Ionizable lipids comprising thioamides, preparation thereof, uses thereof, and compositions thereof

Abstract

The present invention relates to the field of ionizable lipids and compositions thereof. More particularly, the present invention relates to ionizable lipids or pharmaceutically acceptable salts thereof comprising a moiety, the moiety comprising an ionizable nitrogen attached adjacent to a thioamide group. The present invention further relates to a method for preparing said ionizable lipids, lipid nanoparticles comprising said ionizable lipids, and medical uses thereof (e.g, to deliver biologically active agents, such as nucleic acids, to cells and tissues).

Description

[0001] IONIZABLE LIPIDS COMPRISING THIOAMIDES, PREPARATION THEREOF, USES THEREOF, AND COMPOSITIONS THEREOF FIELD OF THE INVENTION

[0002] The present invention relates to the field of ionizable lipids and compositions thereof. More particularly, the present invention relates to ionizable lipids or pharmaceutically acceptable salts thereof comprising an ionizable nitrogen. The present invention further relates to a method for preparing said ionizable lipids, lipid nanoparticles comprising said ionizable lipids, and uses thereof.

[0003] BACKGROUND OF THE INVENTION

[0004] Delivery systems of biologically active agents are vital for ensuring the efficient and safe delivery of said compounds to target cells and tissues. Among these systems, lipid nanoparticles (LNPs) have emerged as a leading technology for delivering of said agents, such as messenger ribonucleic acid (mRNA), in therapeutic and vaccine applications. For instance, LNPs can be designed to encapsulate mRNA, protect it from degradation, and enable its delivery into the cytoplasm of cells, where it can be translated into functional proteins.

[0005] Ionizable lipids are key components of LNPs, as their pH-dependent charge-switching properties facilitate encapsulation of compounds and enable endosomal escape. Importantly, the ionizable head group of the lipids may significantly influence the performance of LNPs, including their stability, biodistribution, and transfection efficiency. Despite advancements in LNP technology, challenges remain in designing ionizable lipids with optimal physicochemical properties to achieve efficient and targeted delivery of therapeutic agents.

[0006] In view of the above, there remains a need for novel ionizable lipids and lipid nanoparticle compositions having improved physicochemical properties, preferably including reduced cytotoxicity, enhanced transfection efficiency, and improved tissue-specific delivery, when compared to conventional ionizable lipids.

[0007] SUMMARY OF THE INVENTION

[0008] It has now been found that some or all of the above challenges can be addressed, and objectives can be achieved, either individually or in any combination, by using the ionizable lipids, methods of their preparation, and compositions as defined herein.The present invention is, at least in part, based on the finding that the molecular structure of ionizable lipids may be designed to tune the apparent pKa of the resulting lipid nanoparticles (LNPs), which allows to balance transfection efficiency and cytotoxicity of the LNP. More specifically, it has been found herein that an ionizable head group of the lipid comprising a thioamide in close proximity to an ionizable nitrogen can effectively be tailored to create a more or less polarized structure, thereby modulating its affinity for hydrophobic environments.

[0009] For instance, an ionizable lipid comprising the ionizable head group as described herein can be used to decrease the apparent pKa of the resulting LNP, which may advantageously avoid premature charge activation, thereby improving delivery efficiency and suppressing off-target transfection.

[0010] Another advantage of the present ionizable lipids is that by offering control over the apparent pKa of the LNP compositions they can allow to reduce non-specific interactions with serum proteins and other cellular components, thereby improving biocompatibility and reducing systemic toxicity. In other words, the key structural motif of the present invention may increase the likelihood of LNPs to reach target tissues or cells.

[0011] As observed herein, and further demonstrated in the experimental section, LNPs as described herein can further advantageously exhibit distinct tissue tropism (e.g., increased tissue or organ targeting). Accordingly, an aspect of the present invention relates to an ionizable lipid or a pharmaceutically acceptable salt thereof, comprising a moiety, the moiety comprising:

[0012] an ionizable nitrogen selected from a secondary or tertiary amine functional group, and

[0013] a thioamide group, wherein the thioamide group is separated from the ionizable nitrogen by a chain of 2 to 10 atoms.

[0014] In particular embodiments, the moiety comprises:

[0015] an ionizable nitrogen selected from a secondary or tertiary amine functional group, and

[0016] a secondary thioamide group, wherein the thioamide group is separated from the ionizable nitrogen by a chain of 2 to 10 atoms.

[0017] In particular embodiments, the ionizable lipid or a pharmaceutically acceptable salt thereof may comprise one or more hydrophobic tails, each independently comprising a linear or branched aliphatic chain having 8 to 36 carbon atoms.In particular embodiments, the ionizable lipid or a pharmaceutically acceptable salt thereof may provide that the hydrophobic tails are covalently attached to the thioamide group by means of a linker structure comprising ester, amide, carbamate, carbonate, disulfide, or thioester linkages, preferably ester linkages.

[0018] In particular embodiments, the moiety as described herein may be represented by the general formula (I) or (II):

[0019]

[0020] wherein

[0021] R1is selected from optionally substituted C2-10alkyl, optionally substituted C3-10alkenyl, optionally substituted C3-10alkynyl, optionally substituted C3-10cycloalkyl, optionally substituted C3-10cycloalkenyl, optionally substituted C2-10heteroalkyl, C2-10haloalkyl;

[0022] R2and R3are each independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heteroalkyl, haloalkyl, optionally substituted heterocyclyl, or hydrogen, provided that at least one is not hydrogen; or

[0023] R2and R3together with the atom to which they are attached form a 3-7-membered saturated, or partially saturated heterocyclyl, optionally comprising at least one further N, O, and / or S; and

[0024] R4is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heteroalkyl, optionally substituted aryl, optionally substituted arylalkyl, or optionally substituted heterocyclyl.

[0025] In particular embodiments, the ionizable lipid or a pharmaceutically acceptable salt thereof may be a compound of Formula (Illa), (II lb), and / or (lllc):

[0026]

[0027] wherein

[0028] X1, X2, X3, X4are each independently selected from -O, -NH, or -S, preferably -O;

[0029] R5, R6, R7are each independently selected from C8-36alkyl or C8-36alkenyl;

[0030] A is a branched or linear aliphatic chain, preferably comprising 3 to 30 carbon atoms; and R1, R2, R3, and R4are as defined herein above.

[0031] In particular embodiments, the ionizable lipid or a pharmaceutically acceptable salt thereof may be a compound of Formula (IVa), (IVb), and / or (IVc):

[0032]

[0033] wherein

[0034] R8, R9is selected from optionally substituted alkyl, hydrogen, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted heteroalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, or optionally substituted aryl, optionally substituted arylalkyl; and

[0035] R1, R2, R3, R4, R5, R6, R7X1, X2, X3, X4are as defined herein above.

[0036] In particular embodiments, the ionizable lipid or a pharmaceutically acceptable salt thereof may be selected from the group consisting of:

[0037]

[0038] 

[0039]

[0040]

[0041] A further aspect provides a method for preparing the ionizable lipid according to an aspect of the present invention or a pharmaceutically acceptable salt thereof. The method comprises the steps of:

[0042] a) providing a lipid precursor molecule comprising a terminal alkyne;

[0043] b) contacting the lipid precursor molecule with an amine compound, comprising a primary amine separated by a chain of 2 to 10 atoms from an ionizable nitrogen selected from a secondary or tertiary amine functional group, in the presence of elemental sulfur, and optionally a base, thereby forming an ionizable lipid comprising a thioamide group separated from the ionizable nitrogen by a chain of 2 to 10 atoms.In particular embodiments, the lipid precursor molecule may comprise a structure that is either: configured to connect one or more hydrophobic tails upon deprotection and subsequent connection; or

[0044] comprises one or more hydrophobic tails, thereby forming a partially assembled lipid precursor molecule;

[0045] wherein each hydrophobic tail independently comprises a linear or branched aliphatic chain having 8 to 36 carbon atoms.

[0046] In particular embodiments, the structure comprises one or more reactive functional groups, or protected forms thereof, suitable for covalent attachment of hydrophobic tails. Such a structure is configured to connect one or more hydrophobic tails upon (optional) deprotection and subsequent connection.

[0047] A further aspect provides a method for modifying the apparent pKa of a lipid nanoparticle composition. The method comprises the step of incorporating at least one ionizable lipid according to an aspect of the invention or a pharmaceutically acceptable salt thereof into the lipid nanoparticle composition.

[0048] In particular embodiments, the lipid nanoparticle composition has a modified apparent pKa between 5.8 and 7.8, or between 5.9 and 7.8, or between 6.0 and 7.8, or between 6.0 and 7.7, or between 6.0 and 7.6, or between 6.0 and 7.5, or between 6.0 and 7.4, or between 6.0 and 7.3, or between 6.0 and 7.2, or between 6.0 and 7.1, more preferably between 6.0 and 7.0.

[0049] It should be noted that (preferred) embodiments of the ionizable lipid according to an aspect of the present invention or a pharmaceutically acceptable salt thereof or the method of its preparation are also (preferred) embodiments of the method for modifying the apparent pKa of a lipid nanoparticle composition as described herein.

[0050] A further aspect provides a lipid nanoparticle composition comprising at least one ionizable lipid according to an aspect of the invention or a pharmaceutically acceptable salt thereof.

[0051] In particular embodiments, the lipid nanoparticle composition further comprises at least one lipid selected from the group consisting of phospholipids, structural lipids, PEG-lipids, and mixtures thereof. In particular 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, at physiological pH.

[0052] In particular embodiments, the nucleic acid is at least any one selected from the group consisting of mRNA, siRNA, rRNA, DNA, aptamer, tRNA, antisense oligonucleotide, shRNA, miRNA, sgRNA, tracrRNA, gRNA, ribozyme, PNA, DNAzyme, and mixtures thereof.

[0053] In particular embodiments, the lipid nanoparticle composition may further comprise one or more trans-cyclooctene (TCO) moieties.

[0054] It should be noted that (preferred) embodiments of the ionizable lipid according to an aspect of the present invention or a pharmaceutically acceptable salt thereof, the method of its preparation; or the method for modifying the apparent pKa of a lipid nanoparticle composition are also (preferred) embodiments of the method for modifying the apparent pKa of a lipid nanoparticle composition as described herein.

[0055] Another aspect provides the lipid nanoparticle composition according to an aspect of the invention for use in medicine.

[0056] Another aspect provides a lipid nanoparticle composition according to an aspect of the invention for use in delivering a cargo compound to a cell, such as for transfecting a cell. The lipid nanoparticle composition may for example carry a nucleic acid molecule or construct that imparts a medical benefit on the organism that harbors the transfected cells, for example the nucleic acid molecule may be therapeutically useful or may encode an expression product that is therapeutically useful.

[0057] In particular embodiments, the cell is a mammalian cell, more preferably an immune cell, a cancer cell, a progenitor cell, or a structural cell in vivo.

[0058] It should be noted that (preferred) embodiments of the ionizable lipid according to an aspect of the present invention or a pharmaceutically acceptable salt thereof or the method of its preparation; are also (preferred) embodiments of the use thereof in medicine, such as for transfecting cells in vivo. 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 according to an aspect of the invention, preferably wherein the cell is a mammalian cell, more preferably an immune cell, a cancer cell, a progenitor cell, or a structural cell in vivo.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.

[0059] DESCRIPTION OF THE FIGURES

[0060] 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.

[0061] FIG. 1A-B: Transfection efficiency of the LNP compositions analyzed by flow cytometry analysis.

[0062] CT26 and HEK293T cells were treated for 24h with LNPs containing mRNA encoding for eGFP and PE-Cy5, at an mRNA dose of 50 and 200 ng / well, respectively (n=3).

[0063] FIG. 2: Quantification of in vivo bioluminescence (whole body) after 4 hours and after 24 hours post intravenous administration of LNPs. Data are presented as the mean luciferase radiance ± standard deviation, n=3. Statistical significance was calculated with one-way ANOVA (ns: non-significant; **p < 0.01; ***p < 0.001; ****p < 0.0001).

[0064] FIG. 3: Luciferase expression in liver and spleen after 24 hours. Data are presented as the mean luciferase radiance ± standard deviation, n=3. Statistical significance was calculated with one-way ANOVA (ns: non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

[0065] FIG. 4: Flow cytometry analysis of TdTomato expression in splenic macrophages (n=3, mean ± SD).

[0066] FIG. 5: Cy5 fluorescence measured using flow cytometry. eGFP mRNA LNPs were added to CD8+ T cells in a final concentration of 2 ng / μL. Cells were incubated for 24 hours at 37°C.

[0067] FIG. 6: eGFP fluorescence measured using flow cytometry. eGFP mRNA LNPs were added to CD8+ T cells in a final concentration of 2 ng / μL. Cells were incubated for 24 hours at 37°C.

[0068] FIG. 7: Cy5 fluorescence measured using flow cytometry in blood (A), liver (B) and spleen (C). anti-CD8 Nb-Tz was administered intravenously to WT C57BI / 6 mice. One hour later, Cy5 labeled LNPs with 1.5% TCO were injected.

[0069] FIG. 8: Transfection of activated human CD8+ T cells. Flow cytometry analysis of activated CD8+ T cells treated with 1 ng / μL SAM-3-APyr eGFP mRNA LNPs, showing quantification of the MFI of eGFP. All data are presented as the means ± standard deviation (SD) for three technical replicates (n = 3).

[0070] DETAILED DESCRIPTION OF THE INVENTIONWhen describing the invention, the terms used are to be construed in accordance with the following definitions, unless a context dictates otherwise.

[0071] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

[0072] The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims. 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.

[0073] As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a step" means one step or more than one step.

[0074] The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements, or method steps. The terms also encompass "consisting of" and "consisting essentially of", which enjoy well-established meanings in patent terminology.

[0075] Whereas the terms "one or more" or "at least one", such as one or more members or at least one member 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. In another example, "one or more" or "at least one" may refer to 1, 2, 3, 4, 5, 6, 7 or more.

[0076] The terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the disclosure described herein are capable of operation in other sequences than described or illustrated herein.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 two or 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.

[0077] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" or "in a particular embodiment" 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 certain 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.

[0078] The recitation of numerical ranges by endpoints includes all intervening values between the lower and upper endpoints, as well as the recited endpoints. Intervening values may be integers or, where applicable, fractions, i.e., more broadly any real numbers such as any rational numbers. This applies to numerical ranges irrespective of whether they are introduced by the expression "from... to..." or the expression "between... and..." or another expression. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, each sub-range between any stated value in a stated range and any other stated value in that stated range is also specifically disclosed. Each sub-range between any stated value in a stated range and either the lower endpoint or the upper endpoint of the stated range is also specifically disclosed. The stated value may be an isolated value or an endpoint of a range subsumed by or overlapping with the stated range. For example, for a stated range with lower endpoint LI and upper endpoint U1 (i.e., stated range Ll-Ul) and a stated sub-range nested within the stated range with lower endpoint L2 and upper endpoint U2 (i.e., stated sub-range L2-U2), also specifically disclosed are the subranges L1-L2, L1-U2, L2-U1, and U2-U1.

[0079] As used herein, the terms "about" or "approximately" are used to provide flexibility to a numerical value or range endpoint by providing that a given value may be "a little above" or "a little below" saidvalue or endpoint, depending on the specific context. Hence, the terms "about" or "approximately" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value or endpoint, such as variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention.

[0080] Unless otherwise stated, use of the terms "about" or "approximately" in accordance with a specific number or numerical range should also be understood to provide support for such numerical terms or range without the term "about". For example, the recitation of "about 30" should be construed as not only providing support for values a little above and a little below 30, but also for the actual numerical value of 30 as well.

[0081] As used herein, the term "substantially" refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, an object that is "substantially" enclosed would mean that the object is either completely enclosed or nearly completely enclosed. The exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context. However, generally speaking the nearness of completion will be so as to have the same overall result as if absolute and total completion were obtained. The use of "substantially" is equally applicable when used in a negative connotation to refer to the complete or near complete lack of an action, characteristic, property, state, structure, item, or result. 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.

[0082] Whenever the term "substituted" is used in the present invention, it 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. Where groups can be substituted, such groups may be substituted with one or more, and preferably one, two or three substituents.

[0083] For instance, a "substituted" alkyl, alkenyl, alkynyl, alkoxyl, aryl cycloalkyl, heteroalkyl, or heterocyclyl refers to an alkyl, alkenyl, alkynyl, alkoxyl, aryl cycloalkyl, heteroalkyl, or heterocyclyl group as defined herein having one or more substituent(s) (for example 1, 2 or 3 substituent(s), or 1 to 2 substituent(s)), at any available point of attachment. Preferred substituents may be selected from but not limited to, for example, a substituent selected from hydroxyl groups, alkoxy groups, alkyl groups, halogen groups,carboxyl groups, cyano groups, amino groups, and mono-or dialkylamino groups. More preferably, a substituted alkyl, alkenyl, alkynyl, alkoxyl, aryl cycloalkyl, heteroalkyl, or heterocyclyl may refer to an alkyl, alkenyl, alkynyl, alkoxyl, aryl cycloalkyl, heteroalkyl, or heterocyclyl group with one or more hydroxyl group(s) and / or alkoxyl group(s) as substituents.

[0084] Substituents optionally are designated with or without bonds. Regardless of bond indications, if a substituent is polyvalent (based on its position in the structure referred to), then any and all possible orientations of the substituent are intended.

[0085] The term "halo" or "halogen" as a group or part of a group is generic for fluoro, chloro, bromo, iodo. The term "hydroxyl" or "hydroxy" as used herein refers to the group -OH.

[0086] The term "amino" as used herein refers to the -NH2group.

[0087] The term "cyano" as used herein refers to the group -CEN.- The term "carboxy" or "carboxyl" or "hydroxycarbonyl" as used herein refers to the group -CO2H. The term "alkyl" as a group or part of a group, refers to a hydrocarbyl group of formula CnH2n+1wherein n is a number greater than or equal to 1, with no site of unsaturation. Alkyl groups may be linear or branched and may be substituted as indicated herein. When a subscript is used herein following a carbon atom, the subscript refers to the number of carbon atoms that the named group may contain. For example, the term " Ci.galkyl", as a group or part of a group, refers to a hydrocarbyl group of formula CnH2n+1wherein n is a number ranging from 1 to 6. Thus, for example, " Ci.gal kyl" includes all linear or branched alkyl groups with between 1 and 6 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. For example, C1-4alkyl includes all linear or branched alkyl groups having 1 to 4 carbon atoms, and thus includes for example methyl, ethyl, n-propyl, / -propyl, 2-methyl-ethyl, butyl, and its isomers (e.g., n-butyl, / -butyl, and t-butyl), and the like. Non-limiting examples of alkyl include methyl, ethyl, 1-propyl (n-propyl), 2-propyl ( / Pr), 1-butyl, 2-methyl-l-propyl(i-Bu), 2-butyl (s-Bu), 2-dimethyl-2-propyl (t-Bu), 1-pentyl (n-pentyl), 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-l-butyl, 2-methyl-l-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl, and n-icosyl.The term "alkenyl" as a group or part of a group, refers to an unsaturated hydrocarbyl group which may be linear, or branched, comprising one or more with at least one site (usually 1 to 3, preferably 1) of unsaturation, namely at least one sp2carbon-sp2carbon double bond. When a subscript is used herein following a carbon atom, the subscript refers to the number of carbon atoms that the named group may contain. Examples of C2-6alkenyl 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.

[0088] The term "alkynyl" as a group or part of a group, refers to a branched or straight chain hydrocarbon comprising at least one site (usually 1 to 3, preferably 1) of unsaturation, namely a sp1carbon-sp1carbon triple bond. In particular embodiments, the term alkynyl refers to hydrocarbons with at least one site (usually 1 to 3, preferably 1) of unsaturation, namely at least one sp1carbon-sp1carbon triple bond. Examples of alkynyl include but are not limited to: ethynyl (-C≡CH), 3-ethyl-cyclohept-1-ynylene, and 1-propynyl (propargyl, -CH₂C≡CH).

[0089] The term "cycloalkyl", as a group or part of a group, refers to a cyclic alkyl group, that is a monovalent, saturated, hydrocarbyl group having 1 or more cyclic structure, and comprising from 3 to 20 carbon atoms, more preferably from 3 to 10 carbon atoms, more preferably from 3 to 8 carbon atoms; more preferably from 3 to 6 carbon atoms. Cycloalkyl includes all saturated hydrocarbon groups containing one or more rings, including monocyclic, bicyclic groups or tricyclic. For example, cycloalkyl comprises a C3-10 monocyclic or C7-18 polycyclic saturated hydrocarbon, such as for instance cyclopropyl, cyclobutyl, cyclopentyl, cyclopropylethylene, methylcyclopropylene, cyclohexyl, cycloheptyl, cyclooctyl, cyclooctylmethylene, norbornyl, fenchyl, trimethyltricycloheptyl, decalinyl, adamantyl and the like. The further rings of multi-ring cycloalkyls may be either fused, bridged and / or joined through one or more spiro atoms. When a subscript is used herein following a carbon atom, the subscript refers to the number of carbon atoms that the named group may contain. For example, the term " C3-locycloalkyl", refers to a cyclic alkyl group comprising from 3 to 10 carbon atoms. For example, the term " Cs-gcycloalkyl", refers to a cyclic alkyl group comprising from 3 to 8 carbon atoms. For example, the term " Cs-ecycloalkyl", refers to a cyclic alkyl group comprising from 3 to 6 carbon atoms. For the avoidance of doubt, fused systems of a cycloalkyl ring with a heterocyclic ring are considered as heterocycle irrespective of the ring that is bound to the core structure. Fused systems of a cycloalkyl ring with an aryl ring are considered as aryl irrespective of the ring that is bound to the core structure. Fused systems of a cycloalkyl ring with a heteroaryl ring are considered as heteroaryl irrespective of the ring that is bound to the core structure.The term "cycloalkenyl" as a group or part of a group, refers to a non-aromatic cyclic alkenyl group, with at least one site (usually 1 to 3, preferably 1) of unsaturation, namely a sp2carbon-sp2carbon double bond; preferably from 5 to 18 carbon atoms, more preferably from 5 to 10 carbon atoms, more preferably from 5 to 6 carbon atoms. Cycloalkenyl includes all unsaturated hydrocarbon groups containing one or more rings, including monocyclic, bicyclic, or tricyclic groups. For example, cycloalkenyl can comprise C5-10 monocyclic or C7-18 polycyclic hydrocarbon. The further rings may be either fused, bridged and / or joined through one or more spiro atoms. When a subscript is used herein following a carbon atom, the subscript refers to the number of carbon atoms that the named group may contain. For example, the term " C5-iocycloalkenyl", refers to a cyclic alkenyl group comprising from 5 to 10 carbon atoms. For example, the term " C5.gcycloalkenyl", refers to a cyclic alkenyl group comprising from 5 to 8 carbon atoms. For example, the term " C5-6cycloalkyl", refers to a cyclic alkenyl group comprising from 5 to 6 carbon atoms. Examples include but are not limited to: cyclobutenyl, cyclopentenyl (-C5H7), cyclopentenylpropylene, methylcyclohexenylene, and cyclohexenyl (-C6H9). The double bond may be in the cis or trans configuration. For the avoidance of doubt, fused systems of a cycloalkenyl ring with a heterocyclic ring are considered as heterocycle irrespective of the ring that is bound to the core structure. Fused systems of a cycloalkenyl ring with an aryl ring are considered as aryl irrespective of the ring that is bound to the core structure. Fused systems of a cycloalkenyl ring with a heteroaryl ring are considered as heteroaryl irrespective of the ring that is bound to the core structure.

