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 should be apparent that the described embodiments are only some of the embodiments of the present invention, and not all of them. 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 of 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 the use or term used in the present application, the use or term used in 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 throughout this specification is intended to imply that the elements listed before any such word or phrase are not in the order of magnitude of the word or phrase, but rather the word or phrase is not necessarily limited to the particular elements listed. 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 mammal in which the disease or condition is to be prevented or treated therewith.
The term "subject" or "patient" in this application includes mammals and non-mammals. Mammals include, but are not limited to: humans, orangutans, apes, monkeys, cows, horses, sheep, pigs, rabbits, dogs, cats, and mice, and the like. Non-mammals include, but are not limited to, birds, fish, and the like. In some embodiments, a "subject" or "patient" is a mammal, e.g., a human.
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 and a solvent, such as ethanol or water. It will be appreciated that any solvate of a compound of formula (I) used in the treatment of a disease or condition, although potentially offering different properties, including pharmacokinetic properties, will result, once absorbed into a subject, in a compound of formula (I) such that use of the 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, the 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 the relatively non-toxic, inorganic or organic acid addition salts of the compounds of the present invention. See, e.g., 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, HCl (or hydrochloric acid), HBr (or hydrobromic acid solution), methanesulfonic acid, sulfur can be usedThe acid, tartaric acid or fumaric acid forms a pharmaceutically acceptable salt with the compound shown in the formula (I).
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). Thus, the compounds claimed herein include not only the nitrogen-containing compounds shown in the structural formula, but also N-oxide derivatives thereof, as the valence 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 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 as2 H (deuterium),3 H (tritium),11 C、13 C、14 C、15 N、17 O and18 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" as used herein is meant to include a compound havingBranched and straight chain saturated aliphatic divalent hydrocarbon groups of specified carbon number. Cn~m Is meant to include groups having n to m carbon atoms. E.g. C2~5 Alkylene radicals including C2 Alkylene radical, C3 Alkylene radical, C4 Alkylene radical, C5 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 is controlling.
It will 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 a representative cationic lipid structure, has high encapsulation efficiency, drug-loading concentration and total RNA concentration, high transfection efficiency and low cytotoxicity, and has high expression and sustained expression in mice. The inventors have discovered that certain compounds, such as YK-201, YK-202, and YK-209, among others, are capable of delivering nucleic acids with significantly improved encapsulation efficiency, drug loading concentration, and total RNA concentration, significantly improved intracellular transfection efficiency, significantly reduced cytotoxicity, and significantly improved expression levels and duration in animals, as compared to the widely different chemical structures of cationic lipids of the prior art. The present invention is based on at least the following findings:
1. a series of compounds were designed, including YK-201, YK-202 and YK-209, and YK-206 and YK-207, with significant differences in the chemical structure of YK-009, G, from the prior art representative cationic lipids, such as SM-102 (compound 25 disclosed in WO2017049245 A2), compound 21 and compound 23 disclosed in WO2021055833A1, HHMA (compound 1 disclosed in CN 112979483B), ALC-0315 (compound 3 disclosed in CN 108368028B), and compound YK-009 disclosed in CN114044741B3 The groups are completely different, and other positions are also different, so that the polarity, the acid-base property, the hydrophilicity and the like are also greatly different. 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 series of designed compounds, LNP preparations prepared from YK-201, YK-202 and YK-209 have obviously improved encapsulation efficiency, drug-loading concentration and total RNA concentration, obviously improved cell transfection activity, 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 YK-209 can be improved by 41 percent compared with the compound 23, the drug-loading concentration YK-209 can reach 2 times of that of the compound 23, and the total RNA concentration YK-201 can reach 1.5 times of that of the compound 21; the cell transfection activity YK-202 can reach 18 times of SM-102, 21 times of compound 21 and 22 times of compound 23; the survival rate of the cells YK-202 is 26.85 percent higher than that of ALC-0315, 8.26 percent higher than that of SM-102 and 11.25 percent higher than that of HHMA; the expression level of mRNA in mice can reach 24 times of that of SM-102, 25 times of that of compound 21 and 23 times of that of compound 23 by YK-202.
In a series of compounds with small difference in chemical structure, LNP preparations prepared from YK-201, YK-202 and YK-209 have remarkably improved cell transfection activity, remarkably reduced cytotoxicity and remarkably improved expression amount and duration of mRNA in mice compared with other compounds.
Compared with YK-201, YK-202 and YK-209, the series of compounds have the structure that only the individual groups have 1-2C differences or G differences1 Reduction of 2C and R1 Decrease by 1C, G3 By introduction of ether bonds, ester bonds, or sulfur atoms, or G3 In which the hydroxy group is changed to methylamino, or G3 Hydroxyl and methyl connected with N are removed, but the transfection activity of YK-201, YK-202 and YK-209 cells can reach more than 1000 times of YK-221 and YK-225, the cytotoxicity can be reduced by 55 percent compared with YK-221, and the expression quantity of mRNA in a mouse body can reach more than 1000 times of YK-223.
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. Compounds with little structural variation are likely to vary greatly in transfection efficiency and/or toxicity to cells, intracellular expression.
For example, YK-204 is G only, as compared to YK-2013 1 more C is connected with the group and N, other structures are completely the same, but the cell transfection efficiency YK-201 is 78 times of YK-204, the toxicity to the transfected cells YK-201 is reduced by 30 percent compared with YK-204, and the expression YK-201 of mRNA in a mouse body can reach 380 times of YK-204; YK-225 is G only, in contrast to YK-2093 In which the hydroxy group is changed to methylamino, R1 And R2 The single chain of the group has 1C each, each single chain in the double chain has 2C less each, other structures are completely the same, but the cell transfection activity YK-209 is 800 times of YK-225, and the toxicity YK-209 to the transfected cells is reduced by 30 percent compared with YK-225.
Therefore, it is very difficult to screen suitable cationic lipid compounds, which can simultaneously have high encapsulation efficiency, drug loading concentration and total RNA concentration, high transfection efficiency and low toxicity to cells, and high and continuous expression of mRNA in mice, and a lot of creative labor is required.
4. Through unique design and extensive screening, the invention discovers that some compounds, such as YK-201, YK-202, YK-209, YK-206 and YK-207, 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 achieves unexpected technical effects.
The method comprises the following specific steps:
1. there are significant differences in chemical structure compared to prior art representative cationic lipids, e.g., SM-102, ALC-0315, compound 21, compound 23, YK-009, and HHMA
Representative cationic lipids of the prior art, such as SM-102, ALC-0315, compound 21, compound 23, YK-009, and HHMA, compared to the series of compounds designed:
a. the HHMA structure has the biggest difference, and the group connected with the central N atom of the HHMA has only 1 side chain similar to 1 side chain of the series structure, and the other parts are completely different.
b. Other cationic lipids of the prior art, such as SM-102, ALC-0315, compound 21, compound 23, and YK-0093 The groups are completely different. Due to G3 The groups have different structures, so that the polarity, the acid-base property, the hydrophilicity and the like of the groups can be greatly different.
c. SM-102, ALC-0315, compound 21, compound 23, and G of YK-0091 、R1 、G2 And R2 The groups also differ significantly.
2. The encapsulation efficiency, the drug loading concentration and the total RNA concentration are obviously improved compared with the prior art which represents cationic lipid
1) Compared with the cationic lipid in the prior art, the LNP preparation prepared from YK-201, YK-202, YK-206, YK-207 and YK-209 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-209 can be improved by 41 percent compared with that of the compound 23, and the drug-loading concentration can reach 2 times of that of the compound 23; the total RNA concentration of YK-201 can reach 1.5 times of that of compound 21.
2) The LNP preparation prepared from different designed compounds has the encapsulation efficiency and the drug loading rate which are greatly different, the range of the encapsulation efficiency of the different compounds is 55% -99%, the drug loading concentration is 25-45 mug/mL, and the total RNA concentration is 25-50 mug/mL.
3. The transfection efficiency of the in vitro cells is obviously improved compared with the prior art that the representative cationic lipid and the compound with similar structure
1) LNP preparations prepared from YK-201, YK-202 and YK-209 have the highest cell transfection efficiency, and are remarkably improved in activity compared with representative cationic lipids in the prior art. For example, YK-202 can be 18 times as high as SM-102, 21 times as high as compound 21, and 22 times as high as compound 23.
2) YK-201, YK-202 and YK-209 are most similar in the structure we have designed, only G1 、G2 、G3 、R1 Or R2 A series of compounds with groups differing by 1-2CThe transfection efficiency of the medium cells is highest. YK-202 may be up to 80 times that of other compounds, such as YK-204.
3) YK-201, YK-202 and YK-209 have some minor differences from the individual radicals only, e.g. G1 Reduction of 2C, R1 Reduction of 1C, G3 The transfection efficiency was highest compared with compounds in which ether bond, ester bond or sulfur atom was introduced. YK-202 may be more than 400 times higher than other compounds, such as YK-217.
4) YK-201, YK-202 and YK-209 with G only3 The transfection efficiency of cells was highest when the hydroxyl group in the group was changed to methylamino group, or when the hydroxyl group was removed from the compound having a methyl group bonded to N. For example, YK-201 and YK-202 can increase the cell transfection efficiency by more than 1000 times compared to YK-221 and YK-225.
5) There is no correlation between the structure of the compound and the intracellular transfection efficiency, and compounds with small structural differences also have a high possibility of very large differences in transfection efficiency. Therefore, screening of cationic lipid compounds with high transfection efficiency requires various designs and much creative work.
4. The cytotoxicity is obviously reduced compared with that of the representative cationic lipid and the compound with similar structure in the prior art
1) LNP formulations prepared from YK-201, YK-202, and YK-209 were minimally cytotoxic and significantly improved in survival over representative cationic lipid cells of the prior art. For example, cell viability YK-202 could be 26.85% higher than ALC-0315, 8.26% higher than SM-102, and 11.25% higher than HHMA.
2) YK-201, YK-202 and YK-209 are most similar in the structure we have designed, i.e. only G1 、G2 、R1 Or R2 Among the compounds with 1-2C difference in groups, cytotoxicity was the lowest. The cell survival rate of the three compounds is improved by 40 percent compared with that of other compounds, such as YK-203.
3) YK-201, YK-202 and YK-209 were the least cytotoxic compared to compounds that were only slightly different in structure by individual groups. The cell survival rate of the three compounds is improved by 50 percent compared with that of other compounds, such as YK-214.
4) YK-201, YK-202 and YK-209 and in structuresAbove is only G3 Compounds in which the hydroxyl group of the group is changed to a methylamino group, or the hydroxyl group is removed and the methyl group attached to the N is the least cytotoxic. For example, the cell survival rates of YK-201, YK-202 and YK-209 are all improved by 55 percent compared with that of YK-221.
5) There is no correlation between the structure and cytotoxicity of compounds, and even compounds with small structural differences are likely to have very large differences in cytotoxicity. Therefore, the cytotoxicity of the compound cannot be predicted from the chemical structure, and screening of a cationic lipid compound having low cytotoxicity is very difficult and requires much creative work.
5. The expression quantity and duration of mRNA in animal body are obviously improved compared with the prior art that the representative cationic lipid and the compound with similar structure
1) The LNP preparation prepared from YK-201, YK-202 and YK-209 has high and continuous mRNA expression in mice, and is obviously improved compared with the representative cationic lipid in the prior art. For example, YK-202 can be up to 24 times that of SM-102, 25 times that of compound 21, and 23 times that of compound 23.
2) YK-201, YK-202 and YK-209 are most similar in the structure we have designed, only G1 、G2 、G3 、R1 Or R2 In a series of compounds with groups differing by 1-2C, mRNA was expressed in the highest amount and for the longest duration in mice. For example, YK-202 can be more than 400 times as high as YK-204 in 24 hours and still can be 40 times as high as YK-204 in 7 days.
3) YK-201, YK-202 and YK-209 are only G1 、G2 、G3 、R1 Or R2 In a series of compounds with slightly different groups, mRNA was expressed in the mice in the highest amount and for the longest duration. For example, YK-202 may be improved by a factor of 800 over YK-217.
4) YK-201, YK-202 and YK-209 with G only3 In a series of compounds in which the hydroxyl group in the group is changed into a methylamino group, or the hydroxyl group and the methyl group connected with the N are removed, the mRNA is expressed in the mouse in the highest amount and lasts for the longest time. For example, the expression level of the three mRNAs is improved by more than 1000 times compared with that of YK-223.
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 even with cationic lipid compounds that differ only slightly in structure, there is a high probability that the mRNA in LNP formulations prepared therefrom will differ 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.
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
G1 Is C1~6 Alkylene, preferably unsubstituted C2~5 Alkylene, more preferably unsubstituted C5 Alkylene or unsubstituted C3 An alkylene group;
G2 is C2~8 Alkylene, preferably unsubstituted C4~7 Alkylene, more preferably unsubstituted C7 Alkylene or unsubstituted C5 Alkylene or unsubstituted C4 An alkylene group;
G3 comprises the following steps: HO (CH)2 )2 N(CH3 )(CH2 )2 -，HO(CH2 )2 N(CH2 CH3 )(CH2 )2 -，(HO(CH2 )2 ) 2 N(CH2 )2 -，CH3 O(CH2 )2 N(CH3 )(CH2 )2 -，(CH3 )2 N(CH2 )3 SC(O)O(CH2 )2 -，(CH3 )2 N(CH2 )3 SC(O)-，CH3 NH(CH2 )2 N(CH3 )(CH2 )2 -or CH3 CH2 NH(CH2 )2 Preferably HO (CH)2 )2 N(CH3 )(CH2 )2 -。
R1 Is C6~20 Straight or branched alkyl, preferably unsubstituted C8~12 Straight-chain alkyl or unsubstituted C18 Branched alkyl, unsubstituted C17 Branched alkyl or unsubstituted C15 Branched alkyl, more preferably unsubstituted C11 Straight chain alkyl or unsubstituted C10 A linear alkyl group;
R2 is C12~25 Branched alkyl, preferably unsubstituted C14~22 Branched alkyl, more preferably unsubstituted C17 Branched alkyl or unsubstituted C18 Branched alkyl or unsubstituted C15 A branched alkyl group;
in one embodiment, G1 Is unsubstituted C5 Alkylene radicals, e.g., - (CH)2 )5 -。
In one embodiment, G1 Is unsubstituted C3 Alkylene radicals, e.g., - (CH)2 )3 -。
In one embodiment, G2 Is unsubstituted C7 Alkylene radicals, e.g., - (CH)2 )7 -。
In one embodiment, G2 Is unsubstituted C5 Alkylene radicals, e.g., - (CH)2 )5 -。
In one embodiment, G2 Is unsubstituted C4 Alkylene radicals, e.g., - (CH)2 )4 -。
In one embodiment, G3 Is HO (CH)2 )2 N(CH3 )(CH2 )2 -。
In one embodiment, G3 Is HO (CH)2 )2 N(CH2 CH3 )(CH2 )2 -。
In one embodiment, G3 Is (HO (CH)2 )2 ) 2 N(CH2 )2 -。
In one embodiment, G3 Is CH3 O(CH2 )2 N(CH3 )(CH2 )2 -。
In one embodiment, G3 Is (CH)3 )2 N(CH2 )3 SC(O)O(CH2 )2 -。
In one embodiment, G3 Is (CH)3 )2 N(CH2 )3 SC(O)-。
In one embodiment, G3 Is CH3 NH(CH2 )2 N(CH3 )(CH2 )2 -。
In one embodiment, G3 Is CH3 CH2 NH(CH2 )2 -。
In one embodiment, R1 Is unsubstituted C8~12 Straight chain alkyl, preferably unsubstituted C11 Straight chain alkyl radicals, i.e., - (CH)2 )10 CH3 。
In one embodiment, R1 Is unsubstituted C8~12 Straight chain alkyl, preferably unsubstituted C10 Straight chain alkyl radicals, i.e., - (CH)2 )9 CH3 。
In one embodiment, R1 Is unsubstituted C18 Branched alkyl, unsubstituted C17 Branched alkyl or unsubstituted C15 A branched alkyl group. For example, R1 Comprises the following steps:、or。
In one embodiment, R2 Is unsubstituted C14~22 Branched alkyl, preferably unsubstituted C17 Branched alkyl or unsubstituted C18 Branched alkyl or unsubstituted C15 A branched alkyl group. For example, R2 Comprises the following steps:、、or。
In one embodiment, G1 Is- (CH)2 )5 -，G2 Is- (CH)2 )7 -，G3 Is HO (CH)2 )2 N(CH3 )(CH2 )2 -，R1 Is- (CH)2 )10 CH3 ，R2 Comprises the following steps:。
in one embodiment, G1 Is- (CH)2 )5 -，G2 Is- (CH)2 )7 -，G3 Is HO (CH)2 )2 N(CH3 )(CH2 )2 -，R1 Is- (CH)2 )10 CH3 ，R2 Comprises the following steps:。
in one embodiment, G1 Is- (CH)2 )5 -，G2 Is- (CH)2 )5 -，G3 Is HO (CH)2 )2 N(CH3 )(CH2 )2 -，R1 Comprises the following steps:，R2 comprises the following steps:。
in one embodiment, G1 Is- (CH)2 )5 -，G2 Is- (CH)2 )7 -，G3 Is HO (CH)2 )2 N(CH3 )(CH2 )2 -，R1 Is- (CH)2 )10 CH3 ，R2 Comprises the following steps:。
in one embodiment, G1 Is- (CH)2 )3 -，G2 Is- (CH)2 )5 -，G3 Is HO (CH)2 )2 N(CH3 )(CH2 )2 -，R1 Is- (CH)2 )9 CH3 ，R2 Comprises the following steps:。
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 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 260nm; the polydispersity of the nanoparticle preparation is less than or equal to 50%, preferably less than or equal to 40%, and 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-201, YK-202, YK-203, YK-204, YK-205, YK-206, YK-207, YK-208, YK-209, YK-210, YK-211, YK-212, YK-213, YK-214, YK-215, YK-216, YK-217, YK-218, YK-219, YK-220, YK-221, YK-222, YK-223, YK-224, YK-225 or YK-226. In a preferred embodiment, the cationic lipid is compound YK-201; in another preferred embodiment, the cationic lipid is compound YK-202; in another preferred embodiment, the cationic lipid is compound YK-209; in another preferred embodiment, the cationic lipid is compound YK-206; in another preferred embodiment, the cationic lipid is compound YK-207.
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 ALC-0315 (Compound 3 in CN 108368028B), may also be Compound 21 or Compound 23 in WO2021055833, and may also be HHMA (Compound 1 in CN 112979483B).
In one embodiment, the molar ratio of the cationic lipid to the carrier is from 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. Such nucleic acids include, but are not limited to, single-stranded DNA, double-stranded DNA, and RNA. Suitable RNAs include, but are not limited to, small interfering RNAs (siRNAs), asymmetric interfering RNAs (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 in the present invention to mean auxiliary lipids which are uncharged at the chosen pH value or which are present in zwitterionic form. The neutral lipids may regulate nanoparticle mobility into lipid bilayer structures and increase efficiency by promoting lipid phase transition, while also possibly affecting target organ specificity.
