Cyclohexane-based lipids for nucleic acid transfection and uses thereof

Abstract

This invention relates to lipids of general formula I and their pharmaceutically acceptable salts, addition salts, and solvates, wherein X is selected from -C(=O)NH-, -C(=O)O-, -C(=S)O-, -C(=O)S-, -C(=S)S-, -C(=O)NHNH-, -CH2-, -O-, -OC(=O)-, -S-, -SC(=O)-, -NH-, -NHNH-, -NHC(=O)-, -NHNHC(=O)-, -C≡C-, -CH=CH-, a five-membered heterocycle containing at least two nitrogen atoms, -CH2C(=O)NH-, -CH2C(=S)O-, -CH2C(=S)S-, -CH2C(=O)NHNH-, -N=CH-, -CH=N-, -NH-N=CH-, and -CH=N-NH-; and Y is an alkylene C2-C 10 Chain; R 1 Selected from alkyl C1-C 46 Alkenyl C2-C 46 , alkynyl C2-C 46 Z is selected from H, -OH, -CH3, -CH2OH, -NH2, -C(=O)NH2, -CONH(CH2)2OH, -CON[(CH2)2OH]2, -CONHCH(CH2OH)2, -CONHCH2CH(-OH)CH2OH, -CONH(CH2)2C(=O)NH2, -CON[CH2C(=O)NH2]2, -CONH(CH2)2NHC(=O)NH2. -CONH(CH2)3-N+(CH3)2-(CH2)2-SO3-, -CONH(CH2)3-N+(CH3)2-(CH2)2-COO-, -COO(CH2)2-O-P(=O)(O-)-O(CH2)2-N+(CH3)3, -N+(CH3)2-(CH2)3-SO3-, -N+(CH3)2-(CH2)2-COO-, Formula (II), Formula (III), where R 2 Independently selected from hydrogen and -CH3; E independently selected from O and S atoms; n is an integer in the range of 1 to 5; and T is selected from -X-Y-N(R 1)2、-C(=O)O(C1-C3 alkyl), -C(=O)OCH2CH2OH, Formula (IV), Formula (V), Formula (VI), -C(=O)OH, -CONH(CH2)2OH, -CON[(CH2)2OH]2, -CONHCH(CH2OH)2, CONH(CH2)2C(=O)NH2, -CON[CH2C(=O)NH2]2, -CONHCH[C(=O)NH2]2, -CONH(CH2)2NHC(=O)NH2, -C(=O)NH2, -CONH(CH2)3-N+(CH3)2-(CH2)2-SO3-, -CONH(CH2)3-N+(CH3)2-(C H2)2‑COO‑,‑NH2,‑NHC(=O)CH3,‑COO(CH2)2‑O‑P(=O)(O‑)‑O(CH2)2‑N+(CH3)3,‑OH,‑O(C1‑C3 alkyl),‑NHC(=O)NH(CH3),‑NHC(=S)N(CH3)2,‑NHC(=S)NH( CH3),‑NHC(=N‑CN)NH2,‑NHC(=N‑CN)NH(CH3),‑NHC(=N‑CN)N(CH3)2,‑NHC[=N‑S(=O)2NH2]NH2,‑N+(CH3)2‑(CH2)3‑SO3‑,‑N+(CH3)2‑(CH2)2‑COO‑, where R 2 E and n are as defined above; and / or if Z is -OH or -CH2OH, and T is -C(=O)OH, then Z, together with T and the three carbon atoms therebetween, can form a cyclic lactone containing 4 to 5 carbon atoms. This type of lipid can be used as a transfection agent. The present invention also describes transfection reagents containing this type of lipid, transfection particles, and their uses.

Description

Technical Field

[0001] This invention relates to novel ionizable lipids and the use of these compounds for transfecting and administering nucleotides and nucleic acids and their synthetic analogues into cells and tissues. Background Technology

[0002] In recent years, the development of nucleic acid (NA)-based therapies has experienced an unprecedented resurgence. Progress is now being made due to the higher efficacy and lower risk of adverse side effects compared to previously tested therapeutic deoxyribonucleic acid (DNA) therapies. Several such drugs have entered clinical use, such as partecillin for hereditary transthyretin amyloidosis, edipulson for certain types of Duchenne muscular dystrophy, and nusinersen for spinal muscular atrophy. All of these diseases are life-threatening, and there are no alternative treatments. Potential drugs targeting ribonucleic acid (RNA), or their uses, can be categorized into three classes based on whether they target NA or proteins or encode proteins. The first class includes single-stranded antisense oligonucleotides (nusinersen, edipulson) that block the translation of messenger RNA (mRNA) or RNA splicing of 13–25 nucleotides (nt); and small interfering RNAs (siRNA, 21–23 nt) that degrade mRNA (partecillin). Therapeutic RNA molecules that target proteins use a class of molecules called RNA aptamers. It is designed to regulate the function of specific proteins. An example of such a drug is pilgatanib, used to treat neovascular age-related macular degeneration, which was the first of its kind approved in 2004. Therapies utilizing mRNA are primarily used to develop so-called personalized vaccines against cancer or vaccines against infectious diseases such as Zika virus. In viral diseases, mRNA-based candidate prophylactic vaccines against rabies and pandemic influenza have been shown to induce safe antibodies in healthy volunteers. Protein-alternative mRNA therapies are also in preclinical development, for example, for the treatment of hemophilia.

[0003] Molecular technologies capable of direct genome editing, particularly those based on the CRISPR-Cas9 system, are rapidly developing. CRISPR technology is a tool that allows alteration of DNA sequences and modification of gene function. Potential applications include repairing genetic defects, treating and preventing the spread of diseases, and improving crops. The CRISPR-Cas9 system has been tested in numerous preclinical and clinical studies, including HIV treatment, treatment of hematologic malignancies, and genetic conditions, including sickle cell disease and β-thalassemia. RNA editing is then enabled via the ADAR system (an adenosine deaminase that acts on RNA), which appears to be safer from a clinical perspective to date. Molecular technologies for direct genome editing can be delivered to the site of action in the form of mRNA encoding the appropriate enzyme responsible for editing.

[0004] The key factor enabling the safe use of all these technologies (or other NA-based technologies) is their safe and efficient delivery to the site of action. A crucial step is the crossing of the cell's phospholipid membrane by negatively charged NA; the process of intentionally introducing NA into eukaryotic cells is called transfection. In recent decades, carriers (so-called transporters) have been extensively developed to efficiently transport NA across cell membranes while protecting it from in vivo degradation (Stewart, MP: Chem. Rev. 2018, 118, 7409-7531).

[0005] Both viral and non-viral (physical and chemical) vectors can be used for NA transfection. Although approximately 70% of clinical trials in the field of gene therapy to date have used viral vectors, this approach carries numerous risks (carcinogenicity, induction of immune responses, tissue nonspecificity, limited NA incorporation capacity, and manufacturing complexity). Physical methods (such as electroporation) are difficult to use systematically in human medicine.

[0006] In contrast, synthetic chemical carriers typically exhibit lower immunogenicity, are capable of transporting larger amounts of genetic material, and, due to their well-defined molecular composition, their structure can be tailored to enhance efficiency and suppress toxicity. Cationic polymers or cationic lipids are used as chemical carriers, forming complexes with negatively charged norepinephrine (NA). This complex can penetrate cell membranes while protecting NA from degradation in the extracellular environment.

[0007] From a structural perspective, so-called lipid nanoparticles (LNPs) represent the most promising and clinically advanced form of these complexes. In them, cationic lipids are typically formulated with PEGylated lipids to prevent aggregation, influence particle size and transfection efficiency, and auxiliary lipids and cholesterol essential for stable NA encapsulation, as illustrated in siRNA transfection systems (Kulkarni, J.: Nanoscale, 2019, 11, 21733-21739). LNPs can accommodate NA molecules ranging in size from a few nucleotide units to millions of nucleotide units.

[0008] Synthetic cationic lipids and lipid-like molecules (similar to synthetic lipid molecules, but with a greater number of hydrophobic chains) are formed from both cationic and hydrophobic domains. To date, a large number of these substances with high structural variability in both domains have been developed through targeted design and testing of libraries generated by combining both.

[0009] Lipids and lipid-like substances (e.g., D-Lin-MC3-DMA, C12-200, cKK-E12, SA2-SC8, etc.) have been specifically developed for siRNA transfection (Dong, Y.: Adv. Drug Deliv. Rev. 2019, 144, 133-147). A formulation containing D-Lin-MC3-DMA was recently (August 2018) introduced into clinical practice under the name Onpattro (formerly known as partecillan), making it the first siRNA drug to be approved in history (Zhang, X.: J. Clin. Pharmacol. 2020, 60(1), 37-49). However, formulations developed for siRNA may be ineffective against mRNA, thus requiring targeted optimization (Cullis, P.: Mol. Ther. 2017, 25(7), 1467-1475).

[0010] Ionizable lipids and lipid-like substances (e.g., D-Lin-MC3-DMA, C12-200, cKK-E12, and TT3) have been used for mRNA transfection (Zhong, Z.: Nano Today 2018, 23, 16-39; Kowalski, P.: Mol. Ther. 2019, 27(4), 1-19; Li, B.: Nano Lett. 2015, 15, 8099-8107). Ionizable lipids can also be used for DNA transfection. Again, it should be emphasized that transfection systems optimized for small molecule (siRNA) transfection are not always suitable for DNA transfection, and even formulations developed for mRNA may be ineffective for DNA (Buck, J.: ACS Nano 2019, 13, 3754-3782).

[0011] Because, despite their enormous therapeutic potential, few synthetic carriers based on ionizable lipids have entered the clinical use stage to date, it is necessary to develop new systems with higher efficiency and extremely low in vivo toxicity. Summary of the Invention

[0012] This invention provides solutions to the efficiency problems of transfecting and targeted delivery of nucleotides and nucleic acids and their synthetic analogues using ionizable (cationic) lipids, as well as the toxicity of these lipids to target organisms or cells. Surprisingly, it was found that using cyclohexane as the central core of ionizable lipids significantly improves transfection efficiency compared to previously known solutions. Simultaneously, these cyclohexane-containing lipids exhibit extremely low cytotoxicity at relevant doses. The unique properties of the cyclohexane core used as the central structural part of the novel ionizable lipids compared to those known to date are the spatial complexity and extensive substitutions in its vicinity.

[0013] The objective of this invention is ionizable lipids of general formula I.

[0014]

[0015] in

[0016] X is selected from the group consisting of: -C(=O)NH-, -C(=O)O-, -C(=S)O-, -C(=O)S-, -C(=S)S-, -C(=O)NHNH-, -CH2-, -O-, -OC(=O)-, -S-, -SC(=O)-, -NH-, -NHNH-, -NHC(=O)-, -NHNHC(=O)-, -C≡C-, -CH=CH-, five-membered heterocycles containing at least two nitrogen atoms, -CH2C(=O)NH-, -CH2C(=O)O-, -CH2C(=S)O-, -CH2C(=S)S-, -CH2C(=O)NHNH-, -N=CH-, -CH=N-, -NH-N=CH- and -CH=N-NH-;

[0017] Y is selected from the group consisting of alkylene C2-C. 10 A chain, wherein in the alkylene chain, one or more -CH2- groups may optionally be replaced by one or more O and / or S atoms; preferably, Y is selected from the group consisting of alkylene C2-C 10 Chains, especially alkylene C3-C6 chains, preferably propylene (-(CH2)3-), pentylene (-(CH2)5-), and hexylene (-(CH2)6-);

[0018] R 1 Whether they are the same or different, each R 1 Independently selected from the group consisting of alkyl C1-C 46 Alkenyl C2-C 46 , alkynyl C2-C 46 The alkyl, alkenyl, or alkynyl group may be linear or branched, and one or more -CH2- groups in the alkyl, alkenyl, or alkynyl group may be optionally replaced by one or more groups selected from the following: -CH(OH)-, -OC(=O)-, -C(=O)O-, -SS-, -C(=O)NH-, -NHC(=O)-, -O-, and -S-;

[0019] If the alkyl, alkenyl, or alkynyl group is branched, one or more >CH- groups may be optionally replaced by >C(OH)- groups;

[0020] And if R 1 If it is an alkyl C1-C4 group, then one hydrogen atom from the terminal -CH3 group can be substituted by Z;

[0021] The condition is that at least one R 1 Contains at least 8 carbon atoms, preferably at least 10 carbon atoms, more preferably at least 12 carbon atoms; even more preferably, at least two R atoms. 1 The substituents contain at least 8 carbon atoms, preferably at least 10 carbon atoms, and more preferably at least 12 carbon atoms;

[0022] Z may be the same as or different from each other, and each Z is independently selected from the group consisting of: hydrogen, -OH, -CH3, -CH2OH, -NH2, -C(=O)NH2, -CONH(CH2)2OH, -CON[(CH2)2OH]2, -CONHCH(CH2OH)2, -CONHCH2CH(-OH)CH2OH, -CONH(CH2)2C(=O)NH2, -CON[CH2C(=O)NH2]2, -CONHCH[C(=O)NH2]2, -CONH(CH2)2NHC(=O)NH2, -CONH(CH2)3-N + (CH3)2-(CH2)2-SO3 - -CONH(CH2)3-N + (CH3)2-(CH2)3-SO3 - -CONH(CH2)3-N + (CH3)2-(CH2)2-COO - -COO(CH2)2-OP(=O)(O - )-O(CH2)2-N + (CH3)3、-N + (CH3)2-(CH2)3-SO3 - -N + (CH3)2-(CH2)2-COO - ,

[0023] in

[0024] R 2 Independently selected from hydrogen and -CH3;

[0025] E is independently selected from O and S atoms;

[0026] n is an integer in the range of 1 to 5;

[0027] And T are either the same as or different from each other, each T is independently selected from the following group: -XYN(R 1 )2, -C(=O)O(C1-C3 alkyl), -C(=O)OCH2CH2OH, -C(=O)NH2, -C(=O)OH, -CONH(CH2)2OH, -CON[(CH2)2OH]2, -CONHCH(CH2OH)2, -CONHCH2CH(-OH)CH2OH, -CONH(CH 2)2C(=O)NH2, -CON[CH2C(=O)NH2]2, -CONHCH[C(=O)NH2]2, -CONH(CH2)2NHC(=O)NH2, -CONH(CH2)3-N + (CH3)2-(CH2)2-SO3 - -CONH(CH2)3-N + (CH3)2-(CH2)3-SO3 - -CONH(CH2)3-N + (CH3)2-(CH2)2-COO - -COO(CH2)2-OP(=O)(O - )-O(CH2)2-N + (CH3)3、 -OH, -O(C1-C3 alkyl), -NH2, -NHC(=O)CH3, -NHS(=O)2CH3, -NHC(=O)N(CH3)2, -NHC(=O)NH(CH3), -NHC(=S)N(CH3)2 , -NHC(=S)NH(CH3), -NHC(=N-CN)NH2, -NHC(=N-CN)NH(CH3), -NHC(=N-CN)N(CH3)2, -NHC[=NS(=O)2NH2]NH2, -N + (CH3)2-(CH2)3-SO3 - -N + (CH3)2-(CH2)2-COO - ,

[0028] Where R 2 E and n are as defined above;

[0029] And / or if Z is -OH or -CH2OH, and T is -C(=O)OH, then Z together with T and the three carbon atoms therebetween can form a cyclic lactone containing 4 to 5 carbon atoms. This invention also covers pharmaceutically acceptable salts, addition salts, and solvates of lipids as defined above.

[0030] The term "alkyl" refers to a saturated hydrocarbon chain, which can be straight, branched, cyclic, or cyclic, and is derived from an alkane by removing a hydrogen atom.

[0031] The term "alkene" refers to a hydrocarbon chain containing at least one double bond between carbon atoms, and is derived from alkenes by removing one hydrogen atom. The hydrocarbon chain can be straight, branched, cyclic, or cyclic.

[0032] The term "alkynyl" refers to a hydrocarbon chain containing at least one triple bond between carbon atoms and optionally one or more double bonds between carbon atoms, and is derived from an alkyne by removing one hydrogen atom. The hydrocarbon chain can be straight, branched, cyclic, or cyclic.

[0033] The term "alkylene" refers to a divalent saturated hydrocarbon chain, which can be linear, branched, cyclic, or cyclic, preferably linear. This chain has two chemical valences, meaning it is derived from alkanes by removing two hydrogen atoms from different carbon atoms, and it is bonded via two single bonds as joints or bridges.

[0034] The terms “branched alkyl”, “branched alkenyl”, and “branched alkynyl” refer to alkyl, alkenyl, or alkynyl groups that contain 1 to 5 branches (hydrocarbon chains) attached to the main hydrocarbon chain.

[0035] When the molecule of general formula I has a positive charge, the compound contains a counterion (which can be a pharmaceutically acceptable anion of an organic or inorganic acid) to form a pharmaceutically acceptable salt. Such anions can be selected from, for example, the group including: acetate, aspartate, benzenesulfonate, benzoate, besylate, bicarbonate, tartrate, bromide, camphorsulfonate, carbonate, chloride, citrate, decanoate, edetate, ethanesulfonate, fumarate, gluconate, gluconate, glutamate, glycolate, hexanoate, iodide, lactate, malate, maleate, mandelic acid, methanesulfonate, methylsulfate, naphthalenesulfonate, nitrate, caprylate, oleate, palmitate, pantothenate, phosphate, polygalacturonic acid, propionate, salicylate, stearate, succinate, sulfate, tartrate, and toluenesulfonate.

[0036] When a compound of Formula I contains a chiral center, then Formula I includes pure enantiomers and mixtures of enantiomers, including racemic compounds.

[0037] Formula I includes compounds of Formula I in their free form, as well as in the form of salts, addition salts (with acids or bases), and / or solvates, including hydrates or alcohol solvates.

[0038] In one embodiment, X is selected from the group consisting of: -C(=O)NH-, -C(=O)O-, -C(=O)NHNH-, -OC(=O)-, -O-, -NHC(=O)-, -NHNHC(=O)-, and a five-membered heterocycle containing at least two nitrogen atoms. Preferably, X is -C(=O)NH- or -NHC(=O)-.

[0039] R 1 Chains can be identical or different from one another throughout the molecule, or R 1 The chains are identical for a single nitrogen atom, but not identical throughout the molecule. Preferably, for ease of synthesis, R... 1 The chain is identical throughout the molecule, or all nitrogen atoms are substituted by the same two R atoms. 1 Or by two different R 1 At least one R 1 The substituent is an aliphatic chain with at least 8 carbon atoms, and R 1 Selected from the group consisting of alkyl C1-C 46 Alkenyl C2-C 46 , alkynyl C2-C 46 The alkyl, alkenyl, or alkynyl group may be linear or branched, and one or more -CH2- groups in the alkyl, alkenyl, or alkynyl group may be optionally replaced by one or more groups selected from the following: -CH(OH)-, -OC(=O)-, -C(=O)O-, -SS-, -C(=O)NH-, -NHC(=O)-, -O-, and -S-;

[0040] If the alkyl, alkenyl, or alkynyl group is branched, one or more >CH- groups may be optionally replaced by >C(OH)- groups;

[0041] Where R 1 If it is an alkyl C1-C4, then one hydrogen from the terminal -CH3 group can be substituted by Z.

