Chloroquine structure-containing compound, composition containing same, and application thereof

Abstract

Disclosed are a chloroquine structure-containing compound, a composition containing same, and an application thereof. The present invention provides a compound as represented by formula (I) or a pharmaceutically acceptable salt thereof. The chloroquine structure-containing compound provided by the invention is a nano material obtained from the synthesis of chloroquine, a derivative thereof, and a lipid tail. The nano material can be applied to gene therapy, drug delivery, and the like. A lipid nanoparticle (LNP) formed from the nano material and mRNA has a novel nanoscale spatial structure different from that of a traditional LNP, and compared to a traditional LNP, the nano material has better pharmaceutical properties. The LNP exhibits an immunosuppressive function, avoids triggering an excessive inflammatory response, and has a higher safety profile.

Description

Compound containing chloroquine structure, composition containing same and application thereof

[0001] This application claims priority to Chinese patent application 2024106568712 with a filing date of May 24, 2024. This application incorporates the entirety of the aforementioned Chinese patent application. TECHNICAL FIELD

[0002] The present application relates to compounds, compositions and methods for delivering therapeutic, diagnostic or prophylactic drugs, such as nucleic acids. In particular, a compound containing a chloroquine structure, a composition containing same and application thereof. BACKGROUND

[0003] mRNA-based therapies show broad application prospects in the fields of prophylactic vaccines against infectious diseases, tumor therapeutic vaccines, protein supplements, protein replacements, etc. Although mRNA vaccines based on lipid nanoparticle delivery systems (LNPs) have been successful in the prevention of infectious diseases, the inflammatory side effects induced by LNP-mRNA vaccines in clinical applications are worrisome. However, in the application scenarios of mRNA, such as protein supplements, protein replacements, and combination with other therapies, the inflammatory reactions induced by mRNA and LNP themselves are redundant, and such inflammatory side effects are particularly a limiting factor for the application of mRNA therapies in other fields. Therefore, the development of a delivery system without inflammatory side effects is particularly important for the improvement of the safety of mRNA drugs and the expansion of application scenarios. SUMMARY

[0004] The technical problem to be solved by the present application is that the existing technology based on lipid nanoparticle delivery systems is prone to induce inflammation. To this end, the present application provides a compound containing a chloroquine structure, a composition containing same and application thereof. The compound containing a chloroquine structure provided by the present application is a nanomaterial synthesized from chloroquine and its derivatives and a lipid tail, which can be used in gene therapy, drug delivery, etc. The lipid nanoparticle (LNP) formed by the nanomaterial and mRNA has a novel nano-space structure different from traditional LNPs, and has better formulation properties than traditional LNPs. The LNP has an immunosuppressive function, can avoid inducing redundant inflammatory reactions, and has higher safety.

[0005] The present application provides a compound as shown in formula I or a pharmaceutically acceptable salt thereof,

[0006] wherein:

[0007] T is C 1-30 alkyl, C 2-30 alkenyl, C 2-30 alkynyl or

[0008] X is a single bond, O, S, or N(R) x ); R x For H or C 1-10 alkyl;

[0009] L 1 It is a single bond, -(CH2) n1 -、 X and L 1 Not both are single bonds; A 1 Terminal A is connected to X. 2 and A 3 The end is connected to the Y-axis;

[0010] A 1 -(CH2) n2 -;

[0011] A 2 and A 3 Independently a single bond or -(CH2) n2 -;

[0012] M 1 and M 2 Independently O or NH;

[0013] M 3 for O, S or N(R) x );

[0014] Y is N or CR Y ;R Y For H, C 1-30 Alkyl, C 2-30 alkenyl, C 2-30 acetylinyl

[0015] R 1 and R 2 Independently for C 1-30 Alkyl, C 2-30 alkenyl, C 2-30 acetylinyl

[0016] D 1 D 2 and D 3 Independently O or NH;

[0017] B 1 and B 3 Independently -(CH2) n3 -;

[0018] B 2 Independently for C 1-30alkyl, C 2-30 alkenyl or C 2-30 alkynyl;

[0019] L 2 is K 1 is 2 is 1 connected to Z;

[0020] K 1 and K 3 are independently -(CH2) n4 -;

[0021] K 2 are independently a single bond or -(CH2) n4 -;

[0022] H 1 , H 2 , H 3 , H 4 and H 5 are independently O or NH;

[0023] H 6 are independently O, S or NH;

[0024] Z 1 is independently N or CH;

[0025] R 3 and R 4 are independently C 1-30 alkyl, C 2-30 alkenyl or C 2-30 alkynyl;

[0026] n1, n2, n3 and n4 are independently an integer from 1 to 10.

[0027] In a certain preferred embodiment, in the compound of formula I, or a pharmaceutically acceptable salt thereof, certain groups are defined as follows, and the definition of groups not mentioned is as described in any of the embodiments of the present application (hereinafter this paragraph is referred to as "in a certain embodiment").

[0028] In a certain embodiment, in T, R Y , B 2 , R 1 , R 2 , R 3 and R 4 , the C 1-30 alkyl is independently a linear alkyl or a branched alkyl, preferably a linear alkyl.

[0029] In certain embodiments, T, R Y , B 2 , R 1 , R 2 , R 3 , and R 4 , the C 1-30 alkyl is independently C 4-20 alkyl, for example C 4-18 alkyl, and further for example C 4-10 alkyl.

[0030] In certain embodiments, T, R Y , B 2 , R 1 , R 2 , R 3 , and R 4 , the C 1-30 alkyl is independently

[0031] In certain embodiments, T, R Y , B 2 , R 1 , R 2 , R 3 , and R 4 , the number of alkenyl linkages in the C 2-30 alkenyl is independently 1, 2, 3, 4, or 5, for example 1, 2, or 3.

[0032] In certain embodiments, T, R Y , B 2 , R 1 , R 2 , R 3 , and R 4 , the C 2-30 alkenyl is independently a straight chain alkenyl or a branched chain alkenyl, preferably a straight chain alkenyl.

[0033] In certain embodiments, T, R Y , B 2 , R 1 , R 2 , R 3 , and R 4 , the C 2-30 alkenyl is independently C 4-20 alkenyl, for example C 4-20 alkenyl, and further for example C 8-20 alkenyl, and yet further for example

[0034] In certain embodiments, T, R Y , B 2 , R1 , R 2 , R 3 , and R 4 , the C 2-30 alkynyl is independently a straight chain alkynyl or a branched chain alkynyl, the C 2-30 number of alkynyl bonds in the C 4-20 straight chain alkynyl.

[0035] In a certain embodiment, R x , the C 1-10 alkyl is C 1-6 alkyl, for example methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, or t-butyl.

[0036] In a certain embodiment, T, the C 2-30 alkenyl is C 4-20 straight chain alkenyl, the C 4-20 number of alkenyl bonds in the C

[0037] In a certain embodiment, R 1 and R 2 , the C 1-30 alkyl is independently C 4-20 straight chain alkyl.

[0038] In a certain embodiment, R 1 and R 2 , the C 2-30 alkenyl is independently C 4-20 straight chain alkenyl, the C 4-20 number of alkenyl bonds in the C

[0039] In a certain embodiment, R 3 and R 4 , the C 1-30 alkyl is independently C 4-20 straight chain alkyl, for example C 4-10 straight chain alkyl.

[0040] In a certain embodiment, R Y , B 2 , the C 2-30 alkenyl is C 4-20 straight chain alkenyl, the C 4-20 number of alkenyl bonds in the C

[0041] In a certain embodiment, R 1 and R 2 , B 2 , the C 1-30alkyl is independently C 4-20 linear alkyl.

[0042] In certain embodiments, R 1 and R 2 B 2 is independently C 2-30 alkenyl is independently C 4-20 alkenyl, e.g., C 4-20 linear alkenyl, the number of alkenyl groups in said C 4-20 linear alkenyl is 1, 2, or 3.

[0043] In certain embodiments, T is C 2-30 alkenyl or preferably, T is C 4-20 linear alkenyl or the number of alkenyl groups in said C 4-20 linear alkenyl is 1, 2, or 3; more preferably, T is

[0044] In certain embodiments, T is C 1-30 alkyl, C 2-30 alkenyl, or C 2-30 alkynyl.

[0045] In certain embodiments, X is a single bond or O; preferably, X is a single bond.

[0046] In certain embodiments, L 1 is a single bond, -(CH2) n1 -, preferably, L 1 is

[0047] In certain embodiments, n1 and n2 are independently 1, 2, 3, 4, 5, 6, 7, or 8; preferably, n1 and n2 are independently 1, 2, 3, or 4.

[0048] In certain embodiments, M 1 is O.

[0049] In certain embodiments, M 3 is independently or N(R x ).

[0050] In certain embodiments, R x is H.

[0051] In certain embodiments, R Y is H, preferably, H preferably, D 2O; more preferably, R Y is H.

[0052] In certain embodiments, R Y is H. 2 is independently C 2-30 alkyl; preferably, B 2 is independently C 4-20 linear alkyl; preferably, B 4-20 the number of alkenyl groups in said C

[0053] In certain embodiments, R 1 and R 2 are independently C 1-30 alkyl, C 2-30 alkenyl, or, R 1 and R 2 are independently C 1-30 alkyl, C 2-30 alkenyl, C 2-30 alkynyl, preferably, R 1 and R 2 are independently C 4-20 linear alkyl, C 4-20 linear alkenyl, more preferably, R 1 and R 2 are independently

[0054] In certain embodiments, D 1 , D 2 , and D 3 are O.

