WO2017010573A1

WO2017010573A1 - β2GPI GENE-SILENCING RNAi MEDICINE COMPOSITION

Info

Publication number: WO2017010573A1
Application number: PCT/JP2016/071076
Authority: WO
Inventors: 一剛長友; 和宏桝田; 美菜子神田; 陽史山田; 宏徒岩井; 智幸直井
Original assignee: 協和発酵キリン株式会社
Priority date: 2015-07-15
Filing date: 2016-07-15
Publication date: 2017-01-19
Also published as: JP2018150239A; TW201705965A

Abstract

The invention provides lipid particles, etc., containing a double-stranded nucleic acid as a drug, and a cationic lipid represented by formula (A), etc., the double-stranded nucleic acid comprising a sense strand and an antisense strand including a double-stranded region of at least 11 base pairs and is complementary to a target β2GPI mRNA sequence selected from the group listed in Table 2-1 to Table 2-16 in an oligonucleotide strand of a strand length of 17-30 nucleotides in the antisense strand.

Description

β2GPI gene expression-suppressing RNAi pharmaceutical composition

The present invention relates to a lipid particle containing a double-stranded nucleic acid used for suppression of β2GPI gene expression, a composition containing the lipid particle, a medicine, and the like.

β2-Glycoprotein 1 (β2GPI, also known as apolipoprotein H (apoH)) is a soluble glycoprotein composed of 326 amino acids and is produced mainly in the liver (“International Journal of Clinical and Laboratory Research (International Journal of Clinical and Laboratory Research) ”, 1992, Vol. 21, pp. 256-263). β2GPI is considered to have various physiological actions, and has been reported to be involved in platelet aggregation, coagulation / fibrinolysis, and uptake of oxidized LDL into macrophages (Non-patent Document 1).

Regarding its relationship to diseases, β2GPI is known to be the main anti-phospholipid antibody antigen that appears in autoimmune diseases such as antiphospholipid antibody syndrome (APS) and systemic lupus erythematosus (SLE) (non-patented) Reference 2). Anti-β2GPI antibodies are also deeply involved in the pathogenesis of diseases, and complexes formed by β2GPI and anti-β2GPI antibodies are found on the membranes of various cells such as vascular endothelial cells, monocytes, platelets, and trophoblasts. It has become clear from studies using animal models and clinical studies that an activation signal can be generated at the receptor, resulting in pathological conditions characteristic of APS such as thrombosis and pregnancy abnormalities (Non-patent Document 3). ). By specifically inhibiting the formation of immune complexes consisting of β2GPI and anti-β2GPI antibodies, it can be expected that the disease can be prevented or treated, but β2GPI is present in blood at a relatively high concentration of 50-500 μg / mL. Therefore, it is not easy to continue to inhibit all of these β2GPIs using, for example, general antibody drugs (Non-patent Document 4).

On the other hand, as a method for suppressing the expression of a target gene, for example, a method using RNA interference (hereinafter referred to as "RNAi") is known, and specifically, a gene targeted in a nematode It has been reported that the expression of the target gene is specifically suppressed by introducing a double-stranded RNA having the same sequence as (Nature), 1998, 391Vol.391, 6669, pp.806-811). In addition, it was found that the expression of the target gene is suppressed by introducing a double-stranded RNA having a length of 21-23 bases instead of a long double-stranded RNA in Drosophila. It is named RNA (siRNA) (see WO 01/75164).

RNAi has been extensively verified in in vivo tests, with effects in fetal animals using siRNAs of 50 base pairs or less (see US Patent Application Publication No. 2002/132788) and effects in adult mice (See International Publication No. 03/10180). In addition, when siRNA is administered intravenously to mouse fetuses, the effect of suppressing the expression of specific genes has been confirmed in the kidney, spleen, lung, pancreas, and liver organs (“Nature Genetics”, (See 2002, Vol. 32, Vol. 1, No. 1, p. 107-108). Furthermore, it has been reported that the expression of specific genes is also suppressed in brain cells by direct administration of siRNA (“Nature Biotechnology”, 2002, Volume 20, No.10, p .1006-1010).

Patent Documents

1 and 2 disclose a part of siRNA sequences targeting the human β2GPI gene, but do not disclose that the siRNA sequence suppresses the expression of the human β2GPI gene.

In addition, pharmaceuticals containing siRNAs are described in, for example, Patent Documents 3 to 5 and the like.
Patent Document 3 includes siRNAs and, for example,

2,2-dilinoleyl-4- (2-dimethylaminoethyl)-[1,3] -dioxolane: DLin-KC2- A medicine containing DMA) or the like is disclosed.
Patent Document 4 includes siRNAs and, for example,

(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) butanoate: DLin-MC3-DMA) and the like are disclosed.
Patent Document 5 discloses a composition containing a cationic lipid and a nucleic acid, a method for introducing a nucleic acid into a cell using the composition, and the like.

International Publication No. 2005/116204 International Publication No. 2008/043561 International Publication No. 2010/042877 International Publication No. 2010/054401 International Publication No. 2014/007398

An object of the present invention is to provide a lipid particle containing a double-stranded nucleic acid, a composition containing the lipid particle, a medicine, and the like.

The present invention relates to the following (1) to (61).
(1) A double-stranded nucleic acid consisting of a sense strand and an antisense strand, and comprising a double-stranded region of at least 11 base pairs, the length of which is 17 to 30 nucleotides in the antisense strand A double-stranded nucleic acid as a drug that is complementary to a target β2GPI mRNA sequence selected from the group described in Tables 2-1 to 2-16,
Formula (A)

(Wherein R ¹ is 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 linear, branched or cyclic alkyl having 1 to 3 carbon atoms, or together, they form alkylene having 2 to 8 carbon atoms, or R ³ together with R ⁵ forms an alkylene having 2 to 8 carbon atoms,
R ⁵ is a hydrogen atom, linear, branched or cyclic alkyl having 1 to 6 carbon atoms, linear or branched alkenyl having 3 to 6 carbon atoms, amino, monoalkylamino, ammonio, mono Alkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl or the same or different 1 to 3 amino, monoalkylamino, ammonio, monoalkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl or dialkylcarbamoyl A linear, branched or cyclic alkyl group having 1 to 6 carbon atoms or a linear or branched alkenyl group having 3 to 6 carbon atoms, or carbon together with R ³ Forming alkylene of 2 to 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 together with R ³ forms an alkylene of 2 to 8 carbon atoms, X ² is a single bond, or is methylene or ethylene),
Formula (B)

(Wherein R ⁶ is 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 a formula (C)

(Wherein R ⁸ is 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 lipid particle containing a cationic lipid represented by a hydrogen atom or a linear, branched or cyclic alkyl having 1 to 3 carbon atoms.
(2) R ¹ , R ² , R ⁶ , R ⁷ , R ⁸ and R ⁹ are tetradecyl, hexadecyl, (Z) -tetradec-9-enyl, (Z) -hexadeca-9-enyl, (Z), respectively. -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 lipid particle according to the above (1), which is an enil.
(3) R ¹ , R ² , R ⁶ , R ⁷ , R ⁸ and R ⁹ are (Z) -octadeca-9-enyl, (9Z, 12Z) -octadeca-9,12-dienyl or (11Z, The lipid particle according to (1), which is 14Z) -icosa-11,14-dienyl.
(4) The lipid particle according to any one of (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 lipid particle according to any one of (1) to (4), wherein X ³ is methylene or ethylene.
(6) The lipid particle 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 -hexylene.
(7) The lipid according to any one of (1) to (5) above, wherein R ³ and R ⁵ together form n-propylene or n-butylene, and R ⁴ is methyl or ethyl. particle.
(8) The lipid particle according to any one of (1) to (6), wherein R ⁵ and R ¹⁰ are each a hydrogen atom or methyl.
(9) The double-stranded region is a double-stranded region comprising 11 to 27 base pairs and is complementary to a target β2GPI mRNA sequence selected from the group described in Tables 2-1 to 2-16. The lipid particle according to any one of (1) to (8), wherein the second nucleotide from the 5 ′ end of an antisense strand is complementary to the second deoxyribonucleotide from the 3 ′ end of the target β2GPI mRNA sequence. .
(10) The lipid particle according to any one of (1) to (9), wherein the sense strand has a length of 21 nucleotide chains, and the antisense strand has a length of 21 nucleotide chains.
(11) The double-stranded nucleic acid in which the sense strand has a length of 21 nucleotides and the antisense strand has a length of 21 nucleotides includes a 19-base pair double-stranded region (1) ) To (10).
(12) The lipid particle according to any one of (1) to (9), wherein the 3 ′ end of the sense strand and the 5 ′ end of the antisense strand form a blunt end.
(13) The lipid particle according to any one of (1) to (12), wherein the double-stranded nucleic acid contains 2′-O-methyl modified nucleotides.
(14) The lipid particle according to (13), wherein 40 to 65% of the nucleotides in the double-stranded region are 2′-O-methyl modified nucleotides.
(15) The lipid particle according to any one of (1) to (14), wherein the antisense strand comprises a sequence selected from the antisense strand group described in Tables 4-1 to 4-5.
(15-1) (Hereinafter, in the description of (15), it also means that (15-1) is also included) It consists of a sense strand and an antisense strand, and contains at least 11 base pair duplex region A double-stranded nucleic acid as a drug, wherein the antisense strand comprises a sequence selected from the antisense strand group described in Tables 4-1 to 4-5;
Formula (A)

(Wherein R ⁸ is 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 lipid particle containing a cationic lipid represented by a hydrogen atom or a linear, branched or cyclic alkyl having 1 to 3 carbon atoms.
(16) The lipid particle according to any one of (1) to (14), wherein the sense strand comprises a sequence selected from the sense strand group described in Tables 4-1 to 4-5.
(16-1) (Hereinafter, in the description of (16), it also means that (16-1) is included.) A double-stranded nucleic acid as a drug, wherein the sense strand comprises a sequence selected from the group of sense strands described in Tables 4-1 to 4-5;
Formula (A)

(Wherein R ⁸ is 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 lipid particle containing a cationic lipid represented by a hydrogen atom or a linear, branched or cyclic alkyl having 1 to 3 carbon atoms.
(17) The above (1) to (14), comprising a pair of sense / antisense strand sequences selected from the group consisting of the sense strand / antisense strand described in Table 4-1 to Table 4-5 The lipid particle according to any one of the above.
(17-1) (Hereinafter, in the description of (17), it also means that (17-1) is included) It consists of a sense strand and an antisense strand, and contains at least 11 base pair duplex region A pair of sense strand / antisense, wherein the sense strand / antisense strand is a double-stranded nucleic acid selected from the group consisting of the sense strand / antisense strand group described in Table 4-1 to Table 4-5 A double-stranded nucleic acid as a drug comprising a sequence of strands;
Formula (A)

(Wherein R ⁸ is 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 lipid particle containing a cationic lipid represented by a hydrogen atom or a linear, branched or cyclic alkyl having 1 to 3 carbon atoms.
(18) A double-stranded nucleic acid comprising a sense strand and an antisense strand, and comprising a double-stranded region of at least 11 base pairs, wherein the strand length of 17 to 30 nucleotides in the antisense strand In which the antisense strand is complementary to a target β2GPI mRNA sequence selected from the group described in Tables 2-1 to 2-16, and the antisense strand is from Table 4-1 to Table 4-5. Or a sequence selected from the antisense strands described in Table 4-1 to Table 4-5, or a sequence selected from the antisense strands described in Table 4-1. A double-stranded nucleic acid as a drug comprising a pair of sense / antisense strand sequences selected from the group consisting of the sense strand / antisense strand described in 4-1 to Table 4-5;
Formula (A)

(Wherein R ⁸ is 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 lipid particle containing a cationic lipid represented by a hydrogen atom or a linear, branched or cyclic alkyl having 1 to 3 carbon atoms.
Note that (15-1), (16-1), (17-1) and (18) may include the matters described in any of (2) to (16) above.
(19) The cationic lipid forms a complex with a double-stranded nucleic acid, or forms a complex with a combination of a neutral lipid and / or a polymer and a double-stranded nucleic acid. (1) The lipid particle according to any one of (18).
(20) A cationic lipid forms a complex with a double-stranded nucleic acid, or a complex of a combination of a neutral lipid and / or a polymer with a double-stranded nucleic acid. The lipid particle according to any one of (1) to (19), wherein the particle is a lipid particle composed of the complex and a lipid membrane encapsulating the complex.
(21) A lipid particle-containing composition comprising the lipid particle according to any one of (1) to (20).
(22) A method for suppressing the expression of β2GPI gene, comprising introducing a double-stranded nucleic acid into a cell using the composition according to (21).
(23) The method according to (22) above, wherein the cell is a cell in a mammalian liver.
(24) The method according to the above (22) or (23), wherein the method of introducing into cells is a method of introducing into cells by intravenous administration.
(25) A method for treating a β2GPI-related disease, comprising administering the composition according to (21) to a mammal.
(26) The method according to (25) above, wherein the β2GPI-related disease is an autoimmune disease or thrombosis.
(27) The method according to (25) or (26) above, wherein the method of administration is intravenous administration.
(28) A medicament for use in the treatment of β2GPI-related diseases, comprising the composition according to (20).
(29) The medicament according to (28), wherein the β2GPI-related disease is an autoimmune disease or thrombosis.
(30) The medicament according to (28) or (29), which is for intravenous administration.
(31) A therapeutic agent for autoimmune disease or thrombosis comprising the composition according to (20).
(32) The therapeutic agent for autoimmune disease or thrombosis according to (31), which is for intravenous administration.
(33) A double-stranded nucleic acid comprising a sense strand and an antisense strand, and comprising a double-stranded region of at least 11 base pairs, wherein the strand length of 17 to 30 nucleotides in the antisense strand A double-stranded nucleic acid as a drug that is complementary to a target β2GPI mRNA sequence selected from the group described in Tables 2-1 to 2-16,
Formula (A)

(Wherein R ¹ is 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 linear, branched or cyclic alkyl having 1 to 3 carbon atoms, or together, they form alkylene having 2 to 8 carbon atoms, or R ³ together with R ⁵ forms an alkylene having 2 to 8 carbon atoms,
R ⁵ is a hydrogen atom, linear, branched or cyclic alkyl having 1 to 6 carbon atoms, linear or branched alkenyl having 3 to 6 carbon atoms, amino, monoalkylamino, ammonio, mono Alkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl or the same or different 1 to 3 amino, monoalkylamino, ammonio, monoalkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl or dialkylcarbamoyl A linear, branched or cyclic alkyl group having 1 to 6 carbon atoms or a linear or branched alkenyl group having 3 to 6 carbon atoms, or carbon together with R ³ Forming alkylene of 2 to 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 together with R ³ forms an alkylene of 2 to 6 carbon atoms, X ² is a single bond, or is methylene or ethylene),
Formula (B)

(Wherein R ⁸ is 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 lipid particle-containing composition containing a cationic lipid represented by a hydrogen atom or a linear, branched or cyclic alkyl having 1 to 3 carbon atoms.
(34) R ¹ , R ² , R ⁶ , R ⁷ , R ⁸ and R ⁹ are tetradecyl, hexadecyl, (Z) -tetradec-9-enyl, (Z) -hexadeca-9-enyl, (Z), respectively. -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 composition according to (33) above, which is an enyl.
(35) R ¹ , R ² , R ⁶ , R ⁷ , R ⁸ and R ⁹ are (Z) -octadeca-9-enyl, (9Z, 12Z) -octadeca-9,12-dienyl or (11Z, The composition according to the above (33), which is 14Z) -icosa-11,14-dienyl.
(36) The composition according to any one of (33) to (35), wherein X ¹ is alkylene having 1 to 3 carbon atoms, and X ² is a single bond or methylene.
(37) The composition according to any one of (33) to (36), wherein X ³ is methylene or ethylene.
(38) The composition according to any one of (33) to (37), wherein R ³ and R ⁴ are the same or different and are methyl or ethyl, or together, form n-pentylene or -hexylene.
(39) The composition according to any one of (33) to (37), wherein R ³ and R ⁵ together form n-propylene or n-butylene, and R ⁴ is methyl or ethyl. object.
(40) The composition according to any one of (33) to (38), wherein R ⁵ and R ¹⁰ are each a hydrogen atom or methyl.
(41) The double-stranded region is a double-stranded region comprising 11 to 27 base pairs and is complementary to a target β2GPI mRNA sequence selected from the group described in Tables 2-1 to 2-16 The composition according to any one of (33) to (40), wherein the second nucleotide from the 5 ′ end of an antisense strand is complementary to the second deoxyribonucleotide from the 3 ′ end of the target β2GPI mRNA sequence. .
(42) The composition according to any one of (33) to (41), wherein the sense strand has a length of 21 nucleotide chains, and the antisense strand has a length of 21 nucleotide chains.
(43) The double-stranded nucleic acid in which the sense strand has a length of 21 nucleotides and the antisense strand has a length of 21 nucleotides includes a 19-base pair double-stranded region (33) ) To (42).
(44) The composition according to any one of (33) to (41), wherein the 3 ′ end of the sense strand and the 5 ′ end of the antisense strand form a blunt end.
(45) The composition according to any one of (33) to (44), wherein the double-stranded nucleic acid comprises 2′-O-methyl modified nucleotides.
(46) The composition according to the above (45), wherein 40 to 65% of the nucleotides in the double-stranded region are 2′-O-methyl modified nucleotides.
(47) The composition according to any one of (33) to (46), wherein the antisense strand comprises a sequence selected from the group of antisense strands described in Tables 4-1 to 4-5.
(48) The composition described in any of (33) to (46) above, wherein the sense strand comprises a sequence selected from the sense strand group described in Tables 4-1 to 4-5.
(49) The above (33) to (46), comprising a pair of sense strand / antisense strand sequences selected from the group consisting of the sense strand / antisense strand described in Table 4-1 to Table 4-5 The composition in any one of.
(50) The cationic lipid forms a complex with a double-stranded nucleic acid, or forms a complex with a combination of a neutral lipid and / or a polymer and a double-stranded nucleic acid, (33) The composition according to any one of (49).
(51) A cationic lipid forms a complex with a double-stranded nucleic acid, or a complex of a combination of a neutral lipid and / or a polymer with a double-stranded nucleic acid. The composition according to any one of (33) to (50), wherein the particle is a lipid particle composed of the complex and a lipid membrane encapsulating the complex.
(52) A method for suppressing the expression of β2GPI gene, comprising introducing a double-stranded nucleic acid into a cell using the composition according to any one of (33) to (51).
(53) The method according to (52) above, wherein the cell is a cell in a mammalian liver.
(54) The method according to the above (52) or (53), wherein the method of introducing into cells is a method of introducing into cells by intravenous administration.
(55) A method for treating a β2GPI-related disease, comprising administering the composition according to any one of (33) to (51) to a mammal.
(56) The method according to (55) above, wherein the β2GPI-related disease is an autoimmune disease or thrombosis.
(57) The method according to (55) or (56) above, wherein the method of administration is intravenous administration.
(58) A medicament for use in the treatment of β2GPI-related diseases, comprising the composition according to any of (33) to (51).
(59) The medicament according to (58), wherein the β2GPI-related disease is an autoimmune disease or thrombosis.
(60) The medicament according to the above (58) or (59), which is for intravenous administration.
(61) A therapeutic agent for autoimmune disease or thrombosis comprising the composition according to any of (33) to (51).
(62) The therapeutic agent for autoimmune disease or thrombosis according to (61), which is for intravenous administration.

For example, the composition containing the lipid particles of the present invention can be administered to a mammal to suppress β2GPI gene expression in vivo and treat β2GPI-related diseases.

The diluted Russell snake venom time (dRVVT) measured using the plasma collected from mice after administration of the preparation 6 or physiological saline obtained in Example 2 to male Balb / c mice is shown. The vertical axis shows the time (seconds) until blood clotting is observed after adding LA test “Gladipore” Reagent 1 (manufactured by MBL) as a measuring reagent, and the horizontal axis shows the individual number. Show. Individual numbers 1 to 2 are individuals to which physiological saline was administered, and individual numbers 3 to 5 were individuals to which preparation 6 was administered.

The β2GPI gene (gene encoding β2GPI) targeted by the nucleic acid of the present invention is a gene that produces β2GPI full-length mRNA corresponding to β2GPI cDNA (SEQ ID NO: 3541) registered as Genbank AccessionNo.NM_000042 Is mentioned.

