Polyalkylene glycol lipid having azide group, and substrate for lipid nanoparticle

Abstract

Provided is a polyalkylene glycol lipid represented by formula (1). (R1 and R2 are each independently a hydrogen atom, an aliphatic hydrocarbon group having 15-22 carbon atoms or an acyl group having 15-22 carbon atoms. k, l and m are numbers that satisfy the relationships 6≥k≥0, 6≥l≥0, 6≥m≥0 and k+l+m≥2. A is an oxygen atom, an ester bond, a carbonate bond or a carbamate bond. B is a divalent hydrocarbon group having 1-4 carbon atoms, n is 21-226, C is a divalent hydrocarbon group having 1-4 carbon atoms, and D is an atomic group represented by formula (2a) or formula (2b) shown in the description.

Description

Azide-containing polyalkylene glycol lipids and lipid nanoparticle substrates

[0001] This invention relates to polyalkylene glycol lipids having azide groups and substrates for lipid nanoparticles.

[0002] In the field of drug delivery systems, research on lipid nanoparticles (LNPs) containing nucleic acids is widespread. These LNPs are microparticles composed of phospholipids, cholesterol, polyethylene glycol (PEG) lipids, and ionized lipids. Because their surface is covered with PEG, they help protect nucleic acids from enzymatic degradation. As a result, LNPs exhibit high blood retention and accumulate by interacting with various cells in the liver (hepatocytes, endothelial cells, B cells, and Kupffer cells), making them suitable for nucleic acid delivery targeting hepatocytes. However, because of these characteristics, although there are examples of LNP delivery to the liver, in order to deliver nucleic acids to a wider variety of organs, it is necessary to introduce ligands that recognize cell surface receptors, such as antibodies, aptamers, peptides, and glycans, to the PEG terminus of the LNP surface to improve targeting ability to target organs.

[0003] To immobilize PEG lipids on the LNP surface, long-chain aliphatic hydrocarbon groups or acyl groups are introduced into the PEG lipids. However, if the number of carbon atoms in the aliphatic hydrocarbon group or acyl group is small, the PEG lipids are not sufficiently immobilized on the LNP surface, and as a result, the PEG lipids are rapidly released into the bloodstream, leading to low blood retention of LNPs and accumulation in the liver, as has been reported (Non-Patent Literature 1). For this reason, PEG lipids are undesirable for giving LNPs organ-specific targeting other than the liver.

[0004] Since the PEG on the surface of LNPs is derived from PEG lipids, which are components of LNPs, if a reactive group that can bind a ligand can be introduced to the PEG terminus of the PEG lipid, the ligand can be immobilized on the LNP surface, thereby conferring targeting ability to target organs. For this purpose, it is important to select an appropriate reactive group that reacts with the ligand. Examples of reactive sites in antibodies include the thiol group of the cysteine ​​side chain and the amino group of the lysine side chain, and since the thiol group is known to react readily with the maleimide group, maleimide PEG lipids have been reported (Patent Document 1). However, since the aforementioned amino acid residues are widely present in antibodies, it is difficult to control the amount introduced.

[0005] A highly specific coupling method is known as the click reaction between an azide group and an alkyne group, and coupling between azide PEG phospholipids and proteins has been reported (Non-Patent Literature 2).

[0006] International Publication No. 2022 / 065263

[0007] Mol Ther Nucleic Acids. 2013; 2(12): e139.Science Advances. 2024 10 (22)

[0008] However, because PEG phospholipids have an electric charge in their phosphate groups, they are not particularly suitable as a base for LNPs that encapsulate nucleic acids. In other words, it has become clear that PEG lipids are needed as a base for LNPs that are organ-specific to organs other than the liver.

[0009] The object of the present invention is to provide a PEG lipid that possesses both sufficient immobilization ability on the LNP surface and highly specific functional groups.

[0010] The inventors of the present invention conducted extensive research to solve the above problems and, as a result, discovered a PEG lipid that does not have a phosphate group, but has a lipid moiety into which a hydrocarbon group or acyl group having 15 to 22 carbon atoms is introduced, and also has an azide group at the end of the PEG chain, thereby completing the present invention.

[0011] In other words, the present invention is as follows: [1] A polyalkylene glycol lipid characterized by being represented by the following formula (1). (In Formula (1), R １ and R ２ are each independently a hydrogen atom, an aliphatic hydrocarbon group having 15 to 22 carbon atoms, or an acyl group having 15 to 22 carbon atoms; k, l and m are numbers satisfying 6≧k≧0, 6≧l≧0, 6≧m≧0 and k+l+m≧2; A is an oxygen atom, an ester bond, a carbonate bond, or a carbamate bond; B is a divalent hydrocarbon group having 1 to 4 carbon atoms; n is 21 to 226; C is a divalent hydrocarbon group having 1 to 4 carbon atoms; and D is an atomic group represented by the following formula (2a) or formula (2b). (In formula (2a), the dashed line represents a bonding site to the hydrocarbon group represented by C; in formula (2b), the dashed line represents a bonding site to the hydrocarbon group represented by C; E is an ester bond, a carbonate bond, a carbamate bond, a urea bond, or an amide bond; and F is a divalent hydrocarbon group having 1 to 4 carbon atoms.)

[0012] [2] The polyalkylene glycol lipid according to [1], wherein in formula (1), D is an atomic group represented by said formula (2a).

[0013] [3] The polyalkylene glycol lipid according to [1], wherein in formula (1), D is an atomic group represented by said formula (2b), and E is an amide bond or a carbamate bond in formula (2b).

[0014] [4] In formula (1), R １ and R ２ are each independently a linear and saturated acyl group having 15 to 20 carbon atoms, the polyalkylene glycol lipid according to any one of [1] to [3].

[0015] [5] The polyalkylene glycol lipid according to any one of [1] to [4], wherein in formula (1), k is 1, m is 1, and l is 0. [6] The polyalkylene glycol lipid according to any one of [1] to [5], wherein in formula (1), A is an oxygen atom.

[0016] [7] The polyalkylene glycol lipid according to any one of [1] to [6], wherein in formula (1), B is a divalent hydrocarbon group having 2 carbon atoms.