[0055] In certain embodiments, n3 is 1, 2, 3, 4, 5, 6, 7, or 8.

[0056] In certain embodiments, R 1 and R 2 , B 2 is independently C 1-30 alkyl or C 2-30 alkenyl; preferably, B 2 is independently C 4-20 linear alkyl or C 4-20 linear alkenyl; preferably, B 4-20 the number of alkenyl groups in said C

[0057] In certain embodiments, L 2 is independently

[0058] In one embodiment, H 1 , H 2 , H 3 , and H 5 are O.

[0059] In one embodiment, H 6 are independently or NH.

[0060] In one embodiment, K 2 is a single bond.

[0061] In one embodiment, n4 is independently 1, 2, 3, 4, 5, 6, 7, or 8; preferably, n4 is independently 1, 2, 3, or 4.

[0062] In one embodiment, Z 1 is CH.

[0063] In one embodiment, R 3 and R 4 are independently C 1-30 alkyl; preferably, R 3 and R 4 are independently C 4-20 straight chain alkyl, for example C 4-10 straight chain alkyl.

[0064] In one embodiment, L 1 is a single bond, preferably, the "1" position is attached to X, the "2" position is attached to Y, and the "3" position is attached to A 3 ; more preferably, L 1 is the "1" position is attached to X, and the "2" position is attached to Y.

[0065] In one embodiment, -X-L 1 - is a single bond, O, preferably, the "2" position is attached to Y; more preferably, -X-L 1 - is the "2" position is attached to Y.

[0066] In one embodiment, R Y is H or

[0067] In one embodiment, B 2 is independently

[0068] In certain embodiments, K 1 is independently a single bond,

[0069] In certain embodiments, K 2 is independently a single bond,

[0070] In certain embodiments, K 3 is independently a single bond,

[0071] In certain embodiments, L 2 is independently a single bond, Preferably, "1" is attached to Y and "2" is attached to Z 1 ; more preferably, L 2 is "1" is attached to Y and "2" is attached to Z 1 .

[0072] In certain embodiments, R 3 and R 4 are independently More preferably, R 3 and R 4 are

[0073] In certain embodiments, R 1 and R 2 are independently Preferably, R 1 and R 2 are independently

[0074] In certain embodiments, the compound of Formula I is selected from any one of the following schemes:

[0075] Scheme 1 : the compound of Formula I is a compound of Formula I-1 :

[0076] R 1 and R 2 are independently C 1-30 alkyl, C 2-30 alkenyl, C 2-30 alkynyl, X, L 1 , Y, D1 , D 2 , D 3 , B 1 , B 2 , and B 3 are as described in any embodiment of this application;

[0077] Scheme 2: the compound of Formula I is a compound of Formula I-2:

[0078] X, L 1 , Y, L 2 , Z 1 , R 3 , and R 4 are as described in any embodiment of this application;

[0079] Scheme 3: the compound of Formula I is a compound of Formula I-3:

[0080] R Y , R 1 , and R 2 are independently C 1-30 alkyl, C 2-30 alkenyl, C 2-30 alkynyl, X, L 1 , D 1 , D 2 , D 3 , B 1 , B 2 , and B 3 are as described in any embodiment of this application.

[0081] In one embodiment, the compound of Formula I is a compound of Formula I-2:

[0082] X is a single bond;

[0083] L 1 is

[0084] A 1 is -(CH2) n2 -; A 2 is -(CH2) n2 -;

[0085] n2 is independently 1, 2, 3, or 4;

[0086] M 1 is O;

[0087] Y is N or CH;

[0088] L 2 independently

[0089] K 1 and K 3 independently -(CH2) n4 -;

[0090] K 2 independently a single bond or -(CH2) n4 -;

[0091] n4 is independently 1, 2, 3 or 4;

[0092] H 1 , H 3 and H 4 is O;

[0093] Z 1 is CH;

[0094] R 3 and R 4 are independently C 4-10 straight chain alkyl.

[0095] In one embodiment, the compound of formula I is any one of the following:

[0096] The present application also provides a lipid carrier comprising a substance Z, the substance Z being a compound of formula I as described in any one of the present application or a pharmaceutically acceptable salt thereof.

[0097] In one preferred embodiment, the lipid carrier further comprises a diluent. The diluent is a conventional diluent in the art, for example the diluent is a citrate buffer or ethanol.

[0098] In one preferred embodiment, the lipid carrier further comprises a phospholipid. The phospholipid is a conventional phospholipid in the art, which is an amphiphilic auxiliary molecule that facilitates fusion of the lipid particle and the cell membrane. The phospholipid can be a phospholipid class of molecules with a charged polar end and a nonpolar end of a fatty chain, for example distearoylphosphatidylcholine (DSPC).

[0099] In a preferred embodiment, the lipid carrier further comprises a PEG lipid (polyethylene glycol modified lipid). The PEG lipid is a lipid molecule modified with a polyethylene glycol hydrophilic end. The PEG lipid is preferably selected from one or more of PEG modified phosphatidylethanolamine, PEG modified phosphatidic acid, PEG modified ceramide, PEG modified dialkylamine, PEG modified diacylglycerol and PEG modified dialkylglycerol, such as PEG modified dimyristoylglycerol (DMG PEG2000), and the like.

[0100] In a preferred embodiment, the lipid carrier further comprises a sterol. The sterol is a conventional sterol in the art, and the sterol includes animal, plant or fungal sterols. Preferably, the sterol is selected from one or more of cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid and alpha-tocopherol, such as cholesterol, and the like.

[0101] In a preferred embodiment, the lipid carrier comprises the diluent, the substance Z, the phospholipid, the PEG lipid and the sterol.

[0102] In a preferred embodiment, the molar ratio of the substance Z to the volume of the diluent is (5-25) mmol: 1 L; for example (10-15) mmol: 1 L; and for example 12 mmol: 1 L.

[0103] In a preferred embodiment, the molar content of the substance Z is 30 mol% to 60 mol%, based on the total molar mass of the substance Z, the phospholipid, the PEG lipid and the sterol as 100%; for example 40 mol% to 55 mol%; and for example 50 mol%.

[0104] In a preferred embodiment, the molar ratio of the substance Z to the phospholipid in the lipid carrier is 1-25: 1, preferably 2-10: 1, for example 5: 1.

[0105] In a preferred embodiment, the molar ratio of the substance Z to the sterol in the lipid carrier is (0.5-3): 1, preferably (0.5-3): 1, for example 1.3: 1.

[0106] In a preferred embodiment, the molar ratio of the substance Z to the PEG lipid in the lipid carrier is 20-50: 1, preferably 20-40: 1, for example 33: 1.

[0107] In the present application, the molar content means the percentage of a substance in the total amount of substances in the lipid carrier, and the sum of the molar contents of the components in the lipid carrier is not more than 100 mol%.

[0108] In a preferred embodiment, the lipid carrier consists of the substance Z, the diluent, the phospholipid, the PEG lipid and the sterol.

[0109] In a preferred embodiment, the lipid carrier consists of the substance Z, the phospholipid, the PEG lipid and the sterol.

[0110] The present application also provides a lipid nanoparticle comprising a therapeutic and / or prophylactic agent and the aforementioned lipid carrier.

[0111] In a preferred embodiment, the therapeutic and / or prophylactic agent is one or more than two nucleic acids. The nucleic acid is a conventional nucleic acid in the art. The therapeutic and / or prophylactic agent can be a single-stranded deoxyribonucleic acid (DNA), double-stranded DNA, small interfering RNA (siRNA), asymmetric double-stranded small interfering RNA (aiRNA), microRNA (miRNA), small hairpin RNA (shRNA), circular RNA (circRNA), transfer RNA (tRNA), messenger RNA (mRNA) and other forms of nucleic acid molecules known in the art, preferably mRNA, such as firefly luciferase (Fluc) mRNA, unmodified U synthetic Luc mRNA or polyinosinic-polycytidylic acid (poly(I:C)).

[0112] In a preferred embodiment, the therapeutic and / or prophylactic agent is VZV gE mRNA (varicella-zoster virus vaccine RNA), preferably having a nucleic acid sequence as shown in SEQ ID NO: 01.

[0113] The present application also provides a composition comprising a substance Z, the substance Z being a compound as shown in Formula I or a pharmaceutically acceptable salt thereof as previously described.

[0114] In a preferred embodiment, the composition further comprises one or more of a diluent, a phospholipid, a PEG lipid, a sterol and a therapeutic and / or prophylactic agent.

[0115] In a preferred embodiment, the diluent, the phospholipid, the PEG lipid, the sterol and the therapeutic and / or prophylactic agent in the composition are as previously described.

[0116] In a preferred embodiment, the substance Z in the composition forms a lipid carrier as previously described with one or more of the diluent, the phospholipid, the PEG lipid and the sterol.

[0117] In a preferred embodiment, the lipid carrier in the composition forms a lipid nanoparticle as previously described with the therapeutic and / or prophylactic agent.

[0118] In a preferred embodiment, the encapsulation efficiency of the therapeutic and / or prophylactic agent in the composition is at least 94.5%, preferably at least 95%, such as 95.6%.