The present invention provides a double-stranded nucleic acid having the ability to reduce or stop the expression of β2GPI gene as a drug, and a lipid particle containing a cationic lipid.
In addition, a composition containing the lipid particles (a composition containing lipid particles) is provided, and the composition is administered to a mammal to suppress β2GPI gene expression and treat β2GPI-related diseases in vivo. A method is also provided.
Furthermore, the present invention also provides methods for treating or preventing disorders associated with anti-β2GPI antibodies.

The lipid particles in the present invention are
Formula (A)

(Wherein R ⁸ is 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 contains a cationic lipid represented by a hydrogen atom or a linear, branched or cyclic alkyl having 1 to 3 carbon atoms.
Hereinafter, the compound represented by formula (A) may be referred to as compound (A), the compound represented by formula (B) as compound (B), and the compound represented by formula (C) as compound (C). . The same applies to the 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 straight-chain or branched alkenyl having 8 to 24 carbon atoms may be a group containing one or more double bonds in straight-chain or branched alkyl having 8 to 24 carbon atoms. ) -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, (2E, 6E) -3,7,11-trimethyldodeca-2,6,10-trienyl, (E) -3,7,11,15-tetra Methylhexadec-2-enyl and (Z) -docosa-13-enyl, etc., preferably (Z) -hexadec-9-enyl, (Z) -octadec-6-enyl, (Z) -octadeca- 9-enyl, (9Z, 12Z) -octadeca-9,12-dienyl, (Z) -icosa-11-enyl, (11Z, 14Z) -icosa-11,14-die Le or (Z) - docos-13-enyl.
The linear or branched alkynyl having 8 to 24 carbon atoms may be a group containing one or more triple bonds in linear or branched alkyl having 8 to 24 carbon atoms. For example, dodeca- Examples include 11-ynyl, tetradec-6-ynyl, hexadec-7-ynyl, hexadec-5,7-diynyl, and octadec-9-ynyl.

The alkyl moiety in alkoxyethyl and alkoxypropyl may be, for example, the linear or branched alkyl having 8 to 24 carbon atoms, specifically, the linear or branched alkyl having 8 to 24 carbon atoms. Examples include the groups exemplified as the branched alkyl.
The alkenyl moiety in alkenyloxyethyl and alkenyloxypropyl may be, for example, the above linear or branched alkenyl having 8 to 24 carbon atoms, specifically, the above linear chain having 8 to 24 carbon atoms. Or the group etc. which were illustrated as branched alkenyl are mentioned.
The alkynyl moiety in alkynyloxyethyl and alkynyloxypropyl may be, for example, a linear or branched alkynyl group having 8 to 24 carbon atoms, specifically, a linear chain having 8 to 24 carbon atoms. Or the group etc. which were illustrated as branched alkynyl are mentioned.

R ¹ and R ² are preferably the same or different and are linear or branched alkyl or alkenyl having 8 to 24 carbon atoms, and are the same or different and are linear or branched having 8 to 24 carbon atoms. The alkenyl is more preferably a linear alkenyl having the same or different carbon number of 8 to 24. R ¹ and R ² are preferably the same, and in this case, they are preferably straight-chain or branched alkyl, alkenyl or alkynyl having 12 to 24 carbon atoms, More preferably, it is 24 linear alkenyl.

R ¹ and R ^2, when different, R ¹ is a 16 to 24 carbon atoms linear or branched alkyl, alkenyl or alkynyl, straight-chain R ² is 8 to 12 carbon atoms like or Branched alkyl, alkenyl or alkynyl is also a preferred form of the invention. In this case, R ¹ is preferably straight-chain alkenyl having 16 to 24 carbon atoms, R ² is preferably straight-chain alkyl having 8 to 12 carbon atoms, and R ¹ is (Z) -octadeca-9 More preferably, it is -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 straight-chain 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 a preferred form of the present invention. In this case, R ¹ is preferably straight-chain alkenyl having 16 to 24 carbon atoms, R ² is preferably alkenyloxyethyl, and R ¹ is (Z) -octadeca-9-enyl, (9Z , 12Z) -octadeca-9,12-dienyl or (11Z, 14Z) -icosa-11,14-dienyl, 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 R ¹ is (9Z, 12Z) -octadeca-9,12- More preferably, it is dienyl and R ² is (9Z, 12Z) -octadeca-9,12-dienyloxyethyl.

When R ¹ and R ² are the same or different and are linear or branched alkyl or alkenyl having 8 to 24 carbon atoms, they are the same or different, 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)- It is preferably icosa-11,14-dienyl or (Z) -docosa-13-enyl, the same or different hexadecyl, (Z) -hexadec-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 More preferably, same or different (Z) More preferred are -octadeca-9-enyl, (9Z, 12Z) -octadeca-9,12-dienyl or (11Z, 14Z) -icosa-11,14-dienyl, and identically (9Z, 12Z) -octadeca Even more preferred is -9,12-dienyl.

R ⁶ and R ⁷ are synonymous with R ¹ and R ² , respectively. Provided that when R ⁷ is straight or branched alkyl, alkenyl or alkynyl having 16 to 24 carbon atoms, R ⁶ and R ⁷ are the same as (9Z, 12Z) -octadeca-9,12- Dienyl is preferred.

R ⁸ and R ⁹ are synonymous with R ¹ and R ² , respectively. However, R ⁸ and R ⁹ are preferably straight or branched alkyl, alkenyl or alkynyl having 16 to 24 carbon atoms, and are identically (9Z, 12Z) -octadeca-9,12- More preferred is dienyl.

Examples of the linear, branched or cyclic alkyl having 1 to 3 carbon atoms in R ³ and R ⁴ include methyl, ethyl, propyl, isopropyl and cyclopropyl, preferably methyl or ethyl, More preferred is methyl.

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

R ³ may be methyl or ethyl, together with R ⁴ form an alkylene of 5 to 7 carbons, or together with R ⁵ form an alkylene of 3 to 5 carbons preferable. However, when R ^{3 and} R ⁴ do not form an alkylene having 5 to 7 carbon atoms, R ⁴ is preferably methyl or ethyl, and more preferably methyl.
R ³ may be methyl, combined with R ⁴ to form n-pentylene or n-hexylene, or R ³ together with R ⁵ to form ethylene, n-propylene. preferable. However, if the R ³ do not form together with R ⁴ n- pentylene or n- hexylene, R ⁴ is preferably methyl.

Examples of the linear, branched or cyclic alkyl having 1 to 6 carbon atoms for R ⁵ include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, And cyclopropylmethyl, pentyl, isopentyl, sec-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-pentyl, neopentyl or hexyl, more preferably methyl, ethyl or propyl.

Examples of the linear or branched alkenyl having 3 to 6 carbon atoms in R ⁵ include allyl, propenyl, butenyl, pentenyl, hexenyl and the like, and preferably allyl.

The monoalkylamino in R ⁵ may be any amino substituted with one, for example, linear, branched or cyclic alkyl having 1 to 6 carbon atoms (as defined above), such as methylamino, Examples include ethylamino, propylamino, butylamino, pentylamino, hexylamino, and the like, preferably methylamino or ethylamino.
In R ⁵ , amino and monoalkylamino may each be coordinated by a hydrogen ion to a lone pair of electrons on the nitrogen atom to form ammonio and monoalkylammonio. Each include ammonio and monoalkylammonio.
In the present invention, ammonio and monoalkylammonio 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 hydroxy substituted with linear, branched or cyclic alkyl having 1 to 6 carbon atoms (as defined above), for example, methoxy, ethoxy, propyloxy, butyloxy , Pentyloxy, hexyloxy and the like, preferably methoxy or ethoxy.

The monoalkylcarbamoyl and dialkylcarbamoyl in R ⁵ are each substituted with one and the same or different two, for example, a linear, branched or cyclic alkyl having 1 to 6 carbon atoms (as defined above). For example, methylcarbamoyl, ethylcarbamoyl, propylcarbamoyl, butylcarbamoyl, pentylcarbamoyl, hexylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl, ethylmethylcarbamoyl, methylpropylcarbamoyl, butylmethylcarbamoyl, methylpentylcarbamoyl and hexylmethyl Carbamoyl and the like can be mentioned, and methylcarbamoyl, ethylcarbamoyl or dimethylcarbamoyl is preferred.

Straight chain of 1 to 6 carbon atoms substituted with 1 to 3 amino, monoalkylamino, ammonio, monoalkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl or dialkylcarbamoyl, the same or different in R ⁵ A branched or cyclic alkyl or a straight or branched alkenyl having 3 to 6 carbon atoms includes a straight, branched or cyclic alkyl having 1 to 6 carbon atoms (as defined above) or 1 to 3 amino, monoalkylamino, ammonio, monoalkylammonio, hydroxy, alkoxy, carbamoyl, mono- or straight-chain or branched alkenyl having 3 to 6 carbon atoms (as defined above) Substituted with alkylcarbamoyl or dialkylcarbamoyl (each as defined above) Any group may be used.

Examples of the alkylene having 2 to 8 carbon atoms formed by R ⁵ and R ³ together include, for example, ethylene, n-propylene, n-butylene, n-pentylene, n-hexylene, n-heptylene and n-octylene. N-propylene, n-butylene or n-pentylene is preferred, n-propylene or n-butylene is more preferred, and n-propylene is still more preferred.

R ⁵ represents a hydrogen atom, straight-chain, branched or cyclic alkyl having 1 to 6 carbon atoms, monoalkylamino, hydroxy, alkoxy, or the same or different 1 to 3 amino, monoalkylamino, hydroxy or alkoxy It is preferably a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms substituted with or a group having 2 to 6 carbon atoms together with R ³ to form a hydrogen atom, Methyl, amino, methylamino, hydroxy, methoxy, or methyl that is the same or different and substituted with 1 to 3 amino or hydroxy, or together with R ³ forms an alkylene of 3 to 5 carbon atoms more preferably, form a hydrogen atom, an alkyl having 1 to 3 carbon atoms or hydroxy or where taken together with R ³ n- propylene or n- butylene, Rukoto more preferably, it is more preferred more that taken together form n- propylene hydrogen atom or R ^3.

Examples of the linear, branched or cyclic alkyl having 1 to 3 carbon atoms in R ¹⁰ include methyl, ethyl, propyl, isopropyl and cyclopropyl, preferably methyl, ethyl or isopropyl, and more. Preferred is methyl or ethyl.
R ¹⁰ is preferably a hydrogen atom or methyl, more preferably a hydrogen atom.

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

X ¹ is preferably alkylene having 1 to 3 carbon atoms, more preferably methylene or ethylene, and X ² is preferably a single bond, methylene or ethylene, and is a single bond or methylene. Is more preferable.
The sum of the carbon number of X ¹ and X ² is preferably 1 to 3, and more 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 Methyl substituted with one amino or hydroxy, or R ³ and R ⁴ together form an alkylene having 5 to 7 carbon atoms, and R ⁵ is a hydrogen atom, methyl, amino, methylamino, hydroxy, Methoxy, or methyl identically or differently substituted with 1 to 3 amino or hydroxy, or R ³ and R ⁵ together form an alkylene of 3 to 5 carbon atoms and R ⁴ is methyl or Preferably it is ethyl, 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 in X ³ include methylene, ethylene and n-propylene, and preferably methylene or ethylene.

Compounds (A), (B) and (C) may form a salt with a pharmaceutically acceptable anion when a hydrogen ion is coordinated to a lone pair of electrons on any nitrogen atom. Good.
In the present invention, examples of the pharmaceutically acceptable anion include 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 citrate ion, benzoate ion and methanesulfonate ion.

Next, a method for producing compounds (A), (B) and (C) will be described. In the production method shown below, when a defined group changes under the conditions of the production method or is inappropriate for carrying out the production method, a method for introducing and removing a protective group commonly used in organic synthetic chemistry [ For example, the method described in Protective 第 Groups (in Organic Synthesis, third edition, TWGreene, John Wiley & Sons Inc. (1999) etc.] is used. Thus, the target compound can be produced. Further, the order of reaction steps such as introduction of substituents can be changed as necessary.

Manufacturing method 1
Compound (A) can be produced by the following method.

(Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , X ¹ and X ² are as defined above, Y is a chlorine atom, bromine atom, iodine atom, trifluoromethanesulfonyloxy, methane (Representing a leaving group such as sulfonyloxy, benzenesulfonyloxy or p-toluenesulfonyloxy, Ar represents a substituted or unsubstituted phenyl group such as p-nitrophenyl, o-nitrophenyl or p-chlorophenyl)

Step

1 and 2
Compound (IIa) is a mixture of ammonia and compound (IIIa) in the absence of 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. for 5 minutes to 100 hours. It can be produced by reacting.
Compound (IIb) is obtained by reacting Compound (IIa) and Compound (IIIb) in the absence of a 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. for 5 minutes. It can be produced by reacting for ~ 100 hours.
Examples of the solvent include methanol, ethanol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, 1,4-dioxane, N, N-dimethylformamide. , N, N-dimethylacetamide, N-methylpyrrolidone, pyridine, water and the like, and these may be used alone or in combination.
Examples of the base include cesium carbonate, potassium carbonate, potassium hydroxide, sodium hydroxide, sodium methoxide, potassium tert-butoxide, triethylamine, diisopropylethylamine, N-methylmorpholine, pyridine and 1,8-diazabicyclo [5.4.0]. -7-Undecene (DBU).
Compound (IIIa) is a commercially available product or a known method (for example, 5th edition, Experimental Chemistry Lecture 13, Synthesis of Organic Compounds I ”, 5th edition, p.374, Maruzen (2005)) or a method analogous thereto. Obtainable.
Compound (IIIb) is a commercially available product or a known method (e.g., 5th edition Experimental Chemistry Course 13 Synthesis of organic compounds I, 5th edition, p. 374, Maruzen (2005)) or a method analogous thereto, Or it can obtain with the below-mentioned manufacturing method.
Compound (IIb) in the case where R ¹ and R ² are the same can be obtained by using 2 equivalents or more of compound (IIIa) in Step 1.

Process 3
Compound (VI) is compound (IVa) with compound (Va) in the absence of solvent 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 prepared by reacting in the presence of an equivalent amount of base at a temperature between −20 ° C. and 150 ° C. for 5 minutes to 72 hours.
Examples of the solvent include dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, 1,4-dioxane, N, N-dimethylformamide, N, N -Dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide and the like can be mentioned, and these can be used alone or in combination.
Examples of the additive include 1-hydroxybenzotriazole and 4-dimethylaminopyridine.
Examples of the base include those exemplified in

Steps

1 and 2.
Compound (Va) can be obtained as a commercial product.
Compound (IVa) is a commercially available product or a known method (for example, 5th edition Experimental Chemistry Course 14 Synthesis of organic compounds II, 5th edition, p.1, Maruzen (2005)) or a method analogous thereto. Obtainable.

Process 4
Compound (A) is compound (IIb), compound (VI), and in the absence of solvent 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 prepared by reacting in the presence of an equivalent amount of base at a temperature between −20 ° C. and 150 ° C. for 5 minutes to 72 hours.
Examples of the solvent and the additive include those exemplified in Step 3.
Examples of the base include those exemplified in

Steps

1 and 2.

Manufacturing method 2
Among the compounds (IIIb), R ² is linear or branched alkoxyethyl, alkoxypropyl, alkenyloxyethyl, alkenyloxypropyl, alkynyloxyethyl or alkynyloxypropyl having 8 to 24 carbon atoms, and Y is Compound (IIIc), which is methanesulfonyloxy, can be produced by the following method.

(Wherein Y is as defined above, R ¹¹ is linear or branched alkyl, alkenyl or alkynyl having 8 to 24 carbon atoms, X ⁴ represents ethylene or propylene, and Ms represents methanesulfonyl. Represents a group)

Process 5
Compound (IVd) is obtained by reacting Compound (IVb) with Compound (IVc) in the absence of 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 from 100 minutes to 100 minutes.
Examples of the solvent include dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, 1,4-dioxane, N, N-dimethylformamide, N, N -Dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide and the like can be mentioned, and these can be used alone or in combination.
Examples of the base include those exemplified in

Steps

1 and 2.
Compound (IVb) is the same as compound (IIIa) described in Production Method 1.
Compound (IVc) can be obtained as a commercial product.

Process 6
Compound (IIIc) is obtained by reacting Compound (IVd) for 5 minutes at a temperature between -20 ° C. and 150 ° C. in the absence of a mesylating reagent and in the presence of a solvent, preferably in the presence of 1 to 10 equivalents of a base. It can be produced by reacting for ~ 72 hours.
Examples of the solvent include those exemplified in Step 5.
Examples of the base include those exemplified in

Steps

1 and 2.
Examples of the mesylation reagent include methanesulfonic acid anhydride and methanesulfonic acid chloride.

Manufacturing method 3
Compound (IIb) can also be produced by the following method.

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

Process 7
Compound (IVe) comprises compound (Vb) and compound (IIIa) in the absence of solvent or in a solvent, preferably in the presence of 1 to 10 equivalents of an additive, and / or preferably 1 to 10 equivalents if necessary. In the presence of a base at a temperature between room temperature and 200 ° C. for 5 minutes to 100 hours.
Examples of the solvent and the base include those exemplified in

Steps

1 and 2, respectively.
Examples of the additive include n-tetrabutylammonium iodide and sodium iodide.
Compound (Vb) can be obtained as a commercial product.

Process 8
Compound (IVf) is obtained by adding Compound (IVe) at a temperature of −20 ° C. and 150 ° C. with 1 equivalent to a large excess of acid, without solvent or in a solvent, and preferably in the presence of 1 to 10 equivalents of an additive. It can be produced by treating at a temperature between 5 minutes and 72 hours.
Examples of the solvent include those exemplified in

Steps

1 and 2.
Examples of the acid include hydrochloric acid, sulfuric acid, phosphoric acid and trifluoroacetic acid.
Examples of the additive include thioanisole, dimethyl sulfide and triisopropylsilane.

Step 9
Compound (IVg) is compound (IIIb) and compound (IVf), without a solvent or in a solvent, preferably in the presence of 1 to 10 equivalents of an additive, and / or preferably 1 to 10 equivalents if necessary. In the presence of a base at a temperature between room temperature and 200 ° C. for 5 minutes to 100 hours.
Examples of the solvent and the base include those exemplified in

Steps

1 and 2, respectively.
Examples of the additive include those exemplified in Step 7.

Process 10
Compound (IIb) is obtained by reacting compound (IVg) and thiol compound in the absence of 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. for 5 minutes to 100 minutes. It can manufacture by making it react for time.
Examples of the solvent and the base include those exemplified in

Steps

1 and 2, respectively.
Examples of the thiol compound include methanethiol, ethanethiol, dodecanethiol, thiophenol and mercaptoacetic acid.

Compound (B) can be produced by applying the same reaction reagents and reaction conditions as the production method of compound (IIb) shown in

production method

1 or 3.

Manufacturing method 4
Compound (C) can be produced by the following method.

(Wherein R ⁸ , R ⁹ , R ¹⁰ , X ³ and Y are as defined above)

Step 11
Compound (C) comprises compound (IIc) and compound (Vc) in the absence of a solvent or in a solvent, preferably in the presence of 1 to 10 equivalents of an additive, and / or preferably 1 to 10 equivalents if necessary. In the presence of a base at a temperature between room temperature and 200 ° C. for 5 minutes to 100 hours.
Examples of the solvent and the base include those exemplified in

Steps

1 and 2, respectively.
Examples of the additive include those exemplified in Step 7.
Compound (IIc) can be produced by applying the same reaction reagent and reaction conditions as the production method of compound (IIb) shown in the above production method 1 or 3.Compound (Vc) is a commercially available product or It can be obtained by a known method (for example, 5th edition, Experimental Chemistry Lecture 13, Synthesis of Organic Compounds I, 5th edition, p. 374, Maruzen (2005)) or a method analogous thereto.

Manufacturing method 5
Compound (C) can also be produced by the following method.

(Wherein R ⁸ , R ⁹ , R ¹⁰ , X ³ and Y are as defined above)

Steps 12 and 13
Compound (IVh) can be produced by reacting compound (Vd) and compound (IIId) under the same conditions as in Step 1 of Production Method 1. Furthermore, compound (C) can be produced by reacting compound (IVh) and compound (IIIe) under the same conditions as in step 2 in production method 1.
Compound (Vd) is commercially available or obtained by a known method (for example, 5th edition, Experimental Chemistry Lecture 13, Synthesis of Organic Compounds I ”, 5th edition, p.374, Maruzen (2005)) or a method analogous thereto. be able to.
Compound (IIId) and Compound (IIIe) are the same as Compound (IIIa) and Compound (IIIb) described in Production Method 1, respectively.
Compound (C) when R ⁸ and R ⁹ are the same can be obtained by using 2 equivalents or more of compound (IIId) in Step 12.

