Compound for delivery system and application thereof

Abstract

The present application provides a compound for a delivery system and uses thereof, and provides an ionizable lipid having a hydrogen bond providing group.

Description

A compound for delivery system and use thereof Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a compound for a delivery system and its use. Background Art

[0002] Therapies based on the delivery of translatable nucleotides have enormous potential for clinical application. The therapeutic process is to produce selectable nucleic acid expression products - protein sequences through synthetic constructs. Regardless of whether the sequence is inherent to the system, it can achieve the effect of increasing the existing level of protein, replacing the missing or non-functional form of protein, and introducing new proteins and their related biological functions into cells or organisms. Therapeutic nucleic acids include small interfering RNA (siRNA), messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNA enzymes, plasmids, immunostimulatory nucleic acids, antagomirs, antimiRs, mRNA mimics, supermir.UI adapters and aptamers. Their expression in specific cells is crucial in treating diseases such as those associated with protein or enzyme deficiency. Therefore, the effective delivery of these therapeutic nucleic acids to target sites in cells or organisms is a key challenge in achieving nucleic acid-based therapies.

[0003] The use of oligonucleotide constructs in the current therapeutic environment mainly faces two problems. First, the structural instability of free RNA itself makes it extremely easy to be rapidly degraded by various enzymes in the body during delivery; second, the ability of free RNA to enter the intracellular compartment where the relevant translation system is located is limited. This limitation is specifically manifested in the following ways: first, the negatively charged RNA skeleton and the negative charge on the cell membrane surface produce electrostatic repulsion, resulting in a membrane barrier; second, after the construct enters the cell through endocytosis generated by electrostatic binding to the cell membrane, it will be encapsulated in the endosome. The mature endosome will then fuse with the lysosome containing specific digestive enzymes. Therefore, the construct needs to escape from the endosome and / or lysosome to prevent the degradation of the payload nucleic acid triggering the construct. Lipid nanoparticles (LNPs) co-formulated with lipids and other lipid components (e.g., neutral lipids, steroids, PEGylated lipids) for loading oligonucleotides have been used to block RNA degradation in vivo and promote cellular uptake of delivered oligonucleotides. However, there is still a need to study the optimized lipids and LNPs for delivering oligonucleotides to improve their encapsulation efficiency, the integrity of the content drug, the escape rate, and the safety of LNP-related drug formulations.

[0004] Summary of the Invention

[0005] The present application provides a compound for a delivery system, including its stereoisomers, pharmaceutically acceptable salts, prodrugs or tautomers; and its uses, including using the compound alone, or in combination with other lipid components such as neutral lipids, charged lipids, steroids and / or their analogs, and / or polymer-conjugated lipids to form lipid nanoparticles for delivering therapeutic agents. The compound of the present application is designed with a substituent group (e.g., urea group) that can form an intermolecular hydrogen bond with the RNA base at the position of 2-6 atoms near the head group (e.g., tertiary amine) that can be protonated in response to changes in pH. The weak interaction force between the nucleic acid formed by this design and the electrostatic effect synergistically load RNA, which can improve the encapsulation efficiency (Encapsulation efficiency) of LNP or liposomes, while improving the integrity (Lipid and cargo integrity) of lipids and loaded drugs. In addition, compared with existing technologies (such as ALCO315), ionizable lipids with urea groups are more conducive to accelerating the degradation of LNPs in the body, thereby improving the safety of formulations containing such LNPs, as the electrophilicity of the hydroxyl groups is neutralized by the amines on both sides and they do not form intramolecular six-membered or five-membered rings with tertiary amines.

[0006] In one aspect, the present application provides a compound, or a pharmaceutically acceptable salt thereof, comprising a cation providing group and a hydrogen bond providing group, wherein the cation providing group and the hydrogen bond providing group contain 2 to 6 atoms.

[0007] In some embodiments, the compound comprises a cationic lipid.

[0008] In some embodiments, the compound comprises an ionizable lipid.

[0009] In some embodiments, the cation-donating group comprises a group that is positively charged at a pH of about 7.0 or less.

[0010] In some embodiments, the cation-donating group comprises a group that is positively charged at a pH of about 4.0 or less.

[0011] In some embodiments, the cation-providing group comprises a tertiary amine group.

[0012] In some embodiments, the hydrogen bond providing group comprises a urea group.

[0013] In some embodiments, the compound comprises a tail that is hydrophobic.

[0014] In some embodiments, the tail comprises a saturated and / or unsaturated fatty chain.

[0015] In some embodiments, the tail is substituted on one or both nitrogen atoms of the urea group.

[0016] In some embodiments, the backbone chain of the tail is 1 to 20 atoms in length.

[0017] In some embodiments, the compound comprises the following structure (I):

[0018] Wherein, L1 is a hydrogen bond providing group, G1 is a linking group with a chain length of 2 to 6 atoms, said L1 is optionally substituted, and said G1 is optionally substituted.

[0019] In some embodiments, the compound comprises the following structure (II):

[0020] Wherein, G1 is a linking group with a chain length of 2 to 6 atoms, said G1 is optionally substituted, R1, R 2a and R 2b are each independently selected from optionally substituted substituents.

[0021] In some embodiments, the compound comprises the following structure (IIIa):

[0022] wherein G1 and G2 are each independently a linking group having a chain length of 2 to 6 atoms, said G1 and G2 are each independently optionally substituted, R1, R 2a 、R 2b , R3, R 4a and R 4b are each independently selected from optionally substituted substituents.

[0023] In some embodiments, the compound comprises the following structure (IIIb):

[0024] wherein G1 is a linking group having a chain length of 2 to 6 atoms, and G1 is optionally substituted, and R1, R 2a 、R 2b , R5 and R6 are each independently selected from optionally substituted substituents.

[0025] In some embodiments, the compound comprises the following structure (IIIc):

[0026] wherein G1 and G2 are each independently a linking group having a chain length of 2 to 6 atoms, said G1 and G2 are each independently optionally substituted, R1, R 2a 、R 2b , R7, R 8a 、R8b and R9 are each independently selected from optionally substituted substituents.

[0027] In some embodiments, the compound comprises the following structure (IIId):

[0028] wherein G1, G2, G3 and G4 are each independently a linking group having a chain length of 2 to 6 atoms, said G1, G2, G3 and G4 are each independently optionally substituted, said H1 and H2 are each independently optionally substituted, R1, R 2a 、R 2b , R7, R 8a 、R 8b 、R 10 、R 11a 、R 11b 、R 12 、R 13a and R 13b are each independently selected from optionally substituted substituents.

[0029] In some embodiments, G1, G2, G3 and G4 are each independently a hydrocarbon group, an alicyclic group, and / or an alicyclic heterocyclic group.

[0030] In some embodiments, G1, G2, G3, and G4 are each independently alkyl, alkenyl, and / or alkynyl.

[0031] In some embodiments, one atom in G1, G2, G3, and G4 is each independently replaced by N, O, or S.

[0032] In some embodiments, H1 and H2 are each independently a hydrocarbon group, an alicyclic group, and / or an alicyclic heterocyclic group.

[0033] In some embodiments, H1 and H2 are each independently alkyl, alkenyl, and / or alkynyl.

[0034] In some embodiments, one atom in H1 and H2 is each independently replaced by N, O, or S.

[0035] In some embodiments, the R1, R 2a 、R 2b , R3, R 4a 、R 4b , R5, R6, R7, R 8a 、R 8b , R9, R 10 、R 11a 、R 11b 、R 12 、R 13a , and R 13bEach is independently a hydrocarbon group, an ester group, an amide group, an N'-alkyl-N-alkenyl-urea group, an alicyclic group, an alicyclic heterocyclic group, an aryl group, and / or a heteroaryl group.

[0036] In some embodiments, the R1, R 2a 、R 2b , R3, R 4a 、R 4b , R5, R6, R7, R 8a 、R 8b , R9, R 10 、R 11a 、R 11b 、R 12 、R 13a , and R 13b Each independently is C1-C 20 Alkyl, C2-C 40 Alkenyl, C2-C 45 Ester group and / or C2-C 20 Alkynyl.

[0037] In some embodiments, the R1, R 2a 、R 2b , R3, R 4a 、R 4b , R5, R6, R7, R 8a 、R 8b , R9, R 10 、R 11a 、R 11b 、R 12 、R 13a , and R 13b Each is independently branched or unbranched.

[0038] In some embodiments, the R1, R 2a 、R 2b , R3, R 4a 、R 4b , R5, R6, R7, R 8a 、R 8b , R9, R 10 、 R 11a 、R 11b 、R 12 、R 13a , and R 13b Each independently contains 0 to 4 unsaturated bonds.

[0039] In some embodiments, the R1, R 2a 、R 2b , R3, R 4a 、R 4b , R5, R6, R7, R 8a 、R8b , R9, R 10 、R 11a 、R 11b 、R 12 、R 13a , and R 13b Each is independently substituted by Rx, wherein Rx is an optionally substituted substituent. X C1-C 20 Alkyl, C2-C 25 Ester group, C2-C 20 alkenyl, amide, urea, hydroxyl, thiol or amino.

[0040] In another aspect, the present application provides a compound, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the structures provided in the present application.

[0041] In another aspect, the present application provides a composition comprising the compound and a therapeutic agent.

[0042] In some embodiments, the composition further comprises a substance selected from the group consisting of a neutral lipid, a steroid, and a polymer-conjugated lipid.

[0043] In some embodiments, the composition comprises liposomes.

[0044] In some embodiments, the composition is about 500 nm or less in diameter.

[0045] In some embodiments, the steroid comprises cholesterol.

[0046] In some embodiments, the polymer-conjugated lipid comprises a PEGylated lipid.

[0047] In some embodiments, the molar ratio of the compound to the neutral lipid is from about 2:1 to about 8:1.

[0048] In some embodiments, the molar ratio of the compound to the steroid is about 5:1 to 1:1.

[0049] In some embodiments, the molar ratio of the compound to the polymer-conjugated lipid is from about 100:1 to about 20:1.

[0050] In some embodiments, the therapeutic agent comprises a nucleic acid.

[0051] In some embodiments, the therapeutic agent comprises a component of a CRISPR system and / or a nucleic acid encoding a component of a CRISPR system.

[0052] In some embodiments, the therapeutic agent comprises a guide RNA and / or a nucleic acid encoding a Cas enzyme.

[0053] In another aspect, the present application provides a cell comprising the compound and / or the composition.

[0054] On the other hand, the present application provides a kit comprising the compound, the composition and / or the cell.

[0055] On the other hand, the present application provides a method for administering a therapeutic agent, which comprises providing the compound, the composition, the cell and / or the kit, wherein the therapeutic agent is mixed with the compound, or the therapeutic agent is present in the composition, the cell and / or the kit.

[0056] On the other hand, the present application provides a method for delivering a nucleic acid, which comprises providing the compound, the composition, the cell and / or the kit, wherein the nucleic acid is mixed with the compound, or the nucleic acid is present in the composition, the cell and / or the kit.

[0057] On the other hand, the present application provides a use of the compound, the composition, the cell and / or the kit in preparing a medicament, wherein the medicament is used in a method for treating and / or preventing a disease or condition.

[0058] Those skilled in the art will readily appreciate other aspects and advantages of the present application from the detailed description below. The detailed description below only illustrates and describes exemplary embodiments of the present application. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are intended to be exemplary only and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0060] FIG1 shows the expression effect of the delivery substance of the present application on Luciferase in mouse liver.

[0061] FIG2 shows the evaluation of the liver transfection effect of the delivery material of the present application in adult Ai9 transgenic mice by intravenous injection.

[0062] FIG3 shows the evaluation of the PCSK9 gene editing effect of the delivery material of the present application in vivo in mice. DETAILED DESCRIPTION

[0063] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0064] Definition of terms

[0065] In this application, the term "compound" generally refers to a substance having two or more different elements. For example, the compound of the present application may be an organic compound, for example, the compound of the present application may be a compound with a molecular weight of less than 500, a compound with a molecular weight of less than 1000, a compound with a molecular weight of more than 1000, or a compound with a molecular weight of more than 1000 or more than 10000. In this application, a compound may also refer to a compound connected by chemical bonds, for example, a compound in which one or more molecules with a molecular weight of less than 1000 are connected to a biomacromolecule by a chemical bond, and the biomacromolecule may be a polysaccharide, protein, nucleic acid, polypeptide, etc. For example, the compound of the present application may include a compound in which a protein is connected to one or more molecules with a molecular weight of less than 1000, a compound in which a protein is connected to one or more molecules with a molecular weight of less than 10000, or a compound in which a protein is connected to one or more molecules with a molecular weight of less than 10000.

[0066] As used herein, the term "compound of the present application" refers to the compound of the present application. The term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compound of the present application. Those skilled in the art will also recognize that "prodrug" generally refers to the prodrug substance that can be metabolized to form a substance having the structure of the compound of the present application after administration to a subject, and "metabolite" generally refers to the substance obtained by the metabolism of the compound of the present application after administration to a subject, and the prodrugs and metabolites contained in such derivatives are included within the scope of the present invention.