[0119] In a preferred embodiment, the particle size of the therapeutic and / or prophylactic agent in the composition is 50-70 nm, such as 50-60 nm, such as 55 nm or 59 nm.

[0120] The present application provides the use of a compound of Formula I, or a pharmaceutically acceptable salt thereof, or the lipid carrier for the preparation of a medicament for the prevention and / or treatment of inflammation, such as an infectious inflammation.

[0121] The present application provides the use of a compound of Formula I, or a pharmaceutically acceptable salt thereof, or the lipid carrier for the preparation of a medicament for the prevention and / or treatment of inflammation caused by the nucleic acid.

[0122] The present application provides the use of a compound of Formula I, or a pharmaceutically acceptable salt thereof, or the lipid carrier for the preparation of a medicament for the prevention and / or treatment of inflammation caused by an elevated expression level of MCP-1, IL-6, ISG54 or NF-κΒ.

[0123] In a preferred embodiment, the elevated expression level of MCP-1, IL-6, ISG54 or NF-κΒ is an elevated expression level of MCP-1, IL-6, ISG54 or NF-κΒ induced by the nucleic acid.

[0124] The present application provides a method for the prevention and / or treatment of inflammation, comprising administering (a therapeutically effective amount of) a compound of Formula I, or a pharmaceutically acceptable salt thereof, or the lipid carrier to a subject.

[0125] As used herein, the terms have the following meanings:

[0126] The term "pharmaceutically acceptable" means relatively non-toxic, safe, and suitable for use with patients.

[0127] The term "pharmaceutically acceptable salt" means a salt of a compound with a pharmaceutically acceptable acid or base.

[0128] In structural fragments, means that the structural fragment is attached to the rest of the molecule through the indicated site. For example, means n-hexyl.

[0129] The term "alkyl" means a straight, branched, or cyclic hydrocarbon chain having the indicated number of carbon atoms, for example, C 1-30Alkyl groups are saturated, linear or branched, and include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc.

[0130] The term "alkylene" refers to a divalent group that is attached to the rest of the molecule by two single bonds, and the rest of the definition is the same as that of the term "alkyl".

[0131] The term "alkenyl" refers to a group having at least two carbon atoms (e.g., C4, C5, C6, C7, C8, C9 ... 2-30 An unsaturated monovalent hydrocarbon group, which is straight-chain or branched, contains one or more carbon-carbon double bonds and no carbon-carbon triple bonds, wherein the one or more carbon-carbon double bonds may be located at the end of the group or inside the group.

[0132] The term "alkynyl" refers to a group having at least two carbon atoms (e.g., C32-C4 ... 2-30 An unsaturated monovalent hydrocarbon group, which is straight-chain or branched, contains one or more carbon-carbon triple bonds and has no carbon-carbon double bonds, wherein the one or more carbon-carbon triple bonds may be located at the end of the group or inside the group.

[0133] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0134] The reagents and raw materials used in this invention are all commercially available.

[0135] The positive and progressive effects of this invention are as follows: the lipid nanoparticles (LNPs) formed by the nanomaterials and mRNA provided in this application are a novel nanostructure that differs from traditional LNPs, and possess one or more superior formulation properties compared to traditional LNPs:

[0136] (1) High drug loading capacity;

[0137] (2) The formulation has strong stability;

[0138] (3) High expression level in vivo;

[0139] (4) Effectively inhibits the decrease in mRNA expression caused by repeated administration;

[0140] (5) It has immunosuppressive function and significantly inhibits the release of inflammatory factors induced by mRNA LNP, thus avoiding redundant inflammatory responses and having higher safety. For example, it can inhibit the release of MCP-1 and IL-6; it can also inhibit the expression of ISG54 and NF-κB. Attached Figure Description

[0141] Figure 1 shows a gel electrophoresis diagram of the freeze-thaw process of HL-3LNP of Luc mRNA.

[0142] Figure 2 is an in vivo fluorescence imaging diagram of Luc mRNA's HL-3 LNP and Luc mRNA's HL-6 LNP.

[0143] Figure 3 is an in vivo fluorescence imaging diagram of Luc mRNA's HL-3 LNP 4 times administration experiment with 3 days interval.

[0144] Figure 4 is a relative percentage of in vivo fluorescence amount of Luc mRNA's HL-3 LNP 4 times administration experiment with 3 days interval relative to the fluorescence amount of Luc mRNA's SM-102 LNP. DETAILED DESCRIPTION

[0145] The present application is further illustrated by the following examples without limiting the present application to the described examples. The experimental methods in the following examples without specific conditions are selected according to the conventional methods and conditions, or according to the product instructions.

[0146] Example 1, synthesis of Lipid 1

[0147] The synthesis route of Lipid 1 is as follows:

[0148] Hydroxychloroquine (HCQ) 335.9 mg (1 mmol) and isostearic acid (h1) 284.5 mg (1 mmol) were weighed and dissolved in 20 mL of anhydrous dichloromethane (DCM), and dicyclohexyl carbodiimide (DCC) 309.5 mg (1.5 mmol) and 4-dimethylaminopyridine (DMAP) 24.4 mg (0.2 mmol) were added, and the reaction was carried out at room temperature for 6 h. TLC was used to monitor the reaction, and the developing agent was DCM:MeOH (V / V) = 20:1.

[0149] After the reaction was completed, the insoluble matter in the reaction solution was filtered out, and the reaction solvent was evaporated under reduced pressure. 15 mL of water and 15 mL of ethyl acetate (EA) were added, extracted, and separated by standing. The organic phase was dried over anhydrous sodium sulfate and concentrated. The product was separated by column chromatography, and the eluent was DCM:MeOH (V / V) = 50:1. The solvent was evaporated under reduced pressure to obtain 447.8 mg of light yellow oily liquid, which was Lipid 1, with a yield of 74.3%.

[0150] H-NMR characterization is as follows: δ H(600 MHz, CDC13) 8.52-8.50 (1H, m, J 5.4), 7.94 (1H, d, J 2.2), 7.70 (1H, d, J 9.0), 7.34 (1H, dd, J 9.0, 2.2), 6.42 (1H, d, J 5.5), 4.14 (2H, t, J 6.2), 3.75-3.65 (1H, m), 2.73-2.46 (7H, m), 2.26 (1H, t, J 7.6), 1.62-1.54 (4H, m, J 13.7, 7.1), 1.31 (3H, t, J 6.2), 1.29-1.14 (32H, m), 1.00 (3H, t, J 7.1), 0.91-0.79 (6H, m, J 12.4, 10.7, 9.5, 6.2).

[0151] Synthesis of Lipid 6

[0152] The synthesis route of Lipid 6 is as follows:

[0153] (1) Synthesis of Compound 1:

[0154] Seventeen-9-ol (h2) 1.2823 g (5 mmol), succinic anhydride (h3) 1.0007 g (10 mmol) and DMAP 0.1222 g (1 mmol) were weighed and dissolved in 20 mL of DCM, and reacted at room temperature for 12 h.

[0155] After the reaction was completed, the reaction solvent was removed by evaporation under reduced pressure, and the product was separated by column chromatography with PE (petroleum ether): EA (V / V) = 1:1 as the eluent. The solvent was removed by evaporation under reduced pressure to obtain 1.6846 g of product Compound 1 with a yield of 94.5%.

[0156] (2) Synthesis of Lipid 6:

[0157] HCQ 335.9 mg (1.5 mmol) and Compound 1 356.5 mg (1 mmol) were dissolved in 20 mL of anhydrous DCM, and DCC 309.5 mg (1.5 mmol) and DMAP 24.4 mg (0.2 mmol) were added. The reaction was carried out at room temperature for 6 h. The reaction was monitored by TLC with DCM: MeOH (V / V) = 20:1 as the developing agent.

[0158] After the reaction was completed, the insoluble matter in the reaction solution was filtered off, the reaction solvent was evaporated under reduced pressure, 15 mL of water and 15 mL of EA were added, extracted, and separated by standing. The organic phase was dried over anhydrous sodium sulfate and concentrated. The product was separated by column chromatography, eluent was DCM:MeOH (V / V) = 45:1, and the solvent was evaporated under reduced pressure to obtain 485.6 mg of a light yellow oily liquid, which was Lipid 6, with a yield of 72.0%.

[0159] H-NMR characterization is as follows: H (600 MHz, CDC13) 8.51 (1H, d, J 5.4), 7.95 (1H, d, J 2.1), 7.71 (1H, d, J 9.0), 7.35 (1H, dd, J 8.9, 2.2), 6.42 (1H, d, J 5.5), 4.90-4.82 (1H, m), 4.16 (2H, t), 3.75-3.65 (1H, m), 2.71-2.46 (11H, m), 1.54-1.45 (4H, m), 1.32 (3H, d, J 6.3), 1.30-1.20 (28H, m), 1.00 (3H, t, J 7.1), 0.87 (6H, td, J 7.0, 4.0).

[0160] Example 3, synthesis of Lipid 10

[0161] The synthesis route of Lipid 10 is as follows:

[0162] (1) Synthesis of Compound 2:

[0163] Take 2-decyl-1-tetradecanol (h4) 1.0639 g (3 mmol), h3 0.6004 g (6 mmol) and DMAP 0.1222 g (1 mmol), dissolve in 20 mL of DCM, and react at room temperature for 12 h.

