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CN118125947A - Low-toxicity spleen-targeted cationic lipid compound containing carbamate structure, composition containing low-toxicity spleen-targeted cationic lipid compound and application of low-toxicity spleen-targeted cationic lipid compound - Google Patents

Low-toxicity spleen-targeted cationic lipid compound containing carbamate structure, composition containing low-toxicity spleen-targeted cationic lipid compound and application of low-toxicity spleen-targeted cationic lipid compound Download PDF

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CN118125947A
CN118125947A CN202410249824.6A CN202410249824A CN118125947A CN 118125947 A CN118125947 A CN 118125947A CN 202410249824 A CN202410249824 A CN 202410249824A CN 118125947 A CN118125947 A CN 118125947A
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compound
composition
lipid
glycero
formula
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于飞
宋更申
张宏雷
刘洋健
李雨晴
张万年
张国亮
李静
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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 present invention provides a compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, as well as compositions comprising the foregoing compounds and their use for delivering therapeutic or prophylactic agents.

Description

Low-toxicity spleen-targeted cationic lipid compound containing carbamate structure, composition containing low-toxicity spleen-targeted cationic lipid compound and application of low-toxicity spleen-targeted cationic lipid compound
Technical Field
The invention belongs to the field of medicines, and particularly relates to a cationic lipid compound, a composition containing the cationic lipid compound and application of the cationic lipid compound.
Background
Efficient targeted delivery of biologically active substances such as small molecule drugs, polypeptides, proteins and nucleic acids, especially nucleic acids, is a persistent medical challenge. Nucleic acid therapeutics face significant challenges due to low cell permeability and high sensitivity to degradation by certain nucleic acid molecules, including RNA.
Compositions, liposomes and liposome complexes (lipoplex) containing cationic lipids have been demonstrated to be effective as transport vehicles for transporting biologically active substances such as small molecule drugs, polypeptides, proteins and nucleic acids into cells and/or intracellular compartments. These compositions generally comprise one or more "cationic" and/or amino (ionizable) lipids, including neutral lipids, structural lipids, and polymer conjugated lipids. Cationic and/or ionizable lipids include, for example, amine-containing lipids that can be readily protonated. While a variety of such lipid-containing nanoparticle compositions have been shown, safety, efficacy and specificity remain to be improved. Notably, the increased complexity of lipid nanoparticles (Lipid Nanoparticle, LNP) complicates their production and may increase their toxicity, a major concern that may limit their clinical use. For example, LNP siRNA particles (e.g., patisiran) require the prior use of steroids and antihistamines to eliminate the unwanted immune response (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.). and thus, there is a need to develop improved cationic lipid compounds that facilitate the delivery of therapeutic and/or prophylactic agents, such as nucleic acids, to cells, and compositions comprising the same.
Disclosure of Invention
The present invention provides a tertiary carbamate-based cationic lipid compound, including pharmaceutically acceptable salts thereof and stereoisomers or tautomers thereof. Enriches the variety of cationic lipid compounds and provides more choices for the effective delivery of nucleic acid drugs, genetic vaccines, small molecule drugs, polypeptides or protein drugs. When formed into lipid nanoparticles with other lipid components, can effectively deliver mRNA or drug molecules into cells to perform biological functions.
In a first aspect, the present disclosure provides a cationic lipid compound that is a compound of formula (I)Or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein:
G 1 is C 2～10 alkylene;
g 2 is C 2～10 alkylene;
g 3 is
R 1 is unsubstituted C 6～25 straight or branched alkyl;
R 2 is unsubstituted C 6～25 straight or branched alkyl;
m and n are 1 or 0, n is 1 when m is 0, and n is 0 when m is 1.
An alternative compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 1 is C 5、C7 or C 3 alkylene.
An alternative compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 2 is C 5、C3 or C 7 alkylene.
An alternative compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 1 is unsubstituted C 8～22 straight chain alkyl.
An alternative compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 1 is unsubstituted C 10、C11、C8、C9 or C 12 straight chain alkyl.
An alternative compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 1 is unsubstituted C 20、C15、C17 branched alkyl.
An alternative compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 1 is
An alternative compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 2 is unsubstituted C 8～22 straight chain alkyl.
An alternative compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 2 is unsubstituted C 10、C11、C8、C9 or C 12 straight chain alkyl.
An alternative compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 2 is unsubstituted C 20、C15、C17 branched alkyl.
An alternative compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 2 is
An alternative compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) has one of the following structures:
An alternative compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) is compound YK-803 having the structure:
An alternative compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) is compound YK-810 having the structure:
An alternative compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) is compound YK-813 having the structure:
In a second aspect, the present invention provides a composition comprising a carrier comprising a cationic lipid comprising a compound of formula (I) as described in any one of the preceding first aspects or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof.
Further compositions wherein the cationic lipid comprises from 25% to 75% by mole of the carrier.
A further composition, wherein the carrier further comprises a neutral lipid.
Further compositions wherein the molar ratio of the cationic lipid to the neutral lipid is from 1:1 to 15:1, preferably 4.5:1.
Further compositions, wherein the neutral lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols, and derivatives thereof.
A further composition, 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 (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphorylcholine (C), 1, 2-dioleoyl-s-glycero-3-phosphorylcholine (Lyso PC), 1, 2-dioleoyl-2-glycero-3-phosphorylcholine, 1-dioleoyl-s-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-glycero-s-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-docosahexaenoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-dioleoyl-sn-glycero-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.
Further compositions wherein the neutral lipid is 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and/or 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
Further compositions, wherein the carrier further comprises a structural lipid.
Further compositions, wherein the molar ratio of the cationic lipid to the structural lipid is from 0.6:1 to 3:1.
A further composition, 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.
A further composition, wherein the structural lipid is cholesterol.
Further compositions, wherein the carrier further comprises a polymer conjugated lipid.
Further compositions wherein the polymer conjugated lipid comprises 0.5% to 10%, preferably 1.5% of the carrier by mole.
A further composition, 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.
A further composition, 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).
Further compositions, wherein the carrier comprises a cationic lipid, a neutral lipid, a structural lipid, and a polymer conjugated lipid. The cationic lipid: neutral lipids: structural lipids: the molar ratio of the conjugated polymer lipid is (25-75): 5-25): 15-65): 0.5-10.
A further composition, wherein the cationic lipid: neutral lipids: structural lipids: the molar ratio of the conjugated polymer lipid is (35-49): 7.5-15): 35-55): 1-5.
A further composition, wherein the cationic lipid: neutral lipids: structural lipids: the molar dosage ratio of the polymer conjugated lipid is 45:10:43.5:1.5.
A further composition, wherein the composition is a nanoparticle formulation having an average particle size of 10nm to 210nm; the polydispersion coefficient (PDI) of the nanoparticle preparation is less than or equal to 50 percent.
A further composition, wherein the nanoparticle formulation has an average particle size of 100nm to 205nm; the polydispersion coefficient (PDI) of the nanoparticle preparation is less than or equal to 30 percent.
Further compositions, wherein the cationic lipid further comprises one or more other ionizable lipid compounds.
Further compositions, wherein a therapeutic or prophylactic agent is also included.
Further composition, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 10:1 to 30:1.
Further composition, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 12.5:1 to 25:1.
Further compositions, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 15:1.
Further compositions, wherein the therapeutic or prophylactic agent comprises one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.
A further composition, wherein the therapeutic or prophylactic agent is a vaccine or compound capable of eliciting an immune response.
Further compositions, wherein the therapeutic or prophylactic agent is a nucleic acid.
Further compositions, wherein the therapeutic or prophylactic agent is ribonucleic acid (RNA).
Further compositions, wherein the therapeutic or prophylactic agent is deoxyribonucleic acid (DNA).
A further composition, 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.
Further compositions, wherein the RNA is mRNA.
Further compositions, wherein the compositions further comprise one or more pharmaceutically acceptable excipients or diluents.
In a third aspect, the present invention provides the use of a compound of formula (I) as described in any one of the preceding first aspects or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof or a composition as described in any one of the preceding second aspects in the manufacture of a nucleic acid medicament, a genetic vaccine, a small molecule medicament, a polypeptide or a protein medicament.
In a fourth aspect, the present invention provides the use of a compound of formula (I) as described in any one of the preceding first aspects or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof or a composition as described in any one of the preceding second aspects in the manufacture of a medicament for the treatment of a disease or condition in a mammal in need thereof.
Preferred uses, wherein the disease or condition is characterized by dysfunctional or abnormal protein or polypeptide activity.
A preferred use, 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.
Preferred uses, wherein the infectious disease is selected from: diseases caused by coronavirus, influenza virus or HIV virus, pediatric pneumonia, rift valley fever, yellow fever, rabies, or various herpes.
Preferred uses wherein the subject to which the medicament is administered is a human.
Preferred uses, wherein the route of administration of the drug is intravenous, intramuscular, intradermal, subcutaneous, intranasal or inhalation.
Preferred uses, wherein the route of administration of the drug is subcutaneous.
Preferably, the medicine is applied in the dosage of 0.001-10 mg/kg.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the following brief description of the drawings of the embodiments will make it apparent that the drawings in the following description relate only to some embodiments of the present disclosure, not to limit the present disclosure.
FIG. 1 shows the results of cell transfection experiments of LNP preparations of Fluc-mRNA prepared based on YK-803, YK-810, YK-813, SM-102, P-76 and Compound 13, wherein: a is YK-803, b is YK-810, c is YK-813, d is SM-102, e is P-76, and f is Compound 13.
FIG. 2 shows the results of cell transfection fluorescence detection of LNP preparations of Fluc-mRNA prepared from different cationic lipids (YK-801、YK-802、YK-803、YK-804、YK-805、YK-806、YK-807、YK-808、YK-809、YK-810、YK-811、YK-812、YK-813、YK-814、SM-102、MC3、HHMA、P-76、, compound 13 and Lipofectamine 3000).
FIG. 3 shows cell viability after addition of LNP preparations of Fluc-mRNA prepared from different cationic lipids (YK-801、YK-802、YK-803、YK-804、YK-805、YK-806、YK-807、YK-808、YK-809、YK-810、YK-811、YK-812、YK-813、YK-814、SM-102、MC3、HHMA、P-76、, compound 13 and Lipofectamine 3000) to cell culture broth for 24 h.
FIG. 4 shows LNP preparations of Fluc-mRNA prepared from different cationic lipids (SM-102, YK-803, YK-810, YK-813) expressed in the liver, spleen, lung, heart and kidney of mice.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present disclosure more apparent, the technical solutions of the embodiments of the present disclosure will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present disclosure. It will be apparent that the described embodiments are some, but not all, of the embodiments of the present disclosure. All other embodiments, which can be made by one of ordinary skill in the art without the benefit of the present disclosure, are intended to be within the scope of the present invention based on the described embodiments.
The present invention may be embodied in other specific forms without departing from its essential attributes. It is to be understood that any and all embodiments of the invention may be combined with any other embodiment or features of multiple other embodiments to yield yet further embodiments without conflict. The invention includes additional embodiments resulting from such combinations.
All publications and patents mentioned in this disclosure are incorporated herein by reference in their entirety. If a use or term used in any of the publications and patents incorporated by reference conflicts with the use or term used in the present disclosure, the use or term of the present disclosure controls.
The section headings used herein are for purposes of organizing articles only and should not be construed as limiting the subject matter.
Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning in the art to which the claimed subject matter belongs. In case there are multiple definitions for a term, the definitions herein control.
Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of quantitative quality such as doses stated in the specification and claims are to be understood as being modified in all instances by the term "about". It should also be understood that any numerical range recited herein is intended to include all sub-ranges within that range and any combination of the individual endpoints of that range or sub-range.
The use of the terms "comprising," "including," or "containing," and the like, in this disclosure, are intended to cover an element listed after that term and its equivalents, but do not exclude the presence of other elements. The terms "comprising" or "including" as used herein, can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …", or "consisting of …".
The term "pharmaceutically acceptable" in the present 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 both humans and mammals in the present 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 the present application refers to a complex formed by combining a compound of formula (I) or a pharmaceutically acceptable salt thereof and a solvent (e.g. ethanol or water). It will be appreciated that any solvate of a compound of formula I used in the treatment of a disease or condition, although potentially providing different properties (including pharmacokinetic properties), will result in a compound of formula I upon absorption into a subject such that the use of a compound of formula I encompasses the use of any solvate of a compound of formula I, respectively.
The term "hydrate" refers to the case where the solvent in the above term "solvate" is water.
It is further understood that the compound of formula I or a pharmaceutically acceptable salt thereof may be isolated in the form of a solvate, and thus any such solvate is included within the scope of the present invention. For example, a compound of formula I or a pharmaceutically acceptable salt thereof may exist in unsolvated forms as well as solvated forms with pharmaceutically acceptable solvents (such as water, ethanol, and the like).
The term "pharmaceutically acceptable salt" refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the present disclosure. See, for example, S.M. Bere 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 disclosure may be converted to N-oxides by treatment with an oxidizing agent (e.g., m-chloroperoxybenzoic acid, hydrogen peroxide, ozone). Thus, the compounds claimed in the present application include not only nitrogen-containing compounds of the formula but also N-oxide derivatives thereof, under conditions of valence and structural permission.
Certain compounds of the present disclosure may exist in the form of one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers and enantiomers. Thus, the presently claimed compounds also include racemic mixtures, single stereoisomers, and optically active mixtures. It will be appreciated by those skilled in the art that one stereoisomer may have better efficacy and/or lower side effects than the other stereoisomers. The single stereoisomers and the mixture with optical activity can be obtained by chiral source synthesis methods, chiral catalysis methods, chiral resolution methods and the like. The racemate can be chiral resolved by chromatographic resolution or chemical resolution. For example, separation can be performed by adding chiral acid resolving agents such as chiral tartaric acid, chiral malic acid, and the like to form salts with the compounds of the present disclosure, utilizing differences in the physicochemical properties of the products, such as solubility.
The invention also includes all suitable isotopic variations of the compounds of the present disclosure. Isotopic variations are defined as compounds in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found or predominantly present in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, and oxygen, such as 2 H (deuterium), 3 H (tritium), 11C、13C、14C、15N、17 O, and 18 O, respectively.