[0067] In the present application, the compound of the present application comprises tautomers, mesomorphs, racemates, enantiomers, and / or diastereomers of the compound. In the present application, the term "diastereomer" generally refers to a stereoisomer with two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers can have different physical properties, such as, melting point, boiling point, spectral properties and reactivity. In the present application, the terms "tautomer" or "tautomeric form" are used interchangeably, generally referring to structural isomers of different energies that can be mutually converted by low energy barriers (low energy barrier). For example, proton tautomers (protontautomers) (also referred to as prototropic tautomers (prototropic tautomers)) include the mutual conversions carried out by proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers (valence tautomers) include the mutual conversions carried out by the reorganization of some bonding electrons. In this application, the term "meso" generally refers to a molecule containing asymmetric atoms but having symmetry factors that result in a total optical rotation of zero. The term "racemate" or "racemic mixture" refers to a composition composed of equimolar amounts of two enantiomeric substances.

[0068] Unless otherwise specified, all compounds mentioned in this application are intended to include all possible optical isomers, such as single chiral compounds, or mixtures of various chiral compounds (i.e., racemates). In all compounds of this application, each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R and S configurations.

[0069] In the present application, some atoms of the compounds of the present application may appear in more than one isotopic form. For example, hydrogen may appear in the form of protium ( 1 H), deuterium ( 2 H) and tritium ( 3 H), carbon can exist in three different isotopes ( 12 C. 13 C and 14 C) naturally occurring. Examples of isotopes that can be incorporated into the compounds of the present application also include, but are not limited to 15 N. 18 O. 17 O. 18 F. 32 P. 33 P. 129 I. 131 I. 123 I. 124 I. 125I, or similar isotopes. Therefore, relative to the natural abundance of these isotopes, the compounds of the present application may be enriched in one or more of these isotopes. As known to those skilled in the art, such isotopically enriched compounds can be used for a variety of purposes. For example, with heavy isotopes such as deuterium ( 2 Deuterium (H) substitution may offer certain therapeutic advantages, possibly due to greater metabolic stability. 2 The natural abundance of deuterium (H) is about 0.015%. Therefore, there is one deuterium atom for approximately every 6500 hydrogen atoms in nature. Therefore, the deuterium-containing compounds of the present application have a deuterium abundance greater than 0.015% at one or more positions (as the case may be). Unless otherwise indicated, the structures described herein may also include compounds that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds whose remaining portions are consistent with the present structure except that hydrogen atoms are replaced by deuterium or tritium, or carbon atoms are replaced by carbon 13 or carbon 14, are within the scope of the present application.

[0070] In this application, the term "pharmaceutically acceptable" refers to substances that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. In this application, the term "pharmaceutically acceptable salt" refers to a salt of the compound as defined above that is pharmaceutically acceptable and has the desired pharmacological activity, including acid addition salts and base addition salts of the compound. The term "pharmaceutically acceptable acid addition salt" refers to a salt which retains the biological efficacy and properties of the free base, is not biologically or otherwise undesirable, and is soluble in water and / or in a solvent. The salt is soluble in water and / or in a solvent. The salt is soluble in water and / or in a solvent. The pharmaceutically acceptable base addition salts include salts formed from hydroxybenzoic acid, ... These salts are prepared by adding an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, diarnol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrazine, choline, betaine, pheniramine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like.

[0071] In this application, the terms "groups containing 2 to 6 atoms" and "linking groups with a chain length of 2 to 6 atoms" refer to 2, 3, 4, 5 or 6 atoms connected to each other via continuous covalent bonds to form a chain-like molecular fragment or linking group, wherein the atoms can be single atoms (such as but not limited to carbon atoms, nitrogen atoms, oxygen atoms and sulfur atoms, etc.), central atoms contained in any substituents (such as but not limited to the central carbon atom of an alkyl, alkenyl or alkynyl group and the central nitrogen atom of an amine group, etc.) and / or atoms in any substituent that participate in forming the main chain (such as but not limited to the carbon atom and oxygen atom connected by a single bond in an ester group, etc.). In certain embodiments, the groups may contain multiple atoms forming a cyclic structure or the linking group between the groups is a cyclic group. In this case, the number of atoms separated or the chain length is the number of atoms forming the shortest distance between the two atoms connected to the main chain on the cyclic structure (including the atoms at both ends).

[0072] In this application, the term "ionizable lipid" refers to a lipid having at least one protonatable or deprotonatable group, such as a cationic lipid, such that the lipid is positively charged at a pH equal to or lower than physiological pH (e.g., pH 7.4) and neutral at a second pH (preferably equal to or higher than physiological pH). One of ordinary skill in the art will understand that the addition or removal of protons depending on pH is an equilibrium process, and reference to charged or neutral lipids refers to the properties of the primary species and does not require that all lipids exist in a charged or neutral form. Typically, the pKa of the protonatable group of an ionizable lipid ranges from about 4 to about 7, and its ionization reaction at different pHs affects the surface charge of the LNP, a charge state that can affect plasma protein absorption, blood clearance, and tissue distribution, which are critical for intracellular delivery of nucleic acids, as well as the ability to form endosomal non-bilayer structures.

[0073] In this application, the term "cation providing group" generally refers to a head group with a positive charge in an ionizable lipid, and its group size and charge density have an impact on the efficiency of encapsulating nucleic acids, stabilizing LNPs, interacting with cell membranes, and promoting processes such as endosome escape. Common ionizable lipids contain only one head group, and some also contain several head groups. Non-limiting examples of cation providing groups include amines (primary amines, secondary amines, tertiary amines, quaternary amines), guanidines, heterocyclic groups, etc. For example, the ionizable lipids DLin-MC3-DMA, SM-102, and ALC-0315 used clinically contain tertiary amine heads, which can undergo pH-dependent cationization. The cation providing group can be substituted or unsubstituted.

[0074] In this application, the term "hydrogen bond providing group" generally refers to a linking group or a group that is part of a linking group in an ionizable lipid that can serve as a hydrogen bond donor to form weak intermolecular interactions with nucleic acid bases. Non-limiting examples of hydrogen bond providing groups include urea, amide, carboxyl, and divalent transition metals. The hydrogen bond providing group may be substituted or unsubstituted.

[0075] In this application, the term "hydrophobic tail" refers to a tail group having a non-polar group, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups optionally substituted with one or more aromatic, cycloaliphatic, or heterocyclic groups. Preferred examples include but are not limited to diacylglycerols, dialkylglycerols, NN-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0076] In this application, the term "main chain" generally refers to the carbon chain with the most carbon atoms, or the longest carbon chain containing functional groups, when referring to a group. In certain embodiments, when a group has chains of the same length that can serve as the main chain, the carbon chain with the most branches (or the simplest branches) is selected as the main chain.

[0077] As used herein, the terms "optionally substituted substituents" and "optionally substituted" mean that the referenced group may be unsubstituted or substituted by one or more additional groups individually and independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, hydroxy, alkoxy, mercapto, cyano, halogen, carbonyl, thiocarbonyl, isocyanato, thiocyanato, isothiocyanato, nitro, perhaloalkyl, perfluoroalkyl, and amino including mono- and di-substituted amino groups, and protected derivatives thereof. Non-limiting examples of optional substituents include halogen, -CN, =O, =N-OH, =N-OR, =NR, OR, -C(O)R, -C(O)OR, -OC(O)R, -OC(O)OR, -C(O)NHR, -C(O)NR2, -OC(O)NHR, -OC(O)NR2, -SR-, -S(O)R, -S(O)2R, -NHR, -N(R)2, -NHC(O)R, -NRC(O)R, -NHC(O)OR, -NRC(O)OR, S(O)2NHR , -S(O)2N(R)2, -NHS(O)2NR2, -NRS(O)2NR2, -NHS(O)2R, -NRS(O)2R, C1-C8 alkyl, C1-C8 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C8 alkyl, and halogen-substituted C1-C8 alkoxy, wherein each R is independently selected from H, halogen, C1-C8 alkyl, C1-C8 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C8 alkyl, and halogen-substituted C1-C8 alkoxy. The position and number of such substituent groups are determined by the valence restrictions well known to each group, for example, =0 is a suitable substituent for alkyl but not for aryl.

[0078] In the present application, the term "alkyl" generally refers to a residue derived from an alkane by removing a hydrogen atom. An alkyl group can be substituted or non-substituted, substituted or non-substituted. The term "alkyl" generally refers to a saturated straight or branched aliphatic hydrocarbon group having a residue derived from the same carbon atom or two different carbon atoms of the parent alkane by removing a hydrogen atom, and can be a straight or branched group containing 1 to 20 carbon atoms, for example, a chain alkyl group containing 1 to 12 carbon atoms, for example, 1 to 6 carbon atoms. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, propyl, butyl, etc. The alkyl group may be substituted or unsubstituted, substituted or unsubstituted, for example, when substituted, the substituent may be substituted at any available point of attachment, and the substituent may be independently selected from one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo, for example, hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H or C 1-6 Aliphatic group.

[0079] In this application, the term "alkenyl" generally refers to a straight or branched hydrocarbon group containing one or more double bonds. Illustrative examples of alkenyl include allyl, homoallyl, vinyl, crotyl, butenyl, pentenyl, and hexenyl. C 2-6 Illustrative examples of alkenyl groups include butadienyl, pentadienyl, hexadienyl and hexatrienyl and their branched forms. The position of the unsaturated bond (double bond) can be at any position of the carbon chain. The alkenyl group can be substituted or unsubstituted.

[0080] In the present application, the term "alkynyl" generally refers to an unsaturated straight-chain or branched alkynyl group, such as ethynyl, 1-propynyl, propargyl, butynyl, etc. Alkynyl groups can be substituted or unsubstituted.

[0081] In this application, the term "hydrocarbyl" generally refers to a straight chain, branched chain, or cyclic hydrocarbon radical or a combination thereof consisting solely of carbon and hydrogen atoms, which may be fully saturated, monounsaturated, or polyunsaturated, and may include divalent and polyvalent radicals, having the specified number of carbon atoms (i.e., C1-C 10represents one to ten carbon atoms). Examples of saturated hydrocarbon radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, n-pentyl, n-hexyl, n-heptyl, and homologs and isomers of n-octyl. Unsaturated hydrocarbon groups are those having one or more double bonds or triple bonds. Examples of unsaturated hydrocarbon groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers. The term "hydrocarbon group" is also meant to include those hydrocarbon derivatives defined in detail below, such as "heterohydrocarbon groups," unless otherwise noted. Hydrocarbon groups limited to hydrocarbon groups are also referred to as "homohydrocarbon groups."

[0082] As used herein, the term "alicyclic group" generally refers to a residue derived by removing a hydrogen atom from the same carbon atom or multiple different carbon atoms of an aliphatic ring. The term "cycloalkane" generally refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon, wherein the carbocyclic ring contains 3 to 20 carbon atoms, may contain 3 to 12 carbon atoms, may contain 3 to 10 carbon atoms, or may contain 3 to 8 carbon atoms. Non-limiting examples of alicyclic groups include cyclopropane, cyclobutane, cyclopentane, cyclopentenyl, cyclohexane, cyclohexenyl, cyclohexadienyl, cycloheptane, cycloheptatrienyl, cyclooctanyl, and the like; polycyclic carbocyclic rings may include spiro, fused, and bridged carbocyclic rings. Alicyclic groups may be substituted or unsubstituted. As used herein, the term "carbocyclic group" generally refers to a residue derived by removing a hydrogen atom from a carbon atom of a carbocyclic ring. The term "carbocycle" generally refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon ring, wherein the carbocycle contains 3 to 20 carbon atoms, may contain 3 to 12 carbon atoms, may contain 3 to 10 carbon atoms, and may contain 3 to 8 carbon atoms. Non-limiting examples of monocyclic carbocycles include cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexene, cyclohexadiene, cycloheptane, cycloheptatriene, cyclooctane, and the like; polycyclic carbocycles may include spirocyclic, fused, and bridged carbocycles. Carbocyclyl groups may be substituted or unsubstituted. In some cases, alicyclic and carbocyclic rings may be used interchangeably.

[0083] As used herein, the term "partially unsaturated" generally refers to a cyclic structure containing at least one double or triple bond between the ring molecules. The term "partially unsaturated" encompasses cyclic structures with multiple unsaturations but is not intended to include aromatic or heteroaromatic rings as defined herein. The term "unsaturated" refers to a moiety having one or more degrees of unsaturation.

