Compounds, lipid nanoparticles comprising the same and uses thereof

Abstract

Disclosed herein is directed to lipids and lipid nanoparticles (LNPs) comprising the same for encapsulating and delivering therapeutic agents (e.g., nucleic acids) into cells. The lipid has the structure of formula (I), wherein, X and W are independently O or -NH; Y is O, S, or -HN; m and o are independently an integer between 1 and 5; n is an integer between 1 and 7; R1 is C1-C20 linear or branched alkyl, or linear or branched alkenyl; R2 is C1-C20 linear or branched alkyl, linear or branched alkenyl, or -C(Ra)((CH2)-Z-C(=O)Rb)2, wherein Ra is C1-C6 alkyl, Rb is C1-C20 linear or branched alkyl, or linear or branched alkenyl, and Z is O or -NH; and R3 is C1-C6 alkyl.

Description

Attorney Docket No. 4404-0140PWO1 COMPOUNDS, LIPID NANOPARTICLES COMPRISING THE SAME AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATIONThis application claims the benefit of U. S. Application No. 63 / 747,631, filed January 21, 2025, the entirety of which is incorporated by reference herein.BACKGROUND OF THE INVENTION

[0001] 1. FIELD OF THE INVENTION

[0002] The present disclosure relates to novel lipids and their uses for enhancing the delivery of therapeutic agents. More particularly, the present disclosure relates to lipids, lipid nanoparticles (LNPs) comprising the same, and their uses for encapsulating and delivering nucleic acids into cells.

[0003] 2. DESCRIPTION OF RELATED ART

[0004] Among the various reagents used to transfect cells with bioactive agents, such as nucleic acids, lipid nanoparticle (LNP)-based delivery systems, including liposomes, are widely regarded as the most effective. As the most advanced non-viral gene delivery system in clinical use, lipid nanoparticles (LNPs) are artificially engineered spherical vesicles composed of a lipid bilayer. This structure allows them to encapsulate an aqueous core within a hydrophobic membrane, preventing hydrophilic solutes from freely passing through. Additionally, the lipid bilayer may fuse with other membranes, such as a cell membrane, facilitating the LNPs to delivery of their contents into the cell.

[0005] With the emergence of pandemics and the development of novel RNA vaccines, a variety of lipid nanoparticles have been explored and optimized for nucleic acid (e.g., mRNA) delivery, providing valuable insights for the future design of mRNA therapeutics. However, unresolved challenges remain. Due to their temperature sensitivity, the low thermal stability of mRNA vaccines poses significant limitations on their storage and distribution.

[0006] Although many lipid nanoparticle-mRNA formulations have been developed and are under clinical evaluation for the prevention and treatment of viral infections, cancer, and genetic diseases, the development of lipid nanoparticles with improved stability, higher loading capacity, and enhanced safety remains a key focus in this field.Attorney Docket No. 4404-0140PWO1

[0007] In view of the foregoing, there exists in the related art a need for a novel lipid for effectively encapsulating and delivering therapeutic agents into cells.SUMMARY OF THE INVENTION

[0008] The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure, and it does not identify key / critical elements of the present invention or delineate the scope of the present invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

[0009] As embodied and broadly described herein, the purpose of the present disclosure is to provide a novel lipid and lipid nanoparticle comprising the same for encapsulating and delivering nucleic acids into cells.

[0010] The first aspect of the present disclosure pertains to a compound of formula (I),

[0011] In the compound of formula (I),X and W are independently O or -NH;Y is O, S, or-HN;m and o are independently an integer between 1 and 5;n is an integer between 1 and 7;Ri is C1-C20 linear or branched alkyl, or linear or branched alkenyl;R2 is C1-C20 linear or branched alkyl, linear or branched alkenyl, or -C(Ra)((CH2)-Z-C(=O)Rb)2, whereinRais C1-C6alkyl,Rbis C1-C20 linear or branched alkyl, or linear or branched alkenyl, andZ is O or -NH; andR3is C1-C6alkyl.Attorney Docket No. 4404-0140PWO1

[0012] According to some embodiments of the present disclosure, in the compound of formula (I), X, Y, and W are independently O, Ri is nonyl; m and o are independently an integer between 1 and 5; n is an integer between 1 and 7; RR2is 7-pentadecyl; and R3is methyl. In one working example, in the formula (I), m is 3, n is 5, and o is 3. In other working example, in the formula (I), m is 1, n is 5, and o is 3. In another working example, in the compound of formula (I), m is 5, n is 5, and o is 3. In still another working example, in the formula (I), m is 3, n is 3, and o is 3. In still another working example, in the formula (I), m, n, and o are respectively 3. In still another working example, in the formula (I), m is 2, n is 1, and o is 3. In still another working example, in the formula (I), m is 3, n is 1, and o is 3. In still another working example, in the formula (I), m is 2, n is 5, and o is 3. In still another working example, in the formula (I), m is 4, n is 5, and o is 3. In still another working example, in the formula (I), m is 3, n is 7, and o is 3. In still another working example, in the formula (I), m is 3, n is 7, and o is 5.

[0013] According to some embodiments of the present disclosure, in the compound of formula (I), X, Y, and W are independently O, Ri is nonyl; R2 is 7-pentadecyl; and R3 is ethyl. In one working example, in the formula (I), m is 3, n is 5, and o is 3.

[0014] According to some embodiments of the present disclosure, in the compound of formula (I), X, Y, and W are independently O; m is 3; n is 5; o is 3; Ri is tridecyl or heptadecyl; RR2is 7-pentadecyl; and R3is methyl.

[0015] According to some embodiments of the present disclosure, in the compound of formula (I), X, Y, and W are independently O; m is 3; n is 5; o is 3; Ri is 7-pentadecyl; R2 is C7-C17 linear or branched alkyl; and R3 is methyl. In one working example, R2 is 7-pentadecyl; in another working example, R2 is nonyl.

[0016] According to embodiments of the present disclosure, in the compound of formula (I), X, Y, and W are independently O; m is 3; n is 5; o is 3; Ri is nonyl; R2 is -C(Ra)((CH2)-Z-C(=O)Rb)2, wherein Rais methyl, Rbis nonyl, and Z is O.

[0017] According to embodiments of the present disclosure, in the compound of formula (I), X and Y are independently -NH; W is O; m is 3; n is 5; o is 3; Ri is nonyl; R2is 7-pentadecyl; and R3is methyl.Attorney Docket No. 4404-0140PWO1

[0018] According to embodiments of the present disclosure, in the compound of formula (I), X is O; Y is S; W is O or -NH; m is 3; n is 5; o is 3; Ri is nonyl; RR2is 7-pentadecyl; and R3is methyl. In one working example, W is O. In another working example, W is -NH.

[0019] According to one embodiment of the present disclosure, in the compound of formula (I), X and Y are independently O; W is -NH; m is 3; n is 5; o is 3; Ri is nonyl; RR2is 7-pentadecyl; and R3is methyl.

[0020] According to one embodiment of the present disclosure, in the compound of formula (I), X and W are independently -NH; Y is O; m is 3; n is 5; o is 3; Ri is nonyl; RR2is 7-pentadecyl; and R3is methyl.

[0021] According to one embodiment of the present disclosure, in the compound of formula (I), X, Y, and W are independently -NH; m is 3; n is 5; o is 3; Ri is nonyl; R2 is7-pentadecyl; and R3 is methyl.

[0022] In preferred embodiments of the present disclosure, a subset of the compound of formula (I) is selected from the group consisting of(MPA01),(MPA02),(MPA03),Attorney Docket No. 4404-0140PWO1(MPA06),(MPA09),Attorney Docket No. 4404-0140PWO1(MPA10),(MPA11),(MPA12),(MPA14),(MPA15),Attorney Docket No. 4404-0140PWO1(MPA17),(MPA24), and(MPA25).

[0023] The second aspect of the present disclosure pertains to a lipid nanoparticle (LNP) comprising the compound of formula (I). Specifically, the LNP comprises one hydrophobic core surrounded by an assembly of the compound of aforementioned lipid of formula (I); and / or one or more hydrophilic cores surrounded by a lipid layer formed by the aforementioned lipid of formula (I).Attorney Docket No. 4404-0140PWO1

[0024] According to alternative embodiments of the present disclosure, the LNP further comprises a therapeutic agent accommodated in the hydrophobic core or the hydrophilic core of the LNP. Specifically, the therapeutic agent disposed in the LNP may be a nucleic acid, a peptide, a polypeptide, a protein, a carbohydrate, a proteoglycan, a glycoprotein, or a combination thereof.

[0025] Examples of the nucleic acid suitable for used as the therapeutic agent in the present disclosure include, but are not limited to, a mitochondrial DNA (mtDNA), a chloroplast DNA (cpDNA), a plasmid, a messenger RNA (mRNA), a small interfering RNA (siRNA), a ribosomal RNA (rRNA), a transfer RNA (tRNA), a microRNA (miRNA), an aptamer, and a combination thereof.

[0026] In some embodiments of the present disclosure, the protein suitable for used as the therapeutic agent in the LNP may be a viral protein.

[0027] In one working example, the nucleic acid is preferably an mRNA encoding the spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. In an alternative working example, the nucleic acid is an mRNA encoding an envelope protein of Dengue virus.

[0028] Additionally or optionally, the LNP of the present disclosure further comprises a cationic lipid, a steroid, a phospholipid, a PEGylated lipid, a PEGylated phospholipid, and a combination thereof.

[0029] According to some embodiments of the present disclosure, the lipid is present in the LNP at a concentration of 40 to 50 mol%.

[0030] According to some embodiments of the present disclosure, the molar ratio of the lipid to the therapeutic agent is about 2:1 to 8:1.

[0031] By virtue of the above features, the lipid described in the present disclosure, together with the LNP comprising it, is capable of forming a stable bilayer structure, thereby effectively encapsulating therapeutic agents and facilitating their delivery into cells.

[0032] The attendant features and advantages of the present disclosure will become better understood with reference to the following detailed description considered in connection with the accompanying drawings.Attorney Docket No. 4404-0140PWO1 BRIEF DESCRIPTION OF THE DRAWING

[0033] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawing(s). For the purposes of illustrating the invention, shown in the drawing(s) are embodiments which are presently preferred.

[0034] Fig. 1 illustrates the functional delivery efficiency (relative light units (RLU / mg protein)) of the selected ionizable lipids in Table 3.DETAILED DESCRIPTION OF THE INVENTION

[0035] The detailed description provided below in connection with the appended drawings is intended as a description of examples and is not intended to represent the only forms in which the example may be constructed or utilized. The description sets forth the functions of the example and the sequence of steps for constructing and operating the example. However, the same or equivalent functions and sequences may be accomplished by different examples.

[0036] 1. Definition

[0037] For convenience, certain terms employed in the specification, examples and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of the ordinary skills in the art to which this invention belongs. Unless otherwise specified, the methods or approaches pertaining to the field of biotechnology used herein, including but not limited to, protein chemistry, biochemistry, molecular biology, genetic engineering, and pharmacology, are employed and performed using conventional and well-established practices in the field, as documented in existing academic literature or textbooks.

[0038] The singular forms "a", "and", and "the" are used herein to include plural referents unless the context clearly dictates otherwise.

[0039] The recitation of ranges of values herein is merely intended as a shorthand method of individually referring to each separate value within the range. Unless otherwise indicated, each individual value is incorporated into the specification as if it were explicitly recited.Attorney Docket No. 4404-0140PWO1

[0040] As used herein, the term "about" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the Examples or elsewhere in the Specification in the context of a particular assay, result or embodiment, the term "about" means within one standard deviation per the practice in the art, or a range of up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

[0041] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any variation thereof are intended to indicate a nonexclusive inclusion, subject to any limitations explicitly stated. For example, a composition, mixture, process, or method that comprises a list of elements is not necessarily limited to only those elements but may also include other elements not expressly listed or inherent to such a composition, mixture, process, or method.

[0042] The transitional phrase "consisting of" excludes any elements, steps, or ingredients not specified. In a claim, this phrase closes the claim to the inclusion of materials other than those explicitly recited, except for impurities ordinarily associated with them. When "consisting of" appears in a clause within the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0043] As used herein, "and / or" specifies that each of the two features or components may be present either individually or together. For example, " X and / or Y" explicitly discloses: (i) X alone, (ii) Y alone, and (iii) both X and Y, as if each scenario were individually set forth herein.