Manufacturing method 6
Among compounds (C), compound (Ca) in which R ¹⁰ is a hydrogen atom can also be produced by the following method.

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

Process 14
Compound (IVi) can be produced by reacting compound (IIc) and compound (Ve) under the same conditions as in Step 11 in Production Method 4.
Compound (IIc) can be produced by applying the same reaction reagent and reaction conditions as the production method of compound (IIb) shown in the above production method 1 or 3.Compound (Ve) is a commercially available product or It can be obtained by a known method (for example, 5th edition, Experimental Chemistry Course 18 Synthesis of Organic Compounds VI, 5th edition, p.171-172, Maruzen (2005)), or a method analogous thereto.

Process 15
Compound (Ca) can be produced by reacting compound (IVi) and a deprotection reagent in the absence of solvent or in a solvent at a temperature between −20 ° C. and 150 ° C. for 5 minutes to 72 hours.
Examples of the solvent include those exemplified in

Steps

1 and 2.
Examples of the deprotecting reagent include tetrabutylammonium fluoride, hydrogen fluoride pyridine complex, fluorine compounds such as hydrofluoric acid, and acids such as acetic acid, trifluoroacetic acid, pyridinium p-toluenesulfonate, hydrochloric acid, and the like. .

Manufacturing method 7
Among compounds (C), compound (Cb) in which R ¹⁰ is a hydrogen atom and X ³ is alkylene having 1 or 2 carbon atoms can also be produced by the following method.

(Wherein R ⁸ and R ⁹ are as defined above, and X ⁵ represents methylene or ethylene)

Step 16
Compound (IVj) is obtained by reacting Compound (IIc) with room temperature and 200 ° C., preferably in the presence of 1 to a large excess of ethyl acrylate, without solvent or in a solvent, and preferably in the presence of 1 to 10 equivalents of a base. By reacting at a temperature between 5 minutes and 100 hours.
Examples of the solvent and the base include those exemplified in

Steps

1 and 2, respectively.
Compound (IIc) can be produced by applying the same reaction reagents and reaction conditions as the production method of compound (IIb) shown in the

above production method

1 or 3.

Step 17
Compound (IVk) can be produced by reacting compound (IIc) with preferably 1 to a large excess of ethyl bromoacetate under the same conditions as in Step 11 in Production Method 4.
Compound (IIc) can be produced by applying the same reaction reagents and reaction conditions as the production method of compound (IIb) shown in the

above production method

1 or 3.

Process 18
Compound (Cb) is compound (IVj) or compound (IVk), preferably at −20 ° C. in the presence of 1 to 10 equivalents of a reducing agent and preferably 1 to 10 equivalents of an additive in a solvent. It can be produced by reacting at a temperature between 150 ° C. for 5 minutes to 72 hours.
Examples of the solvent include tetrahydrofuran, 1,4-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, and borane.
Examples of the additive include aluminum chloride, cerium chloride, iron chloride, acetic acid and hydrochloric acid.

The intermediate and target compound in each of the above production methods are isolated by separation and purification methods commonly used in organic synthetic chemistry, such as filtration, extraction, washing, drying, concentration, recrystallization and / or various chromatography. Can be purified. The intermediate can be subjected to the next reaction without any particular purification.

In the compounds (A), (B) and (C), a hydrogen ion may be coordinated to a lone pair on the nitrogen atom in the structure, and in that case, a pharmaceutically acceptable anion (as described above). And the compounds (A), (B) and (C) also include compounds in which a hydrogen ion is coordinated to a lone electron pair on the nitrogen atom.
Among the compounds (A), (B) and (C), there are compounds in which stereoisomers such as geometric isomers and optical isomers, tautomers and the like may exist, but the compounds (A), ( B) and (C) include all possible isomers and mixtures thereof, including these.
Some or all of each atom in the compounds (A), (B) and (C) may be replaced by the corresponding isotope atoms, respectively, and the compounds (A), (B) and (C) are Also included are compounds substituted with these isotope atoms. For example, some or all of the hydrogen atoms in the compounds (A), (B), and (C) may be hydrogen atoms (deuterium atoms) having an atomic weight of 2.
A compound in which part or all of each atom in the compounds (A), (B) and (C) is replaced with a corresponding isotope atom is the same as in the above production methods using commercially available building blocks. It can be manufactured by the method. A compound in which some or all of the hydrogen atoms in the compounds (A), (B), and (C) are replaced with deuterium atoms can be obtained using, for example, an iridium complex as a catalyst and deuterium as a deuterium source. Using a method of deuterating alcohol or carboxylic acid [see Journal of American Chemical Society (J. Am. Chem. Soc.), Vol. 124, No. 10, 2092 (2002)] It can also be synthesized.

Specific examples of compounds (A), (B) and (C) are shown in Table 1-1 to Table 1-7. However, the compounds (A), (B) and (C) are not limited to these.

In the present invention, a nucleic acid comprising a base sequence complementary to β2GPI mRNA is referred to as an antisense strand nucleic acid, and a nucleic acid comprising a base sequence complementary to the antisense strand nucleic acid is also referred to as a sense strand nucleic acid.

The double-stranded nucleic acid as a drug used in the present invention is a double-stranded nucleic acid having the ability to reduce or stop the expression of β2GPI gene when introduced into a mammalian cell, and comprises a sense strand and an antisense strand. A double-stranded nucleic acid. Also, the sense strand and the antisense strand have at least 11 base pairs, and at least 17 nucleotides and at most 30 in the antisense strand, ie, 17 to 30 nucleotides It is complementary to a target β2GPI mRNA sequence selected from the group described in Tables 2-1 to 2-16 in the oligonucleotide chain of a chain length.

The double-stranded nucleic acid as a drug used in the present invention may be any molecule as long as it is a molecule in which nucleotides or molecules having functions equivalent to the nucleotides are polymerized. For example, RNA that is a polymer of ribonucleotides DNA that is a polymer of deoxyribonucleotides, chimeric nucleic acids composed of RNA and DNA, and nucleotide polymers in which at least one nucleotide of these nucleic acids is substituted with a molecule having a function equivalent to that of the nucleotide. A derivative containing at least one molecule having a function equivalent to a nucleotide in these nucleic acids is also included in the double-stranded nucleic acid as a drug used in the present invention.
Uracil (U) can be uniquely read as thymine (T).

Examples of molecules having functions equivalent to nucleotides include nucleotide derivatives. The nucleotide derivative may be any molecule as long as it is a nucleotide-modified molecule. For example, in order to improve or stabilize nuclease resistance compared to RNA or DNA, affinity for complementary nucleic acid In order to increase cell permeability, to increase cell permeability, or to visualize, a molecule in which ribonucleotides or deoxyribonucleotides are modified is preferably used.

Examples of the molecule in which the nucleotide is modified include a sugar moiety-modified nucleotide, a phosphodiester bond-modified nucleotide and a base-modified nucleotide, and a nucleotide in which at least two of the sugar moiety, phosphodiester bond and base are modified.

The sugar-modified nucleotide may be any nucleotide as long as it is modified or substituted with an arbitrary substituent for a part or all of the chemical structure of the sugar constituting the nucleotide, or substituted with an arbitrary atom. 2'-modified nucleotides are preferably used.

2′-modified nucleotides include, for example, the 2′-OH group of ribose is H, OR, R, R′OR, SH, SR, NH ₂ , NHR, NR ₂ , N ₃ , CN, F, Cl, Br and Substituted with a substituent selected from the group consisting of I (R is alkyl or aryl, preferably alkyl having 1 to 6 carbon atoms, and R ′ is alkylene, preferably alkylene having 1 to 6 carbon atoms) And a nucleotide having a 2′-OH group substituted with H, F or a methoxy group, and more preferably a nucleotide having a 2′-OH group substituted with F or a methoxy group. In addition, the 2′-OH group is a 2- (methoxy) ethoxy group, a 3-aminopropoxy group, a 2-[(N, N-dimethylamino) oxy] ethoxy group, or a 3- (N, N-dimethylamino) propoxy group. 2- [2- (N, N-dimethylamino) ethoxy] ethoxy group, 2- (methylamino) -2-oxoethoxy group, 2- (N-methylcarbamoyl) ethoxy group and 2-cyanoethoxy group Also included are nucleotides substituted with a substituent selected from the group.
The nucleotides within the double-stranded nucleic acid region are preferably 10 to 70%, more preferably 20 to 40%, and more preferably 40 to 65% of 2'-O-methyl modified nucleotides. Is more preferable. Further, the 2′-O-methyl modified nucleotide is preferably contained in an amount of 20 to 40%, more preferably 40 to 60%, and more preferably 60% to 100% with respect to the nucleotide of the sense strand. Further preferred. In addition, the 2′-O-methyl modified nucleotide is preferably contained in an amount of 0 to 40%, more preferably 10 to 20%, and more preferably 20 to 40% with respect to the nucleotide of the antisense strand. Further preferred.

As the sugar moiety-modified nucleotide, a crosslinked nucleic acid (BNA) having two cyclic structures by introducing a crosslinked structure into the sugar moiety is also preferably used. Specifically, Locked Nucleic Acid (LNA) in which 2'-position oxygen atom and 4'-position carbon atom are bridged via methylene ["Tetrahedron Letters", Volume 38 8735, (1997) and “Tetrahedron”, 54, 3607, (1998)], Ethylene bridged nucleic acid (ENA) [Nucleic Acid Research (Nucleic Acid) Research, 32, e175 (2004)], Constrained Ethyl (cEt) [“The Journal of Organic Chemistry” 75, 1569 (2010)], Amido-Bridged Nucleic Acid (AmNA) [“ Chem. Bio. Chem. ”, Vol. 13, 2513 (2012)] and 2'-O, 4'-C-Spirocyclopropylene bridged nucleic acid (scpBNA) [“ Chemical Communications ”, Vol. 51 , 9737 (2015)].
In addition, peptide nucleic acids (PNA) [“ACCOUNTS OF CHEMICAL RESEARCH”, 32, 624 (1999)], oxypeptide nucleic acids (OPNA) [“Journal of American Chemical Society” , 123, 4653 (2001)], peptide ribonucleic acid (PRNA) [“Journal of American Chemical Society”, 122, 6900 (2000)] etc. Can be mentioned.

The phosphodiester bond-modified nucleotide is any nucleotide that has been modified or substituted with an arbitrary substituent for a part or all of the chemical structure of the phosphodiester bond of the nucleotide, or with any atom. For example, a nucleotide in which a phosphodiester bond is replaced with a phosphorothioate bond, a nucleotide in which a phosphodiester bond is replaced with a phosphorodithioate bond, a nucleotide in which a phosphodiester bond is replaced with an alkylphosphonate bond, a phosphate Examples include nucleotides in which a diester bond is substituted with a phosphoramidate bond.

As the base-modified nucleotide, any or all of the nucleotide base chemical structure modified or substituted with an arbitrary substituent or substituted with an arbitrary atom may be used. In which oxygen atoms are substituted with sulfur atoms, hydrogen atoms are substituted with alkyl groups of 1 to 6 carbon atoms or halogen atoms, etc., methyl groups are hydrogen, hydroxymethyl groups or alkyl of 2 to 6 carbon atoms And those in which an amino group is substituted with an alkyl group having 1 to 6 carbon atoms, an alkanoyl group having 1 to 6 carbon atoms, an oxo group, or a hydroxy group.

Nucleotide derivatives include peptides, proteins, sugars, lipids, phospholipids, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, or dyes, either directly or via a linker. Examples include those added to nucleotides. Specifically, 5'-polyamine addition nucleotide derivatives, cholesterol addition nucleotide derivatives, steroid addition nucleotide derivatives, bile acid addition nucleotide derivatives, vitamin addition nucleotide derivatives, Cy5 addition nucleotide derivatives, Cy3 addition Examples include nucleotide derivatives, 6-FAM-added nucleotide derivatives, and biotin-added nucleotide derivatives.
The nucleotide derivative may form a crosslinked structure such as an alkylene structure, a peptide structure, a nucleotide structure, an ether structure, an ester structure, and a structure obtained by combining at least one of these with other nucleotides or nucleotide derivatives in the nucleic acid.

The double-stranded nucleic acid as a drug used in the present invention includes those in which some or all atoms in the nucleic acid molecule are substituted with atoms having different mass numbers (isotopes such as deuterium).

In the present specification, “complementary” means a relationship that allows base pairing between two bases, for example, a moderate hydrogen such as a relationship between adenine and thymine or uracil, and a relationship between guanine and cytosine. It means a double-stranded structure as a whole double-stranded region through a bond.

As used herein, the term “complementary” refers not only to the case where two nucleotide sequences are completely complementary, but also from 0 to 30%, preferably 0 to 20%, more preferably 0 to 10% between the nucleotide sequences. For example, an antisense strand complementary to β2GPI mRNA may contain one or more base substitutions in the base sequence that is completely complementary to the partial base sequence of the mRNA. Means that. Specifically, the antisense strand is 1 to 8, preferably 1 to 6, more preferably 1 to 4, more preferably 1 to 3, and still more preferably 1 to the target sequence of the target gene. It can have 1 to 2 mismatched bases. For example, when the antisense strand is 21 bases long, it may have 1 to 6 mismatch bases with respect to the target sequence of the target gene, and the position of the mismatch is the 5 ′ end of each sequence or It may be the 3 ′ end.
The term “complementary” includes a case where one nucleotide sequence is a sequence in which one or more bases are added and / or deleted in a base sequence that is completely complementary to the other nucleotide sequence. For example, β2GPI mRNA and the antisense strand nucleic acid of the present invention have one or two bulge bases in the antisense strand and / or the target β2GPI mRNA region due to addition and / or deletion of a base in the antisense strand. May be.

The double-stranded nucleic acid as a drug used in the present invention has a nucleic acid containing a base sequence complementary to a partial base sequence of β2GPIGmRNA and / or a base sequence complementary to the base sequence of the nucleic acid. As long as it contains a nucleic acid, it may be composed of any nucleotide or derivative thereof. The double-stranded nucleic acid of the present invention comprises a nucleic acid comprising a base sequence complementary to the target β2GPI mRNA sequence and a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid at least 11 bases. Any length can be used as long as it can form a pair of duplexes, but the length of the sequence capable of forming a duplex is usually 11 to 35 bases, preferably 15 to 30 bases, preferably 17 to 25 bases Is more preferable, 17 to 23 bases are more preferable, and 19 to 23 bases are still more preferable.

As the antisense strand nucleic acid of the present invention, a nucleic acid containing a base sequence complementary to the target β2GPI mRNA sequence is used. Among these nucleic acids, 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 A base deleted, substituted or added may be used.

The nucleic acid that suppresses β2GPI expression is a nucleic acid that includes a base sequence complementary to the target β2GPI mRNA sequence and is complementary to the target β2GPI mRNA sequence. A double-stranded nucleic acid consisting of a nucleic acid containing a basic sequence and a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid and suppressing β2GPI expression is preferably used.

In the present invention, the double-stranded nucleic acid refers to a nucleic acid having two nucleotide chains paired and having a double-stranded region of at least 11 base pairs. A double-stranded region refers to a portion where nucleotides constituting a double-stranded nucleic acid or a derivative thereof constitute a base pair to form a double strand. The double-stranded region is usually 11 to 27 base pairs, preferably 15 to 25 base pairs, more preferably 15 to 23 base pairs, still more preferably 17 to 21 base pairs, and even more preferably 17 to 19 base pairs. .

The single-stranded nucleic acid constituting the double-stranded nucleic acid usually consists of 11 to 30 bases, preferably 15 to 29 bases, more preferably 15 to 27 bases, and 15 to 25 bases. More preferably, it consists of 17 to 23 bases, more preferably 19 to 21 bases.

When the double-stranded nucleic acid as a drug used in the present invention has an additional nucleotide or nucleotide derivative that does not form a duplex on the 3 ′ side or 5 ′ side following the double-stranded region, this is used as an overhang. This is called (overhang). In the case of having an overhang, the nucleotide constituting the overhang may be ribonucleotide, deoxyribonucleotide or a derivative thereof.

As the double-stranded nucleic acid having an overhang, one having an overhang of 1 to 6 bases, preferably 1 to 3 bases at the 3 ′ end or 5 ′ end of at least one strand is used. More preferably, those having a protruding portion consisting of dTdT (dT represents deoxythymidine) or UU (U represents uridine) are used. In the present invention, a double-stranded nucleic acid having a protruding portion on both the antisense strand and the sense strand can be provided on the antisense strand only, only the sense strand, and both the antisense strand and the sense strand. Is preferably used. The antisense strand is selected from the group described in Tables 2-1 to 2-16 in an oligonucleotide chain consisting of 17 to 30 nucleotides, including a double-stranded region followed by a protruding portion. Is sufficiently complementary to the target β2GPI mRNA sequence. Furthermore, as the double-stranded nucleic acid of the present invention, for example, a nucleic acid molecule that generates a double-stranded nucleic acid by the action of a ribonuclease such as Dicer (see International Publication No. 2005/089287), 3 ′ end or 5 ′ It is also possible to use a double-stranded nucleic acid that does not have a terminal overhang and forms a blunt end, a double-stranded nucleic acid in which only the sense strand protrudes (see US Patent Application Publication No. 2012/0040459), and the like.

As the double-stranded nucleic acid as a drug used in the present invention, a nucleic acid having the same sequence as the base sequence of the target gene or its complementary strand may be used, but 5 of at least one strand of the nucleic acid may be used. A double-stranded nucleic acid consisting of a nucleic acid from which 1 to 4 bases have been deleted from the 'terminal or 3' terminal and a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid may be used.

The double-stranded nucleic acid as a drug used in the present invention is a double-stranded RNA (dsRNA) in which RNAs form a double strand, a double-stranded DNA (dsDNA) in which DNAs form a double strand, or RNA And a hybrid nucleic acid in which DNA forms a double strand. Alternatively, one or both of the double strands may be a chimeric nucleic acid of DNA and RNA. Double-stranded RNA (dsRNA) is preferred.

The second nucleotide from the 5 ′ end of the antisense strand of the present invention is preferably complementary to the second deoxyribonucleotide from the 3 ′ end of the target β2GPI mRNA sequence, and 2-7 from the 5 ′ end of the antisense strand. More preferably, the second nucleotide is completely complementary to the second to seventh deoxyribonucleotides from the 3 ′ end of the target β2GPI mRNA sequence, and the second to eleventh nucleotides from the 5 ′ end of the antisense strand are the target β2GPI More preferably, it is completely complementary to the 2-11th deoxyribonucleotide from the 3 ′ end of the mRNA sequence. Further, the 11th nucleotide from the 5 ′ end of the antisense strand in the nucleic acid of the present invention is preferably complementary to the 11th deoxyribonucleotide from the 3 ′ end of the target β2GPI mRNA sequence, and the 5 ′ end of the antisense strand It is more preferable that the 9th to 13th nucleotides are completely complementary to the 9th to 13th deoxyribonucleotides from the 3 ′ end of the target β2GPI mRNA sequence, and the 7th to 15th nucleotides from the 5 ′ end of the antisense strand Is more preferably completely complementary to the 7th to 15th deoxyribonucleotides from the 3 ′ end of the target β2GPI mRNA sequence.

The method for producing a double-stranded nucleic acid as a drug used in the present invention is not particularly limited, and examples thereof include a method using known chemical synthesis, an enzymatic transcription method, and the like. Examples of known chemical synthesis methods include phosphoramidite method, phosphorothioate method, phosphotriester method, CEM method ["Nucleic Acid Research", 35, 3287 (2007)]. For example, it can be synthesized by an ABI3900 high-throughput nucleic acid synthesizer (manufactured by Applied Biosystems). After the synthesis is completed, elimination from the solid phase, deprotection of the protecting group, purification of the target product, and the like are performed. It is desirable to obtain a nucleic acid having a purity of 90% or more, preferably 95% or more by purification. In the case of a double-stranded nucleic acid, the synthesized and purified sense strand and antisense strand are in an appropriate ratio, for example, 0.1 to 10 equivalents, preferably 0.5 to 2 equivalents of sense strand to 1 equivalent of antisense strand. Preferably, 0.9 to 1.1 equivalents, more preferably equimolar amounts are mixed and then annealed, or the mixed product may be used directly without the step of annealing. Annealing may be performed under any conditions that can form a double-stranded nucleic acid, but usually, the sense strand and the antisense strand are mixed in approximately equimolar amounts and then heated at about 94 ° C. for about 5 minutes. Then, it is performed by slowly cooling to room temperature. Examples of the enzymatic transcription method for producing the nucleic acid of the present invention include a method by transcription using a phage RNA polymerase, for example, T7, T3, or SP6 RNA polymerase, using a plasmid or DNA having a target base sequence as a template.

