Compounds, process for their preparation and their use for the preparation of lipid nanoparticles

Abstract

The invention relates to compounds of general formula (I), where X1, X2, X3 and y are defined in the description. The invention further relates to a process for the preparation of said compounds, and to the use of said compounds for the preparation of lipid nanoparticles carrying active ingredients. The invention further relates to a pharmaceutical composition comprising a nanoparticle according to the invention, and to the nanoparticle for use in medicine.

Description

[0001] COMPOUNDS, PROCESS FOR THEIR PREPARATION AND THEIR USE FOR THE PREPARATION OF

[0002] LIPID NANOPARTICLES

[0003] The invention relates to compounds of general formula (I)

[0004] (I), where Xi, X2, X3and y are defined below. The invention further relates to a process for the preparation of said compounds and to the use of said compounds for the preparation of lipid nanoparticles carrying active ingredients.

[0005] STATE OF THE ART

[0006] US Patent No. US10166298B2 discloses lipid nanoparticle formulations for delivery of active ingredients (e.g. oligonucleotides, mRNA), in which formulations the lipid component is a cationic lipid molecule. In addition to cationic lipid molecules, other neutral lipids, cholesterol and polymer- conjugated lipids can be used to form lipid nanoparticles. The cationic lipids according to the cited document have the general formula R1-C(O)-O-G1-N(G3-R3)-G2-O-C(O)-R2, wherein G1, G2 and G3 are unsubstituted alkylene groups, R1 and R2 are alkyl groups, and R3 is OR5, CN, - C(O)OR4, -OC(O)R4 or NR5C(O)R4, where R4 and R5 are hydrogen atoms or alkyl groups. According to the general formula, the cationic lipids of the cited document are essentially tertiary amine compounds, where, as two hydrophobic groups, two fatty acid moieties are linked to the central nitrogen atom by their carboxylic groups, each through an alkylene linker. The structure of the molecules according to the cited document differs significantly from the structure of the lipid molecules according to the present invention.

[0007] US patent US10723692B2 describes molecules with a structure similar to the lipid molecules shown in the previously cited document, which are essentially tertiary amine compounds. The document also discloses lipid molecules in which a polyethylene glycol chain is linked to an amide group, the nitrogen atom of which is substituted by two straight-chain saturated alkyl groups. Similar lipid molecules are also disclosed in US Patent No. US10221127B2 and International Patent Publications No. WO2018081480A1 , WO2018078053 A1 and WO2018078053A1. In the lipids according to US patent US 2019022247 A1 , the hydrophobic chains are linked to a central tertiary carbon atom. Lipids similar to the foregoing are also disclosed in US patent US10369226B2, with the difference that in this case the two hydrophobic chains are linked to a central carboxyl group, oxygen, nitrogen or sulfur atom through a carbon atom. The aforementioned cited documents are substantially different from the lipid molecules according to the present invention.

[0008] International Patent Publication No. WO2020254535A1 discloses an anti-rotavirus composition, wherein according to one embodiment, the active ingredient forms a lipid nanoparticle with a lipid molecule in the composition. According to one embodiment, said lipid molecule is a tertiary amine, wherein a hydroxylbutyl group is attached to the central nitrogen atom and fatty acid moieties with their carboxyl group are attached via an alkylene linker. According to another embodiment, the tertiary amine is substituted with two alkyl groups and a polyethylene glycol chain via a carboxyl group. The lipid molecules according to the cited document are substantially different from the lipid molecules according to the present invention.

[0009] International Patent Publication No. WO2018200892A1 discloses a formulation against Hepatitis C virus, wherein the active ingredient is encapsulated in a lipid nanoparticle. The central part of the lipid molecules constituting the nanoparticle is formed by a tertiary amine or an amide group, to which central part are attached two hydrophobic chains or an alkyl chain and a double - stranded hydrophobic chain through a tertiary carbon atom, where the hydrophobic chains are fatty acid moieties linked through an alkylene group. The cited document also describes lipid molecules containing polyethylene chains.

[0010] US Patent No. US2013123485A1 discloses cationic lipids comprising amino acids and derivatives thereof, and drug delivery systems comprising these lipids. Said lipids are characterized by the following reference formula: wherein n is 1 to 4, R1and R2are alkyl or alkenyl groups of 7 to 24 carbon atoms, B is A-NH, wherein A may be a sugar or a group of formula R4-X-C(O)-CH-(R3)-, wherein X is NH or O, R3is a hydrocarbon having a cationic group derived from an amino acid, for example -(CH2)4-NH2, R4 represents, for example, an alkyl or benzyl group, a sugar, an antibody, a polyethylene glycol. In the central part of the lipids described, asparagine, glutamine, ornithine and lysine can be recognized. The structure of the lipids according to the cited document is different from the structure of the lipids according to the present invention.

[0011] Russian Patent No RU2533554C1 discloses tetrapeptides containing glutamic acid, ornithine and lysine for the preparation of liposomes. The hydrophobic moieties of the disclosed molecules are composed of alcohols with 8 to 16 carbon atoms, which are linked to the amino acids by ester linkages. Regarding the efficiency of tetrapeptides, the cited document states only that their transfection efficiency is 3-5% better than that of dipeptides for a given cell line. The structure of the molecules disclosed in the cited document differs significantly from the structure of the lipids of the invention.

[0012] US patent US7838683B2 discloses cationic amino acid-based lipids, which can be described by the following general formula:

[0013] Reference formula 2 wherein R1together with the C=O group forms an a-amino acid (lysine, arginine or histidine), R2and R3represent alkyl, alkenyl or alkynyl groups of 12 to 30 carbon atoms, A1and A2represent -COO-, -OCO-, -CONH- or -NHCO- groups and n is 2 to 4. Regarding the applicability of the presented lipids, the cited document states that they can be used, for example, for liposomes, cell culture substrates, as emulsifiers, stabilizers or dispersants for medicines, foods or cosmetics. The molecules disclosed in the cited document also have a structure substantially different from that of the lipids according to the invention.

[0014] European patent publication No. EP3686184A42 also describes lipids for the preparation of nanoparticles. In some of the molecules presented, [3-glutamic acid is recognizable in the head fragment, but the amino group of the head fragment is typically substituted by an ω-amino-alkyl- carbonyl group instead of a tertiary amino-alkyl group; and they contain a single-stranded lipophilic chain directly linked to the carboxyl groups of the head fragment by an ester linkage, without a linker, so that the molecules presented herein also differ substantially from the lipids of the invention. International Patent Publication No. W02006098415 41 discloses drug carrier molecules in which e.g. lysine is recognizable as central moiety, and the attached substituents are different from the lipophilic chains found in the lipid molecules of the invention.

[0015] International Patent Publication No. WO19971884042 relates to compounds capable of forming so-called high axial ratio microstructures (e.g. microtubules) to which active molecules (e.g. peptides, nucleic acids) can be attached directly or by polypeptides. According to the document, lipid molecules, such as glutamate-based or polyglutamate-based amphiphilic molecules or ceramide-type compounds are suitable for the formation of the mentioned microstructures, however, the structure of said compounds differs from the molecules according to the present invention. Another important difference is that while the cited document discloses structures on the micrometer scale, the invention relates to nanoparticles.

[0016] International Patent Publication No. WO20100374084.1 discloses an immunostimulant composition comprising a nucleic acid complexed with a cationic or polycationic compound. The cationic or polycationic compounds may be, for example, peptides, proteins, cationic polysaccharides or cationic lipids. However, the cited document does not disclose anything about the structure of cationic lipids and does not mention lipid nanoparticles.

[0017] European Patent No. EP1553109B1 describes polymer gels, one of the constituents of which is a cationic lipid based on glutamic acid, to the two carboxyl groups of which a straight or branched chain alkyl group is connected with an amide bond, and to the amino group, also with an amide bond, an alkyl chain containing a quaternary ammonium group is connected. The structure of the molecules according to the cited document differs from the structure of the lipid molecules according to the invention, and lipid nanoparticles are not disclosed therein.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1: Monitoring the intensity and course of the bioluminescence signal measured in the musculus gastrocnemius of BALB / c mice using the PerkinElmer IVIS imaging system. The translatability profile of the different Luc. mRNA-LNPs can be inferred from the analysis of the bioluminescence signal intensity and course. For comparison, we used Luc. mRNA-LNP containing ALC0315 ionizable lipid as a reference compound. Luc. mRNA-LNPs prepared with the following compounds of the invention: Figure (A): KA453XHCl, KA459XHCl, KA470XHCl, Figure (B): KA417, KA459FB, Figure (C): KA479XHCl and KA481XHCl, Figure (D): KA488XHCl and KA490XHCl. Figure 2: Analysis of the distribution of the total bioluminescence signal from BALB / c mice. PerkinElmer IVIS imaging system was used to investigate the distribution of the bioluminescence signal obtained during whole-body measurement between the liver and the inoculation site (musculus gastronemius). Such studies of the bioluminescence signal allow the determination of the localization patterns of individual Luc. mRNA-LNPs. For comparison, we used a Luc. mRNA-LNP containing ALC0315 ionizable lipid as reference compound.

