CN116082275B

CN116082275B - Spleen high-expression cationic lipid compound, composition containing same and application

Info

Publication number: CN116082275B
Application number: CN202310231430.3A
Authority: CN
Inventors: 宋更申; 张宏雷; 刘洋健; 陈玺朝; 黄华捷; 闫如灿; 李雨晴; 张万年; 张超; 修东辉
Original assignee: Beijing Youcare Kechuang Pharmaceutical Technology Co ltd
Current assignee: Beijing Youcare Kechuang Pharmaceutical Technology Co ltd
Priority date: 2023-03-13
Filing date: 2023-03-13
Publication date: 2023-07-14
Anticipated expiration: 2043-03-13
Also published as: CN116082275A

Abstract

The present invention provides a compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, as well as compositions comprising the foregoing compounds and their use for delivering therapeutic or prophylactic agents.

Description

Spleen high-expression cationic lipid compound, composition containing same and application

Technical Field

The invention belongs to the field of medicines, and particularly relates to a piperazinyl cationic lipid compound, a composition containing the piperazinyl cationic lipid compound and application of the piperazinyl cationic lipid compound.

Background

Efficient targeted delivery of biologically active substances such as small molecule drugs, polypeptides, proteins and nucleic acids, especially nucleic acids, is a persistent medical challenge. Nucleic acid therapeutics face significant challenges due to low cell permeability and high sensitivity to degradation by certain nucleic acid molecules, including RNA.

Compositions, liposomes and liposome complexes (lipoplex) containing cationic lipids have been demonstrated to be effective as transport vehicles for transporting biologically active substances such as small molecule drugs, polypeptides, proteins and nucleic acids into cells and/or intracellular compartments. These compositions generally comprise one or more "cationic" and/or amino (ionizable) lipids, including neutral lipids, structural lipids, and polymer conjugated lipids. Cationic and/or ionizable lipids include, for example, amine-containing lipids that can be readily protonated. While a variety of such lipid-containing nanoparticle compositions have been shown, safety, efficacy and specificity remain to be improved. Notably, the increased complexity of lipid nanoparticles (Lipid Nanoparticle, LNP) complicates their production and may increase their toxicity, a major concern that may limit their clinical use. For example, LNP siRNA particles (e.g., patsiran) require the prior use of steroids and antihistamines to eliminate unwanted immune responses (T. Coelho, D. Adams, A. Silva, et al, safety and efficacy of RNAi therapy for transthyretin amyloidosis, N Engl J Med, 369 (2013) 819-829.). Thus, there is a need to develop improved cationic lipid compounds, and compositions comprising the same, that facilitate the delivery of therapeutic and/or prophylactic agents, such as nucleic acids, to cells.

Disclosure of Invention

The present invention provides a piperazinyl-based cationic lipid compound, including pharmaceutically acceptable salts thereof and stereoisomers or tautomers thereof. Enriches the variety of cationic lipid compounds and provides more choices for the effective delivery of nucleic acid drugs, genetic vaccines, small molecule drugs, polypeptides or protein drugs. When formed into lipid nanoparticles with other lipid components, can effectively deliver mRNA or drug molecules into cells to perform biological functions.

In one aspect, the present disclosure provides a piperazinyl-based cationic lipid compound, which is a compound of formula (I)

Or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein: g ₁ Is C _1~10 An alkylene group; g ₂ Is C _1~10 An alkylene group; l (L) ₁ is-C (O) O-or-C (O) N (R) _a )-；L ₂ is-C (O) O-or-C (O) N (R) _a )-；R ₁ Is unsubstituted C _12~25 Branched alkyl or unsubstituted C _6~15 Straight chain alkyl or R _b -S-R _c Or R is _b -S-S-R _c ；R ₂ Is unsubstituted C _12~25 Branched alkyl or unsubstituted C _6~15 Straight chain alkyl or R _b -S-R _c Or R is _b -S-S-R _c ；R _a Is H or unsubstituted C _5~12 A linear alkyl group; r is R _b Is C _1~10 An alkylene group; r is R _c Is unsubstituted C _1~16 A linear alkyl group.

For example, the compound of formula (I) has one of the following structures:

，

，

，

，

，

，

。

in yet another aspect, the present invention provides a composition comprising a carrier comprising a cationic lipid comprising a compound of formula (I) as described above or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof. In one embodiment, the composition further comprises a therapeutic or prophylactic agent.

Yet another aspect of the present invention provides the above cationic lipid or composition for delivering a therapeutic or prophylactic agent to a patient in need thereof.

In yet another aspect, the invention provides a method of treating or preventing a disease or disorder comprising administering to a patient or subject in need thereof a therapeutically or prophylactically effective amount of the above-described composition.

In a further aspect the present invention provides the use of a compound of formula (I) as defined above, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a composition as defined above, in the manufacture of a nucleic acid medicament, a genetic vaccine, a small molecule medicament, a polypeptide or a protein medicament.

In a further aspect the present invention provides the use of a compound of formula (I) as defined above, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a composition as defined above, in the manufacture of a medicament for the treatment of a disease or condition in a mammal in need thereof.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the following brief description of the drawings of the embodiments will make it apparent that the drawings in the following description relate only to some embodiments of the present disclosure, not to limit the present disclosure.

FIG. 1 shows the results of cell transfection experiments for LNP preparations of eGFP-mRNA prepared based on YK-506, wherein: a is 293T cell bright field map, and b is 293T cell fluorescence map.

FIG. 2 shows the fluorescence absorbance intensities of LNP preparations of Fluc-mRNA prepared from different cationic lipids.

FIG. 3 shows the cell viability of LNP preparations of Fluc-mRNA prepared from different cationic lipids after addition to cell culture broth for 24 h.

FIG. 4 shows the results of in vivo imaging experiments in mice of LNP preparations of Fluc-mRNA prepared based on YK-506, YK-504 and YK-501 cationic lipids, wherein: a is a living mouse of LNP preparation of Fluc-mRNA prepared based on YK-506, YK-504 and YK-501 cationic lipid, b is a mouse of LNP preparation of Fluc-mRNA prepared based on YK-506 with its abdominal cavity opened after euthanasia, c is a fluorescence image of organs (heart, liver, spleen, lung, kidney) of mouse of LNP preparation of Fluc-mRNA prepared based on YK-506.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present disclosure more apparent, the technical solutions of the embodiments of the present disclosure will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present disclosure. It will be apparent that the described embodiments are some, but not all, of the embodiments of the present disclosure. All other embodiments, which can be made by one of ordinary skill in the art without the benefit of the present disclosure, are intended to be within the scope of the present invention based on the described embodiments.

The present invention may be embodied in other specific forms without departing from its essential attributes. It is to be understood that any and all embodiments of the invention may be combined with any other embodiment or features of multiple other embodiments to yield yet further embodiments without conflict. The invention includes additional embodiments resulting from such combinations.

All publications and patents mentioned in this disclosure are incorporated herein by reference in their entirety. If a use or term used in any of the publications and patents incorporated by reference conflicts with the use or term used in the present disclosure, the use or term of the present disclosure controls.

The section headings used herein are for purposes of organizing articles only and should not be construed as limiting the subject matter.

Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning in the art to which the claimed subject matter belongs. In case there are multiple definitions for a term, the definitions herein control.

Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of quantitative quality such as doses stated in the specification and claims are to be understood as being modified in all instances by the term "about". It should also be understood that any numerical range recited herein is intended to include all sub-ranges within that range and any combination of the individual endpoints of that range or sub-range.

The use of the terms "comprising," "including," or "containing," and the like, in this disclosure, are intended to cover an element listed after that term and its equivalents, but do not exclude the presence of other elements. The terms "comprising" or "including" as used herein, can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

The term "pharmaceutically acceptable" in this application means: the compound or composition is chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or with the human or mammal with which the disease or condition is to be prevented or treated.

The term "subject" or "patient" includes humans and mammals in this application.

The term "treatment" as used herein refers to the administration of one or more pharmaceutical substances to a patient or subject suffering from or having symptoms of a disease, to cure, alleviate, ameliorate or otherwise affect the disease or symptoms of the disease. In the context of the present application, the term "treatment" may also include prophylaxis, unless specifically stated to the contrary.

The term "solvate" in this application refers to a complex formed by combining a compound of formula (I) or a pharmaceutically acceptable salt thereof and a solvent (e.g. ethanol or water). It will be appreciated that any solvate of a compound of formula I used in the treatment of a disease or condition, although potentially providing different properties (including pharmacokinetic properties), will result in a compound of formula I upon absorption into a subject such that the use of a compound of formula I encompasses the use of any solvate of a compound of formula I, respectively.

The term "hydrate" refers to the case where the solvent in the above term "solvate" is water.

It is further understood that the compound of formula I or a pharmaceutically acceptable salt thereof may be isolated in the form of a solvate, and thus any such solvate is included within the scope of the present invention. For example, a compound of formula I or a pharmaceutically acceptable salt thereof may exist in unsolvated forms as well as solvated forms with pharmaceutically acceptable solvents (such as water, ethanol, and the like).

The term "pharmaceutically acceptable salt" refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the present disclosure. See, for example, s.m. Berge et al, "Pharmaceutical Salts", J. Pharm. Sci. 1977, 66, 1-19. Among them, inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid, etc.; organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acidLauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) -benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectate acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, and the like. For example, HCl (or hydrochloric acid), HBr (or hydrobromic acid solution), methanesulfonic acid, sulfuric acid, tartaric acid, or fumaric acid may be used to form pharmaceutically acceptable salts with the compounds of formula I.

The nitrogen-containing compounds of formula (I) of the present disclosure may be converted to N-oxides by treatment with an oxidizing agent (e.g., m-chloroperoxybenzoic acid, hydrogen peroxide, ozone). Thus, the compounds claimed herein include not only nitrogen-containing compounds of the formula but also N-oxide derivatives thereof, as valence and structure permit.

Certain compounds of the present disclosure may exist in the form of one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers and enantiomers. Thus, the presently claimed compounds also include racemic mixtures, single stereoisomers, and optically active mixtures. It will be appreciated by those skilled in the art that one stereoisomer may have better efficacy and/or lower side effects than the other stereoisomers. The single stereoisomers and the mixture with optical activity can be obtained by chiral source synthesis methods, chiral catalysis methods, chiral resolution methods and the like. The racemate can be chiral resolved by chromatographic resolution or chemical resolution. For example, separation can be performed by adding chiral acid resolving agents such as chiral tartaric acid, chiral malic acid, and the like to form salts with the compounds of the present disclosure, utilizing differences in the physicochemical properties of the products, such as solubility.

The invention also includes all suitable isotopic variations of the compounds of the present disclosure. Isotopic variations are defined as compounds in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found or predominantly present in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, and oxygen, respectively, for example ² H (deuterium), ³ H (tritium), ¹¹ C、 ¹³ C、 ¹⁴ C、 ¹⁵ N、 ¹⁷ O and ¹⁸ O。

the term "alkyl" is meant in this disclosure to include both branched and straight chain saturated aliphatic monovalent hydrocarbon groups having the specified number of carbon atoms. The term "alkylene" is meant in this disclosure to include both branched and straight chain saturated aliphatic divalent hydrocarbon groups having the specified number of carbon atoms. C (C) _n~m Is meant to include groups having a number of carbon atoms from n to m. For example C _2~5 Alkylene group includes C ₂ Alkylene, C ₃ Alkylene, C ₄ Alkylene, C ₅ An alkylene group.

The alkyl (or alkylene) group may be unsubstituted, or the alkyl (or alkylene) group may be substituted, wherein at least one hydrogen is replaced with another chemical group.

A "therapeutically effective amount" is an amount of a therapeutic agent that, when administered to a patient, ameliorates a disease or condition. A "prophylactically effective amount" is an amount of a prophylactic agent that, when administered to a subject, prevents a disease or condition. The amount of therapeutic agent constituting the "therapeutically effective amount" or the amount of prophylactic agent of the "prophylactically effective amount" varies with the therapeutic agent/prophylactic agent, the disease state and severity thereof, the age, weight, etc. of the patient/subject to be treated/prevented. One of ordinary skill in the art can routinely determine therapeutically effective and prophylactically effective amounts based on their knowledge and disclosure.

In the present application, when the names of the compounds are not identical to the structural formulae, the structural formulae are subject.

It is to be understood that the term "presently disclosed compounds" as used herein may include, depending on the context: a compound of formula (I), an N-oxide thereof, a solvate thereof, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, and mixtures thereof.

The term cationic lipid as used herein refers to a lipid that is positively charged at a selected pH.

