Ionizable phospholipids, preparation thereof, uses thereof, and compositions thereof

Abstract

The present invention pertains to ionizable lipids and their compositions, as well as methods and processes of their preparation and uses thereof. More specifically, the present invention relates to novel ionizable phospholipid compounds or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof. The present invention further relates to lipid nanoparticle compositions comprising said ionizable phospholipid compounds.

Description

[0001] IONIZABLE PHOSPHOLIPIDS, PREPARATION THEREOF, USES THEREOF, AND COMPOSITIONS THEREOF FIELD

[0002] The present invention pertains to ionizable lipids and their compositions, as well as methods and processes of their preparation and uses thereof. More specifically, the present invention relates to novel ionizable phospholipid compounds or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof. The present invention further relates to lipid nanoparticle compositions comprising said ionizable phospholipid compounds.

[0003] BACKGROUND

[0004] In recent years, biologically active agents such as therapeutic nucleic acids are on the rise in pharmaceutical development. Nevertheless, one of the major challenges in developing biologically active agents is their effective and safe delivery into target cells and tissues. For instance, nucleic acids, such as DNA and RNA, are large, negatively charged molecules that cannot easily cross cell membranes on their own. At the same time they are vulnerable to degradation, which limits their stability and effectiveness. To reach the intended cells without triggering undesired immune responses or causing toxicity, advanced delivery systems are required, often tailored to the type and size of the nucleic acid therapeutic. Balancing safety and therapeutic efficacy remains a key hurdle as these nucleic acid therapies move towards clinical applications.

[0005] Among advanced delivery systems, lipid nanoparticles (LNPs) have emerged as a leading technology for delivering biologically active agents, in particular (large) negatively charged molecules such as RNA, through electrostatic complexing of the nucleic acid. Common LNP formulations in the art typically comprise multiple lipidic compounds including an ionizable cationic lipid, phospholipid, sterol and pegylated lipid (for a review see Mendonpa et al. “Design of lipid-based nanoparticles for delivery of therapeutic nucleic acids” (2023) Drug Discovery Today 28(3), 1-17). However, in light of the rapidly evolving field of nucleic acid based biological applications, in particular development of therapeutics, there is an ongoing and persistent need for further diversification of the available delivery vehicles, including a broader range of LNP formulations. Also, the complexity of said formulations may provide for a high variability in performance, physical or chemical instability, cumbersome processing, batch-to-batch variability, and regulatory hurdles.

[0006] W02022002040 relates to phospholipidic compounds wherein no acidic function is left on the phosphate group. Liu S et al. “Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing”Nature Materials (2021) vol.20, 701-710) describes pH switchable zwitterionic phospholipids targeting lung selective delivery. WO2024073678 relates to heterocycle comprising phospholipids also designed for targeting lung-tissue. US2023 / 0295199 relates to charge reversible phospholipids that are not positively charged at a pH of the body fluid.

[0007] SUMMARY OF THE INVENTION

[0008] The present invention addresses challenges and objectives in the art by providing a novel class of ionizable cationic phospholipid compounds, methods of their preparation, their use in cellular delivery of negatively charged cargo such as nucleic acids, lipid nanoparticles comprising such ionizable phospholipids, and compositions as defined herein.

[0009] The present invention is, at least in part, based on the finding that the molecular structure of ionizable lipids may be designed to combine the features of the typical ionisable lipids with the features of the typical phospholipids in a single compound , which can therefore serve the function of ionisable cationic lipid as well as phospholipid in the formation of lipid nanoparticles.

[0010] Accordingly, a first aspect of the present invention relates to a compound according to formula (I)

[0011]

[0012] or a pharmaceutically acceptable salt or isomer thereof,

[0013] wherein:

[0014] m, n and o are integers independently selected from 1 , 2, 3, 4 and 5;

[0015] p is an integer selected from 0 and 1 ;

[0016] - XT and X2are independently selected from O and NH;

[0017] - R1is R2’ or -L1N[R2”]2;

[0018] each R2, R2’ and R2” is independently selected from C4-3o-alkenyl and -L2F1RA; - Z is selected from -NR3R4and -OR5;

[0019] R3, R4and R5are independently selected from hydrogen, Ci.3o-alkyl, C4.3o- alkenyl, -C(=O)-RB, and -L3F2RC,

[0020] each L1, L2and L3is C2.10-alkylene;each F1and F2is independently selected from -O-C(=O)-, -C(=O)-O-, -NH- C(=O)- or-C(=O)-NH-; and,

[0021] each RA, RB, and Rcis independently selected from C4-3o-alkyland C4-3o-alkenyl. In particular embodiments, p is 1 .

[0022] In particular embodiments, RA, RB, and Rcare independently selected from secondary or branched C4.3o-alkyl, and primary C4.3o-alkenyl.

[0023] In particular embodiments,

[0024] m, n and o are integers independently selected from 1 , 2, 3, 4, and 5; p is an integer as defined herein above;

[0025] - XT and X2are as defined herein above;

[0026] - R1is -L1N[L2F1RA]2or -L2F1RA;

[0027] - R2is -L2F1RA;

[0028] - Z is-NR3R4;

[0029] - R3is -L3F2RC;

[0030] R4is hydrogen or -L3F2RC;

[0031] L1, L2and L3are as defined herein above;

[0032] F1and F2are independently selected from -O-C(=O)-, -C(=O)-O-, -NH- C(=O)- or-C(=O)-NH-; and;

[0033] RAand Rcare independently selected from secondary or branched C4.30- alkyl or primary C5.3o-alkenyl.

[0034] In particular embodiments, XT and X2are O.

[0035] In particular embodiments, the present compound may be represented by a compound of formula (II)

[0036] R1

[0037]

[0038] or a pharmaceutically acceptable salt or isomer thereof,

[0039] wherein R1, R2, R3, R4, m, n and o are as defined herein above.

[0040] In a related aspect, a method of manufacture of the compounds of formula (I) and / or (II) as well as precursors for the said manufacture are provided.

[0041] A further related aspect of the present invention relates to the use of the compounds of the invention in the intracellular delivery of (typically negatively charged) cargo, such as negatively charged biologically active compounds, both in vivo or in vitro. Inparticular embodiments, the invention also relates to lipid nanoparticles (LNPs) comprising at least one compound as disclosed herein or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, and, to a composition comprising such LNPs; and preferably said LNPs further comprise one or more lipids selected from the group consisting of phospholipids, structural lipids, PEG-lipids, and mixtures thereof.

[0042] In particular embodiments, the lipid nanoparticle composition further comprises a cargo compound typically being a negatively charged biologically active compound, e.g. selected from the group consisting of nucleic acids, small molecule compounds, peptides, proteins, protein-nucleic acid constructs, and mixtures thereof, particularly wherein the cargo compound is anionic, particularly anionic at formulation pH, such as pH below 6. As used herein, “formulation pH” is the pH of the aqueous phase the cargo is contained in when formulatingthe cargo containing LNP.

[0043] In particular embodiments, the cargo compound is a nucleic acid, such as a ribonucleic acid optionally modified ribonucleic acid. In further embodiments, the cargo is a substantially single stranded ribonucleic acid and / or comprising at least 8000 nucleotides.

[0044] A further related aspect of the present invention relates to a method for manufacturing a lipid nanoparticle (LNP) composition according to a second aspect of the present invention or (preferred) embodiments thereof, the method comprising the steps of:

[0045] (a) providing lipids comprising at least one compound according to the invention as described herein, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, and optionally one or more lipids selected from the group consisting of phospholipids, structural lipids, PEG-lipids, and mixtures thereof;

[0046] (b) dissolving the lipids in an organic solvent, thereby forming an organic phase;

[0047] (c) providing an aqueous phase, preferably comprising one or more cargo compounds;

[0048] (d) forming lipid nanoparticles by mixing the organic phase and aqueous phase under conditions suitable for nanoparticle formation.

[0049] A further related aspect of the present invention relates to the lipid nanoparticle composition as disclosed herein for use in medicine.These and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject-matter of the appended claims is hereby specifically incorporated in this specification.

[0050] DESCRIPTION OF THE FIGURES

[0051] The following description of the figures of specific embodiments of the invention is merely exemplary in nature and is not intended to limit the present teachings, their application or uses.

[0052] FIG. 1 A to F and FIG.2 A and B illustrate the in vivo expression of a bioluminescent marker encoded by an saRNA molecule encapsulated by LNPs formulated with the compounds of the invention as described in the examples.

[0053] DETAILED DESCRIPTION

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0055] “A”, “an” and “the” as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. Byway of example, “an excipient” refers to one or more than one excipients.

[0056] Throughout this disclosure, various publications, patents, and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

[0057] “Comprise”, “comprising”, “comprises” and the like as used herein are synonymous to “include”, “including”, “includes” and the like, or, “contain”, “containing”, “contains” and the like and are inclusive or open-ended terms that specify the presence of what follows and do not exclude the presence of additional, non-recited components, features, elements, members, steps,... known in the art or disclosed herein.

[0058] Whereas the terms “one or more” or “at least one”, such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

[0059] As used herein, the term “and / or” when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of twoor more of the listed items can be employed. For example, if a list is described as comprising group A, B, and / or C, the list can comprise A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.

[0060] Reference throughout this specification to "one embodiment", "an embodiment" or “some embodiments” means that a particular feature, structure or characteristic described in connection with the embodiment(s) is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment", "in an embodiment" or “in some embodiments” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination. The terms “wt%,” “vol%”, or “mol%” refers to a weight percentage of a component, a volume percentage of a component, or molar percentage of a component, respectively, based on the total weight, the total volume of material, or total moles, which includes the component.

[0061] The term "isomer" as used herein means all possible isomeric forms, including tautomeric and stereoisomeric forms, which the compounds of formulae herein may possess, but not including position isomers. Typically, the structures shown herein exemplify only one tautomeric or resonance form of the compounds, but the correspondingalternative configurations are contemplated as well. Unless otherwise stated, the chemical designation of compounds denotes the mixture of all possible stereochemically isomeric forms, said mixtures containing all diastereomers and enantiomers (since the compounds of formulae herein may have at least one chiral centre) of the basic molecular structure, as well as the stereochemically pure or enriched compounds. More particularly, stereogenic centres may have either the R-or S-configu ration, and multiple bonds may have either cis- or trans-configuration. In some embodiments, the term “isomer” refers to all possible tautomers and stereoisomers of the compounds of formulae herein.

[0062] As used herein, the term “pharmaceutically acceptable salt” includes both acid and base addition salts. Examples of pharmaceutically acceptable acid addition salts include, but are not limited to, salts prepared from addition of an inorganic acid ororganic acid to a free basicfunction of the compound of the invention, e.g. a (primary, secondary or tertiary) amine. Examples of pharmaceutically acceptable base addition salts include, but are not limited to, salts prepared from addition of an inorganic base or organic base to a free acid function of the compound of the invention, e.g. the phosphate group.

[0063] As used herein, “pharmaceutically acceptable” means suitable for use in contact with tissues of humans and animals without undue toxicity, irritation, allergic response and the like, commensurate with a reasonable benefit / risk ratio.

[0064] The present description provides ionizable lipids, methods of use of the lipids and lipid nanoparticles (LNPs) which can be used for efficient delivery of cargo to target cells or tissues. The inventors have found that the structural properties of ionizable lipids in which cationic ionizable lipid chemistry is combined with phospholipidic properties allows for the generation of ionizable phospholipid compounds. By combining multiple functionalities in one molecule, production complexity (in particularthe production complexity of the LNPs) can be reduced, while retaining or advantageously even improving transfection efficiency.

[0065] Accordingly, an aspect of the present invention relates to a novel class of phospholipidic compounds. Advantageously, said phospholipidic compounds may be used as ionizable lipids in pharmaceutical applications. The term “ionizable lipid” (or “ionisable cationic lipid”) as used herein refers to a lipid molecule that typically carries a neutral charge at physiological pH (~7.4) but becomes positively charged in acidic environments (e.g., endosomal pH ~5.5).

[0066] In the context of the present invention, the term “ionizable phospholipid” refers to an ionizable lipid molecule composed of a hydrophilic group comprising a phosphate moiety and ionizable nitrogens and hydrophobic tails (derived from fatty acid chains that can be saturated, unsaturated, linear, or branched), wherein said hydrophilic group has variable charge according to the acidity of the environment to which the compound is exposed.

[0067] In particular embodiments, provided herein is a compound of formula (I), a stereoisomeric form, a tautomer, a salt (in particular a pharmaceutically acceptable salt), solvate, hydrate, and / or polymorph thereof,

[0068]

[0069] m, n and o are integers independently selected from 1 , 2, 3, 4 and 5;

[0070] p is an integer selected from 0 and 1 ;

[0071] - XT and X2are independently selected from O and NH;

[0072] - R1is R2’ or -L1N[R2”]2;

[0073] each R2, R2’ and R2” is independently selected from alkenyl and -L2F1RA;

[0074] - Z is selected from -NR3R4and -OR5;

[0075] each R3, R4and R5is independently selected from hydrogen, alkyl, alkenyl, - C(=O)-RB, and -L3F2RC,

[0076] each L1, L2and L3is independently alkylene;

[0077] each F1and F2is independently selected from -O-C(=O)-, -C(=O)-O-, -NH- C(=O)- or-C(=O)-NH-; and,

[0078] each RA, RB, and Rcis independently selected from alkyl and alkenyl.

[0079] Preferably,

[0080] m, n and o are integers independently selected from 1 , 2, 3, 4 and 5;

[0081] p is an integer selected from 0 and 1 ;

[0082] - XT and X2are independently selected from O and NH;

[0083] - R1is R2’ or -L1N[R2”]2;

[0084] each R2, R2’ and R2” is independently selected from C4-3o-alkenyl and -L2F1RA; - Z is selected from -NR3R4and -OR5;

[0085] R3, R4and R5are independently selected from hydrogen, Ci.30-alkyl, C4.30- alkenyl, -C(=O)-RB, and -L3F2RC,

[0086] L1, L2and L3are independently selected from C2.10-alkylene;

[0087] each F1and F2is independently selected from -O-C(=O)-, -C(=O)-O-, -NH- C(=O)- or-C(=O)-NH-; and,

[0088] each RA, RB, and Rcis independently selected from C4.3o-alkyl and C4-30-alkenyl. As used herein, for the terms alkyl and alkenyl or related terms as defined herein below, the notion or pre-fix Ci-i, means thatthe alkyl, alkenyl or related chemicalgroup consists of i to ii carbon atoms composing the alkyl, alkenyl or related chemical group. This does not include the carbon and / or heteroatoms of (optional) substituents.

[0089] The term “alkyl” as used herein as a group or part of a group, refers to primary, secondary, or tertiary, linear or branched saturated hydrocarbon including one or more carbon atoms. As used herein, a branched alkyl is an alkyl group with a nonlinear backbone, i.e. alkyl branches sprout from one or more atoms of the main alkyl chain. A branched alkyl may be a primary, secondary or tertiary alkyl. For example, the term "Cvsoalkyl", as a group or part of a group, refers to a hydrocarbyl group of formula CnH2n+iwherein n is a number ranging from 1 to 30. Thus, for example, “Ci.3Oalkyl” includes all linear or branched alkyl groups with between 1 and 30 carbon atoms, and thus includes methyl, ethyl, n-propyl, / -propyl, butyl, and its isomers (e.g., n-butyl, / -butyl, and t-butyl); pentyl and its isomers, hexyl, and its isomers, etc.

[0090] The term “alkenyl” as used herein as a group or part of a group, refers to primary, secondary or tertiary, linear or branched hydrocarbon including two or more carbon atoms with at least one (usually 1 to 3, preferably 1) site of unsaturation, namely at least one sp2carbon-sp2carbon double bond. Examples of C2.3oalkenyl groups are ethenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl and its isomers, 2-hexenyl and its isomers, 2,4-pentadienyl, and the like. The double bond may be in the cis or trans configuration.

[0091] The term “alkylene” as used herein as a group or part of a group, refers to a divalent hydrocarbon group of formula CnH2nderived from an alkane by the removal of two hydrogen atoms. Alkylene groups may be linear or branched and typically contain 2 to 10 carbon atoms, unless otherwise specified. Examples include ethylene (-CH2CH2-) and propylene (-CH2CH(CH3)-).

[0092] Unless stated otherwise, an alkyl, alkylene or alkenyl may be optionally substituted by one or more (e.g. 1 , 2, 3, 4, or 5) substituents independently selected from halogen (F, Cl, Br, I), oxo, hydroxy, amino, -SH, -OR', -NR’R’, -NHR’, -SR’, wherein R’ is independently selected from C6-alkyl and C3.6-cycloalkyl. The term “substituted” as used herein is meant to indicate that one or more hydrogens on the atom indicated in the expression using“substituted” can be replaced with an indicated group, provided that the indicated atom’s normal valency is not exceeded, and that the substitution results in a chemically stable compound.

[0093] In some embodiments of the compounds described herein, p is 1 .

[0094] In some embodiments of the compounds described herein, XT is O. In some embodiments X2is O. In further embodiments, XT and X2are O.

[0095] In some embodiments of the compounds described herein, R1is R2’. In a further embodiment, R1is -L2F1RA.

[0096] In some embodiments of the compounds described herein, at least one of R2, R2’ and R2” is selected from -L2F1RA. In some embodiments, each R2, R2’ and R2” is -L2F1RA. In a further embodiment, R2, R2’ and R2” are the same.

[0097] In some embodiments of the compounds described herein, Z is -NR3R4. In a further embodiment, Z is -NR3R4wherein R3and R4are independently selected from hydrogen and -L3F2RC. In a further embodiment, Z is -NR3R4wherein R3is -L3F2RCand R4is selected from hydrogen and -L3F2RC. In yet a further embodiment, Z is -NR3R4wherein R3and R4are -L3F2RC.Alternatively, Z is -NR3R4wherein R3and R4are independently selected from hydrogen and Ci.3o-alkyl. In a further embodiment, Z is -NR3R4wherein R3is Ci.4-alkyl and R4is selected from hydrogen and Ci.4-alkyl.

[0098] F1and F2are defined herein as a -C(=O)O- or -OC(=O)- or -NHC(=O)- or -C(=O)NH-. As used herein, these ester or amide functional groups are thus positioned next to the RAgroup, according to: -C(=O)O-RAor -OC(=O)-RAor -NHC(=O)-RAor -C(=O)NH-RArespectively, or, next to the Rcgroup, according to: -C(=O)O-RCor -OC(=O)-RCor -NHC(=O)-RCor -C(=O)NH-RCrespectively. In some embodiments, each F1and F2is independently selected from -O-C(=O)- and -C(=O)-O-.

[0099] In some embodiments of the compounds described herein, RA, RBand Rcare independently selected from secondary or branched C4.3o-alkyl, or, C4.3o-alkenyl, optionally primary C4.30-alkenyl. In a further embodiment all RAare the same, and / or, all Rcare the same. In yet a further embodiment, all RA, RBand Rcare the same. In further embodiments, RA, RBand Rcare independently selected from C4.20-alkyl, C4.18-alkyl, C4.16-alkyl, C4.14-alkyl, C6.3o-alkyl, C6-2o-alkyl, C6-is-alkyl, Ce- -alkyl, C8.3o-alkyl, C8.

[0100] 20-alkyl, C8.18-alkyl, C8.16-alkyl, or, C8.14-alkyl. In some embodiments, the branched alkyl is branched on the carbon atom adjacent to the carbon atom connecting the alkyl radical to the parent molecule. In some embodiments, each RA, RBand Rcis independently selected from secondary or branched C4.30-alkyl, such as C4.20-alkyl, C4.18-alkyl, C4.16-alkyl, C4.14-alkyl, C6-3o-alkyl, C6-2o-alkyl, C6-i8-alkyl, C6-i6-alkyl, C8.30-alkyl, C8.2o-alkyl, C8.18-alkyl, C8.16-alkyl, or, C8.14-alkyl.

[0101] In some embodiments of the compounds described herein, at least one of RA, RBand Rcis selected from C4.30-alkenyl, C4.20- alkenyl, C4.18- alkenyl, C4.16- alkenyl, C4.14-alkenyl, C6.3o- alkenyl, C6-2o- alkenyl, C6-is- alkenyl, C6-16- alkenyl, C8.30- alkenyl, C8.2o-alkenyl, C8.18- alkenyl, C8.16- alkenyl, or, C8.14- alkenyl. In further embodiments, said at least one of RA, RBand Rcis a primary alkenyl.

[0102] In an embodiment of the compounds described herein, when RA, RBor Rcis a C4.30alkyl chain, said RA, RBor Rccan be attached to the rest of the molecule through the third to fifteenth carbon atom, such as the third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth or fifteenth carbon atom, within the alkyl chain.

[0103] In some embodiments of the compounds described herein, each branched alkyl side chain (RA, RB, Rc) is, independently, selected from: henicosan-2-yl, docosan-2-yl, tricosan-2-yl, tetracosan-2-yl, pentacosan-2-yl, hexacosan-2-yl, heptacosan-2-yl, octacosan-2-yl, nonacosan-2-yl, triacontan-2-yl, henicosan-3-yl, docosan-3-yl, tricosan-3-yl, tetracosan-3-yl, pentacosan-3-yl, hexacosan-3-yl, heptacosan-3-yl, octacosan-3-yl, nonacosan-3-yl, triacontan-3-yl, henicosan-4-yl, docosan-4-yl, tricosan-4-yl, tetracosan-4-yl, pentacosan-4-yl, hexacosan-4-yl, heptacosan-4-yl,octacosan-4-yl, nonacosan-4-yl, triacontan-4-yl, henicosan-5-yl, docosan-5-yl, tricosan-5-yl, tetracosan-5-yl, pentacosan-5-yl, hexacosan-5-yl, heptacosan-5-yl, octacosan-5-yl, nonacosan-5-yl, triacontan-5-yl, henicosan-6-yl, docosan-6-yl, tricosan-6-yl, tetracosan-6-yl, pentacosan-6-yl, hexacosan-6-yl, heptacosan-6-yl, octacosan-6-yl, nonacosan-6-yl, triacontan-6-yl, henicosan-7-yl, docosan-7-yl, tricosan-7-yl, tetracosan-7-yl, pentacosan-7-yl, hexacosan-7-yl, heptacosan-7-yl, octacosan-7-yl, nonacosan-7-yl, triacontan-7-yl, henicosan-8-yl, docosan-8-yl, tricosan-8-yl, tetracosan-8-yl, pentacosan-8-yl, hexacosan-8-yl, heptacosan-8-yl, octacosan-8-yl, nonacosan-8-yl, triacontan-8-yl, henicosan-9-yl, docosan-9-yl, tricosan-9-yl, tetracosan-9-yl, pentacosan-9-yl, hexacosan-9-yl, heptacosan-9-yl, octacosan-9-yl, nonacosan-9-yl, triacontan-9-yl, henicosan-10-yl, docosan-10-yl, tricosan-10-yl, tetracosan-10-yl, pentacosan-10-yl, hexacosan-10-yl, heptacosan- 10-yl, octacosan-10-yl, nonacosan-10-yl, triacontan-10-yl, docosan-11-yl, tricosan- 11-yl, tetracosan-11-yl, pentacosan-11-yl, hexacosan-11-yl, heptacosan-11-yl, octacosan-11-yl, nonacosan-11-yl, triacontan-11-yl, tetracosan-12-yl, pentacosan- 12-yl, hexacosan-12-yl, heptacosan-12-yl, octacosan-12-yl, nonacosan-12-yl, triacontan-12-yl, hexacosan-13-yl, heptacosan-13-yl, octacosan-13-yl, nonacosan- 13-yl, triacontan-13-yl, octacosan-14-yl, nonacosan-14-yl, triacontan-14-yl, triacontan-15-yl, butan-2-yl, pentan-2-yl, hexan-2-yl, heptan-2-yl, octan-2-yl, nonan-2-yl, decan-2-yl, hexan-3-yl, heptan-3-yl, octan-3-yl, nonan-3-yl, decan-3-yl, octan-4-yl, nonan-4-yl, decan-4-yl, decan-5-yl, undecan-2-yl, dodecan-2-yl, tridecan-2-yl, tetradecan-2-yl, pentadecan-2-yl, hexadecan-2-yl, heptadecan-2-yl, octadecan-2-yl, nonadecan-2-yl, eicosan-2-yl, undecan-3-yl, dodecan-3-yl, tridecan-3-yl, tetradecan-3-yl, pentadecan-3-yl, hexadecan-3-yl, heptadecan-3-yl, octadecan-3-yl, nonadecan-3-yl, eicosan-3-yl, undecan-4-yl, dodecan-4-yl, tridecan-4-yl, tetradecan-4-yl, pentadecan-4-yl, hexadecan-4-yl, heptadecan-4-yl, octadecan-4-yl, nonadecan-4-yl, eicosan-4-yl, undecan-5-yl, dodecan-5-yl, tridecan-5-yl, tetradecan-5-yl, pentadecan-5-yl, hexadecan-5-yl, heptadecan-5-yl, octadecan-5-yl, nonadecan-5-yl, eicosan-5-yl, dodecan-6-yl, tridecan-6-yl, tetradecan-6-yl, pentadecan-6-yl, hexadecan-6-yl, heptadecan-6-yl, octadecan-6-yl, nonadecan-6-yl, eicosan-6-yl, tetradecan-7 -yl, pentadecan-7 -yl, hexadecan-7 -yl, heptadecan-7 -yl, octadecan-7-yl, nonadecan-7-yl, eicosan-7-yl, hexadecan-8-yl, heptadecan-8-yl, octadecan-8-yl, nonadecan-8-yl, eicosan-8-yl, octadecan-9-yl, nonadecan-9-yl and eicosan-9-yl.

