WO2018225871A1

WO2018225871A1 - Compound serving as cationic lipid

Info

Publication number: WO2018225871A1
Application number: PCT/JP2018/022220
Authority: WO
Inventors: 智幸直井; 慎太郎細江; 泰典植村; 香機八木
Original assignee: 協和発酵キリン株式会社
Priority date: 2017-06-09
Filing date: 2018-06-11
Publication date: 2018-12-13

Abstract

The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof. (In the formula, R1 represents a hydrogen atom or a hydroxymethyl group; n1 represents 1 or 2; A1 and A2 may be the same or different, and each represents a linear C9-C20 alkylene group or the like; M1 and M2 may be the same or different, and each represents -OC(O)-, -C(O)O- or -NHC(O)-; B1 and B2 may be the same or different, and each represents a linear or branched C1-C12 alkyl group or the like; R2 is absent or represents a C1-C3 alkyl group; and in cases where R2 is absent, Y is also absent, while in cases where R2 is a C1-C3 alkyl group, Y is a pharmaceutically acceptable anion.)

Description

Compounds as cationic lipids

The present invention relates to a novel compound as a cationic lipid and a pharmaceutical composition containing the novel compound.

In recent years, nucleic acid drugs have attracted attention as drug formats having a mechanism of action different from existing low-molecular drugs and antibody drugs such as gene therapy, gene expression suppression, and intermolecular interaction inhibition. For example, a short interfering RNA (hereinafter abbreviated as siRNA) has a function of cleaving a target mRNA having its complementary sequence and suppressing the expression of the target protein. In addition, circularly-structured plasmid DNA (pDNA) that exists independently of chromosomal DNA can be introduced into cells to express the target protein. Further, in recent years, a method for expressing a target protein in vivo using artificially synthesized mRNA is known.
In vivo applications of such nucleic acid drugs include administration of nucleic acids alone and administration of nucleic acids utilizing Drug Delivery System (DDS) technology in which nucleic acids are loaded on a carrier for delivery. For example, development of DDS technology using lipid nanoparticles as a carrier for nucleic acid delivery is progressing. In general, lipid nanoparticles for nucleic acid delivery contain a cationic lipid.
Cationic lipids are amphiphilic molecules having a lipophilic region containing one or more hydrocarbon groups and at least one hydrophilic region that can be positively charged. A process in which cationic lipids and macromolecules such as nucleic acids form a positively charged complex that makes it easier for nucleic acids and other macromolecules to pass through the plasma membrane of the cell and enter the cytoplasm. Exists. For this reason, cationic lipids are useful. This process, which can be performed in vitro and in vivo, is also known as transfection.

Patent Documents 1 to 4 describe cationic lipids useful for delivering nucleic acids into cells in vivo and for use in nucleic acid-lipid particle compositions suitable for disease treatment, and cationic lipids. Disclosed are lipid particles comprising.
Patent Document 1 discloses, for example, 2,2-dilinoleyl-4- (2-dimethylaminoethyl)-[1,3] -dioxolane; DLin-KC2-DMA).

Patent Document 2 includes, for example, (6Z, 9Z, 28Z, 31Z) -heptatriconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butanoate; DLin-MC3-DMA) and the like. It is disclosed.

Patent Document 3 discloses, for example, 3- [di [(9Z, 12Z) -octadeca-9,12-dien-1-yl] amino] propan-1-ol.

Patent Document 4 discloses, for example, bis (2-hexyldecyl) 6,6 ′-[(3-hydroxypropyl) azanediyl] dihexanoate.

In addition, Non-Patent Document 1 states that by introducing a biodegradable group into a part of the fatty chain of a cationic lipid, the toxicity in the liver can be reduced while maintaining the ability to deliver nucleic acids to cells in vivo. Disclosed, for example, di [(Z) -non-2-en-1-yl] 9-[[4- (dimethylamino) butanoyl] oxy] heptadecandioate (Di [(Z) -non-2-en Cationic lipids such as 1-yl] 9-[[4- (dimethylamino) butanoyl] oxy] heptadecanedioate) are disclosed.
In recent studies, it has been reported that therapeutic agents of nucleic acid drugs using such lipid nanoparticles exhibit the therapeutic effect of nucleic acid drugs by being delivered to various organs. For example, respiratory diseases targeting the lung include refractory lung diseases such as pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), bronchial asthma and the like. Sufficient therapeutic effect is not obtained by therapy. New therapeutic methods using nucleic acid drugs for such intractable lung diseases are being actively studied.
Non-Patent Document 2 discloses that siRNA can be efficiently delivered to the lung by intravenous administration of liposomes using 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) as a cationic lipid, and It is disclosed that a gene of interest is suppressed in lung epithelial cells. Patent Document 5 discloses that administration of a cationic liposome and a plasmid DNA complex intratracheally increases target gene expression in the human lung and provides a therapeutic effect. Has been. On the other hand, Patent Document 5 discloses a result that intratracheal administration of a cationic liposome and a plasmid DNA complex induces a systemic inflammatory reaction, and Non-Patent Document 3 uses DOTAP. It is disclosed that lipid nanoparticles have a pro-inflammatory effect on various cells. For these reasons, lipid nanoparticles for nucleic acid delivery with higher safety are demanded.

International Publication No. 2010/042877 International Publication No. 2010/054401 International Publication No. 2014/007398 International Publication No. 2016/176330 International Publication No.2013 / 061091

An object of the present invention is to provide, for example, a novel compound as a cationic lipid capable of introducing a nucleic acid into cells and the like, a pharmaceutical composition containing the novel compound, and the like.

The present invention relates to the following (1) to (24).
(1) A compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof.

(Where
R1 is a hydrogen atom or hydroxymethyl,
n1 is 1 or 2,
A1 and A2 are the same or different and are linear C9-C20 alkylene or C9-C20 alkenylene,
M1 and M2 are the same or different and are —OC (O) —, —C (O) O— or —NHC (O) —,
B1 and B2 are the same or different and are linear or branched C1-C12 alkyl or C2-C13 alkenyl,
R2 is absent or is C1-C3 alkyl;
If R2 does not exist, Y does not exist,
When R2 is C1-C3 alkyl, Y is a pharmaceutically acceptable anion. )
(2) The compound according to (1) or a pharmaceutically acceptable salt thereof, wherein B1 and B2 are the same or different and are linear C1-C12 alkyl or C2-C13 alkenyl.
(3) The compound according to (1) or (2) or a pharmaceutically acceptable salt thereof, wherein A1 and A2 are the same or different and are linear C9-C12 alkylene.
(4) The compound according to any one of (1) to (3), or a pharmaceutically acceptable salt thereof, wherein B1-M1-A1 and B2-M2-A2 are the same.
(5) The compound according to any one of (1) to (4) or a pharmaceutically acceptable salt thereof, wherein R1 is a hydrogen atom and n1 is 2.
(6) The compound or a pharmaceutically acceptable salt thereof according to any one of (1) to (4), wherein R1 is hydroxymethyl and n1 is 1.
(7) A compound represented by the following formula (II) or a pharmaceutically acceptable salt thereof.

(Where
n2 is 1 or 2,
L is a linear C12-C24 alkenyl,
A3 is a linear C5-C14 alkylene,
M3 is -OC (O)-, -C (O) O- or -NHC (O)-
B3 is linear or branched C1-C12 alkyl or C2-C13 alkenyl. )
(8) A pharmaceutical composition comprising the compound according to any one of (1) to (7), or a pharmaceutically acceptable salt thereof, and a nucleic acid.
(9) The pharmaceutical composition according to (8), wherein the nucleic acid is a nucleic acid having an action of suppressing the expression of a target gene using RNA interference (RNAi).
(10) The pharmaceutical composition according to (9), wherein the target gene is a gene expressed in the liver, lung, or spleen.
(11) The pharmaceutical composition according to (9), wherein the target gene is a gene expressed in the lung.
(12) The pharmaceutical composition according to any one of (8) to (11), which is for intravenous administration.
(13) The pharmaceutical composition according to any one of (8) to (11), which is for intratracheal administration.
(14) A target gene expression inhibitor using RNA interference (RNAi), comprising the pharmaceutical composition according to any one of (8) to (13).
(15) A therapeutic or prophylactic agent for a disease associated with the liver, lung or spleen, comprising the pharmaceutical composition according to any one of (8) to (13).
(16) A therapeutic or prophylactic agent for diseases related to the lung, comprising the pharmaceutical composition according to any one of (8) to (13).
(17) A method for treating or preventing a disease associated with the liver, lung or spleen, comprising a step of administering the pharmaceutical composition according to any one of (8) to (13) to a subject.
(18) A therapeutic or prophylactic agent for diseases related to the lung, comprising the pharmaceutical composition according to any one of (8) to (13).
(19) A method for treating or preventing a lung-related disease, comprising a step of administering the pharmaceutical composition according to any one of (8) to (13) to a subject.
(20) An antitumor agent comprising the pharmaceutical composition according to any one of (8) to (13).
(21) A method for treating or preventing a malignant tumor, comprising a step of administering the pharmaceutical composition according to any one of (8) to (13) to a subject.
(22) The pharmaceutical composition according to any one of (8) to (13) for use in the treatment or prevention of a disease.
(23) The pharmaceutical composition according to (22) for use in the treatment or prevention of diseases associated with liver, lung or spleen.
(24) The pharmaceutical composition according to (22) for use in the treatment or prevention of diseases related to the lung.

The present invention can provide, for example, a novel compound as a cationic lipid capable of introducing a nucleic acid into a cell or the like, a composition containing the novel compound, and the like.

Formulation 2 and Formulation A-2 obtained in Example 26 and Comparative Example 2 were each administered in normal mice at a dose of 15 μg / mouse and then contained in alveolar lavage fluid (BALF). Cytokine levels were calculated. FIG. 1 shows the results of Keratinocyte chemoattractant (KC). The vertical axis represents the amount of each cytokine (pg / mL), and the horizontal axis represents the formulation number and negative control group (saline). As the amount of cytokine contained in the same BALF as in FIG. 1, FIG. 2 shows the results of Interleukin-6 (IL-6). The vertical axis represents the amount of each cytokine (pg / mL), and the horizontal axis represents the formulation number and negative control group (saline). As the amount of cytokine contained in the same BALF as in FIG. 1, FIG. 3 shows the result of Granulocyte-colony サイトカイン stimulating factorBA (G-CSF). The vertical axis represents the amount of each cytokine (pg / mL), and the horizontal axis represents the formulation number and negative control group (saline). Formulation 2-2, Formulation 3 and Formulation 13 obtained in Example 26 were each intratracheally administered to normal mice at a dose of 20 μg / mouse, and then the amount of each cytokine contained in BALF was calculated. FIG. 4 shows the KC results. The vertical axis represents the amount of each cytokine (pg / mL), and the horizontal axis represents the formulation number and negative control group (saline). FIG. 5 shows the results of IL-6 as the amount of cytokine contained in the same BALF as FIG. The vertical axis represents the amount of each cytokine (pg / mL), and the horizontal axis represents the formulation number and negative control group (saline). FIG. 6 shows the result of G-CSF as the amount of cytokine contained in the same BALF as FIG. The vertical axis represents the amount of each cytokine (pg / mL), and the horizontal axis represents the formulation number and negative control group (saline). Each of Formulation 4, Formulation 6 and Formulation 12 obtained in Example 26 was intratracheally administered to normal mice at a dose of 20 μg / mouse, and then the amount of each cytokine contained in BALF was calculated. FIG. 7 shows the KC results. The vertical axis represents the amount of each cytokine (pg / mL), and the horizontal axis represents the formulation number and negative control group (saline). FIG. 8 shows the result of IL-6 as the amount of cytokine contained in the same BALF as FIG. The vertical axis represents the amount of each cytokine (pg / mL), and the horizontal axis represents the formulation number and negative control group (saline). FIG. 9 shows the results of G-CSF as the amount of cytokine contained in the same BALF as FIG. The vertical axis represents the amount of each cytokine (pg / mL), and the horizontal axis represents the formulation number and negative control group (saline). Formulation 2-3, Formulation 2-4, Formulation A-3 and Formulation A-4 obtained in Example 28, Example 29, Comparative Example 3 and Comparative Example 4, respectively, at a dose of 10 mg / kg, normal Mice were administered intravenously. The amount of each cytokine contained in the blood was calculated 24 hours after administration. FIG. 10 shows the KC results. The vertical axis represents the amount of each cytokine (pg / mL), and the horizontal axis represents the formulation number and negative control group (saline). As the amount of cytokine contained in the same blood as FIG. 10, FIG. 11 shows the results of IL-6. The vertical axis represents the amount of each cytokine (pg / mL), and the horizontal axis represents the formulation number and negative control group (saline). FIG. 12 shows the result of G-CSF as the amount of cytokine contained in the same blood as FIG. The vertical axis represents the amount of each cytokine (pg / mL), and the horizontal axis represents the formulation number and negative control group (saline). Bleomycin (Nippon Kayaku) was intratracheally administered to C57BL / 6J mice under the conditions of 0.012 mg / mouse and obtained in Example 30, Example 31, Comparative Example 5 and Comparative Example 6 after 7, 9 and 13 days. Formulation 2-5, Formulation 2-6, Formulation A-5 and Formulation A-6 were each administered intratracheally under the condition of 1 μg / mouse. BALF and left lung were collected 24 hours after the last dose. FIG. 13 shows the results of quantifying the amount of Tgfb-1 mRNA in the left lung. The amount of mRNA in the specimen was expressed as a relative ratio when the amount of Tgfb-1 mRNA relative to the amount of HPRT1 mRNA was calculated and the value in normal mice was 1. The vertical axis indicates the relative value of the mRNA amount of the sample calculated as described above, and the horizontal axis indicates the formulation number and the negative control group (saline). FIG. 14 shows the results of quantifying the amount of Tgfb-1 mRNA in cells contained in BALF collected simultaneously with the left lung in FIG. The amount of mRNA in the specimen was expressed as a relative ratio when the amount of Tgfb-1 mRNA relative to the amount of HPRT1 mRNA was calculated and the value in normal mice was 1. The vertical axis indicates the relative value of the mRNA amount of the sample calculated as described above, and the horizontal axis indicates the formulation number and the negative control group (saline). FIG. 15 shows the total amount of Tgfb-1 protein contained in the same BALF as FIG. The vertical axis represents the amount of protein (pg / mL) of Tgfb-1, and the horizontal axis represents the formulation number, the negative control group (saline), and normal mice.

The compounds of the present invention
Formula (I) below

(Where
R1 is a hydrogen atom or hydroxymethyl,
n1 is 1 or 2,
A1 and A2 are the same or different and are linear C9-C20 alkylene or C9-C20 alkenylene,
M1 and M2 are the same or different and are —OC (O) —, —C (O) O— or —NHC (O) —,
B1 and B2 are the same or different and are linear or branched C1-C12 alkyl or C2-C13 alkenyl,
R2 is absent or alkyl having 1 to 3 carbon atoms,
If R2 does not exist, Y does not exist,
When R2 is C1-C3 alkyl, Y is a pharmaceutically acceptable anion. Or a compound represented by the following formula (II)

(Where
n2 is 1 or 2,
L is a linear C12-C24 alkenyl,
A3 is a linear C5-C14 alkylene,
M3 is -OC (O)-, -C (O) O- or -NHC (O)-
B3 is linear or branched C1-C12 alkyl or C2-C13 alkenyl. ).
When Y which is an anion is present, a part or the whole of the structure other than Y in the formula (I) exists as a cation, and the anion and the cation form an ionic bond.

The compounds represented by formula (I) and formula (II) have a lipophilic region containing two hydrocarbon groups and a hydrophilic region containing one positively chargeable polar head group, and a cation It has properties as a functional lipid.

Hereinafter, the compound represented by the formula (I) may be referred to as the compound (I). The same applies to the compounds of other formula numbers. Hereinafter, the compounds represented by the formulas (I) and (II), or pharmaceutically acceptable salts thereof may be collectively referred to as “cationic lipids”.

Examples of the linear C9-C20 alkylene include nonylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene, hexadecylene, octadecylene, icosylene and the like.
In the present invention, the case of C9-C20 alkylene will be described as an example. C9-C20 in C9-C20 alkylene means that the alkylene has 9 to 20 carbon atoms.

The linear C9-C20 alkenylene may be a group containing one or more double bonds in the linear C9-C20 alkylene, such as (Z) -tetradec-9-enylene, (Z) -hexadecaylene. -9-Enylene, (Z) -Heptadeca-9-Enylene, (E) -Heptadeca-9-Enylene, (Z) -Heptadeca-11-Enylene, (9Z, 12Z) -Heptadeca-9,12-Dienylene, ( 9Z, 12Z, 15Z) -heptadeca-9,12,15-trienylene, (Z) -octaca-6-enylene, (Z) -octadeca-9-enylene, (E) -octadeca-9-enylene, (Z) -Octadeca-11-enylene, (9Z, 12Z) -octadeca-9,12-dienylene, and (9Z, 12Z, 15Z) -octadeca-9,12,15-trienylene, and their structural isomers. In the description of linear C9-C20 alkenylene, (Z) -tetradec-9-enylene will be described as an example. When 9- is the first carbon atom in A1 and A2 bonded to the nitrogen atom Represents the substitution position in alkenylene which is A1 and A2.

Examples of linear or branched C1-C12 alkyl include methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, hexyl, octyl, nonyl, 2-nonyl, decyl, and dodecyl, and structural isomers thereof. Is mentioned. In the description of branched C1-C12 alkyl, when 2-nonyl is described as an example, 2- is bonded to M1, M2, and M3 among terminal carbon atoms in the alkyl that is B1, B2, and B3. This represents the position of bonding to M1 and M2 in the alkyl as B1, B2 and B3 when the terminal carbon atom closer to the position is the 1st position. Such a description is similarly applied to branched C2-C13 alkenyl described later.

Examples of linear or branched C2-C13 alkyl include ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and tridecyl, and structural isomers thereof.

The linear or branched C2-C13 alkenyl may be a group containing one or more double bonds in the linear or branched C2-C13 alkyl, such as vinyl, allyl, (Z)- But-2-enyl, (Z) -pent-2-enyl, (Z) -hex-2-enyl, (Z) -hept-2-enyl, (Z) -oct-2-enyl, octa-7- Enyl, (E) -3,7-dimethylocta-2,6-dien-1-yl (geranyl), 3,7-dimethyloct-6-en-1-yl (citronellyl), (Z) -nona- 2-enyl, (Z) -nona-3-enyl, (Z) -nona-5-enyl, (E) -nona-2-enyl, nona-8-enyl, (Z) -undec-2-enyl, Examples include undeca-10-enyl, (Z) -dodec-2-enyl and (Z) -tridec-2-enyl, and their structural isomers. In the description of linear or branched C2-C13 as alkenyl, (Z) -but-2-enyl will be described as an example. -2- is the terminal carbon in alkenyl which is B1, B2 and B3. Of the atoms, the position of the double bond in the alkenyl as B1, B2 and B3 when the terminal carbon atom closer to the position of bonding to M1, M2 and M3 is defined as the 1st position.

Examples of C1-C3 alkyl include methyl, ethyl, propyl, isopropyl, cyclopropyl and the like.

Examples of the linear C12-C24 alkyl include dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, docosyl, tetracosyl and the like.

The linear C12-C24 alkenyl may be a group containing one or more double bonds in the linear C12-C24 alkyl, such as (Z) -tetradec-9-enyl, (Z) -hexadeca -9-enyl, (Z) -octadeca-6-enyl, (Z) -octadeca-9-enyl, (E) -octadeca-9-enyl, (Z) -octadeca-11-enyl, (9Z, 12Z) -Octadec-9,12-dienyl, (9Z, 12Z, 15Z) -octadeca-9,12,15-trienyl and the like.

Examples of the linear C5-C14 alkylene include pentylene, hexylene, octylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene and the like.

In the present invention, a linear or branched C2-C13 alkenyl includes a group having a cyclopropane ring in which a methylene biradical is added formally to a double bond of a linear or branched C2-C13 alkenyl. Is done. Furthermore, linear C9-C20 alkenylene and linear C12-C24 alkenyl are also included in linear or branched C2-C13 alkenyl.
Taking (Z) -non-2-enyl as an example, the following groups having a cyclopropane ring are also included in the linear or branched C2-C13 alkenyl in the present invention.