[0042] In one implementation, R 1 Independently selected from the group consisting of alkyl C1-C 46 and alkenyl C2-C 46 In the alkyl or alkenyl group, one or more -CH2- groups may be optionally replaced by one or more groups selected from the following: -CH(OH)-, -OC(=O)-, -C(=O)O-.

[0043] Preferably, R 1 Independently selected from the group consisting of linear or branched alkyl C8-C 20 and linear or branched alkenyl C8-C 20In the alkyl or alkenyl group, one or more -CH2- groups may be optionally replaced by one or more groups selected from the following: -OC(=O)-, -C(=O)O-. More preferably, R 1 Selected from the group consisting of linear or branched alkyl C 10 -C 15 It may be optionally substituted with one or more groups selected from -OC(=O)-, -C(=O)O-; and linear or branched alkenyl C 12 -C 18 It can be optionally replaced by one or more groups selected from -OC(=O)- and -C(=O)O-.

[0044] Preferably, the alkenyl chain contains 1 to 5 carbon-carbon double bonds.

[0045] In one implementation, all R in the molecule 1 They are the same.

[0046] In one embodiment, the molecule contains R 1 Each nitrogen atom of the group is separated by two identical R atoms. 1 The same substitution (but R is bound to different nitrogen atoms in the same type of lipid structure) 1 (May be different).

[0047] In one implementation, two different R 1 It combines with a nitrogen atom.

[0048] At least one R in the lipid structure 1 It must contain at least 8 carbon atoms, preferably at least 10 carbon atoms, and more preferably at least 12 carbon atoms, in order to achieve the lipid-like properties of the molecule. Preferably, the two R atoms in the lipid structure... 1 It contains at least 8 carbon atoms, preferably at least 10 carbon atoms, and more preferably at least 12 carbon atoms.

[0049] In one implementation, a substituent T is -XYN(R) 1 )2.

[0050] In another embodiment, both substituents T are -XYN(R) 1 )2.

[0051] In the latter case, the resulting lipid molecules have the general formula II.

[0052]

[0053] Where X, Y, Z and R 1 As defined above.

[0054] Formula II preferably has the following substituents:

[0055] X is selected from the group including the following: -C(=O)NH-, -C(=O)O-, -C(=S)O-, -C(=O)S-, -C(=S)S-, -C(=O)NHNH-, -CH2-, -O-, -OC(=O)-, -S-, -SC(=O)-, -NH-, -NHNH-, -NHC(=O)-, -NHNHC(=O)-, -C≡C-, -CH=CH-, five-membered heterocycles containing at least two nitrogen atoms, -CH2C(=O)NH-, -CH2C(=O)O-, -CH2C(=S)O-, -CH2C(=S)S-, -CH2C(=O)NHNH-, -N=CH-, -CH=N-;

[0056] Y is an alkylene chain C2-C 10 One or more -CH2- groups may be optionally replaced by one or more O and / or S atoms;

[0057] Z may be the same as or different from each other, and each Z is selected from the following groups: hydrogen atom, -OH, -CH3, -CH2OH, -NH2, -C(=O)NH2, -CONH(CH2)2OH, -CON[(CH2)2OH]2, -CONHCH(CH2OH)2, -CONHCH2CH(-OH)CH2OH, -CONH(CH2)2C(=O)NH2, -CON[CH2C(=O)NH2]2, -CONHCH[C(=O)NH2]2, -CONH(CH2)2NHC(=O)NH2, -N + (CH3)2-(CH2)3-SO3 - -N + (CH3)2-(CH2)2-COO - ,

[0058] And R 1 Whether they are the same or different, each R 1 Select from the group consisting of: alkyl C8-C 20 Alkenyl C8-C 20 , acetylinic C8-C 20 In the alkyl, alkenyl or alkynyl group, one or more -CH2- groups may be optionally replaced by one or more groups selected from the following: -CH(OH)-, -OC(=O)-, -C(=O)O-, -SS-, -C(=O)NH-, -NHC(=O)-, -O-, -S-.

[0059] Linker X is formed by attaching the amine moiety of a molecule to the central cyclohexane core via a reaction. Therefore, depending on the chosen reaction, these can be different linkers, such as those formed via click reactions (e.g., azido-alkynyl cycloaddition) to esters, amides, and the like.

[0060] X is therefore selected from the group consisting of: -C(=O)NH-, -C(=O)O-, -C(=S)O-, -C(=O)S-, -C(=S)S-, -C(=O)NHNH-, -CH2-, -O-, -OC(=O)-, -S-, -SC(=O)-, -NH-, -NHNH-, -NHC(=O)-, -NHNHC(=O)-, -C≡C-, -CH=CH-, a five-membered heterocycle containing at least two nitrogen atoms, -CH2C(=O)NH-, -CH2C(=O)O-, -CH2C(=S)O-, -CH2C(=S)S-, -CH2C(=O)NHNH-, -N=CH-, -CH=N-, -NH-N=CH- and -CH=N-NH-. Preferably, X is selected from -C(=O)NH-, -NHC(=O)-, a five-membered heterocycle containing at least two nitrogen atoms, -OC(=O)-, and -C(=O)O-.

[0061] Linkage Y is an alkylene chain that provides at least a minimum distance between the amine, linker X, and the cyclohexane core. Y is C2-C. 10 Alkylene chain, preferably a C2-C8 alkylene chain, wherein one or more -CH2- groups may be optionally replaced by one or more O and / or S atoms.

[0062] Substituent Z can also modify the properties of compounds of formulas I and II.

[0063] In one embodiment, Z is selected from the group consisting of: hydrogen, -OH, -CH3, -CH2OH, -NH2, -C(=O)NH2, -CONH(CH2)2OH, -CON[(CH2)2OH]2, -CONHCH(CH2OH)2, -CONHCH2CH(-OH)CH2OH, -CONH(CH2)2C(=O)NH2, -CON[CH2C(=O)NH2]2, -CONHCH[C(=O)NH2]2, -CONH(CH2)2NHC(=O)NH2, -N + (CH3)2-(CH2)3-SO3 - -N + (CH3)2-(CH2)2-COO - ,

[0064] Where R 2E and n are as defined above; preferably, Z is H, -CH3, -CH2OH, or if Z is -OH or -CH2OH and T is -C(=O)OH, then Z together with T and the three carbon atoms therebetween can form a cyclic lactone containing 4 to 5 carbon atoms.

[0065] The substituent T can also modify the properties of compounds of formulas I and II. In one embodiment, T is selected from the group consisting of: -XYN(R 1 )2, -C(=O)O(C1-C3 alkyl), -C(=O)OCH2CH2OH, -C(=O)NH2, -C(=O)OH, -CONH(CH2)2OH, -CON[(CH2)2OH]2, -CONHCH(CH2OH)2, -CONHCH2CH(-OH)CH2OH, -CONH(CH 2)2C(=O)NH2, -CON[CH2C(=O)NH2]2, -CONHCH[C(=O)NH2]2, -CONH(CH2)2NHC(=O)NH2, -CONH(CH2)3-N + (CH3)2-(CH2)2-SO3 - -CONH(CH2)3-N + (CH3)2-(CH2)3-SO3 - -CONH(CH2)3-N + (CH3)2-(CH2)2-COO - -COO(CH2)2-OP(=O)(O - )-O(CH2)2-N + (CH3)3、

[0066] Where R 2 E and n are as defined above; more preferably, T is selected from the group consisting of: -XYN(R 1 )2、-C(=O)O(C1-C3 alkyl), -C(=O)OH. More preferably, T is selected from the group consisting of: -XYN(R 1 )2、-C(=O)OCH3、 -C(=O)OH.

[0067] In one specific embodiment, the lipids of Formula I have Z selected from the group consisting of: hydrogen, -OH, -CH3, -CH2OH;

[0068] And T can freely choose from the following groups: -XYN(R) 1)2、-C(=O)O(C1-C3 alkyl), -C(=O)OH;

[0069] And / or if Z is -OH or -CH2OH, and T is -C(=O)OH, then Z, together with T and the three carbon atoms therebetween, can form a cyclic lactone containing 4 to 5 carbon atoms. Preferably, the lipid of formula I has the following substituents:

[0070] X is -C(=O)NH- or -NHC(=O)-;

[0071] Y is selected from -(CH2)3-, -(CH2)4-, -(CH2)5-, and -(CH2)6-;

[0072] R 1 Selected from the group consisting of linear or branched alkyl C 10 -C 15 It may be optionally substituted with one or more groups selected from -OC(=O)-, -C(=O)O-; and linear or branched alkenyl C 12 -C 18 It may be optionally replaced by one or more groups selected from -OC(=O)- and -C(=O)O-;

[0073] T is selected from -XYN(R) 1 )2、-C(=O)OCH3,、 and -C(=O)OH;

[0074] Z is selected from H, -CH3, and -CH2OH.

[0075] Specifically, the following lipids are preferred:

[0076] cis, cis-N 1 N 3 N 5 -tris(6-(bis(dodecylamino)hexyl)cyclohexane-1,3,5-tricarboxamide(3);

[0077] cis, cis-N 1 N 3 N 5 -tris(6-(di((hexyloxycarbonyl)butyl)amino)hexyl)cyclohexane-1,3,5-tricarboxamide(7);

[0078] cis, cis-N 1 N 3 N 5 -tris(6-(bis(dodecylamino)hexyl)-1,3,5-trimethylcyclohexane-1,3,5-tricarboxamide(8);

[0079] cis, cis-N 1 N 3 N 5 -tris(3-(bis(dodecylamino)propyl)-1,3,5-trimethylcyclohexane-1,3,5-tricarboxamide(11);

[0080] cis, cis-N 1 N 3 N 5 -tris(6-(di((9Z,12Z)-octadec-9,12-dien-1-yl)amino)hexyl)-1,3,5-trimethylcyclohexane-1,3,5-tricarboxamide(15);

[0081] Hexa(octane-2-yl)cis,cis-6,6',6”,6”',6””,6””-((((cyclohexane-1,3,5-tricarbonyl)tri(azanediyl))tri(hexane-6,1-diyl))tri(azanetriyl))hexahexanoate (19);

[0082] Hexa(3-methylhexyl)cis,cis-7,7',7”,7”',7””,7””-((((cyclohexane-1,3,5-tricarbonyl)tri(azanediyl))tri(hexane-6,1-diyl))tri(azanetriyl))hexaheptanate (23);

[0083] Hexa((E)-3,7-dimethyloctyl-2,6-dien-1-yl)cis,cis-5,5',5”,5”',5””,5””-((((cyclohexane-1,3,5-tricarbonyl)tri(azanediyl))tri(hexane-6,1-diyl))tri(azanetriyl))hexavalerate (27);

[0084] Hexa((Z)-3,7-dimethyloctyl-2,6-dien-1-yl)cis,cis-5,5',5”,5”',5””,5””-((((cyclohexane-1,3,5-tricarbonyl)tri(azanediyl))tri(hexane-6,1-diyl))tri(azanetriyl))hexavalerate (31);

[0085] Tetra(octane-2-yl)cis,cis-6,6',6”,6”'-((((5-(methoxycarbonyl)cyclohexane-1,3-dicarbonyl)bis(azanediyl))bis(hexane-6,1-diyl))bis(azanetriyl))tetrahexanoate (32);

[0086] Tetra(3-methylhexyl)cis,cis-7,7',7”,7”'-((((5-(methoxycarbonyl)cyclohexane-1,3-dicarbonyl)bis(azanediyl))bis(hexane-6,1-diyl))bis(azanetriyl))tetraheptanoate (33);

[0087] cis,cis-3,5-bis((6-(bis(6-(octane-2-yloxy)-6-oxohexyl)amino)hexyl)carbamoyl)cyclohexane-1-carboxylic acid (35);

[0088] Tetra(octane-2-yl)cis,cis-6,6',6”,6”'-((((5-(pyrrolidine-1-carbonyl)cyclohexane-1,3-dicarbonyl)bis(azanediyl))bis(hexane-6,1-diyl))bis(azanetriyl))tetrahexanoate (36);

[0089] Hexa(3-methylhexyl)cis,cis-7,7',7”,7”',7””,7””-((((1,3,5-trimethylcyclohexane-1,3,5-tricarbonyl)tri(azinaldiyl))tri(hexane-6,1-diyl))tri(azinaltriyl))hexaheptanate (40);

[0090] cis,cis-6,6',6”,6”',6””,6””-((((1,3,5-trimethylcyclohexane-1,3,5-tricarbonyl)tri(azanediyl))tri(hexane-6,1-diyl))tri(azanetriyl))hexahexanoate (41);

[0091] cis, cis-3,5-bis((6-(bis(6-(octane-2-yloxy)-6-oxohexyl)amino)hexyl)carbamoyl)-1,3,5-trimethylcyclohexane-1-carboxylic acid (42);

[0092] Tetra(octane-2-yl)cis,cis-6,6',6”,6”'-((((1,3,5-tris((benzyloxy)methyl)-5-(bis(6-(octane-2-yloxy)-6-oxohexyl)carbamoyl)cyclohexane-1,3-dicarbonyl)bis(azanediyl))bis(hexane-6,1-diyl))bis(azanetriyl))tetrahexanoate (45);

[0093] cis,cis-1,3,5-tris((benzyloxy)methyl)-3,5-bis((6-(bis(6-(octane-2-yloxy)-6-oxohexyl)amino)hexyl)carbamoyl)cyclohexane-1-carboxylic acid (46);

[0094] Hexa(octane-2-yl)cis,cis-6,6',6”,6”',6””,6””-((((1,3,5-tris(hydroxymethyl)cyclohexane-1,3,5-tricarbonyl)tris(azanediyl))tris(hexane-6,1-diyl))tris(azanetriyl))hexahexanoate (47);

[0095] cis,cis-3,5-bis((6-(bis(6-(octane-2-yloxy)-6-oxohexyl)amino)hexyl)carbamoyl)-1,3,5-tris(hydroxymethyl)cyclohexane-1-carboxylic acid (48);

[0096] Tetra(octane-2-yl)cis,cis-6,6',6”,6”'-((((1,3,5-tris((benzyloxy)methyl)-5-(pyrrolidine-1-carbonyl)cyclohexane-1,3-dicarbonyl)bis(azanediyl))bis(hexane-6,1-diyl))bis(azanetriyl))tetrahexanoate (49);

[0097] Tetra(octane-2-yl)cis,cis-6,6',6”,6”'-((((1,3,5-tris(hydroxymethyl)-5-(pyrrolidine-1-carbonyl)cyclohexane-1,3-dicarbonyl)bis(azanediyl))bis(hexane-6,1-diyl))bis(azanetriyl))tetrahexanoate (50);

[0098] cis, cis-N 1 N 7 -bis(6-(bisdodecylamino)hexyl)-5,7-bis(hydroxymethyl)-4-oxo-3-oxabicyclo[3.3.1]nonane-1,7-dicarboxamide (52);

[0099] cis,cis-N,N',N”-(cyclohexane-1,3,5-triyl)tris(6-(bisdodecylamino)hexamamide)(56).

[0100] Compounds of formulas I and II are obtained by using cyclohexane precursors substituted with Z groups at positions 1, 3, and 5, and cyclohexane precursors substituted with X linker groups at positions 1, 1, and 3, or 1, 3, and 5, with the general formula X′-YN(R) 1 The tertiary amine of 2 is prepared by the corresponding reaction, wherein X′ is the precursor group of the linker X. Preferably, X′ is -NH2 or an activated carboxyl group. General formula X′-YN(R) 1 Tertiary amines of 2 can be prepared by reactions and procedures known to those skilled in the art, and some suitable amines are also commercially available.

[0101] In one embodiment, the compound of formula I is preferably obtained by using the compound of formula III.

[0102]

[0103] Where A is H, halogen, or benzyl.

[0104] It is prepared by reacting with a diamine of formula IV.

[0105]

[0106] Optionally in the presence of an alcohol selected from MeOH and BnOH, and optionally in the presence of a pyrrolidine, a condensing agent, and / or a base, optionally after or before hydrogenolysis. In formulas III and IV, R 1 Y and Z are as described in Equation I above.

[0107] In more specific instances, compounds of formula II are preferably prepared by reacting a compound of formula III (where A is hydrogen) with a diamine of formula IV in the presence of a condensing agent and a base, or by reacting a compound of formula III (where A is a halogen) with a diamine of formula IV in the presence of a base. In formulas III and IV, R... 1 Y and Z are as described in Equation II above.

[0108] Another object of the present invention is a transfection agent comprising at least one lipid of general formula I or II and at least one auxiliary lipid. The transfection agent can be prepared in solution form by dissolving and mixing the components, or it can be prepared in particulate form using techniques used in conventional nanoparticle technology (e.g., microfluidic mixing). Particulates are generally understood to mean nanoparticles with a size in the range of 1 to 500 nm. Preferably, the size of the nanoparticles is in the range of 30 to 250 nm, and even more preferably in the range of 40 to 150 nm.

[0109] In one embodiment, the transfection agent contains 10 to 50 mol.% of at least one lipid of formula I and 50 to 90 mol.% of at least one auxiliary lipid. Preferably, the transfection agent contains 15 to 40 mol.% of at least one lipid of formula I and 60 to 85 mol.% of at least one auxiliary lipid. In some preferred embodiments, the transfection agent contains 15 to 40 mol.% of at least one lipid of formula I, 30 to 55 mol.% of cholesterol, and 20 to 50 mol.% of at least one other auxiliary lipid.

[0110] In a particularly preferred embodiment, the transfection agent contains 15 to 40 mol.% of at least one lipid of general formula I, 30 to 55 mol.% of cholesterol, 20 to 45 mol.% of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, and 0.5 to 5 mol.% of 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000.

[0111] The present invention also relates to a transfection particle comprising at least one lipid of formula I, at least one nucleic acid and / or a portion thereof and / or a nucleic acid derivative thereof, and preferably further comprising at least one auxiliary lipid. For example, the transfection particle can be prepared by mixing a solution of a lipid of formula I (optionally containing an auxiliary lipid) with a solution of a nucleic acid and / or a portion thereof and / or a nucleic acid derivative thereof. The mixing can be performed using techniques used in conventional nanoparticle technology, such as microfluidic mixing.

[0112] The weight ratio of the total amount of nucleic acid and / or portions thereof and / or nucleic acid derivatives in the transfection particles to the total amount of lipids and accessory lipids of general formula I is preferably in the range of 1:2 to 1:500, more preferably in the range of 1:5 to 1:100. Specifically, and for illustration, in the particles prepared in the examples below, this ratio is about 1:9 for mRNA and about 1:68 for siRNA.

[0113] Transfected particles are typically nanoparticles, which are generally understood to mean particles with a size ranging from 1 to 500 nm. Typically, the size of transfected nanoparticles is in the range of 50 to 250 nm, more preferably in the range of 40 to 150 nm.

[0114] The structure of the transfected particles was observed using low-temperature transmission electron microscopy, and the results showed that the transfected particles were dense layered lipid nanoparticles containing nucleic acids.

[0115] The auxiliary lipids in transfection reagents and transfection particles are mainly neutral lipids, sterols, or lipid conjugates of lipids and hydrophilic polymers.

[0116] Neutral lipids have zero net charge under physiological conditions and can exist in either an uncharged or electrically neutral zwitterionic form. Neutral lipids can be selected from, for example, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), and 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine-N-(Cyanine 5).