[0020] Figure 3: Ionizable lipid toxicity study in BALB / cOlaHsd mice. For comparison, an empty LNP containing ALC0315 ionizable lipid as reference compound was used. The mean of the clinical scores for each immunized group is shown in the figure. LNPs prepared with the following compounds of the invention: Figure (A): KA417, KA453XHCl, Figure (B): KA459XHCl, KA459FB, Figure (C): KA470XHCl, KA479XHCl and Figure (D): KA481 XHCl, KA488XHCl and KA490XHCl.

[0021] Figure 4: Study of the adjuvant effect of ionizable lipids using PR8 HA specific ELISA. For comparison, an empty LNP containing ALC0315 ionizable lipid as reference compound was used. Naive: group of non-immunized animals. No significant difference between immunized groups, with the exception of KA479. All immunized groups showed a significant difference from the naive group, the significance level being: p<0.0001. Statistical analysis used: one-way ANOVA, Bonferroni's multiple comparisons test.

[0022] Figure 5: Toxicity assay of empty LNPs containing ionizable cationic lipids in HepG2 liver cells [figure (a)] and HEK293 kidney cells [figure (b)] by XTT assay. With the exception of LNPs containing cationic lipids KA479 and KA481, the new lipids did not show toxicity even at high concentrations, similar to the LNP containing the lipid ALC0315 used as reference compound.

[0023] THE TECHNICAL PROBLEM TO BE SOLVED BY THE INVENTION

[0024] The technical problem to be solved by the invention is the provision of cationic lipid molecules for the preparation of lipid nanoparticles carrying active ingredients.

[0025] THE INSIGHT ON WHICH THE INVENTION IS BASED

[0026] In order to achieve said technical goal, we have carried out systematic experimental work, which has resulted in our invention. During our experiments, surprisingly, we have found the following: a) If the head group of the lipid molecule is formed by a trifunctional central molecular part, which is a dicarboxylic amino acid (L-aspartic acid or L-glutamic acid), then the modular synthesis of cationic lipids can be ensured. The two lipid chains required to form the LNP structure are attached to the two functional groups of the dicarboxylic amino acid, while the cationic group capable of ionic interaction with anionic polymers (e.g. RNA, DNA) is attached to the third functional group of the amino acid. This combinatorial synthesis approach results in a simple and high degree of variability of the head group. b) The lipid molecules according to the invention are biocompatible and have a preferable (low) toxicity. c) The lipid molecules of the invention ensure a more specific targeting of the lipid nanoparticles compared to the lipids known from the state of the art (i.e. less or no lipid appears in the liver). d) The lipid molecules according to the invention have a high adjuvant activity. e) LNPs comprising lipid molecules according to the invention have a translatability equal to or better than that of prior art LNPs.

[0027] BRIEF DESCRIPTION OF THE INVENTION

[0028] The invention relates to a compound of the following general formula (I): wherein: y is 1 or 2,

[0029] X1represents a group of formula Alk-L-,

[0030] X2represents a group of formula Alk-L-, where Alk represents a straight or branched chain alkyl group of 10 to 24 carbon atoms,

[0031] L represents a group of formula

[0032] #-C(O)-O-(CH2)n-O-;

[0033] #-C(O)-O-(CH2)n-NH- or

[0034] #-CH2-O-C(O)-(CH2)n-i-NH-; where # indicates the part attached to the Alk- group, and where n is an integer from 2 to 8, X3represents a group selected from glycyl, L-alanyl, L-valyl, L-leucyl, L-isoleucyl, L-seryl, L- threonyl, L-cysteinyl, L-methionyl, L-phenylalanyl, L-tyrosyl, L-tryptophanyl, L-prolyl, L- asparaginyl, L-glutaminyl, L-asparagyl, L-glutamyl, L-lysyl, L-histidyl, L-arginyl, the carbon atom in the CH* group has the L configuration, or a pharmaceutically acceptable salt thereof.

[0035] The invention further relates to a process for the preparation of a compound of general formula (I) or a pharmaceutically acceptable salt thereof, comprising the steps of: a) reacting a compound of formula 1-1 with a diol of formula I-la under ester-forming conditions thereby obtaining a compound of formula 1-2 b) esterifying the compound of formula I-2a with the compound of formula 1-2 under similar conditions as in the previous step then removing the PG1protecting group, thereby obtaining a compound of formula 1-3; c) N-acylating the compound of formula 1-3 with a compound of formula I-3a then removing the PG2protecting group, thereby obtaining a compound of formula 1-4 wherein Alk, n, and y are as defined in claim 1, PG1and PG2are amino-protecting groups, and R, taken together with the group -CO-CH(NH2)- to which it is attached, has the same meaning as X3defined in claim 1; or a) reacting a compound of formula 1-1 with a compound of formula Il-la under ester-forming conditions then removing the PG1protecting group, thereby obtaining a compound of formula II-2 b) N-acylating the compound of formula II-2 with a compound of formula II-2a then removing the PG2protecting group, thereby obtaining a compound of formula II-3 c) N-acylating the compound of formula II -3 with a compound of formula II-3a ll-3a then removing the PG3protecting group, thereby obtaining a compound of formula II-4 wherein Alk, n, and y are as defined in connection with general formula (I), PG1, PG2and PG3are amino-protecting groups, and R, taken together with the group -CO-CH(NH2)- to which it is attached, has the same meaning as X3defined in connection with general formula (I); or a) reacting an alcohol of formula III-l with a compound of formula Ill-la under ester-forming conditions then removing the PG1protecting group, thereby obtaining a compound of formula III-2 b) N-acylating the compound of formula III-2 with a compound of formula III-2a lll-2a then removing the PG2protecting group, thereby obtaining a compound of formula III-3 c) N-acylating the compound of formula III-3 with a compound of formula III-3a lll-3a then removing the PG3protecting group, thereby obtaining a compound of formula III-4 ? wherein Alk, n, and y are as defined in connection with general formula (I), PG1, PG2and PG3are amino-protecting groups, and R, taken together with the group -CO-CH(NH2)- to which it is attached, has the same meaning as X3defined in connection with general formula (I).

[0036] The invention further relates to a nanoparticle comprising the compound of general formula (I) or a pharmaceutically acceptable salt thereof.

[0037] The invention further relates to the use of a compound of general formula (I) or a pharmaceutically acceptable salt thereof for the preparation of a nanoparticle carrying an active ingredient.

[0038] The invention also relates to a pharmaceutical composition comprising a nanoparticle according to the invention.

[0039] The invention also relates to a nanoparticle according to the invention for use in medicine.

[0040] DETAILED DESCRIPTION OF THE INVENTION

[0041] In this description, we use the following acronyms, which have the indicated meanings, the knowledge of which can be expected from a skilled person:

[0042] LNP = lipid nanoparticle; EDC = 1-ethyl-3-(3-dimethylaminopropyl) -carbodiimide;

[0043] DMAP = 4-dimethylamino-pyridine;

[0044] GDI = 1,1'-carbonyldiimidazole;

[0045] HATU = 1 - [bis (dimethylamino) methylene] -1 H-1 ,2, 3-triazolo [4,5-b] pyridinium-3-oxide hexafluorophosphate;

[0046] TBTU = 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate;

[0047] DSCP = 1 ,2-distearoyl-sn-glycero-3-phosphocholine;

[0048] PBS = phosphate -buffered saline;

[0049] DIEA = N,N-diisopropylethylamine;

[0050] XTT = 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium

[0051] PEG = poly (ethylene glycol).

[0052] DMG-PEG = 1 ,2-dimiristol-rac-glycero-3-methoxypolyethylene glycol-2000.

[0053] It is known from the state of the art that lipid nanoparticle (LNP) based technologies use phospholipids, cholesterol, polyethylene glycol lipids and ionizable lipids to prepare nanoparticles that serve as carriers for therapeutic agents. Modification of the ionizable lipid component allows tunability of the properties of the LNP system.

[0054] The novel lipids according to the present invention allow the incorporation of therapeutically active molecules to be delivered (e.g. nucleic acids, polypeptides, proteins, small molecules, active pharmaceutical ingredients, lipids), targeting moieties or small molecule reporters into LNP and the introduction of the resulting LNP into cells, while preserving the stability and homogeneity of the LNP.

[0055] The biological efficiency of mRNA-LNPs, besides the mRNA, depends largely on the ionizable lipid components of the LNPs. Ionizable lipids affect the distribution of mRNA-LNPs within the cell and the body, act as adjuvants, and reduce the toxic effects of LNPs. This nature of ionizable lipids has facilitated the widespread adoption of procedures based on mRNA-LNP technology. The further development of efficient ionizable lipids with less toxicity and diverse organ-specific distribution has become an intensively developing area of lipid research. Our research has led to the preparation and characterization of a completely new family of ionizable lipids. The adjuvant effect and the effect on mRNA translatability of these synthesized lipids, their distribution within the body, and their toxicity were determined.