[0090] The term "cycloalkynyl" as a group or part of a group, to a non-aromatic hydrocarbon group preferably having from 8 to 18 carbon atoms with at least one site (usually 1 to 3, preferably 1) of unsaturation, namely a sp1carbon-sp1carbon triple bond and consisting of or comprising a C8-10monocyclic or C10-18polycyclic hydrocarbon. Examples include but are not limited to: cyclooctynyl, cyclononynyl, and the like. In particular embodiments, the term cycloalkynyl refers to C8-12cycloalkynyl, preferably to C8-10cycloalkynyl, yet more preferably to C8-9cycloalkynyl as further defined herein above with at least one site (preferably 1) of unsaturation, namely a sp1carbon-sp1carbon triple bond. For the avoidance of doubt, fused systems of a cycloalkynyl ring with a heterocyclic ring are considered as heterocycle irrespective of the ring that is bound to the core structure. Fused systems of a cycloalkynyl ring with an aryl ring are considered as aryl irrespective of the ring that is bound to the core structure. Fused systems of a cycloalkynyl ring with a heteroaryl ring are considered as heteroaryl irrespective of the ring that is bound to the core structure.

[0091] The term "alkoxy" or "alkyloxy" or "alkoxyl", as a group or part of a group, refers to a group of formula -ORawherein Rais alkyl as defined herein. Non-limiting examples of suitable Ci.galkoxy includemethoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, and hexyloxy.

[0092] The term "haloalkyl", as a group or part of a group, refers to an alkyl group having the meaning as defined herein, wherein one or more hydrogen atoms are each replaced with a halogen as defined herein. Non-limiting examples of such haloalkyl groups include chloromethyl, 1-bromoethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1,1,1-trifluoroethyl and the like.

[0093] The term "heteroalkyl" as a group or part of a group, refers to an alkyl group having the meaning as defined herein, wherein one or more carbon atoms in the carbon chain are replaced by heteroatoms such as nitrogen, oxygen, or sulfur. Non-limiting examples of heteroalkyl groups include methoxyethyl -CH2CHOCH2-, thiomethyl (-CH2S-), and aminopropyl (-CH2CH2CH2NH2).

[0094] The term "aryl", as a group or part of a group, refers to a polyunsaturated, aromatic hydrocarbyl group having a single ring (i.e. phenyl) or multiple aromatic rings fused together (e.g. naphthyl), or linked covalently, typically comprising 6 to 12 carbon atoms; wherein at least one ring is aromatic, preferably comprising 6 to 10 carbon atoms, wherein at least one ring is aromatic. The aromatic ring may optionally include one to two additional rings (either cycloalkyl, heterocyclyl or heteroaryl) fused thereto. Examples of suitable aryl include C6-12aryl, preferably C6-10aryl, more preferably C6-8aryl. Nonlimiting examples of aryl comprise phenyl, biphenylyl, biphenylenyl, or 1-or 2-naphthanelyl; 5- or 6-tetralinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-azulenyl, 4-, 5-, 6 or 7-indenyl, 4- or 5-indanyl, 5-, 6-, 7- or 8- The term "arylalkyl", as a group or part of a group, means a alkyl as defined herein, wherein at least one hydrogen atom is replaced by at least one aryl as defined herein. Non-limiting examples of arylalkyl group include benzyl, phenethyl, dibenzylmethyl, methylphenylmethyl, 3-(2-naphthyl)-butyl, and the like.

[0095] The terms "heterocyclyl" or "heterocycloakyl" or "heterocyclo", as a group or part of a group, refer to non-aromatic, fully saturated or partially unsaturated cyclic groups (for example, 3 to 7 member monocyclic, 7 to 11 member bicyclic, or comprising a total of 3 to 10 ring atoms) which have at least one heteroatom in at least one carbon atom-containing ring; wherein said ring may be fused to an aryl, cycloalkyl, heteroaryl or heterocyclyl ring. Each ring of the heterocyclyl group containing a heteroatom may have 1, 2, 3 or 4 heteroatoms selected from N, O and / or S, where the N and S heteroatoms may optionally be oxidized and the N heteroatoms may optionally be quaternized, and wherein at least one carbon atom of heterocyclyl can be oxidized to form at least one C=O. The heterocyclic group may be attached at any heteroatom or carbon atom of the ring or ring system,where valence allows. The rings of multi-ring heterocycles may be fused, bridged and / or joined through one or more spiro atoms.

[0096] Non limiting exemplary heterocyclic groups include aziridinyl, oxiranyl, thiiranyl, piperidinyl, azetidinyl, oxetanyl, pyrrolidinyl, thietanyl, 2-imidazolinyl, pyrazolidinyl imidazolidinyl, isoxazolinyl, oxazolidinyl, isoxazolidinyl, thiazol idinyl, isothiazol idinyl, succinimidyl, 3H-indolyl, indolinyl, chromanyl (also known as 3,4-dihydrobenzo[b]pyranyl), isoindolinyl, 2H-pyrrolyl, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, 4H-quinolizinyl, 2-oxopiperazinyl, piperazinyl, homopiperazinyl, 2-pyrazolinyl, 3-pyrazolinyl, tetrahydro-2H-pyranyl, 2H-pyranyl, 4H-pyranyl, 3,4-dihydro-2H-pyranyl, 3-dioxolanyl, 1,4-dioxanyl, 2,5-dioximidazolidinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, indolinyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydroquinolinyl, tetrahydroisoquinolin-l-yl, tetrahydroisoquinolin-2-yl, tetrahydroisoquinolin-3-yl, tetrahydroisoquinolin-4-yl, thiomorpholin-4-yl, thiomorpholin-4-ylsulfoxide, thiomorpholin-4-ylsulfone, 1, 3-dioxolanyl, 1,4-oxathianyl, 1,4-dithianyl, 1,3,5-trioxanyl, lH-pyrrolizinyl, tetrahydro-1, 1-dioxothiophenyl, N- formylpiperazinyl, and morpholin-4-yl. The term "aziridinyl" as used herein includes aziridin-l-yl and aziridin-2-yl. The term "oxyranyl" as used herein includes oxyranyl-2-yl. The term "thiiranyl" as used herein includes thiiran-2-yl. The term "azetidinyl" as used herein includes azetidin-l-yl, azetidin-2-yl and azetidin-3-yl. The term "oxetanyl" as used herein includes oxetan-2-yl and oxetan-3-yl. The term "thietanyl" as used herein includes thietan-2-yl and thietan-3-yl. The term "pyrrolidinyl" as used herein includes pyrrolidin-l-yl, pyrrolidin-2-yl and pyrrolidin-3-yl. The term "tetrahydrofuranyl" as used herein includes tetrahydrofuran-2-yl and tetrahydrofuran-3-yl. The term "tetrahydrothiophenyl" as used herein includes tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl. The term "succinimidyl" as used herein includes succinimid-l-yl and succininmid-3-yl. The term "dihydropyrrolyl" as used herein includes 2,3-dihydropyrrol-l-yl, 2,3-dihydro-lH-pyrrol-2-yl, 2,3-dihydro-lH-pyrrol-3-yl, 2,5-dihydropyrrol-l-yl, 2,5-dihydro-lH-pyrrol-3-yl and 2,5-dihydropyrrol-5-yl. The term "2H-pyrrolyl" as used herein includes 2H-pyrrol-2-yl, 2H-pyrrol-3-yl, 2H-pyrrol-4-yl and 2H-pyrrol-5-yl. The term "3H-pyrrolyl" as used herein includes 3H-pyrrol-2-yl, 3H-pyrrol-3-yl, 3H-pyrrol-4-yl and 3H-pyrrol-5-yl. The term "dihydrofuranyl" as used herein includes 2,3-dihydrofuran-2-yl, 2,3-dihydrofuran-3-yl, 2,3-dihydrofuran-4-yl, 2,3-dihydrofuran-5-yl, 2,5-dihydrofuran-2-yl, 2,5-dihydrofuran-3-yl, 2,5-dihydrofuran-4-yl and 2,5-dihydrofuran-5-yl. The term "dihydrothiophenyl" as used herein includes 2,3-dihydrothiophen-2-yl, 2,3-dihydrothiophen-3-yl, 2,3-dihydrothiophen-4-yl, 2,3-dihydrothiophen-5-yl, 2,5-dihydrothiophen-2-yl, 2,5-dihydrothiophen-3-yl, 2,5-dihydrothiophen-4-yl and 2,5-dihydrothiophen-5-yl. The term "imidazolidinyl" as used herein includes imidazolidin-l-yl, imidazolidin-2-yl and imidazolidin-4-yl. The term "pyrazolidinyl" as used herein includes pyrazolidin-1-yl, pyrazolidin-3-yl and pyrazolidin-4-yl. The term "imidazolinyl" as used herein includes imidazolin- 1-yl, imidazolin-2-yl, imidazolin-4-yl and imidazolin-5-yl. The term "pyrazolinyl" as used herein includes l-pyrazolin-3-yl, l-pyrazolin-4-yl, 2-pyrazolin-l-yl, 2-pyrazolin-3-yl, 2-pyrazolin-4-yl, 2-pyrazolin-5-yl, 3-pyrazolin-l-yl, 3-pyrazolin-2-yl, 3-pyrazolin-3-yl, 3-pyrazolin-4-yl and 3-pyrazolin-5-yl. The term "dioxolanyl" also known as "1,3-dioxolanyl" as used herein includes dioxolan-2-yl, dioxolan-4-yl and dioxolan-5-yl. The term "dioxolyl" also known as "1,3-dioxolyl" as used herein includes dioxol- 2-yl, dioxol-4-yl and dioxol-5-yl. The term "oxazolidinyl" as used herein includes oxazolidin-2-yl, oxazolidin-3-yl, oxazolidin-4-yl and oxazolidin-5-yl. The term "isoxazolidinyl" as used herein includes isoxazolidin-2-yl, isoxazolidin-3-yl, isoxazolidin-4-yl and isoxazolidin-5-yl. The term "oxazolinyl" as used herein includes 2-oxazolinyl-2-yl, 2-oxazolinyl-4-yl, 2-oxazolinyl-5-yl, 3-oxazolinyl-2-yl, 3-oxazolinyl-4-yl, 3-oxazolinyl-5-yl, 4-oxazolinyl-2-yl, 4-oxazolinyl-3-yl, 4-oxazolinyl-4-yl and 4-oxazolinyl-5-yl. The term "isoxazolinyl" as used herein includes 2-isoxazolinyl-3-yl, 2-isoxazolinyl-4-yl, 2-isoxazolinyl-5-yl, 3-isoxazolinyl-3-yl, 3-isoxazolinyl-4-yl, 3-isoxazolinyl-5-yl, 4-isoxazolinyl-2-yl, 4-isoxazolinyl-3-yl, 4-isoxazolinyl-4-yl and 4-isoxazolinyl-5-yl. The term "thiazolidinyl" as used herein includes thiazolidin-2-yl, thiazolidin-3-yl, thiazolidin-4-yl and thiazolidin-5-yl. The term "isothiazolidinyl" as used herein includes isothiazolidin-2-yl, isothiazolidin-3-yl, isothiazolidin-4-yl and isothiazolidin-5-yl. The term "chromanyl" as used herein includes chroman-2-yl, chroman-3-yl, chroman-4-yl, chroman-5-yl, chroman-6-yl, chroman-7-yl and chroman-8-yl. The term "thiazolinyl" as used herein includes 2-thiazolinyl-2-yl, 2-thiazolinyl-4-yl, 2-thiazolinyl-5-yl, 3-thiazolinyl-2-yl, 3-thiazolinyl-4-yl, 3-thiazolinyl-5-yl, 4-thiazolinyl-2-yl, 4-thiazolinyl-3-yl, 4-thiazolinyl-4-yl and 4-thiazolinyl-5-yl. The term "isothiazolinyl" as used herein includes 2-isothiazolinyl-3-yl, 2-isothiazolinyl-4-yl, 2-isothiazolinyl-5-yl, 3-isothiazolinyl-3-yl, 3-isothiazolinyl-4-yl, 3-isothiazolinyl-5-yl, 4-isothiazolinyl-2-yl, 4-isothiazolinyl-3-yl, 4-isothiazolinyl-4-yl and 4-isothiazolinyl-5-yl. The term "piperidyl" also known as "piperidinyl" as used herein includes piperid-l-yl, piperid-2-yl, piperid-3-yl and piperid-4-yl. The term "dihydropyridinyl" as used herein includes 1,2-dihydropyridin-l-yl, 1,2-dihydropyridin-2-yl, l,2-dihydropyridin-3-yl, l,2-dihydropyridin-4-yl, l,2-dihydropyridin-5-yl, 1,2-dihydropyridin-6-yl, 1,4-dihydropyridin-l-yl, l,4-dihydropyridin-2-yl, l,4-dihydropyridin-3-yl, 1,4-dihydropyridin-4-yl, 2,3-dihydropyridin-2-yl, 2,3-dihydropyridin-3-yl, 2,3-dihydropyridin-4-yl, 2,3-dihydropyridin-5-yl, 2,3-dihydropyridin-6-yl, 2,5-dihydropyridin-2-yl, 2,5-dihydropyridin-3-yl, 2,5-dihydropyridin-4-yl, 2,5-dihydropyridin-5-yl, 2,5-dihydropyridin-6-yl, 3,4-dihydropyridin-2-yl, 3,4-dihydropyridin-3-yl, 3,4-dihydropyridin-4-yl, 3,4-dihydropyridin-5-yl and 3,4-dihydropyridin-6-yl. The term "tetrahydropyridinyl" as used herein includes 1,2,3,4-tetrahydropyridin-l-yl, 1, 2,3,4-tetrahydropyridin-2-yl, l,2,3,4-tetrahydropyridin-3-yl, l,2,3,4-tetrahydropyridin-4-yl, 1, 2,3,4-tetrahydropyridin-5-yl, l,2,3,4-tetrahydropyridin-6-yl, 1,2,3,6-tetrahydropyridin-l-yl, 1, 2,3,6-tetrahydropyridin-2-yl. l,2,3,6-tetrahydropyridin-3-yl, l,2,3,6-tetrahydropyridin-4-yl, 1, 2,3,6-tetrahydropyridin-5-yl, l,2,3,6-tetrahydropyridin-6-yl, 2,3,4,5-tetrahydropyridin-2-yl, 2, 3,4,5-tetrahydropyridin-3-yl, 2,3,4,5-tetrahydropyridin-3-yl, 2,3,4,5-tetrahydropyridin-4-yl, 2, 3,4,5-tetrahydropyridin-5-yl and 2,3,4,5-tetrahydropyridin-6-yl. The term "tetrahydropyranyl" also known as "oxanyl" or "tetrahydro-2H-pyranyl", as used herein includes tetrahydropyran-2-yl, tetrahydropyran-3-yl and tetrahydropyran-4-yl. The term "2H-pyranyl" as used herein includes 2H-pyran-2-yl, 2H-pyran-3-yl, 2H-pyran-4-yl, 2H-pyran-5-yl and 2H-pyran-6-yl. The term "4H-pyranyl" as used herein includes 4H-pyran-2-yl, 4H-pyran-3-yl and 4H-pyran-4-yl. The term "3,4-dihydro-2H-pyranyl" as used herein includes 3,4-dihydro-2H-pyran-2-yl, 3,4-dihydro-2H-pyran-3-yl, 3,4-dihydro-2H-pyran-4-yl, 3,4-dihydro-2H-pyran-5-yl and 3,4-dihydro-2H-pyran-6-yl. The term "3,6-dihydro-2H-pyranyl" as used herein includes 3,6-dihydro-2H-pyran-2-yl, 3,6-dihydro-2H-pyran-3-yl, 3,6-dihydro-2H-pyran-4-yl, 3,6-dihydro-2H-pyran-5-yl and 3,6-dihydro-2H-pyran-6-yl. The term "tetrahydrothiophenyl", as used herein includes tetrahydrothiophen-2-yl, tetrahydrothiophenyl -3-yl and tetrahydrothiophenyl -4-yl. The term "2H-thiopyranyl" as used herein includes 2H-thiopyran-2-yl, 2H-thiopyran-3-yl, 2H-thiopyran-4-yl, 2H-thiopyran-5-yl and 2H-thiopyran-6-yl. The term "4H-thiopyranyl" as used herein includes 4H-thiopyran-2-yl, 4H-thiopyran-3-yl and 4H-thiopyran-4-yl. The term "3,4-dihydro-2H-thiopyranyl" as used herein includes 3,4-dihydro-2H-thiopyran-2-yl, 3,4-dihydro-2H-thiopyran-3-yl, 3,4-dihydro-2H-thiopyran-4-yl, 3,4-dihydro-2H-thiopyran-5-yl and 3,4-dihydro-2H-thiopyran-6-yl. The term "3,6-dihydro-2H-thiopyranyl" as used herein includes 3,6-dihydro-2H-thiopyran-2-yl, 3,6-dihydro-2H-thiopyran-3-yl, 3,6-dihydro-2H-thiopyran-4-yl, 3,6-dihydro-2H-thiopyran-5-yl and 3,6-dihydro-2H-thiopyran-6-yl. The term "piperazinyl" also known as "piperazidinyl" as used herein includes piperazin-l-yl and piperazin-2-yl. The term "morpholinyl" as used herein includes morpholin-2-yl, morpholin-3-yl and morpholin-4-yl. The term "thiomorpholinyl" as used herein includes thiomorpholin-2-yl, thiomorpholin-3-yl and thiomorpholin-4-yl. The term "dioxanyl" as used herein includes l,2-dioxan-3-yl, l,2-dioxan-4-yl, l,3-dioxan-2-yl, l,3-dioxan-4-yl, l,3-dioxan-5-yl and l,4-dioxan-2-yl. The term "dithianyl" as used herein includes l,2-dithian-3-yl, 1,2-dithian-4-yl, l,3-dithian-2-yl, l,3-dithian-4-yl, l,3-dithian-5-yl and l,4-dithian-2-yl. The term "oxathianyl" as used herein includes oxathian-2-yl and oxathian-3-yl. The term "trioxanyl" as used herein includes l,2,3-trioxan-4-yl, l,2,3-trioxay-5-yl, l,2,4-trioxay-3-yl, l,2,4-trioxay-5-yl, 1,2,4-trioxay-6-yl and l,3,4-trioxay-2-yl. The term "azepanyl" as used herein includes azepan-l-yl, azepan-2-yl, azepan-l-yl, azepan-3-yl and azepan-4-yl. The term "homopiperazinyl" as used herein includes homopiperazin-l-yl, homopiperazin-2-yl, homopiperazin-3-yl and homopiperazin-4-yl. The term "indolinyl" as used herein includes indolin-l-yl, indolin-2-yl, indolin-3-yl, indolin-4-yl, indolin-5-yl, indolin-6-yl, and indolin-7-yl. The term "quinolizinyl" as used herein includes quinolizidin-l-yl,quinol izidin-2-yl, quinolizidin-3-yl and quinol izidin-4-yl. The term "isoindol inyl" as used herein includes isoindolin-l-yl, isoindolin-2-yl, isoindolin-3-yl, isoindolin-4-yl, isoindolin-5-yl, isoindolin-6-yl, and isoindolin-7-yl. The term "3H-indolyl" as used herein includes 3H-indol-2-yl, 3H-indol-3-yl, 3H-indol-4-yl, 3H-indol-5-yl, 3H-indol-6-yl, and 3H-indol-7-yl. The term "quinolizinyl" as used herein includes quinolizidin-l-yl, quinolizidin-2-yl, quinolizidin-3-yl and quinolizidin-4-yl. The term "quinolizinyl" as used herein includes quinolizidin-l-yl, quinolizidin-2-yl, quinolizidin-3-yl and quinolizidin-4-yl. The term "tetrahydroquinolinyl" as used herein includes tetrahydroquinolin-l-yl, tetrahydroquinolin-2-yl, tetrahydroquinolin-3-yl, tetrahydroquinolin-4-yl, tetrahydroquinolin-5-yl, tetrahydroquinolin-6-yl, tetrahydroquinolin-7-yl and tetrahydroquinolin-8-yl. The term "tetrahydroisoquinolinyl" as used herein includes tetrahydroisoquinolin-l-yl, tetrahydroisoquinolin-2-yl, tetrahydroisoquinolin-3-yl, tetrahydroisoquinolin-4-yl, tetrahydroisoquinolin-5-yl, tetrahydroisoquinolin-6-yl, tetrahydroisoquinolin-7-yl and tetrahydroisoquinolin-8-yl. The term "lH-pyrrolizine" as used herein includes lH-pyrrolizin-l-yl, lH-pyrrolizin-2-yl, lH-pyrrolizin-3-yl, lH-pyrrolizin-5-yl, lH-pyrrolizin-6-yl and lH-pyrrolizin-7-yl. The term "3H-pyrrolizine" as used herein includes 3H-pyrrolizin-l-yl, 3H-pyrrolizin-2-yl, 3H-pyrrolizin-3-yl, 3H-pyrrol izin-5-yl, 3H-pyrrolizin-6-yl and 3H-pyrrol izin-7-yl.

[0097] Any reference to "an ionizable lipid according to the invention", or "an ionizable lipid being a compound of formula (l)-(IVc)" also includes isomers such as stereoisomers and tautomers, salts such as pharmaceutically and / or physiologically acceptable salts, hydrates, solvates, polymorphs of such compounds unless expressly indicated otherwise.

[0098] The term "isomers" as used herein means all possible isomeric forms, including tautomeric and stereochemical forms, which the ionizable lipids of formulae herein may possess, but not including position isomers. Typically, the structures shown herein exemplify one tautomeric or resonance form of the ionizable lipids, but the corresponding alternative configurations are contemplated as well. Depending on its substitution pattern, the ionizable lipids described herein may or may not have one or more optical stereocenters and may or may not exist as different enantiomers or diastereomers. Any such enantiomers, diastereomers or other optical isomers are encompassed by the scope of the invention. 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 center) of the basic molecular structure, as well as the stereochemically pure or enriched lipid compounds.

[0099] The term "salts" relates to any salts that the ionizable lipids may form. They can be pharmaceutically acceptable. Therefore, the ionizable lipids optionally comprise salts of the ionizable lipid herein,especially pharmaceutically acceptable non-toxic salts containing, for example, HO', Cl', Br, I' and OAc. Such salts may include those derived by combination of appropriate cations such as alkali and alkaline earth metal ions or ammonium and quaternary amino ions with an acid anion moiety, typically a carboxylic acid. The ionizable lipids may bear multiple positive charges. The net charge of the ionizable lipids may be positive. Any associated counter ions are typically dictated by the synthesis and / or isolation methods by which the ionizable lipids are obtained. Typical counter ions include, but are not limited to hydroxides, halides, acetates, trifluoroacetates, etc., and mixtures thereof. It will be understood that the identity of any associated counter ion is not a critical feature of the invention, and that the invention encompasses the ionizable lipids in association with any type of counter ion. Moreover, as the ionizable lipids can exist in a variety of different forms, the invention is intended to encompass not only forms of the ionizable lipids that are in association with counter ions (e.g., dry salts), but also forms that are not in association with counter ions (e.g., aqueous or organic solutions). Furthermore, this term also includes the solvates which the ionizable lipids of formulae herein as well as their salts are able to form, such as for example hydrates, alcoholates and the like. Finally, it is to be understood that the lipid nanoparticle compositions described herein can comprise ionizable lipids as defined herein in their unionized, as well as zwitterionic form, and combinations with stoichiometric amounts of water as in hydrates.