[0044] The expressions "in one embodiment," "in some embodiments," and similar phrases as used herein generally indicate that the particular feature, structure, or characteristic described following the phrase may be included in at least one embodiment of the present disclosure and potentially in multiple embodiments (notably, such phrases do not necessarily refer to the same embodiment). The use of examples or exemplary language (e.g., 'such as') provided herein is intended solely to illustrate the present disclosure and does not limit its scope as otherwise claimed. No language in the specification should be construed as indicating that any nonclaimed element is essential to the practice of the present disclosure.Attorney Docket No. 4404-0140PWO1

[0045] All methods described herein may be performed in any suitable order unless explicitly stated otherwise or clearly contradicted by the context.

[0046] The term "alkyl," as used herein, refers to a straight or branched hydrocarbon group containing 1 to 20 carbon atoms, such as, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms, which can be abbreviated as C1-C20 alkyl. Examples include alkyl groups with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms, as well as specific ranges such as C1-C18 alkyl, C1-C16 alkyl, C1-C14 alkyl, C1-C12 alkyl, C1-C10 alkyl, C1-C8 alkyl, C1-C6 alkyl, C1-C4 alkyl, C4-C20 alkyl, C6-C20 alkyl, C8-C20 alkyl, C10-C20 alkyl, C12-C20 alkyl, C14-C20 alkyl, C16-C20 alkyl, C18-C20 alkyl, C12-C18 alkyl, C14-C18 alkyl, C16-C18 alkyl, C11-C13 alkyl, C13-C15 alkyl, and C15-C19 alkyl. Unless specified otherwise, the term "alkyl' used herein encompasses both linear and branched alkyl groups. Exemplary linear alkyl groups suitable for use in the present application include methyl, ethyl, propyl, iso-propyl (also called 2-propyl or 1-methylethyl), butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1,2-dimethylpropyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethylhexyl, n-nonyl (or nonanyl), n-decyl (ordecanyl), n-undecyl, n-dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, or the like. Exemplary branched alkyl groups suitable for use in the present application include hexyl-nonyl, hexyl-decyl, hexyldecanyl, or the like.

[0047] The term "linear alkenyl," as used herein, refers to a straight hydrocarbon group containing 1 to 20 carbon atoms and one or more double bonds. The term "branched alkenyl," as used herein, refers to hydrocarbon chains that include one or more branches (alkyl substituents) along the main chain, with a total of 1 to 20 carbons. This can be abbreviated as C1-C20 alkenyl. Examples include alkenyl groups with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms, as well as specific ranges such as C1-C18 alkenyl, C1-C16 alkenyl, C1-C14 alkenyl, C1-C12 alkenyl, C1-C10 alkenyl, C1-C8 alkenyl, C1-C6 alkenyl, C1-C4 alkenyl, C4-C20 alkenyl, C6-C20 alkenyl, C8-C20 alkenyl, C10-C20 alkenyl, C12-C20 alkenyl, C14-C20 alkenyl, C16-C20 alkenyl, C18-C20 alkenyl, C12-C18 alkenyl, C14-C18 alkenyl, C16-C18 alkenyl, C11-C13 alkenyl, C13-C15 alkenyl, and C15-C19 alkenyl. Generally, the total number of carbon atoms (C) and double bonds (D) in alkenes is represented by the numerical symbol of " C: D", such as 14:1, 16:1, 18:1, 18:2, 18:3, 20:4, or 20:5. Exemplary linear alkenyl groups suitable for use in the present application include methylidene, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl, octadecenyl, nonadecenyl, eicosenyl,Attorney Docket No. 4404-0140PWO1 tetradecenyl (14:1), octadecatrienyl (18:3), octadecadienyl (18:2), eicosatetraenyl (20:4), eicosapentaenyl (20:5), or the like. Exemplary branched alkenyl groups suitable for use in the present application include 2-methyl-l-propenyl (C4), 3-methyl-l-butenyl (C5), 2-ethyl-1-butenyl (C6), 3,3-dimethyl-1-pentenyl (C8), 4-methyl-5-dodecenyl (C12), 6-methyl-9-octadecenyl (C18), or the like.

[0048] The term "cationic lipid" as used herein refers to lipids having one or more fatty acid or fatty alkyl or alkenyl chains and attaining a positive charge through one or more amines present in the polar head group. The cationic lipid is typically protonated (i.e., positively charged) at a pH below its pKa. The ionizable property of a cationic lipid can enhance efficacy by aiding in endosomal escape and reducing toxicity. Depending on the pH of the composition in which the cation ical ly ionizable lipid is dissolved, the lipid is either positively charged or neutral. The cationically ionizable lipid comprises a head group that includes at least one nitrogen atom (N), which is positively charged or capable of being protonated under certain conditions, such as physiological conditions.

[0049] The term "helper lipid" as used herein is typically non-ionic or neutral lipids and known to stabilize the structure of LNP, which in turn enhances the delivery of active pharmaceutical ingredients (APIs) ortherapeutic agents carried by the LNPs. A helper lipid also helps optimize the encapsulation of APIs (e.g., nucleic acids), protect them from degradation, and facilitate their transport into target cells. According to embodiments of the present disclosure, the helper lipids include a steroid, a phospholipid, a PEGylated lipid, a PEGylated phospholipid, and a combination thereof.

[0050] The term "nucleic acid" comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term includes genomic DNA, cDNA, mRNA, and recombinantly produced and chemically synthesized molecules. Nucleic acid may be present as a single-stranded or double-stranded molecule, and it may be linear or covalently circular. Nucleic acid can be isolated. In the context of the present disclosure, "isolated nucleic acid" refers to nucleic acid that has been amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, or produced recombinantly by cloning, or purified, for example by cleavage and separation by gel electrophoresis, or synthesized, for example by chemical synthesis.Attorney Docket No. 4404-0140PWO1

[0051] The term "microRNA" or "miRNA" used herein refers to a class of non-coding RNAs that play roles in regulating gene expression. Most miRNAs are transcribed from DNA sequences into primary miRNAs (pri-miRNAs) and processed into precursor miRNAs (pre-miRNAs) and finally mature miRNAs. Accordingly, the term miRNA used herein refers to all non-coding RNAs involved in miRNA processing and maturation, preferably including precursor miRNAs and mature miRNAs.

[0052] The terms "encoding," "encode," or the like, as used herein, refer to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, cDNA, or RNA (preferably mRNA), to serve as templates for the synthesis of other polymers and macromolecules in biological processes (e.g., transcription or translation). These processes result in either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties arising therefrom. Thus, the final product encoded by a nucleic acid is a protein if the transcription and translation of RNA (e.g., mRNA) corresponding to that nucleic acid produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the RNA sequence and is typically provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0053] The term "subject" or "patient" is used interchangeably herein and is intended to mean a mammal including the human species that is treatable by the API within the cationic lipids. The term "mammal" refers to all members of the class Mammalia, including humans, primates (e.g., monkey, and chimpanzee), domestic and farm animals, such as rabbit, pig, goat, sheep, and cattle; as well as zoo, sports or pet animals (e.g., a horse, a dog, a cat and etc); and rodents, such as mouse, rat, guinea pig, and hamster. In a working example, the subject is a human. Further, the term "subject" or "patient" intended to refer to both the male and female gender unless one gender is specifically indicated.

[0054] 2. Description of the invention

[0055] The present disclosure is based, at least in part, on the discovery that some novel lipids containing a dimethylolpropionic acid tail may act as vehicles forthe delivery of polynucleotides into cells. Accordingly, the present disclosure provides novel lipids and compositions thereof that serve as a vehicle forthe delivery of therapeutic agents.Attorney Docket No. 4404-0140PWO1

[0056] 2.1 Lipids of the Present Disclosure

[0057] The present disclosure aims at providing novel lipids and lipid nanoparticles comprising the same for delivering therapeutic agents e.g., mRNA. In one aspect, the present invention relates to a compound of formula (I),

[0058] In the compound of formula (I),X and W are independently O or -NH;Y is O, S, or -NH; m and o are independently an integer between 1 and 5;n is an integer between 1 and 7;Ri is C1-C20 linear or branched alkyl, or linear or branched alkenyl;R2 is C1-C20 linear or branched alkyl, linear or branched alkenyl, or -C(Ra)((CH2)-Z-C(=O)Rb)2, whereinRais C1-C6 alkyl,Rbis C1-C20 linear or branched alkyl, or linear or branched alkenyl, andZ is O or -NH; andR3 is C1-C6 alkyl.

[0059] In some embodiments of the present disclosure, in the compound of formula (I), X, Y, and W are independently O; Ri is C10-C20 alkyl; R2 is C14-C18 alkyl; and R3 is C1-C4 alkyl.

[0060] In some preferred embodiments, in the compound of formula (I), X, Y, and W are independently O; Ri is nonyl; RR2is 7-pentadecyl; and R3is methyl.

[0061] In one working embodiment, in the compound of formula (I), X, Y, and W are independently O; Ri is nonyl; R2 is 7-pentadecyl; R3 is methyl; and m is 3, n is 5, and o is 3. In another working embodiment, X, Y, and W are independently O; Ri is nonyl; R2 is 7-pentadecyl; R3 is methyl; and m is 1, n is 5, and o is 3. In another working embodiment, X, Y, and W are independently O; Ri is nonyl; R2 is 7-pentadecyl; R3 is methyl; and m, n, and o are independently 3. In yet another working embodiment, X, Y, and W are independently O; Ri is nonyl; R2 is 7-Attorney Docket No. 4404-0140PWO1 pentadecyl; R3 is methyl; and m is 5, n is 5, and o is 3. In a further working embodiment, X, Y, and W are independently O; Ri is nonyl; R2 is 7-pentadecyl; R3 is methyl; and m is 2, n is 1, and o is 3. In yet another working embodiment, X, Y, and W are independently O; Ri is nonyl; R2 is 7-pentadecyl; R3 is methyl; and m is 3, n is 1, and o is 3. In a further working embodiment, X, Y, and W are independently O; Ri is nonyl; R2 is 7-pentadecyl; R3 is methyl; and m is 2, n is 5, and o is 3. In another working embodiment, X, Y, and W are independently O; Ri is nonyl; R2 is 7-pentadecyl; R3 is methyl; and m is 4, n is 5, and o is 3. In an alternative working embodiment, X, Y, and W are independently O; Ri is nonyl; R2 is 7-pentadecyl; R3 is methyl; and m is 3, n is 7, and o is 3. In another working embodiment, X, Y, and W are independently O; Ri is nonyl; R2 is 7-pentadecyl; R3 is methyl; and m is 3, n is 7, and o is 5.

[0062] According to the present disclosure, in an alternative preferred embodiment, in the compound of formula (I), X, Y, and W are independently O; m is 3, n is 5, and o is 3; Ri is nonyl; R2 is 7-pentadecyl; and R3 is ethyl.

[0063] Alternatively, in some embodiments of the present disclosure, in the compound of formula (I), X, Y, and W are independently O; m is 3; n is 5; o is 3; Ri is tridecyl or heptadecyl; RR2is 7-pentadecyl; and R3is methyl.

[0064] In another alternative embodiments, in the compound of formula (I), X, Y, and W are independently O; m is 3; n is 5; o is 3; Ri is 7-pentadecyl; R2 is 7-pentadecyl or nonyl; and R3 is methyl.

[0065] In a further alternative embodiment, in the compound of formula (I), X, Y, and W are independently O; m is 3; n is 5; o is 3; Ri is nonyl; R2 is -C(Ra)((CH2)-Z-C(=O)Rb)2, wherein Rais methyl, Rbis nonyl, and Z is O.

[0066] In a yet alternative embodiment, in the compound of formula (I), X and Y are independently -NH; W is O; m is 3; n is 5; o is 3; Ri is nonyl; RR2is 7-pentadecyl; and R3is methyl.

[0067] In alternative embodiments of the present disclosure, in the compound of formula (I), X is O; Y is S; W is O or -NH; m is 3; n is 5; o is 3; Ri is nonyl; RR2is 7-pentadecyl; and R3is methyl.

[0068] In yet another embodiment of the present disclosure, in the compound of formula (I), X and Y are independently O; W is -NH; m is 3; n is 5; o is 3; Ri is nonyl; RR2is 7-pentadecyl; and R3is methyl.Attorney Docket No. 4404-0140PWO1

[0069] In yet another embodiment of the present disclosure, in the compound of formula (I), X and W are independently -NH; Y is O; m is 3; n is 5; o is 3; Ri is nonyl; RR2is 7-pentadecyl; and R3is methyl.

[0070] In one alternative embodiment of the present disclosure, in the compound of formula (I), X, Y, and W are independently -NH; m is 3; n is 5; o is 3; Ri is nonyl; RR2is 7-pentadecyl; and R3is methyl.

