Quinoline lipid derivative and use thereof

Abstract

Disclosed in the present invention are a quinoline lipid derivative and the use thereof. The general formula of the structure thereof is as shown in formula I. A lipid nanoparticle containing the quinoline ionizable lipid compound provided in the present invention has high in-vitro and in-vivo mRNA delivery efficiency, and the delivery efficiency thereof at the cellular level is better than that of commercial agents, i.e. Lipofectamine 2000 and Lipofectamine MessengerMAX. The lipid nanoparticle containing the quinoline ionizable lipid compound is intramuscularly administrated, which can realize efficient local expression of mRNA.

Description

Quinoline lipid derivatives and uses thereof

[0001] This application claims priority to Chinese patent application No. 202410213298.8, entitled Quinoline lipid derivatives and their uses, filed by the applicant Shenzhen People's Hospital on February 27, 2024. The entire contents of the application are incorporated herein by reference. Technical Field

[0002] The present disclosure belongs to the field of medicine, and in particular relates to quinoline lipid derivatives and uses thereof. Background Art

[0003] Nucleic acid drugs, such as messenger RNA (mRNA), antisense oligonucleotides, and small interfering RNA (siRNA), have broad application prospects, but the development of safe and effective nucleic acid delivery systems is a challenge that restricts the development of such drugs. Lipid nanoparticles (LNPs) are an advanced nucleic acid drug delivery vehicle. Currently widely used LNPs are mainly composed of ionizable lipids and auxiliary lipids (such as phospholipids, structural lipids, polyethylene glycol lipids, etc.). LNPs bind to nucleic acids through electrostatic interactions to form stable nanoparticles, protecting the nucleic acid molecules encapsulated in the LNPs from degradation by nucleases and recognition by the body's immune system. Finally, the exogenous nucleic acid molecules are internalized by the LNPs and escape from endosomal endosomes to exert their functional activity within the cell. LNPs have advantages in delivery efficiency, targeting, stability, etc., and their safety and effectiveness have also been verified in clinical applications. Therefore, the development of new LNPs based on ionizable lipids has broad application prospects.

[0004] SUMMARY OF THE INVENTION

[0005] The present invention provides a novel class of ionizable lipid compounds containing a quinoline nucleus. The side chain at the 4-position of the quinoline ring is connected to a structural segment containing an ionizable nitrogen atom (called a linker arm), which is then linked to several hydrocarbon groups to form a tertiary amine. When these ionizable lipid compounds are combined with other auxiliary lipid components to form lipid nanoparticles, they can deliver mRNA to cells and efficiently translate it into the target protein. Their in vitro delivery performance far surpasses that of commercial reagents such as Lipofectamine 2000 and Lipofectamine MessengerMAX. Furthermore, these quinoline lipid nanoparticles can achieve efficient nucleic acid delivery in vivo. Technical issues

[0006] Develop novel structural quinoline-based ionizable lipid compounds, and new LNPs based on such ionizable lipid compounds have high nucleic acid delivery efficiency. Technical Solutions

[0007] The quinoline ionizable lipid compound provided by the present invention has a general structural formula as shown in Formula I:

[0008] In Formula I, R 1 represents the substituent on the quinoline ring, which can be single-substituted or multi-substituted, R 1 Each is independently selected from the group consisting of: deuterium, tritium, halogen (F, Cl, Br, I), halogen-substituted C1-C6 alkyl, C1-C6 alkoxy, aromatic-substituted C1-C6 alkyl, aromatic-substituted C1-C6 alkoxy, and an aromatic group formed between two adjacent carbon atoms on a quinoline ring, wherein the aromatic group is a substituted or unsubstituted aromatic group, and the H on at least one carbon atom on the aromatic ring of the substituted aromatic group is substituted by deuterium, tritium, halogen (F, Cl, Br, I), halogen-substituted C1-C6 alkyl, or C1-C6 alkoxy;

[0009] The aromatic group may specifically be phenyl or pyridyl;

[0010] More specifically,

[0011] Contains R 1 The quinoline ring can be selected from any one of the following groups:

[0012] R 2 Each independently selected from hydrogen, deuterium, tritium, C4-C 22 Alkyl or alkenyl, wherein the alkyl and alkenyl may introduce one or more groups selected from halogen, hydroxy, amino, oxo, alkoxycarbonyl, acylamino, alkylacylamino, dialkylacylamino, nitro, amino, alkylamino, dialkylamino, carboxyl, thio and thioalkyl groups;

[0013] Specifically, R 2 Can be H, C8~C 22 alkyl or alkenyl;

[0014] m and n are each independently selected from integers of 1 to 8;

[0015] x is an integer selected from 0 to 8. When x is an integer greater than or equal to 2, the number of methylene groups in each repeating unit is equal or different.

[0016] The present disclosure also provides isotope substitutions of the aforementioned compounds or their salts. Preferably, the isotope substitutions are deuterium atom substitutions.

[0017] Optionally, the quinoline ionizable lipid compound of the present invention is selected from any of the following compounds. The general naming convention for quinoline ionizable lipid compounds is: the abbreviation of the substituent at position 7 of the quinoline nucleus, followed by the number of nitrogen atoms in the linker arm and the number of carbon atoms between the nitrogen atoms, followed by the length and degree of unsaturation of the carbon chain attached to the nitrogen atom. If the carbon chain attached to the nitrogen atom contains a hydroxyl group, the number of hydroxyl groups is added to the end of the name.

[0018] The above-mentioned quinoline ionizable lipid compound is prepared by a method comprising the following steps: allowing the compound represented by formula A to undergo a substitution reaction with the compound represented by formula B to obtain the compound represented by formula C, and allowing the compound represented by formula C to undergo a substitution reaction with the compound represented by formula D to obtain the quinoline ionizable lipid compound represented by formula I, wherein R 2 Does not contain hydroxyl groups;

[0019] In formula A, R 1 Same as R in Formula Ⅰ 1 Definition of;

[0020] In Formula B, the definitions of m, n, and x are the same as those of m, n, and x in Formula I;

[0021] In formula C, R 1 The definitions of m, n, and x are the same as those of R in Formula Ⅰ. 1 , m, n, x definitions;

[0022] In formula D, R 2 The definition of R is the same as that of Formula Ⅰ 2 Definition of .

[0023] R 2 C8~C 22 Alkyl or alkenyl, so that the compound represented by formula C is combined with 1,2-epoxy substituted hydrocarbon group ( wherein R3-CH2-CH2=R2) undergoes a substitution reaction to obtain a quinoline ionizable lipid compound represented by formula I.