The term "alkyl" is meant in this disclosure to include both branched and straight chain saturated aliphatic monovalent hydrocarbon groups having the specified number of carbon atoms. The term "alkylene" is meant in this disclosure to include both branched and straight chain saturated aliphatic divalent hydrocarbon groups having the specified number of carbon atoms. C n～m is meant to include groups having a number of carbon atoms from n to m. For example, C 2～5 alkylene includes C 2 alkylene, C 3 alkylene, C 4 alkylene, C 5 alkylene.
The alkyl (or alkylene) group may be unsubstituted, or the alkyl (or alkylene) group may be substituted, wherein at least one hydrogen is replaced with another chemical group.
A "therapeutically effective amount" is an amount of a therapeutic agent that, when administered to a patient, ameliorates a disease or condition. A "prophylactically effective amount" is an amount of a prophylactic agent that, when administered to a subject, prevents a disease or condition. The amount of therapeutic agent constituting the "therapeutically effective amount" or the amount of prophylactic agent of the "prophylactically effective amount" varies with the therapeutic agent/prophylactic agent, the disease state and severity thereof, the age, weight, etc. of the patient/subject to be treated/prevented. One of ordinary skill in the art can routinely determine therapeutically effective and prophylactically effective amounts based on their knowledge and disclosure.
In the present application, when the names of the compounds are not identical to the structural formulae, the structural formulae are determined.
It is to be understood that the term "presently disclosed compounds" as used herein may include, depending on the context: a compound of formula (I), an N-oxide thereof, a solvate thereof, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, and mixtures thereof.
The term cationic lipid as used herein refers to a lipid that is positively charged at a selected pH.
Cationic liposomes readily bind to negatively charged nucleic acids, i.e., interact with negatively charged phosphate groups present in the nucleic acids by electrostatic forces, forming Lipid Nanoparticles (LNPs). LNP is one of the currently mainstream delivery vehicles.
The inventors found that, when screening a large number of compounds, it is very difficult to screen out a suitable cationic lipid compound satisfying the following conditions: the structural difference of the cationic lipid is huge compared with the prior art, and the cationic lipid has extremely high transfection efficiency, extremely low cytotoxicity, high expression and continuous expression in mice. By unique design, the present disclosure finds that some compounds, such as YK-803, YK-810 and YK-813, can deliver nucleic acids with significantly improved intracellular transfection efficiency, significantly reduced cytotoxicity, and significantly improved expression levels and durations in animals compared to cationic lipids with greatly different chemical structures in the prior art, thereby achieving rapid induction of immune responses and antibody production in mRNA vaccines. The vaccine composition has the significant clinical significance and can obviously improve the prevention effect under the condition of not changing the vaccine components.
Briefly, the present invention is based on at least the following findings:
1. A series of cationic lipid compounds were designed, including YK-803, YK-810 and YK-813, with a vast difference in chemical structure from the prior art cationic lipids, such as SM-102, DLin-MC3-DMA (MC 3) and HHMA; some of the chemical structures are similar, for example compound 13.
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 Alamer pharmaceutical (Alnylam Pharmaceuticals, inc.).
HHMA is a cationic lipid compound disclosed in CN112979483B (page 7 of the specification) by the company of ebb biotechnology, su.
P-76 is a cationic lipid compound disclosed in CN113402405B (page 46 of the specification) by Xiaomenobang Biotech Co.
Compound 13 is a cationic lipid compound disclosed in CN114728016a (page 37 of the specification) of fuji film co.
Representative cationic lipids and structurally similar cationic lipids of the prior art have the following chemical structures:
(WO 20170409245A2, page 29 of the specification);
(CN 102625696B, page 6 of the specification, compound of formula I.)/> (CN 112979483B, description page 12)
(CN 113402405B, page 46 of the specification);
(CN 114728016a, page 37 of the specification);
2. in the designed series of compounds, LNP preparations prepared from YK-803, YK-810 and YK-813 have significantly improved cell transfection efficiency, significantly reduced cytotoxicity, significantly improved expression level and duration of mRNA in mice, significantly improved expression level in the spleen of mice and significantly reduced expression level in the liver compared with the typical and structurally similar cationic lipids in the prior art. For example, cell transfection efficiency YK-810 can be up to 5.17 times that of SM-102, 51.99 times that of MC3, 7.97 times that of HHMA, 52.56 times that of P-76, 189.53 times that of Compound 13, and 11.23 times that of Lipofectamine 3000; cell viability YK-810 may be 8% higher than SM-102, 12% higher than MC3, 23% higher than HHMA%, 15% higher than P-76, 26% higher than Compound 13, 60% higher than Lipofectamine 3000; mRNA expression in mice can reach 5.78 times of SM-102, 26.19 times of P-76 and 124.86 times of compound 13 in 24 hours, and can reach 14.93 times of SM-102, 59.48 times of P-76 and 215.00 times of compound 13 in 7 days; mRNA expression level YK-810 in spleen can reach 15.02 times of SM-102, and expression level YK-810 in liver is only 0.08 times of SM-102.
3. In a series of compounds with small chemical structure difference, LNP preparations prepared from YK-803, YK-810 and YK-813 have significantly improved cell transfection efficiency and significantly reduced cytotoxicity compared with other compounds; the expression amount and duration of mRNA in mice are obviously improved. For example, the present series of compounds structurally differ only slightly in part from YK-803, YK-810 and YK-813, but YK-810 cells have a transfection activity as high as 2020 times that of YK-814 and 1390 times that of YK-805; cytotoxicity YK-810 can be reduced by 39% compared to YK-814; mRNA expression in mice can reach 200 times YK-802.
The structure and intracellular transfection efficiency of the cationic lipid compound, toxicity to cells and no obvious correspondence between high and continuous expression of mRNA in animals in LNP preparations prepared from the cationic lipid compound. Compounds with small structural differences are highly likely to differ greatly in transfection efficiency and/or cytotoxicity, as well as intracellular expression. Therefore, screening for suitable cationic lipid compounds can simultaneously have high cell transfection efficiency, low cytotoxicity, high and sustained expression of mRNA in animals, and high expression in the spleen of animals and low expression in the liver, which is a very difficult task and requires a great deal of creative labor.
4. Through unique design and screening, the present disclosure discovers that some compounds, such as YK-803, YK-810 and YK-813, can significantly improve cell transfection efficiency, significantly reduce cytotoxicity, significantly improve expression level and expression time in animals, significantly improve expression level in the spleen of animals and significantly reduce expression level in the liver, improve delivery efficiency, and achieve unexpected technical effects compared with other compounds similar in structure in the prior art.
5. Through unique design and screening, the present disclosure finds that some compounds, such as YK-803, YK-810 and YK-813, can target mRNA to the spleen without expression in other organs, such as the lung, heart and kidney, and with small expression in the liver under the premise of ensuring high efficiency and low toxicity. The low immune response induction efficiency of the vaccine is the reason that the existing tumor therapeutic vaccine cannot exert the maximum curative effect, the spleen is the largest secondary lymphoid organ in the body, the LNP tumor vaccine targeting the spleen can effectively excite the immune response, the curative effect is obviously improved, and the vaccine has important clinical application significance in cancer treatment.
In summary, the present disclosure, through unique design and screening, has discovered compounds such as YK-803, YK-810, and YK-813. These compounds, whether of a large chemical structure, such as SM-102, MC3 and HHMA, or of a smaller structure, such as P-76 and compound 13, are capable of delivering nucleic acids with significantly improved cell transfection efficiency, significantly reduced cytotoxicity, significantly improved expression levels and duration in animals, significantly improved mRNA expression levels in the spleen and significantly reduced expression levels in the liver, compared to the prior art representative cationic lipids. Unexpected technical effects are achieved.
The method comprises the following steps:
1. The chemical structure of the compounds designed by the application is greatly different from that of the typical cationic lipids in the prior art, such as SM-102, MC3 and HHMA; there is little difference, such as compound 13.
This series of compounds was designed in comparison to prior art representative cationic lipids, such as SM-102, MC3, HHMA, P-76 and compound 13:
1) The chemical structure of the compounds designed according to the application is significantly different compared to SM-102, MC3 and HHMA. From the chemical structural formula, the series of compounds of the application are introduced into carbamate-OC (O) N (G 1) -, and are connected with the head part of tertiary amine structure and the long alkyl straight-chain or branched-chain tail part containing ester group through formate, while SM-102, MC3 and HHMA have no carbamate structure; the amino head structure of SM-102 is of an ethanolamine type with a simple structure, the amino head of MC3 is of a dimethyl tertiary amine structure, the amino head structure of HHMA is of a methyl tertiary amine structure, the hydroxyl structure is positioned at the 2-position of a fatty chain, and the series of compounds provided by the application have tertiary amine head groups with rich structure types.
2) Both P-76 and Compound 13 and the contemplated compounds of the present application comprise a urethane structure, wherein P-76 contains two urethane groups and the urethane positions are in the hydrophobic tail branches; compound 13 is very similar in structure to the series of compounds of the present application, each containing a carbamate structure, each containing two hydrophobic tails, and each containing an ester linkage.
2. The transfection efficiency of cells in vitro is obviously improved compared with the typical cationic lipid and the compound with similar structure in the prior art.
1) Of the designed series of compounds, LNP formulations prepared from YK-803, YK-810 and YK-813 have the highest cell transfection efficiency, and compared with the representative cationic lipids in the prior art, the cell transfection efficiency is remarkably improved whether the structure is greatly different (such as SM-102, MC3 and HHMA) or the structure is slightly different (such as compound 13). For example, YK-810 can be transfected 5.17 times, HHMA times, 7.97 times, 52.56 times, 189.53 times of compound 13, and 11.23 times of Lipofectamine 3000 for SM-102.
2) A series of compounds having an ester linkage of similar structure, a carbamate structure, linked to a G 1 or G 2 group, including YK-801, YK-802, and YK-803, were compared. The urethane linkages of these compounds are linked to the G 1 or G 2 groups, with the other structures differing only slightly from the individual groups. Cell transfection results show that the activity of the series of compounds is very different, and the cell transfection efficiency of YK-803 is highest. The cell transfection efficiency of YK-803 can reach 35.36 times of YK-801 and 93.24 times of YK-802.
3) A series of compounds having an ester linkage of similar structure, a carbamate structure, and a G 3 group, including YK-804, YK-805, YK-806, YK-807, YK-808, YK-809, YK-810, YK-811, YK-812, YK-813, and YK-814, were compared. The urethane linkages of these compounds are each linked to a G 3 group, with the other structures differing only slightly from each other. Cell transfection results show that the activity of the series of compounds is very different, and the cell transfection efficiency of YK-810 and YK-813 is highest. For example, the cell transfection efficiencies of YK-810 and YK-813 can be 257.28 times, 1390.52 times and 597.75 times, 2020.10 times and 868.39 times, respectively, that of YK-804, 598.49 times that of YK-805.
4) There is no correspondence between the structure of the compound and the transfection efficiency of cells, and there is a great possibility that there is a great difference in the transfection efficiency of cells in the case of compounds having a small difference in structure. Therefore, screening for cationic lipids with high cell transfection efficiency is a very difficult task, requiring a great deal of creative effort.
3. Cytotoxicity is significantly reduced over the typical cationic lipids and structurally similar compounds of the prior art.
1) The chemical structures of the series of compounds designed by the application, including YK-803, YK-810 and YK-813, differ greatly from those of the prior art representative cationic lipids, such as SM-102, MC3 and HHMA; there are structures similar, for example, to compound 13. LNP formulations prepared from YK-803, YK-810 and YK-813 have minimal cytotoxicity and significantly improved cell viability compared to the typical cationic lipids of the prior art. For example, cell viability YK-810 may be 8% higher than SM-102, 12% higher than MC3, 23% higher than HHMA%, 15% higher than P-76, 26% higher than Compound 13, 60% higher than Lipofectamine 3000.
2) A series of compounds having an ester linkage of similar structure, a carbamate structure, linked to a G 1 or G 2 group, including YK-801, YK-802, and YK-803, were compared. These compounds differ only slightly in the individual groups. The result shows that YK-803 has the lowest cytotoxicity and the cell survival rate is obviously improved. For example, YK-803 may have a cell viability 18% higher than YK-801.
3) A series of compounds having an ester linkage of similar structure, a carbamate structure, and a G 3 group, including YK-804, YK-805, YK-806, YK-807, YK-808, YK-809, YK-810, YK-811, YK-812, YK-813, and YK-814, were compared. The results show that YK-810 and YK-813 have the lowest cytotoxicity, and the cell survival rate is remarkably improved. For example, the cell viability of YK-810 and YK813 was 38% and 35% higher than YK-804, 39% and 36% higher than YK-814, respectively.
4) There is no correspondence between the structure of the compound and cytotoxicity, and even compounds with very small structural differences are very likely to have a large difference in cytotoxicity. Therefore, it is very difficult to screen cationic lipids having low cytotoxicity, which cannot be predicted from chemical structures, and a lot of creative effort is required.
MRNA expression levels and duration in animals were significantly increased over the typical cationic lipids and structurally similar compounds of the prior art and showed targeting to the spleen.
1) LNP formulations prepared from YK-803, YK-810 and YK-813 significantly increased mRNA expression levels and durations in mice compared to the prior art representative cationic lipids, and at 6h, 24h, 48h and 7d compared to the prior art representative cationic lipids. For example, YK-810 may be up to 5.78 times SM-102, 26.19 times P-76, and 124.86 times compound 13 at 24 hours, YK-810 may be up to 7.97 times SM-102, 23.17 times P-76, and 123.44 times compound 13 at 48 hours, and YK-810 may be up to 14.93 times SM-102, 59.48 times P-76, and 215.00 times compound 13 at 7 days.
2) Compared with the compound YK-811 with similar structure and slightly different allopatric groups, the LNP preparation prepared from YK-803, YK-810 and YK-813 has obviously improved expression quantity and duration of mRNA in mice. For example, YK-810 may be 130.17 times YK-802 at 24 hours, 200.74 times at 48 hours, and 110.86 times at 7 days.
3) LNP formulations prepared from YK-803, YK-810 and YK-813 showed significantly improved expression levels of mRNA in the mouse spleen compared to the representative cationic lipid SM-102 of the prior art. For example, YK-803, YK-810 and YK-813 expressed 8.61-fold, 15.02-fold and 7.58-fold, respectively, in spleen as compared to SM-102. mRNA was consistent with the results of cell transfection in example 6 in terms of mouse spleen expression. YK-803, YK-810 and YK-813 are very weak in liver expression, and the expression amounts are 0.02 times, 0.08 times and 0.04 times that of SM-102, respectively. The proportion of the expressed amount of the delivered mRNA in spleen and liver was only 0.09 times, that of YK-803 was 47.44 times, that of YK-810 was 16.50 times, and that of YK-813 was 17.25 times.