Cationic liposomes readily bind to negatively charged nucleic acids, i.e., interact with negatively charged phosphate groups present in the nucleic acids by electrostatic forces, forming Lipid Nanoparticles (LNPs). LNP is one of the currently mainstream delivery vehicles.

Screening for suitable cationic lipid compounds, which have both high transfection efficiency and low toxicity to cells, is a very difficult task to express in the spleen of mice. By unique design, the present disclosure finds that certain compounds, such as YK-506 and YK-504, are capable of delivering nucleic acids with significantly improved cell transfection efficiency, significantly reduced cytotoxicity and significantly improved expression levels in the animal spleen relative to structurally similar cationic lipids in the prior art, such as MC3, C12-200, MIC3 and PPZ-A12, thereby achieving rapid induction of immune responses in vivo and antibody production in mRNA vaccines. The vaccine composition has the significant clinical significance and can obviously improve the prevention effect under the condition of not changing the vaccine components. Has good targeting effect on developing and treating diseases caused by spleen damage or abnormality such as lymphoma, leukemia and the like.

Briefly, the present invention is based on at least the following findings:

the cationic lipid compounds of the present disclosure can be used to deliver nucleic acid molecules, small molecule compounds, polypeptides, or proteins. Compared with the known cationic lipid compounds, the cationic lipid compounds disclosed by the invention have the advantages of higher transfection efficiency, smaller cytotoxicity, remarkably improved expression quantity in animal spleen, improved delivery efficiency and important clinical significance.

1. A series of cationic lipid compounds were designed, including YK-506 and YK-504, with a large difference in chemical structure compared to the prior art representative cationic lipids, such as MC3 (Dlin-MC 3-DMA); there are structural similarities, for example, C12-200, MIC3 and PPZ-A12.

MC3 is a cationic lipid compound disclosed in CN102625696B (page 6 of the specification) by alimer pharmaceutical company (Alnylam Pharmaceuticals, inc.).

C12-200 is disclosed in WO2010/053572A2 (page 157 of the specification) by the institute of technology (MIT) of Massachu Medica.

MIC3 is a cationic lipid reported by university of fort Song Xiangrong et al (Kepan Chen, xiangrong Song, et al mRNA Vaccines Against SARS-CoV-2 Variants Delivered by Lipid Nanoparticles Based on Novel Ionizable Lipids, adv Funct Mat, 2022,2204692 text page 3).

PPZ-A12 is a cationic lipid reported by James Dahlman et al, university of George, U.S.A. (Huanzhen Ni, james E. Dahlman, et al, piperazine-derived lipid nanoparticles deliver mRNA to immune cells in vivo, nat. Commun.2022, 13:4766 text page 3).

Therefore, it is not possible to infer the cell transfection efficiency, cytotoxicity, and expression in the spleen of animals of LNP formulations prepared from this series of compounds designed in this application from the cationic lipid compounds disclosed in the above prior art.

A series of cationic lipid compounds and other representative cationic lipid chemical structures of the prior art contemplated herein are as follows:

table 1 representative cationic lipids designed and prior art in the present application

2. In the designed series of compounds, LNP preparations prepared from YK-506 and YK-504 have significantly improved cell transfection efficiency, significantly reduced cytotoxicity and significantly improved mRNA expression in mouse liver and spleen compared with the typical and structurally similar cationic lipids in the prior art. For example, cell transfection efficiency YK-506 can be up to 4.83 times MC3, 2.94 times C12-200, 4.00 times MIC3 and 3.73 times PPZ-A12; cell viability YK-506 may be 25% higher than MC3, 18% higher than C12-200, 33% higher than MIC3, 41% higher than PPZ-A12; mRNA was expressed in the spleen of mice, YK-506 was 5.94 times that of C12-200, and MC3 was not expressed in the spleen.

3. In a series of compounds with small chemical structure difference, LNP preparation prepared from YK-506 and YK-504 has obviously improved cell transfection efficiency, obviously reduced cytotoxicity and obviously improved expression quantity of mRNA in mouse spleen compared with other compounds. For example, YK-506 cells can be transfected with 100 times YK-501 and 130 times YK-503, cytotoxicity can be reduced by 52% compared with YK-505, and mRNA expression amount in mouse spleen can be more than 200 times YK-501.

4. Through unique design and screening, the present disclosure finds that some compounds, such as YK-506 and YK-504, can be delivered with significantly improved cell transfection efficiency, significantly reduced cytotoxicity and significantly improved expression in the spleen of animals, with improved delivery efficiency, and unexpected technical effects relative to other compounds of similar prior art structures. By increasing the expression level of the spleen (the largest secondary lymphoid organ in the body) in the body, the mRNA vaccine can induce immune response in the body and generate antibodies. The vaccine composition has the significant clinical significance and can obviously improve the prevention effect under the condition of not changing the vaccine components. Has good targeting effect on developing and treating diseases caused by spleen damage or abnormality such as lymphoma, leukemia and the like.

In summary, the present disclosure, through unique design and screening, has discovered compounds such as YK-506 and YK-504. Compared with the cationic lipid with representativeness and similar structure in the prior art, the compounds have the advantages of remarkably improved cell transfection efficiency, remarkably reduced cytotoxicity, remarkably improved expression quantity in the spleen of animals and improved delivery efficiency.

The method comprises the following steps:

1. the chemical structures of the compounds designed in this application are greatly different from the typical cationic lipids of the prior art, such as MC3; there are structural similarities, for example, C12-200, MIC3 and PPZ-A12.

1) Compared with the representative cationic lipid MC3 in the prior art, the chemical structure of the compound designed by the application is completely different and has great difference. MC3 is piperazine-free, and the compounds contemplated by the application all have piperazine groups; MC3 has only 1 branched hydrophobic tail, while the compound designed by the application has 2 hydrophobic tails; the tail structure of MC3 contains double bond, while the tail structure of the compound designed by the application has no double bond, and other groups have larger difference.

2) The chemical structures of the compounds contemplated herein are similar with only slight differences in individual groups compared to prior art piperazinyl containing cationic lipids, such as C12-200, MIC3 and PPZ-A12.

2. The transfection efficiency of cells in vitro is obviously improved compared with the typical cationic lipid and the compound with similar structure in the prior art.

1) LNP formulations prepared from YK-506 and YK-504 have the highest cell transfection efficiency and significantly improved activity compared to the typical and structurally similar cationic lipids of the prior art. For example, YK-506 cells can be transfected 4.83 times as efficiently as MC3, 2.94 times as much as C12-200, 4.00 times as much as MIC3 and 3.73 times as much as PPZ-A12.

2) Similar to the structure, L ₁ And L ₂ The cell transfection efficiency of YK-506 and YK-504 is highest compared with the compounds with the groups of-C (O) O-. For example, YK-506 can be transfected with up to 100 times YK-501, 40 times YK-502 and YK-505.

3) Similar to the structure, L ₁ The radicals being-C (O) O-, L ₂ The radical being-C (O) N ((CH) ₂ ) ₉ CH ₃ ) The transfection efficiency of YK-506 and YK-504 cells is significantly improved compared with the compounds. For example, YK-506 cells can be transfected up to 130 times YK-503.

4) Similar to the structure, L ₁ And L ₂ The radicals are-C (O) NH-, R ₁ And R is ₂ Compared with the compounds both containing the-S-S-group, the transfection efficiency of YK-506 and YK-504 cells is obviously improved. For example, YK-506 cells can be transfected up to 100 times YK-507.

3. Cytotoxicity is significantly reduced over the typical cationic lipids and structurally similar compounds of the prior art.

1) LNP formulations prepared from YK-504, YK-506 have minimal cytotoxicity and significantly improved cell viability compared to the cationic lipids typical of the prior art. For example, YK-506 may have a cell viability 25% higher than MC3, 18% higher than C12-200, 33% higher than MIC3, 41% higher than PPZ-A12.

2) Similar to the structure, L ₁ And L ₂ Compared with the compounds with the groups of-C (O) O-, the cytotoxicity of YK-506 and YK-504 is the lowest, and the cell survival rate is obviously improved. For example, YK-506 may have a cell viability that is 35% higher than YK-501, 39% higher than YK-502, and 52% higher than YK-505.

3) Similar to the structure, L ₁ The radicals being-C (O) O-, L ₂ The radical being-C (O) N ((CH) ₂ ) ₉ CH ₃ ) Compared with the compounds, the cytotoxicity of YK-506 and YK-504 is the lowest, and the cell survival rate is obviously improved. For example, YK-506 and YK-504 have 44% and 28% higher cell viability than YK-503, respectively.

4) Similar to the structure, L ₁ And L ₂ The radicals are-C (O) NH-, R ₁ And R is ₂ Compared with the compounds containing the-S-S-group, the compounds with the cytotoxicity of YK-506 and YK504 are the lowest, and the cell survival rate is obviously improved. For example, YK-506 and YK-504 have 40% and 24% higher cell viability than YK-507, respectively.

mRNA expression levels in the animal spleen are significantly increased over the prior art for representative cationic lipids and structurally similar compounds.

1) Compared with the representative cationic lipid in the prior art, the LNP preparation prepared by YK-506 and YK-504 has obviously improved expression level of mRNA in the spleen of the mouse. For example, MC3 is not expressed in the spleen, whereas YK-506 and YK-504 are both expressed in large amounts, YK-506 can be expressed in the spleen in an amount up to 5.94 times C12-200.

2) LNP preparations prepared from YK-506 and YK-504 showed the highest expression intensity of mRNA in the spleen of mice compared to the compounds having similar structures and slightly different individual groups. For example, YK-506 can be expressed in spleen in an amount of 200 times or more than YK-501.

By increasing the expression level of spleen (the largest secondary lymphoid organ in vivo) in animals, the mRNA vaccine can induce immune response in vivo and generate antibodies. The vaccine composition has the significant clinical significance and can obviously improve the prevention effect under the condition of not changing the vaccine components. Has good targeting effect on developing and treating diseases caused by spleen damage or abnormality such as lymphoma, leukemia and the like.

In one aspect, the present disclosure provides a novel cationic lipid compound for delivering a therapeutic or prophylactic agent. The cationic lipid compounds of the present disclosure can be used to deliver nucleic acid molecules, small molecule compounds, polypeptides, or proteins. Compared with the known cationic lipid compounds, the cationic lipid compounds of the present disclosure exhibit higher transfection efficiency, lower cytotoxicity and high expression in animal spleen, improving delivery efficiency and safety.

The present disclosure provides a cationic lipid that is a compound of formula (I)

Or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein

G ₁ Is C _1~10 An alkylene group;

G ₂ is C _1~10 An alkylene group;

L ₁ is-C (O) O-or-C (O) N (R) _a )-；

L ₂ is-C (O) O-or-C (O) N (R) _a )-；

R ₁ Is unsubstituted C _12~25 Branched alkyl or unsubstituted C _6~15 Straight chain alkyl or R _b -S-R _c Or R is _b -S-S-R _c ；

R ₂ Is unsubstituted C _12~25 Branched alkyl or unsubstituted C _6~15 Straight chain alkyl or R _b -S-R _c Or R is _b -S-S-R _c ；

R _a Is H or unsubstituted C _5~12 A linear alkyl group;

R _b is C _1~10 An alkylene group;

R _c is unsubstituted C _1~16 A linear alkyl group.

In one embodiment, G ₁ Is unsubstituted C ₅ Alkylene or C ₂ Alkylene or C ₇ Alkylene or C ₃ An alkylene group. For example, G ₁ Is unsubstituted C ₅ An alkylene group. Also for example, G ₁ Is unsubstituted C ₇ An alkylene group.

In one embodiment, G ₂ Is unsubstituted C ₅ Alkylene or C ₂ Alkylene or C ₇ Alkylene or C ₃ An alkylene group. For example, G ₂ Is unsubstituted C ₂ An alkylene group. Also for example, G ₂ Is unsubstituted C ₃ An alkylene group.

In one embodiment, G ₁ Is unsubstituted C ₅ Alkylene group, G ₂ Is unsubstituted C ₂ An alkylene group. In another embodiment, G ₁ Is unsubstituted C ₇ Alkylene group, G ₂ Is unsubstituted C ₃ An alkylene group.

In one embodiment, L ₁ is-C (O) O-, -C (O) NH-or-C (O) N ((CH) ₂ ) ₉ CH ₃ ) -. For example, L ₁ is-C (O) O-.

In one embodiment, L ₂ is-C (O) O-, -C (O) NH-or-C (O) N ((CH) ₂ ) ₉ CH ₃ ) -. For example, L ₂ is-C (O) NH-.