In the present invention, when R2 is C1-C3 alkyl, Y is a pharmaceutically acceptable anion. Examples of the pharmaceutically acceptable anion include chloride ion, bromide ion, and nitrate ion. And inorganic acid ions such as sulfate ion and phosphate ion, and organic acid ions such as acetate ion, oxalate ion, maleate ion, fumarate ion, citrate ion, benzoate ion, and methanesulfonate ion.

N1 is preferably 2 when R1 is a hydrogen atom.

N1 is preferably 1 when R1 is hydroxymethyl.

A1 and A2 are the same or different and are linear C9-C20 alkylene or C9-C20 alkenylene, preferably linear C9-C20 alkylene, preferably linear C9-C15 alkylene. More preferably, it is more preferably a linear C9-C12 alkylene.
A1 and A2 are preferably nonylene, undecylene, tridecylene, or pentadecylene, and more preferably nonylene or undecylene.
A1 and A2 are preferably the same.

M1 and M2 are the same or different and are —OC (O) —, —C (O) O— or —NHC (O) —, and are —OC (O) — or —C (O) O—. And M1 and M2 are more preferably the same.
In the description of M1 and M2, in the case where M1 and M2 are —OC (O) —, for example, —OC (O) — represents B1-OC (O) -A1 or B2-OC (O ) -A2 means binding.

B1 and B2 are the same or different and are linear or branched C1-C12 alkyl or C2-C13 alkenyl, preferably linear C1-C12 alkyl or C2-C13 alkenyl, and are the same More preferably, they are linear C1-C12 alkyl or C2-C13 alkenyl, more preferably the same, linear C2-C13 alkenyl, and the same, linear C5-C13 alkenyl. It is even more preferred that they are C11 alkenyl, and even more preferred that they are the same and linear C7-C10 alkenyl.
B1 and B2 are preferably (Z) -hept-2-enyl, (Z) -hept-3-enyl, hepta-8-enyl, (Z) -non-2-enyl, (Z) -nona-3 -Enyl, nona-8-enyl, more preferably (Z) -non-2-enyl,
(Z) -non-3-enyl and non-8-enyl.

B1-M1-A1 and B2-M2-A2 are preferably the same. B1-M1-A1 and B2-M2-A2 are the same or different and are preferably the following structures (1) to (6), preferably the same and the following structures (1) to (6) Are more preferably the same and more preferably the following structures (1) to (4).

In the structures (1) to (6), the wavy bond is a bond to the nitrogen atom in the formula (I).
n3 is preferably an integer of 2 to 10, more preferably an integer of 2 to 5, and further preferably 2, 4 or 5.

It is preferable that R2 does not exist and Y does not exist.

Examples of the pharmaceutically acceptable salt of the compound represented by the formula (I) of the present invention include hydrochloride, odorate, nitrate, sulfate, phosphate, acetate, oxalate, maleate, Examples include fumarate, citrate, benzoate, and methanesulfonate.

N2 is preferably 2.

L is linear C12-C24 alkenyl, preferably linear C14-C18 alkenyl, more preferably linear C16-C18 alkenyl, and linear C18 More preferably, it is alkenyl.

A3 is a linear C5-C14 alkylene, preferably a linear C5-C10 alkylene, and more preferably a linear C5-C7.

M3 is -OC (O)-, -C (O) O- or -NHC (O)-, preferably -OC (O)-or -C (O) O-, O)-is more preferable.

B3 is linear or branched C1-C12 alkyl or C2-C13 alkenyl, preferably linear C1-C12 alkyl or C2-C13 alkenyl, and is linear C2-C13 alkenyl. More preferably.

B3-M3-A3 preferably has the following structures (1) to (6), and more preferably has the following structures (1) to (4).

In the structures (7) to (12), the wavy bond is a bond to the nitrogen atom in the formula (II).
n4 is preferably an integer of 1 to 8, more preferably an integer of 1 to 5, still more preferably an integer of 1 to 3, and even more preferably 1 or 3.

A method for producing the compounds represented by the formulas (I) and (II) of the present invention, or pharmaceutically acceptable salts thereof (hereinafter collectively referred to as cationic lipids) will be described.
In the production method shown below, when the defined group changes under the conditions of the production method or is inappropriate for carrying out the production method, a method for introducing and removing a protective group commonly used in organic synthetic chemistry [ For example, use the method described in Protective Groups in Organic Synthesis, third edition, TW Greene, John Wiley & Sons Inc. (1999), etc.] Thus, the target compound can be produced. Further, the order of reaction steps such as introduction of substituents can be changed as necessary.

Production method 1
Compound (Ia) or compound (Ib) can be produced, for example, by the following method.

(Wherein, A1, A2, M1, M2, B1, B2, n1, R1 and Y are as defined above, R2 ′ represents C1-C3 alkyl, and X1, X2 and X3 are the same or different, (Representing leaving groups such as chlorine atom, bromine atom, iodine atom, trifluoromethanesulfonyloxy, methanesulfonyloxy, benzenesulfonyloxy, p-toluenesulfonyloxy, etc.)

Process 1-3
Compound (IIIf) is compound (IIIa) and compound (IIIb), compound (IIIc) and compound (IIId), or compound (IIIa) and compound (IIIe), each without solvent or in a solvent. It can be produced by reacting with 10 equivalents of a condensing agent in the presence of 1 to 10 equivalents of base at room temperature to 200 ° C. for 5 minutes to 50 hours.

Examples of the solvent include dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, 1,4-dioxane, N, N-dimethylformamide, N, N -Dimethylacetamide, N-methylpyrrolidone, pyridine and the like can be mentioned, and these can be used alone or in combination.

Examples of the condensing agent include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, N, N′-dicyclohexylcarbodiimide, 4- (4,6-dimethoxy-1,3,5-triazine-2- ジン yl ) -4-Methylmorpholinium chloride n hydrate, 1H-benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate, O- (7-azabenzotriazol-1-yl) -N , N, N ′, N ′,-tetramethyluronium hexafluorophosphate and the like.

Examples of the base include potassium carbonate, cesium carbonate, triethylamine, diisopropylethylamine, N-methylmorpholine, pyridine and the like.

Compound (IIIa) is a commercially available product or a known method [for example, “New Experimental Chemistry Lecture 14 Synthesis and Reaction of Organic Compounds (II)”, first edition, Maruzen (1977), “March's Advanced Organic Chemistry: Reactions, Mechanisms, And It can be obtained by combining the methods described in “Structure, 7 ^th Edition”] or a method according thereto.

Compound (IIIb), compound (IIIc), compound (IIId) and compound (IIIe) can be obtained as commercial products.

Step 4 and Step 5
Compound (IVb) is produced by reacting compound (IVa) and compound (IIIf) in the presence of 1 to 500 equivalents of a base without solvent or in a solvent at room temperature to 200 ° C. for 5 minutes to 50 hours. can do. Further, compound (Ia) is obtained by reacting compound (IVb) and compound (IIIg) without solvent or in the presence of 1 to 500 equivalents of a base at room temperature to 200 ° C. for 5 minutes to 50 hours. Can be manufactured.

Examples of the solvent include ethanol, toluene, acetonitrile, tetrahydrofuran, 1,2-dimethoxyethane, 1,4-dioxane, N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, pyridine and the like. These can be used alone or in combination.

Examples of the base include triethylamine, diisopropylethylamine, N-methylmorpholine, pyridine, 1,8-diazabicyclo [5.4.0] -7-undecene (DBU), and the like.

Compound (Ia) in the case where A1 and A2 and B1 and B2 are the same can be produced by using 2 equivalents or more of compound (IIIf) in Step 4.

Compound (IIIg) can be produced in the same manner as Compound (IIIf).

Compound (IVa) can be obtained as a commercial product

Process 6
Compound (Ib) can be produced by reacting compound (Ia) with one or more equivalents of compound (IIIh) without solvent or in a solvent at 0 ° C. to 100 ° C. for 5 minutes to 100 hours. If necessary, Y can be changed to a desired pharmaceutically acceptable anion by using an appropriate method such as a commercially available anion exchange resin.

Examples of the solvent include those exemplified in Steps 1 to 3.

Compound (IIIh) can be obtained as a commercial product.

Production method 2
Compound (II) can be produced, for example, by the following method.

(Wherein, L, A3, M3, B3, n2 are as defined above, and X4 and X5 are the same or different, chlorine atom, bromine atom, iodine atom, trifluoromethanesulfonyloxy, methanesulfonyloxy, benzenesulfonyl (Representing a leaving group such as oxy, p-toluenesulfonyloxy)

Step 7 and Step 8
Compound (IVd) is produced by reacting compound (IIIi) and compound (IVc) in the presence of 1 to 500 equivalents of a base without solvent or in a solvent at room temperature to 200 ° C. for 5 minutes to 50 hours. can do. Further, compound (II) is obtained by reacting compound (IVd) and compound (IIIj) in the presence of 1 to 500 equivalents of a base without solvent or in a solvent at room temperature to 200 ° C. for 5 minutes to 50 hours. Can be manufactured.

Examples of the solvent and base include those exemplified in Step 4 and Step 5.

Compound (IIIi) and compound (IVc) can be obtained as commercial products.

Compound (IIIi) can be produced in the same manner as Compound (IIIf).

The intermediates and target compounds in the above production methods should be isolated and purified by separation and purification methods commonly used in organic synthetic chemistry, such as filtration, extraction, washing, drying, concentration, recrystallization, and / or various chromatography. Can do. The intermediate may be subjected to the next reaction without particular purification.

In the compound (I) of the present invention, a hydrogen ion may be coordinated to a lone electron pair on the nitrogen atom in the structure, and in that case, a salt with a pharmaceutically acceptable anion may be formed. In the present invention, a cationic lipid in which a hydrogen ion is coordinated to a lone pair on a nitrogen atom in the structure is also included as a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof. The

Among the compounds (I) and (II) of the present invention, there may be stereoisomers such as geometric isomers or optical isomers or tautomers, and the like. ) And compound (II) include all possible isomers and mixtures thereof, including these.

A part or all of each atom in the compound (I) and compound (II) of the present invention may be replaced by a corresponding isotope atom, respectively, and the compound (I) and the compound (II) are those isotopes. Also includes compounds replaced with atoms. For example, some or all of the hydrogen atoms in the compounds (I) and (II) may be hydrogen atoms having a weight of 2 (deuterium atoms).

Compounds in which some or all of the respective atoms in the compounds (I) and (II) of the present invention are replaced by the corresponding isotope atoms are the same as in the above production methods using commercially available building blocks. It can be manufactured by the method. In addition, compounds in which some or all of the hydrogen atoms in the compounds (I) and (II) are replaced with deuterium atoms include, for example, alcohols, carboxyls using iridium complexes as catalysts and deuterium as a deuterium source. Manufacturing using a method of deuterating acids etc. [Journal of American Chemical Society (J. Am. Chem. Soc.), Vol. 124, No. 10, 2092 (2002)] etc. Can do.

Specific examples of compound (I) and compound (II) of the present invention are shown in Tables 1 to 6. However, the compound (I) and the compound (II) of the present invention are not limited to these.

The nucleic acid used in the present invention may be any molecule as long as it is a molecule obtained by polymerizing nucleotides and / or molecules having functions equivalent to nucleotides.
Examples of the nucleic acid include ribonucleic acid (RNA) which is a polymer of ribonucleotides, deoxyribonucleic acid (DNA) which is a polymer of deoxyribonucleotides, chimeric nucleic acids composed of RNA and DNA, and at least one nucleotide of these nucleic acids. Examples thereof include nucleotide polymers substituted with molecules having functions equivalent to nucleotides.
A nucleic acid used in the present invention also includes a derivative containing at least a part of the structure of a molecule obtained by polymerizing nucleotides and / or molecules having functions equivalent to nucleotides.
In the present invention, uracil U and thymine T can be replaced with each other.

Examples of molecules having functions equivalent to nucleotides include nucleotide derivatives.

The nucleotide derivative may be any molecule as long as it is a modified molecule, for example, in order to improve nuclease resistance or stabilize from other degradation factors compared to RNA or DNA, for example. In order to increase affinity with complementary strand nucleic acid, to increase cell permeability, or to visualize, a molecule in which ribonucleotide or deoxyribonucleotide is modified is preferably used.

Examples of nucleotide derivatives include sugar-modified nucleotides, phosphodiester bond-modified nucleotides, base-modified nucleotides, and the like.

The sugar-modified nucleotide may be any nucleotide as long as part or all of the chemical structure of the sugar of the nucleotide is modified or substituted with any substituent, or substituted with any atom. '-Modified nucleotides are preferably used.

Examples of the modifying group in the sugar moiety-modified nucleotide include 2′-cyano, 2′-alkyl, 2′-substituted alkyl, 2′-alkenyl, 2′-substituted alkenyl, 2′-halogen and 2′-O-cyano. 2'-O-alkyl, 2'-O-substituted alkyl, 2'-O-alkenyl, 2'-O-substituted alkenyl, 2'-S-alkyl, 2'-S-substituted alkyl, 2'-S -Alkenyl, 2'-S-substituted alkenyl, 2'-amino, 2'-NH-alkyl, 2'-NH-substituted alkyl, 2'-NH-alkenyl, 2'-NH-substituted alkenyl, 2'-SO -Alkyl, 2'-SO-substituted alkyl, 2'-carboxy, 2'-CO-alkyl, 2'-CO-substituted alkyl, 2'-Se-alkyl, 2'-Se-substituted alkyl, 2'-SiH ₂ -alkyl, 2'-SiH ₂ -substituted alkyl, 2'-ONO ₂ , 2'-NO ₂ , 2'-N ₃ , 2'-amino acid residue (hydroxyl group removed from carboxylic acid of amino acid) 2'-O-amino acid residues (synonymous with the above amino acid residues) and the like.

Examples of the sugar-modified nucleotide include a crosslinked nucleic acid (BNA) having two cyclic structures by introducing a crosslinked structure into the sugar moiety.
Examples of cross-linked artificial nucleic acids include, for example, Locked Nucleic Acid (LNA) ["Tetrahedron Letters" in which the 2'-position oxygen atom and the 4'-position carbon atom are cross-linked via methylene. , Volume 38, Issue 50, 1997, Pages 8735-8738, and "Tetrahedron", Volume 54, Issue 14, 1998, Pages 3607-3630] and Ethylene bridged nucleic acid (Ethylene bridged nucleic acid) ENA) ["Nucleic Acid Research", 32, e175 (2004)].

Sugar-modified nucleotides include peptide nucleic acids (PNA) [Acc. Chem. Res., 32, 624 (1999)], oxypeptide nucleic acids (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001). ], Peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)] and the like.

Examples of the modifying group in the sugar-modified nucleotide include 2'-cyano, 2'-halogen, 2'-O-cyano, 2'-alkyl, 2'-substituted alkyl, 2'-O-alkyl, 2'-O- Preferred are substituted alkyl, 2′-O-alkenyl, 2′-O-substituted alkenyl, 2′-Se-alkyl, 2′-Se-substituted alkyl, 2′-cyano, 2′-fluoro, 2′-chloro, 2'-bromo, 2'-trifluoromethyl, 2'-O-methyl, 2'-O-ethyl, 2'-O-isopropyl, 2'-O-trifluoromethyl, 2'-O- [2- (Methoxy) ethyl], 2'-O- (3-aminopropyl), 2'-O- [2- (N, N-dimethylaminooxy) ethyl], 2'-O- [3- (N, N -Dimethylamino) propyl], 2'-O- [2- [2- (N, N-dimethylamino) ethoxy] ethyl], 2'-O- [2- (methylamino) -2-oxoethyl], 2 '-Se-methyl is more preferred, 2'-O-methyl, 2'-O-ethyl and 2'-fluoro are more preferred, and 2'-O-methyl and 2'-O-ethyl are even more preferred.

The modifying group in the sugar moiety-modified nucleotide can also define a preferred range from the size of the modifying group, and is preferably a modifying group corresponding to a size from fluoro to -O-butyl, from -O-methyl More preferred is a modifying group corresponding to a size up to -O-ethyl.

Examples of the alkyl in the modifying group in the sugar-modified nucleotide include C1-C6 alkyl, specifically, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, Neopentyl, hexyl and the like can be mentioned.

Examples of alkenyl in the modifying group in the sugar moiety-modified nucleotide include C3-C6 alkenyl, and specific examples include allyl, propenyl, butenyl, pentenyl, hexenyl and the like.

Examples of the halogen in the modifying group in the sugar-modified nucleotide include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

Examples of amino acids in the amino acid residue include aliphatic amino acids (specifically, glycine, alanine, valine, leucine and isoleucine), hydroxy amino acids (specifically, serine and threonine), acidic amino acids (specifically, Is aspartic acid and glutamic acid), acidic amino acid amide (specifically asparagine and glutamine etc.), basic amino acid (specifically lysine, hydroxylysine, arginine and ornithine etc.), sulfur-containing amino acid (specifically Include cysteine, cystine, methionine, etc.), imino acids (specifically, proline, 4-hydroxyproline, etc.) and the like.

Examples of the substituted alkyl and the substituted alkenyl in the modified group in the sugar-modified nucleotide include halogen (as defined above), hydroxy, sulfanyl, amino, oxo, -O-alkyl (the alkyl part of -O-alkyl is the above-mentioned C1. -S6 alkyl), -S-alkyl (the alkyl part of -S-alkyl is the same as C1-C6 alkyl), -NH-alkyl (the alkyl part of -NH-alkyl is the same as C1-C6 alkyl) Dialkylaminooxy (the two alkyl parts of dialkylaminooxy are the same or different and have the same meaning as the C1-C6 alkyl), dialkylamino (the two alkyl parts of dialkylamino are the same or different and are the same as the C1-C6 alkyl), Dialkylaminoalkyleneoxy (the alkyl part of dialkylaminoalkyleneoxy is the same or different, Le and are synonymous, alkylene moiety refers to a structure in which the hydrogen atom from the C1-C6 alkyl is removed one), and the like, the number of substitutions is preferably 1-3.

The phosphodiester bond-modified nucleotide may be any nucleotide as long as part or all of the chemical structure of the phosphodiester bond of the nucleotide is modified or substituted with any substituent, or with any atom. Good.
Examples of the phosphodiester bond-modified nucleotide include nucleotides in which the phosphodiester bond is replaced with a phosphorothioate bond, nucleotides in which the phosphodiester bond is replaced with phosphorodithioate bond, and phosphodiester bond in the alkyl phosphonate bond. And nucleotides in which a phosphodiester bond is substituted with a phosphoramidate bond.

The base-modified nucleotide may be any nucleotide as long as a part or all of the nucleotide base chemical structure is modified or substituted with an arbitrary substituent, or substituted with an arbitrary atom.
Examples of the base-modified nucleotide include those in which the oxygen atom in the base is substituted with a sulfur atom, those in which a hydrogen atom is substituted with C1-C6 alkyl, those in which methyl is substituted with a hydrogen atom or C2-C6 alkyl, Examples include amino protected with a protecting group such as C1-C6 alkyl or C1-C6 alkanoyl.

As a nucleotide derivative, a nucleotide, sugar moiety, nucleotide derivative modified with at least one of phosphodiester bond or base, lipid, phospholipid, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, dye, etc. In addition, for example, 5'-polyamine addition nucleotide derivative, cholesterol addition nucleotide derivative, steroid addition nucleotide derivative, bile acid addition nucleotide derivative, vitamin addition nucleotide derivative, green fluorescent dye (Cy3) addition Examples include nucleotide derivatives, red fluorescent dye (Cy5) -added nucleotide derivatives, fluorescein (6-FAM) -added nucleotide derivatives, and biotin-added nucleotide derivatives.

In the nucleic acid used in the present invention, a nucleotide or a nucleotide derivative is an alkylene structure, a peptide structure, a nucleotide structure, an ether structure and an ester structure, or a combination of these two or more with other nucleotides or nucleotide derivatives in the nucleic acid. A cross-linked structure such as

The nucleic acid used in the present invention is preferably a nucleic acid that suppresses the expression of the target gene, and more preferably a nucleic acid that has an action of suppressing the expression of the target gene using RNA interference (RNAi).

