HK40081634A - Highly efficient and low-toxic cationic lipid compounds for extrahepatic targeting and compositions thereof - Google Patents
Highly efficient and low-toxic cationic lipid compounds for extrahepatic targeting and compositions thereof Download PDFInfo
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Description
Technical Field
The invention belongs to the field of medicine. The invention particularly relates to a cationic lipid compound, a composition containing the same and application thereof.
Background
Efficient targeted delivery of biologically active substances such as small molecule drugs, polypeptides, proteins and nucleic acids, particularly nucleic acids, is a long-standing medical problem. Nucleic acid therapeutics face significant challenges due to low cell permeability and high sensitivity to degradation of certain nucleic acid molecules, including RNA.
Cationic lipid-containing compositions, liposomes and liposome complexes (lipoplex) have been demonstrated to be effective delivery of biologically active substances, such as small molecule drugs, polypeptides, proteins and nucleic acids, into cells and/or intracellular compartments as transport vehicles. These compositions generally comprise one or more "cationic" and/or amino (ionizable) lipids, and may also comprise neutral lipids, structural lipids, and polymer conjugated lipids. Cationic and/or ionizable lipids include, for example, amine-containing lipids that can be easily protonated. Although a variety of such lipid-containing nanoparticle compositions have been demonstrated, safety, efficacy and specificity remain to be improved. Notably, the increased complexity of Lipid Nanoparticles (LNPs) complicates their production and may increase their toxicity, a major concern that may limit their clinical utility. For example, LNP siRNA particles (e.g., patisiran) 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.). Accordingly, 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
In one aspect of the present invention there is provided a novel 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 2~8 An alkylene group;
G 2 is C 2~8 An alkylene group;
L 1 is-C (O) O-or-OC (O) -;
L 2 is-C (O) O-or-OC (O) -;
R 1 is C 6~25 A linear or branched alkyl group;
R 2 is C 6~25 A linear or branched alkyl group;
G 3 is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -；
G 4 Is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -；
L is- (CH) 2 ) 2 -or- (CH) 2 ) 3 -or- (CH) 2 ) 4 -。
For example, the compound of formula (I) has one of the following structures:
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yet another aspect of the 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.
A further aspect of the invention provides the above cationic lipid or composition for use in delivering a therapeutic or prophylactic agent to a patient in need thereof.
Yet another aspect of 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 described above, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a composition as described above, in the manufacture of a nucleic acid medicament, a genetic vaccine, a small molecule medicament, a polypeptide or a protein medicament.
A further aspect of the invention provides the use of a compound of formula (I) as described above or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a composition as described above, in the manufacture of a medicament for the treatment of a disease or condition in a mammal in need thereof.
Drawings
Figure 1 shows the results of cell transfection experiments with different weight ratios of vector to mRNA used in the preparation of LNP formulations, a is vector: mRNA = 5, b is vector: mRNA = 15, c is vector: mRNA = 35; d is blank control.
FIG. 2 shows the results of cell transfection experiments with different molar ratios of cationic lipid to neutral lipid DSPC used in the preparation of LNP formulations, a being 3.5, b being 4:1, c being 4.9:1, d being blank.
FIG. 3 shows the results of cell transfection experiments with different molar ratios of polymer conjugated lipid to vehicle in the preparation of LNP formulations, with a at 1.5%, b at 10%, and c as a blank control.
Fig. 4 shows the results of cell transfection experiments with different ratios of the components cationic lipid, neutral lipid DSPC, structural lipid cholesterol and polymer conjugated lipid DMG-PEG2000 in the vehicle when preparing LNP formulations, a is 35.
FIG. 5 shows the LNP formulation fluorescence absorbance intensity of Fluc-mRNA prepared from different cationic lipids. (a: YK-407, b-YK-401 c.
FIG. 6 shows the LNP formulation fluorescence absorbance intensity of Fluc-mRNA prepared from different cationic lipids. (a: YK-404 b-YK-406 c.
FIG. 7 shows the cell viability after 24 hours of culture when LNP preparations of Fluc-mRNA prepared from different cationic lipids (YK-407, YK-401, YK-402, YK-403, YK-422, YK-423, YK-009, SM-102, ALC-0315, compound 21, compound 23 and HHMA) and Lipofectamine 3000 preparations containing Fluc-mRNA were added to the cell culture broth.
FIG. 8 shows the cell viability after 24 hours of incubation when LNP formulations of Fluc-mRNA and Lipofectamine 3000 formulations containing Fluc-mRNA prepared from different cationic lipids (YK-407, YK-401, YK-402, YK-403, YK-422, YK-423, YK-404, YK-405, YK-406, YK-408, YK-409, YK-410, YK-411, SM-102, ALC-0315, compound 21, compound 23 and HHMA) were added to the cell culture broth.
FIG. 9 shows the cell survival rate after 24 hours of addition of LNP preparations of Fluc-mRNA prepared from different cationic lipids (YK-407, YK-401, YK-402, YK-403, YK-422, YK-423, YK-412, YK-413, YK-414, YK-415, YK-416, YK-417, YK-418, YK-419, YK-420, YK-421, YK-424, SM-102, ALC-0315, compound 21, compound 23 and HHMA) and Lipofectamine 3000 preparations containing Fluc-mRNA to the cell culture broth.
FIG. 10 shows the results of in vivo imaging experiments on mice with LNP formulations of Fluc-mRNA prepared from different cationic lipids (YK-407, YK-401, YK-415, SM-102, ALC-0315, compound 21, compound 23 and HHMA).
FIG. 11 shows the results of in vivo imaging experiments on mice with LNP formulations of Fluc-mRNA prepared from different cationic lipids (YK-402, YK-411, YK-009, SM-102, ALC-0315, compound 21, compound 23 and HHMA).
FIG. 12 shows the results of in vivo imaging experiments on mice with LNP formulations of Fluc-mRNA prepared from different cationic lipids (YK-403, YK-422, SM-102, ALC-0315, compound 21, compound 23 and HHMA).
FIG. 13 shows the results of in vivo imaging experiments on mice with LNP formulations of Fluc-mRNA prepared from different cationic lipids (YK-423, YK-417, SM-102, ALC-0315, compound 21, compound 23 and HHMA).
FIG. 14 shows LNP formulations of Fluc-mRNA prepared from different cationic lipids (SM-102, YK-402, YK-407, YK-411, YK-418, YK-419, and YK-424) with protein expression in mice.
FIG. 15 shows LNP preparations of Fluc-mRNA prepared from different cationic lipids (SM-102, YK-402, YK-407, YK-411, YK-418, YK-419, and YK-424) expressed in mouse liver, spleen, lung, heart, and kidney.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings of the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without any inventive step, are within the scope of protection of the invention.
The present invention may be embodied in other specific forms without departing from its essential attributes. It is to be understood that, without conflict, any and all embodiments of the present invention may be combined with features from any other embodiment or embodiments to arrive at further embodiments. The invention includes additional embodiments resulting from such combinations.
All publications and patents mentioned in this specification are herein incorporated by reference in their entirety. To the extent that a use or term used in any publication or patent incorporated by reference conflicts with a use or term used in the present application, the use or term of the present application shall govern.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood in the art to which the claimed subject matter belongs. In case there are multiple definitions for a term, the definitions herein control.
Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of properties such as dosages set forth 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-ranges.
The use of "including," "comprising," or "containing" and similar words in the context of this invention is intended to indicate that the element(s) listed before that word(s) covers the element(s) listed after that word(s) and their equivalents, without excluding unrecited elements. The term "comprising" or "includes" as used herein can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of or" consisting of 823030A ".
The term "pharmaceutically acceptable" refers herein to: the compound or composition is compatible chemically and/or toxicologically 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" in this application includes humans and mammals.
The term "treatment" as used herein refers to the administration of one or more pharmaceutical substances to a patient or subject suffering from a disease or having symptoms of the disease, for the purpose of curing, alleviating, reducing, ameliorating, or otherwise affecting the disease or symptoms of the disease. In the context of this application, the term "treatment" may also include prophylaxis, unless specifically stated to the contrary.
The term "solvate" refers herein to a complex formed by combining a compound of formula (I) or a pharmaceutically acceptable salt thereof with a solvent, such as ethanol or water. It is to be understood that any solvate of a compound of formula (I) for use in the treatment of a disease or disorder, while potentially offering different properties, including pharmacokinetic properties, will result in a compound of formula (I) once absorbed into a subject, such that use of a compound of formula (I) encompasses use of any solvate of the 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 salts" refers to relatively non-toxic, inorganic or organic acid addition salts of the compounds of the present invention. 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, nitric acid, or the like; organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) -benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, diglucosic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic 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, a pharmaceutically acceptable salt can be formed with a compound of formula (I) using HCl (or hydrochloric acid), HBr (or hydrobromic acid solution), methanesulfonic acid, sulfuric acid, tartaric acid, or fumaric acid.
The nitrogen-containing compounds of formula (I) of the present invention can be converted to the N-oxides by treatment with an oxidizing agent (e.g., m-chloroperoxybenzoic acid, hydrogen peroxide, ozone). Accordingly, the compounds claimed herein include not only nitrogen-containing compounds represented by the structural formula but also N-oxide derivatives thereof, as the valency and structure permit.
Certain compounds of the present invention may exist as one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers and enantiomers. Thus, the presently claimed compounds also include racemic mixtures, individual stereoisomers, and optically active mixtures. It will be appreciated by those skilled in the art that one stereoisomer may have better efficacy and/or fewer side effects than the other. The single stereoisomer and the mixture with optical activity can be obtained by chiral source synthesis, chiral catalysis, chiral resolution and other methods. The racemate can be subjected to chiral resolution by a chromatographic resolution method or a chemical resolution method. For example, the compounds of the present invention can be separated by adding a chiral acid resolving agent such as chiral tartaric acid, chiral malic acid, etc. to form salts, and utilizing the physicochemical properties, e.g., solubility, of the products.
The invention also includes all suitable isotopic variations of the compounds of the invention. 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 or predominantly present in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen and oxygen, such as 2 H (deuterium), 3 H (tritium), 11 C、 13 C、 14 C、 15 N、 17 O and 18 O。
the term "alkyl" is meant herein to include both branched and straight chain saturated aliphatic monovalent hydrocarbon radicals having the specified number of carbon atoms. The term "alkylene" is meant herein to include both branched and straight chain saturated aliphatic divalent hydrocarbon radicals having the specified number of carbon atoms. C n~m Is meant to include groups having n to m carbon atoms. Such as C 2~5 Alkylene radicals including C 2 Alkylene radical, C 3 Alkylene radical, C 4 Alkylene radical, C 5 An alkylene group.
The alkyl (or alkylene) group may be unsubstituted or substituted wherein at least one hydrogen is replaced by another chemical group.
A "therapeutically effective amount" is an amount of a therapeutic agent that ameliorates a disease or condition when administered to a patient. A "prophylactically effective amount" is an amount of a prophylactic agent that prevents a disease or condition when administered to a subject. The amount of the therapeutic agent constituting the "therapeutically effective amount" or the amount of the prophylactic agent constituting the "prophylactically effective amount" varies depending on the therapeutic agent/prophylactic agent, the disease state and its severity, the age, body weight, etc. of the patient/subject to be treated/prevented. The therapeutically effective amount and the prophylactically effective amount can be routinely determined by one of ordinary skill in the art based on his knowledge and the present invention.
In the present application, when the name of the compound is inconsistent with the structural formula, the structural formula controls.
It is to be understood that the term "compounds of the invention" as used herein may include, depending on the context: a compound of formula (I), N-oxides, solvates, pharmaceutically acceptable salts, stereoisomers, and mixtures thereof.
The term cationic lipid as used herein refers to a lipid that is positively charged at a selected pH value.
Cationic liposomes readily bind to negatively charged nucleic acids, i.e., interact with negatively charged phosphate groups present in the nucleic acids via electrostatic forces, to form Lipid Nanoparticles (LNPs). LNP is one of the currently predominant delivery vehicles.
The inventors have found that it is very difficult to screen a suitable cationic lipid compound satisfying the following conditions when screening a large number of compounds: compared with the prior art, the cationic lipid has great structural difference, simultaneously has extremely high transfection efficiency and extremely low cytotoxicity, and has high expression and continuous expression in mice. The inventors have discovered that compounds such as YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423, among others, are capable of delivering nucleic acids with significantly improved intracellular transfection efficiency, significantly reduced cytotoxicity, and significantly improved expression levels and durations in animals, as compared to prior art cationic lipids with widely differing chemical structures.
Briefly, the present invention is based on at least the following findings:
1. a series of compounds designed, including YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, differ greatly from the chemical structures of representative cationic lipids of the prior art, such as SM-102 (compound 25 disclosed in WO2017049245 A2), ALC-0315 (compound 3 disclosed in CN 108368028B), compound 21 and compound 23 disclosed in WO2021055833A1, HHMA (compound 1 disclosed in CN 112979483B) and compound YK-009 disclosed in CN 114470441B: the head structures are obviously different, and the designed head structures of the series of compounds comprise 2 tertiary amine groups, an L group connected with 2 tertiary amine nitrogen atoms and G respectively connected with 2 tertiary amine nitrogen atoms 3 And G 4 While the head structures of SM-102, ALC-0315, compound 21, compound 23, and YK-009, include only 1 tertiary amine group, and HO (CH) attached to the tertiary amine nitrogen atom 2 ) 2 -; since the difference is large in all other sites, the difference is also large in polarity, acidity or basicity, hydrophilicity, and the like.
Therefore, it is impossible to predict the cell transfection efficiency, cytotoxicity and expression profile in animals of LNP preparations prepared from this series of compounds based on the cationic lipid compounds disclosed in the above prior art.
SM-102, ALC-0315, compound 21, compound 23 and HHMA the chemical structures are as follows:
(WO 2017049245A2, page 29 of the description);
(CN 108368028B, page 24 of the description);
(WO 2021055833A1, page 22 of the description);
(WO 2021055833A1, page 22 of the description);
(CN 112979483B, page 12 of the description).
2. In the designed series of compounds, LNP preparations prepared from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 have the advantages that compared with the typical cationic lipid in the prior art, the encapsulation efficiency, the drug loading concentration and the total RNA concentration are obviously improved, the cell transfection efficiency is obviously improved, the cytotoxicity is obviously reduced, the expression amount and the duration of mRNA in a mouse body are obviously improved, the toxicity to liver is reduced or is not toxic, and the mRNA can be directly delivered to spleen.
For example, the encapsulation efficiency of YK-407 can be improved by 29.0% compared with that of compound 23, the drug-loading concentration can reach 1.78 times of that of compound 23, and the total RNA concentration can reach 1.41 times of that of compound 21; YK-407 cell transfection efficiency can reach 12 times of SM-102, 13 times of compound 21 and 15 times of compound 23; the survival rate of YK-401 cells is 28.00 percent higher than that of ALC-0315, 7.31 percent higher than that of SM-102 and 10.94 percent higher than that of HHMA; in the LNP preparation prepared from YK-407, the expression level of mRNA in mice can reach 27 times of SM-102, 22 times of ALC-0315, 28 times of compound 21, 27 times of compound 23 and 27 times of HHMA; LNP formulations prepared from the compounds contemplated herein, which express reduced amounts of the protein of interest in the liver (YK-402), or do not reside in the liver and express the protein of interest (YK-407, YK-411, YK-418, YK-419, and YK-424), have reduced or no toxicity to the liver; YK-407, YK-419 and YK-424 were able to deliver mRNA directly to the spleen, but were not expressed in other organs such as liver, lung, heart and kidney, and were able to significantly improve the prophylactic effect without changing the vaccine components.
The series of compounds designed by the application and having small difference in chemical structures comprises YK-407, YK-401, YK-402,Compared with other compounds, the LNP preparation prepared from YK-403, YK-422 and YK-423 has the advantages that the cell transfection efficiency is obviously improved, the cytotoxicity is obviously reduced, and the expression amount and the duration of mRNA in a mouse body are obviously improved. Compounds of this series being only individual radicals, e.g. G 3 、G 4 Or the difference of L groups is 1-2C, other structures are slightly different, but the transfection efficiency of YK-407 cells can reach 2500 times of YK-404 and YK-411, the cytotoxicity of YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 can be reduced by 50 percent compared with that of YK-411, and the expression amount of mRNA in mice of LNP preparations prepared from YK-407 can reach 1000 times of that of YK-411.
3. There is no obvious correspondence between the structure of the cationic lipid compound and the transfection efficiency in cells, the toxicity to cells and the high and sustained expression of mRNA in LNP preparations prepared therefrom in animals. Even compounds with small structural differences are likely to differ greatly in transfection efficiency and/or toxicity to cells, and intracellular expression.
For example, YK-411 has a very close structure compared to YK-407. YK-411 is G only 1 The radical ratio YK-407 is 2 more C; r 1 1 less C; r is 2 The gene single chain has 2 more C, each single chain in the double chain has 2 less C, but the cell transfection efficiency YK-407 is 2500 times higher than that of YK-411, the toxicity to transfected cells YK-407 is 55% lower than that of YK-411, and the expression of mRNA YK-407 in mouse body can reach 1000 times higher than that of YK-411.
Therefore, it is very difficult to select suitable cationic lipid compounds, which have high transfection efficiency and low toxicity to cells, and high and sustained expression of mRNA in mice, and it requires much creative work.
4. The present invention, through unique design and extensive screening, has discovered that certain compounds, such as YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, are capable of delivering nucleic acids with significantly improved encapsulation efficiency, drug loading concentration and total RNA concentration, significantly improved transfection efficiency of cells, significantly reduced cytotoxicity and significantly improved expression levels and duration in animals, and are less or non-toxic to liver toxicity, and are capable of delivering mRNA directly to the spleen, but not expressed in other organs, such as the liver, lung, heart and kidney, achieving unexpected technical effects relative to other compounds of the prior art.
In conclusion, the present invention has found some compounds, such as YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, through unique design and extensive screening. These compounds are chemically very different from the prior art representative cationic lipids, are capable of delivering nucleic acids with significantly improved cell transfection efficiency, significantly reduced cytotoxicity and significantly improved expression levels and duration in animals, and have reduced or no toxicity to liver, compared to other compounds of the prior art, with unexpected technical effects.
The method comprises the following specific steps:
1. compared to the prior art representative cationic lipids, e.g., SM-102, ALC-0315, compound 21, compound 23, YK-009, and HHMA, the chemical structure is very different.
This series of compounds was designed, in contrast to the prior art representative cationic lipids:
1) The structural difference with HHMA is the greatest, and as can be seen from the chemical structure diagram, the group of HHMA connected with the central N atom has only 1 side chain close to 1 side chain of the compounds of the series, and other parts are obviously different.
2) The head structure is significantly different compared to SM-102, ALC-0315, compound 21, compound 23, and YK-009. The designed head structure of the series of compounds comprises 2 tertiary amine groups, an L group connected with 2 tertiary amine nitrogen atoms and G3 and G4 groups respectively connected with 2 tertiary amine nitrogen atoms, while the head structure of SM-102, ALC-0315, compound 21, compound 23 and YK-009 only comprises 1 tertiary amine group and HO (CH) connected with the tertiary amine nitrogen atoms 2 ) 2 -. Furthermore, this series of compounds G 1 、L 1 、R 1 、G 2 、R 2 The group, also differs significantly from SM-102, ALC-0315, compound 21, compound 23, and YK-009.
3) Due to the great structural difference, the series of compounds also have great difference from SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-009 in polarity, acidity-basicity, hydrophilicity and the like.
2. Compared with the prior art, the compound has the advantages that the encapsulation efficiency, the drug-loading concentration and the total RNA concentration are obviously improved.
Some of the compounds contemplated by the present application, including YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423, produced LNP formulations with significantly improved encapsulation efficiency, drug loading concentration, and total RNA concentration over prior art cationic lipids, such as SM-102, ALC-0315, compound 21, compound 23, HHMA, and YK-009. For example, the encapsulation efficiency of YK-407 can be improved by 29.0 percent compared with that of the compound 23, the drug-loading concentration can reach 1.78 times of that of the compound 23, and the total RNA concentration can reach 1.41 times of that of the compound 21.
3. In vitro cell transfection efficiency is significantly improved over prior art representative cationic lipids and multiple structurally similar compounds designed herein
1) The LNP preparation prepared from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 has the highest cell transfection efficiency, and is obviously improved in activity compared with the representative cationic lipid in the prior art. For example, YK-407 can be up to 12 times that of SM-102, 13 times that of Compound 21, and 15 times that of Compound 23.
2) And G 3 And G 4 The radical is HO (CH) 2 ) 2 -, L radical is- (CH) 2 ) 2 YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, the transfection efficiency of cells was the highest compared to the structurally similar compounds. For example, YK-407 can be 2500 times as much as YK-404 and YK-411, and YK-401, YK-402, YK-403, YK-422 and YK-423 can be more than 600 times as much as YK-404 and YK-411.
3) And G 3 And G 4 Is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -, L is- (CH) 2 ) 2 -、-(CH 2 ) 3 -or- (CH) 2 ) 4 YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 were most efficiently transfected compared to the series of structurally similar compounds. For example, YK-407 may be 170 times as much as YK-417 and 180 times as much as YK-418.
4) There is no correlation between the structure of the compound and the intracellular transfection efficiency, and there is a high possibility that the difference in transfection efficiency is very large regardless of the compound having a small or large difference in structure. Therefore, it is very difficult to select cationic lipid compounds having high transfection efficiency, and much creative work is required.
4. Cytotoxicity is significantly reduced compared to representative cationic lipids of the prior art and a number of structurally similar compounds designed herein
1) LNP formulations prepared from YK-407, YK-401, YK402, YK-403, YK-422, and YK-423 are minimally cytotoxic and significantly improved in survival rate over representative cationic lipid cells of the prior art. For example, YK-401 cell survival rate can be 28.00% higher than ALC-0315, 7.31% higher than SM-102, and 10.94% higher than HHMA.
2) And G 3 And G 4 Is HO (CH) 2 ) 2 -, L is- (CH) 2 ) 2 Compared with a series of compounds with similar structures, YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 have the lowest cytotoxicity and the highest cell survival rate. For example, the cell survival rate of YK-401 is 58.88 percent higher than that of YK-411 and 50.25 percent higher than that of YK-406; the survival rate of YK-407 cells is 55.04 percent higher than that of YK-411 and 46.41 percent higher than that of YK-406.
3) And G 3 And G 4 Is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -, L is- (CH) 2 ) 2 -、-(CH 2 ) 3 -or- (CH) 2 ) 4 A series of structurally similar compounds of (A) with the lowest cytotoxicity compared to YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423. For example, YK-401 is 53.87% and 54.16% higher than YK-417 and YK-418, respectively, and YK-407 is 50.03% and 50.32% higher than YK-417 and YK-418, respectively.
4) There is no correspondence between the structure and cytotoxicity of the compounds, and there is a high possibility that the compounds with small or large structural differences have very large differences in cytotoxicity. Therefore, the cytotoxicity of the lipid compound cannot be predicted from the chemical structure, and it is very difficult to screen out a cationic lipid compound having low cytotoxicity, and much creative work is required.
mRNA expression levels and durations in animals were significantly increased compared to prior art representative cationic lipids and a number of structurally similar compounds designed herein, with reduced or no toxicity to liver, and only mRNA was delivered directly to the spleen.
1) The LNP preparation prepared from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 has high mRNA expression in mice and continuous expression, and is remarkably improved compared with the representative cationic lipid in the prior art. For example, YK-407 can be 27 times as high as SM-102, 22 times as high as ALC-0315, 28 times as high as Compound 21, 27 times as high as Compound 23, and 27 times as high as HHMA.
2) And G 3 And G 4 Is HO (CH) 2 ) 2 -, L is- (CH) 2 ) 2 -compared to LNP preparations prepared from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, the mRNA expression intensity was highest and the duration was longest in mice. For example, YK-407 can be more than 600 times of YK-411 in 6 hours, 1000 times in 24 hours and still 100 times in 7 days.
3) Similar to the structure, G 3 And G 4 The radical is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -, L radical is- (CH) 2 ) 2 -、-(CH 2 ) 3 -or- (CH) 2 ) 4 LNP preparations prepared from YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 showed the highest expression intensity and the longest duration of mRNA in mice compared to compounds with slightly different groups. For example, YK-407 can be 120 times as high as YK-417 at 48h, and still 20 times as high at 7 d.
4) Compared with the prior art representative cationic lipids, such as SM-102, ALC-0315, compound 21, compound 23 and HHMA, liposomes prepared from the compounds contemplated herein, such as YK-402, YK-407, YK-411, YK-418, YK-419 and YK-424, express a reduced amount of the protein of interest in the liver, or do not reside and express the protein of interest in the liver. The LNP formulations prepared with the compounds contemplated herein are therefore less or non-toxic to liver toxicity compared to prior art cationic lipids. Moreover, some compounds designed in the application, such as YK-407, YK-419 and YK-424, can directly deliver mRNA to the spleen, and only express in the spleen, and do not express in other organs, such as liver, lung, heart and kidney, so that the prevention effect can be remarkably improved without changing vaccine components, and the clinical significance is achieved.
5) There is no correspondence between the structure of the cationic lipid and the high and sustained expression of the delivered mRNA in mice, and regardless of the cationic lipid compounds with little or large structural differences, there is a high probability that the mRNA in LNP formulations prepared therefrom will vary greatly in expression in animals. Whether mRNA is highly expressed and continuously expressed in an animal body cannot be predicted according to the chemical structure of the cationic lipid, and screening out the cationic lipid compound with high mRNA expression and continuous expression is very difficult, and a great deal of creative work is required.
In one aspect, the present invention provides a novel cationic lipid compound for use in the delivery of a therapeutic or prophylactic agent. The cationic lipid compounds of the invention are useful for the delivery of nucleic acid molecules, small molecule compounds, polypeptides or proteins. The cationic lipid compounds of the present invention exhibit higher transfection efficiency and less cytotoxicity, improving delivery efficiency and safety, relative to known cationic lipid compounds.
The invention provides a cationic lipid which is a compound of formula (I)
Or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein
G 1 Is C 2~8 Alkylene, preferably unsubstituted C 3~8 An alkylene group;
G 2 is C 2~8 Alkylene, preferably unsubstituted C 3~8 An alkylene group;
L 1 is-C (O) O-or-OC (O) -;
L 2 is-C (O) O-or-OC (O) -;
R 1 is C 6~25 Straight or branched alkyl, preferably unsubstituted C 11 Straight chain alkyl or unsubstituted C 10 Straight chain alkyl or unsubstituted C 8~18 A branched alkyl group;
R 2 is C 6~25 Straight or branched alkyl, preferably unsubstituted C 11 Straight chain alkyl or unsubstituted C 10 Straight chain alkyl or unsubstituted C 8~18 A branched alkyl group;
G 3 is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -；
G 4 Is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -；
L is- (CH) 2 ) 2 -or- (CH) 2 ) 3 -or- (CH) 2 ) 4 -, is preferably- (CH) 2 ) 2 -or- (CH) 2 ) 3 -。
In one embodiment, G 1 Is unsubstituted C 3 Alkylene radicals, e.g., - (CH) 2 ) 3 -。
In one embodiment, G 1 Is unsubstituted C 5 Alkylene radicals, e.g., - (CH) 2 ) 5 -。
In one embodiment, G 1 Is unsubstituted C 6 Alkylene radicals, e.g., - (CH) 2 ) 6 -。
In one embodiment, G 1 Is unsubstituted C 7 Alkylene radicals, e.g., - (CH) 2 ) 7 -。
In one embodiment, G 1 Is unsubstituted C 8 Alkylene radicals, e.g., - (CH) 2 ) 8 -。
In one embodiment, G 2 Is unsubstituted C 3 Alkylene radicals, e.g., - (CH) 2 ) 3 -。
In one embodiment, G 2 Is unsubstituted C 5 Alkylene radicals, e.g., - (CH) 2 ) 5 -。
In one embodiment, G 2 Is unsubstituted C 6 Alkylene radicals, e.g., - (CH) 2 ) 6 -。
In one embodiment, G 2 Is unsubstituted C 7 Alkylene radicals, e.g., - (CH) 2 ) 7 -。
In one embodiment, G 2 Is unsubstituted C 8 Alkylene radicals, e.g., - (CH) 2 ) 8 -。
In one embodiment, L 1 is-C (O) O-.