In one embodiment, R ₁ Is unsubstituted C ₁₅ Branched alkyl or unsubstituted C ₁₈ Branched alkyl groups. For example, R ₁ The method comprises the following steps:

or->

. In another embodiment, R ₁ Is- (CH) ₂ ) ₂ -S-S-(CH ₂ ) ₁₁ CH ₃ 、-(CH ₂ ) ₂ -S-(CH ₂ ) ₃ CH ₃ 、-(CH ₂ ) ₃ -S-S-(CH ₂ ) ₄ CH ₃ Or- (CH) ₂ ) ₉ CH ₃ 。

In one embodiment, R ₂ Is unsubstituted C ₁₅ Branched alkyl or unsubstituted C ₁₈ Branched alkyl groups. For example, R ₂ The method comprises the following steps:

or->

. In another embodiment, R ₂ Is- (CH) ₂ ) ₂ -S-S-(CH ₂ ) ₁₁ CH ₃ 、-(CH ₂ ) ₂ -S-(CH ₂ ) ₃ CH ₃ 、-(CH ₂ ) ₃ -S-S-(CH ₂ ) ₄ CH ₃ Or- (CH) ₂ ) ₉ CH ₃ 。

For example, the compound of formula (I) has one of the following structures:

，

，

，

，

，

，

。

in a preferred embodiment, the compounds of formula (I) are

Or->

。

In a more preferred embodiment, the compound of formula (I) is

。

Yet another aspect of the present disclosure provides a composition comprising a carrier comprising a cationic lipid comprising a compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, as described above.

In some embodiments, the composition is a nanoparticle formulation having an average size of 100nm to 210nm, preferably 140nm to 205nm; the nanoparticle formulation has a polydispersity of 50% or less, preferably 30% or less, more preferably 25% or less.

Cationic lipids

In one embodiment of the composition/carrier of the present disclosure, the cationic lipid is one or more selected from the compounds of formula (I) above or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the cationic lipid is a compound of formula (I) selected from the group consisting of those described above. For example, the cationic lipid is a compound YK-501, YK-502, YK-503, YK-504, YK-505, YK-506 or YK-507. In some preferred embodiments, the cationic lipid is compound YK-506. In other preferred embodiments, the cationic lipid is compound YK-504.

In some embodiments, the cationic lipid comprises 25% -75% of the carrier by mole, e.g., 35%, 45%, 49%, 50%, 51%, 55%, 60%, 65%.

The carrier may be used to deliver an active ingredient such as a therapeutic or prophylactic agent. The active ingredient may be enclosed within a carrier or may be combined with a carrier.

For example, the therapeutic or prophylactic agent includes one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein. Such nucleic acids include, but are not limited to, single-stranded DNA, double-stranded DNA, and RNA. Suitable RNAs include, but are not limited to, small interfering RNAs (sirnas), asymmetric interfering RNAs (airnas), micrornas (mirnas), dicer-substrate RNAs (dsRNA), small hairpin RNAs (shrnas), messenger RNAs (mrnas), and mixtures thereof.

Neutral lipids

The carrier may comprise neutral lipids. Neutral lipids in the present disclosure refer to lipids that are non-charged at a selected pH or that are present as zwitterionic forms that act as a helper. Neutral lipids may modulate nanoparticle mobility into lipid bilayer structures and increase efficiency by promoting lipid phase changes, while also potentially affecting target organ specificity.

In some embodiments, the molar ratio of the cationic lipid to the neutral lipid is 1:1 to 15:1, e.g., 14:1, 13:1, 12:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5.1:1, 5:1, 4.9:1, 4:1, 3:1, 2:1. In some preferred embodiments, the molar ratio of the cationic lipid to the neutral lipid is 4.9:1.

For example, the neutral lipids may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramides, sterols, and derivatives thereof.

The carrier component of the cationic lipid-containing composition may comprise one or more neutral lipid-phospholipids, such as one or more (poly) unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids may include a phospholipid moiety and one or more fatty acid moieties.

The neutral lipid moiety may be selected from the non-limiting group consisting of: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from the non-limiting group consisting of: lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid and docosahexaenoic acid. Non-natural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, the phospholipid may be functionalized with or crosslinked with one or more alkynes (e.g., alkenyl groups with one or more double bonds replaced with triple bonds). Under appropriate reaction conditions, alkynyl groups may undergo copper-catalyzed cycloaddition reactions upon exposure to azide. These reactions can be used to functionalize the lipid bilayer of the composition to facilitate membrane permeation or cell recognition, or to couple the composition with a useful component such as a targeting or imaging moiety (e.g., dye).

Neutral lipids useful in these compositions may be selected from the non-limiting group consisting of: 1, 2-Dioleoyl-sn-glycero-3-phosphorylcholine (DLPC), 1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dioleoyl-sn-glycero-phosphorylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-octadecenyl-sn-glycero-3-phosphorylcholine (18:0 DietherPC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-3-phosphorylcholine (OChems PC), 1-hexadecyl-sn-3-phosphorylcholine (C16), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1-dioleoyl-2-dioleoyl-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-sn-glycero-3-phosphate ethanolamine (DOPE), 1, 2-di-phytanoyl-sn-glycero-3-phosphate ethanolamine (ME 16.0 PE), 1, 2-di-stearoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-oleoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-linolenoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-arachidonoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-docosahexaenoic acyl-sn-glycero-3-phosphate ethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphate sodium salt (DOPG), 1, 2-di-oleoyl-rac-3-phosphate sodium salt (DOPG) dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl-based phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof.

In some embodiments, the neutral lipid comprises DSPC. In certain embodiments, the neutral lipid comprises DOPE. In some embodiments, the neutral lipid comprises both DSPC and DOPE.

Structured lipids

The carrier of the composition comprising the cationic lipid may also comprise one or more structural lipids. Structured lipids refer in the present disclosure to lipids that enhance the stability of the nanoparticle by filling the interstices between the lipids.

In some embodiments, the molar ratio of the cationic lipid to the structural lipid is about 0.6:1-3:1, e.g., about 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1. In some preferred embodiments, the molar ratio of the cationic lipid to the structural lipid is 49:39.5.

The structural lipid may be selected from, but is not limited to, the group consisting of: cholesterol, non-sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycorine, ursolic acid, alpha-tocopherols, corticosteroids, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipids include cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or combinations thereof.

Polymer conjugated lipids

The carrier of the composition comprising the cationic lipid may also comprise one or more polymer conjugated lipids. The polymer conjugated lipid mainly refers to polyethylene glycol (PEG) modified lipid. Hydrophilic PEG stabilizes LNP, regulates nanoparticle size by limiting lipid fusion, and increases nanoparticle half-life by reducing non-specific interactions with macrophages.

In some embodiments, the polymer conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol. The molecular weight of the PEG modified PEG is generally 350-5000 Da.

For example, the polymer conjugated lipid is selected from one or more of the following: distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG 2000), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000) and methoxypolyethylene glycol ditetradecylamide (ALC-0159).

In one embodiment of the composition/carrier of the present disclosure, the polymer conjugated lipid is DMG-PEG2000.

In one embodiment of the composition/carrier of the present disclosure, the carrier comprises neutral lipid, structural lipid, and polymer conjugated lipid, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer conjugated lipid is (25-75): (5-25): (15-65): (0.5-10), e.g., (35-49): (4.5-15): (35-55): (1-5).

In one embodiment of the composition/carrier of the present disclosure, the carrier comprises a neutral lipid, a structural lipid, and a polymer conjugated lipid, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer conjugated lipid being 49:10:39.5:1.5.

Therapeutic and/or prophylactic agent

The composition may include one or more therapeutic and/or prophylactic agents. In some embodiments, the mass ratio of carrier to the therapeutic or prophylactic agent is 10:1 to 30:1, e.g., 12.5:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1.

In some embodiments, the mass ratio of carrier to the therapeutic or prophylactic agent is 12.5:1 to 25:1, preferably 15:1.

The therapeutic or prophylactic agent includes, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.

For example, the therapeutic or prophylactic agent is a vaccine or compound capable of eliciting an immune response.

The vectors of the present disclosure may deliver therapeutic and/or prophylactic agents to mammalian cells or organs, and thus the present disclosure also provides methods of treating a disease or disorder in a mammal in need thereof, comprising administering to the mammal a composition comprising a therapeutic and/or prophylactic agent and/or contacting mammalian cells with the composition.

Therapeutic and/or prophylactic agents include bioactive substances and are alternatively referred to as "active agents". The therapeutic and/or prophylactic agent can be a substance that, upon delivery to a cell or organ, causes a desired change in the cell or organ or other body tissue or system. Such species may be used to treat one or more diseases, disorders or conditions. In some embodiments, the therapeutic and/or prophylactic agent is a small molecule drug that can be used to treat a particular disease, disorder, or condition. Examples of drugs that may be used in the composition include, but are not limited to, antineoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate and streptozotocin), antineoplastic agents (e.g., dactinomycin D (actinomycin D), vincristine, vinblastine, cytosine arabinoside cytosine arabinoside, anthracycline (anthracycline), alkylating agents, platinum compounds, antimetabolites and nucleoside analogs such as methotrexate and purine and pyrimidine analogs, anti-infective agents, local anesthetics (e.g., dibucaine) and chlorpromazine, beta-adrenergic blockers (e.g., streptozotocin), anti-inflammatory agents (e.g., benzodiazepinephrine), and anti-inflammatory agents (e.g., benzodiazepinephrine), antimuscarin (e.g., benzodiazepinephrine), and antimuscarin (e.g., benzodiazepinephrine), antimuscarin (e.g., benzodiazepinephrine (e), and antimuscarin (e.g., benzodiazepinephrine (e), and other drugs (e.g., benzodiazepinephrine), and other drugs (e.g., benzoglibin), which may be used in combination, ciprofloxacin (ciprofloxacin) and cefoxitin), antifungal agents (e.g., miconazole, terconazole, econazole, isoconazole, butoconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B (amphotericin B)), antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitters, antagonists, antiglaucomas, vitamins, sedatives, and imaging agents.

In some embodiments, the therapeutic and/or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, and/or another therapeutic and/or prophylactic agent. Cytotoxins or cytotoxic agents include any agent that is detrimental to cells. Examples include, but are not limited to, taxol (taxol), cytochalasin B (cytochalasin B), gramicidin D (gramicidin D), ethidium bromide (ethidium bromide), emetine (emetine), mitomycin (mitomycin), etoposide (etoposide), teniposide (teniposide), vincristine, vinblastine, colchicine (colchicine), doxorubicin, daunorubicin (daunorubicin), dihydroxyanthracenedione (dihydroxy anthracin dione), mitoxantrone, mithramycin (mithramycin), actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine (procaine), tetracaine (tetracaine), lidocaine (lidocaine), propranolol, puromycin, maytansinoids (maytansinoid) such as maytansinol (maytansine), lanmycin (rachimycin) (CC-1065), and analogs or homologs thereof. Radioions include, but are not limited to, iodine (e.g., iodine 125 or iodine 131), strontium 89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, samarium 153, and praseodymium. Vaccines include compounds and formulations capable of providing immunity against one or more conditions associated with infectious diseases such as influenza, measles, human Papilloma Virus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis and tuberculosis and may include mRNA encoding antigens and/or epitopes that are the source of infectious diseases. Vaccines can also include compounds and formulations that direct immune responses against cancer cells and can include mRNA encoding tumor cell-derived antigens, epitopes, and/or neoepitopes. Compounds that elicit an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species. In some embodiments, a vaccine and/or compound capable of eliciting an immune response is administered intramuscularly through a composition comprising a compound according to formula (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg) or (III) (e.g.,

compound

3, 18, 20, 25, 26, 29, 30, 60, 108-112 or 122). Other therapeutic and/or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), alkylating agents (e.g., nitrogen mustard (mechlorethamine), thiotepa (thiopa), chlorambucil (chloranserine), azithromycin (CC-1065), melphalan (melphalan), carmustine (carmustine, BSNU), robustin (lomustine, CCNU), cyclophosphamide, busulfan (busulfan), dibromomannitol, streptozotocin, mitomycin C, and cisplatin (II) (DDP), cisplatin), anthracyclines (e.g., daunomycin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly dactinomycin), dactinomycin (formenin), and vincristine (AMC), and antimuscarines (e.g., mitomycin, and the like).

In other embodiments, the therapeutic and/or prophylactic agent is a protein. Therapeutic proteins useful in the nanoparticles in the present disclosure include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), factor VIR, luteinizing Hormone Releasing Hormone (LHRH) analogs, interferon, heparin, hepatitis b surface antigen, typhoid vaccine, and cholera vaccine.