[0071] According to the present disclosure, exemplary compounds of formula (I) include,Attorney Docket No. 4404-0140PWO1(MPA08),(MPA09),(MPA10),Attorney Docket No. 4404-0140PWO1(MPA11),(MPA13),(MPA14),(MPA15),(MPA16),Attorney Docket No. 4404-0140PWO1(MPA24), and(MPA25).

[0072] The compounds of the present disclosure may be prepared in accordance with the procedures described in the working examples. Briefly, a derivative of dimethylolpropionic acid is used as a reaction initiator. It reacts through two hydroxyl groups with long-chain fatty acids, which may be either linear or branched, followed by condensation with N, N-diisopropylethylamine (DIPEA) to form the final lipid products of the present invention. The detailed reaction conditions and chemical agents for producing the present lipid compounds can be modified by a person of ordinary skill in the art without departing from the spirit of the present disclosure.

[0073] Examples of fatty acid suitable for use in production of the present formula (I) include, but are not limited to, saturated fatty acids such as, formic acid, acetic acid, propionic acid,Attorney Docket No. 4404-0140PWO1 butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, lauric acid, stearic acid, arachidic acid, myristoleic acid, palmitoleic acid, and 2-hexyldecanoic acid; and unsaturated fatty acids, such as, myristic acid, palmitic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, and arachidonic acid. In working examples, the fatty acid used in the production of the present compound of formula (I) is capric acid or 2-hexyldecanoic acid.

[0074] 2.2 Lipid Nanoparticles

[0075] Also disclosed herein is a novel lipid nanoparticle (LNP) that facilitates the intracellular delivery of molecules, preferably biologically active and therapeutic molecules, into cells.

[0076] According to embodiments of the present disclosure, the lipid nanoparticle (LNP) comprises a compound of formula (I) described above. Specifically, each LNP has one or more hydrophilic cores enclosed by a lipid bilayer, which is primarily formed by the compound of formula (I). Typically, the LNP may be monolamellar or multilamellar structure. In addition, as is well-known in the art, the LNP may optionally also include other cationic lipids and / or helper lipids, which together help form and stabilize the LNP structure. Accordingly, in some embodiments of the present disclosure, the LNPs have a central hydrophilic core in which therapeutic agents that are hydrophilic in nature may be accommodated.

[0077] In other embodiments, instead of having a hydrophilic core enclosed by a lipid bilayer as indicated above, each LNP may have the structure of a micelle with a plurality of smaller inverse micelles enclosed within it. The micelle has a hydrophobic core enclosed by a single layer of lipids (e.g., the lipids of the present disclosure and neutral lipids), with the hydrophobic tails of the lipids extending toward the center, while the hydrophilic head groups of the lipids face outward. Additionally, multiple inverse micelles, which are smaller in size than the micelle, may be enclosed within the hydrophobic core of the micelle. Note that the term "inverse micelle" refers to a micelle in which the head groups of the lipids are at the center, while the hydrophobic tails extend outward. Accordingly, the hydrophilic therapeutic agent (e.g., nucleic acids) may be disposed inside the multiple hydrophilic cores of the inverse micelles.

[0078] According to alternative embodiments of the present disclosure, the LNP may be produced in the form of a micelle with a single layer formed by the compound of formula (I). Typically, a micelle is a spherical structure formed by the self-assembly of lipids in an aqueousAttorney Docket No. 4404-0140PWO1 environment. According to the present disclosure, the hydrophilic heads of the compound of formula (I) face outward toward the water, while the hydrophobic tails face inward, forming a hydrophobic core. Accordingly, the hydrophobic therapeutic agent (e.g., poorly soluble drugs) may be disposed inside the hydrophobic core of the micelle.

[0079] According to embodiments of the present disclosure, the helper lipids suitable for constructing the LNP may include steroids, phospholipids, PEGylated lipids, PEGylated phospholipids, and a combination thereof.

[0080] Examples of steroids suitable for constructing the LNP include, but are not limited to, cholesterol, lanosterol, ergosterol, phytosterol, stigmasterol, and brassicasteroL Preferably, the LNP further comprises cholesterol.

[0081] Examples of phospholipids suitable for constructing the LNP include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins. Particular examples of phospholipids include, distearoylphosphatidylcholine (or l,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), and diphytanoyl-phosphatidylethanolamine (DPyPE). According to embodiments of the present disclosure, the phospholipid comprised in the LNP is distearoylphosphatidylcholine (DSPC).

[0082] Examples of PEGylated lipids suitable for use in the present disclosure include, but are not limited to, PEG conjugated to diethylene glycol (DEG-PEG) (e.g., 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DEG-PEG 2000); PEG conjugated to dialkyloxypropyl (PEG-DAA) (e.g., dioleoyl phosphatidylethanolamine (DOPE)); PEG conjugated to diacylglycerol (PEGDAG) (e.g., l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N[methoxy(polyethylene glycol)-Attorney Docket No. 4404-0140PWO1 2000] (DOPE-PEG 2000); PEG coupled to phosphatidylethanolamine (PEG-PE) (e.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy( Polyethylene Glycol)-2000], DSPE-PEG 2000); and a combination thereof. In one working example, the PEGylated lipid is DEG-PEG 2000.

[0083] Examples of PEGylated phospholipids suitable for constructing the LNP include, but are not limited to, distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), PEG-distearoylphosphatidylcholine (PEG-DSC), octadecanoic acid conjugated PEG (C18-PEG), phosphatidylethanolamine-PEG (PE-PEG), dioleoylphosphatidylethanolamine-polyethylene glycol (DOPE-PEG), dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG), polyethylene glycol-phosphatidylethanolamine (PEG-PE), polyethylene glycol-sphingomyelin (PEG-SM), and the like.

[0084] According to optional or alternative embodiments of the present disclosure, the LNP of the present disclosure may further comprise one or more cationic lipids to assist forming and stabilizing its structure. Examples of additional cationic lipids suitable for constructing the LNP include, but are not limited to, / V, / V-dimethyl-2,3-bis[(Z)-octadec-9-enoxy]propan-l-amine (DODMA), [3-(dimethylamino)-2-[(Z)-octadec-9-enoyl]oxypropyl] (Z)-octadec-9-enoate (1,2-Dioleoyloxy-3-(dimethylamino) propane; DODAP), [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate (DLin-MC3-DMA), 4-((di((9Z,12Z)-octadeca-9,12-dien-l-yl)amino)oxy)-N, N-dimethyl-4-oxobutan-l-amine (DPL-14), 3|3-[N- (N', N'- dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), l,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), l,2-distearyloxy-N, N-dimethyl-3-aminopropane (DMSDMA), l,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), / V, / V-dimethyl-2,3-bis[(9Z,12Z)-octadeca-9,12-dienoxy]propan-l-amine (DLinDMA), / V, / V-dimethyl-2,3-bis[(9Z,12Z,15Z)-octadeca-9,12,15-trienoxy]propan-l-amine (DLenDMA), [S-(dimethylamino)-2-[(9Z,12Z)-octadeca-9,12-dienoyl]oxypropyl] (9Z,12Z)-octadeca-9,12-dienoate (DLinDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]dioxolane (DLin-KC2-DMA), N, N-dimethyl-2,3-bis(dodecyloxy)propan-l-amine (DLDMA), N, N-dimethyl-2,3-bis(tetradecyloxy)propan-l-amine (DMDMA), 2-[8-[[(3S,8S,9S,10 / ?,13 / ?,14S,17 / ?)-10,13-dimethyl-17-[(2 / ?)-6-methylheptan-2-yl]-2,3,4,7,8,9,ll,12,14,15,16,17-dodecahydro-lH-cyclopenta[a]phenanthren-3-yl]oxy]octoxy]- / V, / V-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dienoxy]propan-l-amine (Octyl-CLinDMA), [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)buta noate (DLin-MC3-DMA), heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-Attorney Docket No. 4404-0140PWO1 102), 6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate (ALC-0315), nonyl 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoate (Lipid 5), bis[(Z)-non-2-enyl] 9-[4-(dimethylamino)butanoyloxy]heptadecanedioate (L319), l-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-l-yl]ethyl]amino]dodecan-2-ol (Lipidoid C12-200), 8-methylnonyl 3-[3-[3-[bis[3-(8-methylnonoxy)-3-oxopropyl]amino]propyl-methylamino]propyl-[3-(8-methylnonoxy)-3-oxopropyl]amino]propanoate (306Oi10), and N-Dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl- ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (Lipidoid 98N12-5).

[0085] The LNP can be produced using procedures and tools well known in the art, such as thin-layer hydration, ethanol injection, microfluidic devices, and similar techniques. In these processes, the lipids ( / .e., the compounds of formula (I)), and optionally other cationic lipids, a helper lipid, a steroid, self-assemble to form a lipid bilayer. The therapeutic agents may either be directly encapsulated within the LNP or coupled to the lipids (i.e., the compounds of formula (I)) prior to encapsulation, resulting in a complex in the form of nanoparticles. The therapeutic agents delivered by the LNP, in either liquid or solid form, may include nucleic acids, polypeptides, proteins, antibodies, carbohydrates, proteoglycans, glycoproteins, small molecules, or combinations thereof.

[0086] In some embodiments of the present disclosure, the therapeutic agent is a nucleic acid, which includes but is not limited to, mitochondrial DNA (mtDNA), chloroplast DNA (cpDNA), plasmid, messenger RNA (mRNA), small interfering RNA (siRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), or aptamer. In preferred embodiments, the therapeutic agent is mRNA. In some working examples, the therapeutic agent is an mRNA encoding the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. In other working examples, the therapeutic agent is an mRNA encoding an envelope protein of Dengue virus.

[0087] In some embodiments of the present disclosure, the therapeutic agent is a protein or a peptide, such as an antigen. Examples of an antigen suitable to be accommodated in the LNP include, but are not limited to, antigens derived from pathogens, neoantigens, tumor-derived lysates, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), mucin proteins, epithelial tumor antigens (ETA), tyrosinase, melanoma-associated antigens (MAGE), RAS proteins, tumorAttorney Docket No. 4404-0140PWO1 suppressor proteins, and combinations thereof. In some working examples, the protein is a viral protein, e.g., a spike protein of SARS-CoV-2 virus.

[0088] In alternative embodiments, the therapeutic agent disposed in the LNP may be a poorly soluble therapeutic agents, such as, non-steroidal anti-inflammatory drugs (NSAIDs), and immunosuppressant drugs.

[0089] According to embodiments of the present disclosure, the compound of formula (I) is present in the LNP at a concentration of about 40 to 50 mol%, such as 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 mol%. According to embodiments of the present disclosure, the compound of the formula (I) and the therapeutic agents are present in the LNP at a molar ratio of about 2:1 to 8:1, such as, about 2:1, 2.5:1, 3:1, 3.5: 1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, and 8:1. In some working examples, the lipid (i.e., the compound of formula (I)) and the therapeutic agents (e.g., mRNA) are present in the LNP at a molar ratio of 6.5:1.

[0090] According to embodiments of the present disclosure, the LNP has a diameter of about 50 to 150 nm, such as 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 120, 125, 130, 135, 140, 145, and 150 nm. In some working examples, the LNP has a diameter of about 111 nm. In other working examples, the LNP has a diameter of about 70 nm.

[0091] 2.3 Formulations Comprising the LNP

[0092] The lipid nanoparticles comprisingthe compounds of formula (I) may be formulated into powders, granules, solutions or suspensions, suppositories or patches, and such formulations can be administered to effectively deliver the active ingredients to the appropriate or desired site of action and lesions. According to some embodiments of the present disclosure, the lipid nanoparticle can be formulated as a vaccine to deliver antigens to a subject, enhancing their resistance to pathogens.

[0093] In some embodiments of the present disclosure, the formulation is prepared in a form suitable for storage (e.g., long-term storage). In one embodiment, the formulation is in a lyophilized (freeze-dried) form. In another embodiment, the formulation comprising the LNP is lyophilized and can be stored at a temperature of approximately -90°C or higher, such as between -90°C and -10°C. For example, the lyophilized formulation described herein can beAttorney Docket No. 4404-0140PWO1 stored at a temperature ranging from about -90°C to 0°C, such as, -90°C, -85°C, -80°C, -75°C, -70°C, -65°C, -60°C, -55°C, -50°C, -45°C, -40°C, -35°C, -30°C, -25°C, -20°C, -15°C, -10°C, -5°C, 0°C. In some embodiments, the temperature suitable for storing lyophilized formulation ranges between -80°C and -40°C, -40°C and -25°C, or -25°C and -10°C, or at a specific temperature of approximately -20°C.

[0094] In some embodiments of the present disclosure, the formulation is in liquid form and can be stored at a temperature ranging from approximately 0°C to 20°C, such as, 0°C, 5°C, 10°C, 15°C, and 20°C. For example, the liquid formulation of the present disclosure can be stored at a temperature ranging from about 1°C to about 15°C, such as between 2°C and 10°C, or from about 2°C and 8°C, or at a specific temperature of about 5°C.

