Means. However, one of “— (Y) _m —A” or “— (Y) _m —B” is a hydrogen atom, and in that case, the other is not a hydrogen atom. ). The compound according to claim 1 or 2, wherein the compound represented by the general formula (1) is at least one selected from the group consisting of compounds represented by the following formula:
(1) 2-[{4-[(hydroxyamino) carbonyl] benzyl} (2-naphthylmethyl) amino] ethyl laurate,
(2) 1-cholest-5-en-3-yl 4- {2-[{4-[(hydroxyamino) carbonyl] benzyl} (2-naphthyl
methyl) amino] ethyl} succinate,
(3) cholest-5-en-3-yl 4-{[(4-{[(2-hydroxyethyl) (2-naphthylmethyl) amino] methyl}
benzoyl) amino] oxy} -4-oxobutanoate,
(4) N- (dodecanoyloxy) -4-{[(2-hydroxyethyl) (2-naphthylmethyl) amino] methyl}
benzamide,
(5) 2- (dodecyldisulfanyl) ethyl 2-[{4-[(hydroxyamino) carbonyl] benzyl} (2-naphthyl
methyl) amino] ethyl carbonate, and
(6) cholest-5-en-3-yl (13R) -13-amino-2- {4-[(hydroxyamino) carbonyl] benzyl} -1- (2-
naphthyl) -6-oxo-5,7-dioxa-10,11-dithia-2-azatetradecan-14-oate
(7) 4-{[[3- (dodecyldisulfanyl) propyl] (2-naphthylmethyl) amino] methyl} -N-
hydroxybenzamide.   A gene expression enhancer comprising the compound according to any one of claims 1 to 3.   The gene expression enhancer according to claim 4, comprising the compound according to any one of claims 1 to 3 and a cationic lipid or cholesterol.   The gene expression enhancer according to claim 5, which has a lipid membrane cyclic structure or a granular structure.   A gene introduction kit comprising the gene expression enhancer according to any one of claims 4 to 6.   The gene introduction kit according to claim 7, comprising the gene expression enhancer according to any one of claims 4 to 6 and a gene to be introduced.   A gene-containing composition with enhanced expression, comprising the gene expression enhancer according to any one of claims 4 to 6 and a gene.   A method for enhancing the expression of an endogenous or exogenous gene by introducing the gene expression enhancer according to any one of claims 4 to 6 or the gene-containing composition according to claim 9 into a cell in vitro. .   The method according to claim 10, wherein the cell is a tumor cell.

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