Lipid compounds containing at least one terminal radical of the formula -NH-CX-A or -NH-CX-NH-A, compositions containing them and their uses

Abstract

The present disclosure relates to novel lipid compounds, lipid nanoparticles (LNPs) containing the same, and the use of the lipid compounds or LNPs for the delivery of nucleic acids. The lipid compounds disclosed herein are of the formula (I): *-NH-CX-(NH)nA (I), where - *- represents the radical of formula (I) fused to one C 10 ~C 55 - n is 0 or 1; - X is an oxygen or sulfur atom, and A represents an optionally substituted 5- or 6-membered unsaturated heterocyclic radical or a 5- or 6-membered aromatic heterocyclic radical, both containing at least one nitrogen atom), or one of the pharmaceutically acceptable salts of said radicals of formula (I); said compounds in all possible racemic, enantiomeric and diastereomeric isomeric forms.

Description

[Technical Field]

[0001] The present disclosure is in the field of novel lipid compounds that can be used in combination with other lipid components, such as neutral lipids, steroid alcohols or their esters, and polymer-conjugated lipids, to form lipid nanoparticles for delivery of therapeutic agents, such as nucleic acids. For example, formulations prepared using the lipid compounds disclosed herein can induce an immune response after administration of an antigen-encoding polynucleotide. [Background technology]

[0002] The field of polynucleotide therapeutics has made remarkable progress in recent years. Polynucleotides include various nucleic acid-based compounds, such as messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, or immunostimulatory nucleic acids. Some nucleic acids, such as mRNA, plasmids, and ssDNA, can be used to induce the expression of specific cellular products, useful, for example, for treating diseases associated with protein or enzyme deficiencies or for expressing vaccine antigens to induce specific immune responses. The therapeutic applications of translatable nucleotide delivery are extremely broad, as constructs can be synthesized to produce any selected protein sequence, whether native to the system or not. The expression products of nucleic acids can enhance existing levels of proteins, replace missing or nonfunctional versions of proteins, introduce new proteins and associated functions into cells or organisms, or expose cells to foreign proteins in order to induce specific immune responses.

[0003] However, there are many challenges associated with delivering polynucleotides to affect desired responses in biological systems, and effective delivery of polynucleotides to intracellular sites of action remains a major problem. To be efficiently delivered to their sites of action, polynucleotides must (i) be protected from enzymatic and non-enzymatic degradation, (ii) be appropriately distributed to the desired biological compartment, (iii) be effectively and efficiently internalized by the targeted cells, and then (iv) be delivered to the intracellular compartment where the relevant translation machinery resides.

[0004] Lipid nanoparticles formed from cationic lipids formulated with neutral lipids, cholesterol, and other lipid components such as PEGylated lipids have been used to protect polynucleotides from degradation and promote their cellular uptake.

[0005] Although lipid nanoparticle-based vehicles containing cationic lipid components have shown promising results in terms of encapsulation, stability, and site localization, there remains a great need for improvements in lipid nanoparticle-based delivery systems.

[0006] There remains a need for improved cationic and ionic lipids that exhibit improved pharmacokinetic properties and can deliver various types of polynucleotides to a wide variety of cell types and tissues with increased efficiency. Importantly, there also remains a need for novel cationic and ionic lipids that exhibit reduced toxicity and can efficiently deliver encapsulated polynucleotides to targeted cells, tissues, and organs. Improved cationic lipids and lipid nanoparticles for polynucleotide delivery also provide optimal polynucleotide / lipid ratios, protect polynucleotides from serum degradation and clearance, are suitable for systemic or local delivery, and provide intracellular delivery of polynucleotides. Additionally, lipid-polynucleotide particles should be well tolerated and provide an adequate therapeutic index so that patient treatment with effective doses of polynucleotides is not associated with unacceptable toxicity and / or risks to the patient. Additionally, lipid-nucleic acid particles should be stable as liquid formulations when stored for long periods at 4-8°C in a pharmaceutically acceptable buffer.

[0007] The present disclosure provides these and related advantages. Summary of the Invention [Means for solving the problem]

[0008] Accordingly, one object of the present disclosure is to provide a compound of formula (I): *-NH-CX-(NH) n -A (I) (In the formula, - *- means that the radical of formula (I) is joined to one C 10 ~C 55 represents a single bond directly or indirectly connecting to a lipophilic or hydrophobic tail group of - n is 0 or 1; X is an oxygen or sulfur atom; - A represents an optionally substituted 5- or 6-membered unsaturated heterocyclic radical or a 5- or 6-membered aromatic heterocyclic radical, both containing at least one nitrogen atom), in all possible racemic, enantiomeric and diastereomeric isomeric forms, or one of the pharmaceutically acceptable salts of said radicals of formula (I).

[0009] For example, the lipid compound of the present disclosure is a cationic lipid. Another object of the present disclosure is to provide a lipid compound of formula (II): R1-Z-NH-CX-(NH) n -A (II) (In the formula, - X, n and A are as defined in formula (I); - R1, C 10 ~C 55 is a lipophilic or hydrophobic tail group; -Z is a spacer arm having 2 to 24, such as 2 to 18, such as 4 to 12 carbon atoms in a branched or unbranched straight-chain, saturated or unsaturated hydrocarbon chain, said chain being interrupted by one or more atoms of oxygen and / or a moiety selected from -SS-; (C=O)-O-; -O-(O=C)-; -S-; -NH-, -NH-(O=C)-; -(O=C)-NH- and -NH-(C=O)-O- and / or being terminated by an oxygen atom or a moiety selected from among -NH-(O=C)--O-(O=C)- and -(O=C)- linked to a hydrophobic tail group, - p is 0 or 1), or one of the pharmaceutically acceptable salts of said compound of formula (II); and any of the racemic, enantiomeric and diastereomeric isomeric forms thereof.

[0010] According to one embodiment, the compound of formula (II) is selected from the group consisting of the following compounds: In the following formulae, the secondary amino moiety may be represented interchangeably as -NH- or -N-.

[0011] [ka] [ka] [ka] [ka] [ka] [ka] and their pharmaceutically acceptable salts, as well as their racemic, enantiomeric and diastereomeric isomeric forms.

[0012] For example, the compound of formula (II) may be any one of compounds (III), (IV), (V), (VIII), (IX), (XII), (XVI), (XIX), or (XXII), or any one of compounds (IV), (VIII), (IX), (XII), (XVI), (XIX), or (XXII), or any one of compounds (IV), (IX), (XII), or (XVI), or any one of compounds (IV) or (XII), or for example, formula (III), (IV) or (V), or compound (IV) (also referred to as DOG-IM4), or a salt thereof, or one of their racemic, enantiomeric and diastereomeric isomeric forms.

[0013] Surprisingly, as detailed in the Examples section, the inventors have observed that the novel lipid compounds disclosed herein enable the formulation of improved compositions, such as lipid nanoparticles, for in vitro and in vivo delivery of mRNA and / or other oligonucleotides or oligonucleotides. Furthermore, the compositions so formed may be stored in a stabilized liquid form at temperatures ranging from 4 to 8°C.

[0014] As shown in the Examples section, the lipid nanoparticles of the present invention have proven highly stable at 5°C, 25°C, and 37°C in terms of pH, osmolality, particle size, mRNA encapsulation, and / or mRNA integrity. This strong stability enables a variety of applications for the lipid nanoparticles of the present invention. For example, the lipid nanoparticles of the present invention may enable pharmaceutical compositions such as vaccines to be stored at room temperature rather than at low temperatures.

[0015] The improved lipid nanoparticles are useful for the expression of proteins encoded by mRNA.The lipid nanoparticles disclosed herein can be used to control, up-regulate or down-regulate protein expression by delivering either miRNA or miRNA inhibitors to regulate the expression of endogenous proteins, or mRNA or plasmid for the expression of transgenes.In addition, the lipid nanoparticles disclosed herein can be used to induce the pharmacological effects resulting from the expression of proteins or protection against infection by delivering mRNA encoding suitable antigens or antibodies.

[0016] The lipid nanoparticles disclosed herein can also be used to induce pharmacological effects resulting from the expression of proteins such as erythropoietin, which are useful in the treatment of metabolic diseases or diseases resulting from protein deficiencies.

[0017] Another object of the present disclosure relates to a composition comprising at least one lipid compound disclosed herein and at least one lipid selected from the group consisting of a neutral lipid, a steroid alcohol or its ester, and a PEGylated lipid.

[0018] Another object of the present disclosure relates to lipid nanoparticles comprising at least one lipid compound and at least one nucleic acid as disclosed herein.

[0019] Another object of the present disclosure relates to a pharmaceutical composition comprising: (i) at least one nucleic acid and at least one lipid compound disclosed herein; or (ii) at least one nucleic acid and at least one composition disclosed herein; or (iii) at least one lipid nanoparticle disclosed herein.

[0020] The pharmaceutical composition disclosed herein can be an immunogenic composition.Therefore, another object of the present disclosure relates to an immunogenic composition, comprising: (i) at least one nucleic acid encoding an antigen and at least one lipid compound disclosed herein, or (ii) at least one nucleic acid encoding an antigen and at least one composition disclosed herein, or (iii) at least one lipid nanoparticle disclosed herein, wherein nucleic acid encodes at least one antigen.

[0021] Another object of the present disclosure relates to a composition for use as a medicament, comprising: (i) at least one nucleic acid and at least one lipid compound disclosed herein; or (ii) at least one nucleic acid and at least one composition disclosed herein; or (iii) at least one lipid nanoparticle disclosed herein.

[0022] Another object of the present disclosure relates to a composition for use in a therapeutic method for preventing and / or treating a disease selected from the group consisting of an infectious disease, an allergy, an autoimmune disease, a rare blood disease, a rare metabolic disease, a rare neurological disease, and a tumor or cancer disease, the composition comprising: (i) at least one nucleic acid and at least one lipid compound disclosed herein, or (ii) at least one nucleic acid and at least one composition disclosed herein, or (iii) at least one lipid nanoparticle disclosed herein.

[0023] The term "rare disease" is used herein in accordance with its art-recognized meaning to mean a disease with an average prevalence threshold of between 40-50 cases / 100,000 people (Richter et al., Value Health. 2015 Sep;18(6):906-14).

[0024] Another object of the present disclosure relates to a composition for use as an immunogenic composition, comprising: (i) at least one nucleic acid encoding an antigen and at least one lipid compound disclosed herein; or (ii) at least one nucleic acid encoding an antigen and at least one composition disclosed herein; or (iii) at least one lipid nanoparticle disclosed herein.

[0025] In some embodiments, the present disclosure also relates to the use of a composition for the manufacture of a medicament for preventing and / or treating an infectious disease, an allergy, an autoimmune disease, a rare blood disease, a rare metabolic disease, a rare neurological disease, and a tumor or cancer disease, the composition comprising: (i) at least one nucleic acid encoding an antigen and at least one lipid compound disclosed herein, or (ii) at least one nucleic acid encoding an antigen and at least one composition disclosed herein, or (iii) at least one lipid nanoparticle disclosed herein.

[0026] Another object of the present disclosure relates to a method for preventing and / or treating a disease in an individual in need thereof, wherein the method comprises administering to the individual an effective amount of (i) at least one nucleic acid and at least one lipid compound disclosed herein, or (ii) at least one nucleic acid and at least one composition disclosed herein, or (iii) at least one lipid nanoparticle disclosed herein.The method disclosed herein can be for preventing and / or treating infectious diseases, allergies, autoimmune diseases, rare blood diseases, rare metabolic diseases, rare neurological diseases, and tumor or cancer diseases.

[0027] Another object of the present disclosure relates to a method for transfecting at least one isolated target cell with a nucleic acid, wherein the method comprises contacting at least one target cell with an effective amount of (i) at least one nucleic acid and at least one lipid compound disclosed herein, or (ii) at least one nucleic acid and at least one composition disclosed herein, or (iii) at least one lipid nanoparticle disclosed herein, thereby transfecting the at least one target cell with the nucleic acid.

[0028] Another object of the present disclosure relates to a method for producing a polypeptide in at least one target cell, the method comprising contacting at least one target cell with an effective amount of (i) at least one nucleic acid encoding said polypeptide and at least one compound disclosed herein, or (ii) at least one nucleic acid encoding said polypeptide and at least one composition disclosed herein, or (iii) at least one lipid nanoparticle disclosed herein, wherein the nucleic acid encodes said polypeptide, such that the at least one target cell is transfected with the nucleic acid operably encoding said polypeptide.

[0029] Another object of the present disclosure relates to a method for producing nucleic acid-charged lipid nanoparticles, wherein the method comprises at least the following steps: a) solubilizing at least one lipid compound disclosed herein in a water-miscible organic solvent; b) mixing the organic solvent obtained in step a) with an aqueous solvent containing at least one nucleic acid to be charged; and c) Obtaining the lipid nanoparticles in an aqueous solvent Includes.

[0030] In the description of various embodiments of the present disclosure, various embodiments or individual features have been disclosed. As would be apparent to one skilled in the art, all combinations of such embodiments and features are possible and can result in the practice of the disclosure. While various embodiments and individual features of the present disclosure have been illustrated and described, various other changes and modifications can be made without departing from the spirit and scope of the present disclosure. It is also apparent that all combinations of the embodiments and features taught in the foregoing disclosure are possible and can result in the practice of the disclosure. [Brief explanation of the drawings]

[0031] [Figure 1] Figure 1 shows the mean hemagglutination inhibition antibody titers (HI titers) measured in serum from mice post-1 immunization (at D20) with LNPs L319, LNPs Lip.(III), LNPs Lip.(IV), or LNPs Lip.(V) (prepared using lipid compounds of formula (III), (IV), or (V)), each loaded with mRNA encoding the full-length hemagglutinin (HA) of influenza virus strain A / Netherlands / 602 / 2009 (H1N1). Total injected mRNA was 0.5, 1, 2.5, or 5.0 μg / dose for LNPs L319 and 1 or 5 μg / dose for LNPs Lip.(III), LNPs Lip.(IV), and LNPs Lip.(V). As a negative control, mice were immunized with PBS buffer, and as a positive control, mice received 10 μg of the monovalent influenza vaccine A / California / 07 / 2009 (H1N1) strain from Vaxigrip™. Geometric mean titers and individual HI titers are shown for each group. [Figure 2] Figure 1 shows the mean hemagglutination-inhibition antibody titers (HI titers) measured in serum from mice post-2 immunization (D42) with LNPs L319, LNPs Lip.(III), LNPs Lip.(IV), or LNPs Lip.(V) (prepared using lipid compounds of formulas (III), (IV), or (V)), each loaded with mRNA encoding the full-length hemagglutinin (HA) of influenza virus strain A / Netherlands / 602 / 2009 (H1N1). The total injected mRNA was 0.5, 1, 2.5, or 5.0 μg / dose for LNPs L319 and 1 or 5 μg / dose for LNPs(III), LNPs(IV), and LNPs(V). As a negative control group, mice were immunized with PBS buffer, and as a positive control group, mice received 10 μg of the monovalent influenza vaccine A / California / 07 / 2009 (H1N1) strain from Vaxigrip™. Geometric mean titers and individual HI titers are shown for each group. [Figure 3] This figure shows the hemagglutination-inhibition antibody mean titers (HI titers) measured in serum collected at D42 in mice immunized at D0 and D21 with different LNPs L319 and LNPs Lip. (IV), each containing DOPE as a neutral lipid and loaded with mRNA encoding the full-length hemagglutinin (HA) of influenza virus strain A / Netherlands / 602 / 2009 (H1N1). The mRNA loaded into LNPs Lip. (IV) contained either natural (Nat) or modified uridine bases (Mod). The mRNA loaded into LNPs L319 contained natural uridine bases. The geometric mean titers and individual HI titers are shown for each group. [Figure 4]This figure shows the hemagglutination-inhibition antibody mean titers (HI titers) measured in serum collected at D42 in mice immunized at D0 and D21 with different LNPs L319 and LNPs Lip.(IV). LNPs Lip.(IV) contained either DSPC or DOPE as the neutral lipid. LNPs L319 contained DSPC as the neutral lipid. LNPs were loaded with mRNA containing natural uridine bases encoding the full-length hemagglutinin (HA) of influenza virus strain A / Netherlands / 602 / 2009 (H1N1). Geometric mean titers and individual HI titers are shown for each group. [Figure 5] This figure shows the hemagglutination-inhibition antibody mean titers (HI titers) measured in serum collected at D42 in mice immunized at D0 and D21 with LNPs Lip. (IV) containing DSPC as a neutral lipid and loaded with mRNA containing natural uridine bases encoding the full-length hemagglutinin (HA) of influenza virus strain A / Netherlands / 602 / 2009 (H1N1). LNPs were stored for different periods: 0, 6, and 12 months before use. Three independent experiments were performed over a period ranging from one year. The geometric mean titers and individual HI titers are shown for each group. [Figure 6] Bioluminescence signal acquisition monitoring protein expression at the injection site (quadruplets) after intramuscular administration of LNPs 319 or LNPs Lip. (IV) loaded with 5 µg of luciferase-encoding mRNA (mRNA-Luc) in female BALB / c ByJ mice. Luminescence levels were assessed by ROIs applied to the injection site zone at 6, 24, 48, and 72 h. Results are expressed as total flux (ph / s) as a function of time (h) after injection of LNPs / mRNA-Luc. Buffer PBS was used as a control. [Figure 7] FIG. 1 shows a synthesis scheme of compound (XIII). [Figure 8] FIG. 1 shows a synthesis scheme for compound (XIV). [Figure 9]FIG. 1 shows a synthesis scheme for compound (XVII). [Figure 10] FIG. 1 shows a synthesis scheme for compound (XXI). [Figure 11] FIG. 1 shows a synthesis scheme for compound (XXII). [Figure 12] Chromatogram of LNPs Lip.(IV) / DSPC containing hEPO mRNA recorded as a function of time. [Figure 13] FIG. 1 shows the pH stability of LNPs Lip.(IV) / DSPC over time at different storage temperatures. [Figure 14] FIG. 1 shows the stability of osmolality of LNPs Lip.(IV) / DSPC over time at different storage temperatures. [Figure 15] FIG. 1 shows the particle size stability of LNPs Lip.(IV) / DSPC over time at different storage temperatures. [Figure 16] FIG. 1 shows the stability of mRNA encapsulation rate in LNPs Lip.(IV) / DSPC over time at different storage temperatures. [Figure 17] FIG. 1 shows mRNA integrity in LNPs Lip.(IV) / DSPC over time at different storage temperatures. [Figure 18A] Figure 18 shows the lipid chromatogram stability of LNPs Lip.(IV) / DSPC over time at different storage temperatures. Upper panel: shows LNPs Lip.(IV) / DSPC after 18 weeks at 4°C; lower panel: shows the same LNPs at TO. [Figure 18B] Figure 18 shows the lipid chromatogram stability of LNPs Lip.(IV) / DSPC over time at different storage temperatures. Upper panel: shows LNPs Lip.(IV) / DSPC after 18 weeks at 25°C; lower panel: shows the same LNPs at TO. [Figure 18C]Figure 18 shows the lipid chromatogram stability of LNPs Lip.(IV) / DSPC over time at different storage temperatures. Top panel: LNPs Lip.(IV) / DSPC after 18 weeks at 37°C; bottom panel: the same LNPs at TO. [Figure 19] FIG. 1 shows the stability of hEPO expression from LNPs Lip.(IV) / DSPC over time at different storage temperatures. [Figure 20] FIG. 1 shows the immunogenicity of LNPs containing influenza HA mRNA in cynomolgus macaques immunized twice (D0, D28) at 4-week intervals with 50 μg of mRNA in LNPs injected IM into the biceps in a volume of 500 μl. [Figure 21] This figure shows the mean hemagglutination inhibition antibody titers (HI titers) measured in serum collected at D21 from mice immunized at D0 and D21 with LNPs L319, LNPs Lip.(IV) [DOG-IM4], LNPs Lip.(IX), LNPs Lip.(XII) and LNPs Lip.(XVI), which contain DSPC as a neutral lipid and are loaded with mRNA encoding the full-length hemagglutinin (HA) of influenza virus strain A / Netherlands / 602 / 2009 (H1N1). DETAILED DESCRIPTION OF THE INVENTION

[0032] definition The terms used herein generally have their ordinary meaning in the art, within the context of this disclosure, and in the specific context in which each term is used.Certain terms are discussed below or elsewhere in the specification to provide additional guidance in describing the compositions and methods of the present disclosure, and how to make and use them.The following definitions are provided for this specification, including the claims.

[0033] The term "terminal radical" means that the radical is a head group or a tail group.

[0034] The term "pharmaceutically acceptable salts" includes addition salts of the compounds disclosed herein, for example, derived from the combination of the compounds with non-toxic acid addition salts.