[0104] In some embodiments of the compounds described herein each RA, RBand Rcis, independently, selected from: undecan-5-yl, tridecan-6-yl, pentadecan-7-yl, heptadecan-8-yl, and nonadecan-9-yl. In further embodiments, RA, RBand Rcare, independently, selected from: undecan-5-yl, tridecan-6-yl, pentadecan-7-yl. In an embodiment, each RA, RBand Rcare undecan-5-yl.In some embodiments of the compounds described herein, RA, RBand Rcare unsubstituted.

[0105] It has been found herein that the alkyl and / or alkenyl chains with the length indicated above can provide a sufficiently large apolar zone and good positioning of the ionizable phospholipid compounds forthe encapsulation of linear cargo compounds such as nucleic acids, in particular large nucleic acids.

[0106] In some embodiments, m and n are integers independently selected from 1 , 2 and 3. In further embodiments, m and n are the same.

[0107] In some embodiments, o is an integer selected from 1 , 2 and 3.

[0108] In some embodiments of the compounds described herein, L1, L2, and L3are independently, selected from: ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene and decylene. In particular embodiments, L1, L2, and L3are independently selected from: ethylene, propylene, butylene, pentylene and hexylene.

[0109] In some embodiments of the compounds described herein, L1is ethylene, propylene, butylene. In further embodiments L1is ethylene.

[0110] In some embodiments of the compounds described herein, L2and L3are independently selected from ethylene, propylene, butylene, pentylene, hexylene, heptylene and octylene. In further embodiments, each L2is independently selected from ethylene, propylene, butylene, pentylene and hexylene. In further embodiments, all L2is the same, optionally all L2are butylene, pentylene or hexylene. In further embodiments, each L3is independently selected from ethylene, propylene, butylene, pentylene and hexylene. In further embodiments, all L3are the same, optionally all L3are ethylene, butylene or hexylene. In some embodiments all L2and L3are butylene. In some embodiments, L1, L2and L3are straight chain alkylene. In further embodiments, L1, L2and L3are unsubstituted.

[0111] In some embodiments of the compounds described herein, Z is -NR3R4, in particular when p is 1. In further embodiments, R3and R4are -L3F2RC.

[0112] In some embodiments, p is 1 ; o is 1 , 2 or 3; X2is O; Z is -NR3R4; and R3and R4are -L3F2RC.

[0113] In some embodiments, p is 1 ; o is 1 , 2 or 3; X2is O; Z is -NR3R4; and R3and R4are independently hydrogen or C1.4alkyl.

[0114] In some embodiments, the compound is a compound according to formula (II), a stereo-isomeric form, a tautomer, a salt (in particular a pharmaceutically acceptable salt), solvate, hydrate, and / or polymorph thereof,

[0115]

[0116] wherein R1, R2, R3, R4, m, n, and o are as defined according to any of the embodiments provided herein.

[0117] In some embodiment, compounds of formula (I) and / or (II) are provided, wherein m, n and o are integers independently selected from 1 , 2, 3, 4 or 5;

[0118] - R1is -L1N[L2F1RA]2or -L2F1RA;

[0119] - R2is-L2F1RA;

[0120] - Zis-NR3R4;

[0121] - R3is-L3F2RC;

[0122] R4is hydrogen or -L3F2RC;

[0123] L1, L2and L3are independently selected from C2.10-alkylene;

[0124] each F1and F2are independently selected from -O-C(=O)-, -C(=O)-O-, - NH-C(=O)- or -C(=O)-NH-; and;

[0125] each RAand Rcare independently selected from secondary or branched C4-3o-alkyl or primary C5-3o-alkenyl.

[0126] In further embodiments, compounds of formula (II) are provided, wherein

[0127] m, n and o are integers independently selected from 1 , 2, 3, 4 or 5;

[0128] - R1is -L1N[L2F1RA]2or -L2F1RA, preferably R1is -L2F1RA;

[0129] - R2is-L2F1RA;

[0130] - R3is-L3F2RC;

[0131] R4is hydrogen or -L3F2RC;

[0132] L1, L2and L3are independently selected from C2.10-alkylene;

[0133] each F1and F2are independently selected from -O-C(=O)-, -C(=O)-O-, - NH-C(=O)- or -C(=O)-NH-; and;

[0134] each RAand Rcare independently selected from secondary or branched C4-3o-alkyl.

[0135] In some embodiments, the compound is selected from the group consisting of:

[0136] - 2-(di((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethyl 2-(4-(2-(di((9Z,12Z)- octadeca-9,12-dien-1-yl)amino)ethyl)piperazin-1-yl)ethyl hydrogen phosphate;

[0137] ((2-(4-(10-butyl-4-hydroxy-4,9-dioxo-3,5-dioxa-8-aza-4A5- phosphahexadecan-1 -yl)piperazin-1 -yl)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate);((2-(4-(13-butyl-4-hydroxy-4,12-dioxo-3,5,11-trioxa-8-aza-4A5- phosphanonadecan-1 -yl)piperazin-1 -yl)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate);

[0138] ((2-(4-(15-butyl-4-hydroxy-4,14-dioxo-3,5,13-trioxa-8-aza-4A5- phosphahenicosan-1 -yl)piperazin-1 -yl)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate);

[0139] ((2-(4-(13-butyl-8-(2-((2-butyloctanoyl)oxy)ethyl)-4-hydroxy-4, 12-dioxo- 3,5,11 -trioxa-8-aza-4A5-phosphanonadecan-1 -yl)piperazin-1- yl)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate);

[0140] ((2-(4-(15-butyl-8-(4-((2-butyloctanoyl)oxy)butyl)-4-hydroxy-4,14-dioxo- 3,5,13-trioxa-8-aza-4A5-phosphahenicosan-1 -yl)piperazin-1- yl)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate);

[0141] ((2-(4-(6-hydroxy-2-methyl-6-oxo-5,7-dioxa-2-aza-6A5-phosphanonan-9- yl)piperazin-1-yl)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate); and,

[0142] ((2-((4-((2-butyloctanoyl)oxy)butyl)(2-(4-(6-hydroxy-2-methyl-6-oxo-5,7- dioxa-2-aza-6A5-phosphanonan-9-yl)piperazin-1- yl)ethyl)amino)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate); and mixtures thereof.

[0143] The present invention further encompasses a process for preparing the compound of formula (I) and / or (II) or (preferred) embodiments thereof, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. Schemes 1-23 illustrate the different reagents, precursors, and / or steps that may be used in embodiments of the present process. The identity and preparation of these precursors, as well as preferred process steps, are described in further detail below.

[0144] In some embodiments, the present process provides aldehyde-containing precursors. The term “aldehyde-containing precursor” as used herein refers to a compound comprising at least one aldehyde functional group (-CHO). Preferably, said compound is reactive under reductive amination. Exemplary aldehyde-containing precursors are:

[0145] - R-F-L-C(=O)H;

[0146] - Z-[(CH2)o-CH2-X2]p-P(=O)(OH)-X1-(CH2)m-C(=O)H, or

[0147] - R’-C(=O)H;

[0148] wherein R is RAor Rc; R’ is R1or R2; F is F1or F2; L is L1, L2, or L3; and Z, X1, X2, RA, Rc, R1, R2, F1, F2, L1, L2, L3, o, p, and m are as defined herein for any of the embodiments of compounds of formula (I) and (II).

[0149] In some embodiments, the aldehyde-containing precursor is a fatty acid-derived precursor. Said fatty acid-derived aldehyde-containing precursors refer to aldehyde-containing compounds obtained from fatty acids. These precursors retain structural elements characteristic of fatty acids, such as a C4-3oalkyl or C4-3oalkenyl chain, which may be saturated or unsaturated, linear or branched. Said compound may either be obtained commercially (e.g., fatty aldehydes such as (9Z, 12Z)-octadeca-9,12-dienal, octanal, nonanal, decanal, dodecanal, 2-ethylhexanal, isoheptanal, (2F,6Z)-2,6-nonadienal) or synthesized accordingto synthesis methods already described in the specialist literature (experimental guidelines can be found in the Reaxys® Database or the SciFinder® Database respectively), or can be prepared using the conventional methods known to the person skilled in the art. For example, such compounds may be derived by oxidation of hydroxyesters, hydroxyamides, or other hydroxyfunctionalized fatty acid derivatives.

[0150] As shown in Scheme 1 of the Example section, illustrating a particular embodiment of the present process, a fatty acid compound may first be coupled to the alcohol group or amide group of an alkyl diol or alkanolamine (exemplified by ethylene glycol or 1 ,4-butanediol) in the presence of a coupling agent such as a carbodiimide (DCC, DIC, EDC) or hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU). The resulting hydroxyester or hydroxyamide can subsequently be transformed into an aldehyde-containing ester or amide via oxidation. Oxidation may be carried out by oxidation methods and procedures that are well-known in the art. Non-limiting suitable examples of oxidizing agents include Dess-Martin periodinane (DMP), TEMPO / NaOCl, Swern oxidation reagents, or pyridinium chlorochromate (PCC). In some embodiments, the present process provides other fatty acid-derived precursors, such as fatty acid-derived halide-containing precursors. A fatty acid-derived halide-containing precursor refers to a haloalkyl compound (e.g. a bromoalkyl compound) derived from fatty acid structures. Said precursors may be prepared via halogenation of fatty acids, esters, or alcohols.

[0151] In some embodiments, the present process provides piperazine-containing precursors. The term “piperazine-containing precursor” as used herein refers to a compound incorporating a piperazine moiety (-N(CH2CH2)2N-) within its structure. Exemplary piperazine-containing precursors are:

[0152]

[0153] wherein

[0154] m and n are integers independently selected from 1 , 2, 3, 4 and 5, or as defined herein for any of the embodiments of compounds of formula (I) and (II);

[0155] - X3is OH, NH2or NHPG;

[0156] - X5is OH or NH2;

[0157] R6, R7are each independently selected from hydrogen, C4-3oalkenyl, or -L1NH2; R8, R9are each independently selected from hydrogen, C4-3oalkenyl, or -L1NH2; L1is C2.10alkylene; and

[0158] PG is an amine protecting group, preferably selected from the group consisting of Boc (tert-butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), Cbz (benzyloxycarbonyl), Alloc (allyloxycarbonyl), Trt (trityl), and Ms (methanesulfonyl).

[0159] In some embodiments, the present process provides phosphate- or phosphoramide-containing precursors. The terms “phosphate- or phosphoramide-containing precursor” as used herein refers to compounds containing a phosphoric ester (-0-P(=O)(OH)2) or a phosphoramide (-NH-P(=O)(OH)2or -NH-P(=O)(OH)(NH2)) moiety. These precursors may participate in phosphorylation reactions. Exemplary phosphate- or phosphoramide-containing precursors are:

[0160]

[0161] wherein

[0162] o is an integer selected from 1 , 2, 3, 4 and 5, or as defined herein for any of the embodiments of compounds of formula (I) and (II);p is an integer selected from 0 and 1, or as defined herein for any of the embodiments of compounds of formula (I) and (II);

[0163] - X4is selected from O or NH;

[0164] - X6is selected from OH or NH2;

[0165] - Z is as defined herein for any of the embodiments of compounds of formula (I) and (II); and

[0166] PG is an amine protecting group, preferably selected from the group consisting of Boc (tert-butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), Cbz (benzyloxycarbonyl), Alloc (allyloxycarbonyl), Trt (trityl), and Ms (methanesulfonyl).

[0167] Generally, the present process comprises the sequential functionalization of a piperazine-containing precursor as described herein with one or more fatty acid-derived chains and a phosphate- or phosphoramide moiety via successive reductive amination, deprotection, and / or phosphorylation reactions. The specific sequence of these transformations may vary depending on the selected reagents, reaction conditions, and desired product characteristics (as illustrated herein in Scheme 7-23).

[0168] Reductive amination is a well-known reaction process that converts a carbonyl group (e.g., an aldehyde or ketone group) into an amine through an imine or iminium intermediate, followed by reduction (see e.g., Hitchhiker’s guide to reductive amination, Synthesis, 2019; 51(13): 2667-2677 or Smith, M. B. (2020). March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (8th ed.). Wiley). The reaction is typically carried out in the presence of an amine donor (e.g., a primary or secondary amine-containing precursor having N-H groups), a reducing agent (e.g., sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, or hydrogen gas in the presence of a metal catalyst e.g., platinum, palladium, nickel), and a solvent such as ethanol, methanol, THF, DCM, or DMF under mild to moderate temperatures. It is apparent to the person skilled in the art that a primary amine group (-RNH2) may participate twice in the reductive amination process, due to the presence of two N-H groups, while a secondary amine group (-RNR’H) typically reacts once.

[0169] In particular embodiments of the present process, aldehyde-containing precursors as described herein may participate in reductive amination reactions with piperazine-containing precursors or derivatives thereof and / or phosphate- or phosphoramide-containing precursors or derivatives thereof.

[0170] The protection and deprotection of functional groups is a strategy employed in the present process to block participation of these groups in an ongoing chemical reaction. Such protecting groups are well known in the art and can be readilyremoved, for example, by chemical or enzymatic hydrolysis, reduction or hydrogenation, or by UV irradiation, etc.

[0171] In particular embodiments of the present process, protecting groups may be used to block participation of hydroxy groups and / or amine groups, such as amine groups of the piperazine-containing precursors or phosphate- or phosphoramide-containing precursors.

[0172] Suitable amine-protecting groups may be, for example carbamate groups such as tert-Butoxycarbonyl (Boc), Benzyloxycarbonyl (Cbz), Fluorenylmethoxycarbonyl (Fmoc), Allyloxycarbonyl (Alloc), Trimethylsilylethoxycarbonyl, amide groups such as formyl, acetyl, trifluoroacetyl, phthaloyl, and other groups such as 2,4-Dinitrophenyl, Triphenylmethyl, Methoxycarbonyl.

[0173] Suitable hydroxy-protecting groups may be, for example, acyl groups such as acetyl, trichloroacetyl, phenoxycarbonyl, benzyloxycarbonyl, benzhydryloxycarbonyl, trityloxycarbonyl and 2,2,2-trichloroethoxycarbonyl, ether groups such as methoxymethyl, benzyloxymethyl, allyl, benzyl, p-methoxybenzyl, p-nitrobenzyl, benzhydryl, trityl or triorganosilyl groups such as tri(C,-C.) alkylsilyl (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, isopropyldimethylsilyl, t-butydimethylsilyl, methyldiisopropylsilyl or methyldi-t-butylsilyl), t-butyl-diphenylsilyl, triarylsilyl (e.g., triphenylsilyl, tri-p-xylylsilyl) or triaralkylsilyl (e.g., tribenzylsilyl).

[0174] Examples of these and other suitable protecting groups and methods for their formation and removal are known in the art. See, for example, Protective Groups in Organic Synthesis, second ed., T. W. Greene and P.G.M. Wuts, John Wiley & Sons, New York, 1991, chapter 2 and references therein.

[0175] Phosphorylation, as used in the present invention, refers to the chemical introduction of a phosphate- or phosphoramide-containing moiety onto a piperazine-containing precursor or a derivative thereof. This reaction typically involves esterification or amidation of a hydroxyl or amine group, preferably in the presence of a coupling agent or catalyst (see e.g., Smith, M. B. (2020). March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (8th ed.). Wiley or Montalbetti, C. A. G. N., & Falque, V. (2005), Amide bond formation: Beyond the myth of coupling reagents. Tetrahedron, 61(46), 10827-10852).

[0176] Non-limiting examples of suitable coupling agents include DCC, EDC.HCl, HATU, HBTU, TPSCl, CC13CN, and carbonyl diimidazole.

[0177] In preferred embodiments, phosphorylation in the present process is performed in the presence of HATU or TPSCl.Each reaction step of the present process may further comprise the use of a suitable organic solvent, such as methanol, ethanol, THF, DCM, or DMF. The organic solvent may be selected to be compatible with the components present in each step. Furthermore, each reaction is allowed to proceed at a temperature and for a time sufficient to permit covalent conjugation between the indicated components.

[0178] In exemplary embodiments, the present process may comprise the steps of:

[0179] i) providing a piperazine-precursor of the following structure:

[0180]

[0181] wherein

[0182] n is an integer selected from 1 , 2, 3, 4 and 5;

[0183] R6, R7are each independently selected from hydrogen, C4-3oalkenyl, or -L1NH2; L1is C2-ioalkylene; and

[0184] PG is an amine protecting group, preferably selected from the group consisting of Boc (tert-butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), Cbz (benzyloxycarbonyl), Alloc (allyloxycarbonyl), Trt (trityl), and Ms (methanesulfonyl);

[0185] ii) reacting the piperazine-precursor with a fatty acid-derived aldehyde- containing precursor under reductive amination conditions, the fatty acid-derived aldehyde-containing precursor having a structure selected from R-F-L-C(=O)H or R’-C(=O)H wherein R and R’ are as defined herein;

[0186] iii) deprotecting the amine group of the piperazine moiety; and

[0187] iv) reacting the amine group of the piperazine moiety with an aldehyde- containing precursor under reductive amination conditions, the aldehyde-containing precursor having the following structure Z- [(CH2)o-CH2-X2]p-P(=O)(OH)-Xi-(CH2)m-C(=O)H;

[0188] wherein Z, X1, X2, o, p, and m are as defined herein.

[0189] In exemplary embodiments, the present process may comprise the steps of:

[0190] i) providing a piperazine-precursor of the following structureR8

[0191]

[0192] wherein

[0193] m and n are integers independently selected from 1 , 2, 3, 4 and 5, or as defined herein for any embodiments thereof;

[0194] - X3is OH or NH2;

[0195] R8, R9are each independently selected from hydrogen, C4-3oalkenyl, or -L1NH2; L1is C2-10alkylene; and

[0196] PG is an amine protecting group, preferably selected from the group consisting of Boc (tert-butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), Cbz (benzyloxycarbonyl), Alloc (allyloxycarbonyl), Trt (trityl), and Ms (methanesulfonyl);

[0197] ii) reacting the piperazine-precursor with a fatty acid-derived aldehyde- containing precursor under reductive amination conditions, the fatty acid-derived aldehyde-containing precursor having a structure selected from R-F-L-C(=O)H or R’-C(=O)H wherein R and R’ are as defined herein; and

[0198] iii) reacting the hydroxyl group or amine group of the piperazine moiety with a phosphate- or phosphoramide-containing precursor, preferably in the presence of a coupling agent such as HATU or TPSCl, of the following structure

[0199]

[0200] wherein

[0201] o is an integer selected from 1 , 2, 3, 4 and 5;

[0202] p is an integer selected from 0 and 1 ;

[0203] - X4is selected from O or NH;

[0204] - X6is selected from OH or NH2;

[0205] - Z is as defined herein for any of the embodiments for compounds accordingto formula (I) or (II).

[0206] In exemplary embodiments, the present process may comprise the steps of:i) providing a piperazine-precursor of the following structure

[0207] R8

[0208]

[0209] wherein

[0210] m and n are integers independently selected from 1 , 2, 3, 4 and 5;

[0211] - X3is OH;

[0212] R8, R9are each independently selected from hydrogen, C4-30 alkenyl, or-L1NH2; L1is C2-ioalkylene; and

[0213] PG is an amine protecting group, preferably selected from the group consisting of Boc (tert-butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), Cbz (benzyloxycarbonyl), Alloc (allyloxycarbonyl), Trt (trityl), and Ms (methanesulfonyl);

[0214] ii) reacting the piperazine-precursor with a fatty acid-derived aldehyde- containing precursor under reductive amination conditions, the fatty acid-derived aldehyde-containing precursor having a structure selected from R-F-L-C(=O)H or R’-C(=O)H wherein R and R’ are as defined herein; and

[0215] iii) reacting the hydroxyl group of the piperazine moiety with a phosphate- or phosphoramide-containing precursor, preferably in the presence of a coupling agent such as HATU orTPSCl, of the following structure

[0216]

[0217] wherein

[0218] o is an integer selected from 1 , 2, 3, 4 and 5;

[0219] p is an integer selected from 0 and 1 ;

[0220] - X4is selected from O or NH;

[0221] - X6is selected from OH or NH2;

[0222] PG is an amine protecting group, preferably selected from the group consisting of Boc (tert-butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), Cbz (benzyloxycarbonyl), Alloc (allyloxycarbonyl), Trt (trityl), and Ms (methanesulfonyl);iv) deprotecting the amine group of the phosphate or phosphoramide moiety;

[0223] v) optionally, reacting the amine group of the phosphate or phosphoramide moiety with a fatty acid-derived aldehyde-containing precursor under reductive amination conditions, the fatty acid-derived aldehyde-containing precursor having a structure selected from R-F-L- C(=O)H or R’-C(=O)H wherein R and R’ are as defined herein.