[0017] [8] The polyalkylene glycol lipid according to any one of [1] to [7], wherein in formula (1), C is a divalent hydrocarbon group having 2 carbon atoms.

[0018] [9] The polyalkylene glycol lipid according to any one of [1] to [8], wherein in formula (2b), F is a divalent hydrocarbon group having 4 carbon atoms.

[0019]

[10] A base material for lipid nanoparticles, comprising the polyalkylene glycol lipid according to any one of [1] to [9].

[0020] The azide group-containing polyalkylene glycol lipid of the present invention is useful as a base material for LNP having organ tropism other than liver.

[0021] Embodiments of the present invention are described in detail below. The azide group-containing polyalkylene glycol lipid of the present invention is represented by the following general formula (1). (In formula (1), R １ and R ２ are each independently a hydrogen atom, an aliphatic hydrocarbon group having 15 to 22 carbon atoms, or an acyl group having 15 to 22 carbon atoms; k, l and m are numbers satisfying 6≧k≧0, 6≧l≧0, 6≧m≧0 and k+l+m≧2; A is an oxygen atom, an ester bond, a carbonate bond or a carbamate bond; B is a divalent hydrocarbon group having 1 to 4 carbon atoms; n is 21 to 226; C is a divalent hydrocarbon group having 1 to 4 carbon atoms; D is an atomic group represented by the following formula (2a) or formula (2b). (In formula (2b), E is an ester bond, a carbonate bond, a carbamate bond, a urea bond or an amide bond; F is a divalent hydrocarbon group having 1 to 4 carbon atoms.)

[0022] In formula (1), R １ , R ２Each of these is independently a hydrogen atom, an aliphatic hydrocarbon group having 15 to 22 carbon atoms, or an acyl group having 15 to 22 carbon atoms. The aliphatic hydrocarbon group may be linear or branched, and may be saturated or unsaturated. The acyl group may be linear or branched, and may be saturated or unsaturated. Furthermore, the number of carbon atoms in the aliphatic hydrocarbon group is preferably 16 or more, preferably 20 or less, and particularly preferably 16, 18, or 20. The number of carbon atoms in the acyl group is preferably 16 or more, preferably 20 or less, and particularly preferably 16, 18, or 20. Among these, linear and saturated acyl groups having 16, 18, or 20 carbon atoms are particularly preferred.

[0023] Specific examples of such aliphatic hydrocarbon groups and acyl groups include pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, eicosyl group, pentadecenyl group, hexadecenyl group, heptadecenyl group, octadecenyl group, nonadecenyl group, eicocenyl group, pentadecadienyl group, hexadecadienyl group, heptadecienyl group, octadecienyl group, nonadecienyl group, eicosadienyl group, octadecadienyl group, eicosatrienyl group, eicosatetraenyl group, isostearyl group, pentadecanoyl group, hexadecanoyl group, heptadecanoyl group, octadecanoyl group, nonadecanoyl The following groups are preferred: arachidinoyl group, tetradecenylcarbonyl group, pentadecenylcarbonyl group, hexadecenylcarbonyl group, heptadecenylcarbonyl group, nonadecenylcarbonyl group, tetradecadienylcarbonyl group, pentadedecadienylcarbonyl group, hexadedecadienylcarbonyl group, heptadedecadienylcarbonyl group, octadedecadienylcarbonyl group, nonadedecadienylcarbonyl group, heptadedecatrienylonylcarbonyl group, nonadedecatrienylonylcarbonyl group, nonadedecatetraenylcarbonyl group, and isostearylcarbonyl group. The following groups are particularly preferred: hexadecanoyl group, octadecanoyl group, and arachidinoyl group.

[0024] k, l, and m are numbers that satisfy 6≧k≧0, 6≧l≧0, 6≧m≧0, and k+l+m≧2. It is particularly preferable that k is between 1 and 3 (inclusive). It is even more preferable that m is between 1 and 3 (inclusive). It is even more preferable that l is between 0 and 3 (inclusive). Among these, the combination k=1, l=0, and m=1 is particularly preferred.

[0025] In formula (1), A is a stable bonding mode that does not easily cleave during LNP preparation or in the blood. Specifically, it is an oxygen atom, an ester bond, a carbonate bond, or a carbamate bond. Among these, an oxygen atom, a carbonate bond, or a carbamate bond is preferred, and an oxygen atom is particularly preferred. Note that an ester bond is "-O-C(O)-", a carbonate bond is "-O-C(O)-O-", and a carbamate bond is "-O-C(O)-N-". Furthermore, the direction of each bond constituting A is not particularly limited.

[0026] In formula (1), n ​​is the number of repeating alkylene glycol units, specifically an integer between 21 and 226. n is preferably 27 or greater, and more preferably 33 or greater. Furthermore, n is preferably 182 or less, and more preferably 135 or less.

[0027] In formula (1), B is a divalent hydrocarbon group having 1 to 4 carbon atoms. The hydrocarbon group may be a saturated or unsaturated hydrocarbon group, and may be a linear or branched hydrocarbon group, and is more preferably an alkylene group. A divalent hydrocarbon group having 2 carbon atoms is particularly preferred.

[0028] In formula (1), C is a divalent hydrocarbon group having 1 to 4 carbon atoms. The hydrocarbon group may be a saturated or unsaturated hydrocarbon group, and may be a straight-chain or branched-chain hydrocarbon group, and is more preferably an alkylene group. Among these, a divalent hydrocarbon group having 2 carbon atoms is particularly preferred.

[0029] In formula (1), D is a group having an azide group, as shown in formula (2a) or (2b) below.

[0030]

[0031] Here, the broken line indicates the binding site to the hydrocarbon group represented by C. E is a stable binding mode that does not easily cleave during LNP preparation or in blood. Specifically, it is an ester bond, a carbonate bond, a carbamate bond, a urea bond, or an amide bond. Among these, an amide bond and a carbamate bond are particularly preferable. The ester bond is "-O-C(O)-", the carbonate bond is "-O-C(O)-O-", the carbamate bond is -O-C(O)-N-, the urea bond is -N-C(O)-N-, and the amide bond is "-C(O)-N-". In addition, the orientation of each bond constituting E is not particularly limited.