In one embodiment, the molar ratio of the cationic lipid to the neutral lipid is from about 1 to 15. In a preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 4.5. 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, the phospholipid may be functionalized with or crosslinked with one or more alkynes (e.g., alkenyl with one or more double bonds replaced with triple bonds). Under appropriate reaction conditions, an alkynyl group may undergo a copper-catalyzed cycloaddition reaction upon exposure to an 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-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dilinonoyl-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 phosphatidylethanolamine (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 the stability of the nanoparticle by filling the gap between the 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, tomatidine, 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 molar ratio of the polymer conjugated lipid to the carrier is 0.5% to 10%, for example, 1%, 2%, 3%, 4%, 5%, preferably 1.5%. 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, 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 to 75): (5 to 25): 15 to 65 (0.5 to 10), such as (35 to 49): 7.5 to 15): 35 to 55 (1 to 5).
In one embodiment of the composition/carrier of the present invention, the carrier comprises a neutral lipid, a structural lipid and a polymeric conjugated lipid, the molar ratio of said cationic lipid, said neutral lipid, said structural lipid and said polymeric conjugated lipid is 49.5.
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 used to treat one or more diseases, disorders, or conditions. In some embodiments, the therapeutic and/or prophylactic agent is a small molecule drug that can be used to treat a particular disease, disorder, or condition. Examples of drugs that may be used in the composition include, but are not limited to, antineoplastic agents (e.g., vincristine (vincristine), doxorubicin (doxorubicin), mitoxantrone (mitoxantrone), camptothecin (camptothecin), cisplatin (cispinin), bleomycin (bleomycin), cyclophosphamide (cyclophosphamide), methotrexate and streptozotocin (streptozotocin)), antineoplastic agents (e.g., actinomycin D (actinomycin D), vincristine, vinblastine (vinblastine), cytosine arabinoside (cytarabine), anthracycline (anthracycline), alkylating agents, platinoids, antimetabolites and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs), anti-infective agents, local anesthetics (e.g., dibucaine (dibucaine) and chlorpromazine (chlorpromazine)), beta-adrenergic blockers (e.g., propranolol (propranolol), morolol (timolol) and labetalol (labetalol)), antihypertensive agents (e.g., clonidine and hydralazine (hydralazine)), (e.g., clolidine (clonidine)), and (e.g., chlorpromazine (hydrazine)), and (e.g., chlorpromazine (prochlorethamine)), (e.g., flutolidine (cloropamide)), and (e.g., flutolanilide (prochlorethazine) antidepressants (e.g. imipramine (imipramine), amitriptyline (amitriptyline) and doxepin (doxepin)), antispasmodics (e.g. phenytoin (phenytoin)), antihistamines (e.g. diphenhydramine (diphenhydramine), chlorpheniramine (chlorpheniramine) and promethazine (promethazine)), antibiotics/antibacterials (e.g. gentamicin (gentamycin), ciprofloxacin (ciprofloxacin) and cefoxitin (cefoxitin)), (e.g. fluxol (ciprofloxacin)), and (cefoxitin))), antifungal agents (e.g., miconazole (miconazole), terconazole (terconazole), econazole (econazole), isoconazole (isoconazole), butoconazole (butoconazole), clotrimazole (clotrimazole), itraconazole (itraconazole), nystatin (nystatin), naftifine (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 that encode 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 (mechlorethamine), thiotepa (thiotepa), chlorambucil (chlorembucil), lac (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 doxorubicin), antibiotics (e.g., dactinomycin (dactinomycin) (formerly actinomycin), bleomycin (formerly daunorubicin), vincristomycin (formerly daunorubicin), and vincristin (e.g., antimitomycin), and cisplatin), and anti-mitomycins (e.g., antimitomycins).
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 (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 can 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. shRNA can be produced inside the target cell after 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 multiple 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 dimethyl dioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, azurin (clinodendrum), bromhexine (bromohexine), carbocisteine (carbocistine), eplerenone (epirazone), mesna (snmea), ambroxol (ambroxol), sobrenol (sobreol), dominol (domidol), letostane (letosteine), seteronin (pronin), thiopronin (tiopronin), gelsolin (gelsolin), thymosin beta 4, streptococcal dnase alpha (streptococcal dnase), nekaleidein (nekalefa), and dnase (e), such as erzines) and dnase (erzines). The surface-altering agent can be disposed within and/or on the nanoparticle 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 penetration 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 are for example 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 may 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 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).
The present invention includes, but is not limited to, the following embodiments:
1. a compound of formula (I)
Or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein
G1 Is C1~6 An alkylene group;
G2 is C2~8 An alkylene group;
R1 is C6~20 A linear or branched alkyl group;
R2 is C12~25 A branched alkyl group;
G3 comprises the following steps: HO (CH)2 )2 N(CH3 )(CH2 )2 -，HO(CH2 )2 N(CH2 CH3 )(CH2 )2 -， (HO(CH2 )2 ) 2 N(CH2 )2 -，CH3 O(CH2 )2 N(CH3 )(CH2 )2 -，(CH3 )2 N(CH2 )3 SC(O)O(CH2 )2 -，(CH3 )2 N(CH2 )3 SC(O)-，CH3 NH(CH2 )2 N(CH3 )(CH2 )2 -or CH3 CH2 NH(CH2 )2 -。
2. A compound of formula (I) according to embodiment 1 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G1 Is unsubstituted C2~5 An alkylene group.
3. A compound of formula (I) according to embodiment 2 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G1 Is unsubstituted C5 Alkylene or unsubstituted C3 An alkylene group.
4. A compound of formula (I) according to any one of the preceding embodiments or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G2 Is unsubstituted C4~7 An alkylene group.
5. A compound of formula (I) according to embodiment 4 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G2 Is unsubstituted C7 Alkylene or unsubstituted C5 Alkylene or unsubstituted C4 An alkylene group.
6. A compound of formula (I) according to any one of the preceding embodiments or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R is1 Is unsubstituted C8~12 Straight chain alkyl.
7. A compound of formula (I) according to embodiment 6 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R is1 Is unsubstituted C11 Straight chain alkyl or unsubstituted C10 Straight chain alkyl.
8. A compound of formula (I) according to any one of the preceding embodiments 1-5, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R1 Is unsubstituted C18 Branched alkyl, unsubstituted C17 Branched alkyl or unsubstituted C15 A branched alkyl group.
9. A compound of formula (I) according to embodiment 8 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R is1 Is composed of、Or。
10. A compound of formula (I) according to any one of the preceding embodiments or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R2 Is unsubstituted C14~22 A branched alkyl group.
11. A compound of formula (I) according to embodiment 10 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R is2 Is unsubstituted C17 Branched alkyl or unsubstituted C18 Branched alkyl or unsubstituted C15 A branched alkyl group.
12. A compound of formula (I) according to embodiment 11 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R is2 Comprises the following steps:
、、or。
13. A compound of formula (I) according to embodiment 1 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) has one of the following structures:
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14. the compound of formula (I) according to embodiment 1 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) is compound YK-201 having the structure:
。
15. a compound of formula (I) according to embodiment 1 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) is compound YK-202 having the structure:
。
16. a compound of formula (I) according to embodiment 1 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) is compound YK-209 having the structure:
。
17. the compound of formula (I) according to embodiment 1 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) is compound YK-206 having the structure:
。
18. the compound of formula (I) according to embodiment 1 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) is compound YK-207 having the structure:
。
19. a composition comprising a carrier comprising a cationic lipid comprising a compound of formula (I) according to any one of the preceding embodiments or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof.
20. The composition of embodiment 19, wherein the cationic lipid is present in a carrier at a molar ratio of 25% to 75%.
21. The composition of any one of embodiments 19-20, wherein the carrier further comprises a neutral lipid.
22. The composition according to embodiment 21, wherein the molar ratio of the cationic lipid to the neutral lipid is 1 to 1, preferably 4.5.
23. The composition of any one of embodiments 21-22, wherein the neutral lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols and derivatives thereof.
24. The composition of embodiment 23, wherein the neutral lipids are 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.
25. The composition of embodiment 24, wherein the neutral lipid is DOPE and/or DSPC.
26. The composition of any one of embodiments 19-25, wherein the carrier further comprises a structural lipid.
27. The composition of embodiment 26, wherein the molar ratio of the cationic lipid to the structural lipid is 0.6 to 1 to 3.
28. The composition according to any one of embodiments 26-27, 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.
29. The composition of embodiment 28, wherein the structural lipid is cholesterol.
30. The composition of any one of embodiments 19-29, wherein the carrier further comprises a polymer conjugated lipid.
31. The composition according to embodiment 30, wherein the molar ratio of the polymeric conjugated lipid to the carrier is between 0.5% and 10%, preferably 1.5%.
32. The composition according to any one of embodiments 30-31, 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.
33. The composition of embodiment 32, 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).
34. The composition of any of embodiments 19-33, 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).
35. The composition of embodiment 34, 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).
36. The composition of embodiment 35, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymeric conjugated lipid is 45.
37. The composition of any of the preceding embodiments 19-36, wherein the composition is a nanoparticle formulation having an average particle size of 10nm to 300nm; the polydispersity coefficient of the nano-particle preparation is less than or equal to 50 percent.
38. The composition of embodiment 37, wherein the nanoparticle formulation has an average particle size from 90nm to 260nm; the polydispersity coefficient of the nano-particle preparation is less than or equal to 40 percent.
39. The composition of any one of embodiments 19-38, wherein the cationic lipid further comprises one or more other ionizable lipid compounds.
40. The composition of any one of embodiments 19-39, further comprising a therapeutic or prophylactic agent.
41. The composition of embodiment 40, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 10.
42. The composition according to embodiment 41, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 12.5.
43. The composition according to embodiment 42, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 15.
44. The composition of any one of embodiments 40-43, wherein the therapeutic or prophylactic agent comprises one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.
45. The composition of any one of embodiments 40-44, wherein the therapeutic or prophylactic agent is a vaccine or compound capable of eliciting an immune response.
46. The composition of any one of embodiments 40-43, wherein the therapeutic or prophylactic agent is a nucleic acid.
47. The composition of any one of embodiments 40-43, wherein the therapeutic or prophylactic agent is a ribonucleic acid (RNA).
48. The composition of any one of embodiments 40-43, wherein the therapeutic or prophylactic agent is a deoxyribonucleic acid (DNA).
49. The composition of embodiment 47, wherein the RNA is selected from the group consisting of: small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microrna (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.
50. The composition of embodiment 49, wherein the RNA is mRNA.
51. The composition according to any one of embodiments 19-50, wherein the composition further comprises one or more of a pharmaceutically acceptable excipient or diluent.
52. Use of a compound of formula (I) according to any one of embodiments 1 to 18 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof or a composition according to any one of embodiments 19 to 51 in the manufacture of a nucleic acid medicament, a genetic vaccine, a small molecule medicament, a polypeptide or a protein medicament.
53. Use of a compound of formula (I) as described in any one of embodiments 1-18 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a composition as described in any one of embodiments 19-51, in the manufacture of a medicament for treating a disease or condition in a mammal in need thereof.
54. The use according to any one of embodiments 52-53, wherein the disease or disorder is characterized by a malfunctioning or abnormal protein or polypeptide activity.
55. The use according to embodiment 54, wherein the disease or condition 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.
56. The use of embodiment 55, 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.
57. The use according to any one of embodiments 53-56, wherein the mammal is a human.
58. The use according to any one of embodiments 52-57, wherein the composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation.
59. The use of embodiment 58, wherein said composition is administered subcutaneously.
60. The use according to any one of embodiments 53-59, wherein a dose of about 0.001mg/kg to about 10mg/kg of the therapeutic or prophylactic agent is administered to the mammal.
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 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
Synthesis of YK-201
The synthetic route is as follows:
the method comprises the following steps: synthesis of heptadecan-9-yl-8- ((2-chloroethyl) (6-oxo-6- ((undecyloxy) hexyl) amino) octanoate (YK-201-PM 1):
SM-102 (10.00 g, 0.014 mol) was added to a single neck flask, 100mL SOCl was added2 The reaction was stirred at room temperature for 6 hours. After the reaction, the reaction solution was poured into ice water to quench the reaction, and extracted with ethyl acetate. The organic phase was vacuum-dried to give the product as a colorless oil (9.51 g, 0.013mol, 93.1%)), C44 H86 ClNO4 , MS(ES): m/z(M+H+ )728.5。
Step two: synthesis of heptadecan-9-yl-8- (2- ((2-hydroxyethyl) (methylamino)) ethyl) (6-oxo-6- ((undecyloxy) hexyl) amino) caprylate (YK-201)
YK-201-PM1 (1.00g, 1.37mmol) and 2-methylamino-ethanol (0.31g, 4.11mmol) were dissolved in acetonitrile (20 mL), and potassium carbonate (0.61g, 4.41mmol) and potassium iodide (38mg, 0.23mmol) were added to the above system, heated to 70 deg.C, and the reaction was stirred for 7 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 give YK-201 (559mg, 0.73mmol, 53.3%). C47 H94 N2 O5 , MS(ES): m/z(M+H+ )767.6。
1 H NMR (400 MHz, Chloroform-d) δ 4.05 (t, J = 6.8 Hz, 2H), 3.57 (t, J = 5.1 Hz, 2H), 2.57 – 2.52 (m, 5H), 2.46 (dt, J = 9.8, 5.1 Hz, 4H), 2.33 – 2.25 (m, 7H), 1.68 – 1.56 (m, 6H), 1.47 (dd, J = 13.4, 6.0 Hz, 8H), 1.28 (d, J = 17.7 Hz, 50H), 0.88 (t, J = 6.8 Hz, 9H).
2. Synthesis of YK-202
The synthetic route is as follows:
the method comprises the following steps: synthesis of 2-octyldecyl-8- ((2-chloroethyl) (6-oxo-6- ((undecyloxy) hexyl) amino) caprylate (YK-202-PM 1):
YK-202-SM1 (1.50 g, 2.07 mmol) was added to a single-neck flask, and 10mL SOCl was added2 The reaction was stirred at room temperature for 6 hours. After the reaction, the reaction solution was poured into ice water to quench the reaction, and extracted with ethyl acetate. The organic phase was dried in vacuo to give the product as a colorless oil (1.63 g, 2.07mmol, 100.0%), C45 H88 ClNO4 , MS(ES): m/z(M+H+ )742.6。
Step two: synthesis of 2-octyldecyl-8- ((2- (2-hydroxyethyl (methyl) amino) ethyl) (6-oxo-6- ((undecyloxy) hexyl) amino) caprylate (YK-202)
Using YK-202-PM1 (1.60 g, 2.07 mmol) obtained above and 2-methylamino-ethanol (0.47 g, 6.21 mmol) as starting materials, the desired compound (1.01 g, 1.29mmol, 62.3%) was obtained according to the method for synthesizing YK-201. C48 H96 N2 O5 ，MS(ES):m/z(M+H+ )781.3。
1 H NMR (400 MHz, Chloroform-d) δ 4.05 (t, J = 6.7 Hz, 2H), 3.96 (d, J = 5.7 Hz, 2H), 3.62 (t, J = 4.8 Hz, 2H), 2.72 (d, J = 5.4 Hz, 2H), 2.64 (dd, J = 14.4, 5.3 Hz, 8H), 2.36 (s, 3H), 2.30 (td, J = 7.4, 4.0 Hz, 4H), 1.60 (qd, J = 15.3, 7.7 Hz, 11H), 1.28 (d, J = 14.9 Hz, 52H), 0.88 (t, J = 6.6 Hz, 9H).
3. Synthesis of YK-203
The synthetic route is as follows:
the method comprises the following steps: synthesis of 3-hexylnonyl-8- ((2-chloroethyl) (6-oxo-6- ((undecyloxy) hexyl) amino) octanoate (YK-203-PM 1):
YK-203-SM1 (280mg, 0.41mmol) was mixed with SOCl2 (2 mL) was used as a starting material to obtain YK-203-PM1 (250 mg,0.36mmol, 87.0%) according to the method for synthesizing YK-201-PM 1. C42 H82 ClNO4 , MS(ES): m/z(M+H+ )700.6。
Step two: synthesis of 3-hexylnonyl-8- ((2- (2-hydroxyethyl (methyl) amino) ethyl) (6-oxo-6- ((undecyloxy) hexyl) amino) octanoate (YK-203)
YK-203 (120 mg,0.16mmol, 73.8%) was obtained from YK-203-PM1 (150mg, 0.22mmol) and 2-methylamino-ethanol (75mg, 0.22mmol) as starting materials by the method for synthesizing YK-201. C45 H90 N2 O5 , MS(ES): m/z(M+H+ )739.7。
1 H NMR (400 MHz, Chloroform-d) δ 4.10 (dt,J = 9.2, 7.0 Hz, 4H), 3.73 – 3.63 (m, 2H), 3.51 (s, 3H), 3.11 (s, 4H), 2.96 (t,J = 6.1 Hz, 2H), 2.88 (q,J = 7.8 Hz, 4H), 2.79 (t,J = 6.1 Hz, 2H), 2.70 (t,J = 4.9 Hz, 2H), 2.42 (s, 2H), 2.33 (dt,J = 13.2, 7.4 Hz, 4H), 1.70 – 1.59 (m, 10H), 1.37 – 1.27 (m, 41H), 0.94 – 0.88 (m, 9H).
4. Synthesis of YK-204
The synthetic route is as follows:
the method comprises the following steps: synthesis of heptadecan-9-yl-8- (2- ((2-hydroxyethyl) (ethylamino)) ethyl) (6-oxo-6- ((undecyloxy) hexyl) amino) octanoate (YK-204)
YK-204 (52mg, 0.07mmol, 55.5%) was obtained from YK-201-PM1 (88mg, 0.12mmol) and 2-ethylamino-ethanol (36mg, 0.41mmol) as raw materials by the method for synthesizing YK-201. C48 H96 N2 O5 , MS(ES): m/z(M+H+ )781.7。
1 H NMR (400 MHz, Chloroform-d) δ 5.41 – 5.24 (m, 1H), 4.85 (t,J = 6.2 Hz, 1H), 4.07 (q,J = 7.7, 6.7 Hz, 3H), 3.91 – 3.48 (m, 2H), 3.36 (d,J = 25.5 Hz, 2H), 3.08 (s, 2H), 2.38 – 2.15 (m, 3H), 2.11 – 1.96 (m, 1H), 1.85 (s, 3H), 1.76 – 1.46 (m, 21H), 1.26 (s, 48H), 0.94 – 0.83 (m, 9H).