[0084] In this application, the term "alicyclic group" generally refers to a stable, non-aromatic, 3- to 7-membered monocyclic carbocyclic ring structure, a fused 7- to 10-membered bicyclic heterocyclic ring structure, or a bridged 6- to 10-membered bicyclic heterocyclic ring structure. These ring structures may be saturated or partially saturated and, in addition to carbon atoms, may contain one or more heteroatoms selected from the group consisting of oxygen, sulfur, and nitrogen. For example, these ring structures may contain 1 to 4 heteroatoms as defined above. When used to refer to atoms in an alicyclic heterocyclic ring structure, the term "nitrogen" may include substituted nitrogen. For example, an alicyclic heterocyclic group may include a "heterocycloalkyl" group, which may refer to a stable, non-aromatic, 3- to 7-membered monocyclic carbocyclic ring structure, a fused 7- to 10-membered bicyclic heterocyclic ring structure, or a bridged 6- to 10-membered bicyclic heterocyclic ring structure. In addition to carbon atoms, these ring structures may contain one or more heteroatoms selected from the group consisting of oxygen, sulfur, and nitrogen. For example, these ring structures may contain 1 to 4 heteroatoms as defined above. Heterocycloalkyl groups may be substituted or unsubstituted. Alicyclic heterocyclic groups may be substituted or unsubstituted.

[0085] In this application, the term "aryl" generally refers to a residue derived from an aromatic ring by removing a hydrogen atom. The term "aromatic ring" can refer to a 6- to 14-membered all-carbon monocyclic ring or a fused polycyclic ring (i.e., a ring sharing adjacent pairs of carbon atoms) with a conjugated π electron system, which can be 6- to 10-membered, such as benzene and naphthalene. The aromatic ring can be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, wherein the ring connected to the parent structure is the aryl ring. Aryl groups can be substituted or unsubstituted. When substituted, the substituents can be one or more of the following groups independently selected from the following groups: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio. Aryl groups can be substituted or unsubstituted.

[0086] As used herein, the term "heteroaryl" generally refers to a residue derived from a carbon atom in a heteroaromatic ring by removing a hydrogen atom. The term "heteroaromatic ring" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms may be selected from the group consisting of oxygen, sulfur, and nitrogen. Heteroaryl groups may be 5 to 10-membered, and may be 5- or 6-membered, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the heteroaryl ring. Heteroaryl can be optionally substituted or unsubstituted. When substituted, the substituent can be one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio. Heteroaryl can be substituted or unsubstituted.

[0087] In this application, as known to those skilled in the art, terms such as "alkyl", "alkenyl", "cycloalkyl", etc. may be preceded by an identifier to indicate the number of atoms present in the group in a particular case, for example, C1-C4 alkyl, C3-C7 cycloalkyloxy, C1-C4 alkylcarbonylamino, etc., and the subscript number after "C" indicates the number of carbon atoms present in the group. For example, C3 alkyl refers to an alkyl group with three carbon atoms (e.g., n-propyl, isopropyl); C 1-10 In the examples, the members of the group may have any number of carbon atoms falling within the range of 1-10.

[0088] One or more hydrogen atoms in the group, for example, up to 5, for example, 1-3 hydrogen atoms are replaced by the substituents of corresponding number independently of one another. The substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible replacements without paying too much effort. For example, amino or hydroxyl groups with free hydrogen may be unstable when combined with carbon atoms with unsaturated (such as olefinic) bonds.

[0089] In this application, the term "neutral lipid" refers to any of a variety of lipid substances that exist in the form of uncharged or neutral zwitterions at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphatidylcholine such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamine such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), ceramides, steroids such as sterols and derivatives thereof. Neutral lipids can be synthetic or naturally derived. Herein, the term "steroid" refers to a compound containing the following carbon skeleton:

[0090] Non-limiting examples thereof include cholesterol and the like.

[0091] In this application, the term "polymer-conjugated lipid" refers to a molecule comprising a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule comprising a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG) and the like.

[0092] In this application, the term "liposome" generally refers to a vesicle with an internal space that is separated from the external medium by one or more bilayer, multilayer, any lamellar or solid membranes. For example, the bilayer membrane can be formed by amphiphilic molecules, such as synthetic or natural lipids containing spatially separated hydrophilic and hydrophobic domains; for another example, the bilayer membrane can be formed by amphiphilic polymers and surfactants. Liposomes encapsulate an aqueous phase, which generally contains substances to be delivered to cells, such as nucleic acids. The term "liposome" as used in this application also covers lipid- and polymer-based nanoparticles.

[0093] In this application, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, and includes DNA, RNA and hybrids thereof. DNA can be in the form of antisense molecules, plasmid DNA, cDNA, PCR products or vectors. RNA can be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, Dicer substrate RNA or viral RNA (vRNA) and combinations thereof. Nucleic acid includes backbone residues or linked nucleic acids containing known nucleotide analogs or modifications, which are synthetic, naturally occurring and non-naturally occurring and have binding properties similar to reference nucleic acids. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides and peptide-nucleic acids (PNA). Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides with similar binding properties to reference nucleic acids. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences, as well as the sequences explicitly indicated. A "nucleotide" contains the following sugars: deoxyribose (DNA) or ribose (RNA); a base and a phosphate group. The nucleotides are linked together by the phosphate group. "Bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including but not limited to modifications that place new reactive groups (such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides).

[0094] In this application, the term "administering" refers to introducing a composition or medicament (e.g., nucleic acid) into an individual and includes simultaneously and sequentially introducing one or more compositions or medicaments."Administering" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, placebo, and experimental methods. "Administering" also encompasses in vitro and ex vivo treatments. Introducing a composition or medicament into an individual is by any suitable route, including oral, pulmonary, nasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intratumoral, or local. Administering includes self-administration and administration by another person. Administering can be performed by any suitable route. Suitable routes of administration allow a composition or medicament to perform its intended function. For example, if suitable route is intravenous, then administering a composition or medicament by introducing the vein of an individual.

[0095] In this application, the term "delivery" refers to providing an entity to a target. For example, delivering a therapeutic and / or prophylactic agent to a subject may involve administering a nanoparticle composition comprising the therapeutic and / or prophylactic agent to the subject (e.g., via intravenous, intramuscular, intradermal, or subcutaneous routes). Administering a nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the nanoparticle composition. Wherein, "contact" refers to establishing a physical connection between two or more entities, and methods for contacting cells with external entities in vivo and in vitro are well known in the field of biology.

[0096] As used herein, the term "prevent" or "prevent" encompasses: (1) inhibiting the onset of a disease in a subject or patient who may be at risk for and / or susceptible to the disease but who does not yet experience or display any or all of the symptoms or signs of the disease; and / or (2) slowing the onset of symptoms or signs of a disease in a subject or patient who may be at risk for and / or susceptible to the disease but who does not yet experience or display any or all of the symptoms or signs of the disease.

[0097] As used herein, the term "treating" encompasses (1) inhibiting a disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease (e.g., preventing further development of the symptoms and / or signs), (2) ameliorating a disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease (e.g., reversing the symptoms and / or signs), and / or (3) achieving any measurable reduction in a disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease.

[0098] As used herein, the term "subject" or "patient" refers to any organism to which the compounds and / or compositions described herein can be administered, e.g., for experimental, diagnostic, prophylactic and / or therapeutic purposes, and typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates and humans) and / or plants.

[0099] In this application, the term "comprising" generally refers to including the features specified but not excluding other elements. The terms "above" and "below" generally refer to including the number.

[0100] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0101] Detailed Description of the Invention

[0102] Compound

[0103] On the one hand, the present application provides a compound, or a pharmaceutically acceptable salt thereof, comprising a cation-providing group and a hydrogen bond-providing group, wherein the cation-providing group and the hydrogen bond-providing group contain 2 to 6 atoms, for example, the cation-providing group and the hydrogen bond-providing group of the compound contain 2, 3, 4, 5, or 6 atoms.

[0104] In the present application, the compound comprises a cationic lipid.

[0105] In the present application, the compound comprises an ionizable lipid.

[0106] In the present application, the cation-providing group comprises a group that is positively charged at a pH of about 7.0 or less. For example, the cation-providing group is a group that is positively charged at a pH of about 7.0, about 6.0, about 5.0, about 4.0, about 3.0, about 2.0, about 1.0, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.2, or about 0.1.

[0107] In the present application, the cation-providing group comprises a group that is positively charged at a pH of about 4.0 or less. For example, the cation-providing group is a group that is positively charged at the following pH conditions: about 4.0, about 3.0, about 2.0, about 1.0, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1.

[0108] In the present application, the cation-providing group comprises a tertiary amine group, wherein the three hydrocarbon groups connected to the amine of the trivalent group in the tertiary amine group are independently selected from optionally substituted substituents.

[0109] In the present application, the hydrogen bond providing group comprises a urea group, wherein the two hydrocarbon groups (i.e., a total of four hydrocarbon groups) respectively connected to the two trivalent amines in the urea group are independently selected from optionally substituted substituents.

[0110] In the present application, the compound comprises a tail portion, and the tail portion is hydrophobic. In certain embodiments, the tail portion is substituted on one or two nitrogen atoms of the urea group. In certain embodiments, the tail portion comprises a saturated fatty chain. In certain embodiments, the tail portion comprises an unsaturated fatty chain. In certain embodiments, the tail portion comprises an unsaturated fatty chain, and the unsaturation is partially unsaturated. In certain embodiments, the tail portion comprises an unsaturated fatty chain, and the unsaturation is monounsaturated. In certain embodiments, the tail portion comprises an unsaturated fatty chain, and the unsaturation is polyunsaturated.

[0111] In the present application, the main chain length of the tail is 1 to 20 atoms. For example, the tail has a main chain length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 atoms.

[0112] In the present application, the compound comprises structure (I): Wherein, L1 is the hydrogen bond donating group, and L1 is optionally substituted. G1 is a linking group having a chain length of 2 to 6 atoms, and G1 is optionally substituted. For example, G1 is a linking group having a chain length of 2, 3, 4, 5, or 6 atoms. For example, G1 is selected from any of the following groups or any combination thereof: hydrocarbon group, alicyclic group, alicyclic heterocyclic group, alkyl group, alkenyl group, and alkynyl group. For example, G1 is optionally substituted, and one atom is replaced by N, O, or S.

[0113] For example, in certain embodiments, the structure of the compound is as follows (as described in structure OT13-1 provided herein): The compound comprises a tertiary amine group as a cation providing group and comprises the structure (I), wherein G1 is a C3 alkyl group with a chain length of 3 atoms, L1 is a urea group capable of serving as a hydrogen bond donor, and the amine of a trivalent radical of the urea group is substituted with a branched long-chain alkenyl group, and the molecular formula is CH(C8H 16 CH=CHCH2CH=CHC5H 11 )2, the substituted portion is also a hydrophobic tail as defined in this application.

[0114] In the present application, the compound comprises any one of structure (II) and structure (IIIb): Wherein, G1 is a linking group with a chain length of 2 to 6 atoms, said G1 is optionally substituted, R1, R 2a , R 2b, R5 and R6 are each independently optionally substituted. For example, G1 is a linking group having the following chain lengths: 2, 3, 4, 5, 6 atoms. For example, G1 is selected from any of the following groups or any combination thereof: hydrocarbon group, alicyclic group, alicyclic heterocyclic group, alkyl group, alkenyl group, alkynyl group. For example, G1 is optionally substituted, and one atom is replaced by N, replaced by O, or replaced by S. For example, R1, R 2a , R 2b , R5 and R6 are each independently selected from the following groups or any combination thereof: hydrocarbon group, ester group, amide, N'-alkyl-N-alkenyl-urea group, alicyclic group, alicyclic heterocyclic group, aryl, heteroaryl, C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl, C 15 Alkyl, C 16 Alkyl, C 17 Alkyl, C 18 Alkyl, C 19 Alkyl, C 20 Alkyl, C2 alkenyl, C3 alkenyl, C4 alkenyl, C5 alkenyl, C6 alkenyl, C7 alkenyl, C8 alkenyl, C9 alkenyl, C 10 Alkenyl, C 11 Alkenyl, C 12 Alkenyl, C 13 Alkenyl, C 14 Alkenyl, C 15 Alkenyl, C 16 Alkenyl, C 17 Alkenyl, C 18 Alkenyl, C 19 Alkenyl, C 20 Alkenyl, C2 alkynyl, C3 alkynyl, C4 alkynyl, C5 alkynyl, C6 alkynyl, C7 alkynyl, C8 alkynyl, C9 alkynyl, C 10 Alkynyl, C 11 Alkynyl, C 12 Alkynyl, C 13 Alkynyl, C 14 Alkynyl, C 15 Alkynyl, C 16 Alkynyl, C 17 Alkynyl, C 18 Alkynyl, C 19 Alkynyl, C 20 Alkynyl. For example, R1, R 2a , R 2b , R5 and R6 are each independently branched or unbranched. For example, R1, R 2a , R2b , R5 and R6 each independently contain 0, 1, 2, 3 or 4 unsaturated bonds. For example, R1, R 2a , R 2b , R5 and R6 are each independently optionally substituted by a substituent R X For example, R1, R 2a , R 2b , R5 and R6 are each independently optionally substituted by a substituent R X replaced, and R X Select any of the following groups: C1-C 20 Alkyl, C2-C 25 Ester group, C2-C 20 Alkenyl, amide, urea, hydroxyl, thiol, amino.