[0032] In formula (2b), F is a divalent hydrocarbon group having 1 to 4 carbon atoms. The hydrocarbon group may be a saturated or unsaturated hydrocarbon group, may be a linear or branched hydrocarbon group, and is more preferably an alkylene group. Among these, a divalent hydrocarbon group having 4 carbon atoms is particularly preferable.

[0033] The azide group-containing polyalkylene glycol lipid of the present invention can be basically produced by the following four production methods, and can be produced by any method suitable therefor after appropriate modification depending on the type of substituents and the like. Among these, production method 1, which has the smallest number of steps, is particularly preferable.

[0034] (Production Method 1)

[0035] In the formula, n is 21 to 226, and R is a linear or branched, saturated or unsaturated hydrocarbon group having 14 to 21 carbon atoms.

[0036] Referring to Production Method 1, compound (I) is reacted with compound (II) to obtain compound (III). Compound (III), diphenylphosphoryl azide (DPPA) (IV) and a base (for example, 1,8-diazabicyclo[5.4.0]-7-undecene) are treated to obtain compound (V). Next, compound (V) is reacted with an acid (for example, phosphoric acid (VI)) to obtain compound (VII). Compound (VII) is reacted with RCO ２ H (VIII), DMAP (4-dimethylaminopyridine) and a condensing agent (for example, N,N'-dicyclohexylcarbodiimide) to obtain compound (IX).

[0037] Compounds (I) and (II) used as raw materials may be purchased commercially or manufactured according to methods familiar to those skilled in the art.

[0038] (Method 2)

[0039] In the formula, n is 21 to 226, E is a hydrocarbon group having 1 to 4 carbon atoms, and R is a linear or branched, saturated or unsaturated hydrocarbon group having 14 to 21 carbon atoms.

[0040] Referring to method 2, compound (I) is reacted with compound (II) to obtain compound (III). Compound (III), compound (X), an azo compound (e.g., diisopropyl azodicarboxylic acid), and a phosphine (e.g., triphenylphosphine) are treated. The crude product is treated with hydrazine (XI) to recover compound (XII). Then, compound (XII), compound (XIII), a condensing agent (e.g., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), and DMAP are treated to obtain compound (XIV). Compound (XIV) is reacted with an acid (e.g., phosphoric acid (VI)) to obtain compound (XV). Compound (XV) is subjected to RCO ２ The compound (XVI) is obtained by treatment with H(VIII), DMAP, and a condensing agent (e.g., N,N'-dicyclohexylcarbodiimide).

[0041] Compounds (I) and (II) used as raw materials may be purchased commercially or manufactured according to methods familiar to those skilled in the art.

[0042] (Method 3)

[0043] In the formula, n is 21 to 226, E is a hydrocarbon group having 1 to 4 carbon atoms, and R is a linear or branched, saturated or unsaturated hydrocarbon group having 14 to 21 carbon atoms.

[0044] Referring to method 3, compound (I) is reacted with compound (II) to obtain compound (III). Compound (III), compound (XVII), and a base (e.g., potassium tert-butoxide) are treated. The crude product is reacted with an acid (e.g., phosphoric acid (VI)) to obtain compound (XVIII). Compound (XVIII) is then processed at RCO ２ The compound (XIX) is obtained by treating it with H(VIII), DMAP, and a condensing agent (e.g., N,N'-dicyclohexylcarbodiimide). Then, the compound (XIX) is treated with hydrogen (XX) and a catalyst (e.g., palladium-carbon) to obtain the compound (XXI). The compound (XXI), compound (X), azo compound (e.g., diisopropyl azodicarboxylic acid), and phosphine (e.g., triphenylphosphine) are treated. The crude product is treated with hydrazine (XI) to recover the compound (XXII). The compound (XXII), compound (XIII), a condensing agent (e.g., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), and DMAP to obtain the compound (XVI).

[0045] Compounds (I) and (II) used as raw materials may be purchased commercially or manufactured according to methods familiar to those skilled in the art.

[0046] (Manufacturing method 4)

[0047] In the formula, n is 21 to 226, E is a hydrocarbon group having 1 to 4 carbon atoms, R is a linear or branched, saturated or unsaturated hydrocarbon group having 14 to 21 carbon atoms, and Y is one of the following groups.

[0048]

[0049] Referring to method 4, compound (I) is reacted with compound (II) to obtain compound (III). Compound (III), compound (XVII), and a base (e.g., potassium tert-butoxide) are treated. The crude product is reacted with an acid (e.g., phosphoric acid (VI)) to obtain compound (XVIII). Compound (XVIII) is treated with RCO ２The compound (XIX) is treated with H(VIII), DMAP, and a condensing agent (e.g., N,N'-dicyclohexylcarbodiimide). Then, the compound (XIX) is treated with hydrogen (XX) and a catalyst (e.g., palladium carbon) to obtain compound (XXI). Compound (XXI) is reacted with compound (XXIII) (e.g., N,N'-disuccinimidyl carbonate) to obtain compound (XXIV). Compound (XXIV) is treated with compound (XXV) to obtain compound (XXVI).

[0050] Compounds (I) and (II) used as raw materials may be purchased commercially or manufactured according to methods familiar to those skilled in the art.

[0051] (Example 1) The following compounds were prepared according to the above-described method 1. However, the following compounds belong to formula (IX) of method 1 and also belong to polyoxyalkylene glycol lipids having an azide group of formula (1). However, in formula (1), R １ and R ２ A is a stearoyl group, with k=1, l=0, m=1, k+l+m=2, A is an oxygen atom, B is an ethylene group, n is 76, C is an ethylene group, and D is the azide group of formula (2a).