In one embodiment, L 1 is-OC (O) -.
In one embodiment, L 2 is-C (O) O-. For example, L 1 And L 2 Are all-C (O) O-.
In one embodiment, L 2 is-OC (O) -. For example, L 1 And L 2 Are all-OC (O) -.
In one embodiment, R 1 Is unsubstituted C 11 Straight chain alkyl radicals, i.e., - (CH) 2 ) 10 CH 3 。
In one embodiment, R 1 Is unsubstituted C 10 Straight chain alkyl radicals, i.e., - (CH) 2 ) 9 CH 3 。
In one embodiment, R 1 Is unsubstituted C 8~18 A branched alkyl group. In a preferred embodiment, R 1 Is unsubstituted C 17 Branched alkyl or C 18 Branched alkyl or C 15 Branched alkyl or C 14 Branched alkyl radical C 8 A branched alkyl group. For example, R 1 Comprises the following steps:、、、or. In another preferred embodiment, R 1 Is unsubstituted C 17 Branched alkyl or C 18 Branched alkyl or C 8 A branched alkyl group. For example, R 1 Comprises the following steps:、or are each。
In one embodiment, R 2 Is unsubstituted C 8~18 A branched alkyl group. In a preferred embodiment, R 2 Is unsubstituted C 17 Branched alkyl or C 18 Branched alkyl or C 15 Branched alkyl or C 14 Branched alkyl radical C 8 A branched alkyl group. For example, R 2 Comprises the following steps:、、、or。
In one embodiment, L is- (CH) 2 ) 2 -。
In one embodiment, L is- (CH) 2 ) 3 -。
In one embodiment, L is- (CH) 2 ) 4 -。
In one embodiment, G 1 Is- (CH) 2 ) 3 -，G 2 Is- (CH) 2 ) 5 -，L 1 is-C (O) O-, L 2 is-C (O) O-, R 1 Is- (CH) 2 ) 10 CH 3 ，R 2 Comprises the following steps:，G 3 is HO (CH) 2 ) 2 -，G 4 Is HO (CH) 2 ) 2 -, L is- (CH) 2 ) 2 -。
In one embodiment, G 1 Is- (CH) 2 ) 5 -，G 2 Is- (CH) 2 ) 5 -，L 1 is-C (O) O-, L 2 is-C (O) O-, R 1 Comprises the following steps:，R 2 comprises the following steps:，G 3 is HO (CH) 2 ) 2 -，G 4 Is HO (CH) 2 ) 2 -, L is- (CH) 2 ) 2 -。
In one embodiment, G 1 Is- (CH) 2 ) 6 -，G 2 Is- (CH) 2 ) 6 -，L 1 is-OC (O) -, L 2 is-OC (O) -, R 1 Comprises the following steps:，R 2 comprises the following steps:，G 3 is HO (CH) 2 ) 2 -，G 4 Is HO (CH) 2 ) 2 -, L is- (CH) 2 ) 2 -。
In one embodiment, G 1 Is- (CH) 2 ) 7 -，G 2 Is- (CH) 2 ) 7 -，L 1 is-C (O) O-, L 2 is-C (O) O-, R 1 Comprises the following steps:，R 2 comprises the following steps:，G 3 is HO (CH) 2 ) 2 -，G 4 Is HO (CH) 2 ) 2 -, L is- (CH) 2 ) 2 -。
In one embodiment, G 1 Is- (CH) 2 ) 5 -，G 2 Is- (CH) 2 ) 5 -，L 1 is-C (O) O-, L 2 is-C (O) O-, R 1 Comprises the following steps:，R 2 comprises the following steps:，G 3 is HO (CH) 2 ) 3 -，G 4 Is HO (CH) 2 ) 3 -, L is- (CH) 2 ) 2 -。
In one embodiment, G 1 Is- (CH) 2 ) 5 -，G 2 Is- (CH) 2 ) 5 -，L 1 is-C (O) O-, L 2 is-C (O) O-, R 1 Comprises the following steps:，R 2 comprises the following steps:，G 3 is HO (CH) 2 ) 3 -，G 4 Is HO (CH) 2 ) 3 -, L is- (CH) 2 ) 3 -。
In an exemplary embodiment, the compound is selected from the following compounds or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof:
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yet another aspect of 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 is a nanoparticle formulation having an average size of from 10nm to 300nm, preferably from 90nm to 280nm; the polydispersity of the nanoparticle formulation is less than or equal to 50%, preferably less than or equal to 40%, more preferably less than or equal to 30%.
Cationic lipids
In one embodiment of the composition/carrier of the present invention, the cationic lipid is one or more selected from the compounds of formula (I) described above or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof. In one embodiment, the cationic lipid is a compound of formula (I) selected from those described above. For example, the cationic lipid is compound YK-401, YK-402, YK-403, YK-404, YK-405, YK-406, YK-407, YK-408, YK-409, YK-410, YK-411, YK-412, YK-413, YK-414, YK-415, YK-416, YK-417, YK-418, YK-419, YK-420, YK-421, YK-422, YK-423, YK-424. In a preferred embodiment, the cationic lipid is compound YK-407, in another preferred embodiment, the cationic lipid is compound YK-401, in another preferred embodiment, the cationic lipid is compound YK-402, in another preferred embodiment, the cationic lipid is compound YK-403, in another preferred embodiment, the cationic lipid is compound YK-422, and in another preferred embodiment, the cationic lipid is compound YK-423.
In another embodiment of the composition/carrier of the present invention, the cationic lipid comprises: (a) One or more selected from the compounds of formula (I) as described above or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof; (b) One or more other ionizable lipid compounds different from (a). (b) The cationic lipid compound may be a commercially available cationic lipid, or a cationic lipid compound reported in the literature. For example, (B) the cationic lipid compound may be SM-102 (compound 25 in WO2017049245A 2), may also be compound 21 and compound 23 in WO2021055833, and may also be HHMA (compound 1 in CN 112979483B).
In one embodiment, the cationic lipid is present in a carrier at a molar ratio of 25% to 75%, e.g., 30%, 40%, 49%, 55%, 60%, 65%, 70%.
The carrier can be used to deliver an active ingredient such as a therapeutic or prophylactic agent. The active ingredient may be encapsulated within or associated 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. The 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 (airRNAs), 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 are understood as meaning, in the context of the present invention, auxiliary lipids which are uncharged at the chosen pH value or which are present in zwitterionic form. The neutral lipid may regulate nanoparticle mobility into lipid bilayer structure and improve efficiency by promoting lipid phase transition, and may also affect the specificity of target organs.
In one embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 1 to 15, such as about 14. In a preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 4. In another preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 4.9.
For example, the neutral lipid may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, 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, a phospholipid may comprise 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, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Also encompassed are non-natural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes. For example, a phospholipid may be functionalized with or crosslinked with one or more alkynes (e.g., an alkenyl group with one or more double bonds replaced with a triple bond). Under appropriate reaction conditions, the alkynyl group may undergo a copper-catalyzed cycloaddition reaction upon exposure to the 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., a dye).
Neutral lipids useful in these compositions may be selected from the non-limiting group consisting of: 1, 2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-didecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) 1, 2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18: 0 Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1, 2-dilinolacyl-sn-glycero-3-phosphocholine, 1, 2-dineoyl-sn-glycero-3-phosphocholine, 1, 2-didodecanoyl-sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-Diphytoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dineoyl-sn-glycero-3-phosphoethanolamine, 1, 2-didodecanoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycero) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG) Palmitoyl Oleoyl Phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl 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 lipids include both DSPC and DOPE.
Structural lipids
The carrier of the cationic lipid-containing composition may also include one or more structural lipids. The structural lipid refers to a lipid that enhances stability of the nanoparticle by filling gaps between lipids in the present invention.
In one embodiment, the molar ratio of the cationic lipid to the structural lipid is about 0.6 to 1 to 3, for example, about 1.0.
The structural lipid may be selected from, but is not limited to, the group consisting of: cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, lycopersine, ursolic acid, alpha-tocopherol, 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 (prednisone), dexamethasone, prednisone (prednisone), and hydrocortisone (hydrocortisone), or combinations thereof.
Polymer conjugated lipids
The carrier of the cationic lipid-containing composition may also include one or more polymeric conjugated lipids. The polymer conjugated lipid mainly refers to polyethylene glycol (PEG) modified lipid. Hydrophilic PEG stabilizes LNP, modulates nanoparticle size by limiting lipid fusion, and increases the half-life of the nanoparticle by reducing non-specific interactions with macrophages.
In one embodiment, the polymeric 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, and PEG-modified dialkylglycerol. PEG-modified PEG typically has a molecular weight of 350-5000Da.
For example, the polymeric conjugated lipid is selected from one or more of the following: distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG 2000), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000), and methoxypolyethylene glycol ditetradecylethanolamide (ALC-0159).
In one embodiment of the composition/carrier of the present invention, the polymeric conjugated lipid is DMG-PEG2000.
In one embodiment of the composition/carrier of the present invention, the carrier comprises a neutral lipid, a structural lipid and a polymer conjugated lipid, and 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), for example, (35) - (49): 7.5) - (15): 35) - (55): 1) - (5).
In one embodiment of the composition/carrier of the invention, the carrier comprises a neutral lipid, a structural lipid, and a polymer conjugated lipid, the molar ratio of said cationic lipid, said neutral lipid, said structural lipid, and said polymer conjugated lipid being 49.
Therapeutic and/or prophylactic agent
The composition may include one or more therapeutic and/or prophylactic agents. In one embodiment, the mass ratio of the carrier to the therapeutic or prophylactic agent is 10.
In one embodiment, the mass ratio of carrier to therapeutic or prophylactic agent is 12.5.
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 invention can deliver therapeutic and/or prophylactic agents to a mammalian cell or organ, and thus the invention also provides methods of treating a disease or condition in a mammal in need thereof, comprising administering to the mammal and/or contacting a mammalian cell with a composition comprising a therapeutic and/or prophylactic agent.
Therapeutic and/or prophylactic agents include biologically active substances and are alternatively referred to as "active agents". The therapeutic and/or prophylactic agent can be a substance that causes a desired change in a cell or organ or other body tissue or system upon delivery to the cell or organ. Such species may be useful for treating 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. <xnotran> ( (vincristine), (doxorubicin), (mitoxantrone), (camptothecin), (cisplatin), (bleomycin), (cyclophosphamide), (streptozotocin)), ( D (actinomycin D), , (vinblastine), (cytosine arabinoside), (anthracycline), , , , ), , ( (dibucaine) (chlorpromazine)), β - ( (propranolol), (timolol) (labetalol)), ( (clonidine) (hydralazine)), ( (imipramine), (amitriptyline) (doxepin)), ( (phenytoin)), ( (diphenhydramine), (chlorpheniramine) (promethazine)), / ( (gentamycin), (ciprofloxacin) (cefoxitin)), </xnotran> Antifungal agents (e.g., miconazole (miconazole), terconazole (terconazole), econazole (econazole), isoconazole (isoconazole), butoconazole (butoconazole), clotrimazole (clotrimazole), itraconazole (itraconazole), nystatin (nystatin), netitifen (naftifine), and amphotericin B)), antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, anti-glaucoma agents, 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. A cytotoxin or cytotoxic agent includes any agent that is harmful to a cell. Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emidine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol, labyrin (lacrimycin), and analogs thereof or analogs thereof. Radioactive ions 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 infectious disease-derived antigens and/or epitopes. Vaccines can also include compounds and agents that direct an immune response against cancer cells and can include mrnas encoding tumor cell-derived antigens, epitopes, and/or neo-epitopes. Compounds that elicit an immune response can include vaccines, corticosteroids (e.g., dexamethasone), and other species. In some embodiments, the vaccine and/or compound capable of eliciting an immune response is administered intramuscularly by 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 (dacarbazine)), alkylating agents (e.g., mechlorethamine (mechlororethamine), thiotepa (thiotepa), chlorambucil (chlorembucil), lachnomycin (CC-1065), melphalan (melphalan), carmustine (carmustine, BSNU), lomustine (lomustine, CCNU), cyclophosphamide, busulfan (busulfan), dibromomannitol, streptozotocin, mitomycin C and cisplatin (DDP), anthracyclines (e.g., daunomycin (daunomycin), and cisplatin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin (gentamycin), and vincristine (vincristine), such as antimitols (vincristine ).
In other embodiments, the therapeutic and/or prophylactic agent is a protein. Therapeutic proteins that may be used in the nanoparticles of the present invention 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, interferons, heparin, hepatitis B surface antigen, typhoid and cholera vaccines.
In some embodiments, the therapeutic agent is a polynucleotide or a nucleic acid (e.g., a ribonucleic acid or a deoxyribonucleic acid). The term "polynucleotide" is used in its broadest sense to include any compound and/or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides for use according to the invention include, but are not limited to, one or more of: deoxyribonucleic acid (DNA); ribonucleic acids (RNA), including messenger mRNA (mRNA), hybrids thereof; an RNAi-inducing factor; an RNAi agent; siRNA; shRNA; a miRNA; antisense RNA; a ribozyme; catalytic DNA; inducing triple helix-forming RNA; aptamers, and the like. In some embodiments, the therapeutic and/or prophylactic agent is RNA. RNA 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 can be of any size and can 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. The siRNA is capable of selectively reducing the expression of a gene of interest or down-regulating the expression of the gene. For example, the selection of the siRNA can be such that a gene associated with a particular disease, disorder, or condition is silenced upon 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 a sgRNA and/or cas9 mRNA. sgRNA and/or cas9 mRNA can be used as gene editing tools. For example, sgRNA-cas9 complexes 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 same. The shRNA may be produced inside the target cell upon delivery of the appropriate construct into the nucleus. Constructs and mechanisms associated with shRNA are well known in the relevant art.
Disease or disorder
The compositions/vectors of the invention 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. Therefore, the composition of the invention can be used for preparing nucleic acid drugs, gene vaccines, small molecule drugs, polypeptide or protein drugs. Due to the wide variety of therapeutic or prophylactic agents described above, the compositions of the present invention can be used to treat or prevent a variety of diseases or conditions.
In one embodiment, the disease or disorder is characterized by a malfunctioning 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 one embodiment, the infectious disease is selected from the group consisting of a disease caused by a coronavirus, an influenza virus, or an HIV virus, pediatric pneumonia, rift valley fever, yellow fever, rabies, and various 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 altering agents, or other components. The permeability enhancing molecule can 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 its derivatives and analogs).
Surface-altering agents may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecylammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, azurin (clendodendrom), bromhexine (brohexine), carbocisteine (carbocistine), eplerenone (epizinone), mesna (snmea), ambroxol (ambroxol), sobrenol (sobreol), dominol (domidodol), letosteine (letostesteine), setinin (pronin), tiopronin (tiopronin), gelsolin (gelsolin), thymosin beta 4, dnase alpha (streptokinase), streptococcinease (streptococcinease), and dnase (e), e.g., dnase (e). The surface-altering agent can be disposed within and/or on the surface of the nanoparticles of the composition (e.g., by coating, adsorption, covalent attachment, or other methods).
The composition may also comprise one or more functionalized lipids. For example, lipids may be functionalized with alkynyl groups that may undergo cycloaddition reactions when exposed to azides under appropriate reaction conditions. In particular, the lipid bilayer may be functionalized in this manner with one or more groups effective to promote membrane permeation, cell recognition, or imaging. The surface of the composition may also be conjugated with 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 material useful in pharmaceutical compositions. For example, the composition may include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonizing 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, dicalcium 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, a composition comprising one or more lipids described herein can further comprise one or more adjuvants, such as Glucopyranosyl Lipid Adjuvant (GLA), cpG oligodeoxyribonucleotides (e.g., class a or class B), poly (I: C), aluminum hydroxide, and Pam3CSK4.
The composition of the present invention may be formulated in the form of solid, semisolid, liquid or gas, such as tablet, capsule, ointment, elixir, syrup, solution, emulsion, suspension, injection, aerosol. The compositions of the present invention may be prepared by methods well known in the pharmaceutical arts. For example, sterile injectable solutions can be prepared by incorporating the required amount of the therapeutic or prophylactic agent in the appropriate solvent with various of the other ingredients described above, as required, in an appropriate solvent, such as sterile distilled water, followed by filtered sterilization. Surfactants may also be added to facilitate the formation of a homogeneous solution or suspension.
For example, the compositions of the present invention may be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In one embodiment, the composition is administered subcutaneously.
The compositions of the present invention 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 dosage of about 0.001mg/kg to about 10mg/kg of the therapeutic or prophylactic agent can be administered to a mammal (e.g., a human).
Examples
The present invention will be further described with reference to the following examples. However, the present invention is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not mentioned are conventional conditions in the industry. In the examples of the present invention, the raw materials used are all commercially available. Unless otherwise indicated, percentages in the context are percentages by weight and all temperatures are given in degrees celsius. The technical features according to the embodiments of the present invention may be combined with each other as long as they do not conflict with each other.
Example 1: synthesis of cationic lipid compounds
1. Synthesis of YK-401
The synthetic route is as follows:
synthesis of 6,6' - (ethane-1, 2-diyl-bis ((2-hydroxyethyl) azelidinyl)) dihexanoic acid bis (heptadecan-9-yl) ester (YK-401)
N, N' -bis (hydroxyethyl) ethylenediamine (28mg, 0.19mmol) and heptadecan-9-yl 6-bromohexanoate (200mg, 0.48mmol) were dissolved in acetonitrile (3 mL), and potassium carbonate (79mg, 0.57mmol) was added to the above system, heated to 75 ℃ and stirred for 5 hours. After completion of the reaction, 20mL of water was added to the reaction mixture, followed by extraction with ethyl acetate (20 mL. Times.2), and the organic phases were combined and washed with brine (20 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure in vacuo to remove the solvent. The residue was purified by silica gel chromatography (dichloromethane/methanol) to give the objective compound (62 mg,0.07mmol, 38.2%). C 52 H 104 N 2 O 6 , MS(ES): m/z(M+H + )853.8。
1 H NMR (400 MHz, chloroform-d) δ 4.86 (p, J = 6.1 Hz, 2H), 3.66 (s, 4H), 2.71 (s, 12H), 2.29 (t, J = 7.4 Hz, 4H), 1.67-1.46 (m, 16H), 1.26 (s, 52H), 0.88 (t, J = 6.7 Hz, 12H).
2. Synthesis of YK-402
The synthetic route is as follows:
the method comprises the following steps: synthesis of 2-octyldecanoic acid 6-bromohexyl ester (YK-402-PM 1)
2-Octyldecanoic acid (300 mg, 1.05 mmol) and 6-bromohexanol (202.5 mg, 1.12 mmol) were dissolved in dichloromethane (5 mL), and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (404.4 mg, 2.11mmol) and 4-dimethylaminopyridine (64.4 mg, 0.53mmol) were added to the above solution, and the reaction was stirred at 30-35 ℃ for 5 hours. After the reaction, the reaction mixture was washed with saturated sodium bicarbonate and 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 (ethyl acetate/n-hexane) to give YK-402-PM1 (478.2 mg, 1.06mmol, 100%).
Step two: synthesis of (ethane-1, 2-diyl-bis ((2-hydroxyethyl) azanediyl)) bis (hexane-6, 1-diyl) bis (2-octyldecanoate) (YK-402)
YK-402 (53.10mg, 0.06mmol, 22.3%) and C (C) were obtained by synthesizing YK-401 from YK-402-PM1 (447.5 mg,0.67 mmol) and N, N' -bis (hydroxyethyl) ethylenediamine (39.7mg, 0.27mmol) as raw materials 54 H 108 N 2 O 6 , MS(ES): m/z(M+H + )881.8。
1 H NMR (400 MHz, chloroform-d) δ 4.06 (t, J = 6.7 Hz, 4H), 3.68 (s, 4H), 2.74 (s, 12H), 2.31 (tt, J = 8.8, 5.3 Hz, 2H), 1.59 (dq, J = 22.3, 6.8 Hz, 12H), 1.25 (s, 60H), 0.88 (t, J = 6.7 Hz, 12H).
3. Synthesis of YK-403
The synthetic route is as follows:
the method comprises the following steps: synthesis of heptadecan-9-yl 8-bromooctanoate (YK-403-PM 1)
Using 9-heptadecanol (3.73 g, 14.53 mmol) and 8-bromooctanoic acid (2.70 g, 12.11 mmol) as raw materials, and following the method for synthesizing YK-402-PM1, YK-403-PM1 (4.43g, 9.60mmol, 79.3%) is obtained.
Step two: synthesis of 8,8' - (ethane-1, 2-diyl-bis ((2-hydroxyethyl) azelidinyl)) bis (heptadecan-9-yl) dioctanoate (YK-403)
YK-403 (98.7mg, 0.11mmol, 54.3%) C and YK-403 (233.6 mg, 0.51 mmol) were synthesized from YK-403-PM1 (30.0mg, 0.20mmol) and N, N' -bis (hydroxyethyl) ethylenediamine (30.0mg, 0.20mmol) as raw materials 56 H 112 N 2 O 6 , MS(ES): m/z(M+H + )909.8。
1 H NMR (400 MHz, chloroform-d) δ 4.86 (p, J = 6.2 Hz, 2H), 3.64-3.57 (m, 4H), 2.61 (dd, J = 9.9, 5.1 Hz, 8H), 2.55-2.47 (m, 4H), 2.27 (t, J = 7.5 Hz, 4H), 1.65-1.57 (m, 4H), 1.55-1.44 (m, 12H), 1.28 (d, J = 16.0 Hz, 62H), 0.88 (t, J = 6.8 Hz, 12H).
4. Synthesis of YK-404
The synthetic route is as follows:
the method comprises the following steps: synthesis of 9-bromononanoic acid octane-3-yl ester (YK-404-PM 1)
Using octane-3-ol (65.9 mg, 0.51 mmol) and 9-bromononanoic acid (100 mg, 0.42 mmol) as raw materials, and synthesizing YK-402-PM1 to obtain YK-404-PM1 (114.9mg, 0.33mmol, 78.3%).
Step two: synthesis of 9,9' - (ethane-1, 2-diyl-bis ((2-hydroxyethyl) azanediyl)) dinonylic acid di (octane-3-yl) ester (YK-404)
YK-404 (30.9mg, 0.05mmol, 34.7%) and C-404 (30.9mg, 0.05mmol, C) are obtained by using YK-404-PM1 (114.9 mg,0.33 mmol) and N, N' -bis (hydroxyethyl) ethylenediamine (19.5mg, 0.13mmol) as raw materials according to the method for synthesizing YK-401 40 H 80 N 2 O 6 , MS(ES): m/z(M+H + )685.6。
1 H NMR (400 MHz, chloroform-d) δ 4.85 (p, J = 6.5 Hz, 2H), 3.79 (s, 4H), 2.87 (s, 12H), 2.32 (t, J = 7.5 Hz, 4H), 1.73-1.53 (m, 16H), 1.35-1.29 (m, 28H), 0.94-0.89 (m, 12H).
5. Synthesis of YK-405
The synthetic route is as follows:
the method comprises the following steps: synthesis of decyl 4- ((2-hydroxyethyl) (2- ((2-hydroxyethyl) amino) ethyl) amino) butyrate (YK-405-PM 1)
N, N' -bis (hydroxyethyl) ethylenediamine (434.1mg, 2.93mmol) and N-decyl 4-bromobutyrate (300mg, 0.98mmol) were dissolved in acetonitrile (4 mL), and potassium carbonate (404.8mg, 2.93mmol) was added to the above system, and the reaction was stirred at 70 ℃ for 4 hours. After the reaction, 20mL of water was added to the reaction mixture, followed by extraction with dichloromethane (20 mL. Times.2), and the organic phases were combined, washed with a saturated aqueous solution of sodium bicarbonate (20 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure in vacuo to remove the solvent. The residue was purified by silica gel chromatography (dichloromethane/methanol (2% triethylamine)) to give YK-405-PM1 (179.5 mg,0.48mmol, 48.9%). C 20 H 42 N 2 O 4 , MS(ES): m/z(M+H + )375.3。
Step two: synthesis of 6- ((2-hydroxyethyl) (2- ((2-hydroxyethyl) (4- (decyloxy) -4-oxobutyl) amino) ethyl) amino) heptadecan-9-yl) hexanoate (YK-405)
YK-405 (42.5mg, 0.06mmol, 13.6%) C and 6-bromohexanoic acid-heptadecan-9-yl ester (215.0mg, 0.52mmol) are used as raw materials to obtain YK-405 (42.5mg, 0.06mmol, 13.6%) by a method for synthesizing YK-401 43 H 86 N 2 O 6 , MS(ES): m/z(M+H + )727.6。
1 H NMR (400 MHz, chloroform-d) δ 4.96-4.69 (m, 1H), 4.06 (t, J = 6.8 Hz, 2H), 3.78-3.72 (m, 2H), 3.72-3.65 (m, 2H), 3.45 (s, 4H), 2.88-2.79 (m, 6H), 2.78-2.64 (m, 4H), 2.32 (dt, J = 19.1, 7.2 Hz, 4H), 1.84 (p, J = 7.1 Hz, 2H), 1.63 (dq, J = 13.9, 7.4 Hz, 6H), 1.53-1.44 (m, 4H), 1.26 (s, 40H), 0.88 (t, J = 6.6 Hz, 9H).
6. Synthesis of YK-406
The synthetic route is as follows:
the method comprises the following steps: synthesis of 6- (2-hydroxyethyl) (2- ((2-hydroxyethyl) amino) ethyl) amino) hexyl 3-hexylnonanoate (YK-406-PM 1)
N, N' -bis (hydroxyethyl) ethylenediamine (183.7 mg, 1.24mmol) and 6-bromohexyl 3-hexylnonanoate (200mg, 0.49mmol) were used as raw materials to obtain YK-406-PM1 (134.7 mg,0.28mmol, 58.1%) by the method for synthesizing YK-405-PM 1. C 27 H 56 N 2 O 4 , MS(ES): m/z(M+H + )473.4。
Step two: synthesis of 6- ((2-hydroxyethyl) (2- ((2-hydroxyethyl) (4- (undecyloxy) -4-oxobutyl) amino) ethyl) amino) -hexyl 3-hexylnonanoate (YK-406)
YK-406 (41.5mg, 0.06mmol, 20.8%) C is obtained by using YK-406-PM1 (134.7 mg,0.28 mmol) and 4-bromobutyric acid-n-decyl ester (149.9mg, 0.49mmol) as raw materials according to the method for synthesizing YK-401 42 H 84 N 2 O 6 , MS(ES): m/z(M+H + )713.6。
1 H NMR (400 MHz, chloroform-d) δ 4.05 (q, J = 6.5 Hz, 4H), 3.68-3.50 (m, 4H), 2.76-2.48 (m, 12H), 2.32 (t, J = 7.1 Hz, 2H), 2.22 (d, J = 6.9 Hz, 2H), 1.81 (dt, J = 15.0, 7.2 Hz, 3H), 1.68-1.56 (m, 4H), 1.50 (d, J = 7.3 Hz, 2H), 1.28 (d, J = 15.8 Hz, 40H), 0.88 (t, J = 6.6 Hz, 9H).