[0224] In exemplary embodiments, the present process may comprise the steps of:

[0225] i) providing a piperazine-precursor of the following structure

[0226]

[0227] wherein

[0228] m and n are integers independently selected from 1 , 2, 3, 4 and 5;

[0229] - X3is NHPG;

[0230] R8, R9are each independently selected from hydrogen, C4-3oalkenyl, or -L1NH2; L1is C2-ioalkylene; and

[0231] PG is an amine protecting group, preferably selected from the group consisting of Boc (tert-butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), Cbz (benzyloxycarbonyl), Alloc (allyloxycarbonyl), Trt (trityl), and Ms (methanesulfonyl);

[0232] ii) reacting the piperazine-precursor with a fatty acid-derived aldehyde- containing precursor under reductive amination conditions, the fatty acid-derived aldehyde-containing precursor having a structure selected from R-F-L-C(=O)H or R’-C(=O)H, wherein R and r’ are as defined herein;

[0233] iii) deprotecting the amine group of the piperazine moiety;

[0234] iv) reacting the amine group of the piperazine moiety with a phosphate- or phosphoramide-containing precursor, preferably in the presence of a coupling agent such as HATU orTPSCl, of the following structure

[0235] ,p

[0236] | *6

[0237] OH

[0238]

[0239] wherein

[0240] o is an integer selected from 1 , 2, 3, 4 and 5;

[0241] p is an integer selected from 0 and 1 ;

[0242] - X4is selected from O or NH;

[0243] - X6is selected from OH or NH2;

[0244] - Z is as defined herein for any of the embodiments for compounds accordingto formula (I) or (II).

[0245] In exemplary embodiments, the present process may comprise the steps of:

[0246] i) providing a piperazine-precursor of the following structure

[0247] R8

[0248]

[0249] wherein

[0250] m and n are integers independently selected from 1 , 2, 3, 4 and 5;

[0251] - X3is NHPG;

[0252] R8, R9are each independently selected from hydrogen, C4-3oalkenyl, or -L1NH2; L1is C2-10alkylene; and

[0253] PG is an amine protecting group, preferably selected from the group consisting of Boc (tert-butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), Cbz (benzyloxycarbonyl), Alloc (allyloxycarbonyl), Trt (trityl), and Ms (methanesulfonyl);

[0254] ii) reacting the piperazine-precursor with a fatty acid-derived aldehyde- containing precursor under reductive amination conditions, the fatty acid-derived aldehyde-containing precursor having a structure selected from R-F-L-C(=O)H or R’-C(=O)H wherein R and R’ are as defined herein;

[0255] iii) deprotecting the amine group of the piperazine moiety;

[0256] iv) reacting the amine group of the piperazine moiety with a phosphate- or phosphoramide-containing precursor, preferably in the presence of a coupling agent such as HATU orTPSCl, of the following structure

[0257]

[0258] wherein

[0259] o is an integer selected from 1 , 2, 3, 4 and 5;

[0260] p is an integer selected from 0 and 1 ;

[0261] - X4is selected from O or NH;

[0262] - X6is selected from OH or NH2;

[0263] PG is an amine protecting group, preferably selected from the group consisting of Boc (tert-butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), Cbz (benzyloxycarbonyl), Alloc (allyloxycarbonyl), Trt (trityl), and Ms (methanesulfonyl);

[0264] v) deprotecting the amine group of the phosphate or phosphoramide moiety;

[0265] vi) optionally, reacting the amine group of the phosphate or phosphoramide moiety with a fatty acid-derived aldehyde-containing precursor under reductive amination conditions, the fatty acid-derived aldehyde-containing precursor having a structure selected from R-F-L- C(=O)H or R’-C(=O)H wherein R and R’ are as defined herein.

[0266] In exemplary embodiments, the present process may comprise the steps of:

[0267] i) providing a phosphate- or phosphoramide-containing precursor of the following structure

[0268]

[0269] wherein

[0270] o is an integer selected from 1 , 2, 3, 4 and 5;

[0271] p is an integer selected from 0 and 1 ;

[0272] - X4is selected from O or NH;

[0273] - X6is selected from OH or NH2;

[0274] PG is an amine protecting group, preferably selected from the group consisting of Boc (tert-butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), Cbz (benzyloxycarbonyl), Alloc (allyloxycarbonyl), Trt (trityl), and Ms (methanesulfonyl);

[0275] ii) reacting the hydroxyl group or amine group of the phosphate- or phosphoramide-containing precursor with a piperazine-precursor, preferably in the presence of a coupling agent such as HATU orTPSCl, of the following structure

[0276]

[0277] wherein

[0278] m and n are integers independently selected from 1 , 2, 3, 4 and 5;

[0279] - X5is OH or NH2; and

[0280] PG is an amine protecting group, preferably selected from the group consisting of Boc (tert-butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), Cbz (benzyloxycarbonyl), Alloc (allyloxycarbonyl), Trt (trityl), and Ms (methanesulfonyl);

[0281] iii) deprotecting the amine group of the phosphate or phosphoramide moiety and the piperazine-moiety;

[0282] iv) reacting the deprotected amine groups with a fatty acid-derived aldehyde-containing precursor under reductive amination conditions, the fatty acid-derived aldehyde-containing precursor having a structure selected from R-F-L-C(=O)H or R’-C(=O)H.

[0283] It should be understood that the disclosed synthetic routes, includingthe selection and order of reaction steps, reagents, and conditions, may be adapted or modified by a skilled person without departing from the scope of the invention.

[0284] It will be understood that any and all herein described embodiments of the compound of the present invention, including preferred embodiments thereof, are also to be interpreted as embodiments of the following related aspects of the invention.

[0285] The compounds of the invention are of interest for use in the formulation of a cargo, typically a biologically active compound, in particular for intracellular delivery of cargo, such as biologically active compounds, both in vivo and in vitro. The compounds are of particular interest for use in the formulation of biologically active compounds that are (overall) negatively charged.

[0286] Accordingly, in some embodiments, the compound is for use in methods to deliver a cargo to a cell, preferably a mammalian cell, such as for transfecting such cell, in vivo. In alternative embodiments, the compound is for use in methods to deliver a cargo to a cell, preferably a mammalian cell, such as for transfecting such cell / n vitro.

[0287] In particular embodiments, the use of the compounds in the intracellular delivery of a cargo also encompasses the use of the compounds in the manufacture of lipid nanoparticles for the delivery of cargo to a cell. Accordingly, in a related aspect, thepresent invention provides a lipid nanoparticle (LNP) composition comprising at least one compound of the invention, more particularly a compound of formula (I) and / or (II) or (preferred) embodiments thereof, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. The term “lipid nanoparticle” or “LNP” as used herein refers to a particle having at least one dimension on the order of nanometers (nm), which contains one or more types of lipid molecules of which at least one is a compound of formula (I) and / or (II) as described herein. The LNP provided herein can further contain at least one non-lipid payload molecule also referred to as “cargo”. In some embodiments, the LNP comprises a cargo molecule either partially or completely encapsulated inside the lipid mass.

[0288] In particular embodiments, the LNP composition may comprise a first compound of formula (I) and / or (II) or (preferred) embodiments thereof, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof and a second compound of formula (I) and / or (II) or (preferred) embodiments thereof, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein the first and second compound of formula (I) and / or (II) are different.

[0289] In preferred embodiments, the LNP composition may further comprise one or more lipids selected from the group consisting of alternative phospholipids, structural lipids, PEG-lipids, alternative ionisable lipids, and mixtures thereof.

[0290] Preferably, the LNP composition does not comprise phospholipids other than the compound(s) of formula (I) and / or (II) or (preferred) embodiments thereof. As mentioned above, this advantageously allows to prepare LNP formulations with fewer components, thereby reducing production complexity.

[0291] The term “alternative phospholipid” is used herein to refer to a phospholipid which is different from the compounds of formula (I) and / or (II). Phospholipids are known in the art to be amphipathic molecules typically comprising a hydrophilic phosphate head group and one or more hydrophobic fatty acid chains. Examples of suitable phospholipids for use in the lipid nanoparticles of the present invention include, but are not limited to, phosphatidylcholines, such as dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dilinoleoylphosphatidylcholine, myristoylpalmitoylphosphatidylcholine, myristoylstearoylphosphatidylcholine, and palmitoylstearoylphosphatidylcholine; phosphatidylglycerols, such as dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, dilinoleoylphosphatidylglycerol, myristoylpalmitoylphosphatidylglycerol, myristoylstearoylphosphatidylglycerol, and palmitoylstearoylphosphatidylglycerol;phosphatidylethanolamines, such as dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, dilinoleoylphosphatidylethanolamine, myristoylpahmitoylphosphatidylethanolamine, myristoylstearoylphosphatidylethanolamine, and palmitoylstearoylphosphatidylethanolamine; phosphatidylserine; phosphatidic acid; phosphatidylinositol; sphingomyelin; cardiolipin; egg yolk lecithin; soybean lecithin; and hydrogenated products thereof, or combinations thereof.

[0292] In some embodiments, the LNP compositions do comprise an alternative phospholipid, e.g. in a concentration of 0.2-45 mol%, preferably 0.5-35 mol% of the total amount of lipid in the LNP. In a further embodiment, said alternative phospholipid is DOPE or DSPC.

[0293] The LNP compositions of the present invention may comprise one or more structural lipids. Structural lipids serve to help form and maintain the lipid bilayer and / or morphology and stability of the particle structure. They may also contribute to the encapsulation and delivery of the payload. The one or more structural lipids may be a sterol, such as cholesterol or derivative thereof. In further embodiments, the one or more structural lipids are selected from cholesterol, cholestenol, spinasterol, fecosterol, sitosterol, ergosterol, ergostenol, campesterol, sigmasterol, brassicasterol, tomadine, alpha-tocopherol, cholesteryl hemisuccinate, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol, phytosterol, phytosterol, timosterol, or combinations thereof. In a preferred embodiment, said sterol is cholesterol.

[0294] In some embodiments, the structural lipid, such as cholesterol, is present in the LNP formulation in a concentration of at least 20 mol%, at least 30 mol%, at least 35 mol%, at least 37 mol% or at least 40 mol%, and / or, at most 60 mol%, at most 55 mol%, at most 50 mol% or at most 47 mol%; such as 30-60 mol%, 30-50 mol%, or 40-50 mol% of the total amount of lipid in the LNP.

[0295] In some embodiments, the LNP provided herein further comprises one or more polymer conjugated lipid. For the purpose of the LNP provided herein, typically hydrophilic polymer conjugated to a lipid are contemplated, such as a polyethyleneglycol (PEG) thus providing a PEG-lipid conjugate, also referred to in the art as a pegylated lipid. Such pegylated lipid provides for a PEG coating of the LNPs thereby reducing LNP aggregation, nonspecific binding of proteins and / or nonspecific endocytosis by (immune) cells. In further embodiments, the one or more polymer lipid conjugates are selected from a PEG-dialkyloxypropyl (DAA), a PEG-diacylglycerol (DAG), a PEG-phospholipid, a PEG-ceramide, and a mixture thereof. Infurther embodiments, the PEG-DAG conjugate is one or more of a PEG-dilauroylglycerol (C12), a PEG-dimyristoylglycerol (C14), a PEG-dipalmitoylglycerol (C16), and a PEG-distearoylglycerol (C18); the PEG-DAA conjugate is one or more of a PEG- di lauryloxy propyl (C12), a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), and a PEG-di stearyloxypropyl (C18). In yet further embodiments, the pegylated lipid is selected from 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG or PEG2k-DMG) and 1 ,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DSG). In a preferred embodiment, PEG2K-DMG is selected.

[0296] In some embodiments, the pegylated lipid(s) may be further derivatised to provide for further functionality, such as specific cell targeting, i.e. by introducing cell-specific ligands at the LNP surface through derivatisation of the pegylated lipid. For example, the cell-specific ligand may be an antibody, orthe cell-specific binding moiety thereof (see for example Chen M.Z. et al. “A Versatile Antibody Capture System that Drives Precise In Vivo Delivery of mRNA loaded Lipid Nanoparticles and Enhances Gene Expression” bioRxiv 2024.08.07.607101 ).

[0297] In some embodiments, the polymer conjugated lipid, such as pegylated lipid, is present in the LNP formulation in a concentration of 0.2-5 mol%, such as 0.5-3 mol%, 0.5-2 mol% or 1-2 mol% of the total amount of lipid in the LNP.

[0298] In some embodiments, the LNP further comprises an alternative ionisable lipid other than the / an ionisable cationic phospholipid according to formula (I) or (II) and as described herein. Any ionisable cationic lipid known in the art for the formulation of nucleic acids (DNA or RNA) is contemplated. In further embodiments, the alternative ionisable cationic lipid is select from the compounds as described in WG2023 / 078950, WG2023 / 078954 or WG2023 / 078946. Other non- limiting examples forthe alternative ionizable lipid are N,N-dioleyl-N,N- dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); N-(1-(2,3-dioleoyloxy)propyl)N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), 1,2-dioleoyl- 3-dimethylammonium propane (DODAP), 3-(N,N-dioleylamino)-1 ,2-propanediol (DOAP), N,N-dimethyl-2,3-dioleoyloxy)-propylamine (DODMA), and N-(1 ,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 1 ,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1 ,2-dilinoleyoxy-3-(dimethylamino)-acetoxypropane (DLin-DAC), 1 ,2-dilinoleyoxy- 3morpholinopropane (DLin-MA), 1 ,2-dilinoleoyl-3- dimethylaminopropane (DLinDAP), 1 ,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethyl-aminopropane (DLin-2-DMAP), 1 ,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1 ,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1 ,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 1 ,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1 ,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminebutyrate (DLin-MC3-DMA), di((Z)-non-2-en-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 1 ,1 ‘-((2-(4-(2-((2-(bis (2-hydroxydodecyl)amino)ethyl)-(2-hydroxydodecyl)amino)ethyl)-piperazin-1 -yl)-ethyl)azanediyl)-bis(dodecan-2-ol) (C12-200), 3,6-bis({4-[bis(2-hydroxy-dodecyl)-amino]butyl})piperazine-2, 5-dione (cKK-E12).

[0299] If an alternative ionizable lipid is present in the LNP composition, the total amount of ionizable lipid taken together does not exceed 70 mol% of the total amount of lipid in the LNP.

[0300] In exemplary embodiments, the LNP composition as described herein may comprise:

[0301] • at least one compound of formula (I) and / or (II) or (preferred) embodiments thereof, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof;

[0302] • at least one structural lipid; and

[0303] • at least one PEG-lipid;

[0304] preferably wherein the molar ratio of the compound : structural lipid : PEG- lipid is from 20 to 70 : 20 to 60 : 0.5 to 3.

[0305] In some exemplary embodiments, the LNP composition may comprise:

[0306] • at least one compound of formula (I) and / or (II) or (preferred) embodiments thereof, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof;

[0307] • at least one alternative phospholipid;

[0308] • at least one structural lipid; and

[0309] • at least one PEG-lipid;

[0310] preferably wherein the molar ratio of the compound : phospholipid : structural lipid : PEG-lipid is from 20 to 60 : 5 to 30 : 20 to 60 : 0.5 to 3.

[0311] Because of the compounds of the invention’s net positive charge in an acidic environment (e.g. at pH below 6), the compounds are particularly suited for the formulation of negatively charged cargo .

[0312] In particular embodiments, the lipid nanoparticle composition further comprises an (ionizable) cargo compound which is encapsulated by the LNPs. In the context of the present disclosure, such cargo is typically a negatively charged compound (atphysiological pH), such as a nucleic acid. As used herein, “encapsulation” or related terms refers to full encapsulation (encapsulated entity fully shielded from the LNP environment), partial encapsulation (encapsulated entity partially exposed to the LNP environment), association by ionic or van de Waals forces or combination of any of the aforementioned. In some embodiments, the therapeutic agent is fully encapsulated. Full encapsulation is believed to provide for better protection of the cargo against environmental factors.

[0313] In some embodiments, the lipid nanoparticle composition further comprises an ionizable cargo compound selected from the group consisting of nucleic acids, small molecule compounds, peptides, proteins, protein-nucleic acid constructs, and mixtures thereof, preferably wherein the cargo compound is ionized, more preferably anionic, such as anionic at physiological pH.

[0314] In alternative embodiments, the compound of the invention may be used for the formulation (and (cellular) delivery) of cargo by means otherthan LNP encapsulation, e.g. compounds of the invention may be provided as a coating or otherwise at the surface of, or, (partially or fully) integrated within alternative micro- or nanoparticles such as solid lipid nanoparticles or polymeric particles, and, available for strong (pH) reversible association of negatively charged cargo compounds.

[0315] In particular embodiments, the ionizable cargo compound is a nucleic acid. The terms “nucleic acid” and “polynucleotide” as used herein interchangeably generally refers to a polymer (preferably linear polymer) of any length composed essentially of nucleoside units. It includes both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), in single-stranded (ss) or (partly) double-stranded (ds) forms. In the present context, the term encompasses:

[0316] • Naturally occurring nucleic acids (e.g., genomic DNA, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA));

[0317] • Synthetic or recombinant nucleic acids, including but not limited to cDNA, saRNA, antisense RNA, siRNA, miRNA, and aptamers; and • Linear, circular, or branched nucleic acid structures.

[0318] The term may encompass chemically modified nucleic acids or polynucleotides. A “modified nucleic acid” or “modified polynucleotide” refers to nucleic acids or a polynucleotide which comprises one or more modified nucleosides, one or more modified inter-nucleoside linkages, or a combination thereof. The one or more modified nucleosides may each independently comprise a modified heterocyclic base, a modified sugar moiety, a modified connection between the base and the sugar moiety, or a combination thereof. By means of an example, an RNA polynucleotide may be denoted as “modified” when it comprises one or more nucleosides other than adenosine, guanosine, uridine, and cytidine, one or moreinter-nucleoside linkages other than a phosphodiester bond, or a combination thereof.

[0319] By means of example, a modified RNA polynucleotide may be primarily composed of adenosine, guanosine, uridine, and / or cytidine nucleosides connected by phosphodiester linkages, and may comprise a minority of nucleosides other than adenosine, guanosine, uridine, and cytidine, and / or a minority of inter-nucleoside linkages other than phosphodiester bond. In another example, a modified RNA polynucleotide may be primarily or exclusively composed of nucleosides other than adenosine, guanosine, uridine, and cytidine, and / or may comprise a majority of or may exclusively comprise inter-nucleoside linkages other than phosphodiester bond. In some embodiments, the present LNP composition may encapsulate modified RNA. In this context, nucleotide analogues or modifications are selected from nucleotide analogues, which remain susceptible to transcription and / or translation. In further embodiments, the modified RNA comprises one or more modified nucleotides selected from 6-aza-cytidine, 2-thio-cytidine, a-thio-cytidine, pseudocytidine, pseudo-iso-cytidine, N1-methyl-pseudocytidine, 5-aminoallyl-uridine, 5-iodo-uridine, pseudouridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, a-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, a-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, a-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine and 5-methyl-cytidine. In yet a further embodiment, the RNA comprises one or more modified nucleotides selected from pseudouridine, N1-methyl-pseudouridine, 5-methyl-uridine, pseudocytidine, N1-methyl-pseudocytidine and 5-methyl-cytidine.

[0320] In some embodiments, the ionizable cargo compound is a nucleic acid selected from the group consisting of saRNA, mRNA, siRNA, rRNA, DNA, aptamer, tRNA, antisense oligonucleotide, shRNA, miRNA, sgRNA, tracrRNA, gRNA, ribozyme, DNAzyme, and mixtures thereof.

[0321] In some embodiments, the cargo compound is a ribonucleic acid; preferably selected from the group consisting of self-amplifying RNA (saRNA), long-chain RNA, coding RNA, non-coding RNA, long non-coding RNA, single stranded RNA (ssRNA), double stranded RNA (dsRNA), linear RNA (linRNA), circular RNA (circRNA), messenger RNA (mRNA), Trans amplifying mRNA, RNA oligonucleotides, antisense oligonucleotides, small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR / Cas9 guide RNAs (gRNA), riboswitches, immunostimulating RNA (isRNA), ribozymes, aptamers, ribosomal RNA (rRNA), transferRNA (tRNA), viral RNA (vRNA), retroviral RNA or replicon RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), a Piwi-interacting RNA (piRNA), and mixtures thereof.

[0322] It was found herein that the present LNP composition is particularly suited to be used in combination with nucleic acid, such as RNA, of a specific size. For instance, an advantage of the present LNP compositions is that nucleic acids of a larger size may be more efficiently encapsulated compared to existing LNP compositions described in the art. In particular, saRNA, known to typically be larger than conventional mRNA, is often reported in the art to result in poor encapsulation or sub-optimal intracellular delivery. In contrast, and as demonstrated herein in the Example section, the present LNP compositions are suitable for intracellular delivery of such large RNA constructs, both in vitro as well as in vivo.

[0323] In some embodiments, the LNP comprises a nucleic acid (e.g. any type of RNA optionally including one or more modified nucleotide) that has a length of at least 1000 nucleotides (nt), at least 2000 nt, at least 3000 nt, at least 4000 nt, at least 5000 nt, at least 6000 nt, at least 7000 nt, at least 8000 nt or at least 9000 nt or more. In a particularly preferred embodiment, said RNA is a self-amplifying RNA (saRNA). The size of the RNA, in particular saRNA, can be of at least 5000 nt or more. More preferably the size of said RNA, such as saRNA, can be between 5000 and 20000 nt, preferably between 6000 and 19000 nt, preferably between 7000 and 18000 nt, preferably between 8000 and 17000 nt, more preferably between 8000 and 16000 nt. In some embodiments, the present LNP composition may comprise one or more, such as one or two, saRNA molecules.

[0324] In exemplary embodiments, the LNP composition as described herein may comprise:

[0325] at least one compound of formula (I) and / or (II) or (preferred) embodiments thereof, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof;

[0326] at least one structural lipid;

[0327] at least one PEG-lipid; and

[0328] at least one nucleic acid, such as a nucleic acid havinga size of at least 8000 nt; preferably wherein the weight ratio of the compound to the nucleic acid in the composition is from 1 :1 to 100:1 , or from 5:1 to 80:1 , orfrom 10:1 to 75:1, orfrom 15:1 to 60:1, orfrom 15:1 to 50:1.

[0329] In exemplary embodiments, the LNP composition as described herein may comprise:

[0330] at least one compound of formula (I) and / or (II) or (preferred) embodiments thereof, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof;at least one alternative phospholipid;

[0331] at least one structural lipid;

[0332] at least one PEG-lipid; and

[0333] at least one nucleic acid, such as a nucleic acid having a size of at least 8000 nt;

[0334] preferably wherein the weight ratio of the compound to the nucleic acid in the composition is from 1:1 to 100:1, orfrom 5:1 to 80:1, orfrom 10:1 to 75:1, or from 15:1 to 60:1, orfrom 15:1 to 50:1.

[0335] In exemplary embodiments, the LNP composition as described herein may comprise:

[0336] at least one compound of formula (I) and / or (II) or (preferred) embodiments thereof, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof;

[0337] at least one structural lipid;

[0338] at least one PEG-lipid; and

[0339] at least one nucleic acid, preferably saRNA as described herein; preferably wherein the weight ratio or molar ratio of the compound to the nucleic acid in the composition is from 1:1 to 100:1, or from 5:1 to 80:1, or from 10:1 to 75:1, orfrom 15:1 to 60:1, orfrom 15:1 to 50:1; and

[0340] wherein the molar ratio of the compound : structural lipid : PEG-lipid is from 20 to 60 : 30 to 60 : 1 to 5.