5. Synthesis of YK-205
The synthetic route is as follows:
the method comprises the following steps: synthesis of pentadecan-8-yl-8- ((2-chloroethyl) (6-oxo-6- ((undecyloxy) hexyl) amino) octanoate (YK-205-PM 1):
YK-205-SM1 (400mg, 0.59mmol) and SOCl2 (3 mL) was used as a starting material to obtain YK-205-PM1 (400 mg, 0.57mmol, 96.8%) according to the method for synthesizing YK-201-PM 1. C42 H82 ClNO4 , MS(ES): m/z(M+H+ )700.5。
Step two: synthesis of pentadecan-8-yl-8- ((2- (2-hydroxyethyl (ethyl) amino) ethyl) (6-oxo-6- ((undecyloxy) hexyl) amino) caprylate (YK-205)
YK-205-PM1 (200mg, 0.29mmol) and 2-ethylamino-ethanol (127mg, 1.43mmol) are used as raw materials, and the target compound (160 mg,0.21mmol, 73.2%) is obtained according to the method for synthesizing YK-201. C46 H92 N2 O5 ，MS(ES):m/z(M+H+ )753.7。
1 H NMR (400 MHz, Chloroform-d) δ 4.86 (p,J = 6.2 Hz, 1H), 4.05 (t,J= 6.8 Hz, 2H), 3.57 (t,J = 4.9 Hz, 2H), 2.69 – 2.58 (m, 7H), 2.54 (s, 4H), 2.29 (dt,J = 10.4, 7.5 Hz, 4H), 1.62 (dq,J = 14.0, 7.3 Hz, 6H), 1.49 (d,J= 6.3 Hz, 8H), 1.29 (d,J = 21.1 Hz, 46H), 1.05 (t,J = 7.1 Hz, 3H), 0.92 – 0.84 (m, 9H).
Synthesis of YK-206
The synthetic route is as follows:
synthesis of pentadecan-8-yl-8- ((2- (2-hydroxyethyl (methyl) amino) ethyl) (6-oxo-6- ((undecyloxy) hexyl) amino) octanoate (YK-206)
YK-205-PM1 (200mg, 0.29mmol) and 2-methylamino-ethanol (107mg, 1.43mmol) are used as raw materials, and the target compound (127 mg,0.17mmol, 58.6%) is obtained according to the method for synthesizing YK-201. C45 H90 N2 O5 ，MS(ES):m/z(M+H+ )739.7。
1 H NMR (400 MHz, Chloroform-d) δ 4.86 (p,J = 6.2 Hz, 1H), 4.05 (t,J= 6.8 Hz, 2H), 3.56 (t,J = 5.1 Hz, 2H), 3.28 (s, 2H), 2.92 – 2.71 (m, 1H), 2.54 (d,J = 10.4 Hz, 6H), 2.44 (dt,J = 9.9, 4.8 Hz, 4H), 2.37 – 2.21 (m, 6H), 1.62 (dq,J = 14.5, 7.6 Hz, 6H), 1.46 (dt,J = 27.8, 9.8 Hz, 8H), 1.28 (d,J = 15.4 Hz, 43H), 0.88 (t,J = 6.7 Hz, 9H).
7. Synthesis of YK-207
The synthetic route is as follows:
the method comprises the following steps: synthesis of 2-octyldecyl-6- ((2-chloroethyl) (4-oxo-4- ((decyloxy) butyl) amino) hexanoate (YK-207-PM 1):
YK-207-SM1 (3.0 g, 4.58mmol) and SOCl2 (5 mL) as a raw material, YK-207-PM1 (2.9 g, 4.31mmol, 94.2%) was obtained according to the method for synthesizing YK-201-PM 1. C40 H78 ClNO4 , MS(ES): m/z(M+H+ )672.3。
Step two: synthesis of 2-octyldecyl-6- (2- ((2- (2-hydroxyethyl (methyl) amino) ethyl) (4-oxo-4- ((decyloxy) butyl) amino) hexanoate (YK-207)
YK-207 (50 mg,0.07mmol, 23.4%) was obtained from YK-207-PM1 (200mg, 0.30mmol) and 2-methylamino-ethanol (22mg, 0.29mmol) as raw materials by the method for synthesizing YK-201. C43 H86 N2 O5 , MS(ES): m/z(M+H+ )711.7。
1 H NMR (400 MHz, Chloroform-d) δ 5.43 – 5.15 (m, 1H), 4.07 (t,J = 6.8 Hz, 2H), 3.96 (d,J = 5.8 Hz, 2H), 3.87 (t,J = 4.6 Hz, 1H), 3.34 (d,J = 25.1 Hz, 3H), 3.05 (s, 3H), 2.70 (s, 2H), 2.47 (t,J = 6.5 Hz, 2H), 2.34 (t,J = 7.3 Hz, 2H), 2.01 (d,J = 6.9 Hz, 2H), 1.84 – 1.74 (m, 2H), 1.65 (dp,J = 21.2, 7.1 Hz, 5H), 1.46 – 1.37 (m, 2H), 1.37 – 1.17 (m, 44H), 0.94 – 0.85 (m, 9H).
8. Synthesis of YK-208
The synthetic route is as follows:
synthesis of 2-octyldecyl-6- (2- ((2- (2-hydroxyethyl (ethyl) amino) ethyl) (4-oxo-4- ((decyloxy) butyl) amino) hexanoate (YK-208)
YK-208 (140 mg,0.19mmol, 87.8%) is obtained by using YK-207-PM1 (150mg, 0.22mmol) and 2-ethylamino-ethanol (59mg, 0.66mmol) as raw materials according to the method for synthesizing YK-201. C44 H88 N2 O5 , MS(ES): m/z(M+H+ )725.7。
1 H NMR (400 MHz, Chloroform-d) δ 5.33 (s, 1H), 4.60 (s, 2H), 4.09 (t,J = 6.8 Hz, 2H), 3.99 (d,J = 5.8 Hz, 2H), 3.68 (t,J = 4.9 Hz, 2H), 2.93 – 2.54 (m, 11H), 2.36 (dt,J = 17.0, 7.3 Hz, 4H), 1.93 – 1.78 (m, 2H), 1.76 – 1.48 (m, 7H), 1.45 – 1.21 (m, 43H), 1.15 (t,J = 7.1 Hz, 3H), 1.00 – 0.78 (m, 9H).
9. Synthesis of YK-209
The synthetic route is as follows:
the method comprises the following steps: synthesis of bis (2-octyldecyl) -6, 6' - ((2-hydroxyethyl) azadialkyl) dihexanoate (YK-209-PM 1)
YK-209-SM1 (1.0 g, 2.34mmol) and YK-209-SM2 (1.15g, 2.58mmol) were dissolved in acetonitrile (7 mL), and potassium carbonate (0.97 g, 7.02 mmol) and potassium iodide (38mg, 0.23mmol) were added to the above system, and the reaction was stirred at 70 ℃ for 8 hours. After the reaction is finished, the reaction liquid is cooled to room temperature and then filtered, and the filtrate is subjected to vacuum concentration under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (methanol/dichloromethane) to give YK-209-PM1 (780 mg, 0.98mmol, 41.9%), C50 H99 NO5 , MS(ES): m/z(M+H+ )794.8。
Step two: synthesis of bis (2-octyldecyl) -6, 6' - ((2-chloroethyl) azadialkyl) dihexanoate (YK-209-PM 2):
YK-209-PM1 (760mg, 0.96mmol) and SOCl are mixed2 (5 mL) was used as a starting material to obtain YK-209-PM2 (800 mg, 0.98mmol, 102.5%) according to the method for synthesizing YK-201-PM 1. C50 H98 ClNO4 , MS(ES): m/z(M+H+ )812.7。
Step three: synthesis of bis (2-octyldecyl) -6, 6' - ((2- (2-hydroxyethyl (methyl) amino) ethyl) azadialkyl) dihexanoate (YK-209)
YK-209 (120 mg,0.14mmol, 78.3%) is obtained by using YK-209-PM2 (150mg, 0.18mmol) and 2-methoxy-ethanol (27mg, 0.36mmol) as raw materials according to a method for synthesizing YK-201. C53 H106 N2 O5 , MS(ES): m/z(M+H+ ) 851.4。
1 H NMR (400 MHz, Chloroform-d) δ 3.96 (d, J = 5.8 Hz, 4H), 3.69 – 3.65 (m, 2H), 2.86 (s, 2H), 2.76 (s, 4H), 2.72 – 2.65 (m, 2H), 2.41 (s, 3H), 2.32 (t, J = 7.4 Hz, 4H), 1.66 (dt, J = 15.0, 7.4 Hz, 10H), 1.26 (s, 62H), 0.88 (t, J = 6.8 Hz, 12H).
10. Synthesis of YK-210
The synthetic route is as follows:
the method comprises the following steps: synthesis of bis (heptadecan-9-yl) -6, 6' - ((2-hydroxyethyl) azadialkyl) dihexanoate (YK-210-PM 1)
Heptadecan-9-yl-6-bromohexanoate (700mg, 1.62mmol) and ethanolamine (55mg, 0.74mmol) were dissolved in acetonitrile (5 mL), and potassium carbonate (307 mg, 2.22 mmol) and potassium iodide (12mg, 0.07mmol) were added to the above system, heated to 70 ℃ and stirred for reaction for 8 hours. After the reaction is finished, the reaction liquid is cooled to room temperature and then filtered, and the filtrate is subjected to vacuum concentration under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (methanol/dichloromethane) to give YK-210-PM1 (450 mg,0.66mmol, 79.4%), C48 H95 NO5 , MS(ES): m/z(M+H+ ) 766.7。
Step two: synthesis of bis (heptadecan-9-yl) -6, 6' - ((2-chloroethyl) azadialkyl) dihexanoate (YK-210-PM 2):
YK-210-PM1 (450mg, 0.59mmol) and SOCl2 (3 mL) was used as a starting material to obtain YK-210-PM2 (460 mg,0.59mmol, 100.0%) according to the method for synthesizing YK-201-PM 1. C48 H94 ClNO4 , MS(ES): m/z(M+H+ )784.6。
Step three: synthesis of bis (heptadecan-9-yl) -6, 6' - ((2- (2-hydroxyethyl (methyl) amino) ethyl) azadialkyl) dihexanoate (YK-210)
YK-210 (136 mg,0.17mmol, 86.9%) was obtained from YK-210-PM2 (150mg, 0.19mmol) and 2-methoxy-ethanol (17mg, 0.23mmol) as starting materials by the method for synthesizing YK-201. C51 H102 N2 O5 , MS(ES): m/z(M+H+ ) 823.8。
1 H NMR (400 MHz, Chloroform-d) δ 5.30 (s, 1H), 4.85 (p,J = 6.2 Hz, 2H), 3.96 (s, 2H), 3.72 – 3.65 (m, 2H), 2.97 (s, 1H), 2.90 (s, 3H), 2.81 (s, 2H), 2.75 – 2.67 (m, 2H), 2.42 (s, 2H), 2.30 (t,J = 7.3 Hz, 4H), 1.72 – 1.62 (m, 7H), 1.50 (d,J = 5.6 Hz, 8H), 1.26 (s, 54H), 0.88 (t,J = 6.8 Hz, 12H).
11. Synthesis of YK-211
The synthetic route is as follows:
the method comprises the following steps: synthesis of bis (3-hexylnonyl) -6, 6' - ((2-hydroxyethyl) azadialkyl) dihexanoate (YK-211-PM 1)
Using 6-bromohexanoic acid-3-hexylnonyl ester (828mg, 2.05mmol) and ethanolamine (50mg, 0.82mmol) as raw materials, and according to the method for synthesizing YK-210-PM1, obtaining YK-211-PM1 (470 mg,0.66mmol, 80.8%) and C44 H87 NO5 , MS(ES): m/z(M+H+ )710.5。
Step two: synthesis of bis (3-hexylnonyl) -6, 6' - ((2-chloroethyl) azadialkyl) dihexanoate (YK-211) -PM 2:
YK-211-PM1 (470mg, 0.66mmol) and SOCl are mixed2 (3 mL) was used as a starting material to obtain YK-211-PM2 (420 mg, 0.58mmol, 87.5%) according to the method for synthesizing YK-201-PM 1. C44 H86 ClNO4 , MS(ES): m/z(M+H+ )728.6。
Step three: synthesis of bis (3-hexylnonyl) -6, 6' - ((2- (2-hydroxyethyl (methyl) amino) ethyl) azadialkyl) dihexanoate (YK-211)
YK-211 (57 mg,0.07mmol, 53.1%) was obtained from YK-211-PM2 (100mg, 0.14mmol) and 2-methylamino-ethanol (12mg, 0.11mmol) as raw materials by the method for synthesizing YK-201. C47 H94 N2 O5 , MS(ES): m/z(M+H+ )767.7。
1 H NMR (400 MHz, Chloroform-d) δ 4.08 (t,J = 7.2 Hz, 4H), 3.82 – 3.75 (m, 2H), 3.31 (d,J = 5.6 Hz, 4H), 3.13 (dd,J = 18.9, 10.6 Hz, 5H), 2.87 (s, 2H), 2.56 (s, 2H), 2.33 (t,J = 7.2 Hz, 4H), 1.78 (p,J = 7.9 Hz, 3H), 1.73 – 1.62 (m, 4H), 1.57 (q,J = 7.0 Hz, 4H), 1.43 (dt,J = 14.4, 7.5 Hz, 6H), 1.25 (s, 42H), 0.88 (t,J = 6.7 Hz, 12H).
12. Synthesis of YK-212
The synthetic route is as follows:
synthesis of bis (2-octyldecyl) -6, 6' - ((2- (bis (2-hydroxyethyl) amino) ethyl) azadialkyl) dihexanoate (YK-212)
YK-209-PM2 (100mg, 0.12mmol) and diethanolamine (26mg, 0.24mmol) are used as raw materials, and YK-212 (80 mg, 0.09mmol, 75.6%) is obtained according to the method for synthesizing YK-201. C54 H108 N2 O6 , MS(ES): m/z(M+H+ )881.8。
1 H NMR (400 MHz, Chloroform-d) δ 4.00 (d,J = 5.8 Hz, 4H), 3.69 (dd,J = 5.6, 4.0 Hz, 4H), 3.04 – 2.93 (m, 5H), 2.85 (t,J = 5.4 Hz, 2H), 2.75 (t,J = 4.8 Hz, 4H), 2.36 (t,J = 7.3 Hz, 4H), 1.70 (tq,J = 11.0, 6.7, 5.6 Hz, 10H), 1.30 (s, 63H), 0.95 – 0.89 (m, 12H).
13. Synthesis of YK-213
The synthetic route is as follows:
synthesis of bis (heptadecan-9-yl) -6, 6' - ((2- (bis (2-hydroxyethyl) amino) ethyl) azadialkyl) dihexanoate (YK-213)
YK-213 (50 mg, 0.06mmol, 30.8%) was obtained from YK-210-PM2 (150mg, 0.19mmol) and diethanolamine (21mg, 0.20mmol) as raw materials by the method for synthesizing YK-201. C52 H104 N2 O6 , MS(ES): m/z(M+H+ )853.8。
1 H NMR (400 MHz, Chloroform-d) δ 4.00 (d,J = 5.8 Hz, 4H), 3.69 (dd,J = 5.6, 4.0 Hz, 4H), 3.04 – 2.93 (m, 5H), 2.85 (t,J = 5.4 Hz, 2H), 2.75 (t,J = 4.8 Hz, 4H), 2.36 (t,J = 7.3 Hz, 4H), 1.70 (tq,J = 11.0, 6.7, 5.6 Hz, 10H), 1.30 (s, 63H), 0.95 – 0.89 (m, 12H).
14. Synthesis of YK-214
The synthetic route is as follows:
synthesis of bis (3-hexylnonyl) -6, 6' - ((2- (bis (2-hydroxyethyl) amino) ethyl) azadialkyl) dihexanoate (YK-214)
3-Hexylnonyl 6-bromohexanoate (303mg, 0.75mmol) and N, N-bis (2-hydroxyethyl) ethylenediamine (50mg, 0.34mmol) were dissolved in acetonitrile (2 mL), and potassium carbonate (140 mg, 1.02 mmol) and potassium iodide (5.6mg, 0.034mmol) were added to the above system, and the reaction was stirred at 70 ℃ for 24 hours. After the reaction is finished, the reaction liquid is cooled to room temperature and then filtered, and the filtrate is subjected to vacuum concentration under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (methanol/dichloromethane) to give YK-214 (120 mg,0.15mmol, 44.3%), C48 H96 N2 O6 , MS(ES): m/z(M+H+ )797.8。
1 H NMR (400 MHz, Chloroform-d) δ 4.11 (t,J = 7.2 Hz, 4H), 4.02 – 3.74 (m, 1H), 3.65 (t,J = 4.9 Hz, 4H), 2.74 (dd,J = 12.4, 7.4 Hz, 11H), 2.34 (t,J = 7.4 Hz, 4H), 1.64 (dq,J = 29.0, 7.4 Hz, 11H), 1.50 – 1.20 (m, 49H), 0.94 – 0.89 (m, 12H).
15. Synthesis of YK-215
The synthetic route is as follows:
synthesis of heptadecan-9-yl-8- ((2- (2-methoxyethyl (methyl) amino) ethyl) (6-oxo-6- ((undecyloxy) hexyl) amino) caprylate (YK-215)
The target compound (98 mg,0.12mmol, 96.5%) was obtained according to the method for synthesizing YK-201 using YK-201-PM1 (95mg, 0.13mmol) and 2-methoxy-N-methylethan-1-amine (14mg, 0.111mmol) as starting materials. C48 H96 N2 O5 ，MS(ES):m/z(M+H+ )781.2。
1 H NMR (400 MHz, Chloroform-d) δ 4.86 (t,J = 6.2 Hz, 1H), 4.05 (t,J= 6.8 Hz, 2H), 3.36 (s, 3H), 3.15 (t,J = 6.3 Hz, 3H), 3.01 (dq,J = 11.7, 7.2, 5.6 Hz, 6H), 2.77 (t,J = 5.0 Hz, 2H), 2.45 (s, 2H), 2.30 (dt,J = 17.9, 7.4 Hz, 4H), 1.85 – 1.57 (m, 10H), 1.50 (d,J = 6.3 Hz, 4H), 1.41 – 1.18 (m, 50H), 0.88 (t,J = 6.6 Hz, 9H).
16. Synthesis of YK-216
The synthetic route is as follows:
synthesis of 2-octyldecyl-6- (2- ((2- (2-methoxyethyl (methyl) amino) ethyl) (4-oxo-4- ((decyloxy) butyl) amino) hexanoate (YK-216)
YK-207-PM1 (170mg, 0.25mmol) and 2-methoxy-N-methyl ethane-1-amine (89mg, 0.45mmol) are used as raw materials, and YK-216 (140 mg,0.19mmol, 77.2%))。C44 H88 N2 O5 , MS(ES): m/z(M+H+ )725.6。
1 H NMR (400 MHz, Chloroform-d) δ 4.09 (t,J = 6.7 Hz, 2H), 4.00 (d,J= 5.8 Hz, 2H), 3.58 (t,J = 5.3 Hz, 2H), 3.39 (s, 3H), 2.94 – 2.50 (m, 11H), 2.51 – 2.25 (m, 7H), 1.88 (d,J = 8.2 Hz, 2H), 1.75 – 1.48 (m, 7H), 1.50 – 1.20 (m, 43H), 0.98 – 0.83 (m, 9H).
