CN116535381A - Cationic lipid compound having five-membered ring acetal structure, composition containing same and use - Google Patents

Cationic lipid compound having five-membered ring acetal structure, composition containing same and use Download PDF

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CN116535381A
CN116535381A CN202310821280.1A CN202310821280A CN116535381A CN 116535381 A CN116535381 A CN 116535381A CN 202310821280 A CN202310821280 A CN 202310821280A CN 116535381 A CN116535381 A CN 116535381A
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compound
formula
composition
solvate
pharmaceutically acceptable
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CN116535381B (en
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张宏雷
潘晨
宋更申
高川
陈玺朝
吕小兵
靳立杰
马雨晴
李静
安然
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Beijing Youcare Kechuang Pharmaceutical Technology Co ltd
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Beijing Youcare Kechuang Pharmaceutical Technology Co ltd
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Abstract

The invention provides a cationic lipid compound with a five-membered ring acetal structure, which is a compound shown in a formula (I) or N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof. Also provided are compositions comprising the foregoing compounds and their use for delivering therapeutic or prophylactic agents.

Description

Cationic lipid compound having five-membered ring acetal structure, composition containing same and use
Technical Field
The invention belongs to the field of medicines. The invention particularly relates to a cationic lipid compound with a five-membered ring acetal structure, a composition containing the cationic lipid compound and application thereof.
Background
Efficient targeted delivery of biologically active substances such as small molecule drugs, polypeptides, proteins and nucleic acids, especially nucleic acids, is a persistent medical challenge. Nucleic acid therapeutics face significant challenges due to low cell permeability and high sensitivity to degradation by certain nucleic acid molecules, including RNA.
Compositions, liposomes and liposome complexes (lipoplex) containing cationic lipids have been demonstrated to be effective as transport vehicles for transporting biologically active substances such as small molecule drugs, polypeptides, proteins and nucleic acids into cells and/or intracellular compartments. These compositions generally comprise one or more "cationic" and/or amino (ionizable) lipids, including neutral lipids, structural lipids, and polymer conjugated lipids. Cationic and/or ionizable lipids include, for example, amine-containing lipids that can be readily protonated. While a variety of such lipid-containing nanoparticle compositions have been shown, safety, efficacy and specificity remain to be improved. Notably, the increased complexity of lipid nanoparticles (Lipid Nanoparticle, LNP) complicates their production and may increase their toxicity, a major concern that may limit their clinical use. For example, LNP siRNA particles (e.g., patsiran) require the prior use of steroids and antihistamines to eliminate unwanted immune responses (T. Coelho, D. Adams, A. Silva, et al, safety and efficacy of RNAi therapy for transthyretin amyloidosis, N Engl J Med, 369 (2013) 819-829.). Thus, there is a need to develop improved cationic lipid compounds, and compositions comprising the same, that facilitate the delivery of therapeutic and/or prophylactic agents, such as nucleic acids, to cells.
Disclosure of Invention
In one aspect, the present invention provides a novel cationic lipid compound which is a compound of formula (I)
Or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein
G 1 Is C 1~15 Alkylene or- (CH) 2 ) a C(O)O(CH 2 ) b -, wherein a and b are each an integer of 1 to 5;
G 2 is C 2~8 An alkylene group;
G 3 is C 1~4 An alkylene group;
R 1 is hydrogen atom, C 6~14 Straight chain alkyl or- (CH) 2 ) c C(O)O(CH 2 ) d CH 3 Wherein c is an integer from 3 to 12 and d is an integer from 1 to 4;
R 2 is hydrogen atom, C 6~14 Linear or branched alkyl;
R 3 is unsubstituted, hydrogen atom, C 6~14 Linear or branched alkyl;
R 4 is unsubstituted, hydrogen atom, C 6~14 Linear or branched alkyl;
R 5 is C 6~25 Linear or branched alkyl;
X 1 and X 2 Respectively a methine group or an oxygen atom.
For example, the compound of formula (I) is one having the following structure:
，
，
，
，
，
，
，
，
，
，
，
，
，
，
，
。
a further aspect of the invention provides a composition comprising a carrier comprising a cationic lipid comprising a compound of formula (I) according to any one of the preceding claims or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof.
In a further aspect the present invention provides the use of a compound of formula (I) as defined above, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a composition as defined above, in the manufacture of a nucleic acid medicament, a genetic vaccine, a small molecule medicament, a polypeptide or a protein medicament.
In a further aspect the present invention provides the use of a compound of formula (I) as defined above, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a composition as defined above, in the manufacture of a medicament for the treatment of a disease or condition in a mammal in need thereof.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following brief description of the drawings of the embodiments will make it apparent that the drawings in the following description relate only to some embodiments of the present invention and are not limiting of the present invention.
FIG. 1 shows the results of cell transfection experiments for LNP formulations of eGFP-mRNA prepared based on YK-702, YK-705, YK-712, YK-716, SM-102 and Compound 1, wherein: a is YK-702, b is YK-705, c is YK-712, d is YK-716, e is SM-102, and f is Compound 1.
FIG. 2 shows the fluorescence absorbance intensities of LNP preparations of Fluc-mRNA prepared from different cationic lipids (YK-702, YK-705, YK-712, YK-716, YK-701, YK-703, YK-704, YK-706, YK-707, YK-708, YK-709, YK-710, YK-711, YK-713, YK-714, YK-715, SM-102, MC3, HHMA, DLin-K-C2-DMA, compound 1 and Lipofectamine 3000).
FIG. 3 shows cell viability of LNP preparations of Fluc-mRNA prepared from different cationic lipids (YK-702, YK-705, YK-712, YK-716, YK-701, YK-703, YK-704, YK-706, YK-707, YK-708, YK-709, YK-710, YK-711, YK-713, YK-714, YK-715, SM-102, MC3, HHMA, DLin-K-C2-DMA, compound 1 and Lipofectamine 3000) after addition to cell culture broth for 24 h.
FIG. 4 shows fluorescence images of mouse organs (heart, liver, spleen, lung, kidney) of LNP preparations of Fluc-mRNA prepared based on YK-704, YK-712, YK-716 and SM-102.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without creative efforts, based on the described embodiments of the present invention fall within the protection scope of the present invention.
The present invention may be embodied in other specific forms without departing from its essential attributes. It is to be understood that any and all embodiments of the invention may be combined with any other embodiment or features of multiple other embodiments to yield yet further embodiments without conflict. The invention includes additional embodiments resulting from such combinations.
All publications and patents mentioned in this application are herein incorporated by reference in their entirety. If a use or term in any of the publications and patents incorporated by reference conflicts with the use or term in the present application, the use or term in the present application shall govern.
The section headings used herein are for purposes of organizing articles only and should not be construed as limiting the subject matter.
Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning in the art to which the claimed subject matter belongs. In case there are multiple definitions for a term, the definitions herein control.
Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of quantitative 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-range.
The use of the terms "comprising," "including," or "containing," and the like, in this application, are intended to cover an element listed after that term and its equivalents, without excluding unrecited elements. The terms "comprising" or "including" as used herein, can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….
The term "pharmaceutically acceptable" in this application means: the compound or composition is chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or with the human or mammal with which the disease or condition is to be prevented or treated.
The term "subject" or "patient" includes humans and mammals in this application.
The term "treatment" as used herein refers to the administration of one or more pharmaceutical substances to a patient or subject suffering from or having symptoms of a disease, to cure, alleviate, ameliorate or otherwise affect the disease or symptoms of the disease. In the context of the present application, the term "treatment" may also include prophylaxis, unless specifically stated to the contrary.
The term "solvate" in this application refers to a complex formed by combining a compound of formula (I) or a pharmaceutically acceptable salt thereof and a solvent (e.g. ethanol or water). It will be appreciated that any solvate of the compound of formula (I) used in the treatment of a disease or condition, although potentially providing different properties (including pharmacokinetic properties), will result in the compound of formula (I) once absorbed into a subject, such that the use of the compound of formula (I) encompasses the 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 salt" refers to the relatively non-toxic, inorganic or organic acid addition salts of the compounds of the present invention. See, for example, s.m. Berge et al, "Pharmaceutical Salts",J. Pharm. Sci. 1977, 66, 1-19. Among them, inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid, etc.; organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) -benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectate acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, and the like. For example, HCl (or hydrochloric acid), HBr (or hydrobromic acid solution), methanesulfonic acid, sulfuric acid, tartaric acid, or fumaric acid may be used to form pharmaceutically acceptable salts with the compounds of formula (I).
The nitrogen-containing compounds of formula (I) of the present invention may be converted to N-oxides by treatment with an oxidizing agent (e.g., m-chloroperoxybenzoic acid, hydrogen peroxide, ozone). Thus, the compounds claimed herein include not only nitrogen-containing compounds of the formula but also N-oxide derivatives thereof, as valence and structure permit.
Certain compounds of the invention may exist in the form of one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers and enantiomers. Thus, the presently claimed compounds also include racemic mixtures, single stereoisomers, and optically active mixtures. It will be appreciated by those skilled in the art that one stereoisomer may have better efficacy and/or lower side effects than the other stereoisomers. The single stereoisomers and the mixture with optical activity can be obtained by chiral source synthesis methods, chiral catalysis methods, chiral resolution methods and the like. The racemate can be chiral resolved by chromatographic resolution or chemical resolution. For example, separation can be performed by adding chiral acid resolving agents such as chiral tartaric acid and chiral malic acid to form salts with the compounds of the present invention, and utilizing differences in physicochemical properties such as 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 found 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, respectively, for example 2 H (deuterium), 3 H (tritium), 11 C、 13 C、 14 C、 15 N、 17 O and 18 O。
the term "alkyl" is meant herein to include both branched and straight chain saturated aliphatic monovalent hydrocarbon groups having the specified number of carbon atoms. The term "alkylene" is meant herein to include both branched and straight chain saturated aliphatic divalent hydrocarbon groups having the specified number of carbon atoms. C (C) n~m Is meant to include groups having a number of carbon atoms from n to m. For example C 2~5 Alkylene group includes C 2 Alkylene, C 3 Alkylene, C 4 Alkylene, C 5 An alkylene group.
The alkyl (or alkylene) group may be unsubstituted, or the alkyl (or alkylene) group may be substituted, wherein at least one hydrogen is replaced with another chemical group.
A "therapeutically effective amount" is an amount of a therapeutic agent that, when administered to a patient, ameliorates a disease or condition. A "prophylactically effective amount" is an amount of a prophylactic agent that, when administered to a subject, prevents a disease or condition. The amount of therapeutic agent constituting the "therapeutically effective amount" or the amount of prophylactic agent of the "prophylactically effective amount" varies with the therapeutic agent/prophylactic agent, the disease state and severity thereof, the age, weight, etc. of the patient/subject to be treated/prevented. One of ordinary skill in the art can routinely determine therapeutically effective and prophylactically effective amounts based on their knowledge and the present application.
In the present application, when the names of the compounds are not identical to the structural formulae, the structural formulae are subject.
It is to be understood that the term "compounds of the invention" as used herein may include, depending on the context: a compound of formula (I), an N-oxide thereof, a solvate thereof, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, and mixtures thereof.
The term cationic lipid as used herein refers to a lipid that is positively charged at a selected pH.
Cationic liposomes readily bind to negatively charged nucleic acids, i.e., interact with negatively charged phosphate groups present in the nucleic acids by electrostatic forces, forming Lipid Nanoparticles (LNPs). LNP is one of the currently mainstream delivery vehicles.
The inventors found that, when screening a large number of compounds, it is very difficult to screen out suitable cationic lipid compounds that meet the following conditions: unlike the prior art, the cationic lipid has a typical cationic lipid structure, has extremely high transfection efficiency and extremely low cytotoxicity, and has high and sustained expression in mice. The inventors have found that certain compounds, such as YK-702, YK-705, YK-711, YK-716, and the like, can significantly improve intracellular transfection efficiency, significantly reduce cytotoxicity, significantly improve expression in the spleen of animals, and improve delivery efficiency compared to cationic lipids of which chemical structures are widely different or less different in the prior art.
Briefly, the present invention is based on at least the following findings:
the cationic lipid compounds of the present invention are useful for delivering nucleic acid molecules, small molecule compounds, polypeptides or proteins. Compared with the known cationic lipid compounds, the cationic lipid compounds of the invention have higher transfection efficiency and smaller cytotoxicity, remarkably improve the expression quantity of animal spleens, improve the delivery efficiency and have important clinical significance.
1. A series of cationic lipid compounds were designed, including YK-702, YK-705, YK-712 and YK-716, with tremendous differences in chemical structure compared to the prior art representative cationic lipids, such as SM-102, DLin-MC3-DMA (MC 3) and HHMA; there are structural similarities, for example DLin-K-C2-DMA and Compound 1.
SM-102 is a cationic lipid compound disclosed in WO20170409245A2 (page 29 of the specification) by morgana corporation (Moderna, inc.).
DLin-MC3-DMA (MC 3) is a cationic lipid compound disclosed in CN102625696B (page 6 of the specification) by the company Arilam pharmaceutical (Alnylam Pharmaceuticals, inc.).
HHMA is a cationic lipid compound disclosed in CN112979483B (page 7 of the specification) by Emblica Biotechnology, st.Job.
DLin-K-C2-DMA is a cationic lipid compound disclosed in CN102245590B (page 4 of the specification) by Tami drawing medicine Co.
Compound 1 is a cationic lipid compound disclosed in CN113039174a (page 4 of the specification) by intel-gerbera therapeutic stock.
Representative cationic lipids and structurally similar cationic lipids of the prior art have the following chemical structures:
(WO 2017049245A2, page 29);
(CN 102625696B, page 6 of the specification, compound of formula I);
(CN 112979483B, page 12 of the specification);
(CN 102245590B, page 4 of the specification);
(CN 113039174A, page 4 of the specification).
2. Of this series of compounds designed, LNP formulations prepared from YK-702, YK-705, YK-712 and YK-716 showed significantly improved cell transfection efficiency, significantly reduced cytotoxicity and significantly improved mRNA expression in mouse liver and spleen compared to the cationic lipids typical in the prior art (whether of widely structurally different cationic lipids such as SM-102, MC3 and HHMA, or of structurally similar cationic lipids such as DLin-K-C2-DMA and Compound 1). For example, cell transfection efficiency YK-716 can be up to 6.73 times that of SM-102, 70.91 times that of MC3, 10.10 times that of HHMA, 16.84 times that of DLin-K-C2-DMA, 9.54 times that of Compound 1, and 12.41 times that of Lipofectamine 3000; cell viability YK-705 was 8% higher than SM-102, 14% higher than MC3, 24% higher than HHMA, 21% higher than DLin-K-C2-DMA, 14% higher than Compound 1, 61% higher than Lipofectamine 3000; mRNA was expressed in liver and spleen of mice in 6.34-fold and 6.79-fold amounts of SM-102, YK-716, respectively.
3. In a series of compounds designed by the application, the chemical structure difference is small, LNP preparations prepared from YK-702, YK-705, YK-712 and YK-716 have remarkably improved cell transfection efficiency, remarkably reduced cytotoxicity and remarkably improved expression level of mRNA in liver and spleen of mice compared with other compounds. For example, YK-716 cells can be transfected with 418.45 times of YK-703 and 468.88 times of YK-715, cytotoxicity can be reduced by 43% compared with YK-701, and mRNA expression in liver and spleen of mice can be 242.76 times and 325.87 times of YK-704.