The target gene in the present invention is not particularly limited as long as it is a gene that produces and expresses mRNA, and examples thereof include genes associated with tumors or inflammation.
Specific examples of genes related to tumor or inflammation as target genes include vascular endothelial growth factor receptor, fibroblast growth factor receptor, fibroblast growth factor receptor, and platelet-derived growth. Factor, platelet-derived growth factor receptor, hepatocyte growth factor, hepatocyte growth factor receptor, Kruppel-like factor, express sequence tag (Ets) transcription factor, nuclear factor, hypoxia-inducible factor, cell Examples include genes encoding proteins such as cycle-related factors, chromosome replication-related factors, chromosome repair-related factors, microtubule-related factors, growth signal pathway-related factors, growth-related transcription factors, and apoptosis-related factors. Endothelial growth factor gene, vascular endothelial growth factor receptor gene, fibroblast growth factor gene (e.g., transforming growth factor-β), Fibroblast growth factor receptor gene, platelet-derived growth factor gene, platelet-derived growth factor receptor gene, hepatocyte growth factor gene, hepatocyte growth factor receptor gene, Kruppel-like factor gene, express sequence tag (Ets) transcription Factor gene, nuclear factor gene, hypoxia-inducible factor gene, cell cycle-related factor gene, chromosome replication-related factor gene, chromosome repair-related factor gene, microtubule-related factor gene (for example, CKAP5 gene), growth signal pathway-related factor gene (For example, KRAS gene), growth-related transcription factor gene, and apoptosis-related factor (for example, BCL-2 gene).

The target gene in the present invention is preferably a gene expressed in the liver, lung or spleen. For example, the gene related to tumor or inflammation described above, hepatitis B virus genome, hepatitis C virus genome, apolipoprotein (APO) ), Hydroxymethylglutaryl (HMG) CoA reductase, kexin 9 type serine protease (PCSK9), factor 12, glucagon, glucocorticoid receptor, leukotriene receptor, thromboxane A2 receptor, histamine H1 receptor, carbonic acid Dehydrase, angiotensin converting enzyme, renin, p53, tyrosine phosphatase (PTP), sodium-dependent glucose transporter, tumor necrosis factor, interleukin, hepcidin, transthyretin, antithrombin, protein C and matriptase enzymes (e.g. TMPRSS6 Protein) Gene encoding the like.

As a nucleic acid that suppresses the expression of the target gene, for example, a nucleic acid that includes a base sequence complementary to a partial base sequence of mRNA of a gene encoding the protein (target gene) and suppresses the expression of the target gene If so, for example, using any nucleic acid such as double-stranded nucleic acid such as siRNA (short interference (RNA) and miRNA (micro RNA), shRNA (short hairpin RNA), single-stranded nucleic acid such as antisense nucleic acid and ribozyme, etc. However, double-stranded nucleic acids are preferred.

A nucleic acid containing a base sequence complementary to a part of the base sequence of the target gene mRNA is called an antisense strand nucleic acid, and a nucleic acid containing a base sequence complementary to the base sequence of the antisense strand nucleic acid is a sense strand. Also called nucleic acid. A sense strand nucleic acid refers to a nucleic acid capable of forming a double strand forming part by pairing with an antisense strand nucleic acid, such as a nucleic acid itself consisting of a partial base sequence of a target gene.

A double-stranded nucleic acid refers to a nucleic acid in which two strands are paired and have a duplex forming part. The duplex forming part refers to a part (duplex forming part) in which a nucleotide or a derivative thereof constituting a double-stranded nucleic acid forms a base pair to form a duplex.
The base pair constituting the duplex forming part is usually 15 to 27 base pairs, preferably 15 to 25 base pairs, more preferably 15 to 23 base pairs, further preferably 15 to 21 base pairs, and 15 to 19 base pairs. Even more preferred is base pairing.

As the antisense strand nucleic acid of the duplex forming part, for example, a nucleic acid consisting of a partial sequence of the mRNA of the target gene, or 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 base in the nucleic acid, A nucleic acid that is deleted or added and has an activity of suppressing the expression of the target protein is preferably used. The single-stranded nucleic acid constituting the double-stranded nucleic acid usually consists of a series of 15 to 30 bases (nucleosides), preferably 15 to 29 bases, more preferably 15 to 27 bases, and further preferably 15 to 25 bases. 17 to 23 bases are more preferred, and 19 to 21 bases are particularly preferred.

Either the antisense strand, the sense strand, or both of the nucleic acids constituting the double-stranded nucleic acid have a portion that does not form a duplex on the 3 ′ side or 5 ′ side following the duplex forming portion. Also good. The part that does not form a double chain is also referred to as a protrusion (overhang).

Examples of the double-stranded nucleic acid having a protruding portion include a double-stranded nucleic acid having a protruding portion consisting of 1 to 4 bases, usually 1 to 3 bases at the 3 ′ end or 5 ′ end of at least one strand.
In a double-stranded nucleic acid having a protruding portion, the protruding portion is preferably a protruding portion consisting of two bases, and more preferably a protruding portion consisting of dTdT or UU.
The overhang can be present only in the antisense strand, only in the sense strand, and both in the antisense strand and the sense strand, but a double-stranded nucleic acid having a protrusion in both the antisense strand and the sense strand is preferred.

Sequence that matches part or all of the base sequence of the target gene mRNA following the duplex formation part, or part or all of the base sequence of the complementary strand of the target gene mRNA following the duplex formation part Sequences that match may be used.
Examples of nucleic acids that suppress the expression of the target gene include nucleic acid molecules that generate double-stranded nucleic acids by the action of ribonucleases such as Dicer (International Publication No. 2005/089287), and protrusions at the 3 ′ end and 5 ′ end. A double-stranded nucleic acid or the like that does not exist can also be used.

When the double-stranded nucleic acid is siRNA, the antisense strand preferably has a sequence of at least the 1st to 17th bases (nucleosides) from the 5 ′ end to the 3 ′ end and the target gene mRNA is continuous. It is a base sequence complementary to the 17 base sequence, and more preferably, the antisense strand has the sequence of the 1st to 19th bases from the 5 ′ end to the 3 ′ end, and the target gene mRNA is continuous. Or the base sequence complementary to the 19 base sequence, or the sequence of the 1st to 21st bases from the 5 'end to the 3' end, Complementary base sequence, or the sequence of bases 1 to 25 from the 5 'end to the 3' end are complementary to the base sequence of 25 consecutive bases of the target gene mRNA. It is.

When the nucleic acid used in the present invention is siRNA, preferably 10 to 100%, more preferably 20 to 100%, and still more preferably 40 to 100% of the sugar in the nucleic acid is substituted with a modifying group at the 2 ′ position. Ribose. In the present invention, ribose substituted with a modifying group at the 2′-position means that the hydroxyl group at the 2′-position of ribose is substituted with the modifying group, and has the same configuration as the hydroxyl group at the 2′-position of ribose. Although it may be present or different, the configuration is preferably the same as the hydroxyl group at the 2 ′ position of ribose. Examples of the modifying group in ribose substituted with a modifying group at the 2′-position include those exemplified as the modifying group in the 2′-modified nucleotide in the sugar moiety-modified nucleotide and the hydrogen atom, and 2′-cyano, 2′-halogen 2′-O-cyano, 2′-alkyl, 2′-substituted alkyl, 2′-O-alkyl, 2′-O-substituted alkyl, 2′-O-alkenyl, 2′-O-substituted alkenyl, 2 '-Se-alkyl or 2'-Se-substituted alkyl is preferred, 2'-cyano, 2'-fluoro, 2'-chloro, 2'-bromo, 2'-trifluoromethyl, 2'-O-methyl, 2'-O-ethyl, 2'-O-isopropyl, 2'-O-trifluoromethyl, 2'-O- [2- (methoxy) ethyl], 2'-O- (3-aminopropyl), 2 '-O- [2- (N, N-dimethyl) aminooxy] ethyl, 2'-O- [3- (N, N-dimethylamino) propyl], 2'-O- [2- [2- ( N, N-dimethylamino) ethoxy] ethyl], 2′-O- [2- (methylamino) -2-oxoethyl], 2′-Se-methyl More preferred are til and hydrogen atoms, more preferred are 2'-O-methyl, 2'-O-ethyl, 2'-fluoro and hydrogen atoms, and even more preferred are 2'-O-methyl and 2'-O-fluoro. .

The nucleic acid used in the present invention includes a derivative in which an oxygen atom or the like contained in a phosphoric acid part, an ester part or the like in the structure of the nucleic acid is substituted with another atom such as a sulfur atom.

The sugar that binds to the 5 ′ terminal base of the antisense strand and the sense strand has a 5′-position hydroxyl group, either a phosphate group or the modifying group, or a phosphate group or the modifying group by an in vivo nucleolytic enzyme, etc. It may be modified by a group that is converted into

The sugar that binds to the 3 ′ terminal base of the antisense strand and the sense strand is such that the hydroxyl group at the 3 ′ position is a phosphate group or the modifying group, or a phosphate group or the modifying group by an in vivo nucleolytic enzyme, etc. It may be modified by a group that is converted into

Examples of the single-stranded nucleic acid include 15 to 27 bases (nucleosides) of the target gene, preferably 15 to 25 bases, more preferably 15 to 23 bases, still more preferably 15 to 21 bases, and still more preferably 15 to 25 bases. A nucleic acid comprising a sequence complementary to a sequence consisting of 19 bases, or 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 base substituted or deleted or added in the nucleic acid, and having an activity of suppressing the expression of the target protein Any nucleic acid can be used.
The single-stranded nucleic acid preferably consists of a sequence of 15 to 30 bases (nucleosides), more preferably 15 to 27 bases, still more preferably 15 to 25 bases, and even more preferably 15 to 23 bases.

As the single-stranded nucleic acid, one obtained by linking an antisense strand and a sense strand constituting a double-stranded nucleic acid via a spacer sequence (spacer oligonucleotide) may be used. The spacer oligonucleotide is preferably a 6- to 12-base single-stranded nucleic acid, and its 5 ′ terminal sequence is preferably 2 U. An example of the spacer oligonucleotide is a single-stranded nucleic acid having a UUCAAGAGA sequence. Either the antisense strand or the sense strand connected by the spacer oligonucleotide may be on the 5 ′ side.
Examples of the single-stranded nucleic acid in which the antisense strand and the sense strand constituting the double-stranded nucleic acid are linked via a spacer oligonucleotide include, for example, a single-stranded nucleic acid such as shRNA having a double-stranded forming part by a stem-loop structure. Preferably there is. Single-stranded nucleic acids such as shRNA are usually 50 to 70 bases in length.

A nucleic acid having a length of 70 bases or less, preferably 50 bases or less, more preferably 30 bases or less, designed to generate a single-stranded nucleic acid or a double-stranded nucleic acid by the action of ribonuclease or the like may be used.

The nucleic acid used in the present invention can be produced using known RNA or DNA synthesis methods and RNA or DNA modification methods.

The pharmaceutical composition of the present invention (hereinafter abbreviated as composition) contains the compound represented by formula (I) or formula (II) of the present invention, or a pharmaceutically acceptable salt thereof (cationic lipid) and nucleic acid To do.
The composition of the present invention may be, for example, a complex of the cationic lipid of the present invention and a nucleic acid.
The composition of the present invention is a composition containing the cationic lipid of the present invention, a neutral lipid and / or polymer, and a nucleic acid. For example, the cationic lipid of the present invention, the neutral lipid and / or Alternatively, it may be a complex of a polymer and a nucleic acid.
The composition of the present invention may contain a lipid membrane, and the complex may be encapsulated by the lipid membrane.
The lipid membrane may be a lipid monolayer (lipid monomolecular membrane) or a lipid bilayer membrane (lipid bimolecular membrane). The lipid membrane may contain the cationic lipid, neutral lipid and / or polymer of the present invention.
The complex and / or lipid membrane contains a cationic lipid other than the cationic lipid which is the compound represented by the formula (I) or the formula (II) of the present invention or a pharmaceutically acceptable salt thereof. Also good.

Examples of the composition of the present invention include a complex of a cationic lipid other than the cationic lipid of the present invention and a nucleic acid, or a cationic lipid other than the cationic lipid of the present invention and a neutral lipid and / or a polymer. A composition containing a complex with a nucleic acid and a lipid membrane encapsulating the complex and containing the cationic lipid of the present invention in the lipid membrane is also included. The lipid membrane in this case may also be a lipid monolayer (lipid monomolecular membrane) or a lipid bilayer membrane (lipid bimolecular membrane). The lipid membrane may contain a cationic lipid other than the cationic lipid of the present invention, a neutral lipid and / or a polymer.

In any composition, the lipid membrane may contain a neutral lipid and / or a polymer. Further, the complex and / or the lipid membrane may contain a cationic lipid other than the cationic lipid of the present invention.

Examples of the form of the complex include a complex of a nucleic acid and a membrane composed of a single lipid (single molecule) layer (reverse micelle), a complex of a nucleic acid and a liposome, and a complex of a nucleic acid and a micelle. Is a complex of a membrane composed of a nucleic acid and a lipid monolayer or a complex of a nucleic acid and a liposome.

Examples of the composition containing a lipid membrane that encapsulates the complex include liposomes and lipid nanoparticles that encapsulate the complex with an arbitrary number of lipid membranes.

In the composition of the present invention, one or more kinds of the cationic lipids of the present invention may be used. The cationic lipids of the present invention include, in addition to the cationic lipids of the present invention, other than the cationic lipids of the present invention. The cationic lipid may be mixed.

Examples of the cationic lipid other than the cationic lipid which is the compound represented by the formula (I) or the formula (II) of the present invention or a pharmaceutically acceptable salt thereof include, for example, JP-A-61-161246 (US). No. 5049386), N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA) and N- (2,3-dioxy). -(9- (Z) -octadecenoyloxy))-prop-1-yl-N, N, N-trimethylammonium chloride (DOTAP), etc., WO 91/16024 and WO 97/019675 N- [1- (2,3-dioleyloxypropyl)]-N, N-dimethyl-N-hydroxyethylammonium bromide (DORIE) and 2,3-dioleyloxy-N -[2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetic acid (DOSPA), etc., disclosed in WO 2005/121348, DLinDMA, etc. 2009/086558 (3R2R) -3,4-bis ((Z) -hexadec-9-enyloxy) -1- disclosed in International Publication No. 2011/136368, DLin-K-DMA, etc. Examples include methylpyrrolidine and N-methyl-N, N-bis (2-((Z) -octadec-6-enyloxy) ethyl) amine.
As the cationic lipid other than the cationic lipid of the present invention, preferably DOTMA, DOTAP, DORIE, DOSPA1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4- Cationic lipids with tertiary amine moieties with two unsubstituted alkyl groups such as dimethylaminomethyl- [1,3] -dioxolane (DLin-K-DMA) or quaternary ammonium moieties with three unsubstituted alkyl groups More preferably, it is a cationic lipid having a tertiary amine moiety.
The unsubstituted alkyl group at the tertiary amine moiety and the quaternary ammonium moiety is preferably a methyl group.
The composition of the present invention may contain a compound (for example, a peptide nucleic acid) chemically similar to the nucleic acid in addition to the nucleic acid.

The composition of this invention can be manufactured according to a well-known manufacturing method or it, and may be manufactured by what kind of manufacturing method. For example, a known method for preparing liposomes can be applied to the production of a composition containing liposome, which is one of the compositions.
Known methods for preparing liposomes include, for example, Bangham et al., “Liposome preparation method” (“J. Mol. Biol.”), 1965, Vol. 13, p. 238- 252], ethanol injection method ["J. Cell Biol.", 1975, Vol. 66, pp. 621-634], French press method ["FBS Letters (FEBS Lett.) ", 1979, Vol. 99, p.210-214], freeze-thaw method [" Arch. Biochem. Biophys. ", 1981 Year 212, p.186-194], reverse phase evaporation ["Proceedings of the National Academy of Sciences United States of America" (Proc. Natl. Acad. Sci. USA) ", 1978, Vol. 75, p.4194-4198] and pH gradient method (see, for example, Japanese Patent No. 2572554, Japanese Patent No. 2659136) And the like.
Examples of the solution in which the liposome is dispersed during the production of the liposome include water, acid, alkali, various buffers, physiological saline, and amino acid infusion.
In the production of liposomes, for example, antioxidants such as citric acid, ascorbic acid, cysteine and ethylenediaminetetraacetic acid (EDTA), for example, isotonic agents such as glycerin, glucose and sodium chloride may be added.
The cationic lipid of the present invention, or a mixture of the cationic lipid of the present invention and a cationic lipid other than the cationic lipid of the present invention is dissolved in an organic solvent such as ethanol, and the solvent is distilled off. Liposomes can be formed by adding saline and stirring with shaking.

The composition of the present invention can be prepared, for example, by dissolving a cationic lipid of the present invention or a mixture of the cationic lipid of the present invention and a cationic lipid other than the cationic lipid in chloroform in advance, and then an aqueous nucleic acid solution. And methanol are added and mixed to form a cationic lipid / nucleic acid complex, and the chloroform layer is taken out, and then the polyethylene glycolated phospholipid, neutral lipid, and water are added to the extracted chloroform layer to form a water-in-oil type. (W / O) emulsion is formed and processed by the reverse phase evaporation method (see Japanese Patent Application Publication No. 2002-508765), nucleic acid is dissolved in an acidic electrolyte aqueous solution, for example, the cation of the present invention Or a mixture of the cationic lipid of the present invention and a cationic lipid other than the cationic lipid of the present invention (in ethanol), and the ethanol concentration is lowered to 20 v / v% to encapsulate the nucleic acid. A method in which posomes are prepared, sizing filtered, excess ethanol is removed by dialysis, and then the sample is dialyzed at a higher pH to remove nucleic acids adhering to the surface of the composition (Special Table 2002-501511) No. gazette and Biochimica et Biophysica Acta, 2001, Vol. 1510, p.152-166) and the like.

Among the compositions of the present invention, a complex of the cationic lipid of the present invention and a nucleic acid, or a complex of a neutral lipid and / or polymer and a nucleic acid, and a complex are encapsulated in the cationic lipid of the present invention. The composition containing lipid nanoparticles containing the lipid membrane can be produced, for example, according to the production method described in WO 02/28367 and WO 2006/080118.

When the composition of the present invention is produced according to the production method described in WO 02/28367 and WO 2006/080118, etc., the cationic lipid, nucleic acid, neutral lipid and / or high lipid of the present invention are used. A complex is produced using molecules and components appropriately selected from cationic lipids other than the cationic lipid of the present invention, and the complex is dispersed in water or a 0-40% ethanol aqueous solution without dissolving it (A Separately, the lipid membrane component encapsulating the complex is dissolved in, for example, an ethanol aqueous solution (B solution), and the A and B solutions having a volume ratio of 1: 1 to 10: 1 are mixed, and water is appropriately added. Can be added to produce the composition of the present invention.
As the cationic lipid in the liquid A and the liquid B, one or plural kinds of the cationic lipid of the present invention or a cationic lipid other than the cationic lipid of the present invention may be used. A cationic lipid other than the cationic lipid of the invention may be used in combination.

In the present invention, a complex of the cationic lipid of the present invention and a nucleic acid, or a complex of the cationic lipid of the present invention, a neutral lipid and / or polymer, and a nucleic acid, and a lipid membrane encapsulating the complex A composition comprising a cationic lipid other than the cationic lipid of the present invention and a nucleic acid, or a cationic lipid other than the cationic lipid of the present invention, a neutral lipid and / or a polymer, and a nucleic acid And a lipid membrane encapsulating the complex, and the composition containing the cationic lipid of the present invention in the lipid membrane, and after the production, the nucleic acid in the complex and the cationic property in the lipid membrane The composition of the present invention also includes those in which the structure of the complex and the membrane is mutated due to electrostatic interaction with the lipid or fusion of the cationic lipid in the complex and the cationic lipid in the lipid membrane. .