[0341] In exemplary embodiments, the LNP composition as described herein may comprise:

[0342] at least one compound of formula (I) and / or (II) or (preferred) embodiments thereof, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof;

[0343] at least one alternative phospholipid;

[0344] at least one structural lipid;

[0345] at least one PEG-lipid; and

[0346] at least one nucleic acid, preferably saRNA as described herein; preferably wherein the weight ratio of the compound to the nucleic acid in the composition is from 1:1 to 100:1, orfrom 5:1 to 80:1, orfrom 10:1 to 75:1, or from 15:1 to 60:1, or from 15:1 to 50:1; and wherein the molar ratio of the compound : phospholipid : structural lipid : PEG-lipid is from 20 to 60 : 5 to 30 : 30 to 60 : 1 to 5.

[0347] As mentioned above, LNP compositions typically comprise nanoparticles of a particular size. In some embodiments, the lipid nanoparticles have a mean diameter of at least 50 nm, at least 60 nm, at least 70 nm or at least 80 nm, and / or, at most 220 nm, at most 210 nm, at most 200 nm, at most 190 nm, at most 180 nm, at most 170 nm, at most 160 nm or at most 150 nm. Unless indicated otherwise, all diameter sizesreferred to herein are the average sizes (hydrodynamic diameters) of the fully formed nanoparticle, as measured by dynamic light scattering (e.g., on a Malvern Zetasizer). In particular embodiments, the present LNP composition has a Zeta potential of between -30 mV and +30 mV. The Zeta potential is the difference in potential between the bulk fluid in which a particle is dispersed and the layer of fluid associated with the nanoparticle surface, and, is an indicator of the charge at the surface of the particle, with a large zeta potential (negative or positive) indicating good colloidal stability of the particle dispersion.

[0348] In particular embodiments, the LNP composition has an N / P ratio between the compound of formula (I) and / or (II) or (preferred) embodiments thereof, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof (and other ionizable lipids) and a nucleic acid cargo compound (e.g., saRNA) of between 5:1 and 45:1 , or between 5:1 and 40:1 or between 5:1 and 35:1 , or between 5:1 and 30:1 , or between 10:1 and 30:1 , or between 10:1 and 25:1. These ratios have been found to ensure optimal nucleic acid adsorption to the lipid nanoparticle.

[0349] In another aspect, the present invention further relates to a method for manufacturing a lipid nanoparticle (LNP) composition according to an aspect of the present invention or (preferred) embodiments thereof.

[0350] It should be noted that (preferred) embodiments of the compound according to an aspect of the present invention or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, and, LNP composition according to another aspect of the present invention, are also (preferred) embodiments of the manufacturing method as described herein. For instance, it should be clear that the weight ratios and molar ratios of each component in the resulting LNP composition apply mutatis mutandis to the method for manufacturing said LNP composition as disclosed herein.

[0351] In particular embodiments, the method may comprise the steps of:

[0352] (a) providing lipids comprising at least one compound of formula (I) and / or (II) or (preferred) embodiments thereof, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, and optionally one or more lipids selected from the group consisting of phospholipids, structural lipids, PEG-lipids, and mixtures thereof;

[0353] (b) dissolvingthe lipids in an organic solvent, thereby forming an organic phase; (c) providing an aqueous phase, preferably comprising one or more cargo compounds as described herein;

[0354] (d) forming lipid nanoparticles by mixing the organic phase and aqueous phase under conditions suitable for nanoparticle formation.Organic solvents used in the present method is selected based on their ability to dissolve the lipids as described herein, and be miscible with the aqueous phase. It is apparent to the skilled person that the organic solvent may be varied depending on the nature of lipids, cargo compound, and / or conditions used for forming the lipid nanoparticles.

[0355] Non-limiting examples of suitable organic solvents include alcohols, such as methanol, ethanol, and isopropanol; acetone, dimethyl sulfoxide (DMSO), dichloromethane (DCM), chloroform, ethyl acetate, acetonitrile, tetrahydrofuran (THF), or combinations thereof. In particular embodiments, the organic solvent comprises or is an alcohol.

[0356] In embodiments, the aqueous phase used to dissolved the one or more cargo compounds may comprise one or more buffers. Non-limiting examples of suitable buffers include citrate buffer, Tris-HCl, HEPES, PBS, acetate buffer, TE (tris-EDTA) buffer, MES buffer, or MOPS buffer. It is apparent to the person skilled in the art that the composition of the aqueous phase may be varied depending on the pH stability of the cargo compound. In a particular embodiment, the citrate buffer is used.

[0357] In a particular embodiment, the lipids may be dissolved in an organic solvent comprising or consisting of ethanol, and, the cargo compound(s), in particular when the cargo is a nucleic acid, may be dissolved in an aqueous solvent comprising a citrate buffer.

[0358] It should be clear that the present invention is not particularly limited by the method for forming the lipid nanoparticles (i.e., step d) of the present method) as described herein. These include, but are not limited to, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, simple and complex coacervation, and other methods well known to those of ordinary skill in the art. The conditions used in preparing the particles may be altered to yield particles of a desired size or property (e.g., hydrophobicity, hydrophilicity, external morphology, “stickiness”, shape, etc.). The method of preparing the particle and the conditions (e.g., solvent, temperature, concentration, air flow rate, etc.) used may also depend on the cargo compound being encapsulated.

[0359] In an exemplary embodiment, the present manufacturing method may comprise the steps of:

[0360] (a) providing lipids comprising at least one compound of formula (I) and / or (II) or (preferred) embodiments thereof, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, at least one structural lipid, at least one PEG-lipid, and optionally at least one alternative phospholipid;(b) dissolving the lipids in an organic solvent comprising one or more alcohols, thereby forming an organic phase;

[0361] (c) providing an aqueous phase, preferably comprising one or more cargo compounds as described herein;

[0362] (d) forming lipid nanoparticles by mixing the organic phase and aqueous phase under conditions suitable for nanoparticle formation.

[0363] In a further embodiment, the mixing of step (d) is microfluidic mixing. As used herein, the term “microfluidic mixing” refers to a mixing method wherein the aim is to achieve thorough and rapid mixing of multiple samples (i.e. organic phase and aqueous phase) in a low pressure accurate mixing device. Such sample mixing is typically achieved by enhancing the diffusion effect between the different species flows. Thereto several low pressure accurate mixing devices can be used.

[0364] In embodiments, the total flow rate (TFR) of the microfluidic device is at least 1 mL / min, or at least 2 mL / min, or at least 5 mL / min, or at least 6 mL / min, or at least 6 mL / min, or at least 7 mL / min, or at least 8 mL / min, or at least 9 mL / min, or at least 15 mL / min.

[0365] In embodiments, the flow rate ratio (FRR) between the organic and aqueous phases is between about 5:1 and 1 :5, or between about 4:1 and 1 :5, or between about 3:1 and 1 :5, or between about 2:1 and 1 :5, or between about 2:1 and 1 :4.

[0366] In a further embodiment, the methods of preparation of the LNP composition further includes a processing step (e) to obtain an LNP composition suitable for use in medicine. Step (e) may comprise one or both of the following:

[0367] buffer exchange, and / or,

[0368] sterilisation.

[0369] A buffer exchange allows replacement of any of the excipients (solvents, buffers, etc.) used in the preceding manufacturing steps that may not be desirable in a composition for use in medicine, by pharmaceutically acceptable excipients. Buffer exchange may be achieved by any method known in the art, including but not limited to dialysis or diafiltration. In particular embodiments, the buffer exchange is performed by tangential flow filtration. In particular for the LNP compositions described herein for use in medicine, buffer exchange allows the formulation of the LNPs in a pharmaceutically acceptable excipient e.g. buffered at physiological pH (~7.4) using a pharmaceutically acceptable buffer, optionally including further excipients such as described in further embodiments herein below.

[0370] In some embodiments, sterilisation is achieved by sterile filtration. Suitable filters for sterile filtration of an LNP composition are known to the skilled person in the art, in particular in the mRNA field. Typically, a PES filter may be used with 0.2 pm pore size.Other technologies suitable for preparing the LNP’s of the present invention include dispersing the components in a suitable dispersing medium, for example, aqueous solvent and organic solvent, and applying one or more of the following methods: ethanol dilution method, a simple hydration method, sonication, heating, vortex, an ether injecting method, a French press method, a cholic acid method, a Ca2+fusion method, a freeze-thaw method, a reversed-phase evaporation method, T-junction mixing, Microfluidic Hydrodynamic Focusing, Staggered Herringbone Mixing, and the like.

[0371] The lipid nanoparticle compositions described herein may be used in pharmaceutical formulations for medical applications, such as the in vivo, ex vivo or in vitro delivery of (ionizable) cargo compounds. This delivery may aid in preventing, inhibiting, or treating diseases, conditions, or traits in a cell, tissue, organ, subject, or organism.

[0372] As used herein, the terms “subject” and “patient” are generally interchangeable and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, and most preferably primates, including both human and non-human mammals. Human subjects are particularly preferred.

[0373] The terms “treat” or “treatment” encompass both therapeutic and prophylactic applications. Therapeutic treatment refers to managing an existing disease, disorder, addiction, or overdose, while prophylactic treatment aims to prevent or reduce the likelihood of developing such conditions. This includes preventing onset, progression, or worsening of a disease or disorder.

[0374] Prophylactic treatment includes vaccination, i.e. prophylactic treatment against an infectious agent. Vaccination may aim at preventing infection by said infectious agent, preventing (severe) disease symptoms upon infection by said infectious agent or reducing severity of disease symptoms upon infection by said infectious agent. Therapeutic treatment typically refers to interventions intended to alter a subject's physiological state from an undesired condition, such as a disease or disorder, towards a more favorable state. This may involve amelioration (symptom relief), palliation (reducing severity), stabilization (preventing worsening), or disease regression. A measurable improvement includes any statistically significant reduction in a relevant biomarker or symptom, where statistical significance is defined as a p-value below 0.05, a commonly accepted threshold in scientific analysis. Treatment may be curative, symptom-reducing, progression-slowing, or stabilizing.

[0375] A "therapeutic amount" or "therapeutically effective amount" refers to the quantity of a lipid nanoparticle composition, more particularly determined by the amount of thecargo compound present therein, sufficient to achieve a desired local or systemic effect in treating a disease or disorder. This amount elicits a biological or medicinal response in a tissue, system, animal, or human, as determined by a researcher, veterinarian, medical doctor, or other clinician. The appropriate dosage depends on the specific lipid nanoparticle composition (or the cargo compound present therein) and the severity of the condition and may be determined by a skilled professional through routine experimentation.

[0376] Accordingly, another aspect of the present invention provides the lipid nanoparticle composition according to an aspect of the invention for use in medicine.

[0377] The present invention further encompasses the use of the lipid nanoparticle composition according to an aspect of the invention in the manufacture of a medicament. More particularly, the application envisages methods of treatment wherein the lipid nanoparticle composition comprising a cargo compound as described herein is administered to a subject envisaged to be treated with said cargo compound.

[0378] Another aspect of the invention provides methods of administering a cargo compound to a subject, such as by transfecting a cell of said subject with a cargo compound, said method comprising providing said cargo compound in a lipid nanoparticle composition. For example, the lipid nanoparticle composition may carry, as a cargo compound, a nucleic acid molecule or construct that confers a medical benefit to the organism harboring the transfected cells. This nucleic acid may itself be therapeutically useful or encode an expression product with therapeutic applications.

[0379] In some embodiments, the lipid nanoparticle composition comprises a cargo compound which is or comprises a nucleic acid selected from the group consisting of self-amplifying RNA (saRNA), long-chain RNA, coding RNA, non-coding RNA, long non-coding RNA, single stranded RNA (ssRNA), double stranded RNA (dsRNA), linear RNA (linRNA), circular RNA (circRNA), messenger RNA (mRNA), Trans amplifying mRNA, RNA oligonucleotides, antisense oligonucleotides, small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR / Cas9 guide RNAs (gRNA), riboswitches, im-munostimulating RNA (isRNA), ribozymes, aptamers, ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA (vRNA), retroviral RNA or replicon RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), a Piwi-interacting RNA (piRNA), and mixtures thereof.

[0380] In some embodiments, these nucleic acids encode functional proteins, enzymes, or therapeutic proteins, such as antigens. For instance, when the nucleic acid encodes an antigen, the composition may be used to elicit a targeted immune response in asubject. The antigen may be associated with infectious diseases (e.g., viruses such as HIV, influenza, or SARS-CoV-2) or other pathological conditions.

[0381] In certain embodiments, the lipid nanoparticle composition comprises a nucleic acid or construct encoding an enzyme for use in vaccination, immunotherapy, protein replacement therapy, or gene-editing. In particular embodiments, the target cell is a mammalian cell, such as a human cell.

[0382] Alternatively, when the nucleic acid encodes a functional protein which the subject is otherwise deficient for or expresses a defective form of, the composition may be used for protein replacement therapy.

[0383] The present invention further encompasses a method for delivering a cargo compound to a cell, the method comprising administering a therapeutically effective amount of a lipid nanoparticle composition according to an aspect of the invention comprising the cargo compound to a subject in need thereof.

[0384] The present invention further encompasses the use of the lipid nanoparticle composition according to an aspect of the invention in the manufacture of a medicament. More particularly, the lipid nanoparticle composition is envisaged in combination with a cargo compound for use in the manufacture of a medicament for use in the treatment of a subject envisaged to obtain medical benefit from the administration of said cargo compound.

[0385] A further aspect of the present invention provides a pharmaceutical composition, comprising one or more lipid nanoparticle compositions as described herein and a pharmaceutically acceptable carrier, diluent or excipient. Such pharmaceutical compositions may be particularly suitable for drug delivery applications, including vaccines. In certain embodiments, the pharmaceutical composition is a vaccine or vaccine formulation.

[0386] The term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject. A carrier may be an organic or inorganic ingredient, either natural or synthetic, that facilitates administration by combining with the lipid nanoparticles and any optionalagents. The pharmaceutical composition mayfurther comprise one or more pharmaceutically acceptable ingredients, as known to the skilled person, including, but not limited to polymeric excipients, for instance chitosan derivatives or salts thereof; lipid excipients, such as phospholipids; surfactants; solvents; buffering agents, and the like. The components of the pharmaceutical compositions are commingled in a manner that precludes interaction that would substantially impair their desired pharmaceutical efficiency.The choice of carrier and additional ingredients depends on the intended mode of administration. When systemic delivery is required, the pharmaceutical composition may be formulated for parenteral administration (e.g., injection), including bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form. Pharmaceutical parenteral formulations may include aqueous solutions of the ingredients. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Alternatively, injection suspensions may be prepared as oil-based suspensions such as are known in the art or that will be readily apparent to those of ordinary skill in the art based on this disclosure.

[0387] In the context of the present invention, the term “vaccine” refers to any formulation intended to provide adaptive immunity (antibody and / or T cell response) against an antigen, e.g. to prevent or treat a disease, disorder, or addiction. Vaccines can be prophylactic (e.g., to prevent or ameliorate the effects of future abuse or overdose) or therapeutic (e.g, to actively treat or reduce the symptoms of an ongoing disease or disorder).

[0388] The lipid nanoparticle composition may be formulated as a pharmaceutical composition suitable for therapeutic use. This composition may further include pharmaceutically acceptable excipients, such as buffering agents, stabilizers, cryoprotectants, or tonicity-adjusting agents, depending on the intended route of administration.

[0389] The lipid nanoparticle composition is suitable for administration to a subject in need thereof via various routes. Any route of administration can be used including parenteral administration, such as intravenous, intramuscular, subcutaneous, or intradermal injection, or other routes such as oral, enteral, transdermal / transmucosal, intranasal, or inhalation delivery. The term enteral, as used herein, is a route of administration in which the agent is absorbed through the gastrointestinal tract or oral mucosa, including oral, rectal, and sublingual. The term parenteral, as used herein, describes administration routes that bypass the gastrointestinal tract, and are typically administered by injection or infusion, including intraarterial, intradermal, subdermal, intramuscular, intranasal, intraocular, intraperitoneal, intravenous, subcutaneous, submucosal, intravaginal, intrasternal, intracavernous, intrathecal, intrameatal, and intraurethral injection. The term transdermal / transmucosal, as used herein, is a route of administration in which the agent is administered through or by way of the skin, including topical. The term inhalation encompasses techniques of administration in which an agent is introduced into the pulmonary tree, including intrapulmonary or transpulmonary and includes intranasal administration. In a specific embodiment, the composition is administered via injection. In an alternative embodiment, the composition isadministered via aerosol or nebulization, particularly nebulisation or spraying in a mucosa lined cavity such as respiratory tract (nose, lungs,...) and female genital tract (e.g. vagina).

[0390] In a preferred embodiment, the composition is administered via injection, ensuring efficient delivery to the target tissues or cells. The LNP compositions may be delivered in liquid form, for instance with a dose size ranging from about 100 pL to 1 mL, such as about 100 pL, about 200 pL, about 300 pL, about 400 pL, about 500 pL, about 600 pL, about 700 pL, about 800 pL, about 900 pL, or about 1 mL (comprising an appropriate dose) for a 10-100 kg subject, although larger or smaller doses may be administered as necessary, depending on the patient's body weight, condition, and therapeutic need.

[0391] This LNP-based pharmaceutical composition provides a versatile and efficient delivery platform for compounds suitable for therapeutic and prophylactic applications in treating or preventing diseases such as genetic disorders, cancers, or infectious diseases.

[0392] Another aspect provides a method for delivering a cargo to a cell in vitro, such as for transfecting a cell in vitro, comprising contacting the cell with a lipid nanoparticle composition as disclosed herein, preferably wherein the cell is a mammalian cell.

[0393] EXAMPLES

[0394] The following examples are intended to illustrate the invention and are not to be construed as being limitations thereon.

[0395] A. Synthesis of exemplary compounds

[0396] Temperatures are given in degrees centigrade. If not mentioned otherwise, all evaporative concentrations are performed under reduced pressure, preferably between about 15 mm Hg and 100 mm Hg (= 20-133 mbar). The structure of final products, intermediates and starting materials is confirmed by standard analytical methods, e.g., microanalysis or spectroscopic characteristics, e.g., MS or NMR. Abbreviations used are those conventional in the art, some of which are defined below.

[0397] AbbreviationsACN Acetonitrile

[0398] AcOH Acetic acid

[0399] BOC tert-Butyloxycarbonyl

[0400] DCM Dichloromethane

[0401] DIPEA Di-isopropylethylamine

[0402] DMAP 4-Dimethylaminopyridine

[0403] DMP Dess-Martin periodinane

[0404] DIAD diisopropyl (E)-diazene-1 ,2-dicarboxylate

[0405] EDC 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride ESI Electrospray ionization

[0406] Et3N Triethylamine

[0407] EtOAc Ethyl acetate

[0408] HATU Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium MeOH Methanol

[0409] MS Mass spectrometry

[0410] NMR Nuclear magnetic resonance

[0411] TBAF Tetra-n-butylammonium fluoride

[0412] THF Tetrahydrofuran

[0413] TPSCl 2,4,6-Triisopropylbenzenesulfonyl chloride

[0414] Methodology

[0415] The following describes the materials and methods used for all examples unless otherwise stated.

[0416] Materials

[0417] All chemicals and solvents were used as obtained by their respective suppliers, unless otherwise noted. Ethylene glycol (99.5+%, TCI), DMAP (99+%, TCI), EDC.HCl (98+%, TCI), HATU (98+%, TCI), DMP (95+%, TCI), DABCO (98+%, TCI), 2-butyloctanoic acid (96%, Sigma-Aldrich), 1 ,4-butanediol (99+%, TCI), L-a-glycerylphosphorylcholine (98%, BLD pharma), Sodium periodate (99+%, Chem-Lab), 2-aminoethyl hydrogen phosphate (98+%, TCI), DIPEA (99.5+%, ThermoScientific), di-tert-butyl dicarbonate (95+%, TCI), Sodium cyanoborohydride (95+%, TCI), 2,4,6-triisopropylbenzenesulfonyl chloride (98%, BLD pharma), Triethylamine (99+%, Sigma-Aldrich), DIAD (98%, Sigma-Aldrich), Triphenylphosphane (95+%, TCI), Dry pyridine (99.5%, ThermoScientific), DOPE (99+%, Avanti), DMG-PEG (99+%, Avanti), Cholesterol (99+%, Avanti), C12-200 (98%, Corden Pharma), Acetone (99+%, AnalytiChem), dichloromethane (99.8+%, AnalytiChem), ethyl acetate (99.5+%, AnalytiChem), ethanol (99.8%, AnalytiChem), toluene (99.85%, AnalytiChem), chloroform (99.5+%, AnalytiChem), methanol (99.8+%, AnalytiChem), ammonia (7N in methanol, ThermoScientific) and heptane (99+%, AnalytiChem) were purchased from a commercial source. The millipore water (MILLI-Q IQ 7005 PURIFICATION SYSTEM) was used.All starting materials which are not explicitly described were either commercially available (the details of suppliers such as for example TCI, Fluorochem , Acros Organics, Sigma-Aldrich, ThermoFisher, Combi-Blocks, Enamine, MatrixScientific, Merck, Chempure, Angene, BLDpharm, Apollo Scientific, Nanjing, Avra, etc. can be found in the SciFinder® Database for example) or the synthesis thereof has already been described precisely in the specialist literature (experimental guidelines can be found in the Reaxys® Database or the SciFinder® Database respectively, for example) or can be prepared using the conventional methods known to the person skilled in the art.

[0418] Instrumentation

[0419] Chromatographic purification was performed using an automated flash chromatography NextGen 300+ system having ELSD and UV detectors utilizing commercially available normal phase Silica Flash Cartridges (12, 40 or 80 g) or reverse phase C18 column (30 g). The flow rate was selected according to size of the column. Thin-layer chromatography (TLC) analysis was performed using precoated TLC aluminium sheets (DC Kleselgel 60 F254) / UV254 (layer: 0.20 mm silica gel with fluorescent indicator UV254). Analysis of TLC plates was performed with UV wavelength at 254 nm.

[0420] Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Model Avance II 400 (Deimos) and Bruker Model Avance II 700 (Hera) Fourier transform NMR spectrometer in CDCl3at 303 K (unless stated otherwise). Samples were prepared using ca. 10-30 mg of compound dissolved in 0.6-1.0 mL of an appropriate deuterated solvent. All spectra were referenced to the solvent residual peak (e.g. 5 = 7.26 ppm for1H and 5 = 77.16 ppm for13C in CDCl3) orto the internal standard TMS. Chemical shifts (5) are reported in ppm; coupling constants (J) are reported in Hz; splitting patterns are assigned as singlet (s), broad singlet (br s), doublet (d), triplet (t), broad triplet (br t), quartet (q), quintet (quint), sextet (sext), septet (sept), multiplet (m) or combinations thereof.