[0115] For example, in certain embodiments, the structure of the compound is as follows (as described in structure OT13-3 provided herein): The compound comprises a tertiary amine group as a cation-providing group and comprises the structure (II), wherein G1 is a C3 hydrocarbon group with a chain length of 3 atoms, connected to G1 is a hydrogen bond-providing group urea group, and the two trivalent amines of the urea group are independently substituted, that is, R1 in the structure (II) is a branched alkyl ester substituent, and its molecular formula is CH(C8H 16 COOCH2C6H 13 C4H9)2, R in the structure (II) 2a and R 2b They are hydrogen and C8 alkyl (in any order).

[0116] In the present application, the compound comprises any one of structure (IIIa) and structure (IIIc): wherein G1 and G2 are each independently a linking group having a chain length of 2 to 6 atoms, said G1 and G2 are each independently optionally substituted, R1, R 2a , R 2b , R3, R 4a , R 4b , R7, R 8a , R 8b and R9 are each independently optionally substituted. For example, G1 and G2 are each independently a linking group having a chain length of 2, 3, 4, 5, or 6 atoms. For example, G1 and G2 are each independently selected from any of the following groups or any combination thereof: hydrocarbon, alicyclic, alicyclic, alkyl, alkenyl, or alkynyl. For example, G1 and G2 are each independently optionally substituted, and one atom is replaced by N, O, or S. For example, R1, R2a , R 2b , R3, R 4a , R 4b , R7, R 8a , R 8b and R9 are each independently selected from the following groups or any combination thereof: hydrocarbon group, ester group, amide, N'-alkyl-N-alkenyl-urea group, alicyclic group, alicyclic heterocyclic group, aryl, heteroaryl, C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl, C 15 Alkyl, C 16 Alkyl, C 17 Alkyl, C 18 Alkyl, C 19 Alkyl, C 20 Alkyl, C2 alkenyl, C3 alkenyl, C4 alkenyl, C5 alkenyl, C6 alkenyl, C7 alkenyl, C8 alkenyl, C9 alkenyl, C 10 Alkenyl, C 11 Alkenyl, C 12 Alkenyl, C 13 Alkenyl, C 14 Alkenyl, C 15 Alkenyl, C 16 Alkenyl, C 17 Alkenyl, C 18 Alkenyl, C 19 Alkenyl, C 20 Alkenyl, C2 alkynyl, C3 alkynyl, C4 alkynyl, C5 alkynyl, C6 alkynyl, C7 alkynyl, C8 alkynyl, C9 alkynyl, C 10 Alkynyl, C 11 Alkynyl, C 12 Alkynyl, C 13 Alkynyl, C 14 Alkynyl, C 15 Alkynyl, C 16 Alkynyl, C 17 Alkynyl, C 18 Alkynyl, C 19 Alkynyl, C 20 Alkynyl. For example, R1, R 2a , R 2b , R3, R 4a , R 4b , R7, R 8a , R 8b and R9 are each independently branched or unbranched. For example, R1, R 2a , R 2b , R3, R4a , R 4b , R7, R 8a , R 8b and R9 independently contain 0, 1, 2, 3 or 4 unsaturated bonds. 2a , R 2b , R3, R 4a , R 4b , R7, R 8a , R 8b and R9 are each independently optionally substituted by a substituent R X For example, R1, R 2a , R 2b , R3, R 4a , R 4b , R7, R 8a , R 8b and R9 are each independently optionally substituted by a substituent R X replaced, and R X Select any of the following groups: C1-C 20 Alkyl, C2-C 25 Ester group, C2-C 20 Alkenyl, amide, urea, hydroxyl, thiol, amino.

[0117] For example, in certain embodiments, the structure of the compound is as follows (as described in structure OT13-5 provided herein): The compound comprises two tertiary amine groups as cation providing groups and forming a piperazine ring (p-diazine ring) between each other, and comprises the structure (IIIa), wherein G1 and G2 are both C2 alkyl groups with a chain length of 2 atoms, which are respectively connected to the hydrogen bond providing group urea group, and the two trivalent amines of each urea group are independently substituted, that is, R1 in the structure (IIIa) is a C2 alkyl group with a chain length of 12 atoms. 12 Alkyl, R in the structure (IIIa) 2a and R 2b Hydrogen and C 12 Alkyl (order is not limited), R3 in the structure (IIIa) is C 12 Alkyl, R in the structure (IIIa) 4a and R 4b Hydrogen and C 12 Alkyl groups (in any order).

[0118] For example, in certain embodiments, the structure of the compound is as follows (as described in structure OT13-4 provided herein): The compound comprises a tertiary amine group as a cation providing group and comprises the structure (IIIc), wherein G1 and G2 are both C6 alkyl groups with a chain length of 6 atoms, which are respectively connected to the hydrogen bond providing group urea group, and the trivalent amine directly connected to G1 or G2 of each urea group is not substituted, that is, R1 and R7 in the structure (IIIc) are both hydrogen, and the trivalent amine not directly connected to G1 or G2 of each urea group is independently substituted, that is, R1 in the structure (IIIc) is 2a 、R 2b 、R 8a and R 8b All are C6 alkyl groups, and R9 in the structure (IIIc) is butanol, whose molecular formula is C4H8OH.

[0119] In the present application, the compound comprises structure (IIId): wherein G1, G2, G3 and G4 are each independently a linking group having a chain length of 2 to 6 atoms, said G1, G2, G3 and G4 are each independently optionally substituted, said H1 and H2 are each independently optionally substituted, said R1, R 2a , R 2b , R7, R 8a , R 8b , R 10 , R 11a , R 11b , R 12 , R 13a and R 13b Each is independently optionally substituted. For example, G1, G2, G3 and G4 are each independently a linking group having the following chain lengths: 2, 3, 4, 5, 6 atoms. For example, G1, G2, G3 and G4 are each independently selected from any of the following groups or any combination thereof: hydrocarbon, alicyclic, alicyclic heterocyclic, alkyl, alkenyl, alkynyl. For example, G1, G2, G3 and G4 are each independently optionally substituted, and one atom is replaced by N, replaced by O or replaced by S. For example, H1 and H2 are each independently selected from any of the following groups or any combination thereof: hydrocarbon, alicyclic, alicyclic heterocyclic, alkyl, alkenyl, alkynyl. For example, H1 and H2 are each independently optionally substituted, and one atom is replaced by N, replaced by O or replaced by S. For example, R1, R 2a , R 2b , R7, R 8a , R 8b , R 10 , R 11a , R 11b , R 12 , R 13a and R 13bEach is independently selected from the following groups or any combination thereof: hydrocarbon group, ester group, amide, N'-alkyl-N-alkenyl-urea group, alicyclic group, alicyclic heterocyclic group, aryl, heteroaryl, C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl, C 15 Alkyl, C 16 Alkyl, C 17 Alkyl, C 18 Alkyl, C 19 Alkyl, C 20 Alkyl, C2 alkenyl, C3 alkenyl, C4 alkenyl, C5 alkenyl, C6 alkenyl, C7 alkenyl, C8 alkenyl, C9 alkenyl, C 10 Alkenyl, C 11 Alkenyl, C 12 Alkenyl, C 13 Alkenyl, C 14 Alkenyl, C 15 Alkenyl, C 16 Alkenyl, C 17 Alkenyl, C 18 Alkenyl, C 19 Alkenyl, C 20 Alkenyl, C2 alkynyl, C3 alkynyl, C4 alkynyl, C5 alkynyl, C6 alkynyl, C7 alkynyl, C8 alkynyl, C9 alkynyl, C 10 Alkynyl, C 11 Alkynyl, C 12 Alkynyl, C 13 Alkynyl, C 14 Alkynyl, C 15 Alkynyl, C 16 Alkynyl, C 17 Alkynyl, C 18 Alkynyl, C 19 Alkynyl, C 20 Alkynyl. For example, R1, R 2a , R 2b , R7, R 8a , R 8b , R 10 , R 11a , R 11b , R 12 , R 13a and R 13b Each independently is branched or unbranched. For example, R1, R 2a , R 2b , R7, R 8a , R 8b , R 10, R 11a , R 11b , R 12 , R 13a and R 13b Each independently contains 0, 1, 2, 3 or 4 unsaturated bonds. For example, R1, R 2a , R 2b , R7, R 8a , R 8b , R 10 , R 11a , R 11b , R 12 , R 13a and R 13b The substituents R are each independently optionally substituted X For example, R1, R 2a , R 2b , R7, R 8a , R 8b , R 10 , R 11a , R 11b , R 12 , R 13a and R 13b The substituents R are each independently optionally substituted X replaced, and R X Select any of the following groups: C1-C 20 Alkyl, C2-C 25 Ester group, C2-C 20 Alkenyl, amide, urea, hydroxyl, thiol, amino.

[0120] For example, in certain embodiments, the structure of the compound is as follows (as described in structure OT13-9 provided herein): The compound comprises two tertiary amine groups as cation providing groups, which are connected through a group with a structure of 3,6-dibutylpiperazine-2,5-dione. The compound further comprises the structure (IIId), wherein H1 and H2 are C4 alkyl groups, G1, G2, G3 and G4 are C2 alkyl groups with a chain length of 2 atoms, each of which is connected to a hydrogen bond providing group urea group, and the trivalent amine directly connected to G1, G2, G3 or G4 of each urea group is not substituted, that is, R1, R7, R 10 and R 12 are hydrogen, and each urea group is not directly connected to the trivalent amine G1, G2, G3 or G4 are independently substituted, that is, R in the structure (IIId) 2a 、R 2b 、R 8a 、R 8b 、R11a 、R 11b 、R 13a and R 13b All have the molecular formula C7H 14 CH=CHC7H 15 Long chain alkenyl.

[0121] On the other hand, the present application provides a compound, or a pharmaceutically acceptable salt thereof, wherein the structure of the compound is selected from any one of the structures listed in the following table:

[0122] It will be understood that any embodiment of a compound comprising structures (I), (II), or (IIIa) to (IIId) as shown above, and any specific substituents and / or variables of compound structures (I), (II), or (IIIa) to (IIId) as shown above, can be independently combined with other embodiments and / or substituents and / or variables of compounds comprising structures (I), (II), or (IIIa) to (IIId) to form embodiments of the present application not specifically shown above. In addition, if any specific list of substituents and / or variables for any specific R group, L group, G group, H group, or variable a, x, y, or z is listed in a specific embodiment and / or claim, it will be understood that each individual substituent and / or variable can be deleted from the specific embodiment and / or claim, and the remaining list of substituents and / or variables will be considered to be within the scope of the present application.

[0123] It is understood that in this specification, combinations of substituents and / or variables of the depicted formulas are permissible only if such contributions result in stable compounds.

[0124] Composition

[0125] On the other hand, the application provides a kind of composition, described composition comprises any one or more and therapeutic agent in compound as described in the application.In certain embodiments, described composition comprises any one or more, therapeutic agent and one or more excipients selected from the lipid of neutral lipid, steroid and polymer conjugation in compound as described in the application.For example, a kind of composition of lipid nanoparticle (LNP) is provided, described LNP comprises any one, therapeutic agent and one or more excipients selected from the lipid of neutral lipid, steroid and polymer conjugation in compound as described in the application.In certain embodiments, described composition comprises any one or more, therapeutic agent and liposome in compound as described in the application.In other embodiments, described composition also comprises other pharmaceutically acceptable excipients and / or carriers.

[0126] In certain embodiments, the composition comprises any one or more of the compounds described herein, a therapeutic agent, and one or more excipients selected from a neutral lipid, a steroid, and a polymer-conjugated lipid, wherein the molar ratio of the compound to the neutral lipid is from about 2:1 to about 8:1. For example, a composition comprising any one or more of the compounds described herein, a therapeutic agent, and one or more excipients selected from a neutral lipid, a steroid, and a polymer-conjugated lipid is provided, wherein the compound has a molar ratio to the neutral lipid in the range of about 2:1 to about 3:1, about 2:1 to about 4:1, about 2:1 to about 5:1, about 2:1 to about 6:1, about 2:1 to about 7:1, about 2:1 to about 8:1, about 3:1 to about 4:1, about 3:1 to about 5:1, about 3:1 to about 6:1, about 3:1 to about 7:1, about 3:1 to about 8:1, about 4:1 to about 5:1, about 4:1 to about 6:1, about 4:1 to about 7:1, about 4:1 to about 8:1, about 5:1 to about 6:1, about 5:1 to about 7:1, about 5:1 to about 8:1, about 6:1 to about 7:1, about 6:1 to about 8:1, about 7:1 to about 8:1.

[0127] In certain embodiments, the composition comprises any one or more of the compounds as described herein, a therapeutic agent, and one or more excipients selected from neutral lipids, steroids comprising cholesterol, and polymer-conjugated lipids. In certain embodiments, the composition comprises any one or more of the compounds as described herein, a therapeutic agent, and one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids, and the compound to the steroid has a molar ratio in the following numerical range: about 5:1 to about 4:1, about 5:1 to about 3:1, about 5:1 to about 2:1, about 5:1 to about 1:1, about 4:1 to about 3:1, about 4:1 to about 2:1, about 4:1 to about 1:1, about 3:1 to about 2:1, about 3:1 to about 1:1, about 2:1 to about 1:1.