According to the production method described in WO 02/28367 and WO 2006/080118, etc., a nucleic acid, preferably a double-stranded nucleic acid, the cationic lipid of the present invention and / or the cationic lipid of the present invention A complex with a liposome containing a cationic lipid is produced and dispersed in water or a 0-40% ethanol aqueous solution without dissolving the complex (solution A). Separately, the cationic lipid of the present invention and / or Alternatively, a cationic lipid other than the cationic lipid of the present invention is dissolved in an aqueous ethanol solution (liquid B), and liquid A and liquid B with a volume ratio of 1: 1 to 10: 1 are mixed, or more appropriately. A composition containing the composition of the present invention and a nucleic acid can also be produced by adding water.
The composition obtained by this production method is preferably a composition containing a complex of a cationic lipid and a nucleic acid and a lipid membrane encapsulating the complex, or from a lipid monolayer containing a nucleic acid and a cationic lipid. A composition comprising a membrane (reverse micelle) and a lipid membrane encapsulating the complex. The lipid membrane in the composition may be any of a lipid monolayer (lipid monomolecular membrane), a lipid bilayer membrane (lipid bimolecular membrane), or a multilayer membrane.

The size of the liposome in the complex of nucleic acid and liposome in the present invention is preferably adjusted in advance to an average particle size of preferably 10 nm to 400 nm, more preferably 20 nm to 110 nm, and still more preferably 20 nm to 80 nm. . The complex and / or lipid membrane may contain a neutral lipid and / or a polymer.
As long as the solution A can form a complex of liposomes and nucleic acids, the ethanol concentration may be 20 to 70%.

Instead of mixing equal amounts of liquid A and liquid B, the complex does not dissolve after mixing liquid A and liquid B, and the ratio is such that ethanol concentration does not dissolve the cationic lipid in liquid B. The liquid may be mixed. Instead of mixing the A liquid and the B liquid in such a ratio that preferably the complex does not dissolve, the cationic lipid in the B liquid does not dissolve, and the ethanol concentration is 20 to 60%. Alternatively, after mixing liquid A and liquid B, mix liquid A and liquid B at a ratio that will result in an ethanol concentration that does not dissolve the complex, and then add water to add cationic lipid in liquid B. It is also possible to use an ethanol concentration at which no dissolution occurs.

The composition obtained by this production method is preferably a composition containing a complex of a cationic lipid and a nucleic acid and a lipid membrane encapsulating the complex, or a membrane comprising a lipid monolayer containing a cationic lipid (Reverse micelle) and nucleic acid complex and a lipid membrane that encapsulates the complex, and a lipid membrane containing a cationic lipid, and the productivity (yield and / or uniformity) of this production method Is excellent.

In the composition of the present invention, the total number of molecules of the cationic lipid of the present invention in the complex is preferably 0.5 to 4 times, more preferably 1.5 to 3.5 times the number of phosphorus atoms of the nucleic acid. More preferably, it is 2 to 3 times.
The total number of the cationic lipid molecules of the present invention and the cationic lipid molecules other than the cationic lipid of the present invention in the complex is preferably 0.5 to 4 times the number of phosphorus atoms of the nucleic acid, and preferably 1.5 to 3.5. It is more preferable that the ratio is twice, and it is more preferable that the ratio is 2 to 3 times.

In the composition of the present invention, the total number of molecules of the cationic lipid of the present invention in the composition containing the complex and the lipid membrane encapsulating the complex is 1 to 10 times the number of phosphorus atoms of the nucleic acid. Preferably, it is 2.5 to 9 times, more preferably 3.5 to 8 times.
The total number of the cationic lipid molecules of the present invention and the cationic lipid molecules other than the cationic lipid of the present invention in the composition is preferably 1 to 10 times the number of phosphorus atoms of the nucleic acid, and preferably 2.5 to 9 The ratio is more preferably double, and further preferably 3.5 to 8 times.

The neutral lipid may be any of simple lipids, complex lipids or derived lipids, and examples thereof include phospholipids, glyceroglycolipids, sphingoglycolipids, sphingoids and sterols.

When the composition of the present invention contains neutral lipids, the total number of neutral lipid molecules is based on the total number of cationic lipid molecules of the present invention and cationic lipids other than the cationic lipids of the present invention. The ratio is preferably 0.1 to 2 times, more preferably 0.2 to 1.5 times, and still more preferably 0.3 to 1.2 times.
The composition of the present invention may contain a neutral lipid in a complex, or may be contained in a lipid membrane encapsulating the complex.
The neutral lipid is preferably contained in the lipid membrane encapsulating the complex, and more preferably contained in both the complex and the lipid membrane encapsulating the complex.

Examples of phospholipids in neutral lipids include phosphatidylcholine (specifically soybean phosphatidylcholine, egg yolk phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), dimyristoylphosphatidylcholine). (DMPC) and dioleoylphosphatidylcholine (DOPC), etc.), phosphatidylethanolamine (specifically distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE)), Dimyristoylphosphatidylethanolamine (DMPE), 16-0-monomethylphosphatidylethanolamine, 16-0-dimethylphosphatidylethanolamine 18-1-transphosphatidylethanolamine, palmitoyl oleoyl phosphatidylethanolamine (POPE) and 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), glycerophospholipid (specifically phosphatidylserine, phosphatidic acid, Phosphatidylglycerol, phosphatidylinositol, palmitoyl oleoylphosphatidylglycerol (POPG) and lysophosphatidylcholine), sphingophospholipids (specifically sphingomyelin, ceramide phosphoethanolamine, ceramide phosphoglycerol and ceramide phosphoglycerophosphate, etc.), glycerophosphono Lipids, sphingophosphonolipids, natural lecithins (specifically egg yolk lecithin and soy lecithin) and hydrogenated phospholipids (specifically hydrogen Natural phospholipids or synthetic pressurized soybean phosphatidylcholine, etc.) and the like.

Examples of the glyceroglycolipid in the neutral lipid include sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride and the like.

Examples of the glycosphingolipid in the neutral lipid include galactosyl cerebroside, lactosyl cerebroside, ganglioside and the like.

Examples of the sphingoid in the neutral lipid include sphingan, icosasphingan and sphingosine, and derivatives thereof.
Derivatives include, for example, —NH ₂ such as sphingan, icosasphingan, or sphingosine —NHCO (CH ₂ ) xCH ₃ (wherein x is an integer of 0 to 18, among which 6, 12 or 18 is preferred) And the like converted to.

Examples of sterols in neutral lipids include cholesterol, dihydrocholesterol, lanosterol, β-sitosterol, campesterol, stigmasterol, brassicasterol, ergocasterol, fucosterol, 3β- [N- (N ', N'-dimethyl Aminoethyl) carbamoyl] cholesterol (DC-Chol) and the like.

Examples of the polymer include protein, albumin, dextran, polyfect, chitosan, dextran sulfate, poly-L-lysine, polyethyleneimine, polyaspartic acid, styrene maleic acid copolymer, isopropylacrylamide-acryl pyrrolidone copolymer. Polyethylene glycol modified dendrimer, polylactic acid, polylactic acid polyglycolic acid, polyethylene glycolated polylactic acid and the like.
The polymer may be a micelle composed of one or more of the exemplified polymer salts.

Examples of the polymer salt include metal salts, ammonium salts, acid addition salts, organic amine addition salts, amino acid addition salts, and the like.
Examples of the metal salt include alkali metal salts such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt and zinc salt.
As an ammonium salt, salts, such as ammonium and tetramethylammonium, are mentioned, for example.
Examples of the acid addition salt include inorganic acid salts such as hydrochloride, sulfate, nitrate and phosphate, and organic acid salts such as acetate, maleate, fumarate and citrate.
Examples of organic amine addition salts include addition salts such as morpholine and piperidine.
Examples of amino acid addition salts include addition salts of glycine, phenylalanine, aspartic acid, glutamic acid, lysine, and the like.

The composition of the present invention preferably contains, for example, a lipid derivative or fatty acid derivative of one or more substances selected from sugars, peptides, nucleic acids and water-soluble polymers, or a surfactant, etc., and is contained in the complex. Alternatively, it may be contained in the lipid membrane encapsulating the complex, and more preferably contained in both the complex and the lipid membrane encapsulating the complex.

When the composition of the present invention contains a lipid derivative or fatty acid derivative of one or more substances selected from sugars, peptides, nucleic acids, and water-soluble polymers, they are selected from sugars, peptides, nucleic acids, and water-soluble polymers. The total number of lipid derivatives and fatty acid derivative molecules of one or more substances is preferably 0.01 to 0.3 times the total number of cationic lipid molecules of the present invention and cationic lipids other than the cationic lipids of the present invention. The ratio is more preferably 0.02 to 0.25 times, and further preferably 0.03 to 0.15 times.

The lipid derivative or fatty acid derivative of one or more substances selected from sugars, peptides, nucleic acids, and water-soluble polymers, or the surfactant is preferably a glycolipid or a lipid derivative or fatty acid derivative of a water-soluble polymer. More preferably, it is a lipid derivative or fatty acid derivative of a water-soluble polymer.
A lipid derivative or fatty acid derivative of one or more substances selected from sugars, peptides, nucleic acids, and water-soluble polymers, or a surfactant is a part of the molecule that is, for example, hydrophobic with other components in the composition of the present invention. It has the property of binding by affinity or electrostatic interaction, etc., and other parts have the property of binding to the solvent at the time of production of the composition, for example, hydrophilic affinity or electrostatic interaction, etc. A substance is preferred.

Examples of lipid derivatives or fatty acid derivatives of sugars, peptides or nucleic acids include sugars such as sucrose, sorbitol and lactose, such as casein-derived peptides, egg white-derived peptides, soybean-derived peptides and peptides such as glutathione, or DNA, RNA, plasmids, etc. , A compound in which a nucleic acid such as siRNA and oligodeoxynucleotide (ODN) and a neutral lipid or the cationic lipid of the present invention, or a fatty acid such as stearic acid, palmitic acid, myristic acid, lauric acid, etc. are bound. .

Examples of the sugar lipid derivative or fatty acid derivative include glyceroglycolipid and glycosphingolipid.

Examples of the water-soluble polymer lipid derivative or fatty acid derivative include polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, oligosaccharide, dextrin, water-soluble cellulose, dextran, chondroitin sulfate, polyglycerin, Chitosan, polyvinylpyrrolidone, polyaspartic acid amide, poly-L-lysine, mannan, pullulan or oligoglycerol etc. or their derivatives and neutral lipids or cationic lipids of the present invention or stearic acid, palmitic acid, myristic acid And a compound in which a fatty acid such as lauric acid is bonded, preferably a lipid derivative or a fatty acid derivative of polyethylene glycol or polyglycerol, more preferably A lipid derivative or a fatty acid derivative of polyethylene glycol.
The lipid derivative or fatty acid derivative of the water-soluble polymer may be a salt.

Examples of lipid derivatives or fatty acid derivatives of polyethylene glycol include polyethylene glycolated lipids [specifically, polyethylene glycol-phosphatidylethanolamine (more specifically, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DMPE) Etc.), polyoxyethylene hydrogenated castor oil 60, Cremophor EL, etc., polyethylene glycol sorbitan fatty acid esters (specifically polyoxyethylene sorbitan monooleate, etc.) and polyethylene glycol fatty acid esters, etc. Preferably, it is a polyethylene glycolated lipid.

Examples of the lipid derivative or fatty acid derivative of polyglycerin include polyglycerinized lipid (specifically, polyglycerin-phosphatidylethanolamine) or polyglycerin fatty acid esters, and the polyglycerinized lipid is preferable.

Examples of the surfactant include polyoxyethylene sorbitan monooleate (specifically polysorbate 80 and the like), polyoxyethylene polyoxypropylene glycol (specifically Pluronic (registered trademark) F68 and the like), sorbitan fatty acid ester (specifically Sorbitan monolaurate and sorbitan monooleate), polyoxyethylene derivatives (specifically polyoxyethylene hydrogenated castor oil 60 and polyoxyethylene lauryl alcohol), glycerin fatty acid ester, polyethylene glycol alkyl ether, etc. Preferably, it is polyoxyethylene polyoxypropylene glycol, glycerin fatty acid ester or polyethylene glycol alkyl ether.

The complex and the lipid membrane in the composition of the present invention can be optionally subjected to surface modification with, for example, a water-soluble polymer [Radasic, edited by F. Martin, "Stealth liposome. Stealth Liposomes "(USA), CRC Press Inc., 1995, p. 93-102].
Examples of water-soluble polymers that can be used for surface modification include polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, oligosaccharide, dextrin, water-soluble cellulose, dextran, chondroitin sulfate, and polyglycerin. , Chitosan, polyvinylpyrrolidone, polyaspartic acid amide, poly-L-lysine, mannan, pullulan, oligoglycerol and the like, preferably dextran, pullulan, mannan, amylopectin or hydroxyethyl starch.
For the surface modification, lipid derivatives or fatty acid derivatives of one or more substances selected from sugars, peptides, nucleic acids and water-soluble polymers can be used. The surface modification is a method in which the complex and lipid membrane in the composition of the present invention contain a lipid derivative or fatty acid derivative of one or more substances selected from sugars, peptides, nucleic acids, and water-soluble polymers, or a surfactant. One of them.

The targeting ligand can also be directly bound to the surface of the composition of the present invention by covalently binding to the polar head residue of the lipid component of the composition of the present invention (see International Publication No. 2006/116107).

The average particle size of the complex or the lipid membrane encapsulating the complex in the composition of the present invention can be freely selected as desired.
As a method for adjusting the average particle size, for example, an extrusion method, a method of mechanically pulverizing large multilamellar liposomes (MLV) or the like (specifically using a manton gourin or a microfluidizer, etc.) [Müller (RHMuller ), Edited by S. Benita, B. Bohm, "Emulsion and Nanosuspensions for the Formulation of the Formulation of the Formulation of the Formulation of the Formulation of the Formulation of the Formulation of the Formulation of the Formulation of the Formulation of the Formulation of the Formulation of the Formulation of the Formulation of the Formulation of Poorly Soluble Drugs) ", Scientific Publishers Stuttgart, 1998, p.267-294].

Regarding the size of the composite in the composition of the present invention, the average particle size is preferably 5 nm to 200 nm, more preferably 20 nm to 150 nm, and 20 nm to 80 nm. Further preferred.

Regarding the size of the composition of the present invention (lipid membrane encapsulating the complex), the average particle size is preferably 10 nm to 300 nm, more preferably 30 nm to 200 nm, and 50 nm to 150 nm. More preferably, it is nm.

The average particle size of the complex in the composition of the present invention or the lipid membrane encapsulating the complex can be measured, for example, by a dynamic light scattering method.

The nucleic acid in the composition of the present invention can be introduced into cells by introducing the composition of the present invention into mammalian cells.

The introduction of the composition of the present invention into mammalian cells in vivo may be performed according to known transfection procedures that can be performed in vivo. For example, the composition of the present invention is intravenously administered to a mammal, including a human, so that the composition is delivered to, for example, a tumor or an inflamed organ or site, and the cells of the delivery organ or site are contained in the composition of the present invention. Can be introduced. The organ or site where the tumor or inflammation has occurred is not particularly limited, but for example, the digestive tract such as the stomach and large intestine, the central nervous system such as the liver, lung, spleen, pancreas, kidney, bladder, brain and spinal cord, skin and blood vessels And eyeballs. In addition, the composition of the present invention can be delivered intravenously to mammals including humans, for example, to the liver, lungs, kidneys, gastrointestinal tract, central nervous system and / or spleen. The nucleic acid in the composition of the present invention can be introduced. Liver, lung, kidney, gastrointestinal tract, central nervous system or spleen cells can be normal cells, cells associated with tumors or inflammation or cells associated with other diseases.

If the nucleic acid in the composition of the present invention is a nucleic acid having an inhibitory effect on the expression of a target gene using RNA interference (RNAi), a nucleic acid that suppresses the expression of the target gene in vivo in a mammalian cell. Can be introduced, and the expression of the target gene can be suppressed.
The present invention can be used as a target gene expression inhibitor using RNA interference (RNAi) containing the composition of the present invention.
The administration subject is preferably a human.

When the target gene in the present invention is a gene expressed in, for example, the liver, lung, or spleen, the present invention contains the composition of the present invention as a therapeutic or prophylactic agent for a disease related to the liver, lung, or spleen. It can be used and is preferably used as a therapeutic or prophylactic agent for diseases related to the lung. The present invention can also be used as an antitumor agent containing the composition of the present invention, and is preferably used for malignant tumors, and more preferably used for malignant tumors in the liver, lung or spleen.

Examples of diseases related to the lung include bronchial asthma, chronic obstructive pulmonary disease, idiopathic interstitial pneumonia, hypersensitivity pneumonia, eosinophilic pneumonia, drug-induced pneumonia, allergic bronchopulmonary aspergillosis, sarcoidosis, collagen Diseased lung, emphysema, alveolar proteinosis, pulmonary arterial hypertension, cystic fibrosis, nontuberculous pulmonary mycobacterial disease, pulmonary thromboembolism, eosinophilic polyangiogenic granulomatosis, lung water species , Pleurisy, empyema, pneumothorax, chronic respiratory failure and the like.

Examples of liver-related diseases include cirrhosis, nonalcoholic steatohepatitis, familial hypercholesterolemia, familial amyloidosis, viral hepatitis, hepatic porphyria, primary hyperoxaluria type I, α1 anti Examples include trypsin deficiency, hypertriglyceridemia, and hepatitis B virus infection.

Examples of diseases related to the spleen include hereditary spherocytosis, idiopathic thrombocytopenic purpura, sepsis, idiopathic portal hypertension, spleen hyperfunction and the like.

The present invention also provides a treatment or prevention method including the step of administering the composition of the present invention to a subject. For example, a method of treating a disease associated with the liver, lungs or spleen is provided by administering the composition of the present invention to a mammal. The administration subject is preferably a human, more preferably a patient suffering from a disease related to the liver, lungs or spleen.

The present invention also provides a method for treating or preventing malignant tumors, comprising the step of administering the composition or antitumor agent of the present invention to a subject, and is more preferably used for malignant tumors in the liver, lung or spleen. .

The composition of the present invention can also be used as a tool for verifying the effectiveness of suppressing a target gene in an in vivo drug efficacy evaluation model for a therapeutic or prophylactic agent for diseases related to the liver, lung or spleen. it can.

The composition of the present invention can stabilize nucleic acids in biological components such as blood components (e.g. blood and digestive tract), reduce side effects, or accumulate drugs in tissues or organs containing target gene expression sites. It can also be used as a preparation for the purpose of increase or the like.

When the composition of the present invention is used as a therapeutic or prophylactic agent for diseases related to the liver, lung or spleen of pharmaceuticals, it is desirable to use the most effective route for treatment as the administration route, For example, parenteral or oral administration such as intraoral, intratracheal, rectal, subcutaneous, intramuscular or intravenous administration can be mentioned, and intravenous administration or intratracheal administration is preferable.

The dose of the composition of the present invention varies depending on the disease state, age, route of administration, etc. of the administration subject, but for example, it may be administered such that the daily dose converted to nucleic acid is 0.1 μg to 1000 μg.

As a preparation suitable for intravenous administration or intramuscular administration, for example, an injection is exemplified, and a prepared dispersion of the composition of the present invention is directly used as an injection or the like.
Suitable preparations preferably include preparations obtained by removing the solvent from the dispersion by, for example, filtration or centrifugation, preparations obtained by lyophilizing the dispersion, and excipients such as mannitol, lactose, trehalose, maltose and glycine. It is the formulation which freeze-dried the added dispersion liquid.

In the case of an injection, the dispersion or solvent of the composition of the present invention is removed or lyophilized, for example, water, acid, alkali, various buffers, physiological saline or amino acid infusion, etc. are mixed for injection. Is preferably prepared. For example, an injection can be prepared by adding an antioxidant such as citric acid, ascorbic acid, cysteine and EDTA or an isotonic agent such as glycerin, glucose and sodium chloride. For example, a cryopreservation agent such as glycerin may be added for cryopreservation.

The present invention also provides a pharmaceutical composition for use in the treatment of a disease; the use of a pharmaceutical composition for the treatment of a disease; the use of a cationic lipid or a pharmaceutical composition in the manufacture of a medicament for the treatment of a disease; Provided is a cationic lipid or pharmaceutical composition for use in the manufacture of a therapeutic medicament; a method of treating or preventing a disease comprising administering an effective amount of the pharmaceutical composition to a subject in need thereof.