[0024] The use of the quinoline ionizable lipid compound represented by the above formula I in the preparation of drug delivery carriers - lipid nanoparticles (LNP) also falls within the scope of protection of the present invention.

[0025] The present disclosure also provides a drug delivery carrier, which includes the above-mentioned quinoline ionizable lipid compound, phospholipid, structured lipid, and polyethylene glycol lipid.

[0026] wherein the phospholipid is selected from 1,2-dierucoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, hydrogenated soybean phosphatidylcholine, 1-palmitoyl-2-oleoyl lecithin, 1-stearoyl-2-oleoyl phosphatidylcholine, 1,2-myristoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero Oleyl-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, distearoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, dioleoylphosphatidylglycerol, 1,2-palmitoylphosphatidylglycerol, 1,2-distearoyl-sn-glycero-3-phosphoglycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol, sphingomyelin, ceramide, cerebroside, cerebroside, or diacylglycerol.

[0027] The structured lipid is selected from at least one of cholesterol, 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, coproposterol, brassicasterol, tomatine, ursolic acid, and α-tocopherol.

[0028] The polyethylene glycol lipid is selected from at least one of PEG-modified phosphatidic acid, PEG-modified phosphatidylethanolamine, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified dialkylglycerol, and PEG-modified diacylglycerol. Preferably, the lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000, distearoyl-rac-glycero-polyethylene glycol 2000, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, PEG-diacylglyceramide, and PEG-1,2-dimyristoyloxypropyl-3-amine.

[0029] In the drug delivery carrier, based on molar proportions, the contents of quinoline ionizable lipid compounds are 10 to 80 parts, phospholipids are 10 to 80 parts, structural lipids are 10 to 80 parts, and polyethylene glycol lipids are 0.01 to 20 parts.

[0030] Specifically, it can include 20 parts of quinoline ionizable lipid compound, 10-50 parts of phospholipid, 20-80 parts of structural lipid, and 0.2-1.45 parts of polyethylene glycol lipid.

[0031] Furthermore, the drug delivery carrier is a spherical lipid nanoparticle with a particle size of 100 to 500 nm.

[0032] The use of the above-mentioned quinoline ionizable lipid compounds and drug delivery carriers in the preparation of drug delivery systems also falls within the scope of protection of the present invention.

[0033] The present invention also provides a drug delivery system - lipid nanoparticles encapsulating active ingredients.

[0034] The drug delivery system provided by the present invention - lipid nanoparticles encapsulating active ingredients, includes quinoline-based ionizable lipid compounds, phospholipids, structural lipids, polyethylene glycol lipids and active ingredients;

[0035] Calculated by mole fraction, the drug delivery system - lipid nanoparticles encapsulating the active ingredient, comprises 10 to 80 parts of quinoline ionizable lipid compounds, 10 to 80 parts of phospholipids, 10 to 80 parts of structural lipids, and 0.01 to 20 parts of polyethylene glycol lipids, and the mass ratio of quinoline ionizable lipid compounds to the active ingredient is 1 to 20:1.

[0036] Specifically, it can be 20 parts of quinoline ionizable lipid compound, 10-50 parts of phospholipid, 20-80 parts of structural lipid, 0.2-1.45 parts of polyethylene glycol lipid, and the mass ratio of quinoline ionizable lipid compound to active ingredient is 2-14:1;

[0037] More specifically, the composition includes 20 parts of quinoline ionizable lipid compound, 50 parts of phospholipid, 60 parts of structural lipid, and 1.1 parts of polyethylene glycol lipid, and the mass ratio of quinoline ionizable lipid compound to active ingredient is 4:1.

[0038] The active ingredient is selected from at least one of DNA, RNA (including any at least one of mRNA, siRNA, aiRNA, miRNA, dsRNA, aRNA, and lncRNA), protein (including any at least one of antibodies, enzymes, recombinant proteins, polypeptides, and short peptides), and pharmaceutically active molecules.

[0039] The lipid nanoparticles carrying the active ingredient are prepared by a method comprising the following steps:

[0040] (A) dissolving and mixing a quinoline-based ionizable lipid compound, optionally with polyethylene glycol lipid, structured lipid, and phospholipid in an organic solvent;

[0041] (B) The resulting mixture is mixed with the active ingredient to form lipid nanoparticles encapsulating the active ingredient.

[0042] In step (A), the organic solvent is preferably methanol and / or ethanol.

[0043] In one embodiment, the present invention further discloses a use of lipid nanoparticles in preparing a drug, wherein an active ingredient is encapsulated in the lipid nanoparticles.

[0044] In one embodiment, the present invention further discloses a method for using a drug, wherein the drug is administered to humans or animals by intravenous injection, intramuscular injection, subcutaneous injection, microneedle patch, oral administration, oral and nasal spray, or application. Beneficial effects

[0045] The advantages of this application are:

[0046] 1. The newly synthesized quinoline-based ionizable lipid compounds feature readily available raw materials, a simple synthetic route, and diverse structures. The starting materials are various quinoline ring derivatives, bromo- or epoxy-substituted alkyl chains of varying lengths, and a polyamine linker. These inexpensive, readily available raw materials offer diverse structures. The target product is obtained via a two-step reaction. First, the 4-position of the quinoline ring derivative is linked to one end of the polyamine linker. This reaction proceeds solvent-free to produce an intermediate, which requires no column chromatography purification and can be used in the next reaction after simple water washing. Bromo- or epoxy-substituted alkyl chains of varying lengths are then attached to the intermediate to yield the product.

[0047] 2. Lipid nanoparticles containing quinoline-based ionizable lipid compounds have high mRNA delivery efficiency in vivo and in vitro, and their delivery efficiency at the cellular level is better than that of the commercial reagents Lipofectamine 2000 and Lipofectamine MessengerMAX.

[0048] 3. Lipid nanoparticles containing quinoline-type ionizable lipid compounds can achieve efficient local expression of mRNA through intramuscular administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIG1 is a synthetic route diagram of the quinoline ionizable lipid compound of the present invention.

[0050] Figure 2 shows the relative delivery activities of Examples 162, 190, 191, 194, and 200, respectively, to Lipofectamine 2000 and Lipofectamine MessengerMAX.

[0051] Figure 3 shows the temporal stability of the particle size of the mRNA delivery system prepared in Example 191 under different storage temperature conditions.

