Cyclohexane lipidoid for nucleic acid transfection and method of using the same

Abstract

The present invention relates to lipidoids of general formula I [Formula 1] JPEG2024528684000049.jpg20169 Here, X is -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-, -NHC(=O)-, -NHNHC(=O)-, -C≡C-, -CH=CH-, a 5-membered heterocycle containing at least two nitrogen atoms, -CHC(=O)NH-, -CHC(=S)O-, -CHC(=S)S-, -CHC(=O)NHNH-, -N=CH-, -CH=N-, -NH-N=CH-, and -CH=N-NH-; Y is alkylene C-C 10 Chain;R 1 is 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-OP(=O)(O - )-O(CH2)2-N + (CH3)3, -N + (CH3)2-(CH2)3-SO3 - , -N + (CH3)2-(CH2)2-COO - , [chemical 2] JPEG2024528684000050.jpg18169 and [C3] JPEG2024528684000051.jpg18169 is selected from Here, R 2 is independently selected from hydrogen and -CH; E is independently selected from O and S atoms; n is an integer ranging from 1 to 5; and T is -XYN(R 1 )2, -C(=O)O(C1-C3 alkyl), -C(=O)OCH2CH2OH, [C4] JPEG2024528684000052.jpg13169 [C5] JPEG2024528684000053.jpg18169 [C6] JPEG2024528684000054.jpg18169 -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-(CH2)2-COO - , -NH2, -NHC(=O)CH3, -COO(CH2)2-OP(=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[=NS(=O)2NH2]NH2, -N + (CH3)2-(CH2)3-SO3 - , -N + (CH3)2-(CH2)2-COO - where R 2 and / or, when Z is -OH or -CHOH and T is -C(=O)OH, Z may form a cyclic lactone containing 4-5 carbon atoms together with T and the 3 carbon atoms therebetween; and pharma- ceutically acceptable salts, addition salts and solvates thereof. The lipidoids are useful as transfection agents. The invention further describes transfection reagents, transfection particles comprising the lipidoids, and methods of their use.

Description

Technical Field

[0001] The present invention relates to novel ionizable lipidoids, as well as nucleotides and nucleic acids, and the use of these compounds for transfection and administration to cells and tissues of their synthetic analogs.

Background Art

[0002] The development of nucleic acid (NA)-based therapies has experienced an unprecedented renaissance in recent years. Ribonucleic acid (RNA) therapies are on the rise because they are more effective and have a lower risk of side effects compared to therapeutic deoxyribonucleic acid (DNA) that has been tested so far. Some of these drugs have already reached clinical use, for example, patisiran for hereditary transthyretin amyloidosis, eteplirsen for certain types of Duchenne muscular dystrophy, nusinersen for spinal muscular atrophy, etc. All of these diseases are life-threatening and have no alternative treatments. Ribonucleic acid (RNA) or drugs that may target its use can be divided into three categories depending on whether they target NA or protein or encode protein. The first category includes single-stranded antisense oligonucleotides (13 - 25 nucleotides (nt)) (nusinersen, eteplirsen) that inhibit the translation of messenger RNA (mRNA) or RNA splicing; and short interfering RNA (siRNA, 21 - 23 nt) (patisiran) that degrades mRNA. Therapeutic RNA molecules that target proteins use a type of molecule known as an RNA aptamer. This is designed to modulate the function of specific proteins. An example of such a drug is pegaptanib, which is used to treat neovascular age-related macular degeneration and was first approved as this type of drug in 2004. mRNA-based therapies are mainly used in the preparation of so-called personalized vaccines for cancer and vaccines for infectious diseases (e.g., Zika virus). In viral diseases, mRNA-based prophylactic vaccine candidates for rabies and novel influenza have been shown to induce safe antibody production in healthy volunteers. mRNA therapeutics that replace proteins are also in the preclinical development stage, for example, as a treatment for hemophilia.

[0003] Molecular technologies that enable direct genome editing, especially those based on the CRISPR-Cas9 system, are currently booming. The CRISPR technology is a tool that enables the alteration of DNA sequences and the modification of gene functions. It has the potential to be applied to the repair of gene abnormalities, the treatment and prevention of diseases, and the improvement of agricultural crops. The CRISPR-Cas9 system has been verified in many preclinical and clinical trials, including the treatment of HIV, the treatment of hematological malignancies, and genetic diseases such as sickle cell anemia and β-thalassemia. Subsequently, RNA editing becomes possible with the ADAR system (adenosine deaminase acting on RNA), but so far, from a clinical perspective, the ADAR system seems to be safer. Molecular technologies for directly editing the genome can be delivered to the site of action in the form of mRNA encoding the appropriate enzyme for editing.

[0004] An important factor enabling the safe use of all these technologies (or other NA-based technologies) is the safe and efficient transport to the site of action. The important step is to penetrate the negatively charged NA into the phospholipid membrane of the cell; the process of intentionally introducing NA into eukaryotic cells is called transfection. In the recent decades, carriers (so-called vectors) have been vigorously developed to efficiently transport NA across the cell membrane while protecting it from in vivo degradation (Stewart, M. P.: Chem. Rev. 2018, 118, 7409-7531).

[0005] For the transfection of NA, both viral vectors and non-viral (physical, chemical) vectors are used. Approximately 70% of the clinical trials in the field of gene therapy have been conducted using viral vectors so far, but this approach has many risks (oncogenicity, induction of immune responses, tissue non-specificity, limitations in NA uptake ability, manufacturing complexity). Physical methods (e.g., electroporation) are difficult to use systemically in human medicine.

[0006] In contrast, synthetic chemical vectors are usually less immunogenic, can transport larger amounts of genetic material, and are composed of well-defined molecules, so their structure can be adjusted as needed to enhance efficiency and suppress toxicity. Cationic polymers or cationic lipids are used as chemical vectors and form complexes with negatively charged NA. This complex penetrates the cell membrane while protecting the NA from degradation in the extracellular environment.

[0007] From a structural perspective, so-called lipid nanoparticles (LNPs) are currently the most promising and clinically advanced form among these complexes. Therein, cationic lipids are usually formed together with helper lipids and cholesterol necessary for stable encapsulation of NA, as well as PEGylated lipids that prevent aggregation and affect particle size and transfection efficiency, as shown, for example, 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.

[0008] Synthetic cationic lipids and lipidoids (synthetic molecules similar to lipids that differ in having a larger number of hydrophobic chains) are formed by a cationic domain and a hydrophobic domain. To date, many of these substances have been developed with high structural variability in both domains, by both targeted design and testing of combinatorially generated libraries.

[0009] Lipids and lipidoids such as D-Lin-MC3-DMA, C12-200, cKK-E12, SA2-SC8 have been specially developed for siRNA transfection (Dong, Y.: Adv. Drug Deliv. Rev. 2019, 144, 133-147). Formulations containing D-Lin-MC3-DMA were recently (August 2018) introduced into the clinic under the name Onpattro (formerly Patisiran), becoming the first approved siRNA drug (Zhang, X.: J. Clin. Pharmacol. 2020, 60 (1), 37-49). However, since formulations developed for siRNA are not always effective for mRNA, target-specific optimization is necessary (Cullis, P...: Mol. Ther. 2017, 25 (7), 1467-1475).

[0010] Ionizable lipids and lipidoids such as D-Lin-MC3-DMA, C12-200, cKK-E12, TT3 are used to transfect mRNA (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 are also used for DNA transfection. Here too, it should be emphasized that transfection systems optimized for small molecule (siRNA) transfection are not necessarily suitable for DNA transfection, and formulations developed for mRNA may not be effective for DNA (Buck, J.: ACS Nano 2019, 13, 3754-3782).

[0011] Despite the huge potential for treatment, synthetic vectors based on ionizable lipids have been little used clinically so far, so there is a need to develop new systems that are more efficient and have very low in vivo toxicity. SUMMARY OF THE INVENTION

[0012] The present invention provides solutions to the problems of the efficiency of transfection and targeted delivery of nucleotides and nucleic acids and their synthetic analogs using ionizable (cationic) lipids, as well as the toxicity of these lipids to target organisms or cells. We have found that when cyclohexane is used as the central core of an ionizable lipidoid, the transfection efficiency of such lipidoids is significantly improved compared to previously known solutions. At the same time, these cyclohexane-containing lipidoids exhibit extremely low cytotoxicity at appropriate dosages. The specific properties of the cyclohexane core used as the central structural motif of the new ionizable lipidoids are the steric complexity in its vicinity and the broad range of substitution when compared to previously known ionizable lipids and lipidoids.

[0013] The object of the present invention is an ionizable lipidoid of general formula (I), wherein

Chemical formula

Chem.

Chem.

Chem.

Chem.

[0014] The term "alkyl" means a saturated hydrocarbon chain, which may be linear, branched, cyclic or cycle-containing, and is derived from an alkane by removing one hydrogen atom.

[0015] The term "alkenyl" means a hydrocarbon chain containing at least one double bond between carbon atoms, and is derived from an alkene by removing one hydrogen atom. The hydrocarbon chain may be linear, branched, cyclic or cycle-containing.

[0016] The term "alkynyl" means a hydrocarbon chain containing at least one triple bond between carbon atoms and optionally also containing one or more double bonds between carbon atoms, and is derived from an alkyne by removing one hydrogen atom. The hydrocarbon chain may be linear, branched, cyclic or cycle-containing.

[0017] The term "alkylene" means a divalent saturated hydrocarbon chain, which may be linear, branched, cyclic or cycle-containing, preferably linear. This chain has a valence of 2, that is, it is derived from an alkane by removing two hydrogen atoms from different carbon atoms and is bonded as a linker or crosslink via two single bonds.

[0018] The terms "branched-chain alkyl", "branched-chain alkenyl", "branched-chain alkynyl" mean an alkyl, alkenyl or alkynyl containing 1 to 5 branches (hydrocarbon chains) attached to the main hydrocarbon chain.

[0019] 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 and forms a pharmaceutically acceptable salt. Such anions can be selected, for example, from the group consisting of acetate, aspartate, besylate, benzoate, besylate, bicarbonate, bitartrate, bromide, camsylate, carbonate, chloride, citrate, decanoate, edetate, esylate, fumarate, gluceptate, gluconate, glutamate, glycolate, hexanoate, iodate, lactate, malate, maleate, mandelate, mesylate, methanesulfonate, napsylate, nitrate, octanoate, oleate, palmitate, pantothenate, phosphate, polygalacturonate, propionate, salicylate, stearate, succinate, sulfate, tartrate, and tosylate.

[0020] When the compound of formula I contains a chiral center, formula I includes not only pure enantiomers but also mixtures of enantiomers including racemates.

[0021] Formula I includes the compound of formula I in free form, as well as salts, addition salts (with acids or bases), and / or solvates in the form of hydrates or alcohol solvates.

[0022] 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 5-membered heterocycle containing at least two nitrogen atoms. Preferably, X is -C(=O)NH- or -NHC(=O)-.

[0023] R 1 chains may be the same throughout the molecule or may differ from each other, or the R 1 chains may be the same for one nitrogen atom but not the same throughout the molecule. Preferably, R 1The lock is the same throughout the molecule or, for ease of synthesis, all nitrogen atoms are identically substituted (two identical Rs 1 , or two different Rs 1 ). At least one R 1 substituent is a fatty chain of at least 8 carbon atoms, and R 1 is selected from the group comprising alkyl C 1 -C 46 , alkenyl C 2 -C 46 , alkynyl C 2 -C 46 , where said alkyl, alkenyl or alkynyl may be straight-chain or branched-chain, and where in said alkyl, alkenyl or alkynyl, one or more -CH 2 - groups may optionally be substituted with one or more groups selected from -CH(OH)-, -OC(=O)-, -C(=O)O-, -S-S-, -C(=O)NH-, -NHC(=O)-, -O-, and -S-; Here, when said alkyl, alkenyl or alkynyl is branched-chain, one or more >CH- groups may optionally be substituted with >C(OH)-; Here, when R 1 is alkyl C 1 -C 4 , one hydrogen of the terminal -CH 3 group may be substituted with Z.

[0024] In one embodiment, R 1 is independently selected from the group comprising alkyl C 1 -C 46 , and alkenyl C 2 -C 46 , where in said alkyl or alkenyl, one or more -CH 2 - groups may optionally be substituted with one or more groups selected from -CH(OH)-, -OC(=O)-, -C(=O)O-.

[0025] Preferably, R 1 is straight-chain or branched-chain alkyl C 8 -C20 and straight-chain or branched alkenyl C 8 -C 20 independently selected from the group consisting of, wherein in said alkyl or alkenyl, one or more -CH 2 - groups may optionally be substituted with one or more groups selected from -OC(=O)-, -C(=O)O-. More preferably, R 1 is a straight-chain or branched alkyl C 10 -C 15 optionally substituted with one or more groups selected from -OC(=O)-, -C(=O)O-, and a straight-chain or branched alkenyl C 12 -C 18 selected from the group consisting of.

[0026] Preferably, the alkenyl chain contains from 1 to 5 carbon-carbon double bonds.

[0027] In one embodiment, all R 1 in the molecule are the same.

[0028] In one embodiment, each nitrogen atom in the molecule having an R 1 group is identically substituted by two identical R 1 (however, R 1 bonded to different nitrogen atoms within the same lipidoid structure may not be the same).

[0029] In one embodiment, two different R 1 are bonded to one nitrogen atom.

[0030] To enable the lipid-like properties of the molecule, at least one R 1 in the lipidoid structure must contain at least 8 carbon atoms, preferably at least 10 carbon atoms, more preferably at least 12 carbon atoms. Preferably, two R 1contains at least 8 carbon atoms, preferably at least 10 carbon atoms, more preferably at least 12 carbon atoms.

[0031] In one embodiment, one substituent T is -X-Y-N(R 1 ) 2 is.

[0032] In another embodiment, both substituents T are -X-Y-N(R 1 ) 2 is.

[0033] In the latter case, the resulting lipidoid molecule has the general formula II,

Chemical formula

[0034] The structure of general formula II preferably has the following substituents: X is -C(=O)NH-, -C(=O)O-, -C(=S)O-, -C(=O)S-, -C(=S)S-, -C(=O)NHNH-, -CH 2 -, -O-, -OC(=O)-, -S-, -SC(=O)-, -NH-, -NHNH-, -NHC(=O)-, -NHNHC(=O)-, -C≡C-, -CH=CH-, a 5-membered heterocyclic ring containing at least 2 nitrogen atoms, -CH 2 C(=O)NH-, -CH 2 C(=O)O-, -CH 2 C(=S)O-, -CH 2 C(=S)S-, -CH 2 C(=O)NHNH-, -N=CH-, -CH=N-; Y is an alkylene chain C 2 -C 10 where one or more -CH 2 - groups may optionally be substituted with one or more O and / or S atoms; Z are the same as or different from each other, and each Z is a hydrogen atom, -OH, -CH 3 -, CH 2 OH, -NH 2 , -C(=O)NH 2 , -CONH(CH 2 ) 2 OH, -CON[(CH 2 ) 2 OH] 2 , -CONHCH(CH 2 OH) 2 , -CONHCH 2 CH(-OH)CH 2 OH, -CONH(CH 2 ) 2 C(=O)NH 2 , -CON[CH 2 C(=O)NH 2 2 , -CONHCH[C(=O)NH 2 2 , -CONH(CH 2 ) 2 NHC(=O)NH 2 , -N + (CH 3 ) 2 -(CH 2 ) 3 -SO 3 - , -N + (CH 3 ) 2 -(CH 2 ) 2 -COO - ,

Chem.