[0100] The present invention includes within its scope solvates of the ionizable lipids as defined herein. The term "solvates" refers to crystals formed by an active compound and a second component (solvent) which, in isolated form, is liquid at room temperature. Such solvates may be formed with common organic solvents, e.g., hydrocarbon solvents such as benzene or toluene; chlorinated solvents such as chloroform or dichloromethane; alcoholic solvents such as methanol, ethanol, or isopropanol; ethereal solvents such as diethyl ether or tetrahydrofuran; or ester solvents such as ethyl acetate. Alternatively, the solvates of the ionizable lipids herein may be formed with water, in which case they will be hydrates.

[0101] When referring to compositions and products and the weight percent of the therein comprised ingredients, it is to be understood that according to the present invention the overall amount of ingredients does not exceed 100 % (± 1 % due to rounding).

[0102] In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as beingpreferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0103] As mentioned above, the present description describes technology centered on ionizable lipids and lipid nanoparticles (LNPs) designed for efficient delivery of (ionizable) cargo compounds, such as nucleic acids, to target cells or tissues. The invention leverages the structural properties of ionizable lipids, particularly their head group chemistry, to be able to tune the apparent pKa of LNP formulated therefrom. This tuneability can allow precise control over transfection efficiency and cytotoxicity, optimizing the delivery process for various therapeutic applications.

[0104] In view of these surprising findings, an aspect of the present invention relates to an ionizable lipid or a pharmaceutically acceptable salt thereof, comprising a moiety, the moiety comprising:

[0105] an ionizable nitrogen, preferably selected from a secondary or tertiary amine functional group, and;

[0106] a thioamide group, wherein the thioamide group is separated from the ionizable nitrogen by a chain of 2 to 10 atoms.

[0107] The term "ionizable lipid" has a well-established meaning within the art and is used herein as such. Specifically, the term generally refers to a lipid or amphiphilic molecule comprising a hydrophobic part and at least one ionizable head group that can acquire a positive charge under acidic conditions, such as those encountered in the endosomal environment, while preferably remaining neutral or nearneutral at physiological pH.

[0108] As used herein, the term "moiety" generally refers to a specific part or functional group of a molecule that contributes to its chemical or functional properties. In the context of the present invention, the term specifically refers to the structural or functional groups that form part of the ionizable lipid, and in particular form at least a part of the head group chemistry. It has been found herein that this specific part of the present ionizable lipid(s) comprising an ionizable nitrogen adjacent to a thioamide group plays an important role in modulating the apparent pKa, hydrophobicity, and charge activation properties of a lipid nanoparticle composition comprising said ionizable lipid(s).

[0109] As used herein, the term "ionizable nitrogen" refers to a nitrogen atom within a functional group that can undergo protonation or deprotonation depending on the surrounding pH, thereby acquiring a positive charge or returning to a neutral state. In the present context, the term encompasses secondary amines, tertiary amines, and optionally other nitrogen-containing functional groups capable of reversible ionization under physiological or endosomal conditions.Non-limiting examples of suitable nitrogen-containing functional groups are:

[0110]

[0111] wherein Rc, Rd, Re, Rfare each independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heteroalkyl, haloalkyl, optionally substituted heterocyclyl.

[0112] It should be clear that the present invention also encompasses structures wherein the above-listed nitrogen-containing functional groups may be substituted with one or more alkyl groups, hydroxyl groups, alkoxy groups, and / or halogen groups. For instance, when considering a 5-membered pyrrolidine-ring, non-limiting suitable examples of substituted structures include:

[0113]

[0114] It is apparent to the person skilled in the art that alkyl substituents are not limited to the methyl or ethyl substituents exemplified above for the substituted 5-membered pyrrolidine structure and may also include, among others, propyl or butyl substituents in various isomeric forms. Furthermore, similar considerations apply to any of the suitable nitrogen-containing functional groups or (sub)structures described herein.

[0115] As stated above, the present moiety is characterized in that the ionizable nitrogen is separated from a thioamide group by a chain of 2 to 10 atoms. The term "thioamide" refers to a functional group that is well-known in the art, which is characterized by a sulfur atom doubly bonded to a carbon atom, which is also singly bonded to a nitrogen atom (-C(=S)-NH-). The phrase "separated by a chain of 2 to 10 atoms" indicates that there are 2 to 10 covalently bonded atoms forming a linear or branched chain between the ionizable nitrogen atom and the thioamide group. These atoms in the chain may include carbon, nitrogen, oxygen, or sulfur atoms. The chain may optionally include unsaturated bonds, aromatic rings, or cyclic structures (both saturated or unsaturated), provided that the shortest covalent distance between the thioamide group and the ionizable nitrogen consists of 2 to 10 atoms. In embodiments of the present invention, the moiety comprises:

[0116] an ionizable nitrogen selected from a secondary or tertiary amine functional group, and1

[0117] a secondary thioamide group, wherein the thioamide group is separated from the ionizable nitrogen by a chain of 2 to 10 atoms.

[0118] The term "secondary thioamide group" as used herein refers to a thioamide group where the nitrogen atom is secondary (i.e. bonded to two carbon atoms and bearing one hydrogen).

[0119] In particular embodiments, the present moiety is characterized in that the ionizable nitrogen is separated from a thioamide group by a chain of 2 to 9 atoms, or 2 to 8 atoms, or 2 to 6 atoms, or 2 to 5 atoms. Advantageously, it has been found herein that the specific arrangement of the thioamide and ionizable nitrogen as disclosed herein may provide flexibility in tuning the spatial and electronic properties of the ionizable lipid.

[0120] In particular embodiments, the moiety as described herein may be represented by the general formula (I) or (II):

[0121]

[0122] wherein R1is selected from optionally substituted C2-10alkyl, optionally substituted C3- loalkenyl, optionally substituted C3-10alkynyl, optionally substituted C3-10cycloalkyl, optionally substituted C3-10cycloalkenyl, optionally substituted C2-10heteroalkyl, C2-10haloalkyl;

[0123] R2and R3are each independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heteroalkyl, haloalkyl, optionally substituted heterocyclyl, or hydrogen, provided that at least one is not hydrogen; or

[0124] R2and R3together with the atom to which they are attached form a 3-7-membered saturated, or partially saturated heterocyclyl, optionally comprising at least one further N, O, and / or S; and

[0125] R4is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl,optionally substituted cycloalkynyl, optionally substituted heteroalkyl, optionally substituted aryl, optionally substituted arylalkyl, or optionally substituted heterocyclyl.

[0126] Some non-limiting examples of suitable moiety structures include:

[0127]

[0128]

[0129] In particular embodiments, the moiety as described herein may be represented by the general formula (I) or (II) withR1selected from optionally substituted C2-10alkyl, optionally substituted C3-10alkenyl, optionally substituted C3-10alkynyl, optionally substituted C3-10cycloalkyl, optionally substituted C3-10cycloalkenyl, optionally substituted C2-10heteroalkyl, C2-10haloalkyl;

[0130] R2and R3each independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heteroalkyl, haloalkyl, optionally substituted heterocyclyl, or hydrogen, provided that at least one is not hydrogen; or

[0131] R2and R3together with the atom to which they are attached form a 3-7-membered saturated, or partially saturated heterocyclyl, optionally comprising at least one further N, O, and / or S; and R4selected from optionally substituted C2-10alkyl, optionally substituted C3-10alkenyl, optionally substituted C3-10alkynyl, optionally substituted C3-7cycloalkyl, optionally substituted C3-7cycloalkenyl, optionally substituted Cs-ycycloalkynyl, optionally substituted C2-10heteroalkyl, optionally substituted phenyl, optionally substituted benzyl, or optionally substituted C3-7heterocyclyl.

[0132] In exemplary embodiments, the present moiety may be represented by the general formula (I) or (II) described above with

[0133] R1selected from optionally substituted C2-4alkyl, optionally substituted C3-4alkenyl, optionally substituted C3-4alkynyl, optionally substituted C3-7cycloalkyl, optionally substituted C3-7cycloalkenyl, C2-4heteroalkyl, C2-4haloalkyl;

[0134] R2and R3each independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heteroalkyl, haloalkyl, optionally substituted heterocyclyl, or hydrogen, provided that at least one is not hydrogen; and

[0135] R4selected from optionally substituted C2-10alkyl, optionally substituted C3-10alkenyl, optionally substituted C3-10alkynyl, optionally substituted C3-7cycloalkyl, optionally substituted C3-7cycloalkenyl, optionally substituted Cs-ycycloalkynyl, optionally substituted C2-10heteroalkyl, optionally substituted phenyl, optionally substituted benzyl, or optionally substituted C3-7heterocyclyl.

[0136] In exemplary embodiments, the present moiety may be represented by the general formula (I) or (II) described above withR1selected from optionally substituted C2-6alkyl, optionally substituted C3-6alkenyl, optionally substituted C3-6alkynyl, optionally substituted C3-7cycloalkyl, optionally substituted C3-7cycloalkenyl, C2-6heteroalkyl, C2-6haloalkyl;

[0137] R2and R3together with the atom to which they are attached form a 3-7-membered saturated, or partially saturated heterocyclyl, optionally comprising at least one further N, O, and / or S; and R4selected from optionally substituted C2-10alkyl, optionally substituted C3-10alkenyl, optionally substituted C3-10alkynyl, optionally substituted C3-7cycloalkyl, optionally substituted C3-7cycloalkenyl, optionally substituted Cs-ycycloalkynyl, optionally substituted C2-10heteroalkyl, optionally substituted phenyl, optionally substituted benzyl, or optionally substituted C3-7heterocyclyl.

[0138] Next to the moiety, the ionizable lipid may comprise one or more hydrophobic tails, which constitute the hydrophobic part of the lipid structure. The term "hydrophobic tail (s)" as used herein refers to one or more apolar, hydrophobic chains covalently attached, or attachable, to the moiety (head group) of the present ionizable lipid. These hydrophobic chains typically comprise saturated or unsaturated hydrocarbon chains, optionally branched, with lengths ranging from 8 to 36 carbon atoms.

[0139] In particular embodiments, the one or more hydrophobic tails are connected to the thioamide group via the carbon atom of the thiocarbonyl group (C(=S)). The skilled person understands that, in the present context, said connection may be direct or indirect, such as wherein a linker or linker structure is interposed between the hydrophobic tails and the C of the thioamide.

[0140] In particular embodiments, each hydrophobic tail may independently comprise a linear or branched aliphatic chain having 8 to 36 carbon atoms, or 10 to 36 carbon atoms, or 10 to 34 carbon atoms, or 10 to 32 carbon atoms, or 10 to 30 carbon atoms, or 10 to 28 carbon atoms, or 10 to 26 carbon atoms, preferably 10 to 24 carbon atoms, or 10 to 22 carbon atoms, or 10 to 20 carbon atoms, or 10 to 18 carbon atoms.

[0141] It should be noted that the present invention is not particularly limited by the number or type of hydrophobic tails, provided that the moiety (head group) is attached to a sufficiently hydrophobic structure to constitute an ionizable lipid suitable for assembling lipid nanoparticles. For instance, the ionizable lipid may comprise one, two, three, four, five, six, or more hydrophobic tails.

[0142] When the ionizable lipid comprises two or more hydrophobic tails, each hydrophobic tail may be different or at least a part of the hydrophobic tails may be identical. Put differently, the presentionizable lipids may comprise asymmetric, partially symmetric, or symmetric hydrophobic tail structures.

[0143] In particular embodiments, each hydrophobic tail may comprise any one of the following structures or substructures:

[0144]

[0145] wherein Rbis selected from C8-36alkyl or C8-36alkenyl, or Cio-gealkyl or Cio-gsalkenyl, or Cio-2galkyl or Cio-2galkenyl, or Cio-26alkyl or Cio-26alkenyl, preferably Cio-24alkyl or Cio-24alkenyl.

[0146] In particular embodiments, Rbis selected from 2-hexyldecyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, eicosyl, docosyl, tetracosyl, hexadecenyl, octadecenyl, eicosenyl, docosenyl, octadecadienyl, octadecatrienyl, eicosatetraenyl, eicosapentaenyl, docosahexaenyl, phytanyl, farnesyl, 2-octyldecyl, iso-octyl, iso-decyl, 2,6-10-trimethylundecyl, and squalenyl.

[0147] In an exemplary embodiment, the ionizable lipid comprises two hydrophobic tails comprising two 2-hexyldecyl chains. In another exemplary embodiment, the ionizable lipid comprises two octadecenyl chains.

[0148] In the present context, the one or more hydrophobic tails are covalently connected or connectable to the ionizable head group either directly (e.g., through a single covalent bond) or via a suitable linker. The term "linker" or "linker structure" as used herein generally refers to a molecular unit or group that is positioned between the one or more hydrophobic tails and the ionizable head group of the lipid. Its primary function is to act as a structural spacer that connects the hydrophobic part of the lipid to the hydrophilic ionizable head group.

[0149] The linker may include a hydrocarbon chain as its backbone, which may be linear or branched. The chain may optionally include saturated or unsaturated bonds. As part of its structure, the linker may comprise linkages which covalently connect the one or more hydrophobic tails and the present moiety (ionizable head group) to the linker's structure.

[0150] In particular embodiments, the linker comprises ester, amide, carbamate, carbonate, disulfide, or thioester linkages, preferably ester linkages, for each hydrophobic tail and moiety.

[0151] In particular embodiments, the linker comprises a backbone chain comprising 3 to 30 atoms, preferably carbon atoms, more preferably 3 to 20 carbon atoms, excluding the linkages that connectthe one or more hydrophobic tails and the present moiety (ionizable head group) to the linker's structure.

[0152] In preferred embodiments, the linker comprises a backbone chain comprising 3 to 20 carbon atoms; ester, amide, carbamate, carbonate, disulfide, or thioester linkages, one for each hydrophobic tail and another for the moiety; and the thioamide group is separated from the ester, amide, carbamate, carbonate, disulfide, or thioester linkages that connect the moiety to the linker by an alkyl, alkenyl, alkynyl, cycloalkyl, or heteroalkyl group.

[0153] In more preferred embodiments, the linker comprises a backbone chain comprising 3 to 20 carbon atoms; ester linkages, one for each hydrophobic tail and another for the moiety; and the thioamide group is separated from the ester linkages that connect the moiety to the linker by an alkyl or cycloalkyl group.

[0154] In more preferred embodiments, the linker comprises a linear or branched hydrocarbon chain comprising 3 to 20 carbon atoms; terminal ester linkages, one for each hydrophobic tail and another for the moiety; and the thioamide group is separated from the ester linkages that connect the moiety to the linker by an alkyl group.

[0155] In an exemplary embodiment, the linker has a structure such that two or more hydrophobic tails are connected to one moiety, preferably through ester linkages.

[0156] In an exemplary embodiment, the linker has a structure such that one hydrophobic tail is connected to one moiety, preferably through ester linkages.

[0157] In an exemplary embodiment, the linker has a structure such that three or more hydrophobic tails are connected to one moiety, preferably through ester linkages.

[0158] Non-limiting examples of suitable linkers include:

[0159]

[0160] wherein

[0161] Rg, Rkare each independently selected from optionally substituted alkyl, hydrogen, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted heteroalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted aryl, or optionally substituted arylalkyl; and

[0162] Y1, Y3, Y5, Y7are each independently selected from a direct bond, oxygen, nitrogen, sulfur, or amine; and Y2, Y4, Y6, Y8are each independently selected from a direct bond, sulfur, or C(=O); or

[0163] Y1, Y3, Y5, Y7are each independently selected from a direct bond, sulfur, or C(=O); and Y2, Y4, Y6, Y8are each independently selected from a direct bond, oxygen, nitrogen, sulfur, or amine.

[0164] It is apparent to a person skilled in the art that the structure of the linker may vary depending on the number of hydrophobic tails present, ensuring proper attachment and functionality of the ionizable lipid. For example, in embodiments comprising two hydrophobic tails, the linker may include a branched or linear chain comprising three linkages: a first and a second linkage which connect the two hydrophobic tails, and a third linkage which connects the moiety (head group). Similarly, for ionizablelipids with a single hydrophobic tail, a simpler spacer may suffice to connect the tail to the moiety (head group).

[0165] In particular embodiments, the ionizable lipid or a pharmaceutically acceptable salt thereof may be a compound of Formula (Va), (Vb), or (Vc);

[0166]

[0167] wherein

[0168] L being a single bond or a linker structure comprising ester, amide, carbamate, carbonate, disulfide, or thioester linkages;

[0169] HT is a hydrophobic tail; and each HT comprising a linear or branched aliphatic chain having 8 to 36 carbon atoms;

[0170] R1is selected from optionally substituted C2-10alkyl, optionally substituted Cg-ioalkenyl, optionally substituted Cg-ioalkynyl, optionally substituted Cg-iocycloalkyl, optionally substituted Cg-iocycloalkenyl, optionally substituted C2-10heteroalkyl, Cj-whaloalkyl;

[0171] R2and R3are each independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heteroalkyl, haloalkyl, optionally substituted heterocyclyl, or hydrogen, provided that at least one is not hydrogen; or R2and R3together with the atom to which they are attached form a 3-7-membered saturated, or partially saturated heterocyclyl, optionally comprising at least one further N, O, and / or S.

[0172] In some embodiments, R1is selected from C2-8alkyl, C3-8alkenyl, C3-8alkynyl, C3-8cycloalkyl, C3-8cycloalkenyl, C2-8heteroalkyl, C2-8haloalkyl.

[0173] In some embodiments, R1is selected from C2-8alkyl, C3-8alkenyl, or C2-8heteroalkyl.In some embodiments, R2and R3are each independently selected from C1-10alkyl, C1-10alkenyl, C1-10alkynyl, C1-10alkoxyl, C6-12aryl, C6-12arylalkyl, C3-8cycloalkyl, C3-8cycloalkenyl, C4-8cycloalkynyl, C1-10heteroalkyl, C1-10haloalkyl, C3-8heterocyclyl, or hydrogen, provided that at least one is not hydrogen; or R2and R3together with the atom to which they are attached form a 3-7-membered saturated, or partially saturated heterocyclyl, optionally comprising at least one further N, O, and / or S.

[0174] In some embodiments, R2and R3are each independently selected from C1-10alkyl, C1-10alkenyl, C1-10alkoxyl, C3-8cycloalkyl, C3-8cycloalkenyl, C1-10heteroalkyl, C3-8heterocyclyl; or R2and R3together with the atom to which they are attached form a 3-7-membered saturated, or partially saturated heterocyclyl, optionally comprising at least one further N, O, and / or S.

[0175] In some embodiments, R1is selected from C2-8alkyl, C3-8alkenyl, or C2-8heteroalkyl; and R2and R3are each independently selected from C1-10alkyl, C1-10alkenyl, C1-10alkoxyl, C3-8cycloalkyl, C3-8cycloalkenyl, C1-10heteroalkyl, C3-8heterocyclyl; or R2and R3together with the atom to which they are attached form a 3-7-membered saturated, or partially saturated heterocyclyl, optionally comprising at least one further N, O, and / or S.

[0176] In particular embodiments, the ionizable lipid or a pharmaceutically acceptable salt thereof may be a compound of Formula (IIIa), (IIIb), and / or (IIIc):

[0177]

[0178]

[0179] wherein

[0180] X1, X2, X3, X4are each independently selected from -O, -NH, or -S, preferably -O;

[0181] R5, R6, R7are each independently selected from C8-36alkyl or C8-36alkenyl;

[0182] A is a branched or linear aliphatic chain, preferably comprising 3 to 30 carbon atoms;

[0183] R4is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heteroalkyl, optionally substituted aryl, optionally substituted arylalkyl, or optionally substituted heterocyclyl;

[0184] R1, R2, and R3are as defined herein above.

[0185] In some embodiments, R4is selected from C1-10alkyl, C1-10alkenyl, C1-10alkynyl, C3-8cycloalkyl, C3-8cycloalkenyl, C4-8cycloalkynyl, C1-10heteroalkyl, C6-12aryl, C6-12arylalkyl, or C3-8heterocyclyl.

[0186] In some embodiments, R4is selected from C1-6alkyl, C1-6alkenyl, C3-6cycloalkyl, C3-6cycloalkenyl, C1-6heteroalkyl, C6aryl, or C3-6heterocyclyl.

[0187] In particular embodiments, the ionizable lipid or a pharmaceutically acceptable salt thereof may be a compound of Formula (IVa), (IVb), and / or (IVc):

[0188]

[0189]

[0190] wherein

[0191] R8, R9is selected from optionally substituted alkyl, hydrogen, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted heteroalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, or optionally substituted aryl, optionally substituted arylalkyl; and

[0192] R1, R2, R3, R4, R5, R6, R7X1, X2, X3, X4are as defined herein above.

[0193] In some embodiments, R8, R9are each independently selected from C1-10alkyl, hydrogen, C1-10alkenyl, C1-10alkynyl, C1-10alkoxyl, C6-12aryl, C6-12arylalkyl, C3-8cycloalkyl, C3-8cycloalkenyl, C4-8cycloalkynyl, C1-10heteroalkyl, C1-10haloalkyl.

[0194] In some embodiments, R8, R9are each independently selected from C1-10alkyl, hydrogen, C1-10alkenyl, C1-10alkynyl, C1-10alkoxyl, C3-8cycloalkyl, C3-8cycloalkenyl, C1-10heteroalkyl, C1-10haloalkyl.

[0195] In some embodiments, R8, R9are each independently selected from C1-10alkyl, hydrogen, C1-10alkenyl, C1-10alkynyl, C1-10alkoxyl.In particular embodiments, the ionizable lipid or a pharmaceutically acceptable salt thereof may be selected from the group consisting of:

[0196] o

[0197]

[0198]

[0199]

[0200] A further aspect of the present invention provides a method for preparing the ionizable lipid according to an aspect of the present invention or a pharmaceutically acceptable salt thereof. The method preferably comprises the steps of:

[0201] a) providing a lipid precursor molecule comprising a terminal alkyne;

[0202] b) contacting the lipid precursor molecule with an amine compound, comprising a primary amine separated by 2 to 10 atoms from an ionizable nitrogen selected from a secondary or tertiary amine functional group, in the presence of elemental sulfur, and optionally abase, thereby forming an ionizable lipid comprising a thioamide group separated from the ionizable nitrogen by 2 to 10 atoms.

[0203] It should be noted that (preferred) embodiments of the ionizable lipid according to an aspect of the present invention or a pharmaceutically acceptable salt thereof are also (preferred) embodiments of the method for its preparation as described herein.

[0204] The term "lipid precursor molecule" as used herein generally refers to an intermediate compound comprising at least one terminal alkyne group, which may serve as a reactive site for the subsequent introduction of at least a part of the (head group) moiety as described herein. A "terminal alkyne" as used herein refers to a functional group characterized by a carbon-carbon triple bond (–C≡C–), where one of the carbon atoms is bonded to a hydrogen atom. This structure provides a reactive site suitable for further reaction (e.g., with an amine compound).

[0205] As used herein, the lipid precursor molecule may comprise a structure that comprises one or more reactive functional groups, or protected forms thereof, suitable for covalent attachment of hydrophobic tails. Hence, a structure that is described as being configured to connect one or more hydrophobic tails upon deprotection and subsequent connection is understood to comprise one or more reactive functional groups, or protected forms thereof, which act as functional anchors for attaching one or more hydrophobic tails to the lipid precursor molecule. Alternatively, the structure may already comprise one or more hydrophobic tails, thereby forming a partially assembled lipid structure.

[0206] For instance, suitable reactive functional groups (or protected forms thereof) present on the lipid precursor molecule may include, without limitation, hydroxyl, carboxyl, amine, thiol, alkene, alkyne, halide, azide, activated ester, aldehyde, ketone, phosphate, or epoxide groups, which are capable of undergoing covalent bond formation with complementary functional groups provided on the respective hydrophobic tails.

[0207] Representative compatible reaction pairs include, but are not limited to ester-forming pairs, amide-forming pairs, thioester-or thioether-forming pairs, click-chemistry pairs, olefin-based coupling pairs, or phosphate or phosphoramidate-forming pairs.