[0117] The sterol may be selected, for example, from cholesterol, β-sitosterol, stigmasterol, campesterol, fucosterol, alfalfa sterol, coprosterol, rapeseed sterol, ergosterol, and 9,11-dehydroergosterol. Preferably, the sterol is cholesterol.

[0118] Lipid conjugates having hydrophilic polymers comprise a lipid moiety and a polymer moiety, such as poly(ethylene glycol), poly(2-ethyl-2-oxazoline), poly(2-methyl-2-oxazoline), poly(glycerol), and poly(sarcosine). Preferably, the polymer moiety consists of poly(ethylene glycol) with a molecular weight ranging from about 500 to about 10,000 Da, more preferably from about 1,000 to about 5,000 Da. Lipid conjugates having hydrophilic polymers may, for example, be selected from 1,2-dimyristoyl-rac-glycerol-3-methoxypoly(ethylene glycol)-2000, 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine-poly(ethylene glycol)-2000, 1,2-distearatel-sn-glycerol-3-phosphate ethanolamine-poly(ethylene glycol)-2000, 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-poly(ethylene glycol)-2000, and similar conjugates. The lipid conjugate containing the hydrophilic polymer is preferably 1,2-dimyristoyl-rac-glycerol-3-methoxypoly(ethylene glycol)-2000.

[0119] Nucleic acids, or portions thereof, contain one or more nucleotides and / or deoxynucleotides. Nucleic acids, or portions thereof, can be therapeutic, diagnostic, or preventative agents, or can provide a marker for the cells or tissues they are transfected with. Compounds of Formula I therefore have primary therapeutic or biotechnological uses.

[0120] The term “nucleic acid or a portion thereof” should be understood to mean, preferably, one or more types of nucleic acids selected from: oligonucleotides (1-100 nucleotides, e.g., aptamers), cyclic dinucleotides (e.g., 2',3'-cGAMP), antisense oligonucleotides, deoxyribonucleic acid (single-stranded DNA, double-stranded DNA, cDNA, plasmid DNA encoding one or more genes), ribonucleic acid, typically messenger RNA (mRNA), transfer RNA (tRNA), small interfering RNA (siRNA), double-stranded RNA, microRNA (miRNA), piwi-RNA (piRNA), antisense RNA (asRNA), guide RNA (gRNA) for the CRISPR system, and combinations thereof (typically, e.g., gRNA and mRNA encoding Cas9 nuclease, Cas13a / C2c2 and Cas13b, or similar nucleases, suitable for CRISPR, CRISPRi and other variants, and subsequent modifications to the host cell or tissue genome or host cell or tissue transcriptome). Furthermore, all nucleic acids (NAs) disclosed herein can be formed or modified with synthetic base analogues, for example, to increase their stability in biological systems. Synthesized NA analogs particularly involve the following substitutions: 2'-O-methyl, 2'-O-methoxy-ethyl, 2'-fluorine, a methylene bridge between the 2'-oxygen and 4'-carbon of the pentose ring (so-called locked nucleic acid), phosphorylation at the 5' and / or 3' ends of the chain, boron phosphonate, or thiophosphate.

[0121] The present invention also includes the use of lipids or transfection agents or transfection particles of Formula I for in vitro transfection of cells or tissues with nucleic acids and / or portions thereof and / or nucleic acid derivatives. Additionally, the present invention includes the use of lipids or transfection particles of Formula I for in vivo transfection of cells or tissues with nucleic acids and / or portions thereof and / or nucleic acid derivatives (excluding transfection of human embryos for industrial or commercial use and excluding modified human lines).

[0122] Transfection particles containing lipids of general formula I can be used for many biological applications in basic research, especially for transfecting cell cultures or animals to deliver active nucleic acids and subsequently silence or activate one or more chromosomal genes, genome editing (gene excision, gene insertion, or mutation introduction) or transcriptome editing, or to express a given protein encoded by a nucleic acid inserted via transfection particles, the so-called "trans".

[0123] In veterinary and human medicine, transfection particles containing lipids of general formula I are preferred for therapeutic or preventative purposes. Particles containing therapeutic nucleic acids can be administered to animals or humans to silence or activate one or more chromosomal genes, silence or activate immunogens, inhibit or activate signal transduction pathways, edit the genome (gene excision, gene insertion, or mutation introduction) or transcriptome, or enable the expression of one or more proteins encoded by nucleic acids.

[0124] The present invention also provides the use of lipids or transfectants or transfected particles of general formula I as pharmaceuticals, particularly for gene therapy. Specifically, they are suitable for treating malignant tumors and / or genetic disorders.

[0125] Lipids or transfectants or transfecting particles of general formula (I) according to the present invention are also suitable for use as preventive vaccines, preferably for the prevention of infectious diseases.

[0126] Lipids or transfected particles of general formula I can be formulated in the form of pharmaceutically acceptable excipients for therapeutic, cosmetic, or biotechnological uses. The formulation can be in liquid or solid form. Liquid forms include, for example, solutions, suspensions, dispersants, gels, and ointments suitable for injection or oral administration. Solid forms include, for example, capsules, tablets, coated tablets, powders, suppositories, and other forms.

[0127] Liquid formulations can be nebulized with an inert gas. Nebulized suspensions can be inhaled directly or from a nebulizer, which can be attached to a mask or ventilator. Solid forms can be administered using a dry powder inhaler. Suspensions or dry powder formulations can be administered orally or nasally from a suitable device.

[0128] For application to the skin or mucous membranes, the formulation can also be prepared in the form of creams, gels, ointments, pastes, aromatic resins, liquids, etc., and can be applied directly to the site of action.

[0129] Pharmaceutically acceptable excipients include solvents, solubility controls, pH adjusters, carriers, fillers, binders, flow aids, disintegrants, preservatives, adsorbents, viscosity controls, and agents that affect the sensory properties of the formulation (e.g., taste, odor, or color).

[0130] Furthermore, lipids or transfectants or transfected particles of general formula I are preferably used for cosmetic purposes to deliver active substances to the site of action. Transfected particles containing active substances can be formulated into creams, gels, ointments, pastes, balsams, liquids, etc., and used as cosmetics, hair care products, or personal hygiene products. Attached Figure Description

[0131] Figure 1 Synthetic schemes for lipids 3 and 7.

[0132] Figure 2 Synthetic schemes for lipids 8 and 11.

[0133] Figure 3 Synthesis scheme for lipid 15.

[0134] Figure 4 Synthetic protocols for lipids 19 and 23.

[0135] Figure 5 Synthetic schemes for lipids 27 and 31.

[0136] Figure 6 Synthetic protocols for lipids 32, 33, 35, and 36.

[0137] Figure 7 Synthetic protocols for precursors 39 and 44 and lipids 40-42.

[0138] Figure 8 Synthetic protocols for lipids 47, 48, 50, and 52.

[0139] Figure 9 Synthesis scheme for lipid 56.

[0140] Figure 10 Histological analysis revealed the functional transmission of mRNA-LNP(B40) in mouse liver compared to the baseline LNP(B45).

[0141] Figure 11Functional delivery of mRNA-LNP(B40) in mouse liver compared to the baseline LNP(B45) was determined by PCR amplification analysis of genomic DNA. Lanes labeled 1 to 4 represent B40, and lanes labeled 5 to 8 represent B45. Detailed Implementation

[0142] Example

[0143] List of abbreviations:

[0144] eq. equivalent

[0145] R f Retention factor

[0146] TLC (Thin Layer Chromatography)

[0147] RVE Rotary Vacuum Evaporator

[0148] rt room temperature

[0149] v / v volume / volume

[0150] br s wide signal

[0151] s singlet

[0152] d Double peak

[0153] t triple peak

[0154] m multiplet

[0155] dd double peak

[0156] J interaction constant

[0157] δ chemical shift

[0158] HRMS high-resolution mass spectrometry

[0159] EI electron ionization

[0160] ESI Electrospray Ionization

[0161] MALDI matrix-assisted laser desorption / ionization

[0162] GC-MS (Gas Chromatography-Mass Spectrometry)

[0163] IR infrared spectrum

[0164] NMR (Nuclear Magnetic Resonance)

[0165] CE5 95:5 (v / v) Cyclohexane-ethyl acetate mixture

[0166] CE20 80:20 (v / v) Cyclohexane-ethyl acetate mixture

[0167] CE50 50:50 (v / v) Cyclohexane-ethyl acetate mixture

[0168] D1 75:22:3 (v / v / v) Dichloromethane-Methanol-25% Aqueous NH3 Mixture

[0169] D2 175:22:3 (v / v / v) dichloromethane-methanol-25% aqueous NH3 mixture

[0170] D3 275:22:3 (v / v / v) dichloromethane-methanol-25% aqueous NH3 mixture

[0171] D4 375:22:3 (v / v / v) dichloromethane-methanol-25% aqueous NH3 mixture

[0172] TFA (trifluoroacetic acid)

[0173] DIPEA N,N-Diisopropylethylamine

[0174] DMF N,N-dimethylformamide

[0175] DCM dichloromethane

[0176] ACN Acetonitrile

[0177] DIC diisopropylcarbodiimide

[0178] DMAP 4-Dimethylaminopyridine

[0179] PyBroP Tripyrrolylphosphonium bromide hexafluorophosphate

[0180] TCE 1,1,2,2-Tetrachloroethane

[0181] LNP lipid nanoparticles

[0182] NA nucleic acid

[0183] DNA deoxyribonucleic acid

[0184] RNA (ribonucleic acid)

[0185] mRNA messenger RNA

[0186] siRNA (small interfering RNA)

[0187] tRNA transfection RNA

[0188] miRNA

[0189] ssDNA / RNA Single-stranded DNA / RNA

[0190] dsDNA / RNA Double-stranded DNA / RNA

[0191] DMG-PEG 2000 1,2-Dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000

[0192] DOPE 1,2-Dioleoyl-sn-glycerol-3-phosphate ethanolamine

[0193] DOPC 1,2-Dioleoyl-sn-glycerol-3-phosphocholine

[0194] DSPC 1,2-Distearyl-sn-glycerol-3-phosphocholine

[0195] DOPE-Cy5 1,2-Dioleoyl-sn-glycerol-3-phosphate ethanolamine-N-(Cyanine 5)

[0196] Lip2000 2000 (Invitrogen)

[0197] h hours

[0198] Example 1

[0199] N 1 N 1 -Didodecylethyl-1,6-diamine 2

[0200] N is added to a 500ml round-bottom flask equipped with a calcium chloride drying tube and a magnetic stirrer. 1A solution of 1,6-diaminohexane (5.0 g, 23.11 mmol) in DCM (100 mL) was cooled to 0 °C in an ice bath. Dodecyl aldehyde (15.38 mL, 69.34 mmol, 3 eq.) was added with vigorous stirring, followed by the addition of sodium triacetoxyborohydride (14.70 g, 69.34 mmol, 3 eq.) in three fractions over 10 minutes. The cooling bath was removed, and the reaction mixture was stirred at room temperature for 2 hours. The reaction progress was monitored by TLC (using ninhydrin) on a TLC plate presaturated with ammonia using an 80:20 (v / v) hexane-ethyl acetate mobile phase. After the reaction was complete, an aqueous solution of NaOH (1 M, 200 mL) was added, the reaction mixture was stirred for 15 minutes, then poured into a separatory funnel and diluted with water (300 mL). The product was extracted with DCM (300 ml, 2 x 50 ml), the combined organic phases were washed with brine (50 ml), dried over anhydrous sodium sulfate, filtered through S2 glass frit, and the solvent was evaporated in RVE. The dark oily residue was purified by silica gel column chromatography using a linear gradient (10-30%) of ethyl acetate in hexane. Amine 1 (3.67 g, 28.7%) was given as a pale yellow oil.

[0201] Trifluoroacetic acid (10 ml) was added to a solution of compound 1 in DCM (10 ml), and the mixture was cooled to 0 °C with stirring in an ice bath. The reaction mixture was then left to stand at 0 °C for 3 h. The solution was then poured into a 1 L separatory flask, diluted with 20% Na2CO3 (300 ml) aqueous solution, and the product was extracted with DCM (250 ml, 2 x 50 ml). The combined organic phases were washed with brine (100 ml), dried over anhydrous sodium sulfate, filtered through S2 glass frit, and the solvent was evaporated in RVE. The crude product was purified by silica gel column chromatography using a linear gradient (0-70%) of D1 in DCM. Diamine 2 was given as a pale yellow oil (2.17 g, 72.2% yield; R2 in mobile phase D2 on a TLC plate pre-saturated with ammonia). f 0.31 (detected using ninhydrin).

[0202] 1 H NMR (600MHz, CDCl3): δ=2.73, 2.65, 2.57, 1.56, 1.52, 1.51, 1.36, 1.31, 1.28, 1.25-1.29, 1.24, 0.87ppm. 13C NMR (150.9MHz, CDCl3): δ=53.51,53.20,41.62,32.40,31.89,29.63,29.61 ,29.57,29.44,29.32,27.39,27.09,26.53,25.65,25.02,22.66,14.10ppm. IR(membrane):ν max / cm -1 =3374w and 3294w(νNH2),2797m(ν s N- CH 2),2956s(ν as CH3),2924vs(ν as CH2),2853s(ν s CH2), 1467m and 1455m, sh(β) s CH2 and δ as CH3), 1378w and 1367w (δ) s CH3), 721m(β) as CH2). HRMS(ESI): m / zC 30 H 65 N2[M+H] + Calculated value: 453.51423; Measured value: 453.51340.

[0203] cis, cis-N 1 N 3 N 5 -tris(6-(bis(dodecylamino)hexyl)cyclohexane-1,3,5-tricarboxamide 3

[0204] DMF (2 μl) and thionyl chloride (300 μl) were added to cis,cis-1,3,5-cyclohexanetricarboxylic acid (17 mg, 0.079 mmol), and the suspension was stirred in a sealed vial at 70 °C for 30 minutes, allowing the reaction mixture to gradually clarify into a homogeneous, colorless solution. Excess SOCl2 was removed by a stream of dry nitrogen at 70 °C, and the residue was dried under vacuum (10 minutes) and cooled to room temperature. N 1 N 1A solution of bis(dodecylhexane-1,6-diamine) 2 (142 mg, 0.315 mmol, 4 eq.) and DIPEA (137 μl, 0.786 mmol, 10 eq.) in a mixture of DCM (1.5 mL) and DMF (0.5 mL) was added via a syringe through a diaphragm, and the reaction mixture was stirred for 10 min. Subsequently, the reaction mixture was adsorbed onto chromatographic silica gel (10 g), and the solvent was removed in RVE. The residue was purified by silica gel column chromatography (40 g) using a linear gradient (0-55%) of D1 in DCM. Lipid 3 (81 mg, 67.7% yield; R) was obtained as a pale yellow semi-solid. f 0.36 in mobile phase D2, detected with ninhydrin. 1 H NMR (600MHz, CDCl3): δ=7.80, 3.20, 3.02-2.96, 2.60, 2.07, 1.81-1.74, 1.37, 1.325, 1.28-1.23, 0.87ppm. 13 C NMR (150.9MHz, CDCl3): δ=175.90,52.85,52.22,42.83,39.42,31.87,31.66,29.58 ,29.48,29.43,29.30,29.08,26.84,26.18,26.00,23.50,23.16,22.65,14.09ppm. IR(CCl4):ν max / cm -1 =3293m(νNH),1640s(amide I) and 1550m(amide II),2964s,sh(ν as CH3), 2871m, sh(ν) s CH3),2927vs(ν as CH2), 2856s, sh(ν) s CH2), 1468m and 1460m, 1445w (β) s CH2 and δ as CH3), 1378w(δ) s CH3), 721w(β) as CH2 and δ as CH2); HRMS (MALDI): m / zC 99 H 199 N6O3[M+H] + Calculated value: 1520.5598; Measured value: 1520.5598.

[0205] Example 2

[0206] N1 N 1 -Di((hexyloxycarbonyl)butyl)hexane-1,6-diamine

[0207] 1-Hexanol (2.48 mL, 19.89 mmol, 1.2 eq), DMAP (61 mg, 0.50 mmol, 0.03 eq.), and DIC (3.37 mL, 21.54 mmol, 1.3 eq.) were added to a solution of 5-bromopentanoic acid (3.00 g, 16.57 mmol) in DCM (60 mL), and the reaction mixture was stirred at room temperature for 1 hour. Subsequently, the reaction mixture was adsorbed onto chromatographic silica gel (16 g), the solvent was removed in RVE, and the residue was purified by silica gel column chromatography (80 g) using a linear gradient (0-10%) of ethyl acetate in cyclohexane. Hexyl 5-bromopentanoate 4 (3.938 g, 89.6% yield; R ester in mobile phase CE5) was given as a colorless liquid. f 0.31, for detecting KMnO4).

[0208] Hexyl 5-bromopentanoate 4 (1.53 g, 5.78 mmol, 2.5 eq.) and anhydrous K2CO3 (3.19 g, 23.11 mmol, 10 eq.) were added to N. 1 A solution of 1,6-tert-butyloxycarbonyl-1,6-diaminohexane (0.50 g, 2.31 mmol) in anhydrous ACN (10 mL) was prepared, and the reaction mixture was vigorously stirred at 35 °C for 2 days. Subsequently, the reaction mixture was adsorbed onto silica gel (16 g), the solvent was removed in RVE, and the residue was purified by silica gel column chromatography (40 g) using a linear gradient (0-100%) of ethyl acetate in cyclohexane. Amine 5 (1.080 g, 79.9% yield; R chromatogram on a TLC plate presaturated with ammonia and mobile phase CE50) was obtained. f 0.31 (detected using ninhydrin).

[0209] Trifluoroacetic acid (4 ml) was added to a solution of compound 5 in DCM (4 ml), cooled to 0 °C with stirring in an ice bath, and the reaction mixture was left at 0 °C for 1 hour. The solution was then poured into a 500 ml separatory flask, diluted with 20% aqueous NaHCO3 (200 ml), and extracted with DCM (150 ml, 2 x 50 ml). The combined organic phases were washed with brine (50 ml), dried over anhydrous sodium sulfate, filtered through S2 glass frit, adsorbed onto silica gel (16 g), the solvent was removed in RVE, and the residue was purified by silica gel column chromatography (40 g) using a linear gradient (0-80%) in DCM with D1. Diamine 6 (0.788 g, 86.9% yield; R in mobile phase D2) was obtained as a pale yellow oil. f0.14 (detected using ninhydrin). 1 H NMR (600MHz, CDCl3): δ = 4.05, 3.63, 3.21, 3.15, 2.79, 2.68, 2.60, 2.51, 2.42, 2.32, 1.61, 1.50, 1.36-1.28, 0.88ppm. 13 C NMR (150.9MHz, CDCl3): δ=173.62,64.52,53.68,53.29,41.22,40.26,34.03,31.41,28.58,25.75,25.57,22.80,22.51,13.98ppm. HRMS(ESI):m / z C 28 H 57 O4N2[M+H] + Calculated value: 485.43128; Measured value: 485.43052.