[0164] After the reaction was completed, the insoluble matter in the reaction solution was filtered off, the reaction solvent was evaporated under reduced pressure, 15 mL of water and 15 mL of EA were added, extracted, and separated by standing. The organic phase was dried over anhydrous sodium sulfate and concentrated. The product was separated by column chromatography, eluent was DCM:MeOH (V / V) = 45:1, and the solvent was evaporated under reduced pressure to obtain 485.6 mg of a light yellow oily liquid, which was Lipid 6, with a yield of 72.0%.

[0165] (2) Synthesis of Lipid 10:

[0166] HCQ 335.9 mg (1.5 mmol) and Compound 2 454.7 mg (1 mmol) were dissolved in 20 mL of anhydrous DCM, and DCC 309.5 mg (1.5 mmol) and DMAP 24.4 mg (0.2 mmol) were added, and the reaction was carried out at room temperature for 6 h. The reaction was monitored by TLC, and the developing agent was DCM:MeOH (V / V) = 20:1.

[0167] After the reaction was completed, the insoluble matter in the reaction solution was filtered off, the reaction solvent was evaporated under reduced pressure, 15 mL of water and 15 mL of EA were added, and the extraction was carried out, and the organic phase was dried over anhydrous sodium sulfate and concentrated. The product was separated by column chromatography, and the eluent was DCM:MeOH (V / V) = 45:1, and the solvent was evaporated under reduced pressure to obtain 590.3 mg of a light yellow oily liquid, which was Lipid 10, with a yield of 76.4%.

[0168] H-NMR characterization is as follows: H (600 MHz, CDC13) 8.51 (1H, d, J 5.4), 7.94 (1H, d, J 2.2), 7.70 (1H, d, J 9.0), 7.34 (1H, dd, J 8.9, 2.2), 6.42 (1H, d, J 5.5), 4.16 (2H, t, J 6.2), 3.97 (2H, d, J 5.8), 3.74-3.64 (1H, m), 2.72-2.47 (13H, m), 1.32 (3H, d, J 6.4), 1.30-1.20 (45H, m), 1.00 (3H, t, J 7.1), 0.88 (6H, t, J 7.0).

[0169] Example 4, synthesis of Lipid 21

[0170] The synthesis route of Lipid 21 is as follows:

[0171] (1) Synthesis of Compound 3:

[0172] h5) 0.5306 g (5 mmol), DCC 3.0949 g (15 mmol) and DMAP 0.1222 g (1 mmol) were dissolved in a mixed solvent of anhydrous DCM and N,N-dimethylformamide (DMF) 15 mL (volume ratio 2:1), and 3.5654 g of Compound 1 (10 mmol) was dissolved in 25 mL of anhydrous DCM, and it was slowly added dropwise into the mixed solution of h5, and the reaction was carried out at room temperature for 12 h.

[0173] After the reaction was completed, the insoluble matter in the reaction solution was filtered out, the reaction solvent was evaporated under reduced pressure, and PE was added to precipitate the insoluble impurities. The product was separated by column chromatography, the eluent was PE:EA (V / V) = 7:1, and the solvent was evaporated under reduced pressure to obtain 2.0786 g of colorless liquid, which was Compound 3, with a yield of 53.1%.

[0174] (2) Synthesis of Compound 4:

[0175] Compound 3 1.5664 g (2 mmol), h3 0.6109 g (4 mmol), and DMAP 0.0611 g (0.5 mmol) were weighed and dissolved in 20 mL of DCM, and the reaction was carried out at room temperature for 12 h. After the reaction was completed, the reaction solvent was evaporated under reduced pressure, and the product was separated by column chromatography, the eluent was PE:EA (V / V) = 5:1, and the solvent was evaporated under reduced pressure to obtain 1.5033 g of colorless semisolid, which was Compound 4, with a yield of 85.1%.

[0176] (3) Synthesis of Lipid 21:

[0177] HCQ 335.9 mg (1.5 mmol) and Compound 4 883.3 mg (1 mmol) were dissolved in 20 mL of anhydrous DCM, and DCC 309.5 mg (1.5 mmol) and DMAP 24.4 mg (0.2 mmol) were added, and the reaction was carried out at room temperature for 12 h. The reaction was monitored by TLC, and the developing agent was DCM:MeOH (V / V) = 20:1.

[0178] After the reaction was completed, the insoluble matter in the reaction solution was filtered out, the reaction solvent was evaporated under reduced pressure, and 15 mL of water and 15 mL of EA were added, and the mixture was extracted and allowed to stand to separate. The organic phase was dried over anhydrous sodium sulfate and concentrated. The product was separated by column chromatography, the eluent was DCM:MeOH (V / V) = 60:1, and the solvent was evaporated under reduced pressure to obtain 679.9 mg of yellowish oily liquid, which was Lipid 21, with a yield of 56.6%.

[0179] H-NMR characterization is as follows: δΗ(600 MHz, CDCI3) 8.50 (1 H, d, J 5.5), 7.97 (1 H, d, J 1.9), 7.73 (1 H, d, J 9.0), 7.36 (1 H, dd, J 8.9, 2.1 ), 6.43 (1 H, d, J 5.6), 4.86 (2H, 28,37, J 6.2), 4.19 - 4.12 (8H, m), 3.70 (1 H, d, J 3.2), 2.73 - 2.49 (19H, m), 2.42 - 2.37 (1 H, m, J 6.0), 1.77 - 1.58 (4H, m), 1.50 (8H, d, J 6.0), 1.33 (3H, d, J 6.4), 1.31 - 1.21 (48H, m), 1.01 (3H, t, J 7.1 ), 0.87 (12H, t, J 7.0).

[0180] Example 5, synthesis of Lipid 29

[0181] The synthesis route of Lipid 29 is as follows:

[0182] Synthesis of Compound 5

[0183] h2 2.5647g (10mmol) and triethylamine (TEA) 2.0238g (20mmol) were weighed into 20mL anhydrous DCM and stirred in an ice bath for pre-cooling, and 1.3576g acryloyl chloride (h6) was weighed into 25mL anhydrous DCM and slowly dropped into the mixture of h2 using a dropping funnel, and the ice bath reaction was carried out for 4h.

[0184] After the reaction was completed, the insoluble matter in the reaction solution was filtered out, the reaction solvent was evaporated under reduced pressure, and the precipitated insoluble impurities were redissolved in PE. The product was separated by column chromatography, the eluent was PE: DCM (V / V) = 4:1, and the solvent was evaporated under reduced pressure to obtain 2.7465g of colorless liquid, which was Compound 5, with a yield of 88.4%.

[0185] (2) Synthesis of Compound 6

[0186] Compound 5 1.8361g (6mmol), ethanolamine (h7) 0.1832g (3mmol) and TEA 0.3036g (3mmol) were weighed into 30mL isopropyl alcohol and refluxed at 80°C for 18h.

[0187] After the reaction was completed, the reaction solvent was evaporated under reduced pressure, the product was separated by column chromatography, the eluent was PE: EA (V / V) = 6:1, and the solvent was evaporated under reduced pressure to obtain 1.6842g of light yellow liquid, which was Compound 6, with a yield of 82.3%.

[0188] (3) Synthesis of Compound 7

[0189] Compound 6 1.3643 g (2 mmol), h3 0.6109 g (4 mmol) and DMAP 0.0611 g (0.5 mmol) were weighed and dissolved in 20 mL of DCM, and reacted at room temperature for 12 h. After the reaction was completed, the reaction solvent was removed by evaporation under reduced pressure, and the product was separated by column chromatography with PE:EA (V / V) = 4:1 as the eluent. The solvent was removed by evaporation under reduced pressure to obtain 1.4460 g of white solid, which was Compound 7, with a yield of 92.4%.

[0190] Synthesis of Lipid 29:

[0191] HCQ 335.9 mg (1.5 mmol) and Compound 7 782.2 mg (1 mmol) were dissolved in 20 mL of anhydrous DCM, and DCC 309.5 mg (1.5 mmol) and DMAP 24.4 mg (0.2 mmol) were added. The reaction was carried out at room temperature for 12 h. TLC was used to monitor the reaction, with DCM:MeOH (V / V) = 20:1 as the developing agent.

[0192] After the reaction was completed, the insoluble matter was filtered out, and the reaction solvent was removed by evaporation under reduced pressure. Then 15 mL of water and 15 mL of EA were added for extraction, and the organic phase was dried over anhydrous sodium sulfate and concentrated. The product was separated by column chromatography with DCM:MeOH (V / V) = 60:1 as the eluent. The solvent was removed by evaporation under reduced pressure to obtain 540.1 mg of yellowish oil, which was Lipid 29, with a yield of 49.1%.

[0193] H-NMR characterization is as follows: δH(600MHz, CDCl3) 8.51 (1H, d, J 5.4), 7.95 (1H, d, J 2.1), 7.71 (1H, d, J 9.0), 7.35 (1H, dd), 6.42 (1H, d, J 5.5), 4.89-4.82 (2H, m), 4.19-4.10 (4H, m), 3.70 (1H, d, J 3.0), 2.86-2.39 (21H, m), 1.50 (8H, d, J 6.1), 1.32 (3H, d, J 6.3), 1.30-1.20 (52H, m), 1.00 (3H, t, J 9.1, 5.1), 0.88 (12H, td, J 6.9, 2.2).