[0052] Figure 4 is a transmission electron micrograph of the mRNA delivery system prepared in Example 191, with a scale bar of 500 nm.

[0053] Figure 5 is a bioluminescence image of in vivo and in vitro organ imaging of mice after intramuscular administration of Example 191. Modes for Carrying Out the Invention

[0054] The present invention provides a class of quinoline-based ionizable lipid compounds, the synthesis methods of which are shown in Examples 1 to 62; the nucleic acid delivery activity of the compounds mixed with phospholipids, PEGylated lipids, and cholesterol to form lipid nanoparticle delivery systems is shown in Examples 63 to 213.

[0055] 1. Synthesis of quinoline-based ionizable lipid compounds

[0056] The general synthetic route is shown in Figure 1. Quinoline ring-containing raw materials and polyamine-containing raw materials are reacted through general step a to obtain an intermediate in which a quinoline core is connected to a polyamine linker. This intermediate is then reacted with a side-chain bromoalkyl group through general step b to obtain Examples 1 to 53. These intermediates are then reacted with a 1,2-epoxy-substituted alkyl group through general step c to obtain Examples 54 to 62. Reaction conditions for the general synthetic route are: a, 130°C for 4 hours; b, K₂CO₃, KI, N,N-dimethylformamide, room temperature for 2 days; c, 1,2-epoxy R₂, methanol, heating at reflux for 2-4 days.

[0057] General Step a: Place quinoline ring-containing starting material (1 equivalent) and polyamine starting material (3 equivalents) into a reaction flask, heat to 130°C, and react for 4 hours. After the reaction is complete, extract with ultrapure water and dichloromethane. The organic phase is washed with ultrapure water and saturated sodium chloride solution, respectively. The organic phase is dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The resulting intermediate is used directly in the next reaction.

[0058] General procedure b: Under nitrogen, dissolve the intermediate obtained in step a in anhydrous dimethylformamide. Add potassium carbonate, potassium iodide, and alkyl bromide sequentially. Allow to react at room temperature for 2 days. After completion of the reaction by thin-layer chromatography, dilute with dichloromethane, wash with ultrapure water and saturated sodium chloride solution, dry over anhydrous sodium sulfate, filter, concentrate, and then chromatograph (dichloromethane:methanol = 10:1) to obtain the final product.

[0059] General Step c: Dissolve the intermediate obtained in Step a in anhydrous methanol, add 1,2-epoxy hydrocarbon, and heat under reflux for 2-4 days. After TLC indicates completion of the reaction, dilute with dichloromethane, then wash with ultrapure water and saturated brine, dry over anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography (dichloromethane:methanol = 10:1) to obtain the final product.

[0060] Example 1. Synthesis of CF3-3N6-C8

[0061] Starting materials 1 (1 mmol) and 2 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate CF3-3N6. Intermediate CF3-3N6 (0.1 mmol), 1-bromooctane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction mixture was heated according to general synthetic procedure b to obtain 30 mg of an oily compound. HRMS (ES): m / z (M+H) + 747.6122.

[0062] Example 2: Synthesis of CF3-3N6-C9

[0063] The intermediate CF3-3N6 (0.1 mmol), 1-bromononane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and 31 mg of an oily product was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) + 789.6526.

[0064] Example 3: Synthesis of CF3-3N6-C10

[0065] The intermediate CF3-3N6 (0.1 mmol), 1-bromodecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and 10 mg of an oily compound was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) + 831.7379.

[0066] Example 4. Synthesis of CF3-3N6-C11

[0067] The intermediate CF3-3N6 (0.1 mmol), 1-bromondecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and 15 mg of an oily compound was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) + 873.7458.

[0068] Example 5. Synthesis of CF3-3N6-C12

[0069] The intermediate CF3-3N6 (0.1 mmol), 1-bromododecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and 55 mg of an oily compound was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) + 915.8504.

[0070] Example 6. Synthesis of CF3-3N6-C13

[0071] The intermediate CF3-3N6 (0.1 mmol), 1-bromotridecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the oily compound (21 mg) was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) + 957.8396.

[0072] Example 7: Synthesis of CF3-3N6-C14

[0073] The intermediate CF3-3N6 (0.1 mmol), 1-bromotetradecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and 13 mg of an oily compound was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) + 999.9261.

[0074] Example 8. Synthesis of CF3-3N6-C15

[0075] The intermediate CF3-3N6 (0.1 mmol), 1-bromopentadecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and 12 mg of an oily compound was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) + 1041.9415.

[0076] Example 9: Synthesis of CF3-3N6-C16

[0077] The intermediate CF3-3N6 (0.1 mmol), 1-bromohexadecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and 8 mg of an oily compound was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) 2+542.4943.

[0078] Example 10: Synthesis of CF3-3N6-C17

[0079] The intermediate CF3-3N6 (0.1 mmol), 1-bromoheptadecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and 15 mg of an oily compound was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) + 1126.0673.

[0080] Example 11. Synthesis of CF3-3N6-C18

[0081] The intermediate CF3-3N6 (0.1 mmol), 1-bromooctadecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and 19 mg of an oily compound was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) 2+ 584.5405.

[0082] Example 12: Synthesis of CF3-3N6-UC18

[0083] The intermediate CF3-3N6 (0.1 mmol), 1-bromo-cis-9-octadecene (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the oily compound (11 mg) was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) + 1161.9140.

[0084] Example 13: Synthesis of CF3-3N6-C22

[0085] The intermediate CF3-3N6 (0.1 mmol), 1-bromodocosane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and 9 mg of an oily compound was obtained according to the general synthetic procedure b. HRMS (ES): m / z (M+H) + 1337.3114.

[0086] Example 14: Synthesis of F-3N6-C12

[0087] Starting materials 3 (1 mmol) and 2 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate F-3N6. Intermediate F-3N6 (0.1 mmol), 1-bromododecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction mixture was followed according to general synthetic procedure b to obtain 20 mg of an oily compound. HRMS (ES): m / z (M+H) + 865.8497.

[0088] Example 15: Synthesis of F-3N6-C17

[0089] Intermediate F-3N6 (0.1 mmol), 1-bromoheptadecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and 16 mg of an oily compound was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) + 1076.0897.

[0090] Example 16: Synthesis of F-3N6-UC18

[0091] Intermediate F-3N6 (0.1 mmol), 1-bromo-cis-9-octadecene (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the oily compound (15 mg) was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) + 1112.0895.