[0035] The term "acid addition salts" includes inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid, and organic acids such as acetic acid, citric acid, propionic acid, tartaric acid, glutamic acid, salicylic acid, oxalic acid, methanesulfonic acid, paratoluenesulfonic acid, succinic acid, and benzoic acid, and related inorganic and organic acids.

[0036] Pharmaceutically acceptable salts of the compounds disclosed herein can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, ethyl acetate, etc. Mixtures of such solvates can also be prepared. The source of such solvates can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent. Such solvates are within the scope of the present disclosure.

[0037] In the context of this disclosure, the following chemical terms have the following meanings: - halogen atoms: fluorine, chlorine, bromine or iodine; -C t ~C z : a carbon chain that may have t to z carbon atoms, where t and z may have values ​​from 1 to 7; for example, C1 to C4 is a carbon chain that may have 1 to 4 carbon atoms; - C1-C4 alkyl, as used herein, refers to a C1-C4 linear, secondary or tertiary saturated hydrocarbon, respectively. Non-limiting examples are methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tertbutyl; - C1-C4 alkoxy is intended to mean an -O-(C1-C4) alkyl radical, wherein the C1-C4 alkyl group is as defined above. Non-limiting examples are methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxy or tert-butoxy; heteroatom is understood to mean nitrogen, oxygen or sulfur; - aromatic heterocycle means a 5- or 6-membered aromatic ring containing one or two heteroatoms; - Aromatic ring refers to a monocyclic or polycyclic, e.g., a monocyclic aromatic hydrocarbon radical of 6 to 20 atoms, e.g., 6 atoms, derived by removing one hydrogen from a carbon atom of a parent aromatic ring system. An example of an aromatic ring disclosed herein is a phenyl group; - When n is 0 in formula (I) of the present disclosure, it means that the moiety -NH is absent.

[0038] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0039] As used herein, the term "about" or "approximately" refers to the normal error range for the respective value, which is readily known to one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself. In some embodiments, the term "about" refers to ±10% of a given value. However, whenever the value in question refers to an indivisible entity, i.e., other entities that lose their identity once subdivided, such as nucleotides, "about" refers to ±1 of the indivisible entity.

[0040] The term "antigen" includes any molecule, e.g., a peptide or protein, that elicits an immune response and / or contains at least one epitope against which an immune response is directed. For example, an antigen is a molecule that, optionally after processing, induces, e.g., an immune response specific to the antigen or a cell expressing the antigen. After processing, the antigen is presented by an MHC molecule and reacts specifically with T lymphocytes (T cells). Thus, the antigen or a fragment thereof must be recognizable by a T cell receptor and, in the presence of an appropriate costimulatory signal, must be able to induce clonal expansion of T cells bearing T cell receptors that specifically recognize the antigen or fragment, resulting in an immune response against the antigen or a cell expressing the antigen.

[0041] According to the present disclosure, any suitable antigen that is a candidate for immune response can be envisioned.Antigens can correspond to or be derived from naturally occurring antigens.Such naturally occurring antigens can include or be derived from allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens, or antigens can also be tumor antigens.

[0042] As used herein, the term "aqueous solution" or "aqueous solvent" refers to a composition that includes water.

[0043] Within this disclosure, the term "cationic group" or "cationic ammonium group" refers to an ion or group of ions that has a positive charge and contains at least one ionizable nitrogen atom. The cationic group disclosed herein has the formula (I): -NH-CX-(NH) n It is composed of the radical -A.

[0044] Aspects and embodiments of the disclosure described herein are understood to include aspects and embodiments "having," "comprising," "consisting of," and "consisting essentially of." The words "have" and "comprise," or variations such as "has," "having," "comprises," or "comprising," are understood to mean the inclusion of the stated elements (such as a composition of matter or method step) but not the exclusion of other elements. The term "consisting of" means the inclusion of the stated elements to the exclusion of additional elements. The term "consisting essentially of" means the inclusion of the stated elements and other elements that do not materially affect the basic and novel characteristics of the disclosure. In some contexts, the term "comprise" may specify exactly the stated features, integers, steps, or components, and thus may be replaced with "consist" in such cases.

[0045] The term "charged lipid" refers to any of several lipid species that exist in positively or negatively charged forms within a useful physiological range, e.g., at pH between about 3 and about 9. Charged lipids can be synthetic or naturally derived. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, and dimethylaminoethanecarbamoylsterol (e.g., DC-Choi).

[0046] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including a virus), that can be isolated from a natural source, and that has not been intentionally modified by man in a laboratory is naturally occurring.

[0047] The term "neutral lipid" refers to any of several lipid species that are not ionizable or are neutral zwitterionic compounds at a selected pH, such as physiological pH. Such lipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin (SM), or ceramide. Neutral lipids can be synthetic or naturally derived.

[0048] As used herein, the term "individual" or "subject" refers to a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0049] The term "lipid" refers to a group of organic compounds, including but not limited to esters of fatty acids, which are generally characterized by being poorly soluble in water but soluble in many organic solvents.

[0050] Lipid is a general term that includes fats, fatty oils, essential oils, waxes, phospholipids, glycolipids, sulfolipids, aminolipids, chromolipids (lipochromes), and fatty acids. Within this disclosure, "lipid" includes neutral lipids, steroid alcohols or their esters, and PEGylated lipids.

[0051] The term "lipid nanoparticle" (LNP) refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1000 nm) that can be formulated with at least one lipid compound disclosed herein. In some embodiments, lipid nanoparticles are included in formulations that can be used to deliver active or therapeutic agents, such as nucleic acids, to a desired target site (e.g., a cell, tissue, organ, tumor, etc.). Such lipid nanoparticles typically comprise a lipid compound disclosed herein and at least one component selected from a neutral lipid, a steroid alcohol or its ester, and a polymer-conjugated lipid.

[0052] As used herein, "lipid encapsulation" refers to lipid nanoparticles that provide an active or therapeutic agent, such as a nucleic acid, with complete encapsulation, partial encapsulation, or both. In one embodiment, the polynucleotide is completely encapsulated in the lipid nanoparticle.

[0053] It should be noted that the terms "head group" and "tail group" as used herein describe portions of compounds of the present disclosure, e.g., functional groups of such compounds. They are used to describe the orientation of one or more functional groups relative to other functional groups in the compound. They are both "end groups."

[0054] As used herein, the term "lipophilic or hydrophobic tail group" qualitatively indicates that the tail has an affinity for lipids (is typically lipid-soluble) and avoids water (is typically not water-soluble).

[0055] The term "PEGylated lipid" refers to a molecule containing both a lipid moiety and a polyethylene glycol moiety. PEGylated lipids are known in the art and include, for example, 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG).

[0056] Within this disclosure, the terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably. They refer to a polymeric form of at least two nucleotides, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Nucleic acids can have any three-dimensional structure and can perform any function, known or unknown. They can be linear or circular. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, multiple loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, closed-ended DNA (ceDNA), self-amplifying RNA, stranded DNA (ssDNA), small interfering RNA (siRNA) and microRNA (miRNA), recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure, if present, may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with a labeling component. The term "complement of a polynucleotide" refers to a polynucleotide molecule having a complementary base sequence and reverse orientation compared to a reference sequence such that it can hybridize with complete fidelity to the reference sequence. "Recombinant" as applied to a polynucleotide means that the polynucleotide is the product of various combinations of in vitro cloning, restriction, and / or ligation steps, and other procedures that result in a construct that can potentially be expressed in a host cell.

[0057] The term "steroid alcohol" or "sterol" refers to a group of lipids composed of a sterane core with a hydroxyl moiety. Examples of steroid alcohols include cholesterol, campesterol, sitosterol, stigmasterol, and ergosterol. An ester of a steroid alcohol or sterol refers to an ester of a carboxylic acid with the hydroxyl group of the steroid alcohol. Suitable carboxylic acids contain, in addition to the carboxyl moiety, a saturated or unsaturated, straight-chain or branched-chain alkyl group. In some embodiments, the alkyl group is C1-C 20 It may be an alkyl group. In another embodiment, the carboxylic acid may be a fatty acid.

[0058] As used herein, the terms "prevent," "preventing," or "delay progression of" (and grammatical variations thereof) with respect to a disease or disorder refer to the prophylactic treatment of a disease, e.g., an individual suspected of having the disease or at risk of developing the disease. Prevention includes, but is not limited to, preventing or delaying the onset or progression of a disease and / or maintaining at least one symptom of a disease below a desired or pathological level. The term "prevention" does not necessarily eliminate 100% the likelihood of the possibility or occurrence of an event. Rather, it means that the likelihood of an event occurring is reduced in the presence of a composition or method described herein.

[0059] Within this disclosure, the term "significantly" as used in reference to a change is intended to mean that the change observed is noticeable and / or that it has statistical significance.

[0060] Within this disclosure, the term "substantially" as used in connection with a feature of the disclosure is intended to define a range of embodiments related to this feature that are nearly similar, but not completely similar, to this feature.

[0061] As used herein, "target cell" or "targeted cell" refers to a cell of interest. The cell can be found in vitro, in vivo, in situ, or in the tissue or organ of an organism. The organism can be an animal, e.g., a mammal, e.g., a human, and e.g., a human patient. In some embodiments, the target cell is a cell isolated from an individual.

[0062] The term "treat" or "treatment" or "therapy" herein refers to the administration or consumption of a composition disclosed herein with the intent to cure, heal, alleviate, relieve, alter, cure, ameliorate, improve or affect the symptoms of a disorder, condition, or to prevent or delay the onset of symptoms, complications, or otherwise prevent or inhibit further development of a disorder in a statistically significant manner.

[0063] As used herein, the terms "therapeutically effective amount" and "prophylactically effective amount" refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of the pathological process under consideration. The specific therapeutically effective amount can be readily determined by an ordinary practitioner and may vary depending on factors such as the type and stage of the pathological process under consideration, the patient's medical history and age, and the administration of other therapeutic agents.

[0064] The lists of sources, ingredients, and components set forth below are recited such that combinations and mixtures thereof are also contemplated and within the scope of this specification.

[0065] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification includes every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0066] All lists of items, such as lists of ingredients, are intended and should be construed as Markush groups. Accordingly, all lists may be read and construed as an item selected from the group consisting of "a list of items," and combinations and mixtures thereof.

[0067] References herein may be made to trade names of components, including various ingredients, utilized in this disclosure. The inventors herein do not intend to be limited to materials with any particular trade name. Equivalent materials (e.g., those obtained from different sources under different names or reference numbers) may be substituted for those referenced by trade name in the description herein.

[0068] Further definitions of radicals and lipid compounds of formula (I) As noted above, the lipid compounds disclosed herein are ionic, e.g., cationic lipid compounds.

[0069] The lipid compounds disclosed herein are amine-containing lipid compounds, and therefore, for example, ionic. Such compounds can be easily protonated, and therefore, their pKa changes depending on the pH value. For example, the compounds disclosed herein have a pKa of less than 7, for example, in the range of 4.5 to 6.7.

[0070] The lipid compounds disclosed herein may have asymmetric center, chiral axis and chiral plane (as described in EL Eliel and SH Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pp. 1119-1190), and may exist as racemates, racemic mixtures and individual diastereomers, and all possible isomers and their mixtures, including optical isomers, are included in the present disclosure.In addition, the cationic lipids disclosed herein may exist as tautomers, and even if only one tautomeric structure is shown, both tautomeric forms are intended to be included in the scope of disclosure.

[0071] Pharmaceutically acceptable salts of the compounds disclosed herein generally have one or more physiologically acceptable counterions, such as halides, phosphates, trifluoroacetates, sulfites, nitrates, gluconates, glucuronates, galacturonate radicals, alkylsulfonates, alkylcarboxylates, propionatesulfonates, and methanesulfonates.

[0072] The compounds disclosed herein and their pharmaceutically acceptable salts may also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, ethyl acetate, etc. Mixtures of such solvates may also be prepared. The source of such solvates may be from the solvent of crystallization, may be inherent in the solvent of preparation or crystallization, or may be adventitious to such solvent. Such solvates are within the scope of the present disclosure.

[0073] For example, the lipid compounds disclosed herein have one hydrophilic head group formed by one radical of formula (I), also referred to as a terminal radical to denote the fact that it is directly or indirectly linked to the end of a hydrophobic or lipophilic tail.

[0074] The radicals of formula (I) have the following definitions: *-NH-CX-(NH) n -A (I) (In the formula, - *- means that the radical of formula (I) is joined to one C 10 ~C 55 represents a single bond directly or indirectly connecting to a lipophilic or hydrophobic tail group of - n is 0 or 1; X is an oxygen or sulfur atom, - A represents an optionally substituted 5- or 6-membered unsaturated heterocyclic radical or a 5- or 6-membered aromatic heterocyclic radical, both of which contain at least one nitrogen atom.

[0075] The compound containing at least one radical of formula (I) may also be one of its pharmaceutically acceptable salts; and one of its possible racemic, enantiomeric and diastereomeric isomeric forms.

[0076] The nitrogen atom of amide functional group can be protonated, so the lipid compound disclosed herein can be protonated.Therefore, as mentioned above, the lipid compound disclosed herein has an apparent pKa that can change according to pH value.

[0077] According to one embodiment, the compounds disclosed herein have a pKa of less than 7.

[0078] According to another particular embodiment, the compounds disclosed herein have a pKa in the range of 4.5 to 6.7. Such pKa can be determined by any conventional method.

[0079] According to one embodiment, X is a sulfur atom and A is a pyridinyl radical.

[0080] According to one embodiment, A is a 3-pyridinyl radical.

[0081] According to another embodiment, X is an oxygen atom and A is a 5-membered aromatic heterocycle containing at least one nitrogen atom. According to one embodiment, A is an imidazolyl radical. For example, A can be a 4-imidazolyl radical.

[0082] One radical of formula (I) is directly or indirectly attached (eg, covalently bonded) to the hydrophobic (lipophilic) tail group.

[0083] The hydrophobic or lipophilic tail is generally C 10 ~C 55 is.

[0084] For example, the lipophilic or hydrophobic tail may be an optionally substituted, branched or unbranched, straight chain, saturated or unsaturated C 10 ~C 55 is a hydrocarbon radical, the hydrocarbon backbone of which is optionally interrupted by one or more atoms of oxygen or nitrogen and / or one or more -O-CO- or -CO-O- groups, and the nitrogen atoms, if present in the backbone, can be directly or indirectly bonded to the radical of formula (I).

[0085] For example, the hydrophobic or lipophilic tails may each independently be an optionally substituted C-C 24 , e.g. C 10 ~C 20 , alkyl chains, optionally substituted, variably saturated or unsaturated C to C 24 , e.g. C 10 ~C 20 , alkenyl chains and optionally substituted saturated, variably saturated or unsaturated C8-C 24 , e.g. C 10 ~C 20, and acyl chains, wherein said alkyl, alkenyl or acyl chains may be interrupted by one or more atoms of oxygen or nitrogen and / or one or more moieties such as -O-CO- or -CO-O-, and preferably at least one moiety such as -O-CO- or -CO-O-.

[0086] Each hydrocarbon chain may be substituted by at least one radical selected from -OH and CO2H.

[0087] According to one embodiment, the hydrophobic or lipophilic tail is selected from the group consisting of:

[0088] [ka] [ka] [ka] [ka]

[0089] In one embodiment, the hydrophobic or lipophilic tail of the compound according to the invention contains at least one amino moiety involved in the linkage to the spacer, in this particular embodiment, the hydrophobic or lipophilic tail is selected from among R1g, h, q, r, u, v, w and z.

[0090] In another embodiment, the hydrophobic or lipophilic tail of the compounds according to the invention may also contain at least three or more hydrocarbon chains, such as, for example, hydrophobic or lipophilic tails R1m, p, q, r, s, u, v, w, x, y, and z. Each hydrocarbon chain may be substituted C8-C 24 , e.g. C 10 ~C 20 Alkyl chain and substitution variably saturated or unsaturated C8-C24 , e.g. C 10 ~C 20 The alkyl or alkenyl chain may optionally and preferably be interrupted by one or more moieties such as -O-CO- or -CO-O-.

[0091] In certain embodiments, the hydrophobic or lipophilic tail of the compounds according to the invention is a tail (R1a) or (R1b), also known as a DOG alkyl or DOG ether, respectively.

[0092] According to another embodiment, the cationic and / or ionic lipid compounds disclosed herein have the formula (II): R1-Z-NH-CX-(NH) n -A (II) (In the formula, X, n and A are as previously defined - R1 may be, for example, one C 10 ~C 55 is a lipophilic or hydrophobic tail group; - Z is a spacer arm having 2 to 24, such as 2 to 18, such as 4 to 12, or such as 2 to 12 carbon atoms in a branched or unbranched straight-chain, saturated or unsaturated hydrocarbon chain, said chain being interrupted by one or more atoms of oxygen and / or a moiety selected from -SS-; -(O=C)-; -(C=O)-O-; -O-(O=C)-; -S-; -NH-, -NH-(O=C)-; -(O=C)-NH- and -NH-(C=O)-O-, and preferably by -(C=O)-O-; -O-(O=C)- and -NH-(C=O)-O-, and optionally terminated by an oxygen atom or a moiety selected from among -NH-(O=C)--O-(O=C)-; -(C=O)-O-; and -(O=C)-, which is linked to a hydrophobic tail group; - p is 0 or 1), or one of the pharmaceutically acceptable salts of said compound of formula (II); and any of the racemic, enantiomeric and diastereomeric isomeric forms thereof.

[0093] The spacer arm is the same as that conventionally considered in the field of lipid cationic compounds. Therefore, the selection of such a spacer arm does not cause any difficulty for those skilled in the art. The spacer arm must be inert or not adversely affect the efficiency of the lipid compound.

[0094] Generally, the spacer has 2 to 24, such as 4 to 12, carbon atoms, or such as 2 to 12 carbon atoms, and includes at least one or more ethylene oxide units and optionally one or more moieties previously disclosed.

[0095] Examples of spacer arms that are convenient for disclosure include the following, with the right end connected to a lipophilic or hydrophobic tail group:

[0096] [ka]

[0097] According to one embodiment, the spacer consists of ethylene oxide units and may comprise 1 to 24, such as 2 to 15, such as 3 to 12, such as 4 to 10, and such as 6 to 8 ethylene oxide units.

[0098] According to another particular embodiment, the spacer has formula (A1) [ka] (In the formula, - the right end is linked to a lipophilic or hydrophobic tail group, - l is 0 or 1; - m is in the range of 1 to 24, such as 2 to 15, for example 3 to 12, for example 2, 3, 4, 5, 6, 7, 8, or 9; - p is 0 or 1; - when p is 1, R' may represent one oxygen atom or a moiety selected from -C=O-; -NH-; -O-CH2-; -NH-C(=O)-; -NH-C(=O)-O-CH2-; OC(=O)-; C=O-NH-(CH2)2-; OCH2C(=O)-O-; --C(=O)-O-(CH2)2- and -SS-, and in particular -NH-C(=O)-; -NH-C(=O)-O-CH2-; -OC(=O)-; -C=O-NH-(CH2)2-; and -C(=O)-O-(CH2)2.

[0099] According to another embodiment, the spacer may comprise 1 to 24, such as 2 to 15, for example 3 to 12, for example 4 to 10, and for example 6 to 8 ethylene oxide units, preferably incorporating at least one moiety selected from among -(C=O)-O-, -O-(O=C)-, -NH-(O=C)-; -(O=C)-NH- and -NH-(C=O)-O-, More preferably, at least one -NH-(C=O)-O- is incorporated.

[0100] In one embodiment, in the lipid compound of formula (II), X is a sulfur atom and A is a pyridinyl radical. For example, A can be a 3-pyridinyl radical.

[0101] According to this embodiment, the compound of formula (II) may be, for example, a compound of formula (V) [ka] or one of its salts, or one of its racemic, enantiomeric and diastereomeric isomeric forms.

[0102] For example, the compound (V) or a derivative thereof is not salified, i.e., the compound exists in the form of a free base.

[0103] In another embodiment, in the lipid compound of formula (II), X is an oxygen atom, and A is a 5-membered aromatic heterocyclic radical containing at least one nitrogen atom. Thus, according to one embodiment, A is an imidazolyl radical, for example, A may be a 4-imidazolyl radical.

[0104] According to this embodiment, the compound of formula (II) is selected from, for example, the following compounds (III) to (XXVII), and salts thereof, or their racemic, enantiomeric, and diastereomeric isomers. In the following formulae, the secondary amino moiety may be represented interchangeably as -NH- or -N-.

[0105] [ka] [ka] [ka] [ka] [ka]

[0106] More particularly, compounds (IV), (VIII), (IX), (XII), (XVI), (XIX) and (XXII), in particular compounds (IV), (IX), (XII) or (XVI), more particularly compounds (IV) or (XII), and (all) especially compound (IV), are of interest, as are their salts, or their racemic, enantiomeric and diastereomeric isomeric forms, e.g., they may be in the form of the free base.