[0095] 2.4 Uses of the Lipid Nanoparticles

[0096] Accordingtothe present disclosure, the compounds of formula (I) and lipid nanoparticles thereof are useful for the delivery of biologically active agents and / or therapeutic agents, thus they may be used in the treatment of diseases. The present disclosure thus encompasses a method for treating a disease in a subject in need and a method of enhancing the immunogenicity of a subject in need. The methods mainly include steps of, administering parenterally an effective amount of the composition (i.e., the lipid nanoparticles (LNPs) comprising the compound of formula (I)) to the subject. According to the present disclosure, the lipid nanoparticle can be administered parenterally to the lesions (e.g., a tumor) of the subject. In other embodiments, the lipid nanoparticle can be formulated as a vaccine and administered parenterally to enhance the subject's resistance to pathogens (e.g., the SARS-CoV-2 virus).

[0097] Exemplary suitable parenteral route includes, but is not limited to, transdermal, percutaneous, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, intradermal, subcutaneous, rectal, intravaginal, and intraperitoneal routes. Suitable routes vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the dosage and the nature of active ingredients, genetic factors and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no moreAttorney Docket No. 4404-0140PWO1 than routine experimentation. In general, the most appropriate route of administration depends on a variety of factors including the agent's stability in the environment of the circulatory system, and / or the condition of the subject (e.g., the severity of lung cancer of the subject, or whether the subject can tolerate conventional treatment). According to some embodiments of the present disclosure, the LNP is formulated into injectable formulation for parental administration. According to other embodiments of the present disclosure, the LNP is formulated into patches for transdermal administration.

[0098] The LNP can be administered at a frequency that effectively prevent, inhibit, suppress, or treat diseases, conditions, or traits in the subject. In some embodiments, the lipid nanoparticle can be administered at a frequency of four times a day to once every three months; for example, at a frequency of four times a day, three times a day, twice a day, once a day, once every other day, once every third day, once every week, once every other week, once monthly, twice monthly, thrice monthly, once every other month, or once every three months. Preferably, the lipid nanoparticle is administered to the subject at a frequency of twice per week (once every third or four day). Optionally, the lipid nanoparticle is administered to the subject at a frequency of once a week. Still optionally, the lipid nanoparticle is administered to the subject at a frequency of once every other week.

[0099] According to embodiments of the present disclosure, diseases treatable by the method may be cancers, infectious diseases (including inflammatory diseases), transplant and / or tissue rejection, autoimmune diseases, and the like.

[0100] Examples of infectious diseases that can be prophylaxed by administering formulations comprisingthe LNPs of the present disclosure include, but are not limited to, bacterial, viral, and fungal infections, or combinations thereof. In one preferred embodiment, the LNP encapsulating mRNA encoding the spike protein of SARS-CoV-2 is administered to a subject to prophylax against SARS-CoV-2 viral infection.

[0101] According to the present disclosure, the lipid nanoparticles (LNPs) may be useful for the delivery of biologically active agents into cultivated cells, thus they may be used as a platform for the delivery of therapeutic materials in preparation of cellular therapies. The present disclosure thus encompasses a method of delivering a biologically active agent into a cultivated cell. The method mainly includes contacting the cultivated cell with an effective amount of theAttorney Docket No. 4404-0140PWO1 LNP, wherein the biologically active agent is a nucleic acid, a polypeptide, a protein, a carbohydrate, a proteoglycan, a glycoprotein, or a combination thereof.

[0102] The cultivated cells provided in the embodiments may be isolated or derived from subjects, and potent to differentiate or develop into cellular transplants with therapeutic benefits by receiving genes or gene products delivered via the LNP, such that the cellular transplants are useful in and beneficial to cell therapy adaptive to select fields, including regenerative medicine, immune system disorders, and cancer. Generally, cultivated cells include stem cells and immune cells. Examples of stem cells include, but are not limited to pluripotent stem cells (PSCs; for example, embryonic stem cells (ESCs), epiblast stem cells (EpiSCs), embryonic germ cells (EGCs), and induced pluripotent stem cells (iPSCs)), adult stem cells (ASCs; for example, hematopoietic stem cells (HSCs), skin stem cells (SSCs), neural stem cells (NSCs), and mesenchymal stem cells (MSCs)), and cancer stem cells (CSCs). Examples of immune cells suitable for use include but are not limited to, T cells, dendritic cells (DCs), natural killer (NK) cells, and macrophages. The immune cells express genetically engineered antigen receptors, including engineered T cell receptors (TCRs) and functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs), including activating, stimulatory, and costimulatory CARs, and combinations thereof.

[0103] By virtue of the features described above, the present disclosure provides novel lipids capable of encapsulating and delivering polynucleotides to cells, thereby enabling the effective and efficient treatment or prophylaxis of diseases.

[0104] EXAMPLES

[0105] Materials and methods

[0106] Preparation of Lipid-Nucleic acid Complexes

[0107] To produce the lipid nanoparticles, the lipid (compound MPA01), distearoylphosphatidylcholine (DSPC), cholesterol, and DMG-PEG 2000 were fully mixed in ethanol at a molar ratio of 46.3:9.4:42.7:1.6, and a total lipid concentration was brought to 50 mM, thereby producing a liquid formulation of lipid nanoparticles (LNPs). As for control group, SM-102 and ALC-0315, ionizable amino lipidsthat both conventionally used in LNPs preparation, was mixed with DSPC, cholesterol, and DMG-PEG 2000 at a molar ratio of 50:10:38.5:1.5 and 46.3:9.4:42.7:1.6, respectively. The wild-type SARS-CoV-2 mRNA encoding the full-lengthAttorney Docket No. 4404-0140PWO1 spike protein was dissolved in 50 mM sodium acetate buffer (pH 4.5) at a fixed nanoparticle (NP) lipid-to-mRNA ratio of 6.5. Formulations were prepared using the Ignite NanoAssemblr systems (Precision NanoSystems).

[0108] Forthe Ignite system, a 300-pL aliquot of the organic phase and an 850-pL aliquot of the aqueous phase were mixed, and the LNPs were directly dialyzed against PBS.

[0109] Characterization of the LNPs

[0110] The LNPs were diluted 100-fold in PBS (pH 7.4) and transferred to a 384-well microplate for size and polydispersity index (PDI) measurements using the Wyatt DynaPro Plate Reader III (Malvern Instruments, UK).

[0111] The encapsulation ratio of lipid nanoparticles for encapsulating mRNAs was determined using a fluorescence plate reader (multi-mode / PerkinElmer, EnSpire) with the aid of an RNA quantification kit (Quant-iT™ RiboGreen RNA Assay Kit, Invitrogen, Cat. No. R11490).

[0112] Absorption spectra of mRNAs with known concentrations ( / .e., 100 pg / mL) were first acquired for the construction of a standard curve. The lipid-RNA complexes (typically with a total volume of 50 pL for each sample well) were then mixed with the reagent from the RNA quantification kit in each well. Optionally, the lipid-RNA complexes were subjected to lysis buffer, thereby bringing RNA back into suspension. The amounts of mRNAs were quantified by interpolating their absorption strengths at 535 nm to that of the standard curve, and the encapsulation efficiency (EE) was calculated by the following formula:Encapsulation efficiency EE %) = Wt / Wi) X 100% where Wt is the total amount of mRNAs released in the LNPs lysis suspension, and Wi is the total quantity of mRNAs added initially during preparation.

[0113] Flow Cytometry Analysis

[0114] The mRNA-LNP was transfected into 293T cells by direct addition. One day posttransfection, the cells were harvested into FACS tubes. The cells were washed with staining buffer (PBS + 1% FBS). After washing, the cells were incubated with anti-RBD antibody at 4°C for 30 minutes. The cells were then washed twice and incubated for 30 minutes with PE-goat-anti-mouse IgG (H+L) antibody (Jackson ImmunoResearch, PA, USA) at 4°C. After twoAttorney Docket No. 4404-0140PWO1 additional washes, the cells were resuspended in 300 pL of staining buffer for flow cytometry analysis using an Attune cell analyzer.

[0115] Example 1: Synthesis of the Compounds of Formula (I)

[0116] The compounds of formula (I) were respectively synthesized in accordance with steps as described in Schemes I-XI

[0117] Scheme IHOBnBr, K2CO3Capric acid, EDCI, DMAP DMF DCMO3-hyd roxy-2-(hyd roxymethy I )-2- methylpropanoic acidBr(CH2)6OH EDCI, DMAP, DCM6a, m=3, n=5, R2=7-pentadecyl 6c, m=1, n=5, R2=7-pentadecyl 6d, m=5, n=5, R2=7-pentadecyl 6e, m=3, n=3, R2=7-pentadecyl 6f, m=2, n=1, R2=7-pentadecyl 6g, m=3, n=1, R2=7-pentadecyl 6 i, m=2, n=5, R2=7-pentadecyl 6j, m=4, n=5, R2=7-pentadecyl 6k, m=3, n=7, R2=7-pentadecyl ACN / CHCI3, DIPEA, 85-100 °CMPA01, MPA07, MPA08, MPA09. MPA10, MPA11, MPA13, MPA14, MPA15Attorney Docket No. 4404-0140PWO1

[0118] Scheme IIHOBnBr, K2CO3 2-Hexyldecanoic acid, EDCI, DMAP DMF DCMo 3-hyd roxy-2-(hy d roxy methyl )-2- methylpropanoic acidACN / CHCI3, DIPEA, 90 °CMPA02Attorney Docket No. 4404-0140PWO1

[0119] Scheme IIIHOBnBr, K2CO3R1CO2H, EDCI, DMAPDMF DCMO3-hydroxy-2-(hydroxymethyl)-2-methylpropanoic acidR3Pd / C, H24b, R3= Me, R1=7-pentadecyl Ethyl acetate 4c, R3= Me, R1=C13H273b, R3= Me, R1=7-pentadecyl3c, R3= Me, R1=C13H27Br(CH2)6OHR15b, R3= Me, R1=7-pentadecylEDCI, DMAP, DCM 5c, R3= Me, R1=C13H276a, m=3, n=5, R2=7-pentadecyl ACN / CHCI3, DIPEA, 85-100 °CMPA03 and MPA04Attorney Docket No. 4404-0140PWO1

[0120] Scheme IVBnBr, K2CO3Capric acid, EDCI, DMAP DMF DCM2,2- bis(hydroxymethyl)butanoic acidBr(CH2)6OH EDCI, DMAP, DCMHO'WT mXNHX>< nOV HR26a, m=3, n=5, R2=7-pentadecyl KI, K2CO3, ACN, refluxMPA05Attorney Docket No. 4404-0140PWO1

[0121] Scheme VBnBr, K2CO3R1CO2H, EDCI, DMAPDMF DCM3-hydroxy-2-(hydroxymethyl)-2-2amethylpropanoic acidPd / C, H24e, R1=C17H35Ethyl acetate 3e, R1=C17H35Br(CH2)6OH 5e, R1=C17H35EDCI, DMAP, DCM6a, m=3, n=5, R2=7-pentadecyl ACN / CHCl3, DIPEA, 85-100 °CMPA12Attorney Docket No. 4404-0140PWO1

[0122] Scheme VIHOBnBr, K2CO3Capric acid, EDCI, DMAPDMF DCMO3-hydroxy-2-(hydroxymethyl)-2-methylpropanoic acidBr(CH2)8OH EDCI, DMAP, DCM6k, m=3, n=7, R2=7-pentadecyl ACN / CHCI3, DI PEA, 85-100 °CMPA16Attorney Docket No. 4404-0140PWO1

[0123] Scheme VIIHOBnBr, K2CO3Capric acid, EDCI, DMAP DMF DCMO3-hydroxy-2-(hydroxymethyl)-2- methylpropanoic acidACN / CHCI3, DIPEA, 85-100 °CAttorney Docket No. 4404-0140PWO1

[0124] The methods for preparing the exemplary compounds, as well as the analytical data for the compounds thus prepared, are set forth below:

[0125] 1.1 Synthesis of compounds MPA01, MPA07, MPA08, MPA09, MPA10, MPA11, MPA13, MPA14, and MPA15 via Scheme I

[0126] Compound 2a: Benzyl 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoateo2a

[0127] A mixture of 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoic acid (5.0 g, 37.0 mmol) and K2CO3(6.7 g, 48.4 mmol) in DMF was prepared, followed by the addition of BnBr (8.3 g, 48.4 mmol). After stirring at room temperature for 19 hours, the reaction mixture was extracted with ethyl acetate and brine and dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using EA / Hex (1 / 1), yielding compound 2a (5.0 g, 22.4 mmol) as a white solid. MS (M+l): 225.