Chem.

[0035] Linker X is formed by the attachment reaction of the amine part of the molecule to the central cyclohexane core. Thus these may be different linkers that rely on reactions selected via click reactions (e.g., azide-alkyne cycloaddition reaction), such as the formation of esters, amides and their analogues.

[0036] X is therefore -C(=O)NH-, -C(=O)O-, -C(=S)O-, -C(=O)S-, -C(=S)S-, -C(=O)NHNH-, -CH 2 -, -O-, -OC(=O)-, -S-, -SC(=O)-, -NH-, -NHNH-, -NHC(=O)-, -NHNHC(=O)-, -C≡C-, -CH=CH-, a 5-membered heterocycle containing at least two nitrogen atoms, -CH 2 C(=O)NH-, -CH 2 C(=O)O-, -CH 2 C(=S)O-, -CH 2 C(=S)S-, -CH 2 C(=O)NHNH-, -N=CH-, -CH=N-, -NH-N=CH-, and -CH=N-NH-. Preferably, X is selected from -C(=O)NH-, -NHC(=O)-, a 5-membered heterocycle containing at least two nitrogen atoms, -OC(=O)-, and -C(=O)O-.

[0037] Linker Y is an alkylene chain that provides at least the minimum distance of an amine from Linker X and the cyclohexane core. Y is a C 2 ~C 10 alkylene chain, preferably a C 2 ~C 8 alkylene chain, where one or more -CH 2 - groups may optionally be substituted with one or more O and / or S atoms.

[0038] Substituent Z can further modify the properties of the compounds of Formulas I and II.

[0039] In one embodiment, Z is hydrogen, -OH, -CH 3 , -CH 2 OH, -NH 2 , -C(=O)NH 2 , -CONH(CH 2 ) 2 OH, -CON[(CH 2 ) 2 OH] 2 , -CONHCH(CH 2 OH) 2 , -CONHCH 2 CH(-OH)CH 2 OH, -CONH(CH 2 ) 2 C(=O)NH 2 , -CON[CH 2 C(=O)NH 2 2 , -CONHCH[C(=O)NH 2 2 , -CONH(CH 2 ) 2 NHC(=O)NH 2 , -N + (CH 3 ) 2 -(CH 2 ) 3 -SO 3 - , -N + (CH 3 ) 2 -(CH 2 )​​2 -COO - 、

Chem.

Chem.

[0040] The substituent T can further modify the properties of the compounds of Formulas I and II. In one embodiment, T is -X-Y-N(R 1 ) 2 , -C(=O)O(C 1 -C 3 alkyl), -C(=O)OCH 2 CH 2 OH, -C(=O)NH 2 ,

Chem.

Chem.

Chem.

Chemical formula

[0041] In a specific embodiment, the lipidoid of formula I is hydrogen, -OH, -CH 3 , -CH 2 OH, Z selected from the group consisting of; and -X-Y-N(R 1 ) 2 , -C(=O)O(C 1 ~C 3 alkyl),

Chemical formula

Chemical formula

[0042] Specifically, the following lipidoids are preferred: cis,cis-N 1 ,N 3 ,N 5 -Tris(6-(didodecylamino)hexyl)cyclohexane-1,3,5-tricarboxamide (3); cis,cis-N 1 ,N 3 ,N 5 -Tris(6-(di((hexyloxycarbonyl)butyl)amino)hexyl)cyclohexane-1,3,5-tricarboxamide (7); cis,cis-N 1 ,N 3 ,N 5 -Tris(6-(didodecylamino)hexyl)-1,3,5-trimethylcyclohexane-1,3,5-tricarboxamide (8); cis,cis-N 1 ,N 3 ,N 5 -Tris(3-(didodecylamino)propyl)-1,3,5-trimethylcyclohexane-1,3,5-tricarboxamide (11); cis,cis-N 1 ,N 3 ,N 5-Tris(6-(di((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)hexyl)-1,3,5-trimethylcyclohexane-1,3,5-tricarboxamide(15); Hexa(2-octyl) cis,cis-6,6’,6’’,6’’’,6’’’’,6’’’’’-((((cyclohexane-1,3,5-tricarbonyl)tris(azanediyl))tris(hexane-6,1-diyl))tris(azantriyl))hexahexanoate(19); Hexakis(3-methylhexyl) cis,cis-7,7’,7’’,7’’’,7’’’’,7’’’’’-((((cyclohexane-1,3,5-tricarbonyl)tris(azanediyl))tris(hexane-6,1-diyl))tris(azantriyl))hexaheptanoate(23); Hexakis((E)-3,7-dimethylocta-2,6-dien-1-yl) cis,cis-5,5’,5’’,5’’’,5’’’’,5’’’’’-((((cyclohexane-1,3,5-tricarbonyl)tris(azanediyl))tris(hexane-6,1-diyl))tris(azantriyl))hexapentanoate(27); Hexakis((Z)-3,7-dimethylocta-2,6-dien-1-yl) cis,cis-5,5’,5’’,5’’’,5’’’’,5’’’’’-((((cyclohexane-1,3,5-tricarbonyl)tris(azanediyl))tris(hexane-6,1-diyl))tris(azantriyl))hexapentanoate(31); Tetra(2-octyl) cis,cis-6,6’,6’’,6’’’-((((5-(methoxycarbonyl)cyclohexane-1,3-dicarbonyl)bis(azanediyl))bis(hexane-6,1-diyl))bis(azantriyl))tetrahexanoate(32); Tetrakis(3-methylhexyl) cis,cis-7,7’,7’’,7’’’-((((5-(methoxycarbonyl)cyclohexane-1,3-dicarbonyl)bis(azanediyl)bis(hexane-6,1-diyl)bis(azantriyl))tetraheptanoate(33); cis,cis-3,5-bis((6-(bis(6-(octan-2-yloxy)-6-oxohexyl)amino)hexyl)carbamoyl)cyclohexane-1-carboxylic acid (35); tetra(octan-2-yl) cis,cis-6,6’,6’’,6’’’-((((5-(pyrrolidine-1-carbonyl)cyclohexane-1,3-dicarbonyl)bis(azanediyl)bis(hexane-6,1-diyl)bis(azantriyl))tetrahexanoate (36); hexakis(3-methylhexyl) cis,cis-7,7’,7’’,7’’’,7’’’’,7’’’’’-((((1,3,5-trimethylcyclohexane-1,3,5-tricarbonyl)tris(azanediyl))tris(hexane-6,1-diyl))tris(azantriyl))hexaheptanoate (40); cis,cis-6,6’,6’’,6’’’’,6’’’’’-((((1,3,5-trimethylcyclohexane-1,3,5-tricarbonyl)tris(azanediyl))tris(hexane-6,1-diyl))tris(azantriyl))hexahexanoate (41); cis,cis-3,5-bis((6-(bis(6-(octan-2-yloxy)-6-oxohexyl)amino)hexyl)carbamoyl)-1,3,5-trimethylcyclohexane-1-carboxylic acid (42); tetra(octan-2-yl) cis,cis-6,6’,6’’,6’’’-((((1,3,5-tris((benzyloxy)methyl)-5-(bis(6-(octan-2-yloxy)-6-oxohexyl)carbamoyl)cyclohexane-1,3-dicarbonyl)bis(azanediyl))bis(hexane-6,1-diyl))bis(azantriyl))tetrahexanoate (45); cis,cis-1,3,5-tris((benzyloxy)methyl)-3,5-bis((6-(bis(6-(octan-2-yloxy)-6-oxohexyl)amino)hexyl)carbamoyl)cyclohexane-1-carboxylic acid (46); Hexa(octan-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(azatriyl)) hexahexanoate (47); cis,cis-3,5-bis((6-(bis(6-(octan-2-yloxy)-6-oxohexyl)amino)hexyl)carbamoyl)-1,3,5-tris(hydroxymethyl)cyclohexane-1-carboxylic acid (48); Tetra(octan-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(azatriyl)) tetrahexanoate (49); Tetra(octan-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(azatriyl)) tetrahexanoate (50); cis,cis-N 1 ,N 7 -bis(6-(didodecylamino)hexyl)-5,7-bis(hydroxymethyl)-4-oxo-3-oxabicyclo[3.3.1]nonane-1,7-dicarboxamide (52); cis,cis-N,N’,N’’-(cyclohexane-1,3,5-triyl)tris(6-(didodecylamino)hexanamide) (56).

[0043] The compounds of formula I and II are cyclohexane precursors substituted with precursors of the groups Z at the 1, 3, and 5 positions and the linker X at the 1 position; the 1 and 3 positions; or the 1, 3, and 5 positions, and a general formula X’-Y-N(R 1 ) 2prepared by the corresponding reaction with a tertiary amine, where X’ is a precursor group of linker X. Preferably, X’ is -NH 2 or an activated carboxyl group. The tertiary amine of the general formula X’-Y-N(R 1 ) 2 can be prepared by reactions and procedures known to those skilled in the art, and some suitable amines are also commercially available.

[0044] In one embodiment, the compound of formula I is preferably prepared by reacting a compound of formula III with a diamine of formula IV,

Chemical formula

Chemical formula

[0045] In a more specific example, the compound of formula II is 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 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 above for formula II.

[0046] Another object of the present invention is a transfection agent comprising at least one lipidoid of general formula I or II and at least one helper lipid. The transfection agent can be prepared in the form of a solution by dissolving and mixing the components, or can be prepared in the form of particles by techniques used in conventional nanoparticle technologies such as microfluidic mixing. The particles are generally understood to mean nanoparticles having dimensions in the range of 1 to 500 nm. Preferably, the dimensions of the nanoparticles are in the range of 30 to 250 nm, more preferably in the range of 40 to 150 nm.

[0047] In one embodiment, the transfection agent contains at least one lipidoid of general formula I in an amount of 10 to 50 mol% and at least one helper lipid in an amount of 50 to 90 mol%. Preferably, the transfection agent contains at least one lipidoid of general formula I in an amount of 15 to 40 mol% and at least one helper lipid in an amount of 60 to 85 mol%. In some preferred embodiments, the transfection agent comprises at least one lipidoid of general formula I in an amount of 15 to 40 mol%, cholesterol in an amount of 30 to 55 mol%, and at least one other helper lipid in an amount of 20 to 50 mol%.

[0048] In a particularly preferred embodiment, the transfection agent contains at least one lipidoid of general formula I in an amount of 15 to 40 mol%, cholesterol in an amount of 30 to 55 mol%, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine in an amount of 20 to 45 mol%, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 in an amount of 0.5 to 5 mol%.

[0049] The present invention also relates to transfection particles comprising at least one lipidoid of general formula I, at least one nucleic acid and / or a part thereof and / or a nucleic acid derivative, and preferably further at least one helper lipid. The transfection particles can be prepared, for example, by mixing a solution of a lipidoid of general formula I, optionally containing a helper lipid, with a solution of a nucleic acid and / or a part thereof and / or a nucleic acid derivative. The mixing can be carried out by techniques used in conventional nanoparticle technology, such as microfluidic mixing.

[0050] The weight ratio of the total amount of the nucleic acid and / or a part thereof and / or a nucleic acid derivative to the total amount of the lipidoid of general formula I and the helper lipid in the transfection particles is preferably in the range of 1:2 to 1:500, more preferably in the range of 1:5 to 1:100. Specifically exemplified, in the particles prepared in the following examples, this ratio was about 1:9 for mRNA and about 1:68 for siRNA.

[0051] The transfection particles are usually nanoparticles, which are generally understood to mean particles having dimensions in the range of 1 to 500 nm. Typically, the dimensions of the transfection nanoparticles are in the range of 50 to 250 nm, more preferably 40 to 150 nm.

[0052] When the structure of the transfection particles was observed by cryo-transmission electron microscopy, the transfection particles were compact layered lipid nanoparticles containing nucleic acid inside.

[0053] The helper lipids contained in the transfection reagent and the transfection particles are mainly neutral lipids, sterols, or lipid conjugates of lipids and hydrophilic polymers.

[0054] Neutral lipids can exist in a net charge of zero under physiological conditions and can be in an uncharged form or an electrically neutral zwitterionic form. Neutral lipids can be selected from, for example, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 5).

[0055] Sterols can be selected from, for example, cholesterol, β-sitosterol, stigmasterol, campesterol, fucosterol, avenasterol, fecosterol, brassicasterol, ergosterol, and 9,11-dehydroergosterol. Preferably, the sterol is cholesterol.

[0056] Lipid conjugates having a hydrophilic polymer include a lipid moiety and a polymer moiety such as poly(ethylene glycol), poly(2-ethyl-2-oxazoline), poly(2-methyl-2-oxazoline), poly(glycerol), poly(sarcosine). Preferably, the polymer moiety consists of poly(ethylene glycol) having a molecular weight in the range of about 500 to about 10,000 Da, more preferably about 1,000 to about 5,000 Da. Lipid conjugates with hydrophilic polymers may be selected, for example, from 1,2-dimyristoyl-rac-glycero-3-methoxypoly(ethylene glycol)-2000, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-2000, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine poly(ethylene glycol)-2000, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine poly(ethylene glycol)-2000, and similar conjugates. The lipid conjugate with the hydrophilic polymer may preferably be 1,2-dimyristoyl-rac-glycero-3-methoxypoly(ethylene glycol)-2000.

[0057] A nucleic acid or a part thereof contains one or more nucleotides and / or deoxynucleotides. The nucleic acid or a part thereof may be a therapeutic agent, a diagnostic agent or a prophylactic agent, or may provide a label to the cells or tissues into which they are transfected. The compounds of formula I therefore mainly have therapeutic or biotechnological uses.