[0107] As shown in the Examples section, compounds (IV), (VIII), (IX), (XII), (XVI), (XIX) and (XXII) are effective in formulating stable LNPs (stable in a liquid state at 4-8°C) that, for example, after parenteral administration, can deliver functional mRNA to target tissues and induce expression of proteins such as EPO or an immune response if the delivered mRNA encodes an antigen.

[0108] Preparation of cationic lipids Compounds according to the present disclosure can be readily prepared from commercially available or literature-described starting materials using methods and procedures known to those skilled in the art.

[0109] For example, a lipid compound of formula (II) can be obtained by covalent bonding between a precursor of a radical of formula (I) and a lipid compound or a derivative thereof having a terminal reactive group capable of reacting with said precursor.

[0110] This terminal reactive group can be positioned directly at the end of the hydrophobic or lipophilic portion of the lipid compound to be converted, or at the end of a spacer that is already linked to the hydrophobic or lipophilic portion of the lipid compound.

[0111] The selection of a convenient precursor of the radical of formula (I) intended to react with the lipid compound to form the expected covalent bond is clearly within the capabilities of a person skilled in the art. The precursor only needs to have a group capable of chemically reacting with one of the lipid compounds to form a covalent bond.

[0112] As regards these starting compounds, i.e., the precursors of the radicals of formula (I) and the lipid compounds or derivatives thereof to be converted, they can be easily produced by those skilled in the art, for example, according to the preparation methods presented in the examples below.

[0113] Covalent bonding can also be carried out according to methods known to those skilled in the art regarding the chemical nature of the reactive groups on the precursor of the radical of formula (I) and the reactive groups on the converting lipid compound or derivative.

[0114] Generally, the covalent bond can be formed by esterification, amidation or cabamation.

[0115] Representative of convenient precursors of radicals of formula (I) containing a pyridinyl group A are the corresponding pyridyl isothiocyanates, such as 3-pyridyl isothiocyanates.

[0116] Representative of convenient precursors of radicals of formula (I) containing an imidazolyl group A are the corresponding imidazole carboxylic acids.

[0117] One particular approach to obtain compounds of formula (I) as compounds of formula (IV) is shown in Scheme 1 below.

[0118] [ka]

[0119] Where typical or specific experimental conditions (i.e., reaction temperatures, times, moles of reagents, solvents, etc.) are given, it is understood that other experimental conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art using routine optimization procedures.

[0120] Any salification can be carried out by conventional methods to form the expected cationic form.

[0121] The coupling reaction is advantageously followed by a subsequent step of purification and / or isolation of the resulting final product. Convenient methods of purification are detailed in the examples below. For example, purification of the resulting compound can be carried out by preparative high performance liquid chromatography (HPLC).

[0122] The present disclosure will be better understood from the following examples, all of which are intended for illustrative purposes only and are not meant to limit the scope of the disclosure in any way.

[0123] Compositions, lipid nanoparticles, and manufacturing processes The present disclosure relates to a composition comprising at least one lipid compound disclosed herein as described above.The composition disclosed herein can further comprise at least one lipid selected from the group consisting of neutral lipid, steroid alcohol or its ester, and PEGylated lipid.

[0124] The compositions disclosed herein can be formulated as lipid nanoparticles containing at least one nucleic acid.

[0125] The compositions or lipid nanoparticles disclosed herein may further comprise at least one therapeutic agent, an anionic or polyanionic agent, such as at least one nucleic acid.

[0126] neutral lipid The composition or lipid nanoparticles disclosed herein can contain neutral lipids.The presence of neutral lipids can improve the structural stability of lipid nanoparticles.Neutral lipids can be appropriately selected in consideration of the delivery efficiency of nucleic acids.

[0127] Neutral lipids differ from the lipid compounds disclosed herein in that they are either non-ionizable at a selected pH or are neutral zwitterionic compounds.

[0128] Neutral lipids useful in the present disclosure may be selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and ceramide.

[0129] Phosphatidylcholine and phosphatidylethanolamine are zwitterionic lipids. Sphingomyelin and ceramide are not ionic lipids.

[0130] As examples of phosphatidylcholines useful in the present disclosure, mention may be made of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine).

[0131] As examples of phosphatidylethanolamines useful in the present disclosure, mention may be made of DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DSPE (1,2-distearoyl-s / i-glycero-3-phosphoethanolamine), DLPE (1,2-dilauroyl-SM-glycero-3-phosphoethanolamine), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, or 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE).

[0132] The neutral lipid may be selected from the group consisting of phosphatidylcholines such as DSPC, DPPC, DMPC, POPC, DOPC, phosphatidylethanolamines such as DOPE, DPPE, DMPE, DSPE, DLPE; sphingomyelin; and ceramide.

[0133] In one embodiment, neutral lipids suitable for the present disclosure may be DSPC, DOPC, and DOPE, for example, DSPC or DOPE.

[0134] The neutral lipid may be present in step a) of the method of formulating lipid nanoparticles disclosed herein in a molar amount ranging from about 0% to about 50%, such as from about 5% to about 45%, such as from about 8% to about 40%, and such as from about 10% to about 30%, relative to the total molar amount of lipids and lipid compounds disclosed herein.

[0135] The neutral lipid may be present in the compositions or lipid nanoparticles disclosed herein in a molar amount ranging from about 0% to about 50%, for example, from about 5% to about 45%, for example, from about 8% to about 40%, and for example, from about 10% to about 30%, relative to the total molar amount of the lipids and lipid compounds disclosed herein.

[0136] The neutral lipid may be present in the compositions or lipid nanoparticles disclosed herein in a lipidic compound:neutral lipid molar ratio ranging from about 70:1 to about 1:2, such as from about 30:1 to about 1:1, such as from about 15:1 to about 2:1, such as from about 10:1 to about 4:1, further such as about 5:1.

[0137] steroid alcohol or its ester The compositions or lipid nanoparticles disclosed herein may contain a steroid alcohol (or sterol) or an ester thereof. The presence of a sterol or ester of a sterol can also improve the structural stability of the lipid nanoparticles.

[0138] Sterols or steroid alcohols useful in the disclosure include cholesterol or derivatives thereof, ergosterol, desmosterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigmasterol-5,22-dien-3-ol), lanosterol (8,24-lanostadien-3b-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), zymosterol (5α-cholesta-8,24-dien-3b-ol), erythrosterol (erythrosterol-5,24-dien-3b-ol), erythrosterol (erythrosterol-5,24-dien-3b-ol), erythrosterol-5,24-dien-3b-ol, ... The steroid hormone may be selected from the group consisting of: 5α-cholesten-3β-ol, lathosterol (5α-cholest-7-en-3β-ol), diosgenin ((3β,25R)-spirost-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitostanol, campesterol (campest-5-en-3β-ol), campestanol (5a-campestan-3b-ol), 24-methylenecholesterol (5,24(28)-cholestadien-24-methylene-3β-ol).

[0139] An ester of a steroid alcohol or sterol refers to an ester of a carboxylic acid with a hydroxyl group of a steroid alcohol. Suitable carboxylic acids contain, in addition to the carboxyl moiety, a saturated or unsaturated, straight or branched chain alkyl group. In some embodiments, the alkyl group is C1-C 20 saturated or unsaturated, straight or branched chain alkyl groups, such as C2 to C 18 , for example C4~C 16 , for example C8~C 12 In other embodiments, the carboxylic acid may be a fatty acid. For example, the fatty acid may be caprylic acid, capric acid, lauric acid, stearic acid, margaric acid, oleic acid, linoleic acid, or arachidic acid.

[0140] In one embodiment, the esters of sterols suitable for the present disclosure may be cholesteryl esters.

[0141] Esters of sterols or steroid alcohols useful in the present disclosure may be selected from the group consisting of cholesteryl margarate (cholest-5-en-3β-yl heptadecanoate), cholesteryl oleate, and cholesteryl stearate.

[0142] Sterols or steroid alcohols or esters useful in the present disclosure include cholesterol or derivatives thereof, ergosterol, desmosterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigmasterol-5,22-dien-3-ol), lanosterol (8,24-lanostadien-3b-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), zymosterol (5α-cholesta-8,24-dien-3b-ol), erythrosterol (erythrosterol-5,24-dien-3b-ol), erythrosterol-5,24-dien-3b-ol, ... 3β-ol), lathosterol (5α-cholest-7-en-3β-ol), diosgenin ((3β,25R)-spirost-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitostanol, campesterol (campest-5-en-3β-ol), campestanol (5a-campestan-3b-ol), 24-methylenecholesterol (5,24(28)-cholestadien-24-methylene-3β-ol), cholesteryl margarate (cholest-5-en-3β-yl heptadecanoate), cholesteryl oleate, and cholesteryl stearate.

[0143] Alternatively, the sterol useful in the present disclosure may be a cholesterol derivative, such as oxidized cholesterol.

[0144] The oxidized cholesterol suitable for the present disclosure can be 25-hydroxycholesterol, 27-hydroxycholesterol, 20α-hydroxycholesterol, 6-keto-5α-hydroxycholesterol, 7-keto-cholesterol, 7β,25-hydroxycholesterol, and 7β-hydroxycholesterol. For example, the oxidized cholesterol can be 25-hydroxycholesterol and 20α-hydroxycholesterol, such as 20α-hydroxycholesterol.

[0145] In one embodiment, a sterol or steroid alcohol, or ester thereof, suitable for the present disclosure may be cholesterol, a cholesteryl ester, or a cholesterol derivative, such as oxidized cholesterol. In one embodiment, a sterol or steroid alcohol suitable for the present disclosure may be cholesterol or a cholesteryl ester, such as cholesterol.

[0146] The sterol or steroid alcohol, or ester thereof, may be present in the compositions or lipid nanoparticles disclosed herein in a molar amount ranging from about 0% to about 60%, such as from about 10% to about 50%, and such as from about 20% to about 50%, relative to the total molar amount of lipids and lipid compounds disclosed herein that may be present in the composition or lipid nanoparticle.

[0147] The sterol or steroid alcohol, or ester thereof, may be present in the compositions or lipid nanoparticles disclosed herein in a molar ratio of lipid compound:steroid alcohol, or ester thereof, which may range from about 4:1 to about 1:2, such as from about 3.5:1 to about 1:1.8, such as from about 2:1 to about 1:1.5, such as from about 1.5:1 to about 1:1.2, for example, from about 1.3:1 to about 1:1.3.

[0148] PEGylated lipids The compositions or lipid nanoparticles disclosed herein may include PEGylated (or PEG-) lipids.

[0149] The expected PEG-modified lipids are those with a length of C6 20 Examples of PEG-modified lipids include, but are not limited to, polyethylene glycol chains up to 5 kDa in length covalently attached to lipids having alkyl chains of up to 5 kDa. The addition of PEG-modified lipids to the lipid nanoparticle compositions disclosed herein can prevent complex aggregation, extend circulation life, and also provide a means to increase delivery of the composition or lipid nanoparticle to target cells.

[0150] Suitable PEGylated lipids include, for example, PEGylated diacylglycerols (PEG-DAGs) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (DMG-PEG), PEGylated phosphatidylethanolamine (PEG-PE), PEGylated succinate diacylglycerols (PEG-S-DAGs) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(co-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), PEG The polyethoxylated ceramide (PEG-cer), or a PEG dialkoxypropyl carbamate such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxyl)propyl)carbamate, 2,3-di(tetradecanoxyl)propyl-N-(co-methoxy(polyethoxy)ethyl)carbamate, or mPEG-N,N-ditetradecylacetamide (also known as 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide or ALC-0159).

[0151] In one embodiment, PEGylated lipids suitable for the present disclosure may be selected from the group consisting of PEG-DAG, DMG-PEG, PEG-PE, PEG-S-DAG, PEG-S-DMG, PEG-cer, or mPEG-N,N-ditetradecylacetamide, or PEG-dialkoxypropylcarbamate.

[0152] For example, a PEGylated lipid suitable for the present disclosure may be DMG-PEG, PEG-PE, or mPEG-N,N-ditetradecylacetamide.

[0153] In some embodiments, a PEGylated lipid suitable for the present disclosure may be DMG-PEG or PEG-PE.

[0154] In some embodiments, a PEGylated lipid suitable for the present disclosure may be mPEG-N,N-ditetradecylacetamide.

[0155] The compositions or lipid nanoparticles disclosed herein may comprise PEGylated lipids in a molar amount ranging from about 1% to about 15%, e.g., from about 1% to about 10%, e.g., from about 1% to about 5%, and e.g., from about 1% to about 3.5%, relative to the total molar amount of lipids and lipid compounds.

[0156] The PEGylated lipid and lipid compound may be present in a molar ratio of lipid compound to PEGylated lipid of about 70:1 to about 4:1, such as about 40:1 to about 10:1, such as about 35:1 to about 15:1, and such as about 33:1 or about 14:1.

[0157] In one embodiment, the composition or lipid nanoparticle may comprise, in addition to the lipid compounds described above, at least one neutral lipid, at least one steroid alcohol or ester thereof, and at least one PEGylated lipid.

[0158] The neutral lipid, steroid alcohol or ester thereof, and PEGylated lipid may be as set forth above.

[0159] In one embodiment, the compositions or lipid nanoparticles described herein may comprise the lipid compounds, neutral lipids, steroid alcohols or esters thereof, and PEGylated lipids disclosed herein in molar amounts of about 30% to about 70% lipid compounds, about 0% to about 50% neutral lipids, 20% to about 50% steroid alcohols or esters thereof, and about 1% to about 15% PEGylated compounds, relative to the total amount of lipids and lipid compounds.

[0160] In one embodiment, the compositions or lipid nanoparticles described herein may comprise the lipid compounds, neutral lipids, steroid alcohols or esters thereof, and PEGylated lipids disclosed herein in molar amounts of about 30% to about 60% lipid compounds, about 5% to about 30% neutral lipids, about 30% to about 48% steroid alcohols or esters thereof, and about 1.5% to about 5% PEGylated lipids, relative to the total amount of lipids and lipid compounds.

[0161] In one embodiment, the compositions or lipid nanoparticles described herein may comprise the lipid compounds, neutral lipids, steroid alcohols or esters thereof, and PEGylated lipids disclosed herein in molar amounts of about 35% to about 50% lipid compounds, about 10% to about 16% neutral lipids, about 38.5% to about 46.5% steroid alcohols or esters thereof, and about 1.5% PEGylated lipids, relative to the total amount of lipids and lipid compounds.

[0162] In one embodiment, the composition or lipid nanoparticles disclosed herein may comprise, based on the total amount of lipids and lipid compounds, about 35% of a lipid compound disclosed herein, about 16% of a neutral lipid, about 46.5% of a steroid alcohol or ester thereof, and about 1.5% of a PEGylated compound.

[0163] In another embodiment, the compositions or lipid nanoparticles disclosed herein may comprise about 50% of the lipid compounds disclosed herein, about 10% of neutral lipids, about 38.5% of steroid alcohols or esters thereof, and about 1.5% of PEGylated compounds, based on the total amount of lipids and lipid compounds.

[0164] In one embodiment, the molar ratio of the lipid compounds disclosed herein to the neutral lipid, steroid alcohol or ester thereof, and PEGylated lipid can be about 35 / 16 / 46.5 / 1.5, about 50 / 10 / 38.5 / 1.5, about 57.2 / 7.1 / 34.3 / 1.4, about 40 / 15 / 40 / 5, about 50 / 10 / 35 / 4.5 / 0.5, about 50 / 10 / 35 / 5, about 40 / 10 / 40 / 10; about 35 / 15 / 40 / 10, or about 52 / 13 / 30 / 5.

[0165] In one embodiment, the molar ratio of a lipid compound disclosed herein to a neutral lipid, a steroid alcohol or ester thereof, and a PEGylated lipid can be about 35 / 16 / 46.5 / 1.5 or about 50 / 10 / 38.5 / 1.5.

[0166] In another embodiment, the lipid compound disclosed herein may be any of compounds (III) to (XXVII), or compounds (III), (IV), (V), (VIII), (IX), (XII), (XVI), (XIX), or (XXII), or compounds (IV), (VIII), (IX), (XII), (XVI), (XIX), or (XXII), or compounds (IV), (IX), (XII), or (XVI), or compounds (IV) or (XII), e.g., compound (IV), wherein the neutral lipid may be DSPC or DOPE, the steroid alcohol may be cholesterol, and the PEGylated lipid may be PEG-PE (PEG2000-PE) or PEG-DMG (PEG2000-DMG).

[0167] In another embodiment, the lipid compound disclosed herein may be compound (III), (IV), (V), (VIII), (IX), (XII), (XVI), (XIX), or (XXII), the neutral lipid may be DSPC, the steroid alcohol may be cholesterol, and the PEGylated lipid may be PEG-PE (PEG2000-PE).

[0168] In another embodiment, the lipid compound disclosed herein may be compound (IV), (VIII), (IX), (XII), (XVI), (XIX), or (XXII), the neutral lipid may be DSPC, the steroid alcohol may be cholesterol, and the PEGylated lipid may be PEG-PE (PEG2000-PE).

[0169] In another embodiment, the lipid compound disclosed herein may be compound (IV), (IX), (XII), or (XVI), the neutral lipid may be DSPC, the steroid alcohol may be cholesterol, and the PEGylated lipid may be PEG-PE (PEG2000-PE).

[0170] In another embodiment, the lipid compound disclosed herein may be compound (IV) or (XII), the neutral lipid may be DSPC, the steroid alcohol may be cholesterol, and the PEGylated lipid may be PEG-PE (PEG2000-PE).

[0171] In another embodiment, the lipid compound disclosed herein may be compound (IV), the neutral lipid may be DSPC, the steroid alcohol may be cholesterol, and the PEGylated lipid may be PEG-PE (PEG2000-PE).

[0172] Lipid nanoparticles (LNPs) The present disclosure relates to lipid nanoparticles containing at least one lipid compound and at least one nucleic acid as disclosed herein.

[0173] In one embodiment, the lipid nanoparticles disclosed herein may contain a lipid compound disclosed herein of formula (III)-(XXVII), or compound (III), (IV), (V), (VIII), (IX), (XII), (XVI), (XIX), or (XXII), or compound (IV), (VIII), (IX), (XII), (XVI), (XIX), or (XXII), or compound (IV), (IX), (XII), or (XVI), or compound (IV) or (XII), or examples of formula (III), (IV) or (V), and examples of formula (IV).

[0174] Additionally, the lipid nanoparticles disclosed herein may comprise at least one lipid selected from the group consisting of neutral phospholipids or sphingolipids, steroid alcohols or esters thereof, and PEGylated lipids.

[0175] According to one embodiment, the compositions disclosed herein can be formulated as lipid nanoparticles, as described above.

[0176] The lipid nanoparticles can have a diameter suitable for systemic, e.g., parenteral, or intramuscular, intradermal, or subcutaneous administration. Typically, the lipid nanoparticles have a Z-average size of less than 600 nanometers (nm), e.g., less than 400 nm.

[0177] In one embodiment, the LNP has a Z-average size of less than 200 nm. This size is advantageously compatible with sterile filtration and is optimal for lymphatic transport after intramuscular or subcutaneous administration. This size is also suitable for intravenous administration, as injection of larger particles can induce capillary thrombosis.

[0178] In some embodiments, the lipid nanoparticles can have a Z-average size ranging from about 20 nm to about 300 nm, e.g., from about 20 nm to about 250 nm, e.g., from about 30 nm to about 200 nm, from about 40 nm to about 180 nm, from about 60 nm to about 170 nm, from about 80 nm to about 160 nm, and from about 90 nm to about 150 nm. In one embodiment, the nanoparticles can have a diameter ranging from about 90 nm to about 150 nm.

[0179] The "Z-average size" of lipid nanoparticles can be determined by dynamic light scattering (DLS). The Z-average size or Z-average used in dynamic light scattering is a parameter also known as the cumulant average. It is the primary and most stable parameter generated by this technique. Z-average is defined as the "harmonic intensity average particle size." Z-average size can be measured using a Zetasizer Nano ZS light scattering instrument (Malvern Instruments). To obtain accurate particle sizing with the Nano ZS, the viscosity of the buffer solution and the refractive index of the material must be provided to the instrument software (PBS: v = 1.02 cP, RI = 1.45).

[0180] While slight variations in size may occur during the manufacturing process, variations of up to 20-30% from a given measurement are acceptable and considered to be within a given size range. Alternatively, size can be determined by a filtration screening assay. For example, a particle preparation is below a given size if at least 90%, e.g., at least 95%, e.g., at least 97% of the particles pass through a "screen-type" filter of a given size.

[0181] "Polydispersity index" is a measure of the homogeneous or heterogeneous size distribution of individual lipid nanoparticles in a lipid nanoparticle mixture, and indicates the breadth of particle distribution in the mixture. PI can be determined, for example, as described herein.