[0421] Liquid chromatography-mass spectrometry-charged aerosol detection (LC-MS / CAD) analysis was performed on a 1260 Infinity II LC System from Agilent with Open LAB CDS ChemStation Edition equipped with a single-quadrupole MS Detector and a Thermo Scientific Charged Aerosol Detector (CAD). The LC-MS / CAD samples were prepared by dissolving 0.1-1 mg of the compound in an Acetonitriledsopropanol (1:1) mixture (ca. 0.5 mL-1.5 mL). The analyses were performed with reverse phase using a CSH Phenyl-Hexyl column (130A, 2.5 pm, 2.1 mm x 50 mm). Two types of elution methods were employed (hereafter called method 1 and method 2).

[0422] In method 1: The gradient was initiated with 50% A (0.1% formic acid in H2O), 12.5% B (0.1% formic acid in CH3CN) and 37.5% C (0.1% formic acid in Isopropanol) over 2min followed by a progressive increase to 25% B (0.1% formic acid in CH3CN) and 75% C (0.1 % formic acid in Isopropanol) over 25 min, followed by an isocratic gradient of 25% B (0.1% formic acid in CH3CN) and 75% C (0.1% formic acid in Isopropanol) over 2 min. The eluent system was then switched to the original solvent system consisting of 50% A (0.1% formic acid in H2O), 12.5% B (0.1% formic acid in CH3CN) and 37.5% C (0.1% formic acid in Isopropanol) over 0.1 min. This was followed by an equilibration process with an isocratic gradient of 50% A (0.1% formic acid in H2O), 12.5% B (0.1% formic acid in CH3CN) and 37.5% C (0.1% formic acid in Isopropanol) over 6 min This method 1 is performed with a flow rate of 0.350 mL / min, with a total run time of 33 min.

[0423] In method 2: The gradient was initiated with 100% A (0.1% formic acid in H2O) over 1 min followed by a progressive increase to 100% B (0.1% formic acid in CH3CN) over 10 min. This was followed by an isocratic gradient of 100% B (0.1% formic acid in CH3CN) over 1 min. The eluent system was then switched to the original solvent system consisting of 100% A (0.1% formic acid in H2O) over 10 sec. Next, an equilibration process was performed with an isocratic gradient of 100% A (0.1% formic acid in H2O) over 2 min 50 sec. This method 2 is performed with a flow rate of 0.400 mL / min, with a total run time of 15 min.

[0424]

[0425] is of

[0426] In a first step, a number of fatty acid-derived aldehyde-containing compounds (16- 4a, 16-4b) was prepared as intermediates of the present ionizable phospholipids (See Schemes 1 and 2).

[0427] q = 1 W-j = OH 16-2a q = 3 1 W-, = OH J16-2bif W^OH: EDC, DMAP, DCM if Wi=NH2: HATU, DIPEA, PCM DMP, DCM 16-4 Aik = 5-undecyl J 16-1a

[0428]

[0429] Scheme 1. Synthesis route towards intermediates 16-4. W2: NH or O; Aik: Branched or Linear Alkyl / Alkenyl Chains; q is 1, 2, 3, 4 or 5.if Y^OH: EDC, DMAP, DCM if Y1=NH2: HATU, DIPEA, DCM Alk^YlAlk^^'W™ DCM > Aik^^VyH o o o

[0430]

[0431] 16-5 16-7 16-8 16-9 Scheme 2: Synthesis route towards intermediates 16-9. Y. NH2or OH; Y2: NH or O; Aik: Branched or Linear Alkyl / Alkenyl Chains; q is 1, 2, 3, 4 or 5.

[0432] Synthesis of precursor 16-4a

[0433] Step 1

[0434] Under inert atmosphere (e.g., Ar), ethylene glycol 16-2a (3.0 equiv, 5.02 mL, 89.9 mmol) was dissolved in DCM (100 mL). To this solution were sequentially added 2-butyloctanoic acid 16-1a (1.0 equiv, 6.76 mL, 30.0 mmol), EDC.HCl (1.1 equiv, 6.32 g, 33.0 mmol), and DMAP (0.8 equiv, 2.93 g, 24.0 mmol). The resulting mixture was stirred overnight (18 hours) at room temperature (approx. 20°C). The reaction mixture was then washed twice with -100 mL of an aqueous solution of HCl (0.2M-0.5M), dried over anhydrous magnesium sulphate, filtered, and the filtrate was concentrated in vacuo. The residue was then purified by normal phase flash column chromatography on a NextGen 300+ system (gradient from 100% Heptane to 80% / 20% Heptane / EtOAc), yielding 2-hydroxyethyl 2-butyloctanoate 16-3a (5.31 g, 21.7 mmol, yield 73%) as a transparent oil.

[0435] MS (ESI) m / z found for C14H28O3Na [M+Na]+: 267.2

[0436] 1H NMR (400MHz, CDCU): 50.88 (3H, t, J = 6.9 Hz), 0.89 (3H, t, J = 7.1 Hz), 1.15-1.40 (12H, m), 1.40-1.54 (2H, m), 1.54-1.68 (2H, m), 1.90 (1 H, t, J = 6.1 Hz), 2.36 (1H, tt, J = 13.3, 5.4 Hz), 3.80-3.86 (2H, m), 4.20-4.25 (2H, m) ppm.

[0437] Step 2

[0438] Under inert atmosphere (e.g., Ar), 16-3a (1.0 equiv, 5.31 g, 21.7 mmol) was dissolved in DCM (100 mL). This solution was treated portion wise with Dess-Martin periodinane (1.2 equiv, 11.1 g, 26.1 mmol) and cooled to 0-10°C. The reaction mixture was subsequently allowed to warm to room temperature (20°C) and stirred for 4 hours. The reaction mixture was then treated with a saturated Na2S2O3solution (-100 mL).The aqueous layer was discarded, and the organic layer was washed twice with -100 mL of a saturated aqueous NaHCO3solution. The combined organic layers were dried over anhydrous magnesium sulphate, filtered, and the filtrate was concentrated in vacuo. Next, the crude residue was suspended in heptane and whitesolid was removed by filtration. The filtrate was collected, solvents were evaporated in vacuo and the obtained residue was purified by normal phase flash column chromatography on a NextGen 300+ system (gradient from 100% Heptane to 80% / 20% Heptane / EtOAc), yielding 2-oxoethyl 2-butyloctanoate 16-4a (5.71 g, 12.0 mmol, yield 54%) as a transparent oil.

[0439] MS (ESI) m / z found for C14H26O3Na [M+Na]+: 243.2

[0440] 1H NMR (400MHz, CDCl3): 50.88 (3H, t, J = 5.9 Hz), 0.89 (3H, t, J = 8.0 Hz), 1.16-1.38 (12H, m), 1.42-1.54 (2H, m), 1.57-1.84 (2H, m), 2.46 (1 H, tt, J = 13.2, 5.4 Hz), 4.65 (2H, d, J = 0.6 Hz), 9.61 (1 H, t, J = 0.7 Hz) ppm.

[0441] Synthesis of precursor 16-4b

[0442] The procedure for the preparation of intermediate 16-4a above was repeated, but using 1 ,4-butanediol instead. The synthesis procedure yielded 4-hydroxybutyl 2-butyloctanoate 16-3b (4.81 g, 17.7 mmol, yield 59%) as a transparent oil.

[0443] MS (ESI) m / z found for C16H32O3Na [M+Na]+: 295.2

[0444] 1H NMR (400MHz, CDCl3): 50.87 (3H, t, J = 6.9 Hz), 0.88 (3H, t, J = 7.1 Hz), 1.18-1.34 (12H, m), 1.37 (1H, t, J = 5.3 Hz) 1.39-1.50 (2H, m), 1.53-1.81 (6H, m), 2.30 (1H, tt, J = 13.4, 5.4 Hz), 3.69 (2H, q, J = 5.9 Hz), 4.11 (2H, t, J = 6.4 Hz) ppm.

[0445] After oxidation of 16-3b, 4-oxobutyl 2-butyloctanoate 16-4b (3.82 g, 14.1 mmol, yield 80%) as a transparent oil.

[0446] MS (ESI) m / z found for C16H30O3Na [M+Na]+: 293.2

[0447] 1H NMR (400MHz, CDCl3): 50.87 (3H, t, J = 6.9 Hz), 0.88 (3H, t, J = 7.1 Hz), 1.16-1.38 (12H, m), 1.38-1.51 (2H, m), 1.52-1.67 (2H, m), 1.93-2.03 (2H, m), 2.30 (1 H, tt, J = 13.3, 5.4 Hz), 2.54 (2H, td, J = 7.2, 1.3 Hz), 4.11 (2H, t, J = 6.4 Hz), 9.80 (1 H, t, J = 1.3 Hz) ppm.

[0448] Synthesis of phosphor-containing precursors

[0449] In a second step, a number of phosphor-containing intermediates (16-12a, 16-17a, 16-21a) was prepared using different synthesis procedures as outlined in Schemes 3 to 6 below.

[0450] i ° DABCO, DMF I □ nu NalO4, ethylene glycol, H2O - ~ 155 5 h OH O °C - rt, 18 h OH

[0451] 16-10 16-11 16-12m-.116-11 am-.116-12a

[0452]

[0453] o=T16-10ao=1 I o=1Scheme 3. Synthesis route towards intermediates 16-12. m and o are independently 1, 2, 3, 4 or 5.

[0454] X2= O, W< = NH2: 16-13

[0455] X2= O, W-j = OH: 16-14

[0456] X2= NH, W!' = NH2: 16-15 X2= NH, W1' = 0H: 16-16

[0457] 0 = 1 16-13a X2= 0, W1' = NH2

[0458] .w- Aik' ' OH

[0459] if W1' = OH: EDC, DMAP, DCM

[0460]

[0461] 16-T if W1' = NH2: HATU, DIPEA, DCM

[0462] Aik' = 5-undecyl J 16-1'a X2= O, W2' = NH, o = 1 16-17a Aik' = 5-undecyl

[0463]

[0464] Scheme 4. Synthesis route towards intermediates 16-17. W?’: NH2or OH; X2and W2NH or O; Aik’: Branched or Linear Alkyl / Alkenyl Chains; o is 1, 2, 3, 4 or 5.

[0465] BOC2O, Na2CO3, Water / Dioxane

[0466] 0 °C - rt

[0467] X2= O: 16-13

[0468] X2= NH: 16-15 BOC"NTWT'X2-'%H

[0469] OH

[0470] 16-21

[0471] X2= O, o = 1 ) 16-13a

[0472] X2= o, o = 1 } 16-21 a

[0473] POCI3, Et3N

[0474] DCM, 0 °C - rt

[0475]

[0476] 16-20 H30+

[0477] Scheme 5. Synthesis route towards intermediate 16-21. X: NH2or OH; X2: NH or O; o is 1, 2, 3, 4 or 5.

[0478] o

[0479] r6HOH

[0480] 16-18

[0481] if Y1'=OH: EDC, DMAP, DCM

[0482]

[0483] 16-5' if Y1'=NH2: HATU, DIPEA, DCM 16-19

[0484] Scheme 6. Synthesis route towards intermediate 16-19. Yi’: NH2orOH;X2and Y2are independently NH or O;Alk’: Branched or Linear Alkyl / Alkenyl Chains; o is 1, 2, 3, 4 or 5.

[0485] Synthesis of precursor 16-12a

[0486] Step 1Under inert atmosphere (e.g., Ar), (L-a-glycerylphosphorylcholine 16-10a (1.0 equiv, 2.00 g, 7.78 mmol) was dissolved in 100 mL DMF. DABCO (6.0 equiv, 5.23 g, 46.7 mmol) was then added to the solution. The reaction mixture was heated to reflux (155 °C) and was allowed to stir for 5 hours. The solvent was evaporated in vacuo, yielding (2R)-2,3-dihydroxypropyl 2-(dimethylamino)ethyl hydrogen phosphate 16-11a as a brown oil. The compound was used for the next step without further purification. Step 2

[0487] Under inert atmosphere (e.g., Ar), 16-11a (1.0 equiv, 2.00 g, 8.26 mmol) was dissolved in water (50 mL). The reaction was cooled to 0°C and sodium periodate (2.0 equiv, 3.53 g, 16.5 mmol) was added to the solution. The reaction mixture was allowed to gradually warm to room temperature and was stirred overnight. After 17 hours, ethylene glycol (1.35 equiv, 622 pL, 11.2 mmol) was added drop-wise and the mixture was stirred for 1 hour at room temperature. The solvent was evaporated in vacuo. The obtained residue was dissolved in EtOH (-50.0 mL), stirred for 2 hours and the white precipitate was filtered off. The filtrate was collected, the solvent was evaporated in vacuo and the residue was purified by normal phase flash column chromatography on a NextGen 300+ system (gradient from 100% EtOAc to 100% MeOH), yielding 2-(dimethylamino)ethyl 2-oxoethyl hydrogen phosphate 16-12a (102 mg, 483 pmol, yield over 2 steps 6.2%) as a yellow viscous liquid.

[0488] MS (ESI) m / z found for C6H15NO5P [M+H]+: 212.1

[0489] 1H NMR (400MHz, CD3OD): 52.92 (6H, s), 3.36-3.38 (2H, m), 3.74-3.81 (2H, m), 4.13-4.19 (2H, m), 4.68 (1 H, t, J = 4.95 Hz) ppm.

[0490] 31P NMR (400MHz, CD3OD): 50.104 (s) ppm.

[0491] Synthesis of precursor 16-17a

[0492] Under inert atmosphere (e.g., Ar), 2-butyloctanoic acid 16-1 ’a (1.00 equiv, 1.00 g, 4.99 mmol), 2-aminoethyl dihydrogen phosphate (1.1 equiv, 774 mg 5.49 mmol), and 2-(3H-[1 ,2, 3]triazo lo[4,5-b] pyrid in-3-y l)-1 ,1 ,3,3-tetramethylisouronium hexafluorophosphate(V) (HATU) (1.1 equiv, 2.09 g, 5.49 mmol) were dissolved in a mixture of DMF:water (40:10 mL). To the resulting solution, was added N-ethyl-N-isopropylpropan-2-amine (DIPEA) (2.0 equiv, 1.79 mL, 9.98 mmol) at room temperature (20°C). The reaction mixture was then stirred at room temperature for 16 hours under Ar atmosphere. Water (50 mL) and DCM (50 mL) were then added to the crude mixture. The aqueous phase was collected, was washed two times with DCM (-100 mL) and the aqueous phase then was concentrated in vacuo. The obtained residue was then purified by normal phase flash chromatography on a NextGen 300+ system (gradient from 100% DCM to 50 / 50 DCM / MeOH). The obtained DIPEA salt was then neutralized via an ion exchange methodology. The compound was dissolved inwater and this aqueous phase was washed with EtOAc and filtered through a 8 cm bed of Dowex 50W-X2 (H+form) resin (200-400 mesh). The beads were washed with water and the combined filtrates were evaporated in vacuo to 2-(2-butyloctanamido)ethyl dihydrogen phosphate 16-17a (2.50 g, 7.73 mmol, 60% yield) as a white solid.

[0493] MS (ESI) m / z found for C14H31NO5P [M+H]+: 324.2

[0494] 1H NMR (400MHz, CD3OD): 50.78-0.81 (6H, m), 1.12-1.23 (12H, br s), 1.25-1.33 (2H, m), 1.41-1.50 (2H, m), 2.08 (1 H, sept, J = 4.8 Hz), 3.34 (2H, t, J = 5.7 Hz), 3.90 (2H, dd, J = 7.2, 5.7 Hz,) ppm.

[0495] 31P NMR (165MHz, CDCl3): 50.97 ppm.

[0496] Synthesis of precursor 16-21 a

[0497] Under inert atmosphere (e.g., Ar), 2-aminoethyl dihydrogen phosphate 16-13a (1.0 equiv, 2.00 g, 14.2 mmol) was dissolved in water and 1,4-Dioxane (7.5 mL:15 ml). Sodium carbonate (1.0 equiv, 4.06 g, 14.2 mmol) was added to the reaction mixture and the reaction mixture was cooled to 0 °C. Next, a solution of di-tert-butyl dicarbonate (1.1 equiv, 3.40 g, 15.6 mmol) in 100% 1,4-dioxane (7.5 mL) was added dropwise at 0 °C so that the ratio water / 1 ,4-Dioxane equals 1 / 1 in the reaction mixture. If the reaction mixture remains turbid, additional water is then added until a complete dissolution of all reagents is observed. The reaction mixture was allowed to heat to room temperature (20°C) and was stirred for 18 hours. The solvents were evaporated in vacuo. The residue was then purified by normal phase flash chromatography on a NextGen 300+ system (gradient from 100% Isopropanol to 100% Water), yielding tert-butyl (2-(phosphonooxy)ethyl)carbamate 16-21 a (2.54 g, 10.5 mmol, yield 70%) as a white solid.

[0498] MS (ESI) m / z found for C7H15NO6P [M-H]’ :239.8

[0499] 1H NMR (400MHz, D2O): 51.36 (9H, s), 3.21 (2H, t, J = 5.4 Hz), 3.75 (2H, dd, J = 11.7, 5.5 Hz) ppm.

[0500] 31P NMR (124MHz, D2O): 51.89 ppm.

[0501] Synthesis of ionizable phospholipids

[0502] In a next step, the intermediates described above were used to prepare the ionizable (phosphor)lipid compounds as described herein. Said compounds were prepared using different synthesis procedures as outlined in Schemes 7-12 below.16-4

[0503] W2= O, q = 3 Aik = 5-undecyl 16-22nr =1 }l6-22a AcOH, NaBH3CN, MeOH

[0504] 16-27 q = 3, n = 1 , r = 1 q = 3, n = 1 W2= O M6-23a W2= O Aik = 5-undecyl Aik = 5-undecyl Formic acid Formic acid

[0505] Aik 16-24 16-28 q = 3, n = 1 , r = 1 q = 3, n = 1 W2= O I 16-24a W2= O 16-28a Aik = 5-undecyl Aik = 5-undecyl o 16-12 16-12Hm = 1, o = 1 | 16-12a m = 1, o = 1 J 16-12a AcOH, NaBH3CN, MeOH AcOH, NaBH3CN, MeOH

[0506] 16-25 16-29 q = 3, m,n = 1, o = 1, r = 1 q = 3, m,n = 1 , o = 1

[0507] W2= O

[0508]

[0509] Aik = 5-undecyl Aik = 5-undecyl Scheme 7. Synthesis route towards phospholipids 16-25 and 16-29. W2: NH or O;Alk:

[0510] Branched or Linear Alkyl / Alkenyl Chains ; q, m, n and o are independently 1, 2, 3, 4 or 5 ; r is selected from Ci.9-alkylene.H2N

[0511] 16-37 16-41 q = 3, m, n = 1, W2= O ■ 16-41 a Aik = 5-undecyl

[0512] BOC"N'KOX2" P'OH TPSCI, Pyridine TPSCI, Pyridine Boc'N'WjXX2" |>"OH OH 40 °C, 18h OH 40 °C, 18h 16-21 OR 16-21 OR HATU, PyridineX =HATU, Pyridine 60 °C, 18h 2 ° 1 i6-21a 0 = 1 60 °C, 18h

[0513] 16-42 q = 3, m, n = 1, o=1, X2and W2= O ■ 16-42a Aik = 5-undecyl 4N HCI in Dioxane 4N HCI in Dioxane

[0514] M^NHj

[0515] 16-43 q = 3, m,n = 1, o=1, X2and W2= O ■ 16-43a

[0516]

[0517] Aik = 5-undecyl Scheme 8. Synthesis route towards phospholipids 16-39 and 16-43. W2andX2: NH or O;Alk: Branched or Linear Alkyl / Alkenyl Chains ; q, m, n and o are independently 1, 2, 3, 4 or 5 ; r is selected from Ci.9-alkylene.W2' = O, q' = 1 16-4'a Aik' = 5-undecyl W2' = O, q' = 3 16-4'b Aik' = 5-undecyl X! = O : 16-39 X! = O : 16-43 X-j = NH : 16-48 X-j = NH : 16-53 q = 3, m,n = 1 , o = 1 1 X-j, X2, and W2= O H6-43a Aik = 5-undecyl J AcOH, NaBH3CN, MeOH AcOH, NaBH3CN, MeOH

[0518] 16-68 16-70

[0519] When W2 - W21I 16-70-1 and Aik = Aik' | q = 3, q' = 3; m, n = 1 , o = 1 I XrX2, W2and W2' = O 16-70-l-a Aik, Aik' = 5-undecyl J q = 3, q' = 1 ; m,n = 1, o = 1 1 X1(X2, W2and W2' = O L 16-70-l-b Aik, Aik' = 5-undecyl

[0520] 16-69 16-71 q =3, q' = 3; m,n = 1, o = 1 | XL X2, W2and W2' = O 16-71a Aik and Aik' = 5-undecyl J q =3, q' = 1 ; m,n = 1 , o = 1 ? X1, X2, W2and W2' = O l“l6-71b Aik and Aik' = 5-undecyl Scheme 9. Synthesis route towards phospholipids 16-68, 16-69, 16-70, 16-70-1, and 16-71. 1 / 14, Xi, X2and W2’are independently NH or O ; Aik and Aik’ are independently Branched or Linear Alkyl / Alkenyl Chains ; q, q’, m, n and o are independently 1, 2, 3, 4

[0521]

[0522] or 5 ; ris selected from C1-9-alkylene.- — ^'F^^WAAI^ - 0 16-17 X2= O, W2' = NH, o = 1]16 17aAik' = 5-undecyl J X = OH: 16-37 X = OH: 16-41 X = NH2: 16-46 X = NH2: 16-51 q = 3; m,n = 1 W2= O X = OH Aik = 5-undecyl If X = OH: TPSCI, Pyridine, 40 °C, 18 h If X = OH: TPSCI, Pyridine, 40 °C, 18 h If X = NH2: DIAD, PPh3, DBU, CHCI3, 0 °C - rt If X = NH2: DIAD, PPh3, DBU, CHCI3, 0 °C - rt

[0523] 16-76 16-77 q = 3; m,n = 1 ; o = 1 , ? Xi, X2, w2= o I W2' = NH |16'77aAik and Aik' = 5-undecyl Scheme 10. Synthesis route towards phospholipids 16-76 and 16-77. W2, Xi, X2and are independently NH or O ; Xi is NH2or OH ; Aik and Aik’ are independently Branched or Linear Alkyl / Alkenyl Chains ; q, m, n and o are independently 1, 2, 3, 4 or 5

[0524]

[0525] ; r is selected from Ci.9-alkylene.H2N 16-40 m,n = 1 | 16-40a AcOH, NaBH3CN, MeOH AcOH, NaBH3CN, MeOH

[0526] 16-85

[0527] 2BocxVCX^OH OH TPSCI, Pyridine TPSCI, Pyridine 16-21 40°C, 18h 16-21 40°C, 18h x2= 0=1

[0528] 4N HCI in Dioxane 4N HCI in Dioxane OH

[0529] M°'FrXWNH2o

[0530] 16-87 o O X u Alk'^ H AcOH, NaBH3CN, MeOH Alk'^ H AcOH, NaBH3CN, MeOH 16-84' 16-84'

[0531] N P 'H*2(-')C' N ^'''Al k' Alk^N^W^ ° ^Alk'AlkxJ16-92 When Aik = Aik' } 16-92-1 Aik 16-88

[0532] m,n = 1 ; o = 1 , X2= O Aik and Aik' = 16-92-l-a

[0533]

[0534] Scheme 11. Synthesis route towards phospholipids 16-88 and 16-92. X2is NH or O ;

[0535] Aik and Aik’ are independently Branched or Linear Alkyl / Alkenyl Chains ; m, n and o are independently 1, 2, 3, 4 or 5 ; ris selected from C1.9- alkylene.o Alk'^W2Wc|^0H

[0536] 16-4 W2= O, q = 31 Aik = 5-undecyl J 16-4b

[0537] AcOH, NaBH3CN, MeOH X2= O: 16-13 X2= O, o = 1 } 16-13a

[0538] W2' Alk' O X2= O: 16-108 X2= NH: 16-109 q' = 3; o = 1 'I W2' = O I X = OH: 16-37 X = OH: 16-41 X = NH2: 16-46 X2= O | 16-108a X = NH2: 16-51

[0539] Aik = 5-undecyl q = 3; m,n = 1 W2= O x = NH2Aik = 5-undecyl if X=NH2if X=NH DIAD, PPh3, DBU, CHCI3, 0 °C - rt2

[0540] DIAD, PPh3, DBU, CHCI3, 0 °C - rt

[0541] 16-68 16-70 When W2= W2' I 16-70-1 and Aik = Aik' | q = 3, q' = 3; m, n = 1 , o = 1 X2, W2and W2' = O X! = NH 16-70-1 -c

[0542]

[0543] Aik, Aik' = 5-undecyl Scheme 12. Alternative synthesis route towards phospholipids 16-68 and 16-70. W2, Xi, X2and W2are independently NH or O ; X is NH2or OH ; Aik and Aik’ are independently Branched or Linear Alkyl / Alkenyl Chains ; q, m, n and o are independently 1 , 2, 3, 4 or 5 ; r is selected from Ci.9-alkylene.o 16-9 AcOH, NaBH3CN, MeOH X2= O: 16-13 X2= O, o = 1 } 16-13a X2= O: 16-110 X2= NH: 16-111 X = OH: 16-54 X = OH: 16-57 X = NH2: 16-61 X= NH2: 16-65 if X=NH2if X=NH2DIAD, PPh3, DBU, CHCI3, 0 °C - rt DIAD, PPh3, DBU, CHCI3, 0 °C - rt

[0544]

[0545] Scheme 13. Alternative synthesis route towards phospholipids 16-72 and 16-74. Y2, X2and Y2are independently NH or O ;Xis NH2or OH ; Aik and Aik’ are independently Branched or Linear Alkyl / Alkenyl Chains ; q, q’, m, n and o are independently 1, 2, 3, 4 or 5 ; ris selected from C1-9-alkylene.