[0210] cis, cis-N 1 N 3 N 5 -tris(6-(di((hexyloxycarbonyl)butyl)amino)hexyl)cyclohexane-1,3,5-tricarboxamide7

[0211] Lipid 7 from cis,cis-1,3,5-cyclohexanetricarboxylic acid (21 mg, 0.097 mmol), N 1 N 1 -Di((hexyloxycarbonyl)butyl)hexane-1,6-diamine 6 (188 mg, 389 mmol, 4 eq.) and DIPEA (169 μl, 0.971 mmol, 10 eq.) were prepared according to the procedure described for lipid 3 in Example 1. Lipid 7 (55 mg, 35%; R) was obtained as a pale yellow semi-solid. f 0.42 in mobile phase D2, detected with ninhydrin. 1 H NMR (600MHz, CDCl3): δ=6.23, 4.05, 3.205, 2.65, 2.34, 2.30, 2.085, 1.63-1.60, 1.49, 1.33-1.29, 0.88ppm. 13 C NMR (150.9MHz, CDCl3): δ=174.35,173.37,64.60,53.35,52.88,43.98,39.12,3 3.79,31.94,31.40,29.21,28.57,26.62,26.33,25.56,22.58,22.51,13.98ppm. IR(CCl4):ν max / cm -1=1736vs(ν s C=O), 1173m, 1075w(ν) s CO), 3293m(νNH), 1640s(amide I) and 1551m(amide II), 2955s(νNH) as CH3),2933vs(ν as CH2), 2875m, sh(ν) s CH3), 2860m(ν) s CH2), 1467m and 1460m (β) s CH2 and δ as CH3), 1379w(δ) s CH3), 724w(β) as CH2 and γ as CH2). HRMS (MALDI): m / z C 93 H 175 N6O 15 [M+H] + Calculated value: 1616.3110; Measured value: 1616.3095.

[0212] Example 3

[0213] cis, cis-N 1 N 3 N 5 -tris(6-(bis(dodecylamino)hexyl)-1,3,5-trimethylcyclohexane-1,3,5-tricarboxamide8

[0214] According to the procedure described in Example 1 for lipids 3, cis,cis-1,3,5-trimethyl-1,3,5-cyclohexanetricarboxylic acid (20 mg, 0.077 mmol), N 1 N 1 Lipid 8 was prepared by reacting 1,6-diamine 2 (140 mg, 310 mmol, 4 eq.) with di(dodecyl)hexane-1,6-diamine 2 (140 mg, 310 mmol, 4 eq.) and DIPEA (135 μl, 0.774 mmol, 10 eq.). Lipid 8 (64 mg, 53%; R in mobile phase D2) was obtained as a slightly yellow solid oil. f 0.43 (detected using ninhydrin). IR (CCl4): ν max / cm -1 =3288w,br(νNH),1651m(amide I),1585w,br,sh and1559m,br(amide II),2956s(νNH) as CH3),2927vs(ν as CH2), 2878m, sh(ν)s CH3), 2855s(ν) s CH2), 1468m and 1459m, sh, 1448m, sh(β) s CH2 and δ as CH3), 1378w(δ) s CH3); 721w(β) as CH2 and γ as CH2). HRMS (MALDI): m / z C 102 H 205 N6O3[M+H] + Calculated value: 1562.6068; Measured value: 1562.6011.

[0215] Example 4

[0216] N 1 N 1 -Didodecylpropane-1,3-diamine 10

[0217] Amine 9 is composed of N 1 -tert-butyloxycarbonyl-1,3-diaminopropane (6.0 g, 34.43 mmol), n-dodecylaldehyde (22.91 ml, 103.30 mmol, 3 eq.), and sodium triacetoxyborohydride (21.89 g, 103.3 mmol, 3 eq.) were prepared according to the procedure described for compound 1 in the examples. Amine 9 was obtained as a pale yellow oil (7.72 g, 43.9%).

[0218] Deprotection of amine 9 was performed according to the procedure described for compound 2 in Example 1; diamine 10 (4.26 g, 68.6%) was obtained as a pale yellow oil; R in mobile phase D2 on a TLC plate pre-saturated with ammonia. f 0.35 (detected using ninhydrin). 1 H NMR (600MHz, CDCl3): δ=3.07, 2.70, 2.50, 1.81, 1.46, 1.28, 1.26, 1.25-1.29, 1.24, 0.87ppm. 13 C NMR (150.9MHz, CDCl3): δ=53.70,53.30,41.18,31.90,29.64,29.62,29.60,29.58,29.48,29.33,27.42,25.71,23.87,22.67,14.10ppm. IR(membrane):ν max / cm -1 =3361w and 3274w(νNH2),2803m(ν s N- CH 2),2954s(ν as CH3),2924vs(ν as CH2),2853s(ν s CH2), 1467m and 1456m, sh(β) s CH2 and δ as CH3), 1378w and 1364w (δ) s CH3), 720m(β) as CH2). HRMS(ESI): m / zC 27 H 59 N2[M+H] + Calculated value: 411.46728; Measured value: 411.46652.

[0219] cis, cis-N 1 N 3 N 5 -tris(3-(bis(dodecylamino)propyl)-1,3,5-trimethylcyclohexane-1,3,5-tricarboxamide 11

[0220] According to the procedure described in Example 1 for lipids 3, cis,cis-1,3,5-trimethyl-1,3,5-cyclohexanetricarboxylic acid (20 mg, 0.077 mmol), N 1 N 1 Lipid 11 was prepared by reacting 10 (127 mg, 310 mmol, 4 eq.) with bis(dodecyl)propane-1,3-diamine 10 (127 mg, 310 mmol, 4 eq.) and DIPEA (135 μl, 0.774 mmol, 10 eq.). Lipid 11 (41 mg, 37% yield; R in mobile phase D2) was obtained as a slightly yellow solid oil. f 0.36 (detected using ninhydrin). IR (CCl4): ν max / cm -1 =3303w,vbr(νNH),1679s,sh(amide I),1513w,br,sh(amide II),2956s(νNH) as CH3),2927vs(ν as CH2), 2878m, sh(ν) s CH3), 2855s(ν) s CH2), 1464m, sh(β) s CH2 and δ as CH3), 1380w(δ) s CH3); 721w(β) as CH2 and γ as CH2). HRMS (MALDI): m / z C93 H 187 N6O3[M+H] + Calculated value: 1436.4665; Measured value: 1436.4629.

[0221] Example 5

[0222] Linolealdehyde 12

[0223] Dess-Martin periodane (4.45 g, 10.49 mmol, 1.3 eq.) was added to linoleyl alcohol (2.50 mL, 8.07 mmol) in a solution of DCM (120 mL) cooled to 0 °C in an ice bath, and the mixture was stirred at 0 °C for 4 hours. The reaction mixture was then quenched by adding sodium thiosulfate solution (20 g Na₂S₂O₃·5H₂O / 100 mL H₂O) and saturated sodium bicarbonate aqueous solution (50 mL), and stirred at room temperature for 1 hour until the initial emulsion became clear. The solution was poured into a 1000 mL separatory flask, diluted with water (150 mL), and the product was extracted with DCM (150 mL, 2 x 50 mL). The combined organic phases were washed with brine (150 mL), dried over anhydrous sodium sulfate, filtered through S₂ glass frit, and the solvent was evaporated on an RVE. The crude product was purified by silica gel column chromatography (isocratic conditions, 5% ethyl acetate in cyclohexane). Aldehyde 12 (1.271 g, 59.6% yield; R) was obtained as a colorless oil. f 0.36 in mobile phase CE5, detected with KMnO4.

[0224] N 1 N 1 -Di((9Z,12Z)-octadec-9,12-terpen-1-yl)hexane-1,6-diamine-14

[0225] Amine 13 is composed of N 1 -tert-butyloxycarbonyl-1,6-diaminohexane (0.345 g, 1.59 mmol), aldehyde 12 (1.27 g, 4.78 mmol, 3 eq.), and sodium triacetoxyborohydride (1.01 g, 4.78 mmol, 3 eq.) were prepared according to the procedure described for compound 1 in the examples. Amine 13 (1.08 g, 94.9% yield; R in CE20 mobile phase) was obtained as a pale yellow oil. f 0.18 (detected using ninhydrin).

[0226] Following the procedure described for compound 2 in Example 1, amine 13 was deprotected in a mixture of TFA (4 ml) and DCM (5 ml); diamine 14 (0.594 g, 64.0%) was obtained as a pale yellow oil in mobile phase D2.f 0.13 (detected using ninhydrin). 1 H NMR (600MHz, CDCl3): δ=5.30-5.40,2.765,2.73,2.67,2.59,2.04,1.51,1.385,1.37,1.34,1.295,1.29,1.28-1.34,1.28,0.88ppm. 13 C NMR (150.9MHz, CDCl3): δ=130.19,130.06,127.99,127.89,53.49,53.45,41.67,32.52, 31.50,29.62,29.46,29.20-29.48,27.37,27.20,27.18,26.52,25.61,22.56,14.06ppm. IR(CCl4):ν max / cm -1 =3011s(ν as =CH); 1646-1673m(νC=C); 3455w(ν as NH2); 3394 (ν) s NH2); 1620w(β) s NH2); 1087m(νC-NH2); 2957s,sh(ν as CH3); 2928 vs (ν as CH2); 2873s,sh(ν) s CH3); 2856 vs (ν s CH2); 2801m(ν) s N- CH 2); 1467m and 1457m, sh(β) s CH2 and δ as CH3); 1378m(δ) s CH3); 721m(β) as and γ as CH2). HRMS:m / zC 42 H 81 N2[M+H] + Calculated value: 613.63943; Measured value: 613.63899.

[0227] cis, cis-N 1 N 3 N 5 -tris(6-(di((9Z,12Z)-octadecyl-9,12-dien-1-yl)amino)hexyl)-1,3,5-trimethylcyclohexane-1,3,5-tricarboxamide 15

[0228] According to the procedure described in lipid 3 of the example, cis,cis-1,3,5-trimethyl-1,3,5-cyclohexanetricarboxylic acid (17 mg, 0.066 mmol), N 1 N 1 Lipid 15 was prepared in bis((9Z,12Z)-octadec-9,12-dien-1-yl)hexane-1,6-diamine 14 (161 mg, 273 mmol, 4 eq.) and DIPEA (115 μl, 0.658 mmol, 10 eq.). Lipid 15 (86 mg, 64% yield; R) was obtained as a pale yellow solid. f 0.36 in mobile phase D2 (detected with ninhydrin). IR (CCl4): ν max / cm -1 =3348w,br,sh and 3302w,br(νNH), 1656m and 1635m,sh(amide I + νC = C), 1565w,sh and 1557w,br(amide II), 3011m(νNH) as =CH),2956s(ν as CH3),2929vs(ν as CH2), 2875m, sh(ν) s CH3), 2856s(ν) s CH2), 1467m and 1450m, sh, (β) s CH2 and δ as CH3), 1379w(δ) s CH3); 720w(β) as CH2+γ as CH2+γ=CH). HRMS(MALDI):m / z C 138 H 253 N6O3[M+H] + Calculated value: 2042.9829; Measured value: 2042.9861.

[0229] Example 6

[0230] Compound 18

[0231] DIC (4.41 mL, 28.7 mmol, 1.6 eq.) and DMAP (88 mg, 0.72 mmol, 0.04 eq.) were added to a solution of 6-bromohexanoic acid (3.50 g, 17.9 mmol) and octane-2-ol (3.51 g, 26.9 mmol, 1.5 eq.) in DCM (30 mL), and the mixture was stirred overnight at room temperature. The reaction mixture was then adsorbed onto silica (16 g), and the solvent was evaporated under vacuum. The crude product was purified by rapid silica chromatography (80 g, eluted with a linear gradient of ethyl acetate in cyclohexane, 0–10%) to give compound 16 (4.02 g, 77%; R) as a colorless oil. f 0.41 in CE5 (visualized via KMnO4).

[0232] Bromoester 16 (4.00 g, 13.1 mmol, 2.6 eq.) and potassium carbonate (7.23 g, 52.3 mmol, 10 eq.) were added to a solution of N-Boc-1,6-hexanediamine (1.13 g, 5.23 mmol, 1 eq.) in anhydrous acetonitrile (10 mL), and the mixture was stirred at 40 °C for 3 days. The reaction mixture was then adsorbed onto silica (16 g), and the solvent was evaporated under vacuum. The crude product was purified by rapid silica chromatography (80 g, eluted with a linear gradient of ethyl acetate in cyclohexane, 0-100%) to give compound 17 (2.80 g, 80%; R on CE50 of an NH3-pretreated TLC plate) as a pale yellow oil. f 0.58 (visualized via ninhydrin).

[0233] Hydrochloric acid (4 mL, 4 M) from dioxane was added to a solution of compound 17 (2.75 g, 4.11 mmol) in anhydrous DCM (4 mL), and the reaction mixture was stirred at room temperature for 1 hour. The solution was then poured into a 500 mL separatory funnel, diluted with saturated NaHCO3 aqueous solution (100 mL), and the product was extracted with diethyl ether (100 mL, 2 x 50 mL). The combined organic phases were washed with brine (75 mL), dried over anhydrous sodium sulfate, filtered through S2 sintered glass, and the solvent was evaporated in an RVE. The crude product was purified by silica gel column chromatography using a linear gradient (0–70%) of D1 in DCM. Amine 18 (1.74 g, 74%; R2 on NH3- pretreated TLC plates) was obtained as a pale yellow oil. f 0.25 (visualized via ninhydrin). 1H NMR (600MHz, CDCl3): δ=4.90-4.77(m), 2.72-2.62(m), 2.39-2.29(m), 2.21(t, J=7.5 Hz),1.64-1.44(m),1.47-1.32(m),1.30-1.17(m),1.15-1.08(m),0.86-0.78(m)ppm. 13 C NMR (150.9MHz, CDCl3): δ=173.42,70.83,53.89,41.87,35.98,31.77,29.13,27.41,27.17,26.84,26.69,25.40,25.09,22.60,20.04,14.09ppm. HRMS(ESI):m / zC 34 H 68 N₂O₄[M+H] + Calculated value: 569.5252; Measured value: 569.5247.

[0234] Hexa(octane-2-yl)cis,cis-6,6',6”,6”',6””,6””-((((cyclohexane-1,3,5-tricarbonyl)tri(azanediyl))tri(hexane-6,1-diyl))tri(azanetriyl))hexahexanoate 19

[0235] Thionyl chloride (700 μL) and DMF (6 μL) were added to cis,cis-cyclohexane-1,3,5-tricarboxylic acid (80 mg, 370 μmol), and the suspension was stirred in a sealed vial at 70 °C for 3 hours; during this period, the suspension became a clear solution. Excess SOCl2 was removed at 70 °C with a stream of dry nitrogen, and the residue was dried under vacuum at room temperature (20 min). The obtained solid was dissolved under argon in anhydrous TCE (2.5 mL) and DIPEA (645 μL, 3.70 mmol). Then, amine 18 (1.05 g, 1.85 mmol, 5 eq.) was added to the solution in anhydrous TCE (2.5 mL), and the reaction mixture was stirred at room temperature for 40 min. The reaction mixture was then evaporated under vacuum, redissolved in DCM, adsorbed onto silica (10 g), and the DCM was removed under vacuum. The crude product was purified by rapid silica chromatography (eluting with a linear gradient of D1 in DCM, 5-35%) to give target compound 19 (489 mg, 71%) as a yellow waxy semi-solid. 1H NMR (600MHz, CDCl3): δ = 5.75, 4.88, 3.21, 2.42, 2.27, 2.22, 2.11, 1.62, 1.58, 1.56, 1.47, 1.46, 1.30, 1.28, 1.27, 1.26, 1.19, 0.87ppm. 13 C NMR(150.9MHz, CDCl3):173.88,173.39,70.82,53.87,53.77,44.10,39.44,35.93,34 .67,31.86,31.73,29.51,29.09,27.08,26.74,25.36,25.01,22.56,20.01,14.06ppm. HRMS(MALDI):m / zC 111 H 211 N6O 15 [M+H] + Calculated value: 1868.5927; Measured value: 1868.5906.

[0236] Example 7

[0237] Compound 22

[0238] DIC (3.60 mL, 23.0 mmol, 1.6 eq.) and DMAP (70 mg, 0.58 mmol, 0.04 eq.) were added to a solution of 7-bromoheptanoic acid (3.00 g, 14.4 mmol) and 3-methylhexane-1-ol (2.50 g, 21.5 mmol, 1.5 eq.) in DCM (30 mL), and the mixture was stirred overnight at room temperature. The reaction mixture was then adsorbed onto silica (16 g), and the solvent was evaporated under vacuum. The crude product was purified by rapid silica chromatography (80 g, eluted with a linear gradient of ethyl acetate in cyclohexane, 0–10%) to give compound 20 (3.64 g, 83%; R in CE5) as a colorless oil. f 0.27 (visualized using KMnO4).

[0239] Bromoester 20 (3.63 g, 11.8 mmol, 2.6 eq.) and potassium carbonate (6.29 g, 45.5 mmol, 10 eq.) were added to a solution of N-Boc-hexane-1,6-diamine (0.98 g, 4.55 mmol, 1 eq.) in anhydrous acetonitrile (10 mL), and the mixture was stirred at 40 °C for 3 days. The reaction mixture was then adsorbed onto silica (16 g), and the solvent was evaporated under vacuum. The crude product was purified by rapid silica chromatography (80 g, eluted with a linear gradient of ethyl acetate in cyclohexane, 0-100%) to give compound 21 (2.90 g, 95%; R on CE50 of an NH3-pretreated TLC plate) as a pale yellow oil. f 0.54 (visualized via ninhydrin).

[0240] Hydrochloric acid (4 mL, 4 M) from dioxane was added to a solution of compound 21 (2.85 g, 4.26 mmol) in anhydrous DCM (4 mL), and the reaction mixture was stirred at room temperature for 1 hour. The solution was then poured into a 500 mL separatory funnel, diluted with saturated NaHCO3 aqueous solution (100 mL), and the product was extracted with diethyl ether (100 mL, 2 x 50 mL). The combined organic phases were washed with brine (75 mL), dried over anhydrous sodium sulfate, filtered through S2 sintered glass, and the solvent was evaporated in an RVE. The crude product was purified by silica gel column chromatography using a linear gradient (0–70%) of D1 in DCM. Amine 22 (2.38 g, 96%; R2 on NH3- pretreated TLC plates) was given as a pale yellow oil. f 0.48 (visualized via ninhydrin).

[0241] 1 H NMR (400MHz, CDCl3): δ = 4.08-3.96 (m), 2.63-2.58 (m), 2.33-2.26 (m), 2.21 (t, J = 7.5Hz), 1.6 1-1.50(m),1.49-1.40(m),1.40-1.30(m),1.30-1.14(m),1.14-1.00(m),0.85-0.76(m)ppm. 13 C NMR(150.9MHz, CDCl3):173.85,62.77,54.13,42.19,38.98,35.55,34.35, 33.79,29.56,29.15,27.49,27.29,26.88,25.01,19.95,19.48,14.26ppm. HRMS(ESI):m / z C 34 H 68 N₂O₄[M+H] +Calculated value: 569.5252; Measured value: 569.5250.