[0208] An example of ester-forming pairs includes a hydroxyl group on the lipid precursor molecule and a carboxylic acid, activated ester (e.g., NHS ester), or acid chloride on a hydrophobic tail; or a carboxyl group on the lipid precursor molecule and a hydroxyl-functionalized hydrophobic tail, optionally following activation of the carboxyl group.An example of amide-forming pairs includes an amine group on the lipid precursor molecule and a carboxylic acid, activated ester (e.g., NHS ester), or acid chloride on a hydrophobic tail; or a carboxyl group on the lipid precursor molecule and an amine-functionalized hydrophobic tail, optionally following activation of the carboxyl group.

[0209] In embodiments, where the reaction conditions for contacting the lipid precursor molecule with the amine compound as described herein are considered detrimental to the integrity of hydrophobic tails or other functional groups of the lipid, protective group chemistry may optionally be employed. It is apparent to the skilled person that the use of protective groups may ensure stability of reactive groups or functionalities of the lipid precursor molecule under such conditions. Hence, their application may be limited to cases where it is necessitated by the chemical environment. For instance, in embodiments where one or more hydrophobic tails may comprise unsaturated hydrocarbon chains, protective group chemistry may be used.

[0210] In the event that protective group chemistry is used, the lipid precursor molecule may include a protected structure that is configured to connect one or more hydrophobic tails upon deprotection of reactive groups; and allows subsequent connection of the one or more hydrophobic tails to said reactive groups.

[0211] Non-limiting examples of suitable protecting groups include protecting groups for alcohol (e.g., tertbutyldimethylsilyl, acetyl, acetal), thiol (e.g., acetamidomethyl or trityl), amine (e.g., Boc or Fmoc), and carboxylic acid functional groups (e.g., methyl or benzyl esters). These protective groups can be readily selected and applied by the skilled person based on the specific synthetic route and reaction conditions. It is further apparent to the skilled person that other protecting groups commonly used in organic synthesis may also be employed as needed, depending on the reactivity of the functional groups present and the chemical environment.

[0212] The invention is not limited to specific protecting groups or coupling methodologies, as the skilled person can adapt these features based on routine experimentation and knowledge in the field of lipid synthesis

[0213] For instance, non-limiting suitable lipid precursor molecules may be:

[0214]

[0215] wherein PG1, PG2, PG3are suitable protecting groups as listed above, and L is a suitable linker as defined herein above.

[0216] In exemplary embodiments, the lipid precursor molecule may be represented by a compound of Formula (Via), (Vlb), and / or (Vic),

[0217]

[0218] wherein PG1, PG2, PG3are suitable protecting groups as listed above (e.g., tertbutyldimethylsilyl, acetyl, acetal),

[0219] A is a branched or linear aliphatic chain, preferably comprising 3 to 30 carbon atoms;

[0220] X1is selected from -O, -NH, or -S, preferably -O; and

[0221] R4is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heteroalkyl, optionally substituted aryl, optionally substituted arylalkyl, or optionally substituted heterocyclyl.

[0222] In exemplary embodiments, the lipid precursor molecule may be represented by a compound of Formula (VIIa), (VIIb), and / or (VIIc),

[0223]

[0224] wherein PG1, PG2, PG3are suitable protecting groups as listed above (e.g., tertbutyldimethylsilyl, acetyl, acetal),

[0225] X1, X2, X3, X4is selected from -O, -NH, or -S, preferably -O;

[0226] R4is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heteroalkyl, optionally substituted aryl, optionally substituted arylalkyl, or optionally substituted heterocyclyl; and

[0227] R8, R9is selected from optionally substituted alkyl, hydrogen, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted heteroalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, or optionally substituted aryl, optionally substituted arylalkyl.

[0228] Alternatively, the one or more hydrophobic tails may already be connected to the lipid precursor molecule, thereby forming a partially assembled lipid precursor molecule.

[0229] For instance, non-limiting suitable lipid precursor molecules may be:

[0230]

[0231] wherein HT is a hydrophobic tail and L is a suitable linker as defined herein above.

[0232] In exemplary embodiments, the lipid precursor molecule may be represented by a compound of Formula (VIIIa), (VIIIb), and / or (VIIIc),

[0233]

[0234] wherein HT is a hydrophobic tail as defined herein above,

[0235] A is a branched or linear aliphatic chain, preferably comprising 3 to 30 carbon atoms;

[0236] X1is selected from -O, -NH, or -S, preferably -O; andR4is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heteroalkyl, optionally substituted aryl, optionally substituted arylalkyl, or optionally substituted heterocyclyl.

[0237] In exemplary embodiments, the lipid precursor molecule may be represented by a compound of Formula (IXa), (IXb), and / or (IXc),

[0238] (IXb),

[0239]

[0240] (IXc),

[0241] wherein HT is a hydrophobic tail as defined herein above,

[0242] X1, X2, X3, X4is selected from -O, -NH, or -S, preferably -O;

[0243] R4is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heteroalkyl, optionally substituted aryl, optionally substituted arylalkyl, or optionally substituted heterocyclyl; and

[0244] R8, R9is selected from optionally substituted alkyl, hydrogen, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted heteroalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, or optionally substituted aryl, optionally substituted arylalkyl.

[0245] The "amine compound" as used herein refers to a molecule which comprises a primary amine (-NH2) and at least one ionizable nitrogen selected from a secondary (-NRH) or tertiary amine (-NR2). The primary amine is separated from the ionizable nitrogen by a chain of 2 to 10 atoms.In some embodiments, the amine compound may comprise functional groups that are compatible with the reaction conditions used for coupling with the alkyne group, provided these groups do not interfere with the intended coupling reaction.

[0246] In certain embodiments, the amine compound may comprise functional groups suitable for further modification or attachment to additional molecular components.

[0247] In particular embodiments, the amine compound may be a compound according to Formula (X)

[0248] , R, R‘

[0249]

[0250] wherein R1, R2, and R3are as defined herein above.

[0251] It should be noted that the present synthesis method is not limited to any particular reaction conditions. While specific embodiments are provided to illustrate the method, these examples are not intended to restrict the scope of the invention. An organic chemist skilled in the art will readily understand that the reaction conditions, including but not limited to solvent systems, temperature, reaction time, stoichiometry, and the presence or absence of bases or additives, may be adjusted depending on the specific lipid precursor molecule and amine compound used.

[0252] In particular embodiments, step b) of the present method may comprise contacting 1.0 equivalent of the lipid precursor molecule with between 1.5 and 4.0 equivalents, preferably 2.0 equivalents, of amine compound, optionally in the presence of between 2.0 and 8.0 equivalents, preferably 4.0 equivalents, of elemental sulfur.

[0253] Step b) of the present synthesis method may be carried out in a suitable solvent system, optionally in the presence of a base, and at elevated temperatures for a defined reaction time. In addition, the synthesis may be conducted in a sealed vessel or under inert atmosphere (e.g., nitrogen or argon) to prevent possible side reactions.

[0254] For instance, the step may be conducted in a single solvent or a combination of solvents. Non-limiting examples of suitable solvents include polar aprotic solvents, such as dimethylformamide (DMF), dimethyl sulfoxide (DMSO), or acetonitrile (ACN); polar protic solvents, such as methanol, ethanol, or isopropanol; or non-polar solvents such as toluene or dichloromethane (DCM).Step b) may optionally include the addition of a base to facilitate deprotonation of the amine compound or stabilize intermediates. Suitable bases include, but are not limited to:

[0255] organic bases such as pyridine, triethylamine (Et3N) or other trialkylamines, 1,4- diazabicyclo[2.2.2]octane (DABCO), l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 4- dimethylaminopyridine (DMAP), l,5-diazabicyclo(4.3.0)non-5-ene (DBN), or imidazole;

[0256] inorganic bases such as sodium hydroxide or other alkali metal hydroxides, calcium hydroxide or other alkaline earth metal hydroxides, potassium carbonate or other alkali metal carbonates, calcium carbonate or other alkaline earth metal carbonates. Step b) may comprise heating to a temperature ranging from 60 °C to 150 °C, and is preferably maintained at 100 °C. Heating is allowed to proceed for a period ranging from 2 to 8 hours, preferably 4 hours, with vigorous stirring to ensure proper mixing of all components.

[0257] Step b) may further comprise purifying the obtained crude product and isolating the ionizable lipid formed. Suitable purification methods include chromatography (e.g., silica gel column chromatography or preparative thin-layer chromatography), crystallization or recrystallization, and / or liquid-liquid extraction.

[0258] The present invention also encompasses the ionizable lipid obtained or obtainable by the present method.

[0259] Advantageously, the ionizable lipids as disclosed herein may be designed to facilitate the formation of lipid nanoparticles (LNPs) and to enable efficient encapsulation and delivery of (ionizable) cargo compounds, such as nucleic acids, by mediating cellular uptake, endosomal escape, and cytoplasmic release of the payload.

[0260] Accordingly, a further aspect of the present invention provides a lipid nanoparticle composition comprising at least one ionizable lipid according to an aspect of the invention or a pharmaceutically acceptable salt thereof. The term "lipid nanoparticle composition" as used herein is well-recognized in the art and generally refers to a formulation comprising nanoparticles formed primarily of lipids. Said nanoparticles can include various structural configurations and morphologies, such as bilayer, multilayer, or core-shell structures, and may be tailored to specific applications. In the context of the present invention, the term may encompass a wide range of lipid-based delivery systems, including but not limited to liposomes, solid lipid nanoparticles (SLNs), and nanostructured lipid carriers (NLCs).It should be noted that (preferred) embodiments of the ionizable lipid according to an aspect of the present invention or a pharmaceutically acceptable salt thereof, the method of its preparation; or the method for modifying the apparent pKa of a lipid nanoparticle composition are also (preferred) embodiments of the lipid nanoparticle composition as described herein.

[0261] In particular embodiments, the lipid nanoparticle composition comprises at least one ionizable lipid and at least one other lipid component. Such other lipid components include, but are not limited to, phospholipids, structural lipids, PEG-lipids, and mixtures thereof.

[0262] Phospholipids are well-known compounds in the art that are amphipathic molecules typically comprising a hydrophilic phosphate head group and one or more hydrophobic fatty acid chains. Suitable phospholipids in the present context include, but are not limited to, DSPC, DOPE, POPC, EPC, DOPC, DPPC, DOPG, DPPG, DSPE, DOTAP, phosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, l,2-dioleoyl-sn-glycero-3-phosphate, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, l,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, POPE, DOPS, DLPC, DMPC, DUPC, l,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine, l-hexadecyl-sn-glycero-3-phosphocholine, l,2-distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, sphingomyelin, or combinations thereof.

[0263] Structural lipids are typically neutral or zwitterionic lipids that can help form and maintain the lipid bilayer or particle structure. These lipids can advantageously provide physical stability to the lipid nanoparticle composition and may further improve the encapsulation and delivery of the payload. Suitable structural lipids in the present context include, but are not limited to, cholesterol, cholestenol, spinasterol, fecosterol, sitosterol, ergosterol, ergostenol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, or combinations thereof.

[0264] PEG-lipids are lipid molecules conjugated with polyethylene glycol (PEG) chains. These lipids may improve the pharmacokinetics of lipid nanoparticle compositions. PEG-lipids can advantageously also help stabilize the lipid nanoparticle composition during storage and prevent aggregation. Suitable PEG-lipids in the present context include, but are not limited to, PEG-ceramide, PEG-DMG, PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, and similar derivatives with varying PEG chains lengths or combinations thereof.Unless otherwise indicated, the term " PEG" as used herein means any polyethylene glycol or other polyalkylene ether polymer. Suitable number average molecular weight values range from about 130 to about 50000 g / mol, or from about 150 to about 30000 g / mol. In certain embodiments, the PEG used herein is a " PEG-2K", also termed PEG 2000, which has a number average molecular weight of about 2000 g / mol.

[0265] In some embodiments, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide.

[0266] In some embodiments, PEG is substituted, e.g., by one or more alkyl, alkoxy, hydroxy or aryl groups. In particular embodiments, the lipid nanoparticle composition further comprises an (ionizable) cargo compound.

[0267] Non-limiting examples of suitable cargo compounds include antibodies (e.g., monoclonal, chimeric, humanized, nanobodies, and fragments thereof etc.), cholesterol, hormones, peptides, proteins, chemotherapeutics and other types of antineoplastic agents, low molecular weight drugs, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, antisense DNA or RNA compositions, chimeric DNA: RNA compositions, allozymes, aptamers, ribozyme, decoys and analogs thereof, plasmids and other types of expression vectors, and small nucleic acid molecules, RNAi agents, short interfering nucleic acid (siNA), messenger ribonucleic acid (messenger RNA, mRNA), short interfering RNA (siRNA), doublestranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules, ribosomal RNA (rRNA), transfer RNA (tRNA), Single-Guide RNA (sgRNA), trans-activating CRISPR RNA (tracrRNA), guide RNA (gRNA), peptide nucleic acid (PNA), a locked nucleic acid ribonucleotide (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), sisiRNA (small internally segmented interfering RNA), aiRNA (assymetrical interfering RNA), DNAzyme, and siRNA with 1, 2 or more mismatches between the sense and anti-sense strand to relevant cells and / or tissues, such as in a cell culture, subject or organism.

[0268] Such compounds may be purified or partially purified, and may be naturally occurring or synthetic, and may be chemically modified.

[0269] In preferred 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, at physiological pH.In more preferred embodiments, the nucleic acid is at least any one selected from the group consisting of mRNA, siRNA, rRNA, DNA, aptamer, tRNA, antisense oligonucleotide, shRNA, miRNA, sgRNA, tracrRNA, gRNA, ribozyme, PNA, DNAzyme, and mixtures thereof.

[0270] Various methods for loading (ionizable) cargo compounds into lipid nanoparticle compositions, such as liposomes and solid lipid nanoparticles are available in the art, including both passive and active loading methods. The exact method used may be chosen based on multiple factors that include, but are not limited to, e.g., the (ionizable) cargo compound to be loaded, the storage method to be used once loaded, the size of the resulting particle, and the dosage regimen contemplated. Methods include, e.g., mechanical mixing of the (ionizable) cargo compound and lipids at the time the liposomes are formed or reconstituted, dissolving all components in an organic solvent and concentrating them into a dry film.

[0271] In embodiments, the lipid nanoparticles may have a size of about 1 to about 2500 nm, or about 10 to about 1500 nm, or about 20 to about 1000 nm, or about 50 to about 600 nm, or about 50 to about 400 nm or about 50 to about 250 nm, or about 50 to about 150 nm. Unless indicated otherwise, all sizes referred 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).

[0272] In exemplary embodiments, the lipid nanoparticle composition comprises:

[0273] at least one ionizable lipid as described herein; and

[0274] at least one phospholipid as described herein.

[0275] In exemplary embodiments, the lipid nanoparticle composition comprises:

[0276] at least one ionizable lipid as described herein;

[0277] at least one phospholipid as described herein; and

[0278] at least one structural lipid as described herein.

[0279] In exemplary embodiments, the lipid nanoparticle composition comprises:

[0280] at least one ionizable lipid as described herein; and

[0281] at least one phospholipid as described herein;

[0282] at least one structural lipid as described herein; and

[0283] at least one PEG-lipid as described herein.

[0284] In exemplary embodiments, the lipid nanoparticle composition comprises:

[0285] at least one ionizable lipid as described herein; andat least one phospholipid as described herein;

[0286] at least one structural lipid as described herein;

[0287] at least one PEG-lipid as described herein; and

[0288] at least one (ionizable cargo compound) as described herein.

[0289] In exemplary embodiments, the lipid nanoparticle composition comprises:

[0290] at least one ionizable lipid as described herein; and

[0291] at least one phospholipid as described herein;

[0292] at least one structural lipid as described herein;

[0293] at least one PEG-lipid as described herein; and

[0294] at least one nucleic acid, preferably mRNA or siRNA, as described herein.

[0295] In exemplary embodiments, the lipid nanoparticle composition comprises:

[0296] at least one ionizable lipid as described herein; and

[0297] at least one phospholipid as described herein;

[0298] at least one structural lipid as described herein; and

[0299] at least one PEG-lipid as described herein;

[0300] wherein the molar ratio of ionizable lipid: phospholipid: structural lipid: PEG-lipid is from 20 to 60: 5 to 30: 30 to 60: 1 to 5.

[0301] In exemplary embodiments, the lipid nanoparticle composition comprises:

[0302] at least one ionizable lipid as described herein; and

[0303] at least one phospholipid as described herein;

[0304] at least one structural lipid as described herein;

[0305] at least one PEG-lipid as described herein; and

[0306] at least one nucleic acid, preferably mRNA or siRNA, as described herein; wherein the weight ratio of the ionizable lipid to the nucleic acid in the composition is from 1 to 20.

[0307] In exemplary embodiments, the lipid nanoparticle composition comprises:

[0308] at least one ionizable lipid as described herein; and

[0309] at least one phospholipid as described herein;

[0310] at least one structural lipid as described herein;

[0311] at least one PEG-lipid as described herein; andat least one nucleic acid, preferably mRNA or siRNA, as described herein; wherein the molar ratio of ionizable lipid: phospholipid: structural lipid: PEG-lipid is from 20 to 60: 5 to 30: 30 to 60: 1 to 5; and

[0312] wherein the weight ratio of the ionizable lipid to the nucleic acid in the composition is from 1 to 20.

[0313] In particular embodiments, the present lipid nanoparticle compositions may have an apparent pKaof between 5.8 and 7.8, preferably between 6.0 and 7.0 (determined by means of TNS assay as described herein in the methodology section).

[0314] In particular embodiments, the present lipid nanoparticle composition may provided as lipid nanoparticles having a polydispersity index below 0.4, preferably below 0.2 (determined by means of DLS analysis).

[0315] Optionally, the present lipid nanoparticle composition may further comprise stabilizers, surfactants, or functional additives, which preferably enhance stability, targeting, and / or delivery properties. In particular embodiments, the lipid nanoparticle composition may further comprise one or more trans-cyclooctene (TCO) moieties. The TCO moieties may be present as substituents on one or more lipid components of the lipid nanoparticle, including the at least one ionizable lipid as described herein; the at least one phospholipid as described herein; the at least one structural lipid as described herein; and / or the at least one PEG-lipid as described herein.

[0316] In preferred embodiments, at least one trans-cyclooctene (TCO) moiety is covalently linked to a lipid component as described above via a spacer or linker group, such that the TCO moiety is exposed at or near the surface of the lipid nanoparticle.

[0317] The trans-cyclooctene (TCO) moiety may be configured to participate in biorthogonal reactions, such as (inverse-electron-demand) Diels-Alder reactions with tetrazine-containing compounds, thereby enabling post-assembly functionalization, targeting, labeling, or crosslinking of the lipid nanoparticle composition. The amount of TCO-functionalized lipid component may be selected such that the overall structural integrity and delivery properties of the lipid nanoparticle composition is maintained. The present invention further encompasses methods for preparing the lipid nanoparticle composition as described herein comprising the step of preparing an ethanolic solution comprising at least one ionizable lipid as described herein, and preferably further comprising at least one lipid selected from the group consisting of phospholipids, structural lipids, PEG-lipids, and mixtures thereof.A further aspect of the present invention provides a method for modifying the apparent pKa of a lipid nanoparticle composition. The method preferably comprises the step of incorporating at least one ionizable lipid according to an aspect of the present invention or a pharmaceutically acceptable salt thereof into the lipid nanoparticle composition, preferably wherein the lipid nanoparticle composition has a modified apparent pKa between 5.8 and 7.8, more preferably between 6.0 and 7.0.

[0318] It should be noted that (preferred) embodiments of the ionizable lipid according to an aspect of the present invention or a pharmaceutically acceptable salt thereof or the method of its preparation are also (preferred) embodiments of the method for modifying the apparent pKa of a lipid nanoparticle composition as described herein.

[0319] The present modification method may involve incorporating at least one ionizable lipid according to an aspect of the present invention or a pharmaceutically acceptable salt thereof in any suitable amount sufficient to achieve the desired modification of the apparent pKa of the lipid nanoparticle composition.

[0320] As used herein, the term "modified apparent pKa" may refer to the apparent pKa of a LNP or compound after structural modification compared to its unmodified or reference form (e.g., comprising an amide-containing analog of the ionizable lipid according to the present invention of a pharmaceutically acceptable salt thereof). Unless stated otherwise, the modified apparent pKa is determined under the same experimental conditions as the apparent pKa of the reference compound. In exemplary embodiments, the apparent pKa of a lipid nanoparticle composition may be lowered by at least 0.5, such as by at least 0.6, 0.7, 0.8, 0.9, or 1.0.

[0321] It should be clear that the to be modified lipid nanoparticle composition may already comprise ionizable lipids (other than those according to the present invention) or other lipids such as phospholipids, cholesterol, or PEG-lipids.

[0322] Based on the present application, the skilled person will recognize that various modifications, including the use of different lipid structures, variations in the ionizable groups, or different ratios of lipid components, may be employed to modify the apparent pKa of the lipid nanoparticle composition to the desired range.

[0323] The modification of the apparent pKa may be measured using any suitable assay or technique known in the art, such as by means of TNS assay as described herein in the methodology section.The presently described lipid nanoparticle compositions (optionally obtained by modification) may be useful in pharmaceutical compositions or formulations for use in medicine, such as delivery of (ionizable) cargo compounds in vivo. Delivery of said cargo compounds may be useful in preventing, inhibiting, or treating diseases, conditions, or traits in a cell, subject or organism.

[0324] As used herein, the terms "subject" or "patient" are generally used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, still more preferably primates, and specifically includes human patients and non-human mammals and primates. Preferred subjects or patients are human subjects.

[0325] The terms "treat" or "treatment" encompass both the therapeutic treatment of an already developed disease or condition, such as the therapy of an already developed disease, disorder, addiction or overdose, as well as prophylactic or preventive measures, wherein the aim is to prevent or lessen the chances of incidence of an undesired affliction, such as to prevent occurrence, development and progression of disease, disorder, addiction or overdose.

[0326] As used herein, the terms "therapeutic treatment" or "therapy" and the like, refer to treatments wherein the aim is to change a subjects body or a part of a subjects body from an undesired physiological state, disease or disorder which is caused by an infectious agent, to a desired state, such as a less severe state (e.g., amelioration or palliation), or even back to its normal, healthy state (e.g., restoring the health, the physical integrity and the physical well-being of a subject), to keep it (i.e., not worsening) at said undesired physiological status (e.g., stabilization), or slow down progression to a more severe or worse state compared to said undesired physiological change or disorder. Measurable lessening includes any statistically significant decline in a measurable marker or symptom. Statistically significant as used herein refers to p values below 0.05, which is a commonly accepted cutoff score in statistical analysis as a skilled person appreciates. " Treatment" encompasses both curative treatments and treatments directed to reduce symptoms and / or slow progression and / or stabilize the disease. A 'therapeutic amount' or 'therapeutically effective amount' as used herein refers to the amount of lipid nanoparticle composition effective to treat a disease or disorder in a subject, i.e., to obtain a desired local or systemic effect. The term thus refers to the quantity of lipid nanoparticle composition that elicits the biological or medicinal response in a tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. Such amount will typically depend on the lipid nanoparticle composition and the severity of the disease or disorder, but can be decided by the skilled person, possibly through routine experimentation.Accordingly, another aspect provides the lipid nanoparticle composition according to an aspect of the invention for use in medicine.

[0327] 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.