The double-stranded nucleic acid as a drug used in the present invention may be modified with one or more ligands or fluorophores at the 5 ′ end, 3 ′ end or / and the inside of the sequence. The nucleic acid is also called a conjugated nucleic acid. At the time of extension reaction on the solid phase, the 5 ′ end, 3 ′ end or / and the inside of the sequence can be modified by reacting a modifying agent capable of reacting on the solid phase. Alternatively, a conjugated nucleic acid can be obtained by previously synthesizing and purifying a nucleic acid into which a functional group such as an amino group, a mercapto group, an azide group, or a triple bond has been introduced, and allowing a modifying agent to act on them. The ligand may be any molecule that has affinity for biomolecules.For example, lipids such as cholesterol, fatty acids, tocopherols, retinoids, N-acetylgalactosamine (GalNAc), galactose (Gal), mannose (Man), etc. Sugars, full antibodies, antibodies such as Fab and VHH, low density lipoprotein (LDL), proteins such as human serum albumin, peptides such as RGD, NGR, R9, and CPP, small molecules such as folic acid, synthetic polyamino acids, etc. These synthetic polymers, nucleic acid aptamers, and the like can be used, and these can also be used in combination. Examples of fluorophores include the Cy3 series, the Alexa series, and the black hole quencher.

The antisense strand and sense strand of the present invention can be designed based on the nucleotide sequence (SEQ ID NO: 3541) of cDNA (sense strand) of full-length mRNA of human β2GPI registered as, for example, Genbank Accession No. NM_000042. it can.

The nucleic acid having β2GPI expression suppressing activity includes the antisense strand nucleic acid of the present invention containing a base sequence complementary to β2GPI2mRNA and the nucleic acid of the present invention containing a base sequence complementary to the base sequence of the nucleic acid. Examples thereof include double-stranded nucleic acids consisting of sense strand nucleic acids and having β2GPI expression inhibitory activity. The single-stranded nucleic acid constituting the double-stranded nucleic acid usually consists of 11 to 30 bases, preferably 15 to 29 bases, more preferably 15 to 27 bases, and more preferably 15 to 25 bases. More preferably, it consists of 17 to 23 bases, more preferably 19 to 21 bases. The double-stranded nucleic acid usually consists of 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 even more preferably 15 to 19 base pairs. Has a double-stranded region.

The double-stranded nucleic acid in the present invention is a double-stranded nucleic acid consisting of a sense strand and an antisense strand, and comprising a double-stranded region of at least 11 base pairs. It may be a double-stranded nucleic acid as a drug that is complementary to a target β2GPI mRNA sequence selected from the group set forth in SEQ ID NOs: 2361-3540 in an oligonucleotide chain with a chain length of 30 nucleotides.
In the present invention, the double-stranded nucleic acid is composed of a sense strand and an antisense strand, and includes a double-stranded region of at least 11 base pairs, and contains 17 to 30 nucleotides in the antisense strand. It consists of an antisense strand represented by SEQ ID NOs: 1181 to 2360 as a double-stranded nucleic acid complementary to a target β2GPI mRNA sequence selected from the group described in SEQ ID NOs: 2361 to 3540 in an oligonucleotide chain having a chain length A double-stranded nucleic acid comprising a sequence selected from the group, a double-stranded nucleic acid comprising a sequence selected from the group consisting of the sense strands represented by SEQ ID NOs: 1-1180, or SEQ ID NOs: 1-1180 and SEQ ID NO: A double-stranded nucleic acid comprising a pair of sense / antisense strand sequences selected from the group consisting of sense / antisense strands described as 1181 to 2360 may be used.
The double-stranded nucleic acid in the present invention is composed of a sense strand and an antisense strand, includes a double-stranded region of at least 11 base pairs, and contains 17 to 30 nucleotides in the antisense strand. A double-stranded nucleic acid complementary to a target β2GPI mRNA sequence selected from the group described in SEQ ID NOs: 2361 to 3540 in an oligonucleotide chain having a chain length, consisting of an antisense strand represented by SEQ ID NOs: 3702 to 3661 A double-stranded nucleic acid comprising a sequence selected from the group, a double-stranded nucleic acid comprising a sequence selected from the group consisting of a sense strand represented by SEQ ID NOs: 3542-3701, or SEQ ID NOs: 3542-3701 and SEQ ID NO: A double-stranded nucleic acid comprising a pair of sense strand / antisense strand sequences selected from the group consisting of sense strand / antisense strand described as 3702 to 3861 may be used. The antisense strand in these double stranded nucleic acids is preferably complementary to SEQ ID NOs: 2456, 2459, 2485, 2486, 3053, 3185, 3239, 3303, 3385, 3398, 3399 or 3499.

In the oligonucleotide strand consisting of a sense strand and an antisense strand in the present invention, comprising a double-stranded region of at least 11 base pairs, and having a chain length of 17 to 30 nucleotides in the antisense strand, Listed in Tables 2-1 to 2-18 and Tables 4-1 to 4-5 as double-stranded nucleic acids complementary to target β2GPI mRNA sequences selected from the groups listed in 4-1 to Table 4-16 A double-stranded nucleic acid comprising a sequence selected from the group consisting of the antisense strands selected or selected from the group consisting of the antisense strands listed in Tables 2-1 to 2-18 and Tables 4-1 to 4-5 A pair of sense strands selected from the group consisting of sense strands / antisense strands described in Tables 2-1 to 2-18 and Tables 4-1 to 4-5 / Double-stranded nucleic acid containing the sequence of the antisense strand may be used. Specific examples of the double-stranded nucleic acid constituting the nucleic acid complex used in the present invention are double-stranded consisting of the sense strand and the antisense strand in Tables 2-1 to 2-18 and Tables 4-1 to 4-5. It is a nucleic acid.

The lipid particle in the present invention is a lipid particle containing compound (A), compound (B) or compound (C) and a double-stranded nucleic acid, for example, compound (A), compound (B) or compound ( C) and double-stranded nucleic acid, or compound (A), compound (B) or compound (C) combined with neutral lipid and / or polymer and double-stranded nucleic acid Examples include lipid particles containing a body, lipid particles composed of the complex, and a lipid membrane encapsulating the complex. The lipid membrane may be a lipid monolayer (lipid monomolecular membrane) or a lipid bilayer membrane (lipid bimolecular membrane). The lipid membrane may contain compound (A), compound (B) or compound (C), neutral lipid and / or polymer. Further, the complex and / or the lipid membrane may contain a cationic lipid other than the compound (A), the compound (B) or the compound (C).
As the form of the complex, in any case, a complex of a double-stranded nucleic acid and a membrane composed of a lipid monolayer (reverse micelle), a complex of a double-stranded nucleic acid and a liposome, a double-stranded nucleic acid and a micelle And a complex of a double-stranded nucleic acid and a membrane composed of a lipid monolayer, or a complex of a double-stranded nucleic acid and a liposome.
Examples of the lipid particles composed of the complex and a lipid bilayer encapsulating the complex include liposomes composed of the complex and a lipid bilayer encapsulating the complex.
For the lipid particles in the present invention, one or more of compound (A), compound (B) or compound (C) may be used, and compound (A), compound (B) or compound (C) and Alternatively, a mixture of a cationic lipid other than the compound (A), the compound (B) or the compound (C) may be used.
Examples of the cationic lipid other than the compound (A), the compound (B) or the compound (C) include, for example, DOTMA, DOTAP disclosed in JP-A-61-161246 (US Pat. No. 5,049,386). N- [1- (2,3-dioleyloxypropyl)]-N, N-dimethyl-N-hydroxyethyl as disclosed in WO 91/16024 and WO 97/019675, etc. Ammonium bromide (DORIE), 2,3-dioleyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetic acid (DOSPA), etc. DLinDMA disclosed in 2005/121348, DLin-K-DMA, etc. disclosed in International Publication No. 2009/086558 are mentioned, preferably DOTMA, DOTAP, DORIE, DOSPA, DLinDMA, DLin- A cationic lipid having a tertiary amine moiety having two unsubstituted alkyl groups such as K-DMA or a quaternary ammonium moiety having three unsubstituted alkyl groups, Ri is preferably a cationic lipid having the tertiary amine moiety. The unsubstituted alkyl group of the tertiary amine moiety and the quaternary ammonium moiety is preferably a methyl group.

The lipid particles in the present invention can be produced according to a known production method or similar methods, and may be produced by any production method. For example, a known liposome preparation method can be applied to the production of a liposome, which is one of lipid particles. Known liposome preparation methods include, for example, Bangham et al.'S liposome preparation method ["Journal of Molecular Biology", 1965, Vol. 13, p.238-252] , Ethanol injection method ["Journal of Cell Biol.", 1975, Vol. 66, pp. 621-634], French press method ["FEBS Letters"] , 1979, Vol. 99, pp. 210-214], freeze-thaw method [“Arch. Biochem. Biophys.”, 1981, Vol. 212, p. 186-194 Refer to], reversed-phase evaporation method [see “Procedures of the National Academy of Sciences United States of America” (Proc. Natl. Acad. Sci. USA), 1978, Vol. 75, pp. 4194-4198] or pH gradient method (for example, Japanese Patent No. 2572554, The reference JP etc. 2659136) and the like. As a solution in which the liposome is dispersed in the production of the liposome, for example, water, acid, alkali, various buffers, physiological saline, amino acid infusion, or the like can be used. In the production of liposomes, for example, an antioxidant such as citric acid, ascorbic acid, cysteine or ethylenediaminetetraacetic acid (EDTA), for example, an isotonic agent such as glycerin, glucose or sodium chloride can be added. is there. Liposomes can also be produced by dissolving lipids or the like in an organic solvent such as ethanol and distilling off the solvent, and then adding physiological saline or the like and stirring to form liposomes.

The lipid particles in the present invention may be prepared by, for example, dissolving compound (A), compound (B) or compound (C) in advance in chloroform, then adding an aqueous solution of double-stranded nucleic acid and methanol and mixing them to form a cationic lipid. / Double-stranded nucleic acid complex is formed, the chloroform layer is taken out, and polyethylene glycolated phospholipid, neutral lipid and water are added to this to form a water-in-oil (W / O) emulsion, and the reverse A method of manufacturing by the phase evaporation method (see JP-T-2002-508765), or dissolving a double-stranded nucleic acid in an acidic aqueous electrolyte solution, adding lipid (in ethanol), and adjusting the ethanol concentration to 20 v / v The double-stranded nucleic acid-encapsulating liposome is prepared by lowering the concentration to 2%, and after sizing filtration and removing excess ethanol by dialysis, the pH of the sample is further increased to dialyze the double-stranded nucleic acid attached to the liposome surface. Those who remove and manufacture (Kohyo 2002-501511 JP and Baiokimika eth bio Physica Acta (Biochimica et Biophysica Acta), 2001 years, 1510, Volume, P.152-166 reference) can be prepared by, or the like.

Among the lipid particles in the present invention, a liposome composed of a complex and a lipid bilayer membrane encapsulating the complex is produced, for example, as described in WO 02/28367 and WO 2006/080118 It can be manufactured according to the method.

The present invention also provides a lipid particle-containing composition (hereinafter sometimes simply referred to as “composition”) containing the lipid particle. Among the compositions of the present invention, for example, compound (A), compound (B) or complex of compound (C) and nucleic acid, or compound (A), compound (B) or compound (C) and neutral lipid and A composition comprising a combination of a polymer and a nucleic acid, and a composition containing a lipid membrane encapsulating the complex, a cationic lipid other than the compound (A), the compound (B) or the compound (C) and a nucleic acid Or a complex of a lipid other than compound (A), compound (B) or compound (C) with a neutral lipid and / or polymer and a nucleic acid, and encapsulating the complex And a composition containing the compound (A), the compound (B) or the compound (C) in the lipid membrane is disclosed in WO 02/28367 and WO 2006/080118. According to the production method described, each complex is produced, and water or 0-40% ethanol aqueous solution is produced. In addition, the complex is dispersed without dissolving (Liquid A), and each lipid membrane component is separately dissolved in, for example, an aqueous ethanol solution (Liquid B), and an equivalent amount or a volume ratio of 1: 1 to 7: 3. The liquid A and liquid B can be mixed, and water can be added appropriately. In addition, as the cationic lipid in the liquid A and liquid B, one or more kinds of compound (A), compound (B) or compound (C), or compound (A), compound (B) or compound (C) A cationic lipid other than the above may be used, and the compound (A), the compound (B) or the compound (C) may be combined with a cationic lipid other than the compound (A), the compound (B) or the compound (C). You may mix and use.
In the present invention, compound (A), compound (B) or compound (C) and nucleic acid complex, or compound (A), compound (B) or compound (C) is neutral lipid and / or polymer. A complex comprising a combination and a nucleic acid, a composition containing a lipid membrane encapsulating the complex, a complex of a compound (A), a compound (B) or a cationic lipid other than the compound (C) and a nucleic acid, Or a complex of a combination of a neutral lipid and / or polymer with a cationic lipid other than compound (A), compound (B) or compound (C) and a nucleic acid, and a lipid membrane encapsulating the complex In addition, during and after the production of the composition containing the compound (A), the compound (B) or the compound (C) in the lipid membrane, electrostatic capacitance between the nucleic acid in the complex and the cationic lipid in the lipid membrane For interaction and fusion of cationic lipid in complex with cationic lipid in lipid membrane Thus, the complex and the membrane structure are also displaced, respectively, compound (A), compound (B) or compound (C) and nucleic acid complex, or compound (A), compound (B) or compound ( C) a composition comprising a combination of a neutral lipid and / or polymer and a nucleic acid, and a composition containing a lipid membrane encapsulating the complex, or compound (A), compound (B) or compound (C ) Or a complex of a cationic lipid and a nucleic acid other than compound (A), or a combination of a cationic lipid other than compound (A), compound (B) or compound (C) with a neutral lipid and / or polymer. It includes a complex and a lipid membrane that encapsulates the complex, and the lipid membrane contains a compound (A), compound (B), or compound (C).

According to the production methods described in WO 02/28367 and WO 2006/080118, etc., nucleic acids (as defined above), preferably double-stranded nucleic acids and compound (A), compound (B) or compound (C ) And / or a complex with a liposome containing a cationic lipid other than compound (A), compound (B) or compound (C), and the complex in water or a 0-40% aqueous ethanol solution Dispersing without dissolving (liquid A), separately, compound (A), compound (B) or compound (C) and / or cationic lipid other than compound (A), compound (B) or compound (C) Can be dissolved in an aqueous ethanol solution (solution B), and the same amount or volume ratio of 1: 1 to 7: 3 can be mixed with solution A and solution B, or water can be added as appropriate. A lipid particle-containing composition containing the cationic lipid can be obtained. The composition is preferably a composition comprising a complex of a cationic lipid and a nucleic acid and a lipid membrane encapsulating the complex, or a membrane comprising a lipid monolayer containing the nucleic acid and the cationic lipid It may be a composition containing a complex with (reverse micelle) and a lipid membrane encapsulating the complex. The lipid membrane in these cases may be a lipid monolayer (lipid monomolecular membrane) or a lipid bilayer membrane (lipid bimolecular membrane).
In addition, the liposome in the complex of the nucleic acid of the present disclosure and the liposome is adjusted in advance to an average particle size of 10 nm to 400 nm, preferably 20 nm to 110 nm, more preferably 30 nm to 80 nm. Further, the complex and / or lipid membrane may contain a neutral lipid and / or a polymer. Liquid A is a compound of compound (A), compound (B) or compound (C), and / or a cationic lipid other than compound (A), compound (B) or compound (C) and the nucleic acid. If the body can be formed, the ethanol concentration may be 20 to 70%.
Also, instead of mixing equal amounts of liquid A and liquid B, mix them at a ratio that will result in an ethanol concentration that does not dissolve the complex after mixing liquid A and liquid B, and does not dissolve the cationic lipids in liquid B. Also good. Instead of mixing the A solution and the B solution in such a ratio that preferably the complex does not dissolve, the cationic lipid in the B solution does not dissolve, and the ethanol concentration is 30 to 60% ethanol aqueous solution. Alternatively, after mixing liquid A and liquid B, mix the liquid A and liquid B at a ratio that gives an ethanol concentration that does not dissolve the complex, and then add water to add the cationic lipid in liquid B. It is also possible to make the ethanol concentration at which no dissolution occurs.
The complex of a nucleic acid and a liposome in the liquid A disclosed in the present specification is prepared by mixing the liquid A and the liquid B, and further adding water appropriately, and then starting from a lipid monolayer containing a cationic lipid. The form is changed to a complex of a membrane (reverse micelle) and a nucleic acid. The composition containing the nucleic acid and the cationic lipid obtained by the production method of the present disclosure is preferably a composition containing a complex of a cationic lipid and a nucleic acid and a lipid membrane encapsulating the complex. Alternatively, a composition comprising a membrane (reverse micelle) composed of a lipid monolayer containing a cationic lipid and a nucleic acid, and a lipid membrane encapsulating the complex, the composition containing a cationic lipid in the lipid membrane And its manufacturability (yield and / or uniformity) is excellent.

The total number of molecules of compound (A), compound (B) or compound (C) in the complex is preferably 0.5 to 8 times the number of phosphorus atoms of the double-stranded nucleic acid, It is more preferably 7 times, and further preferably 2 to 5.5 times. In addition, the total number of molecules of the compound (A), the compound (B) or the compound (C), and the cationic lipid other than the compound (A), the compound (B) or the compound (C) in the complex is the two The number is preferably 0.5 to 8 times, more preferably 1.5 to 7 times, and even more preferably 2 to 5.5 times the number of phosphorus atoms in the double-stranded nucleic acid.
When the lipid particle of the present invention is composed of a complex and a lipid membrane encapsulating the complex, the total number of molecules of the compound (A), compound (B) or compound (C) in the lipid particle is The number of phosphorus atoms in the double-stranded nucleic acid is preferably 1 to 15 times, more preferably 2.5 to 12 times, and even more preferably 3.5 to 9 times. Further, the total number of molecules of the compound (A), compound (B) or compound (C), and cationic lipid other than the compound (A), compound (B) or compound (C) in the lipid particles is The number is preferably 1 to 15 times, more preferably 2.5 to 12 times, and still more preferably 3.5 to 9 times the number of phosphorus atoms in the double-stranded nucleic acid.

The neutral lipid may be any of simple lipids, complex lipids or derived lipids, and examples thereof include, but are not limited to, phospholipids, glyceroglycolipids, sphingoglycolipids, sphingoids, and sterols.
When the lipid particles of the present invention contain neutral lipids, the total number of neutral lipid molecules is compound (A), compound (B) or compound (C), and compound (A), compound (B). Alternatively, it is preferably 0.1 to 2 times, more preferably 0.2 to 1.5 times, and still more preferably 0.3 to 1.2 times the total number of cationic lipid molecules other than compound (C). The lipid particles in the present invention may contain a neutral lipid in a complex, may be contained in a lipid membrane encapsulating the complex, or at least contained in a lipid membrane encapsulating the complex. It is preferable that it is contained in both the complex and the lipid membrane encapsulating the complex.

Examples of phospholipids in neutral lipids include phosphatidylcholine (specifically soybean phosphatidylcholine, egg yolk phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), dimyristoylphosphatidylcholine). (DMPC), dioleoylphosphatidylcholine (DOPC), etc.), phosphatidylethanolamine (specifically distearoyl phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE)), Dimyristoylphosphoethanolamine (DMPE), 16-0-monomethyl PE, 16-0-dimethyl PE, 18-1-trans PE, palmitoyl oleoyl-phosphatidylethanolamine ( POPE), 1 -stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), etc.), glycerophospholipids (specifically phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, palmitoyloleoylphosphatidylglycerol (POPG), lyso) Phosphatidylcholine, 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 hydrogenated phospholipids (specifically hydrogenated soybean phosphatidylcholine and the like).

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

Examples of glycosphingolipids in neutral lipids include galactosyl cerebroside, lactosyl cerebroside, and ganglioside.

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

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

The neutral lipid is preferably phospholipid or sterol, more preferably phosphatidylcholine, phosphatidylethanolamine, or cholesterol, and still more preferably phosphatidylethanolamine, cholesterol, or a combination thereof.

Examples of macromolecules include proteins, albumin, dextran, polyfect, chitosan, dextran sulfate, such as poly-L-lysine, polyethyleneimine, polyaspartic acid, styrene maleic acid copolymer, isopropylacrylamide-acrylpyrrolidone copolymer Examples thereof include one or more polymers such as a polymer, a polyethylene glycol-modified dendrimer, polylactic acid, polylactic acid polyglycolic acid or polyethylene glycolated polylactic acid, or a salt thereof.