[0194] Example 6, Synthesis of Lipid 47

[0195] The synthesis route of Lipid 47 is as follows:

[0196] (1) Synthesis of Compound 8

[0197] SM-102 1.4204 g (2 mmol), h3 0.6109 g (4 mmol) and DMAP 0.0611 g (0.5 mmol) were weighed and dissolved in 20 mL of DCM, and reacted at room temperature for 12 h. After the reaction was completed, the reaction solvent was removed by evaporation under reduced pressure, and the product was separated by column chromatography with DCM:MeOH (V / V) = 20:1 as the eluent. The solvent was removed by evaporation under reduced pressure to obtain 1.5087 g of a light yellow oily liquid, which was Compound 8, with a yield of 93.1%.

[0198] (2) Synthesis of Lipid 47

[0199] HCQ 335.9 mg (1.5 mmol) and Compound 8 810.25 mg (1 mmol) were dissolved in 20 mL of anhydrous DCM, and DCC 309.5 mg (1.5 mmol) and DMAP 24.4 mg (0.2 mmol) were added. The reaction was carried out at room temperature for 12 h. The reaction was monitored by TLC with DCM:MeOH (V / V) = 20:1 as the developing agent.

[0200] After the reaction was completed, the insoluble matter was filtered out, and the reaction solvent was removed by evaporation under reduced pressure. 15 mL of water and 15 mL of EA were added, and the mixture was extracted and allowed to stand to separate. The organic phase was dried over anhydrous sodium sulfate and concentrated. The product was separated by column chromatography with DCM:MeOH (V / V) = 35:1 as the eluent. The solvent was removed by evaporation under reduced pressure to obtain 515.6 mg of a light yellow oily liquid, which was Lipid 47, with a yield of 45.7%.

[0201] H-NMR characterization is as follows: δ H (600 MHz, CDCl3) 8.51 (1H, d, J 5.4), 7.94 (1H, d, J 2.1), 7.71 (1H, dd, J 9.0, 4.6), 7.34 (1H, dd, J 8.9, 2.1), 6.42 (1H, d, J 5.5), 4.89-4.83 (1H, m), 4.19-4.09 (4H, m), 4.08-4.03 (2H, m), 3.70 (1H, d, J 2.8), 2.72-2.38 (17H, m), 2.32-2.25 (4H, m), 1.77-1.55 (14H, m), 1.50 (4H, d, J 5.9), 1.33-1.23 (51H, m), 1.00 (3H, t, J 9.1, 5.1), 0.89-0.86 (9H, m, J 7.2, 4.5, 1.9).

[0202] Example 7, Preparation of Luc mRNA HL-3 LNP and Luc mRNA HL-6 LNP

[0203] Lipid 29 (HL-3), DSPC (purchased from Avanti (Shanghai) Pharmaceutical Co., Ltd.), Chol (purchased from Avanti (Shanghai) Pharmaceutical Co., Ltd.), DMG-PEG2000 (purchased from Avanti (Shanghai) Pharmaceutical Co., Ltd.) were dissolved in ethanol with a molar ratio of 50:10:38.5:1.5 to prepare a mixed lipid solution with a HL-3 molar concentration of 12 mM. Luciferase (Luc) mRNA (Shanghai Weiguan Biological Technology Co., Ltd.) was dispersed in citrate buffer to prepare an acidic mRNA solution with a mass concentration of 0.22 mg / mL. Using a microfluidic device, the mRNA solution was mixed with the mixed lipid ethanol solution (HL-3 molecules and mRNA nucleotides molar ratio 6:1) at a flow rate of 12 mL / min and a volume ratio of 3:1 (i.e. mRNA solution: mixed lipid ethanol solution 3:1). The mixture was dialyzed against 0.01M PBS for 12-24h to remove ethanol, and the LNP solution was concentrated by ultrafiltration (MWCO 10KDa) after dialysis, and filtered through a 0.22μm sterile filter, which was Luc mRNA HL-3 LNP.

[0204] The preparation method of Luc mRNA HL-6 LNP, DLin-MC3-DMA (MC3) LNP (referred to as MC3 LNP) and SM-102 LNP is the same as above, and the hydroxychloroquine derivative lipid used for HL-6 LNP is Lipid 21.

[0205] Example 8, Formulation characterization of Luc mRNA HL-3 LNP and Luc mRNA HL-6 LNP

[0206] The LNP formulation was diluted to a mRNA mass concentration of 0.01 mg / mL, and the average particle size and polydispersity index (PDI) of the LNP were determined using a Malvern particle size analyzer, and the encapsulation efficiency (EE) of the LNP was determined using a quantitative ribonucleic acid assay kit (Quant-it Ribogreen RNA), and the results are shown in Table 1.

[0207] Table 1 MC3 LNP, SM-102 LNP, HL-3 LNP and HL-6 LNP formulation characterization parameters

[0208] Example 9, Detection of apparent pKa of Luc mRNA HL-3 LNP and Luc mRNA HL-6 LNP

[0209] To determine the apparent pKa of LNP, LNP was incubated with TNS (2-p-toluidinyl-6-naphthylsulfonic acid) at different pH conditions and the TNS fluorescence signal was detected. For example, in low pH environment, the traditional ionizable lipid dissociates, the LNP surface is positively charged, interacts with negatively charged TNS, and makes it enter the hydrophobic environment to produce fluorescence, while in high pH environment, the LNP is negatively charged, and the fluorescence signal is weakened. Based on this, the pH value at which 50% of the ionizable lipid is charged is defined as the apparent pKa. A series of buffers with pH ranging from 2.0 to 12.0 were prepared by adding 2M sodium hydroxide and 2M hydrochloric acid to the base buffer (10mM sodium phosphate, 10mM sodium borate, 10mM sodium citrate, 150mM sodium chloride). Add 94μL of the above series of buffers with different pH to a black 96-well plate, then add 4μL of LNP solution (diluted with PBS buffer (pH 7.4) to a mRNA mass concentration of 0.05mg / mL) and 2μL of TNS solution (TNS molar concentration is 300μM, solvent is 10% DMSO / water (V / V)). After adding the LNP solution, mix it evenly by gently shaking, and place it in the dark at room temperature for 5min. Use the enzyme marker to detect the fluorescence intensity at 435nm emission wavelength under 325nm excitation wavelength. The fluorescence intensity is the Y axis, and the buffer pH value is the X axis. The LogEC50 value is the apparent pKa of the LNP to be tested.

[0210] The apparent pKa of the SM-102 LNP, HL-3 LNP and HL-6 LNP prepared in Example 7 was detected by the above method, and the results are shown in Table 2.

[0211] Table 2 Apparent pKa test data of SM-102 LNP, HL-3 LNP and HL-6 LNP (n=3)

[0212] The results show that the hydroxychloroquine derivative lipid LNP has a completely opposite pKa curve from the traditional ionizable lipid SM-102-LNP. From the analysis of LNP structure and TNS detection principle, it can be seen that the hydroxychloroquine derivative lipid LNP is a kind of nanoparticle with a new spatial structure different from traditional LNP.

[0213] Example 10 Freeze-thaw stability of HL-3 LNP of Luc mRNA

[0214] Luc mRNA's HL-3 LNP and Luc mRNA's SM-102 LNP freshly prepared according to Example 7 were repeatedly freeze-thawed (FT) 5 times at -20 °C and -80 °C, and the particle size, PDI and encapsulation efficiency were detected, and the results are shown in Table 3, Table 4, Table 5, respectively. The results show that Luc mRNA's HL-3 LNP has more stable formulation properties than Luc mRNA's SM-102 LNP during freeze-thawing, indicating that the nano-space structure of hydroxychloroquine derivative lipid particles is more stable than traditional ionizable lipid SM-102-LNP.

[0215] Table 3 Freeze-thaw stability test of Luc mRNA's HL-3 LNP at -20 °C

[0216] Table 4 Freeze-thaw stability test of Luc mRNA's SM-102 LNP at -20 °C

[0217] Table 5 Freeze-thaw stability test of Luc mRNA's HL-3 LNP at -80 °C

[0218] In the above freeze-thaw experiment, the mRNA integrity was detected synchronously. The LNP freeze-thawed samples were treated with Triton X-100 at 70 °C for 3 min, and the mRNA integrity was detected by Bio-Rad gel electrophoresis, and the results are shown in Figure 1. The results show that Luc mRNA's HL-3 LNP is still not destroyed after Triton treatment, showing the strong stability of hydroxychloroquine derivative lipid LNP.

[0219] The above freeze-thaw stability experiment and apparent pKa experiment both show that the hydroxychloroquine derivative lipid LNP has a new nano-space structure that is different from traditional LNP.

[0220] Example 11, Luc mRNA's HL-3 LNP and Luc mRNA's HL-6 LNP in vivo expression of Luc mRNA

[0221] The HL-3 LNP, HL-6 LNP, DLin-MC3-DMA (MC3) LNP and SM-102 LNP preparations of Luc mRNA were prepared according to the method of Example 7. Each group of 3 male BALB / c mice (purchased from Beijing Huafukang Biosciences Co., Ltd., body weight 20-25 g) were injected with 5 μg of Luc mRNA (100 μL of LNP solution with mRNA mass concentration of 0.05 mg / mL) via the tail vein of each BALB / c mouse, and 4 h later, 3 mg of D-luciferin potassium salt (200 μL of solution with PBS buffer, mass concentration of 15 mg / mL) was injected intraperitoneally, and the bioluminescence signal was detected by the IVIS small animal in vivo imaging system. The control was normal saline. The results are shown in Figure 2 and Table 6.