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. Has good targeting effect on developing and treating diseases caused by spleen damage or abnormality, such as lymphoma, leukemia and the like.
4) There is no correspondence between the structure of cationic lipid compound and the expression amount and duration of mRNA in animals, and there is a great possibility that there is a great difference in expression of mRNA in animals in LNP preparations prepared from the compound. Therefore, the amount and duration of mRNA expression in animals cannot be predicted according to chemical structures, and it is very difficult to screen cationic lipids with high and continuous mRNA expression in animals, and a lot of creative effort is required.
In one aspect, the present disclosure provides a novel cationic lipid compound for delivering a therapeutic or prophylactic agent. The cationic lipid compounds of the present disclosure can be used to deliver nucleic acid molecules, small molecule compounds, polypeptides, or proteins. The cationic lipid compounds of the present disclosure exhibit higher transfection efficiency, lower cytotoxicity and high expression in animals, improving delivery efficiency and safety relative to known cationic lipid compounds.
The present disclosure provides a cationic lipid that is a compound of formula (I)
Or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein:
G 1 is C 2～10 alkylene;
g 2 is C 2～10 alkylene;
g 3 is
R 1 is unsubstituted C 6～25 straight or branched alkyl;
R 2 is unsubstituted C 6～25 straight or branched alkyl;
m and n are 1 or 0, and when m is 0, n is 1, i.e. the nitrogen element in formula (I) is directly connected with G 3 through covalent bond; when m is 1, n is 0, i.e., the nitrogen element in formula (I) is directly covalently linked to G 2.
In one embodiment, G 1 is unsubstituted C 3 alkylene, e.g., - (CH 2)3 -.
In one embodiment, G 1 is unsubstituted C 5 alkylene, e.g., - (CH 2)5 -.
In one embodiment, G 1 is unsubstituted C 7 alkylene, e.g., - (CH 2)7 -.
In one embodiment, G 2 is unsubstituted C 3 alkylene, e.g., - (CH 2)3 -.
In one embodiment, G 2 is unsubstituted C 5 alkylene, e.g., - (CH 2)5 -.
In one embodiment, G 2 is unsubstituted C 7 alkylene, e.g., - (CH 2)7 -.
In one embodiment, G 3 is
In one embodiment, G 3 is
In one embodiment, G 3 is
In one embodiment, G 3 is
In another embodiment, G 3 is
In some preferred embodiments, the compound of formula (I) has one of the following structures:
In a more preferred embodiment, the compound of formula (I) is one of the following structures:
Yet another aspect of the present disclosure provides a composition comprising a carrier comprising a cationic lipid comprising a compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, as described above.
In some embodiments, the composition is a nanoparticle formulation having an average size of 140nm to 210nm, preferably 140nm to 205nm; the nanoparticle formulation has a polydispersity of 50% or less, preferably 30% or less, more preferably 25% or less.
Cationic lipids
In one embodiment of the composition/carrier of the present disclosure, the cationic lipid is one or more selected from the compounds of formula (I) above or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the cationic lipid is a compound of formula (I) selected from the group consisting of those described above. For example, the cationic lipid is a compound YK-801, YK-802, YK-803, YK-804, YK-805, YK-806, YK-807, YK-808, YK-809, YK-810, YK-811, YK-812, YK-813 or YK-814. In a preferred embodiment, the cationic lipid is compound YK-810. In another preferred embodiment, the cationic lipid is compound YK-803. In another preferred embodiment, the cationic lipid is compound YK-813. In another preferred embodiment, the cationic lipid is compound YK-811. In another preferred embodiment, the cationic lipid is compound YK-809. In another preferred embodiment, the cationic lipid is compound YK-807.
In some embodiments, the cationic lipid comprises 25% to 75%, e.g., 35%, 45%, 49%, 50%, 51%, 55%, 60%, 65% of the molar ratio of the carrier.
The carrier may be used to deliver an active ingredient such as a therapeutic or prophylactic agent. The active ingredient may be enclosed within a carrier or may be combined with a carrier.
For example, the therapeutic or prophylactic agent includes one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein. Such nucleic acids include, but are not limited to, single-stranded DNA, double-stranded DNA, and RNA. Suitable RNAs include, but are not limited to, small interfering RNAs (sirnas), asymmetric interfering RNAs (airnas), micrornas (mirnas), dicer-substrate RNAs (dsRNA), small hairpin RNAs (shrnas), messenger RNAs (mrnas), and mixtures thereof.
Neutral lipids
The carrier may comprise neutral lipids. Neutral lipids in the present disclosure refer to lipids that are non-charged at a selected pH or that are present as zwitterionic forms that act as a helper. Neutral lipids may modulate nanoparticle mobility into lipid bilayer structures and increase efficiency by promoting lipid phase changes, while also potentially affecting target organ specificity.
In some embodiments, the molar ratio of the cationic lipid to the neutral lipid is from 1:1 to 15:1, e.g., 14:1, 13:1, 12:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5.1:1, 5:1, 4.9:1, 4.6:1, 4.5:1, 4.4:1, 4:1, 3:1, 2:1. In some preferred embodiments, the molar ratio of the cationic lipid to the neutral lipid is 4.5:1.
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-didecyl-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-cholesterol hemisuccinyl-sn-3-phosphorylcholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphorylcholine (C), 1, 2-dioleoyl-sn-3-phosphorylcholine (Lyso PC), 1, 2-dioleoyl-2-glycero-sn-3-phosphorylcholine (DOPC), 1-dioleoyl-2-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-sn-3-glycero-3-phosphorylcholine (POPC), 1-dioleoyl-2-glycero-phosphorylcholine (DOPC) 1, 2-Diphytanoyl-sn-glycero-3-phosphate ethanolamine (ME 16.0 PE), 1, 2-distearoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-arachidonoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-docosahexaenoic acid-sn-glycero-3-phosphate ethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphate-rac- (1-glycero) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG) palmitoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl-based phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.
In some embodiments, the neutral lipid comprises DSPC. In certain embodiments, the neutral lipid comprises DOPE. In some embodiments, the neutral lipid comprises both DSPC and DOPE.
Structured lipids
The carrier of the composition comprising the cationic lipid may also comprise one or more structural lipids. Structured lipids refer in the present disclosure to lipids that enhance the stability of the nanoparticle by filling the interstices between the lipids.
In some embodiments, the molar ratio of the cationic lipid to the structural lipid is about 0.6:1 to 3:1, e.g., about 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1. In some preferred embodiments, the molar ratio of the cationic lipid to the structural lipid is 45:43.5.
The structural lipid may be selected from, but is not limited to, the group consisting of: cholesterol, non-sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycorine, ursolic acid, alpha-tocopherols, corticosteroids, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipids include cholesterol and corticosteroids such as prednisolone (prednisolone), dexamethasone, prednisone (prednisone), and hydrocortisone (hydrocortisone), or combinations thereof.
Polymer conjugated lipids
The carrier of the composition comprising the cationic lipid may also comprise one or more polymer conjugated lipids. The polymer conjugated lipid mainly refers to polyethylene glycol (PEG) modified lipid. Hydrophilic PEG stabilizes LNP, regulates nanoparticle size by limiting lipid fusion, and increases nanoparticle half-life by reducing non-specific interactions with macrophages.
In some embodiments, the polymer conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol. The PEG modified PEG molecular weight is typically 350-5000 Da.
For example, the polymer conjugated lipid is selected from one or more of the following: distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG 2000), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000) and methoxypolyethylene glycol ditetradecylamide (ALC-0159).
In one embodiment of the composition/carrier of the present disclosure, the polymer conjugated lipid is DMG-PEG2000.
In one embodiment of the composition/carrier of the present disclosure, the carrier comprises neutral lipid, structural lipid, and polymer conjugated lipid, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer conjugated lipid is (25-75): (5-25): (15-65): (0.5-10), e.g., (35-49): (4.5-15): (35-55): (1-5).
In one embodiment of the composition/carrier of the present disclosure, the carrier comprises a neutral lipid, a structural lipid, and a polymer conjugated lipid, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer conjugated lipid being 45:10:43.5:1.5.
Therapeutic and/or prophylactic agent
The composition may include one or more therapeutic and/or prophylactic agents. In some embodiments, the mass ratio of carrier to the therapeutic or prophylactic agent is 10:1 to 30:1, e.g., 12.5:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1.
In some embodiments, the mass ratio of carrier to the therapeutic or prophylactic agent is 12.5:1 to 25:1, preferably 15:1.
The therapeutic or prophylactic agent includes, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.
For example, the therapeutic or prophylactic agent is a vaccine or compound capable of eliciting an immune response.
The vectors of the present disclosure may deliver therapeutic and/or prophylactic agents to mammalian cells or organs, and thus the present disclosure also provides methods of treating a disease or disorder in a mammal in need thereof, comprising administering to the mammal a composition comprising a therapeutic and/or prophylactic agent and/or contacting mammalian cells with the composition.
Therapeutic and/or prophylactic agents include bioactive substances and are alternatively referred to as "active agents". The therapeutic and/or prophylactic agent can be a substance that, upon delivery to a cell or organ, causes a desired change in the cell or organ or other body tissue or system. Such species may be used to treat one or more diseases, disorders or conditions. In some embodiments, the therapeutic and/or prophylactic agent is a small molecule drug that can be used to treat a particular disease, disorder, or condition. Examples of drugs that may be used in the compositions include, but are not limited to, antineoplastic agents (e.g., vincristine, doxorubicin (doxorubicin), mitoxantrone (mitoxantrone), camptothecin, cisplatin (cispratin), bleomycin (bleomycin), cyclophosphamide (cyclophosphamide), methotrexate, and streptozotocin), antineoplastic agents (e.g., actinomycin D (actinomycin D)), Vincristine, vinblastine (vinblastine), cytosine arabinoside (cytosine arabinoside), anthracyclines (anthracyclines), alkylating agents, platinoids, antimetabolites and nucleoside analogues such as methotrexate and purine and pyrimidine analogues), anti-infective agents, local anesthetics (e.g. dibucaine (dibucaine) and chlorpromazine (chlorpromazine)), beta-adrenergic blockers (e.g. propranolol (propranolol), molol (timolol) and labetalol (labetalol)), Antihypertensives (e.g., clonidine and hydralazine (hydralazine)), antidepressants (e.g., imipramine (imipramine), amitriptyline (AMITRIPTYLINE) and doxepin (doxepin)), antispasmodics (e.g., phenytoin (phenytoin)), antihistamines (e.g., diphenhydramine (DIPHENHYDRAMINE), chlorpheniramine (chlorpheniramine) and promethazine (promethazine)), antibiotics/antibacterial agents (e.g., gentamicin (gentamycin), antibiotics/antibacterial agents (e.g., gentamicin), Ciprofloxacin (ciprofloxacin) and cefoxitin (cefoxitin)), antifungal agents (e.g., miconazole (miconazole), terconazole (terconazole), econazole (econazole), econazole (isoconazole), butoconazole (butaconazole), clotrimazole (clotrimazole), itraconazole (itraconazole), nystatin (nystatin), netifen (naftifine) and amphotericin B (amphotericin B)), and pharmaceutical compositions, Antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, anti-glaucoma agents, vitamins, sedatives and imaging agents.
In some embodiments, the therapeutic and/or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, and/or another therapeutic and/or prophylactic agent. 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), canamycin (emetine), mitomycin (mitomycin), etoposide (etoposide), teniposide (teniposide), vincristine, vinblastine, colchicine (colchicine), doxorubicin, daunorubicin (daunorubicin), dihydroxyanthracene dione (dihydroxy anthracin dione), and, Mitoxantrone, mithramycin (mithramycin), actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine (procaine), tetracaine (tetracaine), lidocaine (lidocaine), propranolol, puromycin, maytansinoids (maytansinoids) such as maytansinol (maytansinol), azithromycin (rachelmycin) (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 (thiotepa), chlorambucil (chlorambucil), azithromycin (CC-1065), melphalan (melphalan), carmustine (carmustine, BSNU), robustin (lomustine, CCNU), and combinations thereof, Cyclophosphamide, busulfan (busulfan), dibromomannitol, streptozotocin, mitomycin C and cisplatin (II) (DDP), cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin (daunomycin)) and doxorubicin), antibiotics (e.g., dactinomycin (dactinomycin) (formerly dactinomycin), bleomycin, mithramycin (mithramycin) and aflatoxin (anthramycin, AMC)), and antimitotics (e.g., vincristine, vinblastine, taxol and maytansinoids).
In other embodiments, the therapeutic and/or prophylactic agent is a protein. Therapeutic proteins useful in the nanoparticles in the present disclosure include, but are not limited to, gentamicin, amikacin (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 antigens, 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 disclosure include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA); ribonucleic acids (RNAs), including messenger mrnas (mrnas), hybrids thereof; RNAi-inducing factors; RNAi factor; siRNA; shRNA; a miRNA; antisense RNA; ribozymes; catalytic DNA; RNA that induces triple helix formation; an aptamer, and the like. In some embodiments, the therapeutic and/or prophylactic agent is RNA. The RNAs useful in the compositions and methods described herein may be selected from the group consisting of, but not limited to: shortmer, antagomir, antisense RNA, ribozyme, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In certain embodiments, the RNA is mRNA.
In certain embodiments, the therapeutic and/or prophylactic agent is mRNA. The mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA may be of any size and may have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA may have a therapeutic effect when expressed in a cell.
In other embodiments, the therapeutic and/or prophylactic agent is an siRNA. siRNA is capable of selectively reducing expression of a gene of interest or down-regulating expression of the gene. For example, the siRNA can be selected such that a gene associated with a particular disease, disorder, or condition is silenced after administration of a composition comprising the siRNA to a subject in need thereof. The siRNA may comprise a sequence complementary to an mRNA sequence encoding a gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.
In certain embodiments, the therapeutic and/or prophylactic agent is sgRNA and/or cas9 mRNA. sgRNA and/or cas9 mRNA may be used as a gene editing tool. For example, the sgRNA-cas9 complex can affect mRNA translation of cellular genes.