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

The lipid particles in the present invention preferably further contain a water-soluble polymer lipid derivative or fatty acid derivative. The lipid derivative or fatty acid derivative of the water-soluble polymer may be contained in a complex, may be contained in a lipid membrane encapsulating the complex, and both the complex and the lipid membrane encapsulating the complex It is more preferable to contain.
When the lipid particle of the present invention contains a lipid derivative or fatty acid derivative of a water-soluble polymer, the total number of molecules of the lipid derivative and fatty acid derivative of the water-soluble polymer is compound (A), compound (B) or compound (C), and preferably 0.01 to 0.3 times, more preferably 0.02 to 0.25 times the total number of molecules of the cationic lipid other than compound (A), compound (B) or compound (C). Preferably, it is 0.03 to 0.15 times.

A water-soluble polymer lipid derivative or fatty acid derivative has a property that a part of the molecule binds to other constituents of the lipid particle by, for example, hydrophobic affinity, electrostatic interaction, etc., and the other part is a lipid particle. A substance having a two-sided property, which has a property of binding with a solvent at the time of manufacture, for example, by hydrophilic affinity, electrostatic interaction or the like, is preferable.

Examples of lipid derivatives or fatty acid derivatives of water-soluble polymers include polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, oligosaccharide, dextrin, water-soluble cellulose, dextran, chondroitin sulfate, chitosan, polyvinyl Pyrrolidone, polyaspartic acid amide, poly-L-lysine, mannan, pullulan, oligoglycerol and the like or their derivatives, and the neutral lipids or compounds (A), compounds (B) or Compound (C) or, for example, those formed by bonding with fatty acids such as stearic acid, palmitic acid, myristic acid or lauric acid, salts thereof, and the like, preferably lipids such as polyethylene glycol derivatives and polyglycerin derivatives Invitation A body or fatty acid derivatives and their salts, more preferably, a lipid derivative or fatty acid derivatives and their salts of polyethylene glycol derivatives.

Examples of lipid derivatives or fatty acid derivatives of polyethylene glycol derivatives include polyethylene glycolated lipids (specifically, polyethylene glycol-phosphatidylethanolamine (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 )), Polyoxyethylene hydrogenated castor oil 60, Cremophor EL, etc.), polyethylene glycol sorbitan fatty acid esters (specifically polyoxyethylene sorbitan monooleate, etc.) and polyethylene glycol fatty acid esters, etc. Preferably, it is a polyethylene glycolated lipid, more preferably Or PEG-DSPE or PEG-DMPE.

Examples of lipid derivatives or fatty acid derivatives of polyglycerin derivatives include polyglycerinized lipids (specifically polyglycerin-phosphatidylethanolamine and the like) and polyglycerin fatty acid esters, preferably polyglycerinized lipids. .

The lipid particles in the present invention can be optionally subjected to surface modification with, for example, a water-soluble polymer, a polyoxyethylene derivative, etc. [Radasic, edited by F. Martin, “Stealth Liposomes ( Stealth Liposomes) "(USA), CRC Press Inc., 1995, pp. 93-102]. Examples of the polymer that can be used for the surface modification include dextran, pullulan, mannan, amylopectin, and hydroxyethyl starch. Examples of the polyoxyethylene derivative include polysorbate 80, Pluronic F68, polyoxyethylene hydrogenated castor oil 60, polyoxyethylene lauryl alcohol, and PEG-DSPE. By the surface modification, a lipid derivative or a fatty acid derivative of a water-soluble polymer can be contained in the complex and lipid membrane in the lipid particle.

The average particle size of the lipid particles in the present invention can be freely selected as desired, but is preferably the following average particle size. As a method for adjusting the average particle size, for example, an extrusion method, a method of mechanically crushing large multilamellar liposomes (MLV) or the like (specifically using a manton gourin, a microfluidizer, etc.) [Müller (RH Muller), S. Benita, B. Bohm, “Emulsion and Nanosuspensions for the Soluble Drugs”, Germany, Germany Scientific Publishers Stuttgart (see 1998, pp. 267-294).

In the present invention, the lipid particles preferably have an average particle diameter of about 10 nm to 1000 nm, more preferably about 30 nm to 300 nm, and further preferably about 50 nm to 200 nm.
The average particle diameter of lipid particles in the present invention can be measured by, for example, a dynamic light scattering method.

The double-stranded nucleic acid in the composition can be introduced into cells by administering the lipid particle-containing composition of the present invention to mammalian cells.

The method for introducing the lipid particle-containing composition of the present invention into a mammalian cell in vivo may be performed according to a known transfection procedure that can be performed in vivo. The lipid particle-containing composition of the present invention is intravenously administered to mammals including humans, and delivered to, for example, blood vessels, liver, lungs, spleen and / or kidneys, and the composition of the present invention is delivered into cells of a delivery organ or site. Double stranded nucleic acids in the composition can be introduced.

When a double-stranded nucleic acid in a composition containing lipid particles of the present invention is introduced into a cell of a delivery organ or site, the expression of β2GPI gene in the cell is decreased, and β2GPI-related diseases such as systemic lupus erythematosus ( SLE), antiphospholipid syndrome, hemodialysis complications and arteriosclerosis in patients with end-stage renal failure. The administration target is a mammal, and preferably a human.
The lipid particle-containing composition of the present invention can also be used as a tool for verifying the effectiveness of suppressing the β2GPI gene in an in vivo drug efficacy evaluation model relating to the therapeutic or preventive agent for the above-mentioned diseases. Examples of in vivo drug efficacy evaluation models include the lupus anticoagulant (LA) test. Here, LA is a kind of antiphospholipid antibody similar to the anti-β2GPI antibody, and exhibits an activity to inhibit phospholipid-dependent coagulation reaction of collected blood in vitro. LA appears mainly in diseases such as SLE and APS and has been reported to correlate with the onset or pathology of thrombosis and infertility as well as anti-β2GPI antibodies (“blood”, 2003, 101, No. 5, p1827-1832. And since anti-β2GPI antibody acquires phospholipid binding activity via β2GPI, it has also been reported to exhibit β2GPI-dependent LA activity (“Thrombosis and Haemostasis”, 1998, Vol. 79, No. 1, p79-86). Furthermore, this β2GPI-dependent LA has been reported to correlate strongly with the incidence of pathological conditions (“blood”, 2004, 104, 12, p3598-3602). If LA activity can be canceled by suppressing the expression, it is considered that a novel and effective treatment for β2GPI-related diseases can be provided. In clinical tests, LA can be detected by measuring activated partial thromboplastin time, kaolin clotting time and / or diluted Russell snake venom time (dRVVT).

When the lipid particle-containing composition of the present invention is used as a therapeutic or prophylactic agent for β2GPI-related diseases, the administration route is preferably the most effective administration route, more preferably intravenous administration. Subcutaneous administration or intramuscular administration, more preferably intravenous administration.
The dose varies depending on the medical condition, age, route of administration, etc. of the administration subject, but for example, the dose may be administered so that the daily dose converted to double-stranded nucleic acid is 0.1 μg to 1000 mg. It is preferable to administer 1 to 100 mg.

Examples of preparations suitable for intravenous administration or intramuscular administration include injections, and lipid particle dispersions prepared by the above-described method can be used as they are in the form of injections, for example. The dispersion can be used after removing the solvent, for example, by filtration, centrifugation, etc., or the dispersion can be lyophilized and / or used with an excipient such as mannitol, lactose, trehalose, maltose or glycine. The added dispersion can be lyophilized for use.
In the case of an injection, for example, water, acid, alkali, various buffers, physiological saline or amino acid infusion, etc., are mixed into the lipid particle-containing composition from which the lipid particle dispersion or the solvent has been removed or freeze-dried. It is preferable to prepare an injection. 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. Moreover, it can also be cryopreserved by adding a cryopreservation agent such as glycerin.

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 and test examples.
The proton nuclear magnetic resonance spectrum ( ¹ H NMR) shown in the reference example was measured at 270 MHz, 300 MHz, or 400 MHz, and exchangeable protons may not be clearly observed depending on the compound and measurement conditions. is there. In addition, although what is used normally is used as the notation of the multiplicity of signals, br represents an apparently wide signal.

Reference example 1
Di ((9Z, 12Z) -octadeca-9,12-dienyl) amine (Compound B-1)
Ammonia (Tokyo Chemical Industry Co., Ltd., about 2 mol / L methanol solution, 18.0 mL, 36.0 mmol) was added to (9Z, 12Z) -octadeca-9,12-dienyl methanesulfonate (New Check 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. A saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, 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 crude product obtained was (9Z, 12Z) -octadeca-9,12-dienyl methanesulfonate (Nu-Chek Prep, Inc., 1.24 g, 3.60 mmol) and 50% aqueous sodium hydroxide (1.44 g, 18.0 mmol) was added and stirred on an oil bath at 110 ° C. 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 (CDCl ₃ ) δ: 0.89 (t, J = 6.9Hz, 6H), 1.30 (br s, 33H), 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).

Reference example 2
Di ((Z) -octadeca-9-enyl) amine (Compound B-2)
In the same manner as in Reference Example 1, ammonia (manufactured by Tokyo Chemical Industry Co., Ltd., about 2 mol / L methanol solution, 12.0 mL, 24.0 mmol) and (Z) -octadeca-9-enyl methanesulfonate (Nu-Chek Prep, Inc., 1.87 g, 5.40 mmol) was used to obtain compound B-2 (0.562 g, yield 36.2%).
ESI-MS m / z: 519 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 6.7 Hz, 6H), 1.29 (br s, 45H), 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).

Reference example 3
Di ((Z) -hexadec-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) -hexadec-9-enyl methanesulfonate ( Nu-Chek Prep, Inc., 0.488 g, 1.46 mmol) was used to obtain compound B-3 (0.243 g, yield 36.0%).
¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 6.9 Hz, 6H), 1.24-1.37 (m, 37H), 1.43-1.52 (m, 4H), 1.98-2.05 (m, 8H), 2.58 (t, J = 7.2 Hz, 4H), 5.31-5.38 (m, 4H).

Reference example 4
Di ((11Z, 14Z) -icosa-11,14-dienyl) amine (Compound B-4)
Ammonia (SIGMA-ALDRICH, approximately 7 mol / L methanol solution, 1.60 mL, 11.2 mmol) and (11Z, 14Z) -icosa-11,14-dienyl methanesulfonate (Nu -Chek Prep, Inc., 0.521 g, 1.40 mmol) was used to obtain compound B-4 (0.292 g, yield 36.6%).
¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 6.8 Hz, 6H), 1.24-1.39 (m, 41H), 1.43-1.51 (m, 4H), 2.02-2.08 (m, 8H), 2.58 (t, J = 7.3 Hz, 4H), 2.77 (t, J = 6.7 Hz, 4H), 5.30-5.41 (m, 8H).

Reference Example 5
3- (Dimethylamino) propyl di ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-1)
Compound B-1 (1.35 g, 2.63 mmol) obtained in Reference Example 1 was dissolved in chloroform (18 mL), “Journal of American Chemical Society (J. Am. Chem. Soc.)”, 1981. 3- (dimethylamino) propyl-4-nitrophenyl-carbonate hydrochloride (Compound VI-1) (1.20 g, 3.94 mmol) synthesized by a method according to the method described in Y., Vol. 103, p.4194-4199 And triethylamine (1.47 mL, 10.5 mmol) were added and 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 solution was diluted with chloroform, washed with 1 mol / L aqueous sodium hydroxide solution three times, then with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was dissolved in a small amount of n-hexane / ethyl acetate (1/4), adsorbed on 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 give 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).

Reference Example 6
3- (Dimethylamino) propyl di ((Z) -octadeca-9-enyl) carbamate (Compound A-2)
In the same manner as in Reference Example 5, using Compound B-2 (0.156 g, 0.301 mmol) obtained in Reference Example 2 instead of Compound B-1, Compound A-2 (0.267 g, yield 88.7%) was obtained. Obtained.
ESI-MS m / z: 648 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 6.6 Hz, 6H), 1.28 (br s, 44H), 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).

Reference Example 7
3- (Dimethylamino) propyl di ((Z) -hexadec-9-enyl) carbamate (Compound A-3)
In the same manner as in Reference Example 5, using Compound B-3 (0.164 g, 0.355 mmol) obtained in Reference Example 3 instead of Compound B-1, Compound A-3 (0.116 g, yield 55.2%) was obtained. Obtained.
ESI-MS m / z: 592 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) 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).

Reference Example 8
3- (Dimethylamino) propyl di ((11Z, 14Z) -icosa-11,14-dienyl) carbamate (Compound A-4)
In the same manner as in Reference Example 5, using Compound B-4 (0.288 g, 0.505 mmol) obtained in Reference Example 4 instead of Compound B-1, Compound A-4 (0.290 g, yield 82.2%) was used. Obtained.
ESI-MS m / z: 700 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 6.8 Hz, 6H), 1.21-1.40 (m, 40H), 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).

Reference Example 9
2- (Dimethylamino) ethyl di ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-5)
In the same manner as in Reference Example 5, Compound B-1 (0.215 g, 0.418 mmol) obtained in Reference Example 1 and “Journal of American Chemical Society (J. Am. Chem. Soc.) ”, 1981, Vol. 103, p. 4194-4199, 2- (dimethylamino) ethyl-4-nitrophenyl-carbonate hydrochloride (compound VI-2 ) (0.162 g, 0.557 mmol) was used to obtain compound A-5 (0.184 g, yield 70.0%).
ESI-MS m / z: 630 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 6.8 Hz, 6H), 1.12-1.39 (m, 32H), 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 A-4 2- (1-Methylpyrrolidin-2-yl) ethyl 4-nitrophenyl carbonate hydrochloride (Compound VI-3)
To a solution of 4-nitrophenyl chloroformate (Tokyo Chemical Industry, 1.761 g, 8.56 mmol) in diethyl ether (20 mL), 2- (1-methylpyrrolidin-2-yl) ethanol (Tokyo Chemical Industry, 1.0 mL, 7.13 mmol) in diethyl ether (20 mL) was added, and the mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure, and the resulting 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-d ₆ ) 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, 2H), 8.11 (d, J = 9.2 Hz, 2H)

Reference Example A-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) ) And stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure, and the resulting 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-d ₆ ) δ: 1.28-1.49 (m, 1H), 1.62-1.89 (m, 5H), 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, 1H).

Reference Example A-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 (ABCR, 386 mg, 2.84 mmol) diethyl An ether (10 mL) solution was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was crystallized from ethanol and collected by filtration to obtain Compound VI-5 (498 mg, yield 53%).
¹ H-NMR (DMSO-d ₆ ) 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, 2H), 4,77 (br s, 1H), 6.95 (d, J = 9.2 Hz, 2H), 8.11 (d, J = 9.2 Hz, 2H)

Reference Example 10
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 Reference Example 1 was dissolved in acetonitrile (3.0 mL), and Compound VI-3 (0.156 g, 0.470 mmol) and triethylamine (0.219) obtained in Reference Example A-4 were dissolved. mL, 1.57 mmol) was added, and the mixture was 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 obtained 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, 6H), 1.20-1.40 (m, 32H), 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).

Reference Example 11
3- (Piperidin-1-yl) propyl di ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-7)
In the same manner as in Reference Example 5, compound A-7 (0.387 g, yield 81%) was obtained using compound VI-4 obtained in Reference Example A-5 instead of compound VI-1.
ESI-MS m / z: 684 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) 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, 4H), 4.09 (t, J = 6.4 Hz, 2H), 5.29-5.42 (m, 8H).

Reference Example 12
3- (Pyrrolidin-1-yl) propyl di ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-8)
In the same manner as in Reference Example 10, using Compound VI-5 (0.168 g, 0.508 mmol) obtained in Reference Example A-6 instead of Compound VI-3, Compound A-8 (0.225 g, yield 99%) )
ESI-MS m / z: 670 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 6.9 Hz, 6H), 1.21-1.40 (m, 32H), 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 A-7 2-Nitro-N-((9Z, 12Z) -octadeca-9,12-dienyl) benzenesulfonamide (Compound IVf-1)
To a solution of (9Z, 12Z) -octadeca-9,12-dienyl methanesulfonate (Nu-Chek Prep, Inc, 2.85 g, 8.27 mmol) in acetonitrile (30 ml), cesium carbonate (6.74 g, 20.67 mmol) , Tetrabutylammonium iodide (Tokyo Chemical Industry, 3.05 g, 8.27 mmol) and N- (tert-butoxycarbonyl) -2-nitrobenzenesulfonamide (Tokyo Chemical Industry, 2.50 g, 8.27 mmol) were added, The reaction was carried out under reflux for 3 hours. The reaction mixture 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 was 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, yield 70.5%) was obtained.
To 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) in trifluoroacetic acid (9.63 ml, 126 mmol) was added and stirred at room temperature for 0.5 hour. Dichloromethane and an aqueous sodium hydroxide solution (1 mol / L, 100 mL) were added to the reaction solution, and a 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 / 50-0 / 100) to obtain Compound IVf-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, 3H), 1.22-1.39 (m, 16H), 1.52 (m, 2H), 2.01-2.05 (m, 4H), 2.77 (t, J = 6.6 Hz, 2H), 3.09 (q, J = 6.7 Hz, 2H), 5.23 (m, 1H), 5.31-5.42 (m, 4H ), 7.71-7.76 (m, 2H), 7.78-7.87 (1H), 8.13-8.15 (m, 1H).

Reference Example 13
Dodecyl ((9Z, 12Z) -octadeca-9,12-dien-1-yl) amine (Compound B-5)
To a solution of compound IVf-1 (0.714 g, 1.584 mmol) obtained in Reference Example A-7 and 1-bromododecane (manufactured by Tokyo Chemical Industry Co., Ltd., 0.474 g, 1.90 mmol) in acetonitrile (6 ml) tetrabutylammonium iodide ( Tokyo Chemical Industry 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 mixture, and the mixture was extracted 3 times with n-hexane. The extract was purified by column chromatography (n-hexane / ethyl acetate = 94 / 6-84 / 16) and purified by N-dodecyl-2-nitro-N-((9Z, 12Z) -octadeca-9,12-diene 1-yl) benzenesulfonamide (0.750 g, 76% yield) was 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 Co., Ltd., 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 mixture, and the mixture was 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 obtain compound B-5 (0.534 g, quantitative yield). It was.
ESI-MS m / z: 434 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 6.9 Hz, 3H), 0.89 (t, J = 6.9 Hz, 3H), 1.24 -1.38 (m, 35H), 1.49-1.54 (m, 4H), 2.05 (q, J = 7.0 Hz, 4H), 2.62 (t, J = 7.4 Hz, 4H), 2.77 (t, J = 6.8 Hz, 2H), 5.30-5.41 (m, 4H).

Reference Example 14
Decyl ((9Z, 12Z) -octadeca-9,12-dien-1-yl) amine (Compound B-6)
In the same manner as in Reference Example 13, compound IVf-1 (0.619 g, 1.37 mmol) obtained in Reference Example A-7, and 1-bromodecane instead of 1-bromododecane (manufactured by Tokyo Chemical Industry Co., Ltd., 0.365 g, 1.65 mmol) was used to obtain compound B-6 (0.423 g, yield 76%).
ESI-MS m / z: 406 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 7.1 Hz, 3H), 0.89 (t, J = 6.9 Hz, 3H), 1.25 -1.38 (m, 31H), 1.46-1.50 (m, 4H), 2.05 (q, J = 7.0 Hz, 4H), 2.59 (t, J = 7.2 Hz, 4H), 2.77 (t, J = 6.9 Hz, 2H), 5.31-5.40 (m, 4H).

Reference Example 15
((9Z, 12Z) -Octadeca-9,12-dien-1-yl) (octyl) amine (Compound B-7)
In the same manner as in Reference Example 13, Compound IVf-1 (0.714 g, 1.58 mmol) obtained in Reference Example A-7 and 1-bromooctane (manufactured by Tokyo Chemical Industry Co., Ltd., 0.367 g instead of 1-bromododecane) , 1.90 mmol) was used to obtain compound B-7 (0.519 g, yield 87%).
ESI-MS m / z: 378 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 7.1 Hz, 3H), 0.89 (t, J = 6.9 Hz, 3H), 1.24 -1.39 (m, 27H), 1.48-1.54 (m, 4H), 2.05 (q, J = 7.0 Hz, 4H), 2.62 (t, J = 7.4 Hz, 4H), 2.77 (t, J = 6.6 Hz, 2H), 5.30-5.41 (m, 4H).