In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term "polynucleotide" is intended to include in its broadest sense any compound and/or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA); ribonucleic acids (RNAs), including messenger mrnas (mrnas), hybrids thereof; RNAi-inducing factors; RNAi factor; siRNA; shRNA; a miRNA; antisense RNA; ribozymes; catalytic DNA; RNA that induces triple helix formation; an aptamer, and the like. In some embodiments, the therapeutic and/or prophylactic agent is RNA. The RNAs useful in the compositions and methods described herein may be selected from the group consisting of, but not limited to: shortmer, antagomir antisense RNA, ribozyme, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In certain embodiments, the RNA is mRNA.

In certain embodiments, the therapeutic and/or prophylactic agent is mRNA. The mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA may be of any size and may have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA may have a therapeutic effect when expressed in a cell.

In other embodiments, the therapeutic and/or prophylactic agent is an siRNA. siRNA is capable of selectively reducing expression of a gene of interest or down-regulating expression of the gene. For example, the siRNA can be selected such that a gene associated with a particular disease, disorder, or condition is silenced after administration of a composition comprising the siRNA to a subject in need thereof. The siRNA may comprise a sequence complementary to an mRNA sequence encoding a gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.

In certain embodiments, the therapeutic and/or prophylactic agent is sgRNA and/or cas9 mRNA. sgRNA and/or cas9 mRNA may be used as a gene editing tool. For example, the sgRNA-cas9 complex can affect mRNA translation of cellular genes.

In some embodiments, the therapeutic and/or prophylactic agent is an shRNA or a vector or plasmid encoding the same. shRNA may be produced inside the target cell after delivery of the appropriate construct into the nucleus. Constructs and mechanisms related to shRNA are well known in the relevant arts.

Diseases or conditions

The compositions/carriers of the present disclosure can deliver therapeutic or prophylactic agents to a subject or patient. The therapeutic or prophylactic agent includes, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein. Thus, the compositions of the present disclosure can be used to prepare nucleic acid drugs, genetic vaccines, small molecule drugs, polypeptides or protein drugs. Because of the wide variety of therapeutic or prophylactic agents described above, the compositions of the present disclosure are useful in the treatment or prevention of a variety of diseases or conditions.

In some embodiments, the disease or disorder is characterized by dysfunctional or abnormal protein or polypeptide activity.

For example, the disease or disorder is selected from the group consisting of: infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases and metabolic diseases.

In some embodiments, the infectious disease is selected from the group consisting of a disease caused by coronavirus, influenza virus, or HIV virus, pediatric pneumonia, rift valley fever, yellow fever, rabies, and a variety of herpes.

Other components

The composition may include one or more components other than those described in the preceding section. For example, the composition may include one or more hydrophobic small molecules, such as vitamins (e.g., vitamin a or vitamin E) or sterols.

The composition may also include one or more permeability enhancing molecules, carbohydrates, polymers, surface modifying agents, or other components. The permeability enhancing molecule may be, for example, a molecule described in U.S. patent application publication No. 2005/0222064. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen, and derivatives and analogs thereof).

Surface modifying agents may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyl dioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamers), mucolytics (e.g., acetylcysteine, mugwort, bromelain, papain, dyers woad), bromohexine, carbocistein, eplerenone, mesna, ambroxol, sobrinol, domidol, ritodtein, stenine, tiopronin, gelsin, thymosin beta 4, dnase alpha, neogenin, and dnase, such as dnase. The surface modifying agent may be disposed within and/or on the nanoparticle of the composition (e.g., by coating, adsorption, covalent attachment, or other means).

The composition may further comprise one or more functionalized lipids. For example, the lipid may be functionalized with an alkynyl group that may undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. In particular, lipid bilayers can be functionalized in this manner with one or more groups effective to facilitate membrane permeation, cell recognition, or imaging. The surface of the composition may also be conjugated to one or more useful antibodies. Functional groups and conjugates useful for targeted cell delivery, imaging, and membrane permeation are well known in the art.

In addition to these components, the composition may include any substance useful in pharmaceutical compositions. For example, the composition may include one or more pharmaceutically acceptable excipients, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersing aids, suspending aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonic agents, thickening or emulsifying agents, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, and the like. Excipients such as starch, lactose or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, e.g., remington's The Science and Practice of Pharmacy, 21 st edition, a.r. gennaro; lippincott, williams & Wilkins, baltimore, MD, 2006).

Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, dibasic calcium phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and/or combinations thereof.

In some embodiments, compositions comprising one or more lipids described herein may also comprise one or more adjuvants, such as Glucopyranosyl Lipid Adjuvants (GLA), cpG oligodeoxyribonucleotides (e.g., class a or class B), poly (I: C), aluminum hydroxide, and Pam3CSK4.

The compositions of the present disclosure may be formulated in solid, semi-solid, liquid or gaseous form, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, aerosols. The compositions of the present disclosure may be prepared by methods well known in the pharmaceutical arts. For example, sterile injectable solutions can be prepared by incorporating the therapeutic or prophylactic agent in the required amount with various of the other ingredients described above in the appropriate solvent such as sterile distilled water and then filter-sterilizing. Surfactants may also be added to promote the formation of a uniform solution or suspension.

For example, the compositions of the present disclosure are administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In some embodiments, the composition is administered subcutaneously.

The compositions of the present disclosure are administered in therapeutically effective amounts, which may vary not only with the particular agent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and may ultimately be at the discretion of the attendant physician or clinician. For example, a dose of 0.001 to 10mg/kg of the therapeutic or prophylactic agent may be administered to a mammal (e.g., a human).

Examples

The present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1: synthesis of cationic lipid compounds

1.Synthesis of 2-octyl decyl 6- (4- (4- (decyloxy) -4-oxobutyl) piperazin-1-yl) hexanoate (YK-501)

The synthetic route is as follows:

step one: synthesis of n-decyl 4-bromobutyrate (YK-501-PM 1)

N-decanol (5.00 g,31.59 mmol) and 4-bromobutyric acid (6.33 g,37.91 mmol) are dissolved in methylene chloride (40 mL), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (9.08 g,47.39 mmol) and 4-dimethylaminopyridine (1.93 g,15.80 mmol) are added to the above solutions, and the reaction is stirred at 30-35 ℃ for 24 h. After completion of the reaction, the reaction mixture was washed with a saturated sodium hydrogencarbonate solution and a saturated brine in this order, and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give n-decyl 4-bromobutyrate (6.20 g,20.18 mmol,63.9%).

Step two: synthesis of tert-butyl 4- (4- (decyloxy) -4-oxobutyl) piperazine-1-carboxylate (YK-501-PM 2)

N-decyl 4-bromobutyrate (1.00 g,3.25 mmol) and t-butyl piperazine-1-carboxylate (0.61 g,3.25 mmol) were dissolved in acetonitrile (10 mL), and potassium carbonate (1.35 g,9.75 mmol) and then heating to 60 ℃ to stir reaction 3 h, after the reaction is completed, cooling the reaction liquid to room temperature, filtering, and concentrating the filtrate under vacuum. The residue was purified by silica gel chromatography to give tert-butyl 4- (4- (decyloxy) -4-oxobutyl) piperazine-1-carboxylate (0.80 g,1.94 mmol,59.7%). C (C) ₂₃ H ₄₄ N ₂ O ₄ ，MS(ES): m/z（M+H ⁺ ）413.4。

Step three: synthesis of decyl 4- (piperazin-1-yl) butyrate (YK-501-PM 3)

Tert-butyl 4- (4- (decyloxy) -4-oxobutyl) piperazine-1-carboxylate (0.60 g,1.45 mmol) was dissolved in dichloromethane (6 mL), trifluoroacetic acid (6 mL) was added to the above-mentioned system, and the reaction was stirred at 30 to 35 ℃ for 1 h. After completion of the reaction, the reaction mixture was washed with a saturated sodium hydrogencarbonate solution and a saturated brine in this order, and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give decyl 4- (piperazin-1-yl) butyrate (315 mg,1.00 mmol,69.0%). C (C) ₁₈ H ₃₆ N ₂ O ₂ ，MS(ES): m/z（M+H ⁺ ）313.3。

Step four: synthesis of 2-octyl decyl 6-bromohexanoate (YK-501-PM 4)

Starting from 6-bromohexanoic acid (764 mg,3.92 mmol) and 2-octyldecanol (1.00 g,3.70 mmol), 2-octyldecanol 6-bromohexanoate (1.25 g,2.78 mmol,75.1%) was obtained according to the method for preparing YK-501-PM 1.

Step five: synthesis of 2-octyl decyl 6- (4- (4- (decyloxy) -4-oxobutyl) piperazin-1-yl) hexanoate (YK-501)

2-octyl decyl 6-bromohexanoate (448 mg,1.00 mmol) and decyl 4- (piperazin-1-yl) butyrate (315 g,1.00 mmol) were dissolved in acetonitrile (3 mL), potassium carbonate (414 mg,3.00 mmol) was added to the above system, the reaction was heated to 60℃and stirred for 5-h, after the reaction was completed, the reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give 2-octyldecyl 6- (4- (4- (decyloxy) -4-oxobutyl) piperazin-1-yl) hexanoate (102 mg,0.15 mmol,15.0) %）。C ₄₂ H ₈₂ N ₂ O ₄ ，MS(ES): m/z（M+H ⁺ ）679.7。

¹ H NMR (CDCl ₃ ，400 MHz，298 K) δ 4.04 (t, J = 6.7 Hz, 2H), 3.95 (d, J = 5.8 Hz, 2H), 2.48 (s, 6H), 2.37 – 2.26 (m, 8H), 1.80 (p, J = 7.4 Hz, 2H), 1.69 – 1.55 (m, 5H), 1.50 (dd, J = 15.3, 7.8 Hz, 2H), 1.36 – 1.18 (m, 46H), 0.87 (t, J = 6.7 Hz, 9H)。

Synthesis of 2.6,6' - (piperazine-1, 4-diyl) bis (2-octyldecyl hexanoate) (YK-502)

The synthetic route is as follows:

starting from piperazine (21 mg,0.24 mmol) and 2-octyldecyl 6-bromohexanoate (269 mg,0.60 mmol), the desired product (95 mg,0.12 mmol,50.0%) was obtained according to the method for preparing YK-501. C (C) ₅₂ H ₁₀₂ N ₂ O ₄ ，MS(ES): m/z（M+H ⁺ ）819.9。

¹ H NMR (CDCl ₃ ，400 MHz, 298 K) δ 3.96 (d, J = 5.5 Hz, 4H), 2.62 (s, 5H), 2.42 (s, 3H), 2.30 (t, J = 7.4 Hz, 4H), 1.68 – 1.52 (m, 10H), 1.29 (brs, , 64H), 0.88 (t, J = 6.4 Hz, 12H)。

3.Synthesis of 3-hexyl nonyl 6- (4- (4- (didecylamino) -4-oxobutyl) piperazin-1-yl) hexanoate (YK-503)

The synthetic route is as follows:

step one: synthesis of 4-bromobutyryl chloride (YK-503-PM 1)

4-Bromobutyric acid (1.00 g,5.78 mmol) is dissolved in dichloromethane (10 ml), N-dimethylformamide (100 mg,1.37 mmol) is added dropwise to the solution, the system temperature is maintained at-5 ℃, thionyl chloride (1.39 g,11.68 mmol) is added dropwise, the temperature is naturally raised to room temperature after the dropwise addition, and the reaction is continued to 4 h. After the reaction, the reaction mixture was concentrated under reduced pressure in vacuo to give 4-bromobutyryl chloride (1.07 g,5.78 mmol,100.0%).

Step two: synthesis of 4-bromo-N, N-didecylbutanamide (YK-503-PM 2)

Didecylamine (802 mg,2.70 mmol) and 4-bromobutyryl chloride (600 mg,3.24 mmol) were dissolved in dichloromethane (15 mL), and triethylamine (273 mg,2.70 mmol) was added to the above solution, followed by stirring at 30 to 35℃for 2 h. After the completion of the reaction, the reaction solution was washed with a saturated sodium hydrogencarbonate solution and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give 4-bromo-N, N-didecylbutyramide (1.11 g,2.49 mmol,92.2%).

