A lipid compound and its preparation method and application

Abstract

The present invention discloses a lipid compound and its preparation method and application. The present invention proposes for the first time a cationic lipid compound with a cyclic imide connecting segment, which connects a cationic head with a tertiary amine or a quasi-tertiary amine and a long-chain hydrophobic chain tail, can be effectively loaded with nucleic acid drugs, and improves the degradation effect of lipid molecules. This design not only enriches the chemical types of lipid compounds and solves the limitation of poor designability of lipid molecule chemical structures; and the LNP prepared using the compound has good transfection efficiency and good preparation safety.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a lipid compound and a preparation method and application thereof. Background Art

[0002] Gene therapy is a new technology that combines modern medicine and molecular biology to introduce target genes into patients and achieve target gene products, thereby treating diseases. However, there are many challenges to achieving effective gene therapy. Among them, how to safely and efficiently deliver nucleic acid molecules (such as DNA and RNA) to target cells has become a key issue. Traditional gene delivery systems such as viral vectors are subject to potential safety risks of the viral vectors themselves, such as genetic toxicity risks, and the capacity of viral vectors is limited. These significant shortcomings greatly limit the application of viral vectors in the field of gene therapy. It is in this context that non-viral vectors have been significantly developed.

[0003] As a non-viral carrier, lipid nanoparticles (LNP) have become one of the most commonly used methods for delivering nucleic acid drugs in the field of gene therapy, and have been widely used in the delivery of drugs such as siRNA, mRNA, and DNA. However, traditional LNPs face the disadvantages of low transfection efficiency and high risks of safety of preparations due to the difficulty in degradation of cationic lipids. For example, cationic lipid DODMA (CAS#: 104162-47-2) is difficult to biodegrade due to the lack of easily degradable linking groups. In addition, the current mainstream cationic lipids, such as SM-102 (CAS#: 2089251-47-6), have complex synthetic routes, involving multi-step esterification and amination reactions, resulting in great difficulty in purification and low overall yield. As the most important component in LNP, cationic lipids, especially ionizable lipids, not only face the limitation of complex synthetic routes, but also face the limitation of poor designability of chemical structures. Therefore, it is urgent to develop a safe and effective lipid compound to meet the needs of nucleic acid drug delivery. Summary of the invention

[0004] The first object of the present invention is to provide a lipid compound, in particular a cyclic imide lipid compound, whose structural formula is shown in formula (I):

[0005]

[0006] Wherein, R1 is independently selected from C1-C3 alkyl, C1-C3 alkoxy or C1-C3 hydroxyalkyl;

[0007] X is as shown in formula (II), and R2 and R3 are each independently selected from hydrogen or C8-C 36When R2 and / or R3 are branched hydrocarbon groups, the branched hydrocarbon group includes at least two C4-C 18 A saturated or unsaturated straight-chain hydrocarbon group;

[0008]

[0009] Y is independently selected from a tertiary amine group, a substituted or unsubstituted azaaromatic ring, or a substituted or unsubstituted azaaliphatic ring containing at least one tertiary amine group;

[0010] L is independently selected from C1-C3 straight chain alkyl;

[0011] i is an integer greater than or equal to 0, j is an integer greater than 1, and k is 0 or 1; and

[0012] i+j+k≤3, p=k+1.

[0013] The cyclic imide lipid compound provided by the present invention has a cyclic imide connecting segment, which is used to connect the cationic head with a tertiary amine or a tertiary amine-like cationic head and a long-chain hydrophobic chain tail. Among them, the cationic head with a tertiary amine or a tertiary amine-like cationic head can be effectively loaded with a nucleic acid drug, and the cyclic imide connecting segment can improve the degradation effect of the lipid molecule. This design not only enriches the chemical types of lipid compounds and solves the limitation that the chemical structure of lipid molecules can be poorly designed; and the LNP prepared using the compound has a good transfection efficiency and a good formulation safety.

[0014] Further, R2 is a hydrogen atom, and R3 is selected from C8-C 22 When R3 is a branched hydrocarbon group, the branched hydrocarbon group includes at least two C9-C 11 A saturated or unsaturated straight chain hydrocarbon group.

[0015] Further, R3 is selected from C8-C 18 Straight chain alkyl or C8-C 18 The straight-chain alkenyl group comprises at least one alkenyl group which is no more than 2 carbon atoms away from the cyclic imide nitrogen heterocycle.

[0016] Further, R3 is selected from C 18 -C 22 A branched hydrocarbon group consisting of a tertiary carbon group and two C8-C 11 Straight chain hydrocarbon composition.

[0017] Furthermore, the two C8-C 11 The straight-chain hydrocarbon group contains at least one straight-chain alkenyl group, and the straight-chain alkenyl group contains at least one alkenyl group which is not more than 2 carbon atoms away from the cyclic imide nitrogen heterocycle.

[0018] Further, Y is a substituted or unsubstituted nitrogen heteroaromatic ring. The nitrogen heteroaromatic ring has similar properties to tertiary amine groups, both have lone pairs of electrons, show strong nucleophilicity, and can participate in nucleophilic addition and nucleophilic substitution reactions. The positively charged head is conducive to binding with negatively charged nucleic acids through electrostatic interaction. On the basis of formula (I), i=0, j=1, k=0, p=1, when the nitrogen heteroaromatic ring is substituted, its substituent is independently selected from methyl or phenyl.

[0019] As a further optimization of the present invention, the nitrogen heteroaromatic ring is selected from any one including but not limited to substituted or unsubstituted pyridine, pyrimidine, pyrrole, imidazole, thiophene or thiazine.

[0020] As a preferred embodiment of the present invention, when Y is a substituted or unsubstituted nitrogen aromatic ring, the lipid compound is:

[0021]

[0022] Further, Y is a substituted or unsubstituted azaaliphatic ring containing at least one tertiary amine group, i=0, j=1 or 2, and the substituents of the azaaliphatic ring are independently selected from C1-C3 alkyl groups or C1-C3 alkyl ether groups.

[0023] As a preferred class of compounds, the azaaliphatic ring has a tertiary amine directly connected to the L group. If the tertiary amine on the azaaliphatic ring is not directly connected to the L group, the azaaliphatic ring should have at least one nitrogen atom substituted by a C1-C3 alkyl group to form a tertiary amine. Therefore, in formula (I), Y is selected from, including but not limited to, substituted or unsubstituted azetidine, tetrahydropyrrole, piperidine, piperazine or morpholine.