[0056] The subject of the invention is therefore a compound of the following general formula (I): wherein: y is 1 or 2,

[0057] Xi represents a group of formula Alk-L-,

[0058] X2represents a group of formula Alk-L-, where Alk represents a straight or branched chain alkyl group of 10 to 24 carbon atoms,

[0059] L represents a group of formula

[0060] #-C(O)-O-(CH2)n-O-;

[0061] #-C(O)-O-(CH2)n-NH- or

[0062] #-CH2-O-C(O)-(CH2)n-i-NH-; where # indicates the part attached to the Alk- group, and where n is an integer from 2 to 8, X3represents a group selected from glycyl, L-alanyl, L-valyl, L-leucyl, L-isoleucyl, L-seryl, L- threonyl, L-cysteinyl, L-methionyl, L-phenylalanyl, L-tyrosyl, L-tryptophanyl, L-prolyl, L- asparaginyl, L-glutaminyl, L-asparagyl, L-glutamyl, L-lysyl, L-histidyl, L-arginyl, the carbon atom in the CH* group has the L configuration, or a pharmaceutically acceptable salt thereof.

[0063] In one embodiment, X3is selected from glycyl, L-alanyl, L-valyl, L-leucyl, L-isoleucyl, L-seryl, L-threonyl, L-cysteinyl, L-methionyl, L-phenylalanyl, L-tyrosyl, L-tryptophanyl, L-prolyl, L- asparaginyl, L-glutaminyl, L-lysyl, L-histidyl, In another embodiment, X3is selected from L-alanyl, L-valyl, L-leucyl, L-isoleucyl, L- methionyl, L-phenylalanyl, L- tyrosyl and L-tryptophanyl groups.

[0064] In a further embodiment, X3is selected from L-valyl, L-leucyl, L-methionyl, L-tyrosyl and L- tryptophanyl groups.

[0065] In one embodiment, the compound is selected from the following compounds: KA417, KA449, KA453, KA455, KA470, KA479, KA504, KA505, KA507, KA460, KA459, KA488, KA489, KA490, KA491, KA495, KA497, KA498, KA500, KA506, KA481, and pharmaceutically acceptable salts thereof.

[0066] In a further embodiment, the compound is selected from the following: KA417, KA449X2HCl, KA453XHCl, KA455XHCl, KA470XHCl, KA479XHCl, KA504, KA505, KA507, KA460XHCl, KA459XHCl, KA459, KA488XHCl, KA489XHCl, KA490XHCl, KA491XHCl, KA495XHCl, KA497XHCl, KA498XHCl, KA500XHCl, KA506, KA481XHCl.

[0067] In a further embodiment, the compound is selected from the following compounds: KA417, KA453, KA470, KA479, KA459, KA488, KA490, KA481, and pharmaceutically acceptable salts thereof.

[0068] In a further embodiment, the compound is selected from the following compounds: KA417, KA453XHCl, KA470XHCl, KA479XHCl, KA459XHCl, KA459, KA488XHCl, KA490XHCl, KA481XHCl.

[0069] Pharmaceutically acceptable salts are the addition salts of compounds of general formula (I) formed with pharmaceutically acceptable acids. Examples of suitable acids are hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, trifluoroacetic acid.

[0070] In one embodiment, the salt is a hydrochloride salt.

[0071] We note that the compounds may be isolated as free base or as any pharmaceutically acceptable salt. In this regard, we emphasize that the scope of the invention includes the compounds according to the examples, regardless of the specifically presented form, both in the form of the free base and in the form of any pharmaceutically acceptable salt. . For a full understanding, the general processes for the preparation of the compounds of the invention are described in detail below.

[0072] General reaction scheme I

[0073] General reaction scheme I describes the preparation of molecules according to general formula (I) wherein L is #-C(O)-O-(CH2)n-O- (# denotes the part attached to the alkyl chain) (e.g. compound KA417 (BNL1)). In general reaction scheme I, Alk, n and y are as defined in connection with formula (I), PG1and PG2are amino-protecting groups (e.g. terc-butyloxycarbonyl group), and R denotes the corresponding a-amino acid side chain of each substituent defined for X3in general formula (I).

[0074] General reaction scheme I

[0075] By esterification of 1-1 carboxylic acid with the appropriate diol using methods available in the literature (e.g. in the presence of EDC-HCl and DMAP) 1-2 alcohol can be obtained. By esterification of an amino-protected L-aspartic acid or L-glutamic acid, using conditions similar to the previous step (e.g. in the presence of EDC-HCl and DMAP) and subsequent removal of the amino-protecting group (e.g. with trifluoroacetic acid in the case of a terc-butyloxycarbonyl group) 1-3 can be obtained. By N-acylation of 1-3 amine under standard conditions with an amino acid of choice (e.g. in the presence of HATU) and by removal of the amino-prc 2- its pharmaceutically acceptable salt (e.g. hydrochloride) is obtained. General reaction scheme II

[0076] General reaction scheme II describes the preparation of molecules according to general formula (I) wherein L is a group of formula #-C(O)-O-(CH2)n-NH- (# denotes the part attached to the alkyl chain) (e.g. compound BNL10). In general reaction scheme II, Alk, n and y are as defined in connection with formula (I), PG1, PG2and PG3are amino-protecting groups (e.g. terc- butyloxycarbonyl group), and R denotes the corresponding a-amino acid side chain of each substituent defined for X3in general formula (I).

[0077] 2. Deprotection 2. Deprotection

[0078] 1-1

[0079] General reaction scheme II

[0080] By esterification of 1-1 carboxylic acid with the appropriate amino alcohol having its amino group protected (e.g. by tert-butoxycarbonyl group), using methods available in the literature (e.g. in the presence of EDC-HCl and DMAP) and after removal of the protecting group (e.g. with trifluoroacetic acid in the case of a terc -butyloxycarbonyl group) II-2 amine can be obtained. By amide coupling of an amino-protected L-aspartic acid or L-glutamic acid (e.g. in the presence of HATU) followed by removal of the amino-protecting group (e.g. with trifluoroacetic acid in the case of terc -butyloxycarbonyl group) II-3 can be obtained. By N-acylation of II-3 amine under standard conditions with an amino acid of choice (e.g. in the presence of HATU) and by removal of the amino-protecting group, II-4 lipid or its pharmaceutically acceptable salt (e.g. hydrochloride) is obtained. General reaction scheme III

[0081] General reaction scheme III describes the preparation of molecules according to general formula (I), wherein L is a group of formula #-CH2-O-C(O)-(CH2)n-i-NH- (# denotes the part attached to the alkyl chain) (e.g. compound BNL21). In general reaction scheme III, Alk, n and y are as defined in connection with formula (I), PG1, PG2and PG3are amino-protecting groups (e.g. terc -butyloxycarbonyl group), and R denotes the corresponding a-amino acid side chain of each substituent defined for X3in general formula (I). 111-1 a lll-2a

[0082] Aik OH

[0083] 111-1 2. Deprotection 2. Deprotection

[0084] General reaction scheme III

[0085] By esterification of III-l alcohol with the appropriate ω-amino-carboxylic acid having its amino group protected (e.g. by tert-butoxycarbonyl group), using methods available in the literature (e.g. in the presence of EDC-HCl and DMAP) and after removal of the protecting group (e.g. with trifluoroacetic acid in the case of a terc -butyloxycarbonyl group) III-2 amine can be obtained. By amide coupling of an amino-protected L-aspartic acid or L-glutamic acid (e.g. in the presence of HATU) followed by removal of the amino-protecting group (e.g. with trifluoroacetic acid in the case of terc -butyloxycarbonyl group) III-3 can be obtained. By N-acylation of III-3 amine under standard conditions with an amino acid of choice (e.g. in the presence of HATU) and by removal of the amino-protecting group, III-4 lipid or its pharmacologically acceptable salt (e.g. hydrochloride) is obtained.

[0086] The invention further relates to a nanoparticle comprising the compound according to the invention. The lipid nanoparticle according to the invention comprises the usual ingredients in the field, typically: cationic lipid according to the invention (30-70%), phospholipid (10-40%), sterol (10- 40%) and polymer conjugated lipid (0.5-5%).

[0087] Phospholipids suitable for the preparation of LNPs are known to the skilled person, for example: distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylethanolamine (POPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimiristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethylphosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE).

[0088] Sterols suitable for the preparation of LNPs are known to the skilled person, examples include: cholesterol and its derivatives such as cholestanol, cholestanone, cholestenone.

[0089] Polymer-conjugated lipids suitable for the preparation of LNPs are known to the skilled person, examples include: PEG-lipid conjugates such as dialkyloxypropyl-linked PEG, diacylglycerol-linked PEG, cholesterol-linked PEG, phosphatidylethanolamine-linked PEG, polyamide oligomers (e.g. ATTA-lipid conjugates) .