[0052] (1) Diphenyl phosphoryl azide (DPPA: 3.5 g) and 1,8-diazabicyclo[5.4.0]-7-undecene (1.9 g) were added to a toluene solution of 1-(hydroxypolyoxyethylene)-2,3-isopropylideneglycerol (n=76) (compound (III): 15 g) and stirred at 80°C for 31 hours. After returning to room temperature, toluene (30 g) and tert-butyl methyl ether (90 g) were added. The mixture was cooled to 0°C and crystallization was performed. The precipitated crystals were filtered and dissolved in toluene (189 g). 15% sodium chloride aqueous solution (189 g) was added, the mixture was heated to 55°C and stirred for 10 minutes, and the upper toluene layer was collected. The same procedure was repeated twice, and the collected toluene layers were mixed, silica gel (Fuji Silicia: PSQ60B) (7.5 g) was added, and the mixture was stirred at room temperature for 10 minutes. The silica gel was filtered off and washed with toluene (30 g). The filtrate was concentrated and dissolved in toluene (55 g). Tert-butyl methyl ether (110 g) was added, and the mixture was cooled to 0°C to perform crystallization. The precipitated crystals were filtered off to obtain 7.6 g of 3-[2-azidoethyl poly(oxyethylene)]-1,2-isopropylidene-sn-glycerol (n=76) (compound (V)). The NMR data for 3-[2-azidoethyl poly(oxyethylene)]-1,2-isopropylidene-sn-glycerol is shown below.

[0053] １ H-NMR (CDCl ３ ): δ1.35 (3H, s), 1.41 (3H, s), 3.39 (2H, t, J=5.1Hz), 3.44-3.53 (3H, m) 3.54-3.90 (302H, m), 4.05 (1H, dd, J=6.4Hz, 8.2Hz), 4.24-4.32 (1H, m)

[0054] ​(2) Add water (48 g) to the toluene solution of 3-[2-azidoethylpoly(oxyethylene)]-1,2-isopropylidene-sn-glycerol (n=76) (2.5 g) obtained in item (1) above, and add 85% phosphoric acid (compound (VI): 0.9 g) to make the pH = 1.5. Stir at room temperature for 1 hour. Discard the upper toluene layer and add 400 g / L sodium hydroxide aqueous solution to make the lower layer pH = 6.7. Add sodium chloride (7.5 g) and add 400 g / L sodium hydroxide aqueous solution to make the pH = 7.0. Add chloroform (7.5 g) and stir for 20 minutes, then collect the lower chloroform layer. Repeat the same procedure twice, mix the collected chloroform layers, and concentrate them. Dissolve in ethyl acetate (15 g) and add heptane (2.5 g). Cool to below 10°C and crystallization is performed. The precipitated crystals were filtered to obtain 2.0 g of 3-[2-azidoethyl poly(oxyethylene)]-sn-glycerol (n=76) (compound (VII)). The NMR data for 3-[2-azidoethyl poly(oxyethylene)]-sn-glycerol is shown below.

[0055] １ H-NMR (CDCl ３ ): δ2.66 (1H, t, J=6.2Hz), 3.36-3.44 (3H, m), 3.44-3.50 (2H, m), 3.53-3.90 (303H, m)

[0056] ​(3) The toluene solution of 3-[2-azidoethyl poly(oxyethylene)]-sn-glycerol (n=76) (2.0 g) and stearic acid (compound (VIII): 0.6 g) obtained in the preceding paragraph (2) was heated to 40°C, and N,N'-dicyclohexylcarbodiimide (0.5 g) and 4-dimethylaminopyridine (28 mg) were added. The mixture was stirred at 60°C for 1 hour. The precipitate was filtered off and washed with toluene (2.0 g). The filtrate was concentrated, and acetonitrile (3.0 g) and heptane (6.0 g) were added to make a solution. The upper heptane layer was discarded. Heptane (8.0 g) was added to the acetonitrile layer, and the mixture was stirred at 50°C for 30 minutes, and the upper heptane layer was discarded. The same procedure was repeated three times to concentrate the acetonitrile layer. The compound was dissolved in acetonitrile (12 g), and Kyoward 700 (manufactured by Kyowa Chemical Industry Co., Ltd.) (0.4 g) and Kyoward 1000 (manufactured by Kyowa Chemical Industry Co., Ltd.) (0.4 g) were added. The mixture was stirred at room temperature for 30 minutes. After that, Kyoward 700 and Kyoward 1000 were filtered off and washed with acetonitrile (2.0 g). The filtrate was concentrated to obtain 1.7 g of 3-[2-azidoethyl poly(oxyethylene)]-1,2-di(stearoyl)-sn-glycerol (n=76) (compound (IX)). The NMR data for 3-[2-azidoethyl poly(oxyethylene)]-1,2-di(stearoyl)-sn-glycerol is shown below.

[0057] １ H-NMR (CDCl ３ ): δ0.88 (6H, t, J = 6.8Hz), 1.16-1.38 (56H, m), 1.54-1.68 (4H, m), 2.30 (4H, q, J = 7.3Hz), 3.39 (2H, t, J = 5.0Hz), 3.43-3.50 (2H, m), 3.53-3.90 (302H, m), 4.15 (1H, dd, J = 6.5Hz, 11.9Hz), 4.34 (1H, dd, J = 3.4Hz, 11.9Hz), 5.17-5.25 (1H, m)

[0058] (Example 2) The following compounds were prepared according to Method 1.

[0059] ​(1) Using 1-(hydroxypolyoxyethylene)-2,3-isopropylideneglycerol (n=44), 3-[2-azidoethylpoly(oxyethylene)]-1,2-isopropylidene-sn-glycerol (n=44) (compound (V)) was obtained by the method described in (1) of Example 1 or a similar method. The NMR data for 3-[2-azidoethylpoly(oxyethylene)]-1,2-isopropylidene-sn-glycerol is shown below.

[0060] １ H-NMR (CDCl ３ ): δ1.35 (3H, s), 1.41 (3H, s), 3.39 (2H, t, J=5.1Hz), 3.44-3.53 (3H, m) 3.54-3.90 (174H, m), 4.05 (1H, dd, J=6.4Hz, 8.2Hz), 4.24-4.32 (1H, m)

[0061] (2) Using the 3-[2-azidoethyl poly(oxyethylene)]-1,2-isopropylidene-sn-glycerol (n=44) obtained in the preceding paragraph (1), 3-[2-azidoethyl poly(oxyethylene)]-sn-glycerol (n=44) (compound (VII)) was obtained by the method described in (2) of Example 1 or a similar method. The NMR data for 3-[2-azidoethyl poly(oxyethylene)]-sn-glycerol is shown below.