[0128] Compound 3a: 2-((Benzyloxy)carbonyl)-2-methylpropane-l,3-diyl bis(decanoate)3a

[0129] A mixture of compound 2a (3.2 g, 14.3 mmol) and capric acid (8.8 g, 51.1 mmol) in dichloromethane (DCM) was stirred at 0°C, followed by the addition of EDCI (9.8 g, 51.1 mmol) and DMAP (1.6 g, 12.8 mmol). After stirring at room temperature for 19 hours, the reaction mixture was washed with 2N HCI (aq) and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using ethyl acetate / hexane (EA / Hex) (1 / 10), yielding compound 3a (7.5 g, 14.0 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 0.88 (t, J = 6.91 Hz, 6H), 1.19-1.39 (m, 27H), 1.55 (t, J = 7.15 Hz, 4H), 2.22 (t, J = 7.63 Hz, 4H), 4.15-4.29 (m, 4H), 5.16 (s, 2H), 7.28-7.39 (m, 5H).

[0130] Compound 4a: 3-(Decanoyloxy)-2-((decanoyloxy)methyl)-2-methylpropanoic acidAttorney Docket No. 4404-0140PW014a

[0131] A mixture of compound 3a (7.5 g, 14.0 mmol) and Pd / C (cat.) in ethyl acetate (EA) was stirred at room temperature under a hydrogen (H2) atmosphere overnight. After filtration and evaporation, the desired product compound 4a (6 g, 13.6 mmol, 97%) was obtained as a white solid without further purification. MS (M-1): 441

[0132] Compound 5a: 2-(((6-Bromohexyl)oxy)carbonyl)-2-methylpropane-l,3-diyl bis(decanoate)

[0133] A mixture of compound 4a (6 g, 13.6 mmol) and 6-bromo-l-hexanol (1.6 g, 8.9 mmol) in dichloromethane (DCM) was stirred at 0°C, followed by the addition of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (2.6 g, 13.6 mmol) and 4-dimethylaminopyridine (DMAP) (0.33 g, 2.7 mmol). After stirring at room temperature for 19 hours, the reaction mixture was washed with 2N HCI (aq) and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using ethyl acetate / hexane (EA / Hex) (1 / 10), yielding compound 5a (1.2 g, 1.98 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 0.88 (t, J = 7.15 Hz, 6H), 1.20-1.32 (m, 29H), 1.32-1.43 (m, 2H), 1.43-1.52 (m, 2H), 1.53-1.63 (m, 2H), 1.63-1.68 (m, 2H), 1.81-1.90 (m, 2H), 2.29 (t, J = 7.39 Hz, 4H), 3.40 (t, J = 6.91 Hz, 2H), 4.12 (t, J = 6.68 Hz, 2H), 4.16-4.28 (m, 4H).

[0134] Compound MPA01: 2-(((6-((6-((2-Hexyldecanoyl)oxy)hexyl)(4-hydroxybutyl)amino)hexyl)oxy)carbonyl)-2-methylpropane-l,3-diyl bis(decanoate)Attorney Docket No. 4404-0140PWO1

[0135] A mixture of compound 5a (0.68 g, 1.10 mmol) and compound 6a (0.96 g, 2.20 mmol) in ACN / CHCI3 was treated with DIPEA (0.8 mL, 4.7 mmol) and heated to 90°C for 17 hours. The solvent was removed, and the mixture was extracted with ethyl acetate and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using dichloromethane / methanol (DCM / MeOH) (20 / 1), yielding compound MPA01 (0.38 g, 0.4 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 0.87 (t, J = 6.91 Hz, 12H), 1.22-1.44 (m, 59H), 1.53-1.73 (m, 18H), 2.20-2.35 (m, 6H), 2.52 (brs., 5H), 3.99 - 4.15 (m, 4H), 4.15-4.27 (m, 4H). MS (M+1): 953

[0136] Compound MPA07: 2-(((6-((6-((2-Hexyldecanoyl)oxy)hexyl)(2-hydroxyethyl)amino)hexyl)oxy)carbonyl)-2-methylpropane-l,3-diyl bis(deca noate)MPA07

[0137] A mixture containing compound 5a (0.5 g, 0.83 mmol) and compound 6c (0.28 g, 0.70 mmol) dissolved in ACN was prepared. Potassium carbonate (0.29 g, 2.1 mmol) and potassium iodide (0.06 g, 0.35 mmol) were then added to the reaction mixture. The mixture was heated to reflux for 13 hours. The solvent was removed, and the mixture was extracted with ethyl acetate and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using dichloromethane / methanol (DCM / MeOH) (20 / 1), yielding compound MPA07 (0.30 g, 0.32 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 0.87 (t, J = 6.91 Hz, 12H), 1.18-1.32 (m, 43H), 1.41 (br. s., 2H), 1.44 (br. s., 6H), 1.54-1.63 (m, 12H), 1.63-1.69 (m, 4H), 1.80-1.89 (m, 4H), 2.29 (t, J = 7.63 Hz, 5H), 2.98-3.07 (m, 6H), 3.65 (t, J = 6.20 Hz, 2H), 4.05 (t, J = 6.44 Hz, 2H), 4.10 (t, J=6.68 Hz, 2H), 4.19 (d, J = 10.97 Hz, 2H), 4.24 (d, J = 10.97 Hz, 2H). MS (M+1): 925.

[0138] Compound MPA08: 2-(((6-((6-((2-Hexyldecanoyl)oxy)hexyl)(6-hydroxyhexyl)amino)hexyl)oxy)carbonyl)-2-methylpropane-l,3-diyl bis(deca noate)Attorney Docket No. 4404-0140PWO1MPA08

[0139] A mixture containing compound 5a (0.5 g, 0.83 mmol) and compound 6d (0.32 g, 0.70 mmol) dissolved in ACN was prepared. Potassium carbonate (0.29 g, 2.1 mmol) and potassium iodide (0.06 g, 0.35 mmol) were then added to the reaction mixture. The mixture was heated to reflux for 13 hours. The solvent was removed, and the mixture was extracted with ethyl acetate and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using dichloromethane / methanol (DCM / MeOH) (20 / 1), yielding compound MPA08 (0.34 g, 0.35 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 0.84 (t, J = 6.68 Hz, 12H), 1.22 (t, J = 11.44 Hz, 48H), 1.27-1.41 (m, 15H), 1.46 (br. s., 5H), 1.51-1.57 (m, 8H), 1.57-1.63 (m, 4H), 2.25 (t, J = 7.39 Hz, 5H), 2.45 (br. s., 5H), 3.59 (t, J = 6.44 Hz, 2H), 4.02 (t, J = 6.44 Hz, 2H), 4.07 (t, J = 6.68 Hz, 2H), 4.13-4.23 (m, 4H). MS (M+1): 981.

[0140] Compound MPA09: 2-(((6-((4-((2-Hexyldecanoyl)oxy)butyl)(4-hydroxybutyl)amino)hexyl)oxy)carbonyl)-2-methylpropane-l,3-diyl bis(decanoate)MPA09

[0141] A mixture of compound 5a (0.98 g, 1.6 mmol) and compound 6e (1.0 g, 2.5 mmol) in ACN / CHCI3 was treated with DIPEA (0.5 mL, 3.0 mmol) and heated to 90°C for 17 hours. The solvent was removed, and the mixture was extracted with ethyl acetate and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using dichloromethane / methanol (DCM / MeOH) (20 / 1), yielding compound MPA09 (0.4 g, 0.4 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 0.87 (t, J = 6.91 Hz, 12H), 1.17-1.33 (m, 47H), 1.34-1.47 (m, 6H), 1.57-1.90 (m, 19H), 2.25-2.37 (m, 5H), 2.70-3.1 (m, 5H), 3.65 - 3.75 (m, 2H), 4.08-4.14 (m, 4H), 4.18-4.26 (m, 4H). MS (M+1): 925Attorney Docket No. 4404-0140PWO1

[0142] Compound MPA10: 2-(((6-((2-((2-Hexyldecanoyl)oxy)ethyl)(3-hydroxypropyl)amino)hexyl)oxy)carbonyl)-2-methylpropane-l,3-diyl bis(deca noate)HO'MPA10

[0143] A mixture of compound 5a (0.77 g, 1.2 mmol) and compound 6f (0.7 g, 2.0 mmol) in ACN / CHCl3was treated with DIPEA (0.4 mL, 2.3 mmol) and heated to 90°C for 17 hours. The solvent was removed, and the mixture was extracted with ethyl acetate and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using dichloromethane / methanol (DCM / MeOH) (20 / 1), yielding compound MPA10 (0.1 g, 0.1 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 0.88 (t, J = 6.91 Hz, 12H), 1.18-1.33 (m, 47 H), 1.39-1.46 (m, 5H), 1.53-1.67 (m, 14H), 1.85-2.00 (m, 2H), 2.26-2.37 (m, 5H), 3.11-3.43 (m, 3H), 3.25 (br. s., 1 H), 3.38 (brs., 2H) 3.82 (br. s., 2 H), 4.10 (t, J = 6.68 Hz, 2H), 4.17-4.26 (m, 4H), 4.56 (brs., 1H). MS (M+Na): 905

[0144] Compound MPA11: 2-(((6-((2-((2-Hexyldecanoyl)oxy)ethyl)(4-hydroxybutyl)amino)hexyl)oxy)carbonyl)-2-methylpropane-l,3-diyl bis-(deca noate)MPA-11

[0145] A mixture of compound 5a (0.63 g, 1.0 mmol) and compound 6g (0.6 g, 1.6 mmol) in ACN / CHCl3was treated with DIPEA (0.3 mL, 1.9 mmol) and heated to 90°C for 17 hours. The solvent was removed, and the mixture was extracted with ethyl acetate and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using dichloromethane / methanol (DCM / MeOH) (20 / 1), yielding compound MPA11 (0.08 g, 0.09 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 0.87 (t, J = 7.15 Hz, 12H), 1.25-1.31 (m, 47H),Attorney Docket No. 4404-0140PWO1 1.37-1.45 (m, 5H), 1.57-1.79 (m, 16H), 2.29 (t, J = 7.15 Hz, 5H), 2.50-3.50 (brs., 3H), 3.65 (brs., 1H,) 4.07-4.13 (m, 2H), 4.16-4.26 (m, 4H). MS (M+l): 897.

[0146] Compound MPA13: 2-(((6-((6-((2-Hexyldecanoyl)oxy)hexyl)(3-hydroxypropyl)amino)hexyl)oxy)carbonyl)-2-methylpropane-l,3-diyl bis-(decanoate)MPA13

[0147] A mixture of compound 5a (0.57 g, 0.9 mmol) and compound 6i (0.6 g, 1.5 mmol) in ACN / CHCl3was treated with DIPEA (0.3 mL, 1.7 mmol) and heated to 90°C for 17 hours. The solvent was removed, and the mixture was extracted with ethyl acetate and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using dichloromethane / methanol (DCM / MeOH) (20 / 1), yielding compound MPA13 (0.06 g, 0.06 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 0.80 - 0.93 (m, 12H), 1.21-1.40 (m, 57H), 1.51-1.77 (m, 17H), 2.24-2.33 (m, 5H), 2.55 (brs., 3H), 2.76 (br. s., 2H), 3.80 (t, J = 5.25 Hz, 2H), 4.03-4.13 (m, 4H), 4.13 - 4.27 (m, 4H). MS (M+l): 939.

[0148] Compound MPA14: 2-(((6-((6-((2-Hexyldecanoyl)oxy)hexyl)(5-hydroxypentyl)amino)hexyl)oxy)carbonyl)-2-methylpropane-l,3-diyl bis(deca noate)MPA14

[0149] A mixture of compound 5a (0.8 g, 1.3 mmol) and compound 6j (1.0 g, 2.2 mmol) in ACN / CHCl3was treated with DIPEA (0.7 mL, 4.2 mmol) and heated to 90°C for 17 hours. The solvent was removed, and the mixture was extracted with ethyl acetate and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatographyAttorney Docket No. 4404-0140PWO1 using dichloromethane / methanol (DCM / MeOH) (20 / 1), yielding compound MPA14 (0.38 g, 0.39 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 0.73-0.85 (m, 12 H), 1.05-1.25 (m, 48 H), 1.25-1.40 (m, 10 H), 1.42-1.61 (m, 19 H), 2.14-2.30 (m, 5 H), 2.48 (br. s., 4 H), 3.28 (br. s., 1 H), 3.32-3.43 (m, 1 H), 3.49-3.64 (m, 2 H), 3.91-4.06 (m, 4 H), 4.08-4.22 (m, 4 H). MS (M+l): 967.