17. Synthesis of YK-217
The synthetic route is as follows:
synthesis of 3-hexylnonyl-11- (4- (decyloxy) -4-oxobutyl) -2-methyl-7-oxo-8-oxo-6-thio-2, 11-diazepane-17-carboxylate (YK-217)
YK-217-SM1 (100mg, 0.16mmol), dimethylamino propanethiol (20mg, 0.32mmol) and pyridine (1.2 mL) are dissolved in dichloromethane (5 mL), triphosgene (77 mg, 0.26 mmol) is added to the system in batches under nitrogen protection and ice bath, ice bath is removed, the temperature is naturally increased to room temperature, and the reaction is stirred for 8 hours. After the reaction, a saturated aqueous sodium bicarbonate solution (5 mL) was added dropwise to the reaction mixture, 20mL of dichloromethane was added thereto, the mixture was separated, the organic phase was washed with saturated brine, and the solvent was removed by vacuum concentration. The residue was purified by silica gel chromatography (methanol/dichloromethane) to give YK-217 (60 mg,0.03 mmol, 16.5%), C43 H84 N2 O6 S, MS(ES): m/z(M+H+ )757.2。
1 H NMR (400 MHz, Chloroform-d) δ 4.14 – 4.00 (m, 2H), 3.96 (t,J = 4.7 Hz, 2H), 3.34 (s, 4H), 2.91 (td,J = 7.2, 3.3 Hz, 2H), 2.47 (d,J = 7.9 Hz, 2H), 2.31 (t,J = 7.0 Hz, 8H), 1.95 – 1.78 (m, 4H), 1.62 (d,J = 11.7 Hz, 6H), 1.31 – 1.23 (m, 45H), 0.87 (dt,J = 7.0, 3.4 Hz, 9H).
18. Synthesis of YK-218
The synthetic route is as follows:
synthesis of 2-octyldecyl-11- (4- (decyloxy) -4-oxobutyl) -2-methyl-7-oxo-8-oxo-6-thio-2, 11-diazepan-17-carboxylate (YK-218)
YK-218 (60 mg, 0.08mmol, 50.0%) C was obtained from YK-207-SM1 (100mg, 0.15mmol) and dimethylaminopropylmercaptan (36.5mg, 0.31mmol) as raw materials by the method for synthesizing YK-21746 H90 N2 O6 S, MS(ES): m/z(M+H+ )799.7。
1 H NMR (400 MHz, Chloroform-d) δ 4.23 (t,J = 6.2 Hz, 2H), 4.05 (t,J= 6.8 Hz, 2H), 3.96 (d,J = 5.8 Hz, 2H), 2.89 (t,J = 7.2 Hz, 2H), 2.70 (t,J= 6.2 Hz, 2H), 2.46 (ddd,J = 12.4, 8.0, 6.1 Hz, 5H), 2.31 (d,J = 7.4 Hz, 9H), 1.91 – 1.83 (m, 2H), 1.77 – 1.68 (m, 2H), 1.62 (tt,J = 7.8, 4.5 Hz, 5H), 1.46 – 1.38 (m, 2H), 1.28 (d,J = 14.4 Hz, 46H), 0.88 (t,J = 6.7 Hz, 9H).
19. Synthesis of YK-219
The synthetic route is as follows:
the method comprises the following steps: synthesis of 6- (4- (decyloxy) -4-oxobutyl) -aminocaproic acid-2-octyldecyl ester (YK-219-PM 1)
YK-219-SM1 (459 mg, 1.19 mmol) and YK-219-SM2 (304 mg, 0.99 mmol) are used as raw materials to obtain YK-207-PM1 (310 mg, 0.51mmol, 51.3%) and C according to the method for synthesizing YK-207-PM138 H75 NO4 , MS(ES): m/z(M+H+ )610.5。
Step two: synthesis of 2-octyldecyl-6- (4- (decyloxy) -4-oxobutyl) ((((3- (dimethylamino) propyl) thio) carbonyl) amino) hexanoic acid ester (YK-219)
YK-219-PM1 (310 mg, 0.51 mmol) and bisMethylaminopropanethiol (303 mg, 2.54 mmol) is used as a raw material, and YK-219 (82 mg,0.11mmol, 21.3%) and C are obtained according to the method for synthesizing YK-21744 H86 N2 O5 S, MS(ES): m/z(M+H+ )755.5。
1 H NMR (400 MHz, Chloroform-d) δ 4.10 (q,J = 4.9, 3.5 Hz, 2H), 3.99 (t,J = 4.6 Hz, 2H), 3.37 (s, 3H), 2.95 (td,J = 7.2, 3.4 Hz, 2H), 2.50 (d,J= 7.8 Hz, 2H), 2.36 (d,J = 3.6 Hz, 9H), 2.07 – 1.84 (m, 4H), 1.70 – 1.59 (m, 6H), 1.43 – 1.26 (m, 47H), 0.91 (dt,J = 7.0, 3.4 Hz, 9H).
20. Synthesis of YK-220
The synthetic route is as follows:
the method comprises the following steps: synthesis of 6- (4- (decyloxy) -4-oxobutyl) -aminocaproic acid-3-hexylnonyl ester (YK-220-PM 1)
YK-220-PM1 (249 mg, 0.44mmol, 46.2%) and C are obtained by using YK-220-SM1 (391 mg, 1.15 mmol) and YK-220-SM2 (292 mg, 0.95 mmol) as raw materials and adopting a method for synthesizing YK-210-PM135 H69 NO4 , MS(ES): m/z(M+H+ )568.5。
Step two: synthesis of 3-hexylnonane-6- (4- (decyloxy) -4-oxobutyl) ((((3- (dimethylamino) propyl) thio) carbonyl) amino) hexanoate (YK-220)
YK-220 (76 mg,0.11mmol, 30.4%) C is obtained by using YK-220-PM1 (200 mg,0.35 mmol) and dimethylaminopropylmercaptan (209 mg, 1.75 mmol) as raw materials according to a method for synthesizing YK-21741 H80 N2 O5 S, MS(ES): m/z(M+H+ )713.6。
1 H NMR (400 MHz, Chloroform-d) δ 4.12 – 4.02 (m, 4H), 3.35 (s, 4H), 2.92 (t,J = 7.2 Hz, 2H), 2.49 – 2.40 (m, 2H), 2.30 (s, 8H), 1.60 (dp,J = 20.8, 7.2 Hz, 8H), 1.44 – 1.18 (m, 40H), 0.88 (t,J = 6.8 Hz, 9H).
21. Synthesis of YK-221
The synthetic route is as follows:
the method comprises the following steps: synthesis of 2-octyldecyl-6- (2- ((2- (N-tert-butoxycarbonyl-N-methylamino) ethyl) (4-oxo-4- ((decyloxy) butyl) amino) hexanoate (YK-221-PM 1)
YK-221-PM1 (120mg, 0.15mmol, 66.2%) was obtained by the method for synthesizing YK-201 using YK-207-PM1 (150mg, 0.22mmol) and methyl (2- (methylamino) ethyl) carbamic acid tert-butyl ester (45mg, 0.22mmol) as raw materials. C49 H97 N3 O6 , MS(ES): m/z(M+H+ )824.7。
Step two: synthesis of 2-octyldecyl-6- (2- ((2- (methylamino) ethyl) (4-oxo-4- ((decyloxy) butyl) amino) hexanoate (YK-221)
YK-221-PM1 (120mg, 0.15mmol) is dissolved in 3mL dichloromethane, 0.5mL trifluoroacetic acid is added dropwise, the mixture is stirred for 2h at room temperature, after the reaction is finished, vacuum drying is carried out, 10mL ethyl acetate is added for dissolving, saturated sodium bicarbonate aqueous solution is added for extraction, and the organic phase is concentrated under vacuum and reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (ethyl acetate/n-hexane) to give the title compound (80 mg,0.11mmol, 73.6%). C44 H89 N3 O4 , MS(ES): m/z(M+H+ )724.6。
1 H NMR (400 MHz, Chloroform-d) δ 4.06 (t,J = 6.8 Hz, 2H), 3.96 (d,J= 5.8 Hz, 1H), 3.21 (s, 3H), 3.05 (s, 2H), 2.91 (s, 1H), 2.73 (s, 2H), 2.54 (s, 2H), 2.49 – 2.17 (m, 11H), 2.03 (d,J = 11.0 Hz, 3H), 1.82 (s, 2H), 1.61 (d,J = 7.3 Hz, 4H), 1.53 – 1.40 (m, 2H), 1.26 (d,J = 3.9 Hz, 42H), 0.88 (t,J = 6.7 Hz, 9H).
Synthesis of YK-222
The synthetic route is as follows:
the method comprises the following steps: synthesis of heptadecan-9-yl-8- (2- ((2- (N-tert-butoxycarbonyl-N-methylamino) ethyl) (6-oxo-6- ((undecyloxy) hexyl) amino) caprylate (YK-222-PM 1)
YK-222-PM1 (120mg, 0.14mmol, 97.4%) was obtained by the method for synthesizing YK-201 using YK-201-PM1 (100mg, 0.14mmol) and methyl (2- (methylamino) ethyl) carbamic acid tert-butyl ester (28mg, 0.15mmol) as raw materials. C49 H97 N3 O6 , MS(ES): m/z(M+H+ )880.3。
Step two: synthesis of heptadecan-9-yl-8- (2- ((2- (methylamino) ethyl) (6-oxo-6- ((undecyloxy) hexyl) amino) octanoate (YK-222)
YK-222-PM1 (120mg, 0.14mmol) was dissolved in 1mL of dichloromethane, and 1mL of trifluoroacetic acid was added dropwise thereto to obtain the objective compound (67 mg, 0.09mmol, 61.3%) according to the method for synthesizing YK-221. C48 H97 N3 O4 , MS(ES): m/z(M+H+ )780.4。
1 H NMR (400 MHz, Chloroform-d) δ 4.93 – 4.80 (m, 1H), 4.05 (t,J = 6.8 Hz, 2H), 3.15 (t,J = 5.4 Hz, 4H), 3.02 (s, 3H), 2.83 (s, 1H), 2.73 (s, 2H), 2.37 – 2.21 (m, 7H), 2.01 (d,J = 5.5 Hz, 6H), 1.63 (tt,J = 13.9, 7.4 Hz, 10H), 1.50 (d,J = 6.4 Hz, 4H), 1.46 – 1.15 (m, 49H), 0.93 – 0.85 (m, 9H).
23. Synthesis of YK-223
The synthetic route is as follows:
the method comprises the following steps: synthesis of bis (2-octyldecyl) -6, 6' - ((2- ((2- (N-Boc-N-methylamino) ethyl) (methylamino) ethyl) azadialkyl) dihexanoate (YK-223-PM 1)
YK-209-PM2 (150mg, 0.18mmol) and methyl (2- (methylamino) ethyl) carbamic acid tert-butyl esterButyl ester (45mg, 0.24mmol) was used as the starting material, and YK-223-PM1 (160mg, 0.17mmol, 92.2%) was obtained according to the method for synthesizing YK-201. C59 H117 N3 O6 , MS(ES): m/z(M+H+ )964.9。
Step two: synthesis of bis (2-octyldecyl) -6, 6' - ((2- ((2- (methylamino) ethyl) azadialkyl) dihexanoate (YK-223)
YK-223-PM1 (160mg, 0.17mmol) was dissolved in 1mL of dichloromethane, and 0.5mL of trifluoroacetic acid was added dropwise according to the method for synthesizing YK-221 to obtain the objective compound (120 mg,0.14mmol, 81.7%). C54 H109 N3 O4 , MS(ES): m/z(M+H+ )864.9。
1 H NMR (400 MHz, Chloroform-d) δ 4.12 (q,J = 7.1 Hz, 1H), 3.97 (d,J= 5.8 Hz, 3H), 2.95 (d,J = 4.8 Hz, 1H), 2.88 – 2.40 (m, 10H), 2.38 – 2.25 (m, 6H), 2.05 (s, 2H), 1.74 – 1.12 (m, 74H), 0.88 (d,J = 6.9 Hz, 12H).
24. Synthesis of YK-224
The synthetic route is as follows:
the method comprises the following steps: synthesis of bis (heptadecan-9-yl) -6, 6' - ((2- ((2- (N-Boc-N-methylamino) ethyl) (methylamino) ethyl) azadialkyl) dihexanoate (YK-224-PM 1)
YK-224-PM1 (120mg, 0.13mmol, 55.7%) was obtained by synthesizing YK-201 using YK-210-PM2 (150mg, 0.19mmol) and methyl (2- (methylamino) ethyl) carbamic acid tert-butyl ester (43mg, 0.23mmol) as starting materials. C57 H113 N3 O6 , MS(ES): m/z(M+H+ )936.8。
Step two: synthesis of bis (heptadecan-9-yl) -6, 6' - ((2- ((2- (methylamino) ethyl) azadialkyl) dihexanoate (YK-224)
YK-224-PM1 (100mg, 0.11mmol) was dissolved in 2mL of dichloromethane, and 1mL of trifluoroacetic acid was added dropwise thereto to synthesize YK-221To give the target compound (70 mg, 0.08mmol, 76.1%). C52 H105 N3 O4 , MS(ES): m/z(M+H+ )836.7。
1 H NMR (400 MHz, Chloroform-d) δ 4.86 (q,J = 6.1 Hz, 2H), 4.12 (q,J= 7.1 Hz, 1H), 2.90 – 2.82 (m, 2H), 2.76 – 2.41 (m, 9H), 2.30 (q,J = 8.3, 7.4 Hz, 6H), 2.04 (s, 1H), 1.63 (q,J = 7.4 Hz, 4H), 1.57 – 1.39 (m, 12H), 1.28 (d,J = 15.2 Hz, 56H), 0.88 (t,J = 6.8 Hz, 12H).
Synthesis of YK-225
The synthetic route is as follows:
the method comprises the following steps: synthesis of bis (3-hexylnonyl) -6, 6' - ((2- ((2- (N-Boc-N-methylamino) ethyl) (methylamino) ethyl) azadialkyl) dihexanoate (YK-225-PM 1)
YK-225-PM1 (120mg, 0.14mmol, 64.9%) was obtained by the method for synthesizing YK-201 using YK-211-PM2 (170mg, 0.21mmol) and methyl (2- (methylamino) ethyl) carbamic acid tert-butyl ester (43mg, 0.23mmol) as raw materials. C53 H105 N3 O6 , MS(ES): m/z(M+H+ )880.8。
Step two: synthesis of bis (3-hexylnonyl) -6, 6' - ((2- ((2- (methylamino) ethyl) azadialkyl) dihexanoate (YK-225)
YK-225-PM1 (120mg, 0.14mmol) was dissolved in 2mL of dichloromethane, and 0.5mL of trifluoroacetic acid was added dropwise thereto according to the method for synthesizing YK-221, whereby the objective compound (80 mg, 0.10mmol, 73.2%) was obtained. C48 H97 N3 O4 , MS(ES): m/z(M+H+ )780.7。
1 H NMR (400 MHz, Chloroform-d) δ 4.08 (t,J = 7.1 Hz, 4H), 3.48 (s, 3H), 3.04 – 2.86 (m, 5H), 2.29 (dd,J = 17.2, 9.6 Hz, 5H), 1.61 (ddd,J = 26.8, 14.3, 7.3 Hz, 10H), 1.47 – 1.35 (m, 6H), 1.25 (s, 45H), 0.88 (t,J = 6.1 Hz, 12H).
Synthesis of YK-226
The synthetic route is as follows:
the method comprises the following steps: synthesis of undecyl 6- ((2- (N-Boc-N-ethylamino) ethyl) amino) hexanoate (YK-226-PM 1):
YK-226-SM1 (278 mg, 0.80 mmol) and tert-butyl (2-aminoethyl) (ethyl) carbamate (150 mg, 0.80 mmol) were dissolved in acetonitrile (3 mL), and potassium carbonate (330 mg, 2.38 mmol) and potassium iodide (13 mg, 0.08 mmol) were added to the above system and heated to 70 ℃ for 4 hours with stirring. After the reaction is finished, the reaction liquid is cooled to room temperature and then filtered, and the filtrate is subjected to vacuum concentration under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (methanol/dichloromethane) to give decyl 4- ((2-hydroxybutyl) amino) butyrate (190 mg,0.46mmol, 57.3%), C23 H46 N2 O4 , MS(ES): m/z(M+H+ )415.4。
Step two: synthesis of 2-octyldecyl-6- (6- (undecyloxy) -6-oxohexyl) ((2- (N-Boc-N-ethylamino) ethyl) amino) hexanoate (YK-226-PM 2)
YK-226-PM1 (190mg, 0.42mmol) purified as described above and 2-octyldecyl 6-bromohexanoate (180mg, 0.46mmol) were dissolved in acetonitrile (3 mL), and potassium carbonate (173mg, 1.26mmol) and potassium iodide (7 mg, 0.04mmol) were added to the above system, and the mixture was heated to 70 ℃ and stirred for reaction for 24 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 (281 mg,0.35mmol, 82.7%). C49 H96 N2 O6 ，MS(ES):m/z(M+H+ )809.7。
Step three: synthesis of 2-octyldecyl-6- (6- (undecyloxy) -6-oxohexyl) ((2- (ethylamino) ethyl) amino) hexanoate (YK-226)
YK-226-PM2 (281mg, 0.35mmol) is dissolved in 2mL of dichloromethane, 1mL of trifluoroacetic acid is added dropwise,the target compound (210 mg,0.30mmol, 84.6%) was obtained according to the method for synthesizing YK-221. C44 H88 N2 O4 , MS(ES): m/z(M+H+ )709.6。
1 H NMR (400 MHz, Chloroform-d) δ 4.09 (t,J = 6.8 Hz, 2H), 4.00 (d,J= 5.8 Hz, 2H), 3.40 – 3.18 (m, 1H), 2.86 – 2.73 (m, 4H), 2.64 (t,J = 6.0 Hz, 2H), 2.46 (q,J = 7.1 Hz, 4H), 2.34 (q,J = 7.7 Hz, 4H), 1.73 – 1.58 (m, 7H), 1.54 – 1.42 (m, 4H), 1.37 – 1.20 (m, 49H), 0.97 – 0.89 (m, 9H).
Synthesis of YK-009
YK-009 159mg was obtained according to the method of CN 114044741B.
28.9 Synthesis of heptadecyl-8- (8- ((3-hexylnonyl) oxy) -8-oxooctyl) - ((2-hydroxyethyl) amino) octanoate (Compound 21)
The synthetic route is as follows:
the method comprises the following steps: synthesis of 9-heptadecyl 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 stirred at 30-35 ℃ for 8 hours. After the reaction, the reaction solution was washed with saturated sodium carbonate, saturated brine and Na2 SO4 And (5) drying. The mixture was filtered, and the filtrate was concentrated under reduced pressure in vacuo and purified by silica gel chromatography (ethyl acetate/n-hexane) to give 8-bromooctanoic acid-9-heptadecanoyl ester (1.20g, 2.60mmol, 66.7%).
