JP6495408B2 - Cationic lipid - Google Patents

Description

  The present invention relates to, for example, a cationic lipid which facilitates introduction of a nucleic acid into a cell or the like, a composition containing the cationic lipid, and the like.

Cationic lipids are amphiphilic molecules having a lipophilic region comprising one or more hydrocarbon groups and a hydrophilic region comprising at least one positively charged polar head group. Cationic lipids and macromolecules such as nucleic acids form complexes which are positively charged as a total charge, and thus macromolecules such as nucleic acids can easily pass through the plasma membrane of cells and enter the cytoplasm, and thus they are cationic. Lipids are useful. This process, which can be performed in vitro and in vivo, is known as transfection.
Patent documents 1 and 2 contain cationic lipids and said lipids which are advantageous for delivering nucleic acids into cells in vivo and for use in nucleic acid-lipid particle compositions suitable for the treatment of diseases. Disclosed are lipid particles. In Patent Document 1, for example,

2,2-ジリノレイル-4-(2-ジメチルアミノエチル)-[1,3]-ジオキソラン(2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane;
DLin-KC2-DMA)等、特許文献2には、例えば、

2,2-Dilinoleyl-4- (2-dimethylaminoethyl)-[1,3] -dioxolane (2,2-dilinoleyl-4- (2-dimethylaminoethyl)-[1,3] -dioxolane;
DLin-KC2-DMA) and the like, Patent Document 2 includes, for example,

(6Z,9Z,28Z,31Z)-ヘプタトリアコンタ-6,9,28,31-テトラエン-19-イル 4-(ジメチルアミノ)ブタノアート((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate;DLin-MC3-DMA)等のカチオン性脂質が開示されている。

(6Z, 9Z, 28Z, 31Z) -Heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butanoate ((6Z, 9Z, 28Z, 31Z) -heptatriaconta-6,9 , 28, 31-tetraen-19-yl 4- (dimethylamino) butyrate; DLin-MC 3- DMA) and the like are disclosed.

WO 2010/042877 pamphlet WO 2010/054401 pamphlet

  An object of the present invention is to provide a cationic lipid which facilitates introduction of a nucleic acid into, for example, a cell, a composition containing the cationic lipid, and the like.

The present invention relates to the following (1) to (22).
(1) Formula (A)

(式中、R¹は炭素数8〜24の直鎖状または分枝状のアルキル、アルケニルもしくはアルキニルであり、
R²は炭素数8〜24の直鎖状または分枝状のアルキル、アルケニルもしくはアルキニル、アルコキシエチル、アルコキシプロピル、アルケニルオキシエチル、アルケニルオキシプロピル、アルキニルオキシエチルまたはアルキニルオキシプロピルであり、
R³およびR⁴は、同一または異なって炭素数1〜3のアルキルであるか、または一緒になって炭素数2〜8のアルキレンを形成するか、またはR³はR⁵と一緒になって炭素数2〜8のアルキレンを形成し、
R⁵は、水素原子、炭素数1〜6のアルキル、炭素数3〜6のアルケニル、アミノ、モノアルキルアミノ、ヒドロキシ、アルコキシ、カルバモイル、モノアルキルカルバモイル、ジアルキルカルバモイルまたは同一もしくは異なって1〜3つのアミノ、モノアルキルアミノ、ヒドロキシ、アルコキシ、カルバモイル、モノアルキルカルバモイルもしくはジアルキルカルバモイルで置換された炭素数1〜6のアルキルもしくは炭素数3〜6のアルケニルであるか、またはR³と一緒になって炭素数2〜8のアルキレンを形成し、
X¹は、炭素数1〜6のアルキレンであり、
X²は、単結合であるか、または炭素数1〜6のアルキレンであり、ただし、X¹とX²の炭素数の和は7以下であり、R⁵が、水素原子の場合、X²は単結合であり、R⁵がR³と一緒になって炭素数2〜6のアルキレンを形成する場合、X²は単結合であるか、またはメチレンもしくはエチレンである)、
式(B)

(Wherein, R ¹ represents a linear or branched alkyl, alkenyl or alkynyl having 8 to 24 carbon atoms,
R ² is linear or branched alkyl, alkenyl or alkynyl having 8 to 24 carbon atoms, alkoxyethyl, alkoxypropyl, alkenyloxyethyl, alkenyloxypropyl, alkynyloxyethyl or alkynyloxypropyl,
R ³ and R ⁴ are the same or different and are alkyl having 1 to 3 carbon atoms, or are taken together to form alkylene having 2 to 8 carbon atoms, or R ³ is taken together with R ⁵ Form an alkylene having 2 to 8 carbon atoms,
R ⁵ is a hydrogen atom, alkyl having 1 to 6 carbon atoms, alkenyl having 3 to 6 carbon atoms, amino, monoalkylamino, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl or the same or different 1 to 3 C 1 -C 6 alkyl or C ³ -C 6 alkenyl substituted with amino, monoalkylamino, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl or dialkylcarbamoyl, or carbon together with R ³ Form an alkylene of 2-8,
X ¹ is alkylene having 1 to 6 carbon atoms,
X ² is a single bond or alkylene having 1 to 6 carbon atoms, provided that the sum of the carbon number of X ¹ and X ² is 7 or less, and when R ⁵ is a hydrogen atom, X ² Is a single bond, and when R ⁵ is taken together with R ³ to form an alkylene having 2 to 6 carbon atoms, X ² is a single bond or methylene or ethylene),
Formula (B)

(式中、R⁶は炭素数8〜24の直鎖状または分枝状のアルキル、アルケニルもしくはアルキニルであり、
R⁷は炭素数8〜24の直鎖状または分枝状のアルキル、アルケニルもしくはアルキニル、アルコキシエチル、アルコキシプロピル、アルケニルオキシエチル、アルケニルオキシプロピル、アルキニルオキシエチルまたはアルキニルオキシプロピルである)、または
式(C)

(Wherein, R ⁶ represents a linear or branched alkyl, alkenyl or alkynyl having 8 to 24 carbon atoms,
R ⁷ is linear or branched alkyl, alkenyl or alkynyl having 8 to 24 carbon atoms, alkoxyethyl, alkoxypropyl, alkenyloxyethyl, alkenyloxypropyl, alkynyloxyethyl or alkynyloxypropyl), or (C)

(式中、R⁸は炭素数8〜24の直鎖状または分枝状のアルキル、アルケニルもしくはアルキニルであり、
R⁹は炭素数8〜24の直鎖状または分枝状のアルキル、アルケニルもしくはアルキニル、アルコキシエチル、アルコキシプロピル、アルケニルオキシエチル、アルケニルオキシプロピル、アルキニルオキシエチルまたはアルキニルオキシプロピルであり、
X³は、炭素数1〜3のアルキレンであり、
R¹⁰は、水素原子または炭素数1〜3のアルキルである)で表されるカチオン性脂質。
(2) R¹、R²、R⁶、R⁷、R⁸およびR⁹が、それぞれテトラデシル、ヘキサデシル、(Z)-テトラデカ-9-エニル、(Z)-ヘキサデカ-9-エニル、(Z)-オクタデカ-6-エニル、(Z)-オクタデカ-9-エニル、(E)-オクタデカ-9-エニル、(Z)-オクタデカ-11-エニル、(9Z,12Z)-オクタデカ-9,12-ジエニル、(9Z,12Z,15Z)-オクタデカ-9,12,15-トリエニル、(Z)-イコサ-11-エニル、(11Z,14Z)-イコサ-11,14-ジエニルまたは(Z)-ドコサ-13-エニルである前記(1)記載のカチオン性脂質。
(3) R¹、R²、R⁶、R⁷、R⁸およびR⁹が、それぞれ(Z)-オクタデカ-9-エニル、(9Z,12Z)-オクタデカ-9,12-ジエニルまたは(11Z,14Z)-イコサ-11,14-ジエニルである前記(1)記載のカチオン性脂質。
(4) X¹が、炭素数1〜3のアルキレンであり、X²が、単結合またはメチレンである前記(1)〜(3)のいずれかに記載のカチオン性脂質。
(5) X³が、メチレンまたはエチレンである前記(1)〜(4)のいずれかに記載のカチオン性脂質。
(6) R³およびR⁴が、同一もしくは異なってメチルもしくはエチル、または一緒になってn-ペンチレンまたはn-ヘキシレンを形成する前記(1)〜(5)のいずれかに記載のカチオン性脂質。
(7) R³およびR⁵が、一緒になってn-プロピレンまたはn-ブチレンを形成し、R⁴が、メチルまたはエチルである前記(1)〜(5)のいずれかに記載のカチオン性脂質。
(8) R5およびR10が、それぞれ水素原子またはメチルである前記(1)〜(7)のいずれかに記載のカチオン性脂質。
(9) 前記(1)〜(8)のいずれかに記載のカチオン性脂質および核酸を含有する組成物。
(10) 該カチオン性脂質と該核酸とが複合体を形成しているか、または該カチオン性脂質に中性脂質および/もしくは高分子を組み合わせたものと該核酸とが複合体を形成している、前記(9)記載の組成物。
(11) 該カチオン性脂質と該核酸とが複合体を形成しているか、または該カチオン性脂質に中性脂質および/もしくは高分子を組み合わせたものと該核酸とが複合体を形成しており、該複合体を封入する脂質膜を含有する前記(9)記載の組成物。
(12) 核酸がRNA干渉(RNAi)を利用した標的遺伝子の発現抑制作用を有する核酸である前記(9)〜(11)のいずれかに記載の組成物。
(13) 標的遺伝子が、肝臓、肺、腎臓または脾臓において発現する遺伝子である前記(12)記載の組成物。
(14) 前記(9)〜(13)のいずれかに記載の組成物を用いて該核酸を細胞内に導入する方法。
(15) 細胞が、ほ乳類の肝臓、肺、腎臓または脾臓にある細胞である前記(14)記載の方法。
(16) 細胞内に導入する方法が、該組成物の静脈内投与によって細胞内に導入する方法である前記(14)または(15)に記載の方法。
(17) 前記(13)に記載の組成物を哺乳動物に投与するする工程を含む、肝臓、肺、腎臓または脾臓に関連する疾患の治療方法。
(18) 投与する方法が、静脈内投与である前記(17)記載の方法。
(19) 前記(12)に記載の組成物を含む、疾患の治療に用いるための医薬。
(20) 静脈内投与用である前記(19)記載の医薬。
(21) 前記(13)に記載の組成物を含む、肝臓、肺、腎臓または脾臓に関連する疾患の治療剤。
(22) 静脈内投与用である前記(21)記載の肝臓、肺、腎臓または脾臓に関連する疾患の治療剤。

(Wherein, R ⁸ is a linear or branched alkyl, alkenyl or alkynyl having 8 to 24 carbon atoms,
R ⁹ is linear or branched alkyl, alkenyl or alkynyl having 8 to 24 carbon atoms, alkoxyethyl, alkoxypropyl, alkenyloxyethyl, alkenyloxypropyl, alkynyloxyethyl or alkynyloxypropyl,
X ³ is alkylene having 1 to 3 carbon atoms,
R < ¹⁰ > is a hydrogen atom or a C1-C3 alkyl represented cationic lipid.
(2) R ¹ , R ² , R ⁶ , R ⁷ , R ⁸ and R ⁹ each represent tetradecyl, hexadecyl, (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) -octadeca-9, 12-dienyl (9Z, 12Z, 15Z) -octadeca-9, 12, 15-trienyl, (Z) -icosa-11-enyl, (11Z, 14Z) -icosa-11, 14-dienyl or (Z) -docosa-13 The cationic lipid according to the above (1), which is -enyl.
(3) R ¹ , R ² , R ⁶ , R ⁷ , R ⁸ and R ⁹ each represent (Z) -octadec-9-enyl, (9Z, 12Z) -octadeca-9, 12-dienyl or (11Z, 14 Z) The cationic lipid according to the above (1), which is Icosa-11, 14-dienyl.
(4) The cationic lipid according to any one of the above (1) to (3), wherein X ¹ is alkylene having 1 to 3 carbon atoms, and X ² is a single bond or methylene.
(5) The cationic lipid according to any one of the above (1) to (4), wherein X ³ is methylene or ethylene.
(6) The cationic lipid according to any one of the above (1) to (5), wherein R ³ and R ⁴ are the same or different and are methyl or ethyl, or together form n-pentylene or n-hexylene .
(7) The cationic group according to any one of the above (1) to (5), wherein R ³ and R ⁵ together form n-propylene or n-butylene and R ⁴ is methyl or ethyl. Lipid.
(8) The cationic lipid according to any one of the above (1) to (7), wherein R5 and R10 are each a hydrogen atom or methyl.
(9) A composition comprising the cationic lipid and the nucleic acid according to any one of the above (1) to (8).
(10) The cationic lipid and the nucleic acid form a complex, or a combination of the cationic lipid and a neutral lipid and / or a polymer and the nucleic acid forms a complex , The composition as described in said (9).
(11) The cationic lipid and the nucleic acid form a complex, or a combination of the cationic lipid and a neutral lipid and / or a polymer and the nucleic acid forms a complex The composition according to the above (9), which comprises a lipid membrane encapsulating the complex.
(12) The composition according to any one of the above (9) to (11), wherein the nucleic acid is a nucleic acid having an action of suppressing the expression of a target gene using RNA interference (RNAi).
(13) The composition according to (12) above, wherein the target gene is a gene expressed in liver, lung, kidney or spleen.
(14) A method for introducing the nucleic acid into cells using the composition according to any one of (9) to (13).
(15) The method according to (14) above, wherein the cells are cells in mammalian liver, lung, kidney or spleen.
(16) The method according to (14) or (15) above, wherein the method of introducing into cells is a method of introducing into cells by intravenous administration of the composition.
(17) A method for treating a disease associated with liver, lung, kidney or spleen, which comprises the step of administering the composition according to (13) to a mammal.
(18) The method according to (17) above, wherein the method of administration is intravenous administration.
(19) A medicament for use in the treatment of a disease, comprising the composition according to (12).
(20) The medicament according to (19), which is for intravenous administration.
(21) A therapeutic agent for a disease associated with liver, lung, kidney or spleen, which comprises the composition according to (13).
(22) The agent for treating a disease associated with liver, lung, kidney or spleen according to the above (21), which is for intravenous administration.

  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, for example, into cells or the like.

The preparation obtained in Example 49 (preparation using each of Compound A-1 and 3 to 5) and the preparation obtained in Comparative Example 1 (DLin-KC2-DMA and each of Compounds XI-1 to 3) are used. Fig. 7 shows the expression rate of target gene mRNA after introduction of the preparation (into the human hepatoma-derived cell line HepG2 cells). The ordinate represents the expression rate of the target gene mRNA when the negative control was 1. The abscissa represents the nucleic acid concentration (nM) and the compound number of the cationic lipid used. Regarding the preparation obtained in Example 49 (preparation using each of the compounds A-1 to 5) and the preparation obtained in Comparative Example 1 (preparation using DLin-KC2-MDA), siRNA 0.3 mg for each of the mice It shows the value of serum cholesterol 48 hours after administration of an amount equivalent to 1 kg / kg. The ordinate represents the relative value of serum cholesterol when the physiological saline administration group is 100. Preparation obtained using Example 49 (preparation using each of compound A-1 to 5) and preparation obtained according to comparative example 1 (DLin-KC2-MDA and preparation using each of compound XI-1 to 3) ) Shows the level of cholesterol in serum 48 hours after administration of siRNA 3 mg / kg equivalent to each mouse. The ordinate represents the relative value of serum cholesterol when the physiological saline administration group is 100. For the preparation obtained in Example 50 or 51 (preparation using each of compound A-6, A-1, A-7 and A-8), the equivalent dose of 0.3 mg / kg of siRNA was administered to mice for 48 hours The concentration of Factor VII protein in plasma after is shown. The ordinate represents the relative value of the concentration of Factor VII protein in plasma when the physiological saline administration group is 100. For the preparation obtained in Example 51 (preparation using each of the compounds A-9 to 12, B-1, B-8 and C-1), the equivalent dose of 0.3 mg / kg of siRNA was administered to mice for 48 hours The concentration of Factor VII protein in plasma after is shown. The ordinate represents the relative value of the concentration of Factor VII protein in plasma when the physiological saline administration group is 100. For the preparation obtained in Example 51 (preparation using each of the compounds A-5 and A-13 to 21), an equivalent of 0.3 mg / kg of siRNA was administered to mice for 48 hours after administration of Factor VII protein in plasma Indicates the concentration. The ordinate represents the relative value of the concentration of Factor VII protein in plasma when the physiological saline administration group is 100. The concentrations of Factor VII protein in plasma for 48 hours after administration of 0.3 mg / kg equivalent of siRNA to mice are shown for each of the preparations obtained in Example 51 (preparations using each of the compounds A-28 to 36). The ordinate represents the relative value of the concentration of Factor VII protein in plasma when the physiological saline administration group is 100. The concentrations of Factor VII protein in plasma for 48 hours after administration of 0.3 mg / kg equivalent of siRNA to mice are shown for each of the preparations obtained in Example 51 (preparation using each of the compounds C-2 to 5). The ordinate represents the relative value of the concentration of Factor VII protein in plasma when the physiological saline administration group is 100. For the preparations obtained in Comparative Example 2 (preparations using each of compounds XI-4 to 8), the concentration of Factor VII protein in plasma is shown for 48 hours after administration of 0.3 mg / kg equivalent of siRNA to mice. The ordinate represents the relative value of the concentration of Factor VII protein in plasma when the physiological saline administration group is 100. For each of the preparations obtained in Examples 52 and 53 (formulations using each of Compound A-1, A-6, A-7, A-10, A-12, B-8 and C-1), a mouse was used. Shows the concentration of Factor VII protein in plasma 48 hours after administration of 0.3 mg or 0.1 mg / kg equivalent amount of siRNA. The ordinate represents the relative value of the concentration of Factor VII protein in plasma when the physiological saline administration group is 100. 1 indicates a 0.3 mg / kg administration group, and □ indicates a 0.1 mg / kg administration group. For the preparation obtained in Example 53 (preparation using each of the compounds A-5 and A-13 to 21), Factor VII in plasma at 48 hours after administration of siRNA equivalent of 0.3 or 0.03 mg / kg to mice respectively Indicates the concentration of protein. The ordinate represents the relative value of the concentration of Factor VII protein in plasma when the physiological saline administration group is 100. 1 indicates a 0.3 mg / kg administration group, and □ indicates a 0.03 mg / kg administration group. For the preparation obtained in Example 53 (preparation using each of compounds A-28 to 36), the concentration of Factor VII protein in plasma at 48 hours after administration of siRNA 0.3 or 0.03 mg / kg equivalent amount to mice respectively Show. The ordinate represents the relative value of the concentration of Factor VII protein in plasma when the physiological saline administration group is 100. 1 indicates a 0.3 mg / kg administration group, and □ indicates a 0.03 mg / kg administration group. The preparation obtained in Example 53 or the preparation obtained in the same manner as Example 52 or 53 (preparation using each of compound A-1, A-6, A-10 and C-1-5) and comparison For the preparation obtained in Example 3 (preparation using compound XI-9), the concentration of Factor VII protein in plasma after 48 hours administration to siRNA mice at 0.3 or 0.03 mg / kg equivalent amount, respectively, is shown. The ordinate represents the relative value of the concentration of Factor VII protein in plasma when the physiological saline administration group is 100. 1 indicates a 0.3 mg / kg administration group, and □ indicates a 0.03 mg / kg administration group. For the preparation obtained in Example 54 (preparation using each of the compounds A-1, A-7 and A-10), in the plasma after 48 hours of administration of 0.3 mg or 0.1 mg / kg equivalent of siRNA to mice respectively The concentration of Factor VII protein is shown. The ordinate represents the relative value of the concentration of Factor VII protein in plasma when the physiological saline administration group is 100. 1 indicates a 0.3 mg / kg administration group, and □ indicates a 0.1 mg / kg administration group.

The cationic lipid of the present invention is
Formula (A)

(式中、R¹は炭素数8〜24の直鎖状または分枝状のアルキル、アルケニルもしくはアルキニルであり、
R²は炭素数8〜24の直鎖状または分枝状のアルキル、アルケニルもしくはアルキニル、アルコキシエチル、アルコキシプロピル、アルケニルオキシエチル、アルケニルオキシプロピル、アルキニルオキシエチルまたはアルキニルオキシプロピルであり、
R³およびR⁴は、同一または異なって炭素数1〜3のアルキルであるか、または一緒になって炭素数2〜8のアルキレンを形成するか、またはR³はR⁵と一緒になって炭素数2〜6のアルキレンを形成し、
R⁵は、水素原子、炭素数1〜6のアルキル、炭素数3〜6のアルケニル、アミノ、モノアルキルアミノ、ヒドロキシ、アルコキシ、カルバモイル、モノアルキルカルバモイル、ジアルキルカルバモイルまたは同一もしくは異なって1〜3つのアミノ、モノアルキルアミノ、ヒドロキシ、アルコキシ、カルバモイル、モノアルキルカルバモイルもしくはジアルキルカルバモイルで置換された炭素数1〜6のアルキルもしくは炭素数3〜6のアルケニルであるか、またはR³と一緒になって炭素数2〜8のアルキレンを形成し、
X¹は、炭素数1〜6のアルキレンであり、
X²は、単結合であるか、または炭素数1〜6のアルキレンであり、ただし、X¹とX²の炭素数の和は7以下であり、R⁵が、水素原子の場合、X²は単結合であり、R⁵がR³と一緒になって炭素数2〜6のアルキレンを形成する場合、X²は単結合であるか、またはメチレンもしくはエチレンである)、
式(B)

(Wherein, R ¹ represents a linear or branched alkyl, alkenyl or alkynyl having 8 to 24 carbon atoms,
R ² is linear or branched alkyl, alkenyl or alkynyl having 8 to 24 carbon atoms, alkoxyethyl, alkoxypropyl, alkenyloxyethyl, alkenyloxypropyl, alkynyloxyethyl or alkynyloxypropyl,
R ³ and R ⁴ are the same or different and are alkyl having 1 to 3 carbon atoms, or are taken together to form alkylene having 2 to 8 carbon atoms, or R ³ is taken together with R ⁵ Form alkylene having 2 to 6 carbon atoms,
R ⁵ is a hydrogen atom, alkyl having 1 to 6 carbon atoms, alkenyl having 3 to 6 carbon atoms, amino, monoalkylamino, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl or the same or different 1 to 3 C 1 -C 6 alkyl or C ³ -C 6 alkenyl substituted with amino, monoalkylamino, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl or dialkylcarbamoyl, or carbon together with R ³ Form an alkylene of 2-8,
X ¹ is alkylene having 1 to 6 carbon atoms,
X ² is a single bond or alkylene having 1 to 6 carbon atoms, provided that the sum of the carbon number of X ¹ and X ² is 7 or less, and when R ⁵ is a hydrogen atom, X ² Is a single bond, and when R ⁵ is taken together with R ³ to form an alkylene having 2 to 6 carbon atoms, X ² is a single bond or methylene or ethylene),
Formula (B)

(式中、R⁶は炭素数8〜24の直鎖状または分枝状のアルキル、アルケニルもしくはアルキニルであり、
R⁷は炭素数8〜24の直鎖状または分枝状のアルキル、アルケニルもしくはアルキニル、ア
ルコキシエチル、アルコキシプロピル、アルケニルオキシエチル、アルケニルオキシプロ
ピル、アルキニルオキシエチルまたはアルキニルオキシプロピルである)、または
式(C)

(Wherein, R ⁶ represents a linear or branched alkyl, alkenyl or alkynyl having 8 to 24 carbon atoms,
R ⁷ is linear or branched alkyl, alkenyl or alkynyl having 8 to 24 carbon atoms, alkoxyethyl, alkoxypropyl, alkenyloxyethyl, alkenyloxypropyl, alkynyloxyethyl or alkynyloxypropyl), or (C)

(式中、R⁸は炭素数8〜24の直鎖状または分枝状のアルキル、アルケニルもしくはアルキニルであり、
R⁹は炭素数8〜24の直鎖状または分枝状のアルキル、アルケニルもしくはアルキニル、アルコキシエチル、アルコキシプロピル、アルケニルオキシエチル、アルケニルオキシプロピル、アルキニルオキシエチルまたはアルキニルオキシプロピルであり、
X³は、炭素数1〜3のアルキレンであり、
R¹⁰は、水素原子または炭素数1〜3のアルキルである)で表されるカチオン性脂質である。
以下、式(A)で表される化合物を化合物(A)ということもある。他の式番号の化合物についても同様である。

(Wherein, R ⁸ is a linear or branched alkyl, alkenyl or alkynyl having 8 to 24 carbon atoms,
R ⁹ is linear or branched alkyl, alkenyl or alkynyl having 8 to 24 carbon atoms, alkoxyethyl, alkoxypropyl, alkenyloxyethyl, alkenyloxypropyl, alkynyloxyethyl or alkynyloxypropyl,
X ³ is alkylene having 1 to 3 carbon atoms,
R ¹⁰ is a hydrogen atom or a cationic lipid represented by alkyl having 1 to 3 carbon atoms.
Hereinafter, the compound represented by the formula (A) may be referred to as a compound (A). The same applies to compounds of other formula numbers.