[0242] Hexa(3-methylhexyl)cis,cis-7,7',7”,7”',7””,7””-((((cyclohexane-1,3,5-tricarbonyl)tri(azanediyl))tri(hexane-6,1-diyl))tri(azanetriyl))hexaheptanate 23

[0243] Thionyl chloride (500 μL) and DMF (4 μL) were added to cis,cis-cyclohexane-1,3,5-tricarboxylic acid (60 mg, 278 μmol), and the suspension was stirred in a sealed vial at 70 °C for 3 hours; during this time, the suspension became a clear solution. Excess SOCl2 was removed at 70 °C with a stream of dry nitrogen, and the residue was dried under vacuum at room temperature (20 min). The obtained solid was dissolved under argon in anhydrous TCE (2 mL) and DIPEA (483 μL, 2.78 mmol, 10 eq.). Then, amine 22 (632 mg, 1.11 mmol, 4 eq.) was added to the solution in anhydrous TCE (2 mL), and the reaction mixture was stirred at room temperature for 40 min. The reaction mixture was then evaporated under vacuum, redissolved in DCM, adsorbed onto silica (10 g), and the DCM was removed under vacuum. The crude product was purified by rapid silica chromatography (eluting with a linear gradient of D1 in DCM, 0-46%) to give target compound 23 (346 mg, 67%; R on NH3- pretreated TLC plate, D2) as a yellow waxy semi-solid. f 0.66 (visualized via ninhydrin). 1 H NMR (600MHz, CDCl3): δ=5.75,4.09,3.21,2.41,2.28,2.22,2.11,1.64,1.62,1 .61,1.58,1.54,1.48,1.44,1.42,1.33,1.32,1.29,1.28,1.13,0.89,0.88ppm. 13 CNMR (150.9MHz, CDCl3): δ=173.92,173.87,62.64,53.90,44.10,39.45,39.14,35.51,3 4.23,31.86,29.54,29.52,29.09,27.22,27.13,26.77,24.97,19.94,19.47,14.26ppm. HRMS(MALDI):m / z C 111 H 211 N6O 15 [M+H] + Calculated value: 1868.5927; Measured value: 1868.5905.

[0244] Example 8

[0245] Compound 24

[0246] N,N'-Diisopropylcarbodiimide (4.07 mL, 24.9 mmol, 1.6 eq.) was added to anhydrous DCM (150 mL) in a solution containing 6-bromopentanoic acid (3.00 g, 16.6 mmol), DMAP (81 mg, 0.66 mmol, 0.04 eq.), and geraniol (4.36 mL, 24.9 mmol, 1.5 eq.). The mixture was stirred overnight at room temperature. The reaction mixture was then adsorbed onto silica (20 g), and the solvent was evaporated under vacuum. The crude product was purified by rapid silica chromatography (120 g, eluted with a linear gradient of ethyl acetate in cyclohexane, 0–10%) to give compound 24 (4.80 g, 91%; R) as a pale yellow oil. f 0.39 in CE5 (visualized via KMnO4). 1 H NMR (400MHz, CDCl3) δ5.37-5.30(m,1H),5.11-5.05(m,1H),4.60(d,J=7.1Hz,2H),3.41(t,J=6.6Hz,2H),2.35(t ,J=7.2Hz,2H),2.15-2.01(m,4H),1.95-1.86(m,2H),1.83-1.74(m,2H),1.70(s,3H),1.68(s,3H),1.60(s,3H).

[0247] Compound 26

[0248] Bromoester 24 (4.80 g, 15.1 mmol, 2.3 eq.) and potassium carbonate (7.27 g, 52.3 mmol, 8 eq.) were added to a solution of N-Boc-1,6-hexanediamine (1.42 g, 6.58 mmol, 1 eq.) in anhydrous ACN (30 mL), and the mixture was stirred at 45 °C for 24 hours. The reaction mixture was then adsorbed onto silica (20 g), and the solvent was evaporated under vacuum. The crude product was purified by silica chromatography (120 g column pretreated with gaseous NH3, eluted with a linear gradient of ethyl acetate in cyclohexane, 0-100%) to give compound 25 (2.80 g, 54%; R on CE50 of NH3-pretreated TLC plate) as a pale yellow oil. f 0.58 (visualized using KMnO4).

[0249] Compound 25 (2.80 g, 4.06 mmol) was charged into a flask equipped with a magnetic stir bar, sealed with a rubber diaphragm, and purged with argon. ACN (10 mL) and DCM (20 mL) were added through the diaphragm using a needle and syringe. Stirring was started, and a solution of 4-toluenesulfonic acid monohydrate (2.32 g, 12.2 mmol, 3 eq.) in ACN (20 mL) was added through the diaphragm using a needle and syringe over a 5-minute timeframe at room temperature. The reaction mixture was stirred at room temperature for 6 hours, followed by neutralization with a stream of gaseous ammonia. The reaction mixture was then adsorbed onto silica (20 g), and the crude product was purified by silica gel column chromatography using a linear gradient (10–43%) of D1 in DCM. Amine 26 (810 mg, 34%; R2 in D2 on an NH3-pretreated TLC plate) was obtained as a yellow oil. f 0.45 (visualized via ninhydrin). HRMS(ESI): m / z C 36 H 64 N₂O₄[M+H] + Calculated value: 589.4939; Measured value: 589.4936.

[0250] Hexa((E)-3,7-dimethyloctyl-2,6-dien-1-yl)cis,cis-5,5',5”,5”',5””,5””-((((cyclohexane-1,3,5-tricarbonyl)tri(azanediyl))tri(hexane-6,1-diyl))tri(azanetriyl))hexapentanoate 27

[0251] Thionyl chloride (700 μL) and DMF (6 μL) were added to cis,cis-cyclohexane-1,3,5-tricarboxylic acid (45 mg, 208 μmol), and the suspension was stirred in a sealed vial at 70 °C for 3 hours; during this time, the suspension became a clear solution. Excess SOCl2 was removed at 70 °C with a stream of dry nitrogen, and the residue was dried under vacuum at room temperature (20 min). The obtained solid was dissolved under argon in anhydrous TCE (1.5 mL) and DIPEA (363 μL, 2.08 mmol, 10 eq.). Then, amine 26 (490 mg, 833 μmol, 4 eq.) was added to the solution of anhydrous TCE (1.0 mL), and the reaction mixture was stirred at room temperature for 40 min. The reaction mixture was then evaporated under vacuum, redissolved in DCM, adsorbed onto silica (10 g), and the DCM was removed under vacuum. The crude product was purified by rapid silica chromatography (eluting with a linear gradient of D1 in DCM, 0-31%) to give target compound 27 (262 mg, 65%; R on NH3- pretreated TLC plate D2) as a yellow waxy semi-solid. f 0.59 (visualized via ninhydrin). 1H NMR (600MHz, CDCl3): δ=5.72,5.32,5.08,4.58,3.21,2.44,2.41,2.32,2.22,2 .11,2.10,2.09,2.03,1.69,1.68,1.61,1.60,1.59,1.58,1.47,1.45,1.29ppm. 13 C NMR (150.9MHz, CDCl3): δ=173.85,173.60,142.16,131.80,123.72,118.32,62.25,53.79,53.45 ,44.12,39.52,39.42,34.14,31.85,29.48,27.03,26.72,26.29,25.67,22.90,17.68,16.46ppm. HRMS(MALDI):m / z C 117 H1 99 N6O 15 [M+H] + Calculated value: 1928.4988; Measured value: 1928.4967.

[0252] Example 9

[0253] Compound 28

[0254] N,N'-Diisopropylcarbodiimide (4.07 mL, 24.9 mmol, 1.6 eq.) was added to anhydrous DCM (60 mL) in a solution containing 6-bromopentanoic acid (3.00 g, 16.6 mmol), DMAP (81 mg, 0.66 mmol, 0.04 eq.), and nerol (4.38 mL, 24.9 mmol, 1.5 eq.). The mixture was stirred overnight at room temperature. The reaction mixture was then adsorbed onto silica (20 g), and the solvent was evaporated under vacuum. The crude product was purified by silica rapid chromatography (120 g, eluted with a linear gradient of ethyl acetate in cyclohexane, 0–10%) to give compound 28 (4.75 g, 90%; R) as a pale yellow oil. f 0.39 in CE5 (visualized via KMnO4). 1H NMR(401MHz, CDCl3)δ5.35(t,J=6.5Hz,1H),5.12-5.06(m,1H),4.57(dd,J=7.2,1.1Hz,2H),3.40(t,J=6.6Hz,2H), 2.34(t,J=7.3Hz,2H),2.15-2.03(m,4H),1.94-1.86(m,2H),1.82-1.74(m,5H),1.68(d,J=1.4Hz,3H),1.60(s,3H).

[0255] Compound 30

[0256] Bromoester 28 (4.75 g, 15.0 mmol, 2.3 eq.) and potassium carbonate (7.20 g, 52.1 mmol, 8 eq.) were added to a solution of N-Boc-1,6-hexanediamine (1.41 g, 6.51 mmol, 1 eq.) in anhydrous ACN (45 mL), and the mixture was stirred at 48 °C for 24 hours. The reaction mixture was then adsorbed onto silica (20 g), and the solvent was evaporated under vacuum. The crude product was purified by silica chromatography (120 g column pretreated with gaseous NH3, eluted with a linear gradient of ethyl acetate in cyclohexane, 0-100%) to give compound 29 (3.62 g, 81%; R on CE50 of NH3-pretreated TLC plate) as a pale yellow oil. f 0.54 (visualized via ninhydrin).

[0257] Compound 29 (3.62 g, 5.25 mmol) was charged into a flask equipped with a magnetic stir bar, sealed with a rubber diaphragm, and purged with argon. DCM (10 mL) was added through the diaphragm using a needle and syringe. Stirring was started, and a solution of 4-toluenesulfonic acid monohydrate (2.50 g, 13.1 mmol, 2.5 eq.) in ACN (25 mL) was added through the diaphragm using a needle and syringe over a 5-minute timeframe at room temperature. The reaction mixture was stirred at room temperature for 5 hours, followed by neutralization with a stream of gaseous ammonia. The reaction mixture was then adsorbed onto silica (20 g), and the crude product was purified by silica gel column chromatography using a linear gradient (10–43%) of D1 in DCM. Amine 30 (1.36 g, 44%; R2 in D2 on an NH3-pretreated TLC plate) was obtained as a yellow oil. f 0.46 (visualized using KMnO4). HRMS(ESI): m / z C 36 H 64 N₂O₄[M+H] + Calculated value: 589.4939; Measured value: 589.4937.

[0258] Hexa((Z)-3,7-dimethyloctyl-2,6-dien-1-yl)cis,cis-5,5',5”,5”',5””,5””-((((cyclohexane-1,3,5-tricarbonyl)tri(azanediyl))tri(hexane-6,1-diyl))tri(azanetriyl))hexapentanoate 31

[0259] Thionyl chloride (700 μL) and DMF (6 μL) were added to cis,cis-cyclohexane-1,3,5-tricarboxylic acid (65 mg, 301 μmol), and the suspension was stirred in a sealed vial at 70 °C for 3 h; during this time, the suspension became a clear solution. Excess SOCl2 was removed at 70 °C with a stream of dry nitrogen, and the residue was dried under vacuum at room temperature (20 min). The obtained solid was dissolved under argon in anhydrous TCE (2 mL) and DIPEA (524 μL, 3.01 mmol, 10 eq.). Then, amine 30 (796 mg, 1.35 mmol, 4.5 eq.) was added to a solution of anhydrous TCE (2.0 mL), and the reaction mixture was stirred at room temperature for 30 min. The reaction mixture was then evaporated under vacuum, redissolved in DCM, adsorbed onto silica (10 g), and the DCM was removed under vacuum. The crude product was purified by rapid silica chromatography (eluting with a linear gradient of D1 in DCM, 5-29%) to give target compound 31 (351 mg, 51%) as a yellow waxy semi-solid. 1 H NMR (600MHz, CDCl3): δ=5.69,5.34,5.08,4.55,3.21,2.41,2.38,2.30,2.22,2.10,2.06,1.75,1.67,1.60,1.59,1.58,1.47,1.28ppm. 13 C NMR (150.9MHz, CDCl3): δ=173.81,173.58,142.44,132.12,123.56,119.22,60.96,53.82,53.48 ,44.12,39.44,34.17,32.15,31.85,29.50,27.07,26.75,26.63,25.68,23.50,22.91,17.65ppm. HRMS(MALDI):m / z C 117 H 199 N6O 15 [M+H] + Calculated value: 1928.4988; Measured value: 1928.4963.

[0260] Example 10

[0261] Tetra(octane-2-yl)cis,cis-6,6',6”,6”'-((((5-(methoxycarbonyl)cyclohexane-1,3-dicarbonyl)bis(azanediyl))bis(hexane-6,1-diyl))bis(azanetriyl))tetrahexanoate 32

[0262] Thionyl chloride (700 μL) and DMF (6 μL) were added to cis,cis-cyclohexane-1,3,5-tricarboxylic acid (60 mg, 278 μmol), and the suspension was stirred in a sealed vial at 70 °C for 3 hours; during this time, the suspension became a clear solution. Excess SOCl2 was removed at 70 °C with a stream of dry nitrogen, and the residue was dried under vacuum at room temperature (20 min). The obtained solid was dissolved under argon in anhydrous TCE (2 mL) and DIPEA (483 μL, 2.78 mmol, 10 eq.). Then, amine 18 (632 mg, 1.11 mmol, 4 eq.) was added to a solution of anhydrous TCE (1.0 mL), and the reaction mixture was stirred at room temperature for 40 min. The reaction mixture was then evaporated under vacuum, redissolved in DCM, adsorbed onto silica (10 g), and the DCM was removed under vacuum. The crude product was purified by rapid silica chromatography (eluting with a linear gradient of D1 in DCM, 5-29%) to give compound 32 (242 mg, 65%; R on NH3- pretreated TLC plate, D2) as a yellow oil. f 0.68 (visualized via ninhydrin). Product 32 is the result of incomplete removal of MeOH from amine 18. 1 H NMR (600MHz, CDCl3): δ=5.68,5.29,4.88,3.67,3.22,2.43,2.40,2.27,2.20,2.19,2 .18,2.09,1.62,1.59,1.57,1.56,1.48,1.47,1.46,1.30,1.28,1.27,1.19,0.87ppm. 13 C NMR (150.9MHz, CDCl3): δ=174.64,173.75,173.38,70.83,53.82,53.76,51.84,43.99,42.09,39.43,3 5.93,34.66,31.81,31.73,31.20,29.50,29.09,27.06,26.72,25.36,24.99,22.56,20.00,14.05ppm. HRMS(MALDI):m / z C 78 H 146 N4O 12 [M+H] + Calculated value: 1332.1010; Measured value: 1332.0987.

[0263] Example 11

[0264] Tetra(3-methylhexyl)cis,cis-7,7',7”,7”'-((((5-(methoxycarbonyl)cyclohexane-1,3-dicarbonyl)bis(azanediyl))bis(hexane-6,1-diyl))bis(azanetriyl))tetraheptanoate 33

[0265] Thionyl chloride (700 μL) and DMF (6 μL) were added to cis,cis-cyclohexane-1,3,5-tricarboxylic acid (55 mg, 254 μmol), and the suspension was stirred in a sealed vial at 70 °C for 3 h; during this time, the suspension became a clear solution. Excess SOCl2 was removed with a stream of dry nitrogen at 70 °C, and the residue was dried under vacuum at room temperature (20 min). The obtained solid was dissolved in anhydrous TCE (2 mL) and DIPEA (443 μL, 2.54 mmol, 10 eq.) under argon atmosphere. Then, MeOH (10.3 μL, 254 μmol, 1.0 eq.) was added to a solution of TCE (0.5 mL) at 0 °C. The reaction mixture was warmed to room temperature, and after stirring for 1 hour, amine 22 (376 mg, 661 μmol, 2.6 eq.) was added to anhydrous TCE (2.0 mL), and the reaction mixture was stirred at room temperature for 40 min. The reaction mixture was then evaporated under vacuum, redissolved in DCM, adsorbed onto silica (10 g), and the DCM was removed under vacuum. The crude product was purified by rapid silica chromatography (eluting with a linear gradient of D1 in DCM, 5-29%) to give target compound 33 (87 mg, 26%; R on NH3- pretreated TLC plate, D2) as a pale yellow oil. f 0.75 (visualized via ninhydrin). 1 H NMR (600MHz, CDCl3): δ=5.68,5.29,4.09,3.67,3.22,2.43,2.40,2.28,2.20,2.18,2 .09,1.64,1.61,1.57,1.53,1.48,1.45,1.42,1.34,1.31,1.28,1.13,0.89,0.88ppm. 13C NMR (150.9MHz, CDCl3): δ=174.65,173.91,173.76,62.85,53.85,51.85,43.98,42.09,39.42,39.15,3 5.52,34.22,31.89,31.19,29.55,29.51,29.06,27.20,27.10,26.72,24.95,19.94,19.47,14.26ppm. HRMS(MALDI):m / z C 78 H 147 N4O 12 [M+H] + Calculated value: 1332.1010; Measured value: 1332.0984.

[0266] Example 12

[0267] Compound 34

[0268] Thionyl chloride (1.2 mL) and DMF (10 μL) were added to cis,cis-cyclohexane-1,3,5-tricarboxylic acid (160 mg, 740 μmol), and the suspension was stirred in a sealed vial at 70 °C for 3 hours; during this time, the suspension became a clear solution. Excess SOCl2 was removed with a stream of dry nitrogen at 70 °C, and the residue was dried under vacuum at room temperature (20 min). The obtained solid was dissolved in anhydrous TCE (3 mL) and DIPEA (1.29 mL, 7.40 mmol, 10 eq.) under argon atmosphere. Then, BnOH (76 μL, 740 μmol, 1.0 eq.) was added to the solution of TCE (0.5 mL) at 0 °C. The reaction mixture was warmed to room temperature, and after stirring for 2 hours, amine 18 (1.09 g, 1.92 mmol, 2.6 eq.) was added to anhydrous TCE (3.0 mL), and the reaction mixture was stirred at room temperature for 40 min. The reaction mixture was then evaporated under vacuum, redissolved in DCM, adsorbed onto silica (10 g), and the DCM was removed under vacuum. The crude product was purified by rapid silica chromatography (eluting with a linear gradient of D1 in DCM, 5-29%) to give compound 34 (150 mg, 14%; R in D2 on an NH3-pretreated TLC plate) as a pale yellow oil. f 0.67 (visualized via ninhydrin). HRMS (MALDI): m / z C 84 H 151 N4O 12 [M+H] + Calculated value: 1408.1323; Measured value: 1408.1283.