[0090] The invention also relates to the use of a compound according to the invention for the preparation of a nanoparticle carrying an active ingredient. Examples of said active ingredient include, without limitation, the following: nucleic acids, polypeptides, proteins, small molecules, pharmaceutical agents, lipids.

[0091] In a preferred embodiment of the invention the active ingredient is a nucleic acid, more preferably an mRNA.

[0092] The invention also relates to a pharmaceutical composition comprising the nanoparticle according to the invention and a pharmaceutically acceptable carrier or excipient.

[0093] By pharmaceutically acceptable carriers and excipients substances commonly used in the field, e.g. solvents, dispersants, preservatives, etc. are meant.

[0094] The products are suitable for veterinary or human administration. LNPs and preparations containing them may be administered by different routes, such as in the form of injections, and by different routes of administration, including parenteral or oral administration.

[0095] The invention also relates to a nanoparticle according to the invention for use in medicine. The lipid molecules of the invention have lower toxicity than, for example, lipids with tertian’ amine head group. The LNPs prepared with the lipid molecules of the invention provide greater specificity of targeting and have equal or better translatability compared to prior art LNPs. Furthermore, the lipids of the invention exhibit high adjuvant activity. Thus, LNPs prepared using the cationic lipids disclosed herein can be used in a more optimal and safer manner for targeting various therapeutic cargo-mules (nucleic acids, peptides, lipids) in mammalians or mammalian cells.

[0096] In one embodiment, lipid nanoparticles comprising 30-70% of the lipid of the invention can be used to deliver nucleic acids into mammalian cells with unexpectedly significantly higher efficiency compared to other lipid nanoparticles.

[0097] In a further embodiment, the targeting of the selected molecule to be delivered (nucleic acids, polypeptides, proteins, small molecules, pharmaceutical agents, lipids) can be further optimized by varying the proportions of cationic lipid (30-70%), phospholipid (10-40%)), sterol (10-40%) and polymer conjugated lipid (0.5-5%,) components in a modular manner.

[0098] The reference compound used in the biological studies was ALC0315 (6-[6-(2- hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl-2-hexyldecanoate, CAS 2036272-55-4) (WO 2018 / 081480, Acuitas Therapeutics):

[0099] Reference compound (ALC0315)

[0100] EXAMPLES

[0101] Example 1: Compound KA417 (BNL1) The compound KA417 (BNL1) was prepared according to the general reaction scheme I as follows.

[0102] 2-hexyldecanoic acid (2.57 g, 10.02 mmol), 1,6-hexanediol (3.55 g, 30.04 mmol), triethylamine (35.2 mmol, 3.5 eq.) and 4-dimethylamino-pyridine (1.22 g, 10.00 mmol) were dissolved in a mixture of 10 ml acetonitrile and 10 ml dichloromethane. EDC-HCl was added to the mixture and stirred at 37° C for 18 h using an oil bath. The resulting mixture with white precipitate was evaporated, filtered through a silica gel layer with 50 ml ethyl acetate and evaporated. The resulting colorless oil was purified on silica gel (40 g) with ethyl acetate / hexane 3:1 eluent to give 2.62 g of 1 (73%).

[0103] 1 (709 mg, 1.99 mmol), N-Boc-L-glutamic acid (189 mg, 0.77 mmol), triethylamine (0.53 ml, 1.53 mmol) and N,N-dimethylaminopyridine (187 mg, 1.53 mmol) were dissolved in a mixture of 5 ml dichloromethane and 5 ml acetonitrile. EDC-HCl (381 mg, 1.99 mmol) was added to the resulting mixture and the mixture was stirred at 37 °C for 18 h. After the reaction time, the mixture was evaporated and filtered through a silica gel layer with 30 ml ethyl acetate, the filtrate was evaporated. The resulting material was purified on silica gel (30 g) with a 3:1 eluent of hexane / ethyl acetate. Thus 421 mg (60%) of 2 was obtained.

[0104] 2 (110.2 mg, 0.119 mmol) was dissolved in 2 ml dichloromethane, then 2 ml trifluoroacetic acid was added and stirred for 30 min at room temperature. The mixture was evaporated and then evaporated 3 further times from 5 ml each of dichloromethane and 3 times from 5 ml each of ethanol. This gave 108 mg (96%) of 3 trifluoroacetate.

[0105] 107.5 mg of 3 trifluoroacetate was dissolved in 1 ml of dichloromethane, triethylamine (19 pl, 0.138 mmol) was added. N-Boc-L-tyrosine (35.6 mg, 0.127 mmol) was dissolved in 1 ml acetonitrile, triethylamine (19 pl, 0.138 mmol) and HATU (48.3 mg, 0.127 mmol) were added. The resulting mixture was stirred for 5 min at room temperature, then the mixture of 3 in dichloromethane was added and stirred for 18 h at room temperature. The mixture was evaporated and purified on silica gel (15 g) with a 4:3 eluent of hexane / ethyl acetate. Thus 107 mg (86%) of 4 was obtained.

[0106] 4 (35.6 mg, 32.7 pmol) was dissolved in 700 pl dichloromethane, 70 pl trifluoroacetic acid was added and stirred for 1.5 h at room temperature. The mixture was evaporated and then evaporated 3 further times from 2 ml each of dichloromethane. It was then purified on silica gel (10 g) using 100 % ethyl acetate followed by acetone (+1 % 7 N ammonia in methanol solution) as eluent. Thus 26 mg (81%) of BNL1 was obtained. NMR (500 MHz, CDCl3) δ 7.05 (d, 2H), 6.79 (d, 2H), 4.60 (m, 1H), 4.14 (m, 1H), 4.07 (t, 8H), 3.60 (dd, 1H), 3.06 (dd, 1H), 2.80 (dd, 1H), 2.31 (m, 3H), 1.95 (m, 1H), 1.64 (m, 16H), 1.43 (m, 6H), 1.25 (m, 46 H), 0.87 (t, 12H).

[0107] Example 2: Compound KA449x2HCl (BNL2)

[0108] The compound KA449x2HCl (BNL2) was prepared according to the general reaction scheme I as follows.

[0109] 5 (37 mg, 32.1 pmol) was dissolved in 500 pl dichloromethane, then 500 pl trifluoroacetic acid was added and stirred for 30 min at room temperature. The mixture was evaporated three times from dichloromethane, then the resulting material was dissolved in 1 ml ethyl acetate, and 1 ml HCl (5-6 N) in isopropanol solution was added. The mixture was evaporated and the process was repeated three times to give 32.5 mg (98%) BNL2.1H NMR (500 MHz, MeOD) δ 4.52 (dd, 1H), 4.21-4.12 (m, 2H), 4.11-4.06 (m, 6H), 3.97 (m, 1H), 2.97 (t, 2H), 2.50 (t, 2H), 2.34 (tt, 1H), 2.22 (ddd, 1H), 2.01 (ddd, 2H), 1.94 (ddt, 2H), 1.77-1.38 (m, 26H), 1.36-1.22 (m, 42H) 0.90 (t, 12H).

[0110] Example 3: Compound KA453XHCl (BNL3) The compound KA-453xHCl (BNL3) was prepared according to the general reaction scheme I and the procedure used for BNL2 (Example 2). NMR (500 MHz, MeOD) δ 7.67 (m, 1H), 7.38 (m, 1H), 7.26-7.21 (m, 1H), 7.14 (t, 1H), 7.06 (m, 1H), 4.51 (dd, 1H), 4.14-4.05 (m, 8H), 3.45 (dd, 1H), 3.22 (dd, 1H), 2.44 (t, 2H), 2.33 (tt, 2H), 2.19 (ddd, 1H), 2.00 (m, 1H), 1.70-1.51 (m, 12H), 1.49-1.37 (m, 13H), 1.35-1.21 (m, 40H), 0.89 (t, 12H).

[0111] Example 4: Compound KA455XHCl (BNL4)

[0112] The compound KA455XHCl (BNL4) was prepared according to the general reaction scheme I and the procedure used for BNL2.1H NMR (500 MHz, MeOD) δ 4.52 (dd, 1H), 4.25 (dd, 1H), 4.18-4.06 (m, 8H), 2.93 (dd, 1H), 2.76 (dd, 1H), 2.46 (t, 2H), 2.34 (tt, 2H), 2.20 (ddd, 1H), 1.99 (ddd, 1H), 1.72-1.54 (m, 12H), 1.50-1.39 (m, 12H), 1.36-1.22 (m, 40H), 0.90 (t, 12H).