Examples of linear or branched alkyl having 8 to 24 carbon atoms include octyl, decyl, dodecyl, tridecyl, tetradecyl, 2,6,10-trimethylundecyl, pentadecyl, 3,7,11-trimethyldodecyl, Hexadecyl, heptadecyl, octadecyl, 6,10,14-trimethylpentadecan-2-yl, nonadecyl, 2,6,10,14-tetramethylpentadecyl, icosyl, 3,7,11,15-tetramethylhexadecyl, henicosyl , Docosyl, tricosyl, tetracosyl and the like.
The linear or branched alkenyl having 8 to 24 carbon atoms may be a linear or branched alkenyl having 8 to 24 carbon atoms including one or more double bonds, for example (Z) -Tetradec-9-enyl, (Z) -hexadeca-9-enyl, (Z) -octadec-6-enyl, (Z) -octadec-9-enyl, (E) -octadeca-9-enyl, (Z) -Octadeca-11-enyl, (9Z, 12Z) -octadeca-9, 12-dienyl, (9Z, 12Z, 15Z) -octadeca-9, 12, 15-trienyl, (Z) -icosa-11-enyl, (( 11Z, 14Z) -Icosa-11,14-dienyl, 3,7,11-trimethyldodeca-2,6,10-trienyl, 3,7,11,15-tetramethylhexadec-2-enyl, (Z) And (Z) -hexadeca-9-enyl, (Z) -octadeca-6-enyl, (Z) -octadeca-9-enyl, (9Z, 12Z) -octadeca and the like. -9,12-dienyl, (Z) -icosa-11-enyl, (11Z, 14Z) -icosa-11,14-dienyl, (Z) -docosa-13-enyl and the like And the like.
The linear or branched alkynyl having 8 to 24 carbon atoms may be a linear or branched alkynyl having 8 to 24 carbon atoms including one or more triple bonds, for example, dodeca-11. -Ynyl, tetradec-6-ynyl, hexadeca-7-ynyl, hexadeca-5,7-diynyl, octadeca-9-ynyl and the like.

Examples of the alkyl moiety in alkoxyethyl and alkoxypropyl include the groups mentioned above as examples of the linear or branched alkyl having 8 to 24 carbon atoms.
Examples of the alkenyl moiety in alkenyloxyethyl and alkenyloxypropyl include the groups mentioned above as examples of linear or branched alkenyl having 8 to 24 carbon atoms.
Examples of the alkynyl moiety in alkynyloxyethyl and alkynyloxypropyl include, for example, the groups described above as examples of linear or branched alkynyl having 8 to 24 carbon atoms.

More preferably, R ¹ and R ² are the same or different and are linear or branched alkyl or alkenyl having 8 to 24 carbon atoms, and the same or different, linear having 8 to 24 carbon atoms Or branched alkenyl is more preferable, and linear or same alkenyl having 8 to 24 carbon atoms is further preferable. Further, R ¹ and R ² are more preferably the same, in which case, it is more preferably a linear or branched alkyl, alkenyl or alkynyl having 12 to 24 carbon atoms, More preferably, it is 12 to 24 linear alkenyl.

R ¹ and R ^2, when different, straight-chain or branched alkyl of R ¹ is 16 to 24 carbon atoms, alkenyl or alkynyl, R ² is a linear or having 8 to 12 carbon atoms It is also one of the preferred forms of the present invention that it is branched alkyl, alkenyl or alkynyl. In this case, R ¹ is more preferably a linear alkenyl having 16 to 24 carbon atoms, and R ² is preferably a linear alkyl having 8 to 12 carbon atoms, and R ¹ is (Z) -octadeca- Most preferably, it is 9-enyl or (9Z, 12Z) -octadeca-9,12-dienyl and R ² is octyl, decyl or dodecyl. When R ¹ and R ² are different, R ¹ is linear or branched alkyl, alkenyl or alkynyl having 12 to 24 carbon atoms, and R ² is alkoxyethyl, alkoxypropyl, alkenyl Oxyethyl, alkenyloxypropyl, alkynyloxyethyl or alkynyloxypropyl is also one of the preferred forms of the present invention. In this case, R ¹ is more preferably a linear alkenyl having 16 to 24 carbon atoms, and R ² is alkenyloxyethyl, and R ¹ is (Z) -octadeca-9-enyl ( 9Z, 12Z) -octadeca-9, 12-dienyl or (11Z, 14Z) -icosa-11, 14-dienyl, wherein R 2 is (Z) -octadeca-9-enyloxyethyl, (9Z, 12Z)- More preferably, it is octadeca-9,12-dienyloxyethyl or (11Z, 14Z) -icosa-11,14-dienyloxyethyl, and R1 is (9Z, 12Z) -octadeca-9,12-dienyl Most preferably, R2 is (9Z, 12Z) -octadeca-9,12-dienyloxyethyl.

When R ¹ and / or R ² are the same or different and are linear or branched alkyl or alkenyl having 8 to 24 carbon atoms, they may be the same or different and tetradecyl, hexadecyl, (Z) -tetradeca- 9-enyl, (Z) -hexadeca-9-enyl, (Z) -octadeca-6-enyl, (Z) -octadeca-9-enyl, (E) -octadeca-9-enyl, (Z) -octadeca- 11-enyl, (9Z, 12Z) -octadeca-9, 12-dienyl, (9Z, 12Z, 15Z) -octadeca-9, 12, 15-trienyl, (Z) -icosa-11-enyl, (11Z, 14Z) ) -Icosa-11,14-dienyl or (Z) -docosa-13-enyl, preferably identical or different, hexadecyl, (Z) -hexadeca-9-enyl, (Z) -octadeca-6-enyl , (Z) -octadeca-9-enyl, (9Z, 12Z) -octadeca-9, 12-dienyl, (Z) -icosa-11-enyl or (11Z, 14Z) -icosa-11, 14-dienyl Is more preferably the same or different More preferably (Z) -octadeca-9-enyl, (9Z, 12Z) -octadeca-9, 12-dienyl or (11Z, 14Z) -icosa-11, 14-dienyl, identically (9Z) , 12Z) -octadeca-9,12-dienyl is most preferred.

R ⁶ and R ⁷ are as defined for R ¹ and R ² respectively. However, when R ⁷ is linear or branched alkyl, alkenyl or alkynyl having 16 to 24 carbon atoms, R ⁶ and R ⁷ are the same (9Z, 12Z) -octadeca-9,12- It is preferably dienyl.

R ⁸ and R ⁹ are as defined for R ¹ and R ² respectively. However, R ⁸ and R ⁹ are preferably the same straight or branched alkyl, alkenyl or alkynyl having 16 to 24 carbon atoms, and the same (9Z, 12Z) -octadeca-9, 12- More preferably it is dienyl.

Examples of alkyl having 1 to 3 carbon atoms as R ³ and R ⁴ include methyl, ethyl, propyl, isopropyl and cyclopropyl, preferably methyl and ethyl, and more preferably methyl.

Examples of the alkylene having 2 to 8 carbon atoms formed by R ³ and R ⁴ together include ethylene, n-propylene, n-butylene, n-pentylene, n-hexylene, n-heptylene, n-octylene and the like Are preferably n-pentylene, n-hexylene, n-heptylene and the like, more preferably n-pentylene, n-hexylene and the like, and still more preferably n-hexylene.

R ³ is methyl or ethyl, or is combined with R ⁴ to form an alkylene having 5 to 7 carbon atoms, or R ⁵ is combined with R ⁵ to form an alkylene having 3 to 5 carbon atoms it is preferred, however, if R ³ does not form an alkylene having 5 to 7 carbon atoms together with R ⁴ is preferably R ⁴ is methyl or ethyl, more preferably methyl. Furthermore, R ³ is methyl, or together with R ⁴ forms n-pentylene or n-hexylene, or R ³ together with R ⁵ forms ethylene, n-propylene is more preferable, however, if R ³ do not form together with R ⁴ n- pentylene or n- hexylene more preferably, R ⁴ is methyl.

As the alkyl having 1 to 6 carbon atoms for R ⁵ , for example, methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, cyclopropylmethyl, pentyl, isopentyl, sec- And pentyl, neopentyl, tert-pentyl, cyclopentyl, hexyl, cyclohexyl and the like, preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, sec-pentyl, tert- Examples thereof include pentyl, neopentyl, hexyl and the like, and more preferable are methyl, ethyl, propyl and the like.

Examples of the alkenyl having 3 to 6 carbon atoms for R ⁵ include allyl, 1-propenyl, butenyl, pentenyl, hexenyl and the like, with preference given to allyl and the like.

Monoalkylamino in R ⁵ may be amino substituted with one, for example, 1 to 6 carbon alkyl (as defined above), such as methylamino, ethylamino, propylamino, butylamino, pentyl Amino, hexylamino and the like can be mentioned, with preference given to methylamino, ethylamino and the like.
The amino and monoalkylamino in R ⁵ may have a hydrogen ion coordinated to the lone electron pair on the nitrogen atom to form ammonio and monoalkylammonio, and amino and monoalkylamino are , Ammonio and monoalkylammonio, respectively.
In the present invention, ammonio and monoalkyl ammonio in which a hydrogen ion is coordinated to a lone electron pair on the nitrogen atom of amino and monoalkylamino may form a salt with a pharmaceutically acceptable anion.

The alkoxy in R ⁵ may be, for example, hydroxy substituted with alkyl having 1 to 6 carbon atoms (as defined above), such as methoxy, ethoxy, propyloxy, butyloxy, pentyloxy, hexyloxy and the like. And preferably methoxy, ethoxy and the like.

Monoalkylcarbamoyl and dialkylcarbamoyl in R ⁵ include carbamoyl substituted with one and the same or different two, for example, C 1 to C 6 alkyl (as defined above), and more specifically Are, for example, methylcarbamoyl, ethylcarbamoyl, propylcarbamoyl, butylcarbamoyl, pentylcarbamoyl, hexylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl, ethylmethylcarbamoyl, methylpropylcarbamoyl, butylmethylcarbamoyl, methylpentylcarbamoyl, hexylmethylcarbamoyl and the like, with preference given to Are, for example, methylcarbamoyl, ethylcarbamoyl, dimethylcarbamoyl and the like.

Examples of the alkylene having 2 to 6 carbon atoms formed by R ⁵ and R ³ together include ethylene, n-propylene, n-butylene, n-pentylene, n-hexylene and the like, preferably n- Propylene, n-butylene, n-pentylene and the like can be mentioned, more preferably n-propylene, n-butylene and the like, and still more preferably n-propylene.

R ⁵ is a hydrogen atom, alkyl having 1 to 6 carbon atoms, monoalkylamino, hydroxy, alkoxy or the same or different and 1 to 3 amino, monoalkylamino, or hydroxy or alkoxy substituted carbon atoms 1 It is preferable that it is an alkyl of 6 or 6 together with R ³ to form an alkylene having 2 to 6 carbon atoms, and a hydrogen atom, methyl, amino, methylamino, hydroxy, methoxy or the same or different 1 It is more preferable that it is methyl substituted by ^-3 amino or hydroxy, or R ³ is combined to form an alkylene having 3 to 5 carbon atoms, and a hydrogen atom, an alkyl having 1 to 3 carbon atoms, or hydroxy or where more preferably in together with R ³ to form a n- propylene or n- butylene, hydrogen atom or R ³ It is most preferred that taken together form n- propylene.

Examples of the alkyl having 1 to 3 carbon atoms in R ¹⁰ include methyl, ethyl, propyl, isopropyl and cyclopropyl and the like, preferably methyl, ethyl and isopropyl and the like, and more preferably methyl, ethyl and the like Can be mentioned. As R ¹⁰ , a hydrogen atom or methyl is more preferable, and a hydrogen atom is most preferable.

Examples of the alkylene having 1 to 6 carbon atoms at X ¹ and X ² include methylene, ethylene, n-propylene, n-butylene, n-pentylene, n-hexylene and the like.

Further, X ¹ is preferably alkylene having 1 to 3 carbon atoms, most preferably methylene or ethylene, and X ² is preferably a single bond, methylene or ethylene, a single bond or methylene It is more preferable that The sum of carbon number of X ¹ and X ² is preferably 1 to 3, and most preferably 2. In any of these cases, R ³ and R ⁴ are the same or different and are methyl or ethyl, and R ⁵ is a hydrogen atom, methyl, amino, methylamino, hydroxy, methoxy or the same or different 1 to 3 R ³ and R ⁴ together form an alkylene of 5 to 7 carbon atoms, R ⁵ is a hydrogen atom, methyl, amino, methylamino, hydroxy, Or methoxy, or identical or different methyl substituted by 1 to 3 amino or hydroxy, or R ³ and R ⁵ together form C 3-5 alkylene, R ⁴ is methyl or Preferably, R ³ and R ⁴ are methyl and R ⁵ is a hydrogen atom, or R ³ and R ⁴ together form n-pentylene or n-hexylene, R ⁵ is selected from the group consisting of hydrogen Or a child, or R ³ and R ⁵ form together n- propylene, more preferably R ⁴ is methyl.

Examples of the alkylene having 1 to 3 carbon atoms at X ³ include methylene, ethylene, n-propylene and the like, with preference given to methylene, ethylene and the like.

The oxygen atoms in formula (A) may be replaced by sulfur atoms.
Formula (Aa) in which one of the oxygen atoms in formula (A) is replaced by sulfur atom

(式中、R¹、R²、R³、R⁴、R⁵、X¹およびX²はそれぞれ前記と同義)で表される化合物Aaは、対応する化合物Aにローソン試薬(Lawesson’s Reagent、2,4-ビス(4-メトキシフェニル)-1,3,2,4-ジチアジホスフェタン-2,4-ジスルフィド)等の1,3,2,4-ジチアジホスフェタン-2,4-ジスルフィド誘導体を作用させることで得ることができる。

(Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , and X ¹ and X ² are as defined above, respectively) have the same meanings as in Compound A, except that the corresponding compound A is a Lawesson's Reagent 1,3,2,4-dithiadiphosphetan-2,4 such as 4, 4-bis (4-methoxyphenyl) -1,3,2,4-dithiadiphosphetan-2,4-disulfide) It can be obtained by the action of a disulfide derivative.

The cationic lipid of the present invention may form a salt with a pharmaceutically acceptable anion when a hydrogen ion is coordinated to a lone electron pair on any nitrogen atom.
In the present invention, pharmaceutically acceptable anions include, for example, inorganic ions such as chloride ion, bromide ion, nitrate ion, sulfate ion and phosphate ion, acetate ion, oxalate ion, maleate ion and fumarate ion. And organic acid ions such as citric acid ion, benzoic acid ion and methanesulfonic acid ion.

  Next, the method for producing the cationic lipid of the present invention will be described. In the preparation methods shown below, when the defined group changes under the conditions of the preparation method or is unsuitable for carrying out the preparation method, introduction and removal of protecting groups commonly used in synthetic organic chemistry Methods [for example, methods described in Protective Groups in Organic Synthesis, third edition, by TW Greene, John Wiley & Sons Inc. (1999), etc.], etc. The target compound can be produced by using Moreover, the order of reaction processes, such as substituent introduction, can also be changed as needed.

Production Method Among compounds (A), compound (Ia) in which R ² is a linear or branched alkyl, alkenyl or alkynyl having 8 to 24 carbon atoms can be produced by the following method.

(式中、R¹、R³、R⁴、R⁵、X¹およびX²はそれぞれ前記と同義であり、R¹¹は炭素数8〜24の直鎖状または分枝状のアルキル、アルケニルまたはアルキニルであり、Yは、塩素原子、臭素原子、ヨウ素原子、トリフルオロメタンスルホニルオキシ、メタンスルホニルオキシ、ベンゼンスルホニルオキシ、p-トルエンスルホニルオキシ等の脱離基を表し、Arはp-ニトロフェニル、o-ニトロフェニル、p-クロロフェニル等の置換フェニル基または無置換フェニル基を表す)

Wherein R ¹ , R ³ , R ⁴ , R ⁵ , X ¹ and X ² are as defined above, and R ¹¹ is a linear or branched alkyl, alkenyl or carbon number of 8 to 24 carbon atoms Y is an leaving group such as chlorine atom, bromine atom, iodine atom, trifluoromethanesulfonyloxy, methanesulfonyloxy, benzenesulfonyloxy, p-toluenesulfonyloxy, etc. Ar is p-nitrophenyl, o -Represents a substituted or unsubstituted phenyl group such as nitrophenyl and p-chlorophenyl)

Steps 1 and 2
Compound (IIa) is a mixture of ammonia and compound (IIIa), without solvent or in a solvent, optionally in the presence of 1 to 10 equivalents of a base, at a temperature between room temperature and 200 ° C. for 5 minutes to 100 hours It can be produced by reaction. Furthermore, compound (IIb) can be prepared by adding compound (IIa) and compound (IIIb) without solvent or in a solvent, preferably in the presence of 1 to 10 equivalents of a base, at a temperature between room temperature and 200 ° C. It can be produced by reacting for 5 minutes to 100 hours.
As the solvent, for example, methanol, ethanol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane, N, N-dimethylformamide, N, N Dimethylacetamide, N-methylpyrrolidone, pyridine, water and the like, and these may be used alone or in combination.
As a base, for example, potassium carbonate, potassium hydroxide, sodium hydroxide, sodium methoxide, potassium tert-butoxide, triethylamine, diisopropylethylamine, N-methylmorpholine, pyridine, 1,8-diazabicyclo [5.4.0] -7- Undesen (DBU) etc. are mentioned.
Compound (IIIa) and Compound (IIIb) are commercially available products or known methods (for example, “5th edition Experimental Chemistry Lecture 13 Synthesis of organic compounds I”, 5th edition, p. 374, Maruzen (2005)) Or it can obtain by the method according to it.
The compound (IIb) in the case where R ¹ and R ¹¹ are the same can be obtained by using two or more equivalents of the compound (IIIa) in step 1.

Step 3
Compound (VI) is obtained by treating Compound (IV) with Compound (V) without solvent or in a solvent, preferably in the presence of 1 to 10 equivalents of an additive, and / or preferably, 1 to 10, preferably. It can be produced by reacting for 5 minutes to 72 hours at a temperature between -20 ° C and 150 ° C in the presence of an equivalent of a base.
As the solvent, for example, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane, N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl pyrrolidone, dimethyl sulfoxide and the like can be mentioned, and these can be used alone or in combination.
Examples of the additive include 1-hydroxybenzotriazole, 4-dimethylaminopyridine and the like.
As the base, those exemplified in Steps 1 and 2 can be mentioned.
Compound (IV) can be obtained as a commercial product.
Compound (V) is a commercially available product or a known method (for example, “5th edition Experimental Chemistry Lecture 14 Synthesis of organic compounds II”, 5th edition, p. 1, Maruzen (2005)) or a method according thereto Can be obtained by

Step 4
Compound (Ia) is obtained by treating compound (IIb) with compound (VI) neat or in a solvent, preferably in the presence of 1 to 10 equivalents of an additive, and / or preferably 1 to 10 It can be produced by reacting for 5 minutes to 72 hours at a temperature between -20 ° C and 150 ° C in the presence of an equivalent amount of a base.
As the solvent and the additive, those exemplified in Step 3 can be mentioned.
As the base, those exemplified in Steps 1 and 2 can be mentioned.
Compound (IIb) can also be produced by the following method.

(式中、R¹、R¹¹およびYはそれぞれ前記と同義であり、Bocはtert-ブトキシカルボニル基を示し、Nsは2-ニトロベンゼンスルホニル基を表す)

(Wherein, R ¹ , R ¹¹ and Y are as defined above, Boc is a tert-butoxycarbonyl group, and Ns is a 2-nitrobenzenesulfonyl group)

Step 5
Compound (IIc) is a compound of N- (tert-butoxycarbonyl) -2-nitrobenzenesulfonamide and compound (IIIa) without solvent or in a solvent, preferably in the presence of 1 to 10 equivalents of an additive, and The reaction can be carried out by reacting for 5 minutes to 100 hours at a temperature between room temperature and 200 ° C., preferably in the presence of 1 to 10 equivalents of a base.
As the solvent, those exemplified in Steps 1 and 2 can be mentioned.
Examples of the additive include n-tetrabutylammonium iodide, sodium iodide and the like.
Examples of the base include cesium carbonate, potassium carbonate, potassium hydroxide, sodium hydroxide, sodium methoxide, potassium tert-butoxide, triethylamine, diisopropylethylamine, N-methylmorpholine, pyridine, DBU and the like.

Step 6
Compound (IId) is a compound (IIc) in an amount of 1 equivalent to a large excess of acid, in the absence of solvent or in a solvent, optionally in the presence of 1 to 10 equivalents of an additive, at -20 ° C and 150 ° C. At a temperature between 5 minutes and 72 hours.
As the solvent, those exemplified in Steps 1 and 2 can be mentioned.
Examples of the acid include hydrochloric acid, sulfuric acid, phosphoric acid, trifluoroacetic acid and the like.
Examples of the additive include thioanisole, dimethyl sulfide, triisopropylsilane and the like.
Step 7
Compound (IIe) is a compound (IIIb) and compound (IId), without solvent or in a solvent, optionally in the presence of 1 to 10 equivalents of an additive, and / or optionally, preferably 1 to 10 equivalents The reaction can be carried out by reacting for 5 minutes to 100 hours at a temperature between room temperature and 200 ° C. in the presence of a base.
As the solvent, those exemplified in Steps 1 and 2 can be mentioned.
Examples of the additive and the base include those exemplified in Step 5.
Step 8
Compound (IIb) is a compound (IIe) and a thiol compound, without solvent or in a solvent, optionally and preferably in the presence of 1 to 10 equivalents of a base, at a temperature between room temperature and 200 ° C. for 5 minutes to 100 It can be produced by reacting for time.
As the solvent, those exemplified in Steps 1 and 2 can be mentioned.
Examples of the thiol compound include methanethiol, ethanethiol, dodecanethiol, thiophenol, mercaptoacetic acid and the like.
As the base, those exemplified in Step 5 can be mentioned.

  Among the compounds (A), compound (Ib) wherein R 2 is alkoxyethyl, alkoxypropyl, alkenyloxyethyl, alkenyloxypropyl, alkynyloxyethyl or alkynyloxypropyl can be produced by the following method.

(式中、R¹、R³、R⁴、R⁵、X¹、X²、YおよびArはそれぞれ前記と同義であり、R¹²はアルコキシエチル、アルコキシプロピル、アルケニルオキシエチル、アルケニルオキシプロピル、アルキニルオキシエチルまたはアルキニルオキシプロピルであり、Msはメタンスルホニル基を表す)

(Wherein, R ¹ , R ³ , R ⁴ , R ⁵ , X ¹ , X ² , Y and Ar are as defined above, and R ¹² is alkoxyethyl, alkoxypropyl, alkenyloxyethyl, alkenyloxypropyl, And is an alkynyloxyethyl or alkynyloxypropyl, Ms represents a methanesulfonyl group)

Step 9
Compound (IIf) is a compound (IIa) and compound (IIIc), without solvent or in a solvent, optionally and preferably in the presence of 1 to 10 equivalents of a base, at a temperature between room temperature and 200 ° C. for 5 minutes It can be produced by reacting for 100 hours.
As the solvent, those exemplified in Steps 1 and 2 can be mentioned.
As the base, those exemplified in Steps 1 and 2 can be mentioned.

Step 10
The compound (Ib) is a compound (IIf) together with the compound (VI) neat or in a solvent, preferably in the presence of 1 to 10 equivalents of an additive, and / or more preferably 1 to 10 It can be produced by reacting for 5 minutes to 72 hours at a temperature between -20 ° C and 150 ° C in the presence of an equivalent amount of a base.
As the solvent and the additive, those exemplified in Step 3 can be mentioned.
As the base, those exemplified in Steps 1 and 2 can be mentioned.

  Compound (IIIc) can be produced by the following method.

(式中、R¹³は炭素数8〜24の直鎖状または分枝状のアルキル、アルケニルまたはアルキニルであり、X⁴はエチレンまたはn-プロピレンを表し、Msはメタンスルホニル基を表す)

(Wherein, R ¹³ represents a linear or branched alkyl, alkenyl or alkynyl having 8 to 24 carbon atoms, X ⁴ represents ethylene or n-propylene, and Ms represents a methanesulfonyl group)

Step 11
Compound (VIIb) is a compound (VIIa) together with compound (VIII) without solvent or in a solvent, preferably in the presence of 1 to 10 equivalents of a base, at a temperature between room temperature and 200 ° C. It can be produced by reacting for a minute to 100 hours.
As the solvent, those exemplified in step 3 can be mentioned.
As the base, those exemplified in Steps 1 and 2 can be mentioned.
Compound (VIIa) is a commercially available product or a known method (for example, “5th edition Experimental Chemistry Lecture 14 Synthesis of organic compounds II”, 5th edition, p. 1, Maruzen (2005)) or a method according thereto Can be obtained by
Compound (VIII) can be obtained as a commercial product.

Step 12
Compound (IIIc) is a compound (VIIb), for 5 minutes at a temperature between −20 ° C. and 150 ° C., without solvent or in a solvent, preferably in the presence of 1 to 10 equivalents of a base, with mesylation reagent It can manufacture by making it react for-72 hours.
As the solvent, those exemplified in step 3 can be mentioned.
As the base, those exemplified in Steps 1 and 2 can be mentioned.
Examples of the mesylation reagent include mesylate anhydride, mesyl chloride and the like.
Compound (IIf) can also be produced by the following method.

(式中、R¹、R¹²、Y、NsおよびMsはそれぞれ前記と同義である)

(Wherein, R ¹ , R ¹² , Y, Ns and Ms are each as defined above)

Step 13
Compound (IIg) is a compound (IIIc) and compound (IId), without solvent or in a solvent, optionally in the presence of 1 to 10 equivalents of an additive, and / or optionally, preferably 1 to 10 equivalents The reaction can be carried out by reacting for 5 minutes to 100 hours at a temperature between room temperature and 200 ° C. in the presence of a base.
As the solvent, those exemplified in Steps 1 and 2 can be mentioned.
Examples of the additive and the base include those exemplified in Step 5.
Step 14
Compound (IIf) is a compound (IIg) and a thiol compound, without solvent or in a solvent, optionally and preferably in the presence of 1 to 10 equivalents of a base, at a temperature between room temperature and 200 ° C. for 5 minutes to 100 It can be produced by reacting for time.
As the solvent, those exemplified in Steps 1 and 2 can be mentioned.
Examples of the thiol compound include those exemplified in Step 8.
As the base, those exemplified in Step 5 can be mentioned.

  Formulas (Aa) to (Ac) in which the oxygen atom in formula (A) is a sulfur atom

(式中、R¹、R²、R³、R⁴、R⁵、X¹およびX²はそれぞれ前記と同義である)で表される化合物Aa〜Acは、それぞれ工程4および工程10において、式(VI)における酸素原子が硫黄原子である式(VIa)〜(VIc)

(Wherein, in the formula, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , X ¹ and X ² have the same meanings as described above, respectively) Formulas (VIa) to (VIc) in which the oxygen atom in formula (VI) is a sulfur atom

(式中、R³、R⁴、R⁵、X¹、X²およびArはそれぞれ前記と同義である)で表される化合物VIa〜VIcを用いることで得ることができる。

(Wherein, R ³ , R ⁴ , R ⁵ , X ¹ , X ² and Ar are as defined above, respectively) can be obtained by using the compounds VIa to VIc.