[0058] The term "nucleic acid or a part thereof" preferably means one or more nucleic acid types selected from oligonucleotides (1 to 100 nucleotides, such as aptamers), cyclic dinucleotides (such as 2',3'-cGAMP), antisense oligonucleotides, deoxyribonucleic acids (single-stranded DNA, double-stranded DNA, cDNA, gene or plasmid DNA encoding a gene), ribonucleic acids, typically messenger RNA (mRNA), transfer RNA (tRNA), short interfering RNA (siRNA), double-stranded RNA, microRNA (miRNA), PiwiRNA (piRNA), antisense RNA (asRNA), guide RNA (gRNA) for the CRISPR system, and combinations thereof (typically, for example, gRNA encoding Cas9 nuclease and mRNA, Cas13a / C2c2 and Cas13b, or similar nucleases suitable for use in the modification of the genome of a host cell or tissue or the transcriptome of a host cell or tissue in CRISPR, CRISPRi and other variations and subsequent). Further, all nucleic acids (NA) disclosed herein can be formed or modified with synthetic base analogs, for example, to enhance stability in biological systems. Synthetic NA analogs specifically include the following substitutions: 2'-O-methyl, 2'-O-methoxy-ethyl, 2'-fluoro, a methylene bridge between the 2'-oxygen and 4'-carbon of the pentose ring (so-called locked nucleic acid), phosphorylation at the 5'-end and / or 3'-end of the strand, boranophosphonate, or phosphorothioate.

[0059] The present invention further includes the use of a lipidoid or transfection agent or transfection particle of formula I for transfecting cells or tissues in vitro with a nucleic acid and / or a part thereof and / or a nucleic acid derivative. Further, the present invention includes a lipidoid or transfection particle of formula I for use in transfecting cells or tissues (excluding the transfection of human embryos for industrial or commercial use and excluding the modification of the human germ line) with a nucleic acid and / or a part thereof and / or a nucleic acid derivative in vivo.

[0060] Transfection particles containing a lipidoid of general formula I are useful for many biological applications in basic research, in particular for the transfection of cell cultures or animals for delivering active nucleic acids, and subsequent silencing or activation of chromosomal genes or genes, genome editing (gene excision, gene insertion or mutagenesis) or transcriptome editing, or enabling the expression of a given protein encoded by a nucleic acid inserted by the transfection particle, so-called "in trans".

[0061] In veterinary and human medicine, transfection particles containing a lipidoid of general formula I can preferably be used for therapeutic or prophylactic purposes. Particles containing a therapeutic nucleic acid are administered to an animal or a human to silence or activate a chromosomal gene, to silence or activate an immunogen, to inhibit or activate a signaling pathway, to edit the genome (gene excision, gene insertion or mutagenesis) or to edit the transcriptome, or to enable the expression of a protein encoded by the nucleic acid.

[0062] The present invention also provides a lipidoid or transfection agent or transfection particle of general formula I for use as a medicament, in particular for gene therapy. In particular, these are suitable for use in the treatment of malignant tumors and / or genetic diseases.

[0063] The lipidoid of general formula (I) or the transfection agent or transfection particle according to the invention is also suitable for use as a prophylactic vaccine, preferably a vaccine for the prevention of infectious diseases.

[0064] The lipidoid or transfection particle of general formula I can be formulated for therapeutic, cosmetic or biotechnology use in the form of a formulation with pharmaceutically acceptable excipients. The formulation can be in liquid or solid form. Liquid forms include, for example, solutions, suspensions, dispersions, gels, ointments suitable for injection or oral administration. Solid forms include, for example, capsules, tablets, coated tablets, powders, suppositories and the like.

[0065] Liquid formulations can be nebulized with an inert gas. The nebulized suspension can be inhaled directly or from a nebulizing device, and the nebulizing device can be attached to a face mask or a respirator. Solid formulations can be administered using a dry powder inhaler. Suspensions or dry powder formulations can be administered orally or nasally from a suitable device.

[0066] For application to the skin or mucosa, the formulation can also be prepared in the form of creams, gels, ointments, pastes, balms, liquids, etc. and applied directly to the site of action.

[0067] Pharmaceutically acceptable excipients include solvents, solubility modifiers, pH adjusters, carriers, fillers, binders, lubricants, disintegrants, preservatives, adsorbents, viscosity modifiers, agents that affect the sensory properties such as the taste, odor, color of the formulation, and the like.

[0068] Furthermore, the lipidoid or transfection agent or transfection particle of general formula I can preferably be used for the purposes of the cosmetics industry in order to deliver the active substance to the site of action. Transfection particles having an active substance can be prepared in the form of creams, gels, ointments, pastes, balms, liquids, etc. and can be used as makeup, hair cosmetics or personal hygiene products.

Brief Description of the Drawings

[0069]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Modes for Carrying Out the Invention

[0070] (Examples) List of Abbreviations: eq. equivalent R f Retention factor TLC Thin layer chromatography RVE Rotary vacuum evaporator rt Room temperature v / v Volume / volume br s Broad signal s Singlet d Doublet t Triplet m Multiplet dd Doublet of doublets J Interaction constant δ Chemical shift HRMS High resolution mass spectrometry EI Electron ionization ESI Electrospray ionization MALDI Matrix-assisted laser desorption ionization GC-MS Gas chromatography - mass spectrometry IR Infrared spectroscopy NMR Nuclear magnetic resonance CE5 95:5 (v / v) cyclohexane - ethyl acetate mixture CE20 80:20 (v / v) cyclohexane - ethyl acetate mixture CE50 50:50 (v / v) cyclohexane - ethyl acetate mixture D1 75:22:3 (v / v / v) dichloromethane - methanol - 25% aqueous NH 3 mixture D2 175:22:3 (v / v / v) dichloromethane - methanol - 25% aqueous NH 3 mixture D3 275:22:3 (v / v / v) dichloromethane - methanol - 25% aqueous NH 3 mixture D4 375:22:3 (v / v / v) dichloromethane - methanol - 25% aqueous NH 3 mixture TFA Trifluoroacetic acid DIPEA N,N - Diisopropylethylamine DMF N,N-Dimethylformamide DCM Dichloromethane ACN Acetonitrile DIC Diisopropylcarbodiimide DMAP 4-Dimethylaminopyridine PyBroP Bromotripyrrolidinophosphonium hexafluorophosphate TCE 1,1,2,2-Tetrachloroethane LNP Lipid nanoparticles NA Nucleic acid DNA Deoxyribonucleic acid RNA Ribonucleic acid mRNA Messenger RNA siRNA Small interfering RNA tRNA Transfer RNA miRNA MicroRNA ssDNA / RNA Single-stranded DNA / RNA dsDNA / RNA Double-stranded DNA / RNA DMG-PEG 2000 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 DOPE 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine DOPC 1,2-Dioleoyl-sn-glycero-3-phosphocholine DSPC 1,2-Distearoyl-sn-glycero-3-phosphocholine DOPE-Cy5 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 5) Lip2000 Lipofectamine® 2000 (Invitrogen) h hour Example 1 N 1 ,N 1 -Didodecylethane-1,6-diamine 2 Into a 500 ml round-bottom flask equipped with a calcium chloride drying tube and a magnetic stirrer, N in DCM (100 ml) 1A solution of -tert-butyloxycarbonyl-1,6-diaminohexane (5.0 g, 23.11 mmol) was placed and cooled to 0 °C in an ice bath. While stirring vigorously, n-dodecylaldehyde (15.38 ml, 69.34 mmol, 3 eq.) was added, followed by sodium triacetoxyborohydride (14.70 g, 69.34 mmol, 3 eq.) added in three portions over 10 minutes. The cooling bath was removed and the reaction mixture was stirred at room temperature for 2 hours. The progress of the reaction was monitored by TLC on a TLC plate pre-saturated with ammonia using a hexane-ethyl acetate mobile phase of 80:20 (v / v) (detected with ninhydrin). After completion of the reaction, aqueous NaOH solution (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 × 50 ml), the combined organic phases were washed with brine (50 ml), dried over anhydrous sodium sulfate, filtered through an S2 frit, and the solvent was evaporated by RVE. The dark oily residue was purified by silica gel column chromatography using a linear gradient of ethyl acetate in hexane (10 - 30%). Amine 1 (3.67 g, 28.7%) was obtained as a yellowish oil.

[0071] Trifluoroacetic acid (10 ml) was added to a solution of Compound 1 in DCM (10 ml), cooled to 0 °C while stirring in an ice bath, and the reaction mixture was left at 0 °C for 3 hours. Then the solution was poured into a 1 l separable flask and diluted with 20% aqueous Na 2 CO 3 (300 ml), and the product was extracted with DCM (250 ml, 2 × 50 ml). The combined organic phases were washed with brine (100 ml), dried over anhydrous sodium sulfate, filtered through an S2 frit, and the solvent was evaporated by RVE. The crude product was purified by silica gel column chromatography using a linear gradient of D1 in DCM (0 - 70%). Diamine 2 (2.17 g, yield 72.2%; R f 0.31 on a TLC plate pre-saturated with ammonia with mobile phase D2, detected with ninhydrin) was obtained in the form of a yellowish oil.

[0072] 11H NMR (600 MHz, CDCl 3 ): 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, 0.87 ppm. 13 13C NMR (150.9 MHz, CDCl 3 ): 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, 14.10 ppm. IR (film): ν max / cm -1 = 3374 w and 3294 w (νNH 2 ), 2797 m (ν s N - CH 2 ), 2956 s (ν as CH 3 ), 2924 vs (ν as CH 2 ), 2853 s (ν s CH 2 ), 1467 m and 1455 m, sh (β s CH 2 and δ as CH 3 ), 1378 w and 1367 w (δ s CH 3 ), 721 m (β as CH 2 ). HRMS (ESI): C 30 H 65 N 2 [M + H] + calculated for m / z was 453.51423; found 453.51340.

[0073] cis,cis - N 1 ,N 3 ,N 5 -Tris(6-(dodecylamino)hexyl)cyclohexane - 1,3,5 - tricarboxamide 3 To cis,cis-1,3,5-cyclohexanetricarboxylic acid (17 mg, 0.079 mmol), DMF (2 μl) and thionyl chloride (300 μl) were added, and the suspension was stirred in a sealed vial at 70 °C for 30 minutes, and the reaction mixture was gradually clarified into a homogeneous colorless solution. Excess SOCl 2 was blown off with a stream of dry nitrogen at 70 °C, and the residue was dried under vacuum (for 10 minutes) and cooled to rt. N 1 ,N 1 -Didodecylhexane-1,6-diamine 2 (142 mg, 0.315 mmol, 4 eq.) and a solution of DIPEA (137 μl, 0.786 mmol, 10 eq.) were added via a septum using a syringe into a mixture of DCM (1.5 ml) and DMF (0.5 ml), and the reaction mixture was stirred for 10 minutes. Then, the reaction mixture was adsorbed onto silica gel for chromatography (10 g), and the solvent was removed by RVE. The residue was purified by silica gel column chromatography (40 g) using a linear gradient of D1 in DCM (0 - 55%). Lipidoide 3 was obtained in the form of a yellowish semi-solid (81 mg, yield 67.7%; R f 0.36, detected with ninhydrin). 1 H NMR (600 MHz, CDCl 3 ): 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, 0.87 ppm. 13 C NMR (150.9 MHz, CDCl 3 ): 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, 14.09 ppm. IR (CCl 4 ): ν max / cm -1 = 3293 m (νNH), 1640 s (amide I) and 1550 m (amide II), 2964 s, sh (ν as CH 3 ), 2871 m, sh (ν s CH 3 ), 2927 vs (ν asCH 2 ), 2856s, sh(ν s CH 2 ), 1468m and 1460m, 1445w(β s CH 2 and δ as CH 3 ), 1378w(δ s CH 3 ), 721w(β as CH 2 and δ as CH 2 ); HRMS(MALDI): C 99 H 199 N 6 O 3 [M + H] + The calculated m / z for it was 1520.5598; the measured value found was 1520.5598.

[0074] Example 2 N 1 , N 1 -bis((hexyloxycarbonyl)butyl)hexane-1,6-diamine 6 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 rt for 1 hour. Then, the reaction mixture was adsorbed onto silica gel for chromatography (16 g), the solvent was removed by RVE, and the residue was purified by silica gel column chromatography (80 g) using a linear gradient of ethyl acetate in cyclohexane (0 - 10%). Hexyl 5-bromopentanoate 4 (3.938 g, yield 89.6%; R f 0.31, KMnO 4 detection) was obtained as a colorless liquid.

[0075] Hexyl 5-bromopentanoate 4 (1.53 g, 5.78 mmol, 2.5 eq.) and anhydrous K 2 CO 3 (3.19 g, 23.11 mmol, 10 eq.) were, N1 To a solution of -tert-butyloxycarbonyl-1,6-diaminohexane (0.50 g, 2.31 mmol) in anhydrous ACN (10 ml) was added, and the reaction mixture was vigorously stirred at 35 °C for 2 days. Then, the reaction mixture was adsorbed onto silica gel for chromatography (16 g), the solvent was removed by RVE, and the residue was purified by silica gel column chromatography (40 g) using a linear gradient of ethyl acetate in cyclohexane (0 - 100%). Amine 5 was obtained in the form of a slightly yellowish oil (1.080 g, yield 79.9%; R on the mobile phase CE50 on a TLC plate pre-saturated with ammonia f 0.31, detected with ninhydrin).

[0076] 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. Then, the solution was poured into a 500 ml separable flask and diluted with 20% aqueous NaHCO 3 (200 ml), and the product was extracted with DCM (150 ml, 2 × 50 ml). The combined organic phases were washed with brine (50 ml), dried over anhydrous sodium sulfate, filtered through an S2 frit, adsorbed onto chromatographic silica gel (16 g), the solvent was removed by RVE, and the residue was purified by silica gel column chromatography (40 g) using a linear gradient of D1 in DCM (0 - 80%). Diamine 6 was obtained as a yellowish oil (0.788 g, yield 86.9%; R in the mobile phase D2 f 0.14, detected with ninhydrin). 1 H NMR (600 MHz, CDCl 3 ): 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, 0.88 ppm. 13 C NMR (150.9 MHz, CDCl 3 ): 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, 13.98 ppm. HRMS (ESI): C28 H 57 O 4 N 2 [M+H] + The calculated m / z for it was 485.43128; the measured value was 485.43052.

[0077] cis,cis-N 1 ,N 3 ,N 5 -Tris(6-(di((hexyloxycarbonyl)butyl)amino)hexyl)cyclohexane-1,3,5-tricarboxamide 7 According to the procedure described for lipidoid 3 in Example 1, lipidoid 7 was prepared from cis,cis-1,3,5-cyclohexanecarboxylic 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.). Lipidoid 7 (55 mg, 35%; R f 0.42 in mobile phase D2, detected with ninhydrin) was obtained as a yellowish semi-solid. 1 1H NMR (600 MHz, CDCl 3 ): 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, 0.88 ppm. 13 13C NMR (150.9 MHz, CDCl 3 ): δ = 174.35, 173.37, 64.60, 53.35, 52.88, 43.98, 39.12, 33.79, 31.94, 31.40, 29.21, 28.57, 26.62, 26.33, 25.56, 22.58, 22.51, 13.98 ppm. IR (CCl 4 ): ν max / cm -1 = 1736 vs (ν s C=O), 1173 m, 1075 w (ν s C-O), 3293 m (νNH), 1640 s (amide I) and 1551 m (amide II), 2955 s (ν as CH3 ), 2933 vs (ν as CH 2 ), 2875 m, sh (ν s CH 3 ), 2860 m (ν s CH 2 ), 1467 m and 1460 m (β s CH 2 and δ as CH 3 ), 1379 w (δ s CH 3 ), 724 w (β as CH 2 and γ as CH 2 ). HRMS (MALDI): C 93 H 175 N 6 O 15 [M + H] + calculated m / z for was 1616.3110; found 1616.3095.