[0328] Another aspect provides a lipid nanoparticle composition according to an aspect of the invention for use in delivering a cargo compound to a cell, such as for transfecting a cell. The lipid nanoparticle composition may for example carry a nucleic acid molecule or construct that imparts a medical benefit on the organism that harbors the transfected cells, for example the nucleic acid molecule may be therapeutically useful or may encode an expression product that is therapeutically useful.

[0329] Such use may be a medical use or a non-medical use. In medical uses, the lipid nanoparticle composition carries a cargo compound, such as a nucleic acid molecule or construct, that imparts a medical benefit to an organism harboring the transfected cells, for example by being therapeutically useful or by encoding an expression product that is therapeutically useful. In non-medical uses, the lipid nanoparticle composition may be used, for example, for in vitro, ex vivo, or research applications, including gene expression studies, cell engineering, screening assays, or diagnostic or analytical purposes. Hence, in embodiments, the delivery of the cargo compound to a cell is used for medical applications.

[0330] In some embodiments, the lipid nanoparticle composition comprises a nucleic acid molecule, such as mRNA, siRNA, or plasmid DNA, that encodes the expression of functional proteins, enzymes, or therapeutic proteins such as an antigen or cytokine. For instance, where the nucleic acid encodes an antigen, the composition can be used to elicit a targeted immune response in a subject. The antigen may be associated with infectious diseases (e.g., viruses such as HIV, influenza, or SARS-CoV-2) or other pathological conditions. In another example, therapeutic proteins (e.g., monoclonal antibodies, enzymes) may be useful to address specific deficiencies or disorders.

[0331] In some embodiments, the lipid nanoparticle composition comprises a nucleic acid molecule or construct that encodes the expression of an enzyme for vaccination, immunotherapy, protein replacement therapy, or gene-editing.

[0332] In particular embodiments, the cell is a mammalian cell, more preferably an immune cell, a cancer cell, a progenitor cell, or a structural cell.In some embodiments, the mammalian cell includes liver cells, splenic cells, T lymphocytes, B lymphocytes, granulocytes, phagocytes, natural killer cells, dendritic cells, or other immune cells in vivo.

[0333] In a preferred embodiment, the immune cell is a lymphocyte, more preferably a T lymphocyte (also referred to herein as T cell). In particularly preferred embodiments, the T lymphocyte is a CD8+ T lymphocyte.

[0334] 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.

[0335] 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 for delivering a cargo compound to a cell.

[0336] It should be noted that (preferred) embodiments of the ionizable lipid according to an aspect of the present invention or a pharmaceutically acceptable salt thereof or the method of its preparation; are also (preferred) embodiments of the use thereof in medicine, such as for transfecting cells in vivo. 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 can be particularly suitable as delivery agents, such as vaccines. In particular embodiments, the pharmaceutical composition is a vaccine composition or a vaccine.

[0337] The term "pharmaceutically-acceptable carrier" means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a subject contemplated by the present application. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the lipid nanoparticles and any other optional agent(s) are combined to facilitate administration. The pharmaceutical composition may further 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 type of carrier and other ingredients will vary depending on the mode of administration. When it is desirable to deliver the pharmaceutical compositions as envisaged herein systemically, it may be formulated for parenteral administration by injection, e.g., by 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.

[0338] 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).

[0339] 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. For improved immune responses, the composition may optionally include adjuvants, such as aluminum salts (e.g., aluminum hydroxide or aluminum phosphate), CpG oligonucleotides, or saponins, or oil-in-water emulsions, such as squalene-based adjuvants.

[0340] 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 isintroduced into the pulmonary tree, including intrapulmonary or transpulmonary and includes intranasal administration. Preferably, the composition is administered via injection.

[0341] In a preferred embodiment, the composition is administered via injection, ensuring efficient delivery to the target tissues or cells. The LNPs may be delivered in liquid form, for instance with a dose size ranging from about 100 µL to 1 mL, such as about 100 µl, about 200 µl, about 300 µl, about 400 µl, about 500 µl, about 600 µl, about 700 µl, about 800 µl, about 900 µl, 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.

[0342] For effective therapeutic use, the lipid nanoparticle composition may be administered in a single dose or in multiple doses at appropriate time intervals. The dosing regimen can include primary and booster doses to evoke and maintain a desired therapeutic effect, such as inducing an immune response, transfecting cells with nucleic acids, or delivering proteins or peptides. The specific dose, number of doses, and time intervals between doses may vary based on factors such as the patient's age, weight, and condition, as well as the disease being treated or prevented. These parameters may be determined by a skilled medical professional using routine experimentation or clinical guidelines. The composition may also include co- or post-translational modifications to proteins or peptides encapsulated or delivered by the lipid nanoparticle composition. These modifications can include glycosylation, PEGylation, phosphorylation, or acetylation, enhancing stability, bioavailability, or therapeutic efficacy. Such modifications may occur during production or be introduced after isolation of the proteins or peptides, depending on the intended use.

[0343] This LNP-based pharmaceutical composition provides a versatile and efficient delivery platform for nucleic acids, proteins, and antigens, suitable for therapeutic and prophylactic applications in treating or preventing diseases such as genetic disorders, cancers, or infectious diseases.

[0344] Another aspect provides a method for transfecting a cell in vitro, comprising contacting the cell with a lipid nanoparticle composition according to an aspect of the invention, preferably wherein the cell is a mammalian cell, more preferably an immune cell, a cancer cell, a progenitor cell, or a structural cell.

[0345] In some embodiments, the mammalian cell includes liver cells, splenic cells, T lymphocytes, B lymphocytes, granulocytes, phagocytes, natural killer cells, dendritic cells, or other immune cells in vivo.In a preferred embodiment, the immune cell is a lymphocyte, more preferably a T lymphocyte (also referred to herein as T cell). In particularly preferred embodiments, the T lymphocyte is a CD8+ T lymphocyte.

[0346] EXAMPLES

[0347] The following examples are intended to illustrate the invention and are not to be construed as being limitations thereon. 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, IR, or NMR. Abbreviations used are those conventional in the art, some of which are defined below.

[0348] Abbreviations

[0349] DCM dichloromethane

[0350] DMF dimethylformamide

[0351] mRNA messenger ribonucleic acid

[0352] NMR nuclear magnetic resonance

[0353] Methodology

[0354] The following describes the materials and methods used for all examples unless otherwise stated. Materials

[0355] Unless otherwise stated, all glassware was oven dried before use and all reactions were carried out under an argon atmosphere using standard Schlenk-techniques. Dry solvents were purchased from Across Organics or Sigma Aldrich and used without further purification. All reagents were purchased from commercial sources and were used without further purification unless otherwise stated. Avanti Polar lipids supplied l,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (DMG-PEG2000) and Distearoylphosphatidylcholine (DSPC). DSPE-PEG2000-TCO was obtained from Ruin Biotech. Thermo Fischer supplied DMEM, RPMI, Opti-MEM culture medium, DPBS (lx), penicillin / streptomycin (100 x), sodium pyruvate (100 x), flow cytometry staining buffer, TrypLE Select, and the Quant-iT RiboGreen RNA Assay Kit. Additionally, HEK293T cells (human embryonic kidney cells) and CT26 cells (mouse colon carcinoma cells) were obtained from ATCC and CT26 / GFP cells (CT26 cells expressing greenfluorescent protein) from GenTarget. eGFP mRNA (enhanced green fluorescent protein messenger RNA) was purchased from Cellerna. Turkey red blood cells were obtained from the faculty of veterinary sciences at Ghent University. MC3 (a clinically approved siRNA drug product) was obtained from Sinopeg.

[0356] All starting materials which are not explicitly described were either commercially available (Sigma-Aldrich) 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.

[0357] Instrumentation

[0358] Unless otherwise stated, all glassware was oven dried before use. Dry solvents were purchased from Acros Organics or Sigma Aldrich and used without further purification. All reagents were purchased from commercial sources and were used without further purification unless otherwise stated. Reaction progress was monitored by thin layer chromatography (TLC) performed on aluminum plates coated with Kieselgel F254 with 0.2 mm thickness. Visualization was achieved by ultraviolet light (254 nm) or by staining with potassium permanganate. Flash column chromatography was performed using silica gel 60 (230-400 mesh, Merck ans co.) or on preparative TLC plates. All1H NMR,13C NMR spectra were recorded on a BrukerAV-400 spectrometer in Chloroform-d1 or CD3OD-d4. Chemical shifts are given in parts per million (ppm), referenced to tetramethylsilane using the solvent peak as internal standard (CDCU:1H = 7.26 ppm,13C = 77.16 ppm; CD3OD-d4:1H = 3.31 ppm,13C = 49.00 ppm). Coupling constants were quoted in Hz.1H NMR splitting patterns were designated as singlet (s), broad (brd), doublet (d), triplet (t), quartet (q), pentet (p), sextet (se), septet (sep), octet (o), apparent (app.) or combinations thereof. Splitting patterns that could not be interpreted were designated as multiplet (m). Mass spectra were obtained using a Finnigan MAT 8200 (70 eV), an Agilent 5973 (70 eV), using electrospray ionization (ESI) or electron impact ionization (El). Dynamic light scattering (DLS) and electrophoretic mobility measurements were performed on a Zetasizer Nano ZS equipped with a HeNe laser (X = 633 nm) and detection at a scattering angle of 173°. Flow cytometry analysis was performed on a BD Accuri C6 flowcytometer and data were processed using the FlowJo software package. Bioluminescence and Cy7 fluorescence were measured on a IVIS Lumina II imaging system and quantified using the Living Image 4.4 software.

[0359] RiboGreen assay50 pL TE buffer and 50 pL 2% Triton X-100 (in TE buffer) were added in duplicates to a black 96-well plate. LNP formulations were diluted to 1 pg / mL in TE buffer and 50 pL of each formulation was added to TE buffer and 2% Triton X-100. After incubation time of 10 minutes, RiboGreen reagent was diluted 1:100 in TE buffer and added to each well. Fluorescence was measured by an Ensight Multimode Plate Reader (Ex485 / Em528).

[0360] TNS assay

[0361] A master buffer stock solution was prepared containing 150 mM sodium chloride, 10 mM sodium borate, 10 mM sodium phosphate and 10 mM citrate. IM HCI or IM NaOH was added to adjust the pH in 0.5 pH unit steps to become buffers ranging from pH 4-12. Buffers were added in triplicate to a black 96-well plate. LNP compositions were diluted with HEPES buffer (pH 4, 5 mM) to a concentration of 0.04 mg / mL mRNA and 3.26 pL LNP composition was added to each well. Immediately afterwards, 2 pL of a 300 pM 6-(p-toluidino)-2-naphthalenesulfonic acid sodium salt (TNS reagent) stock solution in DMSO was added to each well. Fluorescence was measured by an Ensight Multimode Plate Reader (Ex322 / Em431). The apparent pKawas calculated by determining the log of the inflection point. Cell culture

[0362] Both CT26 cells and CT26 / GFP cells were cultured in RPMI medium and HEK293T cells were cultured in DMEM medium. Both RPMI and DMEM medium were supplemented with 10 % fetal bovine serum (FBS), antibiotics (50 units / mL penicillin and 50 pg / mL streptomycin), 2 mM L-glutamine and 1 mM sodium pyruvate. Cells were incubated at 37 °C in a controlled and sterile environment of 95 % relative humidity and 5 % CO2.

[0363] In vitro transfection efficiency

[0364] Cells were seeded in 96-well plates at a density of 20000 cells per well in 100 pL cell culture medium and were incubated for 24 hours at 37 °C and 5 % CO2. LNP formulations were diluted with Opti-MEM medium so that 10 pL volume contained 50 or 200 ng eGFP mRNA. Cells were transfected in triplicate with diluted LNP formulations and incubated for 24 hours at 37 °C and 5 % CO2. After 24 hours incubation, the 96-well plates were centrifuged (5 minutes, 300g) and cell culture medium was removed. 30.0 pL TrypLE Select was added to each well followed by incubation for 5 minutes at 37 °C. For flow cytometry analysis, 200.0 pL flow cytometry staining buffer was added per well and measured on a BD Accuri C6 flowcytometer.

[0365] In vitro transfection of human CD8+T cells

[0366] LNPs were formulated through microfluidic mixing using a NanoAssemblr® Spark™ device (Precision Nanosystems, Vancouver, Canada). The lipid composition consisted of ionizable lipid SAM-3-APyr: Cholesterol: DOPE: DMG-PEG2000 at a molar ratio of 50:38.5:10:1. The aforementioned lipids were dissolved in ethanol while eGFP-mRNA (10 pg) was dissolved in sodium acetate (NaOAc) buffer (50 mM, pH 4.0) to obtain a nitrogen-to-phosphate (N: P) ratio of 6.

[0367] LNPs were prepared by injecting 16 pL of the lipid mixture, 32 pL of the mRNA aqueous solution,and 48 pL of PBS (1:1 dilution) into a microfluidic cartridge. Samples were stored at 4 °C until use without additional purification.

[0368] RNA encapsulation efficiency was checked using a Quant-iT™ RiboGreen™ RNA Assay Kit (Invitrogen, Merelbeke, Belgium) according to manufacturer's instructions.

[0369] Primary human CD8+ T cells were activated with human T Cell TransAct™ reagent (CD3 and CD28) (Miltenyi Biotec) according to the manufacturer's protocol. Activated CD8+ T cells were seeded in clear U-bottom 96-well plates at a density of 50.000 cells per well.

[0370] Cells were treated with 1 pg / mL of the SAM-3-APyr LNPs in serum-free TexMACs™ medium (Miltenyi Biotec), supplemented with 1 pg / mL apolipoprotein E (in well concentration). Cells were then incubated at 37°C and 5% CO2 for 24h. Following incubation, the plate was centrifuged (400 g for 5 minutes). The supernatant was removed, and cells were washed with 200 pL PBS supplemented with 1% bovine serum albumin. Cells were then centrifuged again and resuspended in 100 pL of the same medium. Expression of eGFP was checked via flow cytometry using a CytoFLEX S instrument (Beckman Coulter). Untreated activated T cells served as controls. All conditions were tested in triplicate.

[0371] Hemolysis assay

[0372] Turkey red blood cells (RBCs) were washed five times with phosphate buffered saline (PBS). Afterwards, RBCs were suspended to U-bottom 96-well plates in a ten-fold original blood volume of PBS (pH 7.4) and acetate buffer (pH 5.5) adjusted with 0.9 % NaCI. LNP were diluted with PBS and added to the RBC suspension followed by incubation at 37 °C for lh. After lh, the RBC suspensions were centrifuged at 300g for 5 minutes and the supernatans was transferred to a flat-bottom 96-well plate. The hemoglobin contents were evaluated with an Ensight Multimode Plate Reader at a wavelength of 540 nm. The RBC suspension incubated in PBS was set as a negative control, whereas the RBC suspension incubated with Triton X-100 (1%) was set as a positive control.

[0373] Mice

[0374] Female 6 weeks BALB / cJRj mice were purchased from Janvier labs (Le Genest-Saint-lsle, France) and housed in individual ventilated cages under 14 hours light and 10 hours dark cycle. Mice were accommodated for two weeks prior to the experiments.In vivo bioluminescence and biodistribution

[0375] Balb / c mice, aged 7-9 weeks, were used in the experiment. Mice were housed in individual ventilated cages and given ad libitum access to food and water. 100 pL of LNP compositions containing 5 pg of luciferase expressing mRNA were injected intravenously to the mice (n=3). For in vivo imaging of luciferase activity at the given time points (4 h and 24 h) post injection, mice were injected subcutaneously with 200 pL D-luciferin and in vivo luminescence imaging was recorded 12 min later using the IVIS Lumina II imaging system. After 24 h, the organs were dissected from the mice and measured for luminescence by the IVIS Lumina II imaging system. The luminescence was quantified using the Living Image 4.4 software.

[0376] Flow cytometric analysis of immune cell subsets, immune activation and TdTomato expression Single cell suspensions for flow cytometric analysis were obtained by enzymatic digestion of dissected spleens with Liberase (Roche) and DNAsel (Sigma-Aldrich) at 37 °C. Digestion was neutralized by addition of ice-cold PBS and the resulting suspensions were further mashed over 70um nylon meshes. At the end of the isolation procedure, counting beads (ThermoFisher) were added to all samples. Sample aliquots were then stained for 30 minutes at 4 °C with antibody cocktail. Fixable viability dyes were added to allow discrimination and selection of live cells during analysis. Samples were acquired on a Symphony A3 flow cytometry (BD Biosciences) and analyzed in FlowJo (BD Biosciences).

[0377] Statistical analysis

[0378] Statistical analyses were performed with GraphPad Prism software (version 8.3, GraphPad Software Inc., CA, USA). In vitro and in vivo experiments were analyzed using one-way ANOVA. Data in the study are represented as means ± SEM, unless otherwise mentioned. A p-value of below 0.05 is considered statistically significant difference (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0379] Synthesis of example 1 (SAM-3-Adm)

[0380] Preparation of starting material 1

[0381]

[0382] 2-Hexyldecanoic acid (8.80 g, 34.3 mmol, 1.0 equiv.) was dissolved in dry DCM (30 mL), then oxalyl chloride (3.8 mL, 44.6 mmol, 1.3 equiv.) was dropwise added at 0 °C, followed by DMF (cat., 20 pL). The mixture was stirred vigorously at 0 °C for 1 hour and then at room temperature (RT) for another 1 hour. Then the reaction mixture was concentrated by rotavapor under vacuum with a plug of cotton inserted between the flask and the rotavapor. The acid chloride was used in the next step without further purification and stored at -20 °C for future use.

[0383] Trimethylolethane (1.00 g, 8.32 mmol, 1.0 equiv.) was dissolved in dry DCM (50 mL), and then the acid chloride obtained above (4.80 g, 17.5 mmol, 2.1 equiv.) was added at 0 °C, followed by dropwise addition of Et3N (2.9 ml, 20.8 mmol, 2.5 equiv.). The mixture was stirred vigorously at 0 °C and allowed to warm to room temperature over 16 hours. Then the reaction mixture was washed 3 times by sat. NaHCOs (aq.), 1 time by brine and dried over Na2SO4, filtered and concentrated. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 15:1 then 10:1) to deliver starting material 1 as a colorless oil (2.81 g, 57% yield).

[0384] 1H NMR (400 MHz, Chloroform-d) δ 4.06-3.99 (m, 4H, H1, H6), 3.38 (s, 2H, H3), 2.39-2.32 (m, 2H, H15, H28), 1.64-1.55 (m, 4H, H14, H29 or H18, H30), 1.49-1.41 (m, 4H, H14, H29 or H18, H30), 1.25 (brd, 40H), 0.97 (s, 3H, H5), 0.87 (t, J = 6.8 Hz, 12H, H7, H24, H37, H42).13C NMR (100 MHz, Chloroform-d) 6 177.1 (C16, C26), 65.9 (Cl, C6), 64.7 (C3), 46.0 (C15, C28), 40.6 (C2), 32.6 (C14, C18, C29, C30), 32.0, 31.8, 29.7, 29.6, 29.41, 29.35, 27.63, 27.58, 22.81, 22.74, 17.0 (C5), 14.24 (C7, C37 or C24, C42), 14.20 (C7, C37 or C24, C42). LRMS (ESI) (m / z): calculated for [M+H]+(C38H73O5) requires 597.6, found: 597.6. Preparation of intermediate la

[0385]

[0386] 4-pentynoic acid (1.5 mmol, 1.5 equiv.) was dissolved in dry DCM (3 mL), and then Ghosez reagent (1.8 mmol, 1.8 equiv.) was added at room temperature. The mixture was stirred vigorously at room temperature for 30 min. Then to the reaction mixture was added starting material 1 (1.0 mmol, 1.0 equiv.) and Et3N (2.0 mmol, 2.0 equiv.) dropwise at 0 °C and allowed to warm to room temperature over 3 hours. Then the reaction mixture was diluted by DCM, washed 5 times by water, 1 time by brine, dried over Na2SO4, filtered and concentrated. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 20:1) to deliver intermediate la as a colorless oil (60% yield)

[0387] 1H NMR (400 MHz, Chloroform-d) δ 4.04 (s, 2H, H3), 4.00 (s, 4H, H1, H6), 2.59-2.55 (m, 2H, H44), 2.52-2.47 (m, 2H, H45), 2.37-2.30 (m, 2H, H15, H28), 1.98 (t, J = 2.6 Hz, 1H, H48), 1.62-1.54 (m, 4H, H14, H29 or H18, H30, overlapped with water peak), 1.48-1.41 (m, 4H, H14, H29 or H18, H30), 1.25 (brd, 40H), 1.02 (s, 3H, H5), 0.94-0.82 (m, 12H, H7, H24, H37, H42).13C NMR (100 MHz, Chloroform-d) 6 176.3 (Cl, C6), 171.4 (C3), 82.4 (C46), 69.4 (C48), 66.2 (C3), 65.6 (Cl, C6), 45.9 (C15, C28), 38.5 (C2), 33.4 (C44), 32.5 (C14, C18, C29, C30), 32.0, 31.8, 29.7, 29.6, 29.43, 29.35, 27.61, 27.56, 22.81, 22.75, 17.3 (C5), 14.5 (C45), 14.25 (C7, C37 or C24, C42), 14.20 (C7, C37 or C24, C42). LRMS (ESI) (m / z): calculated for [M+H]+(C42H77O6) requires 677.6, found: 677.7.

[0388] Preparation of Example 1

[0389]

[0390] Intermediate la (0.5 mmol, 1.0 equiv.), elemental sulfur (2.0 mmol, 4.0 equiv.) and N, N-dimethylethylenediamine (1.0 mmol, 2.0 equiv.) were mixed in pyridine (0.5 mL, C =1.0 M) in a Schlenk tube and then the tube was purged by flushing with N2 for 2 min. Afterwards, the tube was sealed and the reaction mixture was stirred vigorously at 100 °C for 4 h. The crude residue was then concentrated and purified by silica gel column chromatography (DCM / MeOH = 20:1 then 15:1 then 10:1) to deliver Example 1 as a brown oil (24% yield).

[0391] 1H NMR (400 MHz, Chloroform-d) δ 8.47 (s, 1H, H49), 3.99 (brd, 6H, Hl, H3, H6), 3.74-3.70 (m, 2H, H50), 2.67 (t, J = 7.4 Hz, 2H, H46), 2.62 (t, J = 5.9 Hz, 2H, H51), 2.39 (t, J = 7.2 Hz, 2H, H44), 2.36-2.31 (m, 2H, H15, H28), 2.29 (s, 6H, H53, H55), 2.12 (app. p, J= 7.3 Hz, 2H, H45), 1.62-1.53 (m, 4H, H14, H29 or H18, H30), 1.48-1.40 (m, 4H, H14, H29 or H18, H30), 1.24 (brd, 40H), 1.01 (s, 3H, H5), 0.87 (t, J = 6.7 Hz, 12H, H7, H24, H37, H42).13C NMR (100 MHz, Chloroform-d) 6204.2 (C48), 176.4 (C16, C26), 172.8 (C43), 65.8 (C3), 65.5 (Cl, C6), 56.3 (C51), 45.9 (C15, C28), 45.2 (C46), 44.9 (C53, C55), 43.1 (C50), 38.5 (C2), 32.9 (C44), 32.4 (C14, C18, C29, C30), 32.0, 31.8, 29.7, 29.6, 29.4, 29.3, 27.6, 27.5, 24.5 (C45), 22.8, 22.7, 17.2 (C5), 14.24 (C7, C37 or C24, C42), 14.19 (C7, C37 or C24, C42). LRMS (ESI) (m / z): calculated for [M+H]+(C46H89N2O6S1) requires 797.6, found: 797.6.Synthesis of example 2 (SAM-4-Adm)

[0392] Preparation of intermediate lb

[0393]

[0394] The procedure defined above for Example 1 was repeated, but using 5-hexynoic acid instead as alkynoic acid to produce intermediate lb as a colorless oil (66% yield).