In some embodiments, the therapeutic and/or prophylactic agent is an shRNA or a vector or plasmid encoding the same. shRNA may be produced inside the target cell after delivery of the appropriate construct into the nucleus. Constructs and mechanisms related to shRNA are well known in the relevant arts.
Diseases or conditions
The compositions/carriers of the present disclosure can deliver therapeutic or prophylactic agents to a subject or patient. The therapeutic or prophylactic agent includes, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein. Thus, the compositions of the present disclosure can be used to prepare nucleic acid drugs, genetic vaccines, small molecule drugs, polypeptides or protein drugs. Because of the wide variety of therapeutic or prophylactic agents described above, the compositions of the present disclosure are useful in the treatment or prevention of a variety of diseases or conditions.
In some embodiments, the disease or disorder is characterized by dysfunctional or abnormal protein or polypeptide activity.
For example, the disease or disorder is selected from the group consisting of: infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases and metabolic diseases.
In some embodiments, the infectious disease is selected from the group consisting of a disease caused by coronavirus, influenza virus, or HIV virus, pediatric pneumonia, rift valley fever, yellow fever, rabies, and a variety of herpes.
Other components
The composition may include one or more components other than those described in the preceding section. For example, the composition may include one or more hydrophobic small molecules, such as vitamins (e.g., vitamin a or vitamin E) or sterols.
The composition may also include one or more permeability enhancing molecules, carbohydrates, polymers, surface modifying agents, or other components. The permeability enhancing molecule may be, for example, a molecule described in U.S. patent application publication No. 2005/0222064. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen, and derivatives and analogs thereof).
Surface modifying agents may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyl dioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamers), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain (bromelain), papain, dyers woad (clerodendrum), bromhexine (bromhexine), carbocisteine (carbocisteine), eplerenone (eprazinone), mesna (mesna), ambroxol (ambroxol), sibiriol (sobrerol), polymitols (domiodol), letrostat (letosteine), setronine (stepronin), tiopronin (tiopronin), gelsolin (gelsolin), thymosin (thymosin) β4, streptococcal dnase α (dornase alfa), netilmicin (neltenexine), and polysteine (erdosteine), and dnase (e.g., rhDNA enzymes). The surface modifying agent may be disposed within and/or on the nanoparticle of the composition (e.g., by coating, adsorption, covalent attachment, or other means).
The composition may further comprise one or more functionalized lipids. For example, the lipid may be functionalized with an alkynyl group that may undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. In particular, lipid bilayers can be functionalized in this manner with one or more groups effective to facilitate membrane permeation, cell recognition, or imaging. The surface of the composition may also be conjugated to one or more useful antibodies. Functional groups and conjugates useful for targeted cell delivery, imaging, and membrane permeation are well known in the art.
In addition to these components, the composition may include any substance useful in pharmaceutical compositions. For example, the composition may include one or more pharmaceutically acceptable excipients, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersing aids, suspending aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonic agents, thickening or emulsifying agents, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, and the like. Excipients such as starch, lactose or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, e.g., remington' S THE SCIENCE AND PRACTICE of Pharmacy, 21 st edition, a.r. gennaro; lippincott, williams & Wilkins, baltimore, MD, 2006).
Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, dibasic calcium phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and/or combinations thereof.
In some embodiments, compositions comprising one or more lipids described herein may also comprise one or more adjuvants, such as Glucopyranosyl Lipid Adjuvants (GLA), cpG oligodeoxyribonucleotides (e.g., class a or class B), poly (I: C), aluminum hydroxide, and Pam3CSK4.
The compositions of the present disclosure may be formulated in solid, semi-solid, liquid or gaseous form, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, aerosols. The compositions of the present disclosure may be prepared by methods well known in the pharmaceutical arts. For example, sterile injectable solutions can be prepared by incorporating the therapeutic or prophylactic agent in the required amount with various of the other ingredients described above in the appropriate solvent such as sterile distilled water and then filter-sterilizing. Surfactants may also be added to promote the formation of a uniform solution or suspension.
For example, the compositions of the present disclosure are administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In some embodiments, the composition is administered subcutaneously.
The compositions of the present disclosure are administered in therapeutically effective amounts, which may vary not only with the particular agent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and may ultimately be at the discretion of the attendant physician or clinician. For example, a dose of 0.001 to 10mg/kg of the therapeutic or prophylactic agent may be administered to a mammal (e.g., a human).
Examples
The 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; CDI, carbonyl diimidazole; and rt: room temperature
Example 1: synthesis of cationic lipid compounds
Synthesis of YK-801
The synthetic route is as follows:
Step one: synthesis of 6- (octyloxy) -6-oxohexyl-1H-imidazole-1-carboxylic acid ester (YK-801-PM 1)
Carbonyl diimidazole (563 mg,3.47 mmol) was dissolved in methylene chloride (10 mL), the temperature was lowered to 0℃in an ice-water bath under the protection of nitrogen atmosphere, octyl 6-hydroxyhexanoate (424 mg,1.74 mmol) was added to the above-mentioned system in portions, and the mixture was allowed to stand at room temperature after the addition was completed, followed by stirring for 3 hours. After the reaction, the reaction mixture was washed with saturated brine (10 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent to give YK-801-PM1(525mg,1.55mmol,89.4％).C18H30N2O4,MS(ES):m/z(M+H+)339.2.
Step two: synthesis of tert-butyl (3-chloro-2-hydroxypropyl) carbamate (YK-801-PM 2)
1-Amino-3-chloro-2-propanol hydrochloride (4.0 g,27.39 mmol) was dissolved in dioxane (180 mL) and water (60 mL), the ice water bath was cooled to 0℃and 1M sodium hydroxide solution (55 mL) was added dropwise to the system, after the dropwise addition, the pH of the system was >10, and di-tert-butyl carbonate (6.6 g,30.24 mmol) was added. After the addition was completed, the reaction was stirred at room temperature for 5 hours. After the completion of the reaction, 100mL of water was added to the reaction mixture, followed by extraction with methylene chloride (100 mL. Times.2), and the combined organic phases were washed with saturated brine (50 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (0-30% ethyl acetate/n-hexane) to give YK-801-PM2 (3.8 g,18.12mmol, 66.2%). C 8H16ClNO3,MS(ES):m/z(M+H+) 210.1.
Step three: synthesis of tert-butyl (2-hydroxy-3- ((2-hydroxyethyl) (methyl) amino) propyl) carbamate (YK-801-PM 3)
YK-801-PM2 (3.8 g,18.12 mmol) and 2- (methylamino) ethanol (1.6 g,21.30 mmol) were dissolved in acetonitrile (50 mL), and potassium carbonate (7.5 g,54.27 mmol) and potassium iodide (3.0 g,18.07 mmol) were added to the above system, which was heated to 70℃and stirred for 5 hours. After the reaction was completed, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. Purifying the residue by silica gel chromatography (0-20% dichloromethane/methanol) to obtain YK-801-PM3(3.5g,14.09mmol,77.8％).C11H24N2O4,MS(ES):m/z(M+H+)249.2.
Step four: synthesis of 1-amino-3- ((2-hydroxyethyl) (methyl) amino) propan-2-ol (YK-801-PM 4)
YK-801-PM3 (3.5 g,14.09 mmol) was added to a 4M hydrogen chloride/1, 4-dioxane solution (35 mL) and the reaction stirred at room temperature for 10 hours. After the reaction, the mixture was concentrated, 50mL of methylene chloride and 50mL of saturated aqueous sodium hydrogencarbonate solution were added, the mixture was stirred, methylene chloride (500 mL. Times.2) was added to the mixture to extract the mixture, and the organic phase was combined and washed with saturated brine (50 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. Obtaining the product YK-801-PM4(1.8g,12.14mmol,86.2％).C6H16N2O2,MS(ES):m/z(M+H+)149.1.
Step five: synthesis of 2-octyldodecyl 6- ((2-hydroxy-3- ((2-hydroxyethyl) (methyl) amino) propyl) amino) hexanoate (YK-801-PM 5)
YK-801-PM4 (1.0 g,6.75 mmol) obtained above and 2-octyl-dodecyl 6-bromohexanoate (3.2 g,6.73 mmol) were dissolved in acetonitrile (10 mL), and potassium carbonate (2.8 g,20.26 mmol) was added to the above system and heated to 70℃to stir and react for 7 hours. After the completion of the reaction, 50mL of water was added to the reaction mixture, followed by extraction with ethyl acetate (50 mL. Times.2), and the combined organic phases were washed with saturated brine (50 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. Purifying the residue by silica gel chromatography (0-20% dichloromethane/methanol) to obtain YK-801-PM5(0.8g,1.47mmol,21.9％).C32H66N2O4,MS(ES):m/z(M+H+)543.5;
Step six: synthesis of octyl (6- (((2-hydroxy-3- ((2-hydroxyethyl) (methyl) amino) propyl) (6- ((2-octyldodecyl) oxy) -6-oxohexyl) carbamoyl) oxyhexanoate (YK-801)
YK-801-PM5 (239 mg,0.44 mmol), YK-801-PM1 (74 mg,0.22 mmol), triethylamine (20 mg,0.20 mmol) and potassium carbonate (83 mg,0.60 mmol) were dissolved in tetrahydrofuran (5 mL), heated to 60℃and reacted under stirring for 3 hours. After the completion of the reaction, 10mL of water was added to the reaction mixture, followed by extraction with methylene chloride (10 mL. Times.2), and the combined organic phases were washed with saturated brine (10 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (0-22% dichloromethane/methanol) to give YK-801 (63 mg,0.08mmol, 35.2%). C 47H92N2O8,MS(ES):m/z(M+H+) 813.7.
1H NMR(CDCl3,400MHz,298K)δ4.40(s,1H),4.33(d,J＝4.8Hz,1H),4.15(t,J＝6.7Hz,2H),4.05(t,J＝6.8Hz,2H),3.95(d,J＝5.8Hz,2H),3.27(d,J＝12.3Hz,2H),3.07-2.93(m,4H),2.87(s,2H),2.57(s,3H),2.31(dd,J＝10.0,4.8Hz,4H),1.90(s,1H),1.75-1.52(m,10H),1.50-1.37(m,4H),1.26(s,44H),0.88(t,J＝6.7Hz,9H).
Synthesis of YK-802
The synthetic route is as follows:
step one: synthesis of 6- (dodecyloxy) -6-oxohexyl-1H-imidazole-1-carboxylic acid ester (YK-802-PM 1)
Using carbonyl diimidazole (131 mg,0.81 mmol) and 6-hydroxy caproic acid dodecyl ester (120 mg,0.40 mmol) as raw materials, and obtaining the product according to the method for synthesizing YK-801-PM1 YK-802-PM1(118mg,0.30mmol,75.0％).C22H38N2O4,MS(ES):m/z(M+H+)395.3.
Step two: synthesis of dodecyl (YK-802) oxy hexanoate (6- (((2-hydroxy-3- ((2-hydroxyethyl) (methyl) amino) propyl) (6- ((2-octyldodecyl) oxy) -6-oxohexyl) carbamoyl)
YK-801-PM5 (525 mg,0.97 mmol) and YK-802-PM1 (39 mg,0.10 mmol) were used as raw materials, and YK-802 (65 mg,0.07mmol, 74.8%) was obtained by the method of synthesizing YK-801. C 51H100N2O8,MS(ES):m/z(M+H+) 869.8.
1H NMR(CDCl3,400MHz,298K)δ4.40(s,1H),4.33(d,J＝4.8Hz,1H),4.15(t,J＝6.7Hz,2H),4.05(t,J＝6.8Hz,2H),3.95(d,J＝5.8Hz,2H),3.27(d,J＝12.3Hz,2H),3.07-2.93(m,4H),2.87(s,2H),2.57(s,3H),2.31(dd,J＝10.0,4.8Hz,4H),1.90(s,1H),1.75-1.52(m,10H),1.50-1.37(m,4H),1.26(s,52H),0.88(t,J＝6.7Hz,9H).
Synthesis of YK-803
The synthetic route is as follows:
Step one: synthesis of 6- ((2-octyldodecyl) oxy) -6-oxohexyl-1H-imidazole-1-carboxylate (YK-803-PM 1)
Using carbonyl diimidazole (131 mg,0.81 mmol) and 6-hydroxy caproic acid-2-octyl dodecyl ester (167 mg,0.40 mmol) as raw materials, and obtaining the YK-801-PM1 according to the method YK-803-PM1(182mg,0.36mmol,89.8％).C30H54N2O4,MS(ES):m/z(M+H+)507.4.
Step two: synthesis of 2-octyldodecyl 6- (2-hydroxy-3- ((2-hydroxyethyl) (methyl) amino) propyl) ((((6- ((2-octyldodecyl) oxy) -6-oxohexyl) oxycarbonyl) amino) hexanoate (YK-803)
YK-803 (75 mg,0.08mmol, 86.0%) was obtained by the method of synthesizing YK-801 from YK-801-PM5 (525 mg,0.97 mmol) and YK-803-PM1 (45 mg,0.09 mmol). C 59H116N2O8,MS(ES):m/z(M+H+) 981.9.
1H NMR(CDCl3,400MHz,298K)δ4.23(t,J＝5.2Hz,3H),4.15(t,J＝6.8Hz,2H),3.96(d,J＝5.6Hz,4H),3.25(d,J＝15.0Hz,2H),3.01(d,J＝7.4Hz,2H),2.87-2.68(m,2H),2.65(d,J＝5.3Hz,2H),2.40(s,3H),2.32(t,J＝7.4Hz,4H),1.86(d,J＝7.3Hz,2H),1.67(dt,J＝15.7,7.3Hz,8H),1.49-1.36(m,4H),1.30(d,J＝28.5Hz,64H),0.88(t,J＝6.7Hz,12H).
Synthesis of YK-804
The synthetic route is as follows:
Step one: synthesis of 2-octyldodecyl 8- (6-oxo-6- (undecyloxy) hexyl) aminocaprylate (YK-804-PM 1)
YK-801-PM5 was synthesized from undecyl 6-aminocaproate (2825 mg,9.90 mmol) and 2-octyldodecyl 8-bromooctanoate (4155 mg,8.25 mmol) to give YK-804-PM1 (3060 mg,4.32mmol, 52.4%). C 45H89NO4,MS(ES):m/z(M+H+) 708.7.