7. Synthesis of YK-407
The synthetic route is as follows:
the method comprises the following steps: synthesis of undecyl 4- ((2-hydroxyethyl) (2- ((2-hydroxyethyl) amino) ethyl) amino) butyrate (YK-407-PM 1)
Using N, N' -bis (hydroxyethyl) ethylenediamine (691.9 mg, 4.67 mmol) and 4-bromobutyric acid-undecyl ester (500 mg, 1.56 mmol) as raw materials, according to the method for synthesizing YK-405-PM1, YK-407-PM1 (271 mg, 0.70mmol, 44.7%)。C 21 H 44 N 2 O 4 , MS(ES): m/z(M+H + )389.4。
Step two: synthesis of heptadecan-9-yl 6- ((2-hydroxyethyl) (2- (2-hydroxyethyl) (4-oxo-4- (undecyloxy) butyl) amino) ethyl) amino) hexanoate (YK-407)
YK-407 (129 mg, 0.17mmol, 24.3%) C is obtained by taking YK-407-PM1 (271 mg, 0.70 mmol) and 6-bromohexanoic acid heptadecan-9-yl ester (453.5 mg, 1.05 mmol) as raw materials according to a method for synthesizing YK-401 44 H 88 N 2 O 6 , MS(ES): m/z(M+H + )741.7。
1 H NMR (400 MHz, CDCl3) δ 4.93 – 4.84 (m, 1H), 4.09 (t, J = 6.8 Hz, 2H), 3.74 (s, 4H), 2.80 (s, 10H), 2.39 – 2.30 (m, 4H), 1.89 (s, 2H), 1.67 (dt, J = 14.9, 7.6 Hz, 6H), 1.54 (d, J = 5.3 Hz, 4H), 1.29 (s, 44H), 0.92 (d, J = 6.5 Hz, 9H).
8. Synthesis of YK-408
The synthetic route is as follows:
synthesis of heptadecan-9-yl 4- ((2-hydroxyethyl) (2- ((2-hydroxyethyl) (4- (decyloxy) -4-oxobutyl) amino) ethyl) amino) butyrate (YK-408)
YK-405-PM1 (276 mg,0.74 mmol) and 4-bromobutyric acid heptadecane-9-yl ester (537.8mg, 1.33mmol) are taken as raw materials, and YK-408 (80mg, 0.11mmol, 15.5%) and C are obtained according to the method for synthesizing YK-401 41 H 82 N 2 O 6 , MS(ES): m/z(M+H + )699.6。
1 H NMR (400 MHz, chloroform-d) δ 5.89 (s, 2H), 5.57-5.34 (m, 1H), 4.65 (t, J = 6.7 Hz, 2H), 4.19 (t, J = 4.4 Hz, 4H), 3.20 (td, J = 15.1, 13.0, 6.2 Hz, 12H), 2.90 (q, J = 7.0 Hz, 4H), 2.40 (p, J = 7.1 Hz, 4H), 2.25-2.17 (m, 2H), 2.15-2.06 (m, 4H), 1.85 (s, 36H), 1.47 (t, J = 6.4 Hz, 9H).
9. Synthesis of YK-409
The synthetic route is as follows:
the method comprises the following steps: synthesis of 2-octyldecyl 6- (2-hydroxyethyl) (2- ((2-hydroxyethyl) amino) ethyl) amino) hexanoate (YK-409-PM 1)
Using N, N' -bis (hydroxyethyl) ethylenediamine (298.0 mg, 2.01mmol) and 2-octyldecyl 6-bromohexanoate (300mg, 0.67mmol) as raw materials, YK-409-PM1 (144.7 mg,0.28mmol, 41.9%) was obtained according to the method for synthesizing YK-405-PM 1. C 30 H 62 N 2 O 4 , MS(ES): m/z(M+H + )515.4。
Step two: synthesis of 2-octyldecyl 6- ((2-hydroxyethyl) (2- ((2-hydroxyethyl) (4- (undecyloxy) -4-oxobutyl) amino) ethyl) amino) hexanoate (YK-409)
YK-409-PM1 (144.7 mg,0.28 mmol) and 4-bromobutyric acid-undecyl ester (108.4 mg, 0.34mmol) are used as raw materials to obtain YK-409 (54.6 mg,0.07mmol, 25.8%) and C according to the method for synthesizing YK-401 45 H 90 N 2 O 6 , MS(ES): m/z(M+H + )755.7。
1 H NMR (400 MHz, chloroform-d) δ 4.06 (t, J = 6.8 Hz, 2H), 3.96 (d, J = 5.8 Hz, 2H), 3.65-3.56 (m, 4H), 2.70-2.46 (m, 12H), 2.31 (td, J = 7.3, 3.7 Hz, 4H), 1.81 (p, J = 7.3 Hz, 2H), 1.70-1.56 (m, 5H), 1.51 (p, J = 7.7 Hz, 2H), 1.28 (d, J = 15.2 Hz, 46H), 0.88 (t, J = 6.7 Hz, 9H).
10. Synthesis of YK-410
The synthetic route is as follows:
synthesis of 2-octyldecyl 6- ((2-hydroxyethyl) (2- ((2-hydroxyethyl) (6- (decyloxy) -6-oxohexyl) amino) ethyl) amino) hexanoate (YK-410)
YK-410 (141mg, 0.18mmol, 48.2%) C YK-409-PM1 (195.0 mg, 0.38 mmol) and 6-bromohexanoic acid n-decyl ester (190.5mg, 0.57mmol) are used as raw materials according to the method for synthesizing YK-401 46 H 92 N 2 O 6 , MS(ES): m/z(M+H + )769.7。
1 H NMR (400 MHz, chloroform-d) δ 4.05 (t, J = 6.4 Hz, 2H), 3.96 (d, J = 5.4 Hz, 2H), 3.61 (s, 4H), 2.63 (d, J = 9.0 Hz, 8H), 2.58-2.50 (m, 4H), 2.30 (s, 4H), 1.77-1.57 (m, 8H), 1.55-1.46 (m, 4H), 1.26 (s, 44H), 0.87 (d, J = 6.3 Hz, 9H).
11. Synthesis of YK-411
The synthetic route is as follows:
the method comprises the following steps: synthesis of 3-hexylnonyl 6- (2-hydroxyethyl) (2- ((2-hydroxyethyl) amino) ethyl) amino) hexanoate (YK-411-PM 1)
Using N, N' -bis (hydroxyethyl) ethylenediamine (329.0 mg, 2.22mmol) and 3-hexylnonyl 6-bromohexanoate (300mg, 0.74mmol) as raw materials, YK-411-PM1 (103.1 mg, 0.22mmol, 29.4%) was obtained according to the method for synthesizing YK-405-PM 1. C 27 H 56 N 2 O 4 , MS(ES): m/z(M+H + )473.4。
Step two: synthesis of 3-hexylnonyl 6- ((2-hydroxyethyl) (2- ((2-hydroxyethyl) (6- (decyloxy) -6-oxobutyl) amino) ethyl) amino) hexanoate (YK-411)
YK-411 (68.6 mg,0.09mmol, 42.9%) C and YK-411 (68.6 mg,0.09mmol, 42.9%) are obtained by using YK-411-PM1 (103.1 mg, 0.22 mmol) and 6-bromohexanoic acid n-decyl ester (76.6 mg, 0.23mmol) as raw materials according to the method for synthesizing YK-401 43 H 86 N 2 O 6 , MS(ES): m/z(M+H + )727.7。
1 H NMR (400 MHz, chloroform-d) δ 4.07 (q, J = 7.0 Hz, 4H), 3.64 (t, J = 6.3 Hz, 4H), 2.69 (s, 8H), 2.30 (t, J = 7.0 Hz, 4H), 1.58 (ddp, J = 32.0, 15.4, 7.2 Hz, 13H), 1.28 (d, J = 15.0 Hz, 42H), 0.88 (t, J = 6.4 Hz, 9H).
12. Synthesis of YK-412
The synthetic route is as follows:
the method comprises the following steps: synthesis of 2-octyldecanoic acid-6- (2-hydroxyethyl-amino) hexyl ester (YK-412-PM 1)
YK-412-PM1 (165 mg,0.39mmol, 68.9%) was obtained from 2-octyldecanoic acid-6-bromohexyl ester (250.0 mg, 0.56mmol) and ethanolamine (136.5 mg, 2.23mmol) as starting materials by the method for synthesizing YK-401. C 26 H 53 NO 3 ，MS(ES): m/z(M+H + )428.4。
Step two: synthesis of (propane-1, 3-diyl-bis ((2-hydroxyethyl) azelidinyl)) -bis (hexane-6, 1-diyl) bis (2-octyldecanoate) (YK-412)
YK-412 (94mg, 0.10mmol, 53.8%) and C are obtained by using YK-412-PM1 (165 mg,0.39 mmol) and 1, 3-dibromopropane (42.8mg, 0.21mmol) as raw materials and according to a method for synthesizing YK-401 55 H 110 N 2 O 6 , MS(ES): m/z(M+H + )895.8。
1 H NMR (400 MHz, chloroform-d) δ 4.06 (t, J = 6.6 Hz, 4H), 3.61 (s, 4H), 2.60 (d, J = 15.9 Hz, 8H), 2.51 (s, 4H), 2.32 (dt, J = 9.9, 5.1 Hz, 2H), 1.61 (tt, J = 14.4, 7.0 Hz, 12H), 1.25 (s, 62H), 0.88 (t, J = 6.6 Hz, 12H).
13. Synthesis of YK-413
The synthetic route is as follows:
the method comprises the following steps: synthesis of undecyl 4- ((3- (tert-butoxycarbonyl) amino) propyl) aminobutyric acid (YK-413-PM 1)
YK-413-PM1 (2.00 g, 4.81mmol, 51.5%) was obtained by the method for synthesizing YK-401 using tert-butyl (3-aminopropyl) carbamate (4.88 g, 28.01 mmol) and undecyl 4-bromobutyrate (3.00 g, 9.34 mmol) as the starting materials. C 23 H 46 N 2 O 4 , MS(ES): m/z(M+H + )415.5。
Step two: synthesis of undecyl 4- ((3- (tert-butoxycarbonyl) amino) propyl) (2-hydroxyethyl) amino) butyrate (YK-413-PM 2)
YK-413-PM2 (2.20 g, 4.80mmol, 99.8%) was obtained by using YK-413-PM1 (2.00 g, 4.81 mmol) and bromoethanol (1.80 g, 14.40 mmol) as raw materials according to the method for synthesizing YK-401. C 25 H 50 N 2 O 5 , MS(ES): m/z(M+H + )459.4。
Step three: synthesis of undecyl 4- (3-aminopropyl) ((2-hydroxyethyl) amino) butyrate (YK-413-PM 3)
YK-413-PM2 (2.20 g, 4.80 mmol) is dissolved in tetrahydrofuran (10 mL), the temperature of the system is controlled at 0 ℃, 4M hydrogen chloride dioxane solution (12 mL) is slowly added dropwise, the temperature is slowly raised to room temperature after the dropwise addition, and the reaction is carried out for 2 hours. After the reaction was completed, a saturated sodium bicarbonate solution was added dropwise to adjust the pH to 7 to 8, the aqueous phase was washed with dichloromethane (20 mL. Times.2), separated, dried over anhydrous sodium sulfate for the organic phase, filtered, and the filtrate was concentrated under reduced pressure in vacuo to remove the solvent, whereby YK-413-PM3 (1.09 g, 3.05mmol, 63.5%) was obtained. C 20 H 42 N 2 O 3 , MS(ES): m/z(M+H + )359.3。
Step four: synthesis of 6- ((3- (2-hydroxyethyl) (4-oxo-4- (undecyloxy) butyl) amino) propyl) amino) heptadecan-9-yl hexanoate (YK-413-PM 4)
YK-413-PM4 (398.9 mg,0.56mmol, 41.8%) is obtained by using YK-413-PM3 (480 mg,1.34 mmol) and heptadecan-9-yl 6-bromohexanoate (580.3mg, 1.34mmol) as raw materials according to the method for synthesizing YK-401. C 43 H 86 N 2 O 5 , MS(ES): m/z(M+H + )711.7。
Step five: synthesis of 6- ((2-hydroxyethyl) (3- ((2-hydroxyethyl) (4-oxo-4- (undecyloxy) butyl) amino) propyl) amino) heptadecan-9-yl hexanoate (YK-413)
YK-413-PM4 (398.9 mg,0.56 mmol) and bromoethanol (310.2 mg, 2.48 mmol) are used as raw materials, and YK-401 is synthesized to obtain YK-413 (104.8 mg, 0.14mmol, 24.8%). C 45 H 90 N 2 O 6 , MS(ES): m/z(M+H + )755.8。
1 H NMR (400 MHz, CDCl 3 ) δ 4.90 – 4.81 (m, 1H), 4.06 (t, J = 6.8 Hz, 2H), 3.67 (s, 4H), 2.70 (s, 12H), 2.37 – 2.28 (m, 4H), 1.85 – 1.79 (m, 2H), 1.68 – 1.58 (m, 6H), 1.50 (d, J = 5.3 Hz, 4H), 1.26 (s, 44H), 0.88 (t, J = 6.6 Hz, 9H).
14. Synthesis of YK-414
The synthetic route is as follows:
the method comprises the following steps: synthesis of decyl 4- ((3- (tert-butoxycarbonyl) amino) propyl) aminobutyrate (YK-414-PM 1)
YK-414-PM1 (760 mg, 1.90mmol, 58.4%) was obtained from tert-butyl (3-aminopropyl) carbamate (577.8 mg, 3.32 mmol) and n-decyl 4-bromobutyrate (1.00g, 3.25mmol) as starting materials by the method for synthesizing YK-401. C 22 H 44 N 2 O 4 , MS(ES): m/z(M+H + )401.4。
Step two: synthesis of decyl 4- ((3- (tert-butoxycarbonyl) amino) propyl) (2-hydroxyethyl) amino) butyrate (YK-414-PM 2)
YK-414-PM2 (600 mg, 1.35mmol, 71.0%) was obtained by the method for synthesizing YK-401 using YK-414-PM1 (760 mg, 1.90 mmol) and bromoethanol (284.0 mg, 2.27 mmol) as raw materials. C 24 H 48 N 2 O 5 , MS(ES): m/z(M+H + )445.3。
Step three: synthesis of decyl 4- ((3-aminopropyl) (2-hydroxyethyl) amino) butyrate (YK-414-PM 3)
YK-414-PM3 (465 mg, 1.35mmol, 100%) is obtained by using YK-414-PM2 (600 mg, 1.35 mmol) as a raw material and adopting a method for synthesizing YK-413-PM 3. C 19 H 40 N 2 O 3 , MS(ES): m/z(M+H + )345.3。
Step four: synthesis of decyl 4- ((3- (heptadecan-9-yloxy) -4-oxobutyl) amino) propyl) (2-hydroxyethyl) amino) butyrate (YK-414-PM 4)
YK-414-PM4 (308.0 mg,0.46mmol, 46.0%) was obtained by synthesizing YK-401 using YK-414-PM3 (344.5 mg,1.00 mmol) and 4-bromobutyric acid-heptadecan-9-yl ester (405.5mg, 1.00mmol) as raw materials. C 40 H 80 N 2 O 5 , MS(ES): m/z(M+H + )669.6。
Step five: synthesis of decyl 4- (3- ((4- (heptadecan-9-yloxy) -4-oxobutyl) (2-hydroxyethyl) amino) propyl) (2-hydroxyethyl) aminobutyrate (YK-414)
YK-414 (80.0 mg,0.11mmol, 23.9%) was obtained by the method for synthesizing YK-401 using YK-414-PM4 (308.0 mg,0.46 mmol) and bromoethanol (172.4 mg, 1.38 mmol) as raw materials. C 42 H 84 N 2 O 6 , MS(ES): m/z(M+H + )713.6。
1 H NMR (400 MHz, CDCl 3 ) δ 4.94 – 4.84 (m, 1H), 4.09 (t, J = 6.8 Hz, 2H), 3.74 (s, 4H), 2.79 (s, 12H), 2.38 (d, J = 6.5 Hz, 4H), 1.88 (s, 4H), 1.71 – 1.60 (m, 4H), 1.54 (s, 4H), 1.31 (d, J = 16.1 Hz, 38H), 0.91 (t, J = 6.6 Hz, 9H).
15. Synthesis of YK-415
The synthetic route is as follows:
the method comprises the following steps: synthesis of 6- ((3- (2-hydroxyethyl) (4-oxo-4- (undecyloxy) butyl) amino) propyl) amino) hexanoic acid 2-octyldecyl ester (YK-415-PM 1)
YK-415-PM1 (320.0 mg,0.44mmol, 52.4%) was obtained by synthesizing YK-401 using YK-413-PM3 (300 mg, 0.84 mmol) and 2-octyldecyl 6-bromohexanoate (374.0 mg, 0.84 mmol) as raw materials. C 44 H 88 N 2 O 5 , MS(ES): m/z(M+H + )725.7。
Step two: synthesis of 6- ((2-hydroxyethyl) (3- ((2-hydroxyethyl) (4-oxo-4- (undecyloxy) butyl) amino) propyl) amino) hexanoic acid 2-octyldecyl ester (YK-415)
YK-415 (37.0 mg,0.05mmol, 10.9%) was obtained by synthesizing YK-401 using YK-415-PM1 (320.0 mg,0.44 mmol) and bromoethanol (200.0 mg, 1.60 mmol) as raw materials. C 46 H 92 N 2 O 6 , MS(ES): m/z(M+H + )769.8。
1 H NMR (400 MHz, CDCl 3 ) δ 4.07 (t, J = 6.5 Hz, 4H), 3.96 (d, J = 5.6 Hz, 2H), 3.30 (s, 4H), 3.16 (s, 2H), 2.48 (s, 2H), 2.33 (t, J = 7.0 Hz, 2H), 2.25 – 2.18 (m, 2H), 2.02 (s, 2H), 1.90 (s, 3H), 1.62 (s, 8H), 1.43 (s, 4H), 1.28 (d, J = 15.2 Hz, 48H), 0.87 (d, J = 7.0 Hz, 9H).
16. Synthesis of YK-416
The synthetic route is as follows:
the method comprises the following steps: synthesis of decyl 6- (3- (tert-butoxycarbonyl) amino) propyl) aminocaproate (YK-416-PM 1)
YK-416-PM1 (754 mg, 1.76mmol, 26.9%) was obtained by the method for synthesizing YK-401 using tert-butyl (3-aminopropyl) carbamate (1.14 g, 6.54 mmol) and n-decyl 6-bromohexanoate (2.20 g, 6.56 mmol) as raw materials. C 24 H 48 N 2 O 4 , MS(ES): m/z(M+H + )429.3。
Step two: synthesis of decyl 6- ((3- ((tert-butoxycarbonyl) amino) propyl) (2-hydroxyethyl) amino) hexanoate (YK-416-PM 2)
YK-416-PM2 (620 mg, 1.31mmol, 74.5%) was obtained by synthesizing YK-401 using YK-416-PM1 (754 mg, 1.76 mmol) and bromoethanol (284.0 mg, 2.27 mmol) as raw materials. C 26 H 52 N 2 O 5 , MS(ES): m/z(M+H + )473.4。
Step three: synthesis of decyl 6- ((3-aminopropyl) (2-hydroxyethyl) amino) hexanoate (YK-416-PM 3)
YK-416-PM3 (488 mg, 1.31mmol, 100%) was obtained by using YK-416-PM2 (620 mg, 1.31 mmol) as a raw material and following the method for synthesizing YK-413-PM 3. C 21 H 44 N 2 O 3 , MS(ES): m/z(M+H + )373.3。
Step four: synthesis of decyl 6- ((2-hydroxyethyl) (3- ((6- ((2-octyldecyl) oxy) -6-oxohexyl) amino) propyl) amino) hexanoate (YK-416-PM 4)
YK-416-PM4 (58 mg,0.08mmol, 12.6%) was obtained by synthesizing YK-401 using YK-416-PM3 (244 mg, 0.65 mmol) and 2-octyldecyl 6-bromohexanoate (278.5 mg, 0.62 mmol) as raw materials. C 45 H 90 N 2 O 5 , MS(ES): m/z(M+H + )739.7。
Step five: synthesis of decyl 6- ((2-hydroxyethyl) (3- ((2-hydroxyethyl) (6- ((2-octyldecyl) oxy) -6-oxohexyl) amino) propyl) amino) hexanoate (YK-416)
YK-416 (30 mg, 0.04mmol, 48.8%) was obtained from YK-416-PM4 (58 mg,0.08 mmol) and bromoethanol (19.6 mg, 0.16 mmol) as starting materials by the method for synthesizing YK-401. C 47 H 94 N 2 O 6 , MS(ES): m/z(M+H + )783.7。
1 H NMR (400 MHz, CDCl 3 ) δ 4.05 (t, J = 6.8 Hz, 2H), 3.96 (d, J = 5.8 Hz, 2H), 3.90 (s, 2H), 3.14 (s, 2H), 3.06 (s, 2H), 2.95 (s, 2H), 2.32 (t, J = 7.3 Hz, 4H), 2.26 – 2.16 (m, 2H), 2.02 (s, 1H), 1.73 (s, 4H), 1.69 – 1.59 (m, 8H), 1.44 – 1.36 (m, 4H), 1.28 (d, J = 14.6 Hz, 48H), 0.88 (t, J = 6.7 Hz, 9H).
17. Synthesis of YK-417
The synthetic route is as follows:
the method comprises the following steps: synthesis of decyl 6- ((3- ((6- ((3-hexyl) oxy) -6-oxohexyl) amino) propyl) (2-hydroxyethyl) amino) hexanoate (YK-417-PM 1)
YK-417-PM1 (44.3 mg,0.06mmol, 30.3%) was obtained by using YK-416-PM3 (81.1 mg, 0.22 mmol) and 3-hexylnonyl 6-bromohexanoate (83.9 mg,0.21 mmol) as raw materials according to the method for synthesizing YK-401. C 42 H 84 N 2 O 5 , MS(ES): m/z(M+H + )697.6。
Step two: synthesis of decyl 6- ((3- ((6- ((3-hexyl) oxy) -6-oxohexyl) (2-hydroxyethyl) amino) propyl) (2-hydroxyethyl) amino) hexanoate (YK-417)
YK-417-PM1 (44.3 mg,0.06 mmol) and bromoethanol (15.9 mg,0.13 mmol) were used as raw materials to obtain YK-417 (26.3 mg, 0.04mmol, 59.1%) according to the method for synthesizing YK-401. C 44 H 88 N 2 O 6 , MS(ES): m/z(M+H + )741.7。
1 H NMR (400 MHz, CDCl 3 ) δ 4.06 (dd, J = 15.3, 8.1 Hz, 4H), 3.82 (s, 2H), 2.94 (d, J = 17.7 Hz, 6H), 2.82 (s, 2H), 2.36 – 2.26 (m, 4H), 2.03 (d, J = 11.7 Hz, 4H), 1.61 (ddd, J = 21.2, 14.6, 7.3 Hz, 12H), 1.43 – 1.20 (m, 43H), 0.88 (t, J = 6.4 Hz, 9H).
18. Synthesis of YK-418
The synthetic route is as follows:
the method comprises the following steps: synthesis of 6- ((3- ((4- (decyloxy) -4-oxobutyl) (2-hydroxyethyl) amino) propyl) amino) hexyl 3-hexylnonanoate (YK-418-PM 1)
YK-414-PM3 (231.7 mg,0.67 mmol) and 3-hexylnonanoic acid 6-bromohexyl ester (258.4 mg, 0.64 mmol) are used as raw materials, and YK-418-PM1 (111.5 mg, 0.17mmol, 26.0%) is obtained according to the method for synthesizing YK-401. C 40 H 80 N 2 O 5 , MS(ES): m/z(M+H + )669.6。
Step two: synthesis of 6- ((3- ((4- (decyloxy) -4-oxobutyl) (2-hydroxyethyl) amino) propyl) (2-hydroxyethyl) amino) hexyl 3-hexylnonanoate (YK-418)
YK-418 (26.3 mg, 0.04mmol, 21.7%) was obtained by the method for synthesizing YK-401 using YK-418-PM1 (111.5 mg, 0.17 mmol) and bromoethanol (25.0 mg,0.20 mmol) as raw materials. C 42 H 84 N 2 O 6 , MS(ES): m/z(M+H + )713.6。
1 H NMR (400 MHz, chloroform-d) δ 4.05 (q, J = 6.5 Hz, 4H), 3.68-3.50 (m, 4H), 2.76-2.48 (m, 12H), 2.32 (t, J = 7.1 Hz, 2H), 2.22 (d, J = 6.9 Hz, 2H), 1.81 (dt, J = 15.0, 7.2 Hz, 3H), 1.68-1.56 (m, 6H), 1.50 (d, J = 7.3 Hz, 2H), 1.28 (d, J = 15.8 Hz, 38H), 0.88 (t, J = 6.6 Hz, 9H).
19. Synthesis of YK-419
The synthetic route is as follows:
the method comprises the following steps: synthesis of heptadecan-9-yl 6- ((2-hydroxyethyl) amino) hexanoate (YK-419-PM 1):
the synthesis of YK-401 was carried out using heptadecane-9-ester 6-bromohexanoate (500mg, 1.19mmol) and ethanolamine (291.2mg, 4.77mmol) as starting materials to obtain YK-419-PM1 (459.1 mg, 1.11mmol, 93.3%). C 25 H 51 NO 3 ，MS(ES): m/z(M+H + )414.4。
Step two: synthesis of di (heptadecan-9-yl) 6,6' - (propane-1, 3-diyl-bis ((2-hydroxyethyl) azenediyl)) dihexanoate (YK-419)
YK-419-PM1 (300.0 mg, 0.73 mmol) and potassium carbonate (302.7 mg, 2.19mmol) were dissolved in acetonitrile (3 mL), and 1, 3-dibromopropane (73.2 mg, 0.36mmol) was slowly added to the system, heated to 50 ℃ and stirred for reaction for 6 hours. After completion of the reaction, 20mL of a saturated aqueous solution of sodium hydrogencarbonate was added to the reaction mixture, followed by extraction with dichloromethane (20 mL. Times.2), and the organic phases were combined and washed with brine (20 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure in vacuo to remove the solvent. The residue was purified by silica gel chromatography (dichloromethane/methanol) to give YK-419 (47.5mg, 0.05mmol, 15.2%), C 53 H 106 N 2 O 6 , MS(ES): m/z(M+H + )867.8。
1 H NMR (400 MHz, chloroform-d) δ 4.86 (p, J = 6.2 Hz, 2H), 3.69 (s, 4H), 2.73 (s, 8H), 2.67-2.60 (m, 4H), 2.29 (t, J = 7.3 Hz, 4H), 1.84-1.75 (m, 2H), 1.64 (q, J = 7.6 Hz, 4H), 1.55-1.47 (m, 10H), 1.26 (s, 56H), 0.88 (t, J = 6.6 Hz, 12H).