[0182] In one embodiment, the polydispersity index of the nanoparticles described herein, as measured by dynamic light scattering, is 0.5 or less, such as 0.4 or less, such as 0.3 or less, or even such as 0.2 or less.

[0183] In one embodiment, the lipid nanoparticles are colloidally stable in the sense that no or substantially no aggregation, precipitation, or increase in size and polydispersity index as measured by dynamic light scattering may be observed over a given period of time, e.g., at least 2 hours to several months, e.g., at least 1, 2, 3, 4, 5, 6, or 12 months.

[0184] The lipid nanoparticles disclosed herein have a pKa in the range of 4.5 to 6.7.

[0185] This pKa can be determined using preformed LNPs composed of the fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) and cationic lipid / DOPE / cholesterol / PEG-lipid (35:16:35:2.5 mol%) at a concentration of approximately 6 mM total lipid in PBS. Briefly, TNS is prepared as a 100 μM stock solution in distilled water. LNPs are diluted to 100 μM total lipid in 90 μL of buffer solution (in triplicate) containing 10 mM HEPES, 10 mM 4-morpholineethanesulfonic acid, 10 mM ammonium acetate, and 130 mM NaCl, with a pH ranging from 2.71 to 11.5. Ten microliters of stock TNS is added to the LNP solution and mixed well in a black 96-well plate. Fluorescence intensity is monitored using excitation and emission wavelengths of 321 and 445 nm on a Tecan Pro200 plate reader. The resulting fluorescence values ​​are used to generate a sigmoidal plot of fluorescence versus buffer pH. The logarithm of the inflection point of this curve is the apparent pKa of the LNP formulation. Such methods are described in detail, for example, in Semple, SC et al., "Rational design of cationic lipids for siRNA delivery." Nat. Biotechnol. 28, 172-176 (2010).

[0186] The lipid nanoparticles can contain or encapsulate at least one nucleic acid.

[0187] The nucleic acid can be encapsulated and / or adsorbed to the outer surface of the lipid nanoparticle. The lipid compound can form a complex with the nucleic acid and / or encapsulate the nucleic acid. Alternatively, the lipid compound can be included in a vesicle that encapsulates the nucleic acid.

[0188] Lipid nanoparticles have an overall surface charge, expressed as the zeta potential, which is the sum of the negative and positive charges on the surface of the particle. The zeta potential is the potential difference between the dispersion medium and the immobile layer of fluid attached to the dispersed particle. The zeta potential is widely used to quantify the magnitude of the charge of the bilayer.

[0189] Zeta potential can be calculated using theoretical models and experimentally determined using electrophoretic mobility measurements or dynamic electrophoretic mobility measurements. Electrophoresis can be used to estimate the zeta potential of particles. In practice, the zeta potential of a dispersion can be measured by applying an electric field to the dispersion. Particles in a dispersion with a zeta potential migrate toward an electrode of opposite charge at a velocity proportional to the magnitude of their zeta potential. This velocity can be measured using laser Doppler anemometry techniques. The frequency or phase shift of an incident laser beam caused by these migrating particles can be measured as particle mobility, which can be converted to zeta potential by inputting the viscosity and dielectric constant of the dispersant and applying Smoluchowski theory. Electrophoretic velocity is proportional to electrophoretic mobility, a measurable parameter. There are several theories relating electrophoretic mobility to zeta potential.

[0190] Zeta potential can be determined using suitable systems such as the Nicomp 380 ZLS system or the Malvern nanoZS. These systems typically measure the electrophoretic mobility and stability of charged particles in suspension. These values ​​are predictive of the repulsive forces exerted by particles in suspension and are directly related to the stability of the colloidal system.

[0191] At neutral pH, the zeta potential of the lipid nanoparticles disclosed herein is close to neutral.

[0192] In one advantage, having a near-zero zeta potential facilitates particle movement within the body, reduces opsonization, and increases access to target tissues.

[0193] In one embodiment, at pH 6.0 to 7.5, the zeta potential of the nanoparticles can range from about −30 mV to about +5 mV, such as from about −20 mV to about 0 mV, and such as from about −10 mV to about 0 mV.

[0194] The lipid nanoparticles described herein can be formed, for example, by adjusting the charge ratio of the lipid compounds disclosed herein (cationic charge from the quaternary ammonium of the lipid compound: N) to the nucleic acid (anionic charge from the phosphate: P) during preparation, and then mixing the lipid compounds with the nucleic acid. The charges of the lipid compounds and the nucleic acid are the charges at a selected pH, such as physiological pH, between about 6.5 and about 7.5.

[0195] The + / - (N / P) charge ratio of the lipids disclosed herein to the nucleic acids in the lipid nanoparticles disclosed herein can be calculated by the following equation: (+ / - charge ratio) = [(amount of cationic lipid (mol)) * (total number of positive charges in the cationic lipid)]: [(amount of nucleic acid (mol)) * (total number of negative charges in the nucleic acid)].

[0196] The amounts of nucleic acid and lipid compound can be easily determined by those skilled in the art, taking into consideration the amounts to be loaded when nanoparticles are prepared.

[0197] According to one embodiment, the ratio of positive to negative charges in nanoparticles suitable for the present disclosure is such that the nanoparticles can have an overall negative charge or an overall neutral or near neutral charge.

[0198] In one embodiment, the charge ratio of positive to negative charges on the nanoparticles ranges from about 4:1 to about 15:1, such as from about 5:1 to about 12:1, such as from about 6:1 to about 9:1, such as from about 6:1 to about 8:1.

[0199] In one embodiment, the lipid nanoparticles disclosed herein that encapsulate nucleic acids may have a Z-average size of about 80-200 nm and a charge ratio N / P of about 4-8:1.

[0200] Lipid nanoparticle manufacturing process The present disclosure relates to methods of producing lipid nanoparticles, eg, lipid nanoparticles comprising at least one nucleic acid, using the lipid compounds disclosed herein.

[0201] In one embodiment, the nucleic acid-containing lipid nanoparticles disclosed herein are prepared by at least the following steps: a) solubilizing at least one lipid compound disclosed herein, e.g., as described above, in a water-miscible organic solvent; b) mixing the organic solvent obtained in step a) with an aqueous solvent containing at least one nucleic acid; and c) obtaining the lipid nanoparticles containing the nucleic acid in an aqueous solvent In one embodiment, the method for producing lipid nanoparticles disclosed herein comprises at least the following steps: a) solubilizing at least one lipid compound disclosed herein and at least one lipid selected from the group consisting of neutral lipids, steroid alcohols or esters thereof, and PEGylated lipids in a water-miscible organic solvent; b) mixing the organic solvent obtained in step a) with an aqueous solvent containing at least one nucleic acid; and c) obtaining the lipid nanoparticles containing the nucleic acid in an aqueous solvent may include:

[0202] The lipid compounds disclosed herein can be present in an amount sufficient to structure the lipid nanoparticles and encapsulate any load to be encapsulated.The amount of ionic lipid compounds used in lipid nanoparticles can be determined by those skilled in the art according to any known technique, and is adapted according to the nature and amount of the load and the nature and amount of other lipids that are likely to be present.

[0203] In one embodiment, step a) further comprises solubilizing at least one lipid selected from the group consisting of neutral lipids, steroid alcohols or esters thereof, and PEGylated lipids in an organic solvent.

[0204] The neutral lipids, steroid alcohols or esters thereof, and PEGylated lipids suitable for this disclosure can be as described herein.

[0205] In another embodiment, step a) may further comprise solubilizing at least one neutral lipid, at least one steroid alcohol or ester thereof, and at least one PEGylated lipid in an organic solvent, wherein the lipid compounds, the neutral lipid, the steroid alcohol or ester thereof, and the PEGylated lipid are present in the organic solvent in molar amounts of about 30% to about 70% lipid compounds, about 0% to about 50% neutral lipid, 20% to about 50% steroid alcohol or ester thereof, and about 1% to about 15% PEGylated compounds, relative to the total amount of lipids and lipid compounds.

[0206] Useful water-miscible organic solvent can be any water-miscible organic solvent that can solubilize the lipid compounds disclosed herein and any other added lipids.Suitable organic solvent examples include ethanol or methanol, 1-propanol, isopropanol, t-butanol, THF, DMSO, acetone, acetonitrile, diglyme, DMF, 1-4 dioxane, ethylene glycol, glycerin, hexamethylphosphoramide, hexamethylphosphorus triamide.In one embodiment, the organic solvent can be ethanol and isopropanol.

[0207] Aqueous solvents that can be used in step b) include aqueous buffer solutions.

[0208] As examples of suitable aqueous buffer solutions, mention may be made of acidic buffer solutions such as citrate buffer, sodium acetate buffer, succinate buffer, borate buffer or phosphate buffer, etc. For example, the aqueous buffer solution may be a citrate buffer solution or an acetate buffer solution.

[0209] The pH of the aqueous solvent may be in the range of about 4.5 to about 7.0, for example, about 5.0 to about 6.5, for example, about 5.5 to about 6.0, for example, about 6.5.

[0210] In step b), the organic solvent and aqueous solvent can be mixed in a ratio of organic solvent:aqueous solvent ranging from about 1:1 to about 1:6, in one embodiment, the ratio ranges from about 1:2 to about 1:4, for example, a ratio of about 1:3.

[0211] According to one embodiment, the organic solvent and the aqueous solvent can be mixed in step b) at a flow rate ranging from about 0.01 ml / min to about 12 ml / min. In some embodiments, the flow rate can range from about 0.02 ml / min to about 10 ml / min, from about 0.5 ml / min to about 8 ml / min, from about 1 ml / min to about 6 ml / min, or about 4 ml / min.

[0212] The mixing step can be performed by any method known in the art. For example, both solvents can be mixed in a T-tube or Y-connector. Alternatively, mixing can be performed by laminar flow mixing using a microfluidic micromixer, as described by Belliveau et al. (2012).

[0213] As shown, the aqueous solvent of step b) comprises a nucleic acid. In one embodiment, the nucleic acid may encode at least one antigen. Suitable nucleic acids may be, for example, those described in detail below.

[0214] The method may optionally further comprise the step of increasing the pH from acidic to neutral.

[0215] In a further embodiment, the method may comprise step d) of increasing the pH of the aqueous solvent containing the lipid nanoparticles obtained in step c) to a pH in the range of about 5.5 to about 7.5, such as about 6.0 to about 7.0, and for example about 6.5 to about 7.0.

[0216] The step of increasing the pH can be accomplished by any method known in the art.

[0217] For example, the change in pH can be brought about by a dialysis or diafiltration step.

[0218] According to one embodiment, step d) of the method disclosed herein may further comprise at least one step of dialysis or diafiltration of the lipid nanoparticles, wherein the dialysis or diafiltration step is carried out against an aqueous medium having a pH in the range of about 5.5 to about 7.5, such as about 6.0 to about 7.0, for example about 6.5.

[0219] The aqueous solvent usable in step d) may further contain a carbohydrate to stabilize the lipid nanoparticles and improve the osmolarity of the solution. Suitable carbohydrates may be sucrose, mannitol, glucose, dextrose, or trehalose. The carbohydrate may be present in an amount of about 5% to about 10%, for example about 8%, based on the total amount of the aqueous solvent.

[0220] According to another embodiment, step d) of the method disclosed herein may comprise at least two steps of dialyzing the lipid nanoparticles. The first dialysis step can be performed against a similar aqueous solvent (similar in terms of pH and content) to remove the organic solvent. The second dialysis step can be performed against a different aqueous solvent (different in terms of pH and, in some cases, content). In such cases, the pH of the dialysis solution may range from about 5.5 to about 7.5, e.g., about 6.0 to about 7.0, e.g., about 6.5. The dialysate for the second dialysis may be a buffer, e.g., a phosphate buffer, a TRIS buffer, a Hepes buffer, a histidine buffer, or a glycine buffer. The osmolarity of the buffer may be adjusted with a salt, such as NaCl, or a carbohydrate, such as glycerol, sucrose, mannitol, glucose, dextrose, or trehalose.

[0221] In one embodiment, for injection of an isotonic solution into the body, the osmolarity is adjusted to reach a final osmolarity close to 290 mOsmol / kg.

[0222] In addition to steps c) and / or d), the method may comprise any further steps suitable for recovering, purifying, concentrating and / or sterilizing the lipid nanoparticles and further formulating them as a pharmaceutical composition, e.g., an immunogenic composition.

[0223] According to one embodiment, the present disclosure relates to lipid nanoparticles obtainable according to the manufacturing method disclosed herein.

[0224] According to another embodiment, the present disclosure provides a method for producing a pharmaceutical composition, comprising at least the following steps: i) mixing at least one nucleic acid with at least one lipid compound disclosed herein, or mixing at least one nucleic acid with at least one composition disclosed herein, or producing at least one lipid nanoparticle according to the methods disclosed herein; and ii) combining the mixed nucleic acid and lipid compound disclosed herein, or the mixed nucleic acid and composition disclosed herein, or the lipid nanoparticles obtained in step i) with at least one pharmaceutically acceptable excipient or carrier. The present invention relates to a method comprising:

[0225] According to another embodiment, the present disclosure provides a method for producing an immunogenic composition, comprising at least the following steps: i) mixing at least one nucleic acid with at least one lipid compound disclosed herein, or mixing at least one nucleic acid with at least one composition disclosed herein, or producing at least one nucleic acid-containing lipid nanoparticle according to a method disclosed herein, wherein the nucleic acid encodes at least one antigen; and ii) combining the mixed nucleic acid and lipid compound disclosed herein, or the mixed nucleic acid and composition disclosed herein, or the lipid nanoparticles obtained in step i) with at least one pharmaceutically acceptable excipient or carrier. The present invention relates to a method comprising:

[0226] Pharmaceutical and immunogenic compositions suitable for this disclosure are described in more detail thereafter.

[0227] In one embodiment, the compositions or lipid nanoparticles disclosed herein can be prepared using the lipid compounds disclosed herein of formula (III)-(XXVII), or compounds (III), (IV), (V), (VIII), (IX), (XII), (XVI), (XIX), or (XXII), or compounds (IV), (VIII), (IX), (XII), (XVI), (XIX), or (XXII), or compounds (IV), (IX), (XII), or (XVI), or compounds (IV) or (XII), or compounds of formula (III), (IV) or (V), for example, and for example, compound (V).

[0228] In another embodiment, the lipid nanoparticles disclosed herein can be produced using a neutral lipid that is DSPC or DOPE, a steroid alcohol that is cholesterol, and a PEGylated lipid that is PEG-PE (PEG2000-PE) or DMG-PEG (DMG-PEG2000).

[0229] In another embodiment, the lipid nanoparticles disclosed herein can be produced using a lipid compound of formula (III)-(XXVII), a neutral lipid that is DSPC, a steroid alcohol that is cholesterol, and a PEGylated lipid that is PEG-PE (PEG2000-PE).

[0230] In another embodiment, the lipid nanoparticles disclosed herein can be produced using a lipid compound of formula (III), (IV), (V), (VIII), (IX), (XII), (XVI), (XIX), or (XXII), a neutral lipid that is DSPC, a steroid alcohol that is cholesterol, and a PEGylated lipid that is PEG-PE (PEG2000-PE).

[0231] In another embodiment, the lipid nanoparticles disclosed herein can be produced using a lipid compound of formula (IV), (VIII), (IX), (XII), (XVI), (XIX), or (XXII), a neutral lipid that is DSPC, a steroid alcohol that is cholesterol, and a PEGylated lipid that is PEG-PE (PEG2000-PE).

[0232] In another embodiment, the lipid nanoparticles disclosed herein can be produced using a lipid compound of formula (IV), (IX), (XII), or (XVI), a neutral lipid that is DSPC, a steroid alcohol that is cholesterol, and a PEGylated lipid that is PEG-PE (PEG2000-PE).

[0233] In another embodiment, the lipid nanoparticles disclosed herein can be produced using a lipid compound of formula (IV) or (XII), a neutral lipid that is DSPC, a steroid alcohol that is cholesterol, and a PEGylated lipid that is PEG-PE (PEG2000-PE).

[0234] In another embodiment, the lipid nanoparticles disclosed herein can be produced using a lipid compound of formula (IV), a neutral lipid that is DSPC, a steroid alcohol that is cholesterol, and a PEGylated lipid that is PEG-PE (PEG2000-PE).

[0235] nucleic acid The compositions or lipid nanoparticles disclosed herein can include at least one anionic or polyanionic therapeutic agent. A suitable therapeutic agent for this disclosure can be a nucleic acid.

[0236] The nucleic acids disclosed herein can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), such as RNA, such as in vitro transcribed RNA (IVT RNA) or synthetic RNA.

[0237] Nucleic acids according to the present disclosure include genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded, and can be in the form of linear or covalently closed circular molecules. Nucleic acids can be used in the form of RNA, which can be prepared, for example, by in vitro transcription from a DNA template, for introduction into cells, i.e., cell transfection. Furthermore, RNA can be modified before application by sequence stabilization, capping, and polyadenylation.

[0238] The nucleic acids may be of eukaryotic or prokaryotic origin, for example, human, animal, plant, bacterial, yeast, or viral origin. The nucleic acids may be obtained by any technique known to those skilled in the art, for example, by library screening, by chemical synthesis, or by mixed methods including chemical or enzymatic modification of sequences obtained by library screening. They may be chemically modified.

[0239] The nucleic acid may be contained in a vector. Vectors are known to those skilled in the art, and may include plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or PI artificial chromosomes (PAC). Vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain the desired coding sequence and appropriate DNA sequences necessary for the expression of an operably linked coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify a specific desired DNA fragment and may lack functional sequences necessary for the expression of the desired DNA fragment.

[0240] In one embodiment, the nucleic acid may be selected from the group consisting of double-stranded RNA (dsRNA), single-stranded RNA (ssRNA); double-stranded DNA (dsDNA); single-stranded DNA (ssDNA); and combinations thereof.

[0241] In one embodiment, the nucleic acid may be selected from the group consisting of messenger RNA (mRNA); antisense oligonucleotide (ASO); short interfering RNA (siRNA); self-amplifying RNA (saRNA); microRNA (miRNA); small nuclear RNA (snRNA); small nucleolar RNA (snoRNA); self-amplifying RNA (saRNA); plasmid DNA (pDNA); closed-end DNA (ceDNA), and combinations thereof.

[0242] In another embodiment, the nucleic acid may be selected from the group consisting of messenger RNA (mRNA); antisense oligonucleotide (ASO); short interfering RNA (siRNA); self-amplifying RNA (saRNA); microRNA (miRNA); plasmid DNA (pDNA); and combinations thereof.

[0243] In another embodiment, the nucleic acid may be selected from the group consisting of messenger RNA (mRNA); short interfering RNA (siRNA); self-amplifying RNA (saRNA); microRNA (miRNA); and combinations thereof.

[0244] In another embodiment, the nucleic acid may be messenger RNA (mRNA).

[0245] In one embodiment, the nucleic acid is mRNA. In certain embodiments, the nucleic acid can be RNA encoding a protein or enzyme. Such polynucleotides can be used as therapeutic agents that can be expressed by target cells to promote the production of functional enzymes or proteins. For example, in certain embodiments, the expression of at least one polynucleotide by target cells produces a functional enzyme or protein that the cell or individual lacks.

[0246] Target cells are cells to which the compositions or lipid nanoparticles disclosed herein can be directed or targeted. Target cells can include specific tissues or organs. In some embodiments, target cells can be hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells (e.g., meningeal cells, astrocytes, motor neurons, cells of the dorsal root ganglion, anterior horn motor neurons), photoreceptor cells (e.g., rods and cones), retinal pigment epithelial cells, secretory cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiac muscle cells, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, antigen-presenting cells such as dendritic cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

[0247] mRNA The term "RNA" includes, e.g., refers to a molecule composed entirely or substantially of, ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group.

[0248] The term includes isolated RNA, such as double-stranded RNA, single-stranded RNA, partially purified RNA, essentially pure RNA, synthetic RNA, or recombinantly produced RNA.

[0249] These may be sequences of natural or artificial origin, such as mRNA (messenger RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), siRNA (silencing RNA), miRNA (microRNA), mtRNA (mitochondrial RNA), shRNA (short hairpin RNA), tmRNA (transfer messenger RNA), vRNA (viral RNA), single-stranded, double-stranded and / or base-paired RNA (ssRNA; dsRNA and bpRNA, respectively), blunt-ended or non-blunt-ended RNA, mature and immature mRNA, coding and non-coding RNA, hybrid sequences of oligonucleotides or synthetic or semi-synthetic sequences, modified or otherwise, and mixtures thereof.