Step two: synthesis of 2-octyldodecyl 8- (N- (6-oxo-6- (undecyloxy) hexyl) -1H-imidazole-1-carboxamide) octanoate (YK-804-PM 2)
Using carbonyl diimidazole (284 mg,1.76 mmol) and YK-804-PM1 (500 mg,0.71 mmol) as raw materials, and synthesizing YK-801-PM1 according to the method YK-804-PM2(337mg,0.42mmol,59.2％).C49H91N3O5,MS(ES):m/z(M+H+)802.7.
Step three: synthesis of 2-octyldodecyl 6- (2-hydroxy-3- ((2-hydroxyethyl) (methyl) amino) propyl) ((((6- ((2-octyldodecyl) oxy) -6-oxohexyl) oxycarbonyl) amino) hexanoate (YK-804)
Using YK-804-PM2 (337 mg,0.42 mmol) and N-methyldiethanolamine (200 mg,1.68 mmol) as raw materials, the method for synthesizing YK-801 is carried out YK-804(260mg,0.30mmol,72.5％).C51H100N2O7,MS(ES):m/z(M+H+)853.8.
1H NMR(CDCl3,400MHz,298K)δ4.40(s,2H),4.05(t,J＝6.7Hz,2H),3.96(d,J＝5.8Hz,2H),3.81(s,2H),3.35(dd,J＝15.2,7.6Hz,4H),3.19(s,2H),3.07(d,J＝25.8Hz,2H),2.92(d,J＝34.8Hz,2H),2.66(s,2H),2.30(td,J＝7.3,3.6Hz,3H),2.18(s,1H),1.70-1.56(m,10H),1.50(d,J＝7.1Hz,2H),1.28(d,J＝16.5Hz,54H),0.88(t,J＝6.7Hz,9H).
Synthesis of YK-805
The synthetic route is as follows:
step one: synthesis of bis (2-octyldodecyl) -6,6' -azadiyl dihexanoate (YK-805-PM 1)
Using 6-aminocaproic acid-2-octyldodecyl ester (436 mg,1.06 mmol) and 6-bromohexanoic acid-2-octyldodecyl ester (504 mg,1.06 mmol) as raw materials, YK-801-PM5 was synthesized according to the method to give YK-805-PM1 (436 mg,0.54mmol, 50.9%). C 52H103NO4,MS(ES):m/z(M+H+) 806.8.
Step two: synthesis of bis (2-octyldodecyl) -6,6' - ((1H-imidazole-1-carbonyl) azadiyl) dihexanoate (YK-805-PM 2)
Using carbonyl diimidazole (263 mg,1.62 mmol) and YK-805-PM1 (436 mg,0.54 mmol) as raw materials, and synthesizing YK-801-PM1 according to the method YK-805-PM2(412mg,0.46mmol,84.7％).C56H105N3O5,MS(ES):m/z(M+H+)900.8.
Step three: synthesis of bis (2-octyldodecyl) -6,6' - ((2- ((2-hydroxyethyl) (methyl) amino) ethoxy) carbonyl) azadiyl) dihexanoate (YK-805)
Using YK-805-PM2 (412 mg,0.46 mmol) and N-methyldiethanolamine (437 mg,3.67 mmol) as raw materials, the method for synthesizing YK-801 is carried out YK-805(200mg,0.21mmol,45.7％).C58H114N2O7,MS(ES):m/z(M+H+)951.9.
1H NMR(CDCl3,400MHz,298K)δ4.30(s,2H),3.96(d,J＝5.7Hz,4H),3.70(s,2H),3.19(s,4H),2.92(s,2H),2.80(s,2H),2.51(s,3H),2.30(t,J＝7.5Hz,4H),1.64(dt,J＝14.9,7.3Hz,6H),1.53(s,4H),1.26(s,68H),0.88(t,J＝6.8Hz,12H).
Synthesis of YK-806
The synthetic route is as follows:
step one: synthesis of 3-hexylnonyl 6- (((4- (decyloxy) -4-oxobutyl) amino) hexanoate (YK-806-PM 1)
YK-801-PM5 was synthesized from 6-aminocaproic acid-3-hexylnonyl ester (630 mg,1.84 mmol) and decyl 4-bromobutyrate (560 mg,1.84 mmol) to give YK-806-PM1 (410 mg,0.72mmol, 39.2%). C 35H69NO4,MS(ES):m/z(M+H+) 568.5.
Step two: synthesis of 3-hexylnonyl 6- (N- (4- (decyloxy) -4-oxobutyl) -1H-imidazole-1-carboxamide) hexanoate (YK-806-PM 2)
Using carbonyl diimidazole (234 mg,1.44 mmol) and YK-806-PM1 (410 mg,0.72 mmol) as raw materials, and synthesizing YK-801-PM1 according to the method YK-806-PM2(400mg,0.60mmol,83.9％).C39H71N3O5,MS(ES):m/z(M+H+)662.5.
Step three: synthesis of 3-hexylnonyl 6- ((4- (decyloxy) -4-oxobutyl) (2- ((2-hydroxyethyl) (methyl) amino) ethoxy) carbonyl) amino) hexanoate (YK-806)
YK-806 (90 mg,0.13mmol, 28.0%) was obtained by the method of synthesizing YK-801 starting from YK-806-PM2 (300 mg,0.45 mmol) and N-methyldiethanolamine (432 mg,3.63 mmol). C 41H80N2O7,MS(ES):m/z(M+H+) 713.6.
1H NMR(CDCl3,400MHz,298K)δ4.57(s,2H),4.06(dd,J＝12.4,6.6Hz,4H),3.38(s,2H),3.32-3.16(m,4H),2.91(s,2H),2.63(s,2H),2.30(dd,J＝17.3,7.4Hz,4H),2.18(s,3H),1.85(s,2H),1.65-1.53(m,10H),1.28(d,J＝18.4Hz,35H),0.88(t,J＝6.7Hz,9H).
Synthesis of YK-807
The synthetic route is as follows:
step one: synthesis of 2-octyldodecyl 6- (6-oxo-6- (undecoxy) hexyl) amino) hexanoate (YK-807-PM 1)
YK-801-PM5 was synthesized from undecyl 6-aminocaproate (540 mg,1.89 mmol) and 2-octyldodecyl 6-bromohexanoate (600 mg,1.26 mmol) to give YK-807-PM1 (390 mg,0.59mmol, 46.4%). C 43H85NO4,MS(ES):m/z(M+H+) 680.7.
Step two: synthesis of 2-octyldodecyl 6- (N- (6-oxo-6- (undecyloxy) hexyl) -1H-imidazole-1-carboxamide) hexanoate (YK-807-PM 2)
Using carbonyl diimidazole (185 mg,1.14 mmol) and YK-807-PM1 (3838 mg,0.57 mmol) as raw materials, and synthesizing YK-801-PM1 according to the method YK-807-PM2(374mg,0.48mmol,84.7％).C47H87N3O5,MS(ES):m/z(M+H+)774.7.
Step three: synthesis of 2-octyldodecyl 6- ((2- ((2-hydroxyethyl) (methyl) amino) ethoxy) carbonyl) (6-oxo-6- (undecyloxy) hexyl) amino) hexanoate (YK-807)
YK-807 (71 mg,0.09mmol, 17.9%) was obtained by the method of synthesizing YK-801 from YK-807-PM2 (374 mg,0.48 mmol) and N-methyldiethanolamine (463mg, 3.87 mmol). C 49H96N2O7,MS(ES):m/z(M+H+) 825.7.
1H NMR(CDCl3,400MHz,298K)δ4.24(t,J＝5.5Hz,2H),4.05(t,J＝6.8Hz,2H),3.96(d,J＝5.7Hz,2H),3.64(t,J＝5.0Hz,2H),3.20(d,J＝5.5Hz,4H),2.82(s,2H),2.71(s,2H),2.42(s,3H),2.34-2.26(m,4H),2.18(s,1H),1.70-1.57(m,8H),1.53(s,4H),1.28(d,J＝15.8Hz,50H),0.88(t,J＝6.7Hz,9H).
Synthesis of YK-808
The synthetic route is as follows:
step one: synthesis of 6- (6- (nonyloxy) -6-oxohexyl) amino) hexanoic acid-heptadecane-9-ester (YK-808-PM 1)
YK-801-PM5 was synthesized from nonyl 6-aminocaproate (890mg, 3.46 mmol) and heptanol 9-yl 6-bromohexanoate (1.0 g,2.31 mmol) to give YK-808-PM1 (588 mg,0.96mmol, 41.7%). C 38H75NO4,MS(ES):m/z(M+H+) 610.6.
Step two: synthesis of heptadecane-9-ester of 6- (N- (6- (nonyloxy) -6-oxohexyl) -1H-imidazole-1-carboxamide) hexanoic acid (YK-808-PM 2)
Using carbonyl diimidazole (313 mg,1.93 mmol) and YK-808-PM1 (588 mg,0.96 mmol) as raw materials, and synthesizing YK-801-PM1 according to the method YK-808-PM2(569mg,0.81mmol,84.2％).C42H77N3O5,MS(ES):m/z(M+H+)704.6.
Step three: synthesis of heptadec-9-yl-6- (((2- ((2-hydroxyethyl) (methyl) amino) ethoxy) carbonyl) (6- (nonyloxy) -6-oxohexyl) amino) hexanoate (YK-808)
Using YK-808-PM2 (469 mg,0.67 mmol) and N-methyldiethanolamine (636 mg,5.34 mmol) as raw materials, the method for synthesizing YK-801 is carried out YK-808(135mg,0.18mmol,26.7％).C44H86N2O7,MS(ES):m/z(M+H+)755.7.
1H NMR(CDCl3,400MHz,298K)δ4.85(t,J＝6.3Hz,1H),4.40(s,2H),4.05(t,J＝6.7Hz,2H),3.81(s,2H),3.20(d,J＝6.5Hz,4H),3.10(s,2H),2.96(s,2H),2.65(d,J＝9.4Hz,3H),2.29(dd,J＝12.9,7.4Hz,4H),1.63(dt,J＝15.4,7.6Hz,8H),1.51(s,8H),1.26(s,38H),0.88(t,J＝6.8Hz,9H).
Synthesis of YK-809
The synthetic route is as follows:
Step one: synthesis of 6- (6-oxo-6- (undecoxy) hexyl) amino) hexanoic acid-heptadecane-9-ester (YK-809-PM 1)
YK-801-PM5 was synthesized from undecyl 6-aminocaproate (2240 mg,7.85 mmol) and heptadecane-9-6-bromohexanoate (3403 mg,7.85 mmol) to give YK-809-PM1 (1740 mg,2.73mmol, 34.7%). C 40H79NO4,MS(ES):m/z(M+H+) 638.6.
Step two: synthesis of heptadecane-9-ester of 6- (N- (6-oxo-6- (undecyloxy) hexyl) -1H-imidazole-1-carboxamide) hexanoic acid (YK-809-PM 2)
Using carbonyl diimidazole (864 mg,5.33 mmol) and YK-809-PM1 (1700mg, 2.66 mmol) as raw materials, and synthesizing YK-801-PM1 according to the method YK-809-PM2(1600mg,2.19mmol,82.0％).C44H81N3O5,MS(ES):m/z(M+H+)732.6.
Step three: synthesis of heptadec-9-yl-6- (((2- (bis (2-hydroxyethyl) amino) ethoxy) carbonyl) (6-oxo-6- (undecyloxy) hexyl) amino) hexanoate (YK-809)
Using YK-809-PM2 (600 mg,0.82 mmol) and triethanolamine (978 mg,6.56 mmol) as raw materials, the method for synthesizing YK-801 is adopted YK-809(110mg,0.14mmol,16.5％).C47H92N2O8,MS(ES):m/z(M+H+)813.7.
1H NMR(CDCl3,400MHz,298K)δ4.85(s,1H),4.25(s,2H),4.05(t,J＝6.7Hz,2H),3.64(s,4H),3.19(d,J＝5.9Hz,4H),2.92(dd,J＝18.7,10.9Hz,2H),2.79(s,4H),2.31(dd,J＝13.7,6.9Hz,4H),1.63(dd,J＝15.3,7.7Hz,8H),1.57-1.45(m,8H),1.28(d,J＝16.7Hz,42H),0.88(t,J＝6.7Hz,9H).
Synthesis of YK-810
The synthetic route is as follows:
Step one: synthesis of 2-octyldodecyl 6- (4- (decyloxy) -4-oxobutyl) amino) hexanoate (YK-810-PM 1)
Using decyl 4-bromobutyrate (92mg, 3.00 mmol) and 2-octyldodecyl 6-aminocaproate (1235 mg,3.00 mmol) as raw materials, YK-801-PM5 was synthesized according to the method to give YK-810-PM1 (1116 mg,1.75mmol, 58.3%). C 40H79NO4,MS(ES):m/z(M+H+) 638.6.
Step two: synthesis of 2-octyldodecyl 6- (N- (4- (decyloxy) -4-oxobutyl) -1H-imidazole-1-carboxamide) hexanoate (YK-810-PM 2)
Using carbonyl diimidazole (851 mg,5.25 mmol) and YK-810-PM1 (1116 mg,1.75 mmol) as raw materials, and synthesizing YK-801-PM1 according to the method YK-810-PM2(1180mg,1.61mmol,92.1％).C44H81N3O5,MS(ES):m/z(M+H+)732.6.
Step three: synthesis of 2-octyldodecyl 6- (((2- (bis (2-hydroxyethyl) amino) ethoxy) carbonyl) (4- (decyloxy) -4-oxobutyl) amino) hexanoate (YK-810)
Using YK-810-PM2 (560 mg,0.81 mmol) and triethanolamine (962 mg,6.45 mmol) as raw materials, the method for synthesizing YK-801 is adopted YK-810(131mg,0.16mmol,20.0％).C47H92N2O8,MS(ES):m/z(M+H+)813.7.
1H NMR(CDCl3,400MHz,298K)δ4.35(s,2H),4.06(t,J＝6.6Hz,2H),3.96(d,J＝5.8Hz,2H),3.76(s,4H),3.24(dd,J＝15.7,7.9Hz,4H),3.06(s,2H),2.96(s,4H),2.31(t,J＝7.0Hz,4H),1.85(s,3H),1.69-1.49(m,6H),1.33-1.25(m,48H),0.88(t,J＝6.7Hz,9H).