[0395] 1H NMR (400 MHz, Chloroform-d) δ 4.00 (s, 2H, H3), 3.99 (s, 4H, H1, H6), 2.46 (t, J = 7.5 Hz, 2H, H44), 2.37-2.30 (m, 2H, H15, H28), 2.25 (td, J = 6.9, 2.6 Hz, 2H, H46), 1.96 (t, J = 2.6 Hz, 1H, H49), 1.83 (p, J = 7.2 Hz, 2H, H45), 1.62-1.53 (m, 4H, H14, H29 or H18, H30), 1.48-1.39 (m, 4H, H14, H29 or H18, H30), 1.24 (brd, 40H), 1.01 (s, 3H, H5), 0.88-0.85 (m, 12H, H7, H24, H37, H42).13C NMR (100 MHz, Chloroform-d) 6 176.3 (Cl, C6), 172.7 (C3), 83.1 (C48), 69.4 (C49), 65.9 (C3), 65.6 (Cl, C6), 45.9 (C15, C28), 38.4 (C2), 32.8 (C44), 32.5 (C14, C18, C29, C30), 32.0, 31.8, 29.7, 29.6, 29.4, 29.3, 27.6, 27.5, 23.6 (C45), 22.8, 22.7, 17.9 (C46), 17.3 (C5), 14.23 (C7, C37 or C24, C42), 14.18 (C7, C37 or C24, C42). LRMS (ESI) (m / z): calculated for [M+H]+(C43H79O6) requires 691.6, found: 691.6.

[0396] Preparation of Example 2

[0397]

[0398] Intermediate lb was contacted with elemental sulfur and N, N-dimethylethylenediamine in the presence of pyridine as defined in Example 1, thereby obtaining Example 2 as a brown oil (18% yield).

[0399] 1H NMR (400 MHz, Chloroform-d) δ 8.52 (s, 1H, H50), 3.99 (brd, 2H, H3, small peak), 3.99 (brd, 4H, Hl, H6, big peak), 3.76-3.72 (m, 2H, H51), 2.68-2.64 (m, 4H, H48, H52), 2.37-2.31 (m, 10H, including H15, H28, H44, H54, H56), 1.87-1.79 (m, 2H, H46), 1.70-1.64 (m, 2H, H45), 1.62-1.53 (m, 4H, H14, H29 orH18, H30), 1.48-1.40 (m, 4H, H14, H29 or H18, H30), 1.25 (brd, 40H), 1.01 (s, 3H, H5), 0.89-0.85 (m, 12H, H7, H24, H37, H42).13C NMR (100 MHz, Chloroform-d) 6 205.2 (C49), 176.3 (C16, C26), 173.1 (C43), 65.9 (C3), 65.6 (Cl, C6), 56.4 (C52), 46.2 (C48), 45.9 (C15, C28), 44.7 (C54, C56), 42.7 (C51), 38.5 (C2), 33.9 (C44), 32.5 (C14, C18, C29, C30), 32.0, 31.8, 29.7, 29.6, 29.43, 29.35, 28.9 (C46), 27.59, 27.54, 24.2 (C45), 22.81, 22.75, 17.3 (C5), 14.25 (C7, C37 or C24, C42), 14.21 (C7, C37 or C24, C42). LRMS (ESI) (m / z): calculated for [M+H]+(C47H91N2O6S1) requires 811.7, found: 811.7.

[0400] Synthesis of example 3 (SAM-5-Adm)

[0401] Preparation of Intermediate lc

[0402]

[0403] The procedure defined above for Example 1 was repeated, but using 6-heptynoic acid instead as alkynoic acid to produce intermediate lc as a colorless oil (yield 62%).

[0404] 1H NMR (400 MHz, Chloroform-d) δ 4.00 (s, 2H, H3), 3.99 (s, 4H, H1, H6), 2.37-2.30 (m, 4H, H15, H28, H44), 2.21 (td, J = 7.0, 2.6 Hz, 2H, H48), 1.95 (t, J = 2.7 Hz, 1H, H50), 1.74 (p, J = 7.5 Hz, 2H, H45), 1.62-1.52 (m, 6H, including H46 & H14, H29 or H18, H30 ), 1.48-1.40 (m, 4H, H14, H29 or H18, H30), 1.25 (brd, 40H), 1.01 (s, 3H, H5), 0.89-0.85 (m, 12H, H7, H24, H37, H42).13C NMR (100 MHz, Chloroform-d) 6 176.3 (Cl, C6), 173.0 (C3), 83.1 (C49, deduced from HSQC), 68.9 (C50), 65.8 (C3), 65.6 (Cl, C6), 45.9 (C15, C28), 38.4 (C2), 33.7 (C44), 32.5 (C14, C18, C29, C30), 32.0, 31.8, 29.7, 29.6, 29.43, 29.35, 27.9 (C46), 27.60, 27.55, 24.0 (C45), 22.81, 22.75, 18.2 (C48), 17.3 (C5), 14.25 (C7, C37 or C24, C42), 14.20 (C7, C37 or C24, C42). LRMS (ESI) (m / z): calculated for [M+H]+(C45H81O6) requires 705.6, found: 705.7. Preparation of Example 3

[0405]

[0406] Intermediate lc was contacted with elemental sulfur and N,N-dimethylethylenediamine in the presence of pyridine as defined in Example 1, thereby obtaining Example 3 as a brown oil (20% yield).

[0407] 3H NMR (400 MHz, Chloroform-d) 6 8.18 (s, 1H, H51), 3.99 (brd, 6H, H1, H3, H6), 3.72-3.68 (m, 2H, H53), 2.68-2.59 (m, 4H, H49, H54), 2.37-2.28 (m, 10H, including H15, H28, H44, H56, H57), 1.83-1.78 (m, 2H, H46), 1.66-1.62 (m, 2H, H45), 1.60-1.53 (m, 4H, H14, H29 or H18, H30), 1.49-1.34 (m, 6H, H14, H29 or H18, H30, H48), 1.25 (brd, 40H), 1.01 (s, 3H, H5), 0.89-0.85 (m, 12H, H7, H24, H37, H42).13C NMR (100 MHz, Chloroform-d) 6 204.5 (C50), 176.4 (C16, C26), 173.3 (C43), 65.8 (C3), 65.6 (Cl, C6), 56.4 (C54), 46.6 (C49), 45.9 (C15, C28), 45.0 (C56, C57), 43.0 (C53), 38.4 (C2), 34.0 (C44), 32.5 (C14, C18, C29, C30), 32.0, 31.8, 29.7, 29.6, 29.42, 29.35, 29.1 (C46), 28.5 (C48), 27.60, 27.55, 24.7 (C45), 22.80, 22.74, 17.3 (C5), 14.25 (C7, C37 or C24, C42), 14.20 (C7, C37 or C24, C42). LRMS (ESI) (m / z): calculated for [M+H]+(C48H92N2O6S1) requires 825.7, found: 825.7.

[0408] Synthesis of example 4 (SAM-9-Adm)

[0409] Preparation of Intermediate Id

[0410] 17

[0411] o

[0412]

[0413] The procedure defined above for Example 1 was repeated, but using 9-decynoic acid instead as alkynoic acid to produce intermediate Id as a colorless oil (65% yield).

[0414] 3H NMR (400 MHz, Chloroform-d) 6 3.99 (s, 2H, H3, small peak), 3.99 (s, 4H, Hl, H6, big peak), 2.37-2.31 (m, 2H, H15, H28), 2.30 (t, J = 7.7 Hz, 2H, H44), 2.17 (td, J = 7.1, 2.7 Hz, 2H, H51), 1.93 (t, J = 2.6 Hz, 1H, H53), 1.62-1.50 (m, 6H, including H45 & H14, H29 or H18, H30, overlapped with water peak),1.48-1.35 (m, 6H, including H50 & H14, H29 or H18, H30 ), 1.30-1.25 (m, 46H), 1.01 (s, 3H, H5), 0.89-0.85 (m, 12H, H7, H24, H37, H42).13C NMR (100 MHz, Chloroform-d) 6 176.3 (Cl, C6), 173.5 (C3), 84.8 (C52), 68.2 (C53), 65.72 (C3), 65.65 (Cl, C6), 45.9 (C15, C28), 38.4 (C2), 34.3 (C44), 32.5 (C14, C18, C29, C30), 32.0, 31.8, 29.7, 29.6, 29.43, 29.35, 29.26, 29.1, 28.8, 28.6, 27.60, 27.55, 25.0 (C45), 22.81, 22.75, 18.5 (C51), 17.3 (C5), 14.24 (C7, C37 or C24, C42), 14.20 (C7, C37 or C24, C42). LRMS (ESI) (m / z): calculated for [M+H]+(C47H87O6) requires 747.6, found: 747.7.

[0415] Preparation of Example 4

[0416] 17

[0417]

[0418] Intermediate Id was contacted with elemental sulfur and N, N-dimethylethylenediamine in the presence of pyridine as defined in Example 1, thereby obtaining Example 4 as a yellowish oil (12% yield).

[0419] 3H NMR (400 MHz, Chloroform-d) 69.89 (s, 1H, H55), 4.12 (d, J = 5.3 Hz, 2H, H56), 3.99 (brd, 6H, Hl, H6, H3), 3.38 (t, J = 5.2 Hz, 2H, H57), 2.86 (s, 6H, H59, H61), 2.67-2.64 (m, 2H, H53), 2.37-2.27 (m, 4H, H15, H28, H44), 1.78-1.70 (m, 2H, H52), 1.65-1.53 (m, 6H, including H45, H14, H29 or H18, H30, overlapped with water peak), 1.48-1.40 (m, 4H, H14, H29 or H18, H30 ), 1.25 (brd, 50H), 1.01 (s, 3H, H5), 0.89-0.85 (m, 12H, H7, H24, H37, H42).13C NMR (100 MHz, Chloroform-d) 6 208.8 (C49), 176.3 (C16, C26), 173.6 (C43), 65.72 (C3), 65.67 (Cl, C6), 56.6 (C57), 46.5 (C53), 45.9 (C15, C28), 44.0 (C59, C61), 40.5 (C56), 38.4 (C2), 34.3 (C44), 32.5 (C14, C18, C29, C30), 32.0, 31.8, 29.7, 29.6, 29.4, 29.35, 29.33, 29.30, 29.1 (C52), 27.60, 27.55, 25.0 (C45), 22.81, 22.75, 17.3 (C5), 14.25 (C7, C37 or C24, C42), 14.21 (C7, C37 or C24, C42). LRMS (ESI) (m / z): calculated for [M+H]+(C52H101N2O6S1) requires 881.7, found: 881.7.

[0420] Synthesis of example 5 (SAM-3-Ac7)

[0421] Preparation of Example 517

[0422]

[0423] The procedure defined above for Example 1 was repeated, but using 2-(azepan-l-yl)ethanamine as bisamine, thereby obtaining example 5 as a brown oil (19% yield).

[0424] 3H NMR (400 MHz, Chloroform-d) 68.99 (s, 1H, H49), 3.99 (brd, 6H, Hl, H3, H6), 3.79 (brd, 2H, H50), 2.94 (brd, 2H, H12), 2.88-2.82 (m, 4H, H56, H61), 2.70 (t, J = 7.5 Hz, 2H, H46), 2.42-2.31 (m, 4H, H15, H28), 2.14-2.10 (m, 2H, H44), 1.76 (brd, 4H, H57, H60), 1.71-1.61 (m, 6H, including H45, H58, H59), 1.60-1.53 (m, 4H, H14, H29 or H18, H30), 1.48-1.41 (m, 4H, H14, H29 or H18, H30), 1.25 (brd, 40H), 1.01 (s, 3H, H5), 0.88-0.85 (m, 12H, H7, H24, H37, H42).13C NMR (100 MHz, Chloroform-d) 6 203.2 (C48), 176.3 (C16, C26), 172.8 (C43), 65.9 (C3), 65.6 (Cl, C6), 55.27 (C56, C61), 55.14 (C51), 45.9 (C46), 45.2 (C15, C28), 42.5 (C50), 38.5 (C2), 32.9 (C44), 32.4 (C14, C18, C29, C30), 32.0, 31.8, 29.7, 29.6, 29.41, 29.33, 27.57, 27.52 (C58, C59), 27.0 (C57, C60), 24.3 (C45), 22.79, 22.73, 17.2 (C5), 14.23 (C7, C37 or C24, C42), 14.19 (C7, C37 or C24, C42). LRMS (ESI) (m / z): calculated for [M+H]+(C50H94N2O6S1) requires 851.7, found: 851.7.

[0425] Synthesis of example 6 (SAM-4-Ac7)

[0426] Preparation of Example 6

[0427]

[0428] The procedure defined above for Example 2 was repeated to produce intermediate lb and 2-(azepan-l-yl)ethanamine was used as bisamine, thereby obtaining example 6 as a brown oil (25% yield).3H NMR (400 MHz, Chloroform-d) 68.91 (s, 1H, H50), 3.99 (brd, 6H, Hl, H6, H3), 3.78 (brd, 2H, H51), 2.93 (brd, 2H, H52), 2.88-2.82 (m, 4H, H56, H61), 2.70 (t, J = 7.5 Hz, 2H, H48), 2.37-2.31 (m, 4H,including H15, H28, H44), 1.87-1.79 (m, 2H, H46), 1.76 (brd, 4H, H57, H60), 1.71-1.62 (m, 6H, including H45, H58, H59, overlapped with water peak), 1.60-1.53 (m, 4H, H14, H29 or H18, H30), 1.48-1.41(m, 4H, H14, H29 or H18, H30), 1.25 (brd, 40H), 1.01 (s, 3H, H5), 0.89-0.85 (m, 12H, H7, H24, H37, H42).

[0429] 13C NMR (100 MHz, Chloroform-d) 6 205.1 (C49), 176.3 (C16, C26), 173.1 (C43), 65.9 (C3), 65.6 (Cl, C6), 55.23 (C56, C61), 55.16 (C52), 46.2 (C48), 45.9 (C15, C28), 42.4 (C51), 38.5 (C2), 33.9 (C44), 32.5 (C14, C18, C29, C30), 32.0, 31.8, 29.7, 29.6, 29.43, 29.35, 28.8 (C46), 27.59, 27.54, 27.10 (C58, C59), 27.08 (C57, C60), 24.2 (C45), 22.81, 22.75, 17.3 (C5), 14.25 (C7, C37 or C24, C42), 14.21 (C7, C37 or C24, C42). LRMS (ESI) (m / z): calculated for [M+H]+(C51H97N2O6S1) requires 865.7, found: 865.7.

[0430] Synthesis of example 7 (SAM-5-Ac7)

[0431] Preparation of Example 7

[0432] 17

[0433]

[0434] The procedure defined above for Example 3 was repeated to produce intermediate lc and 2-(azepan-l-yl)ethanamine was used as bisamine, thereby obtaining example 7 as a brown oil (23% yield).3H NMR (400 MHz, Chloroform-d) 6 3.99 (brd, 8H, Hl, H6, H3, H53), 3.03 (brd, 6H, H54, H56, H61), 2.72 (t, J = 7.5 Hz, 2H, H49), 2.37-2.28 (m, 4H, including H15, H28, H44), 1.86-1.77 (brd, 6H, H48, H57, H60), 1.76-1.53 (brd, 12H, including H45, H46, H58, H59, H14, H29 or H18, H30), 1.52-1.37 (m, 4H, H14, H29 or H18, H30), 1.25 (brd, 40H), 1.01 (s, 3H, H5), 0.89-0.85 (m, 12H, H7, H24, H37, H42).13C NMR (100 MHz, Chloroform-d) 6 205.2 (C50), 176.3 (C16, C26), 173.3 (C43), 65.8 (C3), 65.6 (Cl, C6), 55.5 (C56, C61), 55.1 (C54), 46.3 (C49), 45.9 (C15, C28), 42.1 (C53), 38.4 (C2), 34.0 (C44), 32.5 (C14, C18, C29, C30), 32.0, 31.8, 29.7, 29.6, 29.42, 29.34 (C46), 29.0 (C48), 27.59, 27.54, 27.1 (C58, C59), 27.0 (C57, C60), 24.7 (C45), 22.80, 22.74, 17.3 (C5), 14.25 (C7, C37 or C24, C42), 14.20 (C7, C37 or C24, C42). LRMS (ESI) (m / z): calculated for [M+H]+(C52H98N2O6SI) requires 879.7, found: 879.7.

[0435] Synthesis of example 8 (SAM-9-Ac7)

[0436] Preparation of Example 8

[0437]

[0438] The procedure defined above for Example 4 was repeated to produce intermediate Id and 2-(azepan-l-yl)ethanamine was used as bisamine, thereby obtaining example 8 as a yellowish oil (16% yield).

[0439] 3H NMR (400 MHz, Chloroform-d) 63.99 (brd, 6H, Hl, H6, H3), 3.69 (t, J = 6.3 Hz, 2H, H56), 3.03 (brd, 4H, H61, H66), 2.85 (t, J = 6.1 Hz, 2H, H57), 2.72-2.68 (m, 2H, H53), 2.37-2.27 (m, 4H, H15, H28, H44), 1.87-1.52 (m, 16H, including H45, H52, H62, H63, H64, H65, H14, H29 or H18, H30), 1.48-1.40 (m, 4H, H14, H29 or H18, H30), 1.24 (brd, 50H), 1.01 (s, 3H, H5), 0.89-0.85 (m, 12H, H7, H24, H37, H42).13C NMR (100 MHz, Chloroform-d) 6208.0 (C49), 176.3 (C16, C26), 173.5 (C43), 65.72 (C3), 65.65 (Cl, C6), 55.5, 55.4, 55.2, 54.8, 46.7 (C53), 45.9 (C15, C28), 38.4 (C2), 34.3 (C44), 32.5 (C14, C18, C29, C30), 32.0, 31.8, 29.7, 29.6, 29.45, 29.35, 29.0 (C52), 27.59, 27.54, 25.0 (C45), 22.81, 22.75, 17.3 (C5), 14.25 (C7, C37 or C24, C42), 14.20 (C7, C37 or C24, C42). LRMS (ESI) (m / z): calculated for [M+H]+(C56H107N2O6S1) requires 935.8, found: 935.8.

[0440] Synthesis of example 9 (SAM-3-butylAdm)

[0441] Preparation of Example 9

[0442]

[0443] The procedure defined above for Example 1 was repeated, but using 4-dimethylaminobutylamine as bisamine, thereby obtaining example 9 as a brown oil (20% yield).

[0444] 3H NMR (400 MHz, Chloroform-d) 6 9.73 (s, 1H, H49), 3.99 (brd, 6H, Hl, H3, H6), 3.67-3.63 (m, 2H, H50), 2.67 (m, 4H, H46, H56), 2.48 (s, 6H, H58, H59), 2.39 (t, J = 7.2 Hz, 2H, H44), 2.34-2.31 (m, 2H, H15, H28), 2.12 (app. p, J = 7.3 Hz, 2H, H45), 1.80-1.74(m, 4H, H51, H55), 1.58-1.54 (m, 4H, H14, H29 or H18, H30), 1.46-1.41 (m, 4H, H14, H29 or H18, H30), 1.24 (brd, 40H), 1.02 (s, 3H, H5), 0.87 (t, J = 6.7Hz, 12H, H7, H24, H37, H42).13C NMR (100 MHz, Chloroform-d) 6203.8 (C48), 176.5 (C16, C26), 172.9 (C43), 65.8 (C3), 65.5 (Cl, C6), 58.7 (C56), 51.0, 45.9 (C15, C28), 45.5 (C46), 45.3 (C50), 44.4 (C58, C59), 38.6 (C2), 33.0 (C44), 32.4 (C14, C18, C29, C30), 32.0, 31.8, 29.7, 29.6, 29.40, 29.33, 27.57, 27.52, 25.7 (C45), 24.6 (C51, C55), 24.2, 22.78, 22.73, 17.2 (C5), 14.22 (C7, C37 or C24, C42), 14.18 (C7, C37 or C24, C42). LRMS (ESI) (m / z): calculated for [M+H]+(C48H92N2O6S1) requires 825.7, found: 825.7.

[0445] Synthesis of example 10 (SAM-3-butylAc7)

[0446] Preparation of Example 10

[0447] 17

[0448]

[0449] The procedure defined above for Example 1 was repeated, but using 4-(azepan-l-yl)butan-l-amine as bisamine, thereby obtaining example 10 as a brown oil (25% yield).

[0450] 3H NMR (400 MHz, Chloroform-d) 69.42 (s, 1H, H49), 4.00 (brd, 6H, Hl, H3, H6), 3.75 (brd, 2H, H50), 3.25-3.16 (brd, 4H, H58, H63) 2.99 (brd, 2H, H12), 2.70 (t, J = 7.5 Hz, 2H, H46), 2.42-2.31 (m, 4H, H15, H28, H44), 2.17-2.10 (m, 4H, H59, H63), 1.96-1.90 (brd, 6H, H45, H60, H61), 1.83-1.80 (4H, H58, H63), 1.74 (brd, 4H, H51, H55), 1.64-1.53 (m, 6H, H14, H29 or H18, H30), 1.49-1.40 (m, 4H, H14, H29 or H18, H30), 1.25 (brd, 40H), 1.01 (s, 3H, H5), 0.89-0.85 (m, 12H, H7, H24, H37, H42).13C NMR (100 MHz, Chloroform-d) 6 203.5 (C48), 176.5 (C16, C26), 172.9 (C43), 65.8 (C3), 65.5 (Cl, C6), 56.5 (C56, C61), 54.8 (C56), 49.1 (C51, C55), 45.9 (C46), 45.2 (C15, C28), 42.5 (C50), 38.6 (C2), 33.1 (C44), 32.4 (C14, C18, C29, C30), 32.0, 31.8, 29.7, 29.6, 29.43, 29.35, 27.59, 27.54 (C60, C61), 27.0 (C59, C62), 25.25 (C45), 24.7 (C51, C55), 22.81, 22.75, 17.3 (C5), 14.25 (C7, C37 or C24, C42), 14.21 (C7, C37 or C24, C42). LRMS (ESI) (m / z): calculated for [M+H]+(C52H98N2O6S1) requires 879.7, found: 879.7.

[0451] Synthesis of example 11 (SAM-3-Ade)

[0452] Preparation of Example 1117

[0453] o

[0454]

[0455] The procedure defined above for Example 1 was repeated, but using 2-(diethylamino)ethylamine as bisamine, thereby obtaining example 11 as a yellowish oil (20% yield).

[0456] 3H NMR (400 MHz, Chloroform-d) 68.42 (s, 1H, H49), 3.99 (brd, 6H, Hl, H6, H3), 3.70 (brd, 2H, H50), 2.77-2.74 (m, 2H, H51), 2.69 (t, J = 7.4 Hz, 2H, H46), 2.64-2.59 (m, 4H, H53, H55), 2.40 (t, J = 7.2 Hz, 2H, H44), 2.36-2.30 (m, 2H, H15, H28), 2.11 (app. p, J = 7.3 Hz, 2H, H45), 1.62-1.53 (m, 4H, H14, H29 or H18, H30), 1.48-1.40 (m, 4H, H14, H29 or H18, H30), 1.25 (brd, 40H), 1.07 (t, J = 7.1 Hz, 6H, H56, H57), 1.01 (s, 3H, H5), 0.89-0.85 (m, 12H, H7, H24, H37, H42).13C NMR (100 MHz, Chloroform-d) 6 205.2 (C48), 176.3 (C16, C26), 172.8 (C43), 65.8 (C3), 65.5 (Cl, C6), 50.1 (C51), 46.8 (C53, C55), 45.9 (C15, C28), 45.4 (C46), 43.0 (C50), 38.5 (C2), 32.9 (C44), 32.4 (C14, C18, C29, C30), 32.0, 31.8, 29.7, 29.6, 29.4, 29.3, 27.59, 27.54, 24.3 (C45), 22.8, 22.7, 17.3 (C5), 14.25 (C7, C37 or C24, C42), 14.20 (C7, C37 or C24, C42), 11.5 (C56, C57). LRMS (ESI) (m / z): calculated for [M+H]+(C48H93N2O6S1) requires 824.7, found: 824.7.