[0024] It is worth noting that piperazine can form a cationic head containing two tertiary amines, where i=0, j=2, k=0, and p=1, and the two tertiary amine groups can be independently connected to the long hydrophobic chain tail through the L group and the cyclic imide.

[0025] As a preferred embodiment of the present invention, when Y comprises at least one tertiary amine substituted or unsubstituted azaaliphatic ring, the lipid compound is:

[0026]

[0027] Further, Y is a tertiary amine group, and when k=0, i+j=3; or when k=1, i+j=2. Wherein, i+j=3 (and j is an integer greater than 1) means that the tertiary amine group should be connected to at least one cyclic imide with a long-chain hydrophobic chain tail, but not more than 3. When k=0, i=2, j=1, the cationic head with a tertiary amine group is connected to only one cyclic imide with a long-chain hydrophobic chain tail; when k=0, i=1, j=2, the cationic head with a tertiary amine group is connected to two cyclic imides with long-chain hydrophobic chain tails; when k=0, i=0, j=3, the cationic head with a tertiary amine group is connected to three cyclic imides with long-chain hydrophobic chain tails. For another group of cases, when k=1, p=2, i=1, j=1, the cationic head contains two tertiary amine groups, and each tertiary amine group is connected to only one cyclic imide with a long hydrophobic chain tail; when k=1, p=2, i=0, j=2, the cationic head contains two tertiary amine groups, and each tertiary amine group is connected to two cyclic imides with long hydrophobic chain tails.

[0028] As a preferred embodiment of the present invention, when Y is a tertiary amine group, the lipid compound is:

[0029]

[0030]

[0031]

[0032]

[0033] The second object of the present invention is to provide the use of the lipid compound in preparing LNP lipid nanoparticles.

[0034] The third object of the present invention is to provide a LNP lipid nanoparticle, which comprises the lipid compound of the present invention, auxiliary phospholipids, cholesterol and pegylated lipids.

[0035] The auxiliary phospholipid is selected from any one or more combinations of, including but not limited to, DSPC (distearylphosphatidylcholine), DOPC (dioleoylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine) and DOPE (dioleoylphosphatidylethanolamine).

[0036] The PEGylated lipid is selected from the group consisting of, but not limited to, DMG-PEG2000 or DSG-PEG2000.

[0037] The LNP lipid nanoparticles should at least include no less than 30 mol% of lipid compounds. Preferably, the molar percentages of the components in the LNP lipid nanoparticles are: 30-50 mol% of lipid compounds, 8-12 mol% of auxiliary phospholipids, 35-60 mol% of cholesterol, and 1-3 mol% of PEGylated lipids.

[0038] The fourth object of the present invention is to provide an application of LNP lipid nanoparticles in nucleic acid molecule delivery. The LNP lipid nanoparticles provided by the present invention have a particle size of less than 200nm, a PDI of less than 0.2, and can efficiently and stably encapsulate nucleic acid molecules. In addition, the "one pot and two steps" synthesis method of the product is faster than the prior art, such as the synthesis route of at least five steps of SM-102, so the LNP product has certain cost advantages.

[0039] The fifth object of the present invention is to provide a method for preparing the lipid compound, which comprises mixing compound (III) with a portion of the solvent in an ice bath, adding compound (IV) dropwise under the protection of an inert gas and adding the remaining solvent to obtain an intermediate product; adding an organic base and acetic anhydride to the intermediate product for a ring-closure reaction, and obtaining the lipid compound (I) after purification;

[0040]

[0041] Wherein, R1 is independently selected from C1-C3 alkyl, C1-C3 alkoxy or C1-C3 hydroxyalkyl;

[0042] X is as shown in formula (II), and R2 and R3 are each independently selected from hydrogen or C8-C 36 When R2 and / or R3 are branched hydrocarbon groups, the branched hydrocarbon group includes at least two C4-C 18 A saturated or unsaturated straight chain hydrocarbon group; Y is independently selected from a tertiary amine group, a substituted or unsubstituted nitrogen heteroaromatic ring or a substituted or unsubstituted nitrogen heteroaliphatic ring containing at least one tertiary amine group;

[0043] L is independently selected from C1-C3 straight chain alkyl;

[0044] i is an integer greater than or equal to 0, j is an integer greater than 1, and k is 0 or 1; and

[0045] i+j+k≤3, p=k+1.

[0046] Further, the solvent is selected from any one or more combinations of dichloromethane, chloroform, ethyl acetate, N-methyl-2-pyrrolidone, N,N-dimethylformamide, and 1,4-dioxane. Preferably, the solvent is selected from dichloromethane or chloroform.

[0047] Further, the organic base is selected from any one or more combinations of triethylamine, trimethylamine, tributylamine and pyridine. Preferably, the organic base is selected from triethylamine. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is a graph showing the transfection efficiency of IL-X-LNP on DC2.4 cells in Experimental Example 2;

[0049] Figure 2 It is the biodegradation diagram of the cyclic imide lipid compound IL-23;

[0050] Figure 3 This is a graph showing the expression efficiency of IL-X-LNP and SM102 LNP in delivering Fluc-mRNA in vivo in Experimental Example 4. DETAILED DESCRIPTION

[0051] In order to make the technical solution of the present invention clearer, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0052] Example 1

[0053]

[0054] Compound 1-1 (1.66 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 1-2 (5.85 g, 14.33 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 1-3. Triethylamine (1 ml) and acetic anhydride (2.7 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-1 (5.43 g, yield 75%). 1H NMR (400MHz, Chloroform-d) δ3.56-3.60(m,2H),2.62-2.87(m,2H),2.51-2.55(m,3H),2.44-2.48(m,4H),1.25-1.27(m,38H),0.89-0.93(m,12H).

[0055] Example 2

[0056]

[0057] Compound 2-1 (1.86 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced for protection. The mixture was cooled to 0 ° C by ice bath, and then succinic anhydride 1-2 (5.85 g, 14.33 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 2-2. Triethylamine (1 ml) and acetic anhydride (2.7 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified by a silica gel column and finally rotary evaporated to obtain compound IL-2 (5.66 g, yield 76%). 1H NMR(400MHz,Chloroform-d)δ3.16-3.20(t,2H),3.00-3.02(q,4H),2.62-2.87(m,2H),2.51-2.55(q,1H),2.34 -2.38(t,2H),1.73-1.75(m,2H),1.49-1.51(m,1H),1.25-1.27(m,38H),1.13-1.15(m,6H),0.87-0.91(m,6H).