[0250] Thus, they may be messenger RNAs (mRNAs), including mature and immature mRNAs such as mRNA precursors (pre-mRNAs) or heterogeneous nuclear mRNAs (hnRNAs) and mature mRNAs. Thus, the RNA molecules disclosed herein also encompass monocistronic and polycistronic messenger RNAs.

[0251] For clarity, mRNA encompasses any coding RNA molecule that can be translated into a protein by a eukaryotic host. A coding RNA molecule generally refers to an RNA molecule that contains a sequence that encodes a protein of interest and that can be translated by a eukaryotic host, said sequence beginning with a start codon (ATG) and ending with, for example, a stop codon (i.e., TAA, TAG, TGA).

[0252] RNA can be naturally occurring RNA or modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or modification of at least one nucleotide. Such modifications can include, for example, the addition of non-nucleotide material to at least one nucleotide of RNA, for example, to the end or inside of RNA. The nucleotides in RNA molecules can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs can be referred to as analogs or analogs of naturally occurring RNA.

[0253] In one embodiment, the RNA is mRNA (messenger RNA). mRNA is a transcript produced using DNA as a template and may encode a peptide or protein.

[0254] mRNA typically contains a 5' cap, a 5' untranslated region (5-UTR), a protein or peptide coding region, a 3' untranslated region (3'-UTR), and a 3' poly(A) tail. mRNA has a limited half-life within cells and in vitro. For example, mRNA can be produced by in vitro transcription using a DNA template. Alternatively, RNA can be obtained by chemical synthesis. In vitro transcription methodologies are known to those skilled in the art. For example, various in vitro transcription kits are commercially available.

[0255] RNA can be synthesized in vitro in a cell-free system using an appropriate cell extract and an appropriate DNA template. For example, a cloning vector is applied to generate transcripts. The promoter for controlling transcription can be any promoter for any RNA polymerase. Some examples of RNA polymerases include T7, T3, and SP6 RNA polymerase. A DNA template for in vitro transcription can be obtained by cloning a nucleic acid, such as cDNA, and introducing it into an appropriate vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA. For example, a cloning vector is used to generate transcripts, commonly referred to as a transcription vector.

[0256] In one embodiment, the RNA can encode a protein or peptide, i.e., when present in the appropriate environment, e.g., a cell such as an antigen-presenting cell, e.g., a dendritic cell, the RNA can be expressed to produce the protein or peptide that it encodes.

[0257] The stability and translation efficiency of the RNA can be altered as needed. Modification of RNA within this disclosure refers to any modification of RNA that does not naturally occur in said RNA.

[0258] According to a general embodiment, the mRNA disclosed herein has the following general formula: [5' cap]w-[5' UTR]x-[gene of interest]-[3' UTR]y-[poly A]z may comprise or consist of, wherein [5'UTR] and [3'UTR] are untranslated regions (UTRs); wherein [5'UTR] contains a Kozak sequence; where [gene of interest] is any gene encoding a protein of interest; wherein [5' cap] contains a methylguanine nucleotide linked to the mRNA via a 5'-5' linkage; where [polyA] is the poly(A) tail; where w, x, y, and z are the same or different and equal to 0 or 1.

[0259] According to one embodiment, the mRNA disclosed herein has the following general formula: [5' cap]-[5' UTR]-[gene of interest]-[3' UTR]-[polyA] may be composed of In the formula, [5'UTR] and [3'UTR] are untranslated regions, wherein [5'UTR] contains a Kozak sequence; where [gene of interest] is any nucleic acid encoding a protein of interest; wherein [5' cap] contains a methylguanine nucleotide linked to the mRNA via a 5'-5' linkage; where [polyA] is the poly(A) tail.

[0260] It is generally remembered that the Kozak sequence is a consensus sequence that is present on eukaryotic mRNA and plays a key role in the initiation of the translation process. Kozak sequences and Kozak consensus sequences are well known in the art.

[0261] It is also remembered that poly(A) tails are composed of multiple adenosine monophosphates, as is well known in the art. Poly(A) tails are generally generated in a process called polyadenylation, which is a post-translational modification that commonly occurs during the production of mature messenger RNA; such poly(A) tails contribute to the stability and half-life of the mRNA and can be of variable length. For example, poly(A) tails can be equal to or longer than 10A nucleotides, contain equal to or longer than 20A nucleotides, contain equal to or longer than 100A nucleotides, and for example, contain about 120A nucleotides.

[0262] The 3'UTR does not express a protein. The purpose of the 3'UTR is to increase the stability of the mRNA. In one embodiment, the alpha-globin UTR is selected because it is known to be free of instability.

[0263] Advantageously, the sequence corresponding to the gene of interest can be codon-optimized to obtain efficient protein production in the host considered.

[0264] The RNA molecules disclosed herein can be of varying lengths. Thus, they can be short RNA molecules, e.g., RNA molecules shorter than about 100 nucleotides, or long RNA molecules, e.g., longer than about 100 nucleotides, or longer than about 300 nucleotides.

[0265] RNA, such as mRNA, can encompass synthetic or artificial RNA molecules, but can also encompass naturally occurring RNA molecules.

[0266] According to the present disclosure, RNA molecules such as mRNA can include the following species: (i) cap-unmodified RNA molecules; (ii) cap-modified RNA molecules; (iii) uncapped, unmodified RNA molecules; (iv) Uncapped modified RNA molecules.

[0267] Capped and uncapped RNA molecules According to the most common embodiment, a "capped RNA molecule" is an RNA molecule whose 5' end is linked to a guanosine or modified guanosine, e.g., a 7-methylguanosine (m) connected to a 5'-5' triphosphate ester bond. 7 G) or analogs. This definition corresponds to the most widely accepted definition of a 5' cap (e.g., a naturally occurring and / or physiological cap).

[0268] Within the meaning of this disclosure, a "cap analog" includes a 7-methylguanosine (m) attached to a 5'-5' triphosphate bond. 7 G) and therefore also contain a cap that can be substituted without impairing protein expression of the corresponding messenger RNA in a eukaryotic host.

[0269] An example of a cap is m 7 GpppN, m 7 GpppG, m 7 Gpp s pG, m 7 Gpp s p s pG, m 7 Gpp s p s pG, m 7 Gppppm 7 G, m2 7’,3’-O GpppG, m2 7’,2’-O GpppG, m2 7’,2’-O Gpp s p s G, or m2 7’,2’-O Gppp s p s We can mention G.

[0270] Examples of synthetic caps and / or cap analogs include glyceryl, inverted deoxy abasic residues, residue), 4',5' methylene nucleotide, 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thionucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotide, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seconucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moiety, 3'-3'-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2'-inverted abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3' phosphorothioate, phosphorodithioate, or bridged or non-bridged methylphosphonate moiety.

[0271] Other examples of synthetic caps or cap analogs include ARCA cap analogs, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0272] Of note, among the synthetic caps, some of the caps listed above are suitable as analogs, while others are not, potentially interfering with protein expression. Such distinctions are understood by those of skill in the art.

[0273] By way of reference and in a non-limiting manner, anti-reverse cap analog (ARCA) 3'-O-Me-m 7 The structure of the G(5')ppp(5')G cap analog is shown below: [ka]

[0274] The ARCA cap analog is an example of a cap analog used, for example, during in vitro transcription: it converts the 3'OH group (m 7 It is a modified cap in which the 5'-terminal ARCA site (nearest G) is replaced by -OCH3. However, 100% of transcripts synthesized with ARCA at the 5' end are translatable, resulting in a strong stimulatory effect on translation.

[0275] Providing a 5'-cap or 5'-cap analog on RNA can be achieved by in vitro transcription of a DNA template in the presence of the 5'-cap or 5'-cap analog, where the 5'-cap is co-transcriptionally incorporated into the generated RNA strand, or the RNA can be generated, for example, by in vitro transcription, and the 5'-cap can be attached to the RNA post-transcriptionally using a capping enzyme, for example, vaccinia virus capping enzyme.

[0276] "Non-capped RNA molecule" refers to any RNA molecule that does not fall within the definition of "capped RNA molecule."

[0277] Thus, according to a general embodiment, "uncapped mRNA" may refer to an mRNA whose 5' end is not linked to 7-methylguanosine or an analogue as defined above via a 5'-5' triphosphate bond.

[0278] Uncapped RNA molecules, such as messenger RNA, can have a (5')ppp(5'), (5')pp(5'), (5')p(5') or (5')OH terminus. Such RNA molecules are referred to as 5'pppRNA; 5'ppRNA; 5'pRNA; 5' OH For example, the uncapped RNA molecules disclosed herein are messenger 5' ppp RNAs.

[0279] Therefore, if the RNA molecule is a single-stranded RNA molecule, it 5’ppp ssRNA, 5’pp ssRNA;5’p ssRNA; 5’OH They are sometimes abbreviated as ssRNA.

[0280] Thus, if the RNA molecule is a double-stranded RNA molecule, 5’ppp dsRNA; 5’pp dsRNA; 5’p dsRNA; 5’OH They may be abbreviated as dsRNA.

[0281] In one embodiment, the uncapped mRNA disclosed herein is an uncapped single-stranded mRNA.

[0282] According to one embodiment, the uncapped single-stranded mRNA is an uncapped messenger 5’ppp It may be ssRNA.

[0283] In a non-limiting manner, the first base of the uncapped RNA molecule can be either adenosine, guanosine, cytosine, or uridine.

[0284] Thus, the uncapped RNA molecule can be an uncapped RNA molecule with a (5')ppp(5'), (5')pp(5'), (5')p(5'), or even a blunt ended 5' guanosine tail.

[0285] In one embodiment of the present disclosure, the RNA may be free of uncapped 5'-triphosphates. Removal of such uncapped 5'-triphosphates can be achieved by treating the RNA with a phosphatase.

[0286] Modified and unmodified RNA molecules RNA may also comprise further modifications.For example, the further modifications of the RNA used in the present disclosure may be the extension or truncation of naturally occurring poly(A) tail, or the modification of 5' or 3' untranslated region (UTR), for example, the introduction of a UTR that is not related to the coding region of the RNA, for example, the replacement or insertion of at least one, for example, two copies of the 3'-UTR from a globin gene, such as alpha2-globin, alpha1-globin, beta globin, for example beta globin, and for example human beta globin.

[0287] Within this disclosure, a "modified RNA molecule" refers to an RNA molecule containing at least one modified nucleotide, nucleoside, or base, such as a modified purine or modified pyrimidine. The modified nucleoside or base can be any nucleoside or base that is not A, U, C, or G (for nucleosides, adenosine, uridine, cytidine, or guanosine, respectively; when referring to the sugar moiety only, adenine, uracil, cytosine, or guanine).

[0288] Thus, an "unmodified RNA molecule" refers to any RNA molecule that does not correspond to the definition of a modified RNA molecule.

[0289] In the sense of the present disclosure, the terms "modified and unmodified" are considered separate from the terms "capped and uncapped," since the latter, in the sense of the present disclosure, specifically relate to the base at the 5' end of an RNA molecule.

[0290] In one embodiment, the nucleic acid, e.g., RNA, can contain at least one modified nucleotide, e.g., a modified ribonucleotide. The presence of the modified nucleotide can increase the stability and / or reduce cytotoxicity of the nucleic acid.

[0291] The term RNA stability relates to the half-life of RNA, that is, the period required to remove half of the activity, amount, or number of molecules.In the context of the present disclosure, the half-life of RNA is an indicator of the stability of said RNA.The half-life of RNA may affect the expression period of RNA.RNA with a long half-life can be expected to be expressed for a long time.

[0292] According to one embodiment, "modified RNA molecule" refers to an RNA molecule, such as an mRNA, that contains at least one base or sugar modification as described above and at least one base modification, e.g., as described herein.

[0293] For example, in one embodiment, 5-methylcytidine can partially or completely, e.g., completely, replace cytidine in a RNA suitable for the present disclosure. Alternatively or additionally, in one embodiment, 5-methylcytidine can partially or completely, e.g., completely, replace uridine.

[0294] In a non-limiting manner, examples of modified nucleotides, nucleosides, and bases are disclosed in WO2015 / 024667A1.

[0295] Thus, modified RNA molecules may contain modified nucleotides, nucleosides or bases, including backbone modifications, sugar modifications or base modifications.

[0296] Backbone modifications relevant to this disclosure include those in which the backbone phosphates of the nucleotides contained in the RNA molecules as defined herein are chemically modified.

[0297] Sugar modifications relevant to this disclosure include chemical modifications of the sugars of the nucleotides of an RNA molecule as defined herein.

[0298] Base modifications relevant to the present disclosure include chemical modifications of the base moiety of a nucleotide of an RNA. In this context, the nucleotide analog or modification is selected from nucleotide analogs suitable for transcription and / or translation of an RNA molecule in, for example, a eukaryotic cell.

[0299] Sugar modifications consist of substitution or modification of the 2' hydroxy (OH) group, which can be modified or substituted with several different "oxy" or "deoxy" substituents.

[0300] Examples of "oxy"-2' hydroxyl group modifications include, but are not limited to, alkoxy or aryloxy (-OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), -O(CH2CHO)nCH2CH2OR; "locked" nucleic acids (LNA) in which the 2' hydroxyl is linked, e.g., by a methylene bridge, to the 4' carbon of the same ribose sugar; and amino groups (-O-amino, where the amino group, e.g., NRR, can be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxy.

[0301] The "deoxy" modification includes hydrogen, amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or the amino group can be attached to the sugar via a linker, where the linker includes at least one of the atoms C, N, and O.

[0302] The sugar group can also contain at least one carbon that has the opposite stereochemical configuration to the corresponding carbon in ribose. Thus, modified RNAs can include nucleotides that contain, for example, arabinose as the sugar.

[0303] As described herein, the phosphate backbone can be further modified and incorporated into modified RNA molecules.The phosphate group of the backbone can be modified by replacing at least one of the oxygen atoms with different substituents.In addition, modified nucleosides and modified nucleotides can comprise the complete replacement of unmodified phosphate moieties with modified phosphates as described herein.

[0304] Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate, and phosphotriester.Phosphorodithioate has both non-linked oxygens replaced with sulfur.Phosphate linker can also be modified by replacing linking oxygen with nitrogen (bridged phosphoramidate), sulfur (bridged phosphorothioate) and carbon (bridged methylene phosphonate).

[0305] As described herein, the modified nucleoside and nucleotide that can be incorporated into modified RNA molecules can be further modified in nucleobase portion.For example, the nucleoside and nucleotide described herein can be chemically modified on the major groove surface.In some embodiments, the chemical modification of the major groove can comprise an amino group, a thiol group, an alkyl group, or a halo group.

[0306] For example, nucleotide analogs / modifications include 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine-riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methylinosine-5'-triphosphate 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate Phosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate Phosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, or puromycin-5'-triphosphate, and xanthosine-5'-triphosphate.

[0307] In some embodiments, the modified nucleoside is pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4 ...carboxymethyl-uridine, 1-carboxymethyl-uridine, 1- It may be selected from the list consisting of uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine / 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

[0308] In some embodiments, modified nucleosides and nucleotides include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1 2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

[0309] In other embodiments, modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6- (cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

[0310] In other embodiments, modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0311] In some embodiments, the nucleotide can be modified in the major groove face and can include replacing the hydrogen on C-5 of uracil with a methyl or halo group.

[0312] Modified bases and / or modified RNA molecules are known in the art and are further taught, for example, in Warren et al. ("Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA"; Cell Stem Cell; 2010).

[0313] In view of the above, the modified base may be a modified purine base or a modified pyrimidine base.

[0314] In a non-limiting manner, examples of modified purine bases include modified adenosines and / or modified guanosines, such as hypoxanthine; xanthine; 7-methylguanine; inosine; xanthosine and 7-methylguanosine.

[0315] According to some embodiments, the modified RNA molecule or mRNA corresponds to RNA in which each nucleoside corresponding to either uridine, cytidine, adenosine and / or ribothymidine is modified.

[0316] In a non-limiting manner, examples of modified pyrimidine bases include modified cytidines and / or modified uridines, such as 5,6-dihydrouracil; pseudouridine; 5-methylcytidine; 5-hydroxymethylcytidine; dihydrouridine, and 5-methylcytidine.

[0317] In a non-limiting manner, the modified bases disclosed herein can be modified uridine or cytidine, such as pseudouridine and 5-methylcytidine.

[0318] According to some embodiments, the modified RNA corresponds to RNA in which at least one base corresponding to either U (for uracil), C (for cytosine), A (for adenine) and / or T (for thymine) has been modified.

[0319] Examples of modified bases include methyl-5 uridine (m5U), 2-thio-uridine (s2U), 2'-O-methyl-5 uridine (Ome5U), pseudouridine (ψ), methyl-1 pseudouridine (m1ψ), methyl-5 cytosine (m5C), 2'O-methyl-5 cytosine (Om5C), N6-methyl-adenosine (m6A), and N1-methyl-adenosine (m6A).

[0320] According to some embodiments, the modified mRNA may include 2'-O-methyl-5 uridine (Ome5U) or methyl-1 pseudouridine (m1ψ) as modified bases.

[0321] Capped and uncapped mRNAs, whether modified or unmodified, can also be obtained commercially.

[0322] RNA with an unmasked polyA sequence is translated more efficiently than RNA with a masked polyA sequence.

[0323] The term "poly(A) tail" or "poly(A) sequence" refers to a sequence of adenyl (A) residues typically located at the 3' end of an RNA molecule, and "unmasked poly(A) sequence" means that the poly(A) sequence at the 3' end of the RNA molecule ends with the A of the poly(A) sequence and is not followed by any nucleotides other than A located at the 3' end, i.e., downstream of the poly(A) sequence. Furthermore, a long poly(A) sequence of approximately 120 base pairs provides optimal transcript stability and RNA translation efficiency.

[0324] Thus, to increase the stability and / or expression of RNA used in accordance with the present disclosure, the RNA can be modified to include a polyA sequence having, for example, a length of 10-500, for example, 30-300, further for example, 65-200, and for example, 100-150 adenosine residues. In one embodiment, the polyA sequence has a length of approximately 120 adenosine residues. To further increase the stability and / or expression of RNA used in accordance with the present disclosure, the polyA sequence can be unmasked.

[0325] In addition, incorporating a 3' untranslated region (UTR) into the 3' untranslated region of an RNA molecule can improve translation efficiency. By incorporating two or more such 3' untranslated regions, a synergistic effect can be achieved. The 3' untranslated regions can be autologous or heterologous to the RNA into which they are introduced. In one embodiment, the 3' untranslated region is derived from the human β-globin gene.

[0326] The combination of the above modifications, ie, incorporation of a polyA sequence, unmasking of a polyA sequence, and incorporation of at least one 3' untranslated region, has a synergistic effect on increasing RNA stability and translation efficiency.

[0327] To increase expression of RNA used in accordance with the present disclosure, the RNA can be modified within the coding region, i.e., the sequence encoding the expressed peptide or protein, for example, to increase GC content, increase mRNA stability, and perform codon optimization without altering the sequence of the expressed peptide or protein, thus enhancing translation within the cell.

[0328] It is understood that the uncapped RNA molecule can be either a modified RNA molecule or an unmodified RNA molecule.

[0329] Thus, the capped RNA molecule can be either a modified or an unmodified RNA molecule.

[0330] In one embodiment, the RNA molecules disclosed herein are messenger RNA (mRNA).

[0331] The RNA molecules disclosed herein are, for example, uncapped messenger RNA, in modified or unmodified form.

[0332] The RNA molecules disclosed herein are, for example, capped messenger RNAs, in modified or unmodified form.

[0333] In a non-limiting manner, an uncapped RNA molecule, such as a messenger RNA, may be an uncapped RNA molecule having only naturally occurring bases.

[0334] According to the present disclosure, "naturally occurring base" refers to a base that can be naturally incorporated in vivo into an RNA molecule, such as a messenger RNA, by a host. Therefore, a "naturally occurring base" is different from a synthetic base that has no natural equivalent in said host. However, a "naturally occurring base" may or may not be a modified base, and both terms should not be confused in the sense of the disclosure.

[0335] The uncapped messenger RNA may also be a modified messenger RNA that is not capped and therefore contains at least one modified base.

[0336] Thus, the uncapped messenger RNA may be an uncapped modified messenger RNA having a (5')ppp(5') guanosine terminus and containing at least one modified base.

[0337] The uncapped messenger RNA may also be an uncapped modified messenger RNA having a (5')ppp(5') guanosine tail and containing at least one pseudouridine and at least one 5-methylcytosine.

[0338] Capped messenger RNA may be messenger RNA whose 5' end is linked to 7-methylguanosine or an analogue, connected to a 5'-5' triphosphate bond, and containing naturally occurring bases or modified bases such as pseudo-urine or 5-methylcytosine.

[0339] It is also understood that when both modified and unmodified RNA molecules are used within an embodiment of the present disclosure, they may be used as a mixture and / or in purified form.