20. Synthesis of YK-420
The synthetic route is as follows:
the method comprises the following steps: synthesis of (4-aminobutyl) -carbamic acid tert-butyl ester (YK-420-PM 1)
1, 4-butanediamine (10.00g, 113.44mmol) was dissolved in dichloromethane (100 mL) and potassium carbonate (15.67g, 11) was added3.44 mmol), slowly adding di-tert-butyl dicarbonate (6.20g, 28.36mmol) dropwise, slowly heating to room temperature after the dropwise addition, reacting for 2 hours, after the reaction is finished, leaching by using kieselguhr, washing mother liquor twice by using saturated saline solution (100 mL multiplied by 2), separating liquid, drying organic phase anhydrous sodium sulfate, filtering, and removing the solvent by vacuum concentration under reduced pressure of filtrate. Thus obtaining YK-420-PM1 (4616.4 mg, 24.52mmol, 21.6%). C 9 H 20 N 2 O 2 , MS(ES): m/z(M+H + )189.2。
Step two: synthesis of heptadecan-9-yl 6- ((4- (tert-butoxycarbonyl) amino) butyl) amino) hexanoate (YK-420-PM 2)
YK-420-PM2 (1.26 g, 2.33mmol, 52.1%) was obtained by synthesizing YK-401 using YK-420-PM1 (1.04 g, 5.52 mmol) and heptadecan-9-yl 6-bromohexanoate (2.00 g,4.77 mmol) as raw materials. C 32 H 64 N 2 O 4 , MS(ES): m/z(M+H + )541.5。
Step three: synthesis of 6- (((4- (tert-butoxycarbonyl) amino) butyl) (2-hydroxyethyl) amino) heptadecan-9-yl hexanoate (YK-420-PM 3)
YK-420-PM3 (980.4 mg, 1.68mmol, 72.1%) was obtained by using YK-420-PM2 (1.26 g, 2.33 mmol) and bromoethanol (0.35 g, 2.80 mmol) as raw materials according to the method for synthesizing YK-401. C 34 H 68 N 2 O 5 , MS(ES): m/z(M+H + )585.5。
Step four: synthesis of heptadecan-9-yl 6- ((4-aminobutyl) (2-hydroxyethyl) amino) hexanoate (YK-420-PM 4)
YK-420-PM4 (814.5 mg, 1.68mmol, 100%) was obtained by the method for synthesizing YK-413-PM3 using YK-420-PM3 (980.4 mg, 1.68 mmol) as the raw material. C 29 H 60 N 2 O 3 , MS(ES): m/z(M+H + )485.5。
Step five: synthesis of heptadecan-9-yl 6- ((4- ((6- (heptadecan-9-yloxy) -6-oxohexyl) (2-hydroxyethyl) amino) butyl) amino) hexanoate (YK-420-PM 5)
YK-420-PM5 (306.0 mg, 0.37mmol, 22.0％)。C 52 H 104 N 2 O 5 , MS(ES): m/z(M+H + )837.9。
step six: synthesis of bis (heptadecan-9-yl) 6,6' - (butane-1, 4-diylbis ((2-hydroxyethyl) azenediyl)) dihexanoate (YK-420)
YK-420 (130.5 mg, 0.15mmol, 40.5%) was obtained from YK-420-PM5 (306.0 mg, 0.37 mmol) and bromoethanol (54.8mg, 0.44mmol) as starting materials by the method for synthesizing YK-401. C 54 H 108 N 2 O 6 , MS(ES): m/z(M+H + )881.8。
1 H NMR (400 MHz, CDCl 3 ) δ 4.93 – 4.83 (m, 2H), 3.84 (s, 4H), 2.94 (d, J = 24.3 Hz, 9H), 2.33 (t, J = 7.4 Hz, 4H), 1.82 (s, 4H), 1.68 (dd, J = 15.3, 7.6 Hz, 9H), 1.53 (d, J = 5.5 Hz, 8H), 1.43 – 1.37 (m, 5H), 1.29 (s, 51H), 0.91 (t, J = 6.7 Hz, 12H).
21. Synthesis of YK-421
The synthetic route is as follows
The method comprises the following steps: synthesis of decyl 4- ((4- ((tert-butoxycarbonyl) amino) butyl) amino) butyrate (YK-421-PM 1)
YK-420-PM1 (2.00g, 10.62mmol) and decyl 4-bromobutyrate (3.20g, 10.41mmol) are taken as raw materials, and the method for synthesizing YK-401 is carried out to obtain YK-421-PM1 (1300 mg, 3.14mmol, 30.1%). C 23 H 46 N 2 O 4 , MS(ES): m/z(M+H + )415.4。
Step two: synthesis of decyl 4- ((4- ((tert-butoxycarbonyl) amino) butyl) (2-hydroxyethyl) amino) butyrate (YK-421-PM 2)
YK-421-PM2 (1.20 g, 2.62mmol, 83.3%) was obtained by the method for synthesizing YK-401 using YK-421-PM1 (1.30 g, 3.14 mmol) and bromoethanol (469 mg, 3.75 mmol) as raw materials. C 25 H 50 N 2 O 5 , MS(ES): m/z(M+H + )459.4。
Step three: synthesis of decyl 4- ((4-aminobutyl) (2-hydroxyethyl) amino) butyrate (YK-421-PM 3)
YK-421-PM3 (720 mg,2.01mmol, 76.6%) was obtained by the method for synthesizing YK-413-PM3 using YK-421-PM2 (1.20 g, 2.62 mmol) as a raw material. C 20 H 42 N 2 O 3 , MS(ES): m/z(M+H + )359.3。
Step four: synthesis of 6- ((4- ((4- (decyloxy) -4-oxobutyl) (2-hydroxyethyl) amino) butyl) amino) heptadecan-9-yl hexanoate (YK-421-PM 4)
YK-421-PM4 (106 mg, 0.15mmol, 9.7%) was obtained by synthesizing YK-401 using YK-421-PM3 (580 mg, 1.62 mmol) and heptadecan-9-yl 6-bromohexanoate (664.2mg, 1.53mmol) as raw materials. C 43 H 86 N 2 O 5 , MS(ES): m/z(M+H + )711.7。
Step five: synthesis of heptadecan-9-yl 6- ((4- ((4- (decyloxy) -4-oxobutyl) (2-hydroxyethyl) amino) butyl) (2-hydroxyethyl) amino) hexanoate (YK-421)
YK-421-PM4 (106 mg, 0.15 mmol) and bromoethanol (37.5 mg, 0.30 mmol) are used as raw materials to obtain YK-421 (23.1 mg, 0.03mmol, 20.4%) according to the method for synthesizing YK-401. C 45 H 90 N 2 O 6 , MS(ES): m/z(M+H + )755.7。
1 H NMR (400 MHz, CDCl 3 ) δ 4.95 – 4.72 (m, 1H), 4.07 (t, J = 6.8 Hz, 2H), 3.92 (d, J = 24.6 Hz, 2H), 3.08 (d, J = 46.2 Hz, 6H), 2.42 (t, J = 6.3 Hz, 2H), 2.37 – 2.25 (m, 2H), 2.26 – 2.16 (m, 2H), 2.02 (s, 4H), 1.96 – 1.88 (m, 2H), 1.83 (s, 2H), 1.65 (dd, J = 14.3, 7.6 Hz, 8H), 1.50 (s, 4H), 1.26 (s, 42H), 0.88 (t, J = 6.6 Hz, 9H).
22. Synthesis of YK-422
The synthetic route is as follows:
the method comprises the following steps: synthesis of 2-octyldecyl 6- ((3-hydroxypropyl) amino) hexanoate (YK-422-PM 1)
With 6-bromohexanoic acid-2-octyldecyl ester (200mg, 0.46mmol) and 3-aminopropan-1-ol (174.6 mg, 2)24 mmol) as a starting material, according to the method for synthesizing YK-401, YK-422-PM1 (91 mg,0.21mmol, 46.1%) was obtained. C 27 H 55 NO 3 ，MS(ES): m/z(M+H + )442.4。
Step two: synthesis of 6,6' - (ethane-1, 2-diyl-bis ((3-hydroxypropyl) azelidinyl)) bis (2-octyldecyl) dihexanoate (YK-422)
YK-422 (25.1 mg, 0.03mmol, 34.5%) was obtained by synthesizing YK-419 from YK-422-PM1 (71 mg, 0.16 mmol) and 1, 2-dibromoethane (15.1mg, 0.08mmol). C 56 H 112 N 2 O 6 , MS(ES): m/z(M+H + )909.8。
1 H NMR (400 MHz, chloroform-d) δ 3.96 (d, J = 5.8 Hz, 4H), 3.76 (t, J = 5.1 Hz, 4H), 3.54 (q, J = 7.2 Hz, 2H), 3.45 (s, 4H), 3.19 (t, J = 6.7 Hz, 4H), 3.03-2.93 (m, 4H), 2.32 (t, J = 7.2 Hz, 4H), 1.98-1.87 (m, 4H), 1.80-1.54 (m, 12H), 1.26 (s, 58H), 0.87 (d, J = 7.0 Hz, 12H).
23. Synthesis of YK-423
The synthetic route is as follows:
synthesis of 6,6' - (propane-1, 3-diyl-bis ((3-hydroxypropyl) azelidinyl)) bis (2-octyldecyl) dihexanoate (YK-423)
YK-423 (59.9 mg,0.06mmol, 28.2%) was obtained by synthesizing YK-419 from YK-422-PM1 (200 mg, 0.45 mmol) and 1, 3-dibromopropane (45.6 mg, 0.23mmol). C 57 H 114 N 2 O 6 , MS(ES): m/z(M+H + )923.8。
1 H NMR (400 MHz, chloroform-d) δ 3.96 (d, J = 5.7 Hz, 4H), 3.78 (t, J = 5.1 Hz, 4H), 2.75 (s, 4H), 2.57 (s, 8H), 2.31 (t, J = 7.4 Hz, 4H), 1.78 (d, J = 22.1 Hz, 4H), 1.66-1.55 (m, 8H), 1.27 (s, 64H), 0.88 (t, J = 6.7 Hz, 12H).
24. Synthesis of YK-424
The synthetic route is as follows:
the method comprises the following steps: synthesis of 6- (((4- (tert-butoxycarbonyl) amino) butyl) amino) hexanoic acid-2-octyldecyl ester (YK-424-PM 1)
YK-420-PM1 (200 mg, 1.06 mmol) and 6-bromohexanoic acid-2-octyldecyl ester (475.5 mg, 1.06 mmol) are used as raw materials, and YK-424-PM1 (141.8 mg, 0.26mmol, 24.5%) is obtained according to the method for synthesizing YK-401. C 33 H 66 N 2 O 4 , MS(ES): m/z(M+H + )555.6。
Step two: synthesis of 2-octyldecyl 6- ((4- (tert-butoxycarbonyl) amino) butyl) (3-hydroxypropyl) aminocaproate (YK-424-PM 2)
YK-424-PM2 (137.1 mg, 0.22mmol, 84.6%) was obtained by synthesizing YK-401 using YK-424-PM1 (141.8 mg, 0.26 mmol) and 3-bromo-1-propanol (78.1 mg,0.56 mmol) as raw materials. C 36 H 72 N 2 O 5 , MS(ES): m/z(M+H + )613.5。
Step three: synthesis of 2-octyldecyl 6- ((4-aminobutyl) (3-hydroxypropyl) amino) hexanoate (YK-424-PM 3)
YK-424-PM3 (103.3 mg,0.20mmol, 90.9%) was obtained by the method for synthesizing YK-413-PM3 using YK-424-PM2 (137.1 mg, 0.22 mmol) as a raw material. C 31 H 64 N 2 O 3 , MS(ES): m/z(M+H + ) 513.4。
Step four: synthesis of 6- ((3-hydroxypropyl) (4- ((6- (2-octyldecyl) oxy) -6-oxohexyl) amino) butyl) amino) hexanoic acid-2-octyldecyl ester (YK-424-PM 4)
YK-424-PM4 (46.7 mg,0.05mmol, 27.9%) was obtained by synthesizing YK-401 using YK-424-PM3 (103.3 mg,0.20 mmol) and 2-octyldecyl 6-bromohexanoate (85.5 mg, 0.19mmol) as raw materials. C 55 H 110 N 2 O 5 , MS(ES): m/z(M+H + )879.9。
Step five: synthesis of 6,6' - (butane-1, 4-diylbis ((3-hydroxypropyl) azelidinyl)) bis (2-octyldecyl) dihexanoate (YK-424)
Using YK-424-PM4 (46.7 mg,0.05 mmol) and 3-bromo-1-propanol(7.2 mg,0.05 mmol) as a starting material, YK-424 (23.6 mg, 0.03mmol, 50.0%) was obtained according to the method for synthesizing YK-401. C 58 H 116 N 2 O 6 MS(ES): m/z(M+H + )937.9。
1 H NMR (400 MHz, chloroform-d) δ 3.96 (d, J = 5.8 Hz, 4H), 3.76 (t, J = 5.1 Hz, 4H), 3.54 (q, J = 7.2 Hz, 2H), 3.45 (s, 4H), 3.19 (t, J = 6.7 Hz, 4H), 3.03-2.93 (m, 4H), 2.32 (t, J = 7.2 Hz, 4H), 1.98-1.87 (m, 4H), 1.80-1.54 (m, 16H), 1.26 (s, 58H), 0.87 (d, J = 7.0 Hz, 12H).
25. Synthesis of YK-009
YK-009 162mg was obtained according to CN 114044741B.
26.8 Synthesis of (8- ((3-hexylnonyl) oxy) -8-oxooctyl) - ((2-hydroxyethyl) amino) octanoic acid heptadecan-9-yl ester (Compound 21)
The synthetic route is as follows:
the method comprises the following steps: synthesis of heptadecan-9-yl 8-bromooctanoate (Compound 21-PM 1)
9-Heptadecanol (1.00g, 3.90mmol) and 8-bromooctanoic acid (1.04g, 4.66mmol) were dissolved in dichloromethane (10 mL), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.90 g, 4.68 mmol) and 4-dimethylaminopyridine (24 mg,0.20 mmol) were added, and the reaction was stirred at 30-35 ℃ for 8 hours. After the reaction, the reaction solution was washed with saturated sodium carbonate, saturated brine and Na 2 SO 4 And (5) drying. The mixture was filtered, the filtrate was concentrated under reduced pressure in vacuo and purified by silica gel chromatography (ethyl acetate/n-hexane) to give 9-heptadecyl 8-bromooctanoate (1.28g, 2.77mmol, 71.0%).
Step two: synthesis of heptadecan-9-yl 8- ((2-hydroxyethyl) amino) octanoate (Compound 21-PM 2)
Heptadecan-9-yl 8-bromooctanoate (500mg, 1.08mmol) and ethanolamine (119mg, 3.25mmol) were dissolved in acetonitrile (5 mL), potassium carbonate (149 mg,1.08 mmol) was added, and the reaction was heated to 70 ℃ and stirred for 2 hours. Inverse directionAfter the reaction solution is cooled to room temperature, filtering, and vacuum concentrating the filtrate to remove the solvent. The residue was purified by silica gel chromatography (methanol/dichloromethane) to give 9-heptadecyl-8- ((2-hydroxyethyl) amino) octanoate (365 mg, 0.83mmol, 76.9%), C 27 H 55 NO 3 , MS(ES): m/z(M+H + )442.3。
Step three: synthesis of 3-hexylnonyl 8-bromooctanoate (Compound 21-PM 3)
3-Hexylnonanol (1.00g, 4.38mmol) and 8-bromooctanoic acid (1.17g, 5.25mmol) were used as starting materials, and purified by silica gel chromatography (ethyl acetate/n-hexane) according to the method for producing Compound 21-PM1, to give 8-bromooctanoic acid-3-hexylnonanoate (1.57g, 3.62mmol, 82.6%).
Step four: synthesis of 8- (8- ((3-hexylnonyl) oxy) -8-oxooctyl) - ((2-hydroxyethyl) amino) octanoic acid heptadecan-9-yl ester (Compound 21)
Heptadecan-9-yl 8- ((2-hydroxyethyl) amino) octanoate (200mg, 0.46mmol) and 3-hexylnonyl 8-bromooctanoate (336mg, 0.82mmol) were dissolved in acetonitrile (6 mL), potassium carbonate (254mg, 1.84mmol) and potassium iodide (8.3mg, 0.05mmol) were added, and the reaction was stirred at 70 ℃ for 20 hours. The reaction solution was cooled to room temperature and then filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (ethyl acetate/n-hexane) to obtain the objective compound (220mg, 0.28mmol, 60.9%). C 50 H 99 NO 5 ，MS(ES): m/z(M+H + )794.8。
1 H NMR (400 MHz, CDCl 3 ) δ 4.90 (p, J = 6.3 Hz, 1H), 4.21 – 4.02 (m, 2H), 3.66 (s, 2H), 2.73 (s, 2H), 2.60 (s, 4H), 2.43 – 2.20 (m, 4H), 2.12 – 1.99 (m, 1H), 1.75 – 1.49 (m, 13H), 1.48 – 1.39 (m, 2H), 1.42 – 1.15 (m, 56H), 0.92 (td, J = 6.8, 2.2 Hz, 12H).
27.8 Synthesis of bis (3-hexylnonyl) dioctoate (Compound 23) ((2-hydroxyethyl) azanediyl)
The synthetic route is as follows:
8-Bromobiocaprylic acid-3-hexylnonyl ester (710mg, 1.64mmol) and ethanolamine (40mg, 0.66mmol) were dissolved in acetonitrile (10 mL), and potassium carbonate (1.09 g, 7.92 mmol) and potassium iodide (66mg, 0.39mmol) were added to the above system, and the reaction was stirred at 70 ℃ for 20 hours. After the reaction is finished, the reaction liquid is cooled to room temperature and then filtered, and the filtrate is subjected to vacuum decompression concentration to remove the solvent. The residue was purified by silica gel chromatography (methanol/dichloromethane) to give bis (3-hexylnonyl) dioctoate (160 mg,0.21mmol, 31.8%) 8,8' - ((2-hydroxyethyl) azanediyl) dioctoate (C) 48 H 95 NO 5 , MS(ES): m/z(M+H + )766.5。
1 H NMR (400 MHz, CDCl 3 ) δ 4.12 (t, J = 7.1 Hz, 4H), 3.62 (s, 2H), 2.68 (s, 2H), 2.51 (d, J = 25.8 Hz, 4H), 2.32 (t, J = 7.5 Hz, 4H), 1.72 – 1.57 (m, 8H), 1.55 – 1.40 (m, 6H), 1.40 – 1.17 (m, 55H), 0.92 (t, J = 6.8 Hz, 12H).
Example 2: optimization of preparation conditions of nano lipid particles (LNP preparation)
1. Optimization of vector (liposome) to mRNA ratio
The cationic lipid compound YK-407 synthesized in example 1 was dissolved in ethanol at a molar ratio of 49:10:39.5:1.5 with DSPC (avigato (shanghai) pharmaceutical technology co., ltd.), cholesterol (avigato (shanghai) pharmaceutical technology co., ltd.), and DMG-PEG2000, respectively, to prepare an ethanol lipid solution. The ethanoliphatic solution was quickly added to citrate buffer (pH =4 to 5) by ethanol injection method, and vortexed for 30s for use. eGFP-mRNA was diluted in citrate buffer (pH =4 to 5) to obtain an mRNA aqueous solution. Liposomes were prepared from a volume of liposome solution and aqueous mRNA solution at a weight ratio of total lipid to mRNA of 5, 10, 1, 15, 1, 30 and 35. Carrying out ultrasonic treatment at 25 ℃ for 15min (ultrasonic frequency 40kHz, ultrasonic power 800W). The resulting liposomes were diluted to 10 volumes with PBS and then ultrafiltered in 300kDa ultrafilter to remove ethanol. The volume was then brought to volume with PBS to give a LNP formulation that encapsulated eGFP-mRNA using the cationic lipid YK-407/DSPC/cholesterol/DMG-PEG 2000 (mole percent 49: 39.5.
The results of cell transfection experiments show that the vector to mRNA weight ratio is within the range of 10 to 30, and the transfection effect is better in all the ranges of 15. (FIG. 1)
The same results were obtained for LNP formulations prepared using YK-401, YK-402, YK-403, YK422 and YK-423, which are not shown.
2. Optimization of cationic lipid to neutral lipid ratio
LNP formulations encapsulating eGFP-mRNA were prepared according to the method in 1, wherein the molar ratio of cationic lipid YK-407 to neutral lipid DSPC was 1.
As can be seen from cell transfection experiments, the cationic lipid and neutral lipid have a molar ratio of 1 to 15, and the transfection efficiency is 4. (FIG. 2)
The resulting LNP formulations were prepared using YK-401, YK-402, YK-403, YK422, and YK-423 with similar results, not shown.
3. Optimization of proportion of polymer conjugated lipid in carrier (liposome)
LNP formulations encapsulating eGFP-mRNA were prepared according to method 1, with cationic lipid YK-407 in the vehicle, and polymer conjugated lipid DMG-PEG2000 at 0.5%, 1.5%, 3.5%, 5%, 10%, and 15% molar ratios of the vehicle, respectively.
Cell transfection experiment results show that the molar ratio of the polymer conjugated lipid to the carrier is in the range of 0.5-10%, the transfection effect is achieved, the highest transfection efficiency is achieved at 1.5%, and the lowest transfection efficiency is achieved at 10%. (FIG. 3)
The same results were obtained for LNP formulations prepared using YK-401, YK-402, YK-403, YK422 and YK-423, which are not shown.
4. Optimization of proportion of each component in carrier (liposome)
The LNP formulation encapsulating eGFP-mRNA was prepared according to the procedure in 1, wherein the molar ratio of the cationic lipid YK-407, neutral lipid DSPC, structural lipid cholesterol and polymer conjugated lipid DMG-PEG2000.
Through cell transfection experiments, the molar ratios of the cationic lipid, the neutral lipid, the structural lipid and the polymer conjugated lipid are within the following ratio of 75. The transfection efficiency is good in the range of the proportion of (35 to 49): (7.5 to 15): (35 to 55): 1 to 5), wherein the proportion is 40. The molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the polymer conjugated lipid is shown in figure 4, and can be used for preparing LNP preparations within the range of (25 to 75): (5 to 25): (15 to 65): (0.5 to 10), the preferred proportion is (35 to 49): (7.5 to 15): (35 to 55): 1 to 5), wherein the optimal proportion is 48.5.
The resulting LNP formulations were prepared using YK-401, YK-402, YK-403, YK422, and YK-423 with similar results, not shown.
Example 3: LNP preparation cell transfection assay for eGFP-mRNA
Cell recovery and passage: the 293T cells were recovered and cultured in a petri dish for passage to the desired number of cells.
Blank plate: cells in the dish 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 the cells were adherent.
Cell transfection experiments: LNP preparation containing 1.5. Mu.g of eGFP-mRNA prepared in example 2 (cationic lipid in vehicle was YK-407) and Lipofectamin 3000 preparation of eGFP-mRNA were added to the cell culture medium in 12-well plates, and after further culturing for 24 hours, the transfection efficiency of different samples was examined from the fluorescence intensity by observation with a fluorescence microscope.
According to the experimental results, the preparation conditions of the nano-lipide particles (LNP preparation) are finally determined: vector to mRNA weight ratio 15; the molar ratio of the cationic lipid to the neutral lipid is 4.9; the polymer conjugated lipid accounts for 1.5 percent of the liposome molar ratio; the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the polymer conjugated lipid is 49.
Example 4: preparation of Nanolipid particles (LNP formulations)
TABLE 1 cationic lipid Structure
The cationic lipids listed in table 1 were dissolved in ethanol at a molar ratio of 49.5 with DSPC (avigator (shanghai) pharmaceuticals), cholesterol (avigator (shanghai) pharmaceuticals), and DMG-PEG2000, respectively, to prepare an ethanolic lipid solution, which was rapidly added to a citrate buffer (pH =4 to 5) by ethanol injection, and vortexed for 30s for use. eGFP-mRNA (shanghai onset experimental reagents ltd) or Fluc-mRNA (shanghai onset experimental reagents ltd) was diluted in a citrate buffer (pH =4 to 5) to obtain an mRNA aqueous solution. Liposomes were prepared by mixing a volume of liposome solution with an aqueous mRNA solution at a weight ratio of total lipid to mRNA of 15. Carrying out ultrasonic treatment at 25 ℃ for 15min (ultrasonic frequency 40kHz, ultrasonic power 800W). The resulting liposomes were diluted to 10 volumes with PBS and ultrafiltered in a 300kDa ultrafiltration tube to remove ethanol. The volume was then brought to volume with PBS to give LNP formulations that encapsulated eGFP-mRNA or Fluc-mRNA using cationic lipid/DSPC/cholesterol/DMG-PEG 2000 (mole percent 49.
The Lipofectamine 3000 transfection reagent is widely used for cell transfection at present, has very good transfection performance and excellent transfection efficiency, can improve cell activity, and is suitable for cell types difficult to transfect. We selected Lipofectamine 3000 transfection reagent for control, and prepared Lipofectamine 3000 preparation of eGFP-mRNA or Fluc-mRNA according to the method of Lipofectamine 3000 (Yinxie Jie Co., ltd.) instruction manual.
Example 5: measurement of particle diameter and polydispersity index (PDI) of nano-liposome particle
Particle size and polydispersity index (PDI) were determined by dynamic light scattering using a malvern laser particle sizer.
10 μ L of liposome solution was diluted to 1mL with RNase free deionized water and added to the sample cell, and the assay was repeated 3 times for each sample. The measurement conditions were: 90 ℃ scattering angle, 25 ℃. The test results are as follows:
TABLE 2 particle size and polydispersity index (PDI)
The particle size of the nano-lipid particles prepared in example 4 is between 120 and 210nm, and the particle size of the particles prepared from YK-417 is 128nm, and the particle size of the particles prepared from ALC-0315 is 205nm. The polydispersity of all the nano-lipid particles is between 4% and 25%, wherein the minimum is YK-404 and is 4.2%, and the maximum is YK-414 and is 23.9%.
Example 6: encapsulation efficiency, drug loading concentration and total RNA concentration detection
The entrapment rate is a key quality attribute of the liposome, and refers to the percentage of the drug content encapsulated in the lipid bilayer in the total dosage, which can reflect the drug entrapment degree in the liposome. The encapsulation efficiency specified in Chinese pharmacopoeia is generally not lower than 80%.
The drug loading is the ratio of the amount of the liposome traditional Chinese medicine to the total amount of the liposome traditional Chinese medicine and the carrier, and the size of the drug loading directly influences the clinical application dosage of the medicine, so that the larger the drug loading is, the more the clinical requirement can be met. The drug loading concentration is directly proportional to the drug loading amount, and the relative proportion of the drug loading concentration can represent the relative proportion of the drug loading amount. The relative proportion of total RNA concentration can represent the relative proportion of the amount of mRNA carried by the LNP preparation.
Preparation of reagents:
1 XTE buffer, 0.1% Triton X-100 buffer, riboGreen reagent (1: 200) and mRNA standard stock solutions were prepared.
Sample detection:
an appropriate amount of the sample was taken and added to an appropriate amount of 1 XTE buffer solution, and diluted to a solution containing about 2.8. Mu.g per 1 mL. Then adding the sample into a 96-well plate, adding 50 mu L of diluted sample or mRNA standard stock solution into each well, adding 1 XTE buffer solution and 0.1% Triton X-100 buffer solution, incubating the sample at 37 ℃ for 10 minutes, adding 100 mu L RiboGreen reagent (1: 200) into each sample well of the 96-well plate, centrifuging, reading by using a microplate reader, and processing data. The specific data are shown in tables 3 and 4.
The experimental results are as follows:
(1) Compared with the cationic lipid in the prior art, the LNP preparation prepared from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 has the advantages that the encapsulation efficiency, the drug loading concentration and the total RNA concentration are all obviously improved. For example, the encapsulation efficiency of YK-407 can be improved by 29.0% compared with that of compound 23, the drug-loading concentration can reach 1.78 times of that of compound 23, and the total RNA concentration can reach 1.41 times of that of compound 21.
TABLE 3 encapsulation efficiency, drug loading concentration and Total RNA concentration assay results-1
As can be seen from table 3, the encapsulation efficiency of LNP formulations prepared from different compounds varies greatly. YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, compared with SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-009, the encapsulation efficiency is significantly improved.
The encapsulation efficiency of YK-407 is 85.2%, which is 22.3% higher than that of SM-102, 21.9% higher than that of ALC-0315, 27.2% higher than that of compound 21, 29.0% higher than that of compound 23, 25.0% higher than that of HHMA, and 21.8% higher than that of YK-009.
The encapsulation efficiency of YK-401 is 83.6%, which is improved by 20.7% compared with SM-102, 20.3% compared with ALC-0315, 25.6% compared with compound 21, 27.4% compared with compound 23, 23.4% compared with HHMA, and 20.2% compared with YK-009.