[0150] Compound MPA15: 2-(((6-((8-((2-Hexyldecanoyl)oxy)octyl)(4-hydroxybutyl)amino)hexyl)oxy)carbonyl)-2-methylpropane-l,3-diyl bis-(deca noate)

[0151] A mixture of compound 5a (0.4 g, 0.66 mmol) and compound 6k (0.5 g, 1.1 mmol) in ACN / CHCl3was treated with DIPEA (0.3 mL, 1.7 mmol) and heated to 90°C for 17 hours. The solvent was removed, and the mixture was extracted with ethyl acetate and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using dichloromethane / methanol (DCM / MeOH) (20 / 1), yielding compound MPA15 (0.14 g, 0.14 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 0.87 (t, J = 6.91 Hz, 12H), 1.20-1.39 (m, 61H), 1.39-1.70 (m, 18H), 2.22 - 2.36 (m, 6H), 2.49 (brs., 5H), 3.57 (brs., 2H), 4.01-4.12 (m, 4H), 4.15-4.26 (m, 4H). MS (M+1): 981

[0152] 1.2 Synthesis of compound MPA02 via Scheme II

[0153] Compound 3b: 2-((Benzyloxy)carbonyl)-2-methylpropane-l,3-diyl bis(2-hexyldecanoate)

[0154] A mixture of compound 2a (3.0 g, 13.6 mmol) and 2-hexyldecanoic acid (12.5 g, 48.7 mmol) in dichloromethane (DCM) was stirred at 0°C, then treated with 1-Ethy l-3-(3-dimethylaminopropyl) carbodiimide (EDCI) (9.3 g, 48.7 mmol) and 4-dimethylaminopyridineAttorney Docket No. 4404-0140PWO1 (DMAP) (1.2 g, 9.7 mmol). After stirring at room temperature for 19 hours, the reaction mixture was washed with 2N HCI(aq) and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using ethyl acetate / hexane (EA / Hex) (1 / 10), yielding compound 3b (8.7 g, 12.4 mmol) as yellow oil.1H NMR (600 MHz, CDCl3) δ ppm 0.87 (td, J=7.15, 3.81 Hz, 12H), 1.15-1.34 (m, 43H), 1.34-1.47 (m, 4H), 1.47-1.57 (m, 4H), 2.29 (tt, J=8.64, 5.42 Hz, 2H), 4.19-4.29 (m, 4H), 5.14 (s, 2H), 7.29-7.37 (m, 5H).

[0155] Compound 4b: 3-((2-Hexyldecanoyl)oxy)-2-(((2-hexyldecanoyl)oxy)methyl)-2-methylpropanoic acid

[0156] A mixture of compound 3b (8.7 g, 12.4 mmol) and Pd / C (cat.) in ethyl acetate (EA) was stirred at room temperature under a hydrogen (H2) atmosphere overnight. After filtration and evaporation, the desired product, compound 4b (7.1 g, 12.4 mmol, 100%), was obtained as a white solid without further purification. MS (M-l): 609.

[0157] Compound 5b: 2-(((6-Bromohexyl)oxy)carbonyl)-2-methylpropane-l,3-diyl bis(2-hexyldeca noate)

[0158] A mixture of compound 4b (7.1 g, 12.4 mmol) and 6-bromo-l-hexanol (1.5 g, 8.3 mmol) in dichloromethane (DCM) was stirred at 0°C, then treated with EDCI (2.4 g, 12.4 mmol) and DMAP (0.3 g, 2.5 mmol). After stirring at room temperature for 19 hours, the reaction mixture was washed with 2N HCI(aq) and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using ethyl acetate / hexane (EA / Hex) (1 / 10), yielding compound 5b (1.8 g, 2.3 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 0.87 (td, J =Attorney Docket No. 4404-0140PWO1 7.03, 1.67 Hz, 12H), 1.24 (m, 41H), 1.34-1.51 (m, 10H), 1.51-1.62 (m, 4H), 1.62-1.68 (m, 3H), 1.83-1.90 (m, 1H), 2.27-2.35 (m, 2H), 3.40 (t, J = 6.68 Hz, 2H), 4.10 (t, J = 6.68 Hz, 2H), 4.18-4.25 (m, 4H).

[0159] Compound MPA02: 3-((6-((6-(Decanoyloxy)hexyl)(4-hydroxybutyl)amino)hexyl)oxy)-2-(((2-hexylnonanoyl)oxy)methyl)-2-methyl-3-oxopropyl 2-hexyl decanoateMPA02

[0160] A mixture of compound 5b (0.46 g, 0.6 mmol) and 6b (0.4 g, 1.2 mmol) in ACN / CHCl3was treated with DIPEA (0.4 mL, 2.5 mmol) and heated to 90°C for 17 hours. The solvent was removed, and the residue was extracted with ethyl acetate and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using dichloromethane / methanol (DCM / MeOH) (20 / 1) to yield the compound MPA02 (0.1 g, 0.09 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 0.87 (td, J=7.03, 2.15 Hz, 15H), 1.24-1.29 (m, 56H), 1.33-1.49 (m, 11H), 1.52-1.72 (m, 19 H), 2.27-2.34 (m, 5H), 2.40-2.96 (m, 4H), 4.02-4.11 (m, 4H), 4.15-4.26 (m, 4H). MS (M+l): 1037

[0161] 1.3 Synthesis of compounds MPA03 and MPA04 via Scheme III

[0162] Compound 3c: 2-((Benzyloxy)carbonyl)-2-methylpropane-l,3-diyl ditetradecanoate

[0163] A mixture of compound 2a (3.57 g, 15.9 mmol) and myristic acid (13 g, 56.9 mmol) in dichloromethane (DCM) was stirred at 0°C, then l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) (10.9 g, 56.9 mmol) and 4-dimethylaminopyridine (DMAP) (1.39 g, 11.4 mmol) were added. After stirring at room temperature for 19 hours, the reaction mixture was washed with brine and dried over MgSO4. After evaporation, the residue was purified by silicaAttorney Docket No. 4404-0140PWO1 gel column chromatography using ethyl acetate / hexane (EA / Hex) (1 / 10) to yield the compound 3c (7.7 g, 11.9 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ ppm 0.88 (t, J = 7.15 Hz, 6H), 1.19-1.32 (m, 43H), 1.55 (m, 4H), 2.22 (t, J = 7.63 Hz, 4H), 4.19-4.28 (m, 4H), 5.16 (s, 2H), 7.28-7.37 (m, 5H).

[0164] Compound 4c: 2-Methyl-3-(tetradecanoyloxy)-2-((tetradecanoyloxy)methyl)propanoic acid4c

[0165] A mixture of compound 3c (8.7 g, 12.4 mmol) and Pd / C (cat.) in ethyl acetate (EA) was stirred at room temperature under a hydrogen (H2) atmosphere overnight. After filtration and evaporation, the desired product compound 4c (7.1 g, 12.4 mmol, 100%) was obtained as a white solid without further purification.1H NMR (600 MHz, CDCl3) δ ppm 0.87 (t, J = 7.15 Hz, 6H), 1.18-1.34 (m, 43H), 1.54-1.63 (m, 4H), 2.30 (t, J = 7.63 Hz, 4H), 4.19-4.28 (m, 4H). MS (M-1): 554

[0166] Compound 5c: 2-(((6-Bromohexyl)oxy)carbonyl)-2-methylpropane-l,3-diyl ditetradecanoate

[0167] A mixture of compound 4c (6.5 g, 11.7 mmol) and 6-bromo-l-hexanol (1.4 g, 7.8 mmol) in dichloromethane (DCM) was stirred at 0°C, followed by the addition of EDCI (2.25 g, 11.7 mmol) and DMAP (0.3 g, 2.3 mmol). After stirring at room temperature for 19 hours, the reaction mixture was washed with brine and dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using ethyl acetate / hexane (EA / Hex) (1 / 10) to yield the compound 5c (1.5 g, 2.0 mmol) as yellow oil.1H NMR (600 MHz, CDCl3) δ 0.88 (t,Attorney Docket No. 4404-0140PWO1 J=7.15 Hz, 6H), 1.19-1.33 (m, 43 H), 1.33-1.41 (m, 2H), 1.43-1.51 (m, 2H), 1.5 -1.68 (m, 6H), 1.74-1.88 (m, 2H), 2.29 (t, J = 7.39 Hz, 4H) 3.39-3.54 (m, 2H), 4.11 (t, J = 6.68 Hz, 2H) 4.18-4.26 (m, 4H).

[0168] Compound MPA03: 6-((6-((3-((2-Hexyldecanoyl)oxy)-2-(((2-hexylnonanoyl)oxy)methyl)-2-methylpropanoyl)oxy)hexyl)(4-hydroxybutyl)amino) hexyl 2-hexyldecanoateMPA03

[0169] A mixture of compound 5b (0.45 g, 0.6 mmol) and 6a (0.5 g, 1.2 mmol) in ACN / CHCl3was treated with DIPEA (0.4 mL, 2.5 mmol) and heated to 90°C for 17 hours. The solvent was removed, and the residue was extracted with ethyl acetate and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using dichloromethane / methanol (DCM / MeOH) (20 / 1) to yield the compound MPA03 (0.1 g, 0.09 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 0.87 (td, J = 6.91, 1.91 Hz, 18H), 1.15-1.31 (m, 58H), 1.34-1.49 (m, 14H,) 1.49-1.73 (m, 20H), 1.83-1.90 (m, 4H), 2.25-2.35 (m, 3H), 2.94-3.14 (m, 5H), 4.04-4.09 (m, 4H), 4.16-4.25 (m, 4H). MS (M+l): 1121.

[0170] Compound MPA04: 2-(((6-((6-((2-Hexyldecanoyl)oxy)hexyl)(4-hydroxybutyl)amino)hexyl)oxy)carbonyl)-2-methylpropane-l,3-diyl ditetradecanoateMPA04

[0171] A mixture of compound 5c (0.4 g, 0.6 mmol) and 6a (0.5 g, 1.2 mmol) in ACN / CHCl3was treated with DIPEA (0.24 mL, 1.4 mmol) and heated to 90°C for 17 hours. The solvent was removed, and the residue was extracted with ethyl acetate and brine, then dried over MgSO4.Attorney Docket No. 4404-0140PWO1 After evaporation, the residue was purified by silica gel column chromatography using dichloromethane / methanol (DCM / MeOH) (20 / 1) to yield the compound MPA04 (0.1 g, 0.09 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 0.87 (td, J = 7.03, 2.15 Hz, 12H), 1.19-1.33 (m, 61H), 1.34-1.48 (m, 10H), 1.54-1.74 (m, 22H), 2.24-2.34 (m, 5H), 2.91-2.97 (m, 3H), 3.43-3.52 (m, 1H) 3.68 (brs., 1H), 3.83-3.93 (m, 1H), 4.02-4.14 (m, 4H), 4.15-4.26 (m, 4H). MS (M+l): 1065

[0172] 1.4 Synthesis of compound MPA05 via Scheme IV

[0173] Compound 2b: Benzyl 2,2-bis(hydroxymethyl)butanoate

[0174] A mixture of 2,2-bis(hydroxymethyl)butanoic acid (5.0 g, 33.8 mmol) and K2CO3(6.0 g, 43.9 mmol) in DMF was prepared, followed by the addition of BnBr (7.5 g, 43.9 mmol). After stirring at room temperature for 19 hours, the reaction mixture was extracted with ethyl acetate and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using EA / Hex (1 / 1) to yield the compound 2b (8.0 g, 29.4 mmol) as a colorless oil. MS (M+l): 239.

[0175] Compound 3d: 2-((Benzyloxy)carbonyl)-2-ethylpropane-l,3-diyl bis(decanoate)3d

[0176] A mixture of compound 2b (4 g, 16.8 mmol) and capric acid (11.0 g, 63.8 mmol) in dichloromethane (DCM) was stirred at 0°C, followed by the addition of EDCI (12.2 g, 63.8 mmol) and DMAP (1.4 g, 11.8 mmol). After stirring at room temperature for 19 hours, the reaction mixture was washed with 2N HCI(aq) and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using ethyl acetate / hexane (EA / Hex) (1 / 10) to yield the compound 3d (6.4 g, 11.7 mmol) as a yellow oil.XH NMR (600 MHz, CDCh) 6Attorney Docket No. 4404-0140PWO1 0.77-0.84 (m, 6H), 0.88-1.02 (m, 3H), 1.20 (br. s„ 24H), 1.42-1.53 (m, 4H), 1.53-1.61 (m, 2H), 2.09- 2.21 (m, 4H), 4.16-4.28 (m, 4H), 5.04-5.15 (m, 2H), 7.16-7.34 (m, 5H).