[0340] antigen According to one embodiment, the compositions disclosed herein, such as lipid nanoparticles, may be nucleic acid immunogenic compositions or nucleic acid vaccines comprising at least one polynucleotide, e.g., a polynucleotide construct encoding at least one wild-type or engineered antigen.

[0341] The antigen-containing compositions disclosed herein may vary in their valency. Valency refers to the number of antigen components in a composition or polynucleotide (e.g., RNA polynucleotide) or polypeptide. In some embodiments, immunogenic compositions are monovalent. They may also be compositions containing multiple valencies, such as bivalent, trivalent, or multivalent compositions. Multivalent immunogenic compositions or vaccines may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more antigens or antigenic moieties (e.g., antigenic peptides, etc.). The antigenic components may be on a single polynucleotide or on separate polynucleotides.

[0342] The compositions disclosed herein can be used to protect against, treat, or cure infections resulting from contact with infectious agents such as bacteria, viruses, fungi, protozoans, and parasites.

[0343] The compositions disclosed herein can be used to protect against, treat, or cure cancer diseases.

[0344] According to one embodiment, the nucleic acid may encode at least one antigen selected from the group consisting of a bacterial antigen, a protozoan antigen, a viral antigen, a fungal antigen, a parasitic antigen or a tumor antigen.

[0345] bacterial antigen The bacteria described herein can be gram-positive or gram-negative bacteria. Bacterial antigens include Acinetobacter baumannii, Bacillus anthracis, Bacillus subtilis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, and Chlamydophila psittaci. psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Coagulase Negative Staphylococcus, Corynebacterium diphtheria, Enterococcus faecium, Enterococcus faecium, Corynebacterium diphtheria, Escherichia coli (ETEC), Enterotoxigenic Escherichia coli (ETEC), Enterotoxigenic E. coli, E.coli O157:H7, Enterobacter spp., Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Proteus mirabilis mirabilis), Proteus sps.), Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Serratia marcesens, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus It can be obtained from Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholera, and Yersinia pestis.

[0346] Viral antigens Viral antigens include adenovirus, herpes simplex type 1, herpes simplex type 2, encephalitis virus, papillomavirus, varicella-zoster virus, Epstein-Barr virus, human cytomegalovirus, and human herpesvirus type 8. Human papillomavirus; BK virus; JC virus; smallpox; poliovirus, hepatitis B virus; human bocavirus; parvovirus B19; human astrovirus; Norwalk virus; coxsackievirus; hepatitis A virus; poliovirus; rhinovirus; severe acute respiratory syndrome virus; hepatitis C virus; yellow fever virus; dengue virus; West Nile virus; rubella virus; hepatitis E virus; human immunodeficiency virus (HIV); influenza virus, type A or B; Guanarito virus; Junin virus; Lassa virus; Machupo virus; Sabia virus; Crimean-Congo hemorrhagic fever virus; Ebola virus; Marburg virus; measles virus; mumps virus; parainfluenza virus; respiratory syncytial virus; human metapneumovirus; Hendra virus; Nipah virus; rabies virus; hepatitis D; rotavirus; orbivirus; coltivirus; hantavirus, Middle East respiratory coronavirus. It can be derived from SARS-Cov-2 virus; Chikungunya virus; Zika virus; Parainfluenza virus; Human enterovirus; Hantavirus; Japanese encephalitis virus; Varicella virus; Eastern equine encephalitis; or Banna virus.

[0347] In one embodiment, the antigen is derived from a strain of influenza A or influenza B virus or a combination thereof. The influenza A or influenza B strain may be associated with birds, pigs, horses, dogs, humans, or non-human primates.

[0348] The nucleic acid can encode a hemagglutinin protein or a fragment thereof. The hemagglutinin protein can be H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18, or a fragment thereof. The hemagglutinin protein may or may not include a head domain (HA1). Alternatively, the hemagglutinin protein may or may not include a cytoplasmic domain.

[0349] For example, in embodiments, the hemagglutinin protein is a truncated hemagglutinin protein. The truncated hemagglutinin protein may include a portion of the transmembrane domain.

[0350] In some embodiments, the virus may be selected from the group consisting of H1N1, H3N2, H7N9, H5N1 and H10N8 viruses or B strain viruses.

[0351] In another embodiment, the antigen is derived from a coronavirus, such as SARS-Cov-1 virus, SARS-Cov-2 virus, or MERS-Cov virus.

[0352] fungal antigen Fungal antigens include those from the phylum Ascomycota (e.g., Fusarium oxysporum, Pneumocystis jirovecii, Aspergillus spp., Coccidioides immitis / posadasii, Candida albicans), Basidiomycota (e.g., Filobasidiella neoformans, Trichosporon), Microsporidia (e.g., Encephalitozoon cuniculi, Enterocytozoon bienui), and the phylum Enterocytozoon spp. bieneusi), and the subphylum Mucoromycotina (e.g., Mucor circinelloides, Rhizopus oryzae, Lichtheimia corymbifera).

[0353] Protozoan antigens Protozoan antigens can be derived from Entamoeba histolytica, Giardia lamlia, Trichomonas vaginalis, Toxoplasma gondii, Trypanosoma brucei, T. cruzi, Leishmania donovani, Balantidium coli, Toxoplasma gondii, Plasmodium spp, and Babesia microti.

[0354] parasitic antigen Parasitic antigens include Acanthamoeba, Anisakis, Ascaris lumbricoides, botfly, Balantidium coli, bedbugs, tapeworms, chigger mites, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamlia, hookworms, Leishmania, Linguatula serrata, liver fluke, Dirofilaria loa, Paragonimus, pinworms, and Plasmodium falciparum. falciparum, Schistosoma, Strongyloides stercoralis, tapeworm, Toxoplasma gondii, Trypanosoma, Trichuris, and Wuchereria bancrofti.

[0355] tumor antigens In one embodiment, the antigen may be a tumor antigen, i.e., a component of cancer cells, such as a protein or peptide expressed in cancer cells.The term "tumor antigen" refers to, for example, a protein that is specifically expressed in a limited number of tissues and / or organs or at a specific developmental stage under normal conditions, and is expressed or abnormally expressed in at least one tumor or cancer tissue.Tumor antigens include, for example, differentiation antigens, such as cell type-specific differentiation antigens, i.e., proteins that are specifically expressed in a specific cell type at a specific differentiation stage under normal conditions, and germ line-specific antigens.For example, tumor antigens are presented by the cancer cells that express them.

[0356] For example, tumor antigens include carcinoembryonic antigen, 1-fetoprotein, isoferritin, and fetal sulphoglycoprotein, cc2-H-ferroprotein, and gamma-fetoprotein.

[0357] Other examples of tumor antigens that may be useful in the present disclosure include p53, ART-4, BAGE, beta-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, cell surface proteins of the claudin family, such as CLAUDIN-6, CLAUDIN-18.2 and CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap 100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, myosin / m, MUC1 , MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor BCR-abL, Pm1 / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVrVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, and WT, for example, WT-1.

[0358] Adjuvants The nucleic acid-containing compositions or lipid nanoparticles disclosed herein may further comprise or be co-administered with an adjuvant or immune enhancing agent.

[0359] Adjuvants useful in the present disclosure include, but are not limited to, natural or synthetic adjuvants, which may be organic or inorganic.

[0360] Adjuvants can be selected from any of the following classes: (1) inorganic salts, such as aluminum hydroxide and aluminum phosphate or calcium phosphate gels; (2) oil emulsions and surfactant-based formulations, such as microfluidized detergent-stabilized oil-in-water emulsions, emulsions containing purified saponins, oil-in-water emulsions, stabilized water-in-oil emulsions; (3) particulate adjuvants, such as virosomes (unilamellar liposomal vehicles incorporating influenza hemagglutinin), structured complexes of saponins and lipids, polylactide-co-glycolides (PLGs); (4) microbial derivatives; (5) endogenous human immunomodulators; and / or (6) inert vehicles such as gold particles; (7) microbially derived adjuvants; (8) tensioactive compounds; (9) carbohydrates; or combinations thereof.

[0361] The selection of an appropriate adjuvant and an appropriate amount of adjuvant will be apparent to one skilled in the art.

[0362] Specific adjuvants include, but are not limited to, cationic liposome-DNA complex JVRS-100, aluminum hydroxide vaccine adjuvant, aluminum phosphate vaccine adjuvant, aluminum potassium sulfate adjuvant, Alhydrogel, ISCOM™, Freund's complete adjuvant, Freund's incomplete adjuvant, CpG DNA vaccine adjuvant, cholera toxin, cholera toxin B subunit, liposomes, saponin vaccine adjuvant, DDA adjuvant, squalene-based adjuvant, Etx B subunit adjuvant, IL-12 vaccine adjuvant, LTK63 vaccine mutant adjuvant, TiterMax Gold adjuvant, Ribi vaccine adjuvant, and Montanide. ISA720 adjuvant, Corynebacterium-derived (-derb / ed) P40 vaccine adjuvant, MPL™ adjuvant, AS04, AS02, AS01, lipopolysaccharide vaccine adjuvant, muramyl dipeptide adjuvant, CRL1005, killed Corynebacterium parvum vaccine adjuvant, Montanide ISA 51, Bordetella pertussis component vaccine adjuvant, cationic liposomal vaccine adjuvant, adamantylamide dipeptide vaccine adjuvant, Arlacel A, VSA-3 adjuvant, aluminum vaccine adjuvant, Polygen vaccine adjuvant, Adjumer™, algal glucan, Bay R1005, Theramide®, stearyl tyrosine, Specol, Algamulin, Avridine®, calcium phosphate gel, CTA1-DD gene fusion protein, DOC / Alum complex, gamma inulin, Gelb adjuvant, GM-CSF, GMDP, recombinant hIFN-γ / interferon-g, interleukin-1β, interleukin-2, interleukin-7, Sclavo peptide, Rehydragel LV, RehydragelHPA, Loxoribine, MF59, MTP-PE liposome, Murametide, Murapalmitin, D-Murapalmitin, NAGO, Nonionic surfactant vesicle, PMMA, PAA, Protein cholesterol, QS-21, SPT (antigen preparation), Nanoemulsion vaccine adjuvant, AS03, Quil-A vaccine adjuvant, RC529 vaccine adjuvant, LTR192G vaccine adjuvant, E. coli heat-labile toxin, LT, Amorphous aluminum hydroxyphosphate sulfate adjuvant, Calcium phosphate vaccine adjuvant, Seppic Montanide incomplete adjuvant, Imiquimod, Resiquimod, AF03, Flagellin, Poly(LC), ISCOMATRIX®, Abisc Examples of vaccine adjuvants include o-100 vaccine adjuvant, albumin-heparin microparticle vaccine adjuvant, AS-2 vaccine adjuvant, B7-2 vaccine adjuvant, DHEA vaccine adjuvant, immunoliposomes containing antibodies against costimulatory molecules, SAF-1, Sendai proteoliposomes, Sendai-containing lipid matrices, threonylmuramyl dipeptide (TMDP), Ty particle vaccine adjuvant, bupivacaine vaccine adjuvant, DL-PGL (polyester poly(DL-lactide-co-glycolide)) vaccine adjuvant, IL-15 vaccine adjuvant, LTK72 vaccine adjuvant, MPL-SE vaccine adjuvant, the non-toxic mutant E112K of cholera toxin mCT-E112K and / or Matrix-S.

[0363] Protein expression The composition disclosed herein or the lipid nanoparticles disclosed herein that encapsulate at least one nucleic acid can also be used to treat individuals who are deficient in protein.Therefore, lipid nanoparticles can be used in the method for treating individuals who are deficient in protein, comprising administering lipid nanoparticles that contain at least one nucleic acid, for example, mRNA, wherein the nucleic acid encodes the functional protein corresponding to the protein that is deficient in individuals.In embodiments, functional protein is produced following the expression of nucleic acid by target cells.

[0364] The present disclosure also relates to methods for intracellular delivery of nucleic acids that can correct an existing genetic defect and / or provide a beneficial function to at least one target cell. After successful delivery to a target tissue and cell, the compositions and nucleic acids of the present disclosure are transfected into the target cell, and the nucleic acid (e.g., mRNA) can be translated into a gene product of interest (e.g., a functional protein or enzyme) or otherwise modulate or control the presence or expression of the gene product of interest.

[0365] The compositions and methods provided herein are useful for the management and treatment of multiple diseases, for example, diseases caused by protein and / or enzyme deficiency.The individuals suffering from such diseases may have underlying genetic defects that lead to impaired protein or enzyme expression, including, for example, non-protein synthesis, reduced protein synthesis, or synthesis of proteins that lack or have reduced biological activity.

[0366] Alternatively, the nucleic acid can encode a full-length antibody or a smaller antibody (e.g., both heavy and light chains) to confer immunity to a subject. In an alternative embodiment, the compositions of the present disclosure encode antibodies that can be used to produce a transient or long-term functional response in a subject. For example, the mRNA nucleic acids of the present disclosure can encode functional monoclonal or polyclonal antibodies, which, upon translation (and, if applicable, upon systemic excretion from target cells), may be useful for targeting and / or inactivating biological targets (e.g., stimulatory cytokines such as tumor necrosis factor). Similarly, the mRNA nucleic acids of the present disclosure may encode functional anti-nephritic factor antibodies useful, for example, for treating membranoproliferative glomerulonephritis type II or acute hemolytic uremic syndrome, or may encode anti-vascular endothelial growth factor (VEGF) antibodies useful for treating VEGF-mediated diseases such as cancer.

[0367] Pharmaceutical Composition According to some embodiments, the present disclosure relates to pharmaceutical compositions.

[0368] For purposes of administration, the lipid compounds of the present disclosure can be formulated as lipid nanoparticles together with a therapeutic agent, such as a nucleic acid, and administered as a pharmaceutical composition. The pharmaceutical composition of the present disclosure comprises the lipid compounds disclosed herein and, possibly, at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0369] According to some embodiments, pharmaceutical compositions suitable for the present disclosure may comprise an effective amount of (i) at least one nucleic acid and at least one lipid compound disclosed herein, or (ii) at least one nucleic acid and at least one composition described herein, or (iii) at least one nucleic acid containing lipid nanoparticles described herein, and at least one pharmaceutically acceptable excipient.

[0370] The pharmaceutical composition disclosed herein can be an immunogenic composition.The immunogenic composition suitable for the present disclosure can comprise an effective amount of (i) at least one nucleic acid and at least one lipid compound disclosed herein, or (ii) at least one nucleic acid and at least one composition described herein, or (iii) at least one nucleic acid containing lipid nanoparticles described herein, wherein at least one nucleic acid encodes at least one antigen, and at least one pharmaceutically acceptable excipient.In addition, the immunogenic composition can comprise an adjuvant described herein.

[0371] According to some embodiments, the present disclosure relates to a composition for use as a medicament, comprising (i) at least one nucleic acid and at least one lipid compound according to the present disclosure, or (ii) at least one nucleic acid and at least one composition described herein, or (iii) at least one nucleic acid containing at least one lipid nanoparticle described herein. Such a medicament can be used for the prevention and / or treatment of the diseases described herein.

[0372] According to some embodiments, the present disclosure relates to a composition comprising (i) at least one nucleic acid and at least one lipid compound according to the present disclosure, or (ii) at least one nucleic acid and at least one composition described herein, or (iii) at least one nucleic acid containing at least one lipid nanoparticle described herein, for use in a therapeutic method for preventing and / or treating a disease selected from the group consisting of an infectious disease, an allergy, an autoimmune disease, a rare blood disease, a rare metabolic disease, a rare neurological disease, and a tumor or cancer disease.

[0373] According to some embodiments, a composition comprising (i) at least one nucleic acid and at least one lipid compound according to the present disclosure, or (ii) at least one nucleic acid and at least one composition described herein, or (iii) at least one nucleic acid containing at least one lipid nanoparticle described herein, wherein the nucleic acid encodes at least one antigen, may be for use as an immunogenic composition.

[0374] The immunogenic compositions disclosed herein can be used in the prevention and / or treatment of the infectious diseases set forth herein, and may contain nucleic acids encoding the antigens described herein.

[0375] In some embodiments, the lipid compounds of formula (I) may be present in the pharmaceutical or immunogenic composition in an amount effective to form lipid nanoparticles and deliver a therapeutic agent, e.g., a nucleic acid, to treat a disease or condition of interest.

[0376] Appropriate concentrations and dosages can be readily determined by one skilled in the art.

[0377] Administration of the pharmaceutical and immunogenic compositions disclosed herein can be via any of the accepted modes of administration for compositions to provide similar utilities.

[0378] The compositions disclosed herein can be formulated into solid, semi-solid, or liquid forms, such as powder, solution, suspension, or injection.The typical routes of administering such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, and nasal.The term parenteral used herein includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques.

[0379] In some embodiments, the compositions disclosed herein can be administered by transdermal, subcutaneous, intradermal, or intramuscular routes.

[0380] The compositions disclosed herein are formulated so as to allow the active ingredients therein to be bioavailable upon administration of the composition to a patient.

[0381] Actual methods for preparing such dosage forms will be known, or apparent, to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).

[0382] The composition may contain at least one inert diluent or carrier.

[0383] In one embodiment, the composition may be in the form of a liquid, such as a solution, emulsion, or suspension. The liquid may be for delivery by injection. The composition intended for administration by injection may contain at least one of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents. The liquid composition disclosed herein may include at least one of the following: a sterile diluent such as water for injection; a physiological saline solution, such as saline, Ringer's solution, isotonic sodium chloride; a fixed oil such as synthetic mono- or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can serve as a solvent or suspension medium; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate, and an agent for adjusting tonicity such as sodium chloride or dextrose; or an agent that acts as a cryoprotectant such as sucrose or trehalose.

[0384] The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical compositions for injection are, for example, sterile.

[0385] The pharmaceutical compositions and immunogenic compositions disclosed herein can be prepared by methods well known in the pharmaceutical field.For example, pharmaceutical compositions intended for administration by injection can be prepared by combining the lipid nanoparticles disclosed herein with distilled water or other carriers to form a solution.A surfactant may be added to promote the formation of a homogeneous solution or suspension.

[0386] The compositions disclosed herein are administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the particular therapeutic agent used; the metabolic stability and length of action of the therapeutic agent; the age, weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject being treated.

[0387] The compositions disclosed herein can also be administered simultaneously with, before, or after the administration of at least one other therapeutic agent.Such combined therapy includes the administration of the compositions disclosed herein and at least one additional active agent in a single pharmaceutical dosage formulation, and the administration of the compositions disclosed herein and each active agent in their own separate pharmaceutical dosage formulations.When separate dosage formulations are used, the compositions disclosed herein and at least one additional active agent can be administered essentially simultaneously, i.e., simultaneously, or separately with a time lag, i.e., sequentially; combined therapy is understood to include all these regimens.

[0388] Treatment method In some embodiments, the present invention relates to a method for preventing and / or treating a disease in an individual in need thereof, comprising administering to the individual an effective amount of (i) at least one nucleic acid and at least one lipid compound disclosed herein, or (ii) at least one composition described herein containing a nucleic acid, or (iii) at least one lipid nanoparticle described herein containing a nucleic acid. For example, the compositions containing the LNPs disclosed herein may be for use in therapeutic methods for preventing and / or treating infectious diseases, allergies, autoimmune diseases, rare blood diseases, rare metabolic diseases, rare neurological diseases, and tumor or cancer diseases.

[0389] For example, the disease to which the present disclosure relates may be an infectious disease such as a viral infection, a bacterial infection, a fungal infection or a parasitic infection. Also, the disease to which the present disclosure relates may be a cancer or tumor disease.

[0390] Viral infections include acute febrile pharyngitis, pharyngoconjunctival fever, epidemic keratoconjunctivitis, infantile gastroenteritis, Coxsackie disease, infectious mononucleosis, Burkitt's lymphoma, acute hepatitis, chronic hepatitis, liver cirrhosis, hepatocellular carcinoma, primary HSV-1 infection (e.g., gingivostomatitis in children, tonsillitis and pharyngitis, keratoconjunctivitis in adults), latent HSV-1 infection (e.g., herpes labialis and cold sores), primary HSV-2 infection, latent HSV-2 infection, aseptic meningitis, infectious mononucleosis, cytomegalic inclusion disease, Kaposi's sarcoma, and multicentric Castleman disease. , primary effusion lymphoma, AIDS, influenza, Reye's syndrome, measles, post-infectious encephalomyelitis, mumps, hyperplastic epithelial lesions (e.g., common, flat, plantar, and anogenital warts, laryngeal papillomas, epidermodysplasia verruciformis), cervical cancer, squamous cell carcinoma, croup, pneumonia, bronchiolitis, colds, polio, rabies, bronchiolitis, pneumonia, influenza-like syndrome, severe bronchiolitis with pneumonia, rubella, congenital rubella, chickenpox, Covid-19, respiratory syncytial virus (RSV) infection, and shingles.