The encapsulation efficiency of YK-402 is 91.0%, which is improved by 28.1% compared with SM-102, improved by 27.7% compared with ALC-0315, improved by 33.0% compared with compound 21, improved by 34.8% compared with compound 23, improved by 30.8% compared with HHMA, and improved by 27.6% compared with YK-009.
The encapsulation efficiency of YK-403 is 87.9%, which is 25.0% higher than that of SM-102, 24.6% higher than that of ALC-0315, 29.9% higher than that of compound 21, 31.7% higher than that of compound 23, 27.7% higher than that of HHMA, and 24.4% higher than that of YK-009.
The encapsulation efficiency of YK-422 is 92.4%, which is increased by 29.5% compared with SM-102, by 29.1% compared with ALC-0315, by 34.4% compared with compound 21, by 36.2% compared with compound 23, by 32.2% compared with HHMA, and by 29.0% compared with YK-009.
The encapsulation efficiency of YK-423 is 89.7%, which is improved by 26.8% compared with SM-102, 26.4% compared with ALC-0315, 31.7% compared with compound 21, 33.5% compared with compound 23, 29.5% compared with HHMA, and 26.3% compared with YK-009.
In addition, the LNP preparation prepared from different compounds has great difference in drug-loading concentration and total RNA concentration, and the drug-loading concentration and the total RNA concentration are both obviously improved compared with SM-102, ALC-0315, compound 21, compound 23 and YK-009 through YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423.
The YK-407 drug loading concentration is 35.67 mug/mL, the total RNA concentration is 40.77 mug/mL, which are 1.34 times and 1.13 times of SM-102, 1.33 times and 1.08 times of ALC-0315, 1.62 times and 1.41 times of compound 21, 1.78 times and 1.31 times of compound 23, 1.09 times and 1.12 times of HHMA, and 1.30 times and 1.06 times of YK-009.
YK-401 drug loading concentration is 37.16 mug/mL, total RNA concentration is 41.71 mug/mL, which is 1.40 times and 1.16 times of SM-102, 1.38 times and 1.10 times of ALC-0315, 1.69 times and 1.44 times of compound 21, 1.86 times and 1.34 times of compound 23, 1.14 times and 1.14 times of HHMA, and 1.35 times and 1.08 times of YK-009.
The YK-402 drug loading concentration is 37.67 mug/mL, the total RNA concentration is 40.09 mug/mL, which are 1.42 times and 1.11 times of SM-102, 1.40 times and 1.06 times of ALC-0315, 1.71 times and 1.39 times of compound 21, 1.88 times and 1.29 times of compound 23, 1.15 times and 1.10 times of HHMA, and 1.37 times and 1.04 times of YK-009.
The YK-403 drug loading concentration is 34.21 mug/mL, the total RNA concentration is 40.53 mug/mL, which are respectively 1.29 times and 1.12 times of SM-102, 1.27 times and 1.07 times of ALC-0315, 1.56 times and 1.40 times of compound 21, 1.71 times and 1.31 times of compound 23, 1.05 times and 1.11 times of HHMA, and 1.24 times and 1.05 times of YK-009.
The YK-422 drug loading concentration is 45.05 mug/mL, the total RNA concentration is 45.76 mug/mL, which are 1.70 times and 1.27 times of SM-102, 1.68 times and 1.21 times of ALC-0315, 2.05 times and 1.58 times of compound 21, 2.25 times and 1.47 times of compound 23, 1.38 times and 1.26 times of HHMA, and 1.64 times and 1.19 times of YK-009.
YK-423 drug concentration is 37.47 mug/mL, total RNA concentration is 41.00 mug/mL, which are 1.41 times and 1.14 times of SM-102, 1.39 times and 1.08 times of ALC-0315, 1.71 times and 1.42 times of compound 21, 1.87 times and 1.32 times of compound 23, 1.15 times and 1.13 times of HHMA, and 1.36 times and 1.06 times of YK-009.
Data are analyzed by GraphPad Prism software, and any one of YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 is obviously different from SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-009, and the encapsulation efficiency, the drug loading concentration and the total RNA concentration are obviously improved.
And (3) knotting:
some of the compounds contemplated herein, including YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423, produced LNP formulations with significantly improved encapsulation efficiency, drug loading concentration, and total RNA concentration over prior art cationic lipids, such as SM-102, ALC-0315, compound 21, compound 23, HHMA, and YK-009. For example, the encapsulation efficiency of YK-407 can be improved by 29.0 percent compared with that of the compound 23, the drug-loading concentration can reach 1.78 times of that of the compound 23, and the total RNA concentration can reach 1.41 times of that of the compound 21.
(2) The LNP preparation prepared from different designed compounds has great difference between the encapsulation efficiency and the drug loading capacity, the range of the encapsulation efficiency of different compounds is 70% -95%, the drug loading concentration is 20-50 mug/mL, and the total RNA concentration is 25-55 mug/mL.
TABLE 4 encapsulation efficiency, drug loading concentration and Total RNA concentration assay results-2
As can be seen from Table 4, the encapsulation efficiency of the series of designed compounds is 70% -95%, the drug loading concentration is 20-50 mug/mL, and the total RNA concentration is 25-55 mug/mL. The different compounds have great difference, the highest encapsulation efficiency is YK-420 which reaches 94.9 percent, and the lowest encapsulation efficiency is YK-417 which is 74.8 percent; the highest drug loading concentration is YK-408 which is 45.42 mug/mL, and the lowest drug loading concentration is YK-413 which is only 23.63 mug/mL; the highest total RNA concentration is YK-408 which reaches 52.46 mug/mL, and the lowest total RNA concentration is YK-413 which is only 28.36 mug/mL.
And (3) knotting:
the LNP preparation prepared from different designed compounds has large difference between the encapsulation efficiency and the drug loading capacity, the encapsulation efficiency range of different compounds is 70% -95%, the drug loading concentration is 20-50 mug/mL, and the total RNA concentration is 25-55 mug/mL. It is clear that LNP formulations that are not prepared from structurally similar compounds must have similar encapsulation efficiencies and drug loadings.
To summarize:
some of the compounds contemplated herein, including YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423, produced LNP formulations with significantly improved encapsulation efficiency, drug loading concentration, and total RNA concentration over prior art cationic lipids, such as SM-102, ALC-0315, compound 21, compound 23, HHMA, and YK-009. For example, the encapsulation efficiency of YK-407 can be improved by 29.0% compared with that of compound 23, the drug-loading concentration can reach 1.78 times of that of compound 23, and the total RNA concentration can reach 1.41 times of that of compound 21.
Therefore, the carrier for delivering the mRNA is prepared by YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, the encapsulation rate and the drug loading rate are both obviously improved, so that the dosage of the LNP can be obviously reduced, and an LNP-mRNA preparation product with more uniform distribution and more controllable quality can be provided in the application of a multivalent mRNA vaccine.
The designed LNP preparations prepared from different compounds have large differences between the encapsulation efficiency and the drug loading capacity, the encapsulation efficiency ranges from 70% to 95%, the drug loading concentration ranges from 20 to 50 mug/mL, and the total RNA concentration ranges from 25 to 55 mug/mL. It is clear that LNP formulations that are not prepared from structurally similar compounds must have similar encapsulation efficiencies and drug loadings. In contrast, encapsulation efficiency and drug loading will most likely vary significantly and so the encapsulation efficiency and drug loading of LNP formulations prepared therefrom cannot be inferred from the structure of the compound.
Example 7: in vitro validation of the Performance of LNP delivery vectors
Cell recovery and passage: the procedure is as in example 3.
Plate preparation: the procedure is as in example 3.
1. Fluorescent detection of Fluc-mRNA
LNP preparation containing 0.3 μ g Fluc-mRNA (prepared according to example 4, LNP preparation vehicle components were cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid at a molar ratio of 49.10, 39.5, wherein the cationic lipid is the cationic lipid listed in table 1) was added to the cell culture solution in 96-well plates, and after further culturing for 24 hours, the corresponding reagents were added according to the Gaussia Luciferase Assay Kit instructions and the fluorescence expression intensity per well was measured by IVIS fluorescence detection system. The experiment verifies the transfection efficiency of the LNP preparation in cells, and the specific detection results are shown in tables 6-9.
The experimental results are as follows:
(1) The compounds of the present application, including YK-407, YK-401, YK-402, YK-403, 422, and YK-423, are very different in chemical structure from the prior art cationic lipids.
The series of compounds designed by the present application, including YK-407, YK-401, YK-402, YK-403, 422 and YK-423, are very different from the prior art cationic lipid in chemical structure. For example, these compounds are significantly different in structure from HHMA; compared to SM-102, ALC-0315, compound 21, compound 23, and YK-009, the head structure, including G 3 、G 4 And L groups are significantly different, G 1 、L 1 、R 1 、G 2 、L 2 And R 2 The groups also differ significantly. The specific structural comparison is shown in Table 5.
Table 5 shows the comparison of the structures of the compounds designed with the cationic lipids representative of the prior art
As can be seen from Table 5, the series of compounds designed, including YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, are significantly different from the chemical structures of the representative cationic lipids of the prior art. YK-009 is disclosed in CN114044741B (claim 1), SM-102 is compound 25 disclosed in WO2017049245A2 (page 29 of the specification), ALC-0315 is compound 3 disclosed in CN108368028B (page 24 of the specification), compound 21 and compound 23 are disclosed in WO2021055833A1 (page 22 of the specification), HHMA is compound 1 disclosed in CN112979483B (page 12 of the specification).
A series of compounds designed by the present application, in contrast to the prior art representative cationic lipids:
1) The structural difference with HHMA is the greatest, and the chemical structure chart shows that HHMA is only connected with 1 side chain of a group connected with a central N atom, is similar to 1 side chain of the compounds of the series, and other parts are obviously different, and the structural difference is great.
2) The head structure is significantly different compared to SM-102, ALC-0315, compound 21, compound 23, and YK-009. The designed head structure of the series of compounds comprises 2 tertiary amine groups, an L group connecting 2 tertiary amine nitrogen atoms and G respectively connected with 2 tertiary amine nitrogen atoms 3 And G 4 While the head structures of SM-102, ALC-0315, compound 21, compound 23, and YK-009, include only 1 tertiary amine group, and HO (CH) attached to the tertiary amine nitrogen atom 2 ) 2 -. Furthermore, compounds G of this series 1 、L 1 、R 1 、G 2 、L 2 And R 2 A group ofThere were also significant differences in SM-102, ALC-0315, compound 21, compound 23, and YK-009. Because the head structures are very different, the series of compounds have great differences from SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-009 in polarity, acidity-basicity, hydrophilicity and the like.
The method comprises the following specific steps:
I．YK-407
YK-407 has a significant structural difference compared to prior art cationic lipids, e.g., SM-102, ALC-0315, compound 21, compound 23, YK-009, and HHMA.
Compared with SM-102, the head group of YK-407 is significantly different, the head group of YK-407 has 2 tertiary amine groups, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 2 And in the head group of SM-102 there are 1 tertiary amine group, to the tertiary amine nitrogen atom is attached HO (CH) 2 ) 2 -. Other parts also differ: g of YK-407 compared with SM-102 1 The group is 2C; g 2 The number of groups is 2 less.
Compared with ALC-0315, the head group of YK-407 is significantly different, 2 tertiary amine groups are in the head group of YK-407, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 2 And ALC-0315 has 1 tertiary amine group in the head group, and HO (CH) attached to the tertiary amine nitrogen atom 2 ) 4 -a group. Other parts also differ: g of YK-407 compared with ALC-0315 1 3 less C; l is a radical of an alcohol 1 The group is-C (O) O-, and ALC-0315 is-OC (O) -; the R1 group is a straight chain structure, and ALC-0315 is a branched chain structure; l is 2 The group is-C (O) O-and ALC-0315 is-OC (O) -; g 2 1 less C; r 2 The number of 1 single strand in the double chain group is 2.
Compared with the compound 21, the head group of YK-407 is significantly different, 2 tertiary amine groups are arranged in the head group of YK-407, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 2 And the head group of compound 21 has 1 tertiary amine group, to which the tertiary amine nitrogen atom is attached HO (CH) 2 ) 2 -. Other parts also differ: g of YK-407 compared with Compound 21 1 4 or less C; r is 1 The group is a straight chain structure, and the compound 21 is a branched chain structure; g 2 The number of groups is 2 less.
Compared with the compound 23, the head group of YK-407 is significantly different, 2 tertiary amine groups are arranged in the head group of YK-407, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 2 And the head group of compound 23 has 1 tertiary amine group, to which the tertiary amine nitrogen atom is attached HO (CH) 2 ) 2 -. Other parts also differ: g of YK-407 compared with Compound 23 1 4 or less C; r 1 The group is a straight chain structure, and the compound 23 is a branched chain structure; g 2 2 less C; r 2 The number of single-stranded groups is 2 less, and each single-stranded group in the double-stranded group has 2 more C.
Compared with YK-009, YK-407 has a significantly different head group, 2 tertiary amine groups in YK-407, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 2 And YK-009 in the head group has 1 tertiary amine group, to which is attached the nitrogen atom HO (CH) 2 ) 2 -. Other parts also differ: r of YK-407 compared with YK-009 1 1 more C; r 2 The single chain of the group has 1C.
Compared with HHMA, the structure of YK-407 is obviously different, the structure of only 1 side chain connected with N atoms of HHMA is similar to that of 1 side chain of YK-401, and other parts have obvious difference.
II．YK-401
YK-401 has a significant structural difference compared to prior art cationic lipids, e.g. SM-102, ALC-0315, compound 21, compound 23, YK-009, and HHMA.
Compared with SM-102, YK-401 has a significantly different head group, with 2 tertiary amine groups connecting 2 tertiary amine groups in the head group of YK-401The L group of the amine nitrogen atom being- (CH) 2 ) 2 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 2 And in the head group of SM-102 there are 1 tertiary amine group to which the tertiary amine nitrogen atom is attached HO (CH) 2 ) 2 -. Other parts also differ: r of YK-401 compared with SM-102 1 The group is a branched chain structure, and SM-102 is a linear chain structure; g 2 The number of groups is 2 less.
Compared with ALC-0315, YK-401 has significantly different head group, 2 tertiary amine groups in the head group of YK-401, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 2 And ALC-0315 has 1 tertiary amine group in the head group, and HO (CH) attached to the tertiary amine nitrogen atom 2 ) 4 -a group. Other parts also differ: g of YK-401 compared with ALC-0315 1 1 less C; l is 1 The group is-C (O) O-, and ALC-0315 is-OC (O) -; r 1 2 more C in 1 single strand of the group double strand; g 2 1 less C; l is 2 The group is-C (O) O-, and ALC-0315 is-OC (O) -; r 2 The number of 1 single strand in the double chain group is 2.
Compared with compound 21, the head group of YK-401 is significantly different, the head group of YK-401 has 2 tertiary amine groups, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 2 And the head group of compound 21 has 1 tertiary amine group, to which the tertiary amine nitrogen atom is attached HO (CH) 2 ) 2 -. Other parts also differ: g of YK-401 compared with Compound 21 1 2 less C; r 1 The number of single-chain groups is 2 less, and each single-chain in the double-chain has 2 more C; g 2 The number of groups is 2 less.
Compared with the compound 23, the head group of YK-401 is significantly different, 2 tertiary amine groups are arranged in the head group of YK-401, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 2 -, and 1 tertiary amine in the head group of compound 23Radical, bound to the nitrogen atom of the tertiary amine, is HO (CH) 2 ) 2 -. Other parts also differ: g of YK-401 in comparison with Compound 23 1 2 less C; r 1 The number of single-chain groups is 2 less, and each single-chain in the double-chain has 2 more C; g 2 2 less C; r 2 The group is single-stranded with 2 fewer C's, and each single strand in the double strand is double-stranded with 2 more C's.
Compared with YK-009, the head group of YK-401 is significantly different, 2 tertiary amine groups are in the head group of YK-401, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 2 And YK-009 in the head group has 1 tertiary amine group, to which is attached the nitrogen atom HO (CH) 2 ) 2 -. Other parts also differ: g of YK-401 compared with YK-009 1 The radical is 2 more C; r 1 The group is a branched chain structure, and YK-009 is a straight chain structure; r 2 The single chain of the group has 1C.
Compared with HHMA, the structure of YK-401 is obviously different, HHMA has only 1 side chain connected with N atom and similar structure with 1 side chain of YK-401, and other parts have obvious difference.
III．YK-402
YK-402 has a significant difference in structure compared to prior art cationic lipids, such as SM-102, ALC-0315, compound 21, compound 23, YK-009, and HHMA.
Compared with SM-102, YK-402 has a significantly different head group, with 2 tertiary amine groups in the head group of YK-402, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 2 And in the head group of SM-102 there are 1 tertiary amine group, to the tertiary amine nitrogen atom is attached HO (CH) 2 ) 2 -. Other parts also differ: g of YK-402 compared to SM-102 1 1 more C; l is 1 The group is-OC (O) -, and SM-102 is-C (O) O-; r 1 The group is a branched chain structure, and SM-102 is a linear chain structure; g 2 1 less C; l is a radical of an alcohol 2 The group is-OC (O) -, and SM-102 is-C (O) O-.
Compared with ALC-0315The head group of YK-402 is significantly different, the head group of YK-402 has 2 tertiary amine groups, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 2 And ALC-0315 has 1 tertiary amine group in the head group, and HO (CH) is attached to the tertiary amine nitrogen atom 2 ) 4 -a group. Other parts also differ: r of YK-402 compared to ALC-0315 1 2 more C in 1 single strand of the group double strand; r 2 The number of C is more than 2 for 1 single strand in the group double strand.
Compared with compound 21, the head group of YK-402 is significantly different, the head group of YK-402 has 2 tertiary amine groups, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 2 And the head group of compound 21 has 1 tertiary amine group, to which the tertiary amine nitrogen atom is attached HO (CH) 2 ) 2 -. Other parts also differ: g of YK-402 in comparison with Compound 21 1 1 less C; l is a radical of an alcohol 1 The group is-OC (O) -, and compound 21 is-C (O) O-; r 1 The number of single-chain groups is 2 less, and each single-chain in the double-chain group is 2 more; g 2 1 less C; l is 2 The group is-OC (O) -, and compound 21 is-C (O) O-.
Compared with compound 23, the head group of YK-402 is significantly different, the head group of YK-402 has 2 tertiary amine groups, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 2 And the head group of compound 23 has 1 tertiary amine group, to which the tertiary amine nitrogen atom is attached HO (CH) 2 ) 2 -. Other parts also differ: g of YK-402 in comparison with Compound 23 1 1 less C; l is a radical of an alcohol 1 The group is-OC (O) -, and compound 23 is-C (O) O-; r is 1 The number of single-chain groups is 2 less, and each single-chain in the double-chain group is 2 more; g 2 1 less C; l is a radical of an alcohol 2 The group is-OC (O) -, and compound 23 is-C (O) O-; r 2 The group is single-stranded with 2 fewer C's, and each single strand in the double strand is double-stranded with 2 more C's.
Compared with YK-009The head group of YK-402 is significantly different, the head group of YK-402 has 2 tertiary amine groups, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 All radicals are HO (CH) 2 ) 2 And YK-009 in the head group has 1 tertiary amine group, to which is attached the nitrogen atom HO (CH) 2 ) 2 -. Other parts also differ: g of YK-402 compared with YK-009 1 The radical is 3 more C; l is a radical of an alcohol 1 The group is-OC (O) -, and YK-009 is-C (O) O-; r is 1 The group is a branched chain structure, and YK-009 is a straight chain structure; g 1 1 more C; l is a radical of an alcohol 2 The group is-OC (O) -, and YK-009 is-C (O) O-; r is 2 The single chain of the group has 1C.
Compared with HHMA, the structure of YK-402 is obviously different, HHMA has only 1 side chain connected with N atom and similar structure with 1 side chain of YK-402, and other parts have obvious difference.
IV．YK-403
YK-403 has a significant difference in structure compared to prior art cationic lipids, such as SM-102, ALC-0315, compound 21, compound 23, YK-009, and HHMA.
Compared with SM-102, the head group of YK-403 is significantly different, the head group of YK-403 has 2 tertiary amine groups, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 2 And in the head group of SM-102 there are 1 tertiary amine group, to the tertiary amine nitrogen atom is attached HO (CH) 2 ) 2 -. Other parts also differ: g of YK-403 compared with SM-102 1 The radical is 2 more C; r 1 The group is a branched structure, while SM-102 is a linear structure.
Compared with ALC-0315, the head group of YK-403 is significantly different, 2 tertiary amine groups are in the head group of YK-403, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 All radicals are HO (CH) 2 ) 2 And ALC-0315 has 1 tertiary amine group in the head group, and HO (CH) is attached to the tertiary amine nitrogen atom 2 ) 4 -a group. Other parts also differ: andg of YK-403 compared with ALC-0315 1 The radical is more than 1C; l is 1 The group is-C (O) O-, and ALC-0315 is-OC (O) -; r is 1 1 single chain in the group double chain has 2C; g 2 The radical is more than 1C; l is a radical of an alcohol 2 The group is-C (O) O-, and ALC-0315 is-OC (O) -.
Compared with compound 21, the head group of YK-403 is significantly different, the head group of YK-403 has 2 tertiary amine groups, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 2 And the head group of compound 21 has 1 tertiary amine group, to which the tertiary amine nitrogen atom is attached HO (CH) 2 ) 2 -. Other parts also differ: r of YK-403 compared with Compound 21 1 The group is single-stranded with 2 fewer C's, and each single strand in the double strand is double-stranded with 2 more C's.
Compared with the compound 23, the head group of YK-403 is significantly different, 2 tertiary amine groups are arranged in the head group of YK-403, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -, G3 and G4 are each HO (CH) 2 ) 2 And the head group of compound 23 has 1 tertiary amine group, to which the tertiary amine nitrogen atom is attached HO (CH) 2 ) 2 -. Other parts also differ: r of YK-403 compared with Compound 23 1 The number of single-chain groups is 2 less, and each single-chain in the double-chain has 2 more C; r 2 The group is single-stranded with 2 fewer C's, and each single strand in the double strand is double-stranded with 2 more C's.
Compared with YK-009, the head group of YK-403 is significantly different, 2 tertiary amine groups are arranged in the head group of YK-403, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 All radicals are HO (CH) 2 ) 2 And YK-009 in the head group has 1 tertiary amine group, and to the tertiary amine nitrogen atom is attached HO (CH) 2 ) 2 -. Other parts also differ: g of YK-403 compared with YK-009 1 The radical is 4 more C; r 1 The group is a branched chain structure, and YK-009 is a straight chain structure; g 2 The radical is 2 more C; r 2 The single chain of the group has 1C.
Compared with HHMA, the structure of YK-403 is obviously different, HHMA has 1 side chain connected with N atom similar to that of YK-403, and other parts have obvious difference.
V．YK-422
YK-422 has a significant difference in structure compared to prior art cationic lipids, e.g. SM-102, ALC-0315, compound 21, compound 23, YK-009, and HHMA.
Compared with SM-102, the head group of YK-422 is significantly different, the head group of YK-422 has 2 tertiary amine groups, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 All radicals are HO (CH) 2 ) 3 And in the head group of SM-102 there are 1 tertiary amine group to which the tertiary amine nitrogen atom is attached HO (CH) 2 ) 2 -. Other parts also differ: r of YK-422 in comparison with SM-102 1 The group is a branched chain structure, and SM-102 is a linear chain structure; g 2 2 less C; r is 2 The group is single-stranded with 1 more C.
Compared with ALC-0315, the head group of YK-422 is significantly different, 2 tertiary amine groups are in the head group of YK-422, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 3 And ALC-0315 has 1 tertiary amine group in the head group, and HO (CH) is attached to the tertiary amine nitrogen atom 2 ) 2 -. Other parts also differ: g of YK-422 compared with ALC-0315 1 1 less C; l is a radical of an alcohol 1 The group is-C (O) O-, and ALC-0315 is-OC (O) -; r 1 2 more C in 1 single strand of the group double strand; g 2 1 less C; l is 2 The group is-C (O) O-, and ALC-0315 is-OC (O) -; r 2 The number of 1 single strand in the double chain group is 2.
Compared with the compound 21, the head group of YK-422 is significantly different, the head group of YK-422 has 2 tertiary amine groups, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 3 And the head group of compound 21 has 1 tertiary amine group, to which the tertiary amine nitrogen atom is attached HO (CH) 2 ) 2 -. Other parts also haveThe difference is as follows: g of YK-422 in comparison with Compound 21 1 2 less C; r is 1 The number of single-chain groups is 1 less, and each single-chain in the double-chain group has 2 more C; g 2 2 less C; r 2 The group is single-stranded with 1 more C.
Compared with compound 23, YK-422 has significantly different head group, 2 tertiary amine groups in YK-422, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 3 And the head group of compound 23 has 1 tertiary amine group, to which the tertiary amine nitrogen atom is attached HO (CH) 2 ) 2 -. Other parts also differ: g of YK-422 in comparison with Compound 23 1 2 less C; r 1 The number of single-chain groups is 1 less, and each single-chain in the double-chain group has 2 more C; g 2 2 less C; r 2 The number of single-stranded groups is 1 less C, and each single-stranded group in the double-stranded group has 2 more C.
Compared with YK-009, YK-422 has significantly different head group, 2 tertiary amine groups in YK-422 head group, and L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 2 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 3 And YK-009 in the head group has 1 tertiary amine group, and to the tertiary amine nitrogen atom is attached HO (CH) 2 ) 2 -. Other parts also differ: g of YK-422 compared with YK-009 1 The radical is 2 more C; r 1 The group is a branched structure, and YK-009 is a linear structure.
Compared with HHMA, the structure of YK-422 is obviously different, HHMA has only 1 side chain connected with N atom and similar structure with 1 side chain of YK-422, and other parts have obvious difference.
VI．YK-423
YK-423 has a significant structural difference compared to prior art cationic lipids, e.g. SM-102, ALC-0315, compound 21, compound 23, YK-009, and HHMA.
Compared with SM-102, the head group of YK-423 is significantly different, the head group of YK-423 has 2 tertiary amine groups, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 3 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 3 And in the head group of SM-102 there are 1 tertiary amine group, to the tertiary amine nitrogen atom is attached HO (CH) 2 ) 2 -. Other parts also differ: r of YK-423 compared with SM-102 1 The group is a branched chain structure, and SM-102 is a linear chain structure; g 2 2 less C; r 2 The group is single-stranded with 1 more C.
Compared with ALC-0315, YK-423 has significantly different head group, 2 tertiary amine groups in the head group of YK-423, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 3 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 3 And ALC-0315 has 1 tertiary amine group in the head group, and HO (CH) is attached to the tertiary amine nitrogen atom 2 ) 2 -. Other parts also differ: g of YK-423 compared with ALC-0315 1 1 less C; l is 1 The group is-C (O) O-and ALC-0315 is-OC (O) -; r is 1 The single chain of the group has 1C, and the single chain of the double chain has 2C; g 2 1 more C; l is 2 The group is-C (O) O-, and ALC-0315 is-OC (O) -; r 2 The group has 1C in single chain and 2C in 1 single chain in double chain.
Compared with the compound 21, the head group of YK-423 is significantly different, the head group of YK-423 has 2 tertiary amine groups, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 3 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 3 And the head group of compound 21 has 1 tertiary amine group, to which the tertiary amine nitrogen atom is attached HO (CH) 2 ) 2 -. Other parts also differ: g of YK-423 compared with Compound 21 1 2 less C; r 1 The number of single-chain groups is 1 less, and each single-chain in the double-chain group has 2 more C; g 2 2 less C; r 2 The group is single-stranded with 1 more C.
Compared with the compound 23, the head group of YK-423 is significantly different, the head group of YK-423 has 2 tertiary amine groups, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 3 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 3 -, and in the head group of the compound 23Having 1 tertiary amine group, bound to the tertiary amine nitrogen atom, is HO (CH) 2 ) 2 -. Other parts also differ: g of YK-423 compared with Compound 23 1 2 less C; r is 1 The number of single-chain groups is 1 less, and each single-chain in the double-chain group has 2 more C; g 2 2 less C; r is 2 The group is single-stranded with 1C less and each single strand in the double strand is double-stranded with 2C more.