[0062] １ H-NMR (CDCl ３ ): δ2.66 (1H, t, J=6.2Hz), 3.36-3.44 (3H, m), 3.44-3.50 (2H, m), 3.53-3.90 (175H, m)

[0063] (3) Using the 3-[2-azidoethyl poly(oxyethylene)]-sn-glycerol (n=44) obtained in the preceding paragraph (2), 3-[2-azidoethyl poly(oxyethylene)]-1,2-di(stearoyl)-sn-glycerol (n=44) (compound (IX)) was obtained by the method described in (3) of Example 1 or a similar method. The NMR data for 3-[2-azidoethyl poly(oxyethylene)]-1,2-di(stearoyl)-sn-glycerol is shown below.​​

[0064] １ H-NMR (CDCl ３ ): δ0.88 (6H, t, J = 6.8Hz), 1.16-1.38 (56H, m), 1.54-1.68 (4H, m), 2.30 (4H, q, J = 7.3Hz), 3.39 (2H, t, J = 5.0Hz), 3.43-3.50 (2H, m), 3.53-3.90 (174H, m), 4.15 (1H, dd, J = 6.5Hz, 11.9Hz), 4.34 (1H, dd, J = 3.4Hz, 11.9Hz), 5.17-5.25 (1H, m)

[0065] (Example 3) The following compounds were prepared according to Method 1.

[0066] (1) Using 1-(hydroxypolyoxyethylene)-2,3-isopropylideneglycerol (n=112), 3-[2-azidoethylpoly(oxyethylene)]-1,2-isopropylidene-sn-glycerol (n=112) (compound (V)) was obtained by the method described in (1) of Example 1 or a similar method. The NMR data for 3-[2-azidoethylpoly(oxyethylene)]-1,2-isopropylidene-sn-glycerol is shown below.

[0067] １ H-NMR (CDCl ３ ): δ1.35 (3H, s), 1.41 (3H, s), 3.39 (2H, t, J=5.1Hz), 3.44-3.53 (3H, m) 3.54-3.90 (446H, m), 4.05 (1H, dd, J=6.4Hz, 8.2Hz), 4.24-4.32 (1H, m)

[0068] (2) Using the 3-[2-azidoethyl poly(oxyethylene)]-1,2-isopropylidene-sn-glycerol (n=112) obtained in the preceding paragraph (1), 3-[2-azidoethyl poly(oxyethylene)]-sn-glycerol (n=112) (compound (VII)) was obtained by the method described in (2) of Example 1 or a similar method. The NMR data for 3-[2-azidoethyl poly(oxyethylene)]-sn-glycerol is shown below. ​

[0069] １ H-NMR (CDCl ３ ): δ2.66 (1H, t, J=6.2Hz), 3.36-3.44 (3H, m), 3.44-3.50 (2H, m), 3.53-3.90 (447H, m)

[0070] (3) Using the 3-[2-azidoethyl poly(oxyethylene)]-sn-glycerol (n=112) obtained in the preceding paragraph (2), 3-[2-azidoethyl poly(oxyethylene)]-1,2-di(stearoyl)-sn-glycerol (n=112) (compound (IX)) was obtained by the method described in (3) of Example 1 or a similar method. The NMR data for 3-[2-azidoethyl poly(oxyethylene)]-1,2-di(stearoyl)-sn-glycerol is shown below.

[0071] １ H-NMR (CDCl ３ ): δ0.88 (6H, t, J = 6.8Hz), 1.16-1.38 (56H, m), 1.54-1.68 (4H, m), 2.30 (4H, q, J = 7.3Hz), 3.39 (2H, t, J = 5.0Hz), 3.43-3.50 (2H, m), 3.53-3.90 (446H, m), 4.15 (1H, dd, J = 6.5Hz, 11.9Hz), 4.34 (1H, dd, J=3.4Hz, 11.9Hz), 5.17-5.25 (1H, m)

[0072] (Example 4) The following compounds were prepared according to Method 1.

[0073] (1) Using 1-(hydroxypolyoxyethylene)-2,3-isopropylideneglycerol (n=226), 3-[2-azidoethylpoly(oxyethylene)]-1,2-isopropylidene-sn-glycerol (n=226) (compound (V)) was obtained by the method described in (1) of Example 1 or a similar method. The NMR data for 3-[2-azidoethylpoly(oxyethylene)]-1,2-isopropylidene-sn-glycerol is shown below.

[0074] １ H-NMR (CDCl ３ ​​​): δ1.35 (3H, s), 1.41 (3H, s), 3.39 (2H, t, J=5.1Hz), 3.44-3.53 (3H, m) 3.54-3.90 (902H, m), 4.05 (1H, dd, J=6.4Hz, 8.2Hz), 4.24-4.32 (1H, m)

[0075] (2) Using the 3-[2-azidoethyl poly(oxyethylene)]-1,2-isopropylidene-sn-glycerol (n=226) obtained in the preceding paragraph (1), 3-[2-azidoethyl poly(oxyethylene)]-sn-glycerol (n=226) (compound (VII)) was obtained by the method described in (2) of Example 1 or a similar method. The NMR data for 3-[2-azidoethyl poly(oxyethylene)]-sn-glycerol is shown below.

[0076] １ H-NMR (CDCl ３ ): δ2.66 (1H, t, J=6.2Hz), 3.36-3.44 (3H, m), 3.44-3.50 (2H, m), 3.53-3.90 (903H, m)

[0077] (3) Using the 3-[2-azidoethyl poly(oxyethylene)]-sn-glycerol (n=226) obtained in the preceding paragraph, 3-[2-azidoethyl poly(oxyethylene)]-1,2-di(stearoyl)-sn-glycerol (n=226) (compound (IX)) was obtained by the method described in (3) of Example 1 or a similar method. The NMR data for 3-[2-azidoethyl poly(oxyethylene)]-1,2-di(stearoyl)-sn-glycerol is shown below.