4. Through unique design and screening, the invention discovers that certain compounds, such as YK-702, YK-705, YK-712 and YK-716, can be delivered with significantly improved cell transfection efficiency, significantly reduced cytotoxicity and significantly improved expression levels in the liver and spleen of animals, with improved delivery efficiency, and unexpected technical results over the cationic lipids typical of the prior art (whether of greatly structurally different cationic lipids such as SM-102, MC3 and HHMA, or of structurally similar cationic lipids such as DLin-K-C2-DMA and Compound 1).
In summary, the present invention, through unique design and screening, has discovered compounds such as YK-702, YK-705, YK-712 and YK-716. These compounds have significantly improved cell transfection efficiency, significantly reduced cytotoxicity, and significantly improved expression in animal liver and spleen compared to the cationic lipids represented by the prior art, whether they are cationic lipids of greatly different structures such as SM-102, MC3 and HHMA, or similar structures such as DLin-K-C2-DMA and compound 1.
The method comprises the following steps:
1. compounds contemplated herein, including YK-702, YK-705, YK-712, and YK-716, have chemical structures that differ significantly from the prior art cationic lipids, such as SM-102, MC3, and HHMA; there are minor differences, such as DLin-K-C2-DMA and Compound 1.
1) Compared with the typical cationic lipids SM-102, MC3 and HHMA in the prior art, the chemical structure of the compound designed by the application is completely different and has great difference. SM-102, MC3 and HHMA have no acetal structure, and the compounds designed by the application all have five-membered ring acetal structures.
2) The chemical structure of the compounds contemplated herein is less different than prior art cationic lipids comprising acetal structures, such as DLin-K-C2-DMA and compound 1. DLin-K-C2-DMA and the compound of the application all contain five-membered ring acetal structures, and other structures are slightly different; the acetal structure of compound 1 is a common branched structure, but both compound 1 and the compounds contemplated herein contain a hydroxyethylethylene tertiary amine structure.
2. The transfection efficiency of cells in vitro is obviously improved compared with that of the representative cationic lipid compounds in the prior art (whether the structural differences are large or the structures are similar).
1) Of the designed series of compounds, LNP preparations prepared from YK-702, YK-705, YK-712 and YK-716 have the highest cell transfection efficiency, and compared with the representative cationic lipids in the prior art, the LNP preparations have the advantages of greatly different structures (such as SM-102, MC3 and HHMA) or very small structure difference (such as DLin-K-C2-DMA and compound 1), and the cell transfection efficiency is remarkably improved. For example, YK-716 can be up to 6.73 times SM-102, 70.91 times MC3, 10.10 times HHMA, 16.84 times DLin-K-C2-DMA, 9.54 times Compound 1, and 12.41 times Lipofectamine 3000.
2) Will be of similar structure, X 1 =O、X 2 =CH、R 3 =no substituent, R 4 A series of compounds of the formula YK-701, YK-703, YK-704, YK-713, YK-714 and YK-715, which differ structurally only slightly from the individual groups compared to YK-702, YK-705 and YK-712, are shown. Cell transfection results show that the activity of the series of compounds is very different, and the cell transfection efficiency of YK-702, YK-705 and YK-712 is highest. The cell transfection efficiency of YK-702 can reach 19.89 times of YK-701, 225.35 times of YK-703, 157.50 times of YK-704, 103.88 times of YK-713, 119.84 times of YK-714 and 252.51 times of YK-715 respectively. The cell transfection efficiency of YK-705 can reach 23.29 times of YK-701, 263.81 times of YK-703, 184.38 times of YK-704, 121.61 times of YK-713, 140.29 times of YK-714 and 295.60 times of YK-715 respectively. The cell transfection efficiency of YK-712 can reach 33.93 times of YK-701, 384.32 times of YK-703, 268.60 times of YK-704, 177.16 times of YK-713, 204.37 times of YK-714 and 430.63 times of YK-715 respectively, and the transfection efficiency is obviously improved.
3) Similar to the structure, X 1 =O、X 2 =CH、R 2 =H、R 3 =no substituent, R 4 = C 8 Compounds YK-708, YK-709 and YK as straight-chain alkyl groupsCompared with the YK-710 and the YK-711, the transfection efficiency of the YK-716 cells is obviously improved, and the transfection efficiency can reach 5.63 times, 11.97 times, 7.03 times and 44.48 times of YK-708, YK-709, YK-710 and YK-711 respectively.
4) Similar to the structure, X 1 =CH、X 2 =O、R 1 =C 8 Straight chain alkyl or- (CH) 2 ) 7 C(O)OCH 2 CH 3 、R 2 =H、R 3 =C 8 Straight chain alkyl, R 4 Compared with YK-706 and YK-707, the transfection efficiency of YK-716 cells is obviously improved, and the transfection efficiency can reach 8.04 times and 3.92 times of YK-706 and YK-707 respectively.
3. Cytotoxicity is significantly reduced compared to the typical cationic lipids of the prior art (whether of widely differing or similar structure).
1) The chemical structures of the series of compounds contemplated herein, including YK-702, YK-705, YK-712, and YK-716, differ greatly from the prior art cationic lipids, such as SM-102, MC3, and HHMA; there are structural similarities, for example DLin-K-C2-DMA and Compound 1. LNP formulations prepared from YK-705 and YK-716 were minimally cytotoxic and significantly improved in cell viability compared to the typical cationic lipid compounds of the prior art (whether of widely differing or similar structure). For example, YK-705 cell viability was 14% higher than MC3, 24% higher than HHMA, 21% higher than DLin-K-C2-DMA, 14% higher than Compound 1, 61% higher than Lipofectamine 3000. The cytotoxicity of LNP formulations prepared therefrom cannot be speculated on the basis of the structure of cationic lipid compounds, and there is a strong possibility that the cytotoxicity to transfected cells is very different, whether they are structurally different or structurally similar compounds.
2) Similar to the structure, X 1 =O、X 2 =CH、R 3 =no substituent, R 4 A series of compounds of the formula H, for example YK-701, YK-702, YK-703, YK-704, YK-713, YK-714 and YK-715, which differ structurally only slightly from YK-705 in their individual groups, differ very much in their cytotoxicity, with the highest cell viability of YK-705 being 44% higher than YK-701, 10% higher than YK-702 and than YK-7, respectively03 is 24% higher than YK-704, 8% higher than YK-713, 11% higher than YK-715, and the cell survival rate is remarkably improved.
3) Similar to the structure, X 1 =O、X 2 =CH、R 2 =H、R 3 =no substituent, R 4 = C 8 Compared with YK-708, YK-709, YK-710 and YK-711, the linear alkyl compounds have significantly improved YK-716 cell survival rate, which can be 15% higher than YK-708, 8% higher than YK-709, 23% higher than YK-710 and 10% higher than YK-711, respectively.
4) Similar to the structure, X 1 =CH、X 2 =O、R 1 =C 8 Straight chain alkyl or- (CH) 2 ) 7 C(O)OCH 2 CH 3 、R 2 =H、R 3 =C 8 Straight chain alkyl, R 4 Compared with YK-706 and YK-707, the transfection efficiency of YK-705 and YK-716 cells is obviously improved by 24% and 23% respectively higher than that of YK-706 and 31% and 30% higher than that of YK-707.
mRNA expression level in animal spleen is obviously raised compared with that of cationic lipid in available technology.
1) Compared with the representative cationic lipid in the prior art, the LNP preparation prepared by YK-712 and YK-716 has obviously improved expression level of mRNA in the liver and spleen of mice, especially in the spleen. For example, YK-712 and YK-716 can be expressed in spleen up to 6.52-fold and 6.79-fold, respectively, of SM-102. mRNA was consistent with the results of cell transfection in example 6 in terms of mouse liver and spleen expression.
2) LNP preparations prepared from YK-712 and YK-716 showed the highest expression intensity of mRNA in the liver and spleen of mice compared to the compound YK-704, which was similar in structure and slightly different in individual groups. For example, YK-712 can be expressed in liver and spleen in 237.23-fold and 312.86-fold, respectively, and YK-716 can be expressed in liver and spleen in 242.76-fold and 325.87-fold, respectively, of YK-704. mRNA was consistent with cell transfection activity in terms of expression in mice.
In one aspect, the present invention provides a novel cationic lipid compound for delivering a therapeutic or prophylactic agent. The cationic lipid compounds of the present invention are useful for delivering nucleic acid molecules, small molecule compounds, polypeptides or proteins. Compared with the known cationic lipid compounds, the cationic lipid compounds of the invention show higher transfection efficiency and lower cytotoxicity, and improve delivery efficiency and safety.
In one aspect, the present invention provides a novel cationic lipid compound which is a compound of formula (I)
Or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein:
G 1 is C 1~15 Alkylene or- (CH) 2 ) a C(O)O(CH 2 ) b -, wherein a and b are each an integer of 1 to 5;
G 2 is C 2~8 An alkylene group;
G 3 Is C 1~4 An alkylene group;
R 1 is hydrogen atom, C 6~14 Straight chain alkyl or- (CH) 2 ) c C(O)O(CH 2 ) d CH 3 Wherein c is an integer from 3 to 12 and d is an integer from 1 to 4;
R 2 is hydrogen atom, C 6~14 Linear or branched alkyl;
R 3 is unsubstituted, hydrogen atom, C 6~14 Linear or branched alkyl;
R 4 is unsubstituted, hydrogen atom, C 6~14 Linear or branched alkyl;
R 5 is C 6~25 Linear or branched alkyl;
X 1 and X 2 Respectively a methine group or an oxygen atom.
In one embodiment, G 1 Is unsubstituted C 1~12 Alkylene groups, e.g. G 1 Is unsubstituted C 1 Alkylene, C 5 Alkylene, C 8 Alkylene, C 4 Alkylene or C 12 An alkylene group. In another embodiment, G 1 Is- (CH) 2 ) 3 C(O)OCH 2 -。
In one embodiment，G 2 Is C 3~7 Alkylene groups, e.g. G 2 Is C 5 Alkylene or C 7 An alkylene group.
In one embodiment, G 3 Is C 2~3 Alkylene groups, e.g. G 3 Is C 2 An alkylene group.
In one embodiment, R 1 Is C 5-13 Straight-chain alkyl radicals, e.g. R 1 Is C 10 、C 8 、C 7 、C 9 Or C 13 A linear alkyl group. In another embodiment, R 1 Is- (CH) 2 ) 11 C(O)OCH 2 CH 3 Or- (CH) 2 ) 7 C(O)OCH 2 CH 3 。
In one embodiment, R 2 Is a hydrogen atom. In another embodiment, R 2 Is C 10 、C 8 、C 7 Or C 9 A linear alkyl group.
In one embodiment, R 3 Is unsubstituted. In another embodiment, R 3 Is C 7~12 Straight-chain alkyl groups, e.g. R 3 Is C 8 A linear alkyl group.
In one embodiment, R 4 Is a hydrogen atom. In another embodiment, R 4 Is C 7~12 Straight-chain alkyl groups, e.g. R 4 Is C 8 A linear alkyl group. In another embodiment, R 4 Is unsubstituted.
In one embodiment, R 5 Is C 6~15 Straight-chain alkyl groups, e.g. R 5 Is C 11 Or C 10 A linear alkyl group. In another embodiment, R 5 Is C 10~22 Branched alkyl groups, e.g. R 5 Is C 17 Branched alkyl, in particular R 5 Is that。
In one embodiment, X 1 Is a methine group. In another embodiment, X 1 Is an oxygen atom.
In a kind ofIn an embodiment, X 2 Is a methine group. In another embodiment, X 2 Is an oxygen atom.
In exemplary embodiments, the compound is selected from the following compounds or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof:
table 1: comparison of chemical structures of cationic lipids designed according to the present invention
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In yet another aspect, the present invention provides a composition comprising a carrier comprising a cationic lipid comprising a compound of formula (I) as described above or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof.
In one embodiment, the composition is a nanoparticle formulation having an average size of 10nm to 300nm, preferably 90nm to 260nm; the nanoparticle formulation has a polydispersity of 50% or less, preferably 40% or less, more preferably 30% or less.
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) 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 the group consisting of those described above. For example, the cationic lipid is a compound YK-702, YK-705, YK-712 or YK-716. In a preferred embodiment, the cationic lipid is compound YK-702, in a preferred embodiment, the cationic lipid is compound YK-705, in another preferred embodiment, the cationic lipid is compound YK-712, and in another preferred embodiment, the cationic lipid is compound YK-716.
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) 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 in CN102625696B, DLin-K-C2-DMA in CN102245590B, or compound 1 in CN 113039174A.
In one embodiment, the cationic lipid comprises 25% -75% of the carrier by mole, for example 30%, 40%, 50%, 55%, 60%, 65%, 70%.
The carrier may be used to deliver an active ingredient such as a therapeutic or prophylactic agent. The active ingredient may be enclosed within a carrier or may be combined with a carrier.
For example, the therapeutic or prophylactic agent includes one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein. Such nucleic acids include, but are not limited to, single-stranded DNA, double-stranded DNA, and RNA. Suitable RNAs include, but are not limited to, small interfering RNAs (sirnas), asymmetric interfering RNAs (airnas), micrornas (mirnas), dicer-substrate RNAs (dsRNA), small hairpin RNAs (shrnas), messenger RNAs (mrnas), and mixtures thereof.
Neutral lipids
The carrier may comprise neutral lipids. Neutral lipids in the context of the present invention are lipids that are non-charged at a selected pH or that act as a helper in zwitterionic form. The neutral lipids may modulate nanoparticle mobility into lipid bilayer structures and increase efficiency by promoting lipid phase changes, while also potentially affecting target organ specificity.
In one embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 1:1 to 15:1, such as about 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, and 2:1. In a preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 4:1.
For example, the neutral lipids may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramides, sterols, and derivatives thereof.
The carrier component of the cationic lipid-containing composition may comprise one or more neutral lipid-phospholipids, such as one or more (poly) unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids may include a phospholipid moiety and one or more fatty acid moieties.
The neutral lipid moiety may be selected from the non-limiting group consisting of: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from the non-limiting group consisting of: lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid and docosahexaenoic acid. Non-natural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, the phospholipid may be functionalized with or crosslinked with one or more alkynes (e.g., alkenyl groups with one or more double bonds replaced with triple bonds). Under appropriate reaction conditions, alkynyl groups may undergo copper-catalyzed cycloaddition reactions upon exposure to azide. These reactions can be used to functionalize the lipid bilayer of the composition to facilitate membrane permeation or cell recognition, or to couple the composition with a useful component such as a targeting or imaging moiety (e.g., dye).
Neutral lipids useful in these compositions may be selected from the non-limiting group consisting of: 1, 2-Dioleoyl-sn-glycero-3-phosphorylcholine (DLPC), 1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dioleoyl-sn-glycero-phosphorylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-octadecenyl-sn-glycero-3-phosphorylcholine (18:0 DietherPC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-3-phosphorylcholine (OChems PC), 1-hexadecyl-sn-3-phosphorylcholine (C16), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1-dioleoyl-2-dioleoyl-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-sn-glycero-3-phosphate ethanolamine (DOPE), 1, 2-di-phytanoyl-sn-glycero-3-phosphate ethanolamine (ME 16.0 PE), 1, 2-di-stearoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-oleoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-linolenoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-arachidonoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-docosahexaenoic acyl-sn-glycero-3-phosphate ethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphate sodium salt (DOPG), 1, 2-di-oleoyl-rac-3-phosphate sodium salt (DOPG) dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl-based phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof.