Step two: synthesis of 9-heptadecyl-8- ((2-hydroxyethyl) amino) caprylate (compound 21-PM 2)
8-Bromocaprylic acid-9-heptadecyl ester (500mg, 1.08mmol) and ethanolamine (119mg, 3.25mmol) are dissolved in acetonitrile (5 mL), and potassium carbonate (149 mg,1.08 mmol) is added) The reaction mixture was heated to 70 ℃ and stirred for 2 hours. After the reaction is finished, the reaction solution is cooled to room temperature and then filtered, and the filtrate is subjected to vacuum reduced pressure concentration to remove the solvent. The residue was purified by silica gel chromatography (methanol/dichloromethane) to give 9-heptadecyl-8- ((2-hydroxyethyl) amino) octanoate (372 mg, 0.84mmol, 78.0%), C27 H55 NO3 , MS(ES): m/z(M+H+ )442.3。
Step three: synthesis of 8-bromooctanoic acid-3-hexylnonyl ester (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 (1.62g, 3.74mmol, 85.3%).
Step four: synthesis of 9-heptadecyl-8- (8- ((3-hexylnonyl) oxy) -8-oxooctyl) - ((2-hydroxyethyl) amino) octanoate (Compound 21)
9-heptadecyl-8- ((2-hydroxyethyl) amino) octanoate (200mg, 0.46mmol) and 8-bromooctanoic acid-3-hexylnonanoate (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 by heating to 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 (213mg, 0.27mmol, 58.4%). C50 H99 NO5 ，MS(ES): m/z(M+H+ )794.8。
1 H NMR (400 MHz, CDCl3 ) 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).
29. Synthesis of bis (3-hexylnonyl) -8, 8' - ((2-hydroxyethyl) azadialkyl) dicaprylate (Compound 23)
The synthetic route is as follows:
8-Bromooctanoic 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 concentration under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (methanol/dichloromethane) to give bis (3-hexylnonyl) -8, 8' - ((2-hydroxyethyl) azadialkyl) dicaprylate (150 mg,0.20mmol, 29.7%), C48 H95 NO5 , MS(ES): m/z(M+H+ )766.5。
1 H NMR (400 MHz, CDCl3 ) 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-202 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 a 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. 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 a 300kDa ultrafiltration tube to remove ethanol. Then, volume was adjusted to a certain volume with PBS to obtain LNP formulation encapsulating eGFP-mRNA with cationic lipid YK-202/DSPC/cholesterol/DMG-PEG 2000 (mole percent 49.
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 with the LNP formulations prepared using YK-201 and YK-209, 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-202 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, wherein the highest transfection efficiency is 4.5. (FIG. 2)
The same results were obtained with the LNP formulations prepared using YK-201 and YK-209, 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 the cationic lipid being YK-202 in the vehicle, and the polymer conjugated lipid DMG-PEG2000 in the vehicle at 0.5%, 1.5%, 3.5%, 5%, 10% and 15% molar ratios, 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 resulting LNP formulations were prepared using YK-201 and YK-209 with similar results, 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-202, neutral lipid DSPC, structural lipid cholesterol and polymer conjugated lipid DMG-PEG2000.
As can be seen from cell transfection experiments, the molar ratio of the cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid is from the following ratio of 75. 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), so that the LNP preparation can be prepared, wherein the optimal ratio is 45.
The resulting LNP formulations were prepared using YK-201 and YK-209 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 cells attached.
Cell transfection experiments: an LNP preparation containing 1.5. Mu.g of eGFP-mRNA prepared in example 2 (cationic lipid in the vehicle is YK-202) and a Lipofectamin 3000 preparation of eGFP-mRNA were added to the cell culture medium in a 12-well plate, and after further culturing for 24 hours, the transfection efficiency of different samples was examined by fluorescence intensity using a fluorescence microscope.
According to the experimental results, the preparation conditions of the nano-lipide particles (LNP preparation) are finally determined: vector to mRNA 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; the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the polymer conjugated lipid is 49.
Example 4: preparation of Nano lipid particles (LNP formulation) (optimal formulation)
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 obtained liposome was diluted to 10-fold volume with PBS, and then subjected to ultrafiltration in a 300kDa ultrafiltration tube to remove ethanol. Then the volume is adjusted to a certain volume by PBS, so as to obtain the LNP preparation which uses cationic lipid/DSPC/cholesterol/DMG-PEG 2000 (mole percentage is 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 which are 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 the instruction manual of Lipofectamine 3000 (England Weichai Jie Co., ltd.).
Example 5: measurement of particle diameter and polydispersity index (PDI) of nano-liposome particle
The particle size and polydispersity index (PDI) was determined by dynamic light scattering using a malvern laser particle sizer. mu.L of the liposome solution was diluted to 1mL with RNase-free deionized water, 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-liposome particles prepared in the example 4 is 130-250nm, and the particle size of the nano-liposome particles prepared by HMMA is the smallest and 135nm, and the particle size of the nano-liposome particles prepared by YK-226 is the largest and 246nm. The polydispersity of all the nano-lipid particles is between 5% and 35%, the smallest of which is compound 23 and is 8.6%, and the largest is YK-214 and is 31.5%.
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 sample was taken and added to an appropriate amount of 1 XTE buffer solution to dilute the sample to a solution containing about 2.8. Mu.g of sample 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 of 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-201, YK-202, YK-206, YK-207 and YK-209 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-209 can be improved by 41 percent compared with that of the compound 23, and the drug-loading concentration can reach 2 times of that of the compound 23; the total RNA concentration of YK-201 can reach 1.5 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-201, YK-202, YK-206, YK-207 and YK-209, the encapsulation efficiency is significantly improved compared with SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-009.
The encapsulation efficiency of YK-201 is 89.5%, which is improved by 27.20% compared with SM-102, improved by 26.70% compared with ALC-0315, improved by 31.70% compared with compound 21, improved by 34.30% compared with compound 23, improved by 29.00% compared with HHMA, and improved by 26.50% compared with YK-009.
The encapsulation efficiency of YK-202 is 86.3%, which is 24.00% higher than SM-102, 23.50% higher than ALC-0315, 28.50% higher than compound 21, 31.10% higher than compound 23, 25.80% higher than HHMA, and 23.30% higher than YK-009.
The encapsulation efficiency of YK-206 is 91.4%, which is 29.10% higher than that of SM-102, 28.60% higher than that of ALC-0315, 33.60% higher than that of compound 21, 36.20% higher than that of compound 23, 30.90% higher than that of HHMA, and 28.40% higher than that of YK-009.
The encapsulation efficiency of YK-207 is 90.8%, which is improved by 28.50% compared with SM-102, 28.00% compared with ALC-0315, 33.00% compared with compound 21, 35.60% compared with compound 23, 30.30% compared with HHMA, and 27.80% compared with YK-009.
The encapsulation efficiency of YK-209 is 96.2%, which is increased by 33.90% compared with SM-102, 33.40% compared with ALC-0315, 38.40% compared with compound 21, 41.00% compared with compound 23, 35.70% compared with HHMA, and 33.20% 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 of YK-201, YK-202, YK-206, YK-207 and YK-209 are both obviously improved compared with SM-102, ALC-0315, compound 21, compound 23 and YK-009.
YK-201 drug loading concentration is 40.32 mug/mL, total RNA concentration is 45.07 mug/mL, which is 1.58 times and 1.28 times of SM-102, 1.55 times and 1.21 times of ALC-0315, 1.78 times and 1.47 times of compound 21, 1.95 times and 1.39 times of compound 23, 1.32 times and 1.19 times of HHMA, and 1.50 times and 1.17 times of YK-009.
The YK-202 drug loading concentration is 37.08 mug/mL, the total RNA concentration is 42.98 mug/mL, which are respectively 1.46 times and 1.22 times of SM-102, 1.42 times and 1.16 times of ALC-0315, 1.64 times and 1.40 times of compound 21, 1.79 times and 1.32 times of compound 23, 1.21 times and 1.14 times of HHMA, and 1.38 times and 1.11 times of YK-009.
YK-206 drug loading concentration was 38.09 μ g/mL, total RNA concentration was 41.69 μ g/mL, 1.50 and 1.18 times SM-102, 1.46 and 1.12 times ALC-0315, 1.69 and 1.36 times compound 21, 1.84 and 1.28 times compound 23, 1.24 and 1.10 times HHMA, 1.42 and 1.08 times YK-009.
The YK-207 drug loading concentration is 39.60 mug/mL, the total RNA concentration is 43.63 mug/mL which are respectively 1.56 times and 1.24 times of SM-102, 1.52 times and 1.18 times of ALC-0315, 1.75 times and 1.42 times of compound 21, 1.91 times and 1.34 times of compound 23, 1.29 times and 1.15 times of HHMA, and 1.47 times and 1.13 times of YK-009.
The YK-209 drug loading concentration is 41.76 mug/mL, the total RNA concentration is 43.42 mug/mL, which are 1.64 times and 1.23 times of SM-102, 1.60 times and 1.17 times of ALC-0315, 1.85 times and 1.41 times of compound 21, 2.02 times and 1.34 times of compound 23, 1.36 times and 1.15 times of HHMA, and 1.55 times and 1.12 times of YK-009.
Data were analyzed using GraphPad Prism software, where any of YK-201, YK-202, YK-206, YK-207 and YK-209 was significantly different from SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-009, and the encapsulation efficiency, drug loading concentration and total RNA concentration were significantly increased.
And (3) knotting:
some of the compounds we designed, including YK-201, YK-202, YK-206, YK-207, and YK-209, produced LNP formulations with significantly improved encapsulation efficiency, drug loading concentration, and total RNA concentration compared to 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-209 can be improved by 41 percent compared with that of the compound 23, and the drug-loading concentration can reach 2 times of that of the compound 23; YK-201 total RNA concentration can reach 1.5 times of compound 21.
(2) The LNP preparation prepared from different designed compounds has the encapsulation efficiency and the drug loading rate which are greatly different, the range of the encapsulation efficiency of the different compounds is 55% -99%, the drug loading concentration is 25-45 mug/mL, and the total RNA concentration is 25-50 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 55-99%, the drug loading concentration is 25-45 mug/mL, and the total RNA concentration is 25-50 mug/mL. Different compounds have great difference, the highest encapsulation efficiency is YK-224 which reaches 98.7 percent, and the lowest encapsulation efficiency is YK-220 which is only 57.5 percent; the highest drug loading concentration is YK-217 which is 42.19 mug/mL, and the lowest drug loading concentration is YK-220 which is only 25.27 mug/mL; the highest total RNA concentration is YK-219, which reaches 47.23 mug/mL, and the lowest total RNA concentration is YK-222, which is only 27.43 mug/mL.
And (4) summarizing:
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 55% -99%, the drug loading concentration is 25-45 mug/mL, and the total RNA concentration is 25-50 mug/mL. It follows that LNP formulations not prepared from structurally similar compounds will necessarily have similar encapsulation efficiencies and drug loadings.
To summarize:
the LNP formulations made from some of the compounds we designed, including YK-201, YK-202, YK-206, YK-207 and YK-209, were significantly improved in encapsulation efficiency, drug loading concentration and total RNA concentration compared to 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-209 can be improved by 41 percent compared with that of the compound 23, and the drug-loading concentration can reach 2 times of that of the compound 23; YK-201 total RNA concentration can reach 1.5 times of compound 21.
Therefore, the carrier for delivering the mRNA prepared by YK-201, YK-202, YK-206, YK-207 and YK-209 has obviously improved encapsulation efficiency and drug loading capacity, so that the dosage of the LNP can be obviously reduced, and an LNP-mRNA preparation product with more uniform distribution and 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 55% to 99%, the drug loading concentration ranges from 25 to 45 mug/mL, and the total RNA concentration ranges from 25 to 50 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 preparations containing 0.3 μ g Fluc-mRNA (LNP preparation vehicle components were cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid, molar ratio 49.39.5, where cationic lipid is the cationic lipid listed in table 1) were added to the cell culture broth of 96-well plates, and after 24 hours of incubation, the corresponding reagents were added according to the Gaussia Luciferase Assay Kit instructions and the fluorescence expression intensity of each well was measured by IVIS fluorescence detection system. The experiment verifies the transfection efficiency of the LNP preparation in cells, and the specific detection result is shown in tables 6-9.
The experimental results are as follows:
(1) The compounds of the present application, including YK-201, YK-202, YK-206, YK-207, and YK-209, are very different in chemical structure from prior art cationic lipids.
The series of compounds designed in this application, including YK-201, YK-202, YK-206, YK-207, and YK-209, are very different from the prior art cationic lipid chemical structures. For example, these compounds are structurally completely different from HHMA; g compared with SM-102, ALC-0315, compound 21, compound 23, and YK-0093 Radicals completely different, G1 、G2 、R1 And R2 The groups also all differ significantly. The structural details are 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 3, the series of compounds designed, including YK-201, YK-202, YK-206, YK-207 and YK-209, 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).
In contrast to this series of compounds:
a. the HHMA structure has the largest difference, and as can be seen from the chemical structure diagram, the group connected with the central N atom of the HHMA has only 1 side chain which is close to 1 side chain of the series structure, and the other parts are completely different.
b. Other cationic lipids of the prior art, such as SM-102, ALC-0315, compound 21, compound 23, and YK-0093 The groups are completely different. Due to G3 The groups have different structures, so that the polarity, the acidity and the alkalinity, the hydrophilicity and the like of the groups can be greatly different.
c. SM-102, ALC-0315, compound 21, compound 23, and G of YK-0091 、R1 、G2 And R2 Groups also differ significantly.
The method comprises the following specific steps:
I．YK-201
YK-201 has a significant structural difference compared to prior art cationic lipids, such as SM-102, compound 21, compound 23, YK-009, and HHMA.
G of YK-201 compared with SM-1023 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -, G of SM-1023 The radical is HO (CH)2 )2 -。
G of YK-201 compared with ALC-03151 The group is 1C less than ALC-0315; r1 The group is a straight chain structure, and ALC-0315 is a branched chain structure; g2 The radical has 1 more C than ALC-0315; r2 2 more C in 1 single strand of the group double strand; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -. And, G1 Group and R1 Radical, G2 Group and R2 The direction of ester bonds between the groups is also different between YK-201 and ALC-0315.
G of YK-201 compared with Compound 211 2C fewer groups than compound 21; r1 The group is a straight chain structure, and the compound 21 is a branched chain structure; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -。
G of YK-201 in comparison with Compound 231 2C fewer groups than compound 23; r1 The group is a straight chain structure, and the compound 23 is a branched chain structure; r2 The number of single-chain groups is 2 less, and each single-chain in the double-chain group is 2 more; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -。
G of YK-201 compared with YK-0091 The radical ratio YK-009 has 2 more C; r1 1 more C; g2 The radical is 2 more C; r2 1C less single-chain group; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -。
Compared with HHMA, the structure of YK-201 is completely different, HHMA has only 1 side chain connected with N atom and is similar to the structure of 1 side chain of YK-201, and other parts have obvious difference.
II．YK-202
YK-202 has a significant structural difference compared to prior art cationic lipids, such as SM-102, compound 21, compound 23, YK-009, and HHMA.
R of YK-202 compared to SM-1022 The group single chain has 1 more C than SM-102; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -。
G of YK-202 compared with ALC-03151 The radical is 1C less than ALC-0315; r1 The group is a straight chain structure, and ALC-0315 is a branched chain structure; g2 1 more radicalC；R2 The single chain of the group has 1C, and the single chain of the double chain has 2C; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -. And, G1 Group and R1 Group, G2 Group and R2 The ester bond direction between the groups is different between YK-201 and ALC-0315.
G of YK-202 compared with Compound 211 2 less C; r is1 The group is a straight chain structure, and the compound 21 is a branched chain structure; r is2 The group is single-stranded with 1 more C than compound 21; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -。
G of YK-202 compared with Compound 231 2 less C; r1 The group is a straight chain structure, and the compound 23 is a branched chain structure; r is2 The number of single-chain groups is 1 less, and each single-chain in the double-chain group is 2 more than that of the single-chain group; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -。
G of YK-202 compared with YK-0091 The radical is 2 more C; r1 The radical is more than 1C; g2 The radical is 2 more C; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -。
Compared with HHMA, the structure of YK-202 is completely different, and HHMA has only 1 side chain connected to N atom and similar structure to that of YK-202, and other parts have obvious difference.
III．YK-206
YK-206 has a significant structural difference compared to prior art cationic lipids, such as SM-102, compound 21, compound 23, YK-009, and HHMA.
R of YK-206 compared to SM-1022 Each single chain in the group double chain has 1 less C; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -。
G of YK-206 compared with ALC-03151 The radical is 1C less than ALC-0315; r1 The radicals being straight-chainStructure, and ALC-0315 is branched chain structure; g2 1 more C; r2 1 single chain in the group double chain has more than 1C, and 1 single chain has less than 1C; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -. And, G1 Group and R1 Radical, G2 Group and R2 The direction of ester bonds between the groups is also different between YK-201 and ALC-0315.
G of YK-206 compared with Compound 211 2 less C; r1 The group is a straight chain structure, and the compound 21 is a branched chain structure; r is2 Each single chain in the group double chain has 1 less C; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -。
G of YK-206 compared with Compound 231 2 less C; r1 The group is a straight chain structure, and the compound 23 is a branched chain structure; r2 The single chain of the group has 2 less C, and each single chain in the double chain has 1 less C; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -。
G of YK-206 compared with YK-0091 The radical is 2 more C; r1 1 more C; g2 The radical is 2 more C; r2 The single chain of the group has 1C less, and each single chain in the double chain has 1C less; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -。
Compared with HHMA, YK-206 has completely different structure, HHMA has 1 side chain connected to N atom similar to that of YK-206, and other parts have obvious difference.
IV．YK-207
YK-207 has a significant structural difference compared to prior art cationic lipids, such as SM-102, compound 21, compound 23, YK-009, and HHMA.
G of YK-207 compared with SM-1021 2 less C; r1 1 less C; g2 2 less C; r is2 The group is single-stranded with 1 more C; g3 The radicals are completely different from each other,is HO (CH)2 )2 N(CH3 )(CH2 )2 -。
G of YK-207 compared to ALC-03151 The radical is 3C less than ALC-0315; r is1 The group is a straight chain structure, and ALC-0315 is a branched chain structure; g2 1 less C; r is2 The single chain of the group has 1C, and the single chain of the double chain has 2C; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -. And, G1 Group and R1 Group, G2 Group and R2 The direction of ester bonds between the groups is also different between YK-201 and ALC-0315.
G of YK-207 in comparison with Compound 211 4 or less C; r1 The group is a straight chain structure, and the compound 21 is a branched chain structure; g2 2 less C; r is2 The group is single-stranded with 1 more C; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -。
G of YK-207 in comparison with Compound 231 4 or less C; r is1 The group is a straight chain structure, and the compound 23 is a branched chain structure; g2 2 less C; r is2 The number of single-chain groups is 1 less, and each single-chain in the double-chain group is 2 more than that of the single-chain group; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -。
Compared with HHMA, the structure of YK-207 is completely different, HHMA has only 1 side chain connected with N atom and is similar to the structure of 1 side chain of YK-207, and other parts have obvious difference.