Next, the present invention will be specifically described with reference to Examples, Comparative Examples, Reference Examples, and Test Examples. However, the present invention is not limited to these examples, comparative examples, reference examples, and test examples.
The proton nuclear magnetic resonance spectra ( ¹ H NMR) shown in the Examples and Reference Examples are those measured at 400 MHz, and exchangeable protons may not be clearly observed depending on the compound and measurement conditions. is there. In addition, what is normally used is used as the notation of the multiplicity of signals.

Reference Example 1: (Z) -non-2-en-1-yl 10-bromodecanoate 10-bromodecanoic acid (Tokyo Chemical Industry Co., Ltd., 4.00 g, 15.9 mmol) was dissolved in dichloromethane (250 mL). cis-2-nonen-1-ol (manufactured by Tokyo Chemical Industry Co., Ltd., 2.61 g, 18.3 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (manufactured by Tokyo Chemical Industry Co., Ltd., 3.51 g, 18.3 mmol), N, N-dimethyl-4-aminopyridine (2.24 g, 18.3 mmol) was added and stirred at room temperature overnight. A saturated aqueous ammonium chloride solution was added to the reaction solution, and the mixture was extracted with dichloromethane. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (heptane / chloroform = 95/5) to give (Z) -non-2-en-1-yl 10-bromodecanoate (4.88 g, yield 82% )
¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 6.8 Hz, 3H), 1.21-1.48 (m, 18H), 1.57-1.68 (m, 2H), 1.81-1.90 (m, 2H), 2.06 -2.14 (m, 2H), 2.30 (t, J = 7.4 Hz, 2H), 3.40 (t, J = 6.8 Hz, 2H), 4.62 (t, J = 6.8 Hz, 2H), 5.48-5.57 (m, 1H), 5.60-5.69 (m, 1H).

Di ((Z) -non-2-en-1-yl) 10,10 '-((3-hydroxypropyl) azanediyl) dideanoate (compound 1)
Process 1
(Z) -Non-2-en-1-yl 10-bromooctadecanoate (3.72 g, 9.90 mmol) obtained in Reference Example 1 was dissolved in acetonitrile (20 mL), and 2-nitrobenzenesulfonamide ( 800 mg, 3.96 mmol) and cesium carbonate (3.87 g, 11.9 mmol) were added, and the mixture was stirred at 60 ° C. overnight. A saturated aqueous ammonium chloride solution was added to the reaction solution, and the mixture was extracted with heptane. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (heptane / ethyl acetate = 85/15) to obtain di ((Z) -non-2-en-1-yl) 10,10 ′-(((2-nitro A crude product of phenyl) sulfonyl) azanediyl) dicanoate was obtained.
The obtained crude product was dissolved in acetonitrile (25 mL), and 1,8-diazabicyclo [5.4.0] -7-undecene (Tokyo Chemical Industry Co., Ltd., 1.41 mL, 9.43 mmol), 1-dodecanethiol (2.25 mL, 9.43 mmol) was added and the mixture was stirred at 60 ° C. for 3 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off under reduced pressure. The obtained residue was purified by amino silica gel column chromatography (heptane / chloroform = 90 / 10-0 / 100) to obtain di ((Z) -non-2-en-1-yl) 10,10'-azane Irjidecanoate (2.27 g, yield 87%) was obtained.
ESI-MS m / z: 607 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 6.9 Hz, 6H), 1.22-1.51 (m, 40H), 1.57-1.68 ( m, 4H), 2.05-2.12 (m, 4H), 2.30 (t, J = 7.5 Hz, 4H), 2.57 (t, J = 7.3 Hz, 4H), 4.62 (d, J = 6.9 Hz, 4H), 5.46-5.56 (m, 2H), 5.59-5.69 (m, 2H).
Process 2
Di ((Z) -non-2-en-1-yl) 10,10'-azanediyl didecanoate (400 mg, 0.660 mmol) obtained in step 1 was converted to N, N-dimethylformamide (3.5 mL). ), Tert-butyl (3-iodopropoxy) dimethylsilane (38 mg, 0.792 mmol) and cesium carbonate (430 mg, 1.32 mmol) were added, and the mixture was stirred at room temperature overnight. Water was added to the reaction solution and extracted with heptane. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (heptane / ethyl acetate = 80/20) to obtain di ((Z) -non-2-en-1-yl) 10,10 ′-((3-((tert A crude product of -butyldimethylsilyl) oxy) propyl) azanediyl) didecanoate was obtained.
The obtained crude product was dissolved in tetrahydrofuran (8 mL), tetrabutylammonium fluoride (1 mol / L tetrahydrofuran solution, 1.07 mL, 1.07 mmol) was added, and the mixture was stirred at room temperature for 6 hr. A saturated aqueous sodium chloride solution was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The obtained residue was purified by amino silica gel column chromatography (heptane / ethyl acetate = 70/30) to obtain Compound 1 (325 mg, yield 74%).
ESI-MS m / z: 665 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 6.9 Hz, 6H), 1.19-1.72 (m, 47H), 2.07-2.13 ( m, 4H), 2.30 (t, J = 7.5 Hz, 4H), 2.39 (t, J = 7.8 Hz, 4H), 2.63 (t, J = 5.5 Hz, 2H), 3.79 (t, J = 5.3 Hz, 2H), 4.62 (d, J = 6.9 Hz, 4H), 5.48-5.57 (m, 2H), 5.59-5.69 (m, 2H).

Reference Example 2: (Z) -non-2-en-1-yl 12-bromododecanoate 12-bromododecanoic acid (manufactured by sigma-aldrich, 5.00 g, 17.9 mmol) in dichloromethane (40 mL (Z) -non-2-en-1-ol (Tokyo Chemical Industry Co., Ltd., 3.90 mL, 23.3 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (5.15 g, 26.9 mmol) and dimethylaminopyridine (3.28 g, 26.9 mmol) were sequentially added and allowed to react at room temperature overnight. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 95/5) to give (Z) -non-2-en-1-yl 12-bromododecano Art (5.00 g, 69% yield) was obtained.
¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 6.8 Hz, 3H), 1.23-1.46 (m, 22H), 1.57-1.65 (m, 2H), 1.81-1.89 (m, 2H), 2.10 (q, J = 6.8 Hz, 2H), 2.30 (t, J = 7.5 Hz, 2H), 3.41 (t, J = 7.0 Hz, 2H), 4.61-4.63 (m, 2H), 5.48-5.57 (m, 1H), 5.60-5.68 (m, 1H).

Di ((Z) -non-2-en-1-yl) 12,12 '-((3-hydroxypropyl) azanediyl) didodecanoate (compound 2)
3-Aminopropan-1-ol (manufactured by Tokyo Chemical Industry Co., Ltd., 0.152 mL, 2.00 mmol) was dissolved in N, N-dimethylformamide (2 mL), and (Z) -nona-obtained in Reference Example 2 was obtained. 2-en-1-yl 12-bromododecanoate (1.77 g, 4.39 mmol) and N, N-diisopropylethylamine (3.49 mL, 20.0 mmol) were sequentially added and reacted at 120 ° C. for 3 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by NH silica gel column chromatography (hexane / ethyl acetate = 99/1 to 65/35) to obtain compound 2 (0.420 g, yield 29%). It was.
ESI-MS m / z: 720 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 6.8 Hz, 6H), 1.22-1.39 (m, 44H), 1.39-1.48 ( m, 4H), 1.58-1.70 (m, 6H), 2.10 (q, J = 6.8 Hz, 4H), 2.30 (t, J = 7.6 Hz, 4H), 2.36-2.42 (m, 4H), 2.63 (t , J = 5.6 Hz, 2H), 3.80 (t, J = 5.1 Hz, 2H), 4.62 (dt, J = 6.8, 0.5 Hz, 4H), 5.47-5.56 (m, 2H), 5.60-5.68 (m, 2H).

Reference Example 3: Nonyl 12-bromododecanoate In the same manner as Reference Example 2, instead of (Z) -non-2-en-1-ol, nonan-1-ol (manufactured by Tokyo Chemical Industry Co., Ltd.) was used. Used to obtain nonyl 12-bromododecanoate (1.30 g, yield 60%).
¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 6.8 Hz, 3H), 1.24-1.46 (m, 26H), 1.57-1.65 (m, 4H), 1.81-1.89 (m, 2H), 2.29 (t, J = 7.5 Hz, 2H), 3.41 (t, J = 7.0 Hz, 2H), 4.06 (t, J = 6.8 Hz, 2H).

Dinonyl 12,12 '-((3-hydroxypropyl) azanediyl) didodecanoate (compound 3)
In the same manner as in Example 2, using the nonyl 12-bromododecanoate obtained in Reference Example 3 instead of (Z) -non-2-en-1-yl 12-bromododecanoate, 3 (1.30 g, 60% yield) was obtained.
ESI-MS m / z: 724 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 6.8 Hz, 6H), 1.22-1.36 (m, 56H), 1.42-1.51 ( m, 4H), 1.57-1.70 (m, 6H), 2.29 (t, J = 7.5 Hz, 4H), 2.40 (t, J = 7.6 Hz, 4H), 2.63 (t, J = 5.6 Hz, 2H), 3.80 (t, J = 5.1 Hz, 2H), 4.05 (t, J = 6.7 Hz, 4H).

Reference Example 4: (Z) -Non-3-en-1-yl 12-bromododecanoate In the same manner as in Reference Example 1, instead of cis-2-nonen-1-ol, cis-3-nonene (Z) -non-3-en-1-yl 12-bromododecanoate (1.50 g, yield 52%) was obtained using -1-ol (manufactured by Tokyo Chemical Industry Co., Ltd.).
¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 7.0 Hz, 3H), 1.24-1.45 (m, 20H), 1.57-1.65 (m, 2H), 1.81-1.89 (m, 2H), 2.04 (q, J = 6.8 Hz, 2H), 2.29 (t, J = 7.6 Hz, 2H), 2.34-2.40 (m, 2H), 3.41 (t, J = 6.8 Hz, 2H), 4.06 (t, J = 7.0 Hz, 2H), 5.29-5.39 (m, 1H), 5.46-5.55 (m, 1H).

Di ((Z) -non-3-en-1-yl) 12,12 '-((3-hydroxypropyl) azanediyl) didodecanoate (compound 4)
In the same manner as in Example 2, instead of (Z) -non-2-en-1-yl 12-bromododecanoate, (Z) -non-3-ene-1 obtained in Reference Example 4 was used. Compound 4 (0.420 g, 44% yield) was obtained using -yl 12-bromododecanoate.
ESI-MS m / z: 720 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 6.8 Hz, 6H), 1.19-1.42 (m, 44H), 1.42-1.72 ( m, 10H), 2.04 (q, J = 6.8 Hz, 4H), 2.29 (t, J = 7.6 Hz, 4H), 2.34-2.43 (m, 4H), 2.63 (t, J = 5.7 Hz, 2H), 3.80 (t, J = 5.1 Hz, 2H), 4.06 (t, J = 7.0 Hz, 4H), 5.30-5.38 (m, 2H), 5.46-5.55 (m, 2H).

Reference Example 5: Ethyl 12-bromododecanoate 12-bromododecanoic acid (6.50 g, 23.3 mmol) was dissolved in ethanol (70 mL) and a hydrogen chloride-1,4-dioxane solution (Tokyo Chemical Industry Co., Ltd., 4.0 mol / L, 2.91 mL, 11.6 mmol) was added, and the mixture was reacted at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 95/5) to give ethyl 12-bromododecanoate (7.57 g, quantitative yield). It was.

Reference Example 6: 12,12 '-((3-hydroxypropyl) azanediyl) didodecanoic acid step 1
In the same manner as in Example 2, using ethyl 12-bromododecanoate obtained in Reference Example 5 instead of (Z) -non-2-en-1-yl 12-bromododecanoate, diethyl 12,12 ′-((3-hydroxypropyl) azanediyl) didodecanoate (1.23 g, yield 44%) was obtained.
ESI-MS m / z: 528 (M + H) ⁺
Process 2:

Diethyl

12,12 ′-((3-hydroxypropyl) azanediyl) didodecanoate (1.23 g, 2.33 mmol) obtained in step 1 was dissolved in ethanol (5 mL), and lithium hydroxide monohydrate (0.233 g, 9.32 mmol) of an aqueous solution (2 mL) was added and allowed to react at room temperature for 1 hour. The reaction mixture was made weakly acidic with hydrochloric acid, water was further added, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain 12,12 ′-((3-hydroxypropyl) azanediyl) didodecanoic acid (1.00 g, yield 91%).
ESI-MS m / z: 470 (M-H) ^-

Di ((Z) -non-2-en-1-yl) 12,12 '-((3-hydroxypropyl) azanediyl) didodecanoate (Compound 5)
12,12 ′-((3-hydroxypropyl) azanediyl) didodecanoic acid (0.200 g, 0.424 mmol) obtained in Reference Example 6 was dissolved in N, N-dimethylformamide (3 mL), and (E) -nona -2-en-1-ol (Tokyo Chemical Industry Co., Ltd., 0.709 mL, 4.24 mmol), O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium Hexafluorophosphate (HATU, 0.645 g, 1.70 mmol) and N, N-dimethylaminopyridine (0.104 g, 0.848 mmol) were sequentially added and allowed to react at room temperature overnight. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 99/1 to 90/10) to give compound 5 (0.0680 g, yield 22%). .
ESI-MS m / z: 720 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 6.8 Hz, 6H), 1.21-1.51 (m, 48H), 1.57-1.73 ( m, 6H), 2.05 (q, J = 6.8 Hz, 4H), 2.30 (t, J = 7.6 Hz, 4H), 2.37-2.46 (m, 4H), 2.62-2.70 (m, 2H), 3.80 (t , J = 5.1 Hz, 2H), 4.51 (dd, J = 6.6, 1.0 Hz, 4H), 5.51-5.60 (m, 2H), 5.72-5.81 (m, 2H).

Reference Example 7: Nona-8-en-1-yl 12-bromododecanoate In the same manner as Reference Example 1, instead of cis-2-nonen-1-ol, nona-8-en-1-ol (Manufactured by Tokyo Chemical Industry Co., Ltd.) was used to obtain nona-8-en-1-yl 12-bromododecanoate (0.900 g, yield 42%).

Di (non-8-en-1-yl) 12,12 '-((3-hydroxypropyl) azanediyl) didodecanoate (Compound 6)
In the same manner as in Example 2, instead of (Z) -non-2-en-1-yl 12-bromodecanoate, nona-8-en-1-yl 12- obtained in Reference Example 7 was used. Compound 6 (0.110 g, 14% yield) was obtained using bromododecanoate.
ESI-MS m / z: 720 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 1.21-1.50 (m, 48H), 1.55-1.70 (M10H), 2.00-2.08 (m, 4H), 2.29 (t, J = 7.5 Hz, 4H), 2.37-2.42 (m, 4H), 2.63 (t, J = 5.6 Hz, 2H), 3.80 (t, J = 5.1 Hz, 2H), 4.05 (t, J = 6.7 Hz, 4H), 4.90-5.05 (m, 4H), 5.73-5.88 (m, 2H).

Reference Example 8: (Z) -Octa-5-en-1-yl 12-bromododecanoate In the same manner as Reference Example 1, instead of cis-2-nonen-1-ol, (Z) -octa (Z) -oct-5-en-1-yl 12-bromododecanoate (1.80 g, yield 86%) was obtained using -5-en-1-ol (manufactured by Tokyo Chemical Industry Co., Ltd.).

Di ((Z) -octa-5-en-1-yl) 12,12 '-((3-hydroxypropyl) azanediyl) didodecanoate (compound 7)
In the same manner as in Example 2, instead of (Z) -non-2-en-1-yl 12-bromododecanoate, (Z) -oct-5-ene-1 obtained in Reference Example 8 was used. Compound 7 (0.670 g, yield 45%) was obtained using -yl 12-bromododecanoate.
ESI-MS m / z: 692 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.96 (t, J = 7.6 Hz, 6H), 1.22-1.33 (m, 28H), 1.37-1.50 ( m, 8H), 1.57-1.70 (m, 10H), 1.98-2.10 (m, 8H), 2.29 (t, J = 7.5 Hz, 4H), 2.37-2.42 (m, 4H), 2.63 (t, J = 5.6 Hz, 2H), 3.79 (t, J = 5.1 Hz, 2H), 4.07 (t, J = 6.7 Hz, 4H), 5.25-5.43 (m, 4H).

Reference Example 9: (Z) -Undec-2-en-1-yl 12-bromododecanoate Step 1
2-Propin-1-ol (manufactured by Tokyo Chemical Industry Co., Ltd., 0.989 mL, 16.8 mmol) was dissolved in tetrahydrofuran (20 mL). Hexamethylphosphoric triamide (Tokyo Chemical Industry Co., Ltd., 5.00 mL, 28.7 mmol) and n-butyllithium (Tokyo Chemical Industry Co., Ltd., 1.6 mol / L hexane solution, 21.0 mL, 33.7) after cooling to -78 ° C mmol) were sequentially added, and the mixture was stirred at -78 ° C for 1 hour. The temperature was raised to −30 ° C., and 1-bromooctane (2.50 g, 12.9 mmol) was added. Thereafter, the temperature was gradually raised to room temperature, followed by reaction at room temperature overnight. Water was added to the reaction mixture, and the mixture was extracted with hexane. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (hexane / ethyl acetate = 99/1 to 70/30) to give 2-undecin-1-ol (1.64 g, yield 75). %).
ESI-MS m / z: 168 (M + H) ⁺
Process 2
2-Undecin-1-ol (0.300 g, 1.78 mmol) obtained in step 1 is dissolved in ethyl acetate (15 mL), and quinoline (0.317 mL, 2.67 mmol) and chloroform (0.144 mL, 1.78 mmol) are added. Then, the inside of the container was replaced with argon. After adding Linda catalyst (manufactured by Tokyo Chemical Industry Co., Ltd., 0.095 g, 0.891 mmol / palladium amount), the inside of the container was replaced with hydrogen and reacted at room temperature for 2.5 hours. Water was added to the reaction mixture, and the mixture was extracted with hexane. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain (Z) -undec-2-en-1-ol (crude product, 0.32 g, quantitative).
¹ H-NMR (CDCl ₃ ) δ: 0.86-0.92 (m, 3H), 1.19-1.40 (m, 12H), 2.07 (q, J = 6.6 Hz, 2H), 4.20 (t, J = 5.1 Hz, 2H ), 4.26 (s, 1H), 5.49-5.67 (m, 2H).
Process 3
In the same manner as in Reference Example 1, (Z) -undec-2-en-1-ol obtained in Step 2 was used instead of cis-2-nonen-1-ol, and (Z) -undec- 2-En-1-yl 12-bromododecanoate (0.285 g, yield 75%) was obtained.

Di ((Z) -undec-2-en-1-yl) 12,12 '-((3-hydroxypropyl) azanediyl) didodecanoate (Compound 8)
In the same manner as in Example 2, instead of (Z) -non-2-en-1-yl 12-bromododecanoate, (Z) -undec-2-ene-1 obtained in Reference Example 9 was used. Compound 8 (0.095 g, yield 46%) was obtained using -yl 12-bromododecanoate.
ESI-MS m / z: 776 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 7.0 Hz, 6H), 1.24-1.46 (m, 56H), 1.58-1.69 ( m, 6H), 2.09 (q, J = 6.9 Hz, 4H), 2.30 (t, J = 7.6 Hz, 4H), 2.40 (t, J = 7.7 Hz, 4H), 2.64 (t, J = 5.4 Hz, 2H), 3.80 (t, J = 5.1 Hz, 2H), 4.62 (d, J = 6.8 Hz, 4H), 5.47-5.56 (m, 2H), 5.60-5.69 (m, 2H).

Reference Example 10: (Z) -Tridec-2-en-1-yl 12-bromododecanoate In the same manner as in Reference Example 9, using 1-bromodecane instead of 1-bromooctane in Step 1, ( Z) -Tridec-2-en-1-yl 12-bromododecanoate (0.152 g, yield of 3 steps 33%) was obtained.
¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 7.0 Hz, 3H), 1.23-1.46 (m, 30H), 1.58-1.66 (m, 2H), 1.81-1.89 (m, 2H), 2.09 (t, J = 7.0 Hz, 2H), 2.30 (t, J = 7.6 Hz, 2H), 3.41 (t, J = 7.0 Hz, 2H), 4.62 (d, J = 6.8 Hz, 2H), 5.48-5.56 (m, 1H), 5.60-5.68 (m, 1H).