[0058] Example 3

[0059]

[0060] Compound 3-1 (1.46 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced for protection. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 1-2 (5.85 g, 14.33 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 3-2. Triethylamine (1 ml) and acetic anhydride (2.7 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-3 (5.23 g, yield 75%). 1H NMR(400MHz,Chloroform-d)δ3.16-3.20(t,2H),2.62-2.87(m,2H),2.51-2.55(q,1H),2.30-2.32(t, 2H),2.13-2.15(s,6H),1.73-1.75(m,2H),1.49-1.51(m,1H),1.25-1.27(m,38H),0.87-0.91(m,6H).

[0061] Example 4

[0062]

[0063] Compound 4-1 (1.26 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced for protection. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 1-2 (5.85 g, 14.33 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 4-2. Triethylamine (1 ml) and acetic anhydride (2.7 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-4 (5.00 g, yield 73%). 1H NMR(400MHz,Chloroform-d)δ3.56-3.60(t,2H),2.62-2.87(m,2H),2.51-2.55(m, 1H),2.13-2.15(s,6H),1.49-1.51(m,1H),1.25-1.27(m,38H),0.87-0.91(m,6H).

[0064] Example 5

[0065]

[0066] Compound 5-1 (1.72 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 1-2 (5.85 g, 14.33 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 5-2. Triethylamine (1 ml) and acetic anhydride (2.7 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-5 (5.12 g, yield 70%). 1H NMR(400MHz,Chloroform-d)δ4.58-4.62(s,4H),3.56-3.60(t,2H),2.62-2.87(m,2H),2.51 -2.55(m,3H),1.49-1.51(m,1H),1.25-1.27(m,38H),1.03-1.05(m,2H),0.87-0.91(m,6H).

[0067] Example 6

[0068]

[0069] Compound 6-1 (1.86 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced for protection. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 1-2 (5.85 g, 14.33 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 6-2. Triethylamine (1 ml) and acetic anhydride (2.7 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-6 (5.86 g, yield 80%). 1H NMR(400MHz,Chloroform-d)δ3.56-3.58(t,4H),3.52-3.54(t,4H),2.62-2.87(m,2H),2.5 1-2.55(m,3H),2.38-2.44(t,3H)1.49-1.51(m,1H),1.25-1.27(m,38H),0.87-0.91(m,6H).

[0070] Example 7

[0071]

[0072] Compound 7-1 (1.83 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was passed in. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 1-2 (5.85 g, 14.33 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 7-2. Triethylamine (1 ml) and acetic anhydride (2.7 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-7 (5.94 g, yield 80%). 1H NMR(400MHz,Chloroform-d)δ3.56-3.58(t,2H),2.62-2.87(m,2H),2.51-2.55(m,3H),2.3 8-2.44(t,4H)1.49-1.50(m,3H),1.35-1.39(m,2H),1.25-1.27(m,38H),0.87-0.91(m,6H).

[0073] Example 8

[0074]

[0075] Compound 8-1 (1.83 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced for protection. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 1-2 (5.85 g, 14.33 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 8-2. Triethylamine (1 ml) and acetic anhydride (2.7 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-8 (6.18 g, yield 81%). 1H NMR(400MHz,Chloroform-d)δ3.18-3.20(t,2H),2.62-2.87(m,2H),2.41-2.51(m,4H),2.18 -2.20(s,3H),1.50-1.56(m,5H),1.39-1.41(m,1H),1.25-1.27(m,38H),0.87-0.91(m,6H).

[0076] Example 9

[0077]

[0078] Compound 9-1 (1.79 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced for protection. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 1-2 (5.85 g, 14.33 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 9-2. Triethylamine (1 ml) and acetic anhydride (2.7 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-9 (5.53 g, yield 76%). 1H NMR(400MHz,Chloroform-d)δ7.91-7.93(s,1H),7.15-7.17(d,1H),6.76-6.78(d,1H),4.04-4.06(t,2H),3.18 -3.20(t,2H),2.62-2.87(m,2H),2.56-2.58(m,2H),1.50-1.52(m,1H),1.25-1.27(m,38H),0.87-0.91(m,6H).

[0079] Example 10

[0080]

[0081] Compound 7-1 (1.83 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 1-2 (5.85 g, 14.33 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 10-2. Triethylamine (1 ml) and acetic anhydride (2.7 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-10 (5.94 g, yield 80%). 1H NMR(400MHz,Chloroform-d)δ4.18-4.20(s,2H),2.62-2.87(m,2H),2.53-2.54(m,1H),2.30 -2.34(m,8H),2.12-2.16(s,3H),1.50-1.52(m,1H),1.25-1.27(m,38H),0.87-0.91(m,6H).

[0082] Embodiment 11

[0083]

[0084] Compound 11-1 (1.55 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 1-2 (5.85 g, 14.33 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 11-2. Triethylamine (1 ml) and acetic anhydride (2.7 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-11 (5.42 g, yield 76%). 1H NMR(400MHz,Chloroform-d)δ8.41-8.43(s,1H),8.62-8.64(d,1H),7.63-7.65(m,1H),7.28-7.30(t,1H),4.72 -4.74(s,2H),2.62-2.87(m,2H),2.53-2.54(m,1H),1.50-1.52(m,1H),1.25-1.27(m,38H),0.87-0.91(m,6H).

[0085] Example 12

[0086]

[0087] Compound 12-1 (1.75 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 1-2 (5.85 g, 14.33 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 12-2. Triethylamine (1 ml) and acetic anhydride (2.7 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-12 (6.02 g, yield 82%). 1H NMR(400MHz,Chloroform-d)δ8.57-8.59(s,1H),8.62-8.64(d,1H),7.73-7.75(m,1H),7.28-7.30(d,1H),4.72-4.74(s, 2H),2.62-2.87(m,2H),2.53-2.54(m,1H),2.41-2.43(s,3H),1.50-1.52(m,1H),1.25-1.27(m,38H),0.87-0.91(m,6H).