Synthesis of YK-811
The synthetic route is as follows:
Step one: synthesis of decyl 6- ((6- ((3-hexylnonyl) oxy) -6-oxyhexyl) amino) hexanoate (YK-811-PM 1)
YK-811-PM1 (284 mg,0.90mmol, 36.3%) was obtained by the method of synthesizing YK-801-PM5 from decyl 6-aminocaproate (1000 mg,3.68 mmol) and 3-hexylnonyl 6-bromohexanoate (1000 mg,2.47 mmol). C 37H73NO4,MS(ES):m/z(M+H+) 596.6.
Step two: synthesis of decyl 6- (N- (6- ((3-hexylnonyl) oxy) -6-oxohexyl) -1H-imidazole-1-carboxamide) hexanoate (YK-811-PM 2)
Using carbonyl diimidazole (290 mg,1.79 mmol) and YK-811-PM1 (284 mg,0.90 mmol) as raw materials, and obtaining the product according to the method for synthesizing YK-801-PM1 YK-811-PM2(549mg,0.80mmol,88.8％).C41H75N3O5,MS(ES):m/z(M+H+)690.6.
Step three: synthesis of decyl 6- (((2- (bis (2-hydroxyethyl) amino) ethoxy) carbonyl) (6- ((3-hexylnonyl) oxy) -6-oxohexyl) amino) hexanoate (YK-811)
YK-811 (70 mg,0.09mmol, 12.6%) was obtained by the method of synthesizing YK-801 from YK-811-PM2 (499 mg,0.72 mmol) and triethanolamine (862mg, 5.78 mmol). C 44H86N2O8,MS(ES):m/z(M+H+) 771.6.
1H NMR(CDCl3,400MHz,298K)δ4.36(s,2H),4.06(dd,J＝15.6,7.6Hz,4H),3.77(d,J＝5.9Hz,4H),3.20(d,J＝6.8Hz,4H),3.09(s,2H),2.98(s,4H),2.30(s,4H),1.61(ddd,J＝22.4,14.6,7.2Hz,14H),1.28(d,J＝18.4Hz,37H),0.88(t,J＝6.7Hz,9H).
Synthesis of YK-812
The synthetic route is as follows:
Step one: synthesis of 6- (4-oxo-4- (undecoxy) butyl) amino) hexanoic acid-heptadecane-9-ester (YK-812-PM 1)
YK-801-PM5 was synthesized from undecyl 4-bromobutyrate (964 mg,3.00 mmol) and 6-aminocaproic acid-heptadec-9-yl ester (1110 mg,3.00 mmol) to give YK-812-PM1 (1630 mg,2.67mmol, 89.0%). C 38H75NO4,MS(ES):m/z(M+H+) 610.6.
Step two: synthesis of heptadec-9-yl 6- (N- (4-oxo-4- (undecyloxy) butyl) -1H-imidazole-1-carboxamide) hexanoate (YK-812-PM 2)
Using carbonyl diimidazole (4816 mg,3.00 mmol) and YK-812-PM1 (610 mg,1.00 mmol) as raw materials, and synthesizing YK-801-PM1 according to the method YK-812-PM2(319mg,0.45mmol,45.3％).C42H77N3O5,MS(ES):m/z(M+H+)704.6.
Step three: synthesis of heptadec-9-yl-1-hydroxy-3, 6-bis (2-hydroxyethyl) -10-oxo-11- (4-oxo-4- (undecyloxy) butyl) -9-oxo-3,6,11-triaza-heptadec-17-oic acid ester (YK-812)
YK-812 (68 mg,0.08mmol, 17.2%) was obtained by the method of synthesizing YK-801 from YK-812-PM2 (319 mg,0.45 mmol) and ethylenediamine tetraacetic acid (643 mg,2.72 mmol). C 49H97N3O9,MS(ES):m/z(M+H+) 872.7.
1H NMR(CDCl3,400MHz,298K)δ4.84(s,1H),4.10-3.99(m,4H),3.36(s,6H),3.22(s,6H),2.30(dd,J＝17.7,7.0Hz,6H),1.83(s,4H),1.62(s,6H),1.51(s,12H),1.36-1.21(m,40H),0.88(t,J＝6.7Hz,9H).
Synthesis of YK-813
The synthetic route is as follows:
step one: synthesis of 2-octyldodecyl 8- (6-oxo-6- (undecyloxy) hexyl) aminocaprylate (YK-813-PM 1)
YK-801-PM5 was synthesized from undecyl 6-aminocaproate (1865 mg,6.53 mmol) and 2-octyldodecyl 8-bromooctanoate (3290 mg,6.53 mmol) to give YK-813-PM1 (1820 mg,2.57mmol, 39.4%). C 45H89NO4,MS(ES):m/z(M+H+) 708.7.
Step two: synthesis of 2-octyldodecyl 8- (N- (6-oxo-6- (undecyloxy) hexyl) -1H-imidazole-1-carboxamide) octanoate (YK-813-PM 2)
Using carbonyl diimidazole (830 mg,5.12 mmol) and YK-813-PM1 (1820 mg,2.57 mmol) as raw materials, and synthesizing YK-801-PM1 according to the method YK-813-PM2(2000mg,2.49mmol,96.9％).C49H91N3O5,MS(ES):m/z(M+H+)802.7.
Step three: synthesis of 2-octyldodecyl 8- ((2- (bis (2-hydroxyethyl) amino) ethoxy) carbonyl) (6-oxo-6- (undecyloxy) hexyl) amino) octanoate (YK-813)
YK-813 (60 mg,0.07mmol, 10.9%) was obtained by the method of synthesizing YK-801 starting from YK-813-PM2 (500 mg,0.62 mmol) and triethanolamine (930 mg,6.23 mmol). C 52H102N2O8,MS(ES):m/z(M+H+) 883.8.
1H NMR(CDCl3,400MHz,298K)δ4.46(s,2H),4.05(t,J＝6.6Hz,2H),3.96(d,J＝5.8Hz,2H),3.89(s,4H),3.20(s,6H),2.30(s,4H),1.70-1.45(m,17H),1.28(d,J＝16.3Hz,54H),0.88(t,J＝6.8Hz,9H).
Synthesis of YK-814
The synthetic route is as follows:
step one: synthesis of 6- (4- (decyloxy) -4-oxobutyl) amino) hexanoic acid-heptadecane-9-ester (YK-814-PM 1)
YK-814-PM1 (330 mg,0.55mmol, 30.9%) was obtained by the method of synthesizing YK-801-PM5 from decyl 4-bromobutyrate (550 mg,1.79 mmol) and heptadecyl 6-aminocaproate-9-ester (1000 mg,2.68 mmol). C 37H73NO4,MS(ES):m/z(M+H+) 596.6.
Step two: synthesis of heptadecane-9-ester of 6- (N- (4- (decyloxy) -4-oxobutyl) -1H-imidazole-1-carboxamide) hexanoic acid (YK-814-PM 2)
Using carbonyl diimidazole (180 mg,1.11 mmol) and YK-814-PM1 (330 mg,0.55 mmol) as raw materials, and synthesizing YK-801-PM1 according to the method YK-814-PM2(378mg,0.55mmol,98.9％).C41H75N3O5,MS(ES):m/z(M+H+)690.6.
Step three: synthesis of heptadec-9-hexanoate (YK-814) of 6- ((4- (decyloxy) -4-oxobutyl) (3- (4- (3-hydroxypropyl) piperazin-1-yl) propoxy) carbonyl) amino)
YK-814 (53 mg,0.06mmol, 18.5%) was obtained by the method of synthesizing YK-801 starting from YK-814-PM2 (240 mg,0.35 mmol) and 3,3' - (piperazine-1, 4-diyl) bis (propan-1-ol) (830 mg,4.11 mmol). C 48H93N3O7,MS(ES):m/z(M+H+) 824.7.
1H NMR(CDCl3,400MHz,298K)δ4.90-4.80(m,1H),4.12(s,2H),4.05(t,J＝6.9Hz,2H),3.85(s,2H),3.20(s,4H),2.95(s,4H),2.28(t,J＝7.5Hz,8H),2.00(s,2H),1.83(s,4H),1.58(dd,J＝36.3,13.8Hz,16H),1.26(s,38H),0.88(t,J＝6.7Hz,9H).
Synthesis of P-76
The synthetic route is as follows:
Step one: synthesis of Di-tert-butyl (((2- (2-hydroxyethoxy) ethyl) azadiyl) bis (hexane-6, 1-diyl)) dicarbamate (P-76-PM 1)
Diglycolamine (1051 mg,10.00 mmol) and tert-butyl (6-bromohexyl) carbamate (5604 mg,20.00 mmol) were dissolved in acetonitrile (50 mL), potassium carbonate (4146 mg,30.00 mmol) was added to the above system, and the reaction was heated to 70℃with stirring for 5 hours. After the reaction, the reaction mixture was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (0-24% methanol/dichloromethane) to give P-76-PM1(2720mg,5.40mmol,54.0％).C26H53N3O6,MS(ES):m/z(M+H+)504.4.
Step two: synthesis of N 1 - (6-aminohexyl) -N 1 - (2- ((tert-butyldiphenylsilyl) oxy) ethoxy) ethyl) hexane-1, 6-diamine (P-76-PM 2)
P-76-PM1 (2720 mg,5.40 mmol), t-butyldiphenylchlorosilane (460 mg,5.53 mmol) and imidazole (1021 mg,15.00 mmol) were dissolved in N, N-dimethylformamide (50 mL), and after the completion of the reaction, ethyl acetate (100 mL) and water (100 mL) were added thereto, and the organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure. After 50mL of methylene chloride was added to the residue and dissolved, trifluoroacetic acid (30 mL) was slowly added thereto, and the reaction was stirred at room temperature for 10 hours. After the reaction, the mixture was concentrated, 50mL of methylene chloride and 50mL of saturated aqueous sodium hydrogencarbonate solution were added, the mixture was stirred, methylene chloride (50 mL. Times.2) was added to the mixture to extract the mixture, and the organic phase was combined and washed with saturated brine (50 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. Purifying the residue by silica gel chromatography (0-30% methanol/dichloromethane) to obtain P-76-PM2(2568mg,4.74mmol,87.8％).C32H53N3O2Si,MS(ES):m/z(M+H+)542.4.
Step three: synthesis of 2-hexyldecyl (4-nitrophenyl) carbonate (P-76-PM 3)
4-Nitrophenyl chloroformate (806 mg,4.00 mmol) was dissolved in tetrahydrofuran (10 mL), the temperature of the system was controlled to less than 5℃in an ice-water bath, 2-hexyldecan-1-ol (480 mg,4.04 mmol) was added to the above solution, and after the addition was completed, the mixture was warmed to room temperature and reacted under stirring for 1 hour. Ethyl acetate (20 mL) and water (10 mL) were then added, and the organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (0-20% ethyl acetate/n-hexane) to give P-76-PM3 (1378 mg,3.38mmol, 84.5%).
Step four: synthesis of bis (2-hexyldecyl) (((2- (2-hydroxyethoxy) ethyl) azadiyl) bis (hexane-6, 1-diyl)) dicarbamate (P-76)
Compound P-76-PM3 (1378 mg,3.38 mmol), P-76-PM2 (2492 mg,4.60 mmol) and triethylamine (0.5 mL) were dissolved in tetrahydrofuran (15 mL), heated to 50℃and reacted with stirring for 5 hours. The reaction system was cooled to room temperature, then 1M aqueous tetrabutylammonium bromide (10 mL) was added, stirred at room temperature for 2 hours, and ethyl acetate (20 mL) and water (10 mL) were further added, and the organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (0-20% methanol in dichloromethane) to give P-76 (1368 mg,1.63mmol, 48.2%). C 50H101N3O6,MS(ES):m/z(M+H+) 840.8.
1H NMR(CDCl3,400MHz,298K)δ4.90-4.80(m,4H),4.12(s,2H),4.05(t,J＝6.9Hz,4H),3.85(s,4H),3.20(s,4H),2.95(s,2H),1.83(s,2H),1.58(s,4H),1.26(s,60H),0.88(t,J＝6.7Hz,12H).
16. Synthesis of Compound 13
The synthetic route is as follows:
Step one: synthesis of 2- ((2- (dimethylamino) ethyl) (methyl) amino) ethan-1-ol (Compound 13-PM 1)
N 1,N1,N2 -trimethylethane-1, 2-diamine (1022 mg,10.00 mmol) and bromoethanol (625 mg,5.00 mmol) were dissolved in acetonitrile (20 mL), and potassium carbonate (2073 mg,15.00 mmol) was added to the above system, and the reaction was heated to 70℃and stirred for 5 hours. After the reaction was completed, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (0-24% methanol/methylene chloride) to give compound 13-PM1 (560 mg,4.03mmol, 80.7%). C 7H18N2O,MS(ES):m/z(M+H+) 147.1.
Step two: synthesis of 2- ((2- (dimethylamino) ethyl) (methyl) amino) ethyl (4-nitrophenyl) carbonate (Compound 13-PM 2)
4-Nitrophenyl chloroformate (806 mg,4.00 mmol) was dissolved in tetrahydrofuran (10 mL), the temperature was controlled at less than 5℃in an ice-water bath, and Compound 13-PM1 (560 mg,4.03 mmol) was added to the above solution, and after the addition was completed, the mixture was warmed to room temperature and reacted under stirring for 1 hour. Ethyl acetate (20 mL) and water (10 mL) were then added, and the organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure. Purifying the residue by silica gel chromatography (0-50% ethyl acetate/n-hexane) to obtain the compound 13-PM2(1050mg,3.37mmol,84.3％).C14H21N3O5,MS(ES):m/z(M+H+)312.2.
Step three: synthesis of dinonyl 8,8' -azadiyldioctanoate (Compound 13-PM 3)
Using 8-bromooctanoate diester (349 mg,1.00 mmol) and 8-aminocaprylate nonate (571 mg,2.00 mmol) as raw materials, compound 13-PM3 (510 mg,0.92mmol, 92.1%) was obtained according to the method of synthesizing YK-801-PM 5. C 34H67NO4,MS(ES):m/z(M+H+) 554.5.
Step four: synthesis of nonyl-2, 5-dimethyl-10- (8- (nonyloxy) -8-oxooctyl) -9-oxo-8-oxo-2, 5, 10-triazaoctadecan-18-oic acid ester (Compound 13)
Compound 13-PM2 (284 mg,0.92 mmol), compound 13-PM3 (510 mg,0.92 mmol) and triethylamine (0.5 mL) were dissolved in tetrahydrofuran (5 mL), heated to 50℃and reacted under stirring for 5 hours. The reaction system was cooled to room temperature, then ethyl acetate (10 mL) and water (5 mL) were added, and the organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (0-20% methanol in dichloromethane) to give compound 13 (288 mg,0.40mmol, 43.1%). C 42H83N3O6,MS(ES):m/z(M+H+) 726.6.