Among the compounds (B), compounds (Ba) in which R ⁷ is a linear or branched alkyl, alkenyl or alkynyl having 8 to 24 carbon atoms can be obtained by the method for producing the compound (IIb).
Among the compounds (B), compounds (Bc) in which R ⁷ is alkoxyethyl, alkoxypropyl, alkenyloxyethyl, alkenyloxypropyl, alkynyloxyethyl or alkynyloxypropyl can be obtained by the method for producing compound (IIf) it can.

  Compound (C) can be produced by the following method.

(式中、R⁸、R⁹、R¹⁰、X³およびYはそれぞれ前記と同義である)
工程15
化合物(C)は、化合物(IIh)と化合物(Xa)を、無溶媒でまたは溶媒中、必要により好ましくは1〜10当量の添加剤の存在下、および/または必要により好ましくは1〜10当量の塩基の存在下、室温と200℃の間の温度で、5分間〜100時間反応させることにより製造することができる。
溶媒および塩基としては、それぞれ工程1および2で例示したものがあげられる。
添加剤としては、工程5で例示したものがあげられる。
化合物(IIh)は化合物(B)の製造方法で得ることができる。
化合物(Xa)は市販品としてまたは公知の方法(例えば、「第5版 実験化学講座13 有機化合物の合成I」、第5版、p.374、丸善(2005年))もしくはそれに準じた方法で得ることができる。

(Wherein, R ⁸ , R ⁹ , R ¹⁰ , X ³ and Y are each as defined above)
Step 15
The compound (C) is a compound (IIh) and a compound (Xa), without solvent or in a solvent, optionally in the presence of 1 to 10 equivalents of an additive, and / or optionally, preferably 1 to 10 equivalents The reaction can be carried out by reacting for 5 minutes to 100 hours at a temperature between room temperature and 200 ° C. in the presence of a base.
As the solvent and the base, those exemplified in Steps 1 and 2 can be mentioned, respectively.
As the additive, those exemplified in step 5 can be mentioned.
Compound (IIh) can be obtained by the method for producing compound (B).
Compound (Xa) is a commercially available product or can be produced by a known method (for example, “Fifth Edition Experimental Chemistry Lecture 13 Synthesis of Organic Compounds I”, Fifth Edition, p. 374, Maruzen (2005)) or a method analogous thereto You can get it.

  After compound (C), compound (Ca) in which R 10 is a hydrogen atom can also be produced by the following method.

(式中、R⁸、R⁹、X³およびYはそれぞれ前記と同義であり、Proはトリメチルシリル、トリエチルシリル、トリtert-ブチルシリル、tert-ブチルジメチルシリル、tert-ブチルジフェニルシリルまたはトリフェニルシリル等のシリル型保護基を表す)

(Wherein, R ⁸ , R ⁹ , X ³ and Y are as defined above, and Pro is trimethylsilyl, triethylsilyl, tritert-butylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl or triphenylsilyl etc. Represents a silyl type protecting group of

Step 16
The compound (IXa) is a compound (IIh) and the compound (Xb), without solvent or in a solvent, optionally in the presence of 1 to 10 equivalents of an additive, and / or optionally, preferably 1 to 10 equivalents The reaction can be carried out by reacting for 5 minutes to 100 hours at a temperature between room temperature and 200 ° C. in the presence of a base.
As the solvent and the base, those exemplified in Steps 1 and 2 can be mentioned, respectively.
As the additive, those exemplified in step 5 can be mentioned.
Compound (IIh) can be obtained by the method for producing compound (B).
Compound (Xb) is a commercially available product or a known method (for example, “Fifth Edition Experimental Chemistry Lecture 18 Synthesis of Organic Compounds VI”, Fifth Edition, pages 171-172, Maruzen (2005)) or a modification thereof It can be obtained by the method.

Step 17
Compound (Ca) can be produced by reacting compound (IXa) and a deprotecting reagent without solvent or in a solvent at a temperature between −20 ° C. and 150 ° C. for 5 minutes to 72 hours.
As the solvent, those exemplified in Steps 1 and 2 can be mentioned.
Examples of the deprotecting reagent include tetrabutylammonium fluoride, hydrogen fluoride / pyridine complex, fluorine compounds such as hydrofluoric acid, etc., and acids such as acetic acid, trifluoroacetic acid, pyridinium paratoluenesulfonate, hydrochloric acid etc. .

(式中、R⁸およびR⁹はそれぞれ前記と同義である) Among the compounds (C), a compound (Cb) in which R ¹⁰ is a hydrogen atom and X ³ is a C 2 alkylene can also be produced by the following method.

(Wherein, R ⁸ and R ⁹ are each as defined above)

Step 18
Compound (IXb) is compound (IIh), preferably 1 to large excess of ethyl acrylate, without solvent or in a solvent, preferably in the presence of 1 to 10 equivalents of a base at room temperature and 200 ° C. The reaction can be carried out at a temperature between 5 minutes and 100 hours.
As the solvent, those exemplified in step 1 can be mentioned.
Examples of the base include potassium carbonate, potassium hydroxide, sodium hydroxide, sodium methoxide, sodium ethoxide, potassium tert-butoxide, triethylamine, diisopropylethylamine, N-methylmorpholine, pyridine, DBU and the like.

Step 19
Compound (Cb) is a compound (IXb) and preferably 1 to 10 equivalents of a reducing agent in a solvent, optionally and preferably in the presence of 1 to 10 equivalents of an additive, between -20 ° C and 150 ° C. It can be produced by reacting at temperature for 5 minutes to 72 hours.
Examples of the solvent include tetrahydrofuran, dioxane, diethyl ether, dichloromethane, toluene and the like, and these can be used alone or in combination.
Examples of the reducing agent include lithium aluminum hydride, aluminum hydride, diisobutylaluminum hydride, sodium triacetoxyborohydride, sodium cyanoborohydride, borane and the like.
Examples of the additive include aluminum chloride, cerium chloride, iron chloride, acetic acid, hydrochloric acid and the like.

  The intermediates and target compounds in each of the above-mentioned production methods are isolated and purified by separation and purification methods commonly used in organic synthesis chemistry, for example, filtration, extraction, washing, drying, concentration, recrystallization, various chromatography, etc. be able to. In addition, intermediates can be subjected to the next reaction without particular purification.

In the cationic lipid of the present invention, a hydrogen ion may be coordinated to a lone electron pair on the nitrogen atom in the structure, in which case it forms a salt with a pharmaceutically acceptable anion (as defined above) The cationic lipid of the present invention also includes a compound in which a hydrogen ion is coordinated to a lone electron pair on the nitrogen atom.
Although some cationic lipids of the present invention may have stereoisomers such as geometric isomers and optical isomers, tautomers, etc., the cationic lipids of the present invention include these, All possible isomers and their mixtures are included.
A part or all of each atom in the cationic lipid of the present invention may be replaced by the corresponding isotope atom, and the compound (A), the compound (B) or the compound (C) may be these isotope atoms And compounds which are substituted by For example, some or all of the hydrogen atoms in compound (A), compound (B) or compound (C) may be a hydrogen atom having an atomic weight of 2 (deuterium atom).
Compounds in which some or all of the atoms in the cationic lipid of the present invention are replaced with corresponding isotope atoms can be produced using a commercially available building block by the same method as each of the above production methods it can. Further, compounds in which a part or all of hydrogen atoms in the compound (A), the compound (B) or the compound (C) are replaced with a deuterium atom are, for example, iridium complex as a catalyst and heavy water as a deuterium source Method to deuterate alcohol, carboxylic acid, etc. using [see Journal of American Chemical Society (J. Am. Chem. Soc.), Vol. 124, No. 10, 2092 (2002)], etc. It can also be synthesized.

  Specific examples of the cationic lipid of the present invention obtained by the present invention are shown in Tables 1 to 7. However, the cationic lipid of the present invention is not limited to these.

  The nucleic acid used in the present invention may be, for example, any molecule obtained by polymerizing a nucleotide and / or a molecule having a function equivalent to that of a nucleotide, for example, ribonucleic acid which is a polymer of ribonucleotide (RNA), deoxyribonucleic acid (DNA) which is a polymer of deoxyribonucleotide, chimeric nucleic acid consisting of RNA and DNA, and nucleotide in which at least one nucleotide of these nucleic acids is substituted with a molecule having the same function as the nucleotide Polymers and the like can be mentioned. In addition, the nucleic acid of the present invention is also included in the nucleic acid of the present invention, a derivative that contains at least a part of the structure of the nucleotide and / or a molecule having a function equivalent to that of the nucleotide polymerized. In the present invention, uracil U and thymine T can be read respectively.

Examples of molecules having the same function as nucleotides include nucleotide derivatives and the like.
The nucleotide derivative may be any molecule as long as, for example, a nucleotide is modified, for example, to improve nuclease resistance or stabilize from other degradation factors as compared to RNA or DNA. In order to increase the affinity to the complementary strand nucleic acid, to increase the cell permeability, or to visualize the molecule, ribonucleotides or deoxyribonucleotides modified molecules are preferably used.
Examples of the nucleotide derivative include sugar moiety modified nucleotides, phosphodiester bond modified nucleotides, base modified nucleotides and the like.
The sugar moiety-modified nucleotide may be, for example, any part or all of the chemical structure of the nucleotide sugar modified or substituted with any substituent, or any one substituted with any atom, 2'-modified nucleotides are preferably used.

As the modifying group in the sugar moiety-modified nucleotide, for example, 2′-cyano, 2′-alkyl, 2′-substituted alkyl, 2′-alkenyl, 2′-substituted alkenyl, 2′-halogen, 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 (as defined above for the amino acid residues) and the like.
In addition, as a sugar moiety-modified nucleotide, for example, a bridged nucleic acid (BNA) having a structure in which a modification group at the 2 'position is bridged to a carbon atom at the 4' position, more specifically 2 '. Locked Nucleic Acid (LNA) in which the oxygen atom at position 4 and the carbon atom at 4 'position are bridged via methylene, and ethylene bridged nucleic acid (ENA) [Nucleic Acid Research] , 32, e175 (2004)], etc., which are included in 2'-modified nucleotides.
Furthermore, as a sugar moiety modified nucleotide, peptide nucleic acid (PNA) [Acc. Chem. Res., 32, 624 (1999)], oxypeptide nucleic acid (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001) And peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)] and the like.

As a modifying group in sugar moiety modified nucleotide, 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, 2'-Se-substituted alkyl and the like are preferable, and 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 and the like are more preferred, 2'-O-methyl, 2'-O-ethyl, 2'-fluoro and the like are more preferred, 2'-O-methyl and 2'-O-ethyl are most preferred.
In addition, the modification group in the sugar moiety-modified nucleotide can also define a preferable range from its size, and from the size of fluoro, one corresponding to the size of -O-butyl is preferred, and the size of -O-methyl To the size of -O-ethyl are more preferred.

The alkyl in the modifying group in the sugar moiety-modified nucleotide is the same as the alkyl having 1 to 6 carbons in the definition of the cationic lipid of the present invention.
The alkenyl in the modifying group in the sugar moiety-modified nucleotide is exemplified by alkenyl having 3 to 6 carbon atoms, and examples thereof include allyl, 1-propenyl, butenyl, pentenyl and hexenyl.
Examples of the halogen in the modifying group in the sugar moiety-modified nucleotide include a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like.
Examples of amino acids in amino acid residues include aliphatic amino acids (specifically, glycine, alanine, valine, leucine, isoleucine etc.), hydroxy amino acids (specifically, serine, threonine etc.), acidic amino acids (specifically, Are aspartate, glutamate, etc., acidic amino acid amides (specifically, asparagine, glutamine, etc.), basic amino acids (specifically, lysine, hydroxylysine, arginine, ornithine, etc.), sulfur-containing amino acids (specifically Are, for example, cysteine, cystine, methionine etc., imino acids (specifically, proline, 4-hydroxyproline etc.) and the like.
Examples of the substituent on the substituted alkyl and substituted alkenyl in the modifying group of the sugar moiety-modified nucleotide include halogen (as defined above), hydroxy, sulfanyl, amino, oxo, -O-alkyl (the alkyl moiety of the -O-alkyl is The same as the above alkyl), -S-alkyl (wherein the alkyl moiety of the -S-alkyl is the same as the above alkyl), -NH-alkyl (the alkyl moiety of the -NH-alkyl has the same meaning as the above alkyl), dialkylaminooxy ( The two alkyl moieties of the dialkylaminooxy are the same or different and have the same meaning as the above alkyl), dialkylamino (the two alkyl moieties of the dialkylamino are the same or different and have the same meaning as the above alkyl), the dialkylaminoalkyloxy (the dialkylamino) The two alkyl moieties of alkyloxy are the same or different and are the same as the aforementioned alkyl , And the alkylene moiety meaning), and the like what is hydrogen atom is removed one from the alkyl, the number of substituents is preferably from 1 to 3.

As a phosphodiester bond modified nucleotide, any one of those obtained by modifying or substituting a part or all of the chemical structure of phosphodiester bond of the nucleotide with an arbitrary substituent, or substituting with an arbitrary atom For example, a nucleotide in which a phosphodiester bond is substituted with a phosphorothioate bond, a nucleotide in which a phosphodiester bond is substituted with a phosphorodithioate bond, a nucleotide in which a phosphodiester bond is substituted with an alkyl phosphonate bond, phosphoric acid Nucleotides and the like in which the diester bond is substituted with a phosphoroamidate bond can be mentioned.
As the base-modified nucleotide, any part or all of the chemical structure of the base of the nucleotide may be modified or substituted with any substituent, or any one substituted with any atom, for example, a base Wherein the oxygen atom is substituted by a sulfur atom, the hydrogen atom is substituted by an alkyl group having 1 to 6 carbon atoms, the methyl group is substituted by a hydrogen atom or an alkyl group having 2 to 6 carbon atoms, What has an amino group protected by protecting groups, such as a C1-C6 alkyl group and a C1-C6 alkanoyl group, etc. are mention | raise | lifted.
Furthermore, as a nucleotide derivative, a nucleotide or a sugar derivative, a nucleotide derivative in which at least one of a phosphate diester bond or a base is modified is a lipid, a phospholipid, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, Dyes and the like to which other chemical substances are added are also mentioned, and specifically, 5'-polyamine adduct nucleotide derivative, cholesterol adduct nucleotide derivative, steroid adduct nucleotide derivative, bile acid adduct nucleotide derivative, vitamin adduct nucleotide derivative, green The fluorescent dye (Cy3) -added nucleotide derivative, the red fluorescent dye (Cy5) -added nucleotide derivative, the fluorescein (6-FAM) added nucleotide derivative, the biotin-added nucleotide derivative and the like can be mentioned.
Furthermore, in the nucleic acid used in the present invention, the nucleotide or the nucleotide derivative may be the other nucleotide or nucleotide derivative in the nucleic acid and an alkylene structure, a peptide structure, a nucleotide structure, an ether structure, an ester structure, and at least one of them. A crosslinked structure such as a combined structure may be formed.

  The nucleic acid used in the present invention preferably includes a nucleic acid that suppresses expression of a target gene, and more preferably includes a nucleic acid having an expression suppression effect of a 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, for example, genes related to tumor or inflammation are preferable, for example, vascular endothelial growth factor (hereinafter referred to as VEGF) Abbreviated), vascular endothelial growth factor receptor (hereinafter referred to as VEGFR), fibroblast growth factor, fibroblast growth factor receptor, platelet derived growth factor, platelet derived growth factor receptor, hepatocytes Growth factor, hepatocyte growth factor receptor, Kruppel-like factor (hereinafter abbreviated as KLF), expressed sequence tag (Ets) transcription factor, nuclear factor, hypoxia inducible factor, cell cycle related factor, chromosomal replication Proteins such as related factors, chromosomal repair related factors, microtubule related factors, growth signal pathway related factors, growth related transcription factors, apoptosis related factors Genes such as VEGF gene, VEGFR gene, fibroblast growth factor gene, fibroblast growth factor receptor gene, platelet derived growth factor gene, platelet derived growth factor receptor gene, liver Cell growth factor gene, hepatocyte growth factor receptor gene, KLF gene, Ets transcription factor gene, nuclear factor gene, hypoxia inducible factor gene, cell cycle related factor gene, chromosomal replication related factor gene, chromosomal repair related factor gene, micro Vascular related factor genes (for example, CKAP5 gene etc.), growth signal pathway related factor genes (for example, KRAS gene etc.), growth related transcription factor gene, apoptosis related factor (for example, BCL-2 gene etc.) and the like can be mentioned.

  Moreover, as a target gene in the present invention, for example, a gene expressed in liver, lung, kidney or spleen is preferable, a gene expressed in liver is more preferable, for example, a gene related to the above-mentioned tumor or inflammation, hepatitis B virus Genome, Hepatitis C virus genome, Apolipoprotein (APO), Hydroxymethylglutaryl (HMG) CoA reductase, Kexin type 9 serine protease (PCSK9), Factor 12, Glucagon receptor, Glucocorticoid receptor, Leukotriene receptor , Thromboxane A2 receptor, histamine H1 receptor, carbonic anhydrase, angiotensin converting enzyme, renin, p53, tyrosine phosphatase (PTP), sodium-dependent glucose transporter, tumor necrosis factor, interleukin, hepcidin, trans siren, anti Thrombin, protein C, A gene encoding a protein such as tryptase enzyme (eg, TMPRSS6 gene etc.) can be mentioned.

The nucleic acid that suppresses the expression of a target gene includes, for example, a nucleic acid that includes a base sequence complementary to a part of the base sequence of mRNA of a gene encoding a protein etc. (target gene) and that suppresses the expression of the target gene For example, any nucleic acid such as siRNA (short interference RNA), double-stranded nucleic acid such as miRNA (micro RNA), shRNA (short hairpin RNA), antisense nucleic acid, single-stranded nucleic acid such as ribozyme, etc. Although double-stranded nucleic acid is preferable, it is preferable.
A nucleic acid containing a base sequence complementary to a part of the base sequence of mRNA of the target gene is called antisense strand nucleic acid, and a nucleic acid containing a base sequence complementary to the base sequence of antisense strand nucleic acid is a sense strand It is also called nucleic acid. A sense strand nucleic acid refers to a nucleic acid which can form a double strand forming portion 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 to have a double strand forming part. The double strand forming part refers to a part in which a nucleotide forming a double stranded nucleic acid or a derivative thereof forms a base pair to form a double strand. The base pairs constituting the double strand forming portion are usually 15 to 27 base pairs, preferably 15 to 25 base pairs, more preferably 15 to 23 base pairs, still more preferably 15 to 21 base pairs, and 15 to 19 Base pairs are particularly preferred.

As the antisense strand nucleic acid of the double strand forming part, for example, a nucleic acid consisting of a partial sequence of mRNA of a target gene, or 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 base is substituted in the nucleic acid A nucleic acid which is deleted or added and which has a target protein expression suppression activity is preferably used. A single-stranded nucleic acid constituting a double-stranded nucleic acid usually comprises a series of 15 to 30 bases (nucleosides), preferably 15 to 29 bases, more preferably 15 to 27 bases, and still more preferably 15 to 25 bases. 17 to 23 bases are particularly preferred, and 19 to 21 bases are most preferred.
The antisense strand, the sense strand, or both nucleic acids constituting the double-stranded nucleic acid have an additional nucleic acid which does not form a duplex on the 3 'or 5' side following the duplex forming portion You may The portion which does not form this double chain is also called an overhang (overhang).
As a double-stranded nucleic acid having an overhang, for example, one having an overhang consisting of 1 to 4 bases, usually 1 to 3 bases at the 3 'end or 5' end of at least one strand is used, The thing which has a protrusion which consists of is preferably used, and what has a protrusion which consists of dTdT or UU is used more preferably. The overhang can have only the antisense strand, only the sense strand, and both the antisense and sense strands, but a double stranded nucleic acid having an overhang in both the antisense and sense strands is preferably used .
In addition, a sequence in which a part or all of the nucleotide sequence of the target gene's mRNA matches the duplex strand formation part subsequently, or a nucleotide sequence of the complementary strand of the target gene mRNA strand following the duplex strand formation part Alternatively, all matching sequences may be used. Furthermore, as a nucleic acid that suppresses the expression of a target gene, for example, a nucleic acid molecule that produces the above-mentioned double-stranded nucleic acid by the action of ribonuclease such as Dicer (WO 2005/089287 pamphlet), 3 'end or 5'. It is also possible to use a double stranded nucleic acid or the like which does not have a terminal overhang.

  When the double-stranded nucleic acid is an siRNA, preferably, the antisense strand has a sequence of at least the 1st to 17th bases (nucleosides) from the 5 'end toward the 3' end, of the target gene. Preferably, the antisense strand has a sequence of bases 1 to 19 from the 5 'end toward the 3' end, which is a sequence of bases complementary to the continuous 17 base sequence of mRNA. The sequence of bases complementary to the continuous 19 base sequence of the target gene mRNA, or the sequence of the 1st to 21st bases is complementary to the continuous 21 base sequence of the target gene mRNA Or the sequence of the 1st to 25th bases is a sequence of bases complementary to the continuous 25base sequence of the mRNA of the target gene.

  Furthermore, when the nucleic acid used in the present invention is siRNA, preferably 10 to 70%, more preferably 15 to 60%, still more preferably 20 to 50% of the sugars in the nucleic acid are modified at the 2 'position. Is a ribose substituted with 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 The steric configuration may be the same as the hydroxyl group at the 2'-position of ribose, although it may be different or different. Examples of the modifying group in ribose substituted with a modifying group at the 2 ′ position include those exemplified in the definition of the modifying group in 2′-modified nucleotides in sugar moiety modified nucleotides and 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, 2′-Se-substituted alkyl and the like are preferable, and 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- [ 2- (N, N-Dimethylamino) ethoxy] ethyl], 2'-O- [2- (methyl) Amino) -2-oxoethyl], 2′-Se-methyl, hydrogen atom and the like are more preferable, 2′-O-methyl, 2′-O-ethyl, 2′-fluoro, hydrogen atom and the like are further preferable, 2 ′ Most preferred are -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 phosphate moiety, an ester moiety or the like in the structure of the nucleic acid is substituted by another atom such as a sulfur atom.

In addition, in the sugar linked to the base at the 5 'end of the antisense strand and the sense strand, the hydroxyl group at the 5' position is a phosphate group or the above-mentioned modifying group, or a phosphate group or It may be modified by the group converted to the above-mentioned modifying group.
In addition, in the sugar linked to the base at the 3 'end of the antisense strand and the sense strand, the hydroxyl group at the 3' position is a phosphate group or the above-mentioned modifying group, or a phosphate group or It may be modified by the group converted to the above-mentioned modifying group.

The single-stranded nucleic acid is, for example, a continuous 15 to 27 bases (nucleoside) of a target gene, preferably 15 to 25 bases, more preferably 15 to 23 bases, still more preferably 15 to 21 bases, particularly preferably 15 to 21 bases. A nucleic acid consisting of a complementary sequence of a sequence consisting of 19 bases, or 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 base in the nucleic acid is substituted, deleted or added, and the target protein expression suppression activity Any nucleic acid may be possessed. The single-stranded nucleic acid is preferably composed of a series of 15 to 30 bases (nucleosides), more preferably 15 to 27 bases, still more preferably 15 to 25 bases, particularly preferably 15 to 23 bases Nucleic acids are preferably used.
As the single stranded nucleic acid, one obtained by linking the antisense strand and the sense strand constituting the double stranded nucleic acid described above via a spacer sequence (spacer oligonucleotide) may be used. The spacer oligonucleotide is preferably a single-stranded nucleic acid molecule of 6 to 12 bases, and the sequence at the 5 'end thereof is preferably 2 U. An example of the spacer oligonucleotide is a nucleic acid consisting of the sequence of UUCAAGAGA. The order of the antisense strand and the sense strand linked by the spacer oligonucleotide may be 5 '. The single-stranded nucleic acid is preferably, for example, a single-stranded nucleic acid such as shRNA having a duplex forming part by a stem loop structure. Single stranded nucleic acids such as shRNA are usually 50 to 70 bases in length.
Even when using a nucleic acid having a length of 70 bases or less, preferably 50 bases or less, more preferably 30 bases or less, designed to produce the above single-stranded nucleic acid or double-stranded nucleic acid by the action of ribonuclease etc. Good.

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

The composition of the present invention is a composition containing the cationic lipid and nucleic acid of the present invention, for example, a complex of the cationic lipid of the present invention and a nucleic acid, or a neutral lipid and / or cationic lipid of the present invention. Or a composition containing a complex of a combination of a polymer and a nucleic acid, a composition containing the complex and a lipid membrane encapsulating the complex, and the like. The lipid membrane may be a single lipid membrane (single lipid membrane) or a double lipid membrane (bilayer lipid). The lipid membrane may contain the cationic lipid, neutral lipid and / or polymer of the present invention. In addition, the complex and / or the lipid membrane may contain a cationic lipid other than the cationic lipid of the present invention.
Moreover, as the composition of the present invention, for example, 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, neutral lipid and / or polymer And a complex of nucleic acid and a complex thereof, as well as a lipid membrane encapsulating the complex, and a composition containing the cationic lipid of the present invention in the lipid membrane, and the like. The lipid membrane in this case may also be a single lipid membrane (single lipid membrane) or a double lipid membrane (bilayer lipid). In addition, the lipid membrane may contain cationic lipids other than the cationic lipid of the present invention, neutral lipids and / or polymers.