[0128] In certain embodiments, the composition comprises any one or more of the compounds described herein, a therapeutic agent, and one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids including PEGylated lipids. In certain embodiments, the composition comprises any one or more of the compounds described herein, a therapeutic agent, and one or more excipients selected from a neutral lipid, a steroid, and a polymer-conjugated lipid, and the compound to the polymer-conjugated lipid has a molar ratio in the following range: about 100:1 to about 90:1, about 100:1 to about 80:1, about 100:1 to about 70:1, about 100:1 to about 60:1, about 100:1 to about 50:1, about 100:1 to about 40:1, about 100:1 to about 30:1, about 100:1 to about 20:1, about 90:1 to about 80:1, about 90:1 to about 70:1, about 90:1 to about 60:1, about 90:1 to about 50:1, about 90:1 to about 40:1, about 90:1 to about about 30:1, about 90:1 to about 20:1, about 80:1 to about 70:1, about 80:1 to about 60:1, about 80:1 to about 50:1, about 80:1 to about 40:1, about 80:1 to about 30:1, about 80:1 to about 20:1, about 70:1 to about 60:1, about 70:1 to about 50:1, about 70:1 to about 40:1, about 70:1 to about 30 :1, about 70:1 to about 20:1, about 60:1 to about 50:1, about 60:1 to about 40:1, about 60:1 to about 30:1, about 60:1 to about 20:1, about 50:1 to about 40:1, about 50:1 to about 30:1, about 50:1 to about 20:1, about 40:1 to about 30:1, about 40:1 to about 20:1, about 30:1 to about 20:1.

[0129] In the present application, the diameter of the composition is about 500 nm or less. For example, the composition has a diameter of the following values: about 500 nm, about 450 nm, about 400 nm, about 350 nm, about 300 nm, about 250 nm, about 200 nm, about 150 nm, about 100 nm, about 75 nm, about 50 nm, about 25 nm.

[0130] In the present application, the therapeutic agent comprises one or more nucleic acids. It should be understood that the content disclosed in this application is not limited to the specific nucleic acids disclosed in this specification, and is not limited in scope to any specific source, sequence or type of nucleic acid, but those of ordinary skill in the art can easily identify related homologs in various other nucleic acid sources, including nucleic acids from non-human species. It is expected that the nucleic acids used in the content disclosed in this application may include sequences based on naturally occurring sequences. In certain embodiments, the therapeutic agent comprises one or more nucleic acids, and the nucleic acid comprises one or more modified nucleosides (e.g., comprising one or more nucleosides modified with a sugar moiety), in order to obtain compounds containing the nucleic acid that may have desired properties. In certain embodiments, the therapeutic agent comprises one or more nucleic acids, and the nucleic acid is an oligonucleotide comprising a modified nucleoside (e.g., comprising a modified sugar, a modified nucleobase, a modified bond and / or a nucleotide at other positions such as the 3' position of the sugar on the 3' terminal nucleotide and / or the 5' position of the 5' terminal nucleotide), and the oligonucleotide obtained by selecting a specific modification may have desired characteristics.

[0131] In the present application, the therapeutic agent comprises components of the CRISPR system and / or nucleic acids encoding Cas enzymes. The “CRISPR system” may also be referred to as a “CRISPR / Cas system” and is a tool for site-specific genome targeting in an organism. For example, it may be a type II CRISPR / Cas system, which is a prokaryotic adaptive immune response system that uses non-coding RNA to guide Cas nucleases to induce site-specific DNA cleavage, and subsequently triggers cellular DNA repair mechanisms, i.e., repairing DNA damage through the non-homologous end joining DNA repair pathway (NHEJ) or homology-directed repair (HDR) pathway; the system may also be delivered to mammalian cells or eukaryotic cells to mediate genome editing (e.g., gene knockout or knock-in). For example, the therapeutic agent can comprise one or more Cas nucleases, non-limiting examples of which include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4 , Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof or modified forms thereof, these enzymes are known and can be easily found by those skilled in the art from, for example, the SwissProt database. For example, the therapeutic agent may comprise a Cas9 enzyme and / or a mutant form thereof for specific binding and / or site-directed cleavage of a single-stranded or double-stranded target polynucleotide at a target sequence position. For example, the therapeutic agent comprises a nucleic acid encoding a component of the CRISPR system, wherein "encoding" means that the target polynucleotide sequence can be transcribed and / or translated according to a genetic code that functions in a host cell / host animal to produce a corresponding gene product (e.g., a nuclease). For example, the therapeutic agent comprises a guide RNA (gRNA). For example, the therapeutic agent comprises a nucleic acid encoding a Cas enzyme. In certain embodiments, the "nucleic acid encoding a Cas enzyme" comprises DNA (e.g., a plasmid) or RNA (e.g., mRNA).

[0132] Cells and kits

[0133] On the other hand, the present application provides a cell comprising a compound and / or composition as described herein. For example, the cell is a carrier cell comprising a negatively charged cell membrane. For example, the LNP comprising the compound described herein is protonated under a suitable pH environment, and then aggregates with the cell membrane of the carrier cell through electrostatic interaction. For example, the compound and / or composition described herein is protonated under a suitable pH environment, so that the cell membrane of the carrier cell mediates endocytosis or membrane permeation to include the compound and / or composition.

[0134] In another aspect, the present application provides a kit comprising the compounds, compositions, and / or cells described herein. For example, the kit includes at least one vial, tube, flask, bottle, syringe, or other suitable container in which the components may be placed. For example, if more than one component is present in the kit, the kit may also include a second, third, or other additional container in which the additional components may be separately placed. For example, various combinations of components may be included in the container. For example, all LNP components may be combined in a single container. For example, some or all of the LNP components may be provided in separate containers. In certain embodiments, the kit may also include packaging for accommodating the various containers. For example, the kit may include instructions for using the kit components. For example, the instructions may be in a physical paper form and / or in a machine-readable electronic form.

[0135] Methods of administering therapeutic agents

[0136] In another aspect, the present application provides a method of administering a therapeutic agent, comprising providing a compound described herein, a composition described herein, a cell described herein, and / or a kit described herein. In certain embodiments, the method comprises mixing the therapeutic agent described herein with the compound described herein. In other embodiments, the method comprises placing the therapeutic agent described herein in a composition described herein, a cell described herein, and / or a kit described herein.

[0137] Methods for delivering nucleic acids

[0138] On the other hand, the application provides a method for delivering nucleic acid, the method comprising providing compound as described herein, composition as described herein, cell as described herein and / or test kit as described herein. In some embodiments, the method comprises mixing nucleic acid as described herein with compound as described herein. In other embodiments, the method comprises placing nucleic acid as described herein in composition as described herein, cell as described herein and / or test kit as described herein. For example, the method for delivering nucleic acid comprises delivering systemically compound as described herein, composition as described herein, cell as described herein and / or test kit as described herein, so that it is widely exposed to most of the body, and can be carried out by any means known in the art, including but not limited to intravenous, intra-arterial, subcutaneous and intraperitoneal delivery. For example, the method for delivering nucleic acid comprises delivering locally compound as described herein, composition as described herein, cell as described herein and / or test kit as described herein, so that it directly reaches the target site in the organism, and can be carried out by, for example, direct injection into the disease site (such as, tumor or inflammation site) or target organ (such as, liver, heart, pancreas, kidney etc.). For example, the local delivery includes local application or local injection techniques, including but not limited to intramuscular, subcutaneous or intradermal injection.For example, the local delivery does not exclude systemic pharmacological effects.

[0139] Methods of treating and / or preventing diseases or conditions

[0140] In another aspect, the present application provides a pharmaceutical composition comprising a compound, a composition, a cell, and / or a kit as described herein. In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable auxiliary component. In certain embodiments, the pharmaceutical composition is used to treat and / or prevent a disease.

[0141] On the other hand, the present application provides the use of the compounds described herein, the compositions described herein, the cells described herein, the kits described herein and / or the pharmaceutical compositions described herein for preparing drugs for treating or preventing diseases.

[0142] On the other hand, the present application provides the compounds described herein, the compositions described herein, the cells described herein, the kits described herein and / or the pharmaceutical compositions described herein, which are used to treat or prevent diseases.

[0143] On the other hand, the present application provides a method for treating and / or preventing a disease, comprising administering to a subject or patient a suitable dose of the compound, composition, cell and / or kit described herein.

[0144] In the present application, the disease includes cancer.

[0145] In the present application, the diseases include diseases associated with protein or enzyme deficiency.

[0146] In the present application, the diseases include diseases associated with a specific miRNA or a group or more than one group of miRNAs.

[0147] Without intending to be bound by any theory, the following examples are merely for illustrating the compounds, preparation methods and uses of the present application, and are not intended to limit the scope of the present invention.

[0148] Example

[0149] Preparation of the compound of Example 1

[0150] Synthesis steps of OT13-1

[0151] 1. Synthesis of (6E,10E)-18-bromooctadeca-6,9-diene

[0152] Octadeca-9,2-dien-1-yl methanesulfonate and magnesium bromide were dissolved in THF at room temperature, and ether was added, followed by stirring for 12 hours. The organic solvent was removed by rotary evaporation, and the product was washed with water.

[0153] 2. Synthesis of (6E,10E,28E,31E)-heptatriaconta-6,9,28,31-tetraen-19-ol

[0154] Dissolve (6E,9E)-18-bromooctadecane-6,9-diene (the product of step 1) in THF containing magnesium and iodine as the reaction solvent, add ethyl formate, and stir the reaction for 12 hours.

[0155] 3. Synthesis of (6E,10E,28E,31E)-heptatriaconta-6,9,28,31-tetraen-19-amine

[0156] (6E,10E,28E,31E)-heptatriacontac-6,9,28,31-tetraen-19-ol (product from Step 2) and PCC (pyridinium chlorochromate) were reacted in dichloromethane at 0-20°C for two hours. Sodium cyanoborohydride was then added at room temperature and stirring continued for over two hours. The product was filtered through silica gel and washed with hot ethyl acetate.

[0157] 4. Synthesis of OT13-1

[0158] Dissolve (6E,10E,28E,31E)-heptatriacontria-6,9,28,31-tetraen-19-amine (product from Step 3) in THF at room temperature. Add CDI (N,N'-carbonyldiimidazole) and heat to 70°C. Add neopentane and continue stirring for two hours. Wash the product with water and purify it by silica gel chromatography.

[0159] Synthesis steps of OT13-2

[0160] 5. Synthesis of (10E,12E)-octadeca-9,12-dien-1-yl methanesulfonate

[0161] Dissolve MsCl (methanesulfonyl chloride) in dichloromethane at room temperature. Under inert atmosphere, react (10E,12E)-octadeca-9,12-dien-1-ol with methanesulfonyl chloride at 0-20°C with triethylamine as a catalyst, with gentle stirring, to form an ammonium salt. Terminate the reaction with distilled water, and wash the organic layer with saturated halide, dry, and concentrate. Purify by silica gel column chromatography until the purity exceeds 85%.

[0162] 6. Synthesis of 2-[(10E,12E)-octadeca-9,12-dien-1-yl]isoindoline-1,3-dione

[0163] Dissolve (10E,12E)-octadec-9,12-dien-1-yl methanesulfonate (product from Step 5) and potassium phthalimide in dimethylformamide, and add sodium azide at room temperature. Stir the reaction at 70°C for 2 hours, then evaporate under reduced pressure. Extract with dichloromethane, wash with water, and dry over anhydrous sodium sulfate to obtain a crude product, which is then isolated and purified using a chromatographic column.

[0164] 7. Synthesis of (10E,12E)-octadeca-9,12-dien-1-amine

[0165] Aqueous hydrazine was added to ethanol and reacted with 2-[(10E,12E)-octadeca-9,12-dienyl]isoindole-1,3-dione (the product of step 6) under inert gas at 0-20° C. for two hours. The product was dried and purified by silica gel column chromatography.

[0166] 8. Synthesis of di((9E,12E)-octadeca-9,12-dien-1-yl)amine

[0167] Under alkaline conditions (pH between 8-8.5), slowly add (10E,12E)-octadecadien-1-yl methanesulfonate (product from Step 5) until dissolved. Heat to 100°C and continue stirring for 2 hours. Monitor the reaction by chromatography. Cool the reaction to room temperature, add water, and extract the aqueous layer with ethyl acetate. Wash the product with water and then with PBS saline solution, dry over anhydrous NaSO, and concentrate.

[0168] 9. Synthesis of OT13-2

[0169] Dissolve bis[(6E,10E)-octadec-6,9-dien-18-yl]amine (product from Step 8) in THF at room temperature. Add CDI (N,N'-carbonyldiimidazole) and heat to 70°C. Add neopentane and continue stirring for two hours. Wash the product with water and purify it by silica gel chromatography.