[0078] １ H-NMR (CDCl ３ ): δ0.88 (6H, t, J = 6.8Hz), 1.16-1.38 (56H, m), 1.54-1.68 (4H, m), 2.30 (4H, q, J = 7.3Hz), 3.39 (2H, t, J = 5.0Hz), 3.43-3.50 (2H, m), 3.53-3.90 (902H, m), 4.15 (1H, dd, J = 6.5Hz, 11.9Hz), 4.34 (1H, dd, J=3.4Hz, 11.9Hz), 5.17-5.25 (1H, m)

[0079] (Example 5) The following compounds were prepared according to Method 1.

[0080] Using 3-[2-azidoethylpoly(oxyethylene)]-sn-glycerol (n=76) obtained by the method described in Example 1 (2) or a similar method, 3-[2-azidoethylpoly(oxyethylene)]-1,2-di(palmitoyl)-sn-glycerol (n=76) (compound (IX)) was obtained by the method described in Example 1 (3) or a similar method. The NMR data for 3-[2-azidoethylpoly(oxyethylene)]-1,2-di(palmitoyl)-sn-glycerol is shown below.

[0081] １ H-NMR (CDCl ３ ): δ0.88 (6H, t, J = 6.8Hz), 1.16-1.38 (48H, m), 1.54-1.68 (4H, m), 2.30 (4H, q, J = 7.3Hz), 3.39 (2H, t, J = 5.0Hz), 3.43-3.50 (2H, m), 3.53-3.90 (302H, m), 4.15 (1H, dd, J = 6.5Hz, 11.9Hz), 4.34 (1H, dd, J=3.4Hz, 11.9Hz), 5.17-5.25 (1H, m)

[0082] (Example 6) The following compounds were prepared according to Method 1.

[0083] Using 3-[2-azidoethyl poly(oxyethylene)]-sn-glycerol (n=76) obtained by the method described in Example 1 (2) or a similar method, 3-[2-azidoethyl poly(oxyethylene)]-1,2-di(arachidinoyl)-sn-glycerol (n=76) (compound (IX)) was obtained by the method described in Example 1 (3) or a similar method. The NMR data for 3-[2-azidoethyl poly(oxyethylene)]-1,2-di(arachidinoyl)-sn-glycerol is shown below.

[0084] １ H-NMR (CDCl ３ ​): δ0.88 (6H, t, J = 6.8Hz), 1.16-1.38 (64H, m), 1.54-1.68 (4H, m), 2.30 (4H, q, J = 7.3Hz), 3.39 (2H, t, J = 5.0Hz), 3.43-3.50 (2H, m), 3.53-3.90 (302H, m), 4.15 (1H, dd, J = 6.5Hz, 11.9Hz), 4.34 (1H, dd, J=3.4Hz, 11.9Hz), 5.17-5.25 (1H, m)

[0085] (Example 7) The following compounds were prepared according to Method 2. However, the following compounds belong to polyoxyalkylene glycol lipids having an azide group of formula (1). In formula (1), R １ and R ２ A is a stearoyl group, with k=1, l=0, m=1, k+l+m=2, A is an oxygen atom, B is an ethylene group, n is 76, C is an ethylene group, D is the (-E-F-azide group) of formula (2b), E is an amide bond, and F is a butylene group.

[0086] (1) A toluene solution of 1-(hydroxypolyoxyethylene)-2,3-isopropylideneglycerol (n=76) (compound (III): 5.0 g), phthalimide (compound (X): 0.26 g), and triphenylphosphine (1.93 g) was cooled to 10°C, and diisopropyl azodicarboxylic acid (1.49 g) was added. The mixture was stirred at room temperature for 1 hour, and methanol (0.24 g) was added. After concentrating the solution, it was dissolved in toluene (38 g) and methanol (15 g). Ethylenediamine monohydrate (compound (XI): 2.87 g) was added at room temperature, and the mixture was stirred at 40°C for 4 hours. 25% sodium chloride aqueous solution (50 g) was added, and after stirring at 60°C, the upper organic layer was collected. The same procedure was repeated twice on the collected organic layer, and then the organic layer was concentrated. The residue was subjected to silica gel column chromatography (eluent: ethyl acetate / methanol, chloroform / methanol) to obtain 1.5 g of 3-[2-aminoethylpoly(oxyethylene)]-1,2-isopropylidene-sn-glycerol (n=76) (compound (XII)). The NMR data for 3-[2-aminoethylpoly(oxyethylene)]-1,2-isopropylidene-sn-glycerol (n=76) are shown below.

[0087] １ H-NMR (CDCl ３ ): δ1.35 (3H, s), 1.41 (3H, s), 2.89 (2H, t, J=5.2Hz), 3.44-3.53 (3H, m), 3.54-3.90 (302H, m), 4.05 (1H, dd, J=6.4Hz, 8.2Hz), 4.24-4.32 (1H, m)

[0088] ​(2) To a toluene solution of 3-[2-aminoethylpoly(oxyethylene)]-1,2-isopropylidene-sn-glycerol (n=76) (1.0 g) obtained in the preceding paragraph (1), 5-azidopentanoic acid (compound (XIII): 0.13 g), N,N'-dicyclohexylcarbodiimide (0.24 g), and toluene (3.0 g) were added, and the mixture was stirred at room temperature for 2 hours. The precipitate was filtered off and washed with toluene (1.0 g). Hexane (4.0 g) was added to the filtrate, and crystallization was carried out at 20°C. The precipitated crystals were filtered off. The crystals were dissolved in ethyl acetate (8.0 g), and hexane (4.0 g) was added. Crystallization was carried out at 17°C. The precipitated crystals were filtered off. After repeating the same crystallization procedure twice, the recovered crystals were suspended in hexane (4.0 g) and stirred at room temperature. The crystals were filtered to obtain 0.90 g of 3-[5-azidopentanamidoethyl poly(oxyethylene)]-1,2-isopropylidene-sn-glycerol (n=76) (compound (XIV)). The NMR data for 3-[5-azidopentanamidoethyl poly(oxyethylene)]-1,2-isopropylidene-sn-glycerol (n=76) are shown below.