Compared with YK-009, the head group of YK-423 is significantly different, 2 tertiary amine groups are arranged in the head group of YK-423, and the L group connecting 2 tertiary amine nitrogen atoms is- (CH) 2 ) 3 -，G 3 And G 4 The radicals are all HO (CH) 2 ) 3 And YK-009 in the head group has 1 tertiary amine group, to which is attached the nitrogen atom HO (CH) 2 ) 2 -. Other parts also differ: g of YK-423 compared with YK-009 1 The radical is 2 more C; r 1 The group is a branched structure, and YK-009 is a linear structure.
Compared with HHMA, the structure of YK-423 is obviously different, HHMA has only 1 side chain connected with N atom and similar structure with 1 side chain of YK-423, and other parts have obvious difference.
As can be seen from the above comparison, the series of compounds designed, including YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, are very different in chemical structure from prior art cationic lipid compounds, such as SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-009. The series of compounds are remarkably different from HHMA structure; with head groups of SM-102, ALC-0315, compound 21, compound 23, and YK-009, including G 3 、G 4 And L is significantly different, G 1 、L 1 、R 1 、G 2 、L 2 And R 2 The groups also all differ significantly.
Due to the significant difference of chemical structures, the physicochemical properties of the series of compounds, such as polarity, acid-base property, hydrophilicity and the like, are also significantly different from those of SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-009. Therefore, it is impossible to predict the cell transfection efficiency, cytotoxicity, and expression profile in animals of LNP preparations prepared from this series of compounds based on the above-described cationic lipid compounds disclosed in the prior art.
(2) In a series of designed compounds, LNP preparations prepared from YK-407, YK-401, YK-402, YK-422 and YK-423 have the highest cell transfection efficiency, and are obviously improved compared with the representative cationic lipid in the prior art. For example, YK-407 can be up to 12 times that of SM-102, 13 times that of Compound 21, and 15 times that of Compound 23.
TABLE 6 fluorescent detection of Fluc-mRNA-1
Differences in cell transfection efficiency
Table 6 lists the results of fluorescence detection of LNP preparations containing Fluc-mRNA prepared from different cationic lipids. Wherein YK-009 is disclosed in CN114044741B (claim 1), compound 21 and compound 23 are disclosed in WO2021055833A1 (page 22 of the specification), SM-102 is compound 25 disclosed in WO2017049245A2 (page 29 of the specification), ALC-0315 is compound 3 disclosed in CN108368028B (page 24 of the specification), HHMA is compound 1 disclosed in CN112979483B (page 12 of the specification); lipofectamine 3000 is a cell transfection reagent widely used at present, and has good transfection performance.
As can be seen from Table 6 and FIG. 5, LNP preparations containing Fluc-mRNA prepared from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 exhibited the strongest fluorescence absorption and RLU values of 20385506, 5609550, 5044102, 5249304, 6432971 and 6227115, respectively.
YK-407 was 11.96 times that of SM-102, 9.31 times that of ALC-0315, 13.49 times that of Compound 21, 14.53 times that of Compound 23, 9.81 times that of HHMA, 16.98 times that of Lipofectamine 3000, and 3.96 times that of YK-009.
YK-401 can be 3.29 times of SM-102, 2.56 times of ALC-0315, 3.71 times of compound 21, 4.00 times of compound 23, 2.70 times of HHMA, 4.67 times of Lipofectamine 3000 and 1.09 times of YK-009.
YK-402 can reach 2.96 times of SM-102, 2.30 times of ALC-0315, 3.34 times of compound 21, 3.60 times of compound 23, 2.43 times of HHMA, 4.20 times of Lipofectamine 3000, and 0.98 times of YK-009.
YK-403 can be 3.08 times of SM-102, 2.40 times of ALC-0315, 3.47 times of compound 21, 3.74 times of compound 23, 2.52 times of HHMA, 4.37 times of Lipofectamine 3000, and 1.02 times of YK-009.
YK-422 can be 3.77 times of SM-102, 2.94 times of ALC-0315, 4.26 times of compound 21, 4.59 times of compound 23, 3.09 times of HHMA, 5.36 times of Lipofectamine 3000, and 1.25 times of YK-009.
YK-423 can reach 3.65 times of SM-102, 2.84 times of ALC-0315, 4.12 times of compound 21, 4.44 times of compound 23, 3.00 times of HHMA, 5.19 times of Lipofectamine 3000 and 1.21 times of YK-009.
When data are analyzed by GraphPad Prism software, any one of YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 is obviously different from SM-102, ALC-0315, compound 21, compound 23, HHMA and Lipofectamine 3000, YK-407 is obviously different from YK-009, and the transfection efficiency is obviously improved.
And (4) summarizing:
in terms of chemical structure, a series of compounds designed, including YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, are very different compared to prior art cationic lipids. For example, this series of compound head structures, including G, was designed to compare SM-102, ALC-0315, compound 21, compound 23, and YK-009 3 、G 4 The head structure of the series of compounds is significantly different from the L group, and the series of compounds have 2 tertiary amine groups, the 2 tertiary amine nitrogen atoms are connected by the L group, and the head structure also has 2 hydroxyl-containing groups G 3 And G 4 Is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -, are each attached to 2 tertiary amine nitrogen atoms, while the head groups of SM-102, ALC-0315, compound 21, compound 23 and YK-009 contain only 1 tertiary amine group and 1 hydroxyl-containing group HO (CH) 2 ) 2 -. And, G of the series of compounds 1 、L 1 、R 1 、G 2 、L 2 And R 2 The group, too, is very different from SM-102, ALC-0315, compound 21, compound 23 and YK-009. The series of compounds are remarkably different from HHMA structure,the difference is large.
The LNP preparations prepared from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 have the highest cell transfection efficiency, which is significantly higher than the activity of the typical cationic lipids in the prior art, for example, YK-407 can reach 12 times that of SM-102, 13 times that of compound 21 and 15 times that of compound 23.
Meanwhile, the cell transfection activity is not only achieved by the compounds with the chemical structures similar to those of the cationic lipid in the prior art, but on the contrary, the LNP preparation prepared by the compounds with the greatly different structures can have the advantages of remarkably improved transfection efficiency and very strong cell transfection activity.
(3) YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 with G designed in this application 3 And G 4 The radicals are all HO (CH) 2 ) 2 -, L radical is- (CH) 2 ) 2 The transfection efficiency of cells is highest compared to structurally similar compounds. For example, YK-407 may be 2500 times as high as YK-404 and YK-411.
To compare whether structurally similar compounds have similar transfection efficiencies, the G designed in this application 3 And G 4 The radicals are all HO (CH) 2 ) 2 -, L is- (CH) 2 ) 2 Compounds with slightly different groups were compared with YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423. The results show that the activity difference of the series of compounds is very large, wherein the cell transfection efficiency of YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 is highest, YK-407 can reach 2500 times of YK-404 and YK-411, YK-401, YK-402, YK-403, YK-422 and YK-423 can reach more than 600 times of YK-404 and YK-411, and the transfection efficiency is obviously improved.
Chemical structures of compounds designed in Table 7
TABLE 8 Fluorescence assay of Fluc-mRNA-2
a. Difference in cell transfection efficiency
As can be seen from Table 8 and FIG. 6, the RLU values of the LNP formulations prepared from these compounds are very different from those of YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423.
Specifically, the RLU values for YK-404 and YK-411 are the lowest, 7543 and 7976, respectively, and are very different from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423.
YK-407 can be 2702.57 times and 2555.78 times of YK-404 and YK-411.
YK-401 can reach 743.68 times and 703.28 times of YK-404 and YK-411.
YK-402 can reach 668.71 times and 632.39 times of YK-404 and YK-411.
YK-403 can reach 695.92 times and 658.12 times of YK-404 and YK-411.
YK-422 can reach 852.84 times and 806.52 times of YK-404 and YK-411.
YK-423 can reach 825.55 times and 780.71 times of YK-404 and YK-411.
The RLU value of YK-406 is 78251, which is also very different from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423.
YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 are 260.51 times, 71.69 times, 64.46 times, 67.08 times, 82.21 times and 79.58 times of YK-406, respectively.
The RLU values for YK-405, YK-408, YK-409 and YK-410 are 3197780, 1474136, 669807 and 407868, respectively, and are also significantly different from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423.
YK-407 can be 6.37 times of YK-405, 13.83 times of YK-408, 30.43 times of YK-409 and 49.98 times of YK-410.
YK-401 can be 1.75 times of YK-405, 3.81 times of YK-408, 8.37 times of YK-409 and 13.75 times of YK-410.
YK-402 can reach 1.58 times of YK-405, 3.42 times of YK-408, 7.53 times of YK-409 and 12.37 times of YK-410.
YK-403 can reach 1.64 times of YK-405, 3.56 times of YK-408, 7.84 times of YK-409 and 12.87 times of YK-410.
YK-422 can be 2.01 times of YK-405, 4.36 times of YK-408, 9.60 times of YK-409 and 15.77 times of YK-410.
YK-423 can reach 1.95 times of YK-405, 4.22 times of YK-408, 9.30 times of YK-409 and 15.27 times of YK-410.
The activity difference between YK-404, YK-405, YK-406, YK-408, YK-409, YK-410 and YK-411 is also large. YK-405 cell transfection efficiency is stronger than SM-102, which is 1.88 times of SM-102; YK-408 is not much different from SM-102 and is 0.87 times of SM-102; YK-406 is only 0.05 times that of SM-102; YK-404 and YK-411 were the lowest, only 0.004 and 0.005 times that of SM-102, respectively.
When the data are analyzed by GraphPad Prism software, any one of YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 is obviously different from YK-404, YK-405, YK-406, YK-408, YK-409, YK-410 and YK-411, and the transfection efficiency is obviously improved.
b. Difference in chemical structure
The structures of the series of compounds are very similar, and only the individual groups are slightly different. YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 are very close in structure to other compounds, and are very similar to each other.
I. The difference from YK-407 structure
In contrast to YK-407, YK-404 is G only 1 The radical is 5 more C; r 1 The group is a branched chain structure, and YK-407 is a linear chain structure; g 2 The radical is 3 more C; r 2 In the double-stranded group, 3 less C exist in 1 single strand, 6 less C exist in 1 single strand, and other structures are completely the same, but the cell transfection efficiency YK-407 is 2702.57 times of YK-404.
YK-411 is G only 1 The radical is 2 more C; r 1 1 less C; r is 2 The single chain of the group has 2 more C, each single chain in the double chain has 2 less C, other structures are completely the same, but the cell transfection efficiency YK-407 reaches 2555.78 times of YK-411.
YK-406 is G only 2 The radical is more than 1C; l is a radical of an alcohol 2 The radical-OC (O) -, and YK-407is-C (O) O-; r 2 The single chain of the group has 1 more C, each single chain in the double chain has 2 less C, other structures are completely the same, but the cell transfection efficiency YK-407 reaches 260.51 times of YK-406.
Structural distinction from YK-401
In contrast to YK-401, YK-404 is only G 1 And G 2 Each of the groups has 3 more C; r 1 And R 2 In the group double-chain, 1 single-chain has 3 less C,1 single-chain has 6 less C, and other structures are completely the same, but the cell transfection efficiency YK-401 reaches 743.68 times of YK-404.
YK-411 is R only 1 The group is a straight chain structure, and YK-401 is a branched chain structure; r 2 The single chain of the group has 2 more C, each single chain in the double chain has 2 less C, other structures are completely the same, but the cell transfection efficiency YK-401 reaches 703.28 times of YK-411.
YK-410 is R only 1 The group is a straight chain structure, and YK-401 is a branched chain structure; r 2 The single chain of the group has 1C more, other structures are completely the same, but the transfection efficiency of the cell YK-401 reaches 13.75 times of that of YK-410.
Structural distinction from YK-402
In contrast to YK-402, YK-404 is only G 1 And G 2 2C in each case; l is 1 And L 2 The group is-C (O) O-and YK-402 is-OC (O) -; r 1 And R 2 In the double-stranded group, 3 less C exist in 1 single strand, 6 less C exist in 1 single strand, and other structures are completely the same, but the cell transfection efficiency YK-402 reaches 668.71 times of YK-404.
YK-411 is G only 1 And G 2 1 less C in each group; r is 1 The group is a straight chain structure, and YK-402 is a branched chain structure; r 2 The single chain of the group has 2 more C, and each single chain in the double chain has 2 less C; l is a radical of an alcohol 1 And L 2 All the groups are-C (O) O-, YK-402 is-OC (O) -, other structures are completely the same, but the cell transfection efficiency YK-402 reaches 632.39 times of YK-411.
YK-406 is G only 1 3 fewer C groups; l is a radical of an alcohol 1 The group is-C (O) O-and YK-402 is-OC (O) -; r is 1 The group is in a straight chain structure, and YK-402 is in a branched chain structure; r is 2 The group is single-stranded and has 1C more, each of the double strandsThe single chains each have 2 less C, and the other structures are identical, but the cell transfection efficiency YK-402 reaches 64.46 times of YK-406.
Structural difference from YK-403
In contrast to YK-403, YK-404 is G only 1 And G 2 1 more C in each case; r is 1 And R 2 3C are less in 1 single chain in the group double chain, 6C are less in 1 single chain, and other structures are completely the same, but the cell transfection efficiency YK-403 reaches 695.92 times of YK-404.
YK-411 is G only 1 And G 2 Each group has 2 less C; r is 1 The group is in a straight chain structure, and YK-403 is in a branched chain structure; r is 2 The group single chain has 2 more C, each single chain in the double chain has 2 less C, other structures are completely the same, but the cell transfection efficiency YK-403 reaches 658.12 times of YK-411.
YK-406 is G only 1 4 or less C; r 1 The group is in a straight chain structure, and YK-403 is in a branched chain structure; g 2 1 less C; l is 2 The group is-OC (O) -, and YK-403 is-C (O) O-; r 2 The single chain of the group has 1 more C, each single chain in the double chain has 2 less C, other structures are completely the same, but the cell transfection efficiency YK-403 reaches 67.08 times of YK-406.
Structural difference from YK-422
YK-404 is G only, as compared to YK-422 1 And G 2 Each of the radicals is 3 more C; g 3 And G 4 1 less C in each group; r 1 And R 2 The number of the group single chains is 1C less, the number of the 1 single chain in the double chains is 3C less, the number of the 1 single chain in the double chains is 6C less, other structures are completely the same, but the cell transfection efficiency YK-422 reaches 852.84 times of YK-404.
YK-411 being only G 3 And G 4 1 less C in each group; r 1 The group is a straight chain structure, and YK-422 is a branched chain structure; r 2 The single chain of the group has 1 more C, each single chain in the double chain has 2 less C, other structures are completely the same, but the cell transfection efficiency YK-422 reaches 806.52 times of YK-411.
YK-406 is G only 1 2 less C; g 3 And G 4 1 less C in each group; r 1 The group is a straight chain structure, and YK-422 is a branched chain structure；G 2 1 more C; l is 2 The group is-OC (O) -, and YK-422 is-C (O) O-; r 2 Each single chain in the group double chain has 2 less C, other structures are completely the same, but the cell transfection efficiency YK-422 reaches 82.21 times of YK-406.
Structural distinction from YK-423
In contrast to YK-423, YK-404 is G only 1 And G 2 Each of the groups has 3 more C; g 3 And G 4 1 less C in each group; r 1 And R 2 1C is less in group single chain, 3C is less in 1 single chain in the double chain, and 6C is less in 1 single chain; the other structures are identical, but the cell transfection efficiency YK-423 reaches 825.55 times of YK-404.
YK-411 is G only 3 And G 4 1 less C in each group; r 1 The group is in a straight chain structure, and YK-423 is in a branched chain structure; r is 2 The number of the group single chains is 1, each single chain in the double chains has 2 less C, other structures are completely the same, but the cell transfection efficiency YK-423 reaches 780.71 times of YK-411.
YK-406 is G only 1 2 less C; g 3 And G 4 1 less C in each group; r 1 The group is a straight chain structure, and YK-422 is a branched chain structure; g 2 1 more C; l is a radical of an alcohol 2 The group is-OC (O) -, and YK-423 is-C (O) O-; r 2 Each single chain in the group double chain has 2 less C, other structures are completely the same, but the cell transfection efficiency YK-423 reaches 79.58 times of YK-406.
And (3) knotting:
and G 3 And G 4 The radical is HO (CH) 2 ) 2 -, L radical is- (CH) 2 ) 2 YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, the transfection efficiency of cells was the highest compared to the structurally similar compounds. For example, YK-407 can reach 2500 times of YK-404 and YK-411, YK-401, YK-402, YK-403, YK-422 and YK-423 can reach more than 600 times of YK-404 and YK-411, and the transfection efficiency is obviously improved.
Meanwhile, the structure of the compound and the intracellular transfection efficiency are not in corresponding relation, and even a group of compounds with very similar structures has very high possibility of very different cell transfection efficiencies.
Therefore, it is very difficult to select a cationic lipid compound having a high transfection efficiency from a series of compounds having very similar structures, and much creative work is required.
(4) YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 with G designed in this application 3 And G 4 The radical is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -, L radical is- (CH) 2 ) 2 -、-(CH 2 ) 3 -or- (CH) 2 ) 4 The transfection efficiency of cells is highest compared to the structurally similar compounds. For example, YK-407 may be 170 times as much as YK-417 and 180 times as much as YK-418.
Further processing G 3 And G 4 Is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -, L is- (CH) 2 ) 2 -、-(CH 2 ) 3 -or- (CH) 2 ) 4 -in comparison with YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423. The results show that the activity of the series of compounds is very different, and the cell transfection efficiency of YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 is the highest. For example, YK-407 can reach 170 times of YK-417 and 180 times of YK-418, and the transfection efficiency is obviously improved.
Chemical structures of compounds designed in Table 9
TABLE 10 Fluc-mRNA fluorescence detection results-3
a. Differences in cell transfection efficiency
Although other compounds have some minor differences in individual groups compared to YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, the effect on cell transfection efficiency is very large, up to 180-fold.
Specifically, as can be seen from Table 10, YK-417 and YK-418 are very different from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423.
YK-407 can be 173.81 times of YK-417 and 183.24 times of YK-418.
YK-401 can reach 47.83 times of YK-417 and 50.42 times of YK-418.
YK-402 can be 43.01 times of YK-417 and 45.34 times of YK-418.
YK-403 can reach 44.76 times of YK-417 and 47.18 times of YK-418.
YK-422 can reach 54.85 times of YK-417 and 57.82 times of YK-418.
YK-423 can reach 53.09 times of YK-417 and 55.97 times of YK-418.
YK-413, YK-414, YK-415 and YK-421 have much different activities than YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423.
YK-407 can be up to 14.80 times of YK-413, 45.90 times of YK-414, 68.40 times of YK-415 and 43.69 times of YK-421.
YK-401 can reach 4.07 times of YK-413, 12.63 times of YK-414, 18.82 times of YK-415 and 12.02 times of YK-421.
YK-402 can reach 3.66 times of YK-413, 11.36 times of YK-414, 16.93 times of YK-415 and 10.81 times of YK-421.
YK-403 can reach 3.81 times of YK-413, 11.82 times of YK-414, 17.61 times of YK-415 and 11.25 times of YK-421.
YK-422 can reach 4.67 times of YK-413, 14.49 times of YK-414, 21.59 times of YK-415 and 13.79 times of YK-421.
YK-423 can reach 4.52 times of YK-413, 14.02 times of YK-414, 20.89 times of YK-415 and 13.35 times of YK-421.
YK-412, YK-416, YK-419, YK-420 and YK-424, these 5 compounds also have greater differences in activity than YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423.
YK-407 may be 6.05 times that of YK-412, 5.13 times that of YK-416, 6.44 times that of YK-419, 5.07 times that of YK-420 and 7.07 times that of YK-424.
YK-401 can be 1.66 times of YK-412, 1.41 times of YK-416, 1.77 times of YK-419, 1.40 times of YK-420 and 1.95 times of YK-424.
YK-402 can be up to 1.50 times YK-412, 1.27 times YK-416, 1.59 times YK-419, 1.25 times YK-420, and 1.75 times YK-424.
YK-403 can be 1.56 times of YK-412, 1.32 times of YK-416, 1.66 times of YK-419, 1.31 times of YK-420 and 1.82 times of YK-424.
YK-422 can be 1.91 times of YK-412, 1.62 times of YK-416, 2.03 times of YK-419, 1.60 times of YK-420 and 2.23 times of YK-424.
YK-423 can reach 1.85 times of YK-412, 1.57 times of YK-416, 1.97 times of YK-419, 1.55 times of YK-420 and 2.16 times of YK-424.
When the data are analyzed by GraphPad Prism software, any one of YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 is obviously different from other compounds, and the cell transfection efficiency is obviously improved.
b. Difference in chemical structure
Compared with YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, the series of compounds are only slightly different from each other in terms of individual groups. YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 are very close in structure to other compounds, and are very similar to each other.
I. The difference from YK-407 structure
YK-417 is only 1 more C than the L group compared to YK-407; g 1 The radical is 2 more C; r 1 1 less C; r 2 The single chain of the group has 2 more C, each single chain in the double chain has 2 less C, other structures are completely the same, but the transfection efficiency YK-407 of the cell reaches 173.81 times of YK-417.
YK-418 is only an L group with 1 more C; r 1 1 less C; g 2 1 more C; l is 2 The group is-OC (O) -, and YK-407 is-C (O) O-; the R2 group has more than 1C single chain and less than 2C single chains in each double chainThe other structures of the C are identical, but the cell transfection efficiency YK-407 reaches 183.24 times of that of YK-418.
YK-415 is only an L group with 1 more C; r 2 The group is single-chain and has 1C, other structures are completely the same, but the transfection efficiency of the cell YK-407 reaches 68.40 times of that of YK-415.
Structural distinction from YK-401
YK-417 is only 1 more C than YK-401 for the L group; r 1 The group is a straight chain structure, and YK-401 is a branched chain structure; r 2 The single chain of the group has 2 more C, each single chain in the double chain has 2 less C, other structures are completely the same, but the cell transfection efficiency YK-401 reaches 47.83 times of YK-417.
YK-418 is only an L group with 1 more C; g 1 2 less C; r is 1 The group is a straight chain structure, and YK-401 is a branched chain structure; g 2 1 more C; l is 2 The group is-OC (O) -, and YK-401 is-C (O) O-; r 2 The single chain of the group has 1 more C, each single chain in the double chain has 2 less C, other structures are completely the same, but the cell transfection efficiency YK-401 reaches 50.42 times of YK-418.
YK-415 is only an L group with 1 more C; g 1 2 less C; r is 1 The group is a straight chain structure, and YK-401 is a branched chain structure; r is 2 The single chain of the group has 1C more, other structures are completely the same, but the transfection efficiency of the cell YK-401 reaches 18.82 times of that of YK-415.
Structural distinction from YK-402
YK-417 is only 1 more C than YK-402 for the L group; g 1 And G 2 1 less C in each group; l is a radical of an alcohol 1 And L 2 The group is-C (O) O-and YK-402 is-OC (O) -; r 1 The group is in a straight chain structure, and YK-402 is in a branched chain structure; r is 2 The group single chain has 2 more C, each single chain in the double chain has 2 less C, other structures are completely the same, but the cell transfection efficiency YK-402 reaches 43.01 times of YK-417.
YK-418 is only an L group with 1 more C; g 1 3 less C; l is 1 The group is-C (O) O-and YK-402 is-OC (O) -; r 1 The group is in a straight chain structure, and YK-402 is in a branched chain structure; r 2 The group is single-stranded and has 1C in each chainThe single strand has 2 less C, other structures are completely the same, but the cell transfection efficiency YK-402 reaches 45.34 times of YK-418.
YK-415 is only an L group with 1 more C; g 1 3 less C; l is a radical of an alcohol 1 And L 2 The group is-C (O) O-and YK-402 is-OC (O) -; r 1 The group is in a straight chain structure, and YK-402 is in a branched chain structure; r 2 The single chain of the group has 1C more, other structures are completely the same, but the transfection efficiency of the cell YK-402 reaches 16.93 times of that of YK-415.
Structural distinction from YK-403
YK-417 is only 1 more C than YK-403 for the L group; g 1 And G 2 Each group has 2 less C; r 1 The group is a straight chain structure, and YK-403 is a branched chain structure; r 2 The group single chain has 2 more C, each single chain in the double chain has 2 less C, other structures are completely the same, but the cell transfection efficiency YK-403 reaches 44.76 times of YK-417.
YK-418 is only an L group with 1 more C; g 1 4 or less C; r 1 The group is in a straight chain structure, and YK-403 is in a branched chain structure; g 2 1 less C; l is 2 The group is-OC (O) -, and YK-403 is-C (O) O-; r is 2 The single chain of the group has 1 more C, each single chain in the double chain has 2 less C, other structures are completely the same, but the cell transfection efficiency YK-403 reaches 47.18 times of YK-418.
YK-414 is only an L group with 1 more C; g 1 And G 2 Each group has 4 less C; r 1 The group is in a straight chain structure, YK-401 is in a branched chain structure, other structures are completely the same, but the cell transfection efficiency YK-403 reaches 11.82 times of YK-414.
Structural difference from YK-422
YK-417 is only 1 more C than YK-422 for the L group; g 3 And G 4 1 less C in each group; r is 1 The group is a straight chain structure, and YK-422 is a branched chain structure; r 2 The group single chain has 1 more C, each single chain in the double chain has 2 less C, other structures are completely the same, but the cell transfection efficiency YK-422 reaches 54.85 times of YK-417.
YK-418 is only the L group with 1 more C; g 3 And G 4 1 less C in each group; g 1 2 less C; r 1 The group is a straight chain structure, and YK-422 is a branched chain structure; g 2 1 more C; l is 2 The group is-OC (O) -, and YK-422 is-C (O) O-; r 2 Each single strand in the group double strand has 2 less C, other structures are completely the same, but the cell transfection efficiency YK-422 reaches 57.82 times of YK-418.
YK-414 is only an L group with 1 more C; g 3 And G 4 1 less C in each group; g 1 And G 2 Each group has 2 less C; r 1 The group is a straight chain structure, and YK-422 is a branched chain structure; r 2 The number of the single chain of the group is 1C less, other structures are completely the same, but the transfection efficiency of the cell YK-422 reaches 14.49 times of that of YK-414.
Structural distinction from YK-423
YK-417 is, in contrast to YK-423, only G 3 And G 4 1 less C in each group; r 1 The group is in a straight chain structure, and YK-423 is in a branched chain structure; r 2 The group single chain has 1 more C, each single chain in the double chain has 2 less C, other structures are completely the same, but the cell transfection efficiency YK-423 reaches 53.09 times of YK-417.
YK-418 is G only 3 And G 4 1 less C in each group; g 1 2 less C; r 1 The group is in a straight chain structure, and YK-423 is in a branched chain structure; g 2 The radical is more than 1C; l is 2 The group is-OC (O) -, and YK-423 is-C (O) O-; r 2 Each single chain in the group double chain has 2 less C, other structures are completely the same, but the cell transfection efficiency YK-423 reaches 55.97 times of YK-418.
YK-415 is G only 3 And G 4 1 less C in each group; g 1 2 less C; r 1 The group is in a straight chain structure, YK-423 is in a branched chain structure, and other structures are completely the same, but the cell transfection efficiency YK-423 reaches 20.89 times of YK-415.
Moreover, the cell transfection efficiency of the compounds in the series may be very different from one compound to another with very small structural differences. For example, YK-416 is only R, as compared to YK-417 2 The number of the group single chains is 1 less, each group single chain in the double chains has 2 more C, other structures are completely the same, but the cell transfection efficiency YK-416 reaches that of YK-41734 times.