[0088] Example 13

[0089]

[0090] Compound 13-1 (2.08 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced for protection. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 13-2 (6.08 g, 28.66 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 13-3. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-13 (5.96 g, yield 78%). 1H NMR(400MHz,Chloroform-d)δ3.16-3.20(t,4H),2.62-2.87(m,3H),2.34-2.38(t,4H),2.1 8-2.20(s,3H)1.73-1.75(m,4H),1.49-1.51(m,4H),1.25-1.27(m,24H),0.87-0.91(m,6H).

[0091] Embodiment 14

[0092]

[0093] Compound 13-1 (2.08 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced for protection. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 14-1 (6.88 g, 28.66 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 14-2. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-14 (6.41 g, yield 76%). 1H NMR(400MHz,Chloroform-d)δ3.16-3.20(t,4H),2.62-2.87(m,3H),2.34-2.38(t,4H),2.18 -2.20(s,3H),1.73-1.75(m,4H),1.49-1.51(m,4H),1.25-1.27(m,32H),0.87-0.91(m,6H).

[0094] Embodiment 15

[0095]

[0096] Compound 13-1 (2.08 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced. The mixture was cooled to 0°C by ice bath, and then succinic anhydride 15-1 (9.28 g, 28.66 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 15-2. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-15 (9.11 g, yield 84%). 1H NMR(400MHz,Chloroform-d)δ3.16-3.20(t,4H),2.62-2.87(m,3H),2.34-2.38(t,4H),2.18 -2.20(s,3H),1.73-1.75(m,4H),1.49-1.51(m,4H),1.25-1.27(m,56H),0.87-0.91(m,6H).

[0097] Example 16

[0098]

[0099] Compound 13-1 (2.08 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced. The mixture was cooled to 0°C by ice bath, and then succinic anhydride 16-1 (10.08 g, 28.66 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 16-2. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-16 (9.32 g, yield 84%). 1H NMR(400MHz,Chloroform-d)δ3.16-3.20(t,4H),2.62-2.87(m,3H),2.34-2.38(t,4H),2.18 -2.20(s,3H),1.73-1.75(m,4H),1.49-1.51(m,4H),1.25-1.27(m,64H),0.87-0.91(m,6H).

[0100] Embodiment 17

[0101]

[0102] Compound 13-1 (2.08 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced. The mixture was cooled to 0°C by ice bath, and then succinic anhydride 17-1 (10.04 g, 28.66 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 17-2. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-17 (9.27 g, yield 80%). 1H NMR(400MHz,Chloroform-d)δ5.43-5.48(m,4H),3.16-3.20(t,4H),2.62-2.87(m,6H),2.34-2.38(t,4H),2.28 -2.30(m,2H),2.18-2.20(s,3H),1.92-1.96(q,4H),1.73-1.75(m,4H),1.25-1.40(m,48H),0.87-0.91(m,12H).

[0103] Embodiment 18

[0104]

[0105] Compound 13-1 (2.08 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 1-2 (11.69 g, 28.66 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 18-1. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-18 (10.47 g, yield 79%). 1H NMR(400MHz,Chloroform-d)δ3.16-3.20(t,4H),2.62-2.87(m,4H),2.18-2.20(s, 3H),1.73-1.75(m,4H),1.49-1.51(m,2H),1.19-1.26(m,76H),0.87-0.91(m,12H).

[0106] Embodiment 19

[0107]

[0108] Compound 13-1 (2.08 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 19-1 (6.02 g, 28.66 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 19-2. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-19 (6.06 g, yield 80%). The tail of the long hydrophobic chain of the compound is a C8 straight alkenyl group, in which the distance between the alkenyl group and the cyclic imide nitrogen heterocyclic ring is 2 carbon atoms. 1H NMR (400MHz, Chloroform-d) δ5.41-5.43 (m, 2H), 3.16-3.20 (t, 4H), 2.62-2.87 (m, 6H), 2.06-2.31 (m, 4H) 2.01-2.03 (m, 4H), 1.73-1.75 (m, 4H), 1.25-1.40 (m, 12H), 0.87-0.91 (m, 6H).

[0109] Embodiment 20

[0110]

[0111] Compound 13-1 (2.08 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced for protection. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 20-1 (6.82 g, 28.66 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 20-2. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for another 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-20 (7.13 g, yield 85%). The long-chain hydrophobic chain tail of the compound is a C 10Straight chain alkenyl, in which the alkenyl is 2 carbon atoms away from the cyclic imide nitrogen heterocycle. 1H NMR (400MHz, Chloroform-d) δ5.41-5.43 (m, 2H), 3.16-3.20 (t, 4H), 2.62-2.87 (m, 4H), 2.16-2.18 (s, 3H), 2.06-2.31 (m, 8H), 2.01-2.03 (m, 4H), 1.73-1.75 (m, 4H), 1.25-1.40 (m, 20H), 0.87-0.91 (m, 6H).

[0112] Embodiment 21

[0113]

[0114]

[0115] Compound 13-1 (2.08 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced for protection. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 21-1 (7.63 g, 28.66 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 21-2. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for another 20 hours to complete the chemical ring closure reaction. Extraction was then performed and the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was subjected to rotary evaporation to obtain a crude product, which was then purified using a silica gel column and finally dried by rotary evaporation to obtain compound IL-21 (7.44 g, yield 81%). The long-chain hydrophobic chain tail of the compound is a C 12 Straight chain alkenyl, in which the alkenyl is 2 carbon atoms away from the cyclic imide nitrogen heterocycle. 1H NMR (400MHz, Chloroform-d) δ5.41-5.43 (m, 2H), 3.16-3.20 (t, 4H), 2.62-2.87 (m, 4H), 2.16-2.18 (s, 3H), 2.06-2.31 (m, 8H), 2.01-2.03 (m, 4H), 1.73-1.75 (m, 4H), 1.25-1.40 (m, 28H), 0.87-0.91 (m, 6H).