Step three: synthesis of tert-butyl 4- (4- (didecylamino) -4-oxobutyl) piperazine-1-carboxylate (YK-503-PM 3)

Starting from tert-butyl piperazine-1-carboxylate (464 mg,2.49 mmol) and 4-bromo-N, N-didecylbutanamide (1.11 g,2.49 mmol), tert-butyl 4- (4- (didecylamino) -4-oxobutyl) piperazine-1-carboxylate (929 mg,1.68 mmol,67.5%) was obtained according to the method for preparing YK-501-PM 2. C (C) ₃₃ H ₆₅ N ₃ O ₃ ，MS(ES): m/z（M+H ⁺ ）552.5。

Step four: synthesis of N, N-didecyl-4- (piperazin-1-yl) butanamide (YK-503-PM 4)

Using tert-butyl 4- (4- (didecylamino) -4-oxobutyl) piperazine-1-carboxylate (929 mg,1.68 mmol) as a starting material, N-didecyl-4- (piperazin-1-yl) butanamide (604 mg,1.34 mmol,79.8%) was obtained according to the method for preparing YK-501-PM 3. C (C) ₂₈ H ₅₇ N ₃ O，MS(ES): m/z（M+H ⁺ ）452.5。

Step five: synthesis of 3-hexyl nonyl 6-bromohexanoate (YK-503-PM 5)

Starting from 6-bromohexanoic acid (1.03 g,5.28 mmol) and 3-hexylnonanol (1.00 g,4.38 mmol), 3-hexylnonanoate (1.39 g,3.43 mmol,78.3%) was obtained in the same manner as in the preparation of YK-501-PM 1.

Step six: synthesis of 3-hexyl nonyl 6- (4- (4- (didecylamino) -4-oxobutyl) piperazin-1-yl) hexanoate (YK-503)

Starting from 3-hexyl 6-bromohexanoate (496 mg,1.22 mmol) and N, N-didecyl-4- (piperazin-1-yl) butanamide (300 mg,0.66 mmol), the desired product (99 mg,0.13 mmol,19.7%) was obtained according to the method for preparing YK-501. C (C) ₄₉ H ₉₇ N ₃ O ₃ ，MS(ES): m/z（M+H ⁺ ）776.9。

¹ H NMR (CDCl ₃ , 400 MHz, 298 K) δ 4.07 (t, J = 7.1 Hz, 2H), 3.31 – 3.24 (m, 2H), 3.23 – 3.14 (m, 2H), 2.63 – 2.46 (m, 6H), 2.41 (s, 2H), 2.31 (dt, J= 14.8, 8.9 Hz, 6H), 1.89 – 1.81 (m, 2H), 1.57 – 1.47 (m, 8H), 1.25 (brs, 55H), 0.87 (t, J = 5.9 Hz, 12H)。

4.Synthesis of 2- (butylsulfanyl) ethyl 8- (4- (4- (didecylamino) -4-oxobutyl) piperazin-1-yl) octanoate (YK-504)

The synthetic route is as follows:

step one: synthesis of 2- (butylsulfanyl) ethyl 8-bromooctoate (YK-504-PM 1)

Starting from 2- (butylsulfanyl) ethan-1-ol (200 mg,1.49 mmol) and 8-bromooctanoic acid (400 mg,1.79 mmol), 8-bromooctanoic acid 2- (butylsulfanyl) ethyl ester (300 mg,0.88 mmol,59.1%) was obtained according to the method for preparing YK-501-PM 1.

Step two: synthesis of 2- (butylsulfanyl) ethyl 8- (4- (4- (didecylamino) -4-oxobutyl) piperazin-1-yl) octanoate (YK-504)

With 2- (butylsulfanyl) ethyl 8-bromooctoate (300 mg,0.88 mmol) andN,N-didecyl-4- (piperazin-1-yl) butanamide (400 mg,0.88 mmol) as starting material, following the procedure for the preparation of YK-501, the desired product (133 mg,0.19 mmol,21.6%) was obtained. C (C) ₄₂ H ₈₃ N ₃ O ₃ S，MS(ES): m/z（M+H ⁺ ）710.7。

¹ H NMR (CDCl ₃ ,400 MHz, 298 K) δ 4.21 (t, J = 7.0 Hz, 2H), 3.31 – 3.24 (m, 2H), 3.23 – 3.16 (m, 2H), 2.72 (t, J = 7.0 Hz, 2H), 2.60 – 2.50 (m, 6H), 2.36 – 2.27 (m, 6H), 1.89 – 1.81 (m, 2H), 1.56 (ddd, J = 25.0, 16.5, 7.1 Hz, 12H), 1.26 (brs, 40H), 0.93 – 0.85 (m, 9H)。

5.8 Synthesis of 3- (pentyldithio) propyl (YK-505) octanoate of 8- (4- (6- ((3-hexylnonyl) oxy) -6-oxohexyl) piperazin-1-yl)

The synthetic route is as follows:

step one: synthesis of tert-butyl 4- (6- ((3-hexylnonyl) oxy) -6-oxohexyl) piperazine-1-carboxylate (YK-505-PM 1)

Using piperazine-1-carboxylic acid tert-butyl ester (220 mg,1.18 mmol) and 6-bromohexanoic acid 3-hexyl nonyl ester (400 mg,0.98 mmol) as raw materials, tert-butyl 4- (6- ((3-hexylnonyl) oxy) -6-oxohexyl) piperazine-1-carboxylate (278 mg,0.54 mmol,45.8%) was obtained according to the method for preparing YK-501-PM 2. C (C) ₃₀ H ₅₈ N ₂ O ₄ ，MS(ES): m/z（M+H ⁺ ）511.5。

Step two: synthesis of 3-hexyl nonyl 6- (piperazin-1-yl) hexanoate (YK-505-PM 2)

Using tert-butyl 4- (6- ((3-hexylnonyl) oxy) -6-oxohexyl) piperazine-1-carboxylate (278 mg,0.54 mmol) as a starting material, 3-hexylnonyl 6- (piperazin-1-yl) hexanoate (120 mg,0.29 mmol,53.7%) was obtained according to the method for preparing YK-501-PM 3. C (C) ₂₅ H ₅₀ N ₂ O ₂ ，MS(ES): m/z（M+H ⁺ ）411.4。

Step three: synthesis of 3- (pentyldithio) propan-1-ol (YK-505-PM 3)

3-mercaptopropanol (1.00 g,10.85 mmol) and pentane-1-mercaptan (1.13 g,10.85 mmol) are dissolved in a mixed solvent (100 mL) of dichloromethane and methanol (volume ratio is 1:1), pyridine (1.71 g,21.60 mmol) is added under the protection of nitrogen, iodine particles (2.74 g,10.85 mmol) are added into the system in small batches for multiple times, and after the iodine particles are added, the mixture is stirred at 30-35 ℃ for reaction for 20 h. After completion of the reaction, the reaction mixture was washed with saturated brine and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give 3- (pentyldithio) propan-1-ol (860 mg,4.42 mmol,79.7%).

Step four: synthesis of 3- (pentyldithio) propyl 8-bromooctanoate (YK-505-PM 4)

Using 3- (pentyldithio) propan-1-ol and 8-bromooctanoic acid (100 mg,0.51 mmol) as raw materials, 3- (pentyldithio) propyl 8-bromooctanoate (161 mg,0.40 mmol,78.43%) was obtained by the method of preparing YK-501-PM 1.

Step five: synthesis of 3- (pentyldithio) propyl 8- (4- (6- ((3-hexylnonyl) oxy) -6-oxohexyl) piperazin-1-yl) octanoate (YK-505)

Starting from 3- (pentyldithio) propyl 8-bromooctanoate (161 mg,0.40 mmol) and 3-hexylnonyl 6- (piperazin-1-yl) hexanoate (120 mg,0.29 mmol), the title product (48 mg,0.07 mmol,22.8%) was obtained according to the procedure for preparation of YK-501. C (C) ₄₁ H ₈₀ N ₂ O ₄ S ₂ ，MS(ES): m/z（M+H ⁺ ）729.6。

¹ H NMR (CDCl ₃ ，400 MHz, 298 K) δ 4.16 (t, J = 6.2 Hz, 2H), 4.07 (t, J = 7.1 Hz, 2H), 2.70 (dt, J = 15.0, 7.3 Hz, 4H), 2.53 (s, 4H), 2.35 (dd, J = 14.8, 7.9 Hz, 4H), 2.29 (dd, J = 13.5, 6.0 Hz, 4H), 2.06 – 1.99 (m, 2H), 1.70 – 1.48 (m, 12H), 1.41 – 1.20 (m, 37H), 0.89 (dt, J = 13.6, 7.0 Hz, 9H)。

6.Synthesis of 3-hexyl nonyl 6- (4- (3- ((2- (dodecyl disulfide) ethyl) amino) -3-oxopropyl) piperazin-1-yl) hexanoate (YK-506)

The synthetic route is as follows:

step one: synthesis of S- (2-aminoethyl) sulfuric acid (YK-506-PM 1)

2-Chloroethylamine hydrochloride (5.00 g,43.11 mmol) and sodium thiosulfate (10.20 g,64.51 mmol) were dissolved in water (10 mL) and the reaction was stirred at 70℃for 3 h. After the reaction is finished, carrying out suction filtration while the reaction is hot, washing a filter cake by ethanol, collecting filtrate, cooling to 0-5 ℃, stirring and crystallizing 1 h, carrying out suction filtration on the mixture system to obtain a filter cake, and drying to obtain S- (2-aminoethyl) sulfuric acid (3.64 g,23.16 mmol,53.7%). C (C) ₂ H ₇ NO ₃ S ₂ ，MS(ES): m/z（M+Na ⁺ ）180.0。

Step two Synthesis of 2- (dodecyl disulfide) ethane-1-amine (YK-506-PM 2)

S- (2-aminoethyl) sulfuric acid (2.00 g,12.72 mmol) and dodecane-1-thiol (3.86 g,19.07 mmol) were dissolved in methanol (50 mL), sodium hydroxide (760 mg,19.00 mmol) was added to the above system, and the reaction was stirred at 50℃for 3 h. After the reaction is finished, the system is cooled to room temperature and is filtered by suction, the filter cake is leached by methylene dichloride, and the filtrate is concentrated under vacuum and reduced pressure to obtain 2- (dodecyl disulfide) ethane-1-amine (3.00 g,10.81 mmol,85.0%). C (C) ₁₄ H ₃₁ NS ₂ ，MS(ES): m/z（M+H ⁺ ）278.2。

Step three: synthesis of N- (2- (dodecyl disulfide) ethyl) acrylamide (YK-506-PM 3)

2- (dodecyl disulfide) ethane-1-amine (3.00 g,10.81 mmol) and triethylamine (3.28 g,32.41 mmol) are dissolved in dichloromethane (5 mL), acryloyl chloride (1.17 g,12.93 mmol) is dropwise added under the stirring condition of controlling the temperature to-5 ℃, the mixture is naturally warmed to room temperature after the dropwise addition to continue to react for 0.5 h, and after the reaction is finished, the reaction solution is washed with saturated sodium bicarbonate solution and saturated saline water in sequence and is dried through anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give N- (2- (dodecyl) Disulfide) ethyl) acrylamide (2.60 g,7.84 mmol,72.5%). C (C) ₁₇ H ₃₃ NOS ₂ ，MS(ES): m/z（M+H ⁺ ）332.3。

Step four: synthesis of 3-hexyl nonyl 6- (4- (3- ((2- (dodecyl disulfide) ethyl) amino) -3-oxopropyl) piperazin-1-yl) hexanoate (YK-506)

N- (2- (dodecyl disulfide) ethyl) acrylamide (2.58 g,7.78 mmol) and 3-hexyl nonyl 6- (piperazin-1-yl) hexanoate (1.60 g,3.90 mmol) were added directly to the reaction flask and reacted 48 h with stirring at 70 ℃. After the reaction was completed, the above system was purified by silica gel chromatography to give 3-hexylnonyl 6- (4- (3- ((2- (dodecyldithio) ethyl) amino) -3-oxopropyl) piperazin-1-yl) hexanoate (1.02 g,1.37 mmol,35.1%). C (C) ₄₂ H ₈₃ N ₃ O ₃ S ₂ ，MS(ES): m/z（M+H ⁺ ）742.6。

¹ H NMR (CDCl ₃ ，400 MHz, 298 K) δ 8.32 (s, 1H), 4.06 (t, J = 7.1 Hz, 2H), 3.60 (dd, J = 14.6, 7.2 Hz, 8H), 2.77 (dd, J = 11.0, 5.2 Hz, 8H), 2.70 – 2.63 (m, 2H), 2.45 (d, J = 5.4 Hz, 2H), 2.28 (t, J = 7.3 Hz, 2H), 1.24 (m, 49H), 0.86 (t, J = 6.5 Hz, 9H)。

Synthesis of 7.3,3' - (piperazine-1, 4-diyl) bis (N- (2- (dodecyldithio) ethyl) propionamide) (YK-507)

The synthetic route is as follows:

step one: synthesis of 3,3' - (piperazine-1, 4-diyl) bis (N- (2- (dodecyldithio) ethyl) propionamide) (YK-507)