[0089] １ H-NMR (CDCl ３ ): δ1.35 (3H, s), 1.41 (3H, s), 1.61-1.77 (4H, m), 2.18 (2H, t, J=7.2Hz), 3.26-3.42 (4H, m), 3.44-3.53 (3H, m), 3.54-3.90 (302H, m), 4.05 (1H, dd, J=6.4Hz, 8.2Hz), 4.24-4.32 (1H, m)

[0090] (3) Using the 3-[5-azidopentanamidoethyl poly(oxyethylene)]-1,2-isopropylidene-sn-glycerol (n=76) obtained in the preceding paragraph (2), 3-[5-azidopentanamidoethyl poly(oxyethylene)]-sn-glycerol (n=76) (compound (XV)) was obtained by the method described in Example 1 (2) or a similar method. The NMR data for 3-[5-azidopentanamidoethyl poly(oxyethylene)]-sn-glycerol (n=76) is shown below.

[0091] ​​１ H-NMR (CDCl ３ ): δ1.61-1.77 (4H, m), 2.19 (2H, t, J=7.2Hz), 2.66 (1H, t, J=6.2Hz), 3.26-3.44 (7H, m), 3.44-3.53 (2H, m), 3.53-3.90 (303H, m)

[0092] (4) Using the 3-[5-azidopentanamidoethyl poly(oxyethylene)]-sn-glycerol (n=76) obtained in the preceding paragraph (3), 3-[5-azidopentanamidoethyl poly(oxyethylene)]-1,2-di(stearoyl)-sn-glycerol (n=76) (compound (XVI)) was obtained by the method described in (3) of Example 1 or a similar method. The NMR data for 3-[5-azidopentanamidoethyl poly(oxyethylene)]-1,2-di(stearoyl)-sn-glycerol (n=76) is shown below.

[0093] １ H-NMR (CDCl ３ ): δ0.88 (6H, t, J = 6.8Hz), 1.16-1.38 (56H, m), 1.54-1.78 (8H, m), 2.18 (2H, t, J = 7.2Hz), 2.30 (4H, q, J = 7.3Hz), 3.26-3.43 (4H, m), 3.43-3.50 (2H, m), 3.53-3.90 (302H, m), 4.15 (1H, dd, J = 6.5Hz, 11.9Hz), 4.34 (1H, dd, J=3.4Hz, 11.9Hz), 5.17-5.25 (1H, m)

[0094] (Example 8) The following compounds were prepared according to Method 3.

[0095] ​(1) A toluene solution of 3-hydropolyoxyethylene-1,2-di(stearoyl)-sn-glycerol (n=76) (compound (XXI): 5.0 g), phthalimide (compound (X): 0.22 g), and triphenylphosphine (1.63 g), obtained by the method described in International Publication No. 2022 / 065263 or a similar method, was cooled to 10°C, and diisopropyl azodicarboxylic acid (1.25 g) was added. The mixture was stirred at room temperature for 1 hour, and methanol (0.20 g) was added. After concentrating the solution, it was dissolved in toluene (38 g) and methanol (15 g). Ethylenediamine monohydrate (compound (XI): 2.42 g) was added at room temperature, and the mixture was stirred at 40°C for 4 hours. 25% aqueous sodium chloride solution (50 g) was added, and after stirring at 60°C, the upper organic layer was recovered. The same procedure was repeated twice on the recovered organic layer, and then the organic layer was concentrated. The residue was subjected to silica gel column chromatography (eluent: ethyl acetate / methanol, chloroform / methanol) to obtain 1.3 g of 3-[2-aminoethyl poly(oxyethylene)]-1,2-di(stearoyl)-sn-glycerol (n=76) (compound (XXII)). The NMR data for 3-[2-aminoethyl poly(oxyethylene)]-1,2-di(stearoyl)-sn-glycerol (n=76) are shown below.

[0096] １ H-NMR (CDCl ３ ): δ0.88 (6H, t, J = 6.8Hz), 1.16-1.38 (56H, m), 1.54-1.68 (4H, m), 2.30 (4H, q, J = 7.3Hz), 2.88 (2H, t, J = 5.2Hz), 3.43-3.50 (2H, m), 3.53-3.90 (302H, m), 4.15 (1H, dd, J = 6.5Hz, 11.9Hz), 4.34 (1H, dd, J = 3.4Hz, 11.9Hz), 5.16-5.26 (1H, m)

[0097] ​(2) To a chloroform solution of 3-[2-aminoethylpoly(oxyethylene)]-1,2-di(stearoyl)-sn-glycerol (n=76) (1.0 g) obtained in item (1) above, 5-azidopentanoic acid (compound (XIII): 0.053 g), triethylamine (0.050 g), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.12 g) were added and the mixture was stirred at room temperature for 3 hours. Chloroform (5.0 g) was added. 2% citric acid aqueous solution (10 g) was added and the mixture was stirred at 10°C, after which the lower chloroform layer was collected. The same procedure was repeated four times on the collected chloroform layer, and then the chloroform layer was concentrated. The residue was subjected to silica gel column chromatography (eluent: chloroform / methanol) to obtain 0.79 g of 3-[5-azidopentanamidoethyl poly(oxyethylene)]-1,2-di(stearoyl)-sn-glycerol (n=76) (compound (XVI)). The NMR data for 3-[5-azidopentanamidoethyl poly(oxyethylene)]-1,2-di(stearoyl)-sn-glycerol (n=76) are shown below.

[0098] １ H-NMR (CDCl ３ ): δ0.88 (6H, t, J = 6.8Hz), 1.16-1.38 (56H, m), 1.54-1.77 (8H, m), 2.18 (2H, t, J = 7.2Hz), 2.30 (4H, q, J = 7.3Hz), 3.26-3.42 (4H, m), 3.43-3.50 (2H, m), 3.53-3.90 (302H, m), 4.15 (1H, dd, J = 6.5Hz, 11.9Hz), 4.34 (1H, dd, J=3.4Hz, 11.9Hz), 5.17-5.25 (1H, m)

[0099] (Example 9) The following compounds were prepared according to Method 4. However, the following compounds belong to polyoxyalkylene glycol lipids having an azide group of formula (1). In formula (1), R １ and R ２ ​A is a stearoyl group, with k=1, l=0, m=1, k+l+m=2, A is an oxygen atom, B is an ethylene group, n is 76, C is an ethylene group, D is the (-E-F-azide group) of formula (2b), E is a carbamate bond, and F is a pentylene group.