Reference Example 16
3- (Dimethylamino) propyl dodecyl ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-9)
In the same manner as in Reference Example 5, using Compound B-5 (0.260 g, 0.600 mmol) obtained in Reference Example 13 instead of Compound B-1, Compound A-9 (0.309 g, 91% yield) was obtained. Obtained.
ESI-MS m / z: 563 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 6.9 Hz, 3H), 0.89 (t, J = 6.9 Hz, 3H), 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, 2H), 2.77 (t, J = 6.6 Hz, 2H), 3.13-3.22 (m, 4H), 4.10 (t, J = 6.5 Hz, 2H), 5.30-5.41 (m, 4H).

Reference Example 17
3- (Dimethylamino) propyldecyl ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-10)
In the same manner as in Reference Example 5, using Compound B-6 (0.185 g, 0.456 mmol) obtained in Reference Example 14 instead of Compound B-1, Compound A-10 (0.228 g, 93% yield) was obtained. Obtained.
ESI-MS m / z: 535 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 6.8 Hz, 3H), 0.89 (t, J = 6.7 Hz, 3H), 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, 2H), 2.77 (t, J = 6.8 Hz, 2H), 3.14-3.22 (m, 4H), 4.10 (t, J = 6.4 Hz, 2H), 5.31-5.40 (m, 4H).

Reference Example 18
3- (Dimethylamino) propyl ((9Z, 12Z) -octadeca-9,12-dienyl) (octyl) carbamate (Compound A-11)
In the same manner as in Reference Example 5, using Compound B-7 (0.227 g, 0.600 mmol) obtained in Reference Example 15 instead of Compound B-1, Compound A-11 (0.275 g, 90% yield) was obtained. Obtained.
ESI-MS m / z: 507 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 6.9 Hz, 3H), 0.89 (t, J = 6.9 Hz, 3H), 1.22 -1.39 (m, 26H), 1.47-1.54 (m, 4H), 1.76-1.82 (m, 2H), 2.05 (q, J = 6.0 Hz, 4H), 2.23 (s, 6H), 2.34 (t, J = 7.6 Hz, 2H), 2.77 (t, J = 6.6 Hz, 2H), 3.12-3.22 (m, 4H), 4.10 (t, J = 6.5 Hz, 2H), 5.30-5.41 (m, 4H).

Reference Example A-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) were added to (9Z, 12Z) -octadeca-9,12-dien-1-yl methanesulfonate (983 mg, 2.85 mmol), The mixture was stirred for 1 day with heating 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%).
To 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) at 0 ° C. with mesylic anhydride ( SIGMA-ALDRICH, 0.247 ml, 3.19 mmol) was added, and the mixture was stirred at room temperature for 40 minutes. Water was added to the reaction mixture, and the mixture was 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.

Reference Example 19
(9Z, 12Z) -N- (2-((9Z, 12Z) -octadeca-9,12-dienyloxy) ethyl) octadeca-9,12-dien-1-amine (Compound B-8)
In the same manner as in Reference Example 13, compound IVf-1 (0.798 g, 1.77 mmol) obtained in Reference Example A-7 and compound IIIc-1 obtained in Reference Example A-8 instead of 1-bromododecane ( 0.826 g, 2.13 mmol) was used to obtain compound B-8 (0.676 g, yield 68%).
ESI-MS m / z: 558 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) 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, 8H), 2.61 (t, J = 7.3 Hz, 2H), 2.77 (t, J = 5.5 Hz, 6H), 3.42 (t, J = 6.8 Hz, 2H), 3.52 (t, J = 5.4 Hz, 2H), 5.30-5.41 (m, 8H).

Reference Example 20
3- (Dimethylamino) propyl ((9Z, 12Z) -octadeca-9,12-dienyl) (2-((9Z, 12Z) -octadeca-9,12-dienyloxy) ethyl) carbamate (Compound A-12)
In the same manner as in Reference Example 5, using Compound B-8 (0.184 g, 0.330 mmol) obtained in Reference Example 19 instead of Compound B-1, Compound A-12 (0.208 g, 92% yield) was obtained. Obtained.
ESI-MS m / z: 687 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 6.9 Hz, 6H), 1.25-1.38 (m, 32H), 1.50-1.57 ( m, 4H), 1.77-1.83 (m, 2H), 2.05 (q, J = 7.0 Hz, 8H), 2.22 (s, 6H), 2.34 (t, J = 7.4 Hz, 2H), 2.77 (t, J = 6.6 Hz, 4H), 3.23-3.54 (m, 8H), 4.11 (t, J = 6.5 Hz, 2H), 5.30-5.41 (m, 8H).

Reference Example A-11 Ethyl 3- (di ((9Z, 12Z) -octadeca-9,12-dienyl) amino) propionate (Compound IVj-1)
Compound B-1 (0.788 g, 1.53 mmol) obtained in Example 1 was dissolved in ethanol (8 mL), ethyl acrylate (Tokyo Chemical Industry Co., Ltd., 1.67 mL, 15.3 mmol) and sodium ethoxide (Wako Pure Chemical Industries, Ltd.) Yakuhin Kogyo Co., Ltd., 0.0520 g, 0.767 mmol) was added, and the mixture was stirred for 3 hours with heating under reflux. The reaction mixture was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (n-hexane / ethyl acetate = 85/15) to give compound IVj-1 (0.699 g, yield 74%). It was.
ESI-MS m / z: 615 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 6.9 Hz, 6H), 1.21-1.45 (m, 39H), 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).
Reference Example 21
3- (Di ((9Z, 12Z) -octadeca-9,12-dienyl) amino) propan-1-ol (Compound C-1)
Compound IVj-1 (0.199 g, 0.324 mmol) obtained in Reference Example A-11 was dissolved in tetrahydrofuran (2 mL). Under ice-cooling, lithium aluminum hydride (Pure Chemical Co., Ltd., 0.012 g, 0.324 mmol) was added. The mixture was further stirred for 3 hours. Water (0.0600 mL, 3.33 mmol) and sodium fluoride (manufactured by 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 hour. Insolubles were removed by celite filtration. The filtrate was concentrated and 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 (CDCl ₃ ) 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 = 6.7 Hz, 4H), 3.79 (t, J = 5.3 Hz, 2H), 5.30-5.42 (m, 8H).

Reference Example 22
4- (Dimethylamino) butyl di ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-13)
Process 1
To a solution of 4-nitrophenyl chloroformate (0.867 g, 4.21 mmol) in dichloromethane (20 mL), 4- (tert-butyldimethylsilyl) oxy-1-butanol (SIGMA-ALDRICH, 1.0 mL, 4.21 mmol) And triethylamine (0.881 mL, 6.32 mmol) were added, and the mixture was stirred at room temperature for 1 hour. A saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, 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 was 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). 92%).
Process 2
In the same manner as in Reference Example 10, using 4- (tert-butyldimethylsilyloxy) butyl 4-nitrophenyl carbonate (0.640 g, 1.733 mmol) obtained in Step 1 instead of compound VI-3, A crude product of (tert-butyldimethylsilyloxy) butyl di ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate was obtained. The obtained crude 4- (tert-butyldimethylsilyloxy) butyl di ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate was dissolved in tetrahydrofuran (10 mL), and tetrabutylammonium fluoride was dissolved. (Tokyo Chemical Industry Co., Ltd., about 1 mol / L tetrahydrofuran solution, 2.14 mL, 2.14 mmol) was added, and the mixture was 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. Saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, 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 obtained residue was 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 73%).
Process 3
To 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) was added mesylic acid chloride under ice-cooling. (Pure Chemical Co., Ltd., 0.0360 mL, 0.460 mmol) and triethylamine (0.0930 mL, 0.919 mmol) were added and stirred at 0 ° C. for 30 minutes. A saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, and the mixture was extracted twice with chloroform. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. Dissolve the resulting residue in tetrahydrofuran (1 mL), add dimethylamine (manufactured by Aldrich, approximately 2 mol / L tetrahydrofuran solution, 1.53 mL, 3.06 mmol), and use a microwave reactor at 100 ° C for 1 hour. After stirring, the mixture was stirred at 130 ° C. for 1 hour using a microwave reactor. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by amino silica gel column chromatography (n-hexane / ethyl acetate = 80/20) to give compound A-13 (0.159 g, yield 79%). It was.
ESI-MS m / z: 658 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.90 (q, J = 6.5 Hz, 6H), 1.20-1.38 (m, 32H), 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, 2H), 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 A-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) at room temperature. Stir for 2 hours. The precipitated crystals were collected by filtration to obtain 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, 4H), 2.68-2.82 (m, 3H), 3.51-3.62 (m, 5H ), 4.21 (d, J = 6.0 Hz, 2H), 7.38 (d, J = 9.1 Hz, 2H), 8.27 (d, J = 9.1 Hz, 2H), 12.44 (br s, 1H).

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

Reference Example A-16 (1-Methylpiperidin-2-yl) methyl 4-nitrophenyl carbonate hydrochloride (Compound VI-8)
In the same manner as in Reference Example A-14, 1-methyl-2-piperidinemethanol (manufactured by Tokyo Chemical Industry Co., Ltd., 1.0 mL, 7.43 mmol) was used instead of 1-methyl-4-piperidinemethanol, and compound VI-8 (2.37 g, yield 96%) was obtained.
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, 1H), 4.78-4.86 (m, 1H), 7.47 (d, J = 9.1 Hz, 2H), 8.28 (d, J = 9.1 Hz, 2H ), 12.40 (br s, 1H).

Reference Example A-17 3- (Azepan-1-yl) propyl 4-nitrophenyl carbonate hydrochloride (Compound VI-9)
In the same manner as in Reference Example A-14, instead of 1-methyl-4-piperidinemethanol, 3- (azepan-1-yl) propanol (CHEMBRIDGE, 0.700 g, 4.45 mmol) was used. Compound VI-9 (1.47 g, yield 92%) 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, 2H), 8.25-8.32 (m, 2H), 12.48 (br s, 1H).

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

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

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

Reference Example 23
(1-Methylpiperidin-4-yl) methyl di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) carbamate (Compound A-14)
In the same manner as in Reference Example 10, using Compound VI-6 (0.228 g, 0.689 mmol) obtained in Reference Example A-14 instead of Compound VI-3, Compound A-14 (0.258 g, yield 84) %).
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, 4H), 2.85 (d, J = 11.7 Hz, 2H), 3.13-3.23 (m, 4H), 3.92 (d, J = 6.3 Hz, 2H), 5.30-5.42 ( m, 8H).

Reference Example 24
(1-Methylpiperidin-3-yl) methyl di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) carbamate (Compound A-15)
In the same manner as in Reference Example 10, using Compound VI-7 (0.238 g, 0.719 mmol) obtained in Reference Example A-15 instead of Compound VI-3, Compound A-15 (0.239 g, yield 74) %).
ESI-MS m / z: 670 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 6.9 Hz, 6H), 0.92-1.02 (m, 1H), 1.22-1.39 ( m, 32H), 1.46-1.54 (m, 4H), 1.55-1.65 (m, 1H), 1.66-1.74 (m, 3H), 1.82-1.89 (m, 1H), 1.91-2.00 (m, 1H), 2.05 (q, J = 7.0 Hz, 8H), 2.26 (s, 3H), 2.74-2.80 (m, 5H), 2.84-2.89 (m, 1H), 3.12-3.23 (m, 4H), 3.87 (dd, J = 10.7, 7.6 Hz, 1H), 3.97 (dd, J = 10.7, 5.4 Hz, 1H), 5.30-5.41 (m, 8H).

Reference Example 25
(1-Methylpiperidin-2-yl) methyl di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) carbamate (Compound A-16)
In the same manner as in Reference Example 10, using Compound VI-8 (0.256 g, 0.774 mmol) obtained in Reference Example A-16 instead of Compound VI-3, Compound A-16 (0.313 g, yield 91) %).
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, 1H), 4.17 (dd, J = 11.1, 4.9 Hz, 1H), 5.30-5.42 (m, 8H).

Reference Example 26
2- (1-Methylpyrrolidin-2-yl) ethyl di ((Z) -octadeca-9-enyl) carbamate (Compound A-17)
In the same manner as in Reference Example 10, using Compound B-2 (0.300 g, 0.579 mmol) obtained in Reference Example 2 instead of Compound B-1, Compound A-17 (0.360 g, 92% yield) was obtained. Obtained.
ESI-MS m / z: 674 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) 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).

Reference Example 27
(1-Methylpiperidin-3-yl) methyl di ((Z) -octadeca-9-enyl) carbamate (Compound A-18)
In the same manner as in Reference Example 10, compound B-2 (0.300 g, 0.579 mmol) obtained in Reference Example 2 instead of Compound B-1 and Reference Example A-15 instead of Compound VI-3 Compound A-18 (0.390 g, yield 100%) was obtained using Compound 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), 2.83-2.89 (m, 1H), 3.10-3.25 (m, 4H), 3.87 (dd, J = 10.5, 7.3 Hz, 1H), 3.98 (dd, J = 10.5, 5.3 Hz, 1H), 5.30-5.39 (m, 4H).

Reference Example 28
2- (1-Methylpyrrolidin-2-yl) ethyl di ((11Z, 14Z) -icosa-11,14-dienyl) carbamate (Compound A-19)
In the same manner as in Reference Example 10, using Compound B-4 (0.300 g, 0.526 mmol) obtained in Reference Example 4 instead of Compound B-1, Compound A-19 (0.369 g, 97% yield) was obtained. Obtained.
ESI-MS m / z: 726 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) 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).

Reference Example 29
(1-Methylpiperidin-3-yl) methyl di ((11Z, 14Z) -icosa-11,14-dienyl) carbamate (Compound A-20)
In the same manner as in Reference Example 10, compound B-4 (0.300 g, 0.526 mmol) obtained in Reference Example 4 instead of Compound B-1 and Reference Example A-15 instead of Compound VI-3 Compound A-7 (0.374 g, yield 98%) was obtained using Compound 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, 1H), 5.29-5.43 (m, 8H).

Reference Example 30
(1-Methylpyrrolidin-2-yl) methyl di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) carbamate (Compound A-21)
Compound B-1 (0.0831 g, 0.162 mmol) obtained in Reference Example 1 was dissolved in dichloroethane (1 mL), 1,1′-carbonyldiimidazole (manufactured by Nacalai Tesque, 0.0394 g, 0.243 mmol) was added, Stir at room temperature overnight. Iodomethane (manufactured by Tokyo Chemical Industry Co., Ltd., 0.101 mL, 1.62 mmol) was added to the reaction solution, and the mixture was stirred at 60 ° C. overnight. The reaction solution was concentrated under reduced pressure. Dissolve the resulting residue in tetrahydrofuran (1 mL), add 1-methylpyrrolidine-2-methanol (manufactured by Wako Pure Chemical Industries, 0.0372 g, 0.323 mmol) and triethylamine (0.0563 mL, 0.404 mmol) at room temperature. After stirring overnight, the reaction was stirred at 60 ° C. for 3 hours. A saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, 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 obtain 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, 6H), 1.24-1.39 (m, 32H), 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 , 1H), 4.08 (dd, J = 10.5, 5.5 Hz, 1H), 5.29-5.42 (m, 8H).

Reference Example 31
(1-Methylpyrrolidin-3-yl) methyl di (9Z, 12Z) -octadeca-9,12-dienylcarbamate (Compound A-22)
In the same manner as in Reference Example 10, using Compound VI-12 (0.277 g, 0.876 mmol) obtained in Reference Example A-20 instead of Compound VI-3, Compound A-22 (0.263 g, 66% yield) )
ESI-MS m / z: 656 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 6.9 Hz, 6H), 1.20-1.40 (m, 32H), 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, 1H), 4.03 (dd, J = 10.5, 6.4 Hz, 1H), 5.28-5.42 (m, 8H).

Reference Example 32
2- (1-Methylpiperidin-2-yl) ethyl di (9Z, 12Z) -octadeca-9,12-dienylcarbamate (Compound A-23)
Process 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). And stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to obtain a crude product of 2- (1-methylpiperidin-2-yl) ethyl 4-nitrophenyl carbonate hydrochloride.
Process 2
Crude product of 2- (1-methylpiperidin-2-yl) ethyl 4-nitrophenyl carbonate hydrochloride obtained in Step 1 instead of compound VI-3 in the same manner as in Reference Example 10 (0.302 g, 0.876 mmol) was used to obtain compound A-23 (0.299 g, yield 75%).
ESI-MS m / z: 684 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 6.9 Hz, 6H), 1.19-1.40 (m, 32H), 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).

Reference Example 33
3- (Azepan-1-yl) propyl di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) carbamate (Compound A-24)
In the same manner as in Reference Example 10, compound VI-24 (0.136 g, yield 67%) was obtained using compound VI-9 obtained in Reference Example A-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, 2H), 2.62 (t, J = 5.1 Hz, 4H), 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).

Reference Example 34
3- (Piperidin-1-yl) propyl ((9Z, 12Z) -octadeca-9,12-dienyl) (2-((9Z, 12Z) -octadeca-9,12-dienyloxy) ethyl) carbamate (Compound A- 29)
In the same manner as in Reference Example 10, Compound B-8 (0.150 g, 0.269 mmol) obtained in Reference Example 19 instead of Compound B-1 and Reference Example A-5 instead of Compound VI-3 Compound A-4 (0.170 g, yield 87%) was obtained using compound VI-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, 6H), 1.22-1.40 (m, 32H), 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).

Reference Example 35
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 the same manner as in Reference Example 10, using Compound B-8 (0.120 g, 0.215 mmol) obtained in Reference Example 19 instead of Compound B-1, Compound A-30 (0.140 g, 91% yield) Got.
ESI-MS m / z: 714 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 6.8 Hz, 6H), 1.21-1.40 (m, 32H), 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).

Reference Example 36
3- (Azepan-1-yl) propyl ((9Z, 12Z) -octadeca-9,12-dienyl) (2-((9Z, 12Z) -octadeca-9,12-dienyloxy) ethyl) carbamate (compound A- 31)
In the same manner as in Reference Example 10, Compound B-8 (0.150 g, 0.269 mmol) obtained in Reference Example 19 instead of Compound B-1 and Reference Example A-17 instead of Compound VI-3 Compound A-9 (0.170 g, 85% yield) was obtained using Compound VI-9 (0.145 g, 0.403 mmol).
ESI-MS m / z: 742 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 6.8 Hz, 6H), 1.21-1.39 (m, 32H), 1.48-1.65 ( m, 12H), 1.72-1.85 (m, 2H), 2.05 (q, J = 6.6 Hz, 8H), 2.55 (t, J = 7.5 Hz, 2H), 2.61 (t, J = 5.3 Hz, 4H), 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).

Reference Example 37
(1-Methylpiperidin-3-yl) methyl ((9Z, 12Z) -octadeca-9,12-dienyl) (2-((9Z, 12Z) -octadeca-9,12-dienyloxy) ethyl) carbamate (Compound A -32)
In the same manner as in Reference Example 10, compound B-8 (0.150 g, 0.269 mmol) obtained in Reference Example 19 instead of Compound B-1 and obtained in Reference Example A-15 instead of Compound VI-3 Compound A-32 (0.145 g, yield 76%) was obtained using compound VI-7 (0.133 g, 0.403 mmol).
ESI-MS m / z: 714 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.91 (t, J = 6.8 Hz, 6H), 1.23-1.45 (m, 32H), 1.49-1.77 ( m, 9H), 1.83-2.03 (m, 2H), 2.07 (q, J = 6.6 Hz, 8H), 2.28 (s, 3H), 2.76-2.91 (m, 2H), 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 A-21 2-((Z) -octadeca-9-enyloxy) ethyl methanesulfonate (Compound IIIc-2)
In the same manner as in Reference Example 8, instead of (9Z, 12Z) -octadeca-9,12-dien-1-yl methanesulfonate, (Z) -octadeca-9-en-1-yl methanesulfonate (2.00 g, 5.77 mmol) was used to give compound IIIc-2 (1.29 g, 57% yield).
ESI-MS m / z: 391 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 6.6 Hz, 3H), 1.22-1.38 (m, 22H), 1.50-1.62 ( 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, 2H).

Reference Example A-22 2-((Z) -hexadec-9-enyloxy) ethyl methanesulfonate (Compound IIIc-3)
In the same manner as in Reference Example 8, instead of (9Z, 12Z) -octadeca-9,12-dien-1-yl methanesulfonate, (Z) -hexadeca-9-en-1-yl methanesulfonate (2.00 g, 6.28 mmol) was used to give compound IIIc-3 (1.52 g, 67% yield).
ESI-MS m / z: 363 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.90 (t, J = 6.6 Hz, 3H), 1.25-1.38 (m, 18H), 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, 2H).