[0457] Synthesis of example 12 (SAM-3-Amp)

[0458] Preparation of Example 12

[0459] 17

[0460]

[0461] The procedure defined above for Example 1 was repeated, but using 2-(4-methyl-piperazin-l-yl)-ethylamine as bisamine, thereby obtaining example 12 as a yellowish oil (16% yield).

[0462] 3H NMR (400 MHz, Chloroform-d) 68.15 (s, 1H, H49), 3.99 (brd, 6H, Hl, H6, H3), 3.71 (app. q, J = 5.7 Hz, 2H, H50), 2.68 (t, J = 7.4 Hz, 2H, H46), 2.63 (t, J = 6.1 Hz, 2H, H51), 2.54 (brd, 8H, H55, H56, H58, H59), 2.41 (t, J = 7.1 Hz, 2H, H44), 2.38-2.32 (m, 2H, H15, H28), 2.30 (s, 3H, H60), 2.12 (p, J = 7.2 Hz,2H, H45), 1.60-1.55 (m, 4H, H14, H29 or H18, H30), 1.49-1.40 (m, 4H, H14, H29 or H18, H30), 1.25 (brd, 40H), 1.02 (s, 3H, H5), 0.87 (t, J = 6.8 Hz, 12H, H7, H24, H37, H42).13C NMR (100 MHz, Chloroform-d) 6203.9 (C48), 176.4 (C16, C26), 172.9 (C43), 65.8 (C3), 65.5 (Cl, C6), 55.09, 55.06, 52.7, 46.0 (C60), 45.9 (C15, C28), 45.4 (C46), 42.6 (C50), 38.6 (C2), 32.9 (C44), 32.5 (C14, C18, C29, C30), 32.0, 31.8, 29.7, 29.6, 29.4, 29.3, 27.60, 27.55, 24.4 (C45), 22.80, 22.75, 17.3 (C5), 14.25 (C7, C37 or C24, C42), 14.21 (C7, C37 or C24, C42). LRMS (ESI) (m / z): calculated for [M+H]+(C49H94N3O6S1) requires 852.7, found: 852.7.

[0463] Synthesis of example 13 (SAM-3-Apyr)

[0464] Preparation of Example 13

[0465] 17

[0466]

[0467] The procedure defined above for Example 1 was repeated, but using 1-pyrrolidineethanamine as bisamine, thereby obtaining example 13 as a yellowish oil (13% yield).

[0468] 3H NMR (400 MHz, Chloroform-d) 68.72 (s, 1H, H49), 4.00 (s, 6H, Hl, H3, H6), 3.86-3.81 (m, 2H, H50), 2.93 (brd, 2H, H51), 2.79 (brd, 4H, H55, H58), 2.71 (t, J = 7.4 Hz, 2H, H46), 2.40 (t, J = 7.2 Hz, 2H, H44), 2.36-2.30 (m, 2H, H15, H28), 2.13 (p, J = 7.0 Hz, 2H, H45), 1.91 (brd, 4H, H56, H57), 1.62-1.53 (m, 4H, H14, H29 or H18, H30), 1.48-1.40 (m, 4H, H14, H29 or H18, H30), 1.25 (brd, 40H), 1.02 (s, 3H, H5), 0.87 (t, J = 6.7 Hz, 12H, H7, H24, H37, H42).13C NMR (100 MHz, Chloroform-d) 6 176.3, 172.8, 65.9, 65.6, 56.2, 54.3, 54.0, 53.7, 45.9, 45.1, 38.5, 33.0, 32.4, 32.0, 31.8, 29.7, 29.6, 29.4, 29.3, 27.6, 27.5, 24.4, 23.5, 22.8, 22.7, 17.2, 14.25, 14.21. LRMS (ESI) (m / z): calculated for [M+H]+(C48H91N2O6S1) requires 823.7, found: 823.6.

[0469] Synthesis of example 14 (SAMole-3-Adm)

[0470] Preparation of starting material 2

[0471]

[0472] Trimethylolethane (4.81 g, 40 mmol, 1.0 equiv.) was dissolved in dry acetone (30 mL), and then 2,2-dimethoxypropane (4.17 g, 40 mmol, 1.0 equiv.) was added (Note: Excess of 2,2-Dimethoxypropane will result in the formation of triketal side product) followed by camphoric acid (93 mg, 0.4 mmol, 0.01 equiv.). The mixture was stirred vigorously at room temperature for 2 hours. Then potassium carbonate (1.0 g) was added to the reaction mixture, filtered and the filtrate was collected and concentrated to give crude ketal product which was used in the next step without further purification. The crude ketal product (1.28 g, 8.00 mmol, 1.0 equiv.), 4-pentynoic acid (785 mg, 8.00 mmol, 1.0 equiv.) and DMAP (98 mg, 0.80 mmol, 0.1 equiv.) were mixed in DCM (10 mL), then at 0 °C, DCC (1.65 g, 8.00 mmol, 1.0 equiv.) in DCM (5.0 mL) was added. The mixture was stirred vigorously at 0 °C and allowed to warm to room temperature over 16 hours. Then the reaction mixture was filtered, and the filtrate was collected and concentrated. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 5:1 + 0.1 % EtaN) to deliver starting material 2 as a colorless oil (1.63 g, 85% yield).

[0473] 3H NMR (400 MHz, Chloroform-d) 64.08 (s, 2H, H8), 3.56-3.48 (m, 4H, H10, H14), 2.47-2.45 (m, 2H, H4), 2.39-2.37 (m, 2H, H3), 1.91-1.89 (m, 1H, H7), 1.29 (d, J = 19.0 Hz, 6H, H16, H17), 0.74 (s, 3H, H15).

[0474] 13C NMR (100 MHz, Chloroform-d) 6 171.4 (C2), 97.9 (C12), 82.4 (C6), 69.2 (C7), 66.9 (CIO, C14), 66.0 (C8), 33.2 (C3), 26.9 (C9), 20.1 (C16, C17), 17.6 (C15), 14.2 (C4). LRMS (ESI) (m / z): calculated for [M+H]+(C13H20O4) requires 241.1, found: 241.1.

[0475] Preparation of Intermediate 2a

[0476]

[0477] 1612

[0478] Starting material 2 (0.5 mmol, 1.0 equiv.), elemental sulfur (2.0 mmol, 4.0 equiv.) and N, N-dimethylethylenediamine (1.0 mmol, 2.0 equiv.) were mixed in DMF (0.5 mL, C =1.0 M) in a Schlenk tube and then the tube was purged by flushing with N2 for 2 min. Afterwards, the tube was sealed and the reaction mixture was stirred vigorously at 80 °C for 24 h. The crude residue was then concentrated and purified by silica gel column chromatography (DCM / MeOH = 20:1 then 15:1 then 10:1) to deliver intermediate 2a.About 16% yield, brownish oil. The product was not pure; therefore, it was not characterized at this stage. However, intermediate 2a was confirmed on LRMS. LRMS (ESI) (m / z): calculated for [M+H]+(C17H33N2O4S1) requires 361.2, found: 361.2.

[0479] Preparation of Example 14

[0480] 16

[0481]

[0482] Intermediate 2a (0.2 mmol, 1.0 equiv.) was dissolved in a mixed solvent of THF / MeOH (2 / 2 mL) and then pyridinium p-toluenesulfonate (PPTS) (2.0 mmol, 10 equiv.) (Note: using strong acids, such as HCI, leads to the methyl ester product) was added. The mixture was stirred vigorously at room temperature for about 10 hours till the ketal was cleaved (monitored by1H NMR). Then the reaction mixture was concentrated and used in the next step without further purification. To the crude reaction mixture from above was added oleoyl imidazole (0.6 mmol, 6.0 equiv.) (Note: using oleoyl chloride leads to the major side product 14' where three lipid tails are attached on the molecule) and DMF (2.0 mL). The reaction mixture was then stirred vigorously at 80 °C for 24 h. The crude residue was then concentrated and purified by silica gel column chromatography (DCM / MeOH = 20:1 then 15:1 then 10:1) to deliver Example 14 as brownish oil.

[0483] 13% yield over 2 steps, yellowish oil.1H NMR (400 MHz, Methanol-d4) δ 5.36-5.33 (m, 4H, H33, H41, H40, H43), 4.03 (brd, 6H, Hl, H3, H5), 3.85 (t, J = 6.7 Hz, 2H, H51), 2.88 (t, J = 6.7 Hz, 2H, H52, overlapped with DMF), 2.65 (t, J = 7.4 Hz, 2H, H48), 2.52 (s, 6H, H74, H75), 2.41 (t, J = 7.4 Hz, 2H, H46), 2.34 (t, J = 7.4 Hz, 4H, H14, H34), 2.08-1.97 (m, 10H, including H47 and H32, H42, H39, H44), 1.63-1.60 (m, 4H, H27, H35), 1.33-1.29 (m, 40H, lipid-H), 1.04 (s, 3H, H4), 0.90 (t, J = 7.1 Hz, 6H, H65, H73).13C NMR (101 MHz, Methanol-d4) δ 206.5 (C49), 175.0 (C15, C25), 174.3 (C45), 130.9 (alkene-C), 130.8 (alkene-C), 66.9 (C3), 66.7 (Cl, C5), 57.1 (C52), 54.8 (DCM), 45.6 (C48), 45.0 (C57, C58), 43.2 (C51), 39.7 (C2), 35.0, 33.8 (C46), 33.1, 30.87, 30.81, 30.77, 30.6, 30.5, 30.4, 30.3, 30.20, 30.18, 28.16 (C32 or C42), 28.13 (C32 or C42), 26.1 (C27, C35), 25.5 (C47), 23.8, 17.5 (C4), 14.5 (C65, C73). LRMS (ESI) (m / z): calculated for [M+H]+(C50H93N2O6S1) requires 849.7, found: 849.7.

[0484] Synthesis of example 15 (SAMole-3-Ac7)

[0485] Preparation of Intermediate 2b

[0486]

[0487] 1612

[0488] Starting material 2 as defined above (0.5 mmol, 1.0 equiv.), elemental sulfur (2.0 mmol, 4.0 equiv.) and 2-(azepan-l-yl)ethanamine (1.0 mmol, 2.0 equiv.) were mixed in pyridine (0.5 mL, C =1.0 M) in a Schlenk tube and then the tube was purged by flushing with N2for 2 min. Afterwards, the tube was sealed and the reaction mixture was stirred vigorously at 100 °C for 24 h. The crude residue was then concentrated and purified by silica gel column chromatography (DCM / MeOH = 20:1 then 15:1 then 10:1) to deliver intermediate 2b as a brownish oil (14% yield).

[0489] 3H NMR (400 MHz, Chloroform-d) 68.36 (s, 1H, H18), 4.17 (s, 2H, H7), 3.67-3.56 (m, 6H, H9, H13, H19), 2.80-2.71 (m, 8H, H3, H6, H23, H28), 2.44-2.41 (m, 2H, H21), 2.15-2.11 (m, 2H, H4), 1.69-1.61 (brd, 8H, H24, H25, H26, H27), 1.41 (d, J = 17.2 Hz, 6H, H15, H16), 0.84 (s, 3H, H14).13C NMR (100 MHz, Chloroform-d) 6205.7 (C17), 173.7 (C2), 111.5 (CH), 67.0 (C9, C13), 66.4 (C7), 56.2 (C21), 45.3 (C23, C28), 45.1 (C6), 45.0 (C19), 33.0 (C3), 27.2 (C24, C27), 25.9 (C25, C26), 24.6 (C4), 23.9 (C8), 23.5 (C15, C16), 17.9 (C14). LRMS (ESI) (m / z): calculated for [M+H]+(C21H38N2O4S1) requires 415.3, found: 415.3 Preparation of Example 15

[0490]

[0491] Intermediate 2b (0.2 mmol, 1.0 equiv.) was dissolved in a mixed solvent of THF / MeOH (2 / 2 mL) and then pyridinium p-toluenesulfonate (PPTS) (2.0 mmol, 10 equiv.) (Note: using strong acids, such as HCI, leads to the methyl ester product) was added. The mixture was stirred vigorously at room temperature for about 10 hours till the ketal was cleaved (monitored by1H NMR). Then the reaction mixture was concentrated and used in the next step without further purification. To the crude reaction mixture from above was added oleoyl imidazole (0.6 mmol, 6.0 equiv.) (Note: using oleoyl chloride leads to the major side product 14' where three lipid tails are attached on the molecule) and DMF (2.0 mL). The reaction mixture was then stirred vigorously at 80 °C for 24 h. The crude residue was then concentrated and purified by silica gel column chromatography (DCM / MeOH = 20:1 then 15:1 then 10:1) to deliver Example 15 as yellowish oil (15% yield).1H NMR (400 MHz, Methanol-d4) δ 5.35-5.33 (m, 4H, H33, H41, H40, H43), 4.03 (brd, 6H, Hl, H3, H5), 3.89 (t, J = 6.7 Hz, 2H, H51), 3.10-3.06 (m, 4H, H57, H77), 2.65 (t, J = 7.4 Hz, 2H, H48), 2.41 (t, J = 7.4 Hz, 2H, H46), 2.34 (t, J = 7.4 Hz, 4H, H14, H34), 2.22 (m, 2H, H52), 2.08-2.01 (m, 10H, including H47 and H32, H42, H39, H44), 1.82-1.79 (m, 4H, H76, H58), 1.70-1.60 (m, 8H, H27, H35, H74, H75), 1.37-1.29 (m, 40H), 1.03 (s, 3H, H4), 0.90 (t, J = 7.1 Hz, 6H, H65, H73).13C NMR (101 MHz, Methanol-d4) 6 176.8, 174.9, 174.3, 130.9 (alkene-C), 130.8 (alkene-C), 66.9 (C3), 66.7 (Cl, C5), 59.1 (C52), 56.4 (C57, C77), 55.8, 45.6, 42.7, 39.7, 35.0 (C14, C34), 33.8 (C48), 33.1 (C50), 30.87, 30.81, 30.64, 30.56, 30.48, 30.45, 30.38, 30.34, 30.29, 30.20, 28.17 (C32 or C42), 28.14 (C32 or C42), 27.7 (C74, C75), 26.1 (C27, C35), 25.5 (C58, C76), 23.7, 20.6 (C47), 17.5 (C4), 14.5 (C65, C73). LRMS (ESI) (m / z): calculated for [M+H]+(C54H98N2O6S1) requires 903.7, found: 903.7.

[0492] Synthesis of comparative example 1 (0AM-3-Adm)

[0493] 17

[0494]

[0495] Glutaric acid mono-tert-butyl ester (58 mg, 0.31 mmol, 1.0 equiv.) was dissolved in dry DCM (3 mL), and then Ghosez reagent (83 uL, 0.62 mmol, 2.0 equiv.) was added at room temperature. The mixture was stirred vigorously at room temperature for 30 min. Then to the reaction mixture was added starting material 1 as defined above (185 mg, 0.31 mmol, 1.0 equiv.) and Et3N (86 ul, 0.62 mmol, 2.0 equiv.) dropwise at 0 °C and allowed to warm to room temperature over 3 hours. Then the reaction mixture was diluted by DCM, washed 5 times by water, 1 time by brine and dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 10:1) to deliver a colorless oil.

[0496] The colorless oil (0.10 mmol, 1.0 equiv.) of the previous step was stirred in mixed solvent of DCM / TFA (3 / 1 mL) at room temperature for 1 hour to remove the tert-butyl group. Then the mixture was concentrated and used in the next step without further purification. The product containing the deprotected acid group was then dissolved in DMF (1.5 mL). Then Et3N (70 pl, 0.50 mmol, 5.0 equiv.) and HATU (76 mg, 0.20 mmol, 2.0 equiv.) were added. The reaction mixture was stirred at room temperature for 5 min. Then N, N-dimethylethylenediamine (0.30 mmol, 3.0 equiv.) was added and the reaction mixture was stirred at room temperature for 16 h. Then DMF was removed under vacuumand the resulting residue was directly purified by silica gel column chromatography (DCM / MeOH = 20:1 then 15:1 then 10:1) to deliver comparative example 1 as a colorless oil (56% yield).

[0497] 3H NMR (400 MHz, Methanol-d4) 64.03 (brd, 2H, H3), 4.02 (brd, 4H, Hl, H6), 3.36-3.33 (m, 2H, H51), 2.56 (t, J = 6.7 Hz, 2H, H52), 2.42-2.34 (m,10H, including H54, H55, H15, H28 and H44 or H46), 2.25 (t, 7 = 7.4 Hz, 2H, H44 or H46), 1.91 (app. p, J = 7.5 Hz, 2H, H45), 1.64-1.55 (m, 4H, H14, H29 or H18, H30), 1.52-1.43 (m, 4H, H14, H29 or H18, H30), 1.29 (brd, 40H), 1.06 (s, 3H, H5), 0.92-0.88 (m, 12H, H7, H24, H37, H42).13C NMR (100 MHz, Methanol-d4) 6 177.5, 175.3, 174.1, 66.8 (C3), 66.5 (Cl, C6), 59.2 (C52), 47.1 (C15, C28), 45.3 (C54, C55), 39.6 (C2), 37.7 (C51), 36.0 (C44 or C46), 34.1 (C44 or C46), 33.7 (C14, C29 or C18, C30), 33.0, 32.9, 30.7, 30.6, 30.4, 30.3, 28.62, 28.60, 23.75, 23.70, 22.1 (C45), 17.6 (C5), 14.5 (C7 or C24), 14.4 (C7 or C24). LRMS (ESI) (m / z): calculated for [M+H]+(C46H89O7N2) requires 781.7, found: 781.7.

[0498] Synthesis of comparative example 2 (OAM-3-Ade)

[0499]

[0500] The procedure defined above for comparative example 1 was repeated, but using 2- (diethylamino)ethylamine as bisamine, thereby obtaining comparative example 2 as a colorless oil (47% yield).

[0501] 3H NMR (400 MHz, Methanol-c 64.03 (brd, 2H, H3), 4.02 (brd, 4H, Hl, H6), 3.50 (t, J = 6.3 Hz, 2H, H51), 3.21-3.15 (m, 6H, H52, H54, H55), 2.44-2.35 (m, 4H, including H15, H28 & H44 or H46), 2.30 (t, J = 7.4 Hz, 2H, H44 or H46), 1.92 (app. p, J = 7.5 Hz, 2H, H45), 1.64-1.57 (m, 4H, H14, H29 or H18, H30), 1.52-1.42 (m, 4H, H14, H29 or H18, H30), 1.29 (brd, 46H, including H56, H57), 1.06 (s, 3H, H5), 0.92-0.88 (m, 12H, H7, H24, H37, H42).13C NMR (100 MHz, Methanol-d4) 6 177.5, 176.8, 174.0, 66.8 (C3), 66.5 (Cl, C6), 53.2 (C52), 49.0 (C54, C55), 47.1 (C15, C28), 39.6 (C2), 36.4 (C51), 35.6 (C44 or C46), 34.1 (C44 or C46), 33.6 (C14, C29 or C18, C30), 33.0, 32.9, 30.65, 30.57, 30.4, 30.3, 28.61, 28.58, 23.74, 23.69, 21.8 (C45), 17.6 (C5), 14.48 (C7 or C24), 14.45 (C7 or C24), 9.6 (C56, C57). LRMS (ESI) (m / z): calculated for [M+H]+(C48H93O7N2) requires 808.7, found: 808.7.

[0502] Synthesis of comparative example 3 (OAMole-3-Ac7)Preparation of starting material 3

[0503]

[0504] Trimethylolethane (1.00 g, 8.32 mmol, 1.0 equiv.) was dissolved in dry DCM (50 mL), and then oleoyl chloride (5.26 g, 17.5 mmol, 2.1 equiv.) was added at 0 °C, followed by dropwise addition of Et3N (2.9 ml, 20.8 mmol, 2.5 equiv.). The mixture was stirred vigorously at 0 °C and allowed to warm to room temperature over 16 hours. Then the reaction mixture was washed 3 times by sat. NaHCOs (aq.), 1 time by brine, dried over Na2SO4, filtered and concentrated. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 20:1 then 3:1) to deliver starting material 3 (2.70 g, 58% yield) as a colorless oil.

[0505] 3H NMR (400 MHz, Chloroform-d) 65.39-5.30 (m, 4H, H27, H38, H7, H18), 4.02 (s, 4H, Hl, H6), 3.39 (s, 2H, H3), 2.33 (t, J= 7.6 Hz, 4H, H34, H14), 2.04-1.98 (m, 8H, H28, H39, H8, H19), 1.65-1.58 (m, 4H, H33, H13), 1.35-1.26 (m, 40H), 0.95 (s, 3H, H5), 0.89-0.86 (m, 6H, H46, H26).13C NMR (100 MHz, Chloroform-d) 6 174.3 (C35, C15), 130.2 (alkene-C), 129.8 (alkene-C), 66.0 (Cl, C6), 64.7 (C3), 40.5 (C2), 34.4 (C34, C14), 32.0, 29.9, 29.8, 29.7, 29.5, 29.30, 29.28, 29.25, 29.37, 29.31, 27.37 (C28 or C39), 27.31 (C28 or C39), 25.1, 22.8, 16.9 (C5), 14.3 (C46, C26). LRMS (ESI) (m / z): calculated for [M+H]+(C41H77O5) requires 649.6, found: 649.7.

[0506] Preparation of intermediate 3

[0507]

[0508] Glutaric acid mono-tert-butyl ester (58 mg, 0.31 mmol, 1.0 equiv.) was dissolved in dry DCM (3 mL), and then Ghosez reagent (83 pL, 0.62 mmol, 2.0 equiv.) was added at room temperature. The mixture was stirred vigorously at room temperature for 30 min. Then to the reaction mixture was added starting material 3 (200 mg, 0.31 mmol, 1.0 equiv.) and EtaN (86 ul, 0.62 mmol, 2.0 equiv.) dropwise at 0 °C and allowed to warm to room temperature over 3 hours. Then the reaction mixture was diluted by DCM, washed 5 times by water, 1 time by brine, dried over Na2SO4, filtered and concentrated. Theresulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 10:1) to deliver intermediate 3 (210 mg, 84% yield) as a colorless oil.

[0509] 3H NMR (400 MHz, Chloroform-d) 65.38-5.31 (m, 4H, H27, H38, H7, H18), 4.00 (s, 2H, H3), 3.99 (s, 4H, Hl, H6), 2.37 (t, J = 7.5 Hz, 2H, H48 or H50), 2.32-2.25 (m, 6H, including H34, H14 and H48 or H50), 2.04-1.98 (m, 8H, H28, H39, H8, H19), 1.90 (app. p, J = 7.4 Hz, 2H, H49), 1.64-1.57 (m, 4H, overlapped with water peak), 1.44 (s, 9H, H54, H57, H58), 1.34-1.26 (m, 40H), 1.01 (s, 3H, H5), 0.89-0.86 (m, 6H, H46, H26).13C NMR (100 MHz, Chloroform-d) 6 173.6, 172.9, 172.3, 130.2 (alkene-C), 129.9 (alkene-C), 80.6 (C53), 65.9 (C3), 65.7 (Cl, C6), 38.5 (C2), 34.6 (C48 or C50), 34.3 (C34, C14), 33.3 (C48 or C50), 32.0, 29.91, 29.85, 29.7, 29.5, 29.32, 29.27, 28.2 (C54, C57, C58), 27.37 (C28 or C39), 27.32 (C28 or C39), 25.0, 22.8, 20.4 (C49), 17.3 (C5), 14.3 (C46, C26). LRMS (ESI) (m / z): calculated for [M+H]+(CsoHgiOg) requires 818.7, found: 818.8.