V．YK-209
YK-209 has a significant structural difference compared to prior art cationic lipids, such as SM-102, compound 21, compound 23, YK-009, and HHMA.
R of YK-209 compared to SM-1021 The group is a branched chain structure, and SM-102 is a linear chain structure; g of YK-2092 2 less C; r2 The group is single-stranded with 1 more C; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -。
G of YK-209 compared with ALC-03151 The radical is 1C less than ALC-0315; r1 The group is a straight chain structure, and ALC-0315 is a branched chain structure; g2 1 less C; r2 The single chain of the group has 1C, and the single chain of the double chain has 2C; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -. And, G1 Group and R1 Radical, G2 Group and R2 The direction of ester bonds between the groups is also different between YK-201 and ALC-0315.
G of YK-209 compared with Compound 211 2 less C; r is1 The number of single-chain groups is 1 less, and each single-chain in the double-chain group is 2 more than that of the single-chain group; g2 2 less C; r2 The group is single-stranded with 1 more C; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -。
G of YK-209 in comparison with Compound 231 2 less C; r1 The number of single-chain groups is 1 less, and each single-chain in the double-chain group is 2 more than that of the single-chain group; g2 2 less C; r is2 The number of the group single chains is less than 1C, and each single chain in the double chains is more than 2C; g3 The radicals are completely different and are HO (CH)2 )2 N(CH3 )(CH2 )2 -。
Compared with HHMA, the structure of YK-209 is completely different, the HHMA has only 1 side chain connected with N atoms and is similar to the structure of 1 side chain of YK-209, and other parts have obvious difference.
As can be seen from the above comparison, the series of compounds designed, including YK-201, YK-202, YK-206, YK-207 and YK-209, 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 have completely different structures from HHMA; g with SM-102, ALC-0315, compound 21, compound 23, and YK-0093 Radicals completely different, G1 、G2 、R1 And R2 There are also significant differences in groups.
Due to the significant difference of chemical structures, the physicochemical properties of the series of compounds, such as polarity, acid-base property and hydrophilic property, are also significantly different compared with the same compounds 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 cationic lipid compounds disclosed in the prior art.
(2) In a series of designed compounds, LNP preparations prepared from YK-201, YK-202 and YK-209 have the highest cell transfection efficiency, and are obviously improved compared with the representative cationic lipid in the prior art. For example, YK-202 can be up to 18 times that of SM-102, 21 times that of Compound 21, and 22 times that of Compound 23.
TABLE 6 Fluorescently detectable results of Fluc-mRNA-1
Difference 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-201, YK-202 and YK-209 exhibited the strongest fluorescence absorption and RLU values of 28482617, 29957419 and 26861465, respectively.
YK-201 can reach 17.10 times of SM-102, 13.27 times of ALC-0315, 20.04 times of compound 21, 21.38 times of compound 23, 13.56 times of HHMA, 23.71 times of Lipofectamine 3000 and 5.56 times of YK-009.
YK-202 can be 17.98 times of SM-102, 13.95 times of ALC-0315, 21.08 times of compound 21, 22.49 times of compound 23, 14.27 times of HHMA, 24.94 times of Lipofectamine 3000, and 5.84 times of YK-009.
YK-209 can reach 16.12 times of SM-102, 12.51 times of ALC-0315, 18.90 times of compound 21, 20.17 times of compound 23, 12.79 times of HHMA, 22.36 times of Lipofectamine 3000, and 5.24 times of YK-009.
LNP preparations containing Fluc-mRNA prepared from YK-206 and YK-207 also had strong fluorescence absorption, which was 12 times that of SM-102, 9 times that of ALC-0315, 14 times that of Compound 21, 15 times that of Compound 23, 10 times that of HHMA, 17 times that of Lipofectamine 3000, and 4 times that of YK-009.
The data were analyzed using GraphPad Prism software, where any of YK-201, YK-202, YK-206, YK-207 and YK-209 was significantly different from SM-102, ALC-0315, compound 21, compound 23, HHMA, lipofectamine 3000 and YK-009, resulting in significantly improved transfection efficiency.
And (3) knotting:
in terms of chemical structure, the series of compounds designed, including YK-201, YK-202 and YK-209, differ greatly from the cationic lipids of the prior art, e.g., are completely different from HHMA structures; g compared with SM-102, ALC-0315, compound 21, compound 23, and YK-0093 Radicals completely different, G1 、G2 、R1 And R2 The groups also all differ greatly.
LNP formulations prepared from YK-201, YK-202 and YK-209 were most efficient in cell transfection and significantly improved over the prior art representative cationic lipid activities, e.g., 18 times higher for YK-202, 21 times higher for compound 21 and 22 times higher for compound 23.
We have found that rather than having cell transfection activity only with compounds that are close in chemical structure to prior art cationic lipids, LNP formulations made from compounds that differ greatly in structure may have significantly improved transfection efficiency with very strong cell transfection activity.
(3) YK-201, YK-202 and YK-209 are most similar in the structure we have designed, only G1 、G2 、G3 、R1 Or R2 Compounds with groups differing by 1-2CIn this case, the transfection efficiency of the cells was the highest, and YK-202 was 82 times higher than that of other compounds, for example, YK-204.
To compare whether structurally similar compounds have similar transfection efficiencies, compounds structurally closest to YK-201, YK-202 and YK-209 were compared, differing only in G1 、G2 、G3 、R1 Or R2 The groups differ by 1-2C. The results show that the activity of the series of compounds is very different, wherein the cell transfection efficiency of YK-201, YK-202 and YK-209 is the highest and can respectively reach 78 times, 82 times and 57 times of YK-204 with the lowest activity, and the transfection efficiency is obviously improved.
TABLE 7 Fluorescently detectable results of Fluc-mRNA-2
a. Differential efficiency of cell transfection
As can be seen from Table 7 and FIG. 6, the LNP formulations prepared from these compounds have fluorescence absorption values that are very different from those of YK-201, YK-202 and YK-209.
YK-201 can reach 7.36 times of YK-203, 78.25 times of YK-204, 27.68 times of YK-205, 7.38 times of YK-208, 6.15 times of YK-210 and 20.79 times of YK-211.
YK-202 can reach 7.74 times of YK-203, 82.30 times of YK-204, 29.11 times of YK-205, 7.76 times of YK-208, 6.47 times of YK-210 and 21.87 times of YK-211.
YK-209 can reach 5.44 times of YK-203, 57.85 times of YK-204, 20.46 times of YK-205, 5.46 times of YK-208, 4.55 times of YK-210 and 15.37 times of YK-211.
The activity differences among YK-203, YK-204, YK-205, YK-208, YK-210 and YK-211 are also large. LNP preparations prepared from YK-203, YK-208 and YK-210 have cell transfection efficiencies stronger than those of SM-102, which are respectively 2.32 times, 2.32 times and 2.78 times of SM-102; YK-205 and YK-211 are comparable to SM-102, being 0.62 times and 0.82 times, respectively; YK-204 was the least efficient cell transfection, only 0.22 times that of SM-102.
When the data are analyzed by GraphPad Prism software, any one of YK-201, YK-202 and YK-209 has obvious difference with YK-203, YK-204, YK-205, YK-208, YK-210 and YK-211, and the transfection efficiency is obviously improved.
b. Difference in chemical structure
The structures of the series of compounds are very similar, except that G1 、G2 、G3 、R1 Or R2 The groups differ by 1-2C. YK-201, YK-202 and YK-209 have very close structures with other compounds; other compounds are also very similar.
I. Structural difference from YK-201
In contrast to YK-201, YK-204 is only G3 The group is connected with N by more than 1C, other structures are completely the same, but the transfection efficiency of the cell YK-201 is 78.25 times of that of YK-204.
YK-205 is G only3 The radicals being linked to N by more than 1C, R2 Each single strand in the gene double strand has 1 less C, and other structures are completely the same, but the cell transfection efficiency YK-201 is 27.68 times of YK-205.
YK-203 is only R2 The single chain of the gene 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 of the cell YK-201 is 7.36 times of that of YK-203.
Structural distinction from YK-202
In contrast to YK-202, YK-204 is only G3 The radicals being bound to N by more than 1C, R2 The single chain of the group has 1 less C, other structures are completely the same, but the transfection efficiency of the cell YK-202 is 82.30 times of that of YK-204.
YK-205 is G only3 The radicals being bound to N by more than 1C, R2 The number of the single-stranded group is 1 less, each single-stranded group in the double-stranded group is 1 less, and other structures are completely the same, but the cell transfection efficiency YK-202 is 29.11 times of that of YK-205.
YK-203 is R only2 The group single chain has more than 1C, each single chain in the double chain has less than 2C, other structures are completely the same, but the transfection efficiency YK-202 of the cells is 7.74 times of that of YK-203.
Structural distinction from YK-209
In contrast to YK-209, YK-210 is only R1 And R2 The single chain of the group has 1C less each, and the other structures are completely the same, but the cell is transformedThe dyeing efficiency YK-209 is 4.55 times of YK-210.
YK-211 being only R1 And R2 The single chain of the gene has more than 1C, each single chain in the double chain has less than 2C, other structures are completely the same, but the transfection efficiency YK-209 of the cell is 15.37 times of YK-211.
Furthermore, YK-205 is G only, as compared to YK-2063 The group is connected with 1C more than N, other structures are identical, but the transfection efficiency of the cell YK-206 is 20.29 times of that of YK-205. Other compounds are also similar.
And (3) knotting:
a series of structures we have designed are very similar, only G1 、G2 、G3 、R1 Or R2 Among the compounds with 1-2C difference in groups, YK-201, YK-202 and YK-209 were most efficient in cell transfection, and YK-202 was 80 times higher than YK-204.
Meanwhile, the structure of the compound and the intracellular transfection efficiency are not in corresponding relation, and even a group of compounds with the most similar structures have very high possibility of very different cell transfection efficiencies.
Therefore, it is very difficult to select cationic lipid compounds with high transfection efficiency from a series of compounds with very similar structures, and much creative work is required.
(4) YK-201, YK-202 and YK-209 are structurally identical to those of G alone1 、G2 、G3 、R1 Or R2 Transfection efficiency was highest compared to compounds with some minor differences in groups. YK-202 may be more than 400 times higher than other compounds, such as YK-217.
We further compared the differences in cell transfection efficiencies between YK-201, YK-202 and YK-209 and other structurally similar compounds, which differ only slightly in individual groups, e.g., G1 Reduction of 2C and R1 Reduction of 1C, G3 An ether bond or an ester bond or a sulfur atom is introduced. The results show that the series of compounds are obviously different from YK-201, YK-202 and YK-209, and the transfection efficiency of YK-201, YK-202 and YK-209 cells is obviously higher than that of other compounds, even more than 400 times higher.
TABLE 8 Fluorescence detection of Fluc-mRNA-3
a. Differential efficiency of cell transfection
Although the other compounds are G alone, as compared with YK-201, YK-202 and YK-2093 、G1 、R1 、G2 Or R2 The groups have some minor differences, but the influence on the cell transfection efficiency is very large, and the difference can reach more than 400 times.
Specifically, it can be seen from Table 8 that the fluorescence absorbance of LNP formulations prepared from YK-217 and YK-220 differ greatly from the activity of YK-201, YK-202 and YK-209.
YK-201 can reach 396.42 times of YK-217 and 131.62 times of YK-220.
YK-202 can reach 416.94 times of YK-217 and 138.44 times of YK-220.
YK-209 can reach 293.09 times of YK-217 and 97.32 times of YK-220.
YK-213, YK-215, YK-218 and YK-219 have much different activities than YK-201, YK-202 and YK-209.
YK-201 can reach 20.87 times of YK-213, 29.03 times of YK-215, 46.92 times of YK-218 and 18.91 times of YK-219.
YK-202 can reach 21.95 times of YK-213, 30.53 times of YK-215, 49.35 times of YK-218 and 19.89 times of YK-219.
YK-209 can be 15.43 times of YK-213, 21.46 times of YK-215, 34.69 times of YK-218 and 13.98 times of YK-219.
YK-212, YK-214 and YK-216 also have greatly different activities from YK-201, YK-202 and YK-209.
YK-201 can reach 7.28 times of YK-212, 6.22 times of YK-214 and 12.49 times of YK-216.
YK-202 can be 7.66 times of YK-212, 6.54 times of YK-214 and 13.14 times of YK-216.
YK-209 can reach 5.39 times of YK-212, 4.60 times of YK-214 and 9.24 times of YK-216.
When the data are analyzed by GraphPad Prism software, any one of YK-201, YK-202 and YK-209 has obvious difference with other compounds, and the cell transfection efficiency is obviously improved.
b. Difference in chemical structure
Compared with YK-201, YK-202 and YK-209, the series of compounds only have G1 、G2 、G3 、R1 Or R2 The groups are slightly different. YK-201, YK-202 and YK-209 are very close to other compounds in structure; other compounds are also very similar.
I. Structural difference from YK-201
YK-213 is G only, in contrast to YK-2013 Multiple HOCH with radicals bound to N2 -group, R1 The radicals being branched structures, G2 The gene has 2C less, other structures are identical, but the cell transfection efficiency YK-201 is 20.87 times of YK-213.
YK-215 is G only3 The group introduces ether bond, more than 1 methyl is connected with O, other structures are completely the same, but the cell transfection efficiency YK-201 is 29.03 times of YK-215.
YK-217 is G only3 The radical is (CH)3 )2 N(CH2 )3 SC(O)O(CH2 )2 -，G1 2C, R less1 1 less C, G2 2C, R less2 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-201 reaches 396.42 times of YK-217.
YK-220 is G only3 The radical is (CH)3 )2 N(CH2 )3 SC(O)-，G1 2 or less C, R1 1 less C, G2 2C, R less2 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-201 reaches 131.62 times of YK-220.
Structural distinction from YK-202
YK-217 is G only, as compared to YK-2023 The radical is (CH)3 )2 N(CH2 )3 SC(O)O(CH2 )2 -，G1 2C, R less1 The number of the groups is less than 1C,G2 2 or less C, R2 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-202 reaches 416.94 times of YK-217.
YK-219 is G only3 The radical is (CH)3 )2 N(CH2 )3 SC(O)-，G1 2C, R less1 1 less C, G2 The gene has 2C less, other structures are identical, but the transfection efficiency of the cell YK-202 is 19.89 times of that of YK-219.
YK-220 is G only3 The radical is (CH)3 )2 N(CH2 )3 SC(O)-，G1 2C, R less1 1 less C, G2 2 or less C, R2 The group single chain has more than 1C, each single chain in the double chain has less than 2C, other structures are completely the same, but the cell transfection efficiency YK-202 reaches 138.44 times of YK-220.
Structural distinction from YK-209
YK-213 is G only, in contrast to YK-2093 The radicals being bound to the N-multiple HOCH2 A group R1 And R2 The single chains of the group have 1C each, and the other structures are identical, but the transfection efficiency of the cell YK-209 is 15.43 times that of YK-213.
YK-217 is G only3 The radical is (CH)3 )2 N(CH2 )3 SC(O)O(CH2 )2 -，G1 2C, R less1 1 less C, R2 The group single chain has more than 1C, each single chain in the double chain has less than 2C, other structures are completely the same, but the cell transfection efficiency YK-209 reaches 293.09 times of YK-217.
YK-220 is G only3 The radical is (CH)3 )2 N(CH2 )3 SC(O)-，G1 2C, R less1 The radical being of single-chain structure, R2 The group single chain has more than 1C, each single chain in the double chain has less than 2C, other structures are completely the same, but the cell transfection efficiency YK-209 reaches 97.32 times of YK-220.
Furthermore, YK-220 is G only, as compared to YK-2073 The radical is (CH)3 )2 N(CH2 )3 SC(O)-，R2 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 transfection efficiency YK-207 of the cell is 124.13 times of YK-220. Other compounds are also similar.
And (3) knotting:
with some minor difference from the individual radicals only, e.g. G1 Reduction of 2C and R1 Reduction of 1C, G3 YK-201, YK-202 and YK-209 were transfected with the highest efficiency than compounds incorporating ether bonds, ester bonds or sulfur atoms. For example, YK-201 and YK-202 are both 400 times higher than YK-217 and 130 times higher than YK-220.
Meanwhile, the structure of the compound and the intracellular transfection efficiency are not in corresponding relation, and even a group of compounds with small difference in structure has the high possibility of very large difference in the cell transfection efficiency.
Therefore, it is very difficult to select a cationic lipid compound having a high transfection efficiency from a series of compounds having only a small difference in chemical structure, and much creative work is required.
(5) YK-201, YK-202 and YK-209 are structurally identical to G3 The transfection efficiency was highest in compounds in which the hydroxyl group of the group was changed to a methylamino group, or the hydroxyl group was removed and the methyl group attached to N was removed. For example, YK-201 and YK-202 are both more than 1000 times higher than YK-221 and YK-225.
The compounds listed in Table 9 differ from YK-201, YK-202 and YK-209 only by G3 The hydroxyl group is changed to a methylamino group, or the hydroxyl group and the methyl group attached to N are removed, depending on the group. The cell transfection efficiency results show that in the series of compounds with similar structures, the cell transfection efficiency of YK-201, YK-202 and YK-209 is greatly higher than that of other compounds, and both YK-201 and YK-202 are more than 1000 times higher than that of YK-221 and YK-225.
TABLE 9 fluorescent detection of Fluc-mRNA-4
a. Differential efficiency of cell transfection
G of YK-201, YK-202 and YK-2093 The hydroxyl group in the group is changed into methylamino, or the hydroxyl group and the methyl group connected with N are removed, the cell transfection efficiency is greatly reduced, and YK-201 and YK-202 can be improved by more than 1000 times compared with YK-221 and YK-225.
The method comprises the following specific steps:
as can be seen from Table 9, YK-221 and YK-225, these 2 compounds were very different from YK-201, YK-202 and YK-209 in transfection efficiency.
The transfection efficiency of YK-201 cell can reach 1018.69 times of YK-221 and 1089.41 times of YK-225 respectively.
The transfection efficiency of YK-202 cell can reach 1071.44 times of YK-221 and 1145.82 times of YK-225 respectively.
YK-209 cell transfection efficiency can reach 753.17 times of YK-221 and 805.46 times of YK-225 respectively.
YK-223, YK-224 and YK-226, the cell transfection efficiency of the 3 compounds is also quite different from that of YK-201, YK-202 and YK-209.
The transfection efficiency of YK-201 cell can reach 379.71 times of YK-223, 323.59 times of YK-224 and 493.98 times of YK-226 respectively.
The transfection efficiency of YK-202 cell can reach 399.37 times of YK-223, 340.35 times of YK-224 and 519.55 times of YK-226 respectively.
The transfection efficiency of YK-209 cell can reach 280.74 times of YK-223, 239.25 times of YK-224 and 365.22 times of YK-226 respectively.
YK-222 is also much different from YK-201, YK-202 and YK-209 in that the fluorescence absorption values of YK-201, YK-202 and YK-209 are 46.97 times, 49.41 times and 34.73 times of YK-222, respectively.