[0177] Compound 4d: 2,2-Bis((decanoyloxy)methyl)butanoic acid4d

[0178] A mixture of compound 3d (12.4 g, 22.7 mmol) and Pd / C (cat.) in EA was stirred at room temperature under a H2atmosphere overnight. After filtration and evaporation, the desired product compound 4d (8.2 g, 18 mmol) as a yellow oil was obtained without further purification.1H NMR (600 MHz, CDCl3) δ 0.76-0.83 (m, 6H), 0.86 (t, J = 7.39 Hz, 3H), 1.12-1.27 (m, 24H), 1.47-1.58 (m, 4H), 1.58-1.69 (m, 2H), 2.20-2.31 (m, 4H), 4.15-4.28 (m, 4H).

[0179] Compound 5d: 2-(((6-Bromohexyl)oxy)carbonyl)-2-ethylpropane-l,3-diyl bis(decanoate)Br

[0180] A mixture of compound 4d (4 g, 8.8 mmol) and 6-bromo-l-hexanol (1.06 g, 5.9 mmol) in dichloromethane (DCM) was stirred at 0°C, followed by the addition of EDCI (1.68 g, 8.8 mmol) and DMAP (0.21 g, 2.7 mmol). After stirring at room temperature for 19 hours, the reaction solution was washed with 2N HCI(aq) and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography with ethyl acetate / hexane (EA / Hex) (1 / 10) to yield the compound 5d (0.9 g, 1.39 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 0.82-0.92 (m, 9 H), 1.20-1.33 (m, 22H), 1.35-1.42 (m, 3H), 1.42-1.50 (m, 3H), 1.54-1.67 (m, 8H), 1.82-1.90 (m, 2H), 2.23-2.31 (m, 4H), 3.35-3.43 (m, 4H), 4.06 (t, J = 6.68 Hz, 2H), 4.21-4.30 (m, 2H).

[0181] Compound MPA05: 2-Ethyl-2-(((6-((6-((2-hexyldecanoyl)oxy)hexyl)(4-hydroxybutyl)amino)hexyl)oxy)carbonyl)propane-l,3-diyl bis(deca noate)Attorney Docket No. 4404-0140PWO1

[0182] A mixture containing compound 5d (0.9 g, 1.39 mmol) and compound 6a (0.5 g, 1.20 mmol) dissolved in ACN was prepared. Subsequently, potassium carbonate (0.5 g, 3.6 mmol) and potassium iodide (0.07 g, 0.4 mmol) were added to the reaction mixture. The mixture was then heated to reflux for 13 hours. The solvent was removed, and the residue was extracted with ethyl acetate and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (20 / 1) to yield the compound MPA05 (0.08 g, 0.08 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 0.81-0.90 (m, 15H), 1.17-1.32 (m, 43H), 1.36-1.44 (m, 9H), 1.50-1.58 (m, 6H), 1.58-1.67 (m, 6H), 1.67-1.74 (m, 2H), 1.82 (br. s., 4H), 1.92-2.02 (m, 2H), 2.22-2.32 (m, 5H), 3.01-3.08 (m, 4H), 3.14 (t, J = 7.63 Hz, 2H), 3.71 (t, J = 5.48 Hz, 2H), 4.03 (t, J = 6.68 Hz, 2H), 4.09 (t, J = 6.44 Hz, 2H), 4.20 (d, J = 10.97 Hz, 2H), 4.25 (d, J = 11.44 Hz, 2H). MS (M+l): 967.

[0183] Scheme VIIIMPA06Attorney Docket No. 4404-0140PWO1

[0184] Compound MPA06: (((((4-Hydroxybutyl)azanediyl)bis(hexane-6,l-diyl))bis(oxy))bis(carbonyl))bis(2-methylpropane-2,l,3-triyl) tetrakis(deca noate)O O OMPA06

[0185] 1.5 Synthesis of compound MPA06 via Scheme VIII

[0186] A mixture containing compound 6h (0.4 g, 0.70 mmol) and compound 5a (0.5 g, 0.83 mmol) dissolved in ACN was prepared. Subsequently, potassium carbonate (0.3 g, 2.1 mmol) and potassium iodide (0.06 g, 0.35 mmol) were added to the reaction mixture. The mixture was then heated to reflux for 13 hours. The solvent was removed, and the residue was extracted with ethyl acetate and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (20 / 1) to yield the compound MPA06 (0.13 g, 0.11 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 0.75-0.88 (m, 12H), 1.11-1.30 (m, 54H), 1.30-1.37 (m, 6H), 1.55 (br. s„ 12H), 1.57-1.63 (m, 6H), 1.65 (br. s., 4H), 2.19-2.28 (m, 8H), 2.51 (br. s., 6H), 3.50-3.57 (m, 2H), 4.06 (br. s., 4H), 4.11-4.25 (m, 8H). MS (M+l): 1139.

[0187] 1.6 Synthesis of compound MPA12 via Scheme V

[0188] Compound MPA12: 2-(((6-((6-((2-Hexyldecanoyl)oxy)hexyl)(4-hydroxybutyl)amino)hexyl)oxy)carbonyl)-2-methylpropane-l,3-diyl distearateMPA12Attorney Docket No. 4404-0140PWO1

[0189] A mixture of compound 5e (0.62 g, 0.74 mmol) and compound 6a (0.5 g, 1.2 mmol) in ACN / CHCl₃ was prepared. DIPEA (0.25 mL, 1.7 mmol) was then added, and the mixture was heated to 90°C for 17 hours. The solvent was removed, and the residue was extracted with ethyl acetate and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (20 / 1) to yield the compound MPA12 (0.25 g, 0.21 mmol) as a yellow oil.1H NMR (600 MHz, CDCl₃) δ 0.87 (td, J = 6.91, 1.91 Hz, 12H), 1.12-1.32 (m, 89H), 1.32-1.77 (m, 18H), 2.22-2.34 (m, 5H), 2.66 (brs., 6H), 3.62 (brs., 2H), 4.01-4.14 (m, 4 H), 4.14-4.26 (m, 4 H). MS (M+l): 1178

[0190] 1.7 Synthesis of compound MPA16 via Scheme VI

[0191] Compound MPA16: 2-(((8-((8-((2-Hexyldecanoyl)oxy)octyl)(4-hydroxybutyl)amino)octyl)oxy)carbonyl)-2-methylpropane-l,3-diyl bis(decanoate)MPA16

[0192] A mixture of compound 5a (0.45 g, 0.7 mmol) and compound 6k (0.5 g, 1.1 mmol) in ACN / CHCl₃ was prepared. DIPEA (0.25 mL, 1.7 mmol) was then added, and the mixture was heated to 90°C for 17 hours. The solvent was removed, and the residue was extracted with ethyl acetate and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (20 / 1) to yield the compound MPA16 (0.45 g, 0.44 mmol) as a yellow oil.2H NMR (600 MHz, CDCI3) 6 0.87 (t, J = 6.91 Hz, 12H), 1.15-1.41 (m, 65H), 1.59-1.81 (m, 16H), 1.87 (brs., 2 H), 2.22-2.33 (m, 5H), 2.85- 2.91 (brs., 6H), 3.68 (t, J = 5.25 Hz, 2H), 4.02-4.13 (m, 4H), 4.15-4.26 (m, 4H). MS (M+l): 1009

[0193] 1.8 Synthesis of compound MPA17 via Scheme VII

[0194] Compound MPA17: 2-((6-((6-(2-Hexyldecanamido)hexyl)(4-hydroxybutyl)amino)hexyl)carbamoyl)-2-methylpropane-l,3-diyl bis(decanoate)Attorney Docket No. 4404-0140PWO1MPA17

[0195] A mixture of compound 8 (0.46 g, 0.76 mmol) and compound 9 (0.5 g, 1.2 mmol) in ACN / CHCl₃ was prepared. DIPEA (0.3 mL, 1.7 mmol) was then added, and the mixture was heated to 90°C for 17 hours. The solvent was removed, and the residue was extracted with ethyl acetate and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (20 / 1) to yield the compound MPA17 (0.29 g, 0.3 mmol) as a yellow oil.1H NMR (600 MHz, CDCl₃) δ 0.80-0.91 (m, 12H), 1.14-1.43 (m, 58H), 1.43-1.73 (m, 18H), 1.95 (dt, J=9.30, 4.41 Hz, 1H), 2.30 (t, J = 7.39 Hz, 4H), 2.53 (brs., 6H), 3.16-3.28 (m, 4H), 3.57 (d, J = 4.29 Hz, 2H), 4.14-4.27 (m, 4H), 5.68 (br. s., 1H), 6.14-6.23 (m, 1H). MS (M+l): 951

[0196] Scheme IX

[0197] Compound MPA23: 2-((6-((6-((2-Hexyldecanoyl)oxy)hexyl)(4-hydroxybutyl)amino)hexyl)carbamoyl)-2-methylpropane-l,3-diyl bis(decanoate)Attorney Docket No. 4404-0140PWO1

[0198] 1.9 Synthesis of compound MPA23 via Scheme IX

[0199] A mixture of compound 8 (0.64 g, 0.98 mmol) and compound 6a (0.7 g, 1.64 mmol) in ACN / CHCl3was treated with DIPEA (0.8 mL, 4.7 mmol) and heated to 90°C for 17 hours. The solvent was removed, and the mixture was extracted with ethyl acetate and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography using dichloromethane / methanol (DCM / MeOH) (20 / 1), yielding compound MPA23 (0.38 g, 0.4 mmol) as a yellow oil.XH NMR (600 MHz, CDCI3) 56.18 (s, 1H), 4.21 (q, J = 9.1 Hz, 4H), 4.05 (t, J = 6.7 Hz, 2H), 3.58 (s, 2H), 3.24 (q, J = 6.6 Hz, 2H), 2.53 (s, 5H), 2.30 (t, J = 7.5 Hz, 5H), 1.60 (m, 19H), 1.33 (m, 58H), 0.87 (m, 12H). MS (M+l): 952.

[0200] Scheme XBr5dMPA24

[0201] Compound MPA24: (((((4-Hydroxybutyl)azanediyl)bis(hexane-6,l-diyl))bis(oxy))bis(carbonyl))bis(2-ethylpropane-2,l,3-triyl) tetra kis(deca noate)Attorney Docket No. 4404-0140PWO1MPA-24

[0202] 1.10 Synthesis of compound MPA24 via Scheme X

[0203] A mixture containing compound 6i (0.43 g, 0.70 mmol) and compound 5d (0.5 g, 0.83 mmol) dissolved in ACN was prepared. Subsequently, potassium carbonate (0.3 g, 2.1 mmol) and potassium iodide (0.06 g, 0.35 mmol) were added to the reaction mixture. The mixture was then heated to reflux for 13 hours. The solvent was removed, and the residue was extracted with ethyl acetate and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (20 / 1) to yield the compound MPA24 (0.35 g, 0.3 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 4.22 (q, J = 14.1 Hz, 4H), 4.08 (t, J = 6.6 Hz, 4H), 4.02 (t, J = 6.6 Hz, 4H), 3.70 (t, J = 5.6 Hz, 1H), 3.15 (t, J = 4.8 Hz, 2H), 3.06 (m, J = 5.2 Hz, 4H), 2.25 (t, J = 7.6 Hz, 8H), 1.97 (m, J = 7.3 Hz, 3H), 1.83 (s, 4H), 1.69 (m, J = 6.2 Hz, 3H), 1.61 (m, J = 6.3 Hz, 8H), 1.55 (m, J = 7.3 Hz, 6H), 1.39 (s, 6H), 1.24 (q, J = 7.0 Hz, 52H), 0.84 (q, J = 6.8 Hz, 18H). MS (M+l): 1167.

[0204] Scheme XIHN KI, K2CO3, ACN, refluxMPA25Attorney Docket No. 4404-0140PWO1 Compound MPA25: 2-(((6-((6-(2-Hexyldecanamido)hexyl)(4-hydroxybutyl)amino)hexyl)oxy)carbonyl)-2-methylpropane-l,3-diyl bis(deca noate)MPA25

[0205] 1.11 Synthesis of compound MPA25 via Scheme XI

[0206] A mixture of compound 5a (0.46 g, 0.76 mmol) and compound 9 (0.5 g, 1.2 mmol) in ACN / CHCl₃ was prepared. DIPEA (0.3 mL, 1.7 mmol) was then added, and the mixture was heated to 90 °C for 17 hours. The solvent was removed, and the residue was extracted with ethyl acetate and brine, then dried over MgSO4. After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (20 / 1) to yield the compound MPA25 (0.29 g, 0.3 mmol) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 5.68 (t, J = 5.4 Hz, 1H), 4.21 (q, J = 11.7 Hz, 4H), 4.09 (t, J = 6.7 Hz, 2H), 3.58 (s, 2H), 3.24 (q, J = 6.7 Hz, 2H), 2.55 (brs, 5H), 2.28 (t, J = 7.6 Hz, 4H), 1.95 (septet, J = 4.8 Hz, 1H), 1.70-1.44 (m, 19H), 1.40-1.23 (m, 58H), 1.35 (m, 12H). MS (M+l): 951.