[0222] Table 6. In vivo expression detection results of HL-3 LNP, HL-6 LNP, MC3 LNP and SM-102 LNP preparations of Luc mRNA

[0223] The results show that the mRNA in vivo expression of the hydroxychloroquine derivative lipid LNP is significantly better than that of MC3, and is comparable to that of SM-102.

[0224] Example 12, repeated administration test of HL-3 LNP

[0225] The HL-3 LNP, DLin-MC3-DMA (MC3) LNP and SM-102 LNP preparations of Luc mRNA synthesized with unmodified uracil (Unmodified U) were prepared according to the method of Example 7. The administration and detection methods were according to Example 11, and each mouse was administered 4 times with a 3 d-gap between each administration. The bioluminescence signal was detected and the mouse body weight change curve was monitored 4 h after administration, and the results are shown in Table 7, Table 8, Figure 3 and Figure 4.

[0226] Table 7. In vivo bioluminescence signal detection results of repeated administration of preparations

[0227] Table 8. Monitoring results of mouse body weight change after repeated administration of preparations

[0228] The hydroxychloroquine derivative lipid effectively inhibited the decrease in mRNA expression amount after repeated administration, and improved safety, which is related to the inhibition of immune activation and inflammatory response by hydroxychloroquine. The above results suggest that HL-LNP has application prospects in the field of protein supplementation requiring repeated administration.

[0229] Example 13, cytokine detection of HL-3 LNP single administration

[0230] The HL-3 LNP, MC3 LNP and SM-102 LNP formulations of unmodified U synthetic Luc mRNA (Shanghai Generex Biosciences Co., Ltd.) were prepared by the method of Example 7. Six male BALB / c mice (Beijing Huafukang Biosciences Co., Ltd., body weight 20-25 g) in each group were injected intramuscularly with 25 μg of Luc mRNA (250 μL of LNP solution with mRNA mass concentration of 0.1 mg / mL), and the orbital blood of the mice was collected at 6 h and 48 h after administration, respectively, to separate the serum. The concentrations of MCP-1 and IL-6 in the serum of the mice were detected by Luminex Assay multi-factor detection kit (R&D SYSTEMS), and the results are shown in Table 9.

[0231] Table 9 Concentrations of MCP-1 and IL-6 in the serum of mice after single administration of HL-3 LNP

[0232] The results show that the HL-3 lipid has a significant inhibitory effect on the release of inflammatory factors caused by LNP, and the serum levels of MCP-1 and IL-6 are significantly lower than those of the SM-102 group.

[0233] Example 14, Inhibition of HL Lipid on the Intrinsic Immune Response Induced by Poly(I:C) at the Cell Line Level

[0234] The HL-3 LNP, HL-6 LNP and SM-102 LNP formulations of poly(I:C) (Sigma-Aldrich) were prepared by the method of Example 7, and the concentration of poly(I:C) was controlled at 250 ng / μL.

[0235] The suspension of THP1-Dual Cells (InvivoGen) in RPMI 1640 medium (Invitrogen) was plated in a 96-well plate at 50000 cells / 180 μL / well, and then transfected with poly(I:C).

[0236] The LNP formulations of poly(I:C) described above were diluted ten times with 8% sucrose Tris-acetic acid solution to a concentration of 25 ng / μL of poly(I:C), and were ready for use.

[0237] 1 μg of poly(I:C) was added to 20 μL of Opti-MEM (Invitrogen) and mixed, and 4 μL of Lipofectamine 2000 (Invitrogen) was added to 20 μL of Opti-MEM and mixed, and the above Opti-MEM solutions were mixed, shaken gently, and incubated at room temperature for 10 min, and were ready for use.

[0238] The above poly(I:C) sample to be used was added to the THP1-Dual 96-well plate, 20 μL per well, i.e. 500 ng poly(I:C) per well, three replicates per group, and incubated in a 37°C CO2 incubator for 24 h.

[0239] Take 200 ng hairpin RNA (Shanghai Weiguan Biotechnology Co., Ltd.) and mix with 20 μL Opti-MEM, take another 1 μL Lipofectamine 2000 and mix with 20 μL Opti-MEM, mix the above Opti-MEM solutions, shake gently, incubate at room temperature for 10 min, and use. The above hairpin RNA sample to be used was added to the THP1-Dual 96-well plate, 20 μL per well, i.e. 500 ng poly(I:C) per well, three replicates, as the hairpin RNA control group, and incubated in a 37°C CO2 incubator for 24 h.

[0240] Take another 8% sucrose Tris-acetic acid solution and add it to the THP1-Dual 96-well plate, 20 μL per well, three replicates, as the 8% Tris AC control group, and incubate in a 37°C CO2 incubator for 24 h.

[0241] Take another RPMI 1640 medium and add it to the THP1-Dual 96-well plate, 20 μL per well, three replicates, as the untreated control group, and incubate in a 37°C CO2 incubator for 24 h.

[0242] After 24 h, the cell viability was detected using the CellTiter-Glo 3D Cell Viability Assay Kit (Promega (Beijing) Biotechnology Co., Ltd.), and the results are shown in Table 10.

[0243] Take 1.5 mL of QUANTI-Luc 4 Reagent (20X) (InvivoGen) and dilute it 20 times with 2.35 mL of sterile water to obtain the QUANTI-Luc solution. Take 20 μL of the cell supernatant from the 96-well plate and add it to a 96-well white plate, then add 20 μL of the QUANTI-Luc solution, mix gently, and immediately detect the bioluminescence, which is the Lucia luciferase reporter gene controlled by ISG54 activation, to characterize the activation of ISG54, and the results are shown in Table 10.

[0244] Take QUANTI-Blue Reagent (InvivoGen) 1 mL and QUANTI-Blue Buffer (InvivoGen) 1 mL, add 98 mL of sterile water and mix well, which is the QUANTI-Blue solution. Take 20 μL of cell supernatant of 96-well plate and add 180 μL of QUANTI-Blue solution to a 96-well transparent plate, mix gently and immediately detect the absorbance at 620 nm, which is the secreted embryonic alkaline phosphatase (SEAP) reporter gene controlled by NF-κB activation, which is used to characterize the activation of NF-κB, as shown in Table 10.

[0245] Table 10

[0246] The results show that SM-102 LNP loaded poly (I:C) induces high levels of ISG54 and NF-κB, while HL-3 LNP and HL-6 LNP are comparable to the blank control level, indicating that HL lipids have a significant inhibitory effect on the induction of innate immune response by polyinosinic-polycytidylic acid (poly (I:C)) at the cell line level.

[0247] Example 15. HL lipids inhibit the toxicity of poly (I:C) in mice

[0248] Prepare the mixed nucleic acid preparations of HL-6 LNP and SM-102 LNP co-loaded with poly (I:C) (Sigma-Aldrich) and Luc mRNA (Shanghai Weiguan Biotechnology Co., Ltd.) according to the method of Example 7, wherein the mass ratio of poly (I:C) and Luc mRNA is 1:1, and the concentration of poly (I:C) and mRNA is controlled to be 0.05 mg / mL.

[0249] Inject the mixed nucleic acid preparations into the tail vein of male BALB / c mice (Beijing Huafukang Biotechnology Co., Ltd., body weight 20-25 g) at a dose of 5 μg poly (I:C) and 5 μg Luc mRNA (i.e. 100 μL of the above mixed nucleic acid preparation) per mouse, with 5 mice per group. At 4 hours, 24 hours and 48 hours after administration, detect the expression of Luc according to the method of Example 11, and the results are shown in Table 11; at 4 hours after administration, separate the mouse orbital blood to obtain serum, and use the Luminex Assay multi-factor detection kit (R&D SYSTEMS) to detect the concentration of mouse serum cytokines, and the results are shown in Table 12; observe and record the survival status of mice every day, and all mice in the SM-102 group died at 48 hours, and all mice in the HL-6 group survived well at 216 hours.

[0250] Table 11

[0251] Table 12

[0252] The above results show that intravenous injection of poly(I:C) encapsulated in traditional lipid LNP is lethal to mice, while all mice injected intravenously with HL-LNP@poly(I:C) survived, indicating that HL lipid can significantly inhibit the lethal toxicity of poly(I:C) to mice. Monitoring of cytokines showed that poly(I:C) encapsulated in traditional lipid LNP can induce a strong cytokine storm in mice, while the introduction of HL lipid significantly reduced the level of cytokines and protected mice from death.

[0253] Example 16. Application of HL lipid in varicella-zoster virus (VZV) vaccine

[0254] HL-3LNP, HL-6LNP and SM-102LNP of VZV gE mRNA (Shanghai Lanqiao Biomedicine Co., Ltd.) were prepared according to the method of Example 7, and the concentrations of poly(I:C) and VZV gE mRNA were both 0.02 mg / mL. A placebo of 8% sucrose in Tris-acetic acid solution was set up, and the following four groups were set up: placebo, SM-102-gE, HL-3-gE, HL-6-gE, 5 male BALB / c mice (Beijing Huafukang Biosciences Co., Ltd., body weight 20-25 g) in each group, and the immunization dose was 2 μg mRNA (100 μL of the above gE mRNA preparation) by intramuscular injection, and the placebo group was injected with the same volume of 8% sucrose in Tris-acetic acid solution.