In some embodiments, the neutral lipid comprises DSPC. In certain embodiments, the neutral lipid comprises DOPE. In some embodiments, the neutral lipid comprises both DSPC and DOPE.
Structured lipids
The carrier of the composition comprising the cationic lipid may also comprise one or more structural lipids. Structured lipids in the present invention refer to lipids that enhance the stability of the nanoparticle by filling the interstices between the lipids.
In one embodiment, the molar ratio of the cationic lipid to the structural lipid is about 1:1-5:1, e.g., about 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1.
The structural lipid may be selected from, but is not limited to, the group consisting of: cholesterol, non-sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycorine, ursolic acid, alpha-tocopherols, corticosteroids, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipids include cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or combinations thereof.
Polymer conjugated lipids
The carrier of the composition comprising the cationic lipid may also comprise one or more polymer conjugated lipids. The polymer conjugated lipid mainly refers to polyethylene glycol (PEG) modified lipid. Hydrophilic PEG stabilizes LNP, regulates nanoparticle size by limiting lipid fusion, and increases nanoparticle half-life by reducing non-specific interactions with macrophages.
In one embodiment, the polymer conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol. The PEG modified PEG molecular weight is typically 350-5000Da.
For example, the polymer conjugated lipid is selected from one or more of the following: distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG 2000), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000) and methoxypolyethylene glycol ditetradecylamide (ALC-0159).
In one embodiment of the composition/carrier of the present invention, the polymer conjugated lipid is DMG-PEG2000.
In one embodiment of the composition/carrier of the present invention, the carrier comprises neutral lipid, structural lipid and polymer conjugated lipid, and the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the polymer conjugated lipid is (25-75): (5-25): (15-65): (0.5-10), for example (30-49): (7.5-15): (35-55): (1-5).
In one embodiment of the composition/carrier of the invention, the carrier comprises neutral lipids, structural lipids and polymer conjugated lipids, the molar ratio of the cationic lipids, the neutral lipids, the structural lipids and the polymer conjugated lipids being 40:10:48.5:1.5 or 49:10:39.5:1.5.
Therapeutic and/or prophylactic agent
The composition may include one or more therapeutic and/or prophylactic agents. In one embodiment, the mass ratio of carrier to the therapeutic or prophylactic agent is 10:1 to 30:1, e.g., 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1.
In one embodiment, the mass ratio of carrier to the therapeutic or prophylactic agent is 12.5:1 to 20:1, preferably 15:1.
The therapeutic or prophylactic agent includes, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.
For example, the therapeutic or prophylactic agent is a vaccine or compound capable of eliciting an immune response.
The vectors of the present invention can deliver therapeutic and/or prophylactic agents to mammalian cells or organs, and thus the present invention also provides methods of treating a disease or disorder in a mammal in need thereof, comprising administering to the mammal a composition comprising a therapeutic and/or prophylactic agent and/or contacting mammalian cells with the composition.
Therapeutic and/or prophylactic agents include bioactive substances and are alternatively referred to as "active agents". The therapeutic and/or prophylactic agent can be a substance that, upon delivery to a cell or organ, causes a desired change in the cell or organ or other body tissue or system. Such species may be used to treat one or more diseases, disorders or conditions. In some embodiments, the therapeutic and/or prophylactic agent is a small molecule drug that can be used to treat a particular disease, disorder, or condition. Examples of drugs that may be used in the composition include, but are not limited to, antineoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate and streptozotocin), antineoplastic agents (e.g., dactinomycin D (actinomycin D), vincristine, vinblastine, cytosine arabinoside cytosine arabinoside, anthracycline (anthracycline), alkylating agents, platinum compounds, antimetabolites and nucleoside analogs such as methotrexate and purine and pyrimidine analogs, anti-infective agents, local anesthetics (e.g., dibucaine) and chlorpromazine, beta-adrenergic blockers (e.g., streptozotocin), anti-inflammatory agents (e.g., benzodiazepinephrine), and anti-inflammatory agents (e.g., benzodiazepinephrine), antimuscarin (e.g., benzodiazepinephrine), and antimuscarin (e.g., benzodiazepinephrine), antimuscarin (e.g., benzodiazepinephrine (e), and antimuscarin (e.g., benzodiazepinephrine (e), and other drugs (e.g., benzodiazepinephrine), and other drugs (e.g., benzoglibin), which may be used in combination, ciprofloxacin (ciprofloxacin) and cefoxitin), antifungal agents (e.g., miconazole, terconazole, econazole, isoconazole, butoconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B (amphotericin B)), antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitters, antagonists, antiglaucomas, vitamins, sedatives, and imaging agents.
In some embodiments, the therapeutic and/or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, and/or another therapeutic and/or prophylactic agent. Cytotoxins or cytotoxic agents include any agent that is detrimental to cells. Examples include, but are not limited to, taxol (taxol), cytochalasin B (cytochalasin B), gramicidin D (gramicidin D), ethidium bromide (ethidium bromide), emetine (emetine), mitomycin (mitomycin), etoposide (etoposide), teniposide (teniposide), vincristine, vinblastine, colchicine (colchicine), doxorubicin, daunorubicin (daunorubicin), dihydroxyanthracenedione (dihydroxy anthracin dione), mitoxantrone, mithramycin (mithramycin), actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine (procaine), tetracaine (tetracaine), lidocaine (lidocaine), propranolol, puromycin, maytansinoids (maytansinoid) such as maytansinol (maytansine), lanmycin (rachimycin) (CC-1065), and analogs or homologs thereof. Radioions include, but are not limited to, iodine (e.g., iodine 125 or iodine 131), strontium 89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, samarium 153, and praseodymium. Vaccines include compounds and formulations capable of providing immunity against one or more conditions associated with infectious diseases such as influenza, measles, human Papilloma Virus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis and tuberculosis and may include mRNA encoding antigens and/or epitopes that are the source of infectious diseases. Vaccines can also include compounds and formulations that direct immune responses against cancer cells and can include mRNA encoding tumor cell-derived antigens, epitopes, and/or neoepitopes. Compounds that elicit an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species. In some embodiments, a vaccine and/or compound capable of eliciting an immune response is administered intramuscularly through a composition comprising a compound according to formula (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg) or (III) (e.g., compound 3, 18, 20, 25, 26, 29, 30, 60, 108-112 or 122). Other therapeutic and/or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), alkylating agents (e.g., nitrogen mustard (mechlorethamine), thiotepa (thiopa), chlorambucil (chloranserine), azithromycin (CC-1065), melphalan (melphalan), carmustine (carmustine, BSNU), robustin (lomustine, CCNU), cyclophosphamide, busulfan (busulfan), dibromomannitol, streptozotocin, mitomycin C, and cisplatin (II) (DDP), cisplatin), anthracyclines (e.g., daunomycin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly dactinomycin), dactinomycin (formenin), and vincristine (AMC), and antimuscarines (e.g., mitomycin, and the like).
In other embodiments, the therapeutic and/or prophylactic agent is a protein. Therapeutic proteins that may be used in the nanoparticles of the present invention include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), factor VIR, luteinizing Hormone Releasing Hormone (LHRH) analogs, interferons, heparin, hepatitis B surface antigen, typhoid vaccines, and cholera vaccines.
In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term "polynucleotide" is intended to include in its broadest sense any compound and/or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides for use in accordance with the present invention include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA); ribonucleic acids (RNAs), including messenger mrnas (mrnas), hybrids thereof; RNAi-inducing factors; RNAi factor; siRNA; shRNA; a miRNA; antisense RNA; ribozymes; catalytic DNA; RNA that induces triple helix formation; an aptamer, and the like. In some embodiments, the therapeutic and/or prophylactic agent is RNA. The RNAs useful in the compositions and methods described herein may be selected from the group consisting of, but not limited to: shortmer, antagomir antisense RNA, ribozyme, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In certain embodiments, the RNA is mRNA.
In certain embodiments, the therapeutic and/or prophylactic agent is mRNA. The mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA may be of any size and may have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA may have a therapeutic effect when expressed in a cell.
In other embodiments, the therapeutic and/or prophylactic agent is an siRNA. siRNA is capable of selectively reducing expression of a gene of interest or down-regulating expression of the gene. For example, the siRNA can be selected such that a gene associated with a particular disease, disorder, or condition is silenced after administration of a composition comprising the siRNA to a subject in need thereof. The siRNA may comprise a sequence complementary to an mRNA sequence encoding a gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.
In certain embodiments, the therapeutic and/or prophylactic agent is sgRNA and/or cas9 mRNA. sgRNA and/or cas9 mRNA may be used as a gene editing tool. For example, the sgRNA-cas9 complex can affect mRNA translation of cellular genes.
In some embodiments, the therapeutic and/or prophylactic agent is an shRNA or a vector or plasmid encoding the same. shRNA may be produced inside the target cell after delivery of the appropriate construct into the nucleus. Constructs and mechanisms related to shRNA are well known in the relevant arts.
Diseases or conditions
The compositions/carriers of the 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. Thus, the composition of the invention can be used for preparing nucleic acid medicaments, gene vaccines, small molecule medicaments, polypeptides or protein medicaments. Because of the wide variety of therapeutic or prophylactic agents described above, the compositions of the present invention are useful in the treatment or prevention of a variety of diseases or conditions.
In one embodiment, the disease or disorder is characterized by dysfunctional or abnormal protein or polypeptide activity.
For example, the disease or disorder is selected from the group consisting of: infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases and metabolic diseases.
In one embodiment, the infectious disease is selected from the group consisting of a disease caused by coronavirus, influenza virus, or HIV virus, pediatric pneumonia, rift valley fever, yellow fever, rabies, or 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 modifying agents, or other components. The permeability enhancing molecule may be, for example, a molecule described in U.S. patent application publication No. 2005/0222064. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen, and derivatives and analogs thereof).
Surface modifying agents may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyl dioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamers), mucolytics (e.g., acetylcysteine, mugwort, bromelain, papain, dyers woad), bromohexine, carbocistein, eplerenone, mesna, ambroxol, sobrinol, domidol, ritodtein, stenine, tiopronin, gelsin, thymosin beta 4, dnase alpha, neogenin, and dnase, such as dnase. The surface modifying agent may be disposed within and/or on the nanoparticle of the composition (e.g., by coating, adsorption, covalent attachment, or other means).
The composition may further comprise one or more functionalized lipids. For example, the lipid may be functionalized with an alkynyl group that may undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. In particular, lipid bilayers can be functionalized in this manner with one or more groups effective to facilitate membrane permeation, cell recognition, or imaging. The surface of the composition may also be conjugated to one or more useful antibodies. Functional groups and conjugates useful for targeted cell delivery, imaging, and membrane permeation are well known in the art.
In addition to these components, the composition may include any substance useful in pharmaceutical compositions. For example, the composition may include one or more pharmaceutically acceptable excipients or auxiliary ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersing aids, suspending aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonic agents, thickening or emulsifying agents, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, and the like. Excipients such as starch, lactose or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, e.g., remington's The Science and Practice of Pharmacy, 21 st edition, a.r. gennaro; lippincott, williams & Wilkins, baltimore, MD, 2006).
Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, dibasic calcium phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and/or combinations thereof.
In some embodiments, compositions comprising one or more lipids described herein may also comprise one or more adjuvants, such as Glucopyranosyl Lipid Adjuvants (GLA), cpG oligodeoxyribonucleotides (e.g., class a or class B), poly (I: C), aluminum hydroxide, and Pam3CSK4.
The compositions of the present invention may be formulated in solid, semi-solid, liquid or gaseous form as, for example, tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, aerosols. The compositions of the present invention may be prepared by methods well known in the pharmaceutical arts. For example, sterile injectable solutions can be prepared by incorporating the therapeutic or prophylactic agent in the required amount with various of the other ingredients described above in the appropriate solvent such as sterile distilled water and then filter-sterilizing. Surfactants may also be added to promote the formation of a uniform solution or suspension.
For example, the compositions of the present 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 dose of about 0.001mg/kg to about 10mg/kg of the therapeutic or prophylactic agent may 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
G 1 Is C 1~15 Alkylene or- (CH) 2 ) a C(O)O(CH 2 ) b -, wherein a and b are each an integer of 1 to 5;
G 2 is C 2~8 An alkylene group;
G 3 is C 1~4 An alkylene group;
R 1 is hydrogen atom, C 6~14 Straight chain alkyl or- (CH) 2 ) c C(O)O(CH 2 ) d CH 3 Wherein c is an integer from 3 to 12 and d is an integer from 1 to 4;
R 2 is hydrogen atom, C 6~14 Linear or branched alkyl;
R 3 is unsubstituted, hydrogen atom, C 6~14 Linear or branched alkyl;
R 4 is unsubstituted, hydrogen atom, C 6~14 Linear or branched alkyl;
R 5 is C 6~25 Linear or branched alkyl;
X 1 and X 2 Respectively a methine group or an oxygen atom.
2. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to embodiment 1, wherein G 1 Is unsubstituted C 1~12 An alkylene group.
3. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to embodiment 1 or 2, wherein G 1 Is unsubstituted C 1 Alkylene, C 5 Alkylene, C 8 Alkylene, C 4 Alkylene or C 12 An alkylene group.
4. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to embodiment 1, wherein G 1 Is- (CH) 2 ) 3 C(O)OCH 2 -。
5. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of the preceding embodiments, wherein G 2 Is C 3~7 An alkylene group.
6. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to embodiment 5, wherein G 2 Is C 5 Alkylene or C 7 An alkylene group.
7. The compound of formula (I) or N-oxidation thereof according to embodiment 5A solvate, pharmaceutically acceptable salt or stereoisomer, wherein G 3 Is C 2~3 An alkylene group.
8. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to embodiment 7, wherein G 3 Is C 2 An alkylene group.
9. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of the preceding embodiments, wherein R 1 Is C 5-13 A linear alkyl group.
10. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to embodiment 9, wherein R 1 Is C 10 、C 8 、C 7 、C 9 Or C 13 A linear alkyl group.
11. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of embodiments 1-8, wherein R 1 Is- (CH) 2 ) 11 C(O)OCH 2 CH 3 Or- (CH) 2 ) 7 C(O)OCH 2 CH 3 。
12. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of the preceding embodiments, wherein R 2 Is a hydrogen atom.
13. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of embodiments 1-11, wherein R 2 Is C 10 、C 8 、C 7 Or C 9 A linear alkyl group.
14. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of the preceding embodiments, wherein R 3 Is unsubstituted.
15. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of embodiments 1-13A body, wherein R is 3 Is C 7~12 A linear alkyl group.
16. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to embodiment 15, wherein R 3 Is C 8 A linear alkyl group.
17. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of the preceding embodiments, wherein R 4 Is a hydrogen atom.
18. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of embodiments 1-16, wherein R 4 Is C 7~12 A linear alkyl group.
19. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to embodiment 18, wherein R 4 Is C 8 A linear alkyl group.
20. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of embodiments 1-16, wherein R 4 Is unsubstituted.
21. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of the preceding embodiments, wherein R 5 Is C 6~15 A linear alkyl group.
22. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to embodiment 21, wherein R 5 Is C 11 Or C 10 A linear alkyl group.
23. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of embodiments 1-20, wherein R 5 Is C 10~22 Branched alkyl groups.
24. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to embodiment 23, wherein R 5 Is C 17 Branched alkanesA base.
25. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to embodiment 24, wherein R 5 Is that。
26. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of the preceding embodiments, wherein X 1 Is a methine group.
27. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to embodiment 14, wherein X 1 Is an oxygen atom.
28. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of the preceding embodiments, wherein X 2 Is a methine group.
29. The compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to embodiment 20, wherein X 2 Is an oxygen atom.
30. 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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31. 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-702 having the structure:
。
32. 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-705 having the structure:
。
33. 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-712 having the structure:
。
34. 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 a compound YK-716 having the structure:
。
35. 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.
36. The composition of embodiment 35, wherein the cationic lipid comprises 25% -75% of the carrier by mole.
37. The composition of any one of embodiments 35-36, wherein the carrier further comprises a neutral lipid.
38. The composition of embodiment 37, wherein the molar ratio of the cationic lipid to the neutral lipid is 1:1-15:1, preferably 4.5:1.
39. The composition of embodiment 37, wherein the neutral lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramides, sterols, and derivatives thereof.
40. The composition of embodiment 39, wherein the neutral lipid is selected from one or more of the following: 1, 2-Dioleoyl-sn-glycero-3-phosphorylcholine (DLPC), 1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dioleoyl-sn-glycero-phosphorylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-octadecenyl-sn-glycero-3-phosphorylcholine (18:0 DietherPC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-3-phosphorylcholine (OChems PC), 1-hexadecyl-sn-3-phosphorylcholine (C16), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1-dioleoyl-2-dioleoyl-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-sn-glycero-3-phosphate ethanolamine (DOPE), 1, 2-di-phytanoyl-sn-glycero-3-phosphate ethanolamine (ME 16.0 PE), 1, 2-di-stearoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-oleoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-linolenoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-arachidonoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-docosahexaenoic acyl-sn-glycero-3-phosphate ethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphate sodium salt (DOPG), 1, 2-di-oleoyl-rac-3-phosphate sodium salt (DOPG) dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl-based phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof.
41. The composition of embodiment 40, wherein the neutral lipid is DOPE and/or DSPC.
42. The composition of any one of embodiments 35-41, wherein the carrier further comprises a structural lipid.
43. The composition of embodiment 42, wherein the molar ratio of the cationic lipid to the structural lipid is 0.6:1-3:1.
44. The composition of any of embodiments 42-43, wherein the structural lipid is selected from one or more of the following: cholesterol, non-sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycorine, ursolic acid, alpha-tocopherol, corticosteroids.
45. The composition of embodiment 44, wherein the structural lipid is cholesterol.
46. The composition of any of embodiments 35-45, wherein the carrier further comprises a polymer conjugated lipid.
47. The composition of embodiment 46, wherein the molar ratio of the polymer conjugated lipid to the carrier is 0.5% -10%, preferably 1.5%.
48. The composition of any of embodiments 46-47, wherein the polymer conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol.
49. The composition of embodiment 48, wherein the polymer conjugated lipid is selected from one or more of the following: distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG 2000), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000) and methoxypolyethylene glycol ditetradecylamide (ALC-0159).
50. The composition of any of embodiments 35-49, wherein the carrier comprises neutral lipids, structural lipids, and polymer conjugated lipids in a molar ratio of (25-75): 5-25): 15-65): 0.5-10.
51. The composition of embodiment 50, 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.
52. The composition of embodiment 51, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer conjugated lipid is 45:10:43.5:1.5.
53. The composition of any of embodiments 35-52, wherein the composition is a nanoparticle formulation having an average particle size of 10nm to 300nm; the polydispersity of the nanoparticle preparation is less than or equal to 50 percent.
54. The composition of embodiment 53, wherein the composition is a nanoparticle formulation having an average particle size of 90nm to 280nm; the polydispersity of the nanoparticle preparation is less than or equal to 45 percent.
55. The composition of any of embodiments 35-54, wherein the cationic lipid further comprises one or more other ionizable lipid compounds.
56. The composition of any of embodiments 35-55, further comprising a therapeutic or prophylactic agent.
57. The composition of embodiment 56, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 10:1-30:1.
58. The composition of embodiment 57, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 12.5:1-20:1.
59. The composition of embodiment 58, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 15:1.
60. The composition of any of embodiments 56-59, wherein the therapeutic or prophylactic agent comprises one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.
61. The composition of any of embodiments 56-59, wherein the therapeutic or prophylactic agent is a vaccine or compound capable of eliciting an immune response.
62. The composition of embodiment 60, wherein the therapeutic or prophylactic agent is a nucleic acid.
63. The composition of embodiment 62, wherein the therapeutic or prophylactic agent is ribonucleic acid (RNA).
64. The composition of embodiment 62, wherein the therapeutic or prophylactic agent is deoxyribonucleic acid (DNA).
65. The composition of embodiment 63, wherein the RNA is selected from the group consisting of: small interfering RNAs (siRNA), asymmetric interfering RNAs (aiRNA), micrornas (miRNA), dicer-substrate RNAs (dsRNA), small hairpin RNAs (shRNA), messenger RNAs (mRNA), and mixtures thereof.
66. The composition of embodiment 65, wherein the RNA is mRNA.
67. The composition according to any one of the preceding embodiments, wherein the composition further comprises one or more pharmaceutically acceptable excipients.
68. Use of a compound of formula (I) according to any one of embodiments 1-34 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a composition according to any one of embodiments 35-67, for the preparation of a nucleic acid drug, a genetic vaccine, a small molecule drug, a polypeptide or a protein drug.
69. Use of a compound of general formula (I) as described in any one of embodiments 1-34 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a composition as described in any one of embodiments 35-67, in the manufacture of a medicament for treating a disease or disorder in a mammal in need thereof.
70. The use of embodiment 69, wherein the disease or disorder is characterized by dysfunctional or abnormal protein or polypeptide activity.
71. The use of embodiment 69, wherein the disease or disorder is selected from the group consisting of: infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases and metabolic diseases.
72. The use according to embodiment 71, wherein the infectious disease is selected from the group consisting of: diseases caused by coronavirus, influenza virus or HIV virus, pediatric pneumonia, rift valley fever, yellow fever, rabies, or various herpes.
73. The use according to any one of embodiments 69-72, wherein the mammal is a human.
74. The use according to any one of embodiments 69-73, wherein the medicament is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.
75. The use of embodiment 74, wherein the composition is administered subcutaneously.
76. The use of any one of embodiments 69-75 wherein a dose of about 0.001mg/kg to about 10mg/kg of the medicament is administered to the mammal.
Examples
The invention is further described below with reference to examples. 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 which are not noted are conventional conditions in the industry. In the specific examples of the present invention, all the raw materials used are commercially available. Unless otherwise indicated, percentages are by weight in the context, and all temperatures are given in degrees celsius. The technical features of the various embodiments of the present invention may be combined with each other as long as they do not collide with each other.
The following abbreviations represent the following reagents, respectively:
DCM: dichloromethane; EDCI:1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; DMAP: 4-dimethylaminopyridine; DCC: n, N' -dicyclohexylcarbodiimide; EDCI 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; and rt: room temperature
Example 1: synthesis of cationic lipid compounds
1.6 Synthesis of undecyl- ((5- (2, 2-diheptyl-1, 3-dioxolan-4-yl) pentyl) (2-hydroxyethyl) amino) hexanoate (YK-701)
The synthetic route is as follows:
step one: synthesis of 2- (5-bromopentyl) oxirane (YK-701-PM 1)
7-Bromoheptene (3.0 g, 16.9 mmol) was dissolved in dichloromethane (20 mL) and added portionwise with icemCPBA (4.1 g, 23.7 mmol), the ice bath was removed after the addition was complete. The reaction was stirred at room temperature for 20 hours, washed with saturated aqueous sodium hydrogencarbonate (10 mL. Times.3), dried over anhydrous sodium sulfate, and filtered. The solvent was removed by spin-drying under reduced pressure to give YK-701-PM1 (3.17 g,16.4mmol, 97.0%).
Step two: synthesis of 7-bromoheptane-1, 2-diol (YK-701-PM 2)
YK-701-PM1 (3.17 g,16.4 mmol) was dissolved in a mixed solution of DMF (30 mL) and water (30 mL), trifluoroacetic acid (4.1 g,36.1 mmol) was slowly added under ice bath, and after removal of the ice bath, the reaction was carried out at room temperature for 8 hours. After the reaction was completed, the mixture was extracted with ethyl acetate (15 mL. Times.2), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (0-50% ethyl acetate/n-hexane) to give YK-701-PM2 (650 mg, 3.1mmol, 19.0%).
Step three: synthesis of undecyl 6- ((6, 7-dihydroxyheptyl) (2-hydroxyethyl) amino) hexanoate (YK-701-PM 3)
YK-701-PM2 (154 mg, 0.73 mmol) and undecyl 6- ((2-hydroxyethyl) amino) hexanoate (200 mg,0.61 mmol) were dissolved in acetonitrile (2 mL), potassium carbonate (251 mg, 1.82 mmol) and potassium iodide (20 mg,0.12 mmol) were added to the above system, and the reaction was heated to 70℃with stirring. After the reaction, the reaction mixture was filtered, and the filtrateConcentrated under reduced pressure in vacuo and the residue was purified by silica gel chromatography (0-24% methanol/dichloromethane) to give YK-701-PM3 (50 mg, 0.11mmol, 17.8%). C (C) 26 H 53 NO 5 ,MS(ES): m/z(M+H + )460.4。
Step four: synthesis of 8, 8-dimethoxy pentadecane (YK-701-PM 4)
Pentadecan-8-one (500 mg, 2.21 mmol) was dissolved in methanol (10 mL), and pyridine p-toluenesulfonate (56 mg, 0.22 mmol) and trimethyl orthoformate (5 mL) were added to the above system, and the reaction was stirred at room temperature for 10 hours. After completion of the reaction, triethylamine (0.2 mL) and water (20 mL) were added, followed by extraction with ethyl acetate (15X 2), and the organic phases were combined, dried over anhydrous sodium sulfate, and the solution was removed by spin-drying under reduced pressure to give YK-701-PM4 (420 mg, 1.54mmol, 69.7%).
Step five: synthesis of undecyl 6- ((5- (2, 2-diheptyl-1, 3-dioxolan-4-yl) pentyl) (2-hydroxyethyl) amino) hexanoate (YK-701)
YK-701-PM3 (50 mg, 0.11 mmol) and YK-701-PM4 (200 mg,0.61 mmol) were dissolved in dichloromethane (3 mL), and p-toluenesulfonic acid monohydrate (2.1 mg, 0.01 mmol) was added to the above system and reacted at room temperature with stirring for 5 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (0-7% methanol/dichloromethane) to give YK-701 (35 mg, 0.05mmol, 48.1%). C (C) 41 H 81 NO 5 ,MS(ES): m/z(M+H + )668.6。
YK-701: 1 H NMR (400 MHz, CDCl 3 ) δ 4.07–4.00 (m, 3H), 3.84 (s, 2H), 3.47–3.39 (m, 1H), 2.97 (s, 2H), 2.88 (s, 3H), 2.32 (t, J=7.3 Hz, 2H), 1.80 – 1.52 (m, 13H), 1.45–1.19 (m, 46H), 0.88 (t, J=6.7 Hz, 9H).
Synthesis of undecyl 6- (((2, 2-didecyl-1, 3-dioxolan-4-yl) methyl) (2-hydroxyethyl) amino) hexanoate (YK-702) and undecyl 6- ((2-hydroxyethyl) ((2-tridecyl-1, 3-dioxolan-4-yl) methyl) amino) hexanoate (YK-703)
The synthetic route is as follows:
step one: synthesis of 11, 11-dimethoxy eicosane (YK-702-PM 1)
Using 11-di-undecanone (2.0 g, 6.44 mmol) as a starting material, YK-702-PM1 (798 mg, 2.24mmol, 34.8%) was obtained according to the method of YK-701-PM 4.
Step two: synthesis of undecyl 6- ((2, 3-dihydroxypropyl) (2-hydroxyethyl) amino) hexanoate (YK-702-PM 2)
Using undecyl 6- ((2-hydroxyethyl) amino) hexanoate (200 mg,0.61 mmol) and 3-chloro-1, 2-propanediol (81 mg,0.73 mmol) as starting materials, YK-702-PM2 (120 mg, 0.30mmol, 48.7%) was obtained according to the method of YK-701-PM 3. C (C) 22 H 45 NO 5 ,MS(ES): m/z(M+H + )404.3。
Step three: synthesis of undecyl 6- (((2, 2-didecyl-1, 3-dioxolan-4-yl) methyl) (2-hydroxyethyl) amino) hexanoate (YK-702)
YK-702 (40 mg,0.06mmol, 23.0%) was obtained by the method of YK-701 starting with YK-702-PM1 (707 mg,1.98 mmol) and YK-702-PM2 (100 mg, 0.25 mmol). C (C) 43 H 85 NO 5 , MS(ES): m/z(M+H + )696.7。
YK-702: 1 H NMR (400 MHz, CDCl 3 ) δ 4.31 (s, 1H), 4.18 – 3.99 (m, 3H), 3.66 (s, 2H), 3.57 – 3.46 (m, 1H), 2.78 (s, 6H), 2.38 – 2.28 (m, 2H), 1.69 – 1.58 (m, 8H), 1.39 – 1.25 (m, 52H), 0.96 – 0.86 (m, 9H).
Step four: synthesis of undecyl 6- ((2-hydroxyethyl) ((2-tridecyl-1, 3-dioxolan-4-yl) methyl) amino) hexanoate (YK-703)
YK-702-PM2 (100 mg, 0.25 mmol) and tetradecaldehyde (78.8 mg, 0.37 mmol) were used as starting materials to obtain YK-703 (25 mg,0.04mmol, 16.7%) according to the method of YK-701. C (C) 36 H 71 NO 5 , MS(ES): m/z(M+H + )598.5。
YK-703： 1 H NMR (400 MHz, CDCl 3 ) δ 4.95 (dt, J = 32.4, 4.7 Hz, 1H), 4.38 – 3.91 (m, 3H), 3.86 – 3.41 (m, 2H), 2.94 (s, 2H), 2.39 – 2.29 (m, 2H), 1.62 (dd, J = 15.2, 7.7 Hz, 6H), 1.48 – 1.25 (m, 48H), 0.89 (d, J = 6.5 Hz, 6H).
3.6 Synthesis of undecyl caproate (YK-704) - ((5- (2, 2-dinonyl-1, 3-dioxolan-4-yl) pentyl) (2-hydroxyethyl) amino)
The synthetic route is as follows:
step one: synthesis of 10, 10-dimethoxy-nonadecane (YK-704-PM 1)
Using 10-nonadecanone (1.0 g, 3.54 mmol) as a raw material, YK-704-PM1 (1.0 g, 3.04mmol, 86.0%) was obtained according to the method of YK-701-PM 4.