  The composition of the present invention comprises a composition containing the complex of the cationic lipid of the present invention and a nucleic acid, the complex of the cationic lipid of the present invention and a nucleic acid, and a lipid membrane encapsulating the complex. The lipid membrane contains a composition containing the cationic lipid of the present invention, a complex of a cationic lipid other than the cationic lipid of the present invention and a nucleic acid, and a lipid membrane encapsulating the complex, the lipid membrane Further, a composition containing the cationic lipid of the present invention is more preferable, a composition containing the complex of the cationic lipid of the present invention and a nucleic acid, a complex of the cationic lipid of the present invention and a nucleic acid and the complex Further preferred is a composition containing a lipid membrane encapsulating a body, the lipid membrane containing the cationic lipid of the present invention, and a complex of the cationic lipid of the present invention and a nucleic acid and a lipid membrane encapsulating the complex Contains Composition containing a cationic lipid of the present invention the quality film is most preferred. In any case, the lipid membrane may contain neutral lipids and / or polymers. In addition, the complex and / or the lipid membrane may contain a cationic lipid other than the cationic lipid of the present invention.

The form of the complex includes, for example, a complex of a nucleic acid and a membrane consisting of a single lipid (single molecule) layer (reverse micelle), a complex of a nucleic acid and a liposome, a complex of a nucleic acid and a micelle, etc. Examples include complexes of nucleic acids and membranes consisting of lipid monolayers or complexes of nucleic acids and liposomes.
Examples of the composition containing the complex and a lipid membrane encapsulating the complex include the complex and a liposome encapsulating the complex with a lipid bilayer membrane.
In the composition of the present invention, one or more of the cationic lipids of the present invention may be used, and in the cationic lipids of the present invention, cationic lipids other than the cationic lipids of the present invention are mixed. You may
Examples of cationic lipids other than the cationic lipid of the present invention include N- [1- (2,3-dioleyl) disclosed in JP-A-61-161246 (US Pat. No. 5,049,386). Oxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), N- (2,3-di- (9- (Z) -octadecenoyloxy))-prop-1-yl-N, N, N-trimethylammonium chloride (DOTAP), etc., N- [1- (2,3-dioleoyloxypropyl) as disclosed in WO 91/16024 and WO 97/019675. ] -N, N-Dimethyl-N-hydroxyethyl ammonium bromide (DORIE), 2,3-dioleyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propana DLinDMA et al., Disclosed in WO2005 / 121348, and DLin-K-DMA disclosed in WO2009 / 086558. , (3R, 4R) -3,4-bis ((Z) -hexadeca-9-enyloxy) -1-methylpyrrolidine, N-methyl-N, N disclosed in WO 2011/136368. -Bis (2-((Z) -octadec-6-enyloxy) ethyl) amine and the like, preferably DOTMA, DOTAP, DORIE, DOSPA, 1,2-dilinoleyloxy-N, N-dimethylaminopropane Tertiary amine moiety or two unsubstituted alkyl groups with two unsubstituted alkyl groups such as (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl- [1,3] -dioxolane (DLin-K-DMA) A cationic lipid having a quaternary ammonium moiety having a group is mentioned, and more preferably, a cationic lipid having the tertiary amine moiety is mentioned. More preferably, the tertiary amine moiety and the unsubstituted alkyl group of the quaternary ammonium moiety are methyl groups.
The composition of the present invention may contain a nucleic acid, but may also contain a compound chemically similar to the nucleic acid.

  The composition of the present invention can be produced according to a known production method or in accordance therewith, and may be produced by any production method. For example, known methods for preparing liposomes can be applied to the preparation of a composition containing liposomes, which is one of the compositions. As a known method for preparing a liposome, for example, a method for preparing a liposome of Bangham et al. ["Journal of Molecular Biology" (J. Mol. Biol.), 13: 1965, p. 238- 252], ethanol injection method [see “Journal of Cell Biology (J. Cell Biol.)”, 1975, 66, p. 621-634], French press method [“E. Letters (FEBS Lett.), 1979, 99, pp. 210-214], freeze-thaw method ["Archives of biochemistry and biophysics (Arch. Biochem. Biophys.)", 1981]. 212, p. 186-194], reverse phase evaporation method ["Procedings of the National Academy of Science United States of America (Proc. Natl. Acad. Sci. USA) ”, 1978, 75, p. 4194-4198] or pH gradient method (eg, Patent No. 2572554, Patent No. 2659136). Broadcast, etc. reference), and the like. For example, water, acid, alkali, various buffers, physiological saline, or amino acid infusion can be used as a solution for dispersing the liposome in the preparation of the liposome. In addition, in the preparation of liposomes, it is possible to add an antioxidant such as citric acid, ascorbic acid, cysteine or ethylenediaminetetraacetic acid (EDTA), for example, an isotonicity agent such as glycerin, glucose or sodium chloride. is there. Also, for example, 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. Thereafter, physiological saline or the like is added, and the mixture can be shaken and stirred to form liposomes.

  Also, the composition of the present invention may be prepared, for example, by pre-dissolving 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 in chloroform. An aqueous solution and methanol are added and mixed to form a complex of cationic lipid / nucleic acid, the chloroform layer is further taken out, polyethylene glycolated phospholipid, neutral lipid and water are added thereto, and the water-in-oil type (W / O) A method of forming an emulsion and treating it by reverse phase evaporation (see JP-A-2002-508765) or dissolving a nucleic acid in an acidic aqueous electrolyte solution, for example, 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 (in ethanol) to reduce the ethanol concentration to 20 v / v% to prepare the nucleic acid-encapsulated liposome After filtration, sizing filtration and removal of excess ethanol by dialysis, the sample is further raised in pH and dialyzed to remove nucleic acid attached to the surface of the composition for production (Japanese Patent Publication 2002-501511 and BioKymikaka) -Echito Biophysica Actor (Biochimica et Biophysica Acta, 2001, Volume 1510, see p. 152-166) and the like.

  Among the composition of the present invention, a complex of the cationic lipid of the present invention and a nucleic acid, or a complex of a combination of a cationic lipid of the present invention with a neutral lipid and / or a polymer and a nucleic acid A composition containing a liposome containing a lipid bilayer membrane encapsulating a body can be produced, for example, according to the production method described in WO02 / 28367 pamphlet and WO2006 / 080118 pamphlet etc. .

Further, among the composition of the present invention, for example, a complex of the cationic lipid of the present invention and a nucleic acid, or a complex of a combination of a cationic lipid of the present invention and a neutral lipid and / or a polymer and a nucleic acid And a composition containing a lipid membrane encapsulating the complex, 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 A composition containing a complex of a combination of a polymer and a nucleic acid and a lipid membrane encapsulating the complex, the composition containing the cationic lipid of the present invention in the lipid membrane, etc. Each complex is manufactured according to the manufacturing method described in the pamphlet 28367 and WO2006 / 080118 etc., and dispersed without dissolving the complex in water or 0 to 20% aqueous ethanol solution. (A solution) Separately, each lipid membrane component is dissolved in, for example, an aqueous ethanol solution (A solution B), and equal amounts of A solution and B solution are mixed, and further, water may be added as appropriate. it can. As the cationic lipid in the liquid A and the liquid B, one or more of the cationic lipid of the present invention or a cationic lipid other than the cationic lipid of the present invention may be used, and the cationic lipid of the present invention A lipid and a cationic lipid other than the cationic lipid of the present invention may be used in combination as a mixture.
In the present invention, a complex of the cationic lipid of the present invention and a nucleic acid, or a complex of a combination of a cationic lipid of the present invention and a neutral lipid and / or a polymer and a nucleic acid, and the complex A composition comprising an encapsulated lipid membrane, 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, a neutral lipid and / or a polymer A nucleic acid in a complex, which contains a complex of a combination and a nucleic acid, as well as a lipid membrane encapsulating the complex, the lipid membrane containing the cationic lipid of the present invention and the like. The structures of the complex and the membrane are displaced due to the electrostatic interaction between the lipid and the cationic lipid in the lipid membrane, and the fusion of the cationic lipid in the complex and the cationic lipid in the lipid membrane. A complex of the cationic lipid of the present invention with a nucleic acid, or a complex of a combination of a cationic lipid of the present invention with a neutral lipid and / or a polymer and a nucleic acid, and a lipid membrane encapsulating the complex Or a complex of a cationic lipid other than the cationic lipid of the present invention and a nucleic acid, or a combination of a neutral lipid and / or a polymer with a cationic lipid other than the cationic lipid of the present invention and a nucleic acid And a lipid membrane encapsulating the complex, and the lipid membrane is included in a composition containing the cationic lipid of the present invention.

A nucleic acid (as defined above), preferably a double-stranded nucleic acid and the cationic lipid of the present invention and / or the cation of the present invention according to the production method described in WO 02/28367 and WO 2006/080118 etc. A complex with a liposome containing a cationic lipid other than the lipid, and dispersing the complex in water or 0 to 20% aqueous ethanol without dissolving it (Liquid A), separately, according to the present invention Dissolving a cationic lipid and / or a cationic lipid other than the cationic lipid of the present invention in an aqueous ethanol solution (Liquid B), mixing Liquid A and Liquid B in an equal amount or a volume ratio of 1: 1, or A composition containing the nucleic acid and the cationic lipid can also be obtained by further adding water as appropriate. The composition is preferably a complex of a cationic lipid and a nucleic acid and a composition containing a lipid membrane encapsulating the complex, more preferably from a lipid monolayer containing the nucleic acid and the cationic lipid. A complex with the membrane (reverse micelle) and a lipid membrane encapsulating the complex. The lipid membrane in these cases may be a single lipid membrane (single lipid membrane) or a double lipid membrane (bilayer lipid).
In addition, the liposome in the complex of the nucleic acid of the present disclosure and the liposome is preferably a liposome having a size adjusted in advance to an average particle diameter of 10 nm to 400 nm, more preferably 30 nm to 110 nm, still more preferably 40 nm to 80 nm. . In addition, the complex and / or lipid membrane may contain neutral lipids and / or macromolecules. In addition, the concentration of ethanol may be 20 to 40% as long as solution A can form a complex of the liposome and the nucleic acid.
Also, instead of mixing equal amounts of solution A and solution B, the concentration of ethanol in which the complex does not dissolve after mixing solution A and solution B and the cationic lipid in solution B does not dissolve, preferably the complex It may be replaced by mixing solution A and solution B in such a ratio that the solution does not dissolve, the cationic lipid in solution B does not dissolve, and the ethanol concentration becomes an aqueous ethanol solution of 30 to 60%, or Solution A and solution B are mixed at such a ratio that the concentration of ethanol does not dissolve the complex after mixture of solution and solution B, and water is further added, whereby ethanol in which the cationic lipid in solution B does not dissolve The concentration may be used.
The complex of the nucleic acid and the liposome in the solution A of the present disclosure is prepared by mixing the solution A and the solution B, and after adding water appropriately, a membrane consisting of a lipid monolayer containing a cationic lipid ( The form is changed to a complex of reverse micelles and nucleic acid. The composition containing the nucleic acid and the cationic lipid obtained by the production method of the present disclosure is preferably a complex of a cationic lipid and a nucleic acid and a composition containing a lipid membrane encapsulating the complex. More preferably, a composition comprising a membrane (reverse micelle) consisting of a lipid monolayer comprising a cationic lipid and a nucleic acid, and a lipid membrane encapsulating the complex, wherein the lipid membrane contains a cationic lipid And their manufacturability (yield and / or homogeneity) 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, preferably 1.5 to 3.5 times the number of phosphorus atoms of the nucleic acid. Is more preferable, and 2-3 times is more preferable. Further, the total number of the cationic lipid of the present invention and the cationic lipid 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 1.5 times to 3.5 times are more preferable, and 2 to 3 times times are more preferable.
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 with respect to the number of phosphorus atoms of the nucleic acid. It is preferably double, more preferably 2.5 to 9 times, and still more preferably 3.5 to 8 times. In addition, the total number of the cationic lipid of the present invention and the cationic lipid 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. The ratio is preferably 2.5 to 9 times, more preferably 3.5 to 8 times.

The neutral lipid may be any of simple lipids, complex lipids or derivatized lipids such as, but not limited to, phospholipids, glyceroglycolipids, glycosphingolipids, sphingoids or sterols.
When neutral lipids are contained in the composition of the present invention, the total number of neutral lipid molecules is relative to the total number of cationic lipids of the present invention and cationic lipids other than the cationic lipids of the present invention. It is preferably 0.1 to 1.8 times, more preferably 0.3 to 1.2 times, and still more preferably 0.4 to 1.0 times. The composition of the present invention may contain any neutral lipid in the complex, may be contained in the lipid membrane encapsulating the complex, and at least contained in the lipid membrane encapsulating the complex Is more preferably contained in both the complex and the lipid membrane encapsulating the complex.

  Examples of phospholipids in neutral lipids include phosphatidyl choline (specifically, soybean phosphatidyl choline, egg yolk phosphatidyl choline (EPC), distearoyl phosphatidyl choline (DSPC), dipalmitoyl phosphatidyl choline (DPPC), palmitoyl oleoyl phosphatidyl choline (POPC), dimyristoyl phosphatidyl choline (DMPC), dioleoylphosphatidylcholine (DOPC), etc., phosphatidylethanolamine (specifically, distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), Dimyristoyl phosphoethanolamine (DMPE), 16-0-monomethyl-PE, 16-0-dimethyl-PE, 18-1-trans-PE, palmitoyl oleoyl-phosphatidylethanolamine (POP E), 1-stearoyl-2-oleoyl-phosphatidyl ethanolamine (SOPE), etc., glycerophospholipids (specifically, phosphatidyl serine, phosphatidic acid, phosphatidyl glycerol, phosphatidyl inositol, palmitoyl oleoyl phosphatidyl glycerol (POPG), lyso, Phosphatidyl choline etc., Sphingophospholipids (specifically sphingomyelin, ceramide phosphoethanolamine, ceramide phosphoglycerol, ceramide phosphoglycerophosphate etc), glycerophosphonolipids, sphingophosphonolipids, natural lecithin (specifically egg yolk lecithin And natural or synthetic phospholipids such as soybean lecithin etc. or hydrogenated phospholipids (specifically, hydrogenated soybean phosphatidyl choline etc.).

  Examples of glyceroglycolipids in neutral lipids include sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride or glycosyl diglyceride and the like.

  Examples of glycosphingolipids in neutral lipids include galactosylcerebroside, lactosylcerebroside, ganglioside and the like.

  Examples of the sphingoids in neutral lipids include sphingan, icosasphingan, sphingosine or derivatives thereof. As a derivative, for example, -NH2 such as sphingan, icosasphingan or sphingosine was converted to -NHCO (CH2) x CH3 (wherein, x is an integer of 0 to 18, preferably 6, 12 or 18). The ones are listed.

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

  As the polymer, for example, protein, albumin, dextran, polyfect, chitosan, dextran sulfate, such as poly-L-lysine, polyethyleneimine, polyaspartic acid, styrene maleic acid copolymer, isopropylacrylamide-acrylic pyrrolidone copolymer weight And micelles composed of one or more of a polymer such as polyethylene glycol modified dendrimer, polylactic acid, polylactic acid polyglycolic acid or polyethylene glycolated polylactic acid, or a salt thereof.

  Here, salts of polymers include, for example, metal salts, ammonium salts, acid addition salts, organic amine addition salts, amino acid addition salts and the like. Examples of metal salts include alkali metal salts such as lithium salts, sodium salts and potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, aluminum salts or zinc salts, and the like. Examples of ammonium salts include salts such as ammonium or tetramethylammonium. Acid addition salts include, for example, mineral acid salts such as hydrochlorides, sulfates, nitrates or phosphates, and organic acid salts such as acetates, maleates, fumarates or citrates. Organic amine addition salts include, for example, addition salts such as morpholine or piperidine. Examples of amino acid addition salts include addition salts such as glycine, phenylalanine, aspartic acid, glutamic acid or lysine.

In addition, all compositions of the present invention preferably contain, 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 surfactants, etc., and a complex Or the lipid membrane that encapsulates the complex, and it is more preferable to contain both the complex and the lipid membrane that encapsulates 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, it is selected from sugars, peptides, nucleic acids and water-soluble polymers The total number of molecules of lipid derivatives and fatty acid derivatives of one or more substances is preferably 0.05 to 0.3 times the total number of cationic lipids of the present invention and cationic lipids other than the cationic lipids of the present invention. The value is more preferably 0.07 to 0.25, and still more preferably 0.1 to 0.2.

  As a lipid derivative or fatty acid derivative of one or more substances selected from sugars, peptides, nucleic acids and water-soluble polymers, or surfactants, a glycolipid, or a lipid derivative or fatty acid derivative of a water-soluble polymer is preferably mentioned. More preferably, they include lipid derivatives or fatty acid derivatives of water-soluble polymers. Lipid derivatives or fatty acid derivatives of one or more substances selected from sugars, peptides, nucleic acids and water-soluble polymers, or surfactants, for example, hydrophobic affinity, electrostatics, etc. A substance that has the property of binding through chemical interactions and the like, and the other part has a property of binding with a solvent during production of the composition, for example, through hydrophilic affinity, electrostatic interaction, etc. Is preferred.

  Examples of lipid derivatives or fatty acid derivatives of sugars, peptides or nucleic acids include sugars such as sucrose, sorbitol and lactose, for example casein derived peptides, egg white derived peptides, soy derived peptides, peptides such as glutathione, or for example DNA, RNA, plasmids Nucleic acid such as siRNA, ODN, etc., and the neutral lipid mentioned in the definition of the composition or the cationic lipid of the present invention or a fatty acid such as stearic acid, palmitic acid, myristic acid, lauric acid etc. And the like.

  In addition, as lipid derivatives or fatty acid derivatives of sugars, for example, glyceroglycolipids or glycosphingolipids mentioned in the definition of the above composition are also included.

  Examples of lipid derivatives or fatty acid derivatives of water-soluble polymers include polyethylene glycol, polyglycerin, polyethylene imine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, oligosaccharide, dextrin, water-soluble cellulose, dextran, chondroitin sulfate, polyglycerin, Chitosan, polyvinyl pyrrolidone, polyaspartic acid amide, poly-L-lysine, mannan, pullulan, oligoglycerol etc. or derivatives thereof, neutral lipids mentioned in the definition of the composition, or cationic lipids of the present invention, Or fatty acids such as stearic acid, palmitic acid, myristic acid or lauric acid combined, salts thereof and the like, and more preferably lipid derivatives such as polyethylene glycol or polyglycerin Or fatty acid derivatives and their salts can be mentioned, more preferably, a lipid derivative or a fatty acid derivative of a polyethylene glycol and salts thereof.

  Examples of lipid derivatives or fatty acid derivatives of polyethylene glycol include polyethylene glycolated lipids [specifically, polyethylene glycol-phosphatidyl ethanolamine (more specifically, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE), 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.) or polyethylene glycol fatty acid esters etc. And more preferably polyethylene glycolated lipids.

  Examples of lipid derivatives or fatty acid derivatives of polyglycerin include polyglycerinized lipids (specifically, polyglycerin-phosphatidyl ethanolamine etc.), polyglycerin fatty acid esters and the like, and more preferable are polyglycerinated lipids. Be

  As the surfactant, for example, polyoxyethylene sorbitan monooleate (specifically, polysorbate 80 etc.), polyoxyethylene polyoxypropylene glycol (specifically, pluronic F 68 etc.), sorbitan fatty acid ester (specifically, sorbitan fatty acid ester) Monolaurate, sorbitan monooleate, etc., polyoxyethylene derivatives (specifically, polyoxyethylene hydrogenated castor oil 60, polyoxyethylene lauryl alcohol, etc.), glycerin fatty acid ester, polyethylene glycol alkyl ether, etc. Examples thereof include polyoxyethylene polyoxypropylene glycol, glycerin fatty acid ester or polyethylene glycol alkyl ether.

In addition, the complex and the lipid membrane in the composition of the present invention may optionally be surface-modified with, for example, a water-soluble polymer or the like [DDLasic, edited by F. Martin, "Stealth, • Stealth Liposomes ”(US), see CRC Press Inc, 1995, pp. 93-102]. Examples of water-soluble polymers that can be used for surface modification include polyethylene glycol, polyglycerin, polyethylene imine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, oligosaccharide, dextrin, water-soluble cellulose, dextran, chondroitin sulfate, polyglycerin Chitosan, polyvinyl pyrrolidone, polyaspartic acid amide, poly-L-lysine, mannan, pullulan, oligoglycerol and the like, and dextran, pullulan, mannan, amylopectin and hydroxyethyl starch and the like are preferable. In addition, for surface modification, a lipid derivative or fatty acid derivative (as defined above) of one or more substances selected from sugars, peptides, nucleic acids and water-soluble polymers can be used. The surface modification is such that 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 surfactants. It is one of the methods.
Also, the targeting ligand can optionally be directly attached 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 (WO2006 (See pamphlet 116/107).

  The average particle size of the complex in the composition of the present invention or the lipid membrane encapsulating the complex can be freely selected as desired, but it is preferable to use the average particle size described below. As a method of adjusting the average particle size, for example, a method of mechanically pulverizing (specifically, using Manton Goulin, a microfluidizer, etc.) for extrusion, large multilamellar liposome (MLV), etc. [Mueller (RH) Muller), Benita (S. Benita) and B. Bohm, ed., “Emulsion and Nanosuspensions for the Formulation of the Poorable Drugs (Emulsion and Nanosuspensions for the Formulation) of Poorly Soluble Drugs, "" Scientific Publishers Stuttgart, Germany, 1998, pp. 267-294 ", and the like.

The size of the composite in the composition of the present invention is preferably about 5 nm to 200 nm, more preferably about 20 nm to 150 nm, and still more preferably about 30 nm to 100 nm, as the average particle size.
The size of the composition of the present invention (lipid membrane encapsulating the complex) preferably has an average particle size of about 10 nm to 300 nm, more preferably about 30 nm to 200 nm, and about 50 nm to 150 nm. Is more preferred.
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 dynamic light scattering.

  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.

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 or human being, for example, delivered to a tumor or an organ or site where inflammation has occurred, and in the cells of a delivery organ or site. Nucleic acid can be introduced. The organ or site in which the tumor or inflammation has occurred includes, but is not limited to, for example, the stomach, large intestine, liver, lung, spleen, pancreas, kidney, bladder, skin, blood vessels, eyeballs and the like. In addition, the composition of the present invention can be delivered intravenously to mammals including humans, for example, delivered to the liver, lung, spleen and / or kidney, and in the cells of a delivery organ or site in the composition of the present invention Nucleic acid can be introduced. The cells of liver, lung, spleen and / or kidney may be normal cells, cells associated with tumor or inflammation or cells associated with other diseases.
If the nucleic acid in the composition of the present invention is a nucleic acid having the expression suppression effect of a target gene utilizing RNA interference (RNAi), the nucleic acid or the like which suppresses the expression of the target gene in mammalian cells in vivo It can be introduced and can suppress the expression of the target gene. The administration target is preferably a human.
In addition, if the target gene in the present invention is, for example, a gene expressed in liver, lung, kidney or spleen, preferably a gene expressed in liver, the composition of the present invention is related to liver, lung, kidney or spleen It can be used as a therapeutic or prophylactic agent for a disease, preferably a therapeutic or prophylactic agent for a disease associated with the liver.
That is, the present invention also provides a method of treating a disease associated with the liver, lung, kidney or spleen, which comprises administering the composition of the present invention described above to a mammal. The administration subject is preferably a human, more preferably a human suffering from a disease associated with the liver, lung, kidney or spleen.
In addition, the composition of the present invention is a tool for verifying the efficacy of suppressing a target gene in an in vivo efficacy evaluation model for a therapeutic agent or a preventive agent for diseases associated with liver, lung, kidney or spleen It can also be used.

  The composition of the present invention is, for example, the stabilization of the nucleic acid in biological components (eg, blood, digestive tract etc.) such as blood components, reduction of side effects, or drug accumulation in tissues or organs containing the target gene expression site It can also be used as a preparation aiming at the increase of

When the composition of the present invention is used as a therapeutic agent or prophylactic agent for drugs related to liver, lung, kidney or spleen, the administration route is to use the most effective administration route for treatment. Desirable, for example, parenteral administration such as oral cavity, airway, rectum, subcutaneous, intramuscular or intravenous administration or oral administration can be mentioned, preferably intravenous administration or intramuscular administration can be mentioned, more preferably Can be given intravenously.
Although the dose varies depending on the medical condition, age, administration route and the like of the administration subject, it may be administered, for example, such that the daily dose converted to nucleic acid is about 0.1 μg to 1000 mg.

Examples of preparations suitable for intravenous administration or intramuscular administration include injections, and it is possible to use the dispersion of the composition prepared by the above method as it is, for example, in the form of injections, etc. It is also possible to use the dispersion after removing the solvent, for example by filtration, centrifugation etc., lyophilize the dispersion, and / or use an excipient such as mannitol, lactose, trehalose, maltose or glycine. The added dispersion can also be used after lyophilization.
In the case of injection, the dispersion of the composition or the composition obtained by removing or lyophilizing the above-mentioned solvent is mixed and injected with, for example, water, acid, alkali, various buffers, physiological saline or amino acid infusion, etc. It is preferred to prepare an agent. In addition, for example, an injection can be prepared by adding an antioxidant such as citric acid, ascorbic acid, cysteine or EDTA or an isotonic agent such as glycerin, glucose or sodium chloride, or the like. For example, a cryopreservative such as glycerin can be added and cryopreserved.

Next, the present invention will be specifically described by way of examples, reference examples and test examples. However, the present invention is not limited to these examples, reference examples and test examples.
The proton nuclear magnetic resonance spectra ( ¹ H NMR) shown in the examples and reference examples were measured at 270 MHz, 300 MHz, 400 MHz or 500 MHz, and exchangeable protons became clear depending on the compound and measurement conditions. May not be observed. In addition, although what is normally used is used as a description of the multiplicity of a signal, br represents that it is an apparently wide signal.