FIG. 7 is a graph showing fluorescence absorption of LNP formulations prepared from YK-201, YK-202, YK-221, and YK-225, showing that the fluorescence absorption of YK-221 and YK-225 is very weak compared to YK-201 and YK-202.
When the data are analyzed by GraphPad Prism software, any one of YK-201, YK-202 and YK-209 has obvious difference with other compounds, and the transfection efficiency is obviously improved.
b. Difference in chemical structure
The series of compounds have structures of YK-201, YK-202 and YK-209With little difference, only G3 The hydroxyl in the group is changed into methylamino, or the hydroxyl and the methyl connected with N are removed, and other positions are slightly changed; the structural differences between these compounds are also very small.
I. Structural difference from YK-201
In contrast to YK-201, YK-221 is G only3 In which the hydroxy group is changed to methylamino, G1 2 or less C, R1 1 less C, G2 2 or less C, R2 The group single chain has 1 more C, other structures are completely the same, but the cell transfection efficiency YK-201 reaches 1018.69 times of YK-221.
YK-225 is G only3 In which the hydroxy group is changed to methylamino, R1 The group being of branched structure, G2 2C, R less2 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-201 reaches 1089.41 times of YK-225.
YK-226 is G only3 The radicals having the hydroxy group and the methyl group bound to N removed, G2 Having 3 or fewer C, R groups2 The group is single-stranded and has 1C, other structures are completely the same, but the cell transfection efficiency YK-201 reaches 493.98 times of YK-226.
Structural distinction from YK-202
In contrast to YK-201, YK-221 is G only3 In which the hydroxy group is changed to methylamino, G1 2 or less C, R1 1 less C, G2 The gene has 2 less C, other structures are completely the same, but the cell transfection efficiency YK-202 reaches 1071.44 times of YK-221.
YK-225 is G only3 In which the hydroxy group is changed to methylamino, R1 The radicals being branched structures, G2 2 or less C, R2 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-202 reaches 1145.82 times of YK-225.
YK-226 is G only3 The radicals having the hydroxy group and the methyl group bound to N removed, G2 The gene is 3C less, other structures are identical, but the cell transfection efficiency YK-202 reaches 519.5 of YK-2265 times.
Structural distinction from YK-209
YK-221 is G only, as compared to YK-2093 In which the hydroxy group is changed to methylamino, G1 2C, R less1 The group is a branched chain structure, other structures are completely the same, but the cell transfection efficiency YK-209 reaches 753.17 times of YK-221.
YK-223 is G only3 The hydroxyl group in the group is changed into methylamino, other structures are completely the same, but the cell transfection efficiency YK-209 reaches 280.74 times of YK-223.
YK-225 is G only3 In which the hydroxy group is changed to methylamino, R1 And R2 The single chains of the group have more than 1C, each single chain in the double chains has less than 2C, other structures are completely the same, but the cell transfection efficiency YK-209 reaches 805.46 times of YK-225.
Furthermore, YK-221 is only G, as compared to YK-2073 The hydroxyl group in the group is changed into methylamino, other structures are completely the same, but the transfection efficiency of the cell YK-207 is 960.71 times of that of YK-221. Other compounds are also similar.
And (3) knotting:
from the above, with G3 YK-201, YK-202 and YK-209 were most efficiently transfected in a series of compounds in which the hydroxyl group was changed to a methylamino group or the hydroxyl group and the methyl group attached to N were removed, for example, YK-201 and YK-202 were more than 1000 times as high as YK-221 and YK-225.
Meanwhile, we found that the difference of cell transfection efficiency of compounds with similar structures can not be estimated at all according to the structure difference, and even a group of compounds with small difference in structure has very high possibility of having very large difference in cell transfection efficiency.
Therefore, it is very difficult to select a cationic lipid compound having a high transfection efficiency from a series of compounds having similar 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-201, YK-202 and YK-209.
This series of compounds was designed to differ significantly from the prior art representative cationic lipids, e.g., SM-102, compound 21, compound 23, HHMA and YK-009 chemical structure, G3 The groups are completely different, and other parts are also different, so that the polarity, the acid-base property, the hydrophilicity and the like are also greatly different.
2) The LNP preparation prepared by YK-201, YK-202 and YK-209 has the highest cell transfection efficiency, and is obviously improved compared with the activity of the representative cationic lipid in the prior art. For example, YK-202 can be up to 18 times that of SM-102, 21 times that of Compound 21, and 22 times that of Compound 23.
YK-201, YK-202 and YK-209 are most similar in the structure we have designed, only G1 、G2 、G3 、R1 Or R2 Cell transfection efficiency was highest in a series of compounds with groups differing by 1-2C. YK-202 may be up to 80 times that of other compounds, such as YK-204.
YK-201, YK-202 and YK-209 differ somewhat less than in individual radicals, e.g. G1 Reduction of 2C, R1 Decrease by 1C, G3 The transfection efficiency was highest compared with compounds in which ether bond, ester bond or sulfur atom was introduced. YK-202 may be more than 400 times higher than other compounds, such as YK-217.
YK-201, YK-202 and YK-209 with G only3 The compounds in which the hydroxyl group is changed to a methylamino group or the hydroxyl group is removed from the methyl group linked to N have the highest transfection efficiency. For example, YK-201 and YK-202 can both increase cell transfection efficiency by more than 1000-fold compared to YK-221 and YK-225.
3) There is no correlation between the structure of the compound and the intracellular transfection efficiency, and compounds with small structural differences also have a high possibility of very large differences 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 of 96-well plates, after 24 hours of continued culture, 10 μ L CCK-8 solution was added to each well, and after incubating the plates in the incubator for 1 hour, absorbance at 450nm was measured by a microplate reader. The results are shown in tables 10 to 13.
The experimental results are as follows:
(1) In a series of compounds designed, LNP formulations prepared from YK-201, YK-202 and YK-209 were significantly less cytotoxic than representative cationic lipids of the prior art. For example, YK-202 cell survival rate can be 26.85% higher than ALC-0315, 8.26% higher than SM-102, and 11.25% higher than HHMA.
TABLE 10 cell viability-1
a. Differentiation of cell viability
Table 10 lists the results of the LNP formulation cytotoxicity assays 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 10, LNP preparations of Fluc-mRNA prepared from YK-201, YK-202 and YK-209 were the least cytotoxic and had cell viability rates of 75.93%, 76.81% and 76.69%, respectively.
YK-201 is 7.38% higher than SM-102, 25.97% higher than ALC-0315, 6.91% higher than Compound 21, 5.83% higher than Compound 23, 10.37% higher than HHMA, and 52.88% higher than Lipofectamine 3000.
YK-202 is 8.26% higher than SM-102, 26.85% higher than ALC-0315, 7.79% higher than compound 21, 6.71% higher than compound 23, 11.25% higher than HHMA, and 53.76% higher than Lipofectamine 3000.
YK-209 was 8.14% higher than SM-102, 26.73% higher than ALC-0315, 7.67% higher than Compound 21, 6.59% higher than Compound 23, 11.13% higher than HHMA, and 53.64% higher than Lipofectamine 3000.
YK-206 and YK-207 cell survival rates were 74.57% and 73.88%, respectively, and both were higher than prior art cationic lipids, 6.02% and 5.33% higher than SM-102, 24.61% and 23.92% higher than ALC-0315, 5.55% and 4.86% higher than Compound 21, 4.47% and 3.78% higher than Compound 23, 9.01% and 8.32% higher than HHMA, and 51.52% and 50.83% higher than Lipofectamine 3000, respectively. (FIG. 8)
The data were analyzed using GraphPad Prism software, where any of YK-201, YK-202, and YK-209 was significantly different from SM-102, ALC-0315, compound 21, compound 23, and Lipofectamine 3000, with significantly reduced cytotoxicity.
b. Difference in chemical structure
YK-201, YK-202 and YK-209 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, the group of HHMA connected with the central N atom has only 1 side chain close to 1 side chain of the series of structures, and the other parts are completely different. G compared with SM-102, ALC-0315, compound 21, compound 23, and YK-0093 The groups are completely different; g1 、R1 、G2 And R2 The groups also differ significantly.
And (4) summarizing:
in a series of compounds, LNP preparations prepared from YK-201, YK-202 and YK-209 have the lowest cytotoxicity, and the survival rate of the LNP preparations is obviously improved compared with that of the representative cationic lipid in the prior art. For example, YK-202 cell survival rate can be 26.85% higher than ALC-0315, 8.26% higher than SM-102, and 11.25% higher than HHMA. YK-206 and YK-207 are also less cytotoxic than the representative cationic lipids of the prior art.
YK-201, YK-202 and YK-209 have significant differences in chemical structure, G, compared to the prior art representative cationic lipids3 Radicals completely different, G1 、R1 、G2 And R2 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-201, YK-202 and YK-209 are most similar in the structure we have designed, only G1 、G2 、G3 、R1 Or R2 Among the compounds with 1-2C difference in groups, cytotoxicity was the lowest. The cell survival rate of the three compounds can be improved by 40 percent compared with other compounds, such as YK-203.
To compare the differences in cytotoxicity of structurally similar compounds, we compared YK-201, YK-202 and YK-209 with the structurally closest compounds, which were G alone1 、G2 、G3 、R1 Or R2 The groups differ by 1-2C. The results show that the cytotoxicity of the series of compounds is very different. The survival rates of YK-201, YK-202 and YK-209 cells are the highest and reach 75.93%, 76.81% and 76.69% respectively. The three can be improved by 40% compared with other compounds, such as YK-203.
TABLE 11 cell viability-2
a. Differentiation of cell viability
As can be seen from Table 11, the LNP preparations prepared from these compounds have very different cytotoxicity, with YK-201, YK-202 and YK-209 having the lowest toxicity and the highest cell survival rate. The lowest cell viability was YK-203 and YK-210, which were only 35.56% and 38.38%.
YK-201 is 40.37% higher than YK-203 and 37.55% higher than YK-210.
YK-202 is 41.25% higher than YK-203 and 38.43% higher than YK-210.
YK-209 is 41.13% higher than YK-203 and 38.31% higher than YK-210. (FIG. 9)
YK-204, YK-205, YK-208 and YK-211, the cell survival rates were 42.57%, 58.92%, 46.68% and 52.30%, respectively.
YK-201 is 33.36% higher than YK-204, 17.01% higher than YK-205, 29.25% higher than YK-208, and 23.63% higher than YK-211.
YK-202 is 34.24% higher than YK-204, 17.89% higher than YK-205, 30.13% higher than YK-208, and 24.51% higher than YK-211.
YK-209 is 34.12% higher than YK-204, 17.77% higher than YK-205, 30.01% higher than YK-208, and 24.39% higher than YK-211.
The data were analyzed using GraphPad Prism software, where any of YK-201, YK-202, and YK-209 had a significant difference in cytotoxicity from the other compounds, with a significant reduction in cytotoxicity.
b. Difference in chemical structure
The structures of the series of compounds are very similar, and only the individual groups are slightly different. YK-201, YK-202 and YK-209 are very close to other compounds in structure; other compounds are also very similar.
And (3) knotting:
very close in structure, only G1 、G2 、G3 、R1 Or R2 In a group of compounds with 1-2C difference in groups, YK-201, YK-202 and YK-209 have the lowest cytotoxicity, and the cell survival rates of the three compounds are respectively improved by 40.37%, 41.25% and 41.13% compared with the lowest YK-003.
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 different cytotoxicity.
Therefore, it is very difficult to screen 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-201, YK-202 and YK-209 are structurally identical to G1 、G2 、G3 、R1 Or R2 The compounds with some minor differences in groups were the least cytotoxic. The cell survival rate of the three compounds can be improved by 50 percent compared with other compounds, such as YK-214.
We have found thatFurther, the differences in cytotoxicity of YK-201, YK-202 and YK-209 were compared with other structurally similar compounds. The compounds of this series differ only slightly in individual groups, e.g. G1 Reduction of 2C and R1 Decrease by 1C, G3 An ether bond, an ester bond or a sulfur atom is introduced. The results show that the series of compounds have very large cytotoxicity difference, the survival rates of YK-201, YK-202 and YK-209 are highest, and the cell survival rates of the three compounds can be improved by 50% compared with other compounds, such as YK-214.
TABLE 12 cell viability-3
a. Difference in cell viability
Although the other compounds are G alone, as compared with YK-201, YK-202 and YK-2091 、G2 、G3 、R1 Or R2 Some minor differences in groups, but the effect on cytotoxicity is very large. The cell survival rates of YK-201, YK-202 and YK-209 can be up to 50% higher than other compounds.
As can be seen from Table 12, the survival rate of YK-214 cells was the lowest, only 25.58%. YK-201 is 50.35% higher than YK-214, YK-202 is 51.23% higher than YK-214, and YK-209 is 51.11% higher than YK-214.
The cell survival rates of YK-212, YK-213, YK-215, YK-216 and YK-217 are 35.26%, 41.44%, 44.59%, 45.80% and 42.27%, respectively.
YK-201 is 40.67% higher than YK-212, 34.49% higher than YK-213, 31.34% higher than YK-215, 30.13% higher than YK-216, and 33.66% higher than YK-217.
YK-202 is 41.55 percent higher than YK-212, 35.37 percent higher than YK-213, 32.22 percent higher than YK-215, 31.01 percent higher than YK-216 and 34.54 percent higher than YK-217.
YK-209 is 41.43% higher than YK-212, 35.25% higher than YK-213, 32.10% higher than YK-215, 30.89% higher than YK-216, and 34.42% higher than YK-217.
YK-219 and YK-220 were higher at 58.61% and 58.99%, respectively.
YK-201 is 17.32% higher than YK-219, and YK-220 is 16.94% higher.
YK-202 is 18.20% higher than YK-219, and YK-220 is 17.82% higher.
YK-209 is 18.08% higher than YK-219 and YK-220 is 17.70% higher than YK-220.
The survival rate of YK-218 cells is 72.84 percent and is slightly lower than that of YK-201, YK-202 and YK-209. (FIG. 10)
The data were analyzed using GraphPad Prism software, where any one of YK-201, YK-202, and YK-209 had a significant difference in cytotoxicity from YK-212, YK-213, YK-214, YK-215, YK-216, YK-217, YK-219, and YK-220, and the cytotoxicity was significantly reduced.
b. Difference in chemical structure
The compounds of this series differ only slightly in individual groups, e.g. G1 Reduction of 2C and R1 Reduction of 1C, G3 An ether bond, an ester bond or a sulfur atom is introduced.
And (4) summarizing:
with some minor difference from the individual radicals only, e.g. G1 Reduction of 2C and R1 Reduction of 1C, G3 YK-201, YK-202 and YK-209 are the least cytotoxic compared to compounds incorporating ether linkages, ester linkages or sulfur atoms. For example, YK-201, YK-202 and YK-209 all showed 50% higher cell viability than YK-014.
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.
(4) YK-201, YK-202 and YK-209 are structurally identical to G3 The change of the hydroxyl group of the group to a methylamino group, or the removal of the hydroxyl group and the methyl group attached to N, results in significantly reduced cytotoxicity. For example, compared with YK-221, the cell survival rate of the three is improved by 55%.
The cytotoxicity results show that G in YK-201, YK-202 and YK-2093 The hydroxyl group of the group is changed into methylamino, or the hydroxyl group and the methyl group connected with the N are removed, and the cell survival rate is greatly reducedLow. For example, YK-201, YK-202, and YK-209 may be 55% higher than YK-221.
TABLE 13 cell viability-4
a. Differentiation of cell viability
As can be seen from Table 13, G for YK-201, YK-202 and YK-2093 Changing the hydroxyl group in the group to a methylamino group, or removing the hydroxyl group and the methyl group attached to N, results in a significant increase in cytotoxicity. For example, the cell survival rates of YK-201 and YK-202 are improved by 55 percent compared with that of YK-221. (FIG. 11)
Particularly, the cell survival rates of YK-221, YK-224 and YK-226 are the lowest and are all between 20% and 30%, and respectively are 20.95%, 23.47% and 27.90%.
YK-201 is 54.98% higher than YK-221, 52.46% higher than YK-224, and 48.03% higher than YK-226.
YK-202 is 55.86% higher than YK-221, 53.34% higher than YK-224, and 48.91% higher than YK-226.
YK-209 is 55.74% higher than YK-221, 53.22% higher than YK-224, and 48.79% higher than YK-226.
The cell viability for YK-222, YK-223 and YK-225 was 33.75%, 36.64% and 43.41%, respectively.
YK-201 is 42.18% higher than YK-222, 39.29% higher than YK-223, and 32.52% higher than YK-225.
YK-202 is 43.06% higher than YK-222, 40.17% higher than YK-223 and 33.40% higher than YK-225.
YK-209 is 42.94% higher than YK-222, 40.05% higher than YK-223 and 33.28% higher than YK-225.
The data were analyzed using GraphPad Prism software, where any of YK-201, YK-202, and YK-209 was significantly different from YK-221, YK-222, and YK-223, YK-224, YK-225, and YK-226, and cytotoxicity was significantly reduced.
b. Difference in chemical structure
The structural difference of the series of compounds is very small, and compared with YK-201, YK-202 and YK-209, other compounds are only G3 The hydroxyl group in the group is changed into a methylamino group, or the hydroxyl group and the methyl group connected with the N are removed,the other structures are identical.
And (3) knotting:
and G3 YK-201, YK-202 and YK-209 are the least cytotoxic compared to compounds in which the hydroxyl group in the group is changed to a methylamino group, or the hydroxyl group is removed and the methyl group attached to N is removed. For example, the cell survival rates of the three are respectively improved by more than 50 percent compared with that of YK-221 and YK-224.
At the same time, we have found that there is no correspondence between the structure of the compound and cytotoxicity, even if only G3 There are some differences in the group structure and there is a high probability that the cytotoxicity will vary greatly.
Therefore, it is very difficult to screen cationic lipid compounds with low cytotoxicity from a series of compounds with only a few minor differences among individual groups, and much creative work 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-201, YK-202 and YK-209.
This series of compounds was designed to differ significantly from the prior art representative cationic lipids, e.g., SM-102, compound 21, compound 23, HHMA and YK-009 chemical structure, G3 The groups are completely different, and other parts are also different, so that the polarity, the acid-base property, the hydrophilicity and the like are also greatly different.
2) LNP formulations prepared from YK-201, YK-202 and YK-209 were minimally cytotoxic and significantly improved in survival of the representative cationic lipid cells of the prior art. For example, YK-202 may be 26.85% higher than ALC-0315, 8.26% higher than SM-102, and 11.25% higher than HHMA.
YK-201, YK-202 and YK-209 are most similar in the structure we have designed, i.e. only G1 、G2 、R1 Or R2 Among the compounds with 1-2C difference in groups, cytotoxicity was the lowest. The cell survival rate of the three compounds is improved by 40 percent compared with that of other compounds, such as YK-203.
YK-201, YK-202 and YK-209 were the least cytotoxic than compounds that were structurally slightly different from the individual groups alone. The cell survival rate of the three compounds is improved by 50 percent compared with that of other compounds, such as YK-214.