Di ((Z) -tridec-2-en-1-yl) 12,12 '-((3-hydroxypropyl) azanediyl) didodecanoate (Compound 9)
In the same manner as in Example 2, instead of (Z) -non-2-en-1-yl 12-bromododecanoate, (Z) -tridec-2-ene-1 obtained in Reference Example 10 was used. Compound 9 (0.029 g, yield 26%) was obtained using -yl 12-bromododecanoate.
ESI-MS m / z: 832 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 6.8 Hz, 6H), 1.20-1.50 (m, 66H), 1.56-1.70 ( m, 4H), 2.09 (q, J = 6.8 Hz, 4H), 2.30 (t, J = 7.5 Hz, 4H), 2.40 (t, J = 7.7 Hz, 4H), 2.63 (t, J = 5.6 Hz, 2H), 3.79 (t, J = 5.1 Hz, 2H), 4.62 (d, J = 6.8 Hz, 4H), 5.48-5.56 (m, 2H), 5.61-5.67 (m, 2H).

Reference Example 11: (E) -3,7-Dimethylocta-2,6-dien-1-yl 12-bromododecanoate In the same manner as Reference Example 1, instead of cis-2-nonen-1-ol (E) -3,7-dimethylocta-2,6-dien-1-yl 12-bromododecanoate (2.10 g, 94% yield) was obtained using geraniol (manufactured by Tokyo Chemical Industry Co., Ltd.) It was.
¹ H-NMR (CDCl ₃ ) δ: 1.25-1.34 (m, 12H), 1.37-1.45 (m, 2H), 1.58-1.65 (m, 2H), 1.60-1.61 (m, 3H), 1.67-1.69 ( m, 3H), 1.69-1.71 (m, 3H), 1.81-1.89 (m, 2H), 2.01-2.15 (m, 4H), 2.30 (t, J = 7.5 Hz, 2H), 3.41 (t, J = 7.0 Hz, 2H), 4.59 (dd, J = 7.1, 0.8 Hz, 2H), 5.05-5.12 (m, 1H), 5.30-5.37 (m, 1H).

Bis ((E) -3,7-dimethylocta-2,6-dien-1-yl) 12,12 '-((3-hydroxypropyl) azanediyl) didodecanoate (Compound 10)
In the same manner as in Example 2, instead of (Z) -non-2-en-1-yl 12-bromododecanoate, (E) -3,7-dimethylocta- obtained in Reference Example 11 was used. Compound 10 (0.825 g, yield 49%) was obtained using 2,6-dien-1-yl 12-bromododecanoate.
ESI-MS m / z: 744 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 1.23-1.35 (m, 30H), 1.41-1.50 (m, 4H), 1.56-1.67 (m, 4H ), 1.60 (s, 6H), 1.68 (d, J = 1.0 Hz, 6H), 1.70 (d, J = 1.0 Hz, 6H), 2.01-2.14 (m, 8H), 2.29 (t, J = 7.5 Hz , 4H), 2.40 (t, J = 7.7 Hz, 4H), 2.63 (t, J = 5.6 Hz, 2H), 3.79 (t, J = 5.1 Hz, 2H), 4.59 (d, J = 7.1 Hz, 4H ), 5.05-5.11 (m, 2H), 5.31-5.36 (m, 2H).

Reference Example 12: 3,7-dimethyloct-6-en-1-yl 12-bromododecanoate In the same manner as in Reference Example 1, instead of cis-2-nonen-1-ol, 3,7- Using dimethyl-6-octen-1-ol (manufactured by Tokyo Chemical Industry Co., Ltd.), 3,7-dimethyloct-6-en-1-yl 12-bromododecanoate (3.76 g, yield 74%) was obtained. It was.
¹ H-NMR (CDCl ₃ ) δ: 0.91 (d, J = 6.6 Hz, 3H), 1.14-1.24 (m, 1H), 1.25-1.49 (m, 18H), 1.49-1.75 (m, 2H), 1.60 (s, 3H), 1.67-1.69 (m, 3H), 1.81-1.90 (m, 2H), 1.92-2.06 (m, 2H), 2.28 (t, J = 7.6 Hz, 2H), 3.41 (t, J = 7.0 Hz, 2H), 4.05-4.16 (m, 2H), 5.04-5.13 (m, 1H).

Bis (3,7-dimethyloct-6-en-1-yl) 12,12 '-((3-hydroxypropyl) azanediyl) didodecanoate (compound 11)
In the same manner as in Example 2, instead of (Z) -non-2-en-1-yl 12-bromododecanoate, 3,7-dimethyloct-6-ene- 1-yl 12-bromododecanoate was used to obtain Compound 11 (1.62 g, yield 54%).
ESI-MS m / z: 748 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.91 (d, J = 6.6 Hz, 6H), 1.14-1.22 (m, 2H), 1.23-1.51 ( m, 36H), 1.51-1.71 (m, 10H), 1.60 (d, J = 0.8 Hz, 6H), 1.68 (d, J = 1.3 Hz, 6H), 1.90-2.04 (m, 4H), 2.28 (t , J = 7.5 Hz, 4H), 2.37-2.42 (m, 4H), 2.63 (t, J = 5.6 Hz, 2H), 3.79 (t, J = 5.2 Hz, 2H), 4.03-4.17 (m, 4H) , 5.05-5.12 (m, 2H).

Reference Example 13: 12-bromododecyl nonanoate In the same manner as Reference Example 1, instead of cis-2-nonen-1-ol and 10-bromodecanoic acid, 12-bromododecan-1-ol (manufactured by Tokyo Chemical Industry Co., Ltd.) ) And nonanoic acid (manufactured by Tokyo Chemical Industry Co., Ltd.), 12-bromododecyl nonanoate (0.810 g, yield 32%) was obtained.
¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 7.0 Hz, 3H), 1.21-1.37 (m, 24H), 1.37-1.46 (m, 2H), 1.57-1.66 (m, 4H), 1.81 -1.89 (m, 2H), 2.29 (t, J = 7.6 Hz, 2H), 3.41 (t, J = 7.0 Hz, 2H), 4.06 (t, J = 6.8 Hz, 2H).

((3-Hydroxypropyl) azanediyl) bis (dodecane-12,1-diyl) dinonanoate (compound 12)
In the same manner as in Example 2, using 12-bromododecyl nonanoate obtained in Reference Example 13 instead of (Z) -non-2-en-1-yl 12-bromododecanoate, compound 12 ( 0.128 g, 19% yield).
ESI-MS m / z: 724 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 7.0 Hz, 6H), 1.21-1.38 (m, 50H), 1.41-1.75 ( m, 16H), 2.29 (t, J = 7.6 Hz, 4H), 2.43 (brs, 4H), 2.66 (brs, 2H), 3.80 (t, J = 5.2 Hz, 2H), 4.05 (t, J = 6.8 Hz, 4H).

Reference Example 14: 12-bromododecyl non-8-enoate In the same manner as in Reference Example 1, instead of cis-2-nonen-1-ol and 10-bromodecanoic acid, 12-bromododecan-1-ol (Tokyo 12-bromododecyl nona-8-enoate (1.33 g, yield 52%) was obtained using Kasei Kogyo Co., Ltd. and 8-nonenoic acid (Tokyo Kasei Kogyo Co., Ltd.).
¹ H-NMR (CDCl ₃ ) δ: 1.27-1.44 (m, 22H), 1.58-1.67 (m, 4H), 1.81-1.89 (m, 2H), 2.00-2.07 (m, 2H), 2.29 (t, J = 7.6 Hz, 2H), 3.41 (t, J = 6.8 Hz, 2H), 4.05 (t, J = 6.7 Hz, 2H), 4.91-5.03 (m, 2H), 5.74-5.86 (m, 1H).

((3-hydroxypropyl) azanediyl) bis (dodecane-12,1-diyl) bis (nona-8-enoate) (compound 13)
In the same manner as in Example 2, instead of (Z) -non-2-en-1-yl 12-bromododecanoate, 12-bromododecyl nona-8-enoate obtained in Reference Example 13 was used. Compound 13 (0.450 g, 43% yield) was obtained.
ESI-MS m / z: 720 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 1.22-1.51 (m, 48H), 1.57-1.70 (m, 10H), 2.00-2.07 (m, 4H ), 2.29 (t, J = 7.6 Hz, 4H), 2.36-2.43 (m, 4H), 2.63 (t, J = 5.6 Hz, 2H), 3.80 (t, J = 5.1 Hz, 2H), 4.05 (t , J = 6.8 Hz, 4H), 4.90-5.03 (m, 4H), 5.74-5.86 (m, 2H).

Dimethyl 18,18 '-((3-hydroxypropyl) azanediyl) diolate (Compound 14)
Process 1
2-Nitrobenzenesulfonamide (Tokyo Chemical Industry Co., Ltd., 2.13 g, 10.3 mmol) is dissolved in acetonitrile (34 mL), cesium carbonate (10.1 g, 31.0 mmol), 10-bromo-1-decene (Tokyo Chemical Industry Co., Ltd.) Product, 4.59 mL, 21.7 mmol) was added and stirred at 80 ° C. overnight. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane / ethyl acetate = 90/10) to obtain a crude product of N, N-di (9-decen-1-yl) -2-nitrobenzenesulfonamide. .
Tetrahydrofuran (32 mL), water (5 mL), 4-methylmorpholine 4-oxide (Sigma Aldrich, 2.41 g, 20.6 mmol), microencapsulated osmium tetroxide (Wako Pure Chemical Industries, Ltd.) 10 wt%, 0.261 g, 0.103 mmol) was added, and the mixture was stirred at room temperature overnight. Sodium periodate (5.28 g, 24.7 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 5 hours. A saturated aqueous sodium thiosulfate solution was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with a saturated aqueous sodium thiosulfate solution, dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 90/10) to give 2-nitro-N, N-bis (9-oxononyl) benzenesulfonamide (3.11 g, yield 63%). Obtained.
ESI-MS m / z: 483 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 1.19-1.33 (m, 16H), 1.46-1.65 (m, 8H), 2.42 (td, J = 7.4 , 1.9 Hz, 4H), 3.26 (t, J = 7.7 Hz, 4H), 7.60-7.63 (m, 1H), 7.65-7.70 (m, 2H), 7.99-8.02 (m, 1H), 9.76 (t, J = 1.9 Hz, 2H).
Process 2
9-Bromononanoic acid (Tokyo Kasei Kogyo Co., Ltd., 3.04 g, 12.4 mmol) is dissolved in acetonitrile (60 mL), and triphenylphosphine (Kanto Chemical Co., Ltd., 3.26 g, 12.4 mmol) is added to the solution. Stir. The reaction solution was concentrated under reduced pressure to obtain a crude product of (8-carboxyoctyl) triphenylphosphonium bromide.
The obtained crude product of (8-carboxyoctyl) triphenylphosphonium bromide (0.339 g) in tetrahydrofuran (2 mL) was cooled to −18 ° C., hexamethylphosphoric triamide (0.236 mL, 1.36 mmol), lithium Bis (trimethylsilyl) amide (manufactured by Sigma-Aldrich, 1.0 mol / L tetrahydrofuran solution, 1.36 mL, 1.36 mmol) was added, and the mixture was stirred at −18 ° C. for 20 minutes. The reaction solution was cooled to −78 ° C., and a solution of 2-nitro-N, N-bis (9-oxononyl) benzenesulfonamide (0.109 g, 0.226 mmol) obtained in Step 1 in tetrahydrofuran (2 mL) was added. And stirred at room temperature overnight. Sodium hydrogen carbonate (0.285 g, 3.39 mmol) and dimethyl sulfate (0.324 mL, 3.39 mmol) were added to the reaction solution, and the mixture was stirred at room temperature for 2 hours. A saturated aqueous ammonium chloride solution was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane / ethyl acetate = 70/30) to obtain methyl 18,18 '-(((2-nitrophenyl) sulfonyl) azanediyl) diolate (0.172 g, 0.217 mmol, yield). 96%).
ESI-MS m / z: 809 (M + NH ₄ ) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 1.17-1.38 (m, 36H), 1.46-1.55 (m, 4H), 1.58-1.66 (m, 4H), 1.96-2.04 (m, 8H), 2.30 (t, J = 7.6 Hz, 4H), 3.26 (t, J = 7.7 Hz, 4H), 3.66 (s, 6H), 5.29-5.38 (m, 4H ), 7.59-7.69 (m, 3H), 7.99-8.03 (m, 1H).
Process 3
Methyl 18,18 '-(((2-nitrophenyl) sulfonyl) azanediyl) diolate (0.179 g, 0.217 mmol) was dissolved in acetonitrile (2 mL) and 1,8-diazabicyclo [5.4.0] -7-undecene was dissolved. (0.0890 mL, 0.558 mmol) and 1-dodecanethiol (0.142 mL, 0.565 mmol) were added and stirred at 60 ° C. for 2 hours. 1,8-diazabicyclo [5.4.0] -7-undecene (0.0890 mL, 0.558 mmol) and 1-dodecanethiol (0.142 mL, 0.565 mmol) were added to the reaction solution, and the mixture was further stirred at 60 ° C. for 2 hours. A saturated aqueous sodium thiosulfate solution was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with a saturated aqueous sodium thiosulfate solution, dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol = 90/10) to obtain

methyl

18,18′-azanediyldiolate (0.0703 g, yield 51%).
ESI-MS m / z: 607 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 1.23-1.38 (m, 36H), 1.43-1.52 (m, 4H), 1.59-1.65 (m, 4H ), 1.96-2.05 (m, 8H), 2.30 (t, J = 7.6 Hz, 4H), 2.58 (t, J = 7.4 Hz, 4H), 3.66 (s, 6H), 5.29-5.39 (m, 4H) .
Process 4
Methyl 18,18 '-(((2-nitrophenyl) sulfonyl) azanediyl) diolate (0.0489 g, 0.0807 mmol) was dissolved in acetonitrile (1 mL), N, N-diisopropylethylamine (0.0210 mL, 0.121 mmol), 3-Bromo-1-propanol (0.00829 mL, 0.0887 mmol) was added and stirred at room temperature for 3 hours. N, N-diisopropylethylamine (0.0210 mL, 0.121 mmol) and 3-bromo-1-propanol (0.00829 mL, 0.0887 mmol) were added to the reaction solution, and the mixture was stirred at room temperature for 3 hours. N, N-diisopropylethylamine (0.0210 mL, 0.121 mmol) and 3-bromo-1-propanol (0.00829 mL, 0.0887 mmol) were added, and the mixture was stirred at room temperature overnight and then at 50 ° C. for 2 hr. N, N-diisopropylethylamine (0.0210 mL, 0.121 mmol) and 3-bromo-1-propanol (0.00829 mL, 0.0887 mmol) were added, and the mixture was stirred at 50 ° C. for 1 hr. A saturated aqueous sodium chloride solution was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The obtained residue was purified by amino silica gel column chromatography (hexane / ethyl acetate = 70/30) to obtain Compound 14 (0.0345 g, 0.0520 mmol, yield 64%).
ESI-MS m / z: 665 (M + H) +; ¹ H-NMR (CDCl ₃ ) δ: 1.18-1.82 (m, 46H), 1.91-2.06 (m, 9H), 2.30 (t, J = 7.6 Hz, 4H), 2.40-2.86 (m, 6H), 3.66 (s, 6H), 3.80 (t, J = 5.2 Hz, 2H), 5.29-5.39 (m, 4H).

Dibutyl 18,18 '-((3-hydroxypropyl) azanediyl) diolate (Compound 15)
Process 1

Methyl

18,18 ′-(((2-nitrophenyl) sulfonyl) azanediyl) diolate (0.0730 g, 0.0957 mmol) obtained in Step 2 of Example 14 was dissolved in ethanol (1 mL) and sodium hydroxide ( 2.0 mol / L aqueous solution, 0.241 mL, 0.482 mmol) was added, and the mixture was stirred at room temperature for 3 hours. Hydrochloric acid (2.0 mol / L aqueous solution) was added to the reaction solution until the pH became 2 or less, and then concentrated under reduced pressure. The residue was dissolved in 1-butanol (1 mL) solution and O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate (0.110 g, 0.288 mmol) and N, N-dimethyl-4-aminopyridine (1.2 mg, 0.00982 mmol) were added and stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane / ethyl acetate = 80/20) to obtain

butyl

18,18′-azanediyldiolate (0.0593 g, yield 71%).
ESI-MS m / z: 893 (M + NH ₄ ) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.93 (t, J = 7.4 Hz, 6H), 1.19-1.43 (m, 40H), 1.46-1.56 (m, 4H), 1.56-1.65 (m, 8H), 1.95-2.04 (m, 8H), 2.28 (t, J = 7.5 Hz, 4H), 3.26 (t, J = 7.6 Hz, 4H), 4.06 ( t, J = 6.6 Hz, 4H), 5.29-5.39 (m, 4H), 7.58-7.70 (m, 3H), 7.98-8.03 (m, 1H).
Process 2
In the same manner as in Step 3 and Step 4 of Example 14, instead of

methyl

18,18 ′-(((2-nitrophenyl) sulfonyl) azanediyl) diolate, the

butyl

18,18′-acrylate obtained in Step 1 was used. Zandiyl dioleate (0.0510 g, 0.0583 mmol) was used to give compound 15 (0.0115 g, yield 26%).
ESI-MS m / z: 749 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.93 (t, J = 7.4 Hz, 6H), 1.21-1.45 (m, 40H), 1.51-1.66 ( m, 14H), 1.94-2.06 (m, 9H), 2.29 (t, J = 7.6 Hz, 4H), 2.55-2.66 (m, 4H), 2.75-2.86 (m, 2H), 3.81 (t, J = 5.2 Hz, 2H), 4.06 (t, J = 6.7 Hz, 4H), 5.32-5.36 (m, 4H).

Reference Example 15: Nonan-2-yl 12-bromododecanoate In the same manner as in Reference Example 1, instead of cis-2-nonen-1-ol, nonan-2-ol (manufactured by Tokyo Chemical Industry Co., Ltd.) was used. Used to obtain nonan-2-yl 12-bromododecanoate (1.36 g, yield 47%).
¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 7.0 Hz, 3H), 1.19 (d, J = 6.3 Hz, 3H), 1.21-1.37 (m, 22H), 1.37-1.51 (m, 2H ), 1.51-1.65 (m, 4H), 1.79-1.89 (m, 2H), 2.26 (t, J = 7.5 Hz, 2H), 3.41 (t, J = 6.8 Hz, 2H), 4.85-4.94 (m, 1H).

Di (nonan-2-yl) 12,12 '-((3-hydroxypropyl) azanediyl) didodecanoate (Compound 16)
In the same manner as in Example 2, instead of (Z) -non-2-en-1-yl 12-bromododecanoate, nonan-2-yl 12-bromododecanoate obtained in Reference Example 14 was used. To give compound 16 (0.450 g, yield 44%).
ESI-MS m / z: 724 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 6.8 Hz, 6H), 1.19 (d, J = 6.3 Hz, 6H), 1.22 -1.35 (m, 50H), 1.41-1.73 (m, 12H), 2.26 (t, J = 7.5 Hz, 4H), 2.38-2.45 (m, 4H), 2.64 (t, J = 5.4 Hz, 2H), 3.80 (t, J = 4.7 Hz, 2H), 4.84-4.96 (m, 2H).

Di ((Z) -non-2-en-1-yl) 12,12 '-((1,3-dihydroxypropan-2-yl) azanediyl) didodecanoate (compound 17)
In the same manner as in Example 2, 2-aminopropane-1,3-diol (manufactured by Tokyo Chemical Industry Co., Ltd.) was used instead of 3-aminopropan-1-ol, and compound 17 (0.0500 g, yield 6) %).
ESI-MS m / z: 736 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 7.0 Hz, 6H), 1.22-1.48 (m, 48H), 1.58-1.66 ( m, 2H), 2.10 (q, J = 6.8 Hz, 4H), 2.30 (t, J = 7.6 Hz, 4H), 2.53 (t, J = 7.5 Hz, 4H), 2.92-3.00 (m, 4H), 3.59 (d, J = 6.8 Hz, 4H), 4.62 (dt, J = 6.8, 0.5 Hz, 4H), 5.49-5.57 (m, 2H), 5.60-5.68 (m, 2H).