[0092] Example 17: Synthesis of Cl-3N6-C12

[0093] Starting materials 4 (1 mmol) and 2 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate Cl-3N6. Intermediate Cl-3N6 (0.1 mmol), 1-bromododecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction mixture was followed according to general synthetic procedure b to obtain 21 mg of an oily compound. HRMS (ES): m / z (M+H) + 881.8167.

[0094] Example 18: Synthesis of Cl-3N6-C17

[0095] The intermediate Cl-3N6 (0.1 mmol), 1-bromoheptadecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and 19 mg of an oily compound was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) + 1092.0602.

[0096] Example 19: Synthesis of Cl-3N6-UC18

[0097] The intermediate Cl-3N6 (0.1 mmol), 1-bromo-cis-9-octadecene (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and 17 mg of an oily compound was obtained according to the general synthesis procedure 2. HRMS (ES): m / z (M+H) 2+ 564.0189.

[0098] Example 20: Synthesis of Br-3N6-C12

[0099] Starting materials 5 (1 mmol) and 2 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate Br-3N6. Intermediate Br-3N6 (0.1 mmol), 1-bromododecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction mixture was followed according to general synthetic procedure b to obtain 25 mg of an oily compound. HRMS (ES): m / z (M+H) + 927.7717.

[0100] Example 21. Synthesis of Br-3N6-C17

[0101] The intermediate Br-3N6 (0.1 mmol), 1-bromoheptadecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and 19 mg of an oily compound was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) 2+ 569.5075.

[0102] Example 22: Synthesis of Br-3N6-UC18

[0103] The intermediate Br-3N6 (0.1 mmol), 1-bromo-cis-9-octadecene (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the oily compound (21 mg) was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) + 1171.9796.

[0104] Example 23. Synthesis of I-3N6-C12

[0105] Starting materials 6 (1 mmol) and 2 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate I-3N6. Intermediate I-3N6 (0.1 mmol), 1-bromododecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction mixture was followed according to general synthetic procedure b to obtain 22 mg of an oily compound. HRMS (ES): m / z (M+H) + 973.7888.

[0106] Example 24. Synthesis of I-3N6-C17

[0107] Intermediate I-3N6 (0.1 mmol), 1-bromoheptadecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the mixture was prepared according to the general synthesis procedure b to obtain 25 mg of an oily compound. HRMS (ES): m / z (M+H) + 1183.9993.

[0108] Example 25. Synthesis of I-3N6-UC18

[0109] Intermediate I-3N6 (0.1 mmol), 1-bromo-cis-9-octadecene (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the mixture was prepared according to the general synthesis procedure b to obtain 25 mg of an oily compound. HRMS (ES): m / z (M+H) + 1220.0007.

[0110] Example 26: Synthesis of MO-3N6-C12

[0111] Starting materials 7 (1 mmol) and 2 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate MO-3N6. Intermediate MO-3N6 (0.1 mmol), 1-bromododecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction mixture was followed according to general synthetic procedure b to obtain 20 mg of an oily compound. HRMS (ES): m / z (M+H) 2+ 439.4390.

[0112] Example 27: Synthesis of MO-3N6-C17

[0113] Intermediate MO-3N6 (0.1 mmol), 1-bromoheptadecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and 19 mg of an oily compound was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) + 1088.1759.

[0114] Example 28. Synthesis of MO-3N6-UC18

[0115]

[0116] Intermediate MO-3N6 (0.1 mmol), 1-bromo-cis-9-octadecene (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the oily compound (23 mg) was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) 2+ 562.5577.

[0117] Example 29: Synthesis of 2MO-3N6-C12

[0118] Starting materials 8 (1 mmol) and 2 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate 2MO-3N6. Intermediate 2MO-3N6 (0.1 mmol), 1-bromododecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction was continued according to general synthetic procedure b to obtain 31 mg of an oily compound. HRMS (ES): m / z (M+H) + 907.9985.

[0119] Example 30: Synthesis of 2MO-3N6-C17

[0120] Intermediate 2MO-3N6 (0.1 mmol), 1-bromoheptadecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and 18 mg of an oily compound was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) 2+ 559.1627.

[0121] Example 31: Synthesis of 2MO-3N6-UC18

[0122] Intermediate 2MO-3N6 (0.1 mmol), 1-bromo-cis-9-octadecene (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and 21 mg of an oily compound was obtained according to the general synthesis procedure 2. HRMS (ES): m / z (M+H) + 1154.1244.

[0123] Example 32: Synthesis of BMO-3N6-UC18

[0124] Starting materials 9 (1 mmol) and 2 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate BMO-3N6. Intermediate BMO-3N6 (0.1 mmol), 1-bromo-cis-9-octadecene (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction mixture was followed according to general synthetic procedure b to obtain 22 mg of an oily compound. HRMS (ES): m / z (M+H) 2+ 615.5797.

[0125] Example 33: Synthesis of PNT-3N6-C12

[0126] Starting materials 10 (1 mmol) and 2 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate PNT-3N6. Intermediate PNT-3N6 (0.1 mmol), 1-bromododecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction mixture was followed according to general synthetic procedure b to obtain 17 mg of an oily compound. HRMS (ES): m / z (M+H) 2+ 449.9331.

[0127] Example 34: Synthesis of PNT-3N6-C17

[0128] The intermediate PNT-3N6 (0.1 mmol), 1-bromoheptadecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and 19 mg of an oily compound was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) 2+ 555.0518.

[0129] Example 35: Synthesis of PNT-3N6-UC18

[0130] Intermediate PNT-3N6 (0.1 mmol), 1-bromo-cis-9-octadecene (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the oily compound (25 mg) was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) + 1145.1031.

[0131] Example 36: Synthesis of AD-3N6-C12

[0132] Starting materials 11 (1 mmol) and 2 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate AD-3N6. Intermediate AD-3N6 (0.1 mmol), 1-bromododecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction mixture was followed according to general synthetic procedure b to obtain 30 mg of an oily compound. HRMS (ES): m / z (M+H) + 961.8467.

[0133] Example 37. Synthesis of AD-3N6-C17

[0134] Intermediate AD-3N6 (0.1 mmol), 1-bromoheptadecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the oily compound (25 mg) was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) 2+ 586.5296.

[0135] Example 38. Synthesis of AD-3N6-UC18

[0136] Intermediate AD-3N6 (0.1 mmol), 1-bromo-cis-9-octadecene (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the mixture was prepared according to the general synthesis procedure b to obtain 23 mg of an oily compound. HRMS (ES): m / z (M+H) 2+ 604.5448.