[0391] In one embodiment, the disease is influenza, respiratory syncytial virus (RSV) infection, or Covid-19, e.g., influenza.

[0392] Examples of bacterial infections include abscesses, actinomycosis, acute prostatitis, Aeromonas hydrophila infection, annual ryegrass poisoning, anthrax, bacterial hepatic purpura, bacteremia, bacterial gastroenteritis, bacterial meningitis, bacterial pneumonia, bacterial vaginosis, bacterial-associated skin diseases, bartonellosis, BCG-oma, botryomycosis, botulism, Brazilian spotted fever, Brody abscess, brucellosis, Buruli ulcer, Campylobacter infection, caries, Carrion disease, cat scratch disease, cellulitis, chlamydial infection, cholera, chronic bacterial prostatitis, chronic recurrent multifocal osteomyelitis, clostridial necrotizing enteritis, mixed periodontal-endodontic lesions, contagious bovine pleuropneumonia, and diphtheria. ria, diphtheria stomatitis, ehrlichiosis, erysipelas, epiglottitis, erysipelas, Fitz-Hugh-Curtis syndrome, flea-borne spotted fever, footrot (infectious foot dermatitis), Galley's sclerosing osteomyelitis, gonorrhea, inguinal sarcoma, human granulocytic anaplasmosis, human monocytotropic ehrlichiosis, whooping cough, impetigo, late congenital syphilitic eye disease, Legionnaires' disease, Lemierre's syndrome, leprosy, leptospirosis, listeriosis, Lyme disease, lymphadenitis, melioidosis, meningococcal disease, meningococcal septicemia, methicillin-resistant Staphylococcus aureus (MRSA) infection, Mycobacterium avium intracellulare (mycobacterium avium intracellulare (MAI), Mycoplasma pneumonia, necrotizing fasciitis, nocardiosis, waterborne disease (waterborne or gangrenous stomatitis), omphalitis, orbital cellulitis, osteomyelitis, severe post-splenectomy infection (OPSI), ovine brucellosis, pasteurellosis, periorbital cellulitis, whooping cough (pertussis), plague, pneumococcal pneumonia, Pott's disease, proctitis, pseudomonas infection, psittacosis, pyemia, pyomyositis, Q fever, relapsing feverfever) (typhinia), rheumatic fever, Rocky Mountain spotted fever (RMSF), rickettsiosis, salmonellosis, scarlet fever, septicemia, Serratia infection, shigellosis, southern tick-borne rash disease, staphylococcal scalded skin syndrome, streptococcal pharyngitis, Poole's granulomatosis, porcine brucellosis, syphilis, syphilitic aortitis, tetanus, toxic shock syndrome (TSS), trachoma, trench fever, tropical ulcer, tuberculosis, tularemia, typhoid fever, typhus, urogenital tuberculosis, urinary tract infection, vancomycin-resistant Staphylococcus aureus infection, Waterhouse-Friedrichsen syndrome, Yersinia pseudotuberculosis disease, and yersiniosis.

[0393] Parasitic infections may include amebiasis, giardiasis, trichomoniasis, African sleeping sickness, American sleeping sickness, leishmaniasis (kala-azar), balantidiosis, toxoplasmosis, malaria, acanthamoeba keratitis, and babesiosis.

[0394] Fungal infections can be aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis, mycetoma, paracoccidioidomycosis, and tinea pedis. Furthermore, immunocompromised individuals are susceptible to diseases caused by fungal genera such as Aspergillus, Candida, Cryptoccocus, Histoplasma, and Pneumocystis. Other fungi are so-called dermatophytes and keratophytes, which can attack the eyes, nails, hair, and especially the skin, causing a variety of conditions, among which ringworm, such as athlete's foot, is common. Fungal spores are a major cause of allergies, and various fungi belonging to different taxonomic groups can cause allergic reactions in some individuals.

[0395] Cancer or tumor diseases include, for example, melanoma, malignant melanoma, colon cancer, lymphoma, sarcoma, blastoma, renal cancer, gastrointestinal tumors, glioma, prostate tumor, bladder cancer, rectal tumor, stomach cancer, esophageal cancer, pancreatic cancer, liver cancer, breast cancer (= breast cancer), uterine cancer, cervical cancer, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), liver cancer, various virus-induced tumors, such as papillomavirus-induced cancer types (e.g., cervical cancer = cervical cancer), adenocarcinoma, herpes virus-induced tumors (e.g., Burkitt's lymphoma, EBV-induced B-cell lymphoma), hepatitis B-induced tumors (hepatocellular carcinoma), HTLV-1 and HTLV-2-induced lymphoma, acoustic neuroma, lung cancer (= lung cancer = bronchial carcinoma), small cell lung cancer, and the like. Cancer, pharyngeal cancer, anal cancer, glioblastoma, rectal cancer, astrocytoma, brain tumor, retinoblastoma, basal cell tumor, brain metastasis, medulloblastoma, vaginal cancer, pancreatic cancer, testicular cancer, Hodgkin's syndrome, meningioma, Schneeberger's disease, pituitary tumor, mycosis fungoides, carcinoid, schwannoma, squamous cell carcinoma, Burkitt's lymphoma, laryngeal cancer, kidney cancer, thymoma, uterine cancer, bone cancer, non-Hodgkin's lymphoma, urethral cancer, CU The cancer or tumor disease may be selected from P syndrome, head and neck tumor, oligodendroglioma, vulvar cancer, intestinal cancer, colon cancer, esophageal cancer (= esophageal cancer), warts, tumor of the small intestine, craniopharyngioma, ovarian cancer, reproductive organ tumor, ovarian cancer (= ovarian cancer), pancreatic cancer (= pancreatic cancer), endometrial cancer, liver metastasis, penile cancer, tongue cancer, gallbladder cancer, leukemia, plasmacytoma, eyelid tumor, prostate cancer (= prostate tumor).

[0396] Diseases for which the present disclosure may be useful as a therapeutic intervention include, by way of example, SMN1-associated spinal muscular atrophy (SMA); amyotrophic lateral sclerosis (ALS); GALT-associated galactosemia; cystic fibrosis (CF); SLC3A1-associated disorders, including cystinuria; COL4A5-associated disorders, including Alport syndrome; galactocerebrosidase deficiency; X-linked adrenoleukodystrophy and adrenomyeloneuropathy; Friedreich's ataxia; Pelizaeus-Merzbacher disease; TSC1- and TSC2-associated tuberous sclerosis; Sanfilippo B syndrome (MPS). FMR1-related disorders including fragile X syndrome, fragile X-associated tremor / ataxia, and fragile X premature ovarian failure syndrome; Prader-Willi syndrome; hereditary hemorrhagic telangiectasia (AT); Niemann-Pick disease type C1; neuronal ceroid lipofuscinosis-related disorders including juvenile neuronal ceroid lipofuscinosis (JNCL), adolescent Batten disease, Santavoli-Hartzia disease, Jansky-Bielskovskii disease, and PTT-1 and TPP1 deficiency; EIF2B1-, EIF2B2-, EIF2B3-, EIF2B4-, and EIF2B5-related childhood ataxia with central nervous system hypomyelination / white matter loss; CACNA1A- and CACNB4-related disorders These disorders include transient recurrent ataxia type 2; MECP2-related disorders, including classic Rett syndrome, MECP2-related severe neonatal encephalopathy, and PPM-X syndrome; CDKL5-related atypical Rett syndrome; Kennedy disease (SBMA); Notch-3-related cerebral autosomal dominant arteriopathy with cortical infarction and leukoencephalopathy (CADASIL); SCN1A- and SCN1B-related seizure disorders; polymerase G-related disorders, including Alpers-Huttenlocher syndrome, POLG-related sensory ataxic neuropathy, dysarthria and ophthalmoplegia, and autosomal dominant and recessive progressive external ophthalmoplegia with mitochondrial DNA deletions; X-linked adrenal hypoplasia; X-linked agammaglobulinemia; Fabry disease; and Wilson disease.

[0397] In one embodiment, the nucleic acids of the present disclosure, and for example, mRNA, can encode functional proteins or enzymes. For example, the compositions of the present disclosure can include mRNA encoding erythropoietin (EPO), α1-antitrypsin, carboxypeptidase N, alpha-galactosidase (GLA), ornithine carbamoyltransferase (OTC), or human growth hormone (hGH).

[0398] In other embodiments, the present disclosure relates to a method of transfecting at least one isolated target cell with a nucleic acid, the method comprising contacting at least one target cell with an effective amount of at least one nucleic acid polynucleotide and (i) at least one nucleic acid and at least one lipid compound disclosed herein, or (ii) at least one composition described herein containing a nucleic acid, or (iii) at least one lipid nanoparticle containing a nucleic acid described herein, and transfecting the nucleic acid into the at least one target cell.

[0399] Target cells include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells (e.g., meningeal, astrocytes, motor neurons, cells of the dorsal root ganglion, anterior horn motor neurons), photoreceptor cells (e.g., rods and cones), retinal pigment epithelial cells, secretory cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiac myocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, antigen presenting cells such as dendritic cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

[0400] In one embodiment, the targeted cells may be cells of the spleen, liver, lung, heart, and kidney, hi another embodiment, the targeted cells may be spleen and kidney cells, e.g., spleen cells.

[0401] In some embodiments, lipid nanoparticles or compositions disclosed herein that allow for avoidance of liver clearance may be of particular interest.

[0402] For example, following transfection of at least one target cell with a nucleic acid encapsulated in a lipid nanoparticle, the production of a polypeptide or protein encoded by such nucleic acid is, for example, stimulated, and the ability of such target cells to express the nucleic acid and, for example, produce a polypeptide or protein of interest is enhanced. For example, transfection of a target cell with a composition encapsulating mRNA enhances (i.e., increases) the production of a protein or enzyme encoded by such mRNA.

[0403] In other embodiments, the present disclosure relates to a method for producing a polypeptide in at least one target cell, comprising contacting at least one target cell with an effective amount of (i) at least one nucleic acid and at least one lipid compound disclosed herein, or (ii) at least one composition described herein containing a nucleic acid, or (iii) at least one lipid nanoparticle containing a nucleic acid described herein, to transfect the at least one target cell with a nucleic acid operably encoding the polypeptide.

[0404] The present disclosure should be understood to encompass all variations, combinations, and permutations of at least one limitation, element, clause, descriptive term, etc. from at least one of the claims described herein, as introduced into any claim dependent on the same base claim (or any other related claim), unless otherwise indicated or unless a contradiction or inconsistency would be apparent to one skilled in the art. When elements are presented as a list, e.g., a Markush group or similar format, it should be understood that each subgroup of elements is also disclosed, and that any element can be deleted from the group. In general, when the present disclosure or aspects of the disclosure are referred to as including particular elements, features, etc., it should be understood that they also encompass embodiments consisting of, or consisting essentially of, such elements, features, etc. For purposes of brevity, these embodiments are not always specifically described in so many words herein. It should also be understood that any embodiment or aspect of the present disclosure can be explicitly excluded from the claims, regardless of whether a specific exclusion is set forth in the specification. Publications and other reference materials referred to herein are incorporated by reference to describe the background of the disclosure and to provide additional detail regarding its practice.

[0405] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0406] material and method Nuclear magnetic resonance spectroscopy (H,C NMR) -H and C NMR spectra were recorded at room temperature on the following spectrometer: Brucker Advance 400 (NMR H: 400 MHz and NMR C: 75 MHz).

[0407] Recorded shifts are reported in parts per million (δ) and were calibrated using residual deuterium-free 3: H 7.26 ppm; C 77.16 ppm, MeOH H 3.31 ppm; C 49.0 ppm). Data are expressed as chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, and m = multiplet), coupling constant (J in Hz), integration, and assignment.

[0408] NMR spectra were obtained using the commercially available software NMRnotebook.

[0409] High-resolution mass spectra (HRMS) were obtained using an Agilent Q-TOF (time of flight) 6520, low-resolution mass spectra (LCMS) using an Agilent MSD 1200 SL (ESI / APCI) and an Agilent HPLC 1200 SL. [Example]

[0410] Synthesis of N-((Z)-14-(((E)-octadec-9-en-1-yl)oxy)-3,6,9,12,16-pentaoxatetratriacont-25-en-1-yl)-1H-imidazole-4-carboxamide (Compound IV) (also known as DOG-IM4) [ka]

[0411] Compound IV was prepared according to the following synthetic scheme:

[0412] [ka]

[0413] 1.1 Synthesis of DOG-PEG4-NH2, also known as DOGP4NH2 The synthesis scheme is as follows:

[0414] [ka]

[0415] [ka]

[0416] 1.1.1 Synthesis of triphenylmethane-glycerol (1) Glycerol (30.0 g; 325.8 mmol), trityl chloride (22.5 g; 80.7 mmol), and DMAP (225 mg; 1.84 mmol) were dissolved in 60 mL of anhydrous THF. After the addition of triethylamine (13.5 mL; 96.9 mmol), the mixture was vigorously stirred at room temperature for 22 hours. Then, 100 mL of ethyl acetate and 70 mL of HO were added to the solution. The aqueous phase was extracted with 2 x 70 mL of ethyl acetate. The combined organic phases were washed sequentially with 70 mL of 10% (w / v) NaHCO3 and 70 mL of brine, dried over MgSO4, and filtered. The resulting product was further purified by silica gel column chromatography (elution gradient CHCl / MeOH) to give compound 1 as a white solid (15.7 g; yield 58%). RMN 1 H (300 MHz; CDCl3): δ : 7.49-7.29 (m; 15Hf-j), 3.93-3.90 (m; 1Hb), 3.76-3.62 (m; 2Ha), 3.35-3.24 (m; 2Hc). ES-SM (N2) m / z: 357.1589 ([M + Na]+); exact mass: 334.1689 g.mol -1

[0417] 1.1.2 Synthesis of 1-methanesulfonyl-oleyl alcohol (2) Oleic alcohol (45.0 g; 167.6 mmol) and triethylamine (38 mL; 272.0 mmol) were dissolved in 600 mL of dichloromethane (CHCl), and the mixture was stirred at 4 °C. Methanesulfonyl chloride (17 mL; 217.0 mmol) was added dropwise, and the reaction mixture was placed under vigorous stirring at room temperature under argon. After 12 h, 250 mL of H2O was added, and the aqueous phase was extracted with 2 x 250 mL of CHCl. ​​The organic layer was washed sequentially with 250 mL of 1 N HCl, 250 mL of 10% (w / v) NaHCO3, and 250 mL of brine, and dried over MgSO4. The solvent was then evaporated under vacuum. The resulting product was further purified by silica gel column chromatography (elution gradient 10 / 0 to 10 / 1 cyclohexane / AcOEt). Compound 2 was obtained as a yellowish oil (44 g; yield 76%). RMN 1 H (300 MHz; CDCl3): δ : 5.39-5.31 (m; 2H9-10), 4.21 (t; J=6.4 Hz; 2H1), 2.99 (s; 3Ha), 2.14-1.88 (m; 4H8,11), 1.80-1.67 (tt; J=6.8 Hz; 2H2), 1.52-1.14 (m; 22H3-7, 12-17), 0.88 (t; J=6.8 Hz; 3H18). ES-SM (N2) m / z: 385.3969 ([M + K]+); exact mass: 346.2989 g.mol -1

[0418] 1.1.3 Synthesis of triphenylmethane-dioleylglycerol (3) To a suspension of NaH (6.0 g (60% in oil); 149.5 mmol) in 35 mL of anhydrous DMF was added compound 1 (10.0 g; 29.2 mmol) in solution in 145 mL of anhydrous DMF. The mixture was heated under reflux for 15 minutes and cooled to room temperature. Product 2 (25.9 g; 74.8 mmol) in 90 mL of anhydrous DMF was added dropwise to the mixture, which was then heated under reflux for 15 hours. After cooling to room temperature, 120 mL of H2O was added to remove residual NaH. The aqueous layer was extracted with ethyl acetate (2 x 100 mL). The combined organic layers were washed with 2 x 240 mL of 1 N HCl, 2 x 240 mL of 5% (w / v) NaHCO3, and 240 mL of brine, dried over MgSO4, and filtered. The solvent was evaporated under reduced pressure. Compound 3, obtained as a crude yellowish oil, was used without further purification (17 g; 70% yield).

[0419] ES-SM(N2) m / z: 857.5507 ([M+Na]+); Exact mass: 834.5607 g.mol-1 (product detected by MS)

[0420] 1.1.4 Synthesis of dioleylglycerol (4) Compound 3 (16.0 g; 19.5 mmol) and para-toluenesulfonic acid (pTs-OH.HO) (1.2 g; 6.1 mmol) were dissolved in 270 mL of THF / MeOH 1 / 1 and stirred at room temperature for 16 h. Triethylamine (860 μL; 6.1 mmol) was then added to the mixture to remove excess pTsOH.HO, and the solvent was evaporated under reduced pressure. The residual oil was purified by silica gel chromatography (cyclohexane / AcOEt) to give compound 4 as a colorless oil (6.5 g; 57% yield). RMN 1 H: (300 MHz; CDCl3): δ : 5.38-5.32 (m; 4H9-10), 3.76-3.41 (m; 9Hb-ac-1), 2.13-1.89 (m;8H8,11), 1.69-1.48 (m; 4H2), 1.47-1.12 (m; 44H3-7, 12-17), 0.89 (t; J=6.6 Hz; 6H18). ES-SM (N2) m / z: 615.5213 ([M + Na]+); exact mass: 592.5313 g.mol -1

[0421] 1.1.5 Synthesis of methanesulfonyloxy-ethoxy-ethoxy-ethoxy-ethoxy-ethyl-azide (5) Di-mesylate tetraethylene glycol (25.0 g; 71.4 mmol) was heated under reflux in 150 mL of CHCN in the presence of NaN (5.8 g; 89.5 mmol). After 19 h, the mixture was cooled to room temperature, and the precipitate was collected by filtration and purified by silica gel chromatography (cyclohexane / AcOEt (7 / 3 to 3 / 7)) to give compound 5 as a yellow oil (8.7 g; 41% yield). RMN 1 H (200 MHz; CDCl3): δ : 4.26-4.22 (m; 2Hd), 3.66-3.61 (m; 2He), 3.60-3.52 (10Hf-j), 3.26(t; J=5.4 Hz; 2Hk), 2.95 (s; 3H1). ES-SM (N2) m / z : 320.0539 ([M + Na]+); exact mass: 297.0639 g.mol -1

[0422] 1.1.6 Synthesis of dioleylglycero-ethoxy-ethoxy-ethoxy-ethyl-azide (6) To a suspension of NaH (810 mg (60% in oil); 20.2 mmol) in 13 mL of anhydrous THF was added compound 4 (4 g; 6.8 mmol) in a solution in 50 mL of anhydrous THF containing 13 mL of HMPA. The mixture was heated under reflux for 15 min and cooled to room temperature. Compound 5 (4 g; 13.5 mmol) in a solution in 25 mL of anhydrous THF was added dropwise. The resulting mixture was heated under reflux for 15 h, cooled to room temperature, and excess NaH was removed by the addition of 400 mL of HO. The organic phase was collected, and the aqueous phase was extracted with 3 × 400 mL of AcOEt. The organic phases were combined, washed successively with 2 × 400 mL of 1 N HCl, 2 × 400 mL of 5% (w / v) NaHCO and 400 mL of brine, and dried over MgSO. The solvent was evaporated under reduced pressure and the resulting oil was purified on a silica gel column eluted with cyclohexane / AcOEt to give a yellowish oil (4 g; yield 74%). RMN 1 H (300 MHz; CDCl3): δ : 5.39-5.33 (m; 4H9-10), 3.70-3.50 (m; 23Ha-j,1), 3.45-3.39 (t; J=5.3 Hz; 2Hk), 2.05-1.95 (m; 8H8,11), 1.58-1.53 ​​(m; 4H2), 1.43-1.21 (m; 44H3-7,12-17), 0.88 (t; J=6.8 Hz; 6H18) ES-SM (N2) m / z: 816.6715 ([M + Na]+); Exact mass: 793.6815 g.mol -1

[0423] 1.1.7 Synthesis of 2-[2-[2-[2-[2,3-bis[(~{Z})-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethanamine (DOG-PEG4-NH2) (7) Compound 6 (1.8 g; 2.3 mmol) was dissolved in 180 mL of THF and 400 mL of HO in the presence of triphenylphosphine (1.8 g; 6.8 mmol). The mixture was heated under reflux for 15 h, and then the solvent was evaporated under reduced pressure. The residual oil was purified on a silica gel column (elution gradient CHCl / MeOH / NHOH 9 / 0.9 / 0.1) to give compound 7 as a colorless oil (1.5 g; 86% yield). RMN 1 H (300 MHz; CDCl3 / MeOD 1 / 1): δ : 5.35-5.29 (m; 4H9-10), 3.64-3.43 (m; 23Ha-j-1), 2.78- 2.90 (m; 2Hk), 2.06-1.90 (m; 8H8,11), 1.60-1.52 (m; 4H2), 1.40-1.19 (m; 44H3-7, 12-17), 0.86 (t; J=7,1 Hz; 6H18). ES-SM (N2) m / z: 768.6636 ([M]+); exact mass: 768.6636 g.mol -1

[0424] 1.2 Synthesis of N-((Z)-14-(((E)-octadec-9-en-1-yl)oxy)-3,6,9,12,16-pentaoxatetratriacont-25-en-1-yl)-1H-imidazole-4-carboxamide (Compound IV; also known as DOG-IM4) 4-Imidazolecarboxylic acid (50 mg, 446 μmol) was dissolved in 1 mL of oxalyl chloride and one drop of DMF was added to catalyze the reaction. The reaction was stirred at room temperature under a nitrogen atmosphere. After 3 h, the organic phase was evaporated and the remaining yellow solid was dried overnight under vacuum to give the corresponding acid chloride (58 mg, quantitative yield) without further purification.