And (3) knotting:
and G 3 And G 4 Is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -, L is- (CH) 2 ) 2 -、-(CH 2 ) 3 -or- (CH) 2 ) 4 YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 were most efficiently transfected compared to the series of structurally similar compounds. For example, YK-407 can reach 170 times of YK417 and 180 times of YK-418, and the transfection efficiency is obviously improved.
We have found that there is no correlation between the structure of the compounds and the intracellular transfection efficiency, and even a group of compounds with small differences in structure are very likely to have very large differences in the cell transfection efficiency.
Therefore, it is very difficult to select cationic lipid compounds with high transfection efficiency from a series of compounds with only small differences in chemical structures, and much creative work is required.
To summarize:
1) Through various designs and extensive creative efforts on compound structures, we designed and screened cationic lipid compounds with high cell transfection efficiency, such as YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423.
The series of compounds are designed to have significant differences from the chemical structures of the representative cationic lipids in the prior art, such as SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-009, wherein the difference from HHMA is the largest, HHMA has only 1 side chain connected with the central N atom, and the 1 side chain structure of the series of compounds is similar, and other parts are significantly different; compared with SM-102, ALC-0315, compound 21, compound 23 and YK-009, the head structure of the series of compounds is significantly different, and the head structure of the series of compounds comprises 2 tertiary amine groups, an L group connecting 2 tertiary amine nitrogen atoms, and G all containing hydroxyl 3 And G 4 While the head groups of SM-102, ALC-0315, compound 21, compound 23, and YK-009, only include 1 tertiary amine group, and HO (CH) attached to the tertiary amine nitrogen atom 2 ) 2 -. Due to the very large difference in the structure,therefore, compared with SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-009, the series of compounds have great difference in polarity, acidity-basicity, hydrophilicity and the like.
2) LNP preparations prepared from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 have the highest cell transfection efficiency, and are remarkably improved in activity compared with representative cationic lipids in the prior art. For example, YK-407 can be up to 12 times that of SM-102, 13 times that of Compound 21, and 15 times that of Compound 23.
And G 3 And G 4 The radical is HO (CH) 2 ) 2 -, L radical is- (CH) 2 ) 2 YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, the transfection efficiency of cells was the highest compared to the structurally similar compounds. For example, YK-407 can reach 2500 times of YK-404 and YK-411, YK-401, YK-402, YK-403, YK-422 and YK-423 can reach more than 600 times of YK-404 and YK-411, and the transfection efficiency is obviously improved.
And G 3 And G 4 Is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -, L is- (CH) 2 ) 2 -、-(CH 2 ) 3 -or- (CH) 2 ) 4 YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 were most efficiently transfected compared to the series of structurally similar compounds. For example, YK-407 can reach 170 times of YK-417 and 180 times of YK-418, and the transfection efficiency is obviously improved.
3) There is no correlation between the structure of the compound and the intracellular transfection efficiency, and even compounds with little structural difference are highly likely to have a very large difference in transfection efficiency. Therefore, screening of cationic lipid compounds with high transfection efficiency requires various designs and much creative work.
2. Cell viability assay
LNP formulations containing 1.5 μ g Fluc-mRNA (prepared according to example 4, LNP formulation vehicle components were cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid in a molar ratio of 49:10:39.5:1.5, where the cationic lipid is the cationic lipid listed in table 1) and Lipofectamine 3000 formulations were added to the cell culture broth in 96-well plates, after further incubation for 24 hours, 10 μ L CCK-8 solution was added to each well, and after incubation of the plates in the incubator for 1 hour, absorbance at 450nm was measured by a microplate reader. The results are shown in tables 13 to 16.
The experimental results are as follows:
(1) In a series of compounds designed, LNP formulations prepared from YK-407, YK-401, YK402, YK-403, YK-422 and YK-423 were significantly less cytotoxic than representative cationic lipids in the prior art. For example, YK-401 cell survival rate can be 28.00% higher than ALC-0315, 4.31% higher than SM-102, and 10.94% higher than HHMA.
TABLE 11 cell viability-1
a. Difference in cell viability
Table 11 lists the results of the cytotoxicity assays for LNP formulations prepared from different cationic lipid compounds. Wherein YK-009 is disclosed in CN114044741B (claim 1), SM-102 is compound 25 disclosed in WO2017049245A2 (page 29 of the specification), ALC-0315 is compound 3 disclosed in CN108368028B (page 24 of the specification), compound 21 and compound 23 are disclosed in WO2021055833A1 (page 22 of the specification), HHMA is compound 1 disclosed in CN112979483B (page 12 of the specification); lipofectamine 3000 is a cell transfection reagent widely used at present, and has good transfection performance.
As can be seen from Table 11, LNP preparations of Fluc-mRNA prepared from YK-407, YK-401, YK402, YK-403, YK-422 and YK-423 exhibited the lowest cytotoxicity and the cell survival rates reached 71.37%, 75.21%, 72.70%, 70.76%, 70.48% and 71.14%, respectively.
YK-407 was 3.47% higher than SM-102, 24.16% higher than ALC-0315, 3.53% higher than Compound 21, 2.26% higher than Compound 23, 7.10% higher than HHMA, and 49.71% higher than Lipofectamine 3000.
YK-401 is 7.31% higher than SM-102, 28.00% higher than ALC-0315, 7.37% higher than compound 21, 6.10% higher than compound 23, 10.94% higher than HHMA, and 53.55% higher than Lipofectamine 3000.
YK-402 was 4.80% higher than SM-102, 25.49% higher than ALC-0315, 4.86% higher than Compound 21, 3.59% higher than Compound 23, 8.43% higher than HHMA, and 51.04% higher than Lipofectamine 3000.
YK-403 is 2.86% higher than SM-102, 23.55% higher than ALC-0315, 2.92% higher than Compound 21, 1.65% higher than Compound 23, 6.49% higher than HHMA, and 49.10% higher than Lipofectamine 3000.
YK-422 is 2.58% higher than SM-102, 23.27% higher than ALC-0315, 2.64% higher than compound 21, 1.37% higher than compound 23, 6.21% higher than HHMA, and 48.82% higher than Lipofectamine 3000.
YK-423 was 3.24% higher than SM-102, 23.93% higher than ALC-0315, 3.30% higher than Compound 21, 2.03% higher than Compound 23, 6.87% higher than HHMA, and 49.48% higher than Lipofectamine 3000. (FIG. 7)
The data were analyzed using GraphPad Prism software, where any of YK-407, YK-401, YK402, YK-403, YK-422, and YK-423 was significantly different from SM-102, compound 21, compound 23, ALC-0315, HHMA, and Lipofectamine 3000, with significantly reduced cytotoxicity.
b. Difference in chemical structure
YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 have great difference with the chemical structure of the cationic lipid in the prior art, wherein the difference with the HHMA structure is the largest, and as can be seen from the chemical structure diagram, HHMA only has 1 side chain connected with the central N atom similar to 1 side chain of the series of structures, and other parts are obviously different.
Compared with SM-102, ALC-0315, compound 21, compound 23, and YK-009, the designed series of compound head structures, including G 3 、G 4 And the L group are significantly different. The head structure of the series of compounds comprises 2 tertiary amine groups, an L group connecting 2 tertiary amine nitrogen atoms, and G containing hydroxyl 3 And G 4 While the head groups of SM-102, ALC-0315, compound 21, compound 23, and YK-009, only include 1 tertiary amine group, and HO (CH) attached to the tertiary amine nitrogen atom 2 ) 2 -. And, G of the series of compounds 1 、L 1 、R 1 、G 2 、L 2 And R 2 Group, also with SM-102, ALC-0315, compound 21. Compound 23 and YK-009 differed significantly.
And (4) summarizing:
in a series of designed compounds, LNP preparations prepared from YK-407, YK-401, YK402, YK-403, YK-422 and YK-423 have the lowest cytotoxicity, and the survival rate of LNP preparations is obviously improved compared with that of representative cationic lipid cells in the prior art. For example, YK-401 cell survival rate can be 28.00% higher than ALC-0315, 7.31% higher than SM-102, and 10.94% higher than HHMA.
Compared with the representative cationic lipid in the prior art, the YK-407, YK-401, YK402, YK-403, YK-422 and YK-423 have the advantages that the chemical structures are obviously different, the head groups are obviously different, and G is 1 、L 1 、R 1 、G 2 、L 2 And R 2 The groups also differ significantly.
Thus, LNP formulations that are not prepared solely from compounds with similar structure to the cationic lipids of the prior art are less cytotoxic. In contrast, LNP formulations prepared from compounds with significantly different structures are likely to have significantly reduced cytotoxicity.
(2) YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 with G designed in the application 3 And G 4 The radical is HO (CH) 2 ) 2 -, L radical is- (CH) 2 ) 2 -has the lowest cytotoxicity compared to a structurally similar compound. For example, the cell survival rate of YK-401 is 58.88% higher than that of YK-411 and 50.25% higher than that of YK-406; the survival rate of YK-407 cells is 55.04 percent higher than that of YK-411 and 46.41 percent higher than that of YK-406.
To compare the differences in cytotoxicity of structurally similar compounds, G was used 3 And G 4 Is HO (CH) 2 ) 2 -, L is- (CH) 2 ) 2 -as compared to the cell viability of YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423. The results show that the series of compounds have very significant difference in cytotoxicity. YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 have the highest cell viability, for example YK-401 may be 58.88% higher than YK-411 and 50.25% higher than YK-406; YK-407 may be 55.04% higher than YK-411 and 46.41% higher than YK-406.
TABLE 12 cell viability-2
a. Difference in cell viability
As shown in Table 12, the LNP preparations prepared from these compounds have very different cytotoxicity, wherein YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 have the lowest toxicity and the highest cell survival rate. The lowest cell viability was YK-411 and YK-406, which were only 16.33% and 24.96%.
YK-407 is 55.04% higher than YK-411 and 46.41% higher than YK-406.
YK-401 is 58.88% higher than YK-411 and 50.25% higher than YK-406.
YK-402 is 56.37% higher than YK-411 and 47.74% higher than YK-406.
YK-403 is 54.43% higher than YK-411 and 45.80% higher than YK-406.
YK-422 is 54.15% higher than YK-411 and 45.52% higher than YK-406.
YK-423 is 54.81 percent higher than YK-411 and 46.18 percent higher than YK-406.
The cell survival rates of YK-404, YK-405, YK-408, YK409 and YK-410 are 42.12%, 41.86%, 42.31%, 46.11% and 44.92%, respectively.
YK-407 is 29.25% higher than YK-404, 29.51% higher than YK-405, 29.06% higher than YK-408, 25.26% higher than YK-409, and 26.45% higher than YK-410.
YK-401 is 33.09% higher than YK-404, 33.35% higher than YK-405, 32.90% higher than YK-408, 29.10% higher than YK-409, and 30.29% higher than YK-410.
YK-402 is 30.58% higher than YK-404, 30.84% higher than YK-405, 30.39% higher than YK-408, 26.59% higher than YK-409, and 27.78% higher than YK-410.
YK-403 is 28.64% higher than YK-404, 28.90% higher than YK-405, 28.45% higher than YK-408, 24.65% higher than YK-409, and 25.84% higher than YK-410.
YK-422 is 28.36% higher than YK-404, 28.62% higher than YK-405, 28.17% higher than YK-408, 24.37% higher than YK-409, and 25.56% higher than YK-410.
YK-423 is 29.02% higher than YK-404, 29.28% higher than YK-405, 28.83% higher than YK-408, 25.03% higher than YK-409, and 26.22% higher than YK-410. (FIG. 8)
When the data are analyzed by GraphPad Prism software, any one of YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 has obvious difference in cytotoxicity with other compounds, and the cytotoxicity is obviously reduced.
b. Difference in chemical structure
The structures of the series of compounds are very similar, and only the individual groups are slightly different. YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 are very close in structure to other compounds, and are very similar to each other.
And (3) knotting:
and G 3 And G 4 Is HO (CH) 2 ) 2 -, L is- (CH) 2 ) 2 Compared with a series of compounds with similar structures, YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 have the lowest cytotoxicity and the highest cell survival rate. For example, the cell survival rate of YK-401 is 58.88% higher than that of YK-411 and 50.25% higher than that of YK-406; the survival rate of YK-407 cells is 55.04 percent higher than that of YK-411 and 46.41 percent higher than that of YK-406.
Meanwhile, the structure of the compound has no corresponding relation with cytotoxicity, and even a group of compounds with the most similar structures have very high possibility of very different cytotoxicity.
Therefore, it is very difficult to screen out cationic lipid compounds with low cytotoxicity from a series of compounds with only small differences in chemical structures, and much creative work is required.
(3) YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 with G designed in this application 3 And G 4 The radical is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -, L radical is- (CH) 2 ) 2 -、-(CH 2 ) 3 -or- (CH) 2 ) 4 -has the lowest cytotoxicity compared to a structurally similar compound. For example, YK-401 may be 53.87% higher than YK-417 and 54.16% higher than YK-418; YK-407 may be 50.03% higher than YK-417 and 50.32% higher than YK-418.
Further comparing YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 with other structurally similar compoundsDifferences in cytotoxicity. This series of compounds G 3 And G 4 Is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -, L is- (CH) 2 ) 2 -、-(CH 2 ) 3 -or- (CH) 2 ) 4 -, the other groups are slightly different. The results show that the series of compounds have very large difference in cytotoxicity, and the highest cell survival rates of YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423. For example, YK-401 may be 53.87% higher than YK-417 and 54.16% higher than YK-418; YK-407 may be 50.03% higher than YK-417 and 50.32% higher than YK-418.
TABLE 13 cell viability-3
a. Difference in cell viability
As can be seen from Table 13, the cell viability for YK-414, YK-417, YK-418 and YK-424 was the lowest, 28.75%, 21.34%, 21.05% and 27.93%, respectively, all of which were less than 30%.
YK-407 is 42.62% higher than YK-414, 50.03% higher than YK-417, 50.32% higher than YK-418, and 43.44% higher than YK-424.
YK-401 is 46.46% higher than YK-414, 53.87% higher than YK-417, 54.16% higher than YK-418, and 47.28% higher than YK-424.
YK-402 is 43.95% higher than YK-414, 51.36% higher than YK-417, 51.65% higher than YK-418, and 44.77% higher than YK-424.
YK-403 is 42.01% higher than YK-414, 49.42% higher than YK-417, 49.71% higher than YK-418, and 42.83% higher than YK-424.
YK-422 is 41.73% higher than YK-414, 49.14% higher than YK-417, 49.43% higher than YK-418, and 42.55% higher than YK-424.
YK-423 is 42.39% higher than YK-414, 49.80% higher than YK-417, 50.09% higher than YK-418, and 43.21% higher than YK-424.
The cell survival rates of YK-412, YK-413, YK-415, YK-416, YK-419, YK-420 and YK-421 are 54.14%, 47.20%, 61.16%, 40.97%, 38.92%, 35.59% and 35.71%, respectively.
YK-407 is 17.23% higher than YK-412, 24.17% higher than YK-413, 10.21% higher than YK-415, 30.40% higher than YK-416, 32.45% higher than YK-419, 35.78% higher than YK-420, and 35.66% higher than YK-421.
YK-401 is 21.07% higher than YK-412, 28.01% higher than YK-413, 14.05% higher than YK-415, 34.24% higher than YK-416, 36.29% higher than YK-419, 39.62% higher than YK-420, and 39.50% higher than YK-421.
YK-402 is 18.56% higher than YK-412, 25.50% higher than YK-413, 11.54% higher than YK-415, 31.73% higher than YK-416, 33.78% higher than YK-419, 37.11% higher than YK-420, and 36.99% higher than YK-421.
YK-403 is 16.62% higher than YK-412, 23.56% higher than YK-413, 9.60% higher than YK-415, 29.79% higher than YK-416, 31.84% higher than YK-419, 35.17% higher than YK-420, and 35.05% higher than YK-421.
YK-422 is 16.34% higher than YK-412, 23.28% higher than YK-413, 9.32% higher than YK-415, 29.51% higher than YK-416, 31.56% higher than YK-419, 34.89% higher than YK-420, and 34.77% higher than YK-421.
YK-423 is 17.00 percent higher than YK-412, 23.94 percent higher than YK-413, 9.98 percent higher than YK-415, 30.17 percent higher than YK-416, 32.22 percent higher than YK-419, 35.55 percent higher than YK-420 and 35.43 percent higher than YK-421. (FIG. 9)
When the data are analyzed by GraphPad Prism software, any one of YK-412, YK-413, YK-415, YK-416, YK-419, YK-420 and YK-421 has obvious difference in cytotoxicity with other compounds, and the cytotoxicity is obviously reduced.
b. Difference in chemical structure
The structures of the series of compounds are very similar, and only the individual groups are slightly different. YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 are very close in structure to other compounds, and are very similar to each other.
And (4) summarizing:
and G 3 And G 4 Is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -, L is- (CH) 2 ) 2 -、-(CH 2 ) 3 -or- (CH) 2 ) 4 A series of structurally similar compounds of (A) with the lowest cytotoxicity compared to YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423. For example, YK-401 may be 53.87% higher than YK-417 and 54.16% higher than YK-418; YK-407 may be compared with YK-417 is 50.03% higher than YK-418 by 50.32%.
Meanwhile, the structure of the compound has no corresponding relation with cytotoxicity, and the cytotoxicity is very different possibly even if the difference in the structure is small.
Therefore, it is very difficult to screen out a cationic lipid compound with low cytotoxicity from a series of compounds that are only slightly different in individual groups, and much creative effort is required.
To summarize:
1) We performed cell viability assays on LNP preparations prepared from a series of compounds designed to screen out cationic lipid compounds with low cytotoxicity, such as YK-407, YK-401, YK402, YK-403, YK-422 and YK-423.
The series of compounds are designed to have significant differences from the chemical structures of the representative cationic lipids in the prior art, such as SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-009, wherein the difference from HHMA is the largest, the HHMA is connected with a central N atom, only 1 side chain is close to 1 side chain in the series of structures, and other parts are significantly different; compared with SM-102, ALC-0315, compound 21, compound 23, and YK-009, the head structure was significantly different, as were the other groups. Due to the large difference in structure, there are also large differences in polarity, acidity and basicity, hydrophilicity, and the like.
2) LNP formulations prepared from YK-407, YK-401, YK402, YK-403, YK-422, and YK-423 are minimally cytotoxic and significantly improved in survival rate over representative cationic lipid cells of the prior art. . For example, YK-401 cell survival rate can be 28.00% higher than ALC-0315, 7.31% higher than SM-102, and 10.94% higher than HHMA.
And G 3 And G 4 Is HO (CH) 2 ) 2 -, L is- (CH) 2 ) 2 Compared with a series of compounds with similar structures, YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 have the lowest cytotoxicity and the highest cell survival rate. For example, the cell survival rate of YK-401 is 58.88 percent higher than that of YK-411 and 50.25 percent higher than that of YK-406; the survival rate of YK-407 cells is 55.04 percent higher than that of YK-411 and 46.41 percent higher than that of YK-406.
And G 3 And G 4 Is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -, L is- (CH) 2 ) 2 -、-(CH 2 ) 3 -or- (CH) 2 ) 4 A series of structurally similar compounds of (A) with the lowest cytotoxicity compared to YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423. For example, cell viability YK-401 may be 53.87% higher than YK-417 and 54.16% higher than YK-418; YK-407 may be 50.03% higher than YK-417 and 50.32% higher than YK-418.
3) There is no correlation between the structure and cytotoxicity of compounds, and even compounds with little structural difference are likely to have very large differences in cytotoxicity. Therefore, the cytotoxicity of the lipid compound cannot be predicted from the chemical structure, and it is very difficult to screen out a cationic lipid compound having low cytotoxicity, and much creative work is required.
Example 8: in vivo validation of cationic lipid delivery vehicle Performance
In addition, we also demonstrated the protein expression and duration of the designed cationic lipid-delivered mRNA in mice. In vivo experiments further demonstrate that our LNP delivery vectors are able to deliver mRNA efficiently and consistently with high efficiency into the body.
LNP formulations containing 10 μ g Fluc-mRNA (prepared according to example 4) were injected intramuscularly in 4-6 week old female BALB/C mice weighing 17-19g and at specific time nodes (6 h, 24h, 48h and 7 d) after administration were intraperitoneally injected with fluorographic imaging substrate, the mice were freely active for 5 minutes and then the mean radiation intensity (corresponding to the intensity of fluorescent expression) of the proteins expressed in the mice by LNP-carried mRNA was measured by IVIS Spectrum in vivo animal imager.
The experimental results are as follows:
a. expression of mRNA in mice
The results of the measurement of mean radiation intensity of the protein expressed in mice by mRNA in LNP formulations are shown in tables 14-16 and FIGS. 10-13. In a series of designed compounds, the LNP preparation prepared from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 has high and continuous mRNA expression in mice, and is remarkably improved compared with the representative cationic lipid in the prior art. For example, YK-407 can be 27 times as high as SM-102, 22 times as high as ALC-0315, 28 times as high as Compound 21, 27 times as high as Compound 23, and 27 times as high as HHMA. mRNA expression in mice is consistent with cell transfection activity.
TABLE 14 mouse in vivo imaging Experimental data-1
a. Differences in expression in mice
Table 14 lists the strength of mRNA expression at different times in mice in LNP formulations containing Fluc-mRNA prepared from different cationic lipids. Wherein YK-009 is disclosed in CN114044741B (claim 1), SM-102 is compound 25 disclosed in WO2017049245A2 (page 29 of the specification), ALC-0315 is compound 3 disclosed in CN108368028B (page 24 of the specification), compound 21 and compound 23 are disclosed in WO2021055833A1 (page 22 of the specification), and HHMA is compound 1 disclosed in CN112979483B (page 12 of the specification), these cationic lipids can be used to prepare carriers for delivering mRNA.
As is clear from Table 14, the LNP preparation containing Fluc-mRNA prepared from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 was highly expressed and sustained in mice.
The mean radiation intensity of YK-407, at 6h, was 4001500, 5.79 times that of SM-102, 4.76 times that of ALC-0315, 6.66 times that of Compound 21, 6.88 times that of Compound 23, and 6.05 times that of HHMA; 2015870 at 24h, which is 16.34 times that of SM-102, 10.62 times that of ALC-0315, 18.15 times that of compound 21, 19.97 times that of compound 23, and 16.80 times that of HHMA; 885288 at 48h, 26.93 times that of SM-102, 22.06 times that of ALC-0315, 28.07 times that of compound 21, 26.89 times that of compound 23, and 26.55 times that of HHMA; 48410 at 7d, 7.20 times that of SM-102, 6.90 times that of ALC-0315, 7.59 times that of Compound 21, 7.93 times that of Compound 23, and 8.13 times that of HHMA.
The YK-401 average radiation intensity is 1557640 in 6h, which is 2.25 times that of SM-102, 1.85 times that of ALC-0315, 2.59 times that of compound 21, 2.68 times that of compound 23 and 2.36 times that of HHMA; 806240 at 24h, 6.53 times that of SM-102, 4.25 times that of ALC-0315, 7.26 times that of Compound 21, 7.98 times that of Compound 23, and 6.72 times that of HHMA; 393020 at 48h, 11.96 times that of SM-102, 9.79 times that of ALC-0315, 12.46 times that of Compound 21, 11.94 times that of Compound 23, and 11.79 times that of HHMA; 10512 at 7d, 1.56 times that of SM-102, 1.50 times that of ALC-0315, 1.65 times that of Compound 21, 1.72 times that of Compound 23, and 1.76 times that of HHMA.
The mean radiation intensity of YK-402, at 6h, was 1470100, 2.13 times that of SM-102, 1.75 times that of ALC-0315, 2.45 times that of Compound 21, 2.53 times that of Compound 23, and 2.22 times that of HHMA; 798408 at 24h, 6.47 times that of SM-102, 4.21 times that of ALC-0315, 7.19 times that of Compound 21, 7.91 times that of Compound 23, and 6.65 times that of HHMA; 345740 at 48h, 10.52 times as high as SM-102, 8.62 times as high as ALC-0315, 10.96 times as high as Compound 21, 10.50 times as high as Compound 23, and 10.37 times as high as HHMA; 9940 at 7d, 1.48 times that of SM-102, 1.42 times that of ALC-0315, 1.56 times that of compound 21, 1.63 times that of compound 23, and 1.67 times that of HHMA.
The mean radiation intensity of YK-403, 1588400 at 6h, was 2.30 times that of SM-102, 1.89 times that of ALC-0315, 2.65 times that of Compound 21, 2.73 times that of Compound 23, and 2.40 times that of HHMA; 855204 at 24h, 6.93 times that of SM-102, 4.51 times that of ALC-0315, 7.70 times that of Compound 21, 8.47 times that of Compound 23, and 7.13 times that of HHMA; 308540 at 48h, 9.39 times that of SM-102, 7.69 times that of ALC-0315, 9.78 times that of compound 21, 9.37 times that of compound 23, and 9.25 times that of HHMA; 9857 at 7d, 1.47 times that of SM-102, 1.41 times that of ALC-0315, 1.55 times that of Compound 21, 1.61 times that of Compound 23, and 1.65 times that of HHMA.
The YK-422 average radiation intensity, 1329410 at 6h, is 1.92 times that of SM-102, 1.58 times that of ALC-0315, 2.21 times that of Compound 21, 2.29 times that of Compound 23, and 2.01 times that of HHMA; 708120 at 24h, 5.74 times SM-102, 3.73 times ALC-0315, 6.38 times Compound 21, 7.01 times Compound 23, and 5.90 times HHMA; 271372 at 48h, 8.26 times SM-102, 6.76 times ALC-0315, 8.60 times Compound 21, 8.24 times Compound 23, and 8.14 times HHMA; 9476 at 7d is 1.41 times as great as SM-102, 1.35 times as great as ALC-0315, 1.49 times as great as Compound 21, 1.55 times as great as Compound 23, and 1.59 times as great as HHMA.
YK-423 mean radiation intensity at 6h 1627420, 2.35 times SM-102, 1.94 times ALC-0315, 2.71 times compound 21, 2.80 times compound 23, and 2.46 times HHMA; 756472 at 24h, 6.13 times SM-102, 3.99 times ALC-0315, 6.81 times Compound 21, 7.49 times Compound 23, and 6.30 times HHMA; 350120 at 48h, which is 10.65 times that of SM-102, 8.73 times that of ALC-0315, 11.10 times that of Compound 21, 10.64 times that of Compound 23, and 10.50 times that of HHMA; 10093 at 7d, 1.50 times that of SM-102, 1.44 times that of ALC-0315, 1.58 times that of Compound 21, 1.65 times that of Compound 23, and 1.69 times that of HHMA.
When the data are analyzed by GraphPad Prism software, any one of YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 has obvious difference with SM-102, ALC-0315, compound 21, compound 23 and HHMA at each time, and the expression amount and the duration time are obviously improved.
b. Difference in chemical structure
YK-407, YK-401, YK402, YK-403, YK-422 and YK-423 have great chemical structure difference with cationic lipid in the prior art, wherein the structural difference with HHMA is the largest, and the chemical structure chart shows that the group connected with the central N atom of the HHMA is only similar to 1 side chain of the series of structures and the other parts are obviously different. The head structure of this series of compounds is significantly different compared to SM-102, ALC-0315, compound 21, compound 23, and YK-009. The head structure of the series of compounds comprises 2 tertiary amine groups, an L group connecting 2 tertiary amine nitrogen atoms, and G containing hydroxyl 3 And G 4 While the head groups of SM-102, ALC-0315, compound 21, compound 23, and YK-009, comprise only 1 tertiary amine group, and HO (CH) attached to the tertiary amine nitrogen atom 2 ) 2 -. And, G of the series of compounds 1 、L 1 、R 1 、G 2 、L 2 And R 2 The group also differed significantly from SM-102, ALC-0315, compound 21, compound 23, and YK-009.