Starting with piperazine (50 mg,0.58 mmol) and N- (2- (dodecyl disulfide) ethyl) acrylamide (434 mg,1.31 mmol), the title product (154 mg,0.21 mmol,36.2%) was obtained according to the method for preparing YK-506. C (C) ₃₈ H ₇₆ N ₄ O ₂ S ₄ ，MS(ES): m/z（M+H ⁺ ）749.5。

¹ H NMR (CDCl ₃ ，400 MHz, 298 K) δ 8.42 (s,2H), 3.60 (d, J = 5.4 Hz, 4H), 2.78 (t, J = 5.3 Hz, 4H), 2.70 (dd, J = 15.3, 7.5 Hz, 12H), 2.45 (s, 4H), 1.70 – 1.63 (m, 4H), 1.38 – 1.23 (m, 40H), 0.88 (t, J = 6.3 Hz, 6H)。

Synthesis of 8.1,1' - [ [2- [4- [2- [ [2- [ bis (2-hydroxydodecyl) amino ] ethyl ] piperazin-1-yl ] ethyl ] imino ] bis (dodecane-2 alcohol) (C12-200)

The synthetic route is as follows:

step one: synthesis of 2- (4- (2- ((cyanomethyl) amino) ethyl) piperazin-1-yl) acetonitrile (C12-200-PM 1)

1- (2-aminoethyl) piperazine (4.00 g, 30.96 mmol) and chloroacetonitrile (5.10 g,67.75 mmol) were dissolved in absolute ethanol (250 mL), and potassium carbonate (16.86 g,122.00 mmol) was added to the above system and heated to reflux reaction 7 h. After the reaction is finished, the reaction solution is cooled to room temperature and then filtered, and the filtrate is concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography to give 2- (4- (2- ((cyanomethyl) amino) ethyl) piperazin-1-yl) acetonitrile (3.70g,17.85 mmol,57.7%). C (C) ₁₀ H ₁₇ N ₅ ，MS(ES): m/z（M+H ⁺ ）208.2。

Step two: n (N) ¹ Synthesis of- (2- (4- (2-aminoethyl) piperazin-1-yl) ethyl) ethane-1, 2-diamine (C12-200-PM 2)

2- (4- (2- ((cyanomethyl) amino) ethyl) piperazin-1-yl) acetonitrile (0.50 g, 2.41 mmol) was dissolved in a mixed solvent of methanol (25 mL) and aqueous ammonia (3 mL), and Raney-Nickel-2400 type catalyst (250 mg) was added to the above system. The reaction system was maintained at a pressure of 1000 psi in an atmosphere of hydrogen, reaction 6 h. Filtering the reaction solution after the reaction is finished, and concentrating the filtrate under reduced pressure to remove the solvent to obtain N ¹ - (2- (4- (2-aminoethyl) ethyl)) Piperazin-1-yl) ethyl) ethane-1, 2-diamine blue crude (0.48 g,2.23 mmol,92.5%). C (C) ₁₀ H ₂₅ N ₅ ，MS(ES): m/z（M+H ⁺ ）216.2。

Step three: synthesis of 1,1' - [ [2- [4- [2- [ [2- [ bis (2-hydroxydodecyl) amino ] ethyl ] piperazin-1-yl ] ethyl ] imino ] bis (dodecane-2 alcohol) (C12-200)

1, 2-epoxydodecane (4.00 g, 30.96 mmol) and N ¹ - (2- (4- (2-amino ethyl) piperazin-1-yl) ethyl) ethane-1, 2-diamine (0.30 g) are stirred and mixed evenly by magnetic force, and the system is heated to 80 ℃ to react 48 h. After the reaction, the reaction system was purified by silica gel chromatography to give 1,1' - [ [2- [4- [2- [ [2- [ bis (2-hydroxydodecyl) amino ] -]Ethyl group](2-hydroxydodecyl) amino group]Ethyl group]Piperazin-1-yl]Ethyl group]Imino group]Bis (dodecyl-2 alcohol) (668 mg,0.06 mmol,42.4%). C (C) ₇₀ H ₁₄₅ N ₅ O ₅ ，MS(ES): m/z（M+H ⁺ ）1137.1。

¹ H NMR (CDCl ₃ ，400 MHz, 298 K) δ 5.00–4.00 (br s, 5H), 3.62 and 3.55 (apparent br s, 5H), 3.00–2.00 (m, 30H), 1.52–1.25 (m, 90H), 0.87 (t, J = 7.2 Hz, 15H)。

Synthesis of 9.1,1' - (piperazine-1, 4-diyl) bis (3- ((bis (2-hydroxytetradecyl) amino) ethyl) (methyl) amino) propan-1-one) (MIC 3)

The synthetic route is as follows:

step one: synthesis of 1,1' - (piperazine-1, 4-diyl) bis (prop-2-en-1-one) (MIC 3-PM 1)

Dissolving piperazine (2.00 g,23.22 mmol) and triethylamine (6.56 g,64.82 mmol) in dichloromethane (20 mL), dropwise adding acryloyl chloride (5.25 g,58.05 mmol) under stirring at-5-5deg.C, naturally heating to room temperature after dropwise adding, continuously reacting for 0.5 h, and sequentially adding saturated sodium bicarbonate solution and saturated solution And brine, and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give 1,1' - (piperazine-1, 4-diyl) bis (prop-2-en-1-one) (2.80 g,14.42 mmol,62.1%). C (C) ₁₀ H ₁₄ N ₂ O ₂ ，MS(ES): m/z（M+H ⁺ ）195.2。

Step two: synthesis of di-tert-butyl ((piperazine-1, 4-diylbis (3-oxypropane-3, 1-diyl)) bis (methylazadiyl)) dicarbamate (MIC 3-PM 2)

1,1' - (piperazine-1, 4-diyl) bis (prop-2-en-1-one) (1.00 g,5.15 mmol) and tert-butyl (2- (methylamino) ethyl) carbamate (2.70 g,15.50 mmol) were added directly to the reaction flask and reacted 48 h with stirring at 70 ℃. After the completion of the reaction, the above-mentioned system was purified by silica gel chromatography to obtain di-tert-butyl ((piperazine-1, 4-diylbis (3-oxypropane-3, 1-diyl)) bis (methylazadiyl)) dicarbamate (1.12 g,2.06 mmol,40.0%). C (C) ₂₆ H ₅₀ N ₆ O ₆ ，MS(ES): m/z（M+H ⁺ ）543.3。

Step three: synthesis of 1,1' - (piperazine-1, 4-diyl) bis (3- ((2-aminoethyl) (methyl) amino) propan-1-one) (MIC 3-PM 3)

Di-tert-butyl ((piperazine-1, 4-diylbis (3-oxypropane-3, 1-diyl)) bis (methylazadiyl)) dicarbamate (1.12 g,2.06 mmol) was dissolved in dichloromethane (12 mL), trifluoroacetic acid (6 mL) was added to the above-mentioned system, and the reaction was stirred at 30 to 35℃for 1 h. After completion of the reaction, the reaction mixture was washed with a saturated sodium hydrogencarbonate solution and a saturated brine in this order, and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give 1,1' - (piperazine-1, 4-diyl) bis (3- ((2-aminoethyl) (methyl) amino) propan-1-one) (310 mg,0.91 mmol,44.2%). C (C) ₁₆ H ₃₄ N ₆ O ₂ ，MS(ES): m/z（M+H ⁺ ）343.3。

Step four: synthesis of 1,1' - (piperazine-1, 4-diyl) bis (3- ((bis (2-hydroxytetradecyl) amino) ethyl) (methyl) amino) propan-1-one) (MIC 3)

Starting with 1,1' - (piperazine-1, 4-diyl) bis (3- ((2-aminoethyl) (methyl) amino) propan-1-one) (310 mg,0.91 mmol) and 2-dodecyloxirane (1.27 g,5.98 mmol), the desired product (554 mg,0.46 mmol,50.5%) was obtained according to the procedure for the preparation of C12-200. C (C) ₇₂ H ₁₄₆ N ₆ O ₆ ，MS(ES): m/z（M+H ⁺ ）1193.1。

¹ H NMR (CDCl ₃ ，400 MHz, 298 K) δ 5.19 (s, 2H), 3.75–3.42 (m, 8H), 3.23 (d, J=5.0, 4H), 2.88–2.16 (m, 30H), 1.47–1.16 (m, 92H), 0.89 (t, J=6.8,12H)。

10. Synthesis of N, N' - (piperazine-1, 4-diylbis (propane-3, 1-diyl)) bis (3- (didodecylamino) propanamide) (PPZ-A12)

The synthetic route is as follows:

step one: synthesis of di-tert-butyl ((piperazine-1, 4-diylbis (propyl-3, 1-diyl)) bis (azadiyl) bis (3-oxapropane-3, 1-diyl)) dicarbamate (PPZ-A12-PM 1)

3- ((tert-Butoxycarbonyl) amino) propionic acid (1.70 g, 8.98 mmol), DIPEA (1.16 g, 8.98 mmol), EDCI (1.73 g, 9.02 mmol) and HOBt (1.22 g, 9.03 mmol) were dissolved in dichloromethane (20 mL) and stirred at room temperature for 10 min, then 3,3' - (piperazine-1, 4-diyl) bis (propan-1-amine) (0.60 g, 3.00 mmol) was added dropwise to the above system. The system was stirred at room temperature for 12 h and was prepared by adding saturated NaHCO ₃ The solution (20 mL) was quenched. The aqueous phase was extracted three times with dichloromethane (20 mL) and the organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give (((piperazine-1, 4-diylbis (propyl-3, 1-diyl)) bis (azadiyl) bis (3-oxapropane-3),1-diyl)) di-tert-butyl dicarbamate (1.26 g,2.32 mmol,77.3%). C (C) ₂₆ H ₅₀ N ₆ O ₆ ，MS(ES): m/z（M+H ⁺ ）543.4。

Step two: synthesis of N, N' - (piperazine-1, 4-diylbis (propane-3, 1-diyl)) bis (3-aminopropionamide) (PPZ-A12-PM 2)

The target product (0.70 g,2.04 mmol,87.9%) C was obtained by the method of preparing MIC3-PM3 starting from di-tert-butyl (((piperazine-1, 4-diylbis (propyl-3, 1-diyl)) bis (azadiyl) bis (3-oxapropane-3, 1-diyl)) dicarbamate (1.26 g,2.32 mmol) ₁₆ H ₃₄ N ₆ O ₂ ，MS(ES): m/z（M+H ⁺ ）343.3。

Step three: synthesis of N, N' - (piperazine-1, 4-diylbis (propane-3, 1-diyl)) bis (3- (didodecylamino) propanamide) (PPZ-A12)

N, N' - (piperazine-1, 4-diylbis (propane-3, 1-diyl)) bis (3-aminopropionamide) (0.70 g,2.04 mmol) and dodecanal (2.26 g,12.26 mmol) were dissolved in dichloromethane (10 ml), sodium borohydride acetate (2.16 g,10.19 mmol) was added to the above system, the suspension was stirred at room temperature for reaction 12 h, and by addition of saturated NaHCO ₃ The solution (10 mL) was quenched. The aqueous phase was extracted three times with dichloromethane (20 mL) and the organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give N, N' - (piperazine-1, 4-diylbis (propane-3, 1-diyl)) bis (3- (didodecylamino) propionamide) (1.22 g,1.20 mmol,58.8%). C (C) ₆₄ H ₁₃₀ N ₆ O ₂ ，MS(ES): m/z（M+H ⁺ ）1016.7。

¹ H NMR (CDCl ₃ ，400 MHz, 298 K) δ 8.50 (t, J = 5.6 Hz, 2H), 3.23 (q, J =

6.7 Hz, 4H), 2.64 (t, J = 6.1 Hz, 4H), 2.59 – 2.20 (m, 24H), 1.65 (p, J = 7.1 Hz, 4H), 1.48 – 1.33 (m, 9H), 1.32 – 1.11 (m, 75H), 0.85 (t, J = 6.9 Hz, 12H)。

Example 2: preparation condition optimization of nano lipid particle (LNP preparation)

1. Vector (liposome) and mRNA ratio optimization

The cationic lipid compound YK-506 synthesized in example 1 was dissolved in ethanol at a molar ratio of 49:10:39.5:1.5 with DSPC (Ai Weita (Shanghai) medical science Co., ltd.), cholesterol (Ai Weita (Shanghai) medical science Co., ltd.) and DMG-PEG2000, respectively, to prepare an ethanol lipid solution. And rapidly adding the ethanol lipid solution into a citrate buffer solution (pH=4-5) by an ethanol injection method, and swirling for 30s for later use. eGFP-mRNA (purchased from Shanghai laboratory reagent limited) was diluted in citrate buffer (ph=4-5) to obtain an aqueous mRNA solution. Liposomes were prepared from a volume of liposome solution and an aqueous solution of mRNA at a weight ratio of total lipid to mRNA of 5:1, 10:1, 15:1, 20:1, 30:1 and 35:1, respectively. Ultrasound was performed at 25℃for 15min (ultrasound frequency 40kHz, ultrasound power 800W). The obtained liposome was diluted to 10 times of volume with PBS, and subjected to ultrafiltration in a 300kDa ultrafiltration tube to remove ethanol. The volume was then fixed to volume with PBS to give LNP formulations encapsulating eGFP-mRNA with cationic lipid YK-506/DSPC/cholesterol/DMG-PEG 2000 (molar ratio 49:10:39.5:1.5).