[0078] Example 3 cis, cis - N 1 , N 3 , N 5 -Tris(6-(didodecylamino)hexyl)-1,3,5-trimethylcyclohexane-1,3,5-tricarboxamide 8 Following the procedure described for lipidoid 3 in Example 1, lipidoid 8 was prepared from cis, cis-1,3,5-trimethyl-1,3,5-cyclohexanetricarboxylic acid (20 mg, 0.077 mmol), N 1 , N 1 -Di(dodecyl)hexane-1,6-diamine 2 (140 mg, 310 mmol, 4 eq.) and DIPEA (135 μl, 0.774 mmol, 10 eq.). Lipidoid 8 (64 mg, 53%; R f 0.43 in mobile phase D2, detected with ninhydrin) was obtained as a slightly yellowish solid oil. IR (CCl 4 ): ν max / cm -1= 3288 w, br(νNH), 1651 m (amide I), 1585 w, br, sh and 1559 m, br (amide II), 2956 s (ν as CH 3 ), 2927 vs (ν as CH 2 ), 2878 m, sh (ν s CH 3 ), 2855 s (ν s CH 2 ), 1468 m, and 1459 m, sh, 1448 m, sh (β s CH 2 and δ as CH 3 ), 1378 w (δ s CH 3 ); 721 w (β as CH 2 and γ as CH 2 ). HRMS (MALDI): C 102 H 205 N 6 O 3 [M + H] + calculated m / z for was 1562.6068; found 1562.6011.

[0079] Example 4 N 1 , N 1 -Didodecylpropane-1,3-diamine 10 Following the procedure described for compound 1 of Example 1, amine 9 was prepared from 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.). Amine 9 was obtained as a yellowish oil (7.72 g, 43.9%).

[0080] Deprotection of amine 9 was carried out following the procedure described for compound 2 of Example 1. Diamine 10 (4.26 g, 68.6%;; R in mobile phase D2 on a TLC plate pre-saturated with ammoniaf 0.35, detected with ninhydrin) was obtained in the form of a yellowish oil. 1 1H NMR (600 MHz, CDCl 3 ): δ = 3.07, 2.70, 2.50, 1.81, 1.46, 1.28, 1.26, 1.25 - 1.29, 1.24, 0.87 ppm. 13 13C NMR (150.9 MHz, CDCl 3 ): 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, 14.10 ppm. IR (film): ν max / cm -1 = 3361 w and 3274 w (νNH 2 ), 2803 m (ν s N - CH 2 ), 2954 s (ν as CH 3 ), 2924 vs (ν as CH 2 ), 2853 s (ν s CH 2 ), 1467 m and 1456 m, sh (β s CH 2 and δ as CH 3 ), 1378 w and 1364 w (δ s CH 3 ), 720 m (β as CH 2 ). HRMS (ESI): C 27 H 59 N 2 [M + H] + calculated for was m / z 411.46728; the measured value was 411.46652.

[0081] cis,cis - N 1 ,N 3 ,N 5 -Tris(3-(didodecylamino)propyl)-1,3,5 - trimethylcyclohexane - 1,3,5 - tricarboxamide 11 According to the procedure described for lipidoid 3 of Example 1, cis,cis-1,3,5-trimethyl-1,3,5-cyclohexanetricarboxylic acid (20 mg, 0.077 mmol), N 1 ,N 1 -di(dodecyl)propane-1,3-diamine 10 (127 mg, 310 mmol, 4 eq.) and DIPEA (135 μl, 0.774 mmol, 10 eq.) were used to prepare lipidoid 11. Lipidoid 11 (41 mg, yield 37%; R f 0.36 in mobile phase D2, detected with ninhydrin) was obtained as a slightly yellowish solid oil. IR (CCl 4 ): ν max / cm -1 = 330 w, vbr (νNH), 1679 s, sh (amide I), 1513 w, br, sh (amide II), 2956 s (ν as CH 3 ), 2927 vs (ν as CH 2 ), 2878 m, sh (ν s CH 3 ), 2855 s (ν s CH 2 ), 1464 m, sh (β s CH 2 and δ as CH 3 ), 1380 w (δ s CH 3 ); 721 w (β as CH 2 and γ as CH 2 ). HRMS (MALDI): C 93 H 187 N 6 O 3 [M + H] + calculated for m / z was 1436.4665; the measured value was 1436.4629.

[0082] Example 5 Linolealdehyde 12 Des-Martin periodinane (4.45 g, 10.49 mmol, 1.3 eq.) was added to a solution of linoleyl alcohol (2.50 ml, 8.07 mmol) in DCM (120 ml) cooled to 0 °C in an ice bath, and the mixture was stirred at 0 °C for 4 h. Then, an aqueous sodium thiosulfate solution (20 g Na 2 S 2 O 3 ·5H 2 O / 100 ml H 2 O) and a saturated aqueous sodium hydrogen carbonate solution (50 ml) were added, and the initially milky solution was stirred at rt for 1 h until it 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 an S2 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, yield 59.6%; R f 0.36 with mobile phase CE5, KMnO 4で detection) was obtained as a colorless oil.

[0083] N 1 ,N 1 -Di((9Z,12Z)-octadeca-9,12-dien-1-yl)hexane-1,6-diamine 14 Following the procedure described for Compound 1 of Example 1, 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 used to prepare amine 13. Amine 13 was obtained as a yellowish oil (1.08 g, yield 94.9%; R f 0.18 with mobile phase CE20, ninhydrin detection).

[0084] The deprotection of amine 13 was carried out according to the procedure described for compound 2 in Example 1 in a mixture of TFA (4 ml) and DCM (5 ml), and diamine 14 (0.594 g, 64.0%; R f 0.13, detected with ninhydrin) was obtained as a yellowish oil. 1 H NMR (600 MHz, CDCl 3 ): 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, 0.88 ppm. 13 C NMR (150.9 MHz, CDCl 3 ): 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, 14.06 ppm. IR (CCl 4 ): ν max / cm -1 = 3011 s (ν as =CH); 1646 - 1673 m (νC=C); 3455 w (ν as NH 2 ); 3394 (ν s NH 2 ); 1620 w (β s NH 2 ); 1087 m (νC-NH 2 ); 2957 s, sh (ν as CH 3 ); 2928 vs (ν as CH 2 ); 2873 s, sh (ν s CH 3 ); 2856 vs (ν s CH 2 ); 2801 m (ν s N-CH 2 ); 1467 m and 1457 m, sh (β s CH 2 and δ as CH 3 ); 1378 m (δ s CH 3 ); 721 m (βas and γ as CH 2 ). HRMS: C 42 H 81 N 2 [M + H] + The calculated m / z for it was 613.63943; the measured value was 613.63899.

[0085] cis,cis-N 1 ,N 3 ,N 5 -Tris(6-(di((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)hexyl)-1,3,5-trimethylcyclohexane-1,3,5-tricarboxamide 15 Lipidoid 15 was prepared from cis,cis-1,3,5-trimethyl-1,3,5-cyclohexanecarboxylic acid (17 mg, 0.066 mmol), N 1 ,N 1 -Di((9Z,12Z)-octadeca-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.) according to the procedure described for lipidoid 3 in Example 1. Lipidoid 15 (86 mg, yield 64%; R f 0.36 in mobile phase D2, detected with ninhydrin) was obtained as a pale yellow solid. IR (CCl 4 ): ν max / cm -1 = 3348 w, br, sh and 3302 w, br (νNH), 1656 m and 1635 m, sh (amide I + νC = C), 1565 w, sh and 1557 w, br (amide II), 3011 m (ν as = C-H), 2956 s (ν as CH 3 ), 2929 vs (ν as CH 2 ), 2875 m, sh (ν s CH 3 ), 2856 s (ν s CH 2 ), 1467 m and 1450 m, sh, (β s CH 2 and δas CH 3 ), 1379w(δ s CH 3 ); 720w(β as CH 2 +γ as CH 2 +γ = C - H). HRMS(MALDI): C 138 H 253 N 6 O 3 [M + H] + The calculated m / z for it was 2042.9829; the measured value was 2042.9861.

[0086] Example 6 Compound 18 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 octan - 2 - ol (3.51 g, 26.9 mmol, 1.5 eq.) in DCM (30 mL). The mixture was stirred overnight at rt. Then, the reaction mixture was adsorbed onto silica (16 g) and the solvent was evaporated in vacuo. The crude product was purified by flash chromatography on silica (80 g, eluting with a linear gradient of ethyl acetate in cyclohexane, 0 - 10%) to give compound 16 (4.02 g, 77%; visualized by R f 0.41, KMnO 4 as a colorless oil.

[0087] 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 ACN (10 mL). The mixture was stirred at 40 °C for 3 days. Then, the reaction mixture was adsorbed onto silica (16 g) and the solvent was evaporated in vacuo. The crude product was purified by flash chromatography on silica (80 g, eluting with a linear gradient of ethyl acetate in cyclohexane, 0 - 100%) to give compound 17 (2.80 g, 80%; NH 3- Pretreatment R in CE50 on TLC plate f 0.58, visualized with ninhydrin) was obtained as a pale yellow oil.

[0088] Hydrochloric acid in dioxane (4 mL, 4 M) 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 rt for 1 hour. The solution was then poured into a 500 mL separatory funnel, diluted with saturated aqueous NaHCO 3 (100 mL), and the product was extracted with diethyl ether (100 mL, 2 × 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 by RVE. The crude product was purified by column chromatography on silica using a linear gradient of D1 in DCM (0 - 70%). Amine 18 (1.74 g, 74%; NH 3 - Pretreatment R in D2 on TLC plate f 0.25, visualized with ninhydrin) was obtained as a pale yellow oil. 1 1H NMR (600 MHz, CDCl 3 ): 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 13C NMR (150.9 MHz, CDCl 3 ): 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, 14.09 ppm. HRMS (ESI): C 34 H 68 N 2 O 4 [M + H] + The calculated m / z for was 569.5252; the measured value was 569.5247.

[0089] Hexa(octan-2-yl) cis,cis-6,6’,6’’,6’’’,6’’’’,6’’’’’-((((cyclohexane-1,3,5-tricarbonyl)tris(azanediyl))tris(hexane-6,1-diyl))tris(azatriyl)) hexahexanoate 19 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 h. Excess SOCl 2 was removed at 70 °C under a stream of dry nitrogen, and the residue was dried in vacuo at rt (20 min). The resulting solid was dissolved under argon in anhydrous TCE (2.5 mL) and DIPEA (645 μL, 3.70 mmol). Then, a solution of amine 18 (1.05 g, 1.85 mmol, 5 eq.) in anhydrous TCE (2.5 mL) was added, and the reaction mixture was stirred at rt for 40 min. The reaction mixture was then evaporated in vacuo, redissolved in DCM, adsorbed onto silica (10 g), and DCM was removed in vacuo. The crude product was purified by flash chromatography on silica (eluting with a linear gradient of D1 in DCM, 5 - 35%) to give the desired compound 19 (489 mg, 71%) as a yellow waxy semi-solid. 1 H NMR (600 MHz, CDCl 3 ): 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, 0.87 ppm. 13 C NMR (150.9 MHz, CDCl 3 ): 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, 14.06 ppm. HRMS (MALDI): C 111 H 211 N 6 O 15 [M + H] +The calculated m / z for it was 1868.5927; the measured value was 1868.5906.

[0090] Example 7 Compound 22 DIC (3.60 mL, 23.0 mmol, 1.6 eq) and DMAP (70 mg, 0.58 mmol, 0.04 eq) were added to 7-bromoheptanoic acid (3.00 g, 14.4 mmol) and 3-methylhexan-1-ol (2.50 g, 21.5 mmol, 1.5 eq.) dissolved in DCM (30 mL), and the mixture was stirred overnight at rt. The reaction mixture was then adsorbed onto silica (16 g), and the solvent was evaporated in vacuo. The crude product was purified by flash chromatography on silica (80 g, eluting with a linear gradient of ethyl acetate in cyclohexane, 0 - 10%) to give Compound 20 (3.64 g, 83%; R f 0.27, visualized by KMnO 4 ) as a colorless oil.

[0091] 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 ACN (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 in vacuo. The crude product was purified by flash chromatography on silica (80 g, eluting with a linear gradient of ethyl acetate in cyclohexane, 0 - 100%) to give Compound 21 (2.90 g, 95%; R 3 - in CE50 on pre-treated TLC plate R f 0.54, visualized by ninhydrin) as a pale yellow oil.

[0092] Hydrochloric acid in dioxane (4 mL, 4 M) 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 rt for 1 h. The solution was then poured into a 500 mL separatory funnel and saturated aqueous NaHCO 3(100 mL) was diluted, and the product was extracted with diethyl ether (100 mL, 2 × 50 mL). The combined organic phases were washed with brine (75 mL), dried over anhydrous sodium sulfate, filtered through sintered glass S2, and the solvent was evaporated using an RVE. The crude product was purified by silica gel column chromatography using a linear gradient of D1 in DCM (0 - 70%). Amine 22 (2.38 g, 96%; NH 3 - Pretreatment R in D2 on TLC plate f 0.48, visualized with ninhydrin) was obtained as a pale yellow oil.

[0093] 1 1H NMR (400 MHz, CDCl 3 ): δ = 4.08 - 3.96 (m), 2.63 - 2.58 (m), 2.33 - 2.26 (m), 2.21 (t, J = 7.5 Hz), 1.61 - 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 13C NMR (150.9 MHz, CDCl 3 ): 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, 14.26 ppm. HRMS (ESI): C 34 H 68 N 2 O 4 [M + H] + The calculated m / z for was 569.5252; the measured value was 569.5250.