[0116] Embodiment 22

[0117]

[0118] Compound 13-1 (2.08 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced for protection. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 22-1 (10.03 g, 28.66 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 22-2. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for another 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-22 (9.97 g, yield 86%). The long-chain hydrophobic chain tail of the compound is a C 18 Straight chain alkenyl, in which the alkenyl is 2 carbon atoms away from the cyclic imide nitrogen heterocycle. 1H NMR (400MHz, Chloroform-d) δ5.41-5.43 (m, 2H), 3.16-3.20 (t, 4H), 2.62-2.87 (m, 6H), 2.36-2.38 (m, 4H), 2.16-2.18 (s, 3H), 2.06-2.31 (m, 8H), 2.01-2.03 (m, 4H), 1.73-1.75 (m, 4H), 1.25-1.40 (m, 48H), 0.87-0.91 (m, 6H).

[0119] Embodiment 23

[0120]

[0121] Compound 13-1 (2.08 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced for protection. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 23-1 (7.62 g, 28.66 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 23-2. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for another 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-23 (7.07 g, yield 77%). The long-chain hydrophobic chain tail of the compound is a C 12Straight chain alkenyl, in which the alkenyl is 1 carbon atom away from the cyclic imide nitrogen heterocycle. 1H NMR (400MHz, Chloroform-d) δ5.57-5.72 (m, 4H), 3.40-3.42 (m, 2H), 3.16-3.20 (t, 4H), 2.62-2.87 (m, 4H), 2.36-2.38 (m, 4H), 2.16-2.18 (s, 3H), 1.93-1.95 (m, 4H), 1.73-1.75 (m, 4H), 1.25-1.40 (m, 32H), 0.87-0.91 (m, 6H).

[0122] Embodiment 24

[0123]

[0124]

[0125] Compound 13-1 (2.08 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced. The mixture was cooled to 0°C by ice bath, and then succinic anhydride 24-1 (10.03 g, 28.66 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 24-2. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-24 (9.74 g, yield 84%). The tail of the long hydrophobic chain of the compound is composed of a tertiary carbon group and two C8 straight-chain hydrocarbon groups to form a branched hydrocarbon group, one of which contains an olefinic group that is 2 carbon atoms away from the cyclic imide nitrogen heterocyclic ring. 1HNMR (400MHz, Chloroform-d) δ5.43-5.48 (m, 4H), 3.16-3.20 (t, 4H), 2.62-2.87 (m, 6H), 2.36-2.38 (m, 4H), 2.26-2.30 (m, 2H), 2.16-2.18 (s, 3H), 1.93-1.95 (m, 4H), 1.73-1.75 (m, 4H), 1.25-1.40 (m, 48H), 0.87-0.91 (m, 12H).

[0126] Embodiment 25

[0127]

[0128] Compound 13-1 (2.08 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced. The mixture was cooled to 0°C by ice bath, and then succinic anhydride 25-1 (7.68 g, 28.66 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 25-2. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-25 (7.39 g, yield 80%). 1H NMR(400MHz,Chloroform-d)δ3.16-3.20(t,4H),2.62-2.87(m,6H),2.34-2.38(t,4H),2.18 -2.20(s,3H),1.73-1.75(m,4H),1.49-1.51(m,4H),1.25-1.27(m,40H),0.87-0.91(m,6H).

[0129] Embodiment 26

[0130]

[0131] Compound 26-1 (1.67 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced. The mixture was cooled to 0°C by ice bath, and then succinic anhydride 25-1 (7.68 g, 28.66 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 26-2. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified by a silica gel column and finally rotary evaporated to obtain compound IL-26 (6.90 g, yield 78%). 1H NMR(400MHz,Chloroform-d)δ3.56-3.58(t,4H),2.62-2.87(m,6H),2.51-2.55(m, 4H),2.18-2.20(s,3H),1.49-1.51(m,4H),1.25-1.27(m,40H),0.87-0.91(m,6H).

[0132] Embodiment 27

[0133]

[0134] Compound 27-1 (2.11 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced. The mixture was cooled to 0°C by ice bath, and then succinic anhydride 25-1 (7.68 g, 28.66 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 27-2. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified by a silica gel column and finally rotary evaporated to obtain compound IL-27 (7.42 g, yield 80%). 1H NMR(400MHz,Chloroform-d)δ3.56-3.58(t,4H),3.41-3.43(t,2H),2.62-2.87(m, 6H),2.53-2.57(m,6H),1.49-1.51(m,4H),1.25-1.27(m,40H),0.87-0.91(m,6H).

[0135] Embodiment 28

[0136]

[0137] Compound 28-1 (2.86 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced. The mixture was cooled to 0°C by ice bath, and then succinic anhydride 25-1 (7.68 g, 28.66 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 28-2. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified by a silica gel column and finally rotary evaporated to obtain compound IL-28 (8.13 g, yield 81%). 1H NMR(400MHz,Chloroform-d)δ3.16-3.19(t,4H),2.62-2.87(m,6H),2.34-2.38(t,4H),2.27 -2.31(s,8H),1.71-1.75(m,4H),1.49-1.51(m,4H),1.25-1.27(m,40H),0.87-0.91(m,6H).

[0138] Embodiment 29

[0139]

[0140] Compound 29-1 (2.10 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced. The mixture was cooled to 0°C by ice bath, and then succinic anhydride 25-1 (11.52 g, 42.99 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 29-2. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified by a silica gel column and finally rotary evaporated to obtain compound IL-29 (10.87 g, yield 86%). 1H NMR (400MHz, Chloroform-d) δ3.56-3.58(t,6H),2.62-2.87(m,9H),2.51-2.53(t,6H),1.49-1.51(m,6H),1.25-1.27(m,60H),0.87-0.91(m,9H).

[0141] Embodiment 30

[0142]

[0143] Compound 30-1 (2.70 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced. The mixture was cooled to 0°C by ice bath, and then succinic anhydride 25-1 (11.52 g, 42.99 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 30-2. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-30 (10.59 g, yield 80%). 1H NMR(400MHz,Chloroform-d)δ3.16-3.20(t,6H),2.62-2.87(m,9H),2.34-2.36(t, 6H),1.71-1.75(m,6H),1.49-1.51(m,6H),1.25-1.27(m,60H),0.87-0.91(m,9H).