Di ((9Z, 12Z) -octadeca-9,12-dienyl) amine (compound B-1)
Ammonia (manufactured by Tokyo Chemical Industry Co., Ltd., about 2 mol / L methanol solution, 18.0 mL, 36.0 mmol) was dissolved in (9Z, 12Z) -octadeca-9,12-dienyl methanesulfonate (Newcheck prep ink (Nu- Chek Prep, Inc. 1.55 g (4.50 mmol) was added, and the mixture was stirred at 130 ° C. for 3 hours using a microwave reactor. To the reaction mixture was added saturated aqueous sodium hydrogen carbonate solution, and the mixture was extracted 5 times with chloroform. The organic layers were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure to obtain a crude product of (9Z, 12Z) -octadeca-9,12-dienylamine.
The obtained crude product was combined with (9Z, 12Z) -octadeca-9,12-dienyl methanesulfonate (manufactured by Nu-Chek Prep, Inc., 1.24 g, 3.60 mmol) and 50% aqueous sodium hydroxide solution (1.44 g, 18.0 mmol) was added and stirred at 110 ° C. on an oil bath for 60 minutes. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate, washed with water and then with saturated brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (chloroform / methanol = 100/0 to 95/5) to give compound B-1 (0.838 g, yield 36.2%).
ESI-MS m / z: 515 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.89 (t, J = 6.9 Hz, 6 H), 1.30 (br s, 33 H), 1.41-1.54 (m, 4H), 2.01-2.09 (m, 8H), 2.59 (t, J = 7.2 Hz, 4H), 2.77 (t, J = 5.6 Hz, 4H), 5.28-5.43 (m, 8H).

Di ((Z) -octadeca-9-enyl) amine (Compound B-2)
Ammonia (manufactured by Tokyo Chemical Industry Co., Ltd., about 2 mol / L methanol solution, 12.0 mL, 24.0 mmol) and (Z) -octadec-9-enyl methanesulfonate (Nu-Chek Prep, Compound B-2 (0.562 g, 36.2% yield) was obtained using Inc. Inc. (1.87 g, 5.40 mmol).
ESI-MS m / z: 519 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.88 (t, J = 6.7 Hz, 6H), 1.29 (br s, 45 H), 1.41-1.52 (m, 4H), 1.97-2.05 (m, 8H), 2.58 (t, J = 7.2 Hz, 4H), 5.28-5.40 (m, 4H).

Di ((Z) -hexadeca-9-enyl) amine (compound B-3)
In the same manner as in Reference Example 1, ammonia (manufactured by SIGMA-ALDRICH, approximately 7 mol / L methanol solution, 1.66 mL, 11.6 mmol) and (Z) -hexadeca-9-enyl methanesulfonate ( Compound B-3 (0.243 g, 36.0% yield) was obtained using Nu-Chek Prep, Inc. 0.488 g (1.46 mmol).
¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 6.9 Hz, 6 H), 1.24-1. 37 (m, 37 H), 1.43-1.52 (m, 4 H), 1. 98-2.05 (m, 8 H), 2.58 (2.51 (m, 8 H) t, J = 7.2 Hz, 4H), 5.31-5.38 (m, 4H).

Di ((11Z, 14Z) -Ikosa-11,14-dienyl) amine (Compound B-4)
In the same manner as in Reference Example 1, ammonia (manufactured by SIGMA-ALDRICH, about 7 mol / L methanol solution, 1.60 mL, 11.2 mmol) and (11Z, 14Z) -icosa-11,14-dienyl methanesulfonate (Nu) -Compound B-4 (0.292 g, 36.6% yield) was obtained using Chek Prep, Inc. 0.521 g (1.40 mmol).
¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 6.8 Hz, 6 H), 1.24-1. 39 (m, 41 H), 1.43-1.51 (m, 4 H), 2.02-2.08 (m, 8 H), 2.58 (m t, J = 7.3 Hz, 4 H), 2. 77 (t, J = 6.7 Hz, 4 H), 5. 30-5. 41 (m, 8 H).

3- (Dimethylamino) propyl di ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (compound A-1)
The compound B-1 (1.35 g, 2.63 mmol) obtained in Example 1 is dissolved in chloroform (18 mL), and the “Journal of American Chemical Society (J. Am. Chem. Soc.)”, 1981 , 3- (Dimethylamino) propyl synthesized by the method according to the method described in p.103, p.
4-Nitrophenyl carbonate hydrochloride (Compound VI-1) (1.20 g, 3.94 mmol) and triethylamine (1.47 mL, 10.5 mmol) were added, and the mixture was stirred at 110 ° C. for 60 minutes using a microwave reactor. Compound VI-1 (200 mg, 0.658 mmol) was added to the reaction solution, and the mixture was stirred at 110 ° C. for 20 minutes using a microwave reactor. Compound VI-1 (200 mg, 0.658 mmol) was added to the reaction solution, and the mixture was stirred at 110 ° C. for 20 minutes using a microwave reactor. Compound VI-1 (200 mg, 0.658 mmol) was added to the reaction solution, and the mixture was stirred at 110 ° C. for 20 minutes using a microwave reactor. The reaction mixture was diluted with chloroform, washed three times with 1 mol / L aqueous sodium hydroxide solution, then washed with saturated brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The resulting residue was dissolved in a small amount of n-hexane / ethyl acetate (1/4), adsorbed onto a pad of amino-modified silica gel, eluted with n-hexane / ethyl acetate (1/4) and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (chloroform / methanol = 100/0 to 95/5) to obtain compound A-1 (1.39 g, yield 82.2%).
ESI-MS m / z: 644 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 6.7 Hz, 6H), 1.29 (br s, 32H), 1.45-1.56 (m, 4H), 1.74-1.85 (m, 2H), 2.00-2.09 (m, 8H), 2.23 (s, 6H), 2.35 (t, J = 7.4 Hz, 2H), 2.77 (t, J = 5.8 Hz, 4H ), 3.13-3.23 (m, 4H), 4.10 (t, J = 6.4 Hz, 2H), 5.28-5.43 (m, 8H).

3- (Dimethylamino) propyl di ((Z) -octadeca-9-enyl) carbamate
(Compound A-2)
In a similar manner to Example 5, a compound A-2 (0.267 g, 88.7% yield) was obtained using the compound B-2 (0.156 g, 0.301 mmol) obtained in Example 2 instead of the compound B-1 Obtained.
ESI-MS m / z: 648 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.88 (t, J = 6.6 Hz, 6 H), 1.28 (br s, 44 H), 1.45-1. 55 (m, 4H), 1.75-1.85 (m, 2H), 1.97-2.04 (m, 8H), 2.23 (s, 6H), 2.34 (t, J = 7.6 Hz, 2H), 3.13-3.24 (m, 4H), 4.10 (t, J = 6.4 Hz, 2H), 5.28-5.40 (m, 4H).

3- (Dimethylamino) propyl di ((Z) -hexadeca-9-enyl) carbamate
(Compound A-3)
In a similar manner to Example 5, a compound A-3 (0.116 g, 55.2% yield) was synthesized using the compound B-3 (0.164 g, 0.355 mmol) obtained in Example 3 instead of the compound B-1. Obtained.
ESI-MS m / z: 592 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.88 (t, J = 6.9 Hz, 6H), 1.21-1.38 (m, 36H), 1.47-1.54 (m , 4H), 1.75-1.83 (m, 2H), 2.00-2.04 (m, 8H), 2.22 (s, 6H), 2.34 (t, J = 7.4 Hz, 2H), 3.11-3.24 (m, 4H), 4.10 (t, J = 6.4 Hz, 2H), 5.30-5.38 (m, 4H).

3- (Dimethylamino) propyl di ((11Z, 14Z) -icosa-11,14-dienyl) carbamate (Compound A-4)
In a similar manner to Example 5, a compound A-4 (0.290 g, yield 82.2%) was obtained using the compound B-4 (0.288 g, 0.505 mmol) obtained in Example 4 instead of the compound B-1 Obtained.
ESI-MS m / z: 700 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 6.8 Hz, 6 H), 1.21-1.40 (m, 40 H), 1.46-1.54 (m , 4H), 1.76-1.83 (m, 2H), 2.02-2.08 (m, 8H), 2.23 (s, 6H), 2.35 (t, J = 7.6 Hz, 2H), 2.77 (t, J = 6.7 Hz, 4H), 3.10-3.24 (m, 4H), 4.10 (t, J = 6.4 Hz, 2H), 5.30-5.41 (m, 8H).

2- (Dimethylamino) ethyl di ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-5)
In a similar manner to Example 5, compound B-1 (0.215 g, 0.418 mmol) obtained in Example 1 and 2- (dimethylamino) ethyl 4-nitrophenyl carbonate hydrochloride (in place of compound VI-1) The compound A-2 (0.184 g, yield 70.0%) was obtained using the compound VI-2) (0.162 g, 0.557 mmol).
ESI-MS m / z: 630 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 6.8 Hz, 6 H), 1.12-1. 39 (m, 32 H), 1.45-1. 54 (m , 4H), 2.00-2.07 (m, 8H), 2.28 (s, 6H), 2.57 (t, J = 7.2 Hz, 2H), 2.77 (t, J = 6.7 Hz, 4H), 3.11-3.24 (m, 4H), 4.17 (t, J = 6.7 Hz, 2H), 5.28-5.41 (m, 8H).

Reference Example 1 5-Amino-N, N-di ((9Z, 12Z) -octadeca-9,12-dienyl) pentanamide (Compound XI-1)
Compound B-1 (150 mg, 0.292 mmol) obtained in Example 1 is dissolved in chloroform (4 mL), and 5- (tert-butoxycarbonylamino) pentanoic acid (manufactured by Tokyo Chemical Industry Co., Ltd., 95 mg, 0.438 mmol) ), Diisopropylethylamine (0.255 mL, 1.46 mmol) and HATU (O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate) (Aldrich) , 222 mg, 0.584 mmol) was added and stirred at room temperature for 4 hours. To the reaction mixture was added saturated aqueous sodium hydrogen carbonate solution, and the aqueous layer was extracted with ethyl acetate. The organic layer was washed with water and saturated brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The resulting residue is purified by silica gel column chromatography (chloroform / methanol = 100/0 to 97/3) to give tert-butyl 5- (di ((9Z, 12Z) -octadeca-9,12-dienyl) amino ) 5-Oxopentyl carbamate was obtained.
Dissolve tert-butyl 5- (di ((9Z, 12Z) -octadeca-9,12-dienyl) amino) -5-oxopentylcarbamate in dichloromethane (4 mL) and add trifluoroacetic acid (0.450 mL, 5.84 mmol) ) Was added and stirred at room temperature for 4 hours. To the reaction mixture was added saturated aqueous sodium hydrogen carbonate solution, and the aqueous layer was extracted with chloroform. The organic layer was washed with brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (chloroform / methanol = 100/0 to 90/10) to obtain compound XI-1 (124 mg, yield 96.1%).
ESI-MS m / z: 614 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 6.8 Hz, 6 H), 1.28-1.38 (m, 32 H), 1.43-1.57 (m , 6H), 1.63-1.73 (m, 2H), 2.05 (q, J = 7.0 Hz, 8 H), 2.30 (t, J = 7.2 Hz, 2 H), 2.71 (t, J = 7.2 Hz, 2 H), 2.77 (t, J = 6.2 Hz, 4 H), 3. 19 (t, J = 7.7 Hz, 2 H), 3. 28 (t, J = 7.7 Hz, 2 H), 5. 28-5. 43 (m, 8 H).

Reference Example 2 5- (Dimethylamino) -N, N-di ((9Z, 12Z) -octadeca-9,12-dienyl) pentanamide (Compound XI-2)
Compound XI-1 (90.0 mg, 0.147 mmol) obtained in Reference Example 1 is dissolved in 1,2-dichloroethane (2 mL) and methanol (2 mL), formaldehyde (0.219 mL, 2.94 mmol) and sodium triacetoxyborohydride Hydride (manufactured by Acros Organics, 311 mg, 1.47 mmol) was added and the mixture was stirred at room temperature for 5 hours. To the reaction solution was added saturated aqueous sodium hydrogen carbonate solution, and the aqueous layer was extracted with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (chloroform / methanol = 100/0 to 75/25) to obtain compound XI-2 (88.2 mg, yield 93.9%).
ESI-MS m / z: 642 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.89 (t, J = 7.0 Hz, 6 H), 1.26-1.38 (m, 32 H), 1.46-1.71 (m , 8H), 2.05 (q, J = 7.0 Hz, 8 H), 2.22 (s, 6 H), 2.29 (q, J = 7.0 Hz, 4 H), 2.77 (t, J = 6.2 Hz, 4 H), 3.19 (t , J = 7.9 Hz, 2H), 3.28 (t, J = 7.7 Hz, 2H), 5.28-5.42 (m, 8H).

Reference Example 3 3-aminopropyl di ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (compound XI-3)
The compound B-1 (146 mg, 0.284 mmol) obtained in Example 1 was dissolved in N, N-dimethylformamide (5 mL), and the “Journal of American Chemical Society (J. Am. Chem. Soc. Tert-Butyl 3-((4-nitrophenoxy) carbonyloxy) propylcarbamate (145 mg, 0.426 mmol) synthesized by the method according to the method described in 1981, Vol. 103, p. ) And triethylamine (0.158 mL, 1.14 mmol) were added and stirred at room temperature overnight. Water was added to the reaction mixture, and the aqueous layer was extracted with ethyl acetate. The organic layer was washed with water and saturated brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The resulting residue is purified by silica gel column chromatography (chloroform / methanol = 100/0 to 98/2) to give 3-((N-butoxycarbonyl) amino) propyl di ((9Z, 12Z) -octadeca-9. , 12-dienyl) carbamate was obtained.
Dissolve 3-((N-butoxycarbonyl) amino) propyl di ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate in dichloromethane (4 mL) and trifluoroacetic acid (0.242 mL, 3.13 mmol) The mixture was further stirred at room temperature for 8 hours. To the reaction mixture was added saturated aqueous sodium hydrogen carbonate solution, and the aqueous layer was extracted with chloroform. The organic layer was washed with brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (NH silica gel, chloroform / methanol = 100/0 to 90/10) to obtain compound XI-3 (75.6 mg, 43.3% yield).
ESI-MS m / z: 616 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 6.8 Hz, 6 H), 1.26-1.38 (m, 32 H), 1.46-1.54 (m , 4H), 1.73-1.82 (m, 2H), 2.05 (q, J = 6.6 Hz, 8 H), 2. 76-2.80 (m, 6 H), 3. 18 (br s, 4 H), 4. 15 (t, J = 6.2 Hz , 2H), 5.29-5.43 (m, 8H).

Reference Example 4 2- (1-Methylpyrrolidin-2-yl) ethyl 4-nitrophenyl carbonate hydrochloride (Compound VI-3)
To a solution of 4-nitrophenyl chloroformate (manufactured by Tokyo Chemical Industry Co., Ltd., 1.761 g, 8.56 mmol) in diethyl ether (20 mL), 2- (1-methylpyrrolidin-2-yl) ethanol (manufactured by Tokyo Chemical Industry Co., Ltd.) A solution of 1.0 mL, 7.13 mmol) in diethyl ether (20 mL) was added and stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and the obtained residue was crystallized from ethanol / diethyl ether (1/1), and collected by filtration to give compound VI-3 (1.27 g, yield 54%).
¹ H-NMR (DMSO-d6) δ: 1.59-1.77 (m, 2H), 1.82-2.09 (m, 3H), 2.15-2.26 (m, 1H), 2.76 (s, 3H), 2.93-3.05 (m , 2H), 3.61-3.20 (m, 3H), 4.80 (br s, 1H), 6.95 (d, J = 9.2 Hz, 2 H), 8.11 (d, J = 9.2 Hz, 2 H)

Reference Example 5 4-Nitrophenyl 3- (piperidin-1-yl) propyl carbonate hydrochloride (Compound VI-4)
To a solution of 4-nitrophenyl chloroformate (1.58 g, 7.67 mmol) in diethyl ether (32 mL), 3- (piperidin-1-yl) propan-1-ol (SIGMA-ALDRICH, 1.00 mL, 6.39 mmol) ) Was added and stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and the obtained residue was crystallized from ethanol and collected by filtration to give compound VI-4 (1.86 g, yield 84%).
ESI-MS m / z: 309 (M + H) ⁺ ; ¹ H-NMR (DMSO-d6) δ: 1.28-1.49 (m, 1 H), 1.62-1.89 (m, 5 H), 2.10-2.26 (m, 2H), 2.76-2.96 (m, 2H), 3.04-3.19 (m, 2H), 3.36-3.49 (m, 2H), 4.33 (t, J = 6.1 Hz, 2H), 7.58 (d, J = 9.2 Hz) , 2H), 8.33 (d, J = 9.2 Hz, 2H), 10.37 (br s, 1 H).

Reference Example 6 4-Nitrophenyl 3- (pyrrolidin-1-yl) propyl carbonate hydrochloride (Compound VI-5)
To a solution of 4-nitrophenyl chloroformate (596 mg, 2.84 mmol) in diethyl ether (10 mL), 3- (pyrrolidin-1-yl) propan-1-ol (manufactured by ABCR, 386 mg, 2.84 mmol) diethyl A solution of ether (10 mL) was added and stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and the obtained residue was crystallized from ethanol and collected by filtration to give compound VI-5 (498 mg, yield 53%).
¹ H-NMR (DMSO-d6) δ: 1.76-1.84 (m, 2H), 1.85-2.00 (m, 4H), 3.11-3.16 (m, 2H), 3.30-3.44 (m, 4H), 3.47 (t) , J = 6.0 Hz, 2 H), 4, 77 (br s, 1 H), 6. 95 (d, J = 9.2 Hz, 2 H), 8.1 1 (d, J = 9.2 Hz, 2 H)

2- (1-Methylpyrrolidin-2-yl) ethyl di ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-6)
Compound B-1 (0.161 g, 0.314 mmol) obtained in Example 1 is dissolved in acetonitrile (3.0 mL), Compound VI-3 (0.156 g, 0.470 mmol) obtained in Reference Example 4 and triethylamine (0.219 mL) 1.57 mmol) was added and stirred at 80 ° C. for 2 hours. The reaction solution was diluted with ethyl acetate, washed with water, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by amino silica gel column chromatography (n-hexane / ethyl acetate = 80/20) to give compound A-6 (0.172 g, yield 82%).
ESI-MS m / z: 670 (M + H) +; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 6.9 Hz, 6 H), 1.20-1. 40 (m, 32 H), 1.45-1. 57 (m , 6H), 1.62-1.83 (m, 2H), 1.94-2.18 (m, 12H), 2.31 (s, 3H), 2.77 (t, J = 6.7 Hz, 4H), 3.03-3.26 (m, 5H), 4.06-4.17 (m, 2H), 5.29-5.42 (m, 8H).

3- (Piperidine-1-yl) propyl di ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (compound A-7)
In the same manner as in Example 5, a compound A-7 (0.387 g, yield 81%) was obtained using the compound VI-4 obtained in Reference Example 5 instead of the compound VI-1.
ESI-MS m / z: 684 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.89 (t, J = 6.9 Hz, 6H), 1.21-1.62 (m, 42H), 1.79-1.86 (m , 2H), 2.02-2.08 (m, 8H), 2.32-2.42 (m, 6H), 2.77 (t, J = 6.7 Hz, 4H), 3.10-3.43 (m, 4 H), 4.09 (t, J = 6.4) Hz, 2H), 5.29-5.42 (m, 8H).

3- (Pyrrolidin-1-yl) propyl di ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (compound A-8)
In a similar manner to Example 10, Compound A-8 (0.225 g, yield 99%) was obtained using Compound VI-5 (0.168 g, 0.508 mmol) obtained in Reference Example 6 instead of Compound VI-3. Obtained.
ESI-MS m / z: 670 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 6.9 Hz, 6 H), 1.21-1.40 (m, 32 H), 1.46-1.55 (m , 4H), 1.76-1.80 (m, 4H), 1.82-1.89 (m, 2H), 2.01-2.08 (m, 8H), 2.47-2.55 (m, 6H), 2.77 (t, J = 6.7 Hz, 4H ), 3.11-3.24 (m, 4H), 4.11 (t, J = 6.4 Hz, 2H)
, 5.29-5.42 (m, 8H).

Reference Example 7 2-Nitro-N-((9Z, 12Z) -octadeca-9,12-dienyl) benzenesulfonamide (Compound IId-1)
Cesium carbonate (6.74 g, 20.67 mmol) in a solution of (9Z, 12Z) -octadeca-9,12-dienyl methanesulfonate (manufactured by Nu-Chek Prep, Inc., 2.85 g, 8.27 mmol) in acetonitrile (30 ml) Tetrabutylammonium iodide (manufactured by Tokyo Chemical Industry Co., Ltd., 3.05 g, 8.27 mmol) and N- (tert-butoxycarbonyl) -2-nitrobenzenesulfonamide (manufactured by Tokyo Chemical Industry Co., Ltd., 2.50 g, 8.27 mmol); The reaction was conducted while heating under reflux for 3 hours. The reaction solution was cooled to room temperature, water was added, and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue is purified by column chromatography (n-hexane / ethyl acetate = 91/9 to 70/30) to give tert-butyl (2-nitrophenyl) sulfonyl ((9Z, 12Z) -octadeca-9,12- Dien-1-yl) carbamate (3.21 g, 70.5% yield) was obtained.
Trifluoroacetic acid (9.63) in a solution of tert-butyl (2-nitrophenyl) sulfonyl ((9Z, 12Z) -octadeca-9,12-dien-1-yl) carbamate (3.21 g, 5.83 mmol) in dichloromethane (22.5 ml) ml, 126 mmol) was added and stirred at room temperature for 0.5 h. Dichloromethane and aqueous sodium hydroxide solution (1 mol / L, 100 mL) were added to the reaction solution, and saturated aqueous sodium hydrogen carbonate solution was further added to adjust the pH of the aqueous layer to 8 or more. The resulting mixture was extracted with dichloromethane, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (n-hexane / chloroform = 50/50 to 0/100) to obtain compound IId-1 (2.48 g, yield 94%).
ESI-MS m / z: 451 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 7.0 Hz, 3 H), 1.22-1.39 (m, 16 H), 1.52 (m, 2 H ), 2.01-2.05 (m, 4H), 2.77 (t, J = 6.6 Hz, 2 H), 3.09 (q, J = 6.7 Hz, 2 H), 5.23 (m, 1 H), 5.31-5.42 (m, 4 H) , 7.71-7.76 (m, 2H), 7.78-7.87 (1 H), 8.13-8.15 (m, 1 H).

Dodecyl ((9Z, 12Z) -octadeca-9,12-dien-1-yl) amine (compound B-5)
Tetrabutylammonium iodide (Tokyo Kasei Kogyo Co., Ltd.) in a solution of compound IId-1 (0.714 g, 1.584 mmol) obtained in Reference Example 7 and 1-bromododecane (manufactured by Tokyo Kasei Kogyo Co., Ltd., 0.474 g, 1.90 mmol) in acetonitrile (6 ml) Industrial Co., Ltd. 0.585 g (1.58 mmol) and cesium carbonate (1.03 g, 3.17 mmol) were added, and the mixture was stirred at 60 ° C. for 1 hour. Water was added to the reaction solution, and extracted three times with n-hexane. The extract is purified by column chromatography (n-hexane / ethyl acetate = 94/6 to 84/16) to give N-dodecyl-2-nitro-N-((9Z, 12Z) -octadeca-9,12-diene -1-yl) benzenesulfonamide (0.7
50 g, yield 76%) were obtained.
1-dodecane in a solution of N-dodecyl-2-nitro-N-((9Z, 12Z) -octadeca-9,12-dien-1-yl) benzenesulfonamide (0.748 g, 1.21 mmol) in acetonitrile (7 ml) Add thiol (manufactured by Tokyo Chemical Industry, 0.611 g, 3.02 mmol) and 1,8-diazabicyclo [5.4.0] -7-undecene (manufactured by Nacalai Tesque, 0.460 g, 3.02 mmol) and stir at 60 ° C. for 1 hour did. Water was added to the reaction solution, and extracted twice with ethyl acetate. The organic layers were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (n-hexane / ethyl acetate = 80: 20, then chloroform / methanol = 100/0 to 88/12) to give compound B-5 (0.534 g, quantitative yield) The
ESI-MS m / z: 434 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.88 (t, J = 6.9 Hz, 3 H), 0.89 (t, J = 6.9 Hz, 3 H), 1.24- 1.38 (m, 35 H), 1.49-1.54 (m, 4 H), 2.05 (q, J = 7.0 Hz, 4 H), 2.62 (t, J = 7.4 Hz, 4 H), 2. 77 (t, J = 6.8 Hz, 2 H ), 5.30-5.41 (m, 4H).

Decyl ((9Z, 12Z) -octadeca-9,12-dien-1-yl) amine (Compound B-6)
In a similar manner to Example 13, compound IId-1 (0.619 g, 1.37 mmol) obtained in Reference Example 7 and 1-bromodecane instead of 1-bromododecane (manufactured by Tokyo Chemical Industry Co., Ltd., 0.365 g, 1.65 mmol) Compound B-6 (0.423 g, 76% yield) was obtained using
ESI-MS m / z: 406 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.88 (t, J = 7.1 Hz, 3 H), 0.89 (t, J = 6.9 Hz, 3 H), 1.25- 1.38 (m, 31 H), 1.46-1.50 (m, 4 H), 2.05 (q, J = 7.0 Hz, 4 H), 2.59 (t, J = 7.2 Hz, 4 H), 2. 77 (t, J = 6.9 Hz, 2 H ), 5.31-5.40 (m, 4H).

((9Z, 12Z) -octadeca-9,12-dien-1-yl) (octyl) amine (compound B-7)
In a similar manner to Example 13, compound IId-1 (0.714 g, 1.58 mmol) obtained in Reference Example 7 and 1-bromooctane (manufactured by Tokyo Chemical Industry Co., Ltd., 0.367 g, 1.90) instead of 1-bromododecane Compound B-7 (0.519 g, yield 87%) was obtained using mmol).
ESI-MS m / z: 378 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.88 (t, J = 7.1 Hz, 3 H), 0.89 (t, J = 6.9 Hz, 3 H), 1.24- 1.39 (m, 27 H), 1.48-1.54 (m, 4 H), 2.05 (q, J = 7.0 Hz, 4 H), 2.62 (t, J = 7.4 Hz, 4 H), 2. 77 (t, J = 6.6 Hz, 2 H ), 5.30-5.41 (m, 4H).