Step two: synthesis of undecyl 6- ((5- (2, 2-dinonyl-1, 3-dioxolan-4-yl) pentyl) (2-hydroxyethyl) amino) hexanoate (YK-704)
YK-701-PM3 (120 mg, 0.26 mmol) and YK-704-PM1 (465 mg, 1.42 mmol) were used as raw materials to obtain YK-703 (30 mg,0.04mmol, 15.9%) according to the method of YK-701. C (C) 45 H 89 NO 5 , MS(ES): m/z(M+H + )724.7。
YK-704: 1 H NMR (400 MHz, Chloroform-d) δ 4.08 – 4.02 (m, 2H), 3.92 (s, 1H), 3.12 – 2.90 (m, 4H), 2.32 (t, J = 7.3 Hz, 2H), 1.79 (s, 2H), 1.60 (ddt, J = 22.5, 14.6, 6.8 Hz, 8H), 1.47 (d, J = 17.2 Hz, 2H), 1.41 – 1.22 (m, 58H), 0.88 (t, J = 6.6 Hz, 9H).
4.6 Synthesis of undecyl caproate (YK-705) - ((5- (2, 2-dioctyl-1, 3-dioxolan-4-yl) pentyl) (2-hydroxyethyl) amino)
The synthetic route is as follows:
step one: synthesis of 9, 9-dimethoxy heptadecane (YK-705-PM 1)
Using 9-heptadecanone (500 mg, 1.96 mmol) as a raw material, YK-705-PM1 (500 mg, 1.66mmol, 84.9%) was obtained according to the method of YK-701-PM 4.
Step two: synthesis of undecyl 6- ((5- (2, 2-dioctyl-1, 3-dioxolan-4-yl) pentyl) (2-hydroxyethyl) amino) hexanoate (YK-705)
YK-705 (50 mg, 0.07mmol, 25.7%) was obtained by the method of YK-701 using YK-701-PM3 (130 mg, 0.28 mmol) and YK-705-PM1 (420 mg, 1.40 mmol) as raw materials. C (C) 43 H 85 NO 5 , MS(ES): m/z(M+H + )696.7。
YK-705: 1 H NMR (400 MHz, CDCl 3 ) δ 4.11 – 4.01 (m, 3H), 3.47 (d, J = 23.2 Hz, 1H), 3.18 (s, 1H), 3.07 (s, 2H), 2.40 – 2.31 (m, 2H), 1.86 (s, 2H), 1.67 – 1.54 (m, 8H), 1.44 – 1.24 (m, 56H), 0.88 (t, J = 6.6 Hz, 9H).
5.Synthesis of ethyl 12- (2- (4- ((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) butyl) -5-octyl-1, 3-dioxolan-4-yl) dodecanoate (YK-706)
The synthetic route is as follows:
step one: synthesis of ethyl 13-docosenoate (YK-706-PM 1)
13-docosanoic acid (2.0 g, 5.91 mmol) was dissolved in dichloromethane (20 mL), EDCI (1.3 g, 6.78 mmol), DMAP (72.2 mg,0.59 mmol) and ethanol (408 mg,8.9 mmol) were added sequentially. The reaction was stirred at room temperature for 8 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (0-5% ethyl acetate/n-hexane) to give YK-706-PM1 (1.5 g,4.09mmol, 69.2%).
Step two: synthesis of ethyl 13, 14-dihydroxybehenate (YK-706-PM 2)
YK-706-PM1 (1.0 g, 2.73 mmol) was dissolved in a mixed solution of THF (15 mL) and water (15 mL), potassium osmium (85 mg, 0.27 mmol), N-methylmorpholine-N-oxide (480 mg, 4.1 mmol) was added to the above solution in this order, and the reaction was stirred at room temperature overnight. After the reaction was completed, it was quenched with saturated aqueous sodium sulfite solution. After dilution with water (30 mL), extraction was performed with methylene chloride (30 mL. Times.3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (0-60% ethyl acetate/n-hexane) to give YK-706-PM2 (600 mg, 1.50mmol, 54.9%).
Step three: synthesis of ethyl 12- (2- (4-chlorobutyl) -5-octyl-1, 3-dioxolan-4-yl) dodecanoate (YK-706-PM 3)
YK-706-PM3 (120 mg,0.24mmol, 95.4%) was obtained by the synthetic method of YK-701 using YK-706-PM2 (100 mg, 0.25 mmol) and 5-chlorovaleraldehyde (30 mg, 0.25 mmol) as raw materials.
Step four: synthesis of ethyl 12- (2- (4- ((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) butyl) -5-octyl-1, 3-dioxolan-4-yl) dodecanoate (YK-706)
YK-706 (24 mg,0.03mmol, 14.3%) was obtained by the synthetic method of YK-701-PM3 starting from YK-706-PM3 (120 mg,0.24 mmol) and undecyl 6- ((2-hydroxyethyl) amino) hexanoate (70 mg, 0.21 mmol). C (C) 48 H 93 NO 7 , MS(ES): m/z(M+H + )796.7。
YK-706: 1 H NMR (400 MHz, CDCl 3 ) δ 4.87 (t, J = 4.3 Hz, 1H), 4.21 – 3.99 (m, 6H), 3.91 (d, J = 8.3 Hz, 2H), 3.28 – 3.03 (m, 5H), 2.31 (dt, J = 20.1, 7.4 Hz, 4H), 1.92 (brs, 4H), 1.72 – 1.57 (m, 8H), 1.55 – 1.36 (m, 10H), 1.34 – 1.20 (m, 46H), 0.88 (t, J = 6.7 Hz, 6H).
6.Synthesis of ethyl 8- (2- (4- ((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) butyl) -5-octyl-1, 3-dioxolan-4-yl) octanoate (YK-707)
The synthetic route is as follows:
step one: synthesis of ethyl oleate (YK-707-PM 1)
Using oleic acid (2.0 g, 7.08 mmol), EDCI (1.6 g, 8.35 mmol), DMAP (86 mg, 0.70 mmol), ethanol (326 mg, 7.0 mmol) as a starting material, YK-707-PM1 (1.9 g,6.12mmol, 86.4%) was obtained according to the synthetic method of YK-706-PM 1.
Step two: synthesis of ethyl 9, 10-dihydroxyoctadecanoate (YK-707-PM 2)
YK-707-PM2 (670 mg, 1.94mmol, 67.1%) was obtained by the synthesis method of YK-706-PM2 using YK-707-PM2 (900 mg, 2.90 mmol) as a raw material.
Step three: synthesis of ethyl 8- (2- (4-chlorobutyl) -5-octyl-1, 3-dioxolan-4-yl) octanoate (YK-707-PM 3)
YK-707-PM3 (180 mg, 0.40mmol, 51.6%) was obtained by the synthetic method of YK-701 using YK-707-PM2 (270 mg, 0.78 mmol) and 5-chlorovaleraldehyde (95 mg, 0.79 mmol) as raw materials.
Step four: synthesis of ethyl 8- (2- (4- ((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) butyl) -5-octyl-1, 3-dioxolan-4-yl) octoate (YK-707)
YK-707 (75 mg, 0.10mmol, 25.3%) was obtained by the synthetic method of YK-701-PM3 starting from YK-707-PM3 (180 mg, 0.40 mmol) and undecyl 6- ((2-hydroxyethyl) amino) hexanoate (224 mg, 0.68 mmol). C (C) 44 H 85 NO 7 ,MS(ES): m/z(M+H + )740.6。
YK-707: 1 H NMR (400 MHz, Chloroform-d) δ 4.86 (t, J = 4.4 Hz, 1H), 4.17 – 3.96 (m, 4H), 3.89 (s, 3H), 3.05 (s, 1H), 2.95 (s, 2H), 2.30 (dt, J = 12.1, 7.4 Hz, 4H), 1.89 – 1.73 (m, 3H), 1.73 – 1.56 (m, 8H), 1.55 – 1.21 (m, 52H), 0.88 (t, J = 6.7 Hz, 6H).
7. Synthesis of heptadec-9-yl-8- ((8- (2-decyl-5-octyl-1, 3-dioxolan-4-yl) octyl) (2-hydroxyethyl) amino) octanoate (YK-708) and heptadec-9-yl-8- ((2-hydroxyethyl) (8- (5-octyl-2-tridecyl-1, 3-dioxolan-4-yl) octyl) amino) octanoate (YK-709)
The synthetic route is as follows:
step one: synthesis of 1-bromooctadeca-9-ene (YK-708-PM 1)
Octadec-9-en-1-ol (3.0 g, 11.17 mmol) and triphenylphosphine (3.5 g, 13.34 mmol) were dissolved in dichloromethane (30 mL) and N-bromosuccinimide (2.4 g, 13.48 mmol) was added in portions to the above system and reacted at room temperature for 5 hours. The reaction solution was concentrated under reduced pressure. The residue was purified by silica gel chromatography (0-6% ethyl acetate/n-hexane) to give YK-708-PM1 (3.0 g, 9.1mmol, 81.0%).
Step two: synthesis of 1-bromooctadecane-9, 10-diol (YK-708-PM 2)
YK-708-PM2 (260 mg, 0.71mmol, 47.1%) was obtained by the synthesis method of YK-706-PM2 using YK-708-PM1 (500 mg, 1.51 mmol) as a raw material.
Step three: synthesis of heptadec-9-yl 8- ((9, 10-dihydroxyoctadecyl) (2-hydroxyethyl) amino) octanoate (YK-708-PM 3)
YK-708-PM3 (170 mg, 0.23mmol, 52.0%) was obtained by the method for synthesizing YK-701-PM3 starting from YK-708-PM2 (200 mg, 0.55 mmol) and 8- ((2-hydroxyethyl) amino) heptadec-9-yl octanoate (197 mg, 0.45 mmol). C (C) 45 H 91 NO 5 , MS(ES): m/z(M+H + )726.7。
Step four: synthesis of heptadec-9-yl-8- ((8- (2-decyl-5-octyl-1, 3-dioxolan-4-yl) octyl) (2-hydroxyethyl) amino) octanoate (YK-708)
YK-708 (45 mg, 0.05mmol, 46.6%) was obtained by the synthetic method of YK-701 starting from YK-708-PM3 (80 mg,0.11 mmol) and undecalaldehyde (29 mg,0.17 mmol). C (C) 56 H 111 NO 5 , MS(ES): m/z(M+H + )878.9。
YK-708: 1 H NMR (400 MHz, CDCl 3 ) δ 4.90 – 4.82 (m, 1H), 3.90 (d, J = 6.8 Hz, 1H), 3.70 (s, 2H), 2.79 (s, 2H), 2.68 (s, 4H), 2.33-2.23(m, 2H), 1.64 – 1.18 (m, 86H), 0.89 – 0.85 (m, 12H).
Step five: synthesis of heptadec-9-yl-8- ((2-hydroxyethyl) (8- (5-octyl-2-tridecyl-1, 3-dioxolan-4-yl) octyl) amino) octanoate (YK-709)
YK-709 (35 mg, 0.04mmol, 34.6%) was obtained by the synthetic method of YK-701 using YK-708-PM3 (80 mg,0.11 mmol) and tetradecaldehyde (36 mg,0.17 mmol) as raw materials. C (C) 59 H 117 NO 5 , MS(ES): m/z(M+H + )920.9。
YK-709: 1 H NMR (400 MHz, CDCl 3 ) δ 4.89 – 4.83 (m, 1H), 3.90 (d, J = 7.6 Hz, 1H), 3.61 (t, J = 5.0 Hz, 2H), 2.68 (t, J = 4.9 Hz, 2H), 2.58 – 2.53 (m, 4H), 2.28 (t, J = 7.5 Hz, 2H), 1.73 – 1.03 (m, 92H), 0.91 – 0.83 (m, 12H).
8. Synthesis of heptadec-9-yl-8- ((12- (2-decyl-5-octyl-1, 3-dioxolan-4-yl) dodecyl) (2-hydroxyethyl) amino) octanoate (YK-710) and heptadec-9-yl-8- ((2-hydroxyethyl) (12- (5-octyl-2-tridecyl-1, 3-dioxolan-4-yl) dodecyl) amino) octanoate (YK-711)
The synthetic route is as follows:
step one: synthesis of 1-bromodocosa-9-ene (YK-710-PM 1)
The synthesis of YK-710-PM1 (1.13 g, 2.92mmol, 94.7%) was performed using docosa-9-en-1-ol (1.0 g, 3.08 mmol) and N-bromosuccinimide (641 mg, 3.60 mmol) as starting materials according to the method for YK-708-PM 1.
Step two: synthesis of 22-bromobehenate-9, 10-diol (YK-710-PM 2)
YK-710-PM2 (750 mg, 1.78mmol, 69.0%) was obtained by the synthesis method of YK-707-PM2 using YK-710-PM1 (1.0 g, 2.58 mmol) as a raw material.
Step three: synthesis of heptadec-9-yl 8- ((13, 14-dihydroxybehenyl) (2-hydroxyethyl) amino) octanoate (YK-710-PM 3)
Using YK-710-PM2 (287 mg, 0.68 mmol) and 8- ((2-hydroxyethyl) amino) heptadec-9-yl octanoate (250 mg, 0.57 mmol) as raw materials, YK-710-PM3 (200 mg, 0.26mmol, 44.9%) was obtained according to the synthetic method of YK-701-PM 3. C (C) 49 H 99 NO 5 ,MS(ES): m/z(M+H + )782.8。
Step four: synthesis of heptadec-9-yl-8- ((12- (2-decyl-5-octyl-1, 3-dioxolan-4-yl) dodecyl) (2-hydroxyethyl) amino) octanoate (YK-710)
YK-710 (89 mg, 0.10mmol, 73.3%) was obtained by the synthesis method of YK-701 starting from YK-710-PM3 (100 mg, 0.13 mmol) and undecalaldehyde (36 mg, 0.21 mmol)。C 60 H 119 NO 5 , MS(ES): m/z(M+H + )934.9。
YK-710: 1 H NMR (400 MHz, CDCl 3 ) δ 4.90 – 4.83 (m, 1H), 3.90 (d, J = 8.4 Hz, 1H), 3.59 (t, J = 5.1 Hz, 2H), 2.65 (t, J = 5.1 Hz, 2H), 2.57 – 2.45 (m, 4H), 2.28 (t, J = 7.5 Hz, 2H), 1.62 – 1.22 (m, 94H), 0.90 – 0.85 (m, 12H).
Step five: synthesis of heptadec-9-yl-8- ((2-hydroxyethyl) (12- (5-octyl-2-tridecyl-1, 3-dioxolan-4-yl) dodecyl) amino) octanoate (YK-711)
YK-711 (130 mg, 0.13mmol, 70.0%) was obtained by the synthesis method of YK-701 starting from YK-710-PM3 (150 mg, 0.19 mmol) and tetradecaldehyde (62 mg, 0.29 mmol). C (C) 63 H 125 NO 5 , MS(ES): m/z(M+H + )977.0。
YK-711: 1 H NMR (400 MHz, CDCl 3 ) δ 4.89 – 4.84 (m, 1H), 3.90 (d, J = 8.5 Hz, 1H), 3.55 (t, J = 5.3 Hz, 2H), 2.60 (t, J = 5.2 Hz, 2H), 2.50 – 2.43 (m, 4H), 2.27 (q, J = 7.6 Hz, 2H), 1.61 – 1.22 (m, 100H), 0.90 – 0.84 (m, 12H).