[0144] Embodiment 31

[0145]

[0146] Compound 31-1 (4.53 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced. The mixture was cooled to 0°C by ice bath, and then succinic anhydride 25-1 (11.52 g, 42.99 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 31-2. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified by a silica gel column and finally rotary evaporated to obtain compound IL-31 (15.38 g, yield 82%). 1H NMR(400MHz,Chloroform-d)δ3.16-3.20(t,8H),2.62-2.87(m,12H),2.34-2.36(m,1 2H),1.73-1.76(m,12H),1.49-1.51(m,8H),1.25-1.27(m,80H),0.87-0.91(m,12H).

[0147] Embodiment 32

[0148]

[0149] Compound 32-1 (1.99 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 1-2 (5.85 g, 14.33 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 9-2. Triethylamine (1 ml) and acetic anhydride (2.7 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-32 (5.53 g, yield 76%). 1H NMR(400MHz,Chloroform-d)δ7.91-7.93(s,1H),7.15-7.17(d,1H),6.76-6.78(d,1H),4.04-4.06(t,2H),3.18-3.20(t, 2H),2.62-2.87(m,2H),2.56-2.58(m,2H),2.51-2.55(m,3H),1.50-1.52(m,1H),1.25-1.27(m,38H),0.87-0.91(m,6H).

[0150] Embodiment 33

[0151]

[0152] Compound 30-1 (2.70 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced for protection. The mixture was cooled to 0°C through an ice bath, and then succinic anhydride 21-1 (11.52 g, 42.99 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 33-2. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for another 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-33 (10.65 g, yield 85%). The compound has three independent C 12The hydrophobic chain tail, in which the alkenyl group is one carbon atom away from the adjacent cyclic imide nitrogen heterocycle. 1H NMR (400MHz, Chloroform-d) δ3.16-3.20 (t, 6H), 2.62-2.87 (m, 9H), 2.34-2.36 (t, 6H), 1.71-1.75 (m, 6H) 1.49-1.51 (m, 6H), 1.25-1.27 (m, 60H), 0.87-0.91 (m, 9H).

[0153] Embodiment 34

[0154]

[0155] Compound 31-1 (4.53 g, 14.33 mmol) and 15 ml of dichloromethane were added to a 100 ml round-bottom flask, plugged with a rubber stopper, and nitrogen was introduced. The mixture was cooled to 0°C by ice bath, and then succinic anhydride 21-1 (11.52 g, 42.99 mmol) and an additional 15 ml of dichloromethane were gradually added dropwise. The solution was then stirred for 24 hours to obtain the amide intermediate 34-2. Triethylamine (2 ml) and acetic anhydride (5.4 ml) were then added and stirred for 20 hours to complete the chemical ring closure reaction. After extraction, the residual water in the dichloromethane solution was removed with anhydrous sodium sulfate. The dried solution was rotary evaporated to obtain a crude product, which was then purified using a silica gel column and finally rotary evaporated to obtain compound IL-34 (15.15 g, yield 80%). 1H NMR(400MHz,Chloroform-d)δ3.16-3.20(t,8H),2.62-2.87(m,12H),2.34-2.36(m,1 2H),1.73-1.76(m,12H),1.49-1.51(m,8H),1.25-1.27(m,80H),0.87-0.91(m,12H).

[0156] Embodiment 35

[0157] This embodiment provides a method for further preparing the cyclic imide lipid compound obtained in Example 1-34 into LNP lipid nanoparticles. The cyclic imide lipid compound, DSPC, cholesterol and DMG-PEG 2000 are dissolved in an ethanol solution according to a molar ratio of 50:10:38.5:1.5, and the total lipid concentration is 12mM. The mRNA containing the firefly luciferase (luciferase) or green fluorescent protein (eGFP) reporter gene is dissolved in a citric acid-sodium citrate buffer at pH=4.0 according to N:P=6:1. The ethanol lipid solution and the mRNA buffer are mixed according to a volume ratio of 1:3 using a microfluidic device. The collected product is placed in a dialysis bag (molecular weight cutoff 100kDa) and dialyzed in PBS at pH=7.4 for 12 hours, then the mRNA-LNP is concentrated with an ultrafiltration tube, and the mRNA is fixed to 100μg / ml with a buffer, and frozen to obtain IL-X-LNP ("X" represents the corresponding embodiment number).

[0158] Test Example 1 Characterization and Determination of LNP Encapsulation Efficiency

[0159] The compound IL-X-LNP was prepared according to some of the above examples, and eGFP mRNA-LNP was prepared by the preparation method provided in Example 35. The LNP encapsulation efficiency (EE%), particle size (nm) and PDI (Polydispersity Index) were determined by the following methods.

[0160] Experimental methods

[0161] 1) Particle size analysis test: Use a quartz cuvette with a specification of 10mm*10mm*40mm as the sample dish, and use a dynamic light scattering (DLS) to test the particle size and distribution of the sample solution. The test is performed twice in a row with an interval of 30s. Finally, the average value of the two tests is taken as the final data, and the particle size and PDI are recorded.

[0162] 2) Determination of mRNA encapsulation efficiency: using Quant-it TM The encapsulation efficiency was determined using the RiboGreen RNA Assay Kit.

[0163] a. Dilute the prepared eGFP mRNA-LNP suspension and PBS (negative control, equal volume of TE buffer) to 4 ng / μl with TE buffer in the kit to obtain eGFP mRNA-LNP working solution.

[0164] b. Continue to use TE buffer (or TE buffer containing 2% Triton-X100) to further dilute the eGFP mRNA-LNP working solution with equal volume, mix well and let it stand at 37°C for 10 minutes (TE buffer without Triton-X100 is used to determine the unencapsulated free mRNA, while TE buffer containing 2% Triton-X100 is used to determine the Total mRNA in the eGFP mRNA-LNP working solution, which includes free mRNA and mRNA encapsulated in lipid nanoparticles).

[0165] c. After calibrating the standard curve of fluorescence intensity: concentration with the standard product, pipette an appropriate amount of Quant-it TM RiboGreen RNA reagent nucleic acid dye was added to each group of samples and stained for 5 minutes. Each group of stained samples was transferred to an ELISA reader for detection, and the mRNA in the samples was accurately quantified using software.

[0166] d. The mRNA encapsulation efficiency in lipid nanoparticles was calculated using the following formula:

[0167] Encapsulation efficiency (%) = [1-m (游离mRNA) / m (总mRNA) ]×100%.