[0269] cis,cis-3,5-bis((6-(bis(6-(octane-2-yloxy)-6-oxohexyl)amino)hexyl)carbamoyl)cyclohexane-1-carboxylic acid 35

[0270] A pear-shaped flask equipped with a magnetic stir bar and loaded with palladium / carbon (10% Pd / C, 25 mg). The flask was sealed with a rubber diaphragm, connected to a Schlenk line via a needle, and the atmosphere in the flask was replaced with argon through three vacuum argon cycles. Subsequently, a solution of compound 34 (150 mg, 107 μmol) in MeO (3 mL) was added through the diaphragm using a needle and syringe, and stirring was started. The flask was connected to a continuous hydrogen flow using a second needle, and the main argon inlet to the Schlenk line was closed (excess hydrogen was continuously released from the flask through a Schlenk line bubbler). The reaction mixture was vigorously stirred at room temperature for 4 hours. The flask was then thoroughly rinsed with argon and filtered through a PTFE syringe filter to remove the carbon / palladium. The volatiles were then removed under vacuum to give product 35 (110 mg, 79%, in D2 on NH3-pretreated TLC plates) as a colorless oil. f 0.27 (visualized via ninhydrin). 1 H NMR (600MHz, CDCl3): δ=6.59,4.88,3.20,3.15,2.71,2.67,2.27,2.24,2.20,2.13,2.02,1 .64,1.63,1.60,1.58,1.57,1.56,1.54,1.47,1.45,1.32,1.28,1.27,1.26,1.18,0.87ppm. 13 C NMR (150.9MHz, CDCl3): δ=180.09,175.19,173.10,70.94,53.01,52.06,44.36,44 .32,39.22,35.91,34.43,32.48,32.16,31.72,29.15,29.08,26.72,26.62,26.30 25.36,24.69,24.11,22.55,19.99,14.06ppm. HRMS(MALDI):m / z C 77 H 145 N4O 12 [M+H] + Calculated value: 1318.0854; Measured value: 1318.0842.

[0271] Tetra(octane-2-yl)cis,cis-6,6',6”,6”'-((((5-(pyrrolidine-1-carbonyl)cyclohexane-1,3-dicarbonyl)bis(azanediyl))bis(hexane-6,1-diyl))bis(azanetriyl))tetrahexanoate 36

[0272] Compound 35 (60 mg, 45.5 μmol) and NH4Cl (12.2 mg, 228 μmol, 5 eq.) were added to a vial equipped with a magnetic stir bar and flushed with argon gas by puncturing the septum with a needle. Subsequently, anhydrous DMF (1.5 mL) and DIPEA (48 μL, 273 μmol, 6 eq.) were added using a needle and syringe. The resulting suspension was stirred at room temperature for 10 minutes, and then a solution of PyBroP (53 mg, 114 μmol, 2.5 eq.) in anhydrous DMF (0.5 mL) was added, and the reaction mixture was stirred at room temperature for 90 minutes. The reaction mixture was then evaporated under vacuum, redissolved in DCM, adsorbed onto silica (10 g), and the DCM was removed under vacuum. The crude product was purified by rapid silica chromatography (eluting with a linear gradient of D1 in DCM, 5-29%) to give compound 36 (18 mg, 29%) as a pale yellow oil; R in D2 on an NH3-pretreated TLC plate. f 0.42 (visible using ninhydrin) was the major product. Compound 36 was a product of pyrrolidine impurities in PyBroP generated by the hydrolysis of PyBroP. 1 H NMR (600MHz, CDCl3): δ=6.08,4.88,3.48,3.43,3.35,3.25,3.26,3.20,3.16,2.72,2.50,2.32,2.28,2 .15,1.98,1.95,1.85,1.69,1.68,1.66,1.65,1.60,1.57,1.50,1.46,1.34,1.28,1.26,1.19,0.88ppm. 13 C NMR (150.9MHz, CDCl3): δ=174.39,173.14,172.79,70.97,53.37,47.44,47.40,47.22,47.19,46.43,46.30,46.27,45.90 ,44.03,41.39,39.09,35.92,34.44,32.08,31.73,31.20,29.10,26.70,26.14,25.37,24.69,24.24,22.57,20.00,14.06. HRMS(MALDI):m / z C 81 H 152 N5O 11 [M+H]+ Calculated value: 1371.1483; Measured value: 1371.1467.

[0273] Example 13

[0274] cis,cis-1,3,5-cyclohexanetrimethyl ester 37

[0275] Thionyl chloride (15.29 mL, 210.8 mmol, 4.6 eq.) was slowly added via syringe through a rubber diaphragm to a solution of cis,cis-1,3,5-cyclohexanetricarboxylic acid (9.90 g, 45.8 mmol) in anhydrous methanol (150 mL). The solution was placed in a round-bottom flask equipped with a calcium chloride drying tube and a magnetic stirrer. After the vigorous exothermic reaction ceased, the solution was stirred at room temperature for 12 hours. The solvent was then removed under vacuum, and the oily residue was poured into a 500 mL separatory flask, diluted with saturated aqueous NaHCO3 (250 mL), and extracted with diethyl ether (2 x 100 mL). The combined organic phases were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered through S2 sintered glass, and the solvent was evaporated in an RVE to give 37, a pale yellow oil. 1 HNMR (400MHz, CDCl3): δ = 3.68 (s, 9H), 2.44-2.32 (m, 3H), 2.31-2.21 (m, 3H), 1.59-1.45 (m, 3H) ppm. HRMS(ESI):m / z C 12 H 19 O6[M+H] + Calculated value: 259.11761; Measured value: 259.11766.

[0276] Trimethylcis,cis-1,3,5-trimethylcyclohexane-1,3,5-tricarboxylic acid ester 38

[0277] Diisopropylamine was distilled from sodium hydroxide before use. A solution of diisopropylamine (2.19 mL, 15.5 mmol, 4 eq.) in anhydrous diethyl ether (10 mL) was added dropwise to a solution of 2.0 M n-butyllithium (7.74 mL, 15.5 mmol, 4 eq.) at 0 °C to generate diisopropylaminolithium in situ. A solution of 37 (1.00 g, 3.87 mmol; 1 eq.) in anhydrous diethyl ether (10 mL) was added dropwise to the reaction solution at 0 °C, and the mixture was stirred at 0 °C for 2 h. Dimethyl sulfate (2.20 mL, 23.2 mmol, 6 eq.) was added, and stirring continued overnight at room temperature. The product was washed with water, 1 M HCl, and brine, dried over sodium sulfate, filtered through S2 sintered glass, and the solvent was evaporated in an RVE. GC-MS analysis showed that the ratio of the two isomers, cis, cis / cis, trans, in the crude product was 7:1. Fractional crystallization in a mixture of diethyl ether and pentane yielded cis-cis isomer 38 (225 mg, 19%) as white crystals. 1 H NMR (400MHz, CDCl3): δ = 3.65 (s, 9H), 2.74 (d, J = 13.4Hz, 3H), 1.21 (s, 9H), 0.97 (d, J = 14.8Hz, 3H) ppm. HRMS(EI):m / zC 15 H 24 O6[M] + Calculated value: 300.1567; Measured value: 300.1565.

[0278] cis,cis-1,3,5-trimethylcyclohexane-1,3,5-tricarboxylic acid 39

[0279] Trimethyl cis,cis-1,3,5-trimethylcyclohexane-1,3,5-tricarboxylate 38 (220 mg, 0.73 mmol, 1 eq.) was dissolved in methanol (3.0 mL). A solution of lithium hydroxide monohydrate (369 mg, 6.59 mmol, 9 eq.) in water (2.0 mL) was added, and the reaction mixture was stirred overnight at room temperature. Methanol was removed by evaporation in a refrigerant ether (RVE). The solution was cooled in an ice bath, and the pH was adjusted to 1 with concentrated HCl. A white precipitate formed immediately and was collected by vacuum filtration to produce 39 (60 mg, 32%). 1 ¹H NMR (400MHz, DMSO-d6): δ=11.96(br s, 3H), 2.52(d, 3H, partially overlapping with solvent signal), 1.20(d, J=14.7Hz, 3H), 1.19(s, 9H)ppm.

[0280] Hexa(3-methylhexyl)cis,cis-7,7',7”,7”',7””,7””-((((1,3,5-trimethylcyclohexane-1,3,5-tricarbonyl)tri(azanediyl))tri(hexane-6,1-diyl))tri(azanetriyl))hexaheptanate 40

[0281] Thionyl chloride (0.50 mL) and DMF (2 μL) were added to cis,cis-1,3,5-trimethylcyclohexane-1,3,5-tricarboxylic acid 39 (34 mg, 0.13 mmol), and the suspension was stirred overnight in a sealed vial at 70 °C. Excess SOCl2 was purged with a stream of dry nitrogen, and the residue was dried under vacuum (10 min). After cooling to room temperature, the residue was dissolved in anhydrous TCE (0.5 mL). Then, amine 22 (337 mg, 0.59 mmol, 4.5 eq.) and DIPEA (229 μL, 1.32 mmol, 10 eq.) were added to a solution of anhydrous TCE (1.00 mL), and the reaction mixture was stirred at room temperature for 15 min. The reaction mixture was then adsorbed onto silica (4 g), and the solvent was evaporated under vacuum. The crude product was purified by rapid silica chromatography (40 g, eluted with a linear gradient of D1 in DCM, 0-30%) to obtain compound 40 (6 mg, 5%; eluted with a linear gradient of D1 in DCM, 0-30%), which was a viscous, light yellow oil. f 0.26 (visualized via ninhydrin). HRMS (MALDI): m / z C 114 H 216 N6O 15 [M+H] + Calculated value: 1910.6396; Measured value: 1910.6368.

[0282] Example 14

[0283] Hexa(octane-2-yl)cis,cis-6,6',6”,6”',6””,6””-((((1,3,5-trimethylcyclohexane-1,3,5-tricarbonyl)tri(azaalkyldiyl))tri(hexane-6,1-diyl))tri(azaalkyltriyl))hexahexanoate 41 and cis,cis-3,5-bis((6-(bis(6-(octane-2-yloxy)-6-oxohexyl)amino)hexyl)carbamoyl)-1,3,5-trimethylcyclohexane-1-carboxylic acid 42

[0284] Thionyl chloride (0.50 mL) and DMF (2 μL) were added to a concentration of 39 g (28 mg, 0.11 mmol), and the suspension was stirred in a sealed vial at 70 °C for 2 h. Excess SOCl2 was purged with a stream of dry nitrogen, and the residue was dried under vacuum (10 min). After cooling to room temperature, the residue was dissolved in anhydrous TCE (0.25 mL). Then, amine 18 (278 mg, 0.49 mmol, 4.5 eq.) and DIPEA (180 μL, 1.08 mmol, 10 eq.) were added to the solution in anhydrous TCE (0.75 mL), and the reaction mixture was stirred at room temperature for 15 min. The reaction mixture was then adsorbed onto silica (4 g), and the solvent was evaporated under vacuum. The crude product was purified by rapid silica chromatography (40 g, eluted with a linear gradient of D1 in DCM, 0-30%) to obtain compound 41 (49 mg, 24%; eluted with a linear gradient of D1 in DCM, 0-30%) as a viscous, pale yellow oil. f 0.30 (visible via ninhydrin) and compound 42 (52 mg, 45%; in D4 R) which is a pale yellow oil. f 0.28 (visualized via ninhydrin).

[0285] 41 1 H NMR (600MHz, CDCl3): δ = 7.98, 4.88, 3.12, 2.88, 2.75, 2.68, 2.27, 1.64, 1.56, 1.46, 1.45, 1.28, 1.27, 1.26, 1.23, 1.19, 1.12, 0.87ppm. 13 C NMR (150.9MHz, CDCl3): δ=184.26,177.45,173.06,70.96,52.45,51.68,43.03,43.00,4 2.38,38.83,35.92,34.43,34.17,33.21,31.72,29.08,28.95,22.56,19.99,14.05ppm. HRMS(MALDI):m / z C 114 H 216 N6O 15 [M+H] + Calculated value: 1910.6396; Measured value: 1910.6364.

[0286] 42HRMS(MALDI):m / z C 80 H 150 N4O 12 [M+H] + Calculated value: 1360.1323; Measured value: 1360.1875.

[0287] Example 15

[0288] Trimethylcis,cis-1,3,5-tris((benzyloxy)methyl)cyclohexane-1,3,5-tricarboxylic acid ester 43

[0289] Diisopropylamine was distilled from sodium hydroxide before use. A solution of diisopropylamine (6.56 mL, 46.5 mmol, 4 eq.) in anhydrous diethyl ether (30 mL) was added dropwise to a solution of 2.0 M n-butyllithium (23.2 mL, 46.5 mmol, 4 eq.) at 0 °C to generate diisopropylaminolithium in situ. A solution of trimethyl cyclohexane-1,3,5-tricarboxylic acid ester 37 (3.00 g, 11.6 mmol; 1 equivalent) in anhydrous diethyl ether (30 mL) was added dropwise to the reaction solution at 0 °C, and the mixture was stirred at 0 °C for 2 h. Benzylchloromethyl ether (9.69 mL, 69.7 mmol, 6 eq.) was added, and stirring continued overnight at room temperature. The product was washed with water, 1 M HCl, and brine, dried over sodium sulfate, filtered through S2 sintered glass, and the solvent was evaporated in an RVE. The crude product was purified by rapid silica chromatography (200 g, eluted with a linear gradient of ethyl acetate in cyclohexane, 0-20%). Fractional crystallization in a mixture of diethyl ether (3.0 mL) and pentane (18.0 mL) gave cis,cis isomer 43 (1.52 mg, 22%) as white crystals. 1 ¹H NMR (400MHz, CDCl₃): δ = 7.36–7.22 (m, 15H, partially overlapping with solvent signal), 4.46 (s, 6H), 3.69 (s, 9H), 3.39 (s, 6H), 2.68 (d, J = 14.2Hz, 3H), 1.18 (d, J = 14.9Hz, 3H) ppm. HRMS (ESI): m / z C 36 H 42 O9[M+Na] + Calculated value: 641.2721; Measured value: 641.2719.

[0290] cis,cis-1,3,5-tris((benzyloxy)methyl)cyclohexane-1,3,5-tricarboxylic acid 44

[0291] Compound 43 (250 mg, 0.40 mmol, 1 eq.) was dissolved in methanol (4.0 mL). A solution of lithium hydroxide monohydrate (152 mg, 3.64 mmol, 9 eq.) in water (2.0 mL) was added, and the reaction mixture was stirred overnight at room temperature. Methanol was removed by evaporation in a refrigerant ether (RVE). The solution was cooled in an ice bath, and the pH was adjusted to 1 with concentrated HCl. A white precipitate formed immediately and was collected by vacuum filtration to produce 44 (200 mg, 86%). 1H NMR (400MHz, DMSO-d6): δ = 12.12 (br s, 3H), 7.37-7.18 (m, 15H), 4.39 (s, 6H), 3.39 (s, 6H), 2.36 (d, J = 14.9Hz, 3H), ppm.

[0292] Tetra(octane-2-yl)cis,cis-6,6',6”,6”'-((((1,3,5-tris((benzyloxy)methyl)-5-(bis(6-(octane-2-yloxy)-6-oxohexyl)carbamoyl)cyclohexane-1,3-dicarbonyl)bis(azanediyl))bis(hexane-6,1-diyl))bis(azanetriyl))tetrahexanoate 45 and cis,cis-1,3,5-tris((benzyloxy)methyl)-3,5-bis((6-(bis(6-(octane-2-yloxy)-6-oxohexyl)amino)hexyl)carbamoyl)cyclohexane-1-carboxylic acid 46

[0293] Thionyl chloride (0.50 mL) and DMF (2 μL) were added to a final volume of 44 (100 mg, 0.17 mmol, 1 eq.), and the suspension was stirred overnight in a sealed vial at 70 °C. Excess SOCl2 was purged with a stream of dry nitrogen, and the residue was dried under vacuum (10 min). After cooling to room temperature, the residue was dissolved in anhydrous TCE (0.5 mL). Then, amine 18 (444 mg, 0.78 mmol, 4.5 eq.) and DIPEA (302 μL, 1.73 mmol, 10 eq.) were added to the solution in anhydrous TCE (1.50 mL), and the reaction mixture was stirred at room temperature for 15 min. The reaction mixture was then adsorbed onto silica (4 g), and the solvent was evaporated under vacuum. The crude product was purified by rapid silica chromatography (80 g, eluted with a linear gradient of D1 in DCM, 0-20%) to give compound 45 (46 mg, 12%; R in D4) as a viscous, pale yellow oil. f 0.46 (visible via ninhydrin) and compound 46 (37 mg, 13%; in D4 R) which is a pale yellow oil. f 0.39 (visualized via ninhydrin).

[0294] 45HRMS(MALDI): m / z C 135 H 234 N6O 18 [M+H] + Calculated value: 2228.7652; Measured value: 2228.7702.

[0295] 46HRMS(MALDI):m / z C 101 H 168 N4O 15[M] + Calculated value: 1677.2506; Measured value: 1677.2479.

[0296] Hexa(octane-2-yl)cis,cis-6,6',6”,6”',6””,6””-((((1,3,5-tris(hydroxymethyl)cyclohexane-1,3,5-tricarbonyl)tris(azanediyl))tris(hexane-6,1-diyl))tris(azanetriyl))hexahexanoate 47

[0297] Following the procedure outlined for 35, target compound 47 was prepared from lipid 45 (20 mg, 0.008 mmol, 1 eq.), palladium / carbon (10% Pd / C, 19 mg, 0.018 mmol, 2 eq.), and acetic acid (80 μL, 1.73 mmol) to obtain lipid 47 (5 mg, 30%) as a thick, pale yellow oil. HRMS (MALDI): m / z C 114 H 216 N6O 18 [M+H] + Calculated value: 1958.6244; Measured value: 1958.6314.

[0298] cis,cis-3,5-bis((6-(bis(6-(octane-2-yloxy)-6-oxohexyl)amino)hexyl)carbamoyl)-1,3,5-tris(hydroxymethyl)cyclohexane-1-carboxylic acid 48

[0299] Following the procedure outlined for 35, target compound 48 was prepared from lipid 46 (15 mg, 0.009 mmol, 1 eq.), palladium on carbon (19 mg, 0.018 mmol, 2 eq.), and acetic acid (80 μL, 1.73 mmol), yielding lipid 48 (7 mg, 59%) as a thick, pale yellow oil. HRMS (MALDI): m / z C 80 H 150 N4O 15 [M] + Calculated value: 1407.1098; Measured value: 1407.1042.

[0300] Example 16

[0301] Tetra(octane-2-yl)cis,cis-6,6',6”,6”'-((((1,3,5-tris((benzyloxy)methyl)-5-(pyrrolidine-1-carbonyl)cyclohexane-1,3-dicarbonyl)bis(azanediyl))bis(hexane-6,1-diyl))bis(azanetriyl))tetrahexanoate 49

[0302] Compound 44 (100 mg, 0.17 mmol, 1 eq.) was dissolved in anhydrous DMF (1.0 mL), and DMAP (3 mg, 0.02 mmol, 0.15 eq.) and DIPEA (450 μL, 2.60 mmol, 10 eq.) were added dropwise. Then, a solution of PyBroP (363 mg, 0.78 mmol, 4.5 eq.) in anhydrous DMF (1.0 mL) was added dropwise, and the reaction mixture was stirred at room temperature under an argon atmosphere for 1.5 hours. Next, a solution of amine 18 (444 mg, 0.78 mmol, 4.5 eq.) in anhydrous DMF (1.0 mL) was added, and the reaction mixture was stirred at room temperature under an argon atmosphere for 1 hour. The reaction mixture was then adsorbed onto silica (4 g), and the solvent was evaporated under vacuum. The crude product was purified by rapid silica chromatography (80 g, eluting with a linear gradient of D1 in DCM, 0-30%) to obtain target compound 49 (146 mg, 51%; R in D3) as a pale yellow oil. f 0.58 (visualized via ninhydrin). HRMS (MALDI): m / z C 105 H 175 N5O 14 [M+Na] + Calculated value: 1753.3028; Measured value: 1753.3044.