[0510] Preparation of comparative example 3

[0511]

[0512] Intermediate 3 (54 mg, 0.066 mmol, 1.0 equiv.) was stirred in mixed solvent of DCM / TFA (3 / 1 mL) at room temperature for 1 hour to remove the tertbutyl group. Then the mixture was concentrated and used in the next step without further purification. The crude deprotected acid was then dissolved in DMF (1.5 mL). Then EtgN (46 ul, 0.33 mmol, 5.0 equiv.) and HATU (50 mg, 0.13 mmol, 2.0 equiv.) were added. The reaction mixture was stirred at room temperature for 5 min. Then 2-(azepan-l-yl)ethanamine (21 uL, 0.15 mmol, 2.2 equiv.) was added and the reaction mixture was stirred at room temperature for 16 h. Then DMF was removed under vacuum, and the resulting residue was directly purified by silica gel column chromatography (DCM / MeOH = 20:1 then 15:1 then 10:1) to deliver comparative example 3 (25 mg, 63% yield) as a colorless oil.

[0513] 3H NMR (400 MHz, Methanol-c 65.39-5.31 (m, 4H, H27, H38, H7, H18), 4.04 (s, 2H, H3), 4.03 (s, 4H, Hl, H6), 3.49 (t, J = 6.1 Hz, 2H, H57), 3.27-3.24 (m, 4H, H60, H65), 3.15 (t, J = 6.1 Hz, 2H, H58), 2.41 (t, J = 7.3 Hz, 2H, H48 or H50), 2.34 (t, J = 7.3 Hz, 4H, H34, H14), 2.30 (t, J = 7.4 Hz, 2H, H48 or H50), 2.06-2.01 (m, 8H, H28, H39, H8, H19), 1.96-1.84 (m, 6H, including H49, H61, H64), 1.74-1.71 (m, 4H, H62, H63), 1.65-1.58 (m, 4H, H33, H13), 1.37-1.30 (m, 40H), 1.04 (s, 3H, H5), 0.92-0.89 (m, 6H, H46, H26).

[0514] 13C NMR (100 MHz, Methanol-d4) 6 176.8, 174.9, 174.2, 130.9 (alkene-C), 130.8 (alkene-C), 66.8 (C3), 66.6 (Cl, C6), 58.7 (C58), 56.5 (C60, C65), 39.7, 36.8, 35.6 (C58), 35.0 (C34, C14), 34.0 (C48 or C50),33.1 (C48 or C50), 30.9, 30.8, 30.6, 30.5, 30.4, 30.3, 30.20, 30.19, 28.16 (C28 or C39), 28.14 (C28 or C39), 27.4 (C62, C63), 26.1 (C33, C13), 25.8 (C61, C64), 23.8, 21.8, 20.4 (C49), 17.5 (C5), 14.5 (C46, C26). LRMS (ESI) (m / z): calculated for [M+H]+(C54H99O7N2) requires 887.7, found: 887.7.

[0515] Lipid nanoparticle (LNP) formulations

[0516] Lipid nanoparticle (LNP) compositions were prepared by an ethanol dilution method. Specifically, an ethanolic lipid solution was rapidly mixed with an aqueous buffer under vortex stirring to achieve uniform particle formation. The ethanolic solution contained the ionizable lipid (Examples 1-15 or Comparative Examples 1-3), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG, 2000) as a PEG-lipid, distearoylphosphatidylcholine (DSPC) as a phospholipid, and cholesterol in a molar ratio of 50:1.5:10:38.5.

[0517] The aqueous solution was prepared by combining 10.0 pL of an mRNA stock solution encoding enhanced green fluorescent protein (eGFP) (1 mg / mL) with 56.7 pL of acetate buffer (5 mM, pH 4). The resulting LNP compositions were used directly without further purification.

[0518] All LNP formulations were characterized for hydrodynamic diameter (size), polydispersity index (PDI), zeta potential, pKa, and mRNA encapsulation efficiency. As summarized in Table 1, dynamic light scattering (DLS) analysis indicated that the hydrodynamic diameter of all LNPs ranged between 90-140 nm, with a PDI of <0.20. Electrophoretic mobility measurements confirmed that the zeta potential of ionizable lipids according to the invention was negative at physiological pH (pH 7.4).

[0519] Table 1. Physicochemical properties of LNPs encapsulating eGFP mRNA measured by Dynamic Light Scattering (DLS), Electrophoretic Light Scattering (ELS), RiboGreen assay and 2-(p-toluidino) naphthalene-6-sulfonic acid (TNS) assay as described in the methodology section above.

[0520] LNP ionizable lipid size PDI zeta-potential encapsulation pKa(nm) (mV) efficiency (%)

[0521] LNP1 Example 1 122 0.16 -1.8 ± 4.4 88 6.65 LNP5 Example s 92 0.15 -6.1 ± 7.1 87 6.46 LNP2 Example 2 110 0.16 -4.2 ± 3.9 89 7.18 LNP6 Example 6 103 0.15 -4.8 ± 6.5 88 6.61 LNP3 Example s 100 0.15 -3.7 ± 6.9 90 6.77LNP7 Example? 106 0.12 -2.9 ± 3.5 88 6.32 LNP4 Example 4 122 0.17 -0.1 ±4.8 87 7.44 LNP8 Examples 114 0.13 -3.4 ± 8.9 89 7.16 LNP9 Example 9 139 0.20 3.3 ± 6.8 90 7.76 LNP10 Example 10 110 0.16 -2.1 ± 9.6 92 7.55 LNP11 Example 11 110 0.16 -4.5 ± 6.3 85 6.29 LNP12 Example 12 99.6 0.20 -9.4 ± 8.7 87 7.12 LNP13 Example 14 105 0.18 -1.1 ± 5.7 87 7.23 LNP14 Example 15 118 0.15 -6.4 ± 5.8 88 5.81 CLNP1 Comparative ex. 1 107 0.16 -0.4 ± 2.5 87 7.95 CLNP2 Comparative ex.2 132 0.16 1.3 ± 1.3 88 7.59 CLNP3 Comparative ex.3 140 0.16 1.6 ± 7.6 89 7.50 Bench MC3 134 0.15 -2.9 ± 5.7 90 6.40

[0522] RiboGreen assays demonstrated high mRNA encapsulation efficiencies, with all LNP compositions achieving encapsulation levels >85%.

[0523] Apparent pKa values were determined using a 6-(p-toluidino)-2-naphthalenesulfonic acid sodium salt (TNS) assay. LNPs comprising ionizable lipids with an azepanyl group (e.g., LNPs 5-8) exhibited lower apparent pKa values compared to those containing a dimethylamine group (e.g., LNPs 1-4). This highlights the ability of the invention to tune the pKa of LNP formulations to desired values, which can optimize endosomal membrane disruption and promote cytosolic delivery of the mRNA payload. When comparing LNPs 9-12, it was observed that elongating the distance between the thioamide group and the ionizable nitrogen in the amine group resulted in a lower apparent pKa. It is hypothesized that increasing this spatial distance reduces the electron-withdrawing effect of the thioamide group, further supporting the pKa-tuning capability of the invention.

[0524] Substitution of the thioamide group with an amide group (e.g., when comparing LNPs 1, 11, and 14 with CLNPs 1, 2, and 3, respectively) significantly increased the apparent pKa of the correspondingLNP compositions. This shift in pKa may lead LNPs to bear a cationic charge at neutral pH, making them prone to aggregation in physiological fluids and causing destabilization of phospholipid cell membranes, thereby inducing toxicity.

[0525] These findings demonstrate that lipids comprising a structural moiety wherein a thioamide is positioned adjacent to an ionizable nitrogen as presented herein can enable precise control over LNP physicochemical properties, enhancing their delivery efficiency while minimizing toxicity.

[0526] In vitro screening procedure for transfection efficiency and cytotoxicity

[0527] An initial screening of LNP compositions according to the invention was conducted to evaluate their transfection efficiency and cytotoxicity in vitro. The synthesis procedure described above was used to prepare LNP formulations encapsulating mRNA encoding enhanced green fluorescent protein (eGFP) and 0.1 mol% l,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated to Cyanine-5 (DSPE-Cy5). Each ionizable lipid above and Comparative Examples 1-3 was used to formulate distinct LNP compositions (LNP 1-14).

[0528] CT26 mouse colon cells and human embryonic kidney HEK293T cells were treated with LNP formulations at mRNA doses of 50 ng / well and 200 ng / well, respectively. After 24 hours of incubation, cellular LNP association and eGFP expression (transfection efficiency) were analyzed using flow cytometry. A commercially available benchmark LNP formulation, referred to herein as MC3-containing, LNP formulation (DLin-MC3-DMA, which is used in the clinically approved siRNA drug product Onpattro®), was used as a reference for comparison.

[0529] Most LNP compositions exhibited modest cytotoxicity, with only LNP 2, 3, 4, 10, and 12 inducing higher toxicity at the maximum tested mRNA dose of 200 ng / well. A correlation between higher cytotoxicity and LNP compositions with a higher apparent pKa was observed.

[0530] LNP compositions prepared with LNP 1, 11, and 14 demonstrated the highest transfection efficiency across both cell lines and at both mRNA doses. On HEK293T cells, the cellular association of these LNPs was comparable to the MC3-containing benchmark formulation at both mRNA doses (FIG. 1A). On CT26 cells, the inventive LNP compositions exhibited similar cellular association to the MC3-containing formulation at the lower mRNA dose (50 ng / well) and significantly higher association at the higher dose (200 ng / well), indicating dose- and cell-type-dependent uptake (FIG. IB).

[0531] The transfection efficiencies of LNP 1, 11, and 14 were compared to their structural analogues comprising Comparative Examples 1, 2, and 3, respectively. The LNP compositions of Comparative Examples 2 and 3 induced significant toxicity at 200 ng / well. In contrast, the inventive LNPcompositions exhibited higher transfection efficiency and lower cytotoxicity compared to their amide-containing counterparts, particularly at the higher dose of 200 ng / well on CT26 cells. A similar trend was observed in HEK293T cells at the lower dose of 50 ng / well.

[0532] These properties highlight the advantages of the LNP compositions according to the invention for efficient and safe mRNA delivery.

[0533] In vivo bioluminescence Imaging with Firefly Luciferase mRNA

[0534] The ability of the lipid nanoparticle (LNP) compositions according to the invention to achieve efficient in vivo mRNA transfection was evaluated in BALB / c mice. LNP formulations encapsulating mRNA encoding Firefly Luciferase (FLuc) were prepared using the same methodology described above, but using Flue encoding mRNA instead of eGFP encoding mRNA. Each mouse received an intravenous (IV) injection of the LNP compositions, delivering a 5 pg mRNA dose. The distribution of FLuc-induced bioluminescence was assessed to determine tissue-specific transfection efficiency.

[0535] In this study, LNP compositions LNP 1, 11, and 14, which were compared to their structural analogs, comprising ionizable lipids of comparative Examples 1, 2, and 3, as well as to the MC3-containing LNP formulation described above.

[0536] Bioluminescence imaging performed 4 hours post-injection revealed a clear signal in the abdominal region of mice treated with the inventive LNP compositions and the MC3-containing formulation. In contrast, the comparative examples produced significantly weaker bioluminescent signals. At 24 hours post-injection, the bioluminescence intensity decreased across all groups (FIG. 2). Imaging of major organs 24 hours post-injection demonstrated that the highest bioluminescence signals were detected in the liver and spleen, with minimal signal observed in the lungs, kidneys, and heart. The inventive LNP compositions consistently outperformed the comparative examples in both liver and spleen transfection. Notably, the LNP composition prepared using Example 1 achieved significantly higher FLuc expression in the spleen compared to the liver, whereas the MC3-containing formulation predominantly transfected liver cells (FIG. 3).

[0537] To further elucidate the tissue and cellular tropism of the LNP composition comprising Example 1, compared to the MC3-containing formulation, Cre recombinase mRNA was encapsulated into the LNPs. To that end, LNP compositions were prepared as described above, using Cre recombinase mRNA. Cre recombinase activity was monitored in vivo using the Rosa26-loxP-STOP-loxP-TdTomato (Ail4) mouse model. Upon recombination, the STOP cassette in the Rosa26 locus is excised, leading to permanent expression of TdTomato fluorescence in Cre-expressing cells.Flow cytometry analysis of TdTomato-positive spleen cells identified macrophages and dendritic cells as the primary cell types transfected by the LNP compositions of the invention (FIG. 4). In contrast, the comparative LNP compositions failed to transfect cells effectively.

[0538] The experimental results demonstrate that the LNP compositions according to the invention achieve superior mRNA transfection efficiency in vivo compared to comparative examples. These compositions, exhibited distinct tissue and cellular tropism, with preferential spleen transfection and effective targeting of macrophages and dendritic cells.

[0539] In vitro transfection of human CD8+T cells

[0540] Primary human CD8+T cells were treated with 1 pg / mLof the SAM-3-APyr LNPs in serum-free medium, supplemented with 1 pg / mL apolipoprotein E. Cells were incubated at 37°C for 24h. Expression of eGFP determined via flow cytometry analysis revealed a significant increase in mean fluoresence intensity by eGFP after transfection, indicating a high expression and thus high transfection efficiency for LNPs with SAM-3-APyr as the ionizable lipid (FIG. 8).

[0541] Targeted delivery of mRNA to primary CD8+ T Cells

[0542] LNP compositions were prepared by mixing an aqueous solution containing mRNA with an ethanolic solution comprising ionizable lipid, phospholipids, cholesterol, l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-2000 trans-cyclooctene (DSPE-PEG2000-TCO). Different ionizable lipids were used to prepare the TCO-containing LNP compositions: ALC-0315, D-Lin-MC3-DMA, Example 13, S-Ac7-DOG, and SM-102. ALC-0315, D-Lin-MC3-DMA, and SM-102 were obtained from BroadPharm, S-Ac7-DOG was synthesized according to De Lombaerde, Emily, et al. " Combinatorial Screening of Biscarbamate Ionizable Lipids Identifies a Low Reactogenicity Lipid for Lipid Nanoparticle MRNA Delivery." ADVANCED FUNCTIONAL MATERIALS, vol. 34, no. 21, 2024, pp.

[0543] 1-16.

[0544] For each ionizable lipid, five LNP formulations were prepared as indicated in Table 2 below.

[0545] Table 2. Formulation LNP compositions comprising trans-cyclooctene (TCO). The contents of each component are indicated in molar ratios.

[0546] Formulation ionizable DMG-PEG DSPC DSPC-Cy5 Cholesterol TCO-PEG2k- lipid 2k DSPE1 50 0.0 9.9 0.1 38.5 1.5

[0547] 2 50 0.5 9.9 0.1 38.5 1.0

[0548] 3 50 1.0 9.9 0.1 38.5 0.5

[0549] 4 50 1.5 9.9 0.1 38.5 0.0

[0550] 5 50 1.5 9.9 0.1 38.5 1.5

[0551] The resulting LNP compositions had a size below 200 nm, determined via dynamic light scattering. The zeta potential of the LNP compositions, measured by electrophoretic mobility, was slightly negative (-25.43 to 1.61 mV).

[0552] Encapsulation efficiency of mRNA was assessed using RiboGreen assay, achieving near-complete encapsulation.

[0553] Mouse anti-CD8 nanobodies were produced by recombinant synthesis in Pichia pastoris according to Van Den Eeckhout, B., et al. Specific targeting of IL-ip activity to CD8+ T cells allows for safe use as a vaccine adjuvant, npj Vaccines 5, 64 (2020); and functionalized with tetrazines (Tz) through two methods: (i) random lysine modification using Tz-PEG5-NHS and (ii) site-specific C-terminal Sortase A-mediated conjugation using TZ-PEG4-NH2.

[0554] Random lysine modification involved functionalizing free lysine residues of the anti-CD8 nanobodies with tetrazine moieties via NHS ester crosslinking. NHS-(PEG)5-Tz (5 eq.) was added to anti-CD8 nanobodies (1 eq.) in NaHCOs (0.1 M, pH 8.3) and stirred for 1.5 h at room temperature. Afterwards, the solution was dialyzed against PBS for 48h using a Pur-A-Lyzer™ Maxi Dialysis Kit (MWCO 3500) according to the manufacturer's protocol (see protocol described in Mu, W et al. HER2 Nanobody-Poly(rhamnose) Conjugates Efficiently Recruit Anti-Rhamnose Antibodies from Serum to the Surface of HER2-Expressing Cells, Chem. Mater., 2024, 36, 20, 10113-10124).

[0555] Site-specific C-terminal Sortase A-mediated conjugation using TZ-PEG4-NH2 was performed as described in Massa, S., et al. Sortase A-mediated site-specific labeling of camelid single-domain antibody-fragments: a versatile strategy for multiple molecular imaging modalities, Contrast Media & Molecular Imaging 2016, 11, 328-339.

[0556] Primary OT-I CD8+ T cells were isolated and incubated with 1 pM of Tz-functionalized anti-CD8 nanobodies (anti-CD8 Nb-Tz) for 1 hour at 37°C to enable specific binding. Subsequently, the LNPcompositions described above, containing TCO-functionalized mRNA encoding eGFP (TCO-LNPs), were added to the cells at a final concentration of 2 ng / pL. The cells were incubated for 24 hours at 37°C. Following incubation, cells were analyzed by flow cytometry to measure Cy5 fluorescence (indicating nanobody binding) and eGFP fluorescence (indicating mRNA transfection). The results demonstrated efficient binding of Tz-functionalized nanobodies to CD8+ T cells and successful click-reaction with fluorescently labeled TCO-LNPs. Notably, as illustrated in FIG.5 and FIG. 6, LNP compositions formulated with ionizable lipids of Example 13 achieved the highest eGFP mean fluorescence intensity (MFI), indicating superior transfection efficiency.

[0557] To assess whether selective binding to primary CD8+T-cells could be achieved in vivo, anti-CD8 Nb-Tz was intravenously administered to WT C57BI / 6 mice. One hour later, Cy5 labeled SAM-3-Apyr LNP with 1.5% TCO was injected. Blood, liver and spleen were isolated to determine the biodistribution on cellular level. Flow cytometry data shows that the anti-CD8 Nb-Tz selectively targets TCO-LNPs to CD8+ T-cells in blood, liver and spleen (FIG. 7A-C). These data demonstrate that the click reaction between Tz on the anti-CD8 Nb-Tz and TCO on the LNP surface also occurs efficiently in vivo.

Claims

CLAIMS1. An ionizable lipid or a pharmaceutically acceptable salt thereof, comprising a moiety, the moiety comprising:an ionizable nitrogen selected from a secondary or tertiary amine functional group, anda secondary thioamide group, wherein the thioamide group is separated from the ionizable nitrogen by a chain of 2 to 10 atoms.

2. The ionizable lipid or a pharmaceutically acceptable salt thereof according to claim 1, comprising one or more hydrophobic tails, each independently comprising a linear or branched aliphatic chain having 8 to 36 carbon atoms.

3. The ionizable lipid or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the hydrophobic tails are covalently attached to the secondary thioamide group by means of a linker structure comprising ester, amide, carbamate, carbonate, disulfide, or thioester linkages, preferably ester linkages.

4. The ionizable lipid or pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the moiety has a structure according to Formula (I) or (II),whereinR1is selected from optionally substituted C2-10alkyl, optionally substituted C3-10alkenyl, optionally substituted C3-10alkynyl, optionally substituted C3-10cycloalkyl, optionally substituted C3-10cycloalkenyl, optionally substituted C2-10heteroalkyl, C2-10haloalkyl;R2and R3are each independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heteroalkyl, haloalkyl, optionally substituted heterocyclyl, or hydrogen, provided that at least one is not hydrogen; orR2and R3together with the atom to which they are attached form a 3-7-membered saturated, or partially saturated heterocyclyl, optionally comprising at least one further N, O, and / or S; andR4is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heteroalkyl, optionally substituted aryl, optionally substituted arylalkyl, or optionally substituted heterocyclyl.

5. The ionizable lipid or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein the ionizable lipid is a compound of Formula (Illa), ( I II b), and / or (lllc):whereinX1, X2, X3, X4are each independently selected from -O, -NH, or -S, preferably -O;R5, R6, R7are each independently selected from C8-36alkyl or C8-36alkenyl;A is a branched or linear aliphatic chain, preferably comprising 3 to 30 carbon atoms;R1, R2, R3, and R4are as defined in claim 4.

6. The ionizable lipid or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein the ionizable lipid is a compound of Formula (IVa), (IVb), and / or (IVc):whereinR8, R9is selected from optionally substituted alkyl, hydrogen, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted heteroalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, or optionally substituted aryl, optionally substituted arylalkyl; andR1, R2, R3, R4, R5, R6, R7X1, X2, X3, X4are as defined in any one of claims 4 or 5.

7. The ionizable lipid or pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein the ionizable lipid is a compound of Formula (IVa), (IVb), and / or (IVc):whereinR8, R9is selected from optionally substituted alkyl, hydrogen, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted heteroalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, or optionally substituted aryl, optionally substituted arylalkyl; andR1, R2, R3, R4, R5, R6, R7X1, X2, X3, X4are as defined in any one of claims 4 to 6.

8. The ionizable lipid or pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein the ionizable lipid is selected from the group consisting ofo9. A method for preparing an ionizable lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, the method comprising the steps of:c) providing a lipid precursor molecule comprising a terminal alkyne;d) contacting the lipid precursor molecule with an amine compound, comprising a primary amine separated by a chain of 2 to 10 atoms from an ionizable nitrogen selected from a secondary or tertiary amine functional group, in the presence of elemental sulfur, and optionally a base, thereby forming an ionizable lipid comprising a secondary thioamide group separated from the ionizable nitrogen by a chain of 2 to 10 atoms.

10. The method according to claim 9, wherein the lipid precursor molecule comprises a structure that either comprises:one or more reactive functional groups, or protected forms thereof, suitable for covalent attachment of hydrophobic tails; orone or more hydrophobic tails, thereby forming a partially assembled lipid precursor molecule;wherein each hydrophobic tail independently comprises a linear or branched aliphatic chain having 8 to 36 carbon atoms.

11. A method for modifying the apparent pKa of a lipid nanoparticle composition, the method comprising the step of incorporating at least one ionizable lipid or pharmaceutically acceptable salt thereof according to any one of claims 1 to 8 into the lipid nanoparticle composition, preferably wherein the lipid nanoparticle composition has a modified apparent pKa between 5.8 and 7.8, more preferably between 6.0 and 7.0.

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

13. The lipid nanoparticle composition according to claim 12, further comprising 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, at physiological pH; optionally wherein the nucleic acid is at least any one selected from the group consisting of mRNA, siRNA, rRNA, DNA, aptamer, tRNA, antisense oligonucleotide, shRNA, miRNA, sgRNA, tracrRNA, gRNA, ribozyme, PNA, DNAzyme, and mixtures thereof.

14. The lipid nanoparticle composition according to any one of claims 12 to 13 for use in medicine; in particular for use in delivering a cargo to a cell, such as for transfecting a cell, preferably a mammalian cell, more preferably an immune cell, a cancer cell, a progenitor cell, or a structural cell in vivo.

15. 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 according to any one of claims 12 to 13, preferably wherein the cell is a mammalian cell, more preferably an immune cell, a cancer cell, a progenitor cell, or a structural cell.

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