[0168] Experimental Results

[0169] As shown in Table 1, lipid nanoparticles containing cyclic imide lipid components have a narrow particle size distribution, and the size is suitable for the delivery of nucleic acid drugs. At the same time, thanks to the fact that ionizable lipids can load mRNA well in acidic buffer, the prepared eGFP mRNA-LNP has a high mRNA encapsulation rate.

[0170] Table 1 Encapsulation efficiency and particle size

[0171]

[0172]

[0173] Experimental Example 2 In vitro mRNA translation expression evaluation experiment

[0174] The compound IL-X-LNP ("X" represents the corresponding example number) was prepared using some of the above examples, and eGFP mRNA-LNP was prepared using the preparation method provided in Example 35. The positive cell ratio of each group was analyzed by in vitro transfection experiments.

[0175] Experimental methods

[0176] DC 2.4 cells were cultured in 1640 medium supplemented with 10% (V / V) fetal bovine serum FBS (Gibco) and 1% (V / V) P / S antibody until the logarithmic growth phase. The cells were seeded into 24-well plates at a number of 100,000 per well and transfected with IL-X-LNP containing 2μg eGFP mRNA. 24 hours after transfection, the cell culture medium was aspirated, and the cells were washed slowly and gently with PBS, and then the positive cell ratio of each group was analyzed by flow cytometry.

[0177] Experimental Results

[0178] Please refer to the results Figure 1 As shown, lipids containing 1 cyclic imide group (IL-2-LNP, IL-5-LNP, IL-8-LNP, IL-9-LNP, IL-12-LNP), lipids containing 2 cyclic imide groups (IL-15-LNP, IL-18-LNP, IL-21-LNP, IL-22-LNP, IL-23-LNP, IL-24-LNP, IL-25-LNP, IL-28-LNP), lipids containing 3 cyclic imide groups (IL-30-LNP, IL-33-LNP), and lipids containing 4 cyclic imide groups (IL-31-LNP) all have better gene expression effects in DC 2.4 cells. From a trend, the lipid transfection effect containing multiple cyclic imide groups seems to be slightly higher than the lipid containing a single cyclic imide group. The above results illustrate that the lipid nanoparticles prepared by the cyclic imide lipids proposed by the present invention have a higher transfection efficiency of mRNA nucleic acid. Experimental Example 3 In vitro cytotoxicity study of cyclic imide lipids

[0179] The compound IL-X-LNP was prepared by the above-mentioned examples, and eGFP mRNA-LNP was prepared by the preparation method provided in Example 35, and its cytotoxicity was evaluated by MTT method. Meanwhile, SM-102LNP (Avito Company, batch number SM220908) was used as a control.

[0180] Experimental methods

[0181] DC 2.4 cells were cultured in 1640 medium supplemented with 10% (V / V) fetal bovine serum (FBS) (Gibco) and 1% (V / V) P / S antibody until the logarithmic growth phase. 4cells / well, 100 μL / well of the cell suspension was inoculated into a 96-well plate. After inoculation, the culture was incubated in a cell culture incubator at 37°C and 4% CO2 for 24 hours. After that, different test samples of 0.1, 0.5 and 2.5 μg / ml were added to each well. The test sample was IL-X-LNP (X=1-34) prepared in Example 35. Four replicates were made in each group. After 24 hours of co-incubation, 25 μL of 5 mg / mL MTT in PBS buffer was added to each well. Incubate for another 2 hours, then discard the mixed solution of the culture medium and MTT buffer, add 150 μL / well of DMSO to dissolve the purple crystals of formazan in living cells, and after sufficient oscillation, test the absorbance at 490 nm with an enzyme marker. Calculate and draw a graph based on the measured absorbance value.

[0182] Experimental Results

[0183] As shown in Table 2, the lipid nanoparticles prepared from the cyclic imide lipid compound proposed in the present invention have a cell survival of greater than 95% under the condition of an mRNA concentration of 0.1 μg / ml. Under the condition of an mRNA concentration of 0.5 μg / ml, the cell survival is greater than 85%. Under the condition of an mRNA concentration of 2.5 μg / ml, the cell survival is greater than 80%. The SM-102 liposomes showed lower cytotoxicity when added at a bottom concentration, and showed higher cytotoxicity when the addition amount reached 2.5 μg / ml. Please refer to the figure, taking the theoretical hydrolysis path of the cyclic imide lipid compound IL-23 as an example, under the action of a hydrolase, the cyclic imide can be hydrolyzed and opened, and the amide bond can be further degraded, indicating that the cyclic imide lipid nanoparticles of the present invention have good formulation safety and biocompatibility.

[0184] Table 2 Cell viability (%)

[0185]

[0186] Experimental Example 4: Expression evaluation experiment of firefly luciferase mRNA in mice

[0187] The compound IL-X-LNP was prepared according to some of the above examples, and luc mRNA-LNP was prepared by the preparation method provided in Example 35, which was used to evaluate the expression of mRNA delivered by LNP in animal models.

[0188] Experimental methods

[0189] BALB / c mice aged 4-6 weeks were subcutaneously injected with 20 μg of Fluc-mRNA delivered by different delivery vectors at the base of the tail. Four hours after administration, 100 μL of luciferin at a concentration of 15 mg / ml was intraperitoneally injected. Ten minutes later, the distribution of fluorescent signals was observed using an IVIS mouse in vivo imager (PerkinElmer).

[0190] Experimental Results

[0191] In order to directly evaluate the translational expression efficiency of different IL-X-LNP delivery systems in mice, different delivery vectors were used to encapsulate luc-mRNA. Figure 3 As shown, the LNP containing cyclic imide lipids proposed by the present invention can help mRNA coding to be efficiently expressed in vivo, and the expression efficiency is better than SM-102. At the same time, the mice survived well and had good safety.

[0192] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A lipid compound, the structural formula of which is shown in formula (I): in, R1 is independently selected from C1-C3 alkyl, C1-C3 alkoxy or C1-C3 hydroxyalkyl; X is as shown in formula (II), and R2 and R3 are each independently selected from hydrogen or C8-C 36 When R2 and / or R3 are branched hydrocarbon groups, the branched hydrocarbon group includes at least two C4-C 18 A saturated or unsaturated straight-chain hydrocarbon group; Y is independently selected from a tertiary amine group, a substituted or unsubstituted azaaromatic ring, or a substituted or unsubstituted azaaliphatic ring containing at least one tertiary amine group; L is independently selected from C1-C3 straight chain alkyl; i is an integer greater than or equal to 0, j is an integer greater than 1, and k is 0 or 1; and i+j+k≤3, p=k+1.