[0170] Synthesis steps of OT13-3

[0171] 10. Synthesis of 19-ethoxy-9,11-bis(ethoxycarbonyl)-10,19-dioxyylidenenonadecanoic acid ethyl ester (tetraethyl 9-oxoheptadecane-1.8,10,17-tetracarboxylate)

[0172] White powdered diethyl 1,3-acetonedicarboxylate was added to pure ethanol containing ethyl 8-bromooctanoate and sodium ethoxide and reacted at 80°C for 48 hours. The product was washed with methanol under ice-cooling conditions and purified by silica gel column chromatography.

[0173] 11. Synthesis of 10-oxononadecanedioic acid

[0174] Ethyl 19-ethoxy-9,11-bis(ethoxycarbonyl)-10,19-dioxylidenenonadecanoate (the product of step 10) was reacted with 12M concentrated hydrochloric acid and acetic acid at 110°C for more than 12 hours. The obtained product was washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate. The remaining solvent was removed by distillation under reduced pressure, and the obtained product was purified by silica gel chromatography.

[0175] 12. Synthesis of bis(2-butyloctyl)10-oxononadecanedioate

[0176] 10-Oxylidenenonadecanedioic acid (product from Step 11) and 2-butyloctanol were esterified in the presence of EDCI, DMAP, and DCM at 30°C for 12 hours. The organic layer was separated and washed with saturated sodium chloride solution. The remaining solvent was removed under reduced pressure to afford the product, which was then purified by silica gel chromatography.

[0177] 13. Synthesis of bis(2-butyloctyl)10-((3-(dimethylamino)propyl)amino)nonadecanedioate

[0178] 2-Butyloctyl 19-[(2-butyloctyl)oxy]-10,19-dioxylidenonadecanoate (product from Step 12) and 3-(dimethylamino)propane-1-amine (N-dimethylpropane-1,3-diamine) were added to ethylene dichloride containing acetic acid and sodium triacetoxyborohydride and stirred at 40°C for 12 hours. After cooling to room temperature, water and ethyl acetate were added, and the product was extracted into ethyl acetate. The organic layer was evaporated under reduced pressure, dried, and concentrated in vacuo.

[0179] 14. Synthesis of OT13-3

[0180] 2-Butyloctyl 19-[(2-Butyloctyl)oxy]-10-{[3-(dimethylamino)propyl]amino}-19-oxyylidenenonadecanoate (product from Step 13) was reacted with 1-octylamine in THF in the presence of neopentane at room temperature under the catalysis of CDI. The reaction was stirred for 2 hours. The product was washed with water and purified by silica gel chromatography.

[0181] Synthesis steps of OT13-4

[0182] 15. Synthesis of 6-bromohexan-1-amine

[0183] Dissolve 6-amino-1-hexanol in hot hydrobromic acid in a round-bottom flask and boil at no higher than 70°C for two hours. Extract with ethyl acetate, remove impurities with sodium carbonate, and then wash with water and dry.

[0184] 16. Synthesis of 1-[(6-bromohexyl)amino]-N,N-dihexylmethaneamide (3-(6-bromohexyl)-1,1-dihexylurea)

[0185] 6-Bromohexan-1-amine (the product of step 15) was reacted with dihexylamine in THF under the catalysis of CDI, heated to 70°C, and then introduced with neopentane. The reaction was shaken for 2 hours. The product was purified by silica gel column chromatography.

[0186] 17. Synthesis of OT13-4

[0187] Under conditions of sodium carbonate and anhydrous ethanol at 25-70°C, 1-[(6-bromohexyl)amino]-N,N-dihexylmethaneamide (product from Step 16) and 4-amino-1-butanol were shaken for 2 hours. The organic solvent was removed by rotary evaporation, and the product was washed with water, dried, and purified by silica gel chromatography. Bis[(6E,10E)-octadec-6,9-dien-18-yl]amine (product from Step 8) was dissolved in THF at room temperature. CDI (N,N'-carbonyldiimidazole) was added, and the mixture was heated to 70°C. Neopentane was introduced and stirred continuously for two hours. The product was washed with water and purified by silica gel chromatography.

[0188] Synthesis steps of OT13-5, OT13-6 and OT13-7

[0189] 18. Synthesis of 1-(dodecylamino)-N-{2-[4-(4-oxyylidene-3,5-diazaheptadecan-1-yl)piperazin-1-yl]ethyl}methaneamide (1,1'-(piperazine-1,4-diylbis(ethane-2,1-diyl)bis(3-dodecylurea))

[0190] 2-[4-(2-Aminoethyl)piperazin-1-yl]ethan-1-amine was reacted with dodecane-1-amine in THF under the catalysis of CDI, heated to 70°C, and then introduced with neopentane. The reaction was shaken for 2 hours. The product was purified by silica gel column chromatography to obtain a light yellow solid.

[0191] 19. Synthesis of OT13-5

[0192] 1-(Dodecylamino)-N-{2-[4-(4-oxyylidene-3,5-diazaheptadecan-1-yl)piperazin-1-yl]ethyl}methaneamide (product from Step 18) and 12-oxirane were mixed and heated at 80°C in THF containing chloroacetonitrile with continuous stirring for 2 days until the desired product was detected by MALDI-TOF mass spectrometry. The crude product was purified by silica gel chromatography and confirmed by nuclear magnetic resonance.

[0193] 20. Synthesis of OT13-6

[0194] OT13-6 was synthesized using the same method described in Step 19. 1-(Dodecylamino)-N-{2-[4-(4-oxyylidene-3,5-diazaheptadecan-1-yl)piperazin-1-yl]ethyl}methaneamide (product from Step 18) and 12-oxirane were mixed and heated at 80°C in THF containing chloroacetonitrile with continuous stirring for 2 days until the desired product was detected by MALDI-TOF mass spectrometry. The crude product was purified by silica gel chromatography and confirmed by nuclear magnetic resonance.

[0195] 21. Synthesis of OT13-7

[0196] OT13-7 was synthesized using the same method as described in Step 19. The oxirane in Step 18 was replaced with the unsaturated 2-[(6E)-dodecan-6-enyl]oxirane. The reaction was heated in a sealed container in THF containing chloroacetonitrile at 80°C with continuous stirring for two days until the desired product was detected by MALDI-TOF mass spectrometry. The crude product was purified by silica gel chromatography and confirmed by nuclear magnetic resonance.

[0197] Synthesis steps of OT13-8

[0198] 22. Synthesis of 2-{[2-(4-{2-[(2-aminoethyl)oxy]ethyl}piperazin-1-yl)ethyl]oxy}ethan-1-amine (2,2'-((piperazine-1,4-diylbis(ethane-2,1-diyl))bis(oxy))bis(ethan-1-amine))

[0199] 1,4-bis(2-hydroxyethyl)piperazine is dissolved in a mixture of DMF and acetonitrile, 2-bromoethylamine hydrobromide is added, and the mixture is reacted at 50-100°C for six hours under acidic conditions and catalyzed by potassium bromide.

[0200] 23. Synthesis of 1-(dodecylamino)-N-[2-({2-[4-(7-oxyylidene-6-aza-3-oxaicostan-1-yl)piperazin-1-yl]ethyl}oxy)ethyl]methaneamide (N-(2-(2-(4-(2-(2-(3-dodecylureido)ethoxy)ethyl)piperazin-1-yl)ethoxy)ethyl)tetradecanamide)

[0201] 2-{[2-(4-{2-[(2-aminoethyl)oxy]ethyl}piperazin-1-yl)ethyl]oxy}ethan-1-amine (product from Step 22) was reacted with dodecan-1-amine in THF under the catalysis of CDI, heated to 70°C, and then stirred with neopentane for 2 hours. The product was purified by silica gel column chromatography to obtain a light yellow solid.

[0202] 24. Synthesis of OT13-8

[0203] 1-(Dodecylamino)-N-[2-({2-[4-(7-oxyylidene-6-aza-3-oxaicosan-1-yl)piperazin-1-yl]ethyl}oxy)ethyl]methaneamide (product from Step 23) was mixed with oxirane, sealed, heated at 80°C in THF containing chloroacetonitrile, and stirred for 2 days until the desired product was detected by MALDI-TOF mass spectrometry. The crude product was purified by silica gel chromatography, and the product was ultimately confirmed by NMR.

[0204] Synthesis steps of OT13-9

[0205] 25. Benzyl {[(9S)-12-(methoxycarbonyl)-9-({[(2-methylpropan-2-yl)oxy]carbonyl}amino)-3,10-dioxyylidene-1-phenyl-4,11-diaza-2-oxahexadecan-16-yl]amino}methane 6 -((benzyloxy)carbonyl)-N 2 -(N 6 Synthesis of -((benzyloxy)carbonyl)-N2-(tert-butoxycarbonyl)lysyl)lysinate)

[0206] 6-{[(Benzyloxy)carbonyl]amino}-2-({[(2-methylprop-2-yl)oxy]carbonyl}amino)hexanoic acid (N 6 -((benzyloxy)carbonyl)-N 2 -(tert-butoxycarbonyl)lysine) and [(5-amino-6-methoxy-6-oxyylidenehexyl)amino]methane acid benzyl ester (methyl N 6 -((benzyloxy)carbonyl)lysinate) was mixed and stirred at 25°C for 12 hours in the presence of EDCI, HOBt, and TEA in the presence of acetonitrile as the solvent. After the reaction, the solvent was evaporated under reduced pressure, and the remaining crude product was purified by silica gel column chromatography.

[0207] 26. Synthesis of benzyl {[9-amino-12-(methoxycarbonyl)-3,10-dioxyylidene-1-phenyl-4,11-diaza-2-oxahexadecan-16-yl]amino}methane (methyl N6-((benzyloxy)carbonyl)-N2-(N6-(benzyloxy)carbonyl)lysyl)lysinate)

[0208] Benzyl {[(9S)-12-(methoxycarbonyl)-9-({[(2-methylpropan-2-yl)oxy]carbonyl}amino)-3,10-dioxyylidene-1-phenyl-4,11-diaza-2-oxahexadecan-16-yl]amino}methane (the product of step 25) was reacted with 1,2-dioxane at 25° C. for 5 hours, and hydrochloric acid was added dropwise to remove the Boc group.

[0209] 27. Synthesis of 2-[(9-amino-3,10-dioxyylidene-1-phenyl-4-aza-2-oxadecan-10-yl)amino]-6-{[(benzyloxy)carbonyl]amino}hexanoic acid (N6-((benzyloxy)carbonyl)-N2-(N6-((benzyloxy)carbonyl)lysyl)lysine)

[0210] Benzyl {[9-amino-12-(methoxycarbonyl)-3,10-dioxyylidene-1-phenyl-4,11-diaza-2-oxahexadecan-16-yl]amino}methane (product from Step 26) was dissolved in methanol and reacted under alkaline conditions (adjusted with sodium hydroxide) at 25°C for 12 hours. The product was washed with water and dried under vacuum.

[0211] 28. Synthesis of benzyl ({4-[3,6-dioxo-5-(3-oxo-1-phenyl-4-aza-2-oxaoct-8-yl)piperazin-2-yl]butyl}amino)methane ester (dibenzyl((3,6-dixopiperazine-2,5-diyl)bis(butane-4,1-diyl))dicarbamate)

[0212] 2-[(9-amino-3,10-dioxyylidene-1-phenyl-4-aza-2-oxadecan-10-yl)amino]-6-{[(benzyloxy)carbonyl]amino}hexanoic acid (product from Step 27) was dissolved in acetonitrile and stirred for 12 hours in the presence of EDCI, HOBt, and TEA for cycloacylation. Water and ethyl acetate were added to terminate the reaction. The organic layer was separated and dried over magnesium sulfate, and the remaining solvent was evaporated under reduced pressure. The product was then purified by silica gel chromatography.

[0213] 29. Synthesis of 3,6-bis(4-aminobutyl)piperazine-2,5-dione

[0214] A dry round-bottom flask was purged with hydrogen. Benzyl ({4-[3,6-dioxyylidene-5-(3-oxyylidene-1-phenyl-4-aza-2-oxaoct-8-yl)piperazin-2-yl]butyl}amino)methane (product from Step 28) was added to a dichloromethane solvent containing acetic acid. The mixture was reacted in an oil bath at 165°C for 12 hours under the catalysis of Pd or C. The reaction was maintained at 500 psi using a stainless steel pressure vessel. The product was filtered and concentrated by rotary evaporation to yield a light yellow oil.

[0215] 30. Synthesis of 3,6-bis{4-[bis(2-aminoethyl)amino]butyl}piperazine-2,5-dione

[0216] 3,6-Bis(4-aminobutyl)piperazine-2,5-dione (product from Step 29) and N-[dimethyl(2-methylprop-2-yl)silyl]methaneamide were reacted in a hydrochloric acid solution containing TEA with shaking at room temperature for 17 hours. The TBS group was reduced with NaBH(OAc). The product was filtered and concentrated by rotary evaporation to yield a light yellow oil.

[0217] 31. Synthesis of OT13-9

[0218] 3,6-Bis{4-[bis(2-aminoethyl)amino]butyl}piperazine-2,5-dione (product from Step 30) was reacted with (8E)-hexadec-8-en-1-amine in THF under the catalysis of CDI, heated to 70°C, and then stirred with neopentane for 2 hours. The product was purified by silica gel chromatography to obtain a light yellow solid.