Dinonyl 12,12 '-((1,3-dihydroxypropan-2-yl) azanediyl) didodecanoate (Compound 18)
In the same manner as in Example 2, instead of 3-aminopropan-1-ol and (Z) -non-2-en-1-yl 12-bromododecanoate, 2-aminopropane-1,3- Using diol and nonyl 12-bromodecanoate obtained in Reference Example 3, compound 18 (0.160 g, yield 20%) was obtained.
ESI-MS m / z: 740 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 7.0 Hz, 6H), 1.20-1.39 (m, 52H), 1.39-1.48 ( m, 4H), 1.56-1.67 (m, 8H), 2.29 (t, J = 7.6 Hz, 4H), 2.54 (t, J = 7.5 Hz, 4H), 2.92-3.00 (m, 1H), 3.59 (d , J = 7.1 Hz, 4H), 4.05 (t, J = 6.7 Hz, 4H).

Di (non-8-en-1-yl) 12,12 '-((1,3-dihydroxypropan-2-yl) azanediyl) didodecanoate (Compound 19)
In the same manner as in Example 2, instead of 3-aminopropan-1-ol and (Z) -non-2-en-1-yl 12-bromododecanoate, 2-aminopropane-1,3- Compound 19 (0.0350 g, yield 3%) was obtained using diol and nona-8-en-1-yl 12-bromodecanoate obtained in Reference Example 7.
ESI-MS m / z: 736 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 1.22-1.49 (m, 48H), 1.53-1.69 (m, 8H), 2.01-2.07 (m, 4H ), 2.29 (t, J = 7.6 Hz, 4H), 2.54 (t, J = 7.4 Hz, 4H), 2.92-2.99 (m, 1H), 3.59 (d, J = 7.1 Hz, 4H), 4.05 (t , J = 6.7 Hz, 4H), 4.90-5.03 (m, 4H), 5.75-5.88 (m, 2H).

Di (undec-10-en-1-yl) 12,12 '-((1,3-dihydroxypropan-2-yl) azanediyl) didodecanoate (compound 20)
In the same manner as in Example 2, instead of 3-aminopropan-1-ol and (Z) -non-2-en-1-yl 12-bromododecanoate, 2-aminopropane-1,3- Compound 20 (0.160 g, 12% yield) was obtained using undeca-10-en-1-yl 12-bromodecanoate obtained in the same manner as in Reference Example 7 using diol.
ESI-MS m / z: 792 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 1.23-1.46 (m, 56H), 1.54-1.70 (m, 8H), 2.00-2.08 (m, 4H ), 2.29 (t, J = 7.5 Hz, 4H), 2.54 (t, J = 7.4 Hz, 4H), 2.92-3.00 (m, 1H), 3.59 (t, J = 3.4 Hz, 4H), 4.05 (t , J = 6.8 Hz, 4H), 4.90-5.04 (m, 4H), 5.75-5.88 (m, 2H).

Reference Example 16: (Z) -Non-2-en-1-yl 8-bromooctanoate In the same manner as in Example 1, instead of 10-bromodecanoic acid, 8-bromooctanoic acid (manufactured by Tokyo Chemical Industry Co., Ltd., (2.00 g, 8.96 mmol) was used to obtain (Z) -non-2-en-1-yl 8-bromooctanate (1.06 g, yield 34%).
¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 6.8 Hz, 3H), 1.24-1.48 (m, 14H), 1.57-1.67 (m, 2H), 1.79-1.89 (m, 2H), 2.05 -2.14 (m, 2H), 2.31 (t, J = 7.6 Hz, 2H), 3.40 (t, J = 7.0 Hz, 2H), 4.62 (d, J = 6.8 Hz, 2H), 5.47-5.56 (m, 1H), 5.58-5.69 (m, 1H).

(Z) -Non-2-en-1-yl 8-((3-hydroxypropyl) ((9Z, 12Z) -octadeca-9,12-dien-1-yl) amino) octanoate (Compound 21)
Process 1
(9Z, 12Z) -Octadeca-9,12-dienyl methanesulfonate (Nu Check Prep, 2.85 g, 8.27 mmol) was dissolved in acetonitrile (30 mL), and cesium carbonate (6.74 g, 20.7 mmol), tetrabutylammonium iodide (3.05 g, 8.27 mmol), N- (tert-butoxycarbonyl) -2-nitrobenzenesulfonamide (Tokyo Chemical Industry Co., Ltd., 2.50 g, 8.27 mmol) were added, The mixture was stirred for 3 hours with heating under reflux. After cooling to room temperature, water was added and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The obtained residue was purified by silica gel column chromatography (heptane / ethyl acetate = 70/30) to give tert-butyl ((2-nitrophenyl) sulfonyl) ((9Z, 12Z) -octadeca-9,12-diene- A crude product of 1-yl) carbamate was obtained.
The obtained crude product was dissolved in dichloromethane (25 mL), trifluoroacetic acid (9.66 mL) was added, and the mixture was stirred at room temperature for 1 hr. The reaction solution was concentrated under reduced pressure. The residue was partitioned between chloroform and saturated aqueous sodium bicarbonate. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and the solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography (heptane / chloroform = 50 / 50-0 / 100) to give 2-nitro-N-((9Z, 12Z) -octadeca-9,12-dien-1-yl ) Benzenesulfonamide (2.34 g, 63% yield) was obtained.
ESI-MS m / z: 451 (M + H) +; ¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 7.0 Hz, 3H), 1.22-1.39 (m, 16H), 1.52 (m, 2H), 2.01-2.05 (m, 4H), 2.77 (dd, J = 6.0, 6.4 Hz, 2H), 3.05-3.13 (m, 2H), 5.23 (m, 1H), 5.31-5.42 (m, 4H) , 7.71-7.76 (m, 2H), 7.78-7.87 (1H), 813-8.15 (m, 1H).
Process 2
2-Nitro-N-((9Z, 12Z) -octadeca-9,12-dien-1-yl) benzenesulfonamide (1.50 g, 3.33 mmol) obtained in step 1 was dissolved in acetonitrile (15 mL). (Z) -non-2-en-1-yl 8-bromooctanato (1.50 g, 4.33 mmol), cesium carbonate (2.17 g, 6.66 mmol), tetrabutylammonium iodide (Reference Example 16) 1.23 g, 3.33 mmol) was added, and the mixture was stirred for 2 hours with heating under reflux. After cooling to room temperature, saturated aqueous ammonium chloride solution was added, and the mixture was extracted with heptane. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (heptane / ethyl acetate = 85/15) to obtain (Z) -non-2-en-1-yl 8-((2-nitro-N-((9Z , 12Z) -octadeca-9,12-dien-1-yl) phenyl) sulfonamido) octanoate crude product was obtained.
The obtained crude product was dissolved in acetonitrile (20 mL), and 1,8-diazabicyclo [5.4.0] -7-undecene (1.12 mL, 7.50 mmol) and 1-dodecanethiol (1.79 mL, 7.50 mmol) were added. In addition, the mixture was stirred at 60 degrees for 3 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol = 80/20) to give (Z) -nona-2-en-1-yl 8-(((9Z, 12Z) -octadeca-9,12- Dien-1-yl) amino) octanoate (1.59 g, yield 90%) was obtained.
ESI-MS m / z: 533 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.79-0.92 (m, 6H), 1.20-1.66 (m, 37H), 2.01-2.14 (m, 6H ), 2.30 (t, J = 7.4 Hz, 2H), 2.55-2.61 (m, 4H), 2.77 (dd, J = 6.4, 6.8 Hz, 2H), 4.62 (d, J = 6.8 Hz, 2H), 5.28 -5.43 (m, 4H), 5.46-5.56 (m, 1H), 5.59-5.69 (m, 1H).
Process 3
In the same manner as in step 2 of Example 1, di ((Z) -non-2-en-1-yl) 10,10′-azanediyl dideanoate was obtained in step 2 (Z ) -Non-2-en-1-yl 8-(((9Z, 12Z) -octadeca-9,12-dien-1-yl) amino) octanoate (350 mg, 0.658 mmol) (252 mg, 65% yield) was obtained.
ESI-MS m / z: 591 (M + H) +; ¹ H-NMR (CDCl ₃ ) δ: 0.83-0.94 (m, 6H), 1.19-1.69 (m, 39H), 2.00-2.14 (m, 6H ), 2.30 (t, J = 7.6 Hz, 2H), 2.34-2.44 (m, 4H), 2.63 (t, J = 5.4 Hz, 2H), 2.73-2.80 (m, 2H), 3.79 (t, J = 5.2 Hz, 2H), 4.62 (d, J = 6.8 Hz, 2H), 5.27-5.42 (m, 4H), 5.47-5.57 (m, 1H), 5.59-5.68 (m, 1H).

Reference Example 17: (Z) -Undec-2-en-1-yl 6-bromohexanoate In the same manner as Reference Example 1, instead of 10-bromodecanoic acid, 6-bromohexanoic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) , 5.11 g, 26.2 mmol), and (Z) -undec-2-en-1-ol (5.35 g, 31.4 mmol) obtained in Step 2 of Reference Example 9 instead of cis-2-nonen-1-ol ) To give (Z) -undec-2-en-1-yl 6-bromohexanoate (8.07 g, yield 89%).

(Z) -Undec-2-en-1-yl 6-((3-hydroxypropyl) ((9Z, 12Z) -octadeca-9,12-dien-1-yl) amino) hexanoate (Compound 22)
Process 1
In the same manner as in Step 2 of Example 21, instead of (Z) -non-2-en-1-yl 8-bromooctanoate, (Z) -undec-2- En-1-yl 6-bromohexanoate (2.50 g, 7.19 mmol) and 2-nitro-N-((9Z, 12Z) -octadeca-9,12-diene obtained in Step 1 of Example 21 1-yl) benzenesulfonamide (2.49 g, 5.53 mmol) was used and (Z) -undec-2-en-1-yl 6-(((9Z, 12Z) -octadec-9,12-diene-1 -Ill) amino) hexanoate (2.05 g, 70% yield) was obtained.
ESI-MS m / z: 533 (M + H) +; ¹ H-NMR (CDCl ₃ ) δ: 0.86-0.92 (m, 6H), 1.21-1.57 (m, 34H), 1.59-1.69 (m, 2H ), 2.01-2.13 (m, 6H), 2.31 (t, J = 7.5 Hz, 2H), 2.55-2.62 (m, 4H), 2.77 (dd, J = 6.4, 6.9 Hz, 2H), 4.61 (d, J = 6.9 Hz, 2H), 5.28-5.42 (m, 4H), 5.48-5.55 (m, 1H), 5.60-5.68 (m, 1H).
Process 2
(Z) -Undec-2-en-1-yl 6-(((9Z, 12Z) -octadeca-9,12-dien-1-yl) amino) hexanoate (350 mg, 0.658 mmol) was dissolved in N, N-dimethylformamide (4 mL) and (3-bromopropoxy) (tert-butyl) dimethylsilane (200 mg, 0.790 mmol), potassium iodide (131 mg, 0.790 mmol), Cesium carbonate (429 mg, 1.32 mmol) was sequentially added, and the mixture was stirred at room temperature overnight. Further, the reaction solution was stirred at 40 ° C. for 9 hours. The reaction mixture was cooled to room temperature, water was added, and the mixture was extracted with heptane. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (heptane / ethyl acetate = 80/20) and ((Z) -undec-2-en-1-yl) 6-((3-((tert-butyldimethyl A crude product of silyl) oxy) propyl) (9Z, 12Z-octadeca-9,12-dien-1-yl) amino) hexanoate was obtained.
The obtained crude product was dissolved in tetrahydrofuran (9 mL), tetrabutylammonium fluoride (1 mol / L tetrahydrofuran solution, 1.10 mL, 1.10 mmol) was added, and the mixture was stirred at room temperature overnight. A saturated aqueous sodium chloride solution was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The obtained residue was purified by amino silica gel column chromatography (heptane / ethyl acetate = 75/25) to obtain Compound 22 (292 mg, yield 75%).
ESI-MS m / z: 591 (M + H) +; ¹ H-NMR (CDCl ₃ ) δ: 0.83-0.92 (m, 6H), 1.20-1.71 (m, 39H), 2.00-2.13 (m, 6H ), 2.32 (t, J = 7.3 Hz, 2H), 2.35-2.43 (m, 4H), 2.63 (t, J = 5.4 Hz, 2H), 2.74-2.80 (m, 2H), 3.79 (t, J = 5.0 Hz, 2H), 4.62 (d, J = 6.8 Hz, 2H), 5.29-5.42 (m, 4H), 5.49-5.56 (m, 1H), 5.59-5.68 (m, 1H).

12,12 '-((3-hydroxypropyl) azanediyl) bis (N- (Z) -non-2-en-1-yl) dodecanamide) (Compound 23)
Process 1
Cis-2-nonen-1-ol (1.17 mL, 7.03 mmol) was dissolved in tetrahydrofuran (10 mL), triphenylphosphine (2.03 g, 7.73 mmol) and diisopropyl azodicarboxylate (Tokyo Chemical Industry Co., Ltd., 40% toluene) Solution, 1.9 mol / L, 4.07 mL, 7.73 mmol) was added at 0 ° C. and stirred at room temperature for 15 minutes. Thereafter, potassium phthalimide (Tokyo Kasei Kogyo Co., Ltd., 1.14 g, 6.14 mmol) was added and allowed to react overnight at room temperature. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (hexane / ethyl acetate = 95/5 to 70/30) to give (Z) -2- (non-2-ene-1 -Ill) isoindoline-1,3-dione (0.524 g, 28% yield) was obtained.
ESI-MS m / z: 272 (M + H) ⁺
Process 2
(Z) -2- (Non-2-en-1-yl) isoindoline-1,3-dione (0.524 g, 1.93 mmol) obtained in step 1 was dissolved in ethanol (5 mL) to give hydrazine 1 Hydrate (manufactured by Tokyo Chemical Industry Co., Ltd., 0.193 g, 3.86 mmol) was added and reacted at 50 ° C. for 1 hour. Water was added to the reaction mixture, and the mixture was extracted with chloroform. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (chloroform / methanol = 99/1 to 85/15) to give (Z) -2-non-2-en-1-amine. (0.117 g, 43% yield) was obtained.
ESI-MS m / z: 141 (M + H) ⁺
Process 3
In the same manner as in Example 5, by using the (Z) -2-non-2-en-1-amine obtained in Step 2 instead of cis-2-nonen-1-ol, compound 23 (0.0450 g, 23% yield) was obtained.
ESI-MS m / z: 718 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 7.0 Hz, 6H), 1.20-1.39 (m, 44H), 1.48-1.56 ( m, 4H), 1.58-1.68 (m, 4H), 1.71-1.78 (M2H), 2.07 (q, J = 7.2 Hz, 4H), 2.16 (t, J = 7.6 Hz, 4H), 2.53 (t, J = 7.1 Hz, 4H), 2.75 (t, J = 5.2 Hz, 2H), 3.80 (t, J = 5.2 Hz, 2H), 3.89 (t, J = 5.7 Hz, 4H), 5.35-5.60 (m, 6H ).

12,12 '-((3-hydroxypropyl) azanediyl) bis (N-nonyldodecanamide) (Compound 24)
In the same manner as in Example 5, compound 24 (0.0200 g, yield 10%) was obtained by using nonylamine (manufactured by Tokyo Chemical Industry Co., Ltd.) instead of cis-2-nonen-1-ol.
ESI-MS m / z: 722 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 7.0 Hz, 6H), 1.21-1.36 (m, 52H), 1.41-1.53 ( m, 8H), 1.57-1.72 (m, 8H), 2.15 (t, J = 7.6 Hz, 4H), 2.40 (t, J = 7.5 Hz, 4H), 2.64 (t, J = 5.6 Hz, 2H), 3.20-3.26 (m, 4H), 5.49 (t, J = 4.9 Hz, 2H).

Using the compound 1 obtained in Example 1, a composition was prepared as follows. The nucleic acid used was a sense strand [5'-rGrCrCrArGrArCrUrUrUrGrUrUrGrGrArUrUrUrGrA-3 '(the sugar attached to the base to which r is attached is ribose)] and an antisense strand [5'-rArAmArUmCrCmArAmCrAmUmUmGrAm Hypoxanthine-guanine phosphoribosyltransferase 1, a sugar that binds to the attached base is ribose and ribose in which the hydroxyl group at the 2 ′ position is substituted with a methoxy group. Anti-HPRT1 siRNA that suppresses gene expression (hereinafter referred to as HPRT1) and was obtained from Gene Design (hereinafter referred to as HPRT1 siRNA). The nucleic acid was prepared to 24 mg / mL with distilled water.
Each sample is weighed so as to be compound 1 / PEG-DMPE Na (manufactured by NOF Corporation) = 57.3 / 5.52 (all numerical units are mmol / L) and suspended in an aqueous solution containing hydrochloric acid and ethanol. The mixture was made turbid and stirred and heated repeatedly with a vortex mixer to obtain a uniform suspension. The suspension was passed through a 0.05 μm polycarbonate membrane filter (manufactured by GE Healthcare Japan) at room temperature to obtain a dispersion of particles of compound 1 / PEG-DMPE Na (liposomes). The average particle size of the liposomes obtained with a particle size measuring device (Zetasizer Nano ZS, manufactured by Malvern) was measured and confirmed to be within the range of 30 nm to 100 nm. The resulting liposome dispersion and the HPRT1 siRNA solution were mixed in a ratio of liposome dispersion: HPRT1 siRNA solution = 3: 1, and further mixed with 29 times the amount of distilled water. -A dispersion of DMPE Na / HPRT1 siRNA complex was prepared.
On the other hand, each sample was weighed so that the compound 1 / PEG-DMPE Na (manufactured by NOF Corporation) / cholesterol (manufactured by NOF Corporation) = 8.947 / 0.147 / 20.336 (all numerical units are mmol / L). And dissolved in ethanol to prepare a solution of lipid membrane constituents.
Add 4-fold amount of ethanol to the resulting lipid membrane component solution, and the lipid membrane component solution and the compound 1 / PEG-DMPE Na / HPRT1 siRNA complex dispersion were in a ratio of 2: 3. The resulting mixture was further mixed with several times the amount of distilled water to obtain a crude preparation.
The obtained crude preparation was concentrated using Amicon Ultra (manufactured by Millipore), diluted with physiological saline, and filtered in a clean bench using a 0.2 μm filter (manufactured by Toyo Roshi Kaisha, Ltd.). The siRNA concentration of the obtained composition was measured, and diluted to an appropriate concentration using physiological saline to obtain Formulation 1 (composition containing Compound 1 and HPRT1 siRNA).

Using compounds 2 to 20 obtained in Examples 2 to 20, preparations 2 to 20% (compositions containing each of compounds 2 to 20 and HPRT1 siRNA) were obtained in the same manner as Example 25. In addition, as a separate lot of Formulation 2, Formulation 2-2 was prepared by the same method.

Using the compound 21 obtained in Example 21, a composition was prepared as follows. The same HPRT1 siRNA as in Example 25 was used as the nucleic acid.
A dispersion of Compound 21 / PEG-DMPE Na particles (liposomes) was obtained in the same manner as in Example 25 except that Compound 21 was changed to Compound 21 in Example 25. The resulting liposome dispersion and the HPRT1 siRNA solution were mixed at a ratio of liposome dispersion: HPRT1 siRNA solution = 3: 1, and further mixed with 59 times the amount of distilled water. -A dispersion of DMPE Na / HPRT1 siRNA complex was prepared.
On the other hand, each sample was weighed and dissolved in ethanol so that the compound 21 / PEG-DMPE Na / cholesterol = 8.947 / 0.147 / 20.336 (all numerical units are mmol / L), and a solution of lipid membrane components Was prepared.
Nine times the amount of ethanol was added to the resulting lipid membrane component solution, and the lipid membrane component solution and the compound 21 / PEG-DMPE Na / HPRT1 siRNA complex dispersion were in a ratio of 2: 3. The mixture was further mixed with several times the amount of distilled water to obtain a crude preparation.
Subsequent operations were carried out in the same manner as in Example 25 to obtain Formulation 21 (a composition containing Compound 21 and HPRT1 siRNA).
The average particle diameter and polydispersity index of the preparations (compositions) obtained in Examples 25 to 27 were measured with a particle diameter measuring apparatus. The results are shown in Table 7.