3- (Dimethylamino) propyl dodecyl ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-9)
In a similar manner to Example 5, a compound A-9 (0.309 g, yield 91%) was obtained using the compound B-5 (0.260 g, 0.600 mmol) obtained in Example 13 instead of the compound B-1 Obtained.
ESI-MS m / z: 563 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.88 (t, J = 6.9 Hz, 3 H), 0.89 (t, J = 6.9 Hz, 3 H), 1.23- 1.38 (m, 34H), 1.48-1.53 (m, 4H), 1.77-1.82 (m, 2H), 2.05 (q, J = 7.1 Hz, 4H), 2.23 (s, 6H), 2.34 (t, J = 7.6 Hz, 2 H), 2. 77 (t, J = 6.6 Hz, 2 H), 3.13-3.22
(m, 4H), 4.10 (t, J = 6.5 Hz, 2H), 5.30-1.41 (m, 4H).

3- (Dimethylamino) propyl decyl ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (compound A-10)
In a similar manner to Example 5, the compound B-6 obtained in Example 14 (0.185 g) was substituted for the compound B-1
The compound A-10 (0.228 g, 93% yield) was obtained using 0.456 mmol).
ESI-MS m / z: 535 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.88 (t, J = 6.8 Hz, 3 H), 0.89 (t, J = 6.7 Hz, 3 H), 1.24- 1.38 (m, 30H), 1.48-1.53 (m, 4H), 1.77-1.82 (m, 2H), 2.05 (q, J = 7.0 Hz, 4H), 2.22 (s, 6H), 2.34 (t, J = 7.5 Hz, 2 H), 2. 77 (t, J = 6.8 Hz, 2 H), 3.14-3.22 (m, 4 H), 4. 10 (t, J = 6.4 Hz, 2 H), 5.31-5. 40 (m, 4 H).

3- (Dimethylamino) propyl ((9Z, 12Z) -octadeca-9,12-dienyl) (octyl) carbamate (Compound A-11)
In a similar manner to Example 5, a compound A-11 (0.275 g, 90% yield) was synthesized using the compound B-7 (0.227 g, 0.600 mmol) obtained in Example 15 instead of the compound B-1. Obtained.
ESI-MS m / z: 507 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.88 (t, J = 6.9 Hz, 3 H), 0.89 (t, J = 6.9 Hz, 3 H), 1.22- 1.39 (m, 26 H), 1.47-1. 54 (m, 4 H), 1. 76-1.82 (m, 2 H), 2.05 (q, J = 6.0 Hz, 4 H), 2.23 (s, 6 H), 2. 34 (t, J = 7.6 Hz, 2 H), 2. 77 (t, J = 6.6 Hz, 2 H), 3. 12-3. 22 (m, 4 H), 4. 10 (t, J = 6.5 Hz, 2 H), 5. 30-5. 41 (m, 4 H).

Reference Example 8 2-((9Z, 12Z) -octadeca-9,12-dienyloxy) ethyl methanesulfonate (compound IIIc-1)
Ethylene glycol (3.16 ml, 57.1 mmol) and 1,4-dioxane (5 ml) are added to (9Z, 12Z) -octadeca-9,12-dien-1-yl methanesulfonate (983 mg, 2.85 mmol), The mixture was heated and stirred for 1 day under reflux. The reaction mixture was cooled to room temperature, aqueous sodium hydroxide solution (0.5 mol / L) was added, and the mixture was extracted twice with n-hexane. The organic layers were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (chloroform 100%) to give 2-((9Z, 12Z) -octadeca-9,12-dienyloxy) ethanol (668 mg, yield 75%).
Mesilic anhydride (0 ° C.) in a solution of 2-((9Z, 12Z) -octadeca-9,12-dienyloxy) ethanol (660 mg, 2.13 mmol) and triethylamine (0.444 mL, 3.19 mmol) in dichloromethane (9 ml) A SIGMA-ALDRICH company product (0.247 ml, 3.19 mmol) was added and stirred at room temperature for 40 minutes. Water was added to the reaction solution, and extracted with chloroform. The organic layer was washed with hydrochloric acid (1 mol / L), saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure to give compound IIIc-1.

(9Z, 12Z) -N- (2-((9Z, 12Z) -octadeca-9,12-dienyloxy) ethyl) octadeca-9,12-dien-1-amine (compound B-8)
In a similar manner to Example 13, Compound IId-1 (0.798 g, 1.77 mmol) obtained in Reference Example 7 and Compound IIIc-1 (0.826 g, 2.13) obtained in Reference Example 8 instead of 1-bromododecane Compound B-8 (0.676 g, 68% yield) was obtained using mmol).
ESI-MS m / z: 558 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.89 (t, J = 6.9 Hz, 6H), 1.27-1.38 (m, 32H), 1.46-1.52 (m , 2H), 1.54-1.60 (m, 3H), 2.05 (q, J = 7.0 Hz, 8 H), 2.61 (t, J = 7.3 Hz, 2 H), 2.77 (t, J = 5.5 Hz, 6 H), 3.42 (t, J = 6.8 Hz, 2 H), 3.52 (t, J = 5.4 Hz, 2 H), 5.30-5.41 (m, 8 H).

3- (Dimethylamino) propyl ((9Z, 12Z) -octadeca-9,12-dienyl) (2-((9Z, 12Z) -octadeca-9,12-dienyloxy) ethyl) carbamate (Compound A-12)
In a similar manner to Example 5, a compound A-12 (0.208 g, 92% yield) was synthesized using the compound B-8 (0.184 g, 0.330 mmol) obtained in Example 19 instead of the compound B-1. Obtained.
ESI-MS m / z: 687 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 6.9 Hz, 6 H), 1.25-1.38 (m, 32 H), 1.50-1. 57 (m , 4H), 1.77-1.83 (m, 2H), 2.05 (q, J = 7.0 Hz, 8 H), 2.22 (s, 6 H), 2.34 (t, J = 7.4 Hz, 2 H), 2.77 (t, J = 6.6 Hz, 4H), 3.23-3.54 (m, 8H), 4.11 (t, J = 6.5 Hz, 2H), 5.30-1.41 (m, 8H).

Reference Example 9 N, N-bis (2-((9Z, 12Z) -octadeca-9,12-dienyloxy) ethyl) amine (Compound XI-4)
After slowly adding a solution of N-benzyldiethanolamine (manufactured by Tokyo Chemical Industry Co., Ltd., 1.65 g, 8.44 mmol) in toluene (10 mL) to sodium hydride (oiliness, 60%, 1.69 g, 42.2 mmol) with stirring. A solution of (9Z, 12Z) -octadeca-9,12-dienyl methanesulfonate (manufactured by Nu-Chek Prep, Inc., 6.69 g, 19.4 mmol) in toluene (10 mL) was dropped. The resulting mixture was stirred under heat reflux for 4 hours. After cooling to room temperature, the reaction was quenched with ethanol. To the resulting mixture was added saturated brine and extracted twice with ethyl acetate. The organic layers were combined, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue is purified by silica gel column chromatography (chloroform / methanol = 100/0 to 99/1) to give N-benzyl-N, N-bis (2-((9Z, 12Z) -octadeca-9,12-dienyloxy). ) Ethyl) amine (4.01 g, 69% yield) was obtained.
Dissolve N-benzyl-N, N-bis (2-((9Z, 12Z) -octadeca-9,12-dienyloxy) ethyl) amine (4.01 g, 5.89 mmol) in 1,2-dichloroethane (29 mL) The reaction mixture was added with 1-chloroethyl chloroformate (1.90 mL, 17.4 mmol, manufactured by Tokyo Chemical Industry Co., Ltd.) and stirred at 130 ° C. for 1 hour. Methanol (29 mL) was added to the reaction solution, and the mixture was further stirred at 130 ° C. for 1 hour. After cooling to room temperature, a saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, and the mixture was extracted twice with chloroform. The organic layers were combined, washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by amino silica gel column chromatography (n-hexane / ethyl acetate = 90/10 to 75/25) to give compound XI-4 (5.56 g, yield 92%).
ESI-MS m / z: 621 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 7.0 Hz, 6 H), 1.27-1.30 (m, 33 H), 1.53-1.59 (m, 4H), 2.05 (q, J = 7.1 Hz, 8H), 2.77 (t, J = 6.8 Hz, 4H), 2.80 (t, J = 5.4 Hz, 4H), 3.42 (t, J = 6.8 Hz, 4H) , 3.53 (t, J = 5.4 Hz, 4 H), 5.30-5.41 (m, 8 H).

Reference Example 10 3- (Dimethylamino) propyl bis (2-((9Z, 12Z) -octadeca-9,12-dienyloxy) ethyl) carbamate
(Compound XI-5)
In a similar manner to Example 5, a compound XI-4 (1.32 g, yield 91%) was synthesized using the compound XI-4 (1.20 g, 1.99 mmol) obtained in Reference Example 9 instead of the compound B-1. Obtained.
ESI-MS m / z: 732 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.89 (t, J = 7.0 Hz, 6 H), 1.27-1.30 (m, 30 H), 1.51-1.57 (m, 4H), 1.77-1.83 (m, 4H), 2.05 (q, J = 7.0 Hz, 8 H), 2.23 (s, 6 H), 2.34 (t, J = 7.5 Hz, 2 H), 2.77 (t, J = 6.7) Hz, 4H), 3.38-3.54 (m, 12H), 4.12 (t, J = 6.5 Hz, 2H), 5.30-5.41 (m, 8H).

Reference Example 11 Ethyl 3- (di ((9Z, 12Z) -octadeca-9,12-dienyl) amino) propionate (Compound XI-6)
Compound B-1 (0.788 g, 1.53 mmol) obtained in Example 1 is dissolved in ethanol (8 mL), ethyl acrylate (manufactured by Tokyo Chemical Industry Co., Ltd., 1.67 mL, 15.3 mmol) and sodium ethoxide (Wako pure) YAGCO (0.0520 g, 0.767 mmol) was added, and the mixture was stirred for 3 hours while heating under reflux. The reaction mixture is concentrated under reduced pressure, and the obtained residue is purified by silica gel column chromatography (n-hexane / ethyl acetate = 85/15) to give compound XI-6 (0.699 g, yield 74%) The
ESI-MS m / z: 615 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 6.9 Hz, 6 H), 1.21-1.45 (m, 39 H), 2.02-2.08 (m , 8H), 2.35-2.44 (m, 6H), 2.75-2.80 (m, 6H), 4.12 (q, J = 7.0 Hz, 2H), 5.30-5.42 (m, 8H).

3- (di ((9Z, 12Z) -octadeca-9,12-dienyl) amino) propan-1-ol (compound C-1)
Compound XI-6 (0.199 g, 0.324 mmol) obtained in Reference Example 11 is dissolved in tetrahydrofuran (2 mL), lithium aluminum hydride (manufactured by Junsei Chemical Co., 0.012 g, 0.324 mmol) is added under ice-cooling, Stir for 3 hours. Water (0.0600 mL, 3.33 mmol) and sodium fluoride (Nacalai Tesque, 0.408 g, 9.72 mmol) were added to the reaction solution, and the mixture was stirred at room temperature for 0.5 hours. The insolubles were removed by celite filtration. The filtrate was concentrated and then purified by silica gel column chromatography (chloroform / methanol = 98/2) to give compound C-1 (0.181 g, yield 98%).
ESI-MS m / z: 573 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.89 (t, J = 6.9 Hz, 6H), 1.21-1.40 (m, 32H), 1.42-1.51 (m , 4H), 1.64-1.71 (m, 2H), 2.02-2.08 (m, 8H), 2.40 (t, J = 7.3 Hz, 4H), 2.64 (t, J = 5.3 Hz, 2H), 2.77 (t, J) J = 6.7 Hz, 4 H), 3.79 (t, J = 5.3 Hz, 2 H), 5.30-5.42 (m, 8 H).

Reference Example 12 3- (di ((9Z, 12Z) -octadeca-9,12-dienyl) amino) propane-1,2-diol (Compound XI-7)
Compound B-1 (0.228 g, 0.444 mmol) obtained in Example 1 is dissolved in dichloroethane (2 mL), methanol (2 mL) and 2,3-dihydroxypropanal (Nacalai Tesque, 0.400 g, 4.44) The mmol) was added and it stirred at room temperature for 0.5 hour. Sodium triacetoxyborohydride (manufactured by Tokyo Chemical Industry Co., Ltd., 0.470 g, 2.22 mmol) was added to the reaction solution, and the mixture was stirred overnight at room temperature.
To the reaction mixture, 2,3-dihydroxypropanal (0.400 g, 4.44 mmol) was added, and the mixture was stirred at room temperature for 3 hours. To the reaction solution was added sodium triacetoxyborohydride (0.470 g, 2.22 mmol), and the mixture was stirred overnight at room temperature. To the reaction mixture was added saturated aqueous sodium hydrogen carbonate solution, and the mixture was extracted twice with chloroform. The organic layers were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The obtained residue was purified by amino silica gel column chromatography (n-hexane / ethyl acetate = 50/50) to give compound XI-7 (0.0449 g, yield 17%).
ESI-MS m / z: 589 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.89 (t, J = 6.9 Hz, 6 H), 1.19-1.51 (m, 36 H), 2.02-2.08 (m , 8H), 2.38-2.62 (m, 6H), 2.77 (t, J = 6.7 Hz, 4H), 3.46-3.50 (m, 1H), 3.69-3.77 (m, 2H), 5.30-5.42 (m, 8H) ).

Reference Example 13 3-((3R, 4R) -3,4-bis ((9Z, 12Z) -octadeca-9,12-dienyloxy) pyrrolidin-1-yl) propan-1-ol (Compound XI-8)
Compound XI-8 was synthesized by the method described in WO 2011/136368.

4- (Dimethylamino) butyl di ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-13)
Step 1
4- (tert-Butyldimethylsilyl) oxy-1-butanol (SIGMA-ALDRICH; 1.0 mL, 4.21 mmol) in a solution of 4-nitrophenyl chloroformate (0.867 g, 4.21 mmol) in dichloromethane (20 mL) And triethylamine (0.881 mL, 6.32 mmol) were added and stirred at room temperature for 1 hour. To the reaction mixture was added saturated aqueous sodium hydrogen carbonate solution, and the mixture was extracted twice with chloroform. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The resulting residue is purified by silica gel column chromatography (n-hexane / ethyl acetate = 90/10) to give 4- (tert-butyldimethylsilyloxy) butyl 4-nitrophenyl carbonate (1.44 g, yield The rate is 92%).
Step 2
In a similar manner to Example 10, 4- (tert-butyldimethylsilyloxy) butyl 4-nitrophenyl carbonate (0.640 g, 1.733 mmol) obtained in Step 1 was used in place of compound VI-3 to give 4- A crudely purified product of (tert-butyldimethylsilyloxy) butyl di ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate was obtained. The crude product of the obtained 4- (tert-butyldimethylsilyloxy) butyl di ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate is dissolved in tetrahydrofuran (10 mL), and the tetrabutylammonium fluoride is dissolved. (The Tokyo Chemical Industry Co., Ltd., about 1 mol / L tetrahydrofuran solution, 2.14 mL, 2.14 mmol) was added, and it stirred at room temperature for 1 hour. Tetra-n-butylammonium fluoride (about 1 mol / L tetrahydrofuran solution, 2.14 mL, 2.14 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 2 hours and then at 50 ° C. for 1 hour. To the reaction mixture was added saturated aqueous sodium hydrogen carbonate solution, and the mixture was extracted twice with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The resulting residue is purified by silica gel column chromatography (chloroform / methanol = 95/5) to give 4-hydroxybutyl di ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (0.652 g, yield The rate is 73%).
Step 3
A solution of 4-hydroxybutyl di ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (0.193 g, 0.306 mmol) obtained in step 2 in dichloromethane (2 mL) under ice-cooling mesylate chloride (Manufactured by Junsei Chemical Co., Ltd., 0.0360 mL, 0.460 mmol) and triethylamine (0.0930 mL, 0.919 mmol) were added, and the mixture was stirred at 0 ° C. for 30 minutes. To the reaction mixture was added saturated aqueous sodium hydrogen carbonate solution, and the mixture was extracted twice with chloroform. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The obtained residue is dissolved in tetrahydrofuran (1 mL), dimethylamine (manufactured by Aldrich, approx. 2 mol / L tetrahydrofuran solution, 1.53 mL, 3.06 mmol) is added, and the mixture is heated at 100 ° C. for 1 hour using a microwave reactor. After stirring, it was stirred at 130 ° C. for 1 hour using a microwave reactor. The reaction mixture is concentrated under reduced pressure, and the obtained residue is purified by amino silica gel column chromatography (n-hexane / ethyl acetate = 80/20) to give compound A-13 (0.159 g, yield 79%) The
ESI-MS m / z: 658 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.90 (q, J = 6.5 Hz, 6 H), 1.20-1.38 (m, 32 H), 1.45-1. 56 (m , 6H), 1.61-1.69 (m, 2H), 2.01-2.08 (m, 8H), 2.21 (s, 6H), 2.28 (t, J = 7.6 Hz, 2 H), 2.77 (t, J = 6.6 Hz, 4H), 3.12-3.23 (m, 4H), 4.07 (t, J = 6.6 Hz, 2H), 5.29-5.42 (m, 8H).

Reference Example 14 (1-Methylpiperidin-4-yl) methyl 4-nitrophenyl carbonate hydrochloride (Compound VI-6)
To a solution of 4-nitrophenyl chloroformate (1.50 g, 7.28 mmol) in tetrahydrofuran (32 mL) was added 1-methyl-4-piperidinemethanol (manufactured by Tokyo Chemical Industry Co., Ltd., 1.0 mL, 7.28 mmol), and the mixture was stirred at room temperature. Stir for 2 hours. The precipitated crystals were collected by filtration to give compound VI-6 (1.55 g, yield 64%).
ESI-MS m / z: 295 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 1.93-2.19 (m, 4 H), 2.68-2.82 (m, 3 H), 3.51-3. 62 (m, 5 H) , 4.21 (d, J = 6.0 Hz, 2 H), 7.38 (d, J = 9.1 Hz, 2 H), 8. 27 (d, J = 9.1 Hz, 2 H), 12.44 (br s, 1 H).

Reference Example 15 (1-Methylpiperidin-3-yl) methyl 4-nitrophenyl carbonate hydrochloride (Compound VI-7)
In a similar manner to Reference Example 14, compound VI-7 (2.32) was prepared using 1-methyl-3-piperidinemethanol (manufactured by Tokyo Chemical Industry Co., Ltd., 1.0 mL, 7.21 mmol) instead of 1-methyl-4-piperidinemethanol g, yield 97%).
ESI-MS m / z: 295 (M + H) +.

Reference Example 16 (1-Methylpiperidin-2-yl) methyl 4-nitrophenyl carbonate hydrochloride (Compound VI-8)
In a similar manner to Reference Example 14, compound VI-8 (2.37) was prepared using 1-methyl-2-piperidinemethanol (manufactured by Tokyo Chemical Industry Co., Ltd., 1.0 mL, 7.43 mmol) in place of 1-methyl-4-piperidinemethanol. g, yield 96%).
¹ H-NMR (CDCl 3) δ: 1.51-1.63 (m, 1H), 1.81-2.38 (m, 5H), 2.85-2.99 (m, 4H), 3.21-3.30 (m, 1H), 3.49-3.60 (m , 1H), 4.66 (dd, J = 13.1, 2.4 Hz, 1 H), 4.78-4.86 (m, 1 H), 7.47 (d, J = 9.1 Hz, 2 H), 8.28 (d, J = 9.1 Hz, 2 H) ), 12.40 (br s, 1 H).

Reference Example 17 3- (Azepan-1-yl) propyl 4-Nitrophenyl Carbonate Hydrochloride (Compound VI-9)
Compound VI was obtained by using 3- (azepan-1-yl) propanol (manufactured by CHEMBRIDGE, 0.700 g, 4.45 mmol) in place of 1-methyl-4-piperidinemethanol in the same manner as in Reference Example 14. -9 (1.47 g, 92% yield) was obtained.
ESI-MS m / z: 323 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 1.60-1.75 (m, 2H), 1.79-1.94 (m, 5H), 2.15-2.27 (m, 2H) , 2.44-2.53 (m, 2H), 2.90-3.02 (m, 2H), 3.14-3.24 (m, 2H), 3.55-3.65 (m, 2H), 4.41 (t, J = 5.9 Hz, 2H), 7.37 -7.43 (m, 2 H), 8.25-8.32 (m, 2 H), 12. 48 (br s, 1 H).

Reference Example 18 1-Methylpiperidin-4-yl 4-nitrophenyl carbonate hydrochloride
(Compound VI-10)
In the same manner as in Reference Example 14, 1-methylpiperidin-4-ol (SIGMA-ALDRICH, 0.300 g, 2.60 mmol) was used instead of 1-methyl-4-piperidinemethanol. Compound VI-10 (0.740 g, 90% yield) was obtained.
ESI-MS m / z: 281 (M + H) ⁺ .

Reference Example 19 1-Methylpiperidin-3-yl 4-nitrophenyl carbonate hydrochloride (Compound VI-11)
In the same manner as in Reference Example 14, 1-methylpiperidin-3-ol (SIGMA-ALDRICH, 0.305 g, 2.65 mmol) was used instead of 1-methyl-4-piperidinemethanol. Compound VI-11 (0.410 g, yield 49%) was obtained.
ESI-MS m / z: 281 (M + H) ⁺ .

Reference Example 20 (1-Methylpyrrolidin-3-yl) methyl 4-nitrophenyl carbonate hydrochloride (Compound VI-12)
In the same manner as in Reference Example 14, (1-methyl-3-pyrrolidinyl) methanol (Matrix Scientific 0.500 g, 7.43 mmol) was used instead of 1-methyl-4-piperidinemethanol. Compound VI-12 (0.943 g, yield 69%) was obtained.
ESI-MS m / z: 281 (M + H) ⁺ .

(1-Methylpiperidin-4-yl) methyl di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) carbamate
(Compound A-14)
In a similar manner to Example 10, compound A-14 (0.258 g, yield 84%) was obtained using Compound VI-6 (0.228 g, 0.689 mmol) obtained in Reference Example 14 instead of compound VI-3. I got
ESI-MS m / z: 670 (M + H) +; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 6.9 Hz, 6H), 1.22-1.39 (m, 32H), 1.46-1.54 (m , 4H), 1.56-1.66 (m, 3H), 1.67-1.74 (m, 2H), 1.88-1.95 (m, 2H), 2.05 (q, J = 6.9 Hz, 8H), 2.26 (s, 3H), 2.77 (t, J = 6.8 Hz, 4 H), 2. 85 (d, J = 11.7 Hz, 2 H), 3.13-3.23 (m, 4 H), 3.92 (d, J = 6.3 Hz, 2 H), 5.30-5.42 (m , 8H).

(1-Methylpiperidin-3-yl) methyl di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) carbamate
(Compound A-15)
In the same manner as in Example 10, compound VI-7 (0.238 g, 0.719 mmol) obtained in Reference Example 15 was used instead of compound VI-3, and compound A-15 (0.239 g, yield 74%) I got
ESI-MS m / z: 670 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 6.9 Hz, 6 H), 0.92-1.02 (m, 1 H), 1.22-1.39 (m , 32H), 1.46-1.54 (m, 4H), 1.55-1.65 (m, 1H), 1.61-1.74 (m, 3H), 1.82-1.89 (m, 1H), 1. 91-2.00 (m, 1H), 2.05 (q, J = 7.0 Hz, 8 H), 2.26 (s, 3 H), 2.74-2.80 (m, 5 H), 2.84-2.89 (m, 1 H), 3.12-3. 23 (m, 4 H), 3.87 (dd, J) = 10.7, 7.6 Hz, 1 H), 3. 97 (dd, J = 10.7, 5.4 Hz, 1 H), 5.30-5.41 (m, 8 H).

(1-Methylpiperidin-2-yl) methyl di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) carbamate
(Compound A-16)
In a similar manner to Example 10, the compound VI-8 (0.256 g, 0.774 mmol) obtained in Reference Example 16 was used instead of the compound VI-3, and compound A-16 (0.313 g, yield 91%) I got
ESI-MS m / z: 670 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 6.9 Hz, 6H), 1.22-1.39 (m, 32H), 1.46-1.64 (m , 8H), 1.71-1.76 (m, 2H), 2.02-2.12 (m, 10H), 2.32 (s, 3H), 2.77 (t, J = 6.8 Hz, 4H), 2.79-2.84 (m, 1H), 3.11-3.24 (m, 4H), 4.05 (dd, J = 11.1, 4.9 Hz, 1 H), 4.17 (dd, J = 11.1, 4.9 Hz, 1 H), 5.30-5.42 (m, 8 H).

2- (1-Methylpyrrolidin-2-yl) ethyl di ((Z) -octadeca-9-enyl) carbamate (Compound A-17)
In a similar manner to Example 10, compound A-17 (0.360 g, yield 92%) was obtained using Compound B-2 (0.300 g, 0.579 mmol) obtained in Example 2 instead of compound B-1 Obtained.
ESI-MS m / z: 674 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.88 (t, J = 6.9 Hz, 6H), 1.21-1.38 (m, 44H), 1.45-1.85 (m , 8H), 1.93-2.18 (m, 12H), 2.32 (s, 3H), 3.03-3.26 (m, 5H), 4.05-4.18 (m, 2H), 5.30-5.39 (m, 4H).

(1-Methylpiperidin-3-yl) methyl di ((Z) -octadeca-9-enyl) carbamate (Compound A-18)
In the same manner as in Example 10, compound B-2 (0.300 g, 0.579 mmol) obtained in Example 2 in place of compound B-1 and the compound obtained in Reference Example 15 in place of compound VI-3 Compound A-18 (0.390 g, 100% yield) was obtained using VI-7 (0.287 g, 0.896 mmol).
ESI-MS m / z: 674 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.85-1.04 (m, 7H), 1.20-1.38 (m, 44H), 1.46-1.74 (m, 8H) , 1.82-2.06 (m, 10H), 2.26 (s, 3H), 2.74-2.82 (m, 1H), 2.83-2 2.89 (m, 1H), 3.10-3.25 (m, 4H), 3.87 (dd, J = 10.5, 7.3 Hz, 1 H), 3. 98 (dd, J = 10.5, 5.3 Hz, 1 H), 5.30-5.39 (m, 4 H).