[0100]

[0101] (1) Di(N-succinimidyl) carbonate (compound (XXIII): 0.48 g) and pyridine (0.20 g) were added to a toluene solution of 3-hydropolyoxyethylene-1,2-di(stearoyl)-sn-glycerol (n=76) (compound (XXI): 5.0 g) and 2,6-di-tert-butyl-p-cresol (5.0 mg) obtained by the method described in International Publication No. 2022 / 065263 or a similar method, and the mixture was stirred at 80°C for 2 hours. The reaction solution was cooled to 40°C, the precipitate was filtered off, and the mixture was washed with toluene (10 g). The filtrate was concentrated and dissolved in chloroform (20 g). 20% aqueous sodium chloride solution (25 g) was added to the solution, and after stirring at room temperature, the lower chloroform layer was collected. The same procedure was repeated once on the recovered chloroform layer, then magnesium sulfate (2.5 g) was added to the chloroform layer and stirred at room temperature for 20 minutes. After that, the magnesium sulfate was filtered off and washed with chloroform (5.0 g). The filtrate was concentrated, and the concentrate was suspended in hexane (40 g) and stirred at room temperature for 20 minutes. After filtering off the crystals, they were washed with hexane (10 g) to obtain 3.7 g of compound (XXIV) 3-succinimidyloxycarbonylpoly(oxyethylene)-1,2-di(stearoyl)-sn-glycerol (n=76). The NMR data for 3-succinimidyloxycarbonylpoly(oxyethylene)-1,2-di(stearoyl)-sn-glycerol (n=76) is shown below.

[0102] １ H-NMR (CDCl ３): δ0.88 (6H, t, J = 6.8Hz), 1.19-1.35 (56H, m), 1.58-1.61 (4H, m), 2.30 (4H, q, J = 7.4Hz), 2.84 (4H, s), 3.45-3.48 (1H, m), 3.57-3.83 (303H, m), 4.15 (1H, dd, J = 6.6Hz, 11.9Hz), 4.34 (1H, dd, J = 3.7Hz, 11.9Hz), 4.45-4.47 (2H, m), 5.18-5.23 (1H, m)

[0103] (2) Add 5-azidopentane-1-amine (compound (XXV): 0.046 g) to a dichloromethane solution of 3-succinimidyloxycarbonylpoly(oxyethylene)-1,2-di(stearoyl)-sn-glycerol (n=76) (1.0 g) obtained in the preceding paragraph (1), and stir at room temperature for 1 hour. Add dichloromethane (3.0 g). Add 2% citric acid aqueous solution (4.0 g), stir at room temperature, and then collect the lower dichloromethane layer. Repeat the same procedure twice on the collected dichloromethane layer, and then concentrate the dichloromethane layer. The residue was subjected to silica gel column chromatography (eluent: chloroform / methanol) to obtain 0.81 g of compound (XXVI) 3-[(5-azidopentylcarbamoyl)]oxaethylpoly(oxyethylene)]-1,2-di(stearoyl)-sn-glycerol (n=76). The NMR data for 3-[(5-azidopentylcarbamoyl)]oxaethylpoly(oxyethylene)]-1,2-di(stearoyl)-sn-glycerol (n=76) are shown below.

[0104] １ H-NMR (CDCl ３ ): δ0.88 (6H, t, J = 6.8Hz), 1.19-1.35 (56H, m), 1.58-1.78 (8H, m), 2.30 (4H, q, J = 7.4Hz), 2.84 (4H, s), 2.96 (2H, t, J = 7.2Hz), 3.27 (2H, t, J = 6.0Hz), 3.45-3.48 (1H, m), 3.57-3.83 (303H, m), 4.08-4.25 (3H, m), 4.34 (1H, dd, J = 3.7Hz, 11.9Hz), 4.45-4.47 (2H, m), 5.18-5.23 (1H, m)

[0105] The polyalkylene glycol lipid having an azide group according to the present invention is useful as a base for LNPs that are organ-specific to organs other than the liver.

[0106] Although the present invention has been described in detail and with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention. This application is based on Japanese Patent Application No. 2025-049303 filed on 25 March 2025, the contents of which are incorporated herein by reference.

Claims

1. A polyalkylene glycol lipid characterized by being represented by the following formula (1). (In formula (1), R １ and R ２ Each of the following is independently a hydrogen atom, an aliphatic hydrocarbon group having 15 to 22 carbon atoms, or an acyl group having 15 to 22 carbon atoms; k, l, and m are numbers satisfying 6≧k≧0, 6≧l≧0, 6≧m≧0, and k+l+m≧2; A is an oxygen atom, an ester bond, a carbonate bond, or a carbamate bond; B is a divalent hydrocarbon group having 1 to 4 carbon atoms; n is 21 to 226; C is a divalent hydrocarbon group having 1 to 4 carbon atoms; and D is the atomic group represented by the following formula (2a) or formula (2b). (In formula (2a), the dashed line represents the bonding site to the hydrocarbon group represented by C; in formula (2b), the dashed line represents the bonding site to the hydrocarbon group represented by C; E is an ester bond, carbonate bond, carbamate bond, urea bond, or amide bond; and F is a divalent hydrocarbon group having 1 to 4 carbon atoms.) 2. The polyalkylene glycol lipid according to claim 1, characterized in that in formula (1), D is the atomic group represented by formula (2a).

3. The polyalkylene glycol lipid according to claim 1, characterized in that in formula (1), D is the atomic group represented by formula (2b), and in formula (2b), E is an amide bond or a carbamate bond.

4. In equation (1), R １ and R ２ The polyalkylene glycol lipid according to any one of claims 1 to 3, characterized in that each is independently a linear and saturated acyl group having 15 to 20 carbon atoms.

5. A polyalkylene glycol lipid according to any one of claims 1 to 3, characterized in that, in formula (1), k is 1, m is 1, and l is 0.

6. A polyalkylene glycol lipid according to any one of claims 1 to 3, characterized in that A in formula (1) is an oxygen atom.

7. A substrate for lipid nanoparticles, characterized by comprising a polyalkylene glycol lipid according to any one of claims 1 to 3.

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