[0137] Example 39. Synthesis of CF3-2N4-UC18

[0138] Starting materials 1 (1 mmol) and 12 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate CF3-2N4. Intermediate CF3-2N4 (0.1 mmol), 1-bromo-cis-9-octadecene (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction mixture was followed according to general synthetic procedure b to obtain 26 mg of an oily compound. HRMS (ES): m / z (M+H) + 784.6671.

[0139] Example 40: Synthesis of CF3-2N6-UC18

[0140] Starting materials 1 (1 mmol) and 13 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate CF3-2N6. Intermediate CF3-2N6 (0.1 mmol), 1-bromo-cis-9-octadecene (0.3 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction mixture was followed according to general synthetic procedure b to obtain 21 mg of an oily compound. HRMS (ES): m / z (M+H) + 812.6930. 1 H NMR (400 MHz, deuterated chloroform) δ 8.62 (d, J = 5.3 Hz, 1H), 8.27 (s, 1H), 7.87 (d, J = 8.7 Hz, 1H), 7.60–7.56 (m, 1H), 6.50 (d, J = 5.3 Hz, 1H), 5.46–5.27 (m, 4H), 5.15 (s, 1H), 3.34 (dd, J = 12.3, 6.9 Hz, 2H), 2.42 (dd, J = 14.7, 7.2 Hz, 6H), 2.05–1.98 (m, 8H), 1.80 (dt, J = 14.3, 7.2 Hz, 2H), 1.54–1.21 (m, 54H), 0.88 (t, J = 6.7 Hz, 6H). 13C NMR (101 MHz, deuterated chloroform) δ 152.22, 149.46, 147.64, 130.35, 130.23, 129.89, 129.76, 127.66, 127.62, 120.66, 119.99, 100.00, 54.12, 53.93, 43.23, 32.55, 31.85, 29.71, 29.56, 29.52, 29.46, 29.26, 29.23, 29.08, 28.76, 27.61, 27.26, 27.15, 27.03, 26.90, 26.78, 22.63, 14.06.

[0141] Example 41: Synthesis of CF3-3N2-C12

[0142] Starting materials 1 (1 mmol) and 14 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate CF3-3N2. Intermediate CF3-3N2 (0.1 mmol), 1-bromododecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction mixture was followed according to general synthetic procedure b to obtain 31 mg of an oily compound. HRMS (ES): m / z (M+H) + 803.7182.

[0143] Example 42: Synthesis of CF3-3N2-C17

[0144] The intermediate CF3-3N2 (0.1 mmol), 1-bromoheptadecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the oily compound (22 mg) was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) + 1013.9530.

[0145] Example 43: Synthesis of CF3-3N2-UC18

[0146] The intermediate CF3-3N2 (0.1 mmol), 1-bromo-cis-9-octadecene (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the oily compound (21 mg) was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) + 1049.9529.

[0147] Example 44. Synthesis of CF3-SPN-C12

[0148] Starting materials 1 (1 mmol) and 15 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate CF3-SPN. Intermediate CF3-SPN (0.1 mmol), 1-bromododecane (0.5 mmol), potassium carbonate (0.6 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction mixture was followed according to general synthetic procedure b to obtain 22 mg of an oily compound. HRMS (ES): m / z (M+H) 2+ 536.0104.

[0149] Example 45: Synthesis of CF3-SPN-C17

[0150] The intermediate CF3-SPN (0.1 mmol), 1-bromoheptadecane (0.5 mmol), potassium carbonate (0.6 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the oily compound (20 mg) was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) + 1351.3264.

[0151] Example 46: Synthesis of CF3-SPN-UC18

[0152] The intermediate CF3-SPN (0.1 mmol), 1-bromo-cis-9-octadecene (0.5 mmol), potassium carbonate (0.6 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the oily compound (23 mg) was obtained according to the general synthesis procedure b. HRMS (ES): m / z (M+H) 2+ 700.6693.

[0153] Example 47: Synthesis of CF3-5N2-C12

[0154] Starting materials 1 (1 mmol) and 16 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate CF3-5N2. Intermediate CF3-5N2 (0.1 mmol), 1-bromododecane (0.6 mmol), potassium carbonate (0.8 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction mixture was followed according to general synthetic procedure b to obtain 20 mg of an oily compound. HRMS (ES): m / z (M+H) 2+ 613.2274.

[0155] Example 48: Synthesis of Br-2N4-UC18

[0156] Starting materials 5 (1 mmol) and 12 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate Br-2N4. Intermediate Br-2N4 (0.1 mmol), 1-bromo-cis-9-octadecene (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction mixture was followed according to general synthetic procedure b to obtain 26 mg of an oily compound. HRMS (ES): m / z (M+H) + 794.5911.

[0157] Example 49: Synthesis of Br-2N6-UC18

[0158] Starting materials 5 (1 mmol) and 13 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate Br-2N6. Intermediate Br-2N6 (0.1 mmol), 1-bromo-cis-9-octadecene (0.3 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction mixture was followed according to general synthetic procedure b to obtain 21 mg of an oily compound. HRMS (ES): m / z (M+H) + 822.6225. 1 H NMR (400 MHz, deuterated chloroform) δ 8.48 (d, J = 5.4 Hz, 1H), 8.12 (d, J = 2.0 Hz, 1H), 7.79 (d, J = 9.0 Hz, 1H), 7.48 (dd, J = 8.9, 2.0 Hz, 1H), 6.40 (d, J = 5.5 Hz, 1H), 5.51 (s, 1H), 5.42–5.25 (m, 4H), 3.32 (dd, J = 12.0, 6.7 Hz, 2H), 2.69–2.57 (m, 6H), 1.99 (dd, J = 13.4, 6.7 Hz, 8H), 1.84–1.71 (m, 2H), 1.67–1.23 (m, 54H), 0.88 (t, J = 6.7 Hz, 6H). 13 C NMR (101 MHz, deuterated chloroform) δ 151.49, 150.08, 131.58, 130.15, 129.94, 129.68, 127.71, 123.14, 121.63, 117.42, 98.89, 53.40, 53.27, 42.90, 32.56, 31.85, 29.70, 29.60, 29.55, 29.46, 29.43, 29.34, 29.26, 29.15, 29.01, 28.37, 27.29, 27.17, 27.12, 26.73, 26.59, 25.53, 25.25, 22.63, 14.07.