[0113] Example 5: Compound KA470xHCl (BNL5)

[0114] The compound KA470XHCl (BNL5) was prepared according to the general reaction scheme I and the procedure used for BNL2 (Example 2).1H NMR (500 MHz, MeOD) δ 4.87 (t, 1H), 4.22- 4.07 (m, 8H), 4.02 (t, 1H), 3.00-2.85 (m, 2H), 2.68-2.55 (m, 2H), 2.34 (tt, 2H), 2.22-2.08 (m, 5H), 1.71-1.63 (m, 8H), 1.63-1.51 (m, 4H), 1.49-1.39 (m, 12H) 1.37-1.20 (m, 40H), 0.90 (t, 12H). Example 6: Compound KA479XHCl (BNL6)

[0115] The compound KA479><HCl (BNL6) was prepared according to the general reaction scheme I and the procedure used for BNL2 (Example 2). NMR (500 MHz, MeOD) δ 4.53 (dd, 1H), 4.20-4.07 (m, 8H), 3.89 (dd, 1H), 2.48 (t, 2H), 2.34 (tt, 2H), 2.21 (ddd, 1H), 2.00 (ddd, 1H), 1.81- 1.73 (m, 2H), 1.72-1.63 (m, 8H), 1.62-1.53 (m, 4H) 1.50-1.39 (m, 12H), 1.35-1.23 (m, 40H), 1.02 (dd, 6H), 0.90 (t, 12H).

[0116] Example 7: Compound KA504 (BNL7)

[0117] 6 (62 mg, 60.5 pmol) was dissolved in 1 ml dichloromethane, then 1 ml trifluoroacetic acid was added and stirred for 30 min at room temperature. The reaction mixture was added to 50 ml cc. NaHCO3(aq) solution, then the aqueous phase was extracted three times with 10 ml dichloromethane. The combined organic phase was washed with 50 ml cc. NaCl (aq), dried with anhydrous Na2SO4, filtered and evaporated to give 54 mg (96%) of BNL7.1H NMR(500 MHz, MeOD) δ 4.50 (dd, 1H), 4.14 (dt, 2H), 4.09 (t, 6H), 3.91 (t, 1H), 3.18 (d, 1H) 2.45 (t, 2H), 2.34 (tt, 2H), 2.19 (ddd, 1H), 1.99 (ddd, 1H), 1.71-1.62 (m, 8H), 1.61-1.53 (m, 4H), 1.49-1.40 (m, 12H), 1.35- 1.25 (m, 40H), 1.23 (d, 3H), 0.90 (t, 12H).

[0118] Example 8: Compound KA505 (BNL8)

[0119] The compound KA505 (BNL8) was prepared following the general reaction scheme I and the procedure used for BNL7 (Example 7).1H NMR (500 MHz, MeOD) δ 4.46 (dd, 1H), 4.37 (brs, 1H), 4.19-4.06 (m, 8H), 4.00-3.90 (m, 1H), 3.09-2.98 (m, 1H) 2.96-2.89 (m, 1H), 2.41 (t 2H), 2.34 (tt, 2H), 2.23-2.15 (m, 2H), 2.00 (ddd, 1H), 1.87 (ddd, 1H), 1.71-1.54 (m, 12H), 1.49-1.39 (m, 12H), 1.36-1.23 (m, 40H), 0.90 (t, 12H).

[0120] Example 9: Compound KA507 (BNL9)

[0121] The compound KA507 (BNL9) was prepared according to the general reaction scheme I and the procedure used for BNL7 (Example 7).1H NMR (500 MHz, MeOD) δ 7.67 (brs, 1H), 6.93 (s, 1H), 4.46 (dd, 1H), 4.17-4.05 (m, 8H), 3.88-3.64 (m, 1H) 3.05 (brd, 1H), 2.91 (brs, 1H), 2.40 (t, 2H), 2.34 (tt, 2H), 2.17 (ddd, 1H), 1.97 (ddd, 1H), 1.70-1.62 (m, 8H), 1.61-1.54 (m, 4H), 1.49-1.38 (m, 12H), 1.35-1.23 (m, 40H), 0.90 (t, 12H). Example 10: Compound KA460xHCl (BNL10)

[0122] The compound KA460xHCl (BNL10) was prepared according to the general reaction scheme II as follows. 2-hexyldecanoic acid (2.31 g, 9.01 mmol), Boc-6-amino-l -hexanol (2.15 g, 9.89 mmol), triethylamine (4.0 ml, 28.80 mmol) and 4-dimethylamino-pyridine (0.66 g, 5.40 mmol) were dissolved in a mixture of 10 ml acetonitrile and 10 ml dichloromethane. To the mixture was added EDC-HCl (2.07 g, 10.79 mmol) and stirred at 37°C for 18 h. The resulting mixture was evaporated, filtered through a silica gel layer with about 50 ml ethyl acetate, then evaporated. The resulting material was purified on silica gel (40 g) with ethyl acetate / hexane 1:3 eluent to give 2.88 g of 7 (70%).

[0123] 7 (1.22 g, 2.68 mmol) was dissolved in 4 ml dichloromethane and 4 ml trifluoroacetic acid was added, then stirred at room temperature for 30 min. The resulting mixture was evaporated, then dissolved in 5 ml dichloromethane, and 5 ml of 5-6 N HCl in isopropanol solution was added and the mixture was evaporated. The latter step was repeated two more times. The resulting mixture was evaporated 3 more times from 20 ml each of dichloromethane to give 0.99 g of 8 hydrochloride (94%).

[0124] 8 hydrochloride (0.99 g, 2.53 mmol) was dissolved in 5 ml dichloromethane and DIEA (529 pl, 3.04 mmol) was added. N-Boc-L-glutamic acid (209 mg, 0.84 mmol) was dissolved in 5 ml acetonitrile, N-methyl-imidazole (202 pl, 2.53 mmol) was added, then TBTU (596 mg, 1.86 mmol) was added and the mixture was stirred for 5 min at room temperature. Then the mixture of 8 in dichloromethane was added and stirred at room temperature for 18 hours. After evaporation, the reaction mixture was purified on silica gel (30 g) with ethyl acetate / hexane 2:1 eluent to give 428 mg of 9 (55%).

[0125] 9 (262 mg, 0.284 mmol) was dissolved in 2 ml dichloromethane and 2 ml trifluoroacetic acid was added, then stirred at room temperature for 30 min. The resulting mixture was evaporated, then dissolved in 10 ml dichloromethane, and 2 ml of 5-6 N HCl in isopropanol solution was added and the mixture was evaporated. The latter step was repeated two more times. The resulting mixture was evaporated three more times from 10 ml each of dichloromethane at 40°C to give 239 mg of 10 hydrochloride (98%).

[0126] 10 hydrochloride (57.4 mg, 66.8 pmol) was dissolved in 1 ml dichloromethane and DIEA (14 pl, 80.2 pmol) was added. N-Boc-L-proline (15.8 mg, 73.5 pmol) was dissolved in 1 ml acetonitrile, DIEA (15.5 pl, 88.2 pmol) was added, then E1ATU (27.9 mg, 73.5 pmol) was added and the mixture was stirred for 5 min at room temperature. After that a mixture of 10 in dichloromethane was added and stirred at room temperature for 18 hours. After evaporation, the reaction mixture was purified on silica gel (15 g) with chloro form / acetone 3:1 eluent to give 48 mg of 11 (71%). 11 (37.2 mg, 36.5 pmol) was dissolved in 1 ml dichloromethane and 1 ml trifluoroacetic acid was added, then stirred at room temperature for 30 min. The resulting mixture was evaporated, then dissolved in 5 ml dichloromethane and 0.5 ml of 5-6 N HCl in isopropanol solution was added and the mixture was evaporated. The latter step was repeated two more times. The resulting oily substance was evaporated 5 more times from 5 ml each of dichloromethane at 40°C to give 31.2 mg of BNL10 hydrochloride (89%).

[0127] Example 11: a) Compound KA459XHCl (BNLlla)

[0128] 10 (42.4 mg, 40.9 pmol) was dissolved in 1 ml dichloromethane, then 1 ml trifluoroacetic acid was added and stirred for 30 min at room temperature. The mixture was evaporated three times from dichloromethane, then the resulting material was dissolved in 1 ml ethyl acetate, and 1 ml HCl (5-6 N) in isopropanol solution was added. The mixture was evaporated and the process was repeated three times to give 36.5 mg (92%) of BNLlla. NMR (500 MHz, MeOD) δ 4.36 (t, 1H), 4.08 (t, 4H), 3.91 (t, 1H), 3.19 (dd, 4H), 2.34 (tt, 2H), 2.28-2.24 (m, 2H), 2.04 (ddd, 1H), 1.96 (ddd, 1H), 1.74 (ddd, 2H), 1.69-1.22 (m, 65H), 1.01 (t, 6H), 0.90 (t, 12H). b) Compound KA459FB (BNLllb)

[0129] The procedure described in example a) was followed, with the difference that after the reaction time the reaction mixture was added to 50 ml cc. NaHCCh (aq) solution, the aqueous phase was extracted three times with 10 ml dichloromethane. The combined organic phase was concentrated with 50 ml cc. NaCl (aq), dried with anhydrous Na2SCfl, filtered and evaporated to give the compound BNLllb in free base form (KA459FB).1H NMR (500 MHz, MeOD) δ 4.33 (dd, 1H), 4.08 (t, 4H), 3.45 (t, 1H), 3.19 (dd, 4H), 2.34 (tt, 2H), 2.27-2.24 (m, 2H), 2.05 (ddd, 1H), 1.93 (ddd, 1H), 1.73 (ddd, 2H), 1.69-1.22 (m, 65H), 0.97 (d, 3H), 0.94 (d, 3H), 0.90 (t, 12H).