[0219] Synthesis steps of OT13-10

[0220] 32. Synthesis of 2-(undec-4-yn-1-yloxy)tetrahydro-2H-pyran

[0221] 2-(Pent-4-ynyloxy)tetrahydropyran was dissolved in THF and HMPA system, reacted with 1-bromohexane under the action of polymerization initiator n-butyl lithium to generate a product, which was purified by column chromatography.

[0222] 33. Synthesis of 2-{[(4Z)-undec-4-enyl]oxy}tetrahydropyran ((Z)-2-(undec-4-en-1-yloxy)tetrahydro-2H-pyran)

[0223] The product of step 32 is hydrogenated in an ethanol reaction system under the catalysis of Lindela's catalyst to produce 2-{[(4Z)-undec-4-enyl]oxy}tetrahydropyran. The product is purified by silica gel column chromatography.

[0224] 34. Synthesis of (4Z)-undec-4-en-1-ol

[0225] p-Toluenesulfonic acid is dissolved in methanol and reacted with the product of step 33 to generate (4Z)-undec-4-en-1-ol. The product is purified by silica gel column chromatography.

[0226] 35. Synthesis of (4Z)-undec-4-en-1-yl methanesulfonate

[0227] At room temperature, MsCl (methanesulfonyl chloride) was dissolved in dichloromethane. Under inert gas conditions, ((4Z)-undec-4-en-1-ol and methanesulfonyl chloride were reacted at a reaction temperature of 0-20°C with triethylamine as a catalyst, with gentle stirring. The reaction was terminated with distilled water, and the total organic layer was washed with saturated halide, dried, and concentrated. Purification was performed by silica gel column chromatography until the purity was greater than 85%.

[0228] 36. Synthesis of (4Z)-1-azidoundec-4-ene

[0229] The product of step 35. is reacted with sodium azide in the presence of DMSO, and the resulting product is purified by silica gel column chromatography.

[0230] 37. Synthesis of (4Z)-undec-4-en-1-amine

[0231] The product of step 36. was subjected to hydrogenation substitution reduction in THF solvent by catalysis of triphenylphosphine hydrate. The product was purified by silica gel chromatography and used for

[0232] 38. Synthesis of 2-methylprop-2-yl[(2,2-dimethyl-4,10-dioxyylidene-5-aza-3-oxatetradec-14-yl)amino]methane (di-tert-butyl(5-oxononane-1,9-diyl)dicarbamate)

[0233] [(4-Bromobutyl)amino]methane acid-2-methylprop-2-yl ester was reacted with 2-oxyylidenehexahydropyridine-1-carboxylic acid-2-methylprop-2-yl ester in a THF reaction solvent containing magnesium powder and iodine, and the product was purified by silica gel column chromatography.

[0234] 39. Synthesis of 2-methylprop-2-yl[(10-amino-2,2-dimethyl-4-oxyylidene-5-aza-3-oxatetradec-14-yl)amino]methane (di-tert-butyl(5-aminononane-1,9-diyl)dicarbamate)

[0235] Sodium cyanoborohydride was added to a mixture of ethanol and ammonium acetate at room temperature and stirred for more than 2 hours. The product was filtered through silica and washed with hot ethyl acetate.

[0236] 40. Synthesis of 2-methylpropane-2-yl{[8-(2,2-dimethyl-4-oxylidene-5-aza-3-oxanonan-9-yl)-2-methyl-6-oxylidene-2,5,7-triazadodec-12-yl]amino}methane (di-tert-butyl(5-(3-(2-(dimethylamino)ethyl)ureido)nonane-1,9-diyl)dicarbamate)

[0237] 2-Methylpropan-2-yl [(10-amino-2,2-dimethyl-4-oxyylidene-5-aza-3-oxatetradec-14-yl)amino]methane (product from Step 39) was reacted with 2-(dimethylamino)ethan-1-amine in THF, heated to 70°C, and then stirred with neopentane for 2 hours. The product was purified by silica gel chromatography to obtain the product.

[0238] 41. Synthesis of OT13-10

[0239] The product from step 40 was deprotected by Boc in an ethyl acetate / hydrochloric acid mixture and purified by silica gel column chromatography to afford the product. The product was then heated to 70°C in THF with (4Z)-undec-4-en-1-amine (product from step 37), and neopentane was introduced with shaking for 2 hours. The product was then purified by silica gel column chromatography to afford OT13-10.

[0240] Synthesis steps of OT13-11

[0241] 42. Synthesis of 2-methylpropane-2-yl[(1-phenyl-7-aza-2-oxatridecan-13-yl)amino]methane (tert-butyl(6-((4-(benzyloxy)butyl)amino)hexyl)carbamate)

[0242] [(6-Aminohexyl)amino]methane acid-2-methylprop-2-yl ester reacts with {[(4-bromobutyl)oxy]methyl}benzene in acetonitrile containing potassium carbonate to produce the product.

[0243] 43. Synthesis of 2-methylpropane-2-yl({12-[4-(benzyloxy)butyl]-2,2-dimethyl-4-oxyidene-5,12-diaza-3-oxaoctadec-18-yl}amino)methane (di-tert-butyl(((4-(benzyloxy)butyl)azanediyl)bis(hexane-6,1-diyl))dicarbamate)

[0244] The product from step 42 was reacted with 2-methylprop-2-yl [(5-formylpentyl)amino]methanoate in methanol at room temperature by adding sodium cyanoborohydride. Stirring was continued for more than 2 hours. The product was filtered through silica and washed with hot ethyl acetate.

[0245] 44. Synthesis of 6-(13-amino-1-phenyl-7-aza-2-oxatridecan-7-yl)hexane-1-amine (N1-(6-aminohexyl)-N1-(4-(benzyloxy)butyl)hexane-1,6-diamine)

[0246] The product of step 43 was de-Boc protected in an ethyl acetate / hydrochloric acid mixture. In addition, heptyl-1-amine and 1-iodohexane were reacted in an acetonitrile solvent containing potassium carbonate to generate the product hexyl (octyl) amine, which was purified by silica gel chromatography and used in the next reaction.

[0247] 45. Synthesis of 1-({7-[4-(benzyloxy)butyl]-15-oxyylidene-16-pentyl-7,14,16-triazatetracosan-1-yl}amino)-N-octyl-N-pentylmethaneamide (1,1'-(((4-(benzyloxy)butyl)azanediyl)bis(hexane-6,1-diyl))bis(3-hexyl-3-octylurea))

[0248] The product obtained after deBoc removal from the product in step 43 was heated to 70°C in THF with hexyl(octyl)amine under the catalysis of CDI, and then introduced with neopentane and shaken for 2 hours. The product was purified by silica gel column chromatography to obtain the product.

[0249] 46. ​​Synthesis of OT13-11

[0250] A dry round-bottom flask was purged with hydrogen. 1-({7-[4-(benzyloxy)butyl]-15-oxyylidene-16-pentyl-7,14,16-triazatetracosan-1-yl}amino)-N-octyl-N-pentylmethaneamide (product from Step 45) was added to an ethanol solvent. The mixture was reacted in an oil bath at 165°C for 12 hours under the catalysis of Pd. The reaction was maintained at 500 psi in a stainless steel pressure vessel. After filtration and concentration by rotary evaporation, OT13-11 was obtained.

[0251] Synthesis steps of OT13-12

[0252] 47. Synthesis of 2-methylprop-2-yl[(1-phenyl-5-aza-2-oxadecan-10-yl)amino]methane (tert-butyl(5-((2-(benzyloxy)ethyl)amino)pentyl)carbamate)

[0253] [(5-Bromopentyl)amino]methane acid-2-methylprop-2-yl ester reacts with 2-(benzyloxy)ethan-1-amine in an acetonitrile solvent containing potassium carbonate at room temperature to generate a product, which is purified by silica gel column chromatography.

[0254] 48. Synthesis of benzyl ({11-[2-(benzyloxy)ethyl]-2,2-dimethyl-4-oxyidene-5,11-diaza-3-oxapentadecan-15-yl}amino)methane ester (benzyl (4-((2-(benzyloxy)ethyl)(5-((tert-butoxycarbonyl)amino)pentyl)amino)butyl)carbamate)

[0255] To the product of step 47, benzyl [(3-formylpropyl)amino]methane was added at room temperature, and the mixture was stirred for more than 2 hours under the catalysis of sodium cyanoborohydride. The product was filtered through silica gel and washed with hot ethyl acetate for later use.

[0256] 49. Synthesis of benzyl (4-((5-aminopentyl)(2-(benzyloxy)ethyl)amino)butyl)carbamate)

[0257] The product of step 48 was de-Boc protected in an ethyl acetate / hydrochloric acid mixture system, and the product was purified by silica gel column chromatography and used in the next reaction.

[0258] 50. Synthesis of 2-octyldecanoic acid

[0259] 1-Bromooctane is reacted with decanoic acid in THF solvent under the catalysis of sodium hydride at room temperature with continuous stirring for two hours to produce 2-octyldecanoic acid. The product is purified by silica gel column chromatography for later use.

[0260] 51. Synthesis of 2-octyldecan-1-ol

[0261] 2-Octyldecanoic acid was dissolved in THF solvent and stirred at room temperature for 2 hours. 2-Octyldecan-1-ol was generated by LaN-catalyzed reduction, and the product was purified by silica gel column chromatography.

[0262] 52. Synthesis of 2-(2-octyldecyl)isoindoline-1,3-dione

[0263] At room temperature, Ms2O (methanesulfonic anhydride) is dissolved in dichloromethane containing TEA and gently stirred with 2-octyldec-1-ol (the product of step 51.) at a reaction temperature of 0-25°C under inert gas conditions to generate the product. The reaction is terminated with distilled water, and the total organic layer is washed, then washed with saturated halide, dried, and concentrated. Purify by silica gel column chromatography until the purity is greater than 85%. Thereafter, potassium phthalimide is dissolved in DMF, and sodium azide is added at room temperature. Stirring is continued at a reaction temperature of 70°C for 2 hours and distilled under reduced pressure. Dichloromethane is added for extraction, washed with water, and dried over anhydrous sodium sulfate to obtain a crude product, which is separated and purified by a chromatographic column.

[0264] 53. Synthesis of 2-octyldecan-1-amine

[0265] The product of step 52 is subjected to Gabriel primary amine reaction in an ethanol-based reaction system, and reacted with hydrazine hydrate at 80° C. to generate 2-octyldec-1-amine. The product is purified by silica gel column chromatography.

[0266] 54. Synthesis of benzyl ({5-[2-(benzyloxy)ethyl]-15-octyl-12-oxyidene-5,11,13-triazatricosan-1-yl}amino)methane ester (benzyl (4-((2-(benzyloxy)ethyl)(5-(3-(2-octyldecyl)ureido)pentyl)amino)butyl)carbamate)

[0267] The product 2-octyldec-1-amine obtained in step 53 and the product in step 49 were heated to 70° C. in THF, and neopentane was introduced. The mixture was shaken and reacted for 2 hours to obtain the product, which was then purified by silica gel column chromatography.

[0268] 55. Synthesis of 1-({5-[(4-aminobutyl)(2-hydroxyethyl)amino]pentyl}amino)-N-(2-octyldecyl)methaneamide (1-(5-((4-aminobutyl)(2-hydroxyethyl)amino)pentyl)-3-(2-octyldecyl)urea)

[0269] In a dry round-bottom flask, purge with hydrogen. Add the product from step 54 to a dichloromethane solvent containing acetic acid. Catalyzed by palladium or carbon, the reaction proceeds at room temperature for 2 hours to achieve debenzenization reduction. This reaction is maintained at 500 psi in a stainless steel pressure vessel. The product is filtered, concentrated by rotary evaporation, and purified by silica gel chromatography.

[0270] 56. Synthesis of OT13-12

[0271] Step 55. The obtained product was reacted with undecane-1-amine in THF at 70°C, and neopentane was introduced, and the reaction was shaken for 2 hours to obtain OT13-12. The product was purified by silica gel column chromatography.

[0272] Example 2

[0273] Preparation and characterization of lipid nanoparticles

[0274] By fully mixing the organic phase and the aqueous phase, LNP nanoparticles are prepared to achieve effective encapsulation of gene editing tools or other nucleic acid drugs.

[0275] The organic phase comprises at least one ionizable lipid (OT13-1 to OT13-9), at least one supporting lipid, at least one amphiphilic block copolymer, and cholesterol, dissolved in a water-miscible organic solvent. The organic solvent is preferably selected from ethanol, acetonitrile, acetone, and the like. In this embodiment, OT13-3, DSPC, PEG-DMG, and cholesterol are used, with the molar ratio of the four components being 50:10:1.5:38.5.

[0276] Aqueous phase: An aqueous solution of the gene editing tool, wherein the nucleic acid material uses yeast-extracted RNA as the template RNA (McLean, log R822593) at a content of 0.5-50% (w / v) (preferably 40% (w / v) in this embodiment), and a pH of about 3.0 to about 7.0 (preferably about 4.0 in this embodiment). The aqueous salt solution can be selected from: citric acid buffer, phosphate buffer, and Tris-HCl buffer system; citric acid buffer is used in this embodiment.