[0159] Example 50: Synthesis of Br-3N2-UC18

[0160] Starting materials 5 (1 mmol) and 14 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate Br-3N2. Intermediate Br-3N2 (0.1 mmol), 1-bromododecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction mixture was followed according to general synthetic procedure b to obtain 31 mg of an oily compound. HRMS (ES): m / z (M+H) + 1059.8663.

[0161] Example 51. Synthesis of MO-2N4-UC18

[0162] Starting materials 7 (1 mmol) and 12 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate MO-2N4. Intermediate MO-2N4 (0.1 mmol), 1-bromo-cis-9-octadecene (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction mixture was followed according to general synthetic procedure b to obtain 26 mg of an oily compound. HRMS (ES): m / z (M+H) + 746.6946.

[0163] Example 52: Synthesis of MO-2N6-UC18

[0164] Starting materials 7 (1 mmol) and 13 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate MO-2N6. Intermediate MO-2N6 (0.1 mmol), 1-bromo-cis-9-octadecene (0.3 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction mixture was followed according to general synthetic procedure b to obtain 21 mg of an oily compound. HRMS (ES): m / z (M+H) + 774.7251.

[0165] Example 53: Synthesis of MO-3N2-UC18

[0166] Starting materials 7 (1 mmol) and 14 (3 mmol) were reacted according to general synthetic procedure a to obtain intermediate MO-3N2. Intermediate MO-3N2 (0.1 mmol), 1-bromododecane (0.4 mmol), potassium carbonate (0.5 mmol), and potassium iodide (catalytic amount) were added to 2 mL of N,N-dimethylformamide and the reaction mixture was followed according to general synthetic procedure b to obtain 31 mg of an oily compound. HRMS (ES): m / z (M+H)+ 1011.9663.

[0167] Example 54: Synthesis of CF3-3N6-C12-3OH

[0168] The intermediate CF3-3N6 (0.1 mmol) and 1,2-epoxydodecane (0.4 mmol) were added to 10 ml of anhydrous methanol and 25 mg of an oily compound was obtained according to the general synthesis procedure c. HRMS (ES): m / z (M+H) + 963.8284.

[0169] Example 55: Synthesis of CF3-3N6-C12-4OH

[0170] The intermediate CF3-3N6 (0.1 mmol) and 1,2-epoxydodecane (0.6 mmol) were added to 10 ml of anhydrous methanol and 27 mg of an oily compound was obtained according to the general synthesis procedure c. HRMS (ES): m / z (M+H) + 1148.0035. 1 H NMR (400 MHz, deuterated chloroform) δ 9.20 (dd, J = 8.1, 5.0 Hz, 1H), 8.55 (d, J = 6.2 Hz, 1H), 7.87 (s, 1H), 7.73 (d, J = 8.0 Hz, 1H), 6.48 (d, J = 7.5 Hz, 1H), 4.59 (d, J = 14.5 Hz, 1H), 4.35 (d, J = 6.3 Hz, 1H), 4.02 (s, 1H), 3.60 (d, J = 5.6 Hz, 3H), 3.47 (d, J = 12.0 Hz, 2H), 3.33 (s, 3H), 2.58–2.28 (m, 12H), 1.77–1.18 (m, 88H), 0.91–0.83 (m, 12H). 13 C NMR (101 MHz, deuterated chloroform) δ 154.94, 148.08, 137.63, 134.80, 134.47, 128.12, 124.19, 122.18, 121.47, 120.76, 113.98, 98.61, 69.43, 68.88, 68.80, 67.77, 66.85, 62.60, 6 1.04,60.73,59.80,55.91,55.14,53.92,43.79,35.08,34.91,31.84,29.84,29.76,29.57,29.28,27.80,27.17,27.01,26.87,26.69,25.69,25.64,22.61,14.05.

[0171] Example 56: Synthesis of CF3-3N6-C14-3OH

[0172] The intermediate CF3-3N6 (0.1 mmol) and 1,2-epoxytetradecane (0.4 mmol) were added to 10 ml of anhydrous methanol and 23 mg of an oily compound was obtained according to the general synthesis procedure c. HRMS (ES): m / z (M+H) + 1047.9177.

[0173] Example 57: Synthesis of CF3-3N6-C16-3OH

[0174] The intermediate CF3-3N6 (0.1 mmol) and 1,2-epoxyhexadecane (0.4 mmol) were added to 10 ml of anhydrous methanol and 22 mg of an oily compound was obtained according to the general synthesis procedure c. HRMS (ES): m / z (M+H) + 1132.0112.

[0175] Example 58: Synthesis of CF3-3N6-C18-3OH

[0176] The intermediate CF3-3N6 (0.1 mmol) and 1,2-epoxyoctadecane (0.4 mmol) were added to 10 ml of anhydrous methanol and 21 mg of an oily compound was obtained according to the general synthesis procedure c. HRMS (ES): m / z (M+H) + 1216.1025.

[0177] Example 59: Synthesis of CF3-2N6-C12-3OH

[0178] The intermediate CF3-2N6 (0.1 mmol) and 1,2-epoxydodecane (0.4 mmol) were added to 10 ml of anhydrous methanol and 17 mg of an oily compound was obtained according to the general synthesis procedure c. HRMS (ES): m / z (M+H) + 864.7118.

[0179] Example 60: Synthesis of CF3-2N6-C14-2OH

[0180] The intermediate CF3-2N6 (0.1 mmol) and 1,2-epoxytetradecane (0.3 mmol) were added to 10 ml of anhydrous methanol and 13 mg of an oily compound was obtained according to the general synthesis procedure c. HRMS (ES): m / z (M+H) + 736.5981.

[0181] Example 61: Synthesis of Br-2N6-C12-3OH

[0182] The intermediate Br-2N6 (0.1 mmol) and 1,2-epoxydodecane (0.4 mmol) were added to 10 ml of anhydrous methanol and 16 mg of an oily compound was obtained according to the general synthesis procedure c. HRMS (ES): m / z (M+H) + 874.6381.

[0183] Example 62: Synthesis of Br-2N6-C14-2OH

[0184] The intermediate Br-2N6 (0.1 mmol) and 1,2-epoxytetradecane (0.3 mmol) were added to 10 ml of anhydrous methanol and 13 mg of an oily compound was obtained according to the general synthesis procedure c. HRMS (ES): m / z (M+H) + 746.5209.