9.8 Synthesis of heptadec-9-yl ester of- ((5- (2-decyl-1, 3-dioxolan-4-yl) pentyl) (2-hydroxyethyl) amino) octanoate (YK-712) and heptadec-9-yl ester of 8- ((2-hydroxyethyl) (5- (2-tridecyl-1, 3-dioxolan-4-yl) amino) octanoate (YK-713)
The synthetic route is as follows:
step one: synthesis of 8- ((6, 7-dihydroxyheptyl) (2-hydroxyethyl) amino) octanoic acid-heptadec-9-yl ester (YK-712-PM 1)
YK-712-PM1 (150 mg, 0.26mmol, 53.5%) was obtained by the synthetic method of YK-701-PM3 starting from YK-701-PM2 (300 mg, 1.42 mmol) and 8- ((2-hydroxyethyl) amino) octanoic acid-heptadec-9-yl ester (215 mg, 0.49 mmol). C (C) 34 H 69 NO 5 ,MS(ES): m/z(M+H + )572.5。
Step two: synthesis of heptadec-9-yl 8- ((5- (2-decyl-1, 3-dioxolan-4-yl) pentyl) (2-hydroxyethyl) amino) octanoate (YK-712)
YK-712 (45 mg, 0.06mmol, 47.8%) was obtained by the synthetic method of YK-701 starting with YK-712-PM1 (75 mg, 0.13 mmol) and undecalaldehyde (34 mg, 0.20 mmol). C (C) 45 H 89 NO 5 , MS(ES): m/z(M+H + )724.7。
YK-712: 1 H NMR (400 MHz, CDCl 3 ) δ 4.89 – 4.82 (m, 1H), 4.34 – 3.84 (m, 2H), 3.78 – 3.70 (m, 1H), 3.52 – 3.36 (m, 1H), 2.88 – 2.66 (m, 4H), 2.32 – 2.22 (m, 2H), 1.62 – 1.19 (m, 68H), 0.89-0.86 (m, 9H).
Step three: synthesis of heptadec-9-yl 8- ((2-hydroxyethyl) (5- (2-tridecyl-1, 3-dioxolan-4-yl) amino) octoate (YK-713)
YK-713 (55 mg, 0.07mmol, 55.2%) was obtained by the synthetic method of YK-701 starting with YK-712-PM1 (75 mg, 0.13 mmol) and tetradecaldehyde (42 mg, 0.20 mmol). C (C) 48 H 95 NO 5 , MS(ES): m/z(M+H + )766.7。
YK-713: 1 H NMR (400 MHz, CDCl 3 ) δ 4.90 – 4.81 (m, 1H), 4.14 – 3.86 (m, 3H), 3.51 – 3.38 (m, 1H), 3.06 – 2.87(m, 4H), 2.32 – 2.23 (m, 2H), 1.88 – 1.00 (m, 74H), 0.89 – 0.86 (m, 9H).
10.8 Synthesis of heptadec-9-yl octanoate (YK-714) of- ((4- ((2-decyl-1, 3-dioxolan-4-yl) methoxy) -4-oxobutyl) (2-hydroxyethyl) amino)
The synthetic route is as follows:
step one: synthesis of ethylene oxide-2-yl-methyl-4-bromobutyrate (YK-714-PM 1)
Using 4-bromobutyric acid (2.0 g, 11.98 mmol) and ethylene oxide-2-yl methanol (806 mg,10.88 mmol) as starting materials, YK-714-PM1 (1.4 g, 6.28mmol, 57.7%) was obtained according to the synthesis of YK-707-PM 1.
Step two: synthesis of heptadec-9-yl 8- ((2-hydroxyethyl) (4- (oxiran-2-ylmethoxy) -4-oxobutyl) amino) octanoate (YK-714-PM 2)
Using YK-714-PM1 (660 mg, 2.96 mmol) and 8- ((2-hydroxyethyl) amino) heptadec-9-yl octanoate (1.2 g, 2.72 mmol) as raw materials, YK-714-PM2 (1.0 g, 1.71mmol, 63.0%) was obtained according to the synthetic method of YK-701-PM 3. C (C) 34 H 65 NO 6 , MS(ES): m/z(M+H + )584.5。
Step three: synthesis of heptadec-9-yl 8- ((4- (2, 3-dihydroxypropoxy) -4-oxobutyl) (2-hydroxyethyl) amino) octanoate (YK-714-PM 3)
YK-714-PM2 (150 mg, 0.26 mmol) was used as a starting material, and YK-714-PM3 (154 mg, 0.26mmol, 98.4%) was obtained according to the method for synthesizing YK-701-PM 2. C (C) 34 H 67 NO 7 , MS(ES): m/z(M+H + ) 602.5。
Step four: synthesis of heptadec-9-yl 8- ((4- ((2-decyl-1, 3-dioxolan-4-yl) methoxy) -4-oxobutyl) (2-hydroxyethyl) amino) octanoate (YK-714)
YK-714 (40 mg, 0.05mmol, 20.4%) was obtained by the synthetic method of YK-701 starting with YK-714-PM3 (154 mg, 0.26 mmol) and undecalaldehyde (73 mg, 0.43 mmol). C (C) 45 H 87 NO 7 ,MS(ES): m/z(M+H + )754.7。
YK-714: 1 H NMR (400 MHz, CDCl 3 ) δ 4.92 – 4.81 (m, 1H), 4.35 – 4.06 (m, 3H), 3.98 – 3.52 (m, 4H), 2.94 – 2.72 (m, 5H), 2.48 – 2.41 (m, 2H), 2.28 (t, J = 6.4 Hz, 2H), 2.05 – 1.89(m, 2H), 1.72 – 1.17 (m, 58H), 0.94 – 0.82 (m, 9H).
11.8 Synthesis of heptadec-9-yl ester of- ((5- (2, 2-diheptyl-1, 3-dioxolan-4-yl) pentyl) (2-hydroxyethyl) amino) octanoate (YK-715)
The synthetic route is as follows:
step one: synthesis of 8, 8-dimethoxy pentadecane (YK-715-PM 1)
YK-715-PM1 (2.7 g,9.91mmol, 97.5%) was obtained by the method for synthesizing YK-701-PM4 starting from pentadecan-8-one (2.3 g, 10.16 mmol).
Step two: synthesis of heptadec-9-yl 8- ((5- (2, 2-diheptyl-1, 3-dioxolan-4-yl) pentyl) (2-hydroxyethyl) amino) octanoate (YK-715)
YK-715 (35 mg, 0.04mmol, 12.1%) was obtained by synthesizing YK-701 from YK-715-PM1 (100 mg,0.37 mmol) and YK-712-PM1 (210 mg,0.37 mmol). C (C) 49 H 97 NO 5 , MS(ES): m/z(M+H + )780.7。
YK-715: 1 H NMR (300 MHz, CDCl 3 ) δ 4.86 (s, 1H), 4.03 (s, 2H), 3.71 (s, 2H), 3.46 (d, J = 13.8 Hz, 2H), 2.74 (d, J = 34.4 Hz, 6H), 2.28 (s, 3H), 1.68 – 1.46 (m, 10H), 1.43 – 1.12 (m, 58H), 0.88 (s, 12H).
12.6 Synthesis of decyl 6- ((8- (2-decyl-5-octyl-1, 3-dioxolan-4-yl) octyl) (2-hydroxyethyl) amino) hexanoate (YK-716)
The synthetic route is as follows:
step one: synthesis of 4- (8-bromooctyl) -2-decyl-5-octyl-1, 3-dioxolane (YK-716-PM 1)
YK-716-PM1 (400 mg, 0.77mmol, 59.0%) was obtained by the synthetic method of YK-701 using YK-708-PM2 (480 mg, 1.31 mmol) and undecalaldehyde (413 mg, 2.43 mmol) as raw materials.
Step two: synthesis of decyl 6- ((8- (2-decyl-5-octyl-1, 3-dioxolan-4-yl) octyl) (2-hydroxyethyl) amino) hexanoate (YK-716)
YK-716 (63 mg, 0.08mmol, 13.3%) was obtained according to the method for synthesizing YK-701-PM3 starting from decyl YK-M-082-PM1 (393 mg, 0.76 mmol) and decyl 6- ((2-hydroxyethyl) amino) hexanoate (200 mg, 0.63 mmol). C (C) 47 H 93 NO 5 ,MS(ES): m/z(M+H + )752.7。
YK-716: 1 H NMR (300 MHz, CDCl 3 ) δ 5.10 (t, J = 4.9 Hz, 1H), 4.06 (t, J= 6.8 Hz, 3H), 3.77 (br s, 4H), 2.74 - 2.90 (m, 6H), 2.32 (br t, J = 7.3 Hz, 2H), 1.60 - 1.69 (m, 10H), 1.47 (br d, J = 14.0 Hz, 10H), 1.23 - 1.35 (m, 48H), 0.88 (br t, J = 6.4 Hz, 9H).
13.8 Synthesis of undecyl- ((8, 8-bis (octyloxy) octyl) (2-hydroxyethyl) amino) octanoate (Compound 1)
The synthetic route is as follows:
step one: synthesis of 8-bromo-1-octanal (Compound 1-PM 1)
To a solution of 8-bromooctan-1-ol (10.0 g, 47.8 mmol) in DCM (200 mL) was added pyridinium chlorochromate (PCC) (15.5 g, 71.7 mmol). After stirring at 15 ℃ for 5 hours, the reaction mixture was filtered and concentrated in vacuo. The crude residue was purified by silica gel chromatography (2-20% EtOAc/petroleum ether) to give compound 1-PM1 (6.7 g, 32.3mmol, 67.7%).
Step two: synthesis of 8-bromo-1, 1-bis (octyloxy) octane (Compound 1-PM 2)
Using 8-bromooctanal (2.0 g, 9.66 mmol) and octan-1-ol (1.89 g, 14.51 mmol) as raw materials, compound 1-PM2 (1.96 g, 4.36mmol, 45.1%) was obtained according to the method of YK-701.
Step three: synthesis of undecyl 8- ((8, 8-bis (octyloxy) octyl) (2-hydroxyethyl) amino) octanoate (Compound 1)
Starting with compound 1-PM2 (1.00 g, 2.22 mmol) and undecyl 8- ((2-hydroxyethyl) amino) octanoate (1.19 g, 3.33 mmol), compound 1 (661 mg, 0.91mmol, 41.0%) was obtained according to the method of synthesis of YK-701-PM 3. C (C) 45 H 91 NO 5 , MS(ES): m/z(M+H + ) 726.7。
Compound 1: 1 H NMR (300 MHz, CDCl 3 ) δ 4.50 (t, J = 5.9 Hz, 1H), 4.06 (t, J = 6.8 Hz, 2H), 3.51-3.55 (br s, 4H), 3.31 – 3.60 (m, 2H), 2.55 (br t, J = 5.4 Hz, 2H), 2.43 (t ,J＝7.4Hz , 4H), 2.25 (t ,J＝7.6Hz ,2H), 1.55-1.65 (br d, J = 14.0 Hz, 10H), 1.23 - 1.45 (m, 53H), 0.88 (br t, J = 6.4 Hz, 9H).
example 2: preparation condition optimization of nano lipid particle (LNP preparation)
1. Vector (liposome) and mRNA ratio optimization
The cationic lipid compounds YK-702, YK-705, YK-712 and YK-716 synthesized in example 1 were dissolved in ethanol at a molar ratio of 49:10:39.5:1.5 with DSPC (Ai Weita (Shanghai) medical science, inc.), cholesterol (Ai Weita (Shanghai) medical science, inc.) and DMG-PEG2000, respectively, to prepare an ethanol lipid solution. And rapidly adding the ethanol lipid solution into a citrate buffer solution (pH=4-5) by an ethanol injection method, and swirling for 30s for later use. eGFP-mRNA (purchased from Shanghai laboratory reagent limited) was diluted in citrate buffer (ph=4-5) to obtain an aqueous mRNA solution. Liposomes were prepared from a volume of liposome solution and an aqueous solution of mRNA at a weight ratio of total lipid to mRNA of 5:1, 10:1, 15:1, 20:1, 30:1 and 35:1, respectively. Ultrasound was performed at 25℃for 15min (ultrasound frequency 40kHz, ultrasound power 800W). The obtained liposome was diluted to 10 times of volume with PBS, and subjected to ultrafiltration in a 300kDa ultrafiltration tube to remove ethanol. And then the volume is fixed to a certain volume by PBS to obtain the LNP preparation which uses the cationic lipid YK-702 (or YK-705, YK-712, YK-716)/DSPC/cholesterol/DMG-PEG 2000 (the molar ratio is 49:10:39.5:1.5) to encapsulate the eGFP-mRNA.
The results of cell transfection experiments show that the weight ratio of the vector to the mRNA is in the range of 10:1-30:1, and the vector has good transfection effect, wherein the transfection effect is preferably 15:1, the transfection effect is poor in the ratios of 5:1 and 35:1, and the mRNA cannot be carried by the ratio.
2. Cationic lipid and neutral lipid ratio optimization
LNP formulations encapsulating eGFP-mRNA were prepared according to the method in 1, with cationic lipid YK-702 (or YK-705, YK-712, YK-716) to neutral lipid DSPC molar ratios of 1:1, 3:1, 3.5:1, 4:1, 4.9:1, 10:1, 15:1 and 20:1, respectively.
As can be seen from the cell transfection experiment, the molar ratio of the cationic lipid to the neutral lipid is 1:1-15:1, and the transfection efficiency is 4.5:1.
3. Optimization of the proportion of Polymer conjugated lipid to Carrier (Liposome)
LNP formulations encapsulating eGFP-mRNA were prepared according to the method 1 with YK-702 (or YK-705, YK-712, YK-716) as the cationic lipid in the carrier, with 0.5%, 1.5%, 2.5%, 3.5%, 5%, 10% and 15% of the polymer conjugated lipid DMG-PEG2000 in the carrier molar ratios, respectively.
Cell transfection experiment results show that the polymer conjugated lipid accounts for 0.5% -10% of the carrier mole ratio, and has the transfection effect, and the transfection efficiency is highest when 1.5% and lowest when 10%.
4. Optimization of the ratio of the ingredients in the Carrier (Liposome)
LNP formulations encapsulating eGFP-mRNA were prepared according to the method in 1, with cationic lipid YK-702 (or YK-705, YK-712, YK-716), neutral lipid DSPC, structured lipid cholesterol, and polymer conjugated lipid DMG-PEG2000 molar ratios of 75:5:15:5, 65:8:25:2, 49:10:39.5:1.5, 45:10:43.5:1.5, 45:25:20:10, 40:10:48.5:1.5, 35:10:53.5:1.5, and 25:5:65:5, respectively.
As shown by cell transfection experiments, the cationic lipid, the neutral lipid, the structural lipid and the polymer conjugated lipid have good transfection effect in the ranges of the molar ratio of (35-49): (7.5-15): (35-55): (1-5), wherein the molar ratio of (45:10:43.5:1.5) and (49:10:39.5:1.5) and (45:10:43.5:1.5.
Example 3: LNP preparation of eGFP-mRNA cell transfection experiments
Cell resuscitating and passaging: 293T cells were resuscitated and passaged in petri dishes for culture to the desired cell numbers.
Seed plate: cells in the dishes were digested and counted, plated in 96-well plates at 1 ten thousand cells per well, plated in 12-well plates at 15 ten thousand cells per well, and cultured overnight until cells attached.
Cell transfection experiments: LNP preparations (cationic lipids in the vector were YK-702, YK-705, YK-712 or YK-716) containing 1.5. Mu.g of the eGFP-mRNA prepared in example 2 were added to the cell culture solution of 12-well plates, and after further culturing for 24 hours, the transfection efficiency of the samples was examined by fluorescence microscopy based on fluorescence intensity.