[0255] The administration and sampling procedures are as follows: Prime immunization was performed on Day 0, blood was collected 6h after administration, and Luminex Assay Multi-Cytokine Detection Kit (R&D SYSTEMS) was used to detect cytokines (Prime-6h), and the results are shown in Table 13; blood was collected on Day 20, and VZV gE binding antibody titers were detected by ELISA (Prime-Week 3), and the results are shown in Table 14; Boost immunization was performed on Day 21, blood was collected 6h after administration to detect cytokines (Boost-6h); blood was collected on Day 35, VZV gE binding antibody titers were detected by ELISA (Boost-Week 2), mice were sacrificed and spleens were collected, and VZV gE-specific cellular responses were detected by ELISpot, and the results are shown in Table 15.

[0256] ELISA for detection of VZV gE binding antibody titers was performed as follows: 50 μL / well of coating buffer was used to dissolve recombinant VZV gE antigen (ACRO Biosystems) to coat 96-well plates at a concentration of 100 ng / well, and incubated at 4°C overnight. Subsequently, 2% bovine serum albumin blocking solution was prepared using PBS containing 0.1% Tween 20 (PBST), and the antigen-coated plates were blocked at room temperature for 1 hour. After the serum samples were initially diluted 250-fold, 11 gradient two-fold serial dilutions were performed using PBS. After washing the plates with PBST, the gradient-diluted serum was added and incubated at room temperature for 2 hours. To detect the level of gE-specific IgG antibodies, HRP-labeled goat anti-mouse IgG secondary antibody (Proteintech) was added and incubated at 37°C for 1 hour, followed by the addition of tetramethylbenzidine (TMB) substrate solution (Invitrogen) for color development. After about 10 minutes, the reaction was stopped by adding 1N sulfuric acid, and the absorbance value was measured at 450 nm wavelength using a microplate reader.

[0257] ELISpot for detection of VZV gE-specific cellular responses was performed as follows: splenocytes were co-stimulated with gE polypeptide library (GenScript Corporation, concentration 2 μg / mL) and anti-mouse CD28 antibody (BD Bioscience) for 1 hour, followed by the addition of protein transport inhibitor containing monensin (BD Bioscience) for continued incubation for 5 hours. The splenocytes stimulated with the polypeptide library were plated at one million cells per well into ELISpot plates (MABTECH) pre-coated with IFN-γ or IL-2-specific antibodies, and incubated at 37°C for 48 hours. Subsequently, the plate wells were washed with PBS, and biotinylated anti-IFN-γ or anti-IL-2 antibodies were added and incubated at room temperature for 2 hours, followed by reaction with streptavidin-alkaline phosphatase (ALP) at room temperature for 1 hour. After the addition of substrate solution to initiate spot color development, the plate wells were washed with deionized water. Finally, the plate was placed in a light-protected environment for natural drying, and spot counting analysis was performed using an ELISpot analyzer (AT-SPOT, SINSAGE).

[0258] Table 13

[0259] Table 14

[0260] Table 15

[0261] As shown in Table 14, the HL-gE vaccine and the SM-102-gE vaccine induced comparable levels of humoral immune responses; as shown in Table 15, the HL-gE vaccine and the SM-102-gE vaccine induced comparable levels of cellular immune responses. In terms of inflammatory cytokines, the HL lipid significantly inhibited the LNP-mRNA-induced cytokines, comparable to the placebo level, while these inflammatory cytokines can induce systemic inflammatory side effects in the application of infectious disease prevention vaccines. The above results show that the mRNA vaccine based on the HL lipid can induce specific immune responses comparable to the positive control level, while inhibiting the activation of innate immunity by LNP-mRNA and reducing the release of pro-inflammatory cytokines, showing good application prospects of the HL lipid in mRNA vaccines.

[0262] VZV gE mRNA sequence (SEQ ID NO: 01):

Claims

1. A compound of Formula I: ###0001### or a pharmaceutically acceptable salt thereof. wherein: T is C 1-30 alkyl, C 2-30 alkenyl, C 2-30 alkynyl or X is a single bond, O, S, or N(R x ); R x is H or C 1-10 alkyl; L 1 is a single bond, -(CH2) n1 -, X and L 1 are not simultaneously a single bond; A 1 is attached to X, A 2 and A 3 is attached to Y; A 1 -CH2-; and n2 -; A 2 and A 3 independently a single bond or -(CH2) n2 -; M 1 and M 2 is independently O or NH; M 3 for O, S or N(R x ) ; Y is N or CR Y ; R Y is H, C 1-30 alkyl, C 2-30 alkenyl, C 2-30 alkynyl, R 1 and R 2 are independently C 1-30 alkyl, C 2-30 alkenyl, C 2-30 alkynyl, D 1 , D 2 and D 3 is independently O or NH; B 1 and B 3 independently -(CH2) n3 -; B 2 independently C 1-30 alkyl, C 2-30 alkenyl or C 2-30 alkynyl; L 2 For K 1 the end connected to Y, K 2 the end connected to Z 1 is connected; K 1 and K 3 independently -(CH2) n4 -; K 2 independently a single bond or -(CH2) n4 -; H 1 , H 2 , H 3 , H 4 and H 5 is independently O or NH; H 6 independently O, S or NH; Z 1 independently N or CH; R 3 and R 4 independently C 1-30 alkyl, C 2-30 alkenyl or C 2-30 alkynyl; n1, n2, n3 and n4 are independently an integer from 1 to 10.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, having Formula I, ###0001### I which satisfies one or more of the following conditions: (1) T, R Y , B 2 , R 1 , R 2 , R 3 , and R 4 , the C 1-30 alkyl groups are independently linear alkyl or branched alkyl, preferably linear alkyl; (2) T, R Y , B 2 , R 1 , R 2 , R 3 , and R 4 , wherein the C 1-30 alkyl groups are independently C 4-20 alkyl, for example C 4-18 alkyl, and further for example C 4-10 alkyl; (3) T, R Y B 2 R 1 R 2 R 3 and R 4 In, the C 2-30 The number of alkenyl bonds in the alkenyl group can be 1, 2, 3, 4 or 5 independently, for example 1, 2 or 3; (4) T, R Y , B 2 , R 1 , R 2 , R 3 and R 4 , wherein the C 2-30 alkenyl is independently a straight chain alkenyl or a branched chain alkenyl, preferably a straight chain alkenyl; (5) T, R Y , B 2 , R 1 , R 2 , R 3 , and R 4 , wherein the C 2-30 alkylene is independently C 4-20 alkylene, e.g., C 4-20 alkylene, further e.g., C 8-20 alkylene; (6) T, R Y , B 2 , R 1 , R 2 , R 3 and R 4 , wherein the C 2-30 alkynyl is independently a straight chain alkynyl or a branched chain alkynyl, the C 2- 30 number of alkynyl bonds in the alkenyl is independently 1, 2, 3, 4, or 5, preferably C 4-20 straight chain alkynyl; and (7) R x In particular, the C 1-10 alkyl is a C 1-6 alkyl, for example methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl or t-butyl; Preferably, the compound of Formula I satisfies one or more of the following conditions: (1) T, wherein said C 2-30 Alkenyl is C 4-20 Straight-chain alkenyl, said C 4-20 The number of alkenyl bonds in straight-chain alkenyl is 1, 2 or 3; (2) R 1 and R 2 , said C 1-30 alkyl is independently C 4-20 linear alkyl; (3) R 1 and R 2 wherein said C 2-30 alkenyl is independently C 4-20 linear alkenyl, said C 4-20 linear alkenyl having one, two, or three alkenyl groups. (4) R 3 and R 4 wherein said C 1-30 alkyl is independently C 4-20 linear alkyl, for example C 4-10 linear alkyl; (5)R Y B in 2 In, the C 2-30 The alkenyl group is C 4-20 Straight-chain alkenyl, the C 4-20 The number of alkene bonds in a straight-chain alkenyl group can be 1, 2, or 3; (6) R 1 and R 2 B 2 wherein said C 1-30 alkyl is independently C 4-20 linear alkyl; and (7)R 1 and R 2 B in 2 In, the C 2-30 The alkenyl group is independently C 4-20 Straight-chain alkenyl, the C 4-20 The number of alkene bonds in a straight-chain alkenyl group can be 1, 2, or 3; Preferably, the compound of Formula I satisfies one or two of the following conditions: (1) T, R Y , B 2 , R 1 , R 2 , R 3 , and R 4 , wherein the C 1-30 alkyl groups are independently and (2) T, R Y , B 2 , R 1 , R 2 , R 3 and R 4 , the C 2-30 alkenyl is independently 3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, having Formula I, wherein which satisfies one or more of the following conditions: (1) T is Scheme 1 or Scheme 2, Scheme 1 : T is C 1-30 alkyl, C 2-30 alkenyl or C 2-30 alkynyl; Scheme 2: T is C 2-30 alkenyl or Preferably, T is C 4-20 linear alkenyl or The C 4-20 The number of ethylenic bonds in the straight-chain alkenyl group is 1, 2 or 3; more preferably T is (2) X is a single bond or O; preferably, X is a single bond; (3) R x is H; (4) L 1 is a single bond, -(CH2) n1 -, Preferably, L 1 To (5) n1 and n2 are independently 1, 2, 3, 4, 5, 6, 7 or 8; preferably, n1 and n2 are independently 1, 2, 3 or 4; (6) M 1 is O; (7) M 3 independently or N(R x ) ; (8) R Y is H, Preferably, R Y is H or More preferably, R Y is H; (9) R Y B 2 independently C 2-30 alkenyl; preferably, B 2 independently C 4-20 straight-chain alkenyl, said C 4-20 the number of alkenyl bonds in said straight-chain alkenyl is 1, 2 or 3; (10) R 1 and R 2 independently C 1-30 alkyl, C 2-30 alkenyl, or R 1 and R 2 independently C 1-30 alkyl, C 2-30 alkenyl, C 2-30 alkynyl, R 1 and R 2 are independently C 4-20 straight-chain alkyl, C 4-20 straight-chain alkenyl, More preferably, R 1 and R 2 are independently (11) D 1 , D 2 and D 3 is O; (12) n3 is 1, 2, 3, 4, 5, 6, 7 or 8; (13) R 1 and R 2 wherein B 2 is independently C 1-30 alkyl or C 2-30 alkenyl; preferably, B 2 is independently C 4-20 straight-chain alkyl or C 4-20 straight-chain alkenyl, the number of alkenyl groups in said C 4-20 straight-chain alkenyl being 1, 2 or 3; (14) L 2 independently are (15) H 1 , H 2 , H 3 and H 5 is O; (16) H 6 independently or NH; (17) K 2 is a single bond; (18) n4 is independently 1, 2, 3, 4, 5, 6, 7 or 8; preferably, n4 is independently 1, 2, 3 or 4; (19) Z 1 is CH; (20) R 3 and R 4 independently are C 1-30 alkyl; preferably, R 3 and R 4 independently are C 4-20 straight chain alkyl, again for example C 4-10 straight chain alkyl.