[0277] Mixing of the organic and aqueous phases can be achieved using microfluidics and impinging stream reactors. The embedding efficiency of the gene editing tool RNA can be optimized by regulating the N / P ratio of the system, ranging from 1:1 to 9:1.

[0278] LNPs were prepared by the method of this example, and the particle size, dispersion index (PDI), and embedding efficiency (EE) were tested. The results are shown in the following table:

[0279] Among them, the embedding efficiency characterization method: the embedding efficiency of the prepared LNP was measured by Quant-iTTM RNA Reagent and Kit. Specific detection method:

[0280] Solution Preparation: Dilute an appropriate amount of 20× TE buffer to 1× with ultrapure water, sufficient for the day's experiments. Dilute the concentrated dye solution with 1× TE buffer at a ratio of 1:200 (for a wide range of 25-1000 ng / ml RNA) or 1:2000 (for a narrow range of 1-50 ng / ml RNA). Wrap in foil or store in a dark place away from light. Dilute Triton X-100 to a 5% concentration with 1× TE buffer and set aside.

[0281] Sample treatment: Set ultrapure water with an RNA concentration of 0 as the blank background, set LNP with TE buffer instead of 5% TritonX-100 as the free RNA measurement sample, and set LNP with 5% TritonX-100 as the total RNA measurement sample. Take the LNP sample to be measured and the blank and add equal volumes of 5% TritonX-100 and 1× TE solution, incubate at 50-60°C for 5-10 minutes. After incubation, dilute the LNP sample with 1× TE buffer to 10-400 times (adjust appropriately according to the RNA concentration of the sample group). After dilution, take 100μl and add it to a 96-well plate. Then, add 100μl of the diluted concentrated dye solution in the dark and measure the fluorescence absorbance within 5 minutes. The measurement conditions are excitation wavelength 480nm and emission wavelength 520nm.

[0282] Calculation method: Embedding efficiency = (total RNA absorbance - free RNA absorbance) / total RNA absorbance × 100%.

[0283] Example 3

[0284] Evaluation of delivery effect in mouse liver

[0285] LNPs were prepared by the method of Example 2, wherein the ionizable lipid was OT13-3 and the embedded nucleic acid was Luciferase mRNA (nucleotide sequence is shown in the table below). The prepared Luc-mRNA-LNPs were injected into 6-8 week old C57BL / 6 mice, and the expression level of Lurciferase in the mouse liver was observed 6 hours after injection using in vivo imaging technology (Figure 1), thereby evaluating the delivery effect of OT13 lipid-formed LNPs in the mouse liver. The results showed that 6 hours after injection, the LNPs containing the ionizable lipid OT13-3 successfully delivered Luc-mRNA to the mouse liver and achieved efficient Luciferase expression.

[0286] Evaluation of the effect of intravenous liver transfection in adult Ai9 transgenic mice

[0287] Using the method of Example 2, LNPs containing OT13-3 ionizable lipids were prepared and simultaneously encapsulated with Cre mRNA (nucleotide sequence shown in the table below). These samples were injected into adult Ai9 transgenic mice. Five days after injection, mouse livers were removed, cryosectioned, and observed using confocal laser scanning microscopy. The results demonstrated that OT13-3-containing LNPs could effectively transfect and edit adult mouse livers (Figure 2).

[0288] Evaluation of PCSK9 gene editing effects in mice in vivo

[0289] PCSK9 is a target gene suitable for analyzing the delivery of functional gene editing tools to the liver. It is produced in liver cells and secreted into the blood circulation, and can therefore be used for in vivo screening of liver-directed lipid molecules. EPIREG-mRNA and gRNA (the complementary nucleotide sequences of their targeting sequences are shown in the table below), gene editing tools with silencing effects for PCSK9, were co-encapsulated into LNPs containing OT13-3 by the method of Example 2 and injected into wild-type 6-8-week-old C57BL / 6 mice at a concentration of 3 mg / kg RNA. After 9 days, PCSK9 was detected using a kit (Figure 3). The results showed that OT13-3-formed LNPs had an effect of nearly 80% epigenetic editing and silencing target genes. The in vivo epigenetic editing efficacy of OT13-3-formed LNPs can be compared with a positive control, which is a commercial LNP formed by ionizable lipids used in clinical practice. The evaluation data of this example proves that the OT13 series of molecules can be used as a nucleic acid delivery system in the field of liver-targeted gene therapy.

[0290] The nucleic acid sequence of this example:

[0291] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments listed in the present application are obvious to those skilled in the art and are intended to be within the scope of the appended claims and their equivalents.

Claims

1. A compound, or a pharmaceutically acceptable salt thereof, comprising a cation-donating group and a hydrogen-bond-donating group, wherein the cation-donating group and the hydrogen-bond-donating group contain 2 to 6 atoms.

2. The compound of claim 1, which is a cationic lipid compound.

3. The compound according to any one of claims 1 to 2, which is an ionizable cationic lipid compound.

4. The compound of any one of claims 1 to 3, wherein the cation-donating group comprises a group that is positively charged at a pH of about 7.0 or less.

5. The compound of any one of claims 1 to 4, wherein the cation-donating group comprises a group that is positively charged at a pH of about 4.0 or less.

6. The compound of any one of claims 1 to 5, wherein the cation-providing group comprises a tertiary amine group.

7. The compound of any one of claims 1 to 6, wherein the hydrogen bond providing group comprises a urea group.

8. The compound of any one of claims 1-7, comprising a tail, wherein the tail is hydrophobic.

9. The compound of claim 8, wherein the tail is substituted on one or two nitrogen atoms of the urea group.

10. The compound of claim 8 or 9, wherein the tail comprises a saturated and / or unsaturated fatty chain.

11. The compound according to any one of claims 8 to 10, wherein the main chain length of the tail is 1 to 20 atoms.

12. The compound of any one of claims 1 to 11, comprising the following structure (I), in, The L1 is a hydrogen bond providing group, the G1 is a linking group with a chain length of 2 to 6 atoms, the L1 is optionally substituted, and the G1 is optionally substituted.

13. The compound of any one of claims 1 to 12, comprising the following structure (II), in, The G1 is a linking group with a chain length of 2 to 6 atoms, the G1 is optionally substituted, the R1, R 2a and R 2b are each independently selected from optionally substituted substituents.

14. The compound of any one of claims 1 to 13, comprising the following structure (IIIa), in, The G1 and G2 are each independently a linking group with a chain length of 2 to 6 atoms, the G1 and G2 are each independently optionally substituted, the R1, R 2a 、R 2b , R3, R 4a and R 4b are each independently selected from optionally substituted substituents.

15. The compound of any one of claims 1 to 13, comprising the following structure (IIIb), in, The G1 is a linking group with a chain length of 2 to 6 atoms, the G1 is optionally substituted, the R1, R 2a 、R 2b , R5 and R6 are each independently selected from optionally substituted substituents.

16. The compound of any one of claims 1 to 13, comprising the following structure (IIIc), in, The G1 and G2 are each independently a linking group with a chain length of 2 to 6 atoms, the G1 and G2 are each independently optionally substituted, the R1, R 2a 、R 2b , R7, R 8a 、R 8b and R9 are each independently selected from optionally substituted substituents.

17. The compound of any one of claims 1 to 13, comprising the following structure (IIId), in, The G1, G2, G3 and G4 are each independently a linking group with a chain length of 2 to 6 atoms, the G1, G2, G3 and G4 are each independently optionally substituted, the H1 and H2 are each independently optionally substituted, the R1, R 2a 、R 2b , R7, R 8a 、R 8b 、R 10 、R 11a 、R 11b 、R 12 、R 13a , and R 13b are each independently selected from optionally substituted substituents.

18. The compound according to any one of claims 12 to 17, wherein G1, G2, G3 and G4 are each independently a hydrocarbon group, an alicyclic group, and / or an alicyclic heterocyclic group.

19. The compound according to any one of claims 12 to 18, wherein G1, G2, G3 and G4 are each independently an alkyl, alkenyl, and / or alkynyl group.

20. The compound of any one of claims 12-19, wherein one atom in G1, G2, G3 and G4 is independently replaced by N, O or S.

21. The compound according to any one of claims 17 to 20, wherein H1 and H2 are each independently a hydrocarbon group, an alicyclic group, and / or an alicyclic heterocyclic group.

22. The compound of any one of claims 17-21, wherein H1 and H2 are each independently alkyl, alkenyl, and / or alkynyl.

23. The compound of any one of claims 17 to 22, wherein one atom in H1 and H2 is independently replaced by N, O or S.

24. The compound of any one of claims 17 to 23, wherein R1, R 2a 、R 2b , R3, R 4a 、R 4b , R5, R6, R7, R 8a 、R 8b , R9, R 10 、R 11a 、R 11b 、R 12 、R 13a , and R 13b Each is independently a hydrocarbon group, an ester group, an amide group, an N'-alkyl-N-alkenyl-urea group, an alicyclic group, an alicyclic heterocyclic group, an aryl group, and / or a heteroaryl group.

25. The compound of any one of claims 17 to 24, wherein R1, R 2a 、R 2b , R3, R 4a 、R 4b , R5, R6, R7, R 8a 、R 8b , R9, R 10 、R 11a 、R 11b 、R 12 、R 13a , and R 13b Each independently is C1-C 20 Alkyl, C2-C 40 Alkenyl, C2-C 45 Ester group and / or C2-C 20 Alkynyl.

26. The compound of any one of claims 17-25, wherein R1, R 2a 、R 2b , R3, R 4a 、R 4b , R5, R6, R7, R 8a 、R 8b , R9, R 10 、R 11a 、R 11b 、R 12 、R 13a , and R 13b Each is independently branched or unbranched.

27. The compound of any one of claims 17-26, wherein R1, R 2a 、R 2b , R3, R 4a 、R 4b , R5, R6, R7, R 8a 、R 8b , R9, R 10 、R 11a 、R 11b 、R 12 、R 13a , and R 13b Each independently contains 0 to 4 unsaturated bonds.

28. The compound of any one of claims 17 to 27, wherein R1, R 2a 、R 2b , R3, R 4a 、R 4b , R5, R6, R7, R 8a 、R 8b , R9, R 10 、R 11a 、R 11b 、R 12 、R 13a , and R 13b are each independently substituted with Rx, wherein Rx is an optionally substituted substituent.

29. The compound of claim 28, wherein Rx is C1-C 20 Alkyl, C2-C 25 Ester group, C2-C 20 alkenyl, amide, urea, hydroxyl, thiol or amino.

30. A compound, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

31. A composition comprising a compound according to any one of claims 1 to 30 and a therapeutic agent.

32. The composition of claim 31 , further comprising a substance selected from the group consisting of a neutral lipid, a steroid, and a polymer-conjugated lipid.

33. The composition of claim 31 or 32, comprising liposomes.

34. The composition of any one of claims 31-33, having a diameter of about 500 nm or less.

35. The composition of any one of claims 32-34, wherein the steroid comprises cholesterol.

36. The composition of any one of claims 32-35, wherein the polymer-conjugated lipid comprises a PEGylated lipid.

37. The composition of any one of claims 32-36, wherein the molar ratio of the compound to the neutral lipid is from about 2:1 to about 8:

1.

38. The composition of any one of claims 32-37, wherein the molar ratio of the compound to the steroid is about 5:1 to 1:

1.

39. The composition of any one of claims 32-38, wherein the molar ratio of the compound to the polymer-conjugated lipid is from about 100:1 to about 20:

1.

40. The composition of any one of claims 31-39, wherein the therapeutic agent comprises a nucleic acid.

41. The composition of any one of claims 31-40, wherein the therapeutic agent comprises a component of a CRISPR system and / or a nucleic acid encoding a component of a CRISPR system.

42. A composition as described in any one of claims 31-41, wherein the therapeutic agent comprises a guide RNA and / or a nucleic acid encoding a Cas enzyme.

43. A cell comprising a compound according to any one of claims 1-30 and / or a composition according to any one of claims 31-42.

44. A kit comprising the compound of any one of claims 1-30, the composition of any one of claims 31-42, and / or the cell of claim 43.

45. A method of administering a therapeutic agent, comprising providing a compound of any one of claims 1-30, a composition of any one of claims 31-42, a cell of claim 43, and / or a kit of claim 44, wherein the therapeutic agent is mixed with the compound or is present in the composition, the cell, and / or the kit.

46. ​​A method of delivering a nucleic acid, the method comprising providing a compound as described in any one of claims 1-30, a composition as described in any one of claims 31-42, a cell as described in claim 43 and / or a kit as described in claim 44, wherein the nucleic acid is mixed with the compound or the nucleic acid is present in the composition, the cell and / or the kit.

47. A method of treating and / or preventing a disease or condition, the method comprising providing a compound according to any one of claims 1-30, a composition according to any one of claims 31-42, a cell according to claim 43, and / or a kit according to claim 44.