Comparative Example 1
Preparation A-1 (Compound A and HPRT1 siRNA) was carried out in the same manner as in Example 25 except that Compound A in Example 25 was changed to Compound A using Compound A synthesized by the method described in International Publication No. 2014/007398. Containing the composition). The structure of Compound A is shown in Table 8 below.

Comparative Example 2
A composition was prepared using Compound A as follows. The same nucleic acid as in Example 25 was used as the nucleic acid.
A dispersion of Compound A / PEG-DMPE Na particles (liposomes) was obtained in the same manner as in Example 25 except that Compound 1 in Example 25 was changed to Compound A.
The resulting liposome dispersion and HPRT1 siRNA solution are mixed in a ratio of liposome dispersion: HPRT1 siRNA solution = 3: 1, and further mixed with 5 times the amount of distilled water. -A dispersion of DMPE Na / HPRT1 siRNA complex was prepared.
On the other hand, each sample was weighed so that compound A / PEG-DMPE Na / DSPC (manufactured by NOF Corporation) / cholesterol = 8.947 / 0.147 / 5.981 / 14.355 (all numerical units are mmol / L) and ethanol. To prepare a solution of lipid membrane constituents.
The resulting lipid membrane component solution and the dispersion of Compound A / PEG-DMPE Na / HPRT1 siRNA complex are mixed in a ratio of 2: 3, and then mixed with several times the amount of distilled water. Thus, a crude preparation was obtained.
Subsequent operations were performed in the same manner as in Example 25 to obtain Formulation A-2 (a composition containing Compound A and HPRT1 siRNA).
The average particle size of the preparations (compositions) obtained in Comparative Examples 1 and 2 was measured with a particle size measuring apparatus. The results are shown in Table 9.

Using the compound 2 obtained in Example 2, a composition was prepared as follows. The nucleic acid used consists of a sense strand (5'-CCGUCGUAUUCGUGAGCAAGA-3 ') and an antisense strand (5'-UUGCUCACGAAUACGACGGUG-3'), an anti-luciferase (hereinafter referred to as Luc) gene that suppresses the expression of the luciferase gene. Luc siRNA was obtained from Gene Design (hereinafter referred to as Luc-1 siRNA). The nucleic acid was prepared to 24 mg / mL with distilled water.
Each sample is weighed so as to be compound 2 / PEG-DSPE Na (manufactured by NOF Corporation) = 57.3 / 5.52 (all numerical units are mmol / L) and suspended in an aqueous solution containing hydrochloric acid and ethanol. The mixture was repeatedly stirred and heated with a vortex mixer to obtain a uniform suspension. This suspension was passed through a 0.05 μm polycarbonate membrane filter at room temperature to obtain a dispersion of Compound 2 / PEG-DSPE Na particles (liposomes). The average particle size of the liposomes obtained with a particle size measuring device was measured and confirmed to be within the range of 30 nm to 100 nm. The resulting liposome dispersion and the Luc-1 siRNA solution are mixed in a ratio of liposome dispersion: Luc-1 siRNA solution = 3: 1, and 17.7 times the amount of distilled water is added and mixed. A dispersion of Compound 2 / PEG-DSPE Na / Luc-1 siRNA complex was prepared.
On the other hand, each sample was weighed and dissolved in ethanol so that the compound 2 / PEG-DSPE Na / DSPC / cholesterol = 8.947 / 0.294 / 2.472 / 17.717 (all numerical units are mmol / L), and lipid membrane A solution of the components was prepared.
An equal amount of ethanol is added to the resulting lipid membrane component solution, and the lipid membrane component and the dispersion of the compound 2 / PEG-DSPE Na / Luc-1 siRNA complex are in a ratio of 3: 7. And then mixed with several times the amount of distilled water to obtain a crude preparation.
The obtained crude preparation was concentrated using Amicon Ultra, diluted with physiological saline, and filtered in a clean bench using a 0.2 μm filter. The siRNA concentration of the obtained composition was measured, and diluted with physiological saline according to the administration concentration to obtain Formulation 2-3 (a composition containing Compound 2 and Luc-1 siRNA).

Using the compound 2 obtained in Example 2, a composition was prepared as follows. The same Luc-1 siRNA as in Example 28 was used as the nucleic acid.
In the same manner as in Example 28, a dispersion of Compound 2 / PEG-DSPE Na / Luc-1 siRNA complex was prepared. On the other hand, each sample was weighed and dissolved in ethanol so that the compound 2 / PEG-DSPE Na / cholesterol = 8.947 / 0.294 / 20.189 (all numerical units are mmol / L), and the solution of lipid membrane constituents Was prepared.
The subsequent operations were carried out in the same manner as in Example 28 to obtain Formulation 2-4 (a composition containing Compound 2 and Luc-1 siRNA).
The average particle size of the preparations (compositions) obtained in Examples 28 and 29 was measured with a particle size measuring device. The results are shown in Table 10.

Comparative Example 3
Using compound A, preparation A-3 (composition containing compound A and Luc-1 siRNA) was obtained in the same manner as in Example 28.

Comparative Example 4
Using compound A, preparation A-4 (composition containing compound A and Luc-1 siRNA) was obtained in the same manner as in Example 29.
The average particle size of the preparations (compositions) obtained in Comparative Examples 3 and 4 was measured with a particle size measuring device. The results are shown in Table 11.

Using the compound 2 obtained in Example 2, a composition was prepared as follows. The nucleic acid used was a sense strand [5'-mCmUmUrAmCrGmCmUrGrArGmUrAmCmUmUmCrGrAdTdT-3 ' Deoxyribose, the bond between deoxyribose that binds to the 20th base and deoxyribose that binds to the 21st base from the 5 'end toward the 3' end is a phosphorothioate bond)] and antisense Chain [5'-rUrCrGrArArGmUrArCrUmCrArGrCrGmUrArArGdTdT-3 ' From the 'terminal side to the 3' terminal side, the deoxyribose binding to the 20th base and the deoxyribose binding to the 21st base is a phosphorothioate bond)] A suppressing anti Luc siRNA expression child, it was obtained from Gene Design Inc. (hereinafter referred to as Luc-2 siRNA). The nucleic acid was prepared to 24 mg / mL with distilled water.
Compound 2-5 (compound 2 and containing Luc-2 siRNA) was prepared in the same manner as in Example 25 except that Compound 1 in Example 25 was changed to Compound 2 and the nucleic acid HPRT1 siRNA used in Example 25 was changed to Luc-2 siRNA. Composition) was obtained.

Using the compound 2 obtained in Example 2, a composition was prepared as follows. The nucleic acid used is a sense strand [5'-mGmCrArArArGrArUrArArCrArArArCrUrCrCrArCrGrUrGmGmA-3 '(the sugars bound to the bases to which r and m are attached are ribose and ribose in which the hydroxyl group at the 2' position is substituted with a methoxy group, respectively) And an antisense strand [5'-rUrCrCrArCrGrUrGrGrArGrUrUrUrGrUrUrArUrCrUrUrUrGrC-3 '(the sugar attached to the base to which r is attached is ribose)], a transforming growth factor-beta1 (hereinafter referred to as Tgfb-1) gene It was an anti-Tgfb-1 siRNA that suppresses the expression of and was obtained from Gene Design (hereinafter referred to as Tgfb-1 siRNA). The nucleic acid was prepared to 24 mg / mL with distilled water.
A preparation 2-6 (composition containing Compound 2 and Tgfb-1 siRNA) was obtained in the same manner as in Example 30, except that the nucleic acid Luc-2 siRNA used in Example 30 was changed to Tgfb-1 siRNA.
The average particle size of the preparations (compositions) obtained in Examples 30 and 31 was measured with a particle size measuring device. The results are shown in Table 12.

Comparative Example 5
Compound A-5 (composition containing Compound A and Luc-2 siRNA) was prepared in the same manner as in Comparative Example 2, except that the nucleic acid HPRT1 siRNA used in Comparative Example 2 was changed to Luc-2 siRNA. Obtained.

Comparative Example 6
A preparation A-6 (composition containing Compound A and Tgfb-1 siRNA) was prepared in the same manner as in Comparative Example 2, except that Compound A was used and the nucleic acid HPRT1 siRNA used in Comparative Example 2 was changed to Tgfb-1 siRNA. Obtained.
The average particle size of the preparations (compositions) obtained in Comparative Examples 5 and 6 was measured with a particle size measuring device. The results are shown in Table 13.

Test Example 1: In vitro activity evaluation test of the preparation in human lung fibroblast cell line Preparations 1-13 and 15-21 obtained in Examples 25-27 and Comparative Examples 1-2, activities of A-1 and A-2 Was evaluated by the method described below.
Human lung fibroblast cell line Nomal Human Lung Fibroblasts (Lonza CC-2512), DMEM medium (Thermo Fischer) containing 15% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Thermo Fischer) ) Was seeded at 4000 cells / 100 μL / well and cultured at 37 ° C. under 5% CO ₂ for 22-24 hours. Thereafter, the various preparations prepared in Examples 25 to 27 were added as siRNA concentrations, and the concentrations were adjusted so that the concentration became 5 nM or 25 nM, and 100 μL was added to the cells. As a negative control, 100 μL of DMEM medium containing 15% FBS and 1% penicillin-streptomycin was added to the cells.
Cells treated with various preparations are cultured for 24 hours in a 5% CO ₂ incubator at 37 ° C, washed with ice-cold PBS, and attached using TaqMan Fast Cells-to-CT kit (manufactured by Thermo Fisher, 4939003) Total RNA was recovered according to the instructions for use to prepare cDNA.
Using the obtained cDNA as a template for PCR reaction, Applied Biosystems QuantStudio 12K Flex (Applied Biosystems QuantStudio 12K Flex), TaqMan Fast Universal PCR Master Mix (2X) (Applied Biosystems, 4352042) and TaqMan probe ( TaqMan (registered trademark) Gene Expression Assays, HPRT1: Hs02800695_m1, PPIA: Hs04194521_s1) were used to perform PCR amplification specific for the HPRT1 gene and the constitutively expressed gene PPIA (peptidylprolyl isomerase A) gene, respectively, and quantify the amount of mRNA. It was. The conditions for the PCR reaction were in accordance with the instruction manual attached to TaqMan Fast Universal PCR Master Mix (2X). The amount of mRNA in the specimen was calculated as a relative ratio when the amount of HPRT1 mRNA relative to the amount of PPIA mRNA was calculated, and the value in the negative control treatment group was 1. The results for the amount of mRNA of HPRT1 are shown in Table 14.

Test Example 2: In vivo irritation evaluation test of preparation by intratracheal administration For each preparation 2, 2-2, 3, 4, 6, 12, 13, and A-2 obtained in Example 26 and Comparative Example 2 The in vivo irritation evaluation test was carried out by the following methods. Each preparation was diluted with physiological saline in accordance with the test.
C57BL / 6J mice (9 weeks old, female, purchased from Charles River, Japan) under isoflurane anesthesia, siRNA weight of 15 μg / mouse each or 20 μg / mouse microspray (Pen-Century, IA -IC-M, FMJ-250). Euthanasia was performed 24 hours after administration, 0.75 mL of PBS was injected into the trachea, and collection was repeated twice to collect alveolar lavage fluid (BALF). The obtained recovered liquid was centrifuged under the conditions of 2000 rpm, 3 min, 4 ° C., and the supernatant was recovered and used for the following analysis.
Using the MILLIPLEX MAP Human Cytokine / Chemokine Magnetic Bead Panel (Merck Millipore) and Bio-Plex 200 system (BioRad), the values of KC, IL-6, and G-CSF in the obtained BALF Quantification was performed according to the method described in the instructions attached to the product.
The concentrations of KC, IL-6, and G-CSF in BALF measured in each test are shown in FIGS.

Test Example 3: In vivo irritation evaluation test of the preparation by intravenous administration Each preparation 2-3, 2-4, A-3 and A obtained in Example 28, Example 29, Comparative Example 3 and Comparative Example 4 -4 was subjected to an in vivo stimulation evaluation test by the following method. Each preparation was diluted with physiological saline in accordance with the test.
Each preparation was intravenously administered to Balb / cA Jcl mice (7 weeks old, male, purchased from CLEA Japan, Inc.) at a siRNA weight of 10 mg / kg. Blood was collected 24 hours after administration into Microtina (BD, 365967), centrifuged at 15000 rpm for 2 minutes at 4 ° C using a small cooling centrifuge (MX-201: TOMY), and serum was collected. Collected.
The values of KC, IL-6, and G-CSF in the obtained serum were measured using the BD CBA Flex Sets (BD CBA Flex Sets), 560152 (G-CSF), 558340 (KC ), 558301 (IL-6), 558267 (buffers)) and BD Fax Bath (BD FACS Vers) using a flow cytometer (BD), according to the method described in the instructions attached to the product. Quantified.
The measured serum KC, IL-6, and G-CSF concentrations are shown in FIGS. 10, 11, and 12, respectively.

Test Example 4: In vivo activity evaluation test of the preparation by intratracheal administration The preparations 2-5, 2-6, A-5 and A- obtained in Example 30, Example 31, Comparative Example 5 and Comparative Example 6 Each of 6 was subjected to in vivo activity evaluation test by the following method. Each preparation was diluted with physiological saline in accordance with the test.
C57BL / 6J mice (9 weeks old, female, purchased from Charles River, Japan) were administered a single dose of bleomycin (manufactured by Nippon Kayaku Co., Ltd.) into the trachea under the condition of 0.012 mg / mouse under isoflurane anesthesia. At 7, 10 and 13 days after bleomycin administration, 1 μg / mouse of each formulation was administered intratracheally using a microsprayer. The day after the last dose, BALF, BALF cells and left lung were collected from euthanized mice.
BALF was collected in the same manner as in Test Example 3.
Collection of the left lung and measurement of mRNA were performed by the following method. Buffer RLT (Qiagen, 25 mL) added with 1 mol / L dithiothreitol solution (1 mL) was used as a homogenization buffer. After collecting the left lung, it was immediately homogenized in the above-mentioned homogenization buffer using Tissue lyser (Qiagen). MRNA was recovered from each homogenate according to the attached protocol of Maxwell RSC simplyRNA Tissue Kit (Promega), and cDNA was prepared according to the attached protocol of SuperScript VILO cDNA Synthesis Kit (Thermo Fisher). Maxwell RSC (Promega) was used for mRNA preparation, and GeneAmp PCR system 9700 (Applied Biosynthesis) was used for cDNA preparation. Thereafter, Tgfb-1 mRNA was quantified in the same manner as in Test Example 1. HPRT1, which is a constitutive expression gene, was used as a correction gene.
Cells in BALF were collected by centrifuging BALF collected in a 1.5 mL Eppendorf tube at 2000 rpm at 4 ° C. for 2 minutes to collect precipitated cells. Measurement of mRNA of the recovered BALF cells was performed by the following method. Buffer RLT (Qiagen, 25 mL) added with 1 mol / L dithiothreitol solution (1 mL) was used as a homogenization buffer. After collecting the cells in BALF, the cells were immediately homogenized with a homogenization buffer. Then, mRNA was recovered from each homogenate according to the protocol attached to Maxwell RSC simplyRNA Cells Kit (Promega), and cDNA was prepared according to the protocol attached to SuperScript VILO cDNA Synthesis Kit (Thermo Fisher). Maxwell RSC (Promega) was used for mRNA preparation, and GeneAmp PCR system 9700 (Applied Biosynthesis) was used for cDNA preparation. Thereafter, Tgfb-1 mRNA was quantified in the same manner as in Test Example 1. HPRT1, which is a constitutive expression gene, was used as a correction gene.
The Tgfb-1 protein in BALF was measured using Mouse TGF-beta 1 DuoSet (R & D Systems).
FIG. 13 shows the quantification result of left lung mRNA, FIG. 14 shows the quantification result of cellular mRNA in BALF, and FIG. 15 shows the Tgfb-1 protein concentration in BALF.

From Test Example 1, it was clarified that the preparation containing the cationic lipid of the present invention can introduce nucleic acid into cells in vitro.
From Test Example 2 (FIGS. 1 to 9), it was clarified that the in vivo irritancy can be reduced more than the preparation containing Compound A by intratracheally administering the preparation containing the cationic lipid of the present invention to normal mice.
From Test Example 3 (FIGS. 10 to 12), it was clarified that the in vivo irritancy can be reduced more than the preparation containing Compound A by intravenously administering the preparation containing the cationic lipid of the present invention to normal mice.
From Test Example 4 (FIGS. 13 to 15), by intratracheally administering a formulation containing the cationic lipid of the present invention to a bleomycin model mouse, it facilitates delivery of nucleic acids to cells in the lung and BALF in vivo. It was clarified that nucleic acid can be introduced into cells.

By administering the composition containing the cationic lipid and nucleic acid of the present invention to a mammal or the like, the nucleic acid can be easily introduced into a cell, for example.

SEQ ID NO: 1: Hypoxanthine-guanine phosphoribosyltransferase (HPRT1) siRNA sense strand SEQ ID NO: 2: Hypoxanthine-guanine phosphoribosyltransferase (HPRT1) siRNA antisense strand SEQ ID NO: 3: Luciferase (Luc-1) siRNA Sense strand SEQ ID NO: 4: Luciferase (Luc-1) siRNA antisense strand SEQ ID NO: 5: Luciferase (Luc-2) siRNA sense strand SEQ ID NO: 6: Luciferase (Luc-2) siRNA antisense strand SEQ ID NO: 7: Transformation growth Factor-beta1 (Tgfb-1) siRNA sense strand SEQ ID NO: 8: Transforming growth factor-beta1 (Tgfb-1) siRNA antisense strand

Claims

A compound represented by the following formula (I), or a pharmaceutically acceptable salt thereof.

(Where
R1 is a hydrogen atom or hydroxymethyl,
n1 is 1 or 2,
A1 and A2 are the same or different and are linear C9-C20 alkylene or C9-C20 alkenylene,
M1 and M2 are the same or different and are —OC (O) —, —C (O) O— or —NHC (O) —,
B1 and B2 are the same or different and are linear or branched C1-C12 alkyl or C2-C13 alkenyl,
R2 is absent or is C1-C3 alkyl;
If R2 does not exist, Y does not exist,
When R2 is C1-C3 alkyl, Y is a pharmaceutically acceptable anion. )
2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein B1 and B2 are the same or different and are linear C1-C12 alkyl or C2-C13 alkenyl.
The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein A1 and A2 are the same or different and are linear C9-C12 alkylene.
The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein B1-M1-A1 and B2-M2-A2 are the same.
The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein R1 is a hydrogen atom and n1 is 2.
The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein R1 is hydroxymethyl and n1 is 1.
A compound represented by the following formula (II) or a pharmaceutically acceptable salt thereof.

(Where
n2 is 1 or 2,
L is a linear C12-C24 alkenyl,
A3 is a linear C5-C14 alkylene,
M3 is -OC (O)-, -C (O) O- or -NHC (O)-
B3 is linear or branched C1-C12 alkyl or C2-C13 alkenyl. )
A pharmaceutical composition comprising the compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, and a nucleic acid.
9. The pharmaceutical composition according to claim 8, wherein the nucleic acid is a nucleic acid having an action of suppressing the expression of a target gene using RNA interference (RNAi).
10. The pharmaceutical composition according to claim 9, wherein the target gene is a gene expressed in the liver, lung, or spleen.
A therapeutic or prophylactic agent for a disease associated with the liver, lung or spleen, comprising the pharmaceutical composition according to claim 10.
A method for treating or preventing a disease associated with the liver, lung or spleen, comprising a step of administering the pharmaceutical composition according to any one of claims 8 to 10 to a subject.
A therapeutic or prophylactic agent for diseases related to the lung, comprising the pharmaceutical composition according to any one of claims 8 to 10.
A method for treating or preventing a lung-related disease, comprising a step of administering the pharmaceutical composition according to any one of claims 8 to 10 to a subject.
An antitumor agent comprising the pharmaceutical composition according to any one of claims 8 to 10.
A method for treating or preventing a malignant tumor, comprising a step of administering the pharmaceutical composition according to any one of claims 8 to 10 to a subject.