The results of cell transfection experiments show that the weight ratio of the vector to the mRNA is in the range of 10:1-30:1, and the vector has good transfection effect, wherein the transfection effect is preferably 15:1, the transfection effect is poor in the ratios of 5:1 and 35:1, and the mRNA cannot be carried by the ratio.

2. Cationic lipid and neutral lipid ratio optimization

LNP formulations encapsulating eGFP-mRNA were prepared according to the method in 1 with molar ratios of cationic lipid YK-506 to neutral lipid DSPC of 1:1, 3:1, 3.5:1, 4:1, 4.9:1, 10:1, 15:1 and 20:1, respectively.

As can be seen from the cell transfection experiment, the molar ratio of the cationic lipid to the neutral lipid is 1:1-15:1, and the transfection efficiency is 4.9:1.

3. Optimization of the proportion of Polymer conjugated lipid to Carrier (Liposome)

LNP formulations encapsulating eGFP-mRNA were prepared according to the method in 1 with a cationic lipid of YK-506 in the carrier, wherein the polymer conjugated lipid DMG-PEG2000 was 0.5%, 1.5%, 2.5%, 3.5%, 5%, 10% and 15% of the carrier, respectively, by mole.

Cell transfection experiment results show that the polymer conjugated lipid accounts for 0.5% -10% of the carrier mole ratio, and has the transfection effect, and the transfection efficiency is highest when 1.5% and lowest when 10%.

4. Optimization of the ratio of the ingredients in the Carrier (Liposome)

LNP formulations encapsulating eGFP-mRNA were prepared according to the method in 1, with cationic lipid YK-506, neutral lipid DSPC, structural lipid cholesterol, and polymer conjugated lipid DMG-PEG2000 molar ratios of 75:5:15:5, 65:8:25:2, 49:10:39.5:1.5, 45:10:43.5:1.5, 45:25:20:10, 40:10:48.5:1.5, 35:10:53.5:1.5, and 25:5:65:5, respectively.

As shown by cell transfection experiments, the cationic lipid, the neutral lipid, the structural lipid and the polymer conjugated lipid have good transfection effect in the ranges of the molar ratio of (35-49): (7.5-15): (35-55): (1-5), wherein the molar ratio of (49:10:39.5:1.5) and the conjugated lipid is 75:5:15:5:8:25:2, 49:10:39.5:1.5, 45:10:43.5:1.5, 45:25:20:10, 40:10:48.5:1.5, 35:10:53.5:1.5 and 25:5:65:5.

Example 3: LNP preparation of eGFP-mRNA cell transfection experiments

Cell resuscitating and passaging: 293T cells were resuscitated and passaged in petri dishes for culture to the desired cell numbers.

Seed plate: cells in the dishes were digested and counted, plated in 96-well plates at 1 ten thousand cells per well, plated in 12-well plates at 15 ten thousand cells per well, and cultured overnight until cells attached.

Cell transfection experiments: LNP preparations containing 1.5. Mu.g of the eGFP-mRNA prepared in example 2 (YK-506 as cationic lipid in the carrier) were added to 12-well plate cell culture medium, and after further culturing for 24 hours, the transfection efficiency of the samples was examined by fluorescence intensity, as observed by a fluorescence microscope.

According to the experimental results, the preparation conditions of the nano lipid particles (LNP preparation) are finally determined: the ratio of the vector to the mRNA is 15:1; the molar ratio of the cationic lipid to the neutral lipid is 4.9:1; the polymer conjugated lipid accounts for 1.5% of the liposome; the molar ratio of cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid was 49:10:39.5:1.5 (fig. 1), and the following experiments produced nanolipid particles (LNP formulation) under this condition.

Example 4: preparation of nanolipid particles (LNP formulation) (optimal formulation)

The cationic lipids listed in table 1 were dissolved in ethanol at a molar ratio of 49:10:39.5:1.5 with DSPC (Ai Weita (Shanghai) pharmaceutical technologies, inc.), cholesterol (Ai Weita (Shanghai) pharmaceutical technologies, inc.) and DMG-PEG2000, respectively, to prepare an ethanol lipid solution, which was rapidly added to citrate buffer (ph=4-5) by ethanol injection, and vortexed for 30s for use. The eGFP-mRNA (purchased from Shanghai laboratory reagent Co., ltd.) or the Fluc-mRNA (purchased from Shanghai laboratory reagent Co., ltd.) was diluted in a citrate buffer (pH=4 to 5) to obtain an aqueous mRNA solution. A liposome was prepared by mixing a volume of liposome solution with an aqueous solution of mRNA at a weight ratio of total lipid to mRNA of 15:1. Ultrasound was performed at 25℃for 15min (ultrasound frequency 40kHz, ultrasound power 800W). The obtained liposome was diluted to 10 times of volume with PBS, and subjected to ultrafiltration in a 300kDa ultrafiltration tube to remove ethanol. The mixture was then sized to volume with PBS to give LNP formulations using cationic lipid/DSPC/cholesterol/DMG-PEG 2000 (mol% 49:10:39.5:1.5) to encapsulate eGFP-mRNA or Fluc-mRNA.

Example 5: determination of nanolipid particle size and polydispersity index (PDI)

Particle size and Polydispersity (PDI) were determined using dynamic light scattering using a malvern laser particle sizer.

10 μl of liposome solution was taken, diluted to 1mL with RNase-free deionized water, and added to the sample cell, and each sample was repeatedly assayed 3 times. The measurement conditions are as follows: 90. scattering angle, 25 ℃. The test results are shown in Table 2:

TABLE 2 particle size and polydispersity index (PDI) of nanolipid particles

As can be seen from table 2, the nano lipid particles prepared in example 4 have a particle size of 140-210 nm, and can be used for delivering mRNA:

wherein, the particle size of the particles prepared by YK-505 is the smallest and is 140.00nm; the particle size of the particles prepared from YK-504 was the largest, 200.95nm.

The polydispersity of all the nano lipid particles is 5% -25%, wherein the minimum is YK-505, which is 5.6%; the maximum is YK-504, 23.8%.

The morphology of particles prepared from YK-506 is also at a better level, with a particle size of 144.66 nm and a polydispersity of 11.9%, and is smaller and more uniform than particles prepared from other structurally similar cationic lipids of the prior art, such as C12-200, MIC3 and PPZ-A12.

Example 6: in vitro validation of LNP delivery vehicle performance

Cell resuscitating and passaging: the procedure is as in example 3.

Seed plate: the procedure is as in example 3.

1. Fluorescence detection of Fluc-mRNA (transfection efficiency)

LNP preparations containing 0.3. Mu.g of Fluc-mRNA (LNP preparation carrier composition: cationic lipid, DSPC, cholesterol and DMG-PEG2000, molar ratio: 49:10:39.5:1.5, wherein cationic lipid is the cationic lipid listed in Table 1) were added to cell culture broth of 96-well plates, and after further incubation for 24 h, the corresponding reagents were added according to Gaussia Luciferase Assay Kit instructions, and the intensity of fluorescence expression per well was detected by IVIS fluorescence detection system. The chemical structures of the designed compounds and the representative cationic lipids of the prior art are shown in Table 1. Transfection efficiency in cells of LNP formulations prepared from a range of cationic lipid compounds designed herein and prior art cationic lipids, including MC3, C12-200, MIC3 and PPZ-A12, are shown in Table 3.

Table 3 shows the results of fluorescence detection of LNP preparations containing Fluc-mRNA prepared from different cationic lipids.

TABLE 3 Fluc-mRNA fluorescence detection results

Analysis of experimental results:

(1) Compounds contemplated herein, including YK-506 and YK-504, have chemical structures that differ significantly from the prior art cationic lipids, such as MC3; there are minor differences such as C12-200, MIC3 and PPZ-A12.

Compared with the representative cationic lipid MC3 in the prior art, the chemical structure of the compound designed by the application is completely different and has great difference. MC3 is piperazine-free, and the compounds contemplated by the application all have piperazine groups; MC3 has only 1 branched hydrophobic tail, while the compound designed by the application has 2 hydrophobic tails; the tail structure of MC3 contains double bond, while the tail structure of the compound designed by the application has no double bond, and other groups have larger difference.

The chemical structures of the compounds contemplated herein are similar with only slight differences in individual groups compared to prior art piperazinyl containing cationic lipids, such as C12-200, MIC3 and PPZ-A12.

(2) Of the designed series of compounds, LNP preparations prepared from YK-506 and YK-504 have the highest cell transfection efficiency, and compared with the representative cationic lipids in the prior art, the LNP preparation has significantly improved cell transfection efficiency whether the structure is greatly different (such as MC 3) or the structure is slightly different (such as C12-200, MIC3 and PPZ-A12). For example, YK-506 can be transfected with 4.83 times MC3, 2.94 times C12-200, 4.00 times MIC3 and 3.73 times PPZ-A12.

MC3, C12-200, MIC3, PPZ-A12 are typical cationic lipids in the prior art, and have good transfection performance.

As can be seen from Table 3 and FIG. 2, LNP preparations containing Fluc-mRNA prepared from YK-506 and YK-504 showed the strongest fluorescence absorption and RLU values of 624617 and 112934, respectively.

YK-506 can reach 4.83 times of MC3, 2.94 times of C12-200, 4.00 times of MIC3 and 3.73 times of PPZ-A12, and the transfection efficiency is obviously improved.

YK-504 can be up to 0.87 times of MC3, 0.53 times of C12-200, 0.72 times of MIC3 and 0.67 times of PPZ-A12, and the transfection efficiency is equivalent.

The cell transfection efficiency of LNP formulations prepared therefrom cannot be deduced from the structure of cationic lipid compounds, and is very likely to be very different, both from structurally different to structurally similar compounds.

(3) Will be of similar structure, L ₁ And L ₂ A series of compounds whose groups are-C (O) O-such as YK-501, YK-502 and YK-505, which differ only slightly in the individual groups compared to YK-506, are shown in Table 1. Cell transfection results show (Table 3 and FIG. 2) that the series of compounds have very large activity differences, wherein the cell transfection efficiency of YK-506 is highest and can reach 103.62 times of YK-501, 46.53 times of YK-502 and 43.98 times of YK-505 respectively, and the transfection efficiency is obviously improved. The cell transfection efficiency of YK-504 is 18.73 times of YK-501, 8.41 times of YK-502 and 7.95 times of YK-505 respectively, and the transfection efficiency is obviously improved.

(4) Similar to the structure, L ₁ The radicals being-C (O) O-, L ₂ The radical being-C (O) N ((CH) ₂ ) ₉ CH ₃ ) Compared with the YK-503 compound, the transfection efficiency of YK-506 and YK-504 cells is obviously improved, and the transfection efficiency can reach 133.29 times and 24.10 times of YK-503 respectively.

(5) Similar to the structure, L ₁ And L ₂ The radicals are-C (O) NH-, R ₁ And R is ₂ Compared with the compound YK-507 containing the-S-S-group, the transfection efficiency of YK-506 and YK-504 cells is obviously improved, and the transfection efficiency can respectively reach 104.80 times and 18.95 times of YK-507.

2. Cell viability assay

LNP preparations containing 1.5. Mu.g of Fluc-mRNA (LNP preparation carrier composition: cationic lipid, DSPC, cholesterol and DMG-PEG2000, molar ratio: 49:10:39.5:1.5, wherein cationic lipid is the cationic lipid listed in Table 1) were added to cell culture broth of 96-well plates, after further culturing for 24 h, 10. Mu.L of CCK-8 solution was added to each well, and after incubating the plates in an incubator for 1 h, absorbance at 450nm was measured by a microplate reader, and cell viability results are shown in Table 4.