[0185] 2. Preparation of quinoline-based ionizable lipid nanoparticles encapsulating mRNA, screening and optimization of nucleic acid delivery activity

[0186] The general steps for the preparation of quinoline lipid nanoparticles and in vitro delivery activity testing are as follows:

[0187] (I) Quinoline ionizable lipid compounds (Examples 1 to 62), phospholipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, abbreviated as DOPE), structural lipid (cholesterol), and polyethylene glycol lipid (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000, abbreviated as DMG-PEG2000) were dissolved in ethanol at specific molar ratios (see Tables 1 to 7). Then, 50 mM citrate buffer (pH 3) was added at a volume ratio (ethanol:citrate buffer) of 9:1, and the mixture was mixed to obtain a mixed phase.

[0188] (II) Luciferase (FLuc) mRNA (DD4511-02, Nanjing Novozymes) was diluted with 50 mM citrate buffer (pH 3) to obtain mRNA working solution.

[0189] (III) The mixed phase obtained in step (I) and the mRNA working solution obtained in step (II) are mixed in equal volumes at a predetermined mass ratio (quinoline ionizable lipid compound:mRNA, see Tables 1 to 9). Finally, phosphate buffered saline (PBS) is added to dilute the mixture at a volume twice that of the mixed phase to obtain the lipid nanoparticles.

[0190] (IV) In a 96-well plate (containing 20,000 293T cells per well, plated overnight), 200 ng of the above-mentioned mRNA preparation was added to each well. After further culturing for 24 hours, the expression level of luciferase protein was measured using a microplate reader.

[0191] The specific steps for preparation, screening and optimization of nucleic acid delivery activity are as follows:

[0192] Step (1): For the quinoline ionizable lipid compounds (Examples 1-13), quinoline ionizable lipid nanoparticles (Examples 63-75) were prepared according to the initial formulation (Table 1). Based on the relative delivery activity (Table 1), the quinoline ionizable lipid compounds CF3-3N6-C12 and CF3-3N6-C17 with the highest delivery activity were screened.

[0193] Table 1. Examples 63-75 Note 1: The name of the LNP formulation is derived from the name of the corresponding quinoline lipid used in the formulation plus LNP, the same below. Note 2: The mass ratio is the mass ratio of the quinoline ionizable lipid to the mRNA used in the formulation; the molar ratio is the molar ratio of the quinoline ionizable lipid, DOPE, cholesterol, and DMG-PEG2000, the same below. Note 3: The relative delivery activity refers to the average delivery activity ratio of each example in the table compared to Example 63. The value after ± represents the standard error. For example, the relative delivery activity of Example 67 is 65.79±1.16, which means that the delivery activity of Example 67 is 65.79 times that of Example 63, the same below.

[0194] Step (2): For the quinoline ionizable lipid compound CF3-3N6-C12, a first round of formulation optimization (Table 2) was performed to configure quinoline ionizable lipid nanoparticles (Examples 76-99). According to the size of the relative delivery activity (Table 2), the formulation with the highest delivery activity (Example 82) was screened out, and its relative delivery activity was 5.67 times that of the initial formulation of CF3-3N6-C12 (Example 67). Subsequently, a second round of formulation optimization (Table 3) was performed on the quinoline ionizable lipid compound CF3-3N6-C12, and quinoline ionizable lipid nanoparticles (Examples 100-124) were configured. According to the size of the relative delivery activity (Table 3), the formulation with the highest delivery activity (Example 104) was screened out, and its relative delivery activity was 1.41 times that of Example 100.

[0195] Table 2. Examples 76-99

[0196] Table 3. Examples 100-124

[0197] Step (3): For the quinoline ionizable lipid compound CF3-3N6-C17, the second round of formulations for CF3-3N6-C12 was used to prepare quinoline ionizable lipid nanoparticles (Examples 125-149). Based on the relative delivery activity (Table 4), the formulation with the highest delivery activity (Example 138) was selected, which showed a relative delivery activity 2.55-fold higher than that of Example 125.

[0198] Table 4. Examples 125-149

[0199] Step (4): The quinoline ionizable lipid compounds (Examples 1-13) were reconfigured according to the formulation of Example 138 to obtain quinoline ionizable lipid nanoparticles (Table 5, Examples 150-161). Based on the relative delivery activity (Table 5), the quinoline ionizable lipid compound with the highest delivery activity was selected as CF3-3N6-UC18.

[0200] Table 5. Examples 150-161

[0201] Step (5): Particle size testing and optimization of Example 160.

[0202] The formulation prepared in Example 160 was diluted five-fold with enzyme-free water, and the hydrated particle size was measured using a nanoparticle size analyzer. The results showed that the particle size of Example 160 was 487 nm. The particle size of Example 160 was subsequently optimized by increasing the molar fraction of polyethylene glycol lipid in the formulation to 1.1 (Table 6), resulting in Example 162, whose particle size was reduced to 155 nm.

[0203] Table 6. Example 162

[0204] Step (6): For the quinoline ionizable lipid compounds (Examples 14-62), the formulation of Example 162 was used to prepare quinoline ionizable lipid nanoparticles (Table 7, Examples 163-213). The relative delivery activities of Examples 190, 191, 194, and 200 were 1.17, 3.55, 1.67, and 3.41 times that of Example 162, respectively (Table 7).

[0205] The relative delivery activity of the better embodiment 162,190,191,194 and 200 was compared with the delivery activity of commercial trial Lipofectamine 2000 and Lipofectamine MessengerMAX. Lipofectamine 2000 and Lipofectamine MessengerMAX were loaded with mRNA of the same quality as the quinoline ionizable lipid nanoparticles according to the commercial reagent instructions, and then the cells were processed according to the same operating steps as the above-mentioned quinoline ionizable lipid nanoparticles. The specific process is as follows: in a 96-well plate (containing 20,000 / well 293T cells, overnight after plating), 200ng mRNA / 0.2ul Lipofectamine 2000 or 200ng mRNA / 0.2ul Lipofectamine MessengerMAX was added to each well. After continuing to cultivate for 24 hours, the expression level of luciferase protein was detected with a microplate reader. As can be seen from Figure 2, the relative delivery activities of Examples 162, 190, 191, 194 and 200 are 5.55, 6.50, 19.68, 9.25 and 18.94 times that of Lipofectamine 2000, respectively; the relative delivery activities of Examples 162, 190, 191, 194 and 200 are 1.27, 1.49, 4.51, 2.12 and 4.34 times that of Lipofectamine MessengerMAX, respectively.