And (3) knotting:
in a series of designed compounds, the LNP preparation prepared from YK-407, YK-401, YK402, YK-403, YK-422 and YK-423 has the highest expression level of mRNA in mice, and can be expressed continuously, and the expression levels at 6h, 24h, 48h and 7d are all obviously improved compared with the expression levels of the representative cationic lipid in the prior art. For example, YK-407 can be 27 times as high as SM-102, 22 times as high as ALC-0315, 28 times as high as Compound 21, 27 times as high as Compound 23, and 27 times as high as HHMA. The expression of mRNA in mice was consistent with the results of the cell transfection experiments in example 7.
And, YK-407, YK-401, YK402, YK-403, YK-422 and YK-423 have significant differences in chemical structure compared to the representative cationic lipids of the prior art, with head groups, including G 3 、G 4 And L groups are significantly different, G 1 、L 1 、R 1 、G 2 、L 2 And R 2 The groups also differ significantly.
Therefore, not only LNP preparations made only from compounds with similar structure to the prior art cationic lipids, but also LNP preparations made from compounds with significantly different structures, mRNA is highly likely to be highly expressed and expressed continuously in mice.
(1) YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 with G designed in this application 3 And G 4 The radical is HO (CH) 2 ) 2 -, L radical is- (CH) 2 ) 2 The structurally similar compounds of (a) have the highest expression level and the longest duration of mRNA expression in mice. YK-407 can be expressed in 1000 times higher than other compounds, such as YK-411. mRNA expression in mice is consistent with cell transfection activity.
To compare the differences in the expression intensity and duration of mRNA delivered in mice with delivery vectors prepared from structurally similar compounds, G was designed 3 And G 4 Is HO (CH) 2 ) 2 -, L is- (CH) 2 ) 2 Compounds with slightly different groups were compared with YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 for cell viability. The results show that the cytotoxicity of the series of compounds is very different.
The expression of mRNA in the LNP preparation prepared by different compounds is very different in mice, wherein the expression levels of YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 are the highest, the duration is the longest, and the expression level of YK-407 can reach 1000 times of that of YK-411.
TABLE 15 mouse in vivo imaging Experimental data-2
a. Differences in expression in mice
As can be seen from Table 15, the correlation between G and G 3 And G 4 Is HO (CH) 2 ) 2 -, L is- (CH) 2 ) 2 The expression level and duration of mRNA in mice were highest in LNP preparations prepared from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 compared to the structurally similar compounds of (E-ll).
YK-407 can be 684.02 times as much as YK-411 in 6h, 996.97 times in 24h, 907.99 times in 48h and 100.85 times in 7 d.
YK-401 can reach 266.26 times of YK-411 in 6h, 398.73 times in 24h, 403.10 times in 48h and 21.90 times in 7 d.
YK-402 can reach 251.30 times of YK-411 in 6h, 394.86 times in 24h, 354.61 times in 48h and 20.71 times in 7 d.
The YK-403 can reach 271.52 times of YK-411 in 6 hours, 422.95 times in 24 hours, 316.45 times in 48 hours and 20.54 times in 7 d.
YK-422 can reach 227.25 times of YK-411 in 6 hours, 350.21 times in 24 hours, 278.33 times in 48 hours and 19.74 times in 7 days.
YK-423 can reach 278.19 times of YK-411 in 6h, 374.12 times in 24h, 359.10 times in 48h and 21.03 times in 7 d.
When the data are analyzed by GraphPad Prism software, any one of YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 has obvious difference with other compounds at each time, and the expression amount and the duration time are obviously improved.
b. Difference in chemical structure
This series of compounds G 3 And G 4 Is HO (CH) 2 ) 2 -, L is- (CH) 2 ) 2 Other groups are slightly different and are very similar in structure to YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423.
And (4) summarizing:
and G 3 And G 4 Is HO (CH) 2 ) 2 -, L is- (CH) 2 ) 2 LNP preparations prepared from YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 showed the highest expression intensity and the longest duration of mRNA in mice compared to compounds with slightly different groups. For example, YK-407 can be more than 600 times of YK-411 in 6 hours, 1000 times in 24 hours and still 100 times in 7 days. The expression of mRNA in mice was consistent with the results of the cell transfection experiments in example 7.
We have also found that there is no correspondence between mRNA expression in mice and cationic lipid structure, even though the structure is very similar, i.e.G only 3 、G 4 LNP preparations prepared from a group of compounds differing from the L group by 1-2C, and slightly differing from the other groups, are highly likely to differ greatly in the degree and duration of mRNA expression in mice.
Therefore, it is very difficult to screen out a cationic lipid compound having high and sustained expression in animals from a series of compounds having the most similar structures, and much creative work is required.
(2) YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 with G designed in this application 3 And G 4 The radical is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -, L radical is- (CH) 2 ) 2 -、-(CH 2 ) 3 -or- (CH) 2 ) 4 The structurally similar compounds of (a) have the highest expression level and the longest duration of mRNA expression in mice. For example, the expression level of YK-407 can be 120 times of that of YK-417. mRNA expression in mice is consistent with cell transfection activity.
LNP preparations containing mRNA prepared from YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 were further compared to G 3 And G 4 The radical is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -, L radical is- (CH) 2 ) 2 -、-(CH 2 ) 3 -or- (CH) 2 ) 4 Differences in expression of the structurally similar compounds of (a) to (b) in mice. The compounds of this series differ only slightly in individual groups, e.g. G 3 、G 4 Or the L groups differ by 1-2C, with slight differences in other structures. The results show that the expression levels of YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 are the highest, the duration is the longest, and the expression levels are obviously higher than those of other compounds. For example, YK-407 can be expressed 120 times as much as YK-417.
TABLE 16 mouse in vivo imaging Experimental data-3
a. Differences in expression in mice
As can be seen from Table 19, in this series of compounds, LNP preparations prepared from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 exhibited the highest expression levels and durations of mRNA in mice.
The expression quantity of YK-407 can be 33.96 times of YK-415 and 66.16 times of YK-417 in 6h, 31.25 times of YK-415 and 100.86 times of YK-417 in 24h, 56.07 times of YK-415 and 125.66 times of YK-417 in 48h, and 11.98 times of YK-415 and 21.20 times of YK-417 in 7 d.
The expression level of YK-401 can reach 13.22 times of YK-415 and 25.75 times of YK-417 in 6h, 12.50 times of YK-415 in 24h, 40.34 times of YK-417 in 24h, 24.89 times of YK-415 and 55.79 times of YK-417 in 48h, and 2.60 times of YK-415 and 4.60 times of YK-417 in 7 d.
The expression level of YK-402 can reach 12.48 times of YK-415 and 24.31 times of YK-417 in 6h, 12.38 times of YK-415 and 39.95 times of YK-417 in 24h, 21.90 times of YK-415 and 49.08 times of YK-417 in 48h, and 2.46 times of YK-415 and 4.35 times of YK-417 in 7 d.
The expression level of YK-403 can reach 13.48 times of YK-415 and 26.26 times of YK-417 within 6h, 13.26 times of YK-415 and 42.79 times of YK-417 within 24h, 19.54 times of YK-415 and 43.80 times of YK-417 within 48h, and 2.44 times of YK-415 and 4.32 times of YK-417 within 7 d.
The expression quantity of YK-422 can reach 11.28 times of YK-415 and 21.98 times of YK-417 in 6 hours, 10.98 times of YK-415 and 35.43 times of YK-417 in 24 hours, 17.19 times of YK-415 and 38.52 times of YK-417 in 48 hours, and 2.34 times of YK-415 and 4.15 times of YK-417 in 7 d.
The expression level of YK-423 can reach 13.81 times of YK-415 and 26.91 times of YK-417 in 6h, 11.73 times of YK-415 in 24h, 37.85 times of YK-417 in 24h, 22.17 times of YK-415 and 49.70 times of YK-417 in 48h, and 2.50 times of YK-415 and 4.42 times of YK-417 in 7 d.
When the data are analyzed by GraphPad Prism software, any one of YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 has obvious difference with other compounds at each time, and the expression brightness and duration are obviously improved.
b. Difference in chemical structure
The compounds of this series differ only slightly in the individual radicals, e.g. G 3 、G 4 Or the difference of L groups is 1-2C, other structures are slightly different, but the expression quantity of mRNA, YK-407 can reach 120 times of YK-417.
And (4) summarizing:
and G 3 And G 4 The radical is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -, L radical is- (CH) 2 ) 2 -、-(CH 2 ) 3 -or- (CH) 2 ) 4 -compared to LNP preparations prepared from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, the mRNA expression intensity was highest and the duration was longest in mice. For example, YK-407 can be up to 120 times YK-417 at 48h and still up to 20 times at 7 d. The expression of mRNA in mice was consistent with the results of the cell transfection experiments described in example 7.
We have also found that there is no correspondence between mRNA expression in mice and cationic lipid structure, even though there is some minor difference in individual groups, such as G 3 、G 4 Or L groups differ by 1-2C, other structures are slightly different, LNP preparation prepared from the compounds, and the expression degree and duration of mRNA in miceIt is also highly likely that the differences are very large.
Therefore, it is very difficult to select a cationic lipid compound having high and sustained expression in animals from a series of compounds having only some small differences among individual groups, and much creative work is required.
b. Distribution of liposomes in mice
The results of in vivo imaging of mice show that the distribution of liposomes prepared from different compounds in mice is greatly different, some of the liposomes have protein expression in the liver, some of the liposomes have no protein expression in the liver, and some of the liposomes have protein expression in the spleen.
The method comprises the following specific steps: at 6h, some cationic lipids, such as SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-402, exhibited protein expression in the liver, and the amount of YK-402 expression was reduced compared to SM-102; while some compounds, such as YK-407, YK-411, YK-418, YK-419 and YK-424, were not protein expressed in the liver (FIG. 14). SM-102, YK-402, YK-407, YK-419 and YK-424 expressed proteins in the spleen, with YK-407, YK-419 and YK-424 expressed only in the spleen, were able to deliver mRNA directly to the spleen, and not in other organs, such as the liver, lung, heart and kidney (FIG. 15). YK-411 and YK-418 have no protein expression in liver, spleen, lung, heart and kidney, indicating that the protein is generally expressed in muscle by the two compounds, and the mRNA vaccine at present. ALC-0315, compound 21, compound 23 and HHMA are similar to SM-102 and are not shown.
It can be seen that, in contrast to the prior art cationic lipids, liposomes prepared from some of the compounds contemplated herein, such as YK-407, YK-411, YK-418, YK-419, and YK-424, do not reside in the liver and express the protein of interest after intramuscular injection. If the mRNA carried in the liposomes is expressed in the liver, the expressed protein is metabolized by the liver, which increases the burden on the liver, and thus some of the compounds contemplated herein may have reduced or no hepatotoxicity of the liposomes in comparison to the cationic lipids representative of the prior art.
In addition, liposomes prepared from some of the compounds contemplated herein, such as YK-407, YK-419, and YK-424, are capable of delivering mRNA directly to the spleen, but are not expressed in other organs, such as the liver, lung, heart, and kidney. The spleen is the largest secondary lymphoid organ in vivo, so that the immune response can be rapidly induced and antibodies can be produced. The prevention effect can be obviously improved under the condition of not changing the components of the vaccine, and the method has important clinical significance. Has good target effect on developing and treating diseases caused by spleen damage or abnormality such as lymphoma, leukemia and the like.
And (3) knotting:
compared with the prior art representative cationic lipids, such as SM-102, ALC-0315, compound 21, compound 23 and HHMA, the liposomes prepared from the contemplated compounds of the present application express a reduced amount of the protein of interest in the liver (YK-402), or do not reside in the liver and express the protein of interest (YK-407, YK-411, YK-418, YK-419, and YK-424), and have reduced or no toxicity to the liver. In addition, some compounds designed in the application, such as liposomes prepared from YK-407, YK-419 and YK-424, can directly deliver mRNA to spleen, but are not expressed in other organs, such as liver, lung, heart and kidney, so that the prevention effect can be remarkably improved without changing vaccine components, and the application has important clinical significance.
To summarize:
1) We performed in vivo animal delivery experiments on LNP preparations prepared from a designed series of compounds and screened cationic lipid compounds such as YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, for which mRNA has high and sustained expression in mice.
The series of compounds are distinguished from the typical cationic lipids in the prior art, such as SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-009, by their chemical structure, the head structure, including G 3 、G 4 The L group is obviously different from the L group, and other parts are also different, so that the polarity, the acidity and the alkalinity, the hydrophilicity and the like are also greatly different.
2) The LNP preparation prepared from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 has high and continuous expression of mRNA in mice, and is remarkably improved compared with the representative cationic lipid in the prior art. For example, YK-407 can be 27 times as high as SM-102, 22 times as high as ALC-0315, 28 times as high as Compound 21, 27 times as high as Compound 23, and 27 times as high as HHMA.
And G 3 And G 4 Is HO (CH) 2 ) 2 -, L is- (CH) 2 ) 2 -compared to LNP preparations prepared from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, the mRNA expression intensity was highest and the duration was longest in mice. For example, YK-407 can be more than 600 times of YK-411 in 6 hours, 1000 times in 24 hours and still 100 times in 7 days.
And G 3 And G 4 The radical is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -, L radical is- (CH) 2 ) 2 -、-(CH 2 ) 3 -or- (CH) 2 ) 4 -compared to LNP formulations prepared from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, the mRNA expression intensity was highest and the duration was longest in mice. For example, YK-407 can be up to 120 times YK-417 at 48h and still up to 20 times at 7 d.
Liposomes prepared from the compounds contemplated herein express reduced amounts of the protein of interest in the liver (YK-402), or do not reside in the liver and express the protein of interest (YK-407, YK-411, YK-418, YK-419, and YK-424), as compared to prior art representative cationic lipids, such as SM-102, ALC-0315, compound 21, compound 23, and HHMA. LNP formulations prepared from the compounds contemplated herein are therefore less or non-toxic to liver toxicity compared to prior art cationic lipids. In addition, some compounds designed in the application, such as YK-407, YK-419 and YK-424, can directly deliver mRNA to spleen, but are not expressed in other organs, such as liver, lung, heart and kidney, and can significantly improve the prevention effect without changing the vaccine components, thereby having important clinical significance.
3) There is no correspondence between the structure of the cationic lipid and the high and sustained expression of the delivered mRNA in mice, and whether it is a compound with little structural variation or a compound with very large structural variation, it is highly likely that the mRNA in LNP formulations prepared therefrom will vary greatly in expression in animals. Whether mRNA is highly expressed and continuously expressed in an animal body cannot be predicted according to the chemical structure of the cationic lipid, and screening of the cationic lipid compound with high mRNA expression and continuous expression is very difficult, and a great deal of creative work is required.
And (4) conclusion:
1. a series of compounds were designed, including YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, with significant differences in chemical structure from prior art cationic lipids, such as SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-009, head structures, including G 3 、G 4 The L group is obviously different from the L group, and other parts are also different, so that the polarity, the acid-base property, the hydrophilicity and the like are also greatly different.
In the series of compounds designed by the application, LNP preparations prepared from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 have obviously improved encapsulation efficiency, drug-loading concentration and total RNA concentration, obviously improved cell transfection efficiency, obviously reduced cytotoxicity and obviously improved expression amount and duration of mRNA in mice compared with the typical cationic lipid in the prior art. For example, the encapsulation efficiency of YK-407 can be improved by 29.0% compared with that of compound 23, the drug-loading concentration can reach 1.78 times of that of compound 23, and the total RNA concentration can reach 1.41 times of that of compound 21; YK-407 cell transfection efficiency can reach 12 times of SM-102, 13 times of compound 21 and 15 times of compound 23; the survival rate of YK-401 cells is 28.00 percent higher than that of ALC-0315, 7.31 percent higher than that of SM-102 and 10.94 percent higher than that of HHMA; the expression level of mRNA of the LNP preparation prepared from YK-407 in mice can reach 27 times of SM-102, 22 times of ALC-0315, 28 times of compound 21, 27 times of compound 23 and 27 times of HHMA.
Also, LNP formulations prepared from the compounds contemplated herein express reduced amounts of the protein of interest in the liver (YK-402), or do not reside in the liver and express the protein of interest (YK-407, YK-411, YK-418, YK-419, and YK-424), with reduced or no toxicity to the liver, as compared to the cationic lipids typically found in the prior art; YK-407, YK-419 and YK-424 were able to deliver mRNA directly to the spleen, but were not expressed in other organs, such as liver, lung, heart and kidney, and were able to significantly improve the prophylactic effect without changing the vaccine components.
In the designed compound, the LNP preparation prepared from YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 has obviously improved cell transfection efficiency, obviously reduced cytotoxicity and obviously improved expression amount and duration of mRNA in a mouse body compared with other compounds.
Other compounds are structurally similar to YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, with only individual groups differing, e.g. G 3 、G 4 Or the L groups differ by 1-2C, and the other structures differ slightly, but the activity differences are very large. For example, YK-407 cells can be transfected with 2500 times higher efficiency than YK-404 and YK-411; the cytotoxicity of YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423 can be reduced by 50 percent compared with that of YK-411; the expression quantity of mRNA of the LNP preparation prepared by YK-407 in a mouse body can reach 1000 times of that of YK-411.
2. There is no obvious correspondence between the structure of the cationic lipid compound and the transfection efficiency in cells, the toxicity to cells and the high and sustained expression of mRNA in LNP preparations prepared therefrom in animals. Compounds with little structural variation are also likely to vary greatly in transfection efficiency and/or toxicity to cells, intracellular expression.
For example, YK-411 has a very close structure compared to YK-407. YK-411 being only G 1 The radical is 2 more C; r 1 1 less C; r is 2 The gene single chain has 2 more C, each single chain in the double chain has 2 less C, but the cell transfection efficiency YK-407 is 2500 times higher than that of YK-411, the toxicity to transfected cells YK-407 is 55% lower than that of YK-411, and the expression of mRNA YK-407 in mouse body can reach 1000 times higher than that of YK-411.
Therefore, it is very difficult to select suitable cationic lipid compounds, which have high transfection efficiency and low toxicity to cells, and high and sustained expression of mRNA in mice, and it requires much creative work.
3. Through unique design and extensive screening, the invention discovers that some compounds, such as YK-407, YK-401, YK-402, YK-403, YK-422 and YK-423, can deliver nucleic acid with remarkably improved encapsulation efficiency, drug loading concentration and total RNA concentration, remarkably improved cell transfection efficiency, remarkably reduced cytotoxicity and remarkably improved expression amount and duration in animals compared with other compounds in the prior art, and achieve unexpected technical effects.
Claims (65)
1. A compound of formula (I)Or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein
G 1 Is C 2~8 An alkylene group;
G 2 is C 2~8 An alkylene group;
L 1 is-C (O) O-or-OC (O) -;
L 2 is-C (O) O-or-OC (O) -;
R 1 is C 6~25 A linear or branched alkyl group;
R 2 is composed of C6~25 A linear or branched alkyl group;
G 3 is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -；
G 4 Is HO (CH) 2 ) 2 -or HO (CH) 2 ) 3 -；
L is- (CH) 2 ) 2 -or- (CH) 2 ) 3 -or- (CH) 2 ) 4 -。
2. A compound of formula (I) according to claim 1 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 1 Is unsubstituted C 3~8 An alkylene group.
3. The method of claim 1 or 2Or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 1 Is unsubstituted C 3 Alkylene or C 5 Alkylene or C 6 Alkylene or C 7 Alkylene or C 8 An alkylene group.
4. A compound of formula (I) according to any one of the preceding claims or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 2 Is unsubstituted C 3~8 An alkylene group.
5. A compound of formula (I) according to any one of the preceding claims or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 2 Is unsubstituted C 3 Alkylene or C 5 Alkylene or C 6 Alkylene or C 7 Alkylene or C 8 An alkylene group.
6. A compound of formula (I) according to any one of the preceding claims or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein L 1 is-C (O) O-.
7. A compound of formula (I) according to any one of the preceding claims or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein L 1 is-OC (O) -.
8. A compound of formula (I) according to any one of the preceding claims or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein L 2 is-C (O) O-.
9. A compound of formula (I) according to any one of the preceding claims or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein L 2 is-OC (O) -.
10. A compound of formula (I) according to any one of the preceding claims or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 1 Is unsubstituted C 11 Straight chain alkyl or unsubstituted C 10 A linear alkyl group.
11. A compound of formula (I) according to any one of the preceding claims 1 to 9 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 1 Is unsubstituted C 8~18 A branched alkyl group.
12. A compound of formula (I) according to claim 11 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 1 Is unsubstituted C 17 Branched alkyl or C 18 Branched alkyl or C 8 A branched alkyl group.
13. A compound of formula (I) according to claim 12 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 1 Comprises the following steps:、or is。
14. A compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any preceding claim, wherein R 2 Is unsubstituted C 8~18 A branched alkyl group.
15. A compound of formula (I) according to claim 14 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 2 Is unsubstituted C 17 Branched alkyl or C 18 Branched alkyl or C 15 Branched alkyl or C 14 Branched alkyl radical C 8 A branched alkyl group.
16. A compound of formula (I) according to claim 15 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 2 Comprises the following steps:、、、or。
17. A compound of formula (I), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, according to any preceding claim, wherein the compound of formula (I) has one of the following structures:
，
，
，
，
，
，
，
，
，
，
，
，
，
，
，
，
，
，
，
，
，
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，
。
18. a compound of formula (I) according to claim 1 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) is compound YK-407 having the structure:
。
19. a compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to claim 1, wherein the compound of formula (I) is compound YK-401 having the structure:
。
20. the compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to claim 1, wherein the compound of formula (I) is compound YK-402 having the structure:
。
21. a compound of formula (I) according to claim 1, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) is compound YK-403 having the structure:
。
22. the compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to claim 1, wherein the compound of formula (I) is compound YK-422 having the structure:
。
23. a compound of formula (I) according to claim 1 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) is compound YK-423 having the structure:
。
24. a composition comprising a carrier comprising a cationic lipid comprising a compound of formula (I) according to any preceding claim or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof.
25. The composition of claim 23, wherein the cationic lipid is present in a carrier at a molar ratio of 25% to 75%.
26. The composition of any one of claims 23-24, wherein the carrier further comprises a neutral lipid.
27. The composition according to claim 26, wherein the molar ratio of the cationic lipid to the neutral lipid is 1 to 1, preferably 4.
28. The composition of any one of the preceding claims 26-27, wherein the neutral lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.
29. The composition of any one of the preceding claims 26-28, wherein the neutral lipid is selected from one or more of the following: 1, 2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-didecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) 1, 2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18: 0 Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1, 2-dilinolacyl-sn-glycero-3-phosphocholine, 1, 2-dineoyl-sn-glycero-3-phosphocholine, 1, 2-didodecanoyl-sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-Diphytoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dineoyl-sn-glycero-3-phosphoethanolamine, 1, 2-didodecanoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycero) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG) Palmitoyl Oleoyl Phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.
30. The composition of any one of the preceding claims 26-29, wherein the neutral lipid is DOPE and/or DSPC.
31. The composition of any one of the preceding claims 24-30, wherein the carrier further comprises a structural lipid.
32. The composition according to claim 31, wherein the molar ratio of the cationic lipid to the structural lipid is 0.6 to 1 to 3.
33. The composition of any one of the preceding claims 31-32, wherein the structural lipid is selected from one or more of the following: cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, lycopersine, ursolic acid, alpha-tocopherol, corticosteroid.
34. The composition of any one of the preceding claims 31-33, wherein the structural lipid is cholesterol.
35. The composition of any one of the preceding claims 24-34, wherein the carrier further comprises a polymer conjugated lipid.
36. The composition according to claim 35, wherein the molar ratio of the polymeric conjugated lipid to the carrier is 0.5% to 10%, preferably 1.5%.
37. The composition of any one of the preceding claims 35-36, wherein the polymeric 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, and PEG-modified dialkylglycerol.
38. The composition of any one of the preceding claims 35-37, wherein the polymeric conjugated lipid is selected from one or more of the following: distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000), dimyristoyl glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000) and methoxypolyethylene glycol ditetradecylethanolamide (ALC-0159).
39. The composition of any of the preceding claims 24-38, wherein the carrier comprises a neutral lipid, a structural lipid, and a polymer conjugated lipid, and 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).
40. The composition of claim 39, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer conjugated lipid is (35-49): (7.5-15): (35-55): (1-5).
41. The composition of claim 40, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymeric conjugated lipid is 40.
42. The composition of any one of the preceding claims 24-41, wherein the composition is a nanoparticle formulation having an average particle size of 10nm to 300nm; the polydispersity of the nano-particle preparation is less than or equal to 50%.
43. The composition of claim 42, wherein the nanoparticle formulation has an average particle size of 90nm to 280nm; the polydispersity index of the nano-particle preparation is less than or equal to 40 percent.
44. The composition of any one of the preceding claims 24-43, wherein the cationic lipid further comprises one or more other ionizable lipid compounds.
45. The composition of any one of the preceding claims 24-44, further comprising a therapeutic or prophylactic agent.
46. The composition according to claim 45, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 10 to 1.
47. The composition according to claim 46, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 12.5 to 1 to 20.
48. The composition according to claim 47, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 15.
49. The composition of any one of the preceding claims 45-48, wherein the therapeutic or prophylactic agent comprises one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.
50. The composition of any one of the preceding claims 45-48, wherein the therapeutic or prophylactic agent is a vaccine or compound capable of eliciting an immune response.
51. The composition of any one of the preceding claims 45-48, wherein the therapeutic or prophylactic agent is a nucleic acid.
52. The composition of any one of the preceding claims 45-48, wherein the therapeutic or prophylactic agent is a ribonucleic acid (RNA).
53. The composition of any one of the preceding claims 45-48, wherein the therapeutic or prophylactic agent is deoxyribonucleic acid (DNA).
54. The composition of any one of the preceding claims 45-48, wherein the RNA is selected from the group consisting of: small interfering RNAs (sirnas), asymmetric interfering RNAs (airnas), micrornas (mirnas), dicer-substrate RNAs (dsrnas), small hairpin RNAs (shrnas), messenger RNAs (mrnas), and mixtures thereof.
55. The composition of claim 54, wherein the RNA is mRNA.
56. The composition of any one of the preceding claims 24-55, wherein the composition further comprises one or more of a pharmaceutically acceptable excipient or diluent.
57. Use of a compound of formula (I) according to any one of the preceding claims 1 to 23 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a composition according to any one of the preceding claims 24 to 56, for the preparation of a nucleic acid medicament, a genetic vaccine, a small molecule medicament, a polypeptide or a protein medicament.
58. Use of a compound of formula (I) as described in any one of claims 1-23 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a composition as described in any one of claims 24-56, in the manufacture of a medicament for treating a disease or condition in a mammal in need thereof.
59. The use of claim 58, wherein the disease or disorder is characterized by a malfunctioning or abnormal protein or polypeptide activity.
60. The use of any one of claims 58-59, wherein 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.
61. The use of claim 60, wherein the infectious disease is selected from the group consisting of: diseases caused by coronavirus, influenza virus or HIV virus, infantile pneumonia, rift valley fever, yellow fever, rabies, or various herpes.
62. The use of any one of the preceding claims 58-61, wherein the mammal is a human.
63. The use of any one of the preceding claims 58-62, wherein the composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.
64. The use of claim 63, wherein the composition is administered subcutaneously.
65. The use of any of the preceding claims 58-64, wherein the therapeutic or prophylactic agent is administered to the mammal at a dose of 0.001mg/kg to 10 mg/kg.
Publications (2)
| Publication Number | Publication Date |
|---|---|
| HK40081634A true HK40081634A (en) | 2023-05-25 |
| HK40081634B HK40081634B (en) | 2023-07-14 |
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