[0094] Hexakis(3-methylhexyl) cis,cis-7,7’,7’’,7’’’,7’’’’,7’’’’’-((((cyclohexane-1,3,5-tricarbonyl)tris(azanediyl))tris(hexane-6,1-diyl))tris(azantriyl))hexaheptanoate 23 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 at 70 °C for 3 h in a sealed vial; during this time the suspension changed to a clear solution. Excess SOCl 2 was removed at 70 °C under a stream of dry nitrogen, and the residue was dried in vacuo at rt (20 min). The resulting solid was dissolved in anhydrous TCE (2 mL) and DIPEA (483 μL, 2.78 mmol, 10 eq.) under argon. Next, a solution of amine 22 (632 mg, 1.11 mmol, 4 eq.) in anhydrous TCE (2 mL) was added, and the reaction mixture was stirred at rt for 40 min. The reaction mixture was then evaporated in vacuo, redissolved in DCM, adsorbed onto silica (10 g), and the DCM was removed in vacuo. The crude product was purified by flash chromatography on silica (eluting with a linear gradient of D1 in DCM, 0 - 46%) to give the title compound 23 (346 mg, 67%; NH 3 - pretreated TLC plate with D2, R f 0.66, visualized with ninhydrin) as a yellow waxy semi-solid. 1 H NMR (600 MHz, CDCl 3 ): 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, 0.88 ppm. 13 C NMR (150.9 MHz, CDCl 3 ): δ = 173.92, 173.87, 62.64, 53.90, 44.10, 39.45, 39.14, 35.51, 34.23, 31.86, 29.54, 29.52, 29.09, 27.22, 27.13, 26.77, 24.97, 19.94, 19.47, 14.26 ppm. HRMS (MALDI): C 111 H 211 N 6 O 15 [M + H] + calculated for was m / z 1868.5927; found 1868.5905.

[0095] Example 8 Compound 24 To 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.) in anhydrous DCM (150 mL) was added N,N'-diisopropylcarbodiimide (4.07 mL, 24.9 mmol, 1.6 eq.). The reaction mixture was adsorbed onto silica (20 g), and the solvent was evaporated in vacuo. The crude product was purified by flash chromatography on silica (120 g, eluting with a linear gradient of ethyl acetate in cyclohexane, 0 - 10%) to give the desired compound 24 (4.80 g, 91%; R f 0.39 in CE5, visualized by KMnO 4 ) as a pale yellow oil. 1 1H NMR (400 MHz, CDCl 3 ) δ 5.37 - 5.30 (m, 1H), 5.11 - 5.05 (m, 1H), 4.60 (d, J = 7.1 Hz, 2H), 3.41 (t, J = 6.6 Hz, 2H), 2.35 (t, J = 7.2 Hz, 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).

[0096] Compound 26 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). Then, the reaction mixture was adsorbed onto silica (20 g), and the solvent was evaporated in vacuo. The crude product was purified by flash chromatography on silica (120 g, a column pretreated with gaseous NH 3 , eluting with a linear gradient of ethyl acetate in cyclohexane, 0 - 100%) to give compound 25 (2.80 g, 54%; R 3 0.58 in CE50 on an NH f -pretreated TLC plate, visualized by KMnO4 It was obtained as a pale yellow oil by visualization).

[0097] Compound 25 (2.80 g, 4.06 mmol) was placed in a flask equipped with a magnetic stir bar, closed with a rubber septum, and washed with argon. ACN (10 mL) and DCM (20 mL) were added through the septum with a needle and syringe, stirring was turned on, and a solution of 4-toluenesulfonic acid monohydrate (2.32 g, 12.2 mmol, 3 eq.) in ACN (20 mL) was added dropwise through the septum over 5 minutes at rt. The reaction mixture was stirred at rt for 6 hours and then neutralized by passing gaseous ammonia through it. The reaction mixture was adsorbed onto silica (20 g), and the crude product was purified by silica gel column chromatography using a linear gradient of D1 in DCM (10 - 43%). Amine 26 (810 mg, 34%; NH 3 - Pretreatment TLC plate in D2 with R f 0.45, visualized with ninhydrin) was obtained as a yellow oil. HRMS (ESI): C 36 H 64 N 2 O 4 [M + H] + The calculated m / z for was 589.4939; the measured value was 589.4936.

[0098] Hexakis((E)-3,7-dimethylocta-2,6-dien-1-yl) cis,cis-5,5’,5’’,5’’’,5’’’’,5’’’’’-((((cyclohexane-1,3,5-tricarbonyl)tris(azanediyl))tris(hexane-6,1-diyl))tris(azatriyl)) hexapentanoate 27 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 changed to a clear solution. Excess SOCl 2It was removed with a dry nitrogen stream at 70 °C, and the residue was dried in vacuo at rt (20 minutes). The resulting solid was dissolved in anhydrous TCE (1.5 mL) and DIPEA (363 μL, 2.08 mmol, 10 eq.) under argon. Subsequently, a solution of amine 26 (490 mg, 833 μmol, 4 eq.) in anhydrous TCE (1.0 mL) was added, and the reaction mixture was stirred at rt for 40 minutes. Then, the reaction mixture was evaporated in vacuo, redissolved in DCM, adsorbed onto silica (10 g), and DCM was removed in vacuo. The crude product was purified by flash chromatography on silica (eluting with a linear gradient of D1 in DCM, 0 - 31%), and the desired compound 27 (262 mg, 65%; NH 3 - R on D2 on the pretreatment TLC plate f 0.59, visualized with ninhydrin) was obtained as a yellow waxy semi-solid. 1 1H NMR (600 MHz, CDCl 3 3): 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, 1.29 ppm. 13 13C NMR (150.9 MHz, CDCl 3 3): 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, 16.46 ppm. HRMS (MALDI): C 117 19 99 H1 6 N 15 [M + H] + calculated m / z for was 1928.4988; found 1928.4967.

[0099] Example 9 Compound 28 N,N’-Diisopropylcarbodiimide (4.07 mL, 24.9 mmol, 1.6 eq.) was added to 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.) in anhydrous DCM (60 mL). The reaction mixture was adsorbed onto silica (20 g), and the solvent was evaporated in vacuo. The crude product was purified by flash chromatography on silica (120 g, eluting with a linear gradient of ethyl acetate in cyclohexane, 0 - 10%) to give the desired compound 28 (4.75 g, 90%; R f 0.39 in CE5, visualized by KMnO 4 ). as a pale yellow oil. 1 1H NMR (401 MHz, CDCl 3 ) δ 5.35 (t, J = 6.5 Hz, 1H), 5.12 - 5.06 (m, 1H), 4.57 (dd, J = 7.2, 1.1 Hz, 2H), 3.40 (t, J = 6.6 Hz, 2H), 2.34 (t, J = 7.3 Hz, 2H), 2.15 - 2.03 (m, 4H), 1.94 - 1.86 (m, 2H), 1.82 - 1.74 (m, 5H), 1.68 (d, J = 1.4 Hz, 3H), 1.60 (s, 3H).

[0100] Compound 30 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). Subsequently, the reaction mixture was adsorbed onto silica (20 g), and the solvent was evaporated in vacuo. The crude product was purified by flash chromatography on silica (120 g, column pretreated with gaseous NH 3 , eluting with a linear gradient of ethyl acetate in cyclohexane, 0 - 100%) to give compound 29 (3.62 g, 81%; R 3 0.54 in CE50 on an NH f -pretreated TLC plate, visualized by ninhydrin). as a pale yellow oil.

[0101] Compound 29 (3.62 g, 5.25 mmol) was placed in a flask equipped with a magnetic stir bar, closed with a rubber septum, and flushed with argon. DCM (10 mL) was added through the septum using a needle and syringe, stirring was turned on, 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 septum over 5 minutes at rt using a needle and syringe. The reaction mixture was stirred at rt for 5 h, then neutralized by passing gaseous ammonia through it. The reaction mixture was adsorbed onto silica (20 g), and the crude product was purified by silica gel column chromatography using a linear gradient of D1 in DCM (10 - 43%). Amine 30 (1.36 g, 44%; NH 3 - visualized in D2 on a pre-treated TLC plate with R f 0.46, KMnO 4 was obtained as a yellow oil. HRMS (ESI): C 36 H 64 N 2 O 4 [M + H] + calculated m / z for was 589.4939; found 589.4937.

[0102] Hexakis((Z)-3,7-dimethylocta-2,6-dien-1-yl) cis,cis-5,5’,5’’,5’’’,5’’’’,5’’’’’-((((cyclohexane-1,3,5-tricarbonyl)tris(azanediyl))tris(hexane-6,1-diyl))tris(azantriyl)) hexapentanoate 31 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. Excess SOCl 2It was removed with a dry nitrogen stream at 70 °C, and the residue was dried in vacuo at rt (20 minutes). The resulting solid was dissolved in anhydrous TCE (2 mL) and DIPEA (524 μL, 3.01 mmol, 10 eq.) under argon. Subsequently, a solution of amine 30 (796 mg, 1.35 mmol, 4.5 eq.) in anhydrous TCE (2.0 mL) was added, and the reaction mixture was stirred at rt for 30 minutes. Then, the reaction mixture was evaporated in vacuo, redissolved in DCM, adsorbed onto silica (10 g), and DCM was removed in vacuo. The crude product was purified by flash chromatography on silica (eluting with a linear gradient of D1 in DCM, 5 - 29%) to give the desired compound 31 (351 mg, 51%) as a yellow waxy semi-solid. 1 H NMR (600 MHz, CDCl 3 ): 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, 1.28 ppm. 13 C NMR (150.9 MHz, CDCl 3 ): 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, 17.65 ppm. HRMS (MALDI): C 117 H 199 N 6 O 15 [M + H] + calculated m / z for was 1928.4988; found 1928.4963.

[0103] Tetra(octan-2-yl) cis,cis-6,6’,6’’,6’’’-((((5-(methoxycarbonyl)cyclohexane-1,3-dicarbonyl)bis(azanediyl))bis(hexane-6,1-diyl))bis(azatriyl))tetrahexanoate 32 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 at 70 °C for 3 h in a sealed vial; during this time the suspension changed to a clear solution. Excess SOCl 2 was removed at 70 °C under a stream of dry nitrogen, and the residue was dried in vacuo at rt (20 min). The resulting solid was dissolved in anhydrous TCE (2 mL) and DIPEA (483 μL, 2.78 mmol, 10 eq.) under argon. Next, a solution of amine 18 (632 mg, 1.11 mmol, 4 eq.) in anhydrous TCE (1.0 mL) was added, and the reaction mixture was stirred at rt for 40 min. Next, the reaction mixture was evaporated in vacuo, redissolved in DCM, adsorbed onto silica (10 g), and DCM was removed in vacuo. The crude product was purified by flash chromatography on silica (eluting with a linear gradient of D1 in DCM, 5 - 29%) to give compound 32 (242 mg, 65%; NH 3 - pre-treated TLC plate with D2, R f 0.68, visualized with ninhydrin) as a yellow oil. Product 32 is the result of incomplete removal of MeOH from amine 18. 1 H NMR (600 MHz, CDCl 3 ): 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, 0.87 ppm. 13 C NMR (150.9 MHz, CDCl 3 ): δ = 174.64, 173.75, 173.38, 70.83, 53.82, 53.76, 51.84, 43.99, 42.09, 39.43, 35.93, 34.66, 31.81, 31.73, 31.20, 29.50, 29.09, 27.06, 26.72, 25.36, 24.99, 22.56, 20.00, 14.05 ppm. HRMS (MALDI): C 78 H 146 N 4 O 12 [M + H] +The calculated m / z for it was 1332.1010; the measured value was 1332.0987.

[0104] Example 11 Tetrakis(3-methylhexyl) cis,cis-7,7’,7’’,7’’’-((((5-(methoxycarbonyl)cyclohexane-1,3-dicarbonyl)bis(azanediyl)bis(hexane-6,1-diyl)bis(azatriyl))tetraheptanoate 33 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 at 70 °C for 3 h in a sealed vial. The excess SOCl 2 was removed at 70 °C under a stream of dry nitrogen, and the residue was dried in vacuo at rt (20 min). The resulting solid was dissolved in anhydrous TCE (2 mL) and DIPEA (443 μL, 2.54 mmol, 10 eq.) under argon. Next, a solution of MeOH (10.3 μL, 254 μmol, 1.0 eq.) in TCE (0.5 mL) was added at 0 °C. The reaction mixture was warmed to room temperature and stirred for 1 h, then amine 22 (376 mg, 661 μmol, 2.6 eq.) in anhydrous TCE (2.0 mL) was added, and the reaction mixture was stirred at rt for 40 min. The reaction mixture was then evaporated in vacuo, redissolved in DCM, adsorbed onto silica (10 g), and DCM was removed in vacuo. The crude product was purified by flash chromatography on silica (eluting with a linear gradient of D1 in DCM, 5 - 29%) to give the desired compound 33 (87 mg, 26%; R 3 0.75 on a D2 pre-treated TLC plate, visualized with ninhydrin) as a pale yellow oil. f H NMR (600 MHz, CDCl 1 ): 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, 0.88 ppm. 3 13 ​13C NMR (150.9 MHz, CDCl 3 ): δ = 174.65, 173.91, 173.76, 62.85, 53.85, 51.85, 43.98, 42.09, 39.42, 39.15, 35.52, 34.22, 31.89, 31.19, 29.55, 29.51, 29.06, 27.20, 27.10, 26.72, 24.95, 19.94, 19.47, 14.26 ppm. HRMS (MALDI): C 78 H 147 N 4 O 12 [M + H] + calculated m / z for was 1332.1010; found 1332.0984.

[0105] Example 12 Compound 34 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 h. Excess SOCl 2 was removed at 70 °C under a stream of dry nitrogen, and the residue was dried in vacuo at rt (20 min). The resulting solid was dissolved in anhydrous TCE (3 mL) and DIPEA (1.29 mL, 7.40 mmol, 10 eq.) under argon. Next, a solution of BnOH (76 μL, 740 μmol, 1.0 eq.) in TCE (0.5 mL) was added at 0 °C. The reaction mixture was warmed to room temperature and stirred for 2 h, then amine 18 (1.09 g, 1.92 mmol, 2.6 eq.) in anhydrous TCE (3.0 mL) was added, and the reaction mixture was stirred at rt for 40 min. The reaction mixture was then evaporated in vacuo, redissolved in DCM, adsorbed onto silica (10 g), and DCM was removed in vacuo. The crude product was purified by flash chromatography on silica (eluting with a linear gradient of D1 in DCM, 5 - 29%), and compound 34 (150 mg, 14%; R 3 0.67 on D2 on NH f - pretreated TLC plates, visualized with ninhydrin) was obtained as a pale yellow oil. HRMS (MALDI): C 84 H 151 N4 O 12 [M+H] + The m / z calculated for O was 1408.1323; the measured value was 1408.1283.