[0303] Tetra(octane-2-yl)cis,cis-6,6',6”,6”'-((((1,3,5-tris(hydroxymethyl)-5-(pyrrolidine-1-carbonyl)cyclohexane-1,3-dicarbonyl)bis(azanediyl))bis(hexane-6,1-diyl))bis(azanetriyl))tetrahexanoate 50

[0304] Following the procedure outlined for 35, target compound 50 was prepared from lipid 49 (70 mg, 0.04 mmol, 1 eq.), palladium on carbon (86 mg, 0.40 mmol, 2 eq.), and acetic acid (80 μL, 1.73 mmol) to obtain lipid 50 (57 mg, 96%) as a thick, pale yellow oil. HRMS (MALDI): m / z C 84 H 157 N5O 14 [M+Na] + Calculated value: 1483.1619; Measured value: 1483.1656.

[0305] Example 17

[0306] cis, cis-N 1 N 7-bis(6-(bisdodecylamino)hexyl)-5,7-bis(hydroxymethyl)-4-oxo-3-oxabicyclo[3.3.1]nonane-1,7-dicarboxamide 52

[0307] PyBroP (0.323 g, 0.694 mmol, 5 eq.), N,N-diisopropylethylamine (DIPEA, 0.483 mL, 2.77 mmol, 20 eq.), and amine 10 (0.314 g, 0.694 mmol, 5 eq.) were added to a solution of triacid 44 (80 mg, 0.139 mmol) in anhydrous TCE (4 mL), and the reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was then adsorbed onto silica (10 g), and the solvent was evaporated under vacuum. The crude product was purified by silica gel column chromatography using a linear gradient (20–80%) of D1 in DCM. Diamide 51 (16 mg, 8.0%; R) was obtained as a viscous, pale yellow oil. f 0.56 in mobile phase D3, detected with ninhydrin.

[0308] Palladium (10%) on carbon was added to a solution of diamide 51 in a methanol-ethyl acetate mixture (10+10 mL) under an argon atmosphere. The reaction mixture was then stirred at room temperature for 12 hours under a hydrogen atmosphere. The suspension was filtered through a diatomaceous earth pad and evaporated to give lipid 52 (5.5 mg, 42.9%, in mobile phase D3) as a colorless oil. f 0.61 (detected with ninhydrin) HRMS (MALDI): m / z C 72 H 141 N4O6[M+H] + Calculated value: 1158.0846; Measured value: 1158.0822.

[0309] Example 18

[0310] 6-(bis(dodecylamino)hexanoic acid) 53

[0311] A solution of 6-aminohexanoic acid (2.00 g, 15.2 mmol) in 150 mL of ACN was packed into a 500 mL round-bottom flask equipped with a calcium chloride drying tube and a magnetic stirrer, and sodium triacetoxyborohydride (12.93 g, 61.0 mmol, 4 eq.) was added. Dodecyl aldehyde (10.16 mL, 45.7 mmol, 3 eq.) was slowly added via a syringe through a rubber diaphragm while stirring vigorously, and the resulting white suspension was stirred at room temperature for 5 days. The reaction mixture was then adsorbed onto silica (25 g), and the solvent was evaporated under vacuum. The crude product was purified by silica gel column chromatography using a linear gradient (0-100%) of D1 in DCM. Amino acid 53 (3.203 g, 44.9%) was obtained as a colorless oil. 1 H NMR (400MHz, CDCl3): δ=2.88-2.84(m,6H),2.24(t,2H),1.62-1.72(m,2H),1.42-1.26(m,44H)0.89(t,6H)ppm. 13 C NMR (150.9MHz, CDCl3): δ=178.65,51.37,36.22,31.90,29.61-29.20,27.07,26.74,25.45,23.78,23.31,22.67,14.10ppm. HRMS(ESI):C 30 H 60 O2N[MH] - Calculated m / z value: 466.46295; Measured value: 466.46286.

[0312] cis,cis-1,3,5-triaminocyclohexane trihydrobromide 55

[0313] The synthesis was performed according to Bowen, T. et al., Bioorganic & Medicinal Chemistry Letters, Vol. 6, No. 7, 1996, 807-810, with slight modifications. Cis,cis-1,3,5-cyclohexanetricarboxylic acid (2.0 g, 9.25 mmol) was suspended in toluene (75 mL), and DIPEA (4.83 mL, 27.75 mmol, 3 eq.) was added, followed by diphenylphosphoazide (5.97 mL, 27.75 mmol, 3 eq.). The mixture was stirred at room temperature for 0.5 h, then heated to 90 °C for 0.5 h. Benzyl alcohol (3.20 mL, 30.81 mmol, 3.33 eq.) was added, and the solution was heated to 90 °C for 18 h. After cooling to room temperature, the product was collected by vacuum filtration, washed with minimal cold toluene, and dried under vacuum to give 0.386 g (7.85%) of cis,cis-1,3,5-tris(N-benzyloxycarbonyl)cyclohexane 54.

[0314] Compound 54 (0.386 g, 0.726 mmol) was suspended in a 33% solution of HBr in acetic acid (5 mL) and stirred at room temperature for 12 hours. The yellow suspension was then diluted with diethyl ether (20 mL) and filtered through an S2 sintered glass filter to give trihydrobromide 55 (0.248 g, 91.8%) as a yellow solid.

[0315] cis,cis-N,N',N”-(cyclohexane-1,3,5-triyl)tris(6-(bis(dodecylamino)hexamethylene)56

[0316] PyBroP (0.752 g, 1.61 mmol, 6 eq.), DIPEA (0.937 mL, 5.38 mmol, 20 eq.), and acid 53 (0.755 g, 1.61 mmol, 6 eq.) were added to a solution of triamine salt 55 (0.100 g, 0.269 mmol) in anhydrous TCE (3 mL) and anhydrous DMF (3 mL), and the reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was then adsorbed onto silica (10 g), and the solvent was evaporated under vacuum. The crude product was purified by silica gel column chromatography using a linear gradient (20–30%) of D1 in DCM. Lipid 56 (113 mg, 28.4%; R) was obtained as a viscous, pale yellow oil. f 0.65 in mobile phase D2 (detected with ninhydrin). HRMS (ESI): m / z C 96 H 193 O6N3[M+H] + Calculated value: 1478.5134; Measured value: 1478.5162.

[0317] Example 19

[0318] Preparation of transfection reagent

[0319] The reagents were prepared by mixing the components listed in Tables 1 through 4. All tables contain the final molar concentrations of the transfection reagents. 5 mM stock solutions of each component in 99.7% ethanol (v / v) were used to prepare A01 through A16, A21, A22, A24, A28, and A29. 5 mM stock solutions of lipids, DOPE, and DMG-PEG2000, and a 10 mM stock solution of cholesterol, were prepared in 99.7% ethanol (v / v) for A17 through A20, A23, and A25 through A27. The DOPE-Cy5 stock solution was prepared at a concentration of 0.79 mM in chloroform. TT3 is the lipid used for comparison in this paper according to WO 2016 / 187531. The benchmark D-Lin-MC3-DMA lipid (MedChemExpress Europe) was also used for comparison.

[0320] Table 1. Composition of transfection reagents A01-A06.

[0321]

[0322] Table 2. Composition of transfection reagents A07-A12.

[0323]

[0324]

[0325] Table 3. Composition of transfection reagents A13-A22.

[0326]

[0327] Table 4. Composition of transfection reagents A23-A29.

[0328]

[0329] Example 20

[0330] Preparation of nucleic acid-containing lipid nanoparticles (LNPs)

[0331] The siRNA-containing LNPs (siRNA-LNPs) were prepared as follows: Using a “Y” microfluidic device with two inputs and one output for sampling, 300 μl solutions of each of the A01-A06 transfection reagents prepared in Example 19 were mixed with a solution of 1.2 nmol siRNA (catalog number 4392420, Ambion) in 300 μl of 10 mM citrate buffer (pH 3.0). The lipid mixture and siRNA solution were injected into each inlet separately using a linear pump at a constant flow rate of 300 μl / min. The resulting 600 μl nanoparticle solution was collected and immediately diluted by adding 600 μl of PBS; thus, the corresponding nanoparticle samples designated B01-B06 were formed from the transfection reagents A01-A06.

[0332] DNA encoding the fluorescent protein mKate2 was amplified from plasmid pmKate2-C (Evrogen) using primers (5'-ATCAACATATGGTGAGCGAGCTG-3' (SEQ ID NO.1); 5'-AAGAATTCCTATCATCTGTGCCCCAG-3' (SEQ ID NO.2)) and cloned into the pET24a vector (Invitrogen) under the T7 promoter. The messenger RNA (mRNA) encoding mKate2 was transcribed in vitro using the Ampliscribe T7-Flash Transcription Kit (Lucigen) according to the manufacturer's protocol. An RNA cap analogue ARCA (Jena Bioscience) was added to the in vitro transcription reaction, and poly(A) ends were synthesized using poly(A) polymerase (New England Biolabs) according to a standard protocol.

[0333] The mRNA-containing LNPs (mRNA-LNPs) were prepared as follows: 300 μl solutions of each of the A07-A29 transfection reagents prepared in Example 19 were mixed with a solution of 120 μg mRNA in 300 μl of 10 mM citrate buffer (pH 3.0), and then a solution similar to siRNA-LNPs was prepared. Therefore, corresponding nanoparticle samples designated B07-B29 were generated from transfection reagents A07-A29. Nanoparticle samples designated B08a and B11a were generated from transfection reagents A02 and A05, respectively.

[0334] Each of the LNP samples (B01-B29) was prepared in triplicate. The hydrodynamic diameter of the newly formed LNPs was measured using dynamic light scattering (NanoZS Zetasizer, Malvern, Worcestershire, UK) at 25°C and a scattering angle of 173°. The hydrodynamic diameters of siRNA-LNPs ranged from 67 to 110 nm, and those of mRNA-LNPs ranged from 82 to 288 nm (Table 5). In this form, the particles were used for subsequent bioassays.

[0335] Table 5. Hydrodynamic diameters of LNPs, including the standard deviation measured by dynamic light scattering.

[0336]

[0337] Example 21

[0338] Efficiency of siRNA and mRNA incorporation into lipid nanoparticles

[0339] The efficiency of packaging the siRNA prepared in Example 20 (which causes degradation of mRNA encoding tyrosyl-DNA phosphodiesterase (TDP2)) into siRNA-LNPs B01-B06 was determined using the Qubit microRNA Assay Kit (Life Technologies) according to the manufacturer's instructions. The efficiency of packaging mRNA (encoding fluorescent protein mKate2) into mRNA-LNPs B07-B29 prepared in Example 20 was determined using the Qubit RNAHS Assay Kit (Life Technologies) according to the manufacturer's protocol. Incorporation efficiency was determined by comparing the concentrations of free available siRNA and mRNA in the nanoparticle solution with the concentrations of siRNA and mRNA released from the nanoparticles after nanoparticle degradation. LNPs were degraded using a buffer containing Triton X-100 (10 mM Tris-HCl, pH 8.0; 0.1 mM EDTA, 2% Triton X-100). High packaging efficiency of siRNA was demonstrated, ranging from 76% to 91%. Packaging efficiency of mRNA ranged from 60% to 96% (Table 6).

[0340] Table 6. siRNA packaging efficiency for tyrosyl-DNA phosphodiesterase 2 (TDP2) and efficiency in packaging mRNA encoding the fluorescent protein mKate2 into LNPs, including standard deviations from triplicate experiments.

[0341]

[0342]

[0343] Example 22

[0344] LNP cytotoxicity

[0345] Human cell line HEK293T and human hepatocellular carcinoma cell line HepG2, derived from embryonic kidney cells expressing the SV40 large T antigen, were cultured in 96-well plates in Durbecoix modified medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2 (100 μl of medium per well containing 5 x 10⁻⁶ cells / well). 4 Cells were incubated with 2 μl of LNP B07-B12 (final total concentration of all lipid components in wells was 20 μM) produced in triplicate as described in Example 20, or 10 μl of LNP (final total concentration of all lipid components in wells was 100 μM) for 24 hours. The cytotoxicity of LNPs was analyzed using a CellTiterGlo 2.0 cell viability assay (Promega, USA). Cell viability was normalized to untransfected cells (control). Results are summarized in Tables 7 and 8.

[0346] For both cell lines used, the new LNPs did not show significant toxicity compared to the 100 μM mixture containing TT3 lipids, with survival rates of HEK293T and HepG2 cells decreasing to only 27% and 15%, respectively (Table 8).

[0347] Table 7. For each cell line type, LNP cytotoxicity is expressed as cell viability (%) after addition of 20 μM transfection mixture B07-B12.

[0348]

[0349] Table 8. For each cell line type, LNP cytotoxicity is expressed as cell viability (%) after addition of 100 μM transfection mixture B07-B12.

[0350]

[0351] Example 23

[0352] siRNA was transfected in vitro using a novel LNP.

[0353] According to Example 20, siRNA-LNP particles containing a small interfering RNA (siRNA, catalog number 4392420, Ambion) that induces the degradation of mRNA encoding tyrosyl-DNA phosphodiesterase 2 (TDP2) were prepared. Lipofectamine RNAiMax (Invitrogen) was used as a control transfection reagent specifically for siRNA transfection. The human cell line HEK293T, which is difficult to transfect using existing transfection reagents, and a cell line derived from human multiple myeloma were used for siRNA-LNP knockdown (Brito JLR, Brown N., Morgan GJ (2010) The transfection of siRNAs in Multiple Myeloma Cell Lines. In: Min WP., Ichim T. (edited) RNA Interference. Methods in Molecular Biology (Methods and Protocols), Vol. 623. Humana Press). Cells were cultured in 96-well plates (100 μl of medium per well, 5 x 10 4 Cells were cultured in DMEM medium supplemented with 10% FBS at 37°C and 5% CO2. Cells were transfected with 2 μl siRNA-LNP (final total concentration of all lipid components was 20 μM, and final siRNA concentration was 16 nM) and incubated for 24 hours. Transfection was performed in triplicate biological assays. RNA was isolated using the RNAeasy Plus Micro Kit (Qiagen). cDNA was prepared using the TATAA GrandScript cDNA Supermix (TATAAbiocenter) according to the manufacturer's recommendations. Quantitative RT-PCR was performed using a LightCycler 480 (Roche Life Science). Primers used to amplify the mRNA encoding TDP2 were: 5'-CGAGAGGAGGGTCTCAAAGAG-3' (SEQ ID NO. 3) and 5'-ATTTCGGGAAGGCTGCTGTC-3' (SEQ ID NO. 4). The mRNA encoding GAPDH was used to normalize the data (primers: 5'-AATCCCATCACCATCTTCCA-3' (SEQ ID NO.5) and 5'-TGGACTCCACGACGTACTCA-3' (SEQ ID NO.6)).

[0354] In all these cases, the novel siRNA-LNP significantly reduced TDP2 mRNA levels in cells compared to the commercially available transfection reagent RNAiMax. In the HEK293T cell line, the novel LNP B02-B05 was nearly 3-fold more efficient than RNAiMax. In the OPM-2 myeloma line, B03 reduced mRNA expression 13-fold better than the commercially available siRNA transfection reagent (Table 9).

[0355] Table 9. Compared with the commercially available transfection reagent RNAiMax, the novel siRNA-LNP (B01-B06) reduced endogenously expressed TDP2 mRNA in HEK293T and OPM-2 cell lines. Statistical data were evaluated using Student's unpaired t-test. P-values ​​are based on the Lipofectamine RNAiMax control transfection mixture; p-values ​​< 0.001 are labeled with the letter "a", and p-values ​​< 0.05 are labeled with the letter "c".

[0356]

[0357] Example 24

[0358] mRNA was transfected in vitro using a novel LNP.

[0359] Human cell lines derived from embryonic kidney cells expressing the large SV40 T antigen (HEK293T), hepatocellular carcinoma cells (HepG2), and human osteosarcoma-derived cell line (U2OS) were cultured in 96-well plates in Durbekojic modified medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2 (100 μl of medium per well containing 5 x 10⁻⁶ cells / well). 4 Cells were transfected with 2 μl of mRNA-LNP B07 to B12 prepared in Example 20 and then incubated for 24 hours. The final total concentration of the lipid components in the wells was 20 μM, and the amount of mRNA encoding the fluorescent protein mKate2 was 100 ng. 2000 was used as a control transfection reagent. Transfection was performed in three bioreplicated assays, each with three technical replicates. Cy5 and mKate2 fluorescence were detected using a BD LSR Fortessa cytometer, and the percentage of Cy5-positive cells (indicating LNP entry into cells), the percentage of cells expressing the fluorescent protein mKate2, and the fluorescence intensity of mKate2 were calculated (Table 10) (Table 11). For fluorescence intensity, data were normalized to commercially available transfection reagents. 2000. Based on Cy5 fluorescence, it is clear that the efficiency of all LNPs entering the cell exceeds 90% (Table 10). Furthermore, the fluorescent mKate2 protein generated from the novel LNP B11 from the transfected mRNA was... The number of cells transfected from the 2000 control group was 2.13 times that of the control group (Table 11).

[0360] Table 10. Transfection efficiency of the novel mRNA-LNP (B07-B12), expressed as the percentage of Cy5-positive cells in cell lines HEK293T and HepG2. The transfection mixture contained lipids labeled with the fluorescent dye Cy5, which allowed for observation of LNP entry into cells.

[0361]

[0362] Table 11. Transfection efficiency of novel mRNA-LNP for the U2OS cell line, expressed as the percentage of cells expressing fluorescent mKate2 protein and the mKate2 fluorescence intensity from particle-transfected mRNA. Statistical data were evaluated using Student's unpaired t-test. p-values ​​are correlated with the control transfection mixture Lipofectamine 2000; p-values ​​< 0.001 are indicated by the letter "a".

[0363]

[0364] Example 25

[0365] mRNA was transfected in vitro using a novel LNP.

[0366] Human cell line (HEK293T) derived from embryonic kidney cells expressing the large SV40T antigen was cultured in 96-well plates in Durbekojic modified medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2 (100 μl of medium per well containing 2.5 x 10⁻⁶ cells / well). 4Cells were transfected with mRNA-LNPs B08a, B11a, B13 to B18, B20 to B25, and B29 prepared in Example 20, and then incubated for 24 hours. The final total concentration of all lipid components in the wells was 20 μM, and the amount of mRNA encoding the fluorescent protein mKate2 was 100 ng. B29 LNP containing the baseline D-Lin-MC3-DMA lipids was used as a control. Transfection was performed in triplicate. The percentage of cells expressing the fluorescent protein mKate2 and the fluorescence intensity of mKate2 were analyzed at wavelengths of 561–610 / 20 nm in a BD LSR Fortessa cytometer (Table 12). For fluorescence intensity, the data were normalized to the baseline D-Lin-MC3-DMA containing LNP (B29). Clearly, all the new LNPs transfected HEK293T cells more efficiently than the benchmark D-Lin-MC3-DMA LNP, as evidenced by a significantly higher percentage of transfected cells and a significant increase in fluorescence intensity. In particular, the fluorescent mKate2 protein produced from the transfected mRNA by the new LNP B22 was 13.66 times greater than that produced by cells transfected with the B29 LNP containing the benchmark D-Lin-MC3-DMA lipid (Table 12).