Reference Example 38
(9Z, 12Z) -N- (2-((Z) -octadeca-9-enyloxy) ethyl) octadeca-9,12-dien-1-amine (Compound B-9)
In the same manner as in Reference Example 13, Compound IVf-1 (0.800 g, 1.78 mmol) obtained in Reference Example A-7 and Compound IIIc-2 (Reference Example A-21 instead of 1-bromododecane) 0.728 g, 1.86 mmol) was used to obtain compound B-9 (0.600 g, yield 60%).
ESI-MS m / z: 560 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.86-0.94 (m, 6H), 1.24-1.39 (m, 40), 1.51-1.62 (m, 2H ), 1.96-2.10 (m, 8H), 2.68 (t, J = 7.3 Hz, 2H), 2.78 (t, J = 6.0 Hz, 2H), 2.85 (t, J = 5.1 Hz, 2H), 3.45 (t , J = 6.8 Hz, 2H), 3.57 (t, J = 5.1 Hz, 2H), 5.30-5.44 (m, 6H).

Reference Example 39
(9Z, 12Z) -N- (2-((Z) -Hexadeca-9-enyloxy) ethyl) octadeca-9,12-dien-1-amine (Compound B-10)
In the same manner as in Reference Example 13, Compound IVf-1 (0.610 g, 1.35 mmol) obtained in Reference Example A-7 and Compound IIIc-3 (Reference Example A-22 instead of 1-bromododecane) ( 0.589 g, 1.62 mmol) was used to obtain compound B-10 (0.550 g, yield 76%).
ESI-MS m / z: 532 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.85-0.93 (m, 6H), 1.23-1.38 (m, 34H), 1.45-1.54 (m, 4H ), 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 = 5.4 Hz, 2H), 5.29-5.44 (m, 6H).

Reference Example 40
3- (Piperidin-1-yl) propyl (2-((Z) -octadeca-9-enyloxy) ethyl) ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-33)
In the same manner as in Reference Example 10, Compound B-9 (0.130 g, 0.232 mmol) obtained in Reference Example 38 instead of Compound B-1 and obtained in Reference Example A-5 instead of Compound VI-3 Compound A-4 (0.137 g, 81% yield) was obtained using Compound VI-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, 6H), 2.77 (t, J = 5.9 Hz, 2H), 3.20-3.31 (m, 2H), 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).

Reference Example 41
2- (1-Methylpyrrolidin-2-yl) ethyl (2-((Z) -octadeca-9-enyloxy) ethyl) ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-34 )
In the same manner as in Reference Example 10, using Compound B-9 (0.130 g, 0.232 mmol) obtained in Reference Example 38 instead of Compound B-1, Compound A-34 (0.131 g, yield 79%) Got.
ESI-MS m / z: 716 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.85-0.92 (m, 6H), 1.20-1.39 (38H, m), 1.47-1.61 (m, 8H ), 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, 1H) , 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).

Reference Example 42
3- (Dimethylamino) propyl (2-((Z) -octadeca-9-enyloxy) ethyl) ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-35)
In the same manner as in Reference Example 10, Compound B-9 (0.130 g, 0.232 mmol) obtained in Reference Example 38 instead of Compound B-1 and Compound VI-1 (0.106 g, instead of Compound VI-3) 0.348 mmol) was used to give compound A-35 (0.100 g, 63% yield).
ESI-MS m / z: 690 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.85-0.92 (m, 6H), 1.20-1.39 (m, 38H), 1.45-1.59 (m, 4H ), 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).

Reference Example 43
3- (Dimethylamino) propyl (2-((Z) -hexadec-9-enyloxy) ethyl) ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-36)
In the same manner as in Reference Example 10, instead of Compound B-1, Compound B-10 (0.150 g, 0.282 mmol) obtained in Reference Example 39 and Compound VI-1 (0.095 g, 0.310 mmol) was used to obtain compound A-36 (0.148 g, yield 79%).
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).

Reference Example 44
3- (Piperidin-1-yl) propyl (2-((Z) -hexadec-9-enyloxy) ethyl) ((9Z, 12Z) -octadeca-9,12-dienyl) carbamate (Compound A-37)
In the same manner as in Reference Example 10, Compound B-10 (0.150 g, 0.282 mmol) obtained in Reference Example 39 instead of Compound B-1 and obtained in Reference Example A-5 instead of Compound VI-3 Compound A-4 (0.148 g, yield 75%) was obtained using Compound VI-4 (0.107 g, 0.310 mmol).
ESI-MS m / z: 702 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.82-0.96 (m, 6H), 1.23-1.39 (m, 34H), 1.39-1.48 (m, 2H ), 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).

Reference Example 45
3- (Di ((Z) -octadeca-9-enyl) amino) propan-1-ol (Compound C-2)
Process 1
In the same manner as in Reference Example A-11, instead of Compound B-1, using Compound B-2 (500 mg, 0.965 mmol) obtained in Reference Example 2, 3- (di ((Z) -octadeca-9 -Ethyl) amino) ethyl propionate (548 mg, 92% yield) was obtained.
Process 2
In the same manner as in Reference Example 21, using ethyl 3- (di ((Z) -octadeca-9-enyl) amino) propionate (548 mg, 0.887 mmol) instead of compound IVj-1, compound C-2 (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, 6H), 1.24-1.42 (m, 44H), 1.42-1.50 ( m, 4H), 1.65-1.70 (m, 2H), 2.01 (q, J = 6.4 Hz, 8H), 2.40 (t, J = 7.5 Hz, 4H), 2.63 (t, J = 5.5 Hz, 2H), 3.79 (t, J = 5.3 Hz, 2H), 5.30-5.39 (m, 4H).

Reference Example 46
3- (Di ((11Z, 14Z) -icosa-11,14-dienyl) amino) propan-1-ol (Compound C-3)
Process 1
In the same manner as in Reference Example A-11, using Compound B-4 (400 mg, 0.702 mmol) obtained in Reference Example 4 instead of Compound B-1, 3- (di ((11Z, 14Z) -icosa -11,14-dienyl) amino) ethyl propionate (548 mg, 90% yield) was obtained.
Process 2
In the same manner as in Reference Example 21, ethyl 3- (di ((11Z, 14Z) -icosa-11,14-dienyl) amino) propionate (424 mg, 0.633 mmol) was used instead of compound IVj-1. Compound C-3 (352 mg, yield 88%) was obtained.
ESI-MS m / z: 629 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) 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, 4H), 2.63 (t, J = 5.3 Hz, 2H), 2.78 (t , J = 6.4 Hz, 4H), 3.79 (t, J = 5.0 Hz, 2H), 5.29-5.42 (m, 8H).

Reference Example 47
2- (di ((9Z, 12Z) -octadeca-9,12-dienyl) amino) ethanol (compound C-4)
Process 1
To a solution of Compound B-1 (600 mg, 1.17 mmol) obtained in Reference Example 1 in 1,2-dichloroethane (2.0 mL) was added potassium carbonate (243 mg, 1.76 mmol) and ethyl bromoacetate (195 μL, 1.76 mmol). The mixture was further stirred at 85 ° C. overnight. Water was added to the resulting mixture, and the mixture was extracted twice with heptane. The organic layers were combined, washed with water, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by amino silica gel column chromatography (heptane / ethyl acetate = 100/0 to 95/5) to give 2- (di ((9Z, 12Z) -octadeca-9,12-dienyl) amino) ethyl acetate ( 527 mg, 75% yield).
Process 2
In the same manner as in Reference Example 21, instead of compound IVj-1, 2- (di ((9Z, 12Z) -octadeca-9,12-dienyl) amino) ethyl acetate (527 mg, 0.878 mmol) was used. C-4 (433 mg, yield 88%) was obtained.
ESI-MS m / z: 559 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) 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, 4H), 2.57 (t, J = 5.3 Hz, 2H), 2.77 (t, J = 6.2 Hz, 4H), 3.52 (t, J = 5.5 Hz, 2H), 5.29-5.41 (m, 8H).

Reference Example 48
4- (Di ((9Z, 12Z) -octadeca-9,12-dienyl) amino) butan-1-ol (Compound C-5)
Process 1
To a solution of Compound B-1 (500 mg, 0.973 mmol) obtained in Reference Example 1 in 1,2-dichloroethane (2.0 mL), potassium carbonate (202 mg, 1.46 mmol) and tert-butyl (4-iodobutoxy) (1) Dimethylsilane (manufactured by SIGMA-ALDRICH, 378 μL, 1.46 mmol) was added and stirred at 85 ° C. for 4 hours. Water was added to the resulting mixture, and the mixture was extracted twice with heptane. The organic layers were combined, washed with water, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was 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.
Process 2
To a solution of (4- (tert-butyldimethylsilyloxy) butyl) di ((9Z, 12Z) -octadeca-9,12-dienyl) amine (233 mg, 0.333 mmol) in tetrahydrofuran (5 mL) was added tetrabutyl fluoride. Ammonium (1 mol / L tetrahydrofuran solution, 0.666 mL, 0.666 mmol) was added, and the mixture was stirred at room temperature overnight. To the obtained mixture was added saturated brine, and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was 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%).
ESI-MS m / z: 587 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) 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, 8H), 2.41-2.45 (m, 6H), 2.77 (t, J = 6.6 Hz, 4H), 3.53-3.56 (m, 2H), 5.29-5.42 (m, 8H).

Reference Example DS-1 Preparation of Double-Stranded Nucleic Acid A sense strand consisting of ribonucleotides shown in SEQ ID NOs: 1-1180, an antisense strand consisting of ribonucleotides shown in SEQ ID NOs: 1181-1360, and double-stranded nucleic acids obtained by annealing them ( A sense strand represented by SEQ ID NO: n (n = 1 to 1180) and an antisense strand represented by SEQ ID NO: [n + 1180] were paired) from Sigma-Aldrich.

Test Example 1 Measurement of β2GPI mRNA Knockdown Activity HepG2 cells (obtained from ATCC, ATCC number: HB-8065), a cell line derived from human liver cancer, are placed in a 96-well culture plate at 5,000 cells / 80 μL / well Sowed. As the medium, a MEM medium (Life Technologies, catalog number 11095-098) containing 10% fetal bovine serum (FBS) was used. Double-stranded nucleic acid and RNAiMax transfection reagent (catalog number: manufactured by Life Technology Co., Ltd.) respectively obtained from the sense strand described in SEQ ID NOs: 1-1180 and the antisense strand described in SEQ ID NOs: 1181-1360, respectively. 1401251) is diluted with Opti-MEM medium (Life Technologies, Catalog No. 11058-021) and 96 μl each of 20 μL of siRNA / RNAiMax mixed solution is used so that the final concentration of double-stranded nucleic acid is 100 pmol / L. And cultured at 37 ° C. under 5% CO ₂ for 24 hours. After that, the cells were washed with PBS (Phosphate buffered saline) and described in the instructions attached to the product from each plate using the Cells-to-Ct kit (Applied Biosystems, catalog number: AM1728). CDNA was synthesized according to the method. Add 5 μL of this cDNA to a MicroAmpOptical 96-well plate (Applied Bio Systems, catalog number 4326659), and add 10 μL of TaqMan Gene Expression Master Mix (Applied Biosystems). (Applied Systems, catalog number 4369016), 3 μL UltraPure Distilled Water (Life Technologies, catalog number: 10977-015), 1 μL human β2GPI probe, 1 μL human A glyceraldehyde 3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate dehydrogenase, hereinafter referred to as gapdh) probe is added, and ABI 7900 HT Fast (ABI7900HT Fast) (manufactured by ABI (ABI)) PCR reaction according to the method described in the attached instruction manual More, the gapdh gene is a human Beta2GPI genes and constitutive expression gene by a PCR reaction to measure mRNA amplification amount, respectively, as an internal control for mRNA amplification amount of gapdh, was calculated quasi quantitative value of mRNA of Beta2GPI. A semi-quantitative value of β2GPI mRNA amount was calculated when HepG2 cells were treated with transfection reagent alone without adding siRNA, and β2GPI mRNA relative expression level was calculated when each siRNA was introduced at 1.0. . This experiment was performed 3 times, and the average values of β2GPI mRNA relative expression levels are shown in Tables 2-1 to 2-16.

Furthermore, in order to select a double-stranded nucleic acid having a high knockdown activity, the final concentration of the double-stranded nucleic acid was reduced to 10 pmol / L, and the relative expression level of the β2GPI gene was calculated in the experimental system. The results are shown in Table 3.

Test Example 2 Knockdown Activity of Chemically Modified Double-stranded Nucleic Acid A sense strand consisting of the ribonucleotide sequence shown in SEQ ID NOs: 3542 to 3701, an antisense strand consisting of the ribonucleotide sequence shown in SEQ ID NOs: 3702 to 3861, and annealing them Double-stranded nucleic acid (AH1181 to AH1340; sense strand shown in SEQ ID NO: n (n = 3542-3701) and antisense strand shown in SEQ ID NO: [n + 160] are paired) from Sigma-Aldrich or Gene Design Obtained (see Tables 4-1 to 4-5; in the table, uppercase letters indicate unmodified RNA, and lowercase letters indicate 2′-O-methyl-modified RNA). Double-stranded nucleic acid numbers AH1181-1120 are the β2GPI partial sequence shown in SEQ ID NO: 2456, AH1205-1233 are the β2GPI partial sequence shown in SEQ ID NO: 2459, AH1234-1243 are the β2GPI partial sequence shown in SEQ ID NO: 2485, AH1244-1253 Is a β2GPI partial sequence shown in SEQ ID NO: 2486, AH1254-1263 is a β2GPI partial sequence shown in SEQ ID NO: 3053, AH1264-2127 is a β2GPI partial sequence shown in SEQ ID NO: 3185, AH1272-11282 is a β2GPI partial sequence shown in SEQ ID NO: 3239, AH1283 ˜1297 is the β2GPI partial sequence shown in SEQ ID NO: 3303, AH1298-1311 is the β2GPI partial sequence shown in SEQ ID NO: 3385, AH1312-1316 is the β2GPI partial sequence shown in SEQ ID NO: 3398, and AH1317-1325 is shown in SEQ ID NO: 3399. 2GPI subsequences, and AH1326 ~ 1340 is a β2GPI partial sequence shown in SEQ ID NO: 3499, respectively to the target sequence.
These double-stranded nucleic acids were introduced into HepG2 cells in the same manner as in Test Example 1, and the relative expression level of β2GPI mRNA was calculated after 24 hours. The results are shown in Tables 4-1 to 4-5.

Example 1 Preparation of a Composition Containing Lipid Particles Containing Compound C-1 as a Cationic Lipid and AH1188, AH1191, AH1213 or AH1218 as Double-Stranded Nucleic Acid 1,2-Dimyristoyl-sn-glycero-3 -Phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000 sodium salt] (PEG-DMPE Na), distearoylphosphatidylcholine (DSPC) and cholesterol were purchased from NOF Corporation.
Each double-stranded nucleic acid was dissolved in distilled water at a concentration of 24 mg / mL to obtain a siRNA solution.
Compound C-1 / PEG-DMPE Na = 57.3 / 5.52 mmol / L Weigh each sample, suspend it in an aqueous solution containing hydrochloric acid and ethanol, and stir and heat repeatedly with a vortex mixer. And a uniform suspension was obtained. The suspension was passed through a 0.05 μm polycarbonate membrane filter at room temperature to obtain a dispersion of compound C-1 / PEG-DMPE Na particles (liposomes). Measure the average particle size of liposomes obtained with a particle size measurement device (Zetasizer Nano ZS, manufactured by Malvern, the same shall apply hereinafter) and confirm that it is within the range of 30 nm to 100 nm. confirmed. The resulting liposome dispersion and each siRNA solution were mixed at a ratio of liposome dispersion: siRNA solution = 3: 1, and further mixed with 3 times the amount of distilled water to obtain compound C-1 / A dispersion of PEG-DMPE Na / siRNA complex was prepared.
On the other hand, each sample was weighed and dissolved in 90 vol% ethanol so that the compound C-1 / PEG-DMPE Na / DSPC / cholesterol = 8.947 / 1.078 / 5.707 / 13.698 mmol / L, and the solution of lipid membrane components Was prepared.
After heating the resulting lipid membrane component solution, the resulting mixture was mixed with the resulting compound C-1 / PEG-DMPE Na / siRNA complex dispersion at a ratio of 1: 1, and then 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 in a clean bench using a 0.2 μm filter (manufactured by Toyo Roshi Kaisha, Ltd.). The obtained composition was diluted with physiological saline after measuring the siRNA concentration, whereby preparations 1 to 4 (compound C-1 was contained as a cationic lipid and AH1188, AH1191, AH1213 or AH1218 A composition containing as a single-stranded nucleic acid) was obtained.
The average particle size of lipid particles in the preparation was measured with a particle size measuring device. The results are shown in Table 5.

Example 2 Preparation of Composition Containing Lipid Particles Containing Compound C-4 as Cationic Lipid and AH1187, AH1191, AH1213, or AH1225 as Double-Stranded Nucleic Acid Was prepared.
Each double-stranded nucleic acid was dissolved in distilled water at a concentration of 12 mg / mL to obtain a siRNA solution.
Compound C-4 / PEG-DMPE Each sample was weighed so that Na = 57.3 / 5.52 mmol / L, suspended in an aqueous solution containing hydrochloric acid and ethanol, and stirred and heated repeatedly with a vortex mixer. And a uniform suspension was obtained. This suspension was passed through a 0.05 μm polycarbonate membrane filter at room temperature to obtain a dispersion of compound C-4 / PEG-DMPE Na particles (liposomes). The average particle size of the liposomes obtained with a particle size measuring device was measured and confirmed to be within the range of 30 nm to 100 nm. The resulting liposome dispersion and each siRNA solution were mixed at a ratio of liposome dispersion: siRNA solution = 3: 1, and further mixed with 11 times the amount of distilled water. A dispersion of PEG-DMPE Na / siRNA complex was prepared.
On the other hand, each sample was weighed and dissolved in 100 vol% ethanol so that the compound C-4 / PEG-DMPE Na / DSPC / cholesterol = 4.470 / 0.074 / 2.990 / 7.182 mmol / L. Was prepared.
The resulting lipid membrane component solution and the resulting compound C-4 / PEG-DMPE Na / siRNA complex dispersion are mixed at a ratio of 2: 3, and then several times the amount of distilled water is mixed. Thus, a crude preparation was obtained.
The obtained crude preparation was concentrated using Amicon Ultra, diluted with physiological saline, and filtered in a clean bench using a 0.2 μm filter. The obtained composition was diluted with physiological saline after measuring the siRNA concentration, whereby preparations 5 to 8 (compound C-4 was contained as a cationic lipid and AH1187, AH1191, AH1213 or AH1225 A composition containing as a single-stranded nucleic acid) was obtained. The average particle size of lipid particles in the preparation was measured with a particle size measuring device. The results are shown in Table 6.

Example 3 Preparation of Composition Containing Lipid Particles Containing Compound C-1 or Compound C-4 as a Cationic Lipid and AH1191 as a Double-Stranded Nucleic Acid A formulation is prepared as follows in the same manner as in Example 1. Prepared.
Each double-stranded nucleic acid was dissolved in distilled water at a concentration of 12 mg / mL to obtain a siRNA solution.
Weigh each sample so that it becomes cationic lipid / PEG-DMPE Na = 57.3 / 5.52 mmol / L, suspend it in an aqueous solution containing hydrochloric acid and ethanol, and stir and heat repeatedly with a vortex mixer. A homogeneous suspension was obtained. This suspension was passed through a 0.05 μm polycarbonate membrane filter at room temperature to obtain a dispersion of cationic lipid / PEG-DMPE Na particles (liposomes). The average particle size of the liposomes obtained with a particle size measuring device was measured and confirmed to be within the range of 30 nm to 100 nm. The resulting liposome dispersion and each siRNA solution are mixed at a ratio of liposome dispersion: siRNA solution = 3: 1, and 11 times the amount of distilled water is added and mixed to mix the cationic lipid / PEG. -A dispersion of DMPE Na / siRNA complex was prepared.
On the other hand, each sample was weighed and dissolved in 100 vol% ethanol so that the cationic lipid / PEG-DMPE Na / DSPC / cholesterol = 4.470 / 0.074 / 2.990 / 7.182 mmol / L. Prepared.
The resulting lipid membrane component solution and the resulting cationic lipid / PEG-DMPE Na / siRNA complex dispersion are mixed at a ratio of 2: 3, and this is added to several times the amount of distilled water. Thus, a crude preparation was obtained.
The obtained crude preparation was concentrated using Amicon Ultra, diluted with physiological saline, and filtered in a clean bench using a 0.2 μm filter. After the siRNA concentration was measured, the obtained composition was diluted with physiological saline to prepare preparations 9 and 10 (compound C-1 or compound C-4 as a cationic lipid and two AH1191s). A composition containing as a strand nucleic acid) was obtained.
The average particle size of lipid particles in the preparation was measured with a particle size measuring device. The results are shown in Table 7.