4. The compound of claim 1, or a pharmaceutically acceptable salt thereof, having Formula I, ###0001### I which satisfies one or more of the following conditions: (1) L 1 is a single bond, Preferably, the "1 " position is attached to X, the "2" position is attached to A 3 and the "3" position is attached to Y; more preferably, L 1 is the "1" position is connected to X, and the "2" position is connected to Y; (2) B 2 independently are (3) K 1 independently are (4) K 2 independently a single bond, (5) K 3 independently are (6) R 3 and R 4 are independently More preferably, R 3 and R 4 are Preferably, the compound of Formula I satisfies one or more of the following conditions: (1) -X-L 1 - is a single bond, O, Preferably, the "2" position and Y end are connected to Y; more preferably, -X-L 1 - is the "2" position is connected to Y; (2) L 2 independently Preferably, the "1" position is attached to Y and the "2" position is attached to Z 1 attached; more preferably, L 2 is "1" position is attached to Y, "2" position is attached to Z 1 connected; (3) R Y H or More preferably, in the compound of Formula I, R 1 and R 2 are independently For example, R 1 and R 2 are independently 5. The compound of claim 1, or a pharmaceutically acceptable salt thereof, having Formula I, ###0001### I the compound of Formula I is selected from any one of the following Schemes: Scheme 1 : the compound as shown in formula I is a compound as shown in formula I-1 : R 1 and R 2 are independently C 1-30 alkyl, C 2-30 alkenyl, C 2-30 alkynyl, X, L 1 Y, D 1 D 2 D 3 B 1 B 2 and B 3 are as defined in any one of claims 1-4; Scheme 2: The compound of Formula I is a compound of Formula I-2: X, L 1 Y, L 2 Z 1 R 3 and R 4 are as defined in any one of claims 1-4; Scheme 3: The compound of Formula I is a compound of Formula I-3: R Y , R 1 , and R 2 are independently C 1-30 alkyl, C 2-30 alkenyl, C 2-30 alkynyl, X, L 1 , D 1 , D 2 , D 3 , B 1 , B 2 and B 3 are as defined in any one of claims 1-4; Preferably, the compound of Formula I is a compound of Formula I-2: X is a single bond; L 1 for A 1 is -(CH2) n2 -; A 2 is -(CH2) n2 -; n2 is independently 1, 2, 3 or 4; M 1 is O; Y is N or CH; L 2 independently K 1 and K 3 independently -(CH2) n4 -; K 2 independently single bond or -(CH2) n4 -; n4 is independently 1, 2, 3 or 4; H 1 , H 3 and H 4 is O; Z 1 is CH; R 3 and R 4 are independently C 4-10 straight chain alkyl.

6. The compound of claim 1, or a pharmaceutically acceptable salt thereof, having Formula I, wherein The compound as shown in Formula I is any one of the following compounds:

7. A lipid carrier comprising a substance Z, the substance Z being a compound of Formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1-6; Preferably, the lipid carrier satisfies one or more of the following conditions: (1) the lipid carrier further comprises a diluent; for example, the diluent is a citrate buffer or ethanol; (2) the lipid carrier further comprises a phospholipid; for example, the phospholipid is distearoylphosphatidylcholine; (3) the lipid carrier further comprises a PEG lipid; for example, the PEG lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol and PEG-modified dialkylglycerol; for another example, the PEG-modified dialkylglycerol is PEG-modified dimyristyl glycerol; (4) the lipid carrier further comprises a sterol; for example, the sterol is selected from animal, plant or fungal sterols; for another example, the sterol is selected from one or more of cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid and a-tocopherol; for a further example, the sterol is cholesterol; Preferably, the lipid carrier comprises the diluent, the substance Z, the phospholipid, the PEG lipid and the sterol; More preferably, the lipid carrier satisfies one or more of the following conditions: (1) the molar content of the substance Z is 30 mol% to 60 mol%, for example 40 mol% to 55 mol%, for another example 50 mol%, based on the total molar mass of the substance Z, the phospholipid, the PEG lipid and the sterol; (2) the molar ratio of the substance Z to phospholipid in the lipid carrier is 1-25:1, preferably 2-10:1, for example 5:1; (3) the molar ratio of the substance Z to the sterol in the lipid carrier is (0.5-3): 1, preferably (0.5-3): 1, for example 1.3: 1; (4) the molar ratio of the substance Z to the PEG lipid in the lipid carrier is 20-50: 1, preferably 20-40: 1, for example 33: 1; (5) the volume ratio of the molar number of the substance Z to the diluent is (5-25) mmol: 1 L; for example (10-15) mmol: 1 L; and further for example 12 mmol: 1 L; (6) the lipid carrier consists of the substance Z, the diluent, the phospholipid, the PEG lipid and the sterol; or the lipid carrier consists of the substance Z, the phospholipid, the PEG lipid and the sterol.

8. A lipid nanoparticle comprising a therapeutic agent and / or a prophylactic agent and the lipid carrier of claim 7; preferably, the therapeutic agent and / or the prophylactic agent is one or more than two nucleic acids; more preferably, the therapeutic agent and / or the prophylactic agent is single-stranded deoxyribonucleic acid, double-stranded DNA, small interfering RNA, asymmetric double-stranded small interfering RNA, microRNA, small hairpin RNA, circular RNA, transfer RNA, messenger RNA (mRNA) and other forms of nucleic acid molecules known in the art, most preferably mRNA, for example firefly luciferase mRNA, unmodified uracil synthesized Luc mRNA, poly I:C or VZV gE mRNA.

9. A composition comprising a substance Z, the substance Z being a compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1-6; Preferably, the composition further comprises one or more of a diluent, a phospholipid, a PEG lipid, a sterol and a therapeutic agent and / or a prophylactic agent; the diluent, the phospholipid, the PEG lipid, the sterol and the therapeutic agent and / or the prophylactic agent are as described in claim 8; More preferably, the lipid carrier satisfies one or more of the following conditions: (1) in the composition, the substance Z forms the lipid carrier of claim 7 with one or more of the diluent, the phospholipid, the PEG lipid and the sterol; preferably, the lipid carrier forms the lipid nanoparticle of claim 8 with the therapeutic agent and / or the prophylactic agent; (2) in the composition, the encapsulation efficiency of the therapeutic agent and / or the prophylactic agent is at least 94.5%, preferably at least 95%, for example 95.6%; (3) in the composition, the particle size of the therapeutic agent and / or the prophylactic agent is 50-70 nm, for example 50-60 nm, and further for example 55 nm or 59 nm.

10. Use of a compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1-6 or a lipid carrier of claim 7 in the preparation of a medicament for the prevention and / or treatment of inflammation; for example, the inflammation is infectious inflammation.

11. Use of a compound of formula I according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, or a lipid carrier according to claim 7, for the manufacture of a medicament for the prevention and / or treatment of inflammation caused by nucleic acids as defined in claim 8.

12. Use of a compound of formula I according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, or a lipid carrier according to claim 7, for the manufacture of a medicament for the prevention and / or treatment of inflammation caused by elevated expression levels of MCP-1, IL-6, ISG54 or NF-κB, such as elevated expression levels of MCP-1, IL-6, ISG54 or NF-κB induced by nucleic acids as defined in claim 8.

Images