[0106] cis,cis-3,5-bis((6-(bis(6-(octan-2-yloxy)-6-oxohexyl)amino)hexyl)carbamoyl)cyclohexane-1-carboxylic acid 35 A magnetic stir bar was attached to a round-bottom flask, and palladium on carbon (10% Pd / C, 25 mg) was loaded. The flask was closed with a rubber septum, connected to a Schlenk line via a needle, and the atmosphere in the flask was exchanged with argon via 3 cycles of vac-Ar. Then, a solution of compound 34 (150 mg, 107 μmol) in MeOH (3 mL) was added from the septum using a needle and syringe, and stirring was started. The flask was connected to a continuous flow of hydrogen with a second injection needle, and the main argon inlet to the Schlenk line was closed (excess hydrogen was continuously released from the flask through the bubbler of the Schlenk line). After the reaction mixture was vigorously stirred at rt for 4 h, the flask was thoroughly washed with argon and filtered through a PTFE syringe filter to remove palladium on carbon. Then, volatile substances were removed in vacuo, and the product 35 (110 mg, 79%, NH 3 - R on D2 on a pre-treated TLC plate f 0.27, visualized with ninhydrin) was obtained as a colorless oil. 1 H NMR (600 MHz, CDCl 3 ): 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, 0.87 ppm. 13 C NMR (150.9 MHz, CDCl 3): 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, 14.06 ppm. HRMS (MALDI): C 77 H 145 N 4 O 12 [M + H] + The calculated m / z for [[M+H]] was 1318.0854; the measured value was 1318.0842.

[0107] Tetra(octan-2-yl) cis,cis-6,6’,6’’,6’’’-((((5-(pyrrolidine-1-carbonyl) cyclohexane-1,3-dicarbonyl) bis(azanediyl) bis(hexane-6,1-diyl) bis(azantriyl)) tetrahexanoate 36 Compound 35 (60 mg, 45.5 μmol) and NH 4 Cl (12.2 mg, 228 μmol, 5 eq.) were placed in a vial equipped with a magnetic stir bar, and the septum was pierced with a needle and washed with argon. Then, 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 rt for 10 min, 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 rt for 90 min. Then, the reaction mixture was evaporated in vacuo, redissolved in DCM, adsorbed onto silica (10 g), and DCM was removed in vacuo. The crude product was purified by flash chromatography on silica (eluting with a linear gradient of D1 in DCM, 5 - 29%) to give compound 36 (18 mg, 29%; NH 3 - R f 0.42 on D2 on a pre-treated TLC plate, visualized with ninhydrin) was obtained. Compound 36 is the product of a pyrrolidine impurity in PyBroP resulting from the hydrolysis of PyBroP. 11H NMR (600 MHz, CDCl 3 ): 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, 0.88 ppm. 13 13C NMR (150.9 MHz, CDCl 3 ): 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): C 81 H 152 N 5 O 11 [M + H] + calculated m / z for was 1371.1483; found 1371.1467.

[0108] Example 13 cis,cis-L,3,5-Cyclohexanetricarboxylic acid trimethyl 37 To a solution of cis,cis-l,3,5-cyclohexanetricarboxylic acid (9.90 g, 45.8 mmol) in anhydrous methanol (150 mL) placed in a round-bottom flask equipped with a calcium chloride drying tube and a magnetic stirrer, thionyl chloride (15.29 mL, 210.8 mmol, 4.6 eq.) was slowly added via syringe through a rubber septum. After the intense exothermic reaction ceased, the solution was stirred at room temperature for 12 h. Then, the solvent was removed in vacuo and the oily residue was poured into a 500 mL separatory flask and saturated aqueous NaHCO 3(250 mL) was diluted, and the product was extracted with diethyl ether (2 × 100 mL). The combined organic phases were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered through sintered glass S2, and the solvent was evaporated with an RVE to obtain 37 as a pale yellow oil. 1 H NMR (400 MHz, CDCl 3 ): 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): C 12 H 19 O 6 [M + H] + The calculated m / z for was 259.11761; the measured value was 259.11766.

[0109] Trimethyl cis,cis - 1,3,5 - trimethylcyclohexane - 1,3,5 - tricarboxylate 38 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 lithium diisopropylamide 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 was continued overnight at rt. The product was washed with water, 1 M hydrochloric acid, brine, dried over sodium sulfate, filtered through sintered glass S2, and the solvent was evaporated with an RVE. As a result of GC - MS analysis, the ratio of the two isomers in the crude product was cis,cis / cis,trans = 7:1. Fractional crystallization with a mixed solvent of diethyl ether and pentane gave the cis,cis isomer 38 (225 mg, 19%) as white crystals. 1 H NMR (400 MHz, CDCl 3): δ = 3.65 (s, 9H), 2.74 (d, J = 13.4 Hz, 3H), 1.21 (s, 9H), 0.97 (d, J = 14.8 Hz, 3H) ppm. HRMS (EI): C 15 H 24 O 6 [M] + The calculated m / z for [M] was 300.1567; the measured value was 300.1565.

[0110] cis,cis-1,3,5-Trimethylcyclohexane-1,3,5-tricarboxylic acid 39 cis,cis-1,3,5-Trimethylcyclohexane-1,3,5-tricarboxylic acid trimethyl 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 rt. Methanol was removed by evaporation on an RVE. The solution was cooled in an ice bath and the pH was adjusted to 1 with concentrated hydrochloric acid. A white precipitate formed immediately and was collected by suction filtration to give 39 (60 mg, 32%). 1 1H NMR (400 MHz, DMSO-d6): δ = 11.96 (br s, 3H), 2.52 (d, 3H, partially overlapping with solvent signal), 1.20 (d, J = 14.7 Hz, 3H), 1.19 (s, 9H) ppm.

[0111] Hexakis(3-methylhexyl) cis,cis-7,7’,7’’,7’’’,7’’’’,7’’’’’-((((1,3,5-trimethylcyclohexane-1,3,5-tricarbonyl)tris(azanediyl))tris(hexane-6,1-diyl))tris(azatriyl))hexaheptanoate 40 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 in a sealed vial at 70 °C overnight. Excess SOCl 2It was blown away with a stream of dry nitrogen, and the residue was dried in vacuo (10 minutes). After cooling to rt, the residue was dissolved in anhydrous TCE (0.5 mL). Subsequently, a solution of amine 22 (337 mg, 0.59 mmol, 4.5 eq.) and DIPEA (229 μL, 1.32 mmol, 10 eq.) in anhydrous TCE (1.00 mL) was added, and the reaction mixture was stirred at rt for 15 minutes. The crude product was purified by flash chromatography on silica (40 g, eluting with a linear gradient of D1 in DCM, 0 - 30%), and the target compound 40 (6 mg, 5%; R in D4 f 0.26, visualized with ninhydrin) was obtained as a viscous pale yellow oil. HRMS (MALDI): C 114 H 216 N 6 O 15 [M + H] + The calculated m / z for was 1910.6396; the measured value was 1910.6368.

[0112] Example 14 Hexa(octan-2-yl) cis,cis-6,6’,6’’,6’’’’,6’’’’’-((((1,3,5-trimethylcyclohexane-1,3,5-tricarbonyl)tris(azanediyl))tris(hexane-6,1-diyl))tris(azatriyl))hexahexanoate 41 and cis,cis-3,5-bis((6-(bis(6-(octan-2-yloxy)-6-oxohexyl)amino)hexyl)carbamoyl)-1,3,5-trimethylcyclohexane-1-carboxylic acid 42 Thionyl chloride (0.50 mL) and DMF (2 μL) were added to 39 (28 mg, 0.11 mmol), and the suspension was stirred in a sealed vial at 70 °C for 2 hours. Excess SOCl 2It was blown away with a stream of dry nitrogen, and the residue was dried in vacuo (10 minutes). After cooling to rt, the residue was dissolved in anhydrous TCE (0.25 mL). Next, a solution of amine 18 (278 mg, 0.49 mmol, 4.5 eq.) and DIPEA (180 μL, 1.08 mmol, 10 eq.) in anhydrous TCE (0.75 mL) was added, and the reaction mixture was stirred at rt for 15 minutes. The crude product was purified by flash chromatography on silica (40 g, eluting with a linear gradient of D1 in DCM, 0 - 30%), and the target compound 41 (49 mg, 24%; R in D4 f 0.30, visualized with ninhydrin) was obtained as a pale yellow viscous oil, and compound 42 (52 mg, 45%; R in D4 f 0.28, visualized with ninhydrin) was obtained as a pale yellow oil.

[0113] 41 1 H NMR (600 MHz, CDCl 3 ): 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, 0.87 ppm. 13 C NMR (150.9 MHz, CDCl 3 ): δ = 184.26, 177.45, 173.06, 70.96, 52.45, 51.68, 43.03, 43.00, 42.38, 38.83, 35.92, 34.43, 34.17, 33.21, 31.72, 29.08, 28.95, 22.56, 19.99, 14.05 ppm. HRMS (MALDI): C 114 H 216 N 6 O 15 [M + H] + calculated m / z for was 1910.6396; the measured value was 1910.6364.

[0114] 42 HRMS (MALDI): C 80 H 150 N 4 O 12 [M + H] +The calculated m / z for it was 1360.1323; the measured value was 1360.1875.

[0115] Example 15 Trimethyl cis,cis-1,3,5-tris((benzyloxy)methyl)cyclohexane-1,3,5-tricarboxylate 43 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 lithium diisopropylamide in situ. A solution of trimethyl cyclohexane-1,3,5-tricarboxylate 37 (3.00 g, 11.6 mmol; 1 equiv.) 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. Benzyl chloromethyl ether (9.69 mL, 69.7 mmol, 6 eq.) was added, and stirring was continued overnight at rt. The product was washed with water, 1 M hydrochloric acid, and brine, dried over sodium sulfate, filtered through sintered glass S2, and the solvent was evaporated using an RVE. The crude product was purified by flash chromatography on silica (200 g, eluting with a linear gradient of ethyl acetate in cyclohexane, 0 - 20%). Fractionation with a mixed solvent of diethyl ether (3.0 mL) and pentane (18.0 mL) gave the cis,cis isomer 43 (1.52 mg, 22%) as white crystals. 1 H NMR (400 MHz, CDCl 3 ): δ = 7.36 - 7.22 (m, 15H, overlapping with solvent signal in part), 4.46 (s, 6H), 3.69 (s, 9H), 3.39 (s, 6H), 2.68 (d, J = 14.2 Hz, 3H), 1.18 (d, J = 14.9 Hz, 3H) ppm. HRMS (ESI): C 36 H 42 O 9 [M+Na] + The calculated m / z for it was 641.2721; the measured value was 641.2719.

[0116] cis,cis-1,3,5-Tris((benzyloxy)methyl)cyclohexane-1,3,5-tricarboxylic acid 44 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 at rt overnight. Methanol was removed by evaporation on an RVE. The solution was cooled in an ice bath, and the pH was adjusted to 1 with concentrated hydrochloric acid. A white precipitate formed immediately and was collected by suction filtration to give 44 (200 mg, 86%). 1 1H NMR (400 MHz, 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 = 14.9 Hz, 3H), ppm.

[0117] Tetra(octan-2-yl) cis,cis-6,6’,6’’,6’’’-((((1,3,5-tris((benzyloxy)methyl)-5-(bis(6-(octan-2-yloxy)-6-oxohexyl)carbamoyl)cyclohexane-1,3-dicarbonyl)bis(azanediyl))bis(hexane-6,1-diyl))bis(azatriyl))tetrahexanoate 45 and cis,cis-1,3,5-tris((benzyloxy)methyl)-3,5-bis((6-(bis(6-(octan-2-yloxy)-6-oxohexyl)amino)hexyl)carbamoyl)cyclohexane-1-carboxylic acid 46 Thionyl chloride (0.50 mL) and DMF (2 μL) were added to 44 (100 mg, 0.17 mmol, 1 eq.), and the suspension was stirred in a sealed vial at 70 °C overnight. Excess SOCl 2It was blown away with a stream of dry nitrogen, and the residue was dried in vacuo (10 minutes). After cooling to rt, the residue was dissolved in anhydrous TCE (0.5 mL). Next, a solution of amine 18 (444 mg, 0.78 mmol, 4.5 eq.) and DIPEA (302 μL, 1.73 mmol, 10 eq.) in anhydrous TCE (1.50 mL) was added, and the reaction mixture was stirred at rt for 15 minutes. The crude product was purified by flash chromatography on silica (80 g, eluting with a linear gradient of D1 in DCM, 0 - 20%), and the target compound 45 (46 mg, 12%; R in D4 f 0.46, visualized with ninhydrin) was obtained as a pale yellow viscous oil, and compound 46 (37 mg, 13%; R in D4 f 0.39, visualized with ninhydrin) was obtained as a pale yellow oil.

[0118] 45 HRMS (MALDI): C 135 H 234 N 6 O 18 [M + H] + The calculated m / z for was 2228.7652; the measured value was 2228.7702.

[0119] 46 HRMS (MALDI): C 101 H 168 N 4 O 15 [M] + The calculated m / z for was 1677.2506; the measured value was 1677.2479.

[0120] Hexa(octan - 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(azantriyl))hexahexanoate 47 Following the procedure outlined for 35, the target compound 47 was prepared from lipidoid 45 (20 mg, 0.008 mmol, 1 eq.), palladium on carbon (10% Pd / C, 19 mg, 0.018 mmol, 2 eq.) and acetic acid (80 μL, 1.73 mmol) to give lipidoid 47 (5 mg, 30%) as a thick pale yellow oil. HRMS (MALDI): C 114 H 216 N 6 O 18 [M+H] + The calculated m / z for was 1958.6244; the measured value was 1958.6314.

[0121] cis,cis-3,5-bis((6-(bis(6-(octan-2-yloxy)-6-oxohexyl)amino)hexyl)carbamoyl)-1,3,5-tris(hydroxymethyl)cyclohexane-1-carboxylic acid 48 Following the procedure outlined for 35, the target compound 48 was prepared from lipidoid 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) to give lipidoid 48 (7 mg, 59%) as a thick pale yellow oil. HRMS (MALDI): C 80 H 150 N 4 O 15 [M] + The calculated m / z for was 1407.1098; the measured value was 1407.1042.

[0122] Example 16 Tetra(octan-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(azantriyl))tetrahexanoate 49 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. 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 rt for 1.5 h under an argon atmosphere. 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 rt for 1 h under an argon atmosphere. Then, the reaction mixture was adsorbed onto silica (4 g), and the solvent was evaporated in vacuo. The crude product was purified by flash chromatography on silica (80 g, eluting with a linear gradient of D1 in DCM, 0 - 30%) to give the desired compound 49 (146 mg, 51%; R f 0.58, visualized with ninhydrin) as a pale yellow oil. HRMS (MALDI): C 105 H 175 N 5 O 14 [M+Na] + The calculated m / z for was 1753.3028; the measured value was 1753.3044.