1H NMR(CDCl3,400MHz,298K)δ4.20-4.01(6H,m),3.24-3.09(4H,m),2.71-2.51(4H,m),2.44-2.38(2H,m),2.31(3H,s),2.26(6H,s),1.79-1.43(12H,m),1.37-1.23(40H,m),0.88(6H,t,J＝6.0Hz).
Example 2: preparation condition optimization of nano lipid particle (LNP preparation)
1. Vector (liposome) and mRNA ratio optimization
The cationic lipid compounds YK-803, YK-810 and YK-813 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 Co., ltd.), cholesterol (Ai Weita (Shanghai) medical science Co., ltd.) and DMG-PEG2000, respectively, to prepare an ethanol lipid solution. The ethanol lipid solution was rapidly added to citrate buffer (ph=4-5) by ethanol injection and vortexed for 30 s. eGFP-mRNA (purchased from Shanghai laboratory reagent limited) was diluted in citrate buffer (ph=4-5) to give an aqueous mRNA solution. Liposomes were prepared from a volume of liposome solution and an aqueous solution of mRNA at a weight ratio of total lipid to mRNA of 5:1, 10:1, 15:1, 20:1, 30:1 and 35:1, respectively. Ultrasound was performed at 25℃for 15min (ultrasound frequency 40kHz, ultrasound power 800W). The obtained liposome was diluted to 10 times of volume with PBS, and subjected to ultrafiltration in a 300kDa ultrafiltration tube to remove ethanol. The volume was then fixed to volume with PBS to give LNP formulations encapsulating eGFP-mRNA with cationic lipid YK-810/DSPC/cholesterol/DMG-PEG 2000 (molar ratio 49:10:39.5:1.5).
The results of cell transfection experiments show that the weight ratio of the vector to the mRNA is in the range of 10:1-30:1, and the vector has good transfection effect, wherein the transfection effect is preferably 15:1, the transfection effect is poor in the ratios of 5:1 and 35:1, and the mRNA cannot be carried by the ratio.
2. Cationic lipid and neutral lipid ratio optimization
LNP formulations encapsulating eGFP-mRNA were prepared according to the method in 1, with molar ratios of cationic lipid YK-810 to neutral lipid DSPC of 1:1, 3:1, 3.5:1, 4:1, 4.5:1, 4.9:1, 10:1, 15:1 and 20:1, respectively.
As can be seen from the cell transfection experiments, 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 in 1 with YK-810 (or YK-803, YK-813) as 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, respectively, by mole.
The cell transfection experiment result shows 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-810 (or YK-803, YK-813), neutral lipid DSPC, structural lipid cholesterol, and polymer conjugated lipid DMG-PEG2000 molar ratios of 75:5:15:5, 65:8:25:2, 49:10:39.5:1.5, 45:10:43.5:1.5, 45:25:20:10, 40:10:48.5:1.5, 35:10:53.5:1.5, and 25:5:65:5, respectively.
As can be seen from cell transfection experiments, the cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid have good transfection effect in the ranges of the molar ratio 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, and the transfection effect is the best in the ranges of the molar ratio of (35-49): (7.5-15): (35-55): (1-5), wherein the molar ratio is 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 (YK-803, YK-810 or YK-813 as cationic lipids in the vector) containing 1.5. Mu.g of the eGFP-mRNA prepared in example 2 were added to a 12-well plate cell culture medium, 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 vector to 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 1 cationic lipid Structure
The cationic lipids listed in table 1 were dissolved in ethanol at a molar ratio of 45:10:43.5:1.5 with DSPC (Ai Weita (Shanghai) pharmaceutical technologies, inc.), cholesterol (Ai Weita (Shanghai) pharmaceutical technologies, inc.) and DMG-PEG2000, respectively, to prepare an ethanol lipid solution, which was rapidly added to citrate buffer (ph=4 to 5) by ethanol injection, and vortexed for 30s for use. Either eGFP-mRNA (purchased from Shanghai starting Experimental reagent Co., ltd.) or Fluc-mRNA (purchased from Shanghai starting Experimental reagent Co., ltd.) was diluted in 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 a300 kDa 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: a scattering angle of 90 deg. and 25 deg.. The test results are shown in Table 2:
TABLE 2 particle size and polydispersity index (PDI) of nanolipid particles
Name of the name Particle size (nm) PDI(％)
YK-801 165 11.6
YK-802 158 17.9
YK-803 143 14.7
YK-804 200 24.2
YK-805 170 15.6
YK-806 164 11.5
YK-807 158 15.9
YK-808 169 17.2
YK-809 155 12.6
YK-810 168 14.9
YK-811 145 14.7
YK-812 205 23.8
YK-813 170 5.6
YK-814 148 13.9
SM-102 188 15.2
MC3 175 14.3
HHMA 163 19.2
P-76 159 18.9
Compound 13 197 17.1
As can be seen from table 2, the nano-lipid particles prepared in example 4, with particle sizes between 140 and 210nm, can be used to deliver mRNA:
wherein, the particle size of the particles prepared by YK-803 is the smallest and is 143nm; the particle size of the particles prepared from YK-812 was 205nm at maximum.
The polydispersity of all nano-lipid particles is between 5% and 25%, with the smallest being YK-813, 5.6%; the maximum is YK-804, 24.2%.
The morphology of the particles prepared from YK-803, YK-810 and YK-813 was 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.
Fluorescence detection of Fluc-mRNA (transfection efficiency)
LNP preparations containing 0.3. Mu.g of Fluc-mRNA (LNP preparation carrier components are cationic lipid, DSPC, cholesterol and DMG-PEG2000, molar ratio is 45:10:43.5:1.5, wherein cationic lipid is cationic lipid listed in Table 1) were added to cell culture solution of 96-well plates, and after further culture for 24 hours, corresponding reagents were added according to Gaussia Luciferase Assay Kit instructions, and fluorescence expression intensity per well was detected by IVIS fluorescence detection system. The chemical structures of the designed compounds and the representative cationic lipids of the prior art are shown in Table 1. The transfection efficiency of LNP formulations prepared from a series of cationic lipid compounds designed according to the application and prior art cationic lipids, including SM-102, MC3, HHMA, P-76, compound 13 and Lipofectamine 3000, in cells is shown in Table 3.
Table 3 shows the results of fluorescence detection of LNP preparations containing Fluc-mRNA prepared from different cationic lipids.
TABLE 3Fluc-mRNA fluorescence detection results
Analysis of experimental results:
(1) A series of cationic lipid compounds designed by the present application, including YK-803, YK-810 and YK-813, have a vast difference in chemical structure from the cationic lipids typical of the prior art, such as SM-102, MC3 and HHMA; some of the chemical structures are similar, for example compound 13.
The chemical structure of the compounds contemplated by the present application is significantly different from that of the prior art representative cationic lipids SM-102, MC3 and HHMA. From the chemical structural formula, the series of compounds of the application creatively introduces carbamate-OC (O) N (G 1) -, is connected with the head part of tertiary amine structure and the long alkyl straight-chain or branched tail part containing ester group through formate, and SM-102, MC3 and HHMA have no carbamate structure; the amino head structure of SM-102 is of an ethanolamine type with a simple structure, the amino head of MC3 is of a dimethyl tertiary amine structure, the amino head structure of HHMA is of a methyl tertiary amine structure, the hydroxyl structure is positioned at the 2-position of a fatty chain, and the series of compounds provided by the application have tertiary amine head groups with rich structure types.
The chemical structures of the compounds contemplated by the present application are similar, both having urethane structures, as compared to the cationic lipids of the prior art comprising urethane structures, such as P-76 and compound 13. P-76 contains 2 urethane groups, and Compound 13 and the series of compounds of the application each have 1 urethane group. In addition, P76, compound 13 and the compound of the present application each have 2 hydrophobic tails, and the hydrophobic tail group of Compound 13 also contains an ester bond.
(2) Of the designed series of compounds, LNP formulations prepared from YK-803, YK-810 and YK-813 have the highest cell transfection efficiency, and compared with the representative cationic lipids in the prior art, the cell transfection efficiency is remarkably improved whether the structure is greatly different (such as SM-102, MC3 and HHMA) or the structure is less different (such as compound 13). For example, YK-810 can be transfected 5.17 times as efficiently as SM-102, 51.99 times as efficient as MC3, 7.97 times as efficient as HHMA, 52.56 times as efficient as P-76, 189.53 times as efficient as Compound 13, and 11.23 times as efficient as Lipofectamine 3000.
As can be seen from Table 3, LNP preparations containing Fluc-mRNA prepared from YK-803, YK-810 and YK-813 had the strongest fluorescence absorption and RLU values of 5879074, 8789452 and 3778372, respectively. (FIGS. 1 and 2)
YK-803 can be up to 3.46 times that of SM-102, 34.77 times that of MC3, 5.33 times that of HHMA, 35.15 times that of P-76, 126.77 times that of Compound 13, and 7.51 times that of Lipofectamine 3000.
YK-810 can be up to 5.17 times that of SM-102, 51.99 times that of MC3, 7.97 times that of HHMA, 52.56 times that of P-76, 189.53 times that of Compound 13, and 11.23 times that of Lipofectamine 3000.
YK-813 can reach 2.22 times that of SM-102, 22.35 times that of MC3, 3.43 times that of HHMA, 22.59 times that of P-76, 81.47 times that of Compound 13, and 4.83 times that of Lipofectamine 3000.
Analysis of the data with GRAPHPAD PRISM software, any of YK-803, YK-810 and YK-813 showed significant differences compared to SM-102, MC3, HHMA, P-76, compound 13 and Lipofectamine 3000, with significant increases in 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-803 cells were most efficient in transfection compared to a series of compounds of similar structure in which the urethane linkage was linked to the G 1 or G 2 group. The cell transfection efficiency of YK-803 can reach 35.36 times of YK-801 and 93.24 times of YK-802.
A series of compounds having an ester linkage of similar structure, a carbamate structure, linked to a G 1 or G 2 group, including YK-801, YK-802, and YK-803, were compared. The urethane linkages of these compounds are linked to the G 1 or G 2 groups, with the other structures differing only slightly from the individual groups.
Cell transfection results show that the activity of the series of compounds is very different, and the cell transfection efficiency of YK-803 is highest. The cell transfection efficiency of YK-803 can reach 35.36 times of YK-801 and 93.24 times of YK-802.
The GRAPHPAD PRISM software is used for analyzing the data, the YK-803 is obviously different from the YK-801 and the YK-802, and the transfection efficiency is obviously improved
(4) The cell transfection efficiency of YK-810 and YK-813 was highest compared to a series of compounds having a similar structure in which an ester bond of a urethane structure was linked to a G 3 group. For example, the cell transfection efficiencies of YK-810 and YK-813 can be 1390.52-fold and 597.75-fold, and 2020.10-fold and 868.39-fold, respectively, of YK-805.
A series of compounds having an ester linkage of similar structure, a carbamate structure, and a G 3 group, including YK-804, YK-805, YK-806, YK-807, YK-808, YK-809, YK-810, YK-811, YK-812, YK-813, and YK-814, were compared. The urethane linkages of these compounds are each linked to a G 3 group, with the other structures differing only slightly from each other.
Cell transfection results show that the activity of the series of compounds is very different, and the cell transfection efficiency of YK-810 and YK-813 is highest.
The cell transfection efficiency of YK-810 was 598.49 times that of YK-804, 1390.52 times that of YK-805, 21.26 times that of YK-806, 9.94 times that of YK-807, 244.42 times that of YK-808, 26.30 times that of YK-809, 138.95 times that of YK-811, 80.64 times that of YK-812, and 2020.10 times that of YK-814, respectively.
The cell transfection efficiency of YK-813 was 257.28 times that of YK-804, 597.75 times that of YK-805, 9.14 times that of YK-806, 4.27 times that of YK-807, 105.07 times that of YK-808, 11.31 times that of YK-809, 59.73 times that of YK-811, 34.67 times that of YK-812, and 868.39 times that of YK-814, respectively.
The data are analyzed by GRAPHPAD PRISM software, the YK-810 and the YK-813 have obvious differences compared with the YK-804, the YK-805, the YK-806, the YK-807, the YK-808, the YK-809, the YK-811, the YK-812, the YK-814 and the YK-802, and the transfection efficiency is obviously improved
The cell transfection efficiency of LNP formulations prepared therefrom cannot be deduced from the structure of cationic lipid compounds, and even a series of compounds having very close structures are highly likely to have a very large difference in cell 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 in a molar ratio of 45:10:43.5:1.5, wherein the cationic lipid is a cationic lipid as listed in Table 1) were added to cell culture broth of 96-well plates, after further culturing for 24 hours, 10. Mu.L of CCK-8 solution was added to each well, and after incubating the plates in an incubator for 1 hour, absorbance at 450nm was measured by a microplate reader, and the cell viability results are shown in Table 4.
TABLE 4 cell survival
Analysis of experimental results:
(1) A series of cationic lipid compounds designed by the present application, including YK-803, YK-810 and YK-813, have a vast difference in chemical structure from the cationic lipids typical of the prior art, such as SM-102, MC3 and HHMA; some of the chemical structures are similar, for example compound 13. LNP formulations prepared from YK-803, YK-810 and YK-813 have minimal cytotoxicity and significantly improved cell viability compared to the typical cationic lipids of the prior art. For example, cell viability YK-810 may be 8% higher than SM-102, 12% higher than MC3, 23% higher than HHMA%, 15% higher than P-76, 26% higher than Compound 13, 60% higher than Lipofectamine 3000. (FIG. 3)
(2) A series of compounds having an ester linkage of similar structure, a carbamate structure, linked to a G 1 or G 2 group, including YK-801, YK-802, and YK-803, were compared. These compounds differ only slightly in the individual groups. The result shows that YK-803 has the lowest cytotoxicity and the cell survival rate is obviously improved. For example, YK-803 may have a cell viability 18% higher than YK-801.