[0367] Table 12. Transfection efficiency of the novel mRNA-LNP in the HEK293T cell line is shown as the percentage of cells expressing fluorescent mKate2 protein from the mRNA delivered by LNP. Statistical data were evaluated using a Student unpaired t-test. p-values ​​are relative to the control D-Lin-MC3-DMA(B29) LNP; p-values ​​< 0.0001 are indicated by the letter "a", p < 0.001 by "b", and p < 0.01 by "c".

[0368]

[0369] Example 26

[0370] Monitoring the transfection efficacy of novel mRNA-LNPs using Cre-based recombination.

[0371] Mouse embryonic fibroblasts (MEFs) derived from a mouse model with global dual Cre reporter genes (Mazumdar, MD: Genesis 2007, 45:593-605) were seeded in 2.5 x 10⁻⁶ cells of medium per well (100 μl per well). 4Cells were cultured in 96-well plates at 37°C and 5% CO2 for 24 hours in DMEM supplemented with 10% FBS. Similar to Example 20, messenger RNA (mRNA) encoding Cre recombinase was prepared in vitro from linearized plasmids and packaged into LNPs as follows: 300 μl of transfection reagents A13 to A25, A02 and A27 and control transfection reagent A29 sample (containing baseline D-Lin-MC3-DMA lipid and 120 μg mRNA in 300 μl of 10 mM citrate buffer (pH 3.0)) were assembled into LNPs using a microfluidic device similar to that of Example 20. The resulting mRNA-LNP was immediately diluted in 600 μl PBS; the corresponding nanoparticles labeled B31 to B43 (formed by transfection reagents A13 to A25), nanoparticle B30 (formed by transfection reagent A02), nanoparticle B44 (formed by transfection reagent A27), and similarly, the baseline nanoparticle labeled B45 was thus formed by transfection reagent A29.

[0372] Cells were transfected with the corresponding mRNA-LNP (final total concentration of all lipid components in the wells was 20 μM) carrying 100 ng of mRNA encoding Cre recombinase, followed by incubation for 48 h. B45 LNPs containing the baseline D-Lin-MC3-DMA lipids were used as a control. Transfection was performed in quadruplicate. The percentage of cells expressing GFP was analyzed using a BD LSR Fortessa cytometer (wavelengths 488–530 / 30 nm). Again, it was evident that all the novel LNPs transfected mT / mG cells more efficiently, with a significantly higher percentage of cells expressing GFP compared to cells transfected with the baseline D-Lin-MC3-DMA LNP (B45). Interestingly, for all the novel LNPs tested (except B44), the percentage of cells expressing GFP was consistently above 95%, while the LNP containing the baseline D-Lin-MC3-DMA lipids showed only 21% recombinant cells (Table 13).

[0373] Table 13. Comparison of the efficacy of the novel Cre mRNA-LNP in converting td Tomato to GFP in MEF derived from mT / mG reporter mouse embryos. Statistical data were evaluated using the Student unpaired t-test. p-values ​​were correlated with control D-Lin-MC3-DMA(B45) LNP; p-values ​​<0.0001 were indicated by the letter "a".

[0374]

[0375] Example 27

[0376] Biodistribution of novel mRNA-LNP in vivo

[0377] Similar to Example 20, messenger RNA (mRNA) encoding Cre recombinase was prepared in vitro from linearized plasmids and packaged into LNPs as follows: 300 μl of transfection reagent A22 and control transfection reagent A29 samples (containing baseline lipid D-Lin-MC3-DMA and 120 μg mRNA in 300 μl of 10 mM citrate buffer (pH 3.0)) were assembled into LNPs using a microfluidic device similar to that of Example 20. The resulting mRNA-LNPs were immediately diluted in 600 μl of PBS; corresponding B40-labeled nanoparticles were formed from transfection reagent A22. Similarly, baseline nanoparticles labeled B45 were thus formed from transfection reagent A29. The mRNA-LNPs were dialyzed and filtered in PBS. Endotoxin levels <2 EU / ml. mRNA-LNPs (B40 and B45) were administered intravenously at a concentration of 2.0 mg mRNA / kg in each case to four mice carrying global dual Cre reporter genes (Mazumdar, MD: Genesis 2007, 45:593-605) (bred BIOCEV, Vestec) to enable analysis of successful recombination. In cells successfully delivered with mRNA-LNPs carrying Cre recombinase mRNA, chromosomal recombination occurred, followed by excision of the membrane red protein gene (so-called red tomato) and “opening” transcription of the membrane green protein (GFP) gene. Mice, including non-particle control mice, were sacrificed 5 days after particle administration, and histological analysis of specific organs (liver, heart, kidney, lung, and spleen) was performed according to a standardized protocol. Histological imaging analysis showed that the B40 nanoparticles were intactly distributed in the liver, resulting in 50-80% conversion of cells expressing red membrane protein to cells expressing green membrane protein 5 days after administration. In the case of LNP B45 containing D-Lin-MC3-DMA lipids, the conversion rate was approximately 20%. Figure 10 ).

[0378] Further histological analysis was conducted to evaluate tdTomato / GFP transformation in the livers of mT / mG mice using genomic DNA (gDNA) PCR analysis. Briefly, 50 ng of gDNA template isolated from each liver sample was used for PCR reactions with subsequent primers: (5'-AACGTGCTGGTTATTGTGCTG-'3(SEQ ID NO.7); 5'-AAGTCGTGCTGCTTCATGTG-'3(SEQ ID NO.8)). Figure 11Electrophoretic analysis of gDNA PCR amplification from liver samples showed that the PCR product was 530 bp in the case of active Cre recombination at the tdTomato / tGFP locus, while the PCR product was 2943 bp (upper band) in the case of no Cre recombination. The specified B40 mRNA-LNP showed complete Cre recombination in all four tested mice, while in the case of the baseline LNP (B45), recombination was completed in only 50% of the tested mice.

[0379] Example 28

[0380] The efficacy of the new mRNA-LNP in vivo

[0381] Similar to Example 20, messenger RNA (mRNA) encoding hEPO (human erythropoietin) was prepared in vitro from linearized plasmids and packaged into LNPs as follows: 300 μl of transfection reagent A23 or control transfection reagent A29 sample (containing baseline lipid D-Lin-MC3-DMA and 120 μg mRNA in 300 μl of 10 mM citrate buffer (pH 3.0)) was assembled into LNPs using a microfluidic device similar to that of Example 20. The resulting mRNA-LNPs were immediately diluted in 600 μl of PBS; corresponding B46-labeled nanoparticles were thus formed using transfection reagent A23. Similarly, baseline nanoparticles labeled B47 were thus formed using transfection reagent A29. The mRNA-LNPs were dialyzed and filtered in PBS. Endotoxin levels <0.3 EU / ml. In each case, mRNA-LNPs (B46 and B47) were administered intravenously to three C57B1 / 6 mice (BIOCEV, Vestec) at a concentration of 0.5 mg mRNA / kg, with three control C57B1 / 6 mice administered PBS. Six hours post-administration, blood was collected from the mice via the tail vein and allowed to coagulate in serum separation tubes at room temperature. The tubes were then centrifuged at 7000 rpm for 7 minutes, and the serum samples were aliquoted and stored at -80°C until analysis. hEPO concentrations were determined using the hEPO ELISA assay (Catalog #DEP00; R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Hematological characteristics were determined using a standardized protocol.

[0382] Compared to the control B47 LNP, the nanoparticles designated as B46 showed higher human EPO protein production at 6 hours post-administration, but this was not statistically significant. Importantly, unlike the control B47 LNP, the LNP containing the novel lipid (B46) did not alter hematological characteristics. Blood tests following administration of the new LNP remained comparable to the PBS control (Table 14).

[0383] Table 14. Comparison of the in vivo efficacy of new human EPO mRNA-LNP protein production within 6 hours after administration.

[0384]

[0385] Example 29

[0386] The efficacy of the new siRNA-LNP in vivo

[0387] Transfection reagents A02, A14, A16, A17, A22, A23, and A29 from Example 19 were used to form siRNA-LNPs named B48 to B54, respectively, where the siRNA targets the mouse apolipoprotein B (ApoB) gene, a gene involved in cholesterol translocation expressed in hepatocytes (catalog number 238055Apob mouse siPOOL-40 kit, siTOOLs Biotech GmbH). Alternatively, siRNA-LNPs (B55 to B60) containing control non-targeting siRNA-LNPs (encapsulated in 238055Apob mouse siPOOL-40 kit, siTOOLs Biotech GmbH) were assembled as described in Example 20. The hydrodynamic diameter range, measured by dynamic light scattering, was 103 nm to 154 nm, and the packaging efficiency of the siRNA library ranged from 59% to 99%. The siRNA-LNPs were dialyzed into PBS. Endotoxin levels were <2 EU / ml. Mice were fasted for 4 hours, and plasma was collected via retro-orbital blood collection. Three C57B1 / 6 mice (BIOCEV, Czech Center of Phenogenomics, Vestec) were intravenously administered 16 μg of ApoB-targeting siRNA-LNP, while three control mice received 16 μg of non-targeting siRNA-LNP, and three other mice received PBS as a control. All mice were sacrificed two days after LNP administration. Plasma levels of cholesterol, triglycerides, and LDL-C were measured using an automated system at the Czech Center of Phenogenomics, following a standardized protocol.

[0388] Compared with control animals, the clinical biochemistry of plasma markers affected by ApoB knockdown (such as total cholesterol, triglycerides and LDL-C) was significantly reduced, thus demonstrating that the new LNP effectively delivers ApoB siRNA to the liver (Table 15).

[0389] Table 15. Clinical biochemistry of plasma markers indicating effective ApoB knockdown in the liver. Statistical data were evaluated using Student's unpaired t-test. p1 values ​​were always relative to control mice injected with PBS; p2 values ​​were always relative to mice injected with the corresponding (B55 to B60) LNPs containing non-targeting siRNA; p values ​​< 0.0001 were marked "a", p values ​​< 0.001 were marked "b", p values ​​< 0.01 were marked "c", and p values ​​< 0.05 were marked "d". Cases not evaluated were marked "ne".

[0390]

[0391]

[0392] The toxicity of the novel siRNA-LNP was further evaluated by analyzing the clinical biochemistry and hematological characteristics of plasma markers indicating organ failure, compared to the control PBS containing injection saline and the control LNP (B54) containing lipid D-Lin-MC3-DMA. In particular, the novel siRNA-LNP, named B49, showed better toxicological characteristics compared to the control B54 LNP (Table 16).

[0393] Table 16. Clinical biochemistry of plasma markers and hematological characteristics of the novel siRNA-LNP designated B49 compared with the control siRNA-LNP (B54) containing the baseline lipid D-Lin-MC3-DMA. Statistical data were evaluated using Student's unpaired t-test. p1 values ​​were always relative to control mice injected with PBS; p2 values ​​were always relative to mice injected with control B54 LNP; p < 0.01 was labeled "c", and p < 0.05 was labeled "d".

[0394]

[0395] Industrial applicability

[0396] The lipid-containing transfection particles of the present invention can be used for a variety of biological applications in basic research, particularly cell cultures or animal transfection, to deliver active NA and subsequently silence or activate one or more chromosomal genes, edit the genome or transcriptome, or allow expression of proteins encoded in NA inserted using the transfection particles.

[0397] In veterinary and human medicine, transfection particles containing lipids of general formula I are preferred for therapeutic or preventative purposes. Particles containing therapeutic NAs can be administered to animals or humans to silence or activate one or more chromosomal genes, silence or activate immunogens, inhibit or activate signal transduction pathways, edit the genome or transcriptome, or allow the expression of one or more NA-encoded proteins.

[0398] Lipids or transfectants or transfected particles of Formula I can also be used as pharmaceuticals, particularly for gene therapy, and are suitable for treating malignant tumors and / or genetic conditions. They can also be formulated for cosmetic or biotechnological uses.

Claims

1. Lipids of general formula (I) or pharmaceutically acceptable salts thereof (I)， in X is selected from the following groups: -C(=O)NH-, -NHC(=O)-, and -CH2C(=O)NH-; Y is selected from the group consisting of: alkylene C2-C8 chains; R 1 Whether they are the same or different, each R 1 Independently selected from the group consisting of: alkyl C 10 -C 20 and alkenyl C 10 -C 20 The alkyl or alkenyl group is linear or branched, and one or more -CH2- groups in the alkyl or alkenyl group are optionally replaced by one or more groups selected from the following: -OC(=O)- and -C(=O)O-; Z may be the same as or different from each other, and each Z is independently selected from the following groups: hydrogen, -OH, -CH3 and -CH2OH; And T are either the same or different from each other, each T is independently selected from the following groups: -XYN(R 1 )2、-C(=O)O(C1-C3 alkyl), and -C(=O)OH, wherein at least one T is -XYN(R 1 )2.

2. The lipid according to claim 1, wherein Z is -OH or -CH2OH, and T is -C(=O)OH, and Z together with T and the three carbon atoms therebetween form a cyclic lactone containing 4 to 5 carbon atoms.

3. The lipid according to claim 1, wherein X is selected from the group consisting of -C(=O)NH- and -NHC(=O)-.

4. The lipid-like substance according to claim 1, wherein R 1 Independently selected from the group consisting of: alkyl C 12 -C 20 and alkenyl C 12 -C 18 In the alkyl or alkenyl group, one or more -CH2- groups are optionally replaced by one or more groups selected from -OC(=O)- and -C(=O)O-.

5. The lipid of claim 1, wherein all R in the molecule 1 Same, or carrying R in the molecule 1 All nitrogen atoms in the group are separated by two identical R atoms. 1 Or by two different R 1 Replace in the same way.

6. The lipid of claim 1, wherein Z is selected from the group consisting of hydrogen, -CH3 and -CH2OH.

7. The lipid according to claim 1, wherein both substituents T are -XYN(R) 1 )2.

8. The lipid-like substance according to claim 1, wherein: X is -C(=O)NH- or -NHC(=O)-; Y is selected from -(CH2)3-, -(CH2)4-, -(CH2)5-, and -(CH2)6-; R 1 Choose from the group consisting of: linear or branched alkyl C 10 -C 15 It is optionally replaced by one or more groups selected from -OC(=O)- and -C(=O)O-; and linear or branched alkenyl C 12 -C 18 It may be optionally replaced by one or more groups selected from -OC(=O)- and -C(=O)O-; T is selected from -XYN(R) 1 )2、-C(=O)OCH3、 and -C(=O)OH; Z is selected from H, -CH3, and -CH2OH.

9. The lipid of claim 1, wherein the lipid of general formula (I) contains at least two R groups, each containing at least 12 carbon atoms. 1 Substituents.

10. A transfection agent comprising at least one lipid of general formula (I) according to any one of claims 1 to 9 and at least one auxiliary lipid, wherein said auxiliary lipid is selected from the group consisting of cholesterol, 1,2-dioleoyl- sn -Glyceryl-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl- sn -Glyceryl-3-phosphate choline (DOPC), 1,2-distearate- sn -Glyceryl-3-phosphatecholine (DSPC), 1,2-dipalmitoyl- sn -Glyceryl-3-phosphocholine (DPPC), 1,2-dimyristic- sn -Glyceryl-3-phosphatecholine (DMPC), 1,2-dimyristoyl- rac -Glycerin-3-methoxypoly(ethylene glycol)-2000, 1,2-dimyristoyl- sn -glycerol-3-phosphate ethanolamine-poly(ethylene glycol)-2000, 1,2-distearate- sn -Glyceryl-3-phosphate ethanolamine poly(ethylene glycol)-2000 and 1,2-dipalmitoyl- sn -Glyceryl-3-phosphate ethanolamine poly(ethylene glycol)-2000.

11. The transfection agent according to claim 10, comprising 10 to 50 mol.% of at least one lipid of general formula (I) according to any one of claims 1 to 9 and 50 to 90 mol.% of at least one auxiliary lipid.

12. The transfection agent according to claim 10, comprising 15 to 40 mol.% of at least one lipid of formula (I) according to any one of claims 1 to 9, 30 to 55 mol.% of cholesterol, and 20 to 50 mol.% of at least one auxiliary lipid, wherein the auxiliary lipid is selected from the group consisting of 1,2-dioleoyl- sn -Glyceryl-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl- sn -Glyceryl-3-phosphate choline (DOPC), 1,2-distearate- sn -Glyceryl-3-phosphatecholine (DSPC), 1,2-dipalmitoyl- sn -Glyceryl-3-phosphocholine (DPPC), 1,2-dimyristic- sn -Glyceryl-3-phosphatecholine (DMPC), 1,2-dimyristoyl- rac -Glycerin-3-methoxypoly(ethylene glycol)-2000, 1,2-dimyristoyl- sn -glycerol-3-phosphate ethanolamine-poly(ethylene glycol)-2000, 1,2-distearate- sn -Glyceryl-3-phosphate ethanolamine poly(ethylene glycol)-2000 and 1,2-dipalmitoyl- sn -Glyceryl-3-phosphate ethanolamine poly(ethylene glycol)-2000.

13. A transfection particle comprising at least one lipid of formula (I) according to any one of claims 1 to 9 and at least one RNA, and further comprising at least one auxiliary lipid.

14. The transfection particle of claim 13, wherein the RNA is selected from the group consisting of mRNA and siRNA.

15. The use of a lipid of formula (I) according to any one of claims 1 to 9, or a transfection agent according to claim 10, 11 or 12, or a transfection particle according to claim 13 or 14 for in vitro transfection of cells or tissues with at least one RNA.

16. The use according to claim 15, wherein the RNA is selected from the group consisting of mRNA and siRNA.

17. The use according to claim 15 or 16, for silencing or activating one or more chromosomal genes, silencing or activating immune genes, or enabling the expression of one or more proteins encoded by nucleic acids.

18. Use of a lipid of formula (I) according to any one of claims 1 to 9, or a transfection agent according to claim 10, 11 or 12, or a transfection particle according to claim 13 or 14 in the preparation of a medicament for transfecting cells or tissues in vivo with at least one RNA, excluding human embryo transfection for industrial or commercial purposes and excluding transfection for modifying human lineages.

19. The use according to claim 18, wherein the RNA is selected from the group consisting of mRNA and siRNA.

20. The use according to claim 18 or 19, wherein the drug is used to silence or activate one or more chromosomal genes, silence or activate immune genes, or enable the expression of one or more proteins encoded by nucleic acids.

21. A medicament comprising a lipid of formula (I) according to any one of claims 1 to 9, or a transfection agent according to claim 10, 11 or 12, or a transfection particle according to claim 13 or 14.

22. The medicament of claim 21, wherein the medicament is a medicament for gene therapy.

23. A prophylactic vaccine for preventing infectious diseases, comprising a lipid of formula (I) according to any one of claims 1 to 9, or a transfection agent according to claim 10, 11 or 12, or a transfection particle according to claim 13 or 14.

24. A cosmetic formulation for delivering an active ingredient to a site of action, comprising a lipid of formula (I) according to any one of claims 1 to 9, or a transfection agent according to claim 10, 11 or 12, or a transfection particle according to claim 13 or 14.

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