2. A lipid compound according to claim 1, characterized in that R2 is a hydrogen atom, and R3 is selected from C8-C 22 When R3 is a branched hydrocarbon group, the branched hydrocarbon group includes at least two C9-C 11 A saturated or unsaturated straight chain hydrocarbon group.

3. A lipid compound according to claim 2, characterized in that, R3 is selected from C8-C 18 Straight chain alkyl or C8-C 18 The straight-chain alkenyl group comprises at least one alkenyl group which is no more than 2 carbon atoms away from the cyclic imide nitrogen heterocycle.

4. A lipid compound according to claim 2, characterized in that, R3 is selected from C 18 -C 22 A branched hydrocarbon group consisting of a tertiary carbon group and two C8-C 11 Straight chain hydrocarbon composition.

5. A lipid compound according to claim 4, characterized in that, The two C8-C 11 The straight-chain hydrocarbon group contains at least one straight-chain alkenyl group, and the straight-chain alkenyl group contains at least one alkenyl group which is not more than 2 carbon atoms away from the cyclic imide nitrogen heterocycle.

6. A lipid compound according to any one of claims 1 to 5, characterized in that Y is a substituted or unsubstituted azaaromatic ring, i=0, j=1, and the substituents of the azaaromatic ring are independently selected from methyl or phenyl.

7. A lipid compound according to any one of claims 1 to 6, characterized in that The azaaromatic ring is selected from substituted or unsubstituted pyridine, pyrimidine, pyrrole, imidazole, thiophene or thiazine.

8. A lipid compound according to any one of claims 1 to 7, characterized in that: The lipid compound is:

9. A lipid compound according to any one of claims 1 to 5, characterized in that: Y is a substituted or unsubstituted azaalicyclic ring containing at least one tertiary amine group, i=0, j=1 or 2, and the substituents of the azaalicyclic ring are independently selected from C1-C3 alkyl groups or C1-C3 alkyl ether groups.

10. A lipid compound according to any one of claims 1 to 5 or 9, characterized in that: The azaaliphatic ring contains a nitrogen heteroatom which is not directly attached to the L group and is substituted by a C1-C3 alkyl group.

11. A lipid compound according to any one of claims 1 to 5 or 9 to 10, characterized in that: Y is selected from substituted or unsubstituted azetidine, pyrrole, piperidine, piperazine or morpholine.

12. A lipid compound according to any one of claims 1-5 or 9-11, characterized in that: The compound is selected from:

13. A lipid compound according to any one of claims 1 to 5, characterized in that: Y is a tertiary amine group, and When k=0, i+j=3; or When k=1, i+j=2.

14. A lipid compound according to any one of claims 1 to 5 or 13, characterized in that The compound is selected from:

15. Use of a lipid compound as described in any one of claims 1 to 14 in preparing LNP lipid nanoparticles.

16. A LNP lipid nanoparticle, characterized in that: The method comprises the lipid compound as described in any one of claims 1 to 14, auxiliary phospholipids, cholesterol and pegylated lipids.

17. Use of a LNP lipid nanoparticle as claimed in claim 16 in nucleic acid molecule delivery.

18. A method for preparing a lipid compound, characterized in that: Compound (III) is mixed with a portion of the solvent and placed in an ice bath, compound (IV) is added dropwise under the protection of an inert gas, and the remaining solvent is added to obtain an intermediate product; an organic base and acetic anhydride are added to the intermediate product to carry out a ring-closure reaction, and the lipid compound (I) is obtained after purification; Wherein, R1 is independently selected from C1-C3 alkyl, C1-C3 alkoxy or C1-C3 hydroxyalkyl; X is as shown in formula (II), and R2 and R3 are each independently selected from hydrogen or C8-C 36 When R2 and / or R3 are branched hydrocarbon groups, the branched hydrocarbon group includes at least two C4-C 18 A saturated or unsaturated straight chain hydrocarbon group; Y is independently selected from a tertiary amine group, a substituted or unsubstituted nitrogen heteroaromatic ring or a substituted or unsubstituted nitrogen heteroaliphatic ring containing at least one tertiary amine group; L is independently selected from C1-C3 straight chain alkyl; i is an integer greater than or equal to 0, j is an integer greater than 1, and k is 0 or 1; and i+j+k≤3, p=k+1.

19. The preparation method according to claim 18, characterized in that: The solvent is selected from any one or more combinations of dichloromethane, chloroform, ethyl acetate, N-methyl-2-pyrrolidone, N,N-dimethylformamide, and 1,4-dioxane.

20. The preparation method according to claim 18 or 19, characterized in that: The organic base is selected from any one or more combinations of triethylamine, trimethylamine, tributylamine and pyridine.

21. The preparation method according to any one of claims 18 to 20, characterized in that: R2 is a hydrogen atom, and R3 is selected from C8-C 22 When R3 is a branched hydrocarbon group, the branched hydrocarbon group includes at least two C9-C 11 A saturated or unsaturated straight chain hydrocarbon group.

22. A lipid compound according to claim 21, characterized in that R3 is selected from C8-C 18 Straight chain alkyl or C8-C 18 The straight-chain alkenyl group comprises at least one alkenyl group which is no more than 2 carbon atoms away from the cyclic imide nitrogen heterocycle.

23. A lipid compound according to claim 21, characterized in that: R3 is selected from C 18 -C 22 A branched hydrocarbon group consisting of a tertiary carbon group and two C8-C 11 Straight chain hydrocarbon composition.

24. A lipid compound according to claim 22, characterized in that: The two C8-C 11 The straight-chain hydrocarbon group contains at least one straight-chain alkenyl group, and the straight-chain alkenyl group contains at least one alkenyl group which is not more than 2 carbon atoms away from the cyclic imide nitrogen heterocycle.

25. A lipid compound according to any one of claims 20 to 24, characterized in that: Y is selected from a tertiary amino group, a substituted or unsubstituted pyridine, pyrimidine, pyrrole, imidazole, thiophene, thiazine, azetidine, tetrahydropyrrole, piperidine, piperazine or morpholine.

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