[0546] By way of illustration, alternative synthesis routes for preparing the ionizable (phosphor)lipid compoundsaccordingtotheinvention are presented in Schemes 14-23 below.16-30 16-33 Formic acid Formic acid

[0547] Aik o 16-31 16-34

[0548] 16-12 16-12 AcOH, NaBH3CN, MeOH AcOH, NaBH3CN, MeOH

[0549] Aik

[0550]

[0551] 16-32 16-35 Scheme 9. Synthesis route towards phospholipids 16-32 and 16-35. Y2: NH or O;Alk:

[0552] Branched or Linear Alkyl / Alkenyl Chains ; q, m, n and o are independently 1, 2, 3, 4 or 5 ; r is selected from Ci.9-alkylene.o

[0553] 'Boc

[0554] 16-44 16-49

[0555] AcOH, NaBH3CN, MeOH AcOH, NaBH3CN, MeOH

[0556] 16-45 16-50

[0557] 16-51

[0558] N P DIAD, PP N P DIAD, PPh Boo" X-)o X2" I'OH h3Boe"3OH DBU, CHCI3V)rx2VOH DBU, CHCI316-21 0 °C - rt 16-21 0 °C - rt

[0559] 16-47 16-52

[0560] 4N HCI in Dioxane 4N HCI in Dioxane

[0561] Aik

[0562]

[0563] 16-48 16-53Scheme 10. Synthesis route towards phospholipids 16-48 and 16-53 ; W2and X2are independently NH or O ; Aik is a Branched or Linear Alkyl / Alkenyl Chain ; q, m, n and o are independently 1, 2, 3, 4 or 5 ; r is selected from Ci.9-alkylene.

[0564] 16-36

[0565] AcOH, NaBH3CN, MeOH

[0566] TPSCI, Pyridine TPSCI, Pyridine 40 °C, 18h 40 °C, 18h N ZK P

[0567] Boe' Ho X2' i OH OR OH OR HATU, Pyridine 16-21 HATU, Pyridine 60 °C, 18h 60 °C, 18h

[0568] Aik 16-55 16-58 4N HCI in Dioxane 4N HCI in Dioxane OH

[0569] Aik

[0570]

[0571] 16-56 16-59 Scheme 11. Synthesis route towards phospholipids 16-56 and 16-59. Y2and X2: NH or O; Aik: Branched or Linear Alkyl / Alkenyl Chains ; q, m, n and o are independently 1, 2, 3, 4or5;r is select

[0572]

[0573] ed from C^-alkylene.H2N 16-9 16-44

[0574] AcOH, NaBH3CN, MeOH

[0575] 16-60 16-64 Formic acid

[0576] 16-65 DIAD, PPh3DIAD, PPh3N _ p N P Boc' DBU, CHCI3Boc' "Wo X2' i''OH DBU, CHCI3

[0577] 0 °C - rt OH 0 °C - rt 16-21 16-21

[0578] N' HBOC

[0579] o 16-62 16-66 4N HCI in Dioxane 4N HCI in Dioxane

[0580] (“CNH2

[0581]

[0582] 16-63 16-67 Scheme 12. Synthesis route towards phospholipids 16-63 and 16-67 ; Y2and X2are independently NH or O ; Aik is a Branched or Linear Alkyl / Alkenyl Chain ; q, m, n and o are independently 1, 2, 3, 4 or 5 ; r is selected from Ci.9-alkylene.x0HV 16-9'

[0583] X! = O : 16-56 X! = O : 16-59 X! = NH : 16-63 X-j = NH : 16-67 AcOH, NaBH3CN, MeOH AcOH, NaBH3CN, MeOH

[0584] 16-74 When Y2= Y2' 116 74and Aik = Aik' J

[0585]

[0586] 16-73 16-75 Scheme 18. Synthesis route towards phospholipids 16-72, 16-73, 16-74 and 16-75. Y2, Xi, X2and Y2are independently NH or O ; Aik and Aik’ are independently Branched or Linear Alkyl / Alkenyl Chains ;q, m, m’ n and o are independently 1, 2, 3, 4 or5;r is selected from Ci.9-alkylene.

[0587] 16-17

[0588] X = OH: 16-54 X = OH: 16-57 X = NH2: 16-61 X = NH2: 16-65 If X = OH: TPSCI, Pyridine, 40 °C, 18 h If X = OH: TPSCI, Pyridine, 40 °C, 18 h If X = NH2: DIAD, PPh3, DBU, CHCI3, 0 °C - rt If X = NH2: DIAD, PPh3, DBU, CHCI3, 0 °C - rt

[0589] o

[0590]

[0591] 16-78 16-79Scheme 19. Synthesis route towards phospholipids 16-78 and 16-79. Y2, Xi, X2and are independently NH or O ; X is NH2or OH ; Aik and Aik’ are independently Branched or Linear Alkyl / Alkenyl Chains ; q, m, n and o are independently 1, 2, 3, 4 or 5 ; r is selected from Ci.9-alkylene.

[0592] X = OH: 16-37 X = OH: 16-41 X = NH2: 16-46 X = NH2: 16-51 If X = OH: TPSCI, Pyridine, 40 °C, 18 h If X = OH: TPSCI, Pyridine, 40 °C, 18 h If X = NH2: DIAD, PPh3, DBU, CHCI3, 0 °C - rt If X = NH2: DIAD, PPh3, DBU, CHCI3, 0 °C - rt

[0593] 16-80 16-81 Scheme 20. : Synthesis route towards phospholipids 16-80 and 16-81. \N2, Xi, X2and Y2are independently NH or O ; X is NH2or OH ; Aik and Aik’ are independently Branched or Linear Alkyl / Alkenyl Chains ; q, m, n and o are independently 1, 2, 3, 4 or 5 ; r is selected from C^-alkylene.

[0594] 16-19

[0595] Aik X = OH: 16-54 X = OH: 16-57 X = NH2: 16-61 X= NH2: 16-65

[0596] If X = OH: TPSCI, Pyridine, 40 °C, 18 h If X = OH: TPSCI, Pyridine, 40 °C, 18 h If X = NH2: DIAD, PPh3, DBU, CHCI3, 0 °C - rt If X = NH2: DIAD, PPh3, DBU, CHCI3, 0 °C - rt

[0597] X OH „ O

[0598] Aik, o

[0599] Aik o

[0600]

[0601] 16-82 16-83Scheme 21. Synthesis route towards phospholipids 16-82 and 16-83. Y2, Xi, X2and Y2are independently NH or O ; X is NH2or OH ; Aik and Aik’ are independently Branched or Linear Alkyl / Alkenyl Chains ; q, m, n and o are independently 1, 2, 3, 4 or 5 ;ris selected from C1-9-alkylene.

[0602] o Alk^H Boc 16-84

[0603] 16-49 AcOH, NaBH3CN, MeOH

[0604] 16-93 16-98

[0605] Formic acid Formic acid

[0606] 16-94 16-99 N P N P

[0607] Boe" V)oXQHOH DIAD, PPh3DIAD, PPh3

[0608] DB Boc" V)oXQHOHU, CHCI3DBU, CHCI30 °C - rt 0 °C - rt 16-21 16-21 H OH

[0609] ,NJ „X2,Boc N Mm P2MO N H OH J 6HMBOCH

[0610] Alk^N^V)nN''^ ° Alk^N^

[0611] 16-95 16-100 Aik 4N HCI in Dioxane 4N HCI in Dioxane H OH

[0612] N ^'MmNPX2rt? NH2H OH , NJ ,X, Mm P2'Mo NH2Alk^N^H'? ° o(~l)rAlk^,N^

[0613] Alk16-96 16-101 o o AcOH, NaBH3CN, MeOH AcOH, NaBH3CN, MeOH Alk'^H Alk'^H 16-84' 16-84'

[0614] H OH M I X; MC'N'^'Alk' N o Alk' Alk N Hn Alk

[0615]

[0616] 16-97 16-102

[0617] When Alk = Alk'} 16-102-1Scheme 22. Synthesis route towards phospholipids 16-97 and 16-102. X2is NH or O ;

[0618] Alk and Alk’ are independently Branched or Linear Alkyl / Alkenyl Chains ; n and p are independently 1, 2, 3, 4 or 5 ; ris selected from C1.9- alkylene.

[0619] X2= O: 16-14

[0620] If X = OH: TPSCI, Pyridine, 40 °C, 18 h

[0621] If X = NH2: DIAD, DBU, PPh3, CHCI3

[0622] X = O: 16-85 X = O: 16-89

[0623] X = NH2: 16-94 If X = OH: TPSCI, Pyridine, 40 °C, 18 h X = NH2: 16-99

[0624] If X = NH2: DIAD, DBU, PPh3, CHCI3

[0625] 16-103 16-106

[0626] Alk'Br

[0627] 16-104

[0628]

[0629] 16-105 16-107 Scheme 23. Synthesis route towards phospholipids 16-105 and 16-107. Xi andX2are independently NH orO;Xis NH2or OH ; Alk and Alk’ are independently Branched or Linear Alkyl / Alkenyl Chains ; m, n and o are independently 1 , 2, 3, 4 or 5 ; r is selected from C1-9- alkylene.

[0630] Synthesis of compound 16-25a

[0631] Step 1

[0632] Under inert atmosphere (e.g., Ar), 4-oxobutyl2-butyloctanoate 16-4b (3.4 equiv, 1.87 g, 6.92 mmol) was dissolved in MeOH (1000 mL). AcOH (18.0 equiv, 2.09 mL, 36.6 mmol, 0.21 vol%) was then added to the solution, followed by tert-butyl 4-(2-((2-aminoethyl)amino)ethyl)piperazine-1-carboxylate 16-22a (1.0 equiv, 554 mg, 2.03 mmol). After one minute, sodium cyanoborohydride (5.0 equiv, 639 mg, 10.2 mmol) was added and the mixture was then allowed to stir at room temperature (20°C) overnight (18 hours). The solvents were evaporated in vacuo. The residue was then purified by normal phase flash chromatography on a NextGen 300+ system (gradient from 100% DCM to 95% / 3.5% / 1.5% DCM / MeOH / NH3 7N in MeOH), yielding intermediate tert-butyl4-(2-((2-(bis(4-((2-butyloctanoyl)oxy)butyl)amino)ethyl)(4-((2-butyloctanoyl)oxy)butyl)amino)ethyl)piperazine-1 -carboxylate 16-23a (1.08 g, 1.04 mmol, yield 51 %) as a brown oil.MS (ESI) m / z found for C6iHi2oN403[M+2H]2+: 518.7

[0633] 1H NMR (300MHz, CDCl3): 50.88 (9H, t, J = 6.69 Hz), 0.88 (9H, t, J = 7.01 Hz), 1.13-1.37 (38H, m), 1.46 (15H, t, J = 6.00 Hz), 1.49-1.75 (17H, m), 2.25-2.38 (3H, m), 2.50-3.11 (18H, m), 3.51 (3H, s), 4.11 (6H, t, J = 5.75 Hz) ppm.

[0634] Step 2

[0635] Under inert atmosphere (e.g., Ar), the BOC-protected intermediate 16-23a (1.0 equiv, 1.08 g, 1.04 mmol) was dissolved in formic acid (15.0 ml). The solution was stirred at room temperature (20°C) overnight (18 hours). The formic acid was then evaporated in vacuo. The residue was washed with a saturated aqueous NaHCO3solution (50 mL) and the aqueous layer was extracted three times with DCM (100 mL). The organic layers were combined and washed with brine while the aqueous layer was extracted three times with DCM (100 mL). The four combined organic layers were dried on anhydrous magnesium sulphate, filtered, and concentrated in vacuo. The residue was then purified by normal phase flash chromatography on a NextGen 300+ system (gradient from 100% DCM to 95% / 3.5% / 1.5% DCM / MeOH / NH37N in MeOH), yielding ((2-((4-((2-butyloctanoyl)oxy)butyl)(2-(piperazin-1-yl)ethyl)amino)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate) 16-24a (527.0 mg, 564 pmol, yield 54%) as a transparent oil.

[0636] MS (ESI) m / z calculated for C56H110N4O6[M+2H]2+: 468.6

[0637] 1H NMR (400MHz, CDCU): 50.88 (18H, dt, J = 6.82 Hz), 1.06-1.36 (36H, m), 1.36-1.55 (14H, m), 1.55-1.69 (1 OH, m), 2.19-2.38 (4H, m), 2.38-2.78 (18H, m), 2.92 (4H, t, J = 4.86 Hz), 4.07 (6H, t, J = 6.56 Hz).

[0638] 13C NMR (101 MHz, CDCU): 514.2, 22.7, 23.8, 26.9, 27.6, 29.4, 29.8, 31.8, 32.3, 32.6, 45.9, 51.9, 52.6, 53.2, 54.2, 54.7, 54.8, 57.4, 64.1 , 176.8.

[0639] Step 3

[0640] Under inert atmosphere (e.g., Ar), 16-12a (1.68 equiv, 94.8 mg, 0.449 mmol) was dissolved in MeOH. AcOH (7.5 equiv, 115 pL, 2.00 mmol, 0.17 vol%) was then added to the solution, followed by intermediate 16-24a (1.0 equiv, 250 mg, 0.267 mmol). After one minute, sodium cyanoborohydride (3.0 equiv, 50.4 mg, 0.802 mmol) was added and the solution was then allowed to stir at room temperature overnight. The solvents were evaporated in vacuo. The residue was then purified by reverse phase flash column chromatography on a NextGen 300+ system (gradient from 100% / 0.1 % Water / Formic Acid to 100% / 0.1% ACN / Formic Acid). The purified product was dissolved in DCM (~20 mL) and washed with a 0.1 M NaHCO3(10.0 equiv) aqueous solution. The organic layer was dried over magnesium sulphate, filtered, and the solvent was removed in vacuo yielding ((2-((4-((2-butyloctanoyl)oxy)butyl)(2-(4-(6-hydroxy-2-methyl-6-oxo-5,7-dioxa-2-aza-6A5-phosphanonan-9-yl)piperazin-1-yl)ethyl)amino)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate) 16-25a (61.0 mg, 0.054 mmol, yield 20%) as a light yellow viscous liquid.

[0641] MS (ESI) m / z found for CezH^NsO P [M+2H]2+: 566.1

[0642] 1H NMR (400MHz, CDCl3): 50.80 (9H, t, J = 7.04 Hz), 0.80 (9H, t, J = 6.84 Hz), 1.12-1.26 (38H, m), 1.33-1.44 (12H, m), 1.47-1.58 (12H, m), 2.23 (3H, septet, J = 4.73 Hz), 2.36-2.42 (8H, m), 2.45-2.53 (8H, m), 2.58-2.65 (11 H, m), 2.98 (2H, br s), 3.98^1.01 (8H, m), 4.11 (2H, br s), 5.27 (2H, br s) ppm.

[0643] 13C NMR (101MHz, CDCU): 5 14.1, 14.2, 22.69, 22.72, 23.6, 23.7, 26.7, 26.8, 27.5, 29.3, 29.7, 31.8, 32.3, 32.6, 44.5, 45.8, 51.9, 52.4, 52.9, 53.2, 53.3, 54.1 , 54.6, 56.6, 58.4, 58.8, 60.8, 62.6, 64.0, 176.8 ppm.

[0644] 31P NMR (162MHz, CDCl3): 5 -0.27 ppm.

[0645] Synthesis of compound 16-29a

[0646] The procedure for the preparation of compound 16-25a above was repeated, but using tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate 16-26a instead. The synthesis procedure yielded tert-butyl 4-(2-(bis(4-((2-butyloctanoyl)oxy)butyl)amino)ethyl)piperazine-1 -carboxylate 16-27a (1.04 g, 1.41 mmol, yield 88%) as a transparent oil.

[0647] MS (ESI) m / z found for C43H84N3O6[M+H]+: 738.6

[0648] 1H NMR (400MHz, CDCl3): 50.88 (6H, t, J = 6.90 Hz), 0.89 (6H, t, J = 7.10 Hz), 1.17-1.36 (24H, m), 1.41-1.50 (13H, m), 1.52-1.63 (4H, m), 1.73 (8H, br s), 2.32 (2H, tt, J = 13.1, 5.4 Hz), 2.50 (4H, t, J = 4.8 Hz), 2.73 (2H, t, J = 5.3 Hz), 3.03 (6H, br s), 3.47 (4H, t, J = 4.8 Hz), 4.11 (4H, t, J = 5.9 Hz) ppm.

[0649] Next, the Boc protecting group was removed from 16-27a (1.0 equiv, 1.0355 g, 0.664 mmol) to afford ((2-(piperazin-1-yl)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate) 16-28a (150 mg, 0.235 mmol, yield 36%) as a transparent oil.

[0650] MS (ESI) m / z found for C38H77N3O4[M+2H]2+: 319.9

[0651] 1H NMR (700MHz, CDCl3): 50.84 (6H, t, J = 7.0 Hz), 0.85 (6H, t, J = 7.2 Hz), 1.15-1.31 (24H, m), 1.36-1.43 (4H, m), 1.43-1.47 (4H, m), 1.51-1.62 (8H, m), 1.94 (1 H, br s), 2.27 (2H, tt, J = 13.5, 5.3 Hz), 2.34-2.45 (1 OH, m), 2.51-2.56 (2H, m), 2.85 (4H, t, J = 4.8 Hz), 4.03 (4H, t, 7 = 6.6 Hz) ppm.

[0652] 13C NMR (176MHz, CDCl3): 513.9, 14.0, 22.5, 22.6, 23.7, 26.7, 27.4, 29.2, 29.6, 31.6, 32.2, 32.5, 45.7, 45.9, 51.2, 54.1 , 55.0, 57.3, 63.9, 176.6 ppm.After deprotecting the N-H group of 16-28a (1.0 equiv, 200.0 mg, 313.0 mmol), the phosphor-containing intermediate 16-12a was attached,yielding((2-(4-(6-hydroxy-2-methyl-6-oxo-5,7-dioxa-2-aza-6A5-phosphanonan-9-yl)piperazin-1-yl)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate) 16-29a (83.0 mg, 99.6 pmol, yield 32%) as a light yellow viscous liquid.

[0653] MS (ESI) m / z found for C44H91N4O8P [M+2H]2+:417.5

[0654] 1H NMR (400MHz, CDCU): 50.80 (12H, distorted t, J = 6.85 Hz), 1.18-1.23 (26H, m), 1.34-1.44 (8H, m), 1.47-1.58 (8H, m), 2.23 (2H, septet, J = 4.7 Hz), 2.38 (6H, distorted t, J = 6.7 Hz), 2.48-2.52 (6H, m), 2.60 (9H, br s), 2.95 (2H, br s), 3.96-4.01 (6H, m), 4.08 (2H, brs), 6.39 (2H, brs) ppm.

[0655] 13C NMR (101MHz, CDCU): 5 14.0, 14.1, 22.60, 22.62, 23.5, 26.7, 27.4, 29.2, 29.6, 31.7, 32.2, 32.5, 44.5, 45.8, 51.4, 53.2, 53.9, 56.4, 58.4, 58.7, 61.0, 62.5, 63.9, 176.7 ppm.

[0656] 31P NMR (162MHz, CDCU): 5 -1.04 ppm.

[0657] Synthesis of compound 16-43a

[0658] Step 1

[0659] Under inert atmosphere (e.g., Ar), 4-oxobutyl2-butyloctanoate 16-4b (2.5 equiv, 2.00 g, 7.40 mmol) was dissolved in MeOH (1.07 L). AcOH (15.0 equiv, 2.54 mL, 44.38 mmol, 0.36 vol%) was then added to the solution, followed by 2-(4-(2-aminoethyl)piperazin-1-yl)ethan-1-ol 16-40a (1.0 equiv, 512.6 mg, 2.96 mmol). After one minute, sodium cyanoborohydride (4.0 equiv, 743 mg, 11.83 mmol) was added and the solution was then allowed to stir at room temperature (20°C) overnight (18 hours). The solvents were evaporated in vacuo. The residue was then purified by normal phase flash chromatography on a NextGen 300+ system (gradient from 100% DCM to 95% / 3.5% / 1.5% DCM / MeOH / NH37N in MeOH), yielding ((2-(4-(2-hydroxyethyl)piperazin-1-yl)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate) 16-41a (1.65 g, 2.41 mmol, yield 82%) as a transparent oil.