According to the experimental results, the preparation conditions of the nano lipid particles (LNP preparation) are finally determined: the ratio of the vector to the mRNA is 15:1; the molar ratio of the cationic lipid to the neutral lipid is 4.5:1; the polymer conjugated lipid accounts for 1.5% of the liposome; the molar ratio of cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid was 45:10:43.5:1.5, and the following experiments produced nanolipid particles (LNP formulation) under this condition.
Example 4: preparation of nanolipid particles (LNP formulation) (optimal formulation)
TABLE 2 cationic lipid Structure
YK-701, YK-702, YK-703, YK-704, YK-705, YK-706, YK-707, YK-708, YK-709, YK-710, YK-711, YK-712, YK-713, YK-714, YK-715, YK-716 were synthesized in example 1.
The cationic lipids listed in Table 2 and YK-701 to YK-716 were dissolved in ethanol with respect to DSPC (Ai Weita (Shanghai) medical science Co., ltd.), cholesterol (Ai Weita (Shanghai) medical science Co., ltd.) and DMG-PEG2000 respectively at a molar ratio of 45:10:43.5:1.5 to prepare an ethanol lipid solution, and the ethanol lipid solution was rapidly added to a citrate buffer (pH=4 to 5) by an ethanol injection method, and vortexed for 30s for use. The eGFP-mRNA (purchased from Shanghai laboratory reagent Co., ltd.) or the Fluc-mRNA (purchased from Shanghai laboratory reagent Co., ltd.) was diluted in a citrate buffer (pH=4 to 5) to obtain an aqueous mRNA solution. Liposomes were prepared by mixing a volume of liposome solution with an aqueous solution of mRNA at a weight ratio of total lipid to mRNA of 15:1. Ultrasound was performed at 25℃for 15min (ultrasound frequency 40kHz, ultrasound power 800W). The obtained liposome was diluted to 10 times of volume with PBS, and subjected to ultrafiltration in a 300kDa ultrafiltration tube to remove ethanol. The mixture was then sized to volume with PBS to give LNP formulations using cationic lipid/DSPC/cholesterol/DMG-PEG 2000 (mol% 45:10:43.5:1.5) to encapsulate eGFP-mRNA or Fluc-mRNA. Lipofectamine 3000 transfection reagent is widely used for cell transfection at present, has very good transfection performance and excellent transfection efficiency, can improve cell activity, and is suitable for cell types difficult to transfect. Lipofectamine 3000 preparation of eGFP-mRNA or Fluc-mRNA was prepared by the method described in Lipofectamine 3000 (Ind. Ipomoea) in the Ind. Of Ultrafrican trade, inc. by selecting Lipofectamine 3000 transfection reagent for comparison.
Example 5: determination of nanolipid particle size and polydispersity index (PDI)
Particle size and Polydispersity (PDI) were determined using dynamic light scattering using a malvern laser particle sizer.
10 μl of liposome solution was taken, diluted to 1mL with RNase-free deionized water, and added to the sample cell, and each sample was repeatedly assayed 3 times. The measurement conditions are as follows: 90. scattering angle, 25 ℃. The test results are shown in Table 3:
TABLE 3 particle size and polydispersity index (PDI) of nanolipid particles
As can be seen from table 3, the nano lipid particles prepared in example 4 have particle diameters of 114-203 nm, and can be used for delivering mRNA:
wherein, the particle size of the particles prepared from YK-703 is the smallest and is 114.56nm; the particle size of the particles prepared from YK-711 was the largest, 203.22nm.
The polydispersity of all the nano lipid particles is between 10% and 25%, wherein the minimum is YK-701, which is 11.2%; the maximum is YK-710, 23.1%.
The morphology of the particles prepared from YK-702, YK-705, YK-712, YK-716 is also at a good level.
Example 6: in vitro validation of LNP delivery vehicle performance
Cell resuscitating and passaging: the procedure is as in example 3.
Seed plate: the procedure is as in example 3.
1. Fluorescence detection of Fluc-mRNA (transfection efficiency)
LNP preparations containing 0.3. Mu.g of Fluc-mRNA (LNP preparation carrier composition: cationic lipid, DSPC, cholesterol and DMG-PEG2000, molar ratio: 45:10:43.5:1.5, wherein cationic lipid is the cationic lipid listed in Table 2) were added to cell culture broth of 96-well plates, and after further incubation for 24 h, the corresponding reagents were added according to Gaussia Luciferase Assay Kit instructions, and the intensity of fluorescence expression per well was detected by IVIS fluorescence detection system. The chemical structures of the designed compounds and the representative cationic lipids of the prior art are shown in Table 2. The transfection efficiency of LNP formulations prepared from a series of cationic lipid compounds designed herein, and prior art cationic lipids, including SM-102, MC3, HHMA, DLin-K-C2-DMA, and Compound 1, in cells is shown in Table 4.
TABLE 4 Fluc-mRNA fluorescence detection results
Analysis of experimental results:
(1) Compounds contemplated herein, including YK-702, YK-705, YK-712, and YK-716, have chemical structures that differ significantly from the prior art cationic lipids, such as SM-102, MC3, and HHMA; there are minor differences, such as DLin-K-C2-DMA and Compound 1.
The chemical structure of the compounds designed in this application is quite different and very different compared to the prior art representative cationic lipids SM-102, MC3 and HHMA (table 2). SM-102, MC3 and HHMA have no acetal structure, and the compounds designed by the application all have five-membered ring acetal structures, and other groups also have larger differences.
The chemical structure of the compounds contemplated herein is less different than prior art cationic lipids comprising acetal structures, such as DLin-K-C2-DMA and compound 1 (table 2). DLin-K-C2-DMA and the compound of the application all contain five-membered ring acetal structures, and other structures are slightly different; the acetal structure of compound 1 is a common branched structure, but both compound 1 and the compounds contemplated herein contain a hydroxyethylethylene tertiary amine structure.
(2) In a series of designed compounds, the LNP preparation prepared by YK-702, YK-705, YK-712 and YK-716 has highest cell transfection efficiency, and compared with the representative cationic lipid in the prior art, the LNP preparation has greatly different structures (SM-102, MC3 and HHMA) or very small structure difference (DLin-K-C2-DMA and compound 1), and the cell transfection efficiency is obviously improved. For example, YK-716 can be up to 6.73 times SM-102, 10.10 times 70.91 times HHMA for MC3, 16.84 times DLin-K-C2-DMA, 9.54 times Compound 1, and 12.41 times Lipofectamine 3000.
SM-102, MC3, HHMA, DLin-K-C2-DMA and Compound 1 are typical cationic lipids in the prior art, with good transfection properties.
As can be seen from Table 4, LNP preparations containing Fluc-mRNA prepared from YK-702, YK-705, YK-712 and YK-716 showed the strongest fluorescence absorption and RLU values of 5563052, 6512323, 9487230 and 10329850, respectively.
As can be seen from fig. 1 and 2, LNP formulations comprising eGFP-mRNA prepared from YK-702, YK-705, YK-712, YK-716 showed significantly enhanced cell transfection effects compared to the prior art representative cationic lipids, such as SM-102, MC3, HHMA, DLin-K-C2-DMA, compound 1 and Lipofectamine 3000.
The cell transfection efficiency of YK-702 can reach 3.63 times of SM-102, 38.19 times of MC3, 5.44 times of HHMA, 9.07 times of DLin-K-C2-DMA, 5.14 times of compound 1 and 6.68 times of Lipofectamine 3000, and the transfection efficiency is obviously improved.
The cell transfection efficiency of YK-705 can reach 4.24 times of SM-102, 44.70 times of MC3, 6.37 times of HHMA, 10.62 times of DLin-K-C2-DMA, 6.02 times of compound 1 and 7.82 times of Lipofectamine 3000, and the transfection efficiency is obviously improved.
The cell transfection efficiency of YK-712 can reach 6.18 times of SM-102, 65.12 times of MC3, 9.27 times of HHMA, 15.47 times of DLin-K-C2-DMA, 8.77 times of compound 1 and 11.40 times of Lipofectamine 3000, and the transfection efficiency is obviously improved.
The cell transfection efficiency of YK-716 can reach 6.73 times of SM-102, 70.91 times of MC3, 10.10 times of HHMA, 16.84 times of DLin-K-C2-DMA, 9.54 times of compound 1 and 12.41 times of Lipofectamine 3000, and the transfection efficiency is obviously improved.
The transfection efficiency of YK-710 cells was 0.96 times that of SM-102, 2.40 times that of DLin-K-C2-DMA and 1.36 times that of Compound 1, and the transfection efficiency was comparable.
The data were analyzed using GraphPad Prism software, and any of YK-702, YK-705, YK-712, and YK-716 was significantly different from SM-102, MC3, HHMA, DLin-K-C2-DMA, and Compound 1 and Lipofectamine 3000, with significantly improved transfection efficiency.
The cell transfection efficiency of LNP formulations prepared therefrom cannot be deduced from the structure of cationic lipid compounds, and is very likely to be very different, both from structurally different to structurally similar compounds.
(3) YK-702, YK-705 and YK-712 are similar to the structures and X 1 =O、X 2 =CH、R 3 =no substituent, R 4 Cell transfection efficiency was significantly improved compared to a series of compounds of =h. For example, YK-712 can be transfected with 380 times as efficiently as YK-703.
Will be of similar structure, X 1 =O、X 2 =CH、R 3 =no substituent, R 4 A series of compounds, e.g., YK-701, YK-703, YK-704, YK-713, YK-714 and YK-715, =H, were structurally different from YK-702, YK-705 and YK-712 only by slightly different individual groups (Table 1). Cell transfection results show that the activity of the series of compounds is very different, wherein the cell transfection efficiency of YK-702, YK-705 and YK-712 is the highest, and the cell transfection efficiency of YK-702 can reach 19.89 times of YK-701, 225.35 times of YK-703, 157.50 times of YK-704, 103.88 times of YK-713, 119.84 times of YK-714 and 252.51 times of YK-715 respectively. The cell transfection efficiency of YK-705 can reach 23.29 times of YK-701, 263.81 times of YK-703, 184.38 times of YK-704, 121.61 times of YK-713, 140.29 times of YK-714 and 295.60 times of YK-715 respectively. The cell transfection efficiency of YK-712 can reach 33.93 times of YK-701, 384.32 times of YK-703, 268.60 times of YK-704, 177.16 times of YK-713, 204.37 times of YK-714 and 430.63 times of YK-715 respectively, and the transfection efficiency is obviously improved.
(4) YK-716 is similar to structure and X 1 =O、X 2 =CH、R 2 =H、R 3 =no substituent, R 4 = C 8 Compared with the linear alkyl compound, the cell transfection efficiency is obviously improved. For example, YK-716 can be transfected up to 44.48 times YK-711.
Similar to the structure, X 1 =O、X 2 =CH、R 2 =H、R 3 =no substituent, R 4 = C 8 Compared with the linear alkyl compounds YK-708, YK-709, YK-710 and YK-711 (table 1), the transfection efficiency of the YK-716 cells is obviously improved, and the transfection efficiency can reach 5.63 times, 11.97 times, 7.03 times and 44.48 times of the transfection efficiency of the YK-708, YK-709, YK-710 and YK-711 respectively.
(5) YK-716 is similar to structure and X 1 =CH、X 2 =O、R 1 =C 8 Straight chain alkyl or- (CH) 2 ) 7 C(O)OCH 2 CH 3 、R 2 =H、R 3 =C 8 Straight chain alkyl, R 4 Cell transfection efficiency was significantly improved compared to the compounds without substituents. For example, YK-716 can be transfected with 8.04 times as much cells as YK-706.
Similar to the structure, X 1 =CH、X 2 =O、R 1 =C 8 Straight chain alkyl or- (CH) 2 ) 7 C(O)OCH 2 CH 3 、R 2 =H、R 3 =C 8 Straight chain alkyl, R 4 Compared with YK-706 and YK-707 (table 1), the transfection efficiency of YK-716 cells is obviously improved and can reach 8.04 times and 3.92 times of YK-706 and YK-707 respectively.
The data were analyzed using GraphPad Prism software, and any of YK-702, YK-705, YK-712, and YK-716 showed significant differences compared to the other compounds, with significantly improved transfection efficiency.
2. Cell viability assay
LNP preparations containing 1.5. Mu.g of Fluc-mRNA (LNP preparation carrier composition: cationic lipid, DSPC, cholesterol and DMG-PEG2000, molar ratio: 45:10:43.5:1.5, wherein cationic lipid is the cationic lipid listed in Table 2) were added to cell culture broth of 96-well plates, after further culturing for 24 h, 10. Mu.L of CCK-8 solution was added to each well, and after incubating the plates in an incubator for 1 h, absorbance at 450nm was measured by a microplate reader, and cell viability results are shown in Table 5.
TABLE 5 cell survival
Analysis of experimental results:
(1) The chemical structures of the series of compounds contemplated herein, including YK-702, YK-705, YK-711 and YK-716, differ greatly from the prior art cationic lipids, such as SM-102, MC3 and HHMA; there are structural similarities, for example DLin-K-C2-DMA and Compound 1 (Table 2). LNP formulations prepared from YK-705 and YK-716 have minimal cytotoxicity and significantly improved cell viability compared to the cationic lipids typical of the prior art. For example, YK-705 cell viability may be 8% higher than SM-102, 14% higher than MC3, 24% higher than HHMA, 21% higher than DLin-K-C2-DMA, 14% higher than Compound 1, 61% higher than Lipofectamine 3000. The cytotoxicity of LNP formulations prepared therefrom cannot be speculated on the basis of the structure of cationic lipid compounds, and there is a strong possibility that the cytotoxicity to transfected cells is very different, whether they are structurally different or structurally similar compounds.
(2) Will be of similar structure, X 1 =O、X 2 =CH、R 3 =no substituent, R 4 A series of compounds, e.g., YK-701, YK-702, YK-703, YK-704, YK-713, YK-714 and YK-715, =H, differing only slightly in their individual groups compared to YK-705 (Table 1), showed a very large difference in cytotoxicity, with YK-705 having the highest cell viability, 44% higher than YK-701, 10% higher than YK-702, 24% higher than YK-703, 11% higher than YK-704, 8% higher than YK-713, 11% higher than YK-715, and significantly improved cell viability.
(4) Similar to the structure, X 1 =O、X 2 =CH、R 2 =H、R 3 =no substituent, R 4 = C 8 Compared with the compounds YK-708, YK-709, YK-710 and YK-711 (table 1) of the linear alkyl, the survival rate of YK-716 cells is obviously improved,can be 15% higher than YK-708, 8% higher than YK-709, 23% higher than YK-710, and 10% higher than YK-711, respectively.
(5) Similar to the structure, X 1 =CH、X 2 =O、R 1 =C 8 Straight chain alkyl or- (CH) 2 ) 7 C(O)OCH 2 CH 3 、R 2 =H、R 3 =C 8 Straight chain alkyl, R 4 Compared with YK-706 and YK-707 (Table 1), the cell survival rates of YK-705 and YK-716 are significantly improved by 24% and 23% respectively, and 31% and 30% respectively, higher than that of YK-706. (FIG. 3)
Example 7: in vivo validation of cationic Lipid (LNP) delivery vehicle performance
It was verified that delivery vectors prepared from cationic lipids designed herein, such as YK-712 and YK-716, are capable of enriching in mouse liver and spleen, and that the delivered mRNA is significantly elevated in mouse liver and spleen protein expression compared to prior art cationic lipids. In vivo experiments further demonstrate that LNP delivery vectors prepared from the cationic lipids contemplated herein can efficiently deliver mRNA into animals and be expressed efficiently in the liver and spleen.