(3) A series of compounds having an ester linkage of similar structure, a carbamate structure, and a G 3 group, including YK-804, YK-805, YK-806, YK-807, YK-808, YK-809, YK-810, YK-811, YK-812, YK-813, and YK-814, were compared. The results show that YK-810 and YK-813 have the lowest cytotoxicity, and the cell survival rate is remarkably improved. For example, the cell viability of YK-810 and YK813 was 38% and 35% higher than YK-804, 39% and 36% higher than YK-814, respectively.
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.
Example 7: in vivo validation of cationic Lipid (LNP) delivery vehicle performance
MRNA expression in mice
Protein expression and duration of designed cationic lipid delivery mRNA in mice was verified. In vivo experiments further demonstrate that LNP delivery vectors prepared from the cationic lipids of the application can efficiently deliver mRNA into animals and be expressed with high efficiency and duration.
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 the tail vein, and the mice were subjected to intraperitoneal injection of fluorography substrates at specific time points (6 h, 24h, 48h and 7 d) after the administration, and the mice were free to move for 5min, and then the average radiation intensity (corresponding to the 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. The average radiation intensity (corresponding to fluorescence expression intensity) of the protein expressed in the mouse by the mRNA carried by LNP was measured by IVIS Spectrum small animal in vivo imager, and the results of the mouse in vivo imaging measurement are shown in Table 5.
TABLE 5 in vivo imaging experimental data for mice
Analysis of experimental results:
(1) LNP formulations prepared from YK-803, YK-810 and YK-813 showed significantly improved and sustained expression of mRNA in mice compared to the representative cationic lipids of the prior art. For example, LNP preparation mRNA prepared from YK-810 can be expressed in animals at 5.78 times that of SM-102, 26.19 times that of P-76 and 124.86 times that of compound 13 in 24 hours, 7.97 times that of SM-102, 23.17 times that of P-76 and 123.44 times that of compound 13 in 48 hours, and 14.93 times that of SM-102, 59.48 times that of P-76 and 215.00 times that of compound 13 in 7 days. mRNA was consistent with cell transfection activity in terms of expression in mice.
(2) The LNP formulations prepared from YK-803, YK-810 and YK-813 showed significantly improved mRNA expression intensity and duration in mice compared to the structurally similar, slightly different compounds YK-802. For example, LNP preparation mRNA prepared from YK-810 can be expressed in animals up to 130.17 times YK-802 at 24 hours, up to 200.74 times YK-802 at 48 hours, and up to 110.86 times YK-810 at 7 days. mRNA was consistent with cell transfection activity in terms of expression in mice.
2. Distribution of liposomes in mice
In addition, it was further verified that the delivery vectors prepared from the cationic lipids designed according to the present application, e.g., YK-803, YK-810 and YK-813, are capable of enriching in the spleen of mice, and that the delivered mRNA is significantly elevated in the spleen protein expression level of mice compared to the prior art cationic lipids, e.g., SM-102, while being poorly expressed in the liver.
The specific experimental process is as follows:
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 the tail vein, and the mice were given intraperitoneal injection of fluorography substrate at a specific time point (6 h) after administration, and were free to move for 5min, 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 visceral organ imaging experimental data at a specific time point (6 h) after mouse administration
Analysis of experimental results:
LNP formulations prepared from YK-803, YK-810 and YK-813 showed significantly improved expression levels of mRNA in the mouse spleen compared to the representative cationic lipid SM-102 of the prior art. For example, YK-803, YK-810 and YK-813 expressed 8.61-fold, 15.02-fold and 7.58-fold, respectively, in spleen as compared to SM-102. mRNA was consistent with the results of cell transfection in example 6 in terms of mouse spleen expression. YK-803, YK-810 and YK-813 are very weak in liver expression, and the expression amounts are 0.02 times, 0.08 times and 0.04 times that of SM-102, respectively. The proportion of the expressed amount of the delivered mRNA in spleen and liver was only 0.09 times, that of YK-803 was 47.44 times, that of YK-810 was 16.50 times, and that of YK-813 was 17.25 times.
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. Has good targeting effect on developing and treating diseases caused by spleen damage or abnormality, such as lymphoma, leukemia and the like.
Furthermore, LNP preparations containing Fluc-mRNA prepared from all compounds showed very large differences in expression in different organs of mice, YK-803, YK-810 and YK-813 were expressed almost exclusively in the spleen, in small amounts in the liver, and none in other organs such as heart, lung and kidney; SM-102 was expressed in liver and spleen, but not in heart, lung and kidney (fig. 4).
In summary, the present application contemplates a series of cationic lipid compounds, e.g., YK-803, YK-810 and YK-813, that have significantly improved cell transfection efficiency, significantly reduced cytotoxicity, and significantly improved mRNA expression and duration in mice.
1. A series of compounds were designed, including YK-803, YK-810 and YK-813, representative cationic lipids of the prior art, with a vast difference in chemical structure, such as SM-102, MC3 and HHMA; some of the chemical structures are similar, for example compound 13.
2. In the designed series of compounds, LNP formulations prepared from YK-803, YK-810 and YK-813 showed significantly improved cell transfection efficiency, significantly reduced cytotoxicity, and significantly improved mRNA expression and duration in mice compared to the prior art representative cationic lipids (whether of widely different structure, e.g., SM-102, MC3 and HHMA, or of closely similar structure, e.g., P-76 and Compound 13). For example, YK-810 can be 5.17 times as efficient as SM-102, 51.99 times as efficient as MC3, 7.97 times as efficient as HHMA, 52.56 times as efficient as P-76, 189.53 times as efficient as Compound 13, and 11.23 times as efficient as Lipofectamine 3000; cell viability YK-810 may be 8% higher than SM-102, 12% higher than MC3, 23% higher than HHMA%, 15% higher than P-76, 26% higher than Compound 13, 60% higher than Lipofectamine 3000; mRNA expression in animals can reach 5.78 times of SM-102, 26.19 times of P-76 and 124.86 times of compound 13 in 24 hours, 14.93 times of SM-102, 59.48 times of P-76 and 215.00 times of compound 13 in 7 days; mRNA expression level YK-810 in spleen can reach 15.02 times of SM-102, and expression level YK-810 in liver is only 0.08 times of SM-102.
3. In a series of compounds with small chemical structure difference, LNP preparations prepared from YK-803, YK-810 and YK-813 have significantly improved cell transfection efficiency, significantly reduced cytotoxicity and significantly improved mRNA expression in mice compared with other compounds. For example, YK-810 cell transfection efficiency can be 2020 times that of YK-814, cytotoxicity can be reduced by 38% compared with YK-804, and mRNA expression in mice can be 200 times that of YK-802.
4. Through unique design and screening, the present disclosure discovers that some compounds, such as YK-803, YK-810 and YK-813, can significantly improve cell transfection efficiency, significantly reduce cytotoxicity, significantly improve expression level and expression time in animals, significantly improve expression level in the spleen of animals and significantly reduce expression level in the liver, improve delivery efficiency, and achieve unexpected technical effects compared with other compounds similar in structure in the prior art. Can realize the rapid induction of immune response and antibody production in mRNA vaccine. The vaccine composition has the significant clinical significance and can obviously improve the prevention effect under the condition of not changing the vaccine components.
5. Through unique design and screening, the present disclosure finds that some compounds, such as YK-803, YK-810 and YK-813, can target mRNA to the spleen without expression in other organs, such as the lung, heart and kidney and with small expression in the liver under the premise of ensuring high efficiency and low toxicity. The low immune response induction efficiency of the vaccine is the reason that the existing tumor therapeutic vaccine cannot exert the maximum curative effect, the spleen is the largest secondary lymphoid organ in the body, the LNP tumor vaccine targeting the spleen can effectively excite the immune response, the curative effect is obviously improved, and the vaccine has important clinical application significance in cancer treatment.
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 (57)

1.A compound of formula (I)
Or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein,
G 1 is C 2～10 alkylene;
g 2 is C 2～10 alkylene;
g 3 is
R 1 is unsubstituted C 6～25 straight or branched alkyl;
R 2 is unsubstituted C 6～25 straight or branched alkyl;
m and n are 1 or 0, n is 1 when m is 0, and n is 0 when m is 1.
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 C 5、C7 or C 3 alkylene.
3. A compound of formula (I) according to claim 1 or 2, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 2 is C 5、C3 or C 7 alkylene.
4. A compound of formula (I) according to any one of the preceding claims, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 1 is unsubstituted C 8～22 straight chain alkyl.
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 R 1 is unsubstituted C 10、C11、C8、C9 or C 12 straight chain alkyl.
6. A compound of formula (I) according to any one of the preceding claims, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 1 is unsubstituted C 20、C15、C17 branched alkyl.
7. A compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of the preceding claims, wherein R 1 is
8. A compound of formula (I) according to any one of the preceding claims, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 2 is unsubstituted C 8～22 straight chain alkyl.
9. A compound of formula (I) according to any one of the preceding claims, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 2 is unsubstituted C 10、C11、C8、C9 or C 12 straight chain alkyl.
10. A compound of formula (I) according to any one of the preceding claims, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 2 is unsubstituted C 20、C15、C17 branched alkyl.
11. A compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of the preceding claims, wherein R 2 is
12. 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 the compound of formula (I) has one of the following structures:
13. 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-803 having the structure:
14. 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-810 having the structure:
15. 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-813 having the structure:
16. a composition comprising a carrier comprising a cationic lipid comprising a compound of formula (I) as defined in any one of the preceding claims or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof.
17. The composition of claim 16, wherein the cationic lipid comprises 25% to 75% of the carrier by mole.
18. The composition of any one of claims 16-17, wherein the carrier further comprises a neutral lipid.
19. Composition according to claim 18, wherein the molar ratio of cationic lipid to neutral lipid is 1:1 to 15:1, preferably 4.5:1.
20. The composition of any of claims 18-19, wherein the neutral lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols, and derivatives thereof.
21. The composition of claims 18-20, 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 (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphorylcholine (C), 1, 2-dioleoyl-s-glycero-3-phosphorylcholine (Lyso PC), 1, 2-dioleoyl-2-glycero-3-phosphorylcholine, 1-dioleoyl-s-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-glycero-s-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-docosahexaenoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-dioleoyl-sn-glycero-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.
22. The composition of claim 20, wherein the neutral lipid is 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and/or 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
23. The composition of any one of claims 16-22, wherein the carrier further comprises a structural lipid.
24. The composition of claim 23, wherein the molar ratio of the cationic lipid to the structural lipid is from 0.6:1 to 3:1.
25. The composition of any one of claims 23-24, 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.
26. The composition of claim 25, wherein the structural lipid is cholesterol.
27. The composition of any one of claims 16-26, wherein the carrier further comprises a polymer conjugated lipid.
28. The composition of claim 27, wherein the molar ratio of the polymer conjugated lipid to the carrier is 0.5-10%, preferably 1.5%.
29. The composition of any of claims 27-28, 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.
30. The composition of claim 29, 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).
31. The composition of any one of claims 16-30, wherein the carrier comprises a cationic lipid, a neutral lipid, a structural lipid, and a polymer conjugated lipid. The cationic lipid: neutral lipids: structural lipids: the molar ratio of the conjugated polymer lipid is (25-75): 5-25): 15-65): 0.5-10.
32. The composition of claim 31, wherein the cationic lipid: neutral lipids: structural lipids: the molar ratio of the conjugated polymer lipid is (35-49): 7.5-15): 35-55): 1-5.
33. The composition of claim 32, wherein the cationic lipid: neutral lipids: structural lipids: the molar dosage ratio of the polymer conjugated lipid is 45:10:43.5:1.5.
34. The composition of any one of claims 16-33, wherein the composition is a nanoparticle formulation having an average particle size of 10nm to 210nm; the polydispersion coefficient (PDI) of the nanoparticle preparation is less than or equal to 50 percent.
35. The composition of claim 34, wherein the nanoparticle formulation has an average particle size of 100nm to 205nm; the polydispersion coefficient (PDI) of the nanoparticle preparation is less than or equal to 30 percent.
36. The composition of any one of claims 16-35, wherein the cationic lipid further comprises one or more other ionizable lipid compounds.
37. The composition of any one of claims 16-36, further comprising a therapeutic or prophylactic agent.
38. The composition of claim 37, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is from 10:1 to 30:1.
39. The composition of claim 38, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 12.5:1 to 25:1.
40. The composition of claim 39, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 15:1.
41. The composition of any one of claims 37-40, wherein the therapeutic or prophylactic agent comprises one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.
42. The composition of any one of claims 37-40, wherein the therapeutic or prophylactic agent is a vaccine or compound capable of eliciting an immune response.
43. The composition of any one of claims 37-42, wherein the therapeutic or prophylactic agent is a nucleic acid.
44. The composition of claim 43, wherein the therapeutic or prophylactic agent is ribonucleic acid (RNA).
45. The composition of claim 43, wherein the therapeutic or prophylactic agent is deoxyribonucleic acid (DNA).
46. The composition of claim 37, 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.
47. The composition of claim 46, wherein the RNA is mRNA.
48. The composition of any one of claims 16-47, wherein the composition further comprises one or more pharmaceutically acceptable excipients or diluents.
49. Use of a compound of formula (I) as defined in any one of claims 1 to 15 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof or a composition as defined in any one of claims 18 to 50 for the preparation of a nucleic acid medicament, a genetic vaccine, a small molecule medicament, a polypeptide or a protein medicament.
50. Use of a compound of formula (I) as defined in any one of claims 1 to 15 or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof or a composition as defined in any one of claims 18 to 50 in the manufacture of a medicament for the treatment of a disease or condition in a mammal in need thereof.
51. The use of claim 50, wherein the disease or disorder is characterized by dysfunctional or abnormal protein or polypeptide activity.
52. The use of claim 50 or 51, 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.
53. The use of claim 52, 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.
54. The use of any one of claims 50-53, wherein the subject to which the medicament is administered is a human.
55. The use of any one of claims 49-54, wherein the route of administration of the medicament is intravenous, intramuscular, intradermal, subcutaneous, intranasal, or inhalation.
56. The use of claim 55, wherein the route of administration of the medicament is subcutaneous.
57. The use of any one of claims 49-56, wherein the medicament is administered at a dose of 0.001-10 mg/kg.
CN202410249824.6A 2024-03-05 2024-03-05 Low-toxicity spleen-targeted cationic lipid compound containing carbamate structure, composition containing low-toxicity spleen-targeted cationic lipid compound and application of low-toxicity spleen-targeted cationic lipid compound Pending CN118125947A (en)

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