[0660] MS (ESI) m / z found for C4oH81N305[M+2H]2+: 341.7

[0661] 1H NMR (400MHz, CDCU): 50.87 (6H, t, J = 6.9 Hz), 0.88 (6H, t, J = 7.1 Hz), 1.16-1.37 (27H, m), 1.37-1.54 (8H, m), 1.54-1.68 (8H, m), 2.30 (2H, tt, J = 13.4, 5.34 Hz), 2.38-2.69 (16H, m), 3.60 (2H, t, J = 5.38 Hz), 4.07 (4H, t, J = 6.6 Hz) ppm.

[0662] 13C NMR (101MHz, CDCU): 5 14.0, 14.1, 22.60, 22.63, 23.7, 26.7, 27.4, 29.2, 29.7, 31.7, 32.2, 32.5, 45.8, 51.4, 52.8, 53.7, 54.1 , 56.7, 57.7, 59.2, 64.9, 176.7 ppm.

[0663] Step 2Under inert atmosphere (e.g., Ar), the intermediate tert-butyl (2-(phosphonooxy)ethyl)carbamate 16-21 a (2.5 equiv, 708.0 mg, 2.93 mmol) was dissolved in 109 mL of anhydrous pyridine (80 vol%). Under Ar, either 2,4,6-triisopropylbenzenesulfonyl chloride (10.0 equiv, 3.55 g, 11.74 mmol) or HATU (5.0 equiv, 2.23 g, 5.872 mmol) was then added. This was followed by the addition of a solution of 16-41a (1.0 equiv, 800.0 mg, 1.174 mmol) in anhydrous pyridine (27 mL) (20vol%).The resulting solution concentration was maintained at 14mM, then stirred at 40°C overnight (18 hours) (in case the reaction is performed with HATU as a coupling agent, the reaction temperature was set at 60 °C instead). The reaction was then quenched with water and the solution was stirred for 1 hour. The solvents were evaporated in vacuo. The residue was then dissolved in diethyl ether (-100 mL) and was washed three times with deionized water (-100 mL). The organic layer was collected, dried on anhydrous magnesium sulphate, filtered, and the solvent was evaporated in vacuo. The obtained residue was then purified by normal phase flash chromatography on a NextGen 300+ system (gradient from 100% DCM to 80% / 18% / 2% DCM / MeOH / 7N NH3in MeOH), yielding ((2-(4-(4-hydroxy-11,11-dimethyl-4,9-dioxo-3,5,10-trioxa-8-aza-4A5-phosphadodecan-1 -yl)piperazin-1-yl)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate) 16-42a (747.0 mg, 1.174 mmol, yield 70% when using TPSCl as coupling agent, yield 21% when using HATU as a coupling agent) as a transparent oil.

[0664] MS (ESI) m / z found for C43H86N4O10P[M-Boc+2H]2+: 403.3

[0665] 1H NMR (400 MHz, CDCl3): 50.85-0.89 (12H, m), 1.20-1.32 (26H, m), 1.42-1.48 (13H, m), 1.52-1.66 (11 H, m), 2.27-2.34 (2H, m), 2.56 (4H, br s), 2.71 (4H, br s), 2.90 (4H, br s), 3.05 (6H, br s), 3.36 (2H, br s), 3.94-3.99 (2H, m), 4.07 (4H, t, J = 6.5 Hz), 4.17 (2H, br s,), 5.60 (1 H, br s,) ppm.

[0666] 13C NMR (101 MHz, CDCU): 5 14.0, 14.1, 22.60, 22.62, 26.6, 27.4, 28.5, 29.2, 29.7, 31.7, 32.2, 32.5, 41.6, 45.7, 51.1, 53.3, 54.7, 58.2, 63.6, 64.9, 79.0, 156.1 , 176.7 ppm.

[0667] 31P NMR (165 MHz, CDCl3): 50.43 ppm.

[0668] Step 3

[0669] Under inert atmosphere (e.g., Ar), the BOC-protected intermediate 16-42a (1.0 equiv, 727.0 mg, 0.803 mmol) was dissolved in DCM (5.0 mL). The solution was cooled to 0 °C and hydrogen chloride 4N in 1,4-Dioxane (10.0 equiv, 2.0 mL, 8.2 mmol) was added. The mixture was gradually heated to room temperature (20°C) and the solution was then stirred for 3 hours. After this reaction time, volatiles were removed under vacuo to yield ((2-(4-(2-(((2-aminoethoxy)(hydroxy)phosphoryl)oxy)ethyl)piperazin-1-yl)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate) hydrochloride 16-43a*HCl (583.0 mg, .7 1 mmol, yield before neutralization 100%) as a light yellow viscous liquid. Optionally, the purity level may be increased by reverse phase flash chromatography on a NextGen 300+ system (gradient from 100% / 0.1 % Water / Formic Acid to 100% / 0.1 % ACN / Formic Acid).

[0670] To neutralize the amine hydrochloride salt, 16-43a-HCl (1.0 equiv, 30.0 mg, 35.6 pmol) was dissolved in THF and the solution was treated with Et3N (4.0 equiv, 19.9 pL, 142.4 pmol). The obtained mixture was allowed to stir for 20 minutes, and the volatiles were evaporated in vacuo. The residue was suspended in THF (10.0 mL), filtered through sintered disc funnel containing filter paper, and the solvent was evaporated in vacuo to yield ((2-(4-(2-(((2-aminoethoxy)(hydroxy)phosphoryl)oxy)ethyl)piperazin-1-yl)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate) 16-43a (6.9 mg, 0.021 mmol, yield of the neutralization step 56%, total yield 56%) as a transparent oil. MS (ESI) m / z found for C42H87N4O8P[M+2H]2+: 403.4

[0671] 1H-NMR for 16-43a-HCl (400MHz, CD3OD) 50.91 (12H, distorted t, J = 6.7 Hz), 1.18-1.40 (24H, m), 1.40-1.54 (4H, m), 1.54-1.66 (4H, m), 1.70-1.82 (4H, m), 1.82-1.94 (4H, m), 2.37 (2H, sept, J = 4.69 Hz), 3.14-3.35 (12H, m), 3.50 (2H, t, J = 5.60 Hz), 3.54-3.70 (6H, m), 4.16 (4H, t, J = 6.36 Hz), 4.26 (2H, q, J = 5.22 Hz), 4.41 (2H, br s,) ppm.

[0672] 1H-NMR for 16-43a (400MHz, CDCU) 50.86 (6H, t, J = 6.8 Hz), 0.87 (6H, t, J = 6.8 Hz), 1.16-1.36 (24H, m), 1.36-1.49 (4H, m), 1.49-1.69 (12H, m), 2.26-2.36 (2H, m), 2.37-3.13 (18H, m), 3.22 (2H, br s), 4.07 (4H, t, J = 6.42 Hz), 4.13 (4H, br s) ppm.

[0673] Synthesis of compound 16-70-l-a

[0674] Under inert atmosphere (e.g., Ar), the intermediate 4-oxobutyl 2-butyloctanoate 16-4b (5.0 equiv, 447 mg, 1.65 mmol) was dissolved in MeOH (100 mL). AcOH (18.0 equiv, 340 pL, 5.95 mmol, 0.34 vol%) was then added to the solution, followed by compound 16-43a-HCl (1.0 equiv, 266 mg, 330 mmol). After one minute, sodium cyanoborohydride (8.0 equiv, 166 mg, 2.64 mmol) was added and the solution was then allowed to stir at room temperature (20°C) overnight (16-18 hours). The solvents were evaporated in vacuo. The residue was then purified by normal phase flash chromatography on a NextGen 300+ system (gradient from 100% DCM to 60% / 40% DCM / MeOH). The purified product was dissolved in DCM (~50 mL) and washed with a 0.1 M NaHCO3(10.0 equiv) aqueous solution. The organic layer was dried over magnesium sulphate, filtered, and the solvent was removed in vacuo yielding compound ((2-(4-(15-butyl-8-(4-((2-butyloctanoyl)oxy)butyl)-4-hydroxy-4,14-dioxo-3,5,13-trioxa-8-aza-4A5-phosphahenicosan-1 -yl)piperazin-1-yl)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate) 16-70-l-a (169 mg, 129 pmol, yield 39%) as a yellow viscous liquid.MS (ESI) m / z found for C74H147N4O12P [M+2H]2+: 657.8

[0675] 1H NMR (400MHz, CDCU): 50.89-0.85 (24H, m), 1.33-1.15 (48H, m), 1.52-1.35 (12H, m), 1.65-1.53 (20H, m), 2.34-2.27 (4H, m), 2.51-2.41 (4H, m), 2.66-2.50 (4H, br m), 2.76 (8H, br s), 2.94 (8H, brs), 4.19-4.01 (12H, m) ppm.

[0676] 13C NMR (101MHz, CDCU): 514.1 , 14.2, 22.3, 22.7, 22.7, 23.4, 26.5, 26.8, 27.6, 29.3, 29.8, 31.8, 32.27, 32.31, 32.58, 32.63, 45.8, 45.9, 51.1 , 52.1 , 52.7, 53.7, 53.8, 55.1, 55.9, 58.8, 61.3, 62.1, 63.5, 63.9, 176.7, 176.8 ppm.

[0677] 31P NMR (165MHz, CDCU): 31.09 ppm

[0678] Synthesis of compound 16-70-l-b

[0679] Under inert atmosphere (e.g., Ar), the intermediate 16-4a (2.5 equiv, 473 mg, 1.95 mmol) was dissolved in 233 mL MeOH instead. AcOH (9.0 equiv, 402 pL, 7.02 mmol, 0.17 vol%) was then added to the solution, followed by compound 16-43a-HCl (1.0 equiv, 650 mg, 780 mmol). After one minute, sodium cyanoborohydride (4.0 equiv, 196 mg, 3.12 mmol) was added and the solution was then allowed to stir at room temperature (20°C) overnight (18 hours). The solvents were evaporated in vacuo. The residue was then purified by reverse phase flash chromatography on a NextGen 300+ system (gradient from 100% / 0.1 % Water / Formic Acid to 100% / 0.1 % ACN / Formic Acid). The purified product was dissolved in DCM (~30 mL) and washed with a 0.1 M NaHCCU (10.0 equiv) aqueous solution. The organic layer was dried over magnesium sulphate, filtered, and the solvent was removed in vacuo yielding compound ((2-(4-(13-butyl-8-(2-((2-butyloctanoyl)oxy)ethyl)-4-hydroxy-4,12-dioxo-3,5,11-trioxa-8-aza-4A5-phosphanonadecan-1 -yl)piperazin-1 -yl)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate) 16-70-l-b (60.0 mg, 48.0 pmol, yield 6.1%) as a light yellow viscous liquid.

[0680] MS (ESI) m / z found for C70H139N4O12P [M+2H]2+: 629.7

[0681] 1H NMR (400MHz, CDCU): 60.89-0.85 (24H, m), 1.30-1.21 (52H, m), 1.52-1.40 (12H, m), 1.63-1.54 (12H, m), 2.34-2.26 (4H, m), 2.50 (4H, br s), 2.67 (4H, br s), 2.88-2.83 (10H, m,), 3.05 (4H, brs), 3.95 (2H, q, J = 6.6 Hz), 4.16-4.05 (10H, m) ppm

[0682] 13C NMR (101MHz, CDCU): 6 14.0, 14.1, 22.6, 23.0, 26.6, 27.42, 27.44, 29.2, 29.3, 29.6, 29.70, 29.71, 31.7, 32.1, 32.2, 32.4, 32.5, 45.70, 45.75, 53.3, 53.4, 55.0, 55.1, 62.3, 63.7, 63.9, 176.5, 176.7 ppm

[0683] 31P NMR (162MHz, CDCU): 50.47 ppm

[0684] Synthesis of compound 16-70-l-c

[0685] Step 1Under inert atmosphere (e.g., Ar), the intermediate 16-4b (2.5 equiv, 575.0 mg, 2.13 mmol) was dissolved in MeOH (427.0 mL). AcOH (15 equiv, 730.0 pL, 12.8 mmol, 0.17 vol%) was then added to the solution, followed by 16-13a (1.0 equiv, 120 mg, 851.0 pmol). After one minute, sodium cyanoborohydride (4.0 equiv, 214.0 mg, 3.40 mmol) was added and the solution was stirred at room temperature (20°C) overnight (18 hours). After stirring overnight, additional intermediate 16-4b (2.5 equiv, 575.0 mg, 2.13 mmol) and sodium cyanoborohydride (4.0 equiv, 214.0 mg, 3.40 mmol) were added to the reaction mixture and the reaction was stirred at room temperature (20°C) for 4 hours. The solvents were evaporated in vacuo. The residue was purified by normal phase flash chromatography on a NextGen 300+ system (gradient from 100% DCM to 70% / 27% / 3% DCM / MeOH / NH37N in MeOH) yielding compound ((2-(phosphonooxy)ethyl)azanediyl)bis(butane-4,1-diyl) bis(2-butyloctanoate) 16-108a (520.0 mg, 851.0 pmol, yield 94%) as a white paste.

[0686] MS (ESI) m / z found for C34H7oN08P [M+H]2: 650.5

[0687] 1H NMR (400MHz, CD3OD): 50.86 (6H, t, J = 6.94 Hz), 0.86 (6H, t, J = 7.10 Hz), 1.16-1.36 (24H, m), 1.36-1.49 (4H, m), 1.49-1.62 (4H, m), 1.62-1.80 (8H, m), 1.86 (4H, s), 2.31 (2H, tt, J = 5.29, 9.01 Hz), 2.97 (4H, t, J = 7.54 Hz), 3.11 (2H, t, J = 4.76 Hz), 3.98-4.07 (2H, m), 4.10 (4H, t, J = 6.28 Hz) ppm

[0688] 31P NMR (162MHz, CD3OD): 53.76 ppm

[0689] Step 2

[0690] Under inert atmosphere (e.g., Ar), tert-butyl (2-(4-(2-aminoethyl)piperazin-1-yl)ethyl)carbamate 16-51a (1.0 equiv, 150.0 mg, 220.2 pmol), the phosphate intermediate 16-108a (1.25 equiv, 178.9 mg, 275.3 pmol) and DBU (2.0 equiv, 67.5 pL, 440.5 pmol) were dissolved in dichloromethane (2.5 mL) and the solution was cooled to 0°C and stirred for 30 minutes. DIAD (4.0 equiv, 173 pL, 880.9 pmol) and TPP (4.0 equiv, 231.0 mg, 880.9 pmol) were dissolved in dichloromethane (2.5 mL) and added dropwise to the reaction mixture. The reaction was allowed to stir at 0 °C for 25 additional minutes. After this reaction time, the reaction mixture was partitioned between sat.aq.NaHCO3solution and DCM. The organic phase was collected and the aqueous phase was washed twice with DCM. The three organic phases were combined and evaporated in vacuo. The obtained residue is then purified by normal phase flash chromatography on NextGen 300+ system (gradient from 100% DCM to 20% of MeOH / NH37N in MeOH (90% / 10%), yielding ((2-(4-(15-butyl-8-(4-((2-butyloctanoyl)oxy)butyl)-4-hydroxy-4,14-dioxo-5,13-dioxa-3,8-diaza-4A5-phosphahenicosan-1-yl)piperazin-1-yl)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate) 16-70-l-c (45 mg, 23% yield, 34.0 pmol) as light yellow liquid.

[0691] MS (ESI) m / z found for C74H148N5O11P [M+2H]+2: 657.21H NMR (400MHz, CDCU): 50.86-0.90 (24H, m), 1.19-1.33 (48H, m), 1.40-1.50 (12H, m), 1.52-1.66 (20H, m), 2.27-2.34 (4H, m), 2.39-2.62 (14H, m), 2.75 (4H, br s), 2.91-2.96 (6H, m), 3.61 (d, J= 10.8 Hz, 2H), 4.00-4.10 (10H, m) ppm.

[0692] 13C NMR (101MHz, CDCU): 5 14.1, 14.2, 22.2, 22.74, 22.75, 23.7, 26.6, 26.8, 27.6, 29.4, 29.8, 31.8, 32.28, 32.34, 32.56, 32.59, 32.65, 45.8, 45.9, 52.9, 53.1, 53.5, 53.69, 53.73, 54.1, 63.5, 64.1, 176.78, 176.8 ppm.

[0693] 31P NMR (165MHz, CDCU): 51.73 ppm

[0694] Synthesis of compound 16-71 a

[0695] Under inert atmosphere (e.g., Ar), the intermediate 16-4b (2.5 equiv, 210.0 mg, 0.78 mmol) was dissolved in MeOH (96.7 mL). AcOH (9.0 equiv, 168.0 mg, 168.0 mmol, 0.17 vol%) was then added to the solution, followed by 16-43a-HCl (1.0 equiv, 250.0 mg, 311.0 mmol). After one minute, sodium cyanoborohydride (4.0 equiv, 78.0 mg, 65.0 mmol) was added and the solution was then allowed to stir at room temperature (20°C) overnight (18 hours). The solvents were evaporated in vacuo. The residue was then purified by normal phase flash chromatography on a NextGen 300+ system (gradient from 100% DCM to 90% / 7% / 3% DCM / MeOH / NH37N in MeOH). The purified product was dissolved in 100% / 0.1 % ACN / Formic Acid solution, then the solvent was evaporated in vacuo. The residue was dissolved in DCM and the solution was washed with a 0.1 M NaHCO3(10.0 equiv) aqueous solution. The organic phase was collected while the aqueous phase was extracted with DCM. The combined organic fractions were dried over magnesium sulphate, filtered, and the volatiles were removed in vacuo yielding compound ((2-(4-(15-butyl-4-hydroxy-4,14-dioxo-3,5,13-trioxa-8-aza-4A5-phosphahenicosan-1-yl)piperazin-1-yl)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate) 16-71a (66 mg, 62 pmol, yield 20%) as an orange viscous liquid. MS (ESI) m / z found for C58H117N4O10P [M+2H]2+: 530.6

[0696] 1H NMR (400MHz, CDCl3): 50.92-0.83 (18H, m), 1.36-1.16 (36H, m), 1.36-1.52 (1 OH, m), 1.66-1.53 (1 OH, m), 1.78-1.67 (2H, m), 1.90-1.78 (2H, m), 2.35-2.24 (3H, m), 2.68-2.36 (16H, m), 2.67 (2H, t, J = 6.0 Hz), 2.91 (2H, t, J = 6.0 Hz), 3.15 (2H, d, J = 4.8 Hz), 4.14-3.94 (8H, m), 4.24 (2H, distorted dd, t, J = 13.1 , 6.1 Hz) ppm.

[0697] 13C NMR(101MHz, CDCU): 14.1 , 14.2, 22.7, 22.8, 23.0, 23.8, 26.0, 26.9, 27.55, 27.57, 29.4, 29.76, 29.79, 29.84, 31.8, 32.2, 32.3, 32.5, 32.6, 45.8, 45.9, 47.8, 49.9, 51.7, 53.5, 53.7, 54.2, 56.8, 58.4, 60.9, 63.1 , 63.8, 64.1 , 176.6, 176.8.

[0698] Synthesis of compound 16-71 bThe procedure for the preparation of compound 16-71a above was repeated, but using intermediate 16-4’a instead. The synthesis procedure yielded ((2-(4-(15-butyl-4-hydroxy-4,14-dioxo-3,5,13-trioxa-8-aza-4A5-phosphahenicosan-1 -yl)piperazin-1-yl)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate) 16-71b (66 mg, 62 pmol, yield 20%) as an orange viscous liquid.

[0699] MS (ESI) m / z found for C58H117N4O10P [M+2H]2+: 530.6

[0700] Synthesis of compound 16-77a

[0701] Under inert atmosphere (e.g., Ar), 2-(2-butyloctanamido)ethyl dihydrogen phosphate 16-17a (3.5 equiv, 83.0 mg, 0.26 mmol) and 2,4,6-triisopropylbenzenesulfonyl chloride (8.0 equiv, 180.0 mg, 0.59 mmol) were dissolved in dry pyridine (3.0 mL) at room temperature (20°C). To the resulting reaction mixture, a solution of ((2-(4-(2-hydroxyethyl)piperazin-1-yl)ethyl)azanediyl)bis(butane-4,1-diyl) bis(2-butyloctanoate) 16-41a (1.0 equiv, 50.0 mg, 73.0 mmol) in dry pyridine (0.5 mL) was added then the flask was flushed with argon and closed. Final concentration of reaction mixture was maintained at 14mM. The reaction mixture was then stirred at 40 °C for 30 minutes. The reaction mixture was then partitioned in water and diethyl ether and the organic layer was collected while the aqueous phase was extracted two times with diethyl ether. The combined organic phases were dried anhydrous magnesium sulphate, filtered, and volatiles were evaporated in vacuo. The residue was then purified by normal flash chromatography on a NextGen 300+ system (gradient from 100% DCM to 70% / 30% DCM / MeOH). The residue was dissolved in DCM and the solution was washed with a 0.1 M NaHCO3(10.0 equiv) aqueous solution. The organic phase was collected while the aqueous phase was extracted with DCM. The combined organic fractions were dried over magnesium sulphate, filtered, and the volatiles were removed in vacuo yielding compound ((2-(4-(10-butyl-4-hydroxy-4,9-dioxo-3,5-dioxa-8-aza-4A5-phosphahexadecan-1 -yl)piperazin-1-yl)ethyl)azanediyl)di(butane-4,1-diyl) bis(2-butyloctanoate) 16-77a (29.6 mg, 30 pmol, yield 34%) as a light yellow viscous liquid.

[0702] MS (ESI) m / z found for C54H107N4O9P [M+2H]2+: 494.6

[0703] 1H NMR (400MHz, CDCU): 50.89-0.84 (18H, br s,), 1.25 (38H, br s), 1.50-1.38 (9H, m), 1.64-1.53 (9H, m), 2.07-2.00 (1 H, m), 2.34-2.27 (2H, m), 2.49-2.48 (4H, m), 2.66 (4H, br s), 2.88 (4H, br s), 3.01 (4H, br s), 3.51-3.50 (2H, m), 4.01-3.96 (2H, m), 4.07 (4H, t, J = 6.6Hz), 4.15 (2H, br s), 7.06 (1 H, br s) ppm.

[0704] 13C NMR (101 MHz, CDCU): 514.0, 14.06, 14.08, 14.12,22.60, 22.61, 22.7,22.8,23.0, 26.6, 27.4, 27.6, 29.2, 29.5, 29.6, 29.8, 31.7, 31.8, 32.2, 32.5, 32.7, 33.0, 40.45, 40.50, 45.7, 47.9, 51.9, 53.5, 55.2, 58.5, 63.7, 64.8, 69.7, 176.4, 176.7 ppm

[0705] 31P NMR (165 MHz, CDCU): 51.01 ppmSynthesis of compound 16-92-l-a

[0706] Step 1

[0707] Under inert atmosphere (e.g., Ar), (9Z,12Z)-octadeca-9,12-dienal 16-84a (2.3 equiv, 702.0 mg, 2.65 mmol) was dissolved in MeOH (383.0 ml). AcOH (10.0 equiv, 660 pL, 11.5 mmol, 0.17 vol%) was then added to the solution, followed by 2-(4-(2-aminoethyl)piperazin-1-yl)ethan-1-ol 16-40 (1.0 equiv, 200.0 mg, 1.15 mmol). After one minute, sodium cyanoborohydride (3.0 equiv, 218.0 mg, 3.46 mmol) was added and the solution was then allowed to stir at room temperature (20°C) for 18 hours. After this reaction time, solvents were evaporated in vacuo. The residue was then partitioned in a mixture of DCM (-100 ml), saturated aqueous NaHCO3solution (~80 mL) and saturated brine solution (~20 mL). The organic layer was collected while the aqueous layer was extracted two times with DCM (-100 mL). The organic layers were combined, dried over anhydrous magnesium sulphate, filtered and evaporated in vacuo. The residue was then purified by normal phase flash chromatography on a NextGen 300+ system (gradient from 100% DCM to 10% / 90% MeOH / DCM) yielding 2-(4-(2-(di((9Z,12Z)-octadeca-9,12-dien-1 -yl)amino)ethyl)piperazin-1 -yl)ethan-1-ol 16-89a (550.0 mg, 0.821 mmol, yield 71%) as a light yellow viscous liquid.