YK-201, YK-202 and YK-209 are structurally identical to those of G alone3 Compounds in which the hydroxyl group of the group is changed to a methylamino group, or the hydroxyl group is removed and the methyl group attached to the N is the least cytotoxic. For example, the cell survival rates of YK-201, YK-202 and YK-209 are all improved by 55 percent compared with that of YK-221.
3) There is no correlation between the structure and cytotoxicity of compounds, and even compounds with small structural differences 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 validated 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 results are shown in tables 14 to 17 and FIGS. 12 to 14.
The experimental results are as follows:
(1) In a series of designed compounds, the LNP preparation prepared from YK-201, YK-202 and YK-209 has high and continuous mRNA expression in mice, and is obviously improved compared with the representative cationic lipid in the prior art. For example, YK-202 can be up to 24 times that of SM-102, 25 times that of Compound 21, and 23 times that of Compound 23. mRNA expression in mice is consistent with cell transfection activity.
TABLE 14 mouse in vivo imaging Experimental data-1
a. In vivo expression differentiation in mice
Table 14 lists the strength of mRNA expression at different times in mice for 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 can be seen from Table 14, LNP preparations containing Fluc-mRNA prepared from YK-201, YK-202 and YK-209 were highly expressed and continuously expressed in mice.
The mean radiation intensity of YK-201, at 6h, is 2858500, which is 4.21 times that of SM-102, 3.39 times that of ALC-0315, 4.76 times that of Compound 21, 4.81 times that of Compound 23, and 4.47 times that of HHMA; 2025680 at 24h, 17.49 times that of SM-102, 11.11 times that of ALC-0315, 18.76 times that of Compound 21, 18.27 times that of Compound 23, and 18.47 times that of HHMA; 730760 at 48h, which is 23.91 times that of SM-102, 19.16 times that of ALC-0315, 24.34 times that of compound 21, 22.78 times that of compound 23, and 24.82 times that of HHMA; 30151 at 7d, 5.11 times that of SM-102, 4.58 times that of ALC-0315, 4.94 times that of compound 21, 5.17 times that of compound 23, and 5.68 times that of HHMA.
YK-202 mean intensity of radiation, 2935500, at 6h, 4.32 times that of SM-102, 3.48 times that of ALC-0315, 4.89 times that of Compound 21, 4.94 times that of Compound 23, and 4.59 times that of HHMA; 2158840 at 24h, 18.64 times that of SM-102, 11.84 times that of ALC-0315, 19.99 times that of Compound 21, 19.47 times that of Compound 23, and 19.69 times that of HHMA; 755000 at 48h, 24.71 times that of SM-102, 19.80 times that of ALC-0315, 25.15 times that of Compound 21, 23.54 times that of Compound 23, and 25.64 times that of HHMA; 33524 at 7d, 5.68 times that of SM-102, 5.09 times that of ALC-0315, 5.49 times that of compound 21, 5.75 times that of compound 23, and 6.31 times that of HHMA.
The YK-209 average radiation intensity is 2524250 at 6h, which is 3.72 times that of SM-102, 3.00 times that of ALC-0315, 4.20 times that of compound 21, 4.25 times that of compound 23 and 3.95 times that of HHMA; 1869220 at 24h, 16.14 times that of SM-102, 10.25 times that of ALC-0315, 17.31 times that of Compound 21, 16.86 times that of Compound 23, and 17.05 times that of HHMA; 629080 at 48h, which is 20.59 times as great as SM-102, 16.50 times as great as ALC-0315, 20.95 times as great as Compound 21, 19.61 times as great as Compound 23, and 21.36 times as great as HHMA; 26361 at 7d, 4.47 times that of SM-102, 4.00 times that of ALC-0315, 4.32 times that of compound 21, 4.52 times that of compound 23, and 4.96 times that of HHMA.
The LNP preparation prepared from YK-206 and YK-207 also showed high and sustained expression of mRNA in mice. For example, YK-206 and YK-207 can each be 16 times as high as SM-102, compound 21, compound 23 and HHMA and 13 times as high as ALC-0315.
When the data are analyzed by GraphPad Prism software, any one of YK-201, YK-202 and YK-209 has obvious difference with SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-009 at each time, and the expression amount and the duration are obviously improved.
b. Difference in chemical structure
YK-201, YK-202 and YK-209 have very different chemical structures compared to prior art cationic lipids, such as SM-102, compound 21, compound 23, HHMA and YK-009. The structural difference with HHMA is the largest, except that 1 side chain connected with a central N atom of the HHMA is similar to 1 side chain of YK-201, YK-202 and YK-209, and other structures of the HHMA are completely different. G of YK-201, YK-202 and YK-209 compared to SM-102, ALC-0315, compound 21, compound 23 and YK-0093 Radicals completely different, G1 、R1 、G2 And R2 The groups also differ greatly.
And (3) knotting:
in a series of designed compounds, the expression level of mRNA of LNP preparations prepared from YK-201, YK-202 and YK-209 in mice is highest and is expressed continuously, and the expression level at 6h, 24h, 48h and 7d is obviously improved compared with that of the representative cationic lipid in the prior art. For example, YK-202 can be up to 24 times that of SM-102, 25 times that of compound 21, and 23 times that of compound 23. The expression of mRNA in mice was consistent with the results of the cell transfection experiments in example 7.
Also, YK-201, YK-202 and YK-209 are very different from the structures of the typical cationic lipids in the prior art, G3 Radicals completely different, G1 、R1 、G2 And R2 The groups also differ significantly.
Thus, rather than LNP formulations prepared only from compounds with similar structure to prior art cationic lipids, which are highly expressed in mice, LNP formulations prepared from compounds with significantly different structures are also highly likely to express mRNA with sustained expression.
(2) YK-201, YK-202 and YK-209 are most similar in the structure we have designed, i.e. G only1 、G2 、G3 、R1 Or R2 Among the compounds with 1-2C difference in groups, mRNA expression was highest and the duration was longest in mice. The expression level of YK-202 can reach more than 400 times of that of other compounds, such as YK-204. The mRNA was consistent with cell transfection activity in mice for in vivo expression.
For comparison, the most closely represented by the structure, i.e. G only1 、G2 、G3 、R1 Or R2 The delivery vectors prepared from compounds with 1-2C difference in groups, the difference in the expression intensity and duration of the delivered mRNA in mice, and YK-201, YK-202 and YK-209 were compared with YK-204. The results show that the expression of mRNA in LNP preparations prepared by the series of compounds is very different in mice, wherein the expression levels of YK-201, YK-202 and YK-209 are the highest, the duration is the longest, and the expression level of YK-202 can reach more than 400 times of that of YK-204.
TABLE 15 mouse in vivo imaging Experimental data-2
a. In vivo expression differentiation in mice
As can be seen from Table 15, the LNP preparations prepared from YK-201, YK-202 and YK-209 exhibited the highest expression level and duration of mRNA in mice among the compounds of the family with the closest structure but slightly different individual group structures.
YK-201 is 81.10 times of YK-204 in 6h, 387.32 times in 24h, 299.49 times in 48h and 39.99 times in 7 d.
YK-202 is 83.29 times of YK-204 in 6h, 412.78 times in 24h, 309.43 times in 48h and 44.46 times in 7 d.
YK-209 is 71.62 times of YK-204 at 6h, 357.40 times at 24h, 257.82 times at 48h and 34.96 times at 7 d.
When data are analyzed by GraphPad Prism software, any one of YK-201, YK-202 and YK-209 has obvious difference with other compounds at each time, and the expression amount and the duration are obviously improved.
b. Difference in chemical structure
The structural difference of the series of compounds is very small, namely G1 、G2 、G3 、R1 Or R2 The groups differ by 1-2C. For example, YK-204 is only G compared to YK-2013 The number of C connected with the group N is 1, and other structures are completely the same, but the expression quantity of mRNA, YK-201 can reach 380 times of YK-204. Other compounds are also similar.
And (3) knotting:
most similar to the structure, i.e. only G1 、G2 、G3 、R1 Or R2 Compared with compounds with 1-2C difference in groups, LNP preparations prepared from YK-201, YK-202 and YK-209 showed the highest mRNA expression intensity and the longest duration in mice. For example, YK-202 can be more than 400 times as high as YK-204 in 24 hours and still can be 40 times as high as YK-204 in 7 days. 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 the most similar is by structure, i.e.G alone1 、G2 、G3 、R1 Or R2 The radicals differing by 1-2The degree and duration of expression of mRNA in mice in LNP preparations prepared from a group of compounds C is also likely to vary greatly.
Therefore, it is very difficult to screen out a series of compounds with the most similar structures for cationic lipid compounds with high and sustained expression in animals, and much creative work is required.
(3) YK-201, YK-202 and YK-209 are structurally identical to those of G alone1 、G2 、G3 、R1 Or R2 The compounds with some minor differences in the groups expressed the highest amount of mRNA in mice and lasted the longest. The expression level of YK-202 can reach more than 800 times of other compounds, such as YK-217. mRNA expression in mice is consistent with cell transfection activity.
We further compared the difference in expression in mice of LNP preparations containing mRNA prepared from YK-201, YK-202, and YK-209 and other structurally similar compounds. The compounds of this series differ only slightly in individual groups, e.g. G1 Reduction of 2C, R1 Decrease by 1C, G3 An ether bond, an ester bond or a sulfur atom is introduced. The results show that YK-201, YK-202 and YK-209 have the highest expression level and the longest duration, and are obviously higher than other compounds. YK-202 can be improved by 800 times compared with YK-217.
TABLE 16 mouse in vivo imaging Experimental data-3
a. In vivo expression differentiation in mice
As can be seen from Table 16, in this series of compounds, the LNP preparations prepared from YK-201, YK-202 and YK-209 exhibited the highest expression level and duration of mRNA in mice.
The expression quantity of YK-201 is 56.53 times of YK-215 and 116.27 times of YK-217 in 6h, 297.24 times of YK-215 and 764.41 times of YK-217 in 24h, 200.76 times of YK-215 and 713.63 times of YK-217 in 48h, and 29.56 times of YK-215 and 57.65 times of YK-217 in 7 d.
The expression quantity of YK-202 is 58.06 times of YK-215 and 119.40 times of YK-217 in 6h, 316.78 times of YK-215 and 814.66 times of YK-217 in 24h, 207.42 times of YK-215 and 737.30 times of YK-217 in 48h, and 32.87 times of YK-215 and 64.10 times of YK-217 in 7 d.
The expression quantity of YK-209 is 49.92 times of YK-215 and 102.67 times of YK-217 in 6h, 274.28 times of YK-215 and 705.37 times of YK-217 in 24h, 172.82 times of YK-215 and 614.34 times of YK-217 in 48h, and 25.84 times of YK-215 and 50.40 times of YK-217 in 7 d.
When the data are analyzed by GraphPad Prism software, any one of YK-201, YK-202 and YK-209 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 individual groups, e.g. G1 Reduction of 2C and R1 Reduction of 1C, G3 An ether bond, an ester bond or a sulfur atom is introduced. In contrast to YK-201, YK-215 is G only3 The ether bond is introduced into the group, more than 1 methyl is connected with O, other structures are completely the same, but the mRNA expression quantity can reach 300 times of YK-215 by YK-201. Other compounds are also similar.
And (3) knotting:
very close to the structure, only G1 、G2 、G3 、R1 Or R2 In comparison to some compounds with minor differences in groups, LNP formulations prepared from YK-201, YK-202, and YK-209 expressed mRNA in mice with the highest intensity and the longest duration. For example, YK-202 can be 800 times as high as YK-217 at 24 hours and still 60 times as high at 7 days. The mRNA expression 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 there is only some minor difference in individual groups, e.g.G1 Reduction of 2C and R1 Reduction of 1C, G3 The degree and duration of expression of mRNA in mice are also likely to vary greatly in LNP formulations prepared from these compounds, incorporating ether linkages, ester linkages or sulfur atoms.
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.
(4) YK-201, YK-202 and YK-209 are structurally identical to G3 Compounds with slightly different groups expressed the highest amount of mRNA in mice and lasted the longest. The expression level of the three compounds can reach more than 1000 times of that of other compounds, such as YK-223. The mRNA was consistent with cell transfection activity in mice for in vivo expression.
The compounds listed in Table 16 differ from YK-201, YK-202 and YK-209 only in G3 The groups are slightly different, such as a change of hydroxyl to methylamino. The transfection result in the body of the mouse shows that the YK-201, YK-202 and YK-209 have the highest expression quantity and the longest duration, which are obviously higher than other compounds, and the three are all more than 1000 times higher than YK-223.
TABLE 17 mouse in vivo imaging Experimental data-4
a. In vivo expression differentiation in mice
As can be seen from Table 17, in this series of compounds, the LNP preparations prepared from YK-201, YK-202 and YK-209 exhibited the highest expression level and duration of mRNA in mice.
YK-201 can reach 276.05 times of YK-223 in 6h, 1134.20 times in 24h, 1077.82 times in 48h and 68.53 times in 7 d.
YK-202 can reach 283.49 times of YK-223 in 6h, 1208.76 times in 24h, 1113.57 times in 48h and 76.19 times in 7 d.
YK-209 can reach 243.77 times of YK-223 in 6h, 1046.60 times in 24h, 927.85 times in 48h and 59.91 times in 7 d.
When the data are analyzed by GraphPad Prism software, any one of YK-201, YK-202 and YK-209 has obvious difference with other compounds at each time, and the expression amount and the duration are obviously improved.
b. Difference in chemical structure
The chemical structure of the series of compounds is only G3 The groups are slightly different. For example, YK-223 is G alone, as compared to YK-2093 The groups are different, namely, hydroxyl in YK-209 is changed into methylamino, other structures are completely the same, but the expression level of YK-209 can reach more than 1000 times of YK-223. Other compounds are also similar.
And (3) knotting:
very close to the structure, only G3 Compared with compounds with slightly different groups, the LNP preparation prepared from YK-201, YK-202 and YK-209 has the highest mRNA expression strength in mice, the longest duration and the expression amount which can be more than 1000 times of that of YK-223. The mRNA expression 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 if the structures are very close, only G3 The change of hydroxyl group in the group into methyl amino group or the removal of hydroxyl group and methyl group connected with N, LNP preparation prepared by these compounds, the expression degree and duration of mRNA in mice are very different.
Therefore, it is very difficult to screen cationic lipid compounds with high and sustained expression in animals from a series of compounds with very close structures, and much creative work is required.
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-201, YK-202 and YK-209 for which mRNA has high and sustained expression in mice.
This series of compounds was designed to differ significantly from the typical cationic lipids of the prior art, e.g., SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-009 in chemical structure, G3 The groups are completely different, and other parts are also different, so that the polarity, the acid-base property, the hydrophilicity and the like are also greatly different.
2) The LNP preparation prepared from YK-201, YK-202 and YK-209 has high and continuous mRNA expression in mice, and is obviously improved compared with the representative cationic lipid in the prior art. For example, YK-202 can be up to 24 times that of SM-102, 25 times that of Compound 21, and 23 times that of Compound 23.
YK-201, YK-202 and YK-209 are most similar in the structure we have designed, only G1 、G2 、G3 、R1 Or R2 In a series of compounds with groups differing by 1-2C, mRNA was expressed in the highest amount and for the longest duration in mice. For example, YK-202 can be more than 400 times as high as YK-204 in 24 hours and still can be 40 times as high as YK-204 in 7 days.
YK-201, YK-202 and YK-209 are only G1 、G2 、G3 、R1 Or R2 In a series of compounds with slightly different groups, mRNA was expressed in the mice in the highest amount and for the longest duration. For example, YK-202 may be increased 800-fold over YK-217.
YK-201, YK-202 and YK-209 are only G3 Among a series of compounds in which the hydroxyl group in the group is changed to a methylamino group, or the hydroxyl group and the methyl group linked to N are removed, mRNA is expressed in the highest amount and for the longest duration in the mouse. For example, the expression level of mRNA of the three is improved by more than 1000 times compared with that of YK-223.
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 even with the cationic lipid compounds having little structural variation, there is a high probability that the mRNA in LNP formulations made 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 designed, including YK-201, YK-202 and YK-209, and YK-206 and YK-207, were significantly different in chemical structure from prior art cationic lipids, such as SM-102, ALC-0315, compound 21, compound 23, HHMA, and YK-009, G3 The groups are completely different, and other positions are also different, so that the polarity, the acid-base property, the hydrophilicity and the like of the modified starch are also greatly different.
In the series of designed compounds, compared with the representative cationic lipid in the prior art, the LNP preparation prepared from YK-201, YK-202 and YK-209 has the advantages that 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, and the expression quantity and the duration of mRNA in a mouse body are obviously improved. For example, the encapsulation efficiency YK-209 can be improved by 41 percent compared with the compound 23, the drug-loading concentration YK-209 can reach 2 times of that of the compound 23, and the total RNA concentration YK-201 can reach 1.5 times of that of the compound 21; the cell transfection efficiency YK-202 can reach 18 times of SM-102, 21 times of compound 21 and 22 times of compound 23; the cell survival rate YK-202 is 26.85 percent higher than ALC-0315, 8.26 percent higher than SM-102 and 11.25 percent higher than HHMA; the expression level of mRNA in mice can reach 24 times of that of SM-102, 25 times of that of compound 21 and 23 times of that of compound 23 by YK-202.
In a series of compounds with small difference in chemical structure, LNP preparations prepared from YK-201, YK-202 and YK-209 have remarkably improved cell transfection efficiency, remarkably reduced cytotoxicity and remarkably improved expression amount and duration of mRNA in mice compared with other compounds.
Compared with YK-201, YK-202 and YK-209, the series of compounds have the structure that only the individual groups have 1-2C differences or G differences1 Reduction of 2C, R1 Decrease by 1C, G3 Introducing ether linkages, ester linkages, or sulfur atoms, or G3 In which the hydroxy group is changed to methylamino, or G3 Hydroxyl and methyl connected with N are removed, but the transfection efficiency of YK-201 and YK-202 cells can reach more than 1000 times of YK-221 and YK-225, the cytotoxicity can be reduced by 55 percent compared with YK-221, and the expression quantity of mRNA in mice can reach more than 1000 times of YK-223.
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 likely to vary greatly in transfection efficiency and/or toxicity to cells, intracellular expression.
For example, YK-204 is G only, as compared to YK-2013 The gene is connected with N and has more than 1C, other structures are completely the same, but the cell transfection efficiency YK-201 is 78 times of YK-204, the toxicity to the transfected cells YK-201 is reduced by 30 percent compared with YK-204, and the expression of mRNA YK-201 in a mouse body can reach 380 times of YK-204; YK-225 is G only, in contrast to YK-2093 In which the hydroxy group is changed to methylamino, R1 And R2 The single chain of the group has 1C, each single chain of the double chain has 2 less C, and other structures are completely the same, but the cell transfection efficiency YK-209 is 800 times of YK-225, and the toxicity YK-209 to the transfected cells is reduced by 30 percent compared with YK-225.
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-201, YK-202, YK-209, YK-206 and YK-207, 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 achieves unexpected technical effects.