2- (1-Methylpyrrolidin-2-yl) ethyl di ((11Z, 14Z) -icosa-11,14-dienyl) carbamate (Compound A-19)
In the same manner as in Example 10, compound A-4 (0.369 g, yield 97%) was synthesized using compound B-4 (0.300 g, 0.526 mmol) obtained in Example 4 instead of compound B-1. Obtained.
ESI-MS m / z: 726 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.89 (t, J = 6.9 Hz, 6H), 1.21-1.40 (m, 40H), 1.44-1.85 (m , 8H), 1.93-2.18 (m, 12H), 2.32 (s, 3H), 2.77 (t, J = 6.4 Hz, 4H), 3.04-3.27 (m, 5H), 4.05-4.18 (m, 2H), 5.29-5.42 (m, 8H).

(1-Methylpiperidin-3-yl) methyl di ((11Z, 14Z) -Icosa-11,14-dienyl) carbamate (Compound A-20)
In a similar manner to Example 10, compound B-4 (0.300 g, 0.526 mmol) obtained in Example 4 in place of compound B-1 and the compound obtained in Reference Example 15 in place of compound VI-3 Compound A-20 (0.374 g, yield 98%) was obtained using VI-7 (0.261 g, 0.789 mmol).
ESI-MS m / z: 726 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.85-1.04 (m, 7H), 1.21-1.40 (m, 40H), 1.45-1.75 (m, 8H) , 1.82-2.09 (m, 10H), 2.26 (s, 3H), 2.74-2.90 (m, 6H), 3.11-3.24 (m, 4H), 3.87 (dd, J = 10.5, 7.5 Hz, 1H), 3.98 (dd, J = 10.5, 5.5 Hz, 1 H), 5.29-5.43 (m, 8 H).

(1-Methylpyrrolidin-2-yl) methyl di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) carbamate (Compound A-21)
The compound B-1 (0.0831 g, 0.162 mmol) obtained in Example 1 is dissolved in dichloroethane (1 mL), and 1,1′-carbonyldiimidazole (Nacalai Tesque, 0.0394 g, 0.243 mmol) is added thereto. Stir at room temperature overnight. To the reaction solution was added iodomethane (manufactured by Tokyo Chemical Industry Co., Ltd., 0.101 mL, 1.62 mmol), and the mixture was stirred overnight at 60 ° C. The reaction solution was concentrated under reduced pressure. The obtained residue is dissolved in tetrahydrofuran (1 mL), 1-methylpyrrolidine-2-methanol (manufactured by Wako Pure Chemical Industries, 0.0372 g, 0.323 mmol) and triethylamine (0.0563 mL, 0.404 mmol) are added, and the mixture is stirred at room temperature. After stirring overnight, the reaction solution was stirred at 60 ° C. for 3 hours. To the reaction mixture was added saturated aqueous sodium hydrogen carbonate solution, and the mixture was extracted twice with n-hexane. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The obtained residue was purified by amino silica gel column chromatography (n-hexane / ethyl acetate = 90/10) to give compound A-21 (0.0318 g, yield 30%).
ESI-MS m / z: 656 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 6.9 Hz, 6 H), 1.24-1. 39 (m, 32 H), 1.46-1.67 (m , 5H), 1.67-1.85 (m, 2H), 1.89-2.00 (m, 1H), 2.05 (q, J = 7.0 Hz, 8H), 2.21-2.30 (m, 1H), 2.42 (s, 3H), 2.43-2.51 (m, 1H), 2.77 (t, J = 6.6 Hz, 4H), 3.03-3.08 (m, 1H), 3.11-3.25 (m, 4H), 4.00 (dd, J = 10.5, 6.0 Hz, 1 H), 4.08 (dd, J = 10.5, 5.5 Hz, 1 H), 5.29-5.42 (m, 8 H).

(1-Methylpyrrolidin-3-yl) methyl di (9Z, 12Z) -octadeca-9,12-dienylcarbamate
(Compound A-22)
In the same manner as in Example 10, compound A-12 (0.263 g, yield 66%) was obtained using Compound VI-12 (0.277 g, 0.876 mmol) obtained in Reference Example 20 instead of compound VI-3. Obtained.
ESI-MS m / z: 656 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 6.9 Hz, 6 H), 1.20-1. 40 (m, 32 H), 1.44-1. 78 (m , 5H), 1.92-2.09 (m, 9H), 2.24 (dd, J = 9.4, 6.2 Hz, 1H), 2.34 (s, 3H), 2.39-2.63 (m, 3H), 2.68-2.80 (m, 5H) ), 3.10-3.25 (m, 4H), 3.95 (dd, J = 10.5, 7.8 Hz, 1 H), 4.03 (dd, J = 10.5, 6.4 Hz, 1 H), 5.28-5.42 (m, 8 H).

2- (1-Methylpiperidin-2-yl) ethyl di (9Z, 12Z) -octadeca-9,12-dienylcarbamate (Compound A-23)
Step 1
To a solution of 4-nitrophenyl chloroformate (0.844 g, 7.19 mmol) in tetrahydrofuran (12 mL) was added 2- (1-methylpiperidin-2-yl) ethanol (Matrix Scientific, 0.500 g, 3.49 mmol) The mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure to give crude 2- (1-methylpiperidin-2-yl) ethyl 4-nitrophenyl carbonate hydrochloride.
Step 2
In a similar manner to Example 10, a crude purified product (0.302 g, 2- (1-methylpiperidin-2-yl) ethyl 4-nitrophenyl carbonate hydrochloride obtained in Step 1) was obtained instead of Compound VI-3. Compound A-23 (0.299 g, yield 75%) was obtained using 0.876 mmol).
ESI-MS m / z: 684 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 6.9 Hz, 6 H), 1.19-1.40 (m, 32 H), 1.45-1.75 (m , 11H), 1.93-2.11 (m, 11H), 2.27 (s, 3H), 2.74-2.86 (m, 5H), 3.10-3.24 (m, 4H), 4.06-4.19 (m, 2H), 5.29-5.42 (m, 8H).

3- (Azepan-1-yl) propyl di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) carbamate
(Compound A-24)
In the same manner as in Example 10, compound A-24 (0.136 g, yield 67%) was obtained using compound VI-9 obtained in Reference Example 17 instead of compound VI-3.
ESI-MS m / z: 698 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.86-0.94 (m, 6H), 1.22-1.41 (m, 36H), 1.45-1.67 (m, 8H) , 1.75-1.85 (m, 2H), 2.00-2.11 (m, 8H), 2.55 (t, J = 7.5 Hz, 2 H), 2.62 (t, J = 5.1 Hz, 4 H), 2.77 (t, J = 5.9) Hz, 4H), 3.13-3.23 (m, 4H), 4.10 (t, J = 6.4 Hz, 2H), 5.28-5.45 (m, 8H).

3- (Piperidin-1-yl) propyl ((9Z, 12Z) -octadeca-9,12-dienyl) (2-((9Z, 12Z) -octadeca-9,12-dienyloxy) ethyl) carbamate (compound A- 29)
In a similar manner to Example 10, compound B-8 (0.150 g, 0.269 mmol) obtained in Example 19 instead of compound B-1 and compound VI obtained in Reference Example 5 instead of compound VI-3 Compound A-29 (0.170 g, yield 87%) was obtained using -4 (0.201 g, 0.672 mmol).
ESI-MS m / z: 728 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 6.8 Hz, 6 H), 1.22-1.40 (m, 32 H), 1.40-1.63 (m , 14H), 1.78-1.87 (m, 2H), 2.05 (q, J = 6.6 Hz, 8H), 2.33-2.41 (m, 6H), 2.77 (t, J = 6.0 Hz, 4H), 3.20-3. 31 ( m, 2H), 3.40 (t, J = 6.6 Hz, 4H), 3.46-3.57 (m, 2H), 4.10 (t, J = 6.4 Hz, 2H), 5.27-5.45 (m, 8H).

2- (1-Methylpyrrolidin-2-yl) ethyl ((9Z, 12Z) -octadeca-9,12-dienyl) (2-((9Z, 12Z) -octadeca-9,12-dienyloxy) ethyl) carbamate ( Compound A-30)
In a similar manner to Example 10, compound A-30 (0.140 g, 91% yield) was obtained using Compound B-8 (0.120 g, 0.215 mmol) obtained in Example 19 instead of Compound B-1. I got
ESI-MS m / z: 714 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 6.8 Hz, 6 H), 1.21-1.40 (m, 32 H), 1.45-1.59 (m , 6H), 1.64-1.82 (m, 2H), 1.93-2.17 (m, 12H), 2.31 (s, 3H), 2.70-2.81 (m, 4H), 3.06 (t, J = 7.8 Hz, 1H), 3.20-3.31 (m, 2H), 3.40 (t, J = 5.9 Hz, 4H), 3.46-3.58 (m, 2H), 4.06-4.17 (m, 2H), 5.28-5.44 (m, 8H).

3- (azepan-1-yl) propyl ((9Z, 12Z) -octadeca-9,12-dienyl) (2-((9Z, 12Z) -octadeca-9,12-dienyloxy) ethyl) carbamate
(Compound A-31)
In a similar manner to Example 10, compound B-8 (0.150 g, 0.269 mmol) obtained in Example 19 instead of compound B-1 and compound VI obtained in Reference Example 17 instead of compound VI-3 Compound A-31 (0.170 g, yield 85%) was obtained using -9 (0.145 g, 0.403 mmol).
ESI-MS m / z: 742 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.89 (t, J = 6.8 Hz, 6 H), 1.21-1.39 (m, 32 H), 1.48-1.65 (m , 12H), 1.72-1.85 (m, 2H), 2.05 (q, J = 6.6 Hz, 8 H), 2.55 (t, J = 7.5 Hz, 2 H), 2.61 (t, J = 5.3 Hz, 4 H), 2.77 (t, J = 5.9 Hz, 4H), 3.21-3.30 (m, 2H), 3.40 (t, J = 6.8 Hz, 4H), 3.46-3.55 (m, 2H), 4.10 (t, J = 6.4 Hz, 2H), 5.26-5.42 (m, 8H).

(1-Methylpiperidin-3-yl) methyl ((9Z, 12Z) -octadeca-9,12-dienyl) (2-((9Z, 12Z) -octadeca-9,12-dienyloxy) ethyl) carbamate (Compound A -32)
In a similar manner to Example 10, compound B-8 (0.150 g, 0.269 mmol) obtained in Example 19 instead of compound B-1 and compound VI obtained in Reference Example 15 instead of compound VI-3 Compound A-32 (0.145 g, yield 76%) was obtained using -7 (0.133 g, 0.403 mmol).
ESI-MS m / z: 714 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.91 (t, J = 6.8 Hz, 6 H), 1.23-1.45 (m, 32 H), 1.49-1 .77 (m , 9H), 1.83-2.03 (m, 2H), 2.07 (q, J = 6.6 Hz, 8 H), 2.28 (s, 3 H), 2. 76-2.91 (m, 2 H), 2. 80 (t, J = 5.9 Hz, 4H), 3.21-3.33 (m, 2H), 3.43 (t, J = 6.4 Hz, 4H), 3.48-3.58 (m, 2H), 3.85-4.05 (m, 2H), 5.30-5.47 (m, 8H) .

Reference Example 21 2-((Z) -Octadeca-9-enyloxy) ethyl methanesulfonate (Compound IIIc-2)
In the same manner as in Example 8, (Z) -octadec-9-en-1-yl methanesulfonate (2.00) was used instead of (9Z, 12Z) -octadeca-9,12-dien-1-yl methanesulfonate. Compound IIIc-2 (1.29 g, yield 57%) was obtained using g, 5.77 mmol).
ESI-MS m / z: 391 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.89 (t, J = 6.6 Hz, 3 H), 1.22-1.38 (m, 22 H), 1.50-1.2 (m , 2H), 1.97-2.05 (m, 4H), 3.06 (s, 3H), 3.48 (t, J = 6.8 Hz, 2H), 3.67-3.72 (m, 2H), 4.36-4.39 (m, 2H), 5.35 (t, J = 5.5 Hz, 2 H).

Reference Example 22 2-((Z) -Hexadeca-9-enyloxy) ethyl methanesulfonate (Compound IIIc-3)
In the same manner as in Example 8, (Z) -hexadec-9-en-1-yl methanesulfonate (2.00) was used instead of (9Z, 12Z) -octadeca-9,12-dien-1-yl methanesulfonate. The compound IIIc-3 (1.52 g, yield 67%) was obtained using g, 6.28 mmol).
ESI-MS m / z: 363 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.90 (t, J = 6.6 Hz, 3 H), 1.25-1.38 (m, 18 H), 1.53-1.62 (m , 2H), 1.98-2.06 (m, 4H), 3.07 (s, 3H), 3.49 (t, J = 6.6 Hz, 2H), 3.68-3.72 (m, 2H), 4.36-4.40 (m, 2H), 5.36 (t, J = 5.5 Hz, 2 H).

(9Z, 12Z) -N- (2-((Z) -octadeca-9-enyloxy) ethyl) octadeca-9,12-dien-1-amine (Compound B-9)
In a similar manner to Example 13, Compound IId-1 (0.800 g, 1.78 mmol) obtained in Reference Example 7 and Compound IIIc-2 (0.728 g, 1.86) obtained in Reference Example 21 instead of 1-bromododecane Compound B-9 (0.600 g, 60% yield) was obtained using mmol).
ESI-MS m / z: 560 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.86-0.94 (m, 6 H), 1.24-1. 39 (m, 40), 1.51-1. 62 (m, 2 H) , 1.96-2.10 (m, 8 H), 2. 68 (t, J = 7.3 Hz, 2 H), 2. 78 (t, J = 6.0 Hz, 2 H), 2. 85 (t, J = 5.1 Hz, 2 H), 3. 45 (t, J J = 6.8 Hz, 2 H), 3.57 (t, J = 5.1 Hz, 2 H), 5.30-5.44 (m, 6 H).

(9Z, 12Z) -N- (2-((Z) -hexadeca-9-enyloxy) ethyl) octadeca-9,12-dien-1-amine (Compound B-10)
In a similar manner to Example 13, Compound IId-1 (0.610 g, 1.35 mmol) obtained in Reference Example 7 and Compound IIIc-3 (0.589 g, 1.62) obtained in Reference Example 22 instead of 1-bromododecane Compound B-10 (0.550 g, 76% yield) was obtained using mmol).
ESI-MS m / z: 532 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.85 to 0.93 (m, 6 H), 1.23-1.38 (m, 34 H), 1.45-1. 54 (m, 4 H) , 1.94-2.11 (m, 8H), 2.60 (t, J = 7.3 Hz, 2H), 2.77 (t, J = 5.4 Hz, 4H), 3.43 (t, J = 6.8 Hz, 2H), 3.53 (t, J J = 5.4 Hz, 2H), 5.29-5.44 (m, 6H).

3- (Piperidin-1-yl) propyl (2-((Z) -octadeca-9-enyloxy) ethyl) ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-33)
In a similar manner to Example 10, compound B-9 (0.130 g, 0.232 mmol) obtained in Example 38 instead of compound B-1 and compound VI obtained in Reference Example 5 instead of compound VI-3 Compound A-33 (0.137 g, yield 81%) was obtained using -4 (0.120 g, 0.348 mmol).
ESI-MS m / z: 729 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.84-0.92 (m, 6H), 1.20-1.36 (m, 38H), 1.40-1.62 (m, 10H) , 1.77-1.87 (m, 2H), 1.96-2.09 (m, 8H), 2.37 (t, J = 7.5 Hz, 6 H), 2.77 (t, J = 5.9 Hz, 2 H), 3.20-3.31 (m, 2 H) ), 3.40 (t, J = 6.6 Hz, 4H), 3.45-3.56 (m, 2H), 4.10 (t, J = 6.4 Hz, 2H), 5.28-5.44 (m, 6H).

2- (1-Methylpyrrolidin-2-yl) ethyl (2-((Z) -octadeca-9-enyloxy) ethyl) ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-34 )
In a similar manner to Example 10, Compound A-34 (0.131 g, yield 79%) was obtained using Compound B-9 (0.130 g, 0.232 mmol) obtained in Example 38 instead of Compound B-1. I got
ESI-MS m / z: 716 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.85 to 0.92 (m, 6 H), 1.20-1. 39 (38 H, m), 1.47-1. 61 (m, 8 H) , 1.66-1.83 (m, 2H), 1.93-2.18 (10H, m), 2.32 (s, 3H), 2.78 (t, J = 5.9 Hz, 2H), 3.07 (t, J = 8.4 Hz, 1 H), 3.21-3.31 (m, 2H), 3.41 (t, J = 6.6 Hz, 4H), 3.47-3.56 (m, 2H), 4.08-4.19 (m, 2H), 5.29-5.43 (m, 6H).

3- (Dimethylamino) propyl (2-((Z) -octadeca-9-enyloxy) ethyl) ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-35)
In a similar manner to Example 10, compound B-9 (0.130 g, 0.232 mmol) obtained in Example 38 instead of compound B-1 and compound VI-1 (0.106 g, instead of compound VI-3) Compound A-35 (0.100 g, 63% yield) was obtained using 0.348 mmol).
ESI-MS m / z: 690 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.85 to 0.92 (m, 6 H), 1.20-1. 39 (m, 38 H), 1.45-1. 59 (m, 4 H) , 1.74-1.84 (m, 2H), 1.96-2.09 (m, 8H), 2.23 (s, 6H), 2.34 (t, J = 7.5 Hz, 2H), 2.77 (t, J = 5.7 Hz, 2H), 3.21-3.30 (m, 2H), 3.35-3.44 (m, 4H), 3.45-3.55 (m, 2H), 4.11 (t, J = 6.4 Hz, 2H), 5.26-5.44 (m, 6H).

3- (Dimethylamino) propyl (2-((Z) -hexadeca-9-enyloxy) ethyl) ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-36)
In a similar manner to Example 10, compound B-10 (0.150 g, 0.282 mmol) obtained in Example 39 instead of compound B-1 and compound VI-1 (0.095 g, instead of compound VI-3) Compound A-36 (0.148 g, yield 79%) was obtained using 0.310 mmol).
ESI-MS m / z: 662 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.85-0.94 (m, 6H), 1.21-1.39 (m, 34H), 1.47-1.59 (m, 4H) , 1.76-1.84 (m, 2H), 1.94-2.09 (m, 8H), 2.23 (s, 6H), 2.34 (t, J = 7.3 Hz, 2H), 2.77 (t, J = 6.3 Hz, 2H), 3.20-3.31 (m, 2H), 3.31-3.45 (m, 4H), 3.45-3.57 (m, 2H), 4.11 (t, J = 6.3 Hz, 2H), 5.27-5.46 (m, 6H).

3- (Piperidin-1-yl) propyl (2-((Z) -hexadeca-9-enyloxy) ethyl) ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-37)
In a similar manner to Example 10, compound B-10 (0.150 g, 0.282 mmol) obtained in Example 39 instead of compound B-1 and compound VI obtained in Reference Example 5 instead of compound VI-3 Compound A-37 (0.148 g, yield 75%) was obtained using -4 (0.107 g, 0.310 mmol).
ESI-MS m / z: 702 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.82 to 0.96 (m, 6 H), 1.23-1.39 (m, 34 H), 1.39-1 to 48 (m, 2 H) , 1.49-1.61 (m, 8H), 1.79-1.86 (m, 2H), 1.95-2.09 (m, 8H), 2.33-2.42 (m, 6H), 2.78 (t, J = 6.8 Hz, 2H), 3.21. -3.32 (m, 2H), 3.34-3.44 (m, 4H), 3.46-3.56 (m, 2H), 4.10 (t, J = 6.3 Hz, 2H), 5.29-5.44 (m, 6H).

3- (Di ((Z) -octadec-9-enyl) amino) propan-1-ol (Compound C-2)
Step 1
In a similar manner to Reference Example 11, the compound B-2 (500 mg, 0.965 mmol) obtained in Example 2 was used instead of compound B-1 to give 3- (di ((Z) -octadeca-9-enyl) ) Ethyl (amino) propionate (548 mg, 92% yield) was obtained.
Step 2
In a similar manner to Example 21, Compound C-2 was obtained using ethyl 3- (di ((Z) -octadeca-9-enyl) amino) propionate (548 mg, 0.887 mmol) instead of compound XI-6. (0.445 g, 87% yield) was obtained.
ESI-MS m / z: 577 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.88 (t, J = 6.9 Hz, 6 H), 1.24-1.42 (m, 44 H), 1.42-1. 50 (m , 4H), 1.65-1.70 (m, 2H), 2.01 (q, J = 6.4 Hz, 8 H), 2.40 (t, J = 7.5 Hz, 4 H), 2.63 (t, J = 5.5 Hz, 2 H), 3.79 (t, J = 5.3 Hz, 2H), 5.30-5.39 (m, 4H).

3- (Di ((11Z, 14Z) -Icosa-11,14-dienyl) amino) propan-1-ol (Compound C-3)
Step 1
In a similar manner to Reference Example 11, a compound B-4 (400 mg, 0.702 mmol) obtained in Example 4 was used instead of the compound B-1 to give 3- (di ((11Z, 14Z) -icosa-11) Ethyl, 14-dienyl) amino) propionate (548 mg, 90% yield) was obtained.
Step 2
In a similar manner to Example 21, ethyl 3- (di ((11Z, 14Z) -icosa-11,14-dienyl) amino) propionate (424 mg, 0.633 mmol) was used instead of compound XI-6 Compound C-3 (352 mg, yield 88%) was obtained.
ESI-MS m / z: 629 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.89 (t, J = 6.9 Hz, 6H), 1.24-1.40 (m, 40H), 1.42-1. 50 (m , 4H), 1.64-1.70 (m, 2H), 2.02-2.08 (m, 8H), 2.40 (t, J = 7.5 Hz, 4 H), 2.63 (t, J = 5.3 Hz, 2 H), 2.78 (t, J = 6.4 Hz, 4H), 3.79 (t, J = 5.0 Hz, 2H), 5.29-5.42 (m, 8H).

2- (di ((9Z, 12Z) -octadeca-9,12-dienyl) amino) ethanol (compound C-4)
Step 1
Potassium carbonate (243 mg, 1.76 mmol) and ethyl bromoacetate (195 μL, 1.76 mmol) were added to a solution of compound B-1 (600 mg, 1.17 mmol) obtained in Example 1 in 1,2-dichloroethane (2.0 mL) In addition, it was stirred at 85 ° C. overnight. To the resulting mixture was added water and extracted twice with heptane. The organic layers were combined, washed with water, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue is purified by amino silica gel column chromatography (heptane / ethyl acetate = 100/0 to 95/5) to give ethyl 2- (di ((9Z, 12Z) -octadeca-9,12-dienyl) amino) acetate ( 527 mg, yield 75%) was obtained.
Step 2
In a similar manner to Example 21, a compound was obtained using ethyl 2- (di ((9Z, 12Z) -octadeca-9,12-dienyl) amino) acetate (527 mg, 0.878 mmol) in place of compound XI-6, C-4 (433 mg, yield 88%) was obtained.
ESI-MS m / z: 559 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.89 (t, J = 6.9 Hz, 6H), 1.24-1.39 (m, 32H), 1.39-1.46 (m , 4H), 2.02-2.08 (m, 8H), 2.43 (t, J = 7.5 Hz, 4 H), 2.57 (t, J = 5.3 Hz, 2 H), 2.77 (t, J = 6.2 Hz, 4 H), 3.52 (t, J = 5.5 Hz, 2H), 5.29-5.41 (m, 8H).

4- (di ((9Z, 12Z) -octadeca-9,12-dienyl) amino) butan-1-ol (compound C-5)
Step 1
Potassium carbonate (202 mg, 1.46 mmol) and tert-butyl (4-iodobutoxy) dimethylsilane in a solution of compound B-1 (500 mg, 0.973 mmol) obtained in Example 1 in 1,2-dichloroethane (2.0 mL) (SIGMA-ALDRICH company make, 378 microliters, 1.46 mmol) was added, and it stirred at 85 degreeC for 4 hours. To the resulting mixture was added water and extracted twice with heptane. The organic layers were combined, washed with water, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue is purified by silica gel column chromatography (heptane / ethyl acetate = 95 / 5-80 / 20) to give (4- (tert-butyldimethylsilyloxy) butyl) di ((9Z, 12Z) -octadeca-9, 12-dienyl) amine (233 mg, 34% yield) was obtained.
Step 2
(4- (tert-Butyldimethylsilyloxy) butyl) di ((9Z, 12Z) -octadeca-9,12-dienyl) amine (233 mg, 0.333 mmol) in tetrahydrofuran (5 mL) solution of tetrabutyl fluoride Ammonium (1 mol / L tetrahydrofuran solution, 0.666 mL, 0.666 mmol) was added, and the mixture was stirred overnight at room temperature. To the resulting mixture was added saturated brine and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue is purified by amino silica gel column chromatography (heptane / ethyl acetate = 90/10) and further purified by silica gel column chromatography (ethyl acetate / methanol = 100/0 to 90/10) to give compound C-5 ( 160 mg (yield 82%) were obtained.
ESI-MS m / z: 587 (M + H) ⁺ ; ¹ H-NMR (CDCl3) δ: 0.89 (t, J = 6.9 Hz, 6H), 1.23-1.40 (m, 32H), 1.43-1.51 (m , 4H), 1.62-1.68 (m, 4H), 2.05 (q, J = 7.0 Hz, 8 H), 2.41-2.45 (m, 6 H), 2.77 (t, J = 6.6 Hz, 4 H), 3.53-3.56 ( m, 2H), 5.29-5.42 (m, 8H).