[0708] MS (ESI) m / z found for C44H85N3O [M+2H]2+: 335.9

[0709] 1H NMR (400MHz, CDCl3): 50.89 (6H, t, J = 6.9 Hz), 1.26-1.39 (32H, m), 1.44-1.47 (4H, m), 2.05 (8H, q, J = 6.7 Hz), 2.39-2.30 (2H, br m,), 2.56-2.47 (14H, m), 2.64 (2H, t, J = 6.3 Hz), 2.77 (4H, t, J = 6.4 Hz,), 3.61 (2H, t, J = 5.4 Hz,), 5.42-5.30 (8H, m,) ppm.

[0710] 13C NMR (101MHz, CDCU): 5 14.1, 22.6, 25.6, 26.6, 27.22, 27.24, 27.5, 29.3, 29.4 , 29.6, 29.7, 31.5 , 51.2, 52.8, 53.7, 54.6, 56.2, 57.7, 59.2, 127.9, 128.0, 130.1, 130.2 PPm

[0711] Step 2

[0712] Under inert atmosphere (e.g., Ar), the BOC-protected (tert-butyl (2-(phosphonooxy)ethyl)carbamate 16-21 a (2.5 equiv, 495.0 mg, 2.05 mmol), 2-(4-(2-(di((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethyl)piperazin-1-yl)ethan-1-ol 16-89a (1.0 equiv, 550.0 mg, 0.821 mmol) and 2,4,6-triisopropylbenzenesulfonyl chloride (10.0 equiv, 249.0 mg, 8.21 mmol) were dissolved in dry pyridine (58 mL) at room temperature (20°C). The final concentration of 16-89a in the reaction solvent was 14.0 mM. Under Ar, the reaction mixture was then stirred at 40 °C for 18 hours. The solvents were evaporated in vacuo, and the residue was dissolved in water (-100 mL) and was then extracted three times with diethyl ether (-100 mL). The combined organicfractions were collected, dried over anhydrous magnesium sulphate, filtered, and volatiles were evaporated in vacuo. The residue was then purified by normal phase flash chromatography on a NextGen 300+ system (gradient from 100% DCM to80% / 18% / 2% DCM / MeOH / 7N NH3in MeOH), yielding compound tert-butyl (2-(((2-(4-(2-(di((9Z,12Z)-octadeca-9,12-dien-1 -yl)amino)ethyl)piperazin-1-yl)ethoxy)(hydroxy)phosphoryl)oxy)ethyl)carbamate 16-90a (136.0 mg, 0.088 mmol, yield 20%, 58% purity) as a light yellow viscous liquid.

[0713] MS (ESI) m / z found for C47H90N4O4P [M+2H-Boc]2+: 397.2

[0714] 1H NMR (400MHz, CDCU): 50.88 (6H, t, J = 6.88 Hz), 1.18-1.39 (32H, m), 1.42 (9H, s), 1.58 (4H, br s), 2.04 (8H, q, J = 6.78 Hz), 2.42-3.09 (22H, m), 3.34 (2H, q, J = 4.44 Hz), 3.88-3.98 (2H, m), 4.04 (2H, q, J = 5.26 Hz), 5.26-5.43 (8H, m), 5.69 (1 H, br s) ppm31P NMR (165 MHz, CDCl3): 50.55 ppm.

[0715] Step 3

[0716] Under inert atmosphere (e.g., Ar), in a 8 ml vial, tert-butyl (2-(((2-(4-(2-(di((9Z,12Z)-octadeca-9,12-dien-1 -yl)amino)ethyl)piperazin-1-yl)ethoxy)(hydroxy)phosphoryl)oxy)ethyl)carbamate 16-90a (1.0 equiv, 136.0 mg, 0.152 mmol) was dissolved in dry DCM (2.0 ml). To the resulting reaction mixture, was added 4N HCl in Dioxane (10.0 equiv, 381.0 mL, 1.52 mmol) at 0 °C. The solution was allowed to stir at 0 °C for 3 hours. Volatiles were evaporated in vacuo, yielding crude compound 2-aminoethyl 2-(4-(2-(di((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethyl)piperazin-1-yl)ethyl hydrogen phosphate 16-91a-HCl (121.0 mg, 0.152 mmol, yield 100%) as a light yellow viscous liquid, used for the next step without further purification.

[0717] MS (ESI) m / z found for C46H91N4O4P [M+2H]2+: 397.4

[0718] Step 4

[0719] Under inert atmosphere (e.g., Ar), (9Z,12Z)-octadeca-9,12-dienal 16-84a (5.0 equiv, 200 mg, 0.756 mmol) was dissolved in MeOH (91 mL). AcOH (18.0 equiv, 156 pL, 2.72 mmol, 0.17 vol%) was then added to the solution, followed by a solution of 2-aminoethyl (2-(4-(2-(di((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethyl)piperazin-1-yl)ethyl) hydrogen phosphate 16-91 a-HCl (1.0 equiv, 120.0 mg, 0.151 mmol) in MeOH was added to above reaction mixture. After one minute, sodium cyanoborohydride (4.0 equiv, 38.0 mg, 0.605 mmol) was added and the solution was then allowed to stir at room temperature (20°C) overnight (16-18 hours). The solvents were evaporated in vacuo. The residue was then partitioned in a mixture of DCM, saturated aqueous NaHCO3solution and saturated brine solution. The organic layer was collected while the aqueous layer was extracted two times with DCM. The organic layers were combined, dried over anhydrous magnesium sulphate, filtered and evaporated in vacuo. The residue was then purified by normal phase flash chromatography on a NextGen 300+ system (gradient from 100% DCM to 10% / 90% MeOH / DCM), yielding2-(di((9Z, 12Z)-octadeca-9, 12-dien-1 -yl)amino)ethyl 2-(4-(2-(di((9Z, 12Z)-octadeca-9,12-dien-1-yl)amino)ethyl)piperazin-1-yl)ethyl hydrogen phosphate 16-92-l-a (63.0 mg, 0.049 mmol, yield 32%) as a light yellow viscous liquid.

[0720] MS (ESI) m / z found for C82H155N4O4P [M+2H]2+: 645.7

[0721] 1H NMR (400MHz, CDCl3): 50.89 (12H, t, J = 6.8 Hz), 1.26-1.37 (64H, m), 1.41-1.48 (4H, m), 1.67 (4H, br s), 2.05 (16H, q, J = 6.8 Hz), 2.47-2.61 (16H, m), 2.69 (2H, t, J = 6.0 Hz ), 2.77 (8H, t, J = 6.4 Hz), 2.84-2.88 (4H, m), 3.06 (2H, br s), 4.05-4.16 (4H, m), 5.29-5.42 (16H, m) ppm.

[0722] 13C NMR (101 MHz, CDCU): 514.2, 22.7, 25.8, 27.1 , 27.3, 27.4, 27.7, 29.3, 29.3, 29.4, 29.5, 29.5, 29.7, 29.7, 29.8, 31.6, 51.2, 53.4, 53.6, 53.8, 54.5, 56.2, 58.5, 58.6, 60.0, 63.2, 128.0, 128.1, 128.1, 128.2, 130.1, 130.2, 130.3, 130.4 ppm.

[0723] 31P NMR (165 MHz, CDCl3): 51.47 ppm

[0724] B. Evaluation of exemplary compounds of the invention for RNA delivery Lipid nanoparticle (LNP) formulation

[0725] Lipid nanoparticles (LNPs) were formulated using the ionizable phospholipid compounds 16-25a, 16-29a, 16 -43a, 16-70-l-a, 16-70-l-b, and 16-77a described above, along with 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and DMG-PEG 2000 lipid. The lipid components were dissolved in ethanol at a molar ratio respectively of 35:20:43.5:1.5 (1) or, alternatively, 55:0:43.5:1.5 (2) (the latter thus not comprising DOPE). In the case of C12-200 as ionisable lipid, a lipid concentration of 100pg / pL of C12-200, 25pg / pL of DOPE, 20pg / pL of cholesterol and 25 pg / pL DMG-PEG 2000 was used.

[0726] Each LNP was prepared by targeting an N / P ratio of approximately 40:1 with selfamplifying RNA (saRNA). Specifically, firefly luciferase saRNA (approx. 9 659 nucleotides) was diluted in 10 mM citrate buffer (pH 4.5). A microfluidic mixer (Ignite, Precision Nanosystems, California, USA) was used to combine the lipid solution with the aqueous saRNA solution at a volumetric ratio of 1 :3 (vol / vol), with total flow rates exceeding 10 mL / min. Following mixing, ethanol was removed, and the external buffer was exchanged with 10 mM Tris-HCl (pH 7.4) via dialysis. The resulting LNP suspension was filtered through a 0.2 pm pore sterile filter.

[0727] LNP characterization & encapsulation efficiency

[0728] The particle size distribution of the LNPs was determined using a Zetasizer (Malvern Panalytical, UK), yielding a diameter range of 50-150 nm with a polydispersity index (PDI) between 0.1-0.4. The surface charge (zeta potential) was also measured using the Zetasizer, with values ranging from +20 mV to -20 mV.The saRNA loading efficiency of LNP formulations was quantified using a Quant-iT RiboGreen assay (Thermo Fisher Scientific, Waltham, Massachusetts, USA). A total of 14 samples were diluted tenfold in 1 * Tris-HCl-EDTA (TE) buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5), with or without 2% (v / v) Triton X-100 (Sigma-Aldrich, Saint Louis, Missouri, USA). Standard solutions were prepared in 1 * TE buffer with and without 2% (v / v) Triton X-100 to account for fluorescence variations. The assay was conducted following the manufacturer's protocol, using a black 96-well plate and a Tecan Infinite® 200 PRO microplate reader with an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The encapsulation efficiency of saRNA in LNP formulations was calculated to be 80-100%.

[0729] These results demonstrate that even the LNP formulations without any additional phospholipid, other than the ionizable lipid compound according to the invention, provided high encapsulation efficiency.

[0730] In vivo study 1

[0731] To determine the transfection efficiency of the presently formulated LNP compositions, female SWISS mice (6 weeks old) were obtained from Janvier Laboratories (Paris, France) and housed in individually ventilated cages, with ad libitum access to food and water. Mice (n=7 per group) were anesthetized using isoflurane (Zoetis, Louvain-La-Neuve, Belgium) at 5% for induction and 2% for maintenance. Each mouse received an intramuscular injection of 1 pg of LNP-formulated luciferase saRNA suspended in 100 pL of 10 mM Tris-HCl buffer (50 pL per leg).

[0732] The expression of luciferase was monitored using non-invasive / n vivo bioluminescent imaging (MS Lumina LT III, Perkin Elmer, Waltham, Massachusetts, USA). Bioluminescence signals were measured 10 minutes after subcutaneous injection of 200 pL D-luciferin (GoldBio, Saint Louis, Missouri, USA, #LUCK-1G). Imaging was performed at day 0 (pre-injection), day 1 , day 3, day 5, day 7, day 10, day 15, and day 20. The bioluminescence as a measure for the protein expression over time are reported in Table 2 and illustrated in Figures 1 A-F.Table 1 : Characteristics of the formulated LNP compositions

[0733] Characterisation of LN Ps

[0734] Lipid Z-average (nm) Polydispersity Index (PI) Encapsulation (%) Zetapotential (mV)

[0735] ±SD ±SD ± SD (%) ±SD C12-200 88.23 6.43 0.23 0.03 98.24 0.81 5.08 6.33 16-25 a (1) 84.72 2.61 0.23 0.03 96.91 9.91 5.12 0.11 16-25 a (2) 237.02 5.81 0.17 0.03 93.30 1.99 19.23 0.64 16-29 a (1) 71.96 1.22 0.22 0.01 95.79 14.06 13.18 0.66 16-29 a (2) 78.43 1.53 0.22 0.01 77.79 14.91 16.44 1.00 16-43 a (1) 112.44 2.10 0.13 0.02 91.77 2.73 -1.29 2.71 16-70-l-a (1) 100.09 0.75 0.21 0.02 95.99 6.40 -6.26 0.36 16-70-l-a (2) 126.20 1.07 0.14 0.03 82.43 9.93 -20.71 1.42 16-70-l-b (1) 97.75 1.94 0.20 0.02 97.67 4.83 -13.84 0.42 16-70-l-b (2) 102.50 1.01 0.15 0.02 92.45 11.04 -23.38 2.54 16-77a(1) 103.10 0.52 0.25 0.01 73.11 17.25 -20.38 1.05

[0736]

[0737] 16-77 a (2) 109.66 1.03 0.15 0.01 56.07 14.79 -18.97 0.11Table 2: Bioluminescence expression

[0738] Bioluminescence expression of LN Ps

[0739] Lipid Day 0 Day 1 Day 3 Day 5 Day 7 Day 10 Day 15 Day 20

[0740] (Before

[0741] injection)

[0742] Control 6.28E+04 9.41E+04 1.90E+05 5.03E+05 3.73E+04 6.64E+04 2.36E+04 6.20E+04 C12-200 5.80E+04 1.34E+08 2.65E+08 4.34E+08 1.93E+08 2.16E+07 2.06E+06 6.79E+05 16-25 a (1) 2.80E+04 2.22E+06 3.69E+07 7.16E+07 7.41E+07 6.02E+06 2.61E+05 6.02E+05 16-25 a (2) 2.13E+04 1.93E+05 3.36E+06 8.85E+06 1.21E+07 9.10E+06 2.19E+06 1.64E+05 16-29 a (1) 2.17E+04 2.41 E+06 5.32E+07 1.16E+08 1.13E+08 5.85E+06 8.74E+05 2.55E+05 16-29 a (2) 1.98E+04 2.18E+06 3.06E+07 7.28E+07 5.84E+07 1.24E+07 9.38E+05 7.68E+05 16-43 a (1) 2.13E+04 5.08E+05 7.63E+06 1.08E+07 9.50E+06 1.23E+07 1.79E+06 1.06E+06 16-70-l-a (1) 1.25E+05 1.59E+08 9.93E+08 1.25E+09 2.38E+08 1.78E+06 2.49E+05 1.16E+05 16-70-l-a (2) 1.17E+05 7.00E+07 9.49E+08 1.16E+09 7.96E+08 1.54E+06 2.74E+05 5.05E+05 16-70-l-b (1) 9.55E+04 5.77E+07 2.78E+08 5.00E+08 6.85E+07 1.77E+06 1.70E+05 4.18E+04 16-70-l-b (2) 1.14E+05 5.21 E+07 3.06E+08 4.80E+08 5.91E+07 1.55E+06 4.15E+05 1.45E+05 16-77a(1) 1.08E+05 4.75E+07 4.43E+08 6.35E+08 2.26E+08 1.98E+06 2.00E+05 5.64E+04

[0743]

[0744] 16-77 a (2) 1.35E+05 5.02E+07 3.72E+08 5.02E+08 6.19E+07 1.62E+06 7.26E+05 3.87E+05Table 3: Weight evolution

[0745] Weight (g)

[0746] Lipid Day 0 Day 1 Day 3 Day 5 Day 7 Day 10 Day 15 Day 20 (Before

[0747] injection)

[0748] ±SD ±SD ±SD ±SD ±SD ±SD ±SD ±SD Control 26.04 1.55 26.10 1.53 26.45 1.59 26.59 1.76 26.81 1.75 26.58 1.48 27.50 1.79 28.00 1.80 C12-200 28.06 3.29 26.36 2.78 27.31 2.51 27.36 2.46 27.84 2.19 27.90 1.90 28.80 2.10 30.26 1.98 16-25 a (1) 26.39 1.19 26.14 1.16 26.66 1.32 26.74 0.93 27.07 1.33 27.49 1.68 27.77 1.34 29.19 1.20 16-25 a (2) 26.69 1.64 26.60 1.65 27.20 1.59 26.87 1.41 27.69 1.39 27.56 1.77 28.17 1.55 29.80 2.16 16-29 a (1) 26.40 1.71 26.70 1.47 26.86 1.75 27.31 1.65 27.14 1.37 27.84 1.82 28.90 1.98 30.30 2.23 16-29a(2) 26.46 1.50 26.33 1.34 26.64 1.87 26.69 1.71 27.51 2.02 27.89 2.30 28.06 1.89 29.14 2.18 16-43a(1) 26.76 2.33 26.86 2.40 26.91 2.00 27.37 2.11 27.71 2.16 27.93 2.48 28.56 2.61 30.26 2.31 16-70-l-a(1) 28.79 1.20 27.61 1.24 28.24 0.92 28.63 1.28 28.79 1.61 28.74 1.40 30.41 1.30 31.64 2.41 16-70-l-a(2) 29.69 2.22 28.60 1.88 28.64 1.76 29.00 1.58 28.69 2.04 29.50 1.57 29.91 1.64 31.14 2.59 16-70-l-b(1) 31.10 2.88 30.21 2.61 29.26 2.13 29.70 2.01 29.73 2.25 29.59 2.43 31.20 3.36 31.63 2.42 16-70-l-b(2) 29.67 3.02 27.89 2.28 27.63 2.25 27.97 2.28 27.63 2.05 27.90 2.08 29.51 2.17 29.20 2.31 16-77 a (1) 28.76 2.73 28.80 2.58 28.87 2.45 28.99 2.65 28.89 2.49 29.64 2.60 31.09 3.03 31.77 2.69

[0749]

[0750] 16-77 a (2) 30.56 2.53 29.47 2.49 28.67 2.33 29.13 2.19 29.00 2.35 29.57 2.50 30.69 3.19 31.07 3.04In vivo study 2

[0751] Lipid nanoparticles (LNPs) were formulated using the ionizable phospholipid compounds 16-70-l-a, cpd 16-71a, cpd 16-92-l-a and cpd 16-70-l-c, along with 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and DMG-PEG 2000 lipid. Lipids were admixed similar to the LNP formulation described above at a molar ratio respectively of 35:20:43.5:1.5 (1) or, alternatively, 55:0:43.5:1.5 (2) (the latter thus not comprising DOPE). Transfection efficiency was measured through luciferase expression after administration. BALB / C mice (n=6 per group) were administered one injection comprising 1 pg of LNP-formulated luciferase saRNA suspended in 50pl. The bioluminescence as a measure for the protein expression over time is reported in Figure 2B, and, the area under the curve in Figure 2A.

Claims

CLAIMS1 . A compound accordingto formula (I)or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:m, n and o are integers independently selected from 1 , 2, 3, 4 and 5; p is an integer selected from 0 and 1 ;XT and X2are independently selected from O and NH;R1is R2’ or -L1N[R2”]2;each R2, R2’ and R2” is independently selected from C4-3o-alkenyl and - L2F1RA;Z is selected from -NR3R4and -OR5;R3, R4and R5are independently selected from hydrogen, Ci-3o-alkyl, C4.30-alkenyl, -C(=O)-RBor -L3F2RC,each L1, L2and L3is independently selected from C2.10-alkylene; each F1and F2is independently selected from -O-C(=O)-, -C(=O)-O-, - NH-C(=O)- or -C(=O)-NH-; and,each RA, RB, and Rcis independently selected from C4.3o-alkyl and C4.30- alkenyl.

2. The compound according to claim 1 or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein p is 1 .

3. The compound according to claim 1 or 2 or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein each RA, RBand Rcare, independently selected from secondary or branched C4.30-alkyl and primary C4.3o-alkenyl.

4. The compound according to any of claims 1 to 3 or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, whereinm, n and o are integers independently selected from 1 , 2, 3, 4, and 5; p is an integer as defined in any one of claims 1 to 3;- XT and X2are as defined in any one of claims 1 to 3;- R1is -L1N[L2F1RA]2or -L2F1RA;- R2is-L2F1RA;- Zis-NR3R4;- R3is-L3F2RC;R4is hydrogen or -L3F2RC;L1, L2and Pare as defined in any one of claims 1 to 3;F1and F2are independently selected from -O-C(=O)-, -C(=O)-O-, -NH- C(=O)-or-C(=O)-NH-; and;RAand Rcare independently selected from secondary or branched C4-30- alkyl or primary C5-3o-alkenyl.

5. The compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein XT and X2are O.

6. The compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein the compound is accordingto formula (II)RI1R3OHI 1o| |D -N' . ( ) 0 O ' 0,0 6(II) or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein R1, R2, R3, R4, m, n and o are as defined in any one of claims 1 to 5.

7. A pharmaceutically acceptable formulation comprising the compound accordingto anyone of the preceding claims and a cargo compound selected from the group consisting of nucleic acids, small molecule compounds, peptides, proteins, protein-nucleic acid constructs, and mixtures thereof, preferably wherein the cargo compound is ionized, more preferably anionic, at physiological pH, .

8. A lipid nanoparticle composition comprising at least one compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof; and preferably further comprising one or more lipids selected from the group consisting of phospholipids, structural lipids, PEG-lipids, and mixtures thereof.

9. The lipid nanoparticle composition according to claim 8 further comprising a cargo compound selected from the group consisting of nucleic acids, small molecule compounds, peptides, proteins, protein-nucleic acid constructs,and mixtures thereof, preferably wherein the cargo compound is ionized, more preferably anionic, at physiological pH.

10. The lipid nanoparticle composition according to claim 9, wherein the cargo compound is a nucleic acid having a length between 8000 and 17000 nt.11.A method for manufacturing a lipid nanoparticle composition accordingto any one of claims 8 to 10, the method comprising the steps of:(a) providing lipids comprising at least one compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, and optionally one or more lipids selected from the group consisting of phospholipids, structural lipids, PEG-lipids, and mixtures thereof;(b) dissolving the lipids in an organic solvent, thereby forming an organic phase;(c) providing an aqueous phase, preferably comprising one or more cargo compounds;(d) forming lipid nanoparticles by mixing the organic phase and aqueous phase under conditions suitable for nanoparticle formation.

12. The method according to claim 11, wherein step d) comprises spray drying, single or double emulsion solvent evaporation, solvent extraction, phase separation, or simple or complex coacervation for the formation of lipid nanoparticles.

13. The lipid nanoparticle composition accordingto any one of claims 8 to 10 for use in medicine.

14. The lipid nanoparticle composition for use in medicine accordingto claim 13, wherein said use involves delivering a cargo to a cell, such as for transfecting a cell, preferably a mammalian cell, in vivo or ex vivo.

15. A method for delivering a cargo to a cell / n vitro, such as for transfecting a cell in vitro, comprising contacting the cell with a lipid nanoparticle composition according to any one of claims 8 to 10, preferably wherein the cell is a mammalian cell.

Images