TABLE 4 cell survival

Analysis of experimental results:

(1) The chemical structures of the series of compounds contemplated herein, including YK-506 and YK-504, differ greatly from the prior art cationic lipids, such as MC3; there are structural similarities, for example, C12-200, MIC3 and PPZ-A12. LNP formulations prepared from YK-506 and YK-504 have minimal cytotoxicity and significantly improved cell viability compared to the cationic lipids typical of the prior art. For example, YK-506 cell viability may be 25% higher than MC3, 18% higher than C12-200, 33% higher than MIC3, 41% higher than PPZ-A12. The cytotoxicity of LNP formulations prepared therefrom cannot be speculated on the basis of the structure of cationic lipid compounds, and there is a strong possibility that the cytotoxicity to transfected cells is very different, whether they are structurally different or structurally similar compounds.

(2) Similar to the structure, L ₁ And L ₂ Compared with the compounds with the groups of-C (O) O-, the cytotoxicity of YK-506 and YK-504 is the lowest, and the cell survival rate is obviously improved. For example, YK-506 may have a cell viability that is 35% higher than YK-501, 39% higher than YK-502, and 52% higher than YK-505. The cell viability of YK-504 was 19% higher than that of YK-501, 23% higher than that of YK-502, and 36% higher than that of YK-505, respectively.

(3) Similar to the structure, L ₁ The radicals being-C (O) O-, L ₂ The radical being-C (O) N ((CH) ₂ ) ₉ CH ₃ ) Compared with the compounds, the cytotoxicity of YK-506 and YK-504 is the lowest, and the cell survival rate is obviously improved. For example, YK-506 and YK-504 have 44% and 28% higher cell viability than YK-503, respectively.

(4) Similar to the structure, L ₁ And L ₂ The radicals are-C (O) NH-, R ₁ And R is ₂ YK-compared with the compounds each containing a-S-group506 and YK504 have the lowest cytotoxicity, and the cell survival rate is obviously improved. For example, YK-506 and YK-504 have 40% and 24% higher cell viability than YK-507, respectively.

Example 7: in vivo validation of cationic Lipid (LNP) delivery vehicle performance

Example 7 demonstrates that delivery vectors prepared from cationic lipids designed herein, such as YK-506 and YK-504, can be enriched in the mouse spleen and that the amount of mRNA delivered is significantly increased in the mouse spleen protein expression compared to prior art cationic lipids. In vivo experiments further demonstrate that our LNP delivery vector is able to efficiently deliver mRNA into animals and is efficiently expressed in the spleen.

LNP preparations containing 10. Mu.g of Fluc-mRNA were injected into female BALB/C mice of 17-19 g weight, 4-6 weeks old, via tail vein, and the mice were given intraperitoneal injection of fluorography substrate at a specific time point (3 h) after administration, and the mice were free to move for 5 min, and then the average radiation intensity (corresponding to fluorescence expression intensity) of the protein expressed in the mice by the mRNA carried by LNP was detected by IVIS Spectrum small animal in vivo imager. After sampling, the mice were euthanized with carbon dioxide, dissected, and the internal organs of the mice were precisely isolated: heart, liver, spleen, lung, kidney. The average radiation intensity (corresponding to fluorescence expression intensity) of the mRNA carried by LNP in the protein expressed by each organ of the mice was measured by IVIS Spectrum small animal in vivo imaging instrument, and the results of the in vivo imaging measurement of the mice are shown in Table 5.

TABLE 5 in vivo imaging experimental data at a specific time point (3 h) after mouse administration

(1) Compared with the representative cationic lipid in the prior art, the LNP preparation prepared by YK-506 and YK-504 has obviously improved expression level of mRNA in the spleen of the mouse. For example, MC3 is not expressed in the spleen, whereas YK-506 and YK-504 are both expressed in large amounts in the spleen, YK-506 can be expressed in the spleen in an amount up to 5.94 times C12-200, YK-504 being 1.16 times C12-200. mRNA was consistent with the results of cell transfection in example 6 in terms of mouse spleen expression.

Furthermore, LNP preparations containing Fluc-mRNA prepared from all compounds were very different in expression in different organs of mice, YK-506, YK-504, YK-501 and C12-200 were expressed in liver and spleen, but not in other organs such as heart, lung and kidney; MC3 was expressed only in the liver, but not in spleen, heart, lung and kidney (FIG. 4).

(2) Compared with the compound YK-501 which has similar structure and slightly different individual groups, the LNP preparation prepared from YK-506 and YK-504 has the highest expression intensity of mRNA in the spleen of the mice. For example, YK-506 can be expressed in spleen in an amount of 207.98 times that of YK-501, and YK-504 can be expressed in spleen in an amount of 40.72 times that of YK-501. mRNA was consistent with cell transfection activity in terms of expression in mice.

In summary, the present application contemplates a range of cationic lipid compounds, e.g., YK-506 and YK-504, that significantly increase cell transfection efficiency, significantly decrease cytotoxicity, and significantly increase mRNA expression in mouse liver and spleen.

1. A series of compounds were designed, including YK-506 and YK-504, with chemical structures that differ greatly from the prior art cationic lipids, such as MC3; there are structural similarities such as C12-200, MIC3 and PPZ-A12.

3. In a series of compounds with small chemical structure difference, LNP preparation prepared from YK-506 and YK-504 has obviously improved cell transfection efficiency, obviously reduced cytotoxicity and obviously improved expression quantity of mRNA in mouse spleen compared with other compounds. For example, YK-506 cells can be transfected with 100 times YK-501 and 130 times YK-503, cytotoxicity can be reduced by 52% compared with YK-505, and mRNA expression amount in mouse spleen can be 200 times YK-501.

4. Through unique design and screening, the present disclosure finds that some compounds, such as YK-506 and YK-504, can be delivered with significantly improved cell transfection efficiency, significantly reduced cytotoxicity and significantly improved expression in the spleen of animals, with improved delivery efficiency, and unexpected technical effects relative to other compounds of similar prior art structures. By increasing the expression level of spleen (the largest secondary lymphoid organ in vivo) in animals, the mRNA vaccine can induce immune response in vivo and generate antibodies. The vaccine composition has the significant clinical significance and can obviously improve the prevention effect under the condition of not changing the vaccine components. Has good targeting effect on developing and treating diseases caused by spleen damage or abnormality such as lymphoma, leukemia and the like.

The present invention has been described in detail with the purpose of enabling those skilled in the art to understand the contents of the present invention and to implement the same, but not to limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit of the present invention should be covered in the scope of the present invention.

Claims

1. A compound of the formula or a pharmaceutically acceptable salt thereof,

。

2. a compound of the formula or a pharmaceutically acceptable salt thereof,

。

3. a composition comprising a carrier comprising a cationic lipid comprising a compound of any one of claims 1-2 or a pharmaceutically acceptable salt thereof.

4. A composition according to claim 3, wherein the cationic lipid comprises 25% -75% of the carrier by mole.

5. The composition of claim 3, wherein the carrier further comprises a neutral lipid.

6. The composition of claim 5, wherein the molar ratio of the cationic lipid to the neutral lipid is 1:1-15:1.

7. The composition of claim 6, wherein the molar ratio of the cationic lipid to the neutral lipid is 4.9:1.

8. The composition of claim 5, wherein the neutral lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramides, sterols, and derivatives thereof.

9. The composition of claim 8, wherein the neutral lipid is selected from one or more of the following: 1, 2-Dioleoyl-sn-glycero-3-phosphorylcholine (DLPC), 1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dioleoyl-sn-glycero-phosphorylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-octadecenyl-sn-glycero-3-phosphorylcholine (18:0 DietherPC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-3-phosphorylcholine (OChems PC), 1-hexadecyl-sn-3-phosphorylcholine (C16), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1-dioleoyl-2-dioleoyl-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-sn-glycero-3-phosphate ethanolamine (DOPE), 1, 2-di-phytanoyl-sn-glycero-3-phosphate ethanolamine (ME 16.0 PE), 1, 2-di-stearoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-oleoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-linolenoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-arachidonoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-docosahexaenoic acyl-sn-glycero-3-phosphate ethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphate sodium salt (DOPG), 1, 2-di-oleoyl-rac-3-phosphate sodium salt (DOPG) dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl-based phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof.

10. The composition of claim 9, wherein the neutral lipid is 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and/or 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

11. The composition of claim 3, wherein the carrier further comprises a structural lipid.

12. The composition of claim 11, wherein the molar ratio of the cationic lipid to the structural lipid is 0.6:1-3:1.

13. The composition of claim 11, wherein the structural lipid is selected from one or more of the following: cholesterol, non-sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycorine, ursolic acid, alpha-tocopherol, corticosteroids.

14. The composition of claim 13, wherein the structural lipid is cholesterol.

15. The composition of claim 3, wherein the carrier further comprises a polymer conjugated lipid.

16. The composition of claim 15, wherein the polymer conjugated lipid comprises 0.5% -10% of the carrier by mole.

17. The composition of claim 16, wherein the polymer conjugated lipid comprises 1.5% of the carrier by mole.

18. The composition of claim 15, wherein the polymer conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol.

19. The composition of claim 18, wherein the polymer conjugated lipid is selected from one or more of the following: distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG 2000), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000) and methoxypolyethylene glycol ditetradecylamide (ALC-0159).

20. The composition of claim 3, wherein the carrier comprises a cationic lipid, a neutral lipid, a structural lipid, and a polymer conjugated lipid.

21. The composition of claim 20, wherein the cationic lipid: neutral lipids: structural lipids: the molar use ratio of the polymer conjugated lipid is (25-75): (5-25): (15-65): (0.5-10).

22. The composition of claim 21, wherein the cationic lipid: neutral lipids: structural lipids: the molar dosage ratio of the polymer conjugated lipid is 49:10:39.5:1.5.

23. The composition of claim 3, wherein the composition is a nanoparticle formulation having an average particle size of 10nm to 210nm; the polydispersion coefficient (PDI) of the nanoparticle preparation is less than or equal to 50 percent.

24. The composition of claim 23, wherein the nanoparticle formulation has an average particle size of 100nm to 205nm; the polydispersion coefficient (PDI) of the nanoparticle preparation is less than or equal to 30 percent.

25. A composition according to claim 3, wherein the cationic lipid further comprises one or more other ionizable lipid compounds.

26. The composition of claim 3, further comprising a therapeutic or prophylactic agent.

27. The composition of claim 26, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 10:1-30:1.

28. The composition of claim 27, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 12.5:1-25:1.

29. The composition of claim 28, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 15:1.

30. The composition of claim 26, wherein the therapeutic or prophylactic agent comprises one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.

31. The composition of claim 26, wherein the therapeutic or prophylactic agent is a vaccine or compound capable of eliciting an immune response.

32. The composition of claim 26, wherein the therapeutic or prophylactic agent is a nucleic acid.

33. The composition of claim 32, wherein the therapeutic or prophylactic agent is ribonucleic acid (RNA).

34. The composition of claim 32, wherein the therapeutic or prophylactic agent is deoxyribonucleic acid (DNA).

35. The composition of claim 33, wherein the RNA is selected from the group consisting of: small interfering RNAs (siRNA), asymmetric interfering RNAs (aiRNA), micrornas (miRNA), dicer-substrate RNAs (dsRNA), small hairpin RNAs (shRNA), messenger RNAs (mRNA), and mixtures thereof.

36. The composition of claim 33, wherein the RNA is mRNA.

37. The composition of any one of claims 3-36, wherein the composition further comprises one or more pharmaceutically acceptable excipients.

38. Use of a compound according to any one of claims 1-2 or a pharmaceutically acceptable salt thereof or a composition according to any one of claims 3-37 in the manufacture of a nucleic acid medicament, a genetic vaccine, a small molecule medicament, a polypeptide or a protein medicament.

39. Use of a compound according to any one of claims 1-2, or a pharmaceutically acceptable salt thereof, or a composition according to any one of claims 3-37, in the manufacture of a medicament for treating a disease or condition in a mammal in need thereof, said disease or condition selected from the group consisting of: infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases and metabolic diseases.

40. The use according to claim 39, wherein the infectious disease is selected from: diseases caused by coronavirus, influenza virus or HIV virus, pediatric pneumonia, rift valley fever, yellow fever, rabies, or herpes.

41. The use of any one of claims 39-40, wherein the subject to which the medicament is administered is a human.

42. The use of any one of claims 39-40, wherein the route of administration of the medicament is intravenous, intramuscular, intradermal, subcutaneous, intranasal, or inhalation.

43. The use according to claim 42, wherein the route of administration of the medicament is subcutaneous.

44. The use of any one of claims 39-40, wherein the medicament is administered at a dose of 0.001-10 mg/kg.