LNP preparations containing 10. Mu.g of Fluc-mRNA were injected into female BALB/C mice of 17-19 g weight, 4-6 weeks old, via tail vein, and the mice were given intraperitoneal injection of fluorography substrate at a specific time point (6 h) after administration, and the mice were free to move for 5 min, and then the average radiation intensity (corresponding to fluorescence expression intensity) of the protein expressed in the mice by the mRNA carried by LNP was detected by IVIS Spectrum small animal in vivo imager. After sampling, the mice were euthanized with carbon dioxide, dissected, and the internal organs of the mice were precisely isolated: heart, liver, spleen, lung, kidney. The average radiation intensity (corresponding to fluorescence expression intensity) of the mRNA carried by LNP in the protein expressed by each organ of the mice was measured by IVIS Spectrum small animal in vivo imaging instrument, and the results of the in vivo imaging measurement of the mice are shown in Table 6.
TABLE 6 in vivo imaging experimental data at a specific time point (6 h) after mouse dosing
Analysis of experimental results:
(1) LNP formulations prepared from YK-712 and YK-716, delivered mRNA levels expressed in the liver and spleen of mice, and in particular in the spleen, were significantly increased compared to the representative cationic lipids of the prior art. For example, YK-712 and YK-716 can be expressed in the spleen of mice up to 6.52-fold and 6.79-fold, respectively, of SM-102. mRNA was consistent with the results of cell transfection in example 6 in terms of mouse liver and spleen expression.
The spleen is the largest secondary lymphoid organ in the animal body, and the mRNA vaccine can rapidly induce immune response and generate antibodies in the body by improving the expression level of the delivered mRNA in the spleen. Can obviously improve the prevention effect without changing the vaccine components, and has important clinical significance.
In addition, LNP preparations containing Fluc-mRNA prepared from all compounds showed very large differences in expression in different organs of mice, YK-712, YK-716 and SM-102 were expressed in the liver and spleen, but not in other organs such as heart, lung and kidney. (FIG. 4).
(2) LNP preparations prepared from YK-712 and YK-716 showed the highest expression intensity of mRNA in the liver and spleen of mice compared to the compound YK-704, which was similar in structure and slightly different in individual groups. For example, YK-712 can be expressed in liver and spleen in 237.23-fold and 312.86-fold, respectively, and YK-716 can be expressed in liver and spleen in 242.76-fold and 325.87-fold, respectively, of YK-704. mRNA was consistent with cell transfection activity in terms of expression in mice.
In summary, the present application contemplates a range of cationic lipid compounds, e.g., YK-702, YK-705, YK-712, and YK-716, with significantly improved cell transfection efficiency, significantly reduced cytotoxicity, and significantly improved mRNA expression in the mouse spleen.
1. A series of compounds were designed, including YK-702, YK-705, YK-712 and YK-716, with chemical structures that were greatly different from the cationic lipids typical of the prior art, such as SM-102, MC3 and HHMA; there are structural similarities, such as DLin-K-C2-DMA and Compound 1.
2. In the designed series of compounds, LNP formulations prepared from YK-702, YK-705, YK-712 and YK-716 have significantly improved cell transfection efficiency, significantly reduced cytotoxicity and significantly improved mRNA expression in mouse liver and spleen compared to the cationic lipids typical in the prior art (including greatly different structures and similar structures). For example, cell transfection efficiency YK-716 can be up to 6.73 times that of SM-102, 70.91 times that of MC3, 10.10 times that of HHMA, 16.84 times that of DLin-K-C2-DMA, 9.54 times that of Compound 1, and 12.41 times that of Lipofectamine 3000; cell viability YK-705 was 8% higher than SM-102, 14% higher than MC3, 24% higher than HHMA, 21% higher than DLin-K-C2-DMA, 14% higher than Compound 1, 61% higher than Lipofectamine 3000; mRNA was expressed in the spleen of the mice, and YK-716 was 6.79 times as much as SM-102.
3. In a series of compounds with small chemical structure difference, LNP preparations prepared from YK-702, YK-705, YK-712 and YK-716 have remarkably improved cell transfection efficiency, remarkably reduced cytotoxicity and remarkably improved mRNA expression level in the spleen of mice compared with other compounds. For example, YK-716 cells can be transfected with 418.45 times of YK-703 and 468.88 times of YK-715, cytotoxicity can be reduced by 43% compared with YK-701, and mRNA expression in spleen of mice can be up to 325.87 times of YK-704.
4. Through unique design and screening, the invention discovers that certain compounds, such as YK-702, YK-705, YK-712 and YK-716, can improve the delivery efficiency with remarkably improved cell transfection efficiency, remarkably reduced cytotoxicity and remarkably improved expression level in the spleen of an animal body compared with other compounds with similar structures in the prior art, and has unexpected technical effects.
The present invention has been described in detail with the purpose of enabling those skilled in the art to understand the contents of the present invention and to implement the same, but not to limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit of the present invention should be covered in the scope of the present invention.

Claims (78)

1. A compound of formula (I)
Or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein
G 1 Is C 1~15 Alkylene or- (CH) 2 ) a C(O)O(CH 2 ) b -, wherein a and b are each an integer of 1 to 5;
G 2 is C 2~8 An alkylene group;
G 3 is C 1~4 An alkylene group;
R 1 is hydrogen atom, C 6~14 Straight chain alkyl or- (CH) 2 ) c C(O)O(CH 2 ) d CH 3 Wherein c is an integer from 3 to 12 and d is an integer from 1 to 4;
R 2 is hydrogen atom, C 6~14 Linear or branched alkyl;
R 3 is unsubstituted, hydrogen atom, C 6~14 Linear or branched alkyl;
R 4 is unsubstituted, hydrogen atom, C 6~14 Linear or branched alkyl;
R 5 is C 6~25 Linear or branched alkyl;
X 1 and X 2 Respectively a methine group or an oxygen atom.
2. A compound of formula (I) according to claim 1, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 1 Is unsubstituted C 1~12 An alkylene group.
3. A compound of formula (I) according to claim 2, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 1 Is unsubstituted C 1 Alkylene, C 5 Alkylene, C 8 Alkylene, C 4 Alkylene or C 12 An alkylene group.
4. A compound of formula (I) according to claim 1, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 1 Is- (CH) 2 ) 3 C(O)OCH 2 -。
5. A compound of formula (I) according to any one of the preceding claims, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 2 Is C 3~7 An alkylene group.
6. A compound of formula (I) according to claim 5, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 2 Is C 5 Alkylene or C 7 An alkylene group.
7. A compound of formula (I) according to claim 5, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 3 Is C 2~3 An alkylene group.
8. A compound of formula (I) according to claim 7, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 3 Is C 2 An alkylene group.
9. A compound of formula (I) according to any one of claims 1 to 4, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 1 Is C 5-13 A linear alkyl group.
10. A compound of formula (I) according to claim 9, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 1 Is C 10 、C 8 、C 7 、C 9 Or C 13 A linear alkyl group.
11. The method according to any one of claims 1-4A compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 1 Is- (CH) 2 ) 11 C(O)OCH 2 CH 3 Or- (CH) 2 ) 7 C(O)OCH 2 CH 3 。
12. A compound of formula (I) according to any one of claims 1 to 4, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 2 Is a hydrogen atom.
13. A compound of formula (I) according to any one of claims 1 to 4, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 2 Is C 10 、C 8 、C 7 Or C 9 A linear alkyl group.
14. A compound of formula (I) according to any one of claims 1 to 4, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 3 Is unsubstituted.
15. A compound of formula (I) according to any one of claims 1 to 4, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 3 Is C 7~12 A linear alkyl group.
16. A compound of formula (I) according to claim 15, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 3 Is C 8 A linear alkyl group.
17. A compound of formula (I) according to any one of claims 1 to 4, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 4 Is a hydrogen atom.
18. A compound of formula (I) or according to any one of claims 1 to 4An N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 4 Is C 7~12 A linear alkyl group.
19. A compound of formula (I) according to claim 18, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 4 Is C 8 A linear alkyl group.
20. A compound of formula (I) according to any one of claims 1 to 4, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 4 Is unsubstituted.
21. A compound of formula (I) according to any one of claims 1 to 4, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 5 Is C 6~15 A linear alkyl group.
22. A compound of formula (I) according to claim 21, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 5 Is C 11 Or C 10 A linear alkyl group.
23. A compound of formula (I) according to any one of claims 1 to 4, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 5 Is C 10~22 Branched alkyl groups.
24. A compound of formula (I) according to claim 23, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 5 Is C 17 Branched alkyl groups.
25. A compound of formula (I) according to claim 24, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 5 Is that。
26. A compound of formula (I) according to any one of claims 1 to 4, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein X 1 Is a methine group.
27. A compound of formula (I) according to claim 14, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein X 1 Is an oxygen atom.
28. A compound of formula (I) according to any one of claims 1 to 4, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein X 2 Is a methine group.
29. A compound of formula (I) according to claim 20, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein X 2 Is an oxygen atom.
30. The compound of formula (I) according to claim 1, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) has one of the following structures:
，
，
，
，
，
，
，
，
，
，
，
，
，
，
，
。
31. the compound of formula (I) according to claim 1, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) is compound YK-702 having the structure:
。
32. the compound of formula (I) according to claim 1, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) is compound YK-705 having the structure:
。
33. the compound of formula (I) according to claim 1, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) is compound YK-712 having the structure:
。
34. the compound of formula (I) according to claim 1, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) is a compound YK-716 having the structure:
。
35. A composition comprising a carrier comprising a cationic lipid comprising a compound of formula (I) according to any one of claims 1-34 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof.
36. The composition of claim 35, wherein the cationic lipid comprises 25% -75% of the carrier by mole.
37. The composition of claim 35, wherein the carrier further comprises a neutral lipid.
38. The composition of claim 37, wherein the molar ratio of the cationic lipid to the neutral lipid is 1:1-15:1.
39. The composition of claim 38, wherein the molar ratio of the cationic lipid to the neutral lipid is 4.5:1.
40. The composition of claim 37, wherein the neutral lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramides, sterols, and derivatives thereof.
41. The composition of claim 40, wherein the neutral lipid is selected from one or more of the following: 1, 2-Dioleoyl-sn-glycero-3-phosphorylcholine (DLPC), 1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dioleoyl-sn-glycero-phosphorylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-octadecenyl-sn-glycero-3-phosphorylcholine (18:0 DietherPC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-3-phosphorylcholine (OChems PC), 1-hexadecyl-sn-3-phosphorylcholine (C16), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1-dioleoyl-2-dioleoyl-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-sn-glycero-3-phosphate ethanolamine (DOPE), 1, 2-di-phytanoyl-sn-glycero-3-phosphate ethanolamine (ME 16.0 PE), 1, 2-di-stearoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-oleoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-linolenoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-arachidonoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-docosahexaenoic acyl-sn-glycero-3-phosphate ethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphate sodium salt (DOPG), 1, 2-di-oleoyl-rac-3-phosphate sodium salt (DOPG) dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl-based phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof.
42. The composition of claim 41, wherein the neutral lipid is DOPE and/or DSPC.
43. The composition of claim 35, wherein the carrier further comprises a structural lipid.
44. The composition of claim 43, wherein the molar ratio of the cationic lipid to the structural lipid is 0.6:1-3:1.
45. The composition of claim 43, wherein the structural lipid is selected from one or more of the following: cholesterol, non-sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycorine, ursolic acid, alpha-tocopherol, corticosteroids.
46. The composition of claim 45, wherein the structural lipid is cholesterol.
47. The composition of claim 35, wherein the carrier further comprises a polymer conjugated lipid.
48. The composition of claim 47, wherein the polymer conjugated lipid comprises 0.5% -10% of the carrier by mole.
49. The composition of claim 48, wherein the polymer conjugated lipid comprises 1.5% of the carrier by mole.
50. The composition of claim 47, wherein the polymer conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol.
51. The composition of claim 50, wherein the polymer conjugated lipid is selected from one or more of the following: distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG 2000), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000) and methoxypolyethylene glycol ditetradecylamide (ALC-0159).
52. The composition of claim 35, wherein the carrier comprises neutral lipids, structural lipids, and polymer conjugated lipids in a molar ratio of (25-75): 5-25): 15-65): 0.5-10.
53. The composition of claim 52, 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.
54. The composition of claim 53, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer conjugated lipid is 45:10:43.5:1.5.
55. The composition of claim 35, wherein the composition is a nanoparticle formulation having an average particle size of 10nm to 300nm; the polydispersity of the nanoparticle preparation is less than or equal to 50 percent.
56. The composition of claim 55, wherein the composition is a nanoparticle formulation having an average particle size of 90nm to 280nm; the polydispersity of the nanoparticle preparation is less than or equal to 45 percent.
57. The composition of claim 35, wherein the cationic lipid further comprises one or more other ionizable lipid compounds.
58. The composition of claim 35, further comprising a therapeutic or prophylactic agent.
59. The composition of claim 58, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 10:1-30:1.
60. The composition of claim 59, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 12.5:1-20:1.
61. The composition of claim 60, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 15:1.
62. The composition of claim 58, wherein the therapeutic or prophylactic agent comprises one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.
63. The composition of claim 58, wherein the therapeutic or prophylactic agent is a vaccine or compound capable of eliciting an immune response.
64. The composition of claim 62, wherein the therapeutic or prophylactic agent is a nucleic acid.
65. The composition of claim 64, wherein the therapeutic or prophylactic agent is ribonucleic acid (RNA).
66. The composition of claim 64, wherein the therapeutic or prophylactic agent is deoxyribonucleic acid (DNA).
67. The composition of claim 65, wherein the RNA is selected from the group consisting of: small interfering RNAs (siRNA), asymmetric interfering RNAs (aiRNA), micrornas (miRNA), dicer-substrate RNAs (dsRNA), small hairpin RNAs (shRNA), messenger RNAs (mRNA), and mixtures thereof.
68. The composition of claim 67, wherein said RNA is mRNA.
69. The composition of any one of claims 35-68, wherein the composition further comprises one or more pharmaceutically acceptable excipients.
70. Use of a compound of formula (I) according to any one of claims 1 to 34 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof or a composition according to any one of claims 35 to 69 in the manufacture of a nucleic acid medicament, a genetic vaccine, a small molecule medicament, a polypeptide or a protein medicament.
71. Use of a compound of general formula (I) as defined in any one of claims 1 to 34 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof or a composition as defined in any one of claims 35 to 69 in the manufacture of a medicament for the treatment of a disease or condition in a mammal in need thereof.
72. The use of claim 71, wherein the disease or disorder is characterized by dysfunctional or abnormal protein or polypeptide activity.
73. The use of claim 71, 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.
74. The use of claim 73, wherein the infectious disease is selected from the group consisting of: diseases caused by coronavirus, influenza virus or HIV virus, pediatric pneumonia, rift valley fever, yellow fever, rabies, or various herpes.
75. The use of any one of claims 71-74, wherein the mammal is a human.
76. The use of any one of claims 71-74, wherein the medicament is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.
77. The use of claim 76, wherein the composition is administered subcutaneously.
78. The use of any one of claims 71-74, wherein a dose of about 0.001mg/kg to about 10mg/kg of the medicament is administered to the mammal.
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