[0123] Tetra(octan-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(azatriyl))tetrahexanoate 50 Following the procedure outlined for 35, the target compound 50 was prepared from lipidoid 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), and lipidoid 50 (57 mg, 96%) was obtained as a pale yellow thick oil. HRMS (MALDI): C 84 H 157 N 5 O 14 [M+Na] +The calculated m / z for it was 1483.1619; the measured value was 1483.1656.

[0124] Example 17 cis,cis-N 1 ,N 7 -Bis(6-(dodecylamino)hexyl)-5,7-bis(hydroxymethyl)-4-oxo-3-oxabicyclo[3.3.1]nonane-1,7-dicarboxamide 52 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). The reaction mixture was stirred at rt for 12 h. The reaction mixture was then adsorbed onto silica (10 g) and the solvent was evaporated in vacuo. The crude product was purified by silica gel column chromatography using a linear gradient of D1 in DCM (20 - 80%). Diamide 51 (16 mg, 8.0%; Rf 0.56 in mobile phase 3, detected by ninhydrin) was obtained in the form of a viscous yellowish oil. f 0.56, detected by ninhydrin) was obtained in the form of a viscous yellowish oil.

[0125] Under an argon atmosphere, palladium on charcoal (10%) was added to a solution of diamide 51 in a methanol - ethyl acetate mixed solvent (10 + 10 mL). The suspension was filtered through a pad of celite and evaporated to give lipidoid 52 as a colorless oil (5.5 mg, 42.9%, Rf 0.61 in mobile phase D3, detected by ninhydrin). HRMS (MALDI): C f 0.61, detected by ninhydrin). HRMS (MALDI): C 72 H 141 N 4 O 6 [M + H] + The calculated m / z for [M + H] was 1158.0846; the measured value was 1158.0822.

[0126] Example 18 6-(Dodecylamino)hexanoic acid 53 A solution of 6-aminohexanoic acid (2.00 g, 15.2 mmol) in ACN (150 mL) was placed in 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. While stirring vigorously, n-dodecylaldehyde (10.16 mL, 45.7 mmol, 3 eq.) was slowly added via syringe through a rubber septum, and the resulting white suspension was stirred at room temperature for 5 days. The crude product was purified by silica gel column chromatography using a linear gradient of D1 in DCM (0 - 100%). Amino acid 53 (3.203 g, 44.9%) was obtained as a colorless oil. 1 H NMR (400 MHz, CDCl 3 ): 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 (150.9 MHz, CDCl 3 ): 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, 14.10 ppm. HRMS (ESI): C 30 H 60 O 2 N[M - H] - The calculated m / z for was 466.46295; the measured value was 466.46286.

[0127] cis,cis-1,3,5-triaminocyclohexane trihydrobromide 55 The synthesis was carried out 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) was added. 25 mmol) was suspended in toluene (75 mL), DIPEA (4.83 mL, 27.75 mmol, 3 eq.) was added, followed by diphenylphosphoryl azide (5.97 mL, 27.75 mmol, 3 eq.). The mixture was stirred at rt for 0.5 h and 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 rt, 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-tri(N-benzyloxycarbonyl)cyclohexane 54.

[0128] Compound 54 (0.386 g, 0.726 mmol) was suspended in a 33% solution of HBr in acetic acid (5 mL) and stirred at rt for 12 h. The yellow suspension was then diluted with diethyl ether (20 mL) and filtered through sintered glass S2 to give trihydrobromide 55 as a yellow solid (0.248 g, 91.8%).

[0129] cis,cis-N,N’,N’’-(Cyclohexane-1,3,5-triyl)tris(6-(didodecylamino)hexanamide) 56 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). It was added to a mixed solution of anhydrous TCE (3 mL) and anhydrous DMF (3 mL) of 269 mmol, and the reaction mixture was stirred at rt for 12 hours. The crude product was purified by silica gel column chromatography using a linear gradient of D1 in DCM (20 - 30%). Lipidoide 56 (113 mg, 28.4%; detected by ninhydrin) was obtained in the form of a viscous yellowish oil. HRMS (ESI): C f 0.65, detected by ninhydrin) was obtained in the form of a viscous yellowish oil. HRMS (ESI): C 96 H 193 O 6 N 3 [M + H] + The calculated m / z for was 1478.5134; the measured value was 1478.5162.

[0130] Example 19 Preparation of Transfection Reagents The reagents were prepared by mixing the components from Table 1 to Table 4. All tables include the final molar concentrations in the transfection reagents. For the preparation of A01 to A16, A21, A22, A24, A28 and A29, stock 5 mM solutions of each component in 99.7% ethanol (v / v) were used. For the preparation of A17 - A20, A23, and A25 - A27, stock 5 mM solutions of lipidoide, DOPE and DMG - PEG2000 in 99.7% ethanol (v / v), and a stock 10 mM solution of cholesterol were used. The concentration of the DOPE - Cy5 stock solution was 0.79 mM and it was prepared in chloroform. TT3 is a lipid according to WO2016 / 187531 and was used herein for comparison. The benchmark D - Lin - MC3 - DMA lipid (MedChemExpress Europe) was also used for comparison.

[0131] [Table 1]

[0132]

Table 2

[0133]

Table 3

[0134]

Table 4

[0135] Example 20 Preparation of Lipid Nanoparticles (LNP) Containing Nucleic Acid The siRNA-containing LNP (siRNA-LNP) was prepared as follows: 300 μl of each solution of the transfection reagents A01 - A06 prepared in Example 19 was mixed with a solution of 1.2 nmol siRNA (catalog number 4392420, Ambion) in 300 μl of 10 mM citrate buffer (pH 3.0) using a "Y" microfluidic device having two inputs and one output for sampling. The lipid mixture and the siRNA solution were separately injected into their respective inlets at a constant flow rate of 300 μl / min by a linear pump. Thus, corresponding nanoparticle samples named B01 - B06 were formed from the transfection reagents A01 - A06.

[0136] DNA encoding the fluorescent protein mKate2 was amplified from plasmid pmKate2-C (Evrogen) using primers (5’-ATCAACATATGGTGAGCGAGCTG-3’ (SEQ ID NO: 1); 5’-AAGAATTCCTATCATCTGTGCCAG-3’ (SEQ ID NO: 2)) and cloned into the pET24a vector (Invitrogen) under the T7 promoter. Messenger RNA (mRNA) encoding mKate2 was transcribed in vitro using the Ampliscribe T7-Flash transcription kit (Lucigen) according to the manufacturer's protocol. RNA cap analog ARCA (Jena Bioscience) was added to the in vitro transcription reaction, and the poly(A) tail was synthesized using poly(A) polymerase (New England Biolabs) according to the standard protocol.

[0137] mRNA-containing LNP (mRNA-LNP) was prepared as follows: 300 μl of each solution of the A07-A29 transfection reagent prepared in Example 19 was mixed with a solution of 120 μg of mRNA in 300 μl of 10 mM citrate buffer (pH 3.0), and the preparation was made similar to siRNA-LNP. In this way, corresponding nanoparticle samples named B07-B29 were prepared from the transfection reagent A07-A29. Nanoparticle samples named B08a and B11a were prepared from the transfection reagents A02 and A05, respectively.

[0138] Each LNP sample (B01 - B29) was prepared in three portions. The hydrodynamic diameter of the freshly formed LNP was measured at 25 °C at a scattering angle of 173° using dynamic light scattering (NanoZS Zetasizer, Malvern, Worcestershire, UK). The hydrodynamic diameter of siRNA-LNP was 67 - 110 nm, and that of mRNA-LNP was 82 - 288 nm (Table 5). Particles of this shape were used in subsequent biological tests.

[0139] [Table 5]

[0140] Example 21 Incorporation efficiency of siRNA and mRNA into lipid nanoparticles The packaging efficiency of siRNA (which causes degradation of mRNA encoding tyrosyl DNA phosphodiesterase (TDP2)) prepared in Example 20 into siRNA-LNPs B01 - B06 was determined using the Qubit microRNA Assay Kit (Life Technologies) according to the manufacturer's protocol. The packaging efficiency of mRNA (encoding fluorescent protein mKate2) prepared in Example 20 into mRNA-LNPs B07 - B29 was determined using the Qubit RNA HS Assay Kit (Life Technologies) according to the manufacturer's protocol. The incorporation efficiency was determined by comparing the concentrations of siRNA and mRNA freely available in the nanoparticle solution with the concentrations of siRNA and mRNA released from the nanoparticles after degradation. The LNPs were degraded with a buffer containing Triton X-100 (10 mM Tris-HCl, pH 8.0; 0.1 mM EDTA, 2% Triton X-100). The packaging efficiency of siRNA was high, ranging from 76% to 91%. The packaging efficiency of mRNA was 60% to 96% (Table 6).

[0141]

Table 6

[0142] Example 22 Cytotoxicity of LNP Human cell lines derived from embryonic kidney cells expressing SV40 large T antigen (HEK293T), and human hepatocellular carcinoma cell line (HepG2) were cultured in Dulbecco's modified medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in 96-well plates (5×10 4 cells per 100 μl of culture medium per well) at 37°C, 5% CO 2They were cultured. The cells were transfected with 2 μl of LNP B07-B12 prepared in triplicate in Example 20 (the final total concentration of the total lipid components in the well was 20 μM) or LNP 10 μl (the final total concentration of the total lipid components in the well was 100 μM), and then incubated for 24 hours. The cytotoxicity of the LNP was analyzed by the CellTiterGlo 2.0 cell viability assay (Promega, USA). The cell viability was normalized to non-transfected cells (control). The results are summarized in Tables 7 and 8.

[0143] For both cell lines used, none of the new LNPs showed significant toxicity compared to the 100 μM mixture with TT3 lipid, and the cell viabilities of HEK293T cells and HepG2 cells decreased to only 27% and 15% (Table 8).

[0144] [Table 7]

[0145] [Table 8]

[0146] Example 23 In vitro siRNA transfection using the new LNP Short interfering RNA (siRNA, catalog number 4392420, Ambion) that causes degradation of mRNA encoding tyrosyl-DNA phosphodiesterase 2 (TDP2) was used to prepare siRNA-LNP particles according to Example 20. Lipofectamine RNAiMax (Invitrogen) was used as a control transfection reagent for siRNA transfection. For siRNA-LNP knockdown, human cell line HEK293T and a cell line derived from human multiple myeloma that is very difficult to transfect with available transfection reagents were used (Brito J.L.R., Brown N., Morgan G.J. (2010) The transfection of siRNAs in Multiple Myeloma Cell Lines.In:Min WP., Ichim T. (eds) RNA Interference.Methods in Molecular Biology (Methods and Protocols), vol 623.Humana Press). Cells were seeded in 96-well plates (5×10 4 cells per well in 100 μl of culture medium) and cultured at 37 °C and 5% CO 2Cells were cultured. Cells were transfected with 2 μl of siRNA-LNP (final total lipid component concentration was 20 μM, final siRNA concentration was 16 nM), and then incubated for 24 hours. Transfection was performed in biological triplicates. 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 the LightCycler 480 (Roche Life Science). Primers for amplifying the mRNA encoding TDP2 were as follows: The mRNA encoding GAPDH was used for data normalization (primers: 5'-AATCCCATCACCATCTTCCA-3' (SEQ ID NO: 5) and 5'-TGGACTCCACGACGTACTCA-3' (SEQ ID NO: 6)).

[0147] In all of these cases, the new siRNA-LNP significantly decreased the level of intracellular TDP2 mRNA compared to the commercially available transfection reagent RNAiMax. In the HEK293T cell line, the novel LNPs B02 - B05 showed an efficiency almost 3-fold higher than RNAiMax. In the OPM-2 myeloma cell line, B03 decreased mRNA expression 13-fold more than the commercially available siRNA transfection reagent (Table 9).

[0148]

Table 9

[0149] Example 24 In vitro mRNA transfection using novel LNPs Human cell lines derived from embryonic kidney cells expressing large SV40 T antigen (HEK293T), hepatocellular carcinoma cells (HepG2), and human osteosarcoma-derived cell line (U2OS) were cultured in Dulbecco's modified medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in 96-well plates (5×10 4cells) at 37°C, 5% CO 2 The cells were transfected with 2 μl of mRNA-LNP B07 - B12 prepared in Example 20 and then incubated for 24 hours. The final total concentration of the lipid component in the well was 20 μM, and the amount of mRNA encoding the fluorescent protein mKate2 was 100 ng. Lipofectamine® 2000 was used as the control transfection reagent. The transfection was performed in three biological replicates, and each biological replicate had three technical replicates. The fluorescence of Cy5 and mKate2 was detected using a BD LSR Fortessa cytometer, and the percentage of Cy5-fluorescent positive cells indicating the entry of LNP into the cells (Table 10), the percentage of cells expressing the fluorescent protein mKate2, and the fluorescence intensity of mKate2 (Table 11) were calculated. For the fluorescence intensity, the data was normalized against the commercially available transfection reagent Lipofectamine® 2000. According to the Cy5 fluorescence, it can be seen that all LNPs were introduced into the cells with an efficiency exceeding 90% (Table 10). Furthermore, the new LNP B11 produced 2.13-fold more fluorescent mKate2 protein from the transfected mRNA compared to the cells transfected with the Lipofectamine® 2000 transfection control (Table 11).

[0150]

Table 10

[0151]

Table 11

[0152] Example 25 In vitro mRNA transfection using a novel LNP A human cell line (HEK293T) derived from embryonic kidney cells expressing large SV40T antigen was cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a 96-well plate (2.5×10 4 cells per well in 100 μl of culture medium) at 37 °C and 5% CO 2 . The cells were transfected with the mRNA-LNPs B08a, B11a, B13 - B18, B20 - B25, and B29 prepared in Example 20 and incubated for 24 hours. The final total concentration of the total lipid components in the well was 20 μM, and the amount of mRNA encoding the fluorescent protein mKate2 was 100 ng. B29 LNP containing the benchmark D-Lin-MC3-DMA lipid 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 (Table 12) were analyzed with a BD LSR Fortessa cytometer at a wavelength of 561 - 610 / 20 nm. For the fluorescence intensity, the data was normalized against the benchmark D-Lin-MC3-DMA-containing LNP (B29). As is clear from the fact that the percentage of transfected cells was significantly higher and the fluorescence intensity was significantly increased compared to the benchmark D-Lin-MC3-DMA LNP, all the new LNPs transfected HEK293T cells more efficiently. In particular, the new LNP B22 produced 13.66-fold more fluorescent mKate2 protein from the transfected mRNA compared to the cells transfected with the B29 LNP containing the benchmark D-Lin-MC3-DMA lipid (Table 12).