[0206] Table 7. Examples 162-213

[0207] 3. Other performance and characterization analyses of nanomedicines are as follows:

[0208] (1) Preparation Stability Test. The preparation prepared in Example 191 was diluted 5-fold with enzyme-free water, and the hydrated particle size was measured using a nanoparticle size analyzer at different times. The results, as shown in Figure 3, showed that the particle size of the preparation was stable over 32 days.

[0209] (2) Morphological Characteristics. 10 μL of the preparation prepared in Example 191 was placed on a copper mesh to dry, and the morphological characteristics of the preparation were observed using a transmission electron microscope. As shown in Figure 4, the preparation was oval in shape, and its particle size was substantially consistent with the hydrated particle size.

[0210] (3) In vivo activity detection of the mRNA delivery system. The preparation prepared in Example 191 was injected intramuscularly into C57BL / 6J mice at a dose of 0.5 mg / kg mRNA. Four hours after administration, luciferase substrate was injected intraperitoneally at a dose of 150 mg / kg; 10 minutes later, the anesthetized mice were imaged using a small animal imager (PerkinElmer, IVIS Lumina LT); after in vivo imaging, the main tissues of the mice were removed for in vitro imaging. The imaging images were quantitatively analyzed using PerkinElmer in vivo imaging software to obtain the overall and individual organ bioluminescence levels. As shown in Figure 5, the mRNA delivery system delivered mRNA to the muscle tissue of mice. Industrial Applicability

[0211] The present invention uses different quinoline ring derivatives, brominated or epoxy-substituted alkyl chains of different lengths, and polyamine linker arms as raw materials, and prepares the target product through a two-step reaction. The prepared lipid nanoparticles containing quinoline ionizable lipid compounds have high in vivo and in vitro mRNA delivery efficiency, and their delivery efficiency at the cellular level is better than that of commercial reagents Lipofectamine 2000 and Lipofectamine MessengerMAX. In addition, the lipid nanoparticles containing quinoline ionizable lipid compounds can achieve efficient local expression of mRNA through intramuscular administration.

Claims

1. A quinoline ionizable lipid compound represented by Formula I or a pharmaceutically acceptable salt thereof: In Formula I, R 1 Represents the substituent on the quinoline ring, which is single-substituted or multi-substituted, R 1 Each is independently selected from the group consisting of: deuterium, tritium, halogen, halogen-substituted C1-C6 alkyl, C1-C6 alkoxy, aromatic-substituted C1-C6 alkyl, aromatic-substituted C1-C6 alkoxy, and an aromatic group formed between two adjacent carbon atoms on a quinoline ring; R 2 Each independently selected from hydrogen, deuterium, tritium, C4-C 22 alkyl or alkenyl; m and n are each independently selected from integers of 1 to 8; x is an integer selected from 0 to 8. When x is an integer greater than or equal to 2, the number of methylene groups in each repeating unit is equal or different.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that The aromatic group is phenyl or pyridyl.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that Containing R 1 The quinoline ring is selected from any one of the following groups:

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that The alkyl and alkenyl groups may include one or more groups selected from halogen, hydroxy, amino, oxo, alkoxycarbonyl, acylamino, alkylacylamino, dialkylacylamino, nitro, amino, alkylamino, dialkylamino, carboxyl, thio and thioalkyl groups.

5. A method for preparing the compound of formula I in claim 1, comprising the steps of: subjecting the compound of formula A to a substitution reaction with the compound of formula B to obtain the compound of formula C; subjecting the compound of formula C to a substitution reaction with the compound of formula D to obtain the quinoline ionizable lipid compound of formula I, wherein R 2 Does not contain hydroxyl groups; In formula A, R 1 Same as R in Formula Ⅰ 1 Definition of; In Formula B, the definitions of m, n, and x are the same as those of m, n, and x in Formula I; In formula C, R 1 The definitions of m, n, and x are the same as those of R in Formula Ⅰ. 1 , m, n, x definitions; In formula D, R 2 The definition of R is the same as that of Formula Ⅰ 2 Definition of; R 2 C8~C 22 Alkyl or alkenyl, so that the compound represented by formula C is combined with 1,2-epoxy substituted hydrocarbon group ( , wherein R3-CH2-CH2=R2) reacts to undergo a substitution reaction to obtain a quinoline ionizable lipid compound shown in formula I.

6. A drug delivery carrier comprising a quinoline ionizable lipid compound represented by formula I in claim 1, a phospholipid, a structured lipid, and a polyethylene glycol lipid. Calculated by mole, the quinoline ionizable lipid compound is 10 to 80 parts, the phospholipid is 10 to 80 parts, the structural lipid is 10 to 80 parts, and the polyethylene glycol lipid is 0.01 to 20 parts.

7. Use of the quinoline ionizable lipid compound represented by formula I in claim 1 or the drug delivery carrier according to claim 6 in the preparation of a drug delivery system.

8. A drug delivery system - lipid nanoparticles encapsulating an active ingredient, comprising a quinoline-based ionizable lipid compound, a phospholipid, a structured lipid, a polyethylene glycol lipid, and an active ingredient; The drug delivery system - lipid nanoparticles encapsulating the active ingredient comprises 10 to 80 parts of a quinoline ionizable lipid compound, 10 to 80 parts of a phospholipid, 10 to 80 parts of a structural lipid, and 0.01 to 20 parts of a polyethylene glycol lipid, in a molar ratio of quinoline ionizable lipid compound to the active ingredient of 1 to 20:

1. Specifically, it can be 20 parts of quinoline ionizable lipid compound, 10-50 parts of phospholipid, 20-80 parts of structural lipid, 0.2-1.45 parts of polyethylene glycol lipid, and the mass ratio of quinoline ionizable lipid compound to active ingredient is 2-14:

1.

9. The drug delivery system - lipid nanoparticles carrying active ingredients according to claim 8, characterized in that: The active ingredient is selected from at least one of DNA, RNA, protein, and pharmaceutically active molecules.

10. A method for preparing the drug delivery system-lipid nanoparticles carrying active ingredients according to claim 8 or 9, comprising the following steps: (A) dissolving and mixing the quinoline ionizable lipid compound represented by formula I in claim 1 with phospholipids, structural lipids and polyethylene glycol lipids in an organic solvent; (B) The resulting mixture is mixed with the active ingredient to form lipid nanoparticles encapsulating the active ingredient.

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