[0130] Example 12: Compound KA488XHCl (BNL12)

[0131] The compound KA488XHCl (BNL12) was prepared according to the general reaction scheme II and the procedure used for BNL10 (Example 10).1H NMR (500 MHz, MeOD) δ 4.31 (t, 1H), 4.08 (t, 4H), 3.23-3.15 (m, 5H), 2.34 (tt, 2H), 2.26 (t, 2H) 2.06 (ddd, 1H), 1.92 (ddd, 1H), 1.75-1.68 (m, 1H), 1.68-1.21 (m, 66H), 0.96-0.87 (m, 18H).

[0132] Example 13: Compound KA489XHCl (BNL13)

[0133] The compound KA489XHCl (BNL13) was prepared according to the general reaction scheme II and the procedure used for BNL10 (Example 10).1H NMR (500 MHz, MeOD) δ 4.31 (dd, 1H), 4.08 (td, 4H), 3.21-3.13 (m, 5H), 2.34 (tt, 2H), 2.26 (t, 2H) 2.04 (ddd, 1H), 2.00-1.87 (m, 2H), 1.75-1.68 (m, 1H), 1.68-1.21 (m, 65H), 0.97 (d, 3H), 0.94-0.87 (m, 15H). Example 14: Compound KA490xHCl (BNL14)

[0134] The compound KA490xHCl (BNL14) was prepared according to the general reaction scheme II and the procedure used for BNL10 (Example 10). NMR (500 MHz, MeOD) δ 4.31 (dd, 1H), 4.08 (t, 4H), 3.47 (t, 1H), 3.18 (dt, 4H), 2.56 (t, 2H) 2.34 (tt, 2H), 2.26 (t, 2H), 2.11-1.87 (m, 6H), 1.81 (q, 1H), 1.68-1.23 (m, 64H), 0.90 (t, 12H).

[0135] Example 15: Compound KA491xHCl (BNL15)

[0136] The compound KA491XHCl (BNL15) was prepared according to the general reaction scheme II and the procedure used for BNL10 (Example 10).1H NMR (500 MHz, MeOD) δ 7.31- 7.26 (m, 2H), 7.25-7.20 (m, 3H), 4.28 (dd, 1H), 4.08 (t, 4H), 3.62 (t, 1H) 3.16 (t, 4H), 3.04 (dd, 1H), 2.84 (d, 1H), 2.33 (tt, 2H), 2.18 (m, 2H), 2.02 (ddd, 1H), 1.86 (ddd, 1H), 1.65 (quin, 4H), 1.60-1.23 (m, 61H), 0.90 (t, 12H). Example 16: Compound KA495XHCl (BNL16)

[0137] The compound KA495XHCl (BNL16) was prepared according to the general reaction scheme II and the procedure used for BNL10 (Example 10). NMR (500 MHz, MeOD) δ 4.70 (t, 1H), 4.08 (t, 4H), 3.75 (dd, 1H), 3.63 (dd, 1H), 3.46 (t, 1H) 3.17 (dd, 4H), 2.68 (t, 2H), 2.34 (tt, 2H), 1.68-1.62 (m, 4H), 1.61-1.53 (m, 4H), 1.52-1.25 (m, 60H), 0.90 (t, 12H).

[0138] Example 17: Compound KA497><HCl (BNL17)

[0139] The compound KA497XHCl (BNL17) was prepared according to the general reaction scheme II and the procedure used for BNL10 (Example 10).1H NMR (500 MHz, MeOD) δ 4.37 (dd, 1H), 4.08-4.04 (m, 5H), 3.72 (d, 1H), 3.22-3.15 (m, 4H), 2.34 (tt, 2H) 2.29 (t, 2H), 2.18 (t, 2H), 2.06 (ddd, 1H), 1.96 (ddd, 1H), 1.67-1.36 (m, 24H), 1.34-1.24 (m, 41H), 0.90 (t, 12H).

[0140] Example 18: Compound KA498xHCl (BNL18) The compound KA498xHCl (BNL18) was prepared according to the general reaction scheme II and the procedure used for BNL10 (Example 10). NMR (500 MHz, MeOD) δ 4.35 (brs, 1H), 4.31 (dd, 1H), 4.08 (t, 4H), 3.91 (t, 1H), 3.24-3.14 (m, 4H) 3.01-2.98 (d, 1H, rotamers), 2.93-2.90 (brs, 1H, rotamers), 2.34 (tt, 2H), 2.22 (t, 2H), 2.16 (dd, 1H), 2.05 (ddd, 1H), 1.93-1.82 (m, 2H), 1.65 (quin, 4H), 1.60-1.36 (m, 20H), 1.34-1.24 (m, 40H), 0.90 (t, 12H).

[0141] Example 19: Compound KA500XHCl (BNL19)

[0142] The compound KA500XHCl (BNL19) was prepared according to the general reaction scheme II and the procedure used for BNL10 (Example 10).1H NMR (500 MHz, MeOD) δ 4.34 (dd, 1H), 4.00 (t, 4H), 3.97 (t, 1H), 3.26-3.16 (m, 4H), 3.24-3.14 (m, 4H) 2.47 (t, 2H), 2.34-2.28 (m, 4H), 2.15-2.02 (m, 3H), 1.96 (ddd, 1H), 1.65 (quin, 4H), 1.60-1.37 (m, 20H), 1.33-1.25 (m, 36H), 0.90 (t, 12H).

[0143] Example 20: Compound KA506 (BNL20)

[0144] Compound KA506 (BNL20) was prepared following the general reaction scheme II and the procedure used for BNL7 (Example 7). NMR (500 MHz, MeOD) δ 7.04 (d, 2H), 6.72 (d, 2H), 4.27 (dd, 1H), 4.08 (t, 4H), 3.55 (t, 1H) 3.20-3.11 (m, 4H), 2.91 (dd, 1H), 2.75 (dd, 1H), 2.33 (tt, 2H), 2.19 (ddd, 2H), 2.08-2.01 (m, 1H), 1.88-1.80 (m, 1H), 1.65 (quin, 4H), 1.61-1.35 (m, 20H), 1.34-1.22 (m, 40H), 0.90 (t, 12H). Example 21: Compound KA481XHCl (BNL21)

[0145] The compound KA481XHCl (BNL21) was prepared according to the general reaction scheme III as follows.

[0146] 2-hexyl-l -decanol (1.05 g, 4.32 mmol), Boc-6-amino-1-hexanoic acid (1.00 g, 4.32 mmol), DIEA (15.12 mmol) and 4-dimethylamino-pyridine (0.53 g, 4.32 mmol) were dissolved in a mixture of 5 ml acetonitrile and 5 ml dichloromethane. To the mixture was added EDC-HCl (0.99 g, 5.18 mmol), then stirred for 18 hours at room temperature. The resulting mixture was evaporated, filtered through a silica gel layer with about 30 ml ethyl acetate, then evaporated. The resulting material was purified on silica gel (30 g) with ethyl acetate / hexane 1:3 eluent to give 1.68 g of 12 (85%).

[0147] 12 (1.68 g, 3.69 mmol) was dissolved in 4 ml dichloromethane and 4 ml trifluoroacetic acid was added, then stirred at room temperature for 50 min. The resulting mixture was evaporated, then dissolved in 2 ml dichloromethane and 2 ml of 5-6 N HCl in isopropanol solution was added and the mixture was evaporated. The latter step was repeated two more times. The resulting oily substance was evaporated 5 more times from 5 ml each of ethyl acetate to give 1.30 g of 13 hydrochloride (90%).

[0148] 13 hydrochloride (303 mg, 0.77 mmol) was dissolved in 3 ml dichloromethane and DIEA (162 pl, 0.93 mmol) was added. N-Boc-L-aspartic acid (64 mg, 0.28 mmol) was dissolved in 3 ml acetonitrile, DIEA (115 pl, 0.66 mmol) was added, then HATU (252 mg, 0.66 mmol) was added and the mixture was stirred for 5 min at room temperature. After that the mixture of 13 in dichloromethane was added, then stirred at room temperature for 18 hours. After evaporation, the reaction mixture was purified on silica gel (25 g) with ethyl acetate / hexane 1:1 eluent to give 222 mg of 14 (87%).