[0153]

Table 12

[0154] Example 26 Transfection Effect of Novel mRNA-LNPs Monitored by Cre-Based Recombination Mouse embryonic fibroblasts (MEFs) derived from a mouse model with a global dual Cre reporter (Mazumdar, M.D.: Genesis 2007, 45:593 - 605) were seeded in 96 - well plates (2.5×10 4 cells per well in 100 μl of culture medium), and cultured at 37°C, 5% CO 2 for 24 hours in DMEM supplemented with 10% FBS. Messenger RNA (mRNA) encoding Cre recombinase was prepared in vitro from a linearized plasmid as in Example 20 and packaged into LNPs as follows: Samples of transfection reagent A13 - A25, A02, and A27, and a control transfection reagent A29 containing the benchmark D - Lin - MC3 - DMA lipid, as well as 120 μg of mRNA in 300 μl of 10 mM citrate buffer (pH 3.0) were incorporated into LNPs using a microfluidic device similar to that in Example 20. The resulting mRNA - LNPs were immediately diluted with 600 μl of PBS; the corresponding nanoparticles labeled B31 - B43 (formed from transfection reagent A13 - A25), nanoparticle B30 (formed from transfection reagent A02), nanoparticle B44 (formed from transfection reagent A27), and likewise, the benchmark nanoparticles labeled B45 were thus formed from transfection reagent A29.

[0155] Cells were transfected with each mRNA-LNP containing 100 ng of mRNA encoding Cre recombinase (final total concentration of total lipid components in the well was 20 μM), and then incubated for 48 hours. B45 LNP containing the benchmark D-Lin-MC3-DMA lipid was used as a control. Transfection was performed in quadruplicate. The percentage of cells expressing GFP was analyzed using a BD LSR Fortessa cytometer (wavelength 488 - 530 / 30 nm). As is clear from the significantly higher percentage of cells expressing GFP compared to cells transfected with the benchmark D-Lin-MC3-DMA LNP, all new LNPs transfected mT / mG cells more efficiently (B45). Interestingly, the percentage of cells expressing GFP was always over 95% for all new LNPs tested (except B44), whereas for the LNP containing the benchmark D-Lin-MC3-DMA lipid, the percentage of recombinant cells was only 21% (Table 13).

[0156]

Table 13

[0157] Example 27 In vivo distribution of novel mRNA-LNPs Messenger RNA (mRNA) encoding Cre recombinase was prepared in vitro from linearized plasmid as in Example 20 and packaged into LNPs as follows: A 300 μl sample of transfection reagent A22, a control transfection reagent A29 containing the benchmark lipid D-Lin-MC3-DMA, and 120 μg of mRNA in 300 μl of 10 mM citrate buffer (pH 3.0) were incorporated into LNPs using a microfluidic device similar to that in Example 20. The resulting mRNA-LNP was immediately diluted with 600 μl of PBS to form the corresponding B40-labeled nanoparticles from transfection reagent A22. Similarly, B45-labeled nanoparticles were formed from transfection reagent A29. The mRNA-LNP was dialyzed and filtered with PBS. The endotoxin level was less than 2 EU / ml. mRNA-LNP (B40 and B45) was intravenously administered to four mice (breeding BIOCEV, Vestec) having a global dual Cre reporter (Mazumdar, M.D.: Genesis 2007, 45:593-605) at a concentration of 2.0 mg mRNA / kg each to enable analysis of successful recombination. In cells where the mRNA-LNP carrying Cre recombinase mRNA was successfully delivered, chromosomal recombination and subsequent excision of the membrane red protein gene (so-called red tomato), and "turning on" of the transcription of the membrane green protein (GFP) gene occurred. Mice including non-particulate control mice were sacrificed 5 days after particulate administration, and histological analysis of specific organs (liver, heart, kidney, lung, and spleen) was performed according to a standardized protocol. From the analysis of histological images, it was revealed that B40 nanoparticles were completely distributed in the liver, and 50-80% of the cells expressing the red membrane protein were converted to cells expressing the green membrane protein 5 days after application. In the case of LNPs B45 containing D-Lin-MC3-DMA lipid, the conversion rate was approximately 20% (Figure 10).

[0158] Histological analysis was further complemented by evaluation of tdTomato / GFP conversion in the livers of mT / mG mice by genomic DNA (gDNA) PCR analysis. Briefly, 50 ng of gDNA template isolated from each liver sample was used in a PCR reaction with the following primers: (5’-AACGTGCTGGTTATTGCTG-’3 (SEQ ID NO: 7); 5’-AAGTCGTGCTGCTTCATGTG-’3 (SEQ ID NO: 8)). In Figure 11, electrophoretic analysis of gDNA PCR amplification of the obtained liver samples showed a 530 bp PCR product when active Cre recombination occurred at the tdTomato / tGFP locus and a 2943 bp PCR product (upper band) when Cre recombination did not occur. The designated B40 mRNA-LNP showed complete Cre recombination in all 4 mice tested, whereas in the case of the benchmark LNP (B45), recombination was complete in only 50% of the mice tested.

[0159] Example 28 In Vivo Efficacy of Novel mRNA-LNPs Messenger RNA (mRNA) encoding hEPO (human erythropoietin) was prepared in vitro from linearized plasmid similar to Example 20 and packaged into LNP as follows: 300 μl of transfection reagent A23, or control transfection reagent A29 containing benchmark lipid D-Lin-MC3-DMA, and 120 μg of mRNA in 300 μl of 10 mM citrate buffer (pH 3.0) were incorporated into LNP using a microfluidic device similar to Example 20. Thus, corresponding B46-labeled nanoparticles were formed from transfection reagent A23. Similarly, B47-labeled nanoparticles were formed from transfection reagent A29. The mRNA-LNP was dialyzed against PBS and filtered. The endotoxin level was less than 0.3 EU / ml. mRNA-LNP (B46 and B47) was intravenously administered to three C57B1 / 6 mice (BIOCEV, Vestec) at a concentration of 0.5 mg mRNA / kg each, and three control C57B1 / 6 mice were administered PBS. Six hours after administration, blood was collected from the mice via the tail vein and placed in serum separation tubes and allowed to clot at room temperature. Then, the tubes were centrifuged at 7000 rpm for 7 minutes, the serum samples were aliquoted, and stored at -80°C until analysis. The hEPO concentration was determined using the hEPO ELISA assay (catalog number DEP00; R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. The hematological profile was determined using a standardized protocol.

[0160] Nanoparticles named B46 showed high production of human EPO protein 6 hours after administration compared to control B47 LNP, but it was not statistically significant. Importantly, LNP containing the new lipidoid (B46) did not change the hematological profile, in contrast to control B47 LNP. Blood tests after administration of the new LNP were equivalent to the PBS control (Table 14).

[0161]

Table 14

[0162] Example 29 In vivo efficacy of novel siRNA-LNP Using the transfection reagents A02, A14, A16, A17, A22, A23, and A29 of Example 19, siRNAs targeting the mouse apolipoprotein B (ApoB) gene, a hepatocyte-expressed gene involved in cholesterol transport, were used to form siRNA-LNPs designated B48 to B54, respectively (Catalog No. 238055 Apob mouse siPOOL-40 kit, siTOOLs Biotech GmbH), and control non-target siRNA-LNP (enclosed with 238055 Apob mouse siPOOL-40 kit, siTOOLs Biotech GmbH) and siRNA-LNP (B55 to B60) were assembled alternately as described in Example 20. The hydrodynamic diameter measured by dynamic light scattering ranged from 103 nm to 154 nm, and the packaging efficiency of the siRNA pool ranged from 59% to 99%. The siRNA-LNP was dialyzed against PBS. The endotoxin level was less than 2 EU / ml. The mice were fasted for 4 hours before plasma collection by retro-orbital bleeding. The siRNA-LNP targeting ApoB was intravenously administered to 3 C57B1 / 6 mice (BIOCEV, Czech Center of Phenogenomics, Vestec) at a siRNA concentration of 16 μg, 16 μg of non-target siRNA-LNP was administered to another 3 controls, and PBS control was administered to another 3. All mice were sacrificed 2 days after LNP application. Plasma levels of cholesterol, triglyceride, and LDL-C were measured using the automated system of the Czech Phenogenomics Center according to a standardized protocol.

[0163] Clinical biochemistry of plasma markers such as total cholesterol, triglyceride, and LDL-C affected by ApoB knockdown was significantly decreased compared to control animals, demonstrating efficient delivery of ApoB siRNA to the liver by the novel LNP (Table 15).

[0164]

Table 15

[0165]

Table 16

Industrial Applicability

[0166] The transfection particles containing the lipidoid of the present invention are useful for various biological applications in basic research, particularly for delivering active NA and then silencing or activating chromosomal genes or genes, editing the genome or transcriptome, or enabling the expression of proteins encoded by the NA inserted using the transfection particles in cell culture or animal transfection.

[0167] In veterinary and human medicine, the transfection particles containing the lipidoid of general formula I can preferably be used for therapeutic or prophylactic purposes. The therapeutic NA-containing particles can be administered to animals or humans to enable silencing or activation of chromosomal genes, silencing or activation of immunogens, inhibition or activation of signal transduction pathways, editing of the genome or transcriptome, or expression of NA-encoded proteins.

[0168] The lipidoid or transfection agent or transfection particle of formula I can also be used as a pharmaceutical, in particular for gene therapy, and is suitable for use in the treatment of malignant tumors and / or genetic diseases. They can also be formulated for cosmetic or biotechnology applications.

Claims

1. A compound of general formula (I), 【Chemical 1】 where X is selected from the group consisting of -C(=O)NH- and -NHC(=O)-; Y is selected from the group consisting of alkylene C ２ -C8 chains; R １ is the same as or different from each other, and each R １ is independently selected from the group consisting of alkyl C10-C20, alkenyl C10-C20, and alkynyl C10-C20, wherein the alkyl, alkenyl, or alkynyl may be straight-chain or branched, and in the alkyl, alkenyl, or alkynyl, one or more -CH ２ - groups may optionally be substituted with one or more groups selected from -OC(=O)- and -C(=O)O-; However, at least one R １ contains at least 10 carbon atoms; Z's are the same as or different from each other, and each Z is independently selected from the group consisting of hydrogen, -CH ３ and CH ２ OH, and T are the same as or different from each other, and each T is -X-Y-N(R １ ) ２ , -C(=O)O(C １ -C ３ alkyl), [Chemical Formula 2] independently selected from the group consisting of -C(=O)OH and -NHC(=O)CH3, where at least one T is X-Y-N(R1)2, and, when Z is -CH ２ OH and T is -C(=O)OH, Z can form a cyclic lactone containing 4 to 5 carbon atoms together with T and the three carbon atoms between them; a compound, or a pharmaceutically acceptable salt, addition salt and solvate thereof.

2. All Rs in the molecule １ The compound according to claim 1, wherein all are the same.

3. Both substituents T are -X-Y-N(R １ ), ２ which is the compound according to Claim 1.

4. Z is selected from the group consisting of hydrogen, -CH ３ and -CH ２ OH; and T is -X-Y-N(R １ ) ２ , -C(=O)O(CH3), [Chemical Formula 3] and selected from the group consisting of -C(=O)OH; and / or, Z is -CH ２ When OH and T is -C(=O)OH, Z may form a cyclic lactone containing 4 to 5 carbon atoms together with T and three carbon atoms between them, the compound according to claim 1.

5. The compound of general formula (I) contains at least two R １ substituents, each of these substituents containing at least 12 carbon atoms, the compound according to claim 1.

6. Comprising at least one compound of general formula (I) according to claim 1 and at least one helper lipid; said helper lipid is cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-rac-glycero-3-methoxypoly(ethylene glycol)-2000, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-2000, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine poly(ethylene glycol)-2000, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine poly(ethylene glycol)-2000, a transfection agent selected from the group consisting of.

7. The transfection agent according to claim 6, comprising at least one compound of general formula (I) according to claim 1 in an amount of 10 to 50 mol% and at least one helper lipid in an amount of 50 to 90 mol%.

8. At least one compound of general formula (I) according to claim 1 in an amount of 15 to 40 mol%, cholesterol in an amount of 30 to 55 mol%, and at least one other helper lipid in an amount of 20 to 50 mol%; said other helper lipid is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-rac-glycero-3-methoxypoly(ethylene glycol)-2000, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-2000, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine poly(ethylene glycol)-2000, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine poly(ethylene glycol)-2000, the transfection agent according to claim 6.

9. A transfection particle comprising at least one compound of general formula (I) according to claim 1, at least one nucleic acid and / or a part thereof and / or a nucleic acid derivative; and at least one helper lipid.

10. A method of using the compound of general formula (I) according to any one of claims 1 to 5, or the transfection agent according to claims 6, 7 or 8, or the transfection particle according to claim 9, for in vitro transfection of cells or tissues with a nucleic acid and / or a part thereof and / or a nucleic acid derivative.

11. The method of use according to claim 10, for silencing or activating chromosomal genes, silencing or activating immune genes, inhibiting or activating signal transduction pathways, editing the genome or transcriptome, or enabling the expression of proteins encoded by nucleic acids.

12. The compound of general formula (I) according to any one of claims 1 to 5, or the transfection agent according to claim 6, 7 or 8, or the transfection particle according to claim 9, for use in the transfection of cells or tissues by nucleic acids and / or a part thereof and / or nucleic acid derivatives in vivo, provided that it is excluded from the transfection of human embryos for industrial or commercial purposes and from the modification of the human germ line, transfection particles.

13. For use in the in vitro transfection of cells or tissues by nucleic acids and / or a part thereof and / or nucleic acid derivatives, which enables the silencing or activation of chromosomal genes, the silencing or activation of immune genes, the inhibition or activation of signal transduction pathways, the editing of the genome or transcriptome, or the expression of proteins (plural) encoded by nucleic acids, the compound of general formula (I) according to any one of claims 1 to 5, or the transfection agent according to claim 6, 7 or 8, or the transfection particle according to claim 9.

14. The compound of general formula (I) according to any one of claims 1 to 5, or the transfection agent according to claim 6, 7 or 8, or the transfection particle according to claim 9, for use as a pharmaceutical.

15. The compound, or transfection agent, or transfection particle according to claim 14, wherein the pharmaceutical is for the treatment of malignant tumors and / or genetic diseases.

16. The compound, or transfection agent, or transfection particle according to claim 14, wherein the pharmaceutical is for gene therapy.

17. The compound of general formula (I) according to any one of claims 1 to 5, or the transfection agent according to claim 6, 7 or 8, or the transfection particle according to claim 9, for use as a prophylactic vaccine.

18. The compound, or transfection agent, or transfection particle according to claim 17, wherein the prophylactic vaccine is for the prevention of infectious diseases.

19. Use of a compound of general formula (I) according to any one of claims 1 to 5, or a transfection agent according to claim 6, 7 or 8, or transfection particles according to claim 9, in a cosmetic preparation for delivering an active ingredient to a site of action.

20. A compound of general formula (I) according to claim 1, selected from the group consisting of: [Chemical Formula 4] 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 【Chem.】 [Chemical]

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