Test example 3
In vivo activity of the preparation in mice (inhibition of blood β2GPI protein expression)
Each formulation obtained in Example 1 was subjected to an in vivo evaluation test by the following method. Each preparation was diluted with physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) according to the test.
The antibody against mouse β2GPI used for measuring blood protein concentration was prepared by immunizing a rabbit with mouse β2GPI. After the rabbit (Japanese white breed) was acclimated and bred, mouse β2GPI recombinant protein mixed with Freund's complete adjuvant was subcutaneously administered to the back. Fourteen days after the first administration, mouse β2GPI recombinant protein mixed with Freund's incomplete adjuvant was administered subcutaneously in the back and in the thigh muscle. Blood was collected 14 days after the second administration, and antibodies were collected from the obtained serum using Mab Select Sure (GE Healthcare, catalog number: 17-0618-02) according to the method described in the product instructions. Purified. The obtained antibody was labeled with HRP using a peroxidase labeling kit-NH2 (manufactured by Dojindo Laboratories, catalog number: LK11) according to the method described in the product instructions.
Mice (Balb / c, obtained from CLEA Japan, Inc.) were acclimated and bred, and then each formulation was intravenously administered to mice at an siRNA concentration of 0.1 mg / kg. Two days after administration, blood was collected in microtina (Becton Dickinson, 365965) coated with heparin lithium, and 8000 rpm, 8 minutes, 4 minutes using a small cooling centrifuge (05PR-22: Hitachi). Plasma was collected by centrifugation at 0 ° C. The β2GPI protein concentration in the obtained plasma was measured by the following method. Dispense anti-mouse / rat apolipoprotein H antibody (manufactured by R & D Systems, catalog: AF6575) at 4 mg / mL into 96-well immunoplates at 96 ° C for 16 hours at 4 ° C did. After removing the antibody solution, 200 ml each of Block Ace (DS Pharma, catalog number: UK-B40) dissolved in sterilized water to 1% (w / v) was dispensed and blocked at room temperature for 1 hour. After removing the Block Ace solution, mouse plasma and mouse β2GPI recombinant protein solution (standard solution) diluted with Dulbecco's phosphate buffered saline (without calcium and magnesium, manufactured by Nacalai Tesque, catalog number: 14249-95) ) Was dispensed in 50 mL portions and allowed to react at room temperature for 1 hour. After washing 3 times with Tween-PBS (Wako, Catalog No. 288-09368), Dulbecco containing Block Ace with a ratio of Block Ace powder mass / DPBS volume of 0.1% (w / v) 50 mL of the HRP-labeled rabbit antibody diluted with phosphate buffered saline was dispensed and reacted at room temperature for 1 hour. After washing 3 times with Tween-PBS, 50 mL of TMB substrate chromogen (TMB + Substrate-Chromogen) (manufactured by Dako, catalog number: S1599) was added, reacted for 15 minutes, and then 0.5 mol / L sulfuric acid ( The reaction was terminated by adding 50 mL of Wako, catalog number: 192-04755). The absorbance of the reaction solution was measured with ARVO-X3 (Perkin Elmer, 450 nm) or EnVision-2102 (Perkin Elmer, 450 nm). A calibration curve was created from the obtained absorbance, and the β2GPI protein concentration in plasma was calculated.
The results of the calculated β2GPI protein concentration in plasma are shown in Table 8 as the inhibition rate with respect to the physiological saline administration group.

Test example 4
In vitro activity of the preparation against HepG2 cells (suppression of β2GPI mRNA expression in the liver)
Each preparation obtained in Example 2 was introduced into a human liver cancer-derived cell line HepG2 cell (HB-8065) by the following method.
After dispensing each preparation diluted with Optimem (Opti-MEM, GIBCO, 31985) so that the final concentration of nucleic acid is 1-10000 pmol / L, it is dispensed 20 μL each into a 96-well culture plate HepG2 cells suspended in Opti-MEM containing 10% fetal bovine serum (FBS, SAFC Biosciences, 12203C) were seeded at a cell number of 12500/80 μL / well, and incubated at 37 ° C. Each formulation was introduced into HepG2 cells by culturing under 5% CO ₂ conditions. In addition, as a negative control group, cells not treated were seeded.
Cells into which each preparation has been introduced are cultured for 24 hours in a 5% CO ₂ incubator at 37 ° C, and washed with ice-cold phosphate buffered saline (PBS, manufactured by Gibco (GIBCO), catalog number 14190). , Super Prep (Registered Trademark) Cellulosis and RT Tea Kit Forkyu PCR (Toyobo Co., Ltd., Catalog No.SCQ-101) was used to collect total RNA according to the method described in the instructions attached to the product. Then, cDNA was prepared by reverse transcription reaction using the obtained total RNA as a template.
Using the obtained cDNA as a template, Tacman (registered trademark) Gene Expression Assays probe (Applied Biosystems) as a probe, ABI7900HT Fast (ABI7900HT Fast) (ABI (ABI)) By using a PCR reaction according to the method described in the attached instruction manual, β2GPI gene and constitutive expression gene glyceraldehyde 3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate dehydrogenase, hereinafter The mRNA amplification amount was measured by PCR reaction of the gene (denoted gapdh), and the semi-quantitative value of β2GPI mRNA was calculated using the gapdh mRNA amplification amount as an internal control. The expression rate of β2GPI mRNA was determined from the semiquantitative value of β2GPI mRNA, with the semiquantitative value of β2GPI mRNA in the negative control measured in the same manner as 1. The results of the expression rate of the obtained β2GPI mRNA are shown in Table 9 as IC50 values.

Test Example 5
In vivo activity of the preparation in mice (inhibition of β2GPI mRNA expression in the liver)
Each formulation obtained in Example 2 was subjected to an in vivo evaluation test by the following method. Each preparation was diluted with physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) according to the test. Mice (Balb / c, obtained from Clea Japan) were acclimated and bred, and then each formulation was intravenously administered to mice at an siRNA concentration of 0.03 mg / kg. Two days after administration, the liver was collected and stored frozen in liquid nitrogen. Liver frozen samples were prepared using Trizol® NA Isolation Regents (Life Technologies, catalog number 15596026) and Magna Pure Cellular NA Volume Volume Kit (Roche), catalog number 05467535001) was used to collect total RNA according to the method described in the instructions attached to the product. Furthermore, reverse transcription using the total RNA obtained as a template according to the method described in the instructions attached to the product using the transcripter first strand CDNA synthesis kit (Roche, catalog number 04897030001). CDNA was prepared by photoreaction. Using the obtained cDNA as a template, Tackman (registered trademark) Gene Expression Assays probe (Applied Biosystems) as a probe, AB I 7900 H Fast ABI 7900HT Fast (ABI) attached, instructions attached PCR reaction according to the method described in the document, the β2GPI gene and the constitutively expressed gene glyceraldehyde 3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate dehydrogenase) The amount of amplified mRNA was measured for each reaction, and the semi-quantitative value of β2GPI mRNA was calculated using the amount of amplified mRNA of gapdh as an internal control. Similarly, the β2GPI mRNA expression rate was determined from the β2GPI mRNA quasi-quantitative value of 1 in the same physiological saline administration group. Table 10 shows the results of the expression suppression rate of the obtained β2GPI mRNA.

Test Example 6
In vivo activity of the preparation in mice (inhibition of blood β2GPI protein expression)
In the same manner as in Test Example 3, preparations 5 to 8 were administered as siRNA concentrations of 0.03 mg / kg to balb / c mice, and blood was collected 7 days after administration to measure the amount of β2GPI protein. Table 11 shows the measured amount of β2GPI protein in the blood as the inhibition rate relative to the physiological saline administration group.

Test Example 7
Evaluation of immunostimulation in mice of preparations Microtina (manufactured by BD Co., Ltd.) was prepared by administering preparations 5 to 8 at a siRNA concentration of 0.3 mg / kg to balb / c mice and applying blood coagulation promoters 24 hours after administration. 365967) and centrifuged at 8000 rpm for 8 minutes at 4 ° C. using a small refrigerated centrifuge (05PR-22: manufactured by Hitachi) to collect serum. Granulocyte-colony stimulating factor (G-CSF), Keratinocyte-derived Cytokine (KC or GRO), Interferon-γ (IFN-g), Tumor Necrosis Factor (TNF), Interleukin-1b (IL-1b) ), Interleukin-10 (IL-10) and Interleukin-6 (IL-6) values were calculated using the BDCB Flex Set (BD Corporation) and BD FACSVers flow cytometer ( BD), and was quantified according to the method described in the instructions attached to the product.
The measured blood G-CSF and KC concentrations are shown in Table 12. For cytokines other than those shown in Table 12 (IFN-g, TNF, IL-1b, IL-10, and IL-6), the lower limit of quantitation (10 pg / mL) or less.

Test Example 8
In vivo activity of the preparation in mice (inhibition of blood β2GPI mRNA expression)
In the same manner as in Test Example 5, preparations 9 and 10 obtained in Example 3 were administered to balb / c mice at 0.01 mg / kg as siRNA concentrations, and the amount of β2GPI mRNA in the liver 2 days after administration was measured. Table 13 shows the measured mRNA amount as the inhibition rate relative to the physiological saline administration group.

Test Example 9
Results of Lupus Anticoagulant Test on Blood of Mice Administered with Formulation Formulation 6 obtained in Example 2 was administered to mice to evaluate whether or not β2GPI-dependent LA can be released.
The formulation 6 obtained in Example 2 was intravenously administered at 0.1 mg / kg in siRNA concentration to acclimated mice (Balb / c, obtained from Clea Japan, Inc.). Saline was administered to the control group of mice. The test was conducted using 2 saline administration groups and 3 preparation 6 administration groups. The saline administration group was assigned individual numbers 1-2, and the preparation 6 administration group was assigned individual numbers 3-5.
Two days after administration, blood was collected so that the volume ratio was 3.2% citrate solution: blood = 1: 9, and the collected blood was collected at 1500 × g (g: centrifugal force) using a small cooling centrifuge (MX305: manufactured by Tommy). ), And centrifuged at room temperature for 15 minutes. The supernatant plasma was centrifuged again under the same conditions, and the plasma on the buffy coat was collected, and this was subjected to LA activity evaluation as double-centrifuged plasma.
90 mL of double-centrifuged plasma obtained from each individual was added to a 1.5 mL Eppendorf tube, where the rabbit-derived anti-β2GPI antibody (30 mg / mL) used in Test Example 2 or phosphate buffered saline (DPBS) Was added for 1 hour at room temperature. This was warmed for 1 minute using a water bath (SM-05N, manufactured by Taitec Co., Ltd.) set at 37 ° C, and 100 uL LA test “Gladipore” reagent 1 (MBL (MBL), catalog) No. 4150) was mixed as a coagulation activation reagent for dRVVT measurement. As an evaluation of LA activity, blood coagulation was determined visually. The clotting time was measured using a stopwatch. For each individual, the clotting time when the anti-β2GPI antibody was added was obtained as β2GPI-dependent LA, and the clotting time when DPBS was added as the clotting time when LA was negative. The test results obtained are shown in FIG. As is clear from FIG. 1, in the mice administered with saline, coagulation time was prolonged in the presence of anti-β2GPI antibody, that is, β2GPI-dependent LA activity was observed, whereas in mice administered with formulation 6, anti-β2GPI antibody was present. There was no extension of the clotting time even if it was below. This indicates that β2GPI-dependent LA can be released by reducing the expression of β2GPI in blood. Therefore, it has been clarified that β2GPI-related diseases can be treated by administering a composition containing lipid particles of the present invention to a mammal and reducing the expression of the β2GPI gene in vivo.

The composition containing the lipid particles of the present invention can be administered to a mammal to suppress β2GPI gene expression and treat β2GPI-related diseases in vivo.

SEQ ID NOs: 1-1180 show the sense strand RNA base sequences of siRNAs for the β2GPI gene.
SEQ ID NOs: 1181 to 2360 show the antisense strand RNA base sequences of siRNA for the β2GPI gene.
SEQ ID NOs: 2361 to 3540 show the target β2GPI gene in terms of DNA base sequence.
SEQ ID NO: 3541 shows the cDNA base sequence of the β2GPI gene.
SEQ ID NOs: 3542 to 3701 show the sense strand RNA base sequences of double-stranded nucleic acids (AH1181 to AH1340).
SEQ ID NOs: 3702 to 3861 show the antisense strand RNA base sequences of double-stranded nucleic acids (AH1181 to AH1340).

Claims

A double-stranded nucleic acid comprising a sense strand and an antisense strand and comprising a double-stranded region of at least 11 base pairs, wherein the oligonucleotide has a length of 17 to 30 nucleotides in the antisense strand A double-stranded nucleic acid as a drug that is complementary in sequence to a target β2GPI mRNA sequence selected from the group described in Table 2-1 to Table 2-16;
Formula (A)

(Wherein R 1 is 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 linear, branched or cyclic alkyl having 1 to 3 carbon atoms, or together, they form alkylene having 2 to 8 carbon atoms, or R 3 together with R 5 forms an alkylene having 2 to 8 carbon atoms,
R 5 is a hydrogen atom, linear, branched or cyclic alkyl having 1 to 6 carbon atoms, linear or branched alkenyl having 3 to 6 carbon atoms, amino, monoalkylamino, ammonio, mono Alkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl or the same or different 1 to 3 amino, monoalkylamino, ammonio, monoalkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl or dialkylcarbamoyl A linear, branched or cyclic alkyl group having 1 to 6 carbon atoms or a linear or branched alkenyl group having 3 to 6 carbon atoms, or carbon together with R 3 Forming alkylene of 2 to 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 together with R 3 forms an alkylene of 2 to 8 carbon atoms, X 2 is a single bond, or is methylene or ethylene),
Formula (B)

(Wherein R 6 is 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 a formula (C)

(Wherein R 8 is 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 lipid particle containing a cationic lipid represented by a hydrogen atom or a linear, branched or cyclic alkyl having 1 to 3 carbon atoms.
R 1 , R 2 , R 6 , R 7 , R 8 and R 9 are 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) -octadec-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 The lipid particle according to claim 1, wherein
R 1 , R 2 , R 6 , R 7 , R 8 and R 9 are (Z) -octadeca-9-enyl, (9Z, 12Z) -octadeca-9,12-dienyl or (11Z, 14Z)- 2. The lipid particle according to claim 1, which is icosa-11,14-dienyl.
The lipid particle according to any one of claims 1 to 3, wherein X 1 is alkylene having 1 to 3 carbon atoms, and X 2 is a single bond or methylene.
The lipid particle according to any one of claims 1 to 4, wherein X 3 is methylene or ethylene.
The lipid particle according to any one of claims 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.
The lipid particle according to any one of claims 1 to 5, wherein R 3 and R 5 together form n-propylene or n-butylene and R 4 is methyl or ethyl.
The lipid particle according to any one of claims 1 to 6, wherein R 5 and R 10 are each a hydrogen atom or methyl.
Antisense complementary to a target β2GPI mRNA sequence selected from the group described in Table 2-1 to Table 2-16, wherein the duplex region is a duplex region comprising 11 to 27 base pairs The lipid particle according to any one of claims 1 to 8, wherein the second nucleotide from the 5 'end of the strand is complementary to the second deoxyribonucleotide from the 3' end of the target β2GPI mRNA sequence.
The lipid particle according to any one of claims 1 to 9, wherein the sense strand has a length of 21 nucleotide chains and the antisense strand has a length of 21 nucleotide chains.
The double-stranded nucleic acid, wherein the sense strand is 21 nucleotides in length and the antisense strand is 21 nucleotides in length, comprises a 19 base pair duplex region. The lipid particle according to any one of the above.
The lipid particle according to any one of claims 1 to 9, wherein the 3 'end of the sense strand and the 5' end of the antisense strand form a blunt end.
The lipid particle according to any one of claims 1 to 12, wherein the double-stranded nucleic acid contains 2'-O-methyl modified nucleotide.
The lipid particle according to claim 13, wherein 40 to 65% of the nucleotides in the double-stranded region are 2'-O-methyl modified nucleotides.
The lipid particle according to any one of claims 1 to 14, wherein the antisense strand comprises a sequence selected from the antisense strand group described in Tables 4-1 to 4-5.
The lipid particle according to any one of claims 1 to 14, wherein the sense strand includes a sequence selected from the sense strand group described in Tables 4-1 to 4-5.
15. The method according to claim 1, comprising a pair of sense / antisense strands selected from the group consisting of the sense strand / antisense strand described in Table 4-1 to Table 4-5. The lipid particles described.
A double-stranded nucleic acid comprising a sense strand and an antisense strand and comprising a double-stranded region of at least 11 base pairs, wherein the oligonucleotide has a length of 17 to 30 nucleotides in the antisense strand The strand is complementary to a target β2GPI mRNA sequence selected from the group listed in Table 2-1 to Table 2-16, and the antisense strand is described in Table 4-1 to Table 4-5 A sequence selected from the group of antisense strands, or the sense strand includes a sequence selected from the group of antisense strands listed in Tables 4-1 to 4-5, or Table 4-1 A double-stranded nucleic acid as a drug comprising a pair of sense / antisense strand sequences selected from the group consisting of the sense / antisense strands listed in Table 4-5;
Formula (A)

(Wherein R 1 is 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 linear, branched or cyclic alkyl having 1 to 3 carbon atoms, or together, they form alkylene having 2 to 8 carbon atoms, or R 3 together with R 5 forms an alkylene having 2 to 8 carbon atoms,
R 5 is a hydrogen atom, linear, branched or cyclic alkyl having 1 to 6 carbon atoms, linear or branched alkenyl having 3 to 6 carbon atoms, amino, monoalkylamino, ammonio, mono Alkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl or the same or different 1 to 3 amino, monoalkylamino, ammonio, monoalkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl or dialkylcarbamoyl A linear, branched or cyclic alkyl group having 1 to 6 carbon atoms or a linear or branched alkenyl group having 3 to 6 carbon atoms, or carbon together with R 3 Forming alkylene of 2 to 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 together with R 3 forms an alkylene of 2 to 8 carbon atoms, X 2 is a single bond, or is methylene or ethylene),
Formula (B)

(Wherein R 6 is 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 a formula (C)

(Wherein R 8 is 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 lipid particle containing a cationic lipid represented by a hydrogen atom or a linear, branched or cyclic alkyl having 1 to 3 carbon atoms.
The cationic lipid forms a complex with a double-stranded nucleic acid, or forms a complex with a combination of a neutral lipid and / or a polymer and a double-stranded nucleic acid. 20. The lipid particle according to any one of 18.
The cationic lipid forms a complex with a double-stranded nucleic acid, or forms a complex with a combination of a neutral lipid and / or a polymer and a double-stranded nucleic acid, and the lipid particles are The lipid particle according to any one of claims 1 to 19, which is a lipid particle composed of a complex and a lipid membrane encapsulating the complex.
A lipid particle-containing composition comprising the lipid particle according to any one of claims 1 to 20.
A method for suppressing β2GPI gene expression, comprising introducing a double-stranded nucleic acid into a cell using the composition according to claim 21.
23. The method of claim 22, wherein the cell is a cell in a mammalian liver.
24. The method according to claim 22 or 23, wherein the method of introduction into cells is a method of introduction into cells by intravenous administration.
A method for treating a β2GPI-related disease, comprising administering the composition according to claim 21 to a mammal.
26. The method according to claim 25, wherein the β2GPI-related disease is an autoimmune disease or thrombosis.
27. The method according to claim 25 or 26, wherein the method of administration is intravenous administration.
A medicament for use in the treatment of β2GPI-related diseases, comprising the composition according to claim 21.
The medicament according to claim 28, wherein the β2GPI-related disease is an autoimmune disease or thrombosis.
30. The medicament according to claim 28 or 29, wherein the medicament is for intravenous administration.
A therapeutic agent for autoimmune disease or thrombosis comprising the composition according to claim 21.
The therapeutic agent for autoimmune disease or thrombosis according to claim 31, which is for intravenous administration.