Reference Example 23 3- (Dimethylamino) propyl 2,3-bis ((9Z, 12Z) -octadeca-9,12-dienyloxy) propyl (methyl) carbamate (Compound XI-9)
Step 1
Dichloromethane (4 of 2,3-bis ((9Z, 12Z) -octadeca-9,12-dienyloxy) propan-1-ol (0.303 g, 0.514 mmol) synthesized by the method described in WO2009 / 129395) Triethylamine (0.108 ml, 0.772 mmol) and mesyl chloride (0.060 ml, 0.772 mmol) were added to the solution at 0 ° C. and stirred at room temperature for 3 hours. Saturated aqueous sodium bicarbonate solution was added to the reaction mixture, and the mixture was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure.
The obtained residue was dissolved in dichloromethane (4 mL), methylamine (7 mol / L methanol solution, 2.20 mL) was added, and the mixture was stirred at 110 ° C. for 5 minutes using a microwave reactor. Water was added to the reaction mixture, and the mixture was extracted with n-hexane, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The resulting residue is purified by amino silica gel column chromatography (n-hexane / ethyl acetate = 95/5 to 70/30) to give N-methyl-2,3-bis ((9Z, 12Z) -octadeca-9 12, 12-dien-1-yloxy) propan-1-amine (0.278 g, 90% yield) was obtained.
ESI-MS m / z: 603 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 7.1 Hz, 6 H), 1.27-1.39 (m, 34 H), 1.51-1.58 (m , 3H), 2.05 (q, J = 7.1 Hz, 8H), 2.44 (s, 3H), 2.64-2.70 (m, 2H), 2.77 (t, J = 6.9 Hz, 4H), 3.41-3.50 (m, 5H), 3.54-3.58 (m, 1H), 3.61-3.65 (m, 1H), 5.30-5.41 (m, 8H).
Step 2
To a suspension of N-methyl-2,3-bis ((9Z, 12Z) -octadeca-9,12-dien-1-yloxy) propane-1-amine (0.220 g, 0.365 mmol) in acetonitrile (2 mL) Compound VI-1 (0.167 g, 0.548 mmol) and triethylamine 0.255 mL, 1.827 mmol) were added and stirred at 80 ° C. overnight. The reaction solution was diluted with saturated aqueous sodium bicarbonate and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by amino silica gel column chromatography (n-hexane / ethyl acetate = 80/20 to 65/35) to give compound XI-9 (0.178 g, yield 67%).
ESI-MS m / z: 732 (M + H) ⁺ ; ¹ H-NMR (CDCl 3) δ: 0.89 (t, J = 6.9 Hz, 6 H), 1.26-1.38 (m, 32 H), 1.51-1.59 (m , 4H), 1.77-1.83 (m, 2H), 2.05 (q, J = 7.0 Hz, 8 H), 2.22 (s, 6 H), 2.34 (t, J = 7.4 Hz, 2 H), 2.77 (t, J = 6.8 Hz, 4 H), 2. 97 (s, 3 H), 3. 9-3. 25 (m, 1 H), 3. 38-3. 65 (m, 8 H), 4.1 2 (t, J = 6.3 Hz, 2 H), 5. 30-5. 41 (m, 8 H) ).

The composition was prepared as follows using the compounds (compounds A-1 to 5) obtained in Examples 5 to 9. The nucleic acid used is a sense strand [5'-rGmUrCrAmUrCrArCrArCrUrGrArAmUrArCrArAmU-3 '(a sugar linked to a base with r and m attached is ribose and ribose wherein the hydroxyl group at the 2' position is substituted with -O-methyl, respectively Apolipoprotein B (Apolipoprotein-B) consisting of an antisense strand and an antisense strand [5'-rArUrUrGrGrUrUrCrGrUrGrUrGrArCrArC-3 '(all sugars binding to the bases are ribose and the 5' end is phosphorylated)] (Hereinafter referred to as apo-b), which is an anti-APO-B siRNA for suppressing the expression of a gene, was obtained from Gene Design (hereinafter referred to as apo-b siRNA).
Each of Compound A-1 to 5 / 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N- (methoxy (polyethylene glycol) -2000) sodium salt (PEG-DMPE Na, N- (carbonyl methoxy) Polyethylene glycol 2000) -1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine sodium salt, manufactured by NOF Corporation) / distearoylphosphatidylcholine (DSPC, 1,2-distearoyl-sn-glycero-3-) Each sample is weighed and dissolved in 90 vol% ethanol so that phosphocholine, cholesterol (manufactured by NOF Corporation) / cholesterol (manufactured by NOF Corporation) = 8.947 / 1.078 / 5.707 / 13.698 mmol / L, and the components of the lipid membrane are obtained. The solution was prepared. On the other hand, apo-b siRNA / distilled water (24 mg / mL) was added to Tris-EDTA buffer (200 mM Tris-HCl, 20 mM EDTA, Invitrogen) and 20 mM citrate buffer (pH 5) And diluted with 1.5 mg / mL of apo-b siRNA aqueous solution (2 mM Tris-EDTA buffer, 20 mM citrate buffer, pH 5.0).
The obtained lipid solution was heated to 37 ° C., and then 500 μL was transferred to a preparation preparation container, and 500 μL of the obtained apo-b siRNA aqueous solution was added under stirring. To 1000 μL of the resulting lipid nucleic acid mixture suspension, 1000 μL of 20 mM citrate buffer (containing 300 mM NaCl, pH 6.0) is added under stirring, and DPBS (Dulbecco's Phosphate-Buffered Saline, Dulbecco's phosphate) is further added. Buffered saline (manufactured by Invitrogen) 3310 μL was added dropwise to obtain a crude preparation. The obtained crude preparation was concentrated using Amicon Ultra (manufactured by Millipore), diluted with DPBS, and filtered using a 0.2 μm filter (manufactured by Toyo Roshi Co., Ltd.) in a clean bench. The composition (containing each of compounds A-1 to 5 and a nucleic acid) is measured by measuring the siRNA concentration of the obtained composition and diluting it with DPBS to 0.3 or 0.03 mg / mL in siRNA concentration The composition was obtained.
The average particle size of the preparation (composition) was measured with a particle size measuring device (Zetasizer Nano ZS, manufactured by Malvern, hereinafter the same). The results are shown in Table 8.

Comparative Example 1
Example 49 is the same as Example 49 except using DLin-KC2-DMA synthesized by the method according to Patent Document 1 and the compounds (Compounds XI-1 to 3) obtained in Reference Examples 1 to 3 The formulation was obtained.
The structural formulas of the compounds (DLin-KC2-DMA and compounds XI-1 to 3) used in the comparative examples are shown in Table 9.
The average particle size of the preparation (composition) was measured with a particle size measuring device. The results are shown in Table 10.

Test Example 1
Each preparation (composition A-1 and 3-5 and each containing a nucleic acid) obtained in Example 49 and each preparation obtained in Comparative Example 1 (DLin-KC2-DMA and Compound XI-1 Each of the compositions (3) to (3) and the nucleic acid-containing composition) was introduced into human hepatoma-derived cell line HepG2 cells (HB-8065) by the following methods.
Each preparation diluted with Optimem (Opti-MEM, GIBCO, 31985) to a final concentration of 3-100 nM of nucleic acid is aliquoted into 96-well culture plates in 20 μl aliquots and then 1.25% HepG2 cells suspended in Opti-MEM containing fetal bovine serum (FBS, SAFC Biosciences (SAFC Biosciences) 12203C) are seeded at a cell number of 6250/80 μL / well, 37 ° C, 5% Each preparation was introduced into HepG2 cells by culturing under CO 2 conditions. In addition, as a negative control group, cells treated with nothing were seeded.
The cells into which each preparation has been introduced are cultured in a 5% CO 2 incubator at 37 ° C. for 24 hours, washed with ice-cold phosphate buffered saline (PBS, GIBCO, 14190), and Cells-to-Ct Kit ( Using Applied Biosciences (ABI) AM 1728, according to the method described in the instruction attached to the product, recovery of total RNA and preparation of cDNA by reverse transcription reaction using the obtained total RNA as a template And did.
Using the obtained cDNA as a template and a universal probe library (Universal Probe Library, Roche Applied Science, 04683633001) as a probe, described in the attached instruction manual using ABI 7900HT Fast (manufactured by ABI) The apo-b gene and the constitutively expressed gene glyceraldehyde 3-phosphate dehydrogenase (hereinafter referred to as gapdh) gene are PCR-reacted by PCR reaction according to the method described above. The amount of amplified mRNA was measured respectively, and the amount of amplified mRNA of gapdh was used as an internal control to calculate the semi-quantitative value of the apo-b mRNA. The expression rate of apo-b mRNA was determined from the semi-quantitative value of apo-b mRNA, where the semi-quantitative value of apo-b mRNA in the negative control similarly measured was 1. The result of the expression rate of the obtained apo-b mRNA is shown in FIG.

  As is clear from FIG. 1, among the preparations obtained in Example 49 (the compounds A-1 and 3-5 and the composition containing the nucleic acid) and the respective preparations obtained in Comparative Example 1, DLin- The composition containing KC2-DMA, compound XI-1 and compound XI-3, and the nucleic acid suppressed the expression of mRNA of the apo-b gene after introduction into human hepatoma cell line HepG2 cells. . On the other hand, among the preparations obtained in Comparative Example 1, the composition containing compound XI-2 and the nucleic acid was used to express the mRNA of the apo-b gene after introduction into human hepatoma-derived cell line HepG2 cells. It did not suppress.

Test example 2
Each preparation obtained in Example 49 (each of compounds A-1 to 5 and a composition containing a nucleic acid) and each preparation obtained in Comparative Example 1 (DLin-KC2-DMA and compounds XI-1 to 3) The in vivo drug efficacy evaluation test was carried out for each of the above, as well as the composition containing the nucleic acid, according to the following method. Each preparation was diluted with DPBS and used according to the test.
After conditioned breeding of mice (Balb / c, obtained from CLEA Japan, Inc.), each preparation was intravenously administered to mice at an siRNA concentration of 3 to 0.3 mg / kg. Blood was collected 48 hours after administration, and the collected blood was centrifuged at 3000 rpm for 20 minutes at 4 ° C. using a small cooling centrifuge (05PR-22: manufactured by Hitachi, Ltd.). Using the Cholesterol Assay Kit (Cayman Chemical, Cat #: 10007640) and following the method described in the product instructions, the fluorescence in the standard solution and serum sample is ARVO (530 nm / 595 nm) or EnVision It measured by (531 nm / 595 nm). A calibration curve was prepared from the obtained fluorescence, and cholesterol concentration in serum was calculated.
The results of the calculated cholesterol concentration in serum are shown in FIGS.

As apparent from FIGS. 2 and 3, the preparation obtained in Example 49 (a composition containing anti-APO-B siRNA for suppressing the expression of the apo-b gene and each of the compounds A-1 to 5) The results of measurement of cholesterol concentration, which were tested for efficacy evaluation in vivo, are lower than those of the compositions containing the compounds XI-1 to 3 and nucleic acid among the preparations obtained in Comparative Example 1, By administering the preparation obtained in Example 22, it is shown that the expression of the apo-b gene is strongly suppressed.
Thus, the composition of the present invention can introduce nucleic acid into cells etc., and the cationic lipid of the present invention should be a cationic lipid that facilitates the delivery of nucleic acid into cells in vivo. It became clear.

Using the compound (compound A-6) obtained in Example 10, a composition was prepared as follows. The nucleic acid used is the sense strand [5'-rGrGrAfUfCrAfUfCfUrFrArArGfUfCfUrAfCdTdT-3 '(r, a sugar that binds to a base attached with r, d and f has a ribose, deoxyribose and hydroxyl group at the 2' position substituted with fluorine respectively A bond between a deoxyribose which is a ribose and is bound to a 20th base from the 5 'end toward a 3' end and a deoxyribose which is bound to a 21st base is a phosphorothioate bond), and an antisense strand [5′-rGfUrArArGrAfCfUfrGrArGrAfUfGfAfUfCfCdTd-3 ′ (r, a sugar linked to a base with r, d and f attached is ribose, deoxyribose and ribose wherein the hydroxyl group at the 2 ′ position is substituted with fluorine, respectively The bond between the deoxyribose bound to the 20th base from the side toward the 3 'end and the deoxyribose bound to the 21st base is a phosphorothioate bond. Liquid coagulation Factor 7 (Coagulation Factor VII, below f7 represents a) a suppressing anti f7 siRNA expression of genes were obtained from Gene Design Inc. (hereinafter referred to f7 siRNA).
Each sample was added so that compound A-6 / PEG-DMPE Na (manufactured by NOF Corporation) / DSPC (manufactured by NOF Corporation) / cholesterol (manufactured by NOF Corporation) = 3.532 / 0.270 / 1.156 / 2.401 mmol / L The mixture was weighed and dissolved in 100 vol% ethanol to prepare a solution of lipid membrane components. On the other hand, f7 siRNA / distilled water (24 mg / mL) was added to Tris-EDTA buffer (200 mM Tris-HCl, 20 mM EDTA, Invitrogen) and 20 mM citrate buffer (pH 4.0) And diluted with 0.375 mg / mL of f7 siRNA aqueous solution (2 mM Tris-EDTA buffer, 20 mM citrate buffer, pH 4.0).
The obtained lipid solution was heated to 37 ° C., 800 μL was transferred to a preparation preparation container, and 800 μL of the obtained aqueous solution of f7 siRNA was added under stirring. To 1600 μL of the obtained lipid nucleic acid mixture suspension, 1600 μL of 20 mM citrate buffer (containing 300 mM NaCl, pH 6.0) is added with stirring, and 7086 μL of DPBS (Invitrogen) is added dropwise to obtain a crude product. The formulation was obtained. The obtained crude preparation was concentrated using Amicon Ultra (manufactured by Millipore), diluted with DPBS, and filtered using a 0.45 μm filter (manufactured by Toyo Roshi Co., Ltd.) in a clean bench. The siRNA concentration of the obtained composition was measured, and diluted with DPBS to a siRNA concentration of 0.03 mg / mL to obtain a preparation (composition containing compound A-6 and nucleic acid).
The average particle size of the preparation (composition) was measured with a particle size measuring device. The results are shown in Table 11.

  Among the compounds obtained in Examples 1 to 48, Compounds A-1, A-5, A-7 to 21, A-28 to 36, B-1, B-8 and C-1 to 5 were used respectively Preparation in the same manner as in Example 50 (Compounds A-1, A-5, A-7 to 21, A-28 to 36, B-1, B-8 and C-1 to 5, and nucleic acids The composition containing The average particle size of the preparation (composition) was measured with a particle size measuring device. The results are shown in Table 11.

Comparative example 2
A formulation was obtained in the same manner as in Example 50 except that the compound A-6 was changed to the compounds (Compounds XI-4 to 8) obtained in Reference Examples 9 to 13.
The structural formulas of the compounds (Compounds XI-4 to 8) used in Comparative Example 2 are shown in Table 12.
The average particle size of the preparation (composition) was measured with a particle size measuring device. The results are shown in Table 13.

Test Example 3
Each preparation obtained in Examples 50 and 51 (Compound A-1, A-5 to 21, A-28 to 36, B-1, B-8 and C-1 to 5, respectively, and a nucleic acid The in vivo drug efficacy evaluation test was carried out for each of the formulations (compositions XI-4 to 8 and the composition containing the nucleic acid) obtained in the composition and the comparative example 2 according to the following method. Each preparation was diluted with DPBS or saline according to the test and used.
After conditioned breeding of mice (Balb / c, obtained from CLEA Japan, Inc.), each preparation was intravenously administered to the mice at an siRNA concentration of 0.3, 0.1 and / or 0.03 mg / kg. Blood was collected 48 hours after administration, and the collected blood was centrifuged at 8000 rpm for 8 minutes at 4 ° C. using a micro high-speed cooling centrifuge (TOMY MX305: manufactured by Tomy Seiko Co., Ltd.). The absorbance in the standard solution and the plasma sample was measured with ARVO (405 nm) using BIOPHEN VII kit (manufactured by ANIARA cat #: A221304) according to the method described in the product instruction. A calibration curve was prepared from the obtained absorbance, and Factor VII protein concentration in plasma was calculated. The number of cases was 3 animals in each group.
The results of the calculated Factor VII protein concentration in plasma are shown in FIGS.

As apparent from FIGS. 4-8, each preparation (anti-Factor VII siRNA which suppresses the expression of Factor VII gene obtained in Examples 50 and 51, and compounds A-6, A-1, A-7-12 Evaluation of in vivo efficacy of compositions containing B-1, B-8, C-1, A-5, A-13-21, A-28-26, and C-2-5, respectively) The measured results of the factor VII protein concentration in the plasma show that administration of each preparation obtained in Examples 50 and 51 strongly suppresses the expression of Factor VII gene.
Thus, the composition of the present invention can introduce nucleic acid into cells etc., and the cationic lipid of the present invention should be a cationic lipid that facilitates the delivery of nucleic acid into cells in vivo. It became clear.

The composition was prepared as follows using the compound A-1 obtained in Example 5.
As the nucleic acid, the same nucleic acid as that used in Example 50 was prepared at 24 mg / mL with distilled water and used.
Each sample is weighed so that compound A-1 / PEG-DMPE Na (manufactured by NOF Corporation) = 57.3 / 5. 2 mmol / L, suspended in an aqueous solution containing hydrochloric acid and ethanol, and stirred with a vortex mixer. And heating was repeated to obtain a uniform suspension. This suspension was passed through a 0.2 μm polycarbonate membrane filter at room temperature, and further through a 0.05 μm polycarbonate membrane filter to obtain a dispersion of particles of Compound A-1 / PEG-DMPE Na (liposomes). The average particle size of the obtained liposomes was measured by the particle size measuring device, and it was confirmed to be in the range of 30 nm to 100 nm. The dispersion solution of the obtained liposome and the f7 siRNA solution are mixed in the ratio of dispersion solution of liposome: f7 siRNA solution = 3: 1, and then 3 times amount of distilled water is added and mixed, thereby compound A-1 A dispersion of the / PEG-DMPE Na / f7 siRNA complex was prepared.
On the other hand, each sample was prepared so that A-1 / PEG-DMPE Na (manufactured by NOF Corporation) / DSPC (manufactured by NOF Corporation) / cholesterol (manufactured by NOF Corporation) = 8.947 / 1.078 / 5.707 / 13.698 mmol / L. Were dissolved in 90 vol% ethanol to prepare a solution of lipid membrane components.
After heating the solution of the obtained lipid membrane component, it is mixed with the obtained dispersion of compound A-1 / PEG-DMPE Na / f7 siRNA complex in a ratio of 1: 1, and further several times the amount Distilled water was mixed to obtain a crude preparation.
The obtained crude preparation was concentrated using Amicon Ultra (manufactured by Millipore), diluted with physiological saline, and filtered using a 0.2 μm filter (manufactured by Toyo Roshi Co., Ltd.) in a clean bench. The composition (composition containing compound A-1 and a nucleic acid) is prepared by measuring the siRNA concentration of the obtained composition and diluting it with physiological saline so that the siRNA concentration will be 0.03, 0.01 or 0.003 mg / mL. Object).
The average particle size of the preparation (composition) was measured with a particle size measuring device. The results are shown in Table 14.

Among the compounds obtained in Examples 1 to 48, compounds A-5 to 7, A-10, A-12 to 21, A-28 to 36, B-8 and C-1 to 5 were used, respectively. A formulation was carried out in the same manner as in Example 52 (Compound A-5 to 7, A-10, A-12 to 21, A-28 to 36, B-8 and C-1 to 5, and a composition containing a nucleic acid Object).
The average particle size of the preparation (composition) was measured with a particle size measuring device. The results are shown in Table 14.

Comparative example 3
A formulation was obtained in the same manner as in Example 52 except that the compound A-1 was changed to the compound XI-9 obtained in Reference Example 23.
The structural formula of the compound (Compound XI-9) used in Comparative Example 3 is shown in Table 12.
The average particle size of the preparation (composition) was measured with a particle size measuring device. The results are shown in Table 13.

Test Example 4
Preparation obtained in each of Examples 52 and 53 or in the same manner as Example 52 or 53 (Compound A-1, A-5 to A-7, A-10, A-12 to 21, A-28 Test Examples for the composition containing each of? 36, B-8 and C-1 to 5 and nucleic acid, and the preparation obtained in Comparative Example 3 (composition containing compound XI-9 and nucleic acid) The in vivo drug efficacy evaluation test was performed by the same method as 3. The results of the calculated Factor VII protein concentration in plasma are shown in FIGS.

The compositions were prepared as follows using the compounds (compounds A-1, A-7 and A-10) obtained in Examples 5, 11 and 17, respectively. As the nucleic acid, the same nucleic acid as used in Example 50 was used.
Each of compound A-1, A-7 or A-10 / PEG-DMPE Na (manufactured by NOF Corporation) / DSPC (manufactured by NOF Corporation) / cholesterol (manufactured by NOF Corporation) = 7.030 / 0.755 / 2.038 / 4.892 mmol Each sample was weighed and dissolved in 100 vol% ethanol so as to be / L to prepare a solution of components of the lipid membrane. On the other hand, f7 siRNA / distilled water (24 mg / mL) was added to Tris-EDTA buffer (200 mM Tris-HCl, 20 mM EDTA, Invitrogen) and 20 mM citrate buffer (pH 4.0) The solution was diluted with the solution to prepare 0.536 mg / mL aqueous solution of f7 siRNA (2 mM Tris-EDTA buffer, 20 mM citrate buffer, pH 4.0).
The obtained lipid solution was heated to 37 ° C., and then 560 μL was transferred to a container for preparation of a preparation, and 560 μL of the obtained aqueous solution of f7 siRNA was added under stirring. 1120 μL of 20 mM citrate buffer (containing 300 mM NaCl, pH 6.0) is added to 1120 μL of the obtained lipid nucleic acid mixed suspension under stirring, and 4960 μL of DPBS (Invitrogen) is added dropwise to give a crude product. The formulation was obtained. The obtained crude preparation was concentrated using Amicon Ultra (manufactured by Millipore), diluted with DPBS, and filtered using a 0.45 μm filter (manufactured by Toyo Roshi Co., Ltd.) in a clean bench. Furthermore, by measuring the siRNA concentration of the obtained composition, and diluting with DPBS so that the siRNA concentration is 0.03 or 0.01 mg / mL, the formulations (compounds A-1, A-7 and A-10) Each and the composition containing a nucleic acid were obtained.
The average particle size of the preparation (composition) was measured with a particle size measuring device. The results are shown in Table 15.

Test Example 5
An in vivo efficacy evaluation test was carried out by the same method as in Test Example 3 for each of the preparations obtained in Example 54 (each of compound A-1, A-7 or A-10 and a composition containing a nucleic acid). . The result of the calculated Factor VII protein concentration in plasma is shown in FIG.

  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, for example, into cells or the like.

SEQ ID NO: 1-apolipoprotein B siRNA sense strand SEQ ID NO: 2-apolipoprotein B siRNA antisense strand SEQ ID NO: 3-blood coagulation factor 7 siRNA sense strand SEQ ID NO: 4- blood coagulation factor 7 siRNA antisense strand

Claims (15)

Formula (C)

(Wherein, R ⁸ is linear alkenyl having 12 to 24 carbon atoms,
R ⁹ is a linear or branched alkyl or alkenyl having 8 to 24 carbon atoms,
X ³ is alkylene having 1 to 3 carbon atoms,
Cationic lipid represented by R ¹⁰ is a hydrogen atom (with the exception of dioleoyl monoethanolamine , dioleylpropanolamine and N-oleyl-N-n-octadecyl-N-2-hydroxyethylamine ) . R ⁸ represents (Z) -tetradec-9-enyl, (Z) -hexadeca-9-enyl, (Z) -octadec-6-enyl, (Z) -octadeca-9-enyl, (E) -octadeca- 9-enyl, (Z) -octadeca-11-enyl, (9Z, 12Z) -octadeca-9, 12-dienyl, (9Z, 12Z, 15Z) -octadeca-9, 12, 15-trienyl, (Z)- The cationic lipid according to claim 1, which is icosa-11-enyl, (11Z, 14Z) -icosa-11, 14-dienyl or (Z) -docosa-13-enyl. R ⁸ is (Z) -hexadec-9-enyl, (Z) -octadec-6-enyl, (Z) -octadec-9-enyl, (9Z, 12Z) -octadeca-9, 12-dienyl, (Z 2. The cationic lipid according to claim 1, which is-) Icosa-11-enyl or (11Z, 14Z) -Icosa-11, 14-dienyl. R ⁹ is tetradecyl, hexadecyl, (Z) -tetradec-9-enyl, (Z) -hexadeca-9-enyl, (Z) -octadec-6-enyl, (Z) -octadeca-9-enyl, (E) -Octadeca-9-enyl, (Z) -octadeca-11-enyl, (9Z, 12Z) -octadeca-9, 12-dienyl, (9Z, 12Z, 15Z) -octadeca-9, 12, 15-trienyl, (( The cationic lipid according to claim 1, which is Z) -icosa-11-enyl, (11Z, 14Z) -icosa-11, 14-dienyl or (Z) -docosa-13-enyl. R ⁹ is hexadecyl, (Z) -hexadec-9-enyl, (Z) -octadec-6-enyl, (Z) -octadec-9-enyl, (9Z, 12Z) -octadeca-9, 12-dienyl ( The cationic lipid according to claim 1, which is Z) -icosa-11-enyl or (11Z, 14Z) -icosa-11, 14-dienyl. The cationic lipid according to any one of claims 1 to 5, wherein X ³ is methylene or ethylene. Formula (C)

(Wherein, R ⁸ is linear or branched alkenyl having 8 to 24 carbon atoms,
R ⁹ is a linear or branched alkyl or alkenyl having 8 to 24 carbon atoms,
X ³ is alkylene having 1 to 3 carbon atoms,
A composition containing a cationic lipid represented by R ¹⁰ is a hydrogen atom and a nucleic acid. The cationic lipid and the nucleic acid form a complex, or a combination of the cationic lipid and a neutral lipid and / or a macromolecule and the nucleic acid form a complex. The composition according to 7. The cationic lipid and the nucleic acid form a complex, or a combination of the cationic lipid and a neutral lipid and / or a polymer and the nucleic acid forms a complex, the complex The composition according to claim 7, comprising a lipid membrane encapsulating a body. The composition according to any one of claims 7 to 9, wherein the nucleic acid is a nucleic acid having an expression suppression effect of a target gene utilizing RNA interference (RNAi). The composition according to claim 10, wherein the target gene is a gene expressed in liver, lung, kidney or spleen. A medicament comprising the composition according to claim 10. The medicament according to claim 12, which is for intravenous administration. A therapeutic agent for a disease associated with liver, lung, kidney or spleen, comprising the composition according to claim 11. The agent for treating a disease associated with liver, lung, kidney or spleen according to claim 14, which is for intravenous administration.

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