[0149] 14 (216 mg, 0.24 mmol) was dissolved in 1 ml dichloromethane and 1 ml trifluoroacetic acid was added, then stirred at room temperature for 35 min. The resulting mixture was evaporated, then dissolved in 2 ml dichloromethane, and 2 ml of 5-6 N HCl in isopropanol solution was added and the mixture was evaporated. The latter step was repeated two more times. The resulting oily substance was evaporated 3 more times from 5 ml each of dichloromethane at 40°C to give 201 mg of 15 hydrochloride (100%).

[0150] 15 hydrochloride (68.9 mg, 81.6 pmol) was dissolved in 1.5 ml dichloromethane and DIEA (17 pl, 97.9 pmol) was added. N-Boc-L-leucine monohydrate (24.4 mg, 97.9 pmol) was dissolved in 1.5 ml acetonitrile, DIEA (17 pl, 97.9 pmol) was added, then EIATU (37.2 mg, 97.9 pmol) was added and the mixture was stirred for 5 min at room temperature. A mixture of 15 in dichloromethane was then added and stirred at room temperature for 18 hours. After evaporation, the reaction mixture was purified on silica gel (15 g) with ethyl acetate / hexane 3:2 eluent to give 58.2 mg of 16 (70%).

[0151] 16 (58.2 mg, 57 pmol) was dissolved in 1 ml dichloromethane and 1 ml trifluoroacetic acid was added, then stirred at room temperature for 30 min. The resulting mixture was evaporated, then dissolved in 1 ml dichloromethane and 0.5 ml of 5-6 N HCl in isopropanol solution was added and the mixture was evaporated. The latter step was repeated two more times. The resulting oily substance was evaporated 5 more times from 5 ml each of dichloromethane at 40°C to give 45.5 mg of BNL21 (83%)?H NMR (500 MHz, MeOD) δ 4.71 (t, 1H), 4.00 (d, 4H), 3.86 (t, 1H), 3.23-3.11 (m, 4H), 2.68 (dd, 1H) 2.60 (dd, 1H), 2.33 (dt, 4H), 1.73 (ddd, 2H), 1.67-1.58 (m, 6H), 1.56-1.47 (m, 4H), 1.41-1.25 (m, 51H), 1.00 (t, 6H), 0.90 (t, 12H).

[0152] Example 22: Preparation, translatability and localization studies of mRNA-LNPs encoding luciferase (Luc.) in BALB / cOlaHsd mice

[0153] For the expression of the luciferase enzyme in mice, the mRNA encoding the enzyme was individually packed into LNPs prepared from cationic lipids ALC0315, KA417, KA453XHCl, KA459XHCl, KA459FB, KA470XHCl, KA479XHCl, KA481 XHCl, KA488XHCl, and KA490XHCl using a mixture of cationic lipid:DSPC:cholesterol:DMG-PEG-lipid = 50:10:38.5:1.5 molar ratio. The cationic lipids were dissolved in methanol. A mixture of DSPC, cholesterol, DMG- PEG-lipid in the desired ratio in chloroform was evaporated with nitrogen and vacuum, and then a solution of the cationic lipid in alcohol was added to adjust the desired molar ratio. The mRNA was dissolved at a final concentration of 0.25 mg / ml in 25mM sodium acetate buffer (pH 4.0). LNPs were formulated in a fishbone microfluidic device using syringe pumps, maintaining a lipid:mRNA molar ratio of 20:1 and a flow ratio of 1:3 at a flow rate of 2 ml / min. For formulation of empty LNPs, sodium acetate buffer without mRNA was used maintaining the same flow ratio and flow rate. In the prepared LNP solution, buffer was replaced by dialysis with PBS and concentrated by centrifugal filtration. The particle size distribution of the final LNP was determined using a Malvern Zetasizer Nano ZS (Malvern, UK) instrument. The measured values are given in the following table.

[0154] From the data in the table it can be concluded that the resulting LNPs fall within the desired size range with the desired size distribution. Furthermore, it can be concluded that they are stable to freezing, i.e. their size does not change during freezing to an extent that would affect their usability. We note that some lipids were isolated in salt form and other lipids in free base form. The lipid KA459 was isolated in both salt form (KA459XHCl) and free base form (KA459FB) and LNP was prepared using both forms. The procedure for the preparation of LNPs is identical for the free base and salt forms.

[0155] Table 1: Particle sizes of the LNPs prepared

[0156] D: average particle size (Z -average)

[0157] PDI: polydispersity index

[0158] FB: free base form

[0159] Mice (Envigo, BALB / cOlaHsd, 6-8 weeks old females) were vaccinated (intramuscular- musculus gastrocnemius) with 5 gg of mRNA-LNP vaccines encoding luciferase and after 1 day D- luciferin substrate (Xenolight, D-Luciferin Potassium Salt, P / N 122799) was administered at an (intraperitoneal) dose of 150 mg / kg. Anesthesia was performed with isoflurane in a 20% 02 / 80% air mixture. The induction concentration of isoflurane was 5%, followed by 2.5-3% for maintenance (PerkinElmer, RAS-4 Rodent Anesthesia System). During anesthesia, animals were placed on the imaging platform while receiving 2% isoflurane through the nasal cone. Bioluminescence imaging was performed using the PerkinElmer In Vivo Imaging System (IVIS). Bioluminescence images were captured 5 min after D-luciferin administration using an automatic exposure time set by the system to ensure that the detected signal was within the effective detection range (above the noise level and below the saturation limit). Measurements were continued for 9 days. The bioluminescence values were evaluated using the software provided by PerkinElmer (PerkinElmer Living image) by measuring the photon flux (total flux, p / s) in the region of interest where the bioluminescence signal originated.

[0160] Based on the translatability assays, Luc. mRNA-LNPs prepared using KA459XHCl, KA459FB, KA481XHCl, KA488XHCl and KA490XHCl ionizable lipids have higher translatability than Luc. mRNA-LNP containing ALC0315 ionizable lipid as reference compound. In turn, Luc. mRNA-LNPs prepared using KA453XHCl and KA479XHCl ionizable lipids approach the bioluminescence intensity obtained during the translatability assay of the mRNA-LNP containing the reference compound (see Figure 1).

[0161] By localization analysis of the bioluminescent signal, we determined the ratio of the distribution of each mRNA-LNP within the organism (see Figure 2). It can be observed that each of the Luc. mRNA-LNPs containing the ionizable lipids of the invention exhibits a different signal intensity in the liver and at the site of inoculation compared to the mRNA-LNP containing the ALC0315 ionizable lipid used as the reference compound. This difference in the distribution pattern allows us to use our ionizable lipids for different res earch / therapeutic applications.

[0162] Example 23: Toxicity and adjuvant effect in BATB / cOlaHsd mice

[0163] Mice (Envigo, BAEB / cOlaHsd, 6-8 weeks old females) were co-immunized under anaesthesia with 10 pig of empty LNPs containing an ionizable lipid identical to the PR8 HA mRNA-LNP vaccine and 10 μg of PR8 HA (Influenza A / PR / 8 / 34 haemagglutinin) protein intramuscularly (site of inoculation: musculus gastrocnemius). The degree of inflammation at the site of vaccination was assessed on days 1, 2, 4, 6 and 7 after vaccination and graded clinically on these days (see Figure 3). Grade 4 is the most severe inflammation, restricting the animal's movements, while grade 1 corresponds to a mild, barely noticeable inflammation, not restricting the animal's movements at all. During scoring, the degree of swelling and its restrictive effect on the animal's movement was examined.

[0164] On the first day after vaccination, LNPs prepared with KA417, KA459XHCl, KA479XHCl and KA481 XHCl lipids caused less inflammation compared to the reference compound (ALC0315), while LNP prepared with the lipid KA459FB caused inflammation equivalent to LNP prepared with the ionizable lipid ALC0315. LNPs prepared with the lipid KA453XHCl, KA470XHCl, KA488XHCl and KA490XHCl according to the invention resulted in more severe, partially movement-limiting inflammation on the first day after vaccination. On day 7, similarly to the reference compound, the inflammation caused by all of our ionizable lipids regressed or was reduced to a degree that did not restrict the animals' movement at all.