[0207] Example 2: The LNPs Encapsulate and Help Delivering Nucleic Acids

[0208] In this example, LNPs comprised of commercially available ionizable lipids were formulated with a total lipid concentration of 25.92 mM. SM-102-LNPs were formulated with SM-102 / DSPC / Cholesterol / DMG-PEG2000 in the molar ratio of 50 / 10 / 38.5 / 1.5. ALC-0315- and MPA-LNPs were formulated in the molar ratio of 46.3 / 9.4 / 42.7 / 1.6, respectively. Each lipid was dissolved in ethanol and mixed according to the specified molar ratios in the organic phase. The Fluc-mRNA was dissolved in 25 mM sodium acetate buffer (pH 4), with the NP lipid:mRNA ratio at a constant value of 6.5, prior to mixing in the Ignite NanoAssmblr (Precision NanoSystems). In the Ignite system, a 600-pL aliquot of the organic phase and an 1800-pL aliquot of the aqueous phase were mixed and then dialyzed and concentrated with Amicon® ultra-15 (MWCO 30 kDa).

[0209] Measurement of LNP size and polydispersity index (PDI)Attorney Docket No. 4404-0140PWO1

[0210] LNPs were diluted 100-fold in PBS (pH 7.4) and transferred into a 384-well microplate for size and polydispersity index (PDI) measurements by Wyatt DynaPro Plate Reader III (Malvern Instruments, UK). Results are summarized in Table 1.

[0211] Table 1. Size and PDI of LNPsNo. Name Size(nm) PDI1 MPA01-LNP 83.56 0.069872 MPA06-LNP 67.64 0.10913 MPA07-LNP 77.96 0.10444 MPA09-LNP 82.07 0.084715 MPA13-LNP 86.83 0.11516 MPA14-LNP 94.82 0.16447 MPA17-LNP 109 0.1648 MPA23-LNP 116 0.082529 MPA25- LNP 94.2 0.134610 ALC-0315-LNP 74.78 0.100311 SM-102-LNP 87.83 0.1547

[0212] Measurement of LNP encapsulation efficiency and concentration

[0213] LNPs were diluted in TE (20-fold) or 1% Triton X-100 / TE (40-fold) for free and total RNA quantification, respectively, and analyzed by RiboGreen assay using a SpectraMax iD3. Results are summarized in Table 2.

[0214] Table 2. Encapsulation Efficiency and Concentration of LNPsNo. Name EE(%) Con.(ug / uL)1 MPA01-LNP 95.22 0.42 MPA06-LNP 94.22 0.393 MPA07-LNP 92.03 0.434 MPA09-LNP 94.34 0.315 MPA13-LNP 91.47 0.416 MPA 14- LNP 87.61 0.277 MPA17-LNP 80.45 0.308 MPA23-LNP 92.85 0.409 MPA25- LNP 85.56 0.4010 ALC-0315-LNP 94.64 0.4211 SM-102-LNP 95.58 0.40

[0215] Fluc-mRNA LNPs Delivery in HEK293T CellsAttorney Docket No. 4404-0140PWO1

[0216] HEK293T Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were seeded into white 96-well plates at a density of approximately 1 x 104cells per well and allowed to adhere overnight. Firefly luciferase mRNA-loaded LNPs were added to the cells at a dose corresponding to approximately 200 ng mRNA per well. The cells were incubated at 37 °C under 5% CO2for 48 hours. Thereafter, luciferase expression was measured using a luminescence assay reagent (for example, One-Gio or an equivalent reagent). Luminescence was detected using a microplate luminometer, and the results were expressed as relative light units (RLU / mg protein) to evaluate the functional delivery efficiency of selected ionizable lipids in Table 3 and Fig.l.

[0217] Table 3. Delivery efficiency of LNPsNo. Name RLU / mg proteins1 MPA01-LNP 1630792602 MPA06-LNP 1769985013 MPA07-LNP 3246617534 MPA09-LNP 1736412915 MPA13-LNP 3374148186 MPA14-LNP 1507217347 MPA17-LNP 495240088 MPA23-LNP 1544923749 MPA25- LNP 11640787810 ALC-0315-LNP 3712432311 SM-102-LNP 140493984

[0218] The experimental results in Table 3 demonstrate that the lipid nanoparticles comprising the cationic lipids of the present invention (e.g., MPA07 and MPA13) exhibit unexpectedly superior transfection efficiency compared to conventional lipid formulations based on ALC-0315 or SM-102. Accordingly, the present invention provides a delivery vehicle suitable for various therapeutic applications. The enhanced potency of the present lipids renders them particularly suitable for use in compositions for preventing or treating infectious diseases, cancers, and genetic disorders.

[0219] Example 3: The LNPs Encapsulate and Help Delivering Nucleic AcidsAttorney Docket No. 4404-0140PWO1

[0220] In this example, the ability for the LNPs for encapsulating nucleic acids was investigated. To this purpose, the compound of Example 1 (i.e., compounds MPA01) was mixed with mRNAs (i.e., SARS-CoV-2 full length spike protein mRNA) by following the preparation procedures described in " Materials and Methods" section, thereby producing lipid nanoparticles (LNPs) containing mRNA therein. The size, polydispersity index (PDI), and encapsulation efficiency (EE) of each LNPs were analyzed in accordance with procedures described in " Materials and Methods" section. Results are summarized in Table 4.

[0221] Table 4. Characterization of the LNPs analyzed by Ignite NanoAssmblrFormulationParameters ALC-0315-LNP MPA01-LNP SM-102-LNPSize (nm) 72 70 86PDI 0.17 0.09 0.08Expression level (%) 88.3 ± 0.14 95.5 ± 0.01 91.6 ± 2.3EE (%) 96.2 98.0 98.0

[0222] The data depicted in Table 4 confirm that the cationic lipid MPA01 can form nanoparticles when combined with helper lipids. The physical characteristics of MPA01-LNP, including size and polydispersity index (PDI), are similar to those of LNPs composed of commercially available cationic lipids (e.g., Ignite). MPA01-LNP effectively delivered mRNA encoding the SARS-CoV-2 spike protein. In terms of mRNA delivery efficiency, MPA01-LNP outperformed other commercial LNP formulations (e.g., SM-102, ALC-0315). These results suggest that MPA01 is a promising cationic lipid for use in LNPs for gene and drug delivery.

[0223] It will be understood that the above description of embodiments is given by way of example only and that various modifications may be made by those with ordinary skill in the art. The above specification, examples, and data provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those with ordinary skill in the art could make numerous alterations to the disclosed embodiments without departing from the spirit or scope of this invention.

Claims

Attorney Docket No. 4404-0140PWO1 WHAT IS CLAIMED IS:

1. A compound of formula (I),wherein,X and W are independently O or -NH;Y is O, S, or -NH;m and o are independently an integer between 1 and 5;n is an integer between 1 and 7;Ri is C1-C20 linear or branched alkyl or linear or branched alkenyl;R2 is C1-C20 linear or branched alkyl, linear or branched alkenyl, or -C(Ra)((CH2)-Z- C(=O)Rb)2, whereinRais C1-C6 alkyl,Rbis C1-C20 linear or branched alkyl, or linear or branched alkenyl, and Z is O or -NH; andR3 is C1-C6 alkyl.

2. The compound of claim 1, wherein X, Y, and W are independently O; Ri is nonyl; RR2is 7-pentadecyl; and R3is methyl.

3. The compound of claim 2, wherein:(a) m is 3, n is 5, and o is 3;(b) m is 1, n is 5, and o is 3;(c) m is 5, n is 5, and o is 3;(d) m is 3, n is 3, and o is 3;(e) m is 2, n is 1, and o is 3;(f) m is 3, n is 1, and o is 3;Attorney Docket No. 4404-0140PWO1 (g) m is 2, n is 5, and o is 3;(h) m is 4, n is 5, and o is 3;(i) m is 3, n is 7, and o is 3; or(j) m is 3, n is 7, and o is 5.

4. The compound of claim 1, wherein X, Y, and W are independently O, Ri is nonyl; R2 is 7-pentadecyl; and R3 is ethyl.

5. The compound of claim 4, wherein m is 3, n is 5, and o is 3.

6. The compound of claim 1, wherein X, Y, and W are independently O; m is 3; n is 5; o is 3; Ri is tridecyl or heptadecyl; RR2is 7-pentadecyl; and R3is methyl.

7. The compound of claim 1, wherein X, Y, and W are independently O; m is 3; n is 5; o is 3; Ri is 7-pentadecyl; R2 is 7-pentadecyl or nonyl; and R3 is methyl.

8. The compound of claim 1, wherein X, Y, and W are independently O; m is 3; n is 5; o is 3; Ri is nonyl; R2 is -C(Ra)((CH2)-Z-C(=O)Rb)2, wherein Rais methyl, Rbis nonyl, and Z is O.

9. The compound of claim 1, wherein X and Y are independently -NH; W is O; m is 3; n is 5; o is 3; Ri is nonyl; RR2is 7-pentadecyl; and R3is methyl.

10. The compound of claim 1, wherein X is O; Y is S; W is O or -NH; m is 3; n is 5; o is 3; Ri is nonyl; RR2is 7-pentadecyl; and R3is methyl.

11. The compound of claim 1, wherein X and Y are independently O; W is -NH; m is 3; n is 5; o is 3; Ri is nonyl; RR2is 7-pentadecyl; and R3is methyl.

12. The compound of claim 1, wherein X and W are independently -NH; Y is O; m is 3; n is 5; o is 3; Ri is nonyl; RR2is 7-pentadecyl; and R3is methyl.

13. The compound of claim 1, wherein X, Y, and W are independently -NH; m is 3; n is 5; o is 3; Ri is nonyl; RR2is 7-pentadecyl; and R3is methyl.

14. The compound of claim 1, wherein the compound is selected from the group consisting of:Attorney Docket No. 4404-0140PWO1(MPA03),(MPA05),(MPA06)Attorney Docket No. 4404-0140PWO1(MPA09),(MPA12),Attorney Docket No. 4404-0140PWO1(MPA15),(MPA16),(MPA17),Attorney Docket No. 4404-0140PWO1(MPA24), and(MPA25).

15. A lipid nanoparticle (LNP) having one or more hydrophilic cores surrounded by a lipid bilayer, wherein the lipid bilayer comprises the compound of claim 1.

16. The LNP of claim 15, further comprising a therapeutic agent accommodated in the hydrophilic core.

17. The LNP of claim 16, wherein the therapeutic agent is a nucleic acid, a peptide, a polypeptide, a protein, a carbohydrate, a proteoglycan, a glycoprotein, or a combination thereof.

18. The LNP of claim 17, wherein the nucleic acid is a mitochondrial DNA (mtDNA), a chloroplast DNA (cpDNA), a plasmid, a messenger RNA (mRNA), a small interfering RNA (siRNA), a ribosomal RNA (rRNA), a transfer RNA (tRNA), a microRNA (miRNA), or an aptamer.

19. The LNP of claim 17, wherein the protein is a viral protein.

20. The LNP of claim 18, wherein the nucleic acid is an mRNA encoding the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus.

21. The LNP of claim 18, wherein the nucleic acid is an mRNA encoding an envelope protein of Dengue virus.

22. The LNP of claim 16, further comprising a cationic lipid, a steroid, a phospholipid, a PEGylated lipid, a PEGylated phospholipid, and a combination thereof.

23. The LNP of claim 22, wherein the lipid is present in the LNP at a concentration of about 40 to 50 mol%.Attorney Docket No. 4404-0140PWO1 24. The LNP of claim 23, wherein the molar ratio of the lipid to the therapeutic agent is about 2:1 to 8:1.

25. A micelle having one hydrophobic core surrounded by an assembly of the compound of claim 1.

26. The micelle of claim 25, further comprising a therapeutic agent accommodated in the hydrophobic core.

27. The micelle of claim 26, wherein the therapeutic agent is a poorly soluble therapeutic agent.

28. The micelle of claim 26, further comprising a cationic lipid, a steroid, a phospholipid, a PEGylated lipid, a PEGylated phospholipid, and a combination thereof.

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