[0165] To investigate the adjuvant effect of each LNP, blood was collected on day 28 after vaccination (retroorbital blood sampling) and sera were collected, and enzyme -linked immunosorbent assay (ELISA) was used to determine the amount of PR8 HA-specific antibodies (see Figure 4). 96-well ELISA plates (Corning, 3590) were coated (0.1 μg protein / well, 100 μl / well) with PR8 HA protein in IX PBS (Corning, 21-031-CV) and then the samples were incubated at 4°C overnight. After three washes with 0.05% Tween 20 (Sigma-Aldrich, P9416-100ML) in IX PBS solution (300 μl / well), samples were incubated in 2% BSA (Bovine Serum Albumin, Sigma, A7030- 100G) in IX PBS solution (200 μl / well) at 25°C for 2 hours. After repeated washing in triplicate, sera from immunized mice were diluted in 2% BSA - IX PBS solution (in one-third dilutions, where the initial dilution was 1:300) and added to the wells (100 μl / well). The plates were stored for 2 h at 25°C, and after washing three times, the samples were incubated for 1 h at 25°C with secondary antibody (goat anti-mouse IgG-HRP, Jackson ImmunoResearch, 115-035-003, 1:10 000 dilution, diluted in 2% BSA - IX PBS, 100 μl / well). 10-15 min after the addition of TMB substrate (LGC Seracare, 5120-0047) (100 μl / well), the developed color reaction was stopped with 2N H2SO4 (50 μl / well). Detection was performed at 450 nm using an ELISA reader. The high PR8 HA specific antibody response induced by the PR8 HA protein as well as by the empty LNPs demonstrates that our ionizable lipids have a high adjuvant effect. With the exception of KA479, all ionizable lipids are able to induce an adjuvant effect in a similar manner to the lipid ALC0315 used as a reference compound.

[0166] Example 24: Evaluation of toxicity by XTT assay on tissue cultures from HepG2 human liver and HEK293 human kidney cells

[0167] The XTT assay is a widely used method for determining cytotoxicity based on the measurement of cell metabolic activity and cell viability. The pale yellow XTT (2,3-bis(2-methoxy-4- nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium) dye is converted by metabolically active cells into an orange, water-soluble form. The reaction requires the presence of NADH produced by the mitochondria and an externally added electron-coupling agent. The amount of formazan formed is proportional to the number of viable cells and their metabolic activity. The absorbance of the final reaction product can be measured by spectrophotometry at 450 nm. The method is suitable for quantitative comparison.

[0168] For the assay, HepG2 and HEK293 cells were grown in DMEM-High Glucose medium containing 10% FBS (Gibco). For the measurements, cells were divided into 96-well plates (100 μl medium and 10 000 cells / well). After 24 h, medium containing empty LNPs without mRNA was added to the cells (half dilution, 100 μl medium / well final volume). After a further 24 h, XTT reagent (Invitrogen CyQUANT™ XTT Cell Viability Assay #X12223) was added to the cells. Samples were incubated for 4 h in the dark at 37°C in a 5% CO2thermostat and their absorbance was measured spectrophotometrically at 450 nm using an ELISA plate -reader. The amount of LNPs added to the cells, consisting of cationic lipid:DSPC:cholesterol:PEG-lipid components, was adjusted to the cholesterol content of the LNPs in each case, according to the following cholesterol concentrations: 15 μg / ml, 30 μg / ml, 60 μg / ml, 120 μg / ml. Each LNP was prepared with one of the following cationic lipids: ALC0315, KA417, KA453XHCl, KA459XHCl, KA459FB, KA470XHCl, KA479XHCl, KA481 XHCl, KA488XHCl and KA490XHCl. As controls, the buffer used for dissolution of LNPs and untreated cells were used. Specific absorbance values for samples containing cells were calculated as follows:

[0169] Specific Absorbance = [Abs450 nm(Test) - Abs450 nm(Blank)]

[0170] Cell viability was expressed as % of untreated control cells: Viability % = [ 100 x (sample spec. Absorbance) / (control spec. Absorbance)]

[0171] The results are shown in Figure 5.

[0172] Values greater than 100% in some samples were caused by increased metabolic activity due to lipid content of the LNPs and the sugar content of the buffer. Most of the LNPs containing the new lipids were not found to be toxic, even when applied at higher concentrations. Exceptions were LNPs containing the cationic lipids KA479 and KA481, but these lipids also had a negative effect on the cells only in larger amounts.

[0173] Note that in the biological studies, LNPs prepared from both the salt form (KA459XHCl) and the free base form (KA459FB) of BNL11 were tested and no significant differences were observed between the salt and free base forms with respect to the resulting LNP and its biological properties. This is in line with our expectation, since the state of the cationic lipid in an LNP in a given medium is essentially determined by the medium, regardless of the starting form: its protonation depends on the pH of the dispersion medium, and its counterion depends on the molar ratio of anions present in the block phase of the dispersion. (Therefore, in the case of the prepared LNPs or in the biological examples, the initial form of the cationic lipid is not always indicated, and if indicated, it does not mean that the lipid remains in this form in the LNP.)

[0174] INDUSTRIAL APPLICABILITY

[0175] The invention can provide cationic lipid molecules for the preparation of lipid nanoparticles carrying active ingredients. The lipid molecules according to the invention have lower toxicity and high adjuvant activity, and as a constituent, provide lipid nanoparticles with higher specificity of targeting and equal or better translatability than lipid molecules known in the prior art.

Claims

Claims1. A compound of the following general formula (I):wherein: y is 1 or 2,Xi represents a group of formula Alk-L-,X2represents a group of formula Alk-L-, where Alk represents a straight or branched chain alkyl group of 10 to 24 carbon atoms,L represents a group of formula#-C(O)-O-(CH2)n-O-;#-C(O)-O-(CH2)n-NH- or#-CH2-O-C(O)-(CH2)n-i-NH-; where # indicates the part attached to the Alk- group, and where n is an integer from 2 to 8, X3represents a group selected from glycyl, L-alanyl, L-valyl, L-leucyl, L-isoleucyl, L-seryl, L- threonyl, L-cysteinyl, L-methionyl, L-phenylalanyl, L-tyrosyl, L-tryptophanyl, L-prolyl, L- asparaginyl, L-glutaminyl, L-asparagyl, L-glutamyl, L-lysyl, L-histidyl, L-arginyl,the carbon atom in the CH* group has the L configuration, or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein X3is selected from glycyl, L-alanyl, L-valyl, L-leucyl, L-isoleucyl, L-seryl, L-threonyl, L-cysteinyl, L- methionyl, L-phenylalanyl, L-tyrosyl, L-tryptophanyl, L-prolyl, L-asparaginyl, L-glutaminyl, L-lysyl, L-histidyl,3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, wherein X3is selected from L-alanyl, L-valyl, L-leucyl, L-isoleucyl, L-mefhionyl, L-phenylalanyl, L-tyrosyl and L- tryptophanyl.

4. The compound according to claim 3 or a pharmaceutically acceptable salt thereof, wherein X3is selected from L-valyl, L-leucyl, L-mefhionyl, L-tyrosyl and L-tryptophanyl.

5. The compound of claim 1, wherein the compound is selected from the group consisting of:in the indicated form, in the form of free base or in the form of any pharmaceutically acceptable salt.

6. A process for the preparation of a compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, comprising the steps of: a) reacting a compound of formula 1-1 with a diol of formula I-la under ester-forming conditionsthereby obtaining a compound of formula 1-2? b) esterifying the compound of formula I-2a with a compound of formula 1-2 under similar conditions as in the previous stepPGiHN OHVdA>H o l-2a then removing the PG1protecting group, thereby obtaining a compound of formula 1-3;c) N-acylating the compound of formula 1-3 with a compound of formula I-3athen removing the PG2protecting group, thereby obtaining a compound of formula 1-4;wherein Alk, n, and y are as defined in claim 1, PG1and PG2are amino-protecting groups, and R, taken together with the group -CO-CH(NH2)- to which it is attached, has the same meaning as X3defined in claim 1; or a) reacting a compound of formula 1-1 with a compound of formula Il-la under ester-forming conditionsthen removing the PG1protecting group, thereby obtaining a compound of formula II-2b) N-acylating the compound of formula II-2 with a compound of formula II-2athen removing the PG2protecting group, thereby obtaining a compound of formula II-3c) N-acylating the compound of formula II -3 with a compound of formula II-3all-3a then removing the PG3protecting group, thereby obtaining a compound of formula II-4wherein Alk, n, and y are as defined in claim 1, PG1, PG2andPG3are amino-protecting groups, and R, taken together with the group -CO-CH(NH2)- to which it is attached, has the same meaning as X3defined in claim 1; or a) reacting an alcohol of formula III-l with a compound of formula Ill-la under ester-forming conditions111-1 lll-1a then removing the PG1protecting group, thereby obtaining a compound of formula III-2? b) N-acylating the compound of formula III-2 with a compound of formula III-2aPG2HN O"V d'OH o lll-2a then removing the PG2protecting group, thereby obtaining a compound of formula III-3c) N-acylating the compound of formula III-3 with a compound of formula III-3alll-3a then removing the PG3protecting group, thereby obtaining a compound of formula III-4wherein Alk, n, and y are as defined in claim 1, PG1, PG2andPG3are amino-protecting groups, and R, taken together with the group -CO-CH(NH2)- to which it is attached, has the same meaning as X3defined in claim 1.

7. Nanoparticle comprising a compound of general formula (I) according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof.

8. Use of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, for the preparation of a nanoparticle carrying an active ingredient.

9. Pharmaceutical composition comprising a nanoparticle according to claim 7 and a pharmaceutically acceptable carrier or excipient.

10. Nanoparticle according to claim 7 for use in medicine.

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