[0425] DOG-PEG 4-NH2 (30 mg, 39 μmol) was dissolved in 5 mL of anhydrous DCM, and the acid chloride (5.6 mg, 43 μmol) and DIPEA (25 μL) in 1.5 mL of anhydrous DMF were added. The mixture was stirred overnight at room temperature under a nitrogen atmosphere. The solvent was evaporated, and the product was purified by flash chromatography (4 g column, DCM / MeOH / NH4OH 9 / 0.9 / 0.1) to give the desired compound (30 mg, 87%). 1 H-NMR (CDCl3, 400 MHz): δ 7.69-7.61 (m, 3H, NH, N=CH-NH, NHCH=C), 5.40-5.29 (m, 4H, 2 x CH=CH), 3.68-3.39 (m, 25H, 12 x OCH 2, 1 x OCH, CH2NHC(O)), 2.06-1.90 (m, 8H, 2 x CH2CH=CHCH2), 1.59-1.49 (m, 4H, 2 x OCH2CH2), 1.39-1.20 (m, 44H, 22 x oleyl-CH2), 0.87 (t, J=6.8, 6H, 2 x CH3) ppm. 13 C-NMR (CDCl3, 75MHz): δ 163.04 (NHC=O), 135.52 (N=CH-NH), 130.53, 130.43, 130.07, 129.97 (2 x CH=CH, NHCH=C, CH =C), 78.06 (OCH), 71.88-70.15 (12 x OCH2), 39.09 (CH2NHC(O)), 32.76-26.23 (oleyl), 22.83 (2 x CH3CH2), 14.25 (2 x CH3) ppm. HR-MS (direct injection, positive ionization): m / z = 884.7039 [M+Na] + (calculated: 884.71) [Example]

[0426] Synthesis of N-((Z)-14-(((E)-octadec-9-en-1-yl)oxy)-3,6,9,12,16-pentaoxatetratriacont-25-en-1-yl)-1H-imidazole-2-carboxamide (Compound III) [ka] It was prepared according to Scheme 1 and the molar amounts considered in Example 1 by using 2-imidazolecarboxylic acid instead of 4-imidazolecarboxylic acid. The product was purified by flash chromatography (4 g column, DCM / MeOH / NH4OH 9 / 0.9 / 0.1) to give the desired compound (45 mg, 65%). 1 H-NMR (CDCl3, 400 MHz): δ 7.15 (m, 1H, N-CH=CH), 7.16 (m, 1H, CH=CH-NH), 5.40-5.29 (m, 4H, 2 x CH=CH), 3.68-3.37 (m, 25H, 12 x OCH 2, 1 x OCH, CH2NHC(O)), 2.2 (br s, 1H, NH signal), 2.1-1.90 (m, 8H, 2 x CH2CH=CHCH2), 1.59-1.49 (m, 4H, 2 x OCH2CH2), 1.37-1.20 (m, 44H, 22 x oleyl-CH2), 0.87 (t, J=6.8, 6H, 2 x CH3) ppm. 13 C-NMR (CDCl3, 75MHz): δ 158.95 (NHC=O), 141.20 (N=CH-NH), 130.52, 130.44, 130.06, 129,98, 129.87 (2 x CH=CH, CH=CH-NH), 119.09 (N-CH=C), 78.06 (OCH), 71.83-69.80 (12 x OCH2), 39.30 (CH2NHC(O)), 32.76-26.23 (oleyl), 22.83 (2 x CH3CH2), 14.25 (2 x CH3) ppm. HR-MS (direct injection, positive ionization): m / z = 884.7057 [M+Na] + (calculated: 884.71) [Example]

[0427] Synthesis of N-((Z)-14-(((E)-octadec-9-en-1-yl)oxy)-3,6,9,12,16-pentaoxatetratriacont-25-en-1-yl)-1H-pyridinyl-3-carboxamide (Compound V) [ka] It was prepared according to the molar amounts considered in Scheme 1, Example 1, and by using 3-pyridylisothiocyanate instead of 4-imidazolecarboxylic acid. 1 H-NMR (CDCl3, 400 MHz): δ 8.79-8.01 (m, 3H, pyridin), 7.26 (m, 1H, pyridin), 5.40-5.29 (m, 4H, 2 x CH=CH), 3.94-3.29 (m, 25H, 12 x OCH 2, 1 x OCH, CH2NHC(S)), 2.1-1.90 (m, 8H, 2 x CH2CH=CHCH2), 1.61-1.45 (m, 4H, 2 x OCH2CH2), 1.42-1.18(m, 44H, 22 x oleyl-CH2), 0.87 (t, 7=6.8, 6H, 2 x CH3) ppm. 1 H-NMR (MeOD, 400 MHz): δ 8.62, 8.29, 8.09, 7.39 (m, 4H, pyridin), 5.42-5.31 (m, 4H, 2 x CH=CH), 3.88-3.39 (m, 25H, 12 x OCH 2,1 x OCH, CH2NHC(S)), 2.08-1.94 (m, 8H, 2 x CH2CH=CHCH2), 1.61-1.49 (m, 4H, 2 x OCH2CH2), 1.40-1.24 (m, 44H, 22 x oleyl-CH2), 0.90 (t, J=6.8, 6H, 2 x CH3) ppm. 13 C-NMR (CDCl3, 75MHz): δ 181.82 (CH2NHC(S)), 145.86, 145.01, 136.18, (3C, pyridin) 131.20-129.84 (1C pyridine, 2 x CH=CH), 123.22 (1C, pyridin), 78.05 (OCH), 77.94, 72.72-70.22 (12 x OCH2), 44.86 (CH2NHC(S)), 32.73-26.24 (oleyl), 22.80 (2 x CH3CH2), 14.23 (2 x CH3) ppm. HR-MS (direct injection, positive ionization): m / z = 904.7166 [M+H] + (calculated: 904.72) [Example]

[0428] Synthesis of N-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethyl]-1H-imidazole-4-carboxamide (Compound IV) [ka] To a mixture of 2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethanamine (1.2 g, 1.56 mmol) in DCM (30 mL) was added a solution of 1H-imidazole-4-carbonyl chloride (0.612 g, 4.69 mmol) and DIEA (1.01 g, 7.81 mmol) in DMF (20 mL). The mixture was stirred at ambient temperature for 16 h. The mixture was concentrated and the residue was purified by column chromatography on silica gel eluting with 0% to 10% MeOH in DCM to give N-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethyl]-1H-imidazole-4-carboxamide (0.504 g, 37.4%) as a yellow oil. 1 H NMR (500 MHz, CDCl3) δ 10.99 (s, 1H), 7.73 - 7.59 (m, 3H), 5.39 - 5.30 (m, 4H), 3.69 - 3.40 (m, 25H), 2.09 - 1.92 (m, 8H), 1.60 - 1.50 (m, 4H), 1.28 (t, J = 14.5 Hz, 44H), 0.88 (t, J = 6.9 Hz, 6H). [Example]

[0429] Synthesis of N-[2-[2-[2-[2-(2,3-dihexadecaoxypropoxy)ethoxy]ethoxy]ethoxy]ethyl]-1H-imidazole-4-carboxamide (Compound VI) [ka] 4-Imidazolecarboxylic acid (2.5 g, 22.3 mmol) was dissolved in 60 mL of oxalyl chloride, and a few drops of DMF were added to catalyze the reaction. The reaction was stirred overnight at room temperature under a nitrogen atmosphere. The organic phase was evaporated, and the remaining yellow solid was dried overnight under a vacuum pump to give the corresponding acid chloride (2.5 g, quantitative) without further purification. 2-[2-[2-[2-(2,3-dihexadecaoxypropoxy)ethoxy]ethoxy]ethoxy]ethanamine (400 mg, 0.5 mmol) was dissolved in 25 mL of anhydrous DCM, and the acid chloride (262 mg, 2 mmol) and DIPEA (0.325 g, 2.5 mmol) in 3 mL of anhydrous DMF were added. The mixture was stirred overnight at room temperature under a nitrogen atmosphere. The solvent was evaporated and the product purified by flash chromatography (12 g column, DCM / MeOH / NHOH 9 / 0.9 / 0.1) to give N-[2-[2-[2-[2-(2,3-dihexadecaoxypropoxy)ethoxy]ethoxy]ethoxy]ethyl]-1H-imidazole-4-carboxamide (250 mg, 0.293 mmol, 58.3% yield) as a yellow solid. 1 H NMR (500 MHz, CDCl3) δ 7.65 (s, 1H), 7.62 (s, 1H), 7.59 (s, 1H), 3.69 - 3.40 (m, 25H), 1.60 - 1.49 (m, 4H), 1.33 - 1.22 (m, 52H), 0.88 (t, J = 6.9 Hz, 6H). [Example]

[0430] Synthesis of N-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propanoylamino]ethoxy]ethoxy]ethoxy]ethyl]-1H-imidazole-4-carboxamide (Compound VII) [ka]

[0431] The compounds were synthesized based on the chemistry shown in Scheme (4). [ka]

[0432] synthesis Step (1) [ka] To a solution of 2,3-bis[(Z)-octadec-9-enoxy]propan-1-ol (1 g, 1.69 mmol) in DCM (20 ml) at 0 °C, Dess-Martin periodinane (841 mg, 1.69 mmol) was added for 5 min. The mixture was then stirred at 25 °C under N for 2 h. After the reaction, the mixture was diluted with DCM (30 ml), washed with NaHCO / NaSO (1 / 1) (50 ml × 3) and brine (50 ml), dried over NaSO, filtered, and concentrated to give 2,3-bis[(Z)-octadec-9-enoxy]propanal (1.2 g, crude) as a yellow oil, which was used directly in the next step. 1 H NMR (400 MHz, CDCl3) δ 9.72 (d, J = 1.4 Hz, 1H), 5.35 (t, J = 5.4 Hz, 4H), 3.84 - 3.79 (m, 1H), 3.74 - 3.56 (m, 5H), 3.44 (ddd, J = 12.6, 9.4, 2.7 Hz, 3H), 2.03 - 1.96 (m, 8H), 1.63 (d, J = 7.2 Hz, 2H), 1.54 (d, J = 6.9 Hz, 2H), 1.26 (d, J = 4.5 Hz, 44H), 0.90 - 0.87 (m, 6H).

[0433] Step (2) [ka] 2,3-Bis[(Z)-octadec-9-enoxy]propanal (1.2 g, 1.62 mmol) was dissolved in t-BuOH:HO (3:1, 20 mL) containing NaHPO.2HO (759 mg, 4.87 mmol), 2-methyl-2-butene (3.4 mL), and sodium chlorite (411 mg, 4.87 mmol). The reaction was stirred at room temperature for 1 h and diluted with ethyl acetate. The aqueous layer was extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated to give 2,3-bis[(Z)-octadec-9-enoxy]propanoic acid (778 mg, 78.9% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 5.40 - 5.31 (m, 4H), 4.04 (dd, J = 5.0, 3.3 Hz, 1H), 3.80 (dd, J = 10.5, 3.2 Hz, 1H), 3.70 (dd, J = 10.5, 5.1 Hz, 1H), 3.62 (q, J = 6.8 Hz, 2H), 3.51 - 3.44 (m, 2H), 2.01 (dd, J = 14.7, 8.9 Hz, 8H), 1.65 - 1.54 (m, 4H), 1.27 (dd, J = 6.7, 2.7 Hz, 44H), 0.88 (t, J = 6.8 Hz, 6H).

[0434] Step (3) [ka] To a solution of 2,3-bis[(Z)-octadec-9-enoxy]propanoic acid (100 mg, 0.165 mmol) in DCM (2 mL) was added tert-butyl N-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl]carbamate (48 mg, 0.165 mmol), 4-dimethylaminopyridine (2 mg, 0.02 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (94 mg, 0.25 mmol), and triethylamine (33 mg, 0.33 mmol). The mixture was stirred at 25 °C for 18 h. After the reaction, the mixture was diluted with DCM (50 mL), washed with water (50 mL × 2), brine (50 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by flash chromatography eluting with 2% to 8% methanol in dichloromethane to give tert-butyl N-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propanoylamino]ethoxy]ethoxy]ethoxy]ethyl]carbamate as a pale yellow oil.

[0435] Step (4) [ka] To a solution of tert-butyl N-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propanoylamino]ethoxy]ethoxy]ethoxy]ethyl]carbamate (226 mg, 0.256 mmol) in DCM (2 ml) was added TFA (0.5 ml). The mixture was stirred at 25° C. for 3 hours. After the reaction, the mixture was concentrated to give tert-butyl N-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propanoylamino]ethoxy]ethoxy]ethoxy]ethyl]carbamate (300 mg, crude). 1H NMR (400 MHz, CDCl3) δ 5.41 - 5.31 (m, 4H), 3.94 (s, 1H), 3.83 - 3.69 (m, 7H), 3.53 (dddd, J = 26.7, 22.9, 11.7, 4.5 Hz, 15H), 2.06 - 1.91 (m, 8H), 1.54 (d, J = 7.0 Hz, 4H), 1.26 (d, J = 4.3 Hz, 44H), 0.88 (t, J = 6.8 Hz, 6H).

[0436] Step (5) [ka] To a solution of N-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl]-2,3-bis[(Z)-octadec-9-enoxy]propanamide (400 mg, 0.512 mmol) in DCM (10 ml) was added N,N-diisopropylethylamine (265 mg, 2.05 mmol) and 1H-imidazole-4-carbonyl chloride (200 mg, 1.54 mmol) in DMF (2 ml). The mixture was stirred at 25°C for 14 hours. After the reaction, the mixture was diluted with EA (100 ml) and washed with water (100 ml x 2) and brine (100 ml). The organics were concentrated and purified by flash (10% MeOH in DCM) to give N-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propanoylamino]ethoxy]ethoxy]ethoxy]ethyl]-1H-imidazole-4-carboxamide (280 mg, 61.2% yield) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 10.35 - 10.08 (m, 1H), 7.64 (s, 1H), 7.59 (s, 1H), 7.48 (s, 1H), 7.05 (s, 1H), 5.44 - 5.31 (m, 4H), 3.89 (dd, J = 5.6, 2.8 Hz, 1H), 3.77 (dd, J = 10.6, 2.6 Hz, 1H), 3.69 - 3.39 (m, 21H), 2.10 - 1.93 (m, 7H), 1.63 - 1.52 (m, 5H), 1.26 (d, J = 4.6 Hz, 44H), 0.88 (t, J = 6.8 Hz, 6H). [Example]

[0437] Synthesis of nonyl 8-[3-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octoxy)propoxy]octanoate (Compound VIII) [ka]

[0438] Compound (VIII) was synthesized based on the chemistry shown in Scheme (5).

[0439] [ka]

[0440] synthesis Step (1) [ka] 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethanol (50 g, 176 mmol) and triethylamine (35.6 g, 352 mmol) in dry dichloromethane (500 mL) under nitrogen were cooled to -5 °C. Methanesulfonyl chloride (30.2 g, 264 mmol) in dry DCM (20 mL) was added dropwise to this solution at 0 °C. The mixture was allowed to warm to room temperature and stirred at room temperature for 18 h. Triethylamine hydrochloride was filtered off, and the DCM solution was washed with 0.1 N HCl and dried over sodium sulfate. The solvent was removed to give 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (69.1 g, 175 mmol, quantitative) as a pale yellow oil, which was used without further purification. 1 H NMR (400 MHz, CDCl3) δ 7.37 - 7.27 (m, 5H), 4.56 (s, 2H), 4.39 - 4.33 (m, 2H), 3.78 - 3.73 (m, 2H), 3.69 - 3.60 (m, 12H), 3.06 (s, 3H).

[0441] Step (2) [ka] To a solution of (2,2-dimethyl-1,3-dioxolan-4-yl)methanol (24.4 g, 175 mmol) in THF (500 mL) was added NaH (14 g, 351 mmol), and the mixture was heated to reflux for 15 minutes. The reaction was then cooled to room temperature, and 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (69.1 g, 175 mmol) was added under nitrogen, and the reaction was heated at 80° C. for 24 hours. TLC indicated that the starting material had been consumed. The reaction was quenched with water and extracted with ethyl acetate. The aqueous layer was extracted again with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluted with 20–50% ethyl acetate in petroleum ether to give 24-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (54.4 g, 70% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.38 - 7.27 (m, 5H), 4.57 (s, 2H), 4.28 (t, J = 5.9 Hz, 1H), 4.05 (dd, J = 8.3, 6.4 Hz, 1H), 3.72 (dd, J = 8.3, 6.4 Hz, 1H), 3.70 - 3.61 (m, 16H), 3.57 (dd, J = 10.0, 5.8 Hz, 1H), 3.49 (dd, J = 10.0, 5.5 Hz, 1H), 1.42 (s, 3H), 1.35 (s, 3H).

[0442] Step (3) [ka] A mixture of 4-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (54.4 g, 123 mmol) and HO (200 mL) in AcOH (200 mL) was stirred at room temperature for 18 h.

[0443] TLC (EA / PE 1 / 1, SM Rf: 0.5; product Rf: 0.1) showed that all starting material had been consumed. The solvent was removed in vacuo and azeotroped several times with toluene. 2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (49 g, 123 mmol, quantitative) was obtained as a pale yellow oil, which was used without further purification. 1 H NMR (400 MHz, CDCl3) δ 7.38 - 7.27 (m, 5H), 4.57 (s, 2H), 3.88 - 3.81 (m, 1H), 3.70 - 3.51 (m, 21H).

[0444] Step (4) [ka] To a solution of 3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]propane-1,2-diol (24 g, 60.3 mmol) in dry DMF (200 mL) under nitrogen was added NaH (9.64 g, 241 mmol), and the mixture was heated at 80° C. for 15 minutes. The reaction was then cooled to room temperature, and 9-bromonona-1-ene (31.9 g, 151 mmol) was added dropwise to the solution. The mixture was stirred at room temperature for 30 minutes, then at 80° C. for 18 hours.

[0445] TLC (EA / PE=1 / 1, Rf: 0.5) showed that a new spot was formed. The reaction was quenched with water (50 mL) and then partitioned between ethyl acetate and water. The aqueous layer was extracted again with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 20% to 50% ethyl acetate in petroleum ether to give 2-[2-[2-[2-[2,3-bis(nona-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (9.3 g, 14.6 mmol, 24.2% yield) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.37 - 7.27 (m, 5H), 5.89 - 5.72 (m, 2H), 5.04 - 4.89 (m, 4H), 4.57 (s, 2H), 3.71 - 3.60 (m, 17H), 3.59 - 3.38 (m, 9H), 2.03 (q, J = 6.7 Hz, 4H), 1.60 - 1.49 (m, 4H), 1.43 - 1.23 (m, 16H).

[0446] Step (5) [ka] To a solution of 2-[2-[2-[2-[2-[2,3-bis(nona-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (9.3 g, 14.6 mmol) in MeCN (80 mL), CCl (80 mL), and water (80 mL) was added NaIO (24.9 g, 116 mmol) and RuCl (656 mg, 2.91 mmol). The reaction mixture was stirred at room temperature for 24 h.

[0447] LCMS showed the title compound as the major product, along with a partial mono-aldehyde product. The reaction was filtered, and the filtrate was diluted with ethyl acetate (800 mL) and washed with 1 N aq. HCl (400 mL). The organic layer was washed with NaSO solution, then dried over sodium sulfate, filtered, and concentrated to give 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (10 g, 12.4 mmol) as a yellow oil, which was used without further purification.

[0448] Step (6) 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(8-oxooctoxy)propoxy]octanoic acid (10 g, 8 mmol) was dissolved in t-BuOH:HO (3:1, 160 mL) containing NaHPO.2HO (3.73 g, 24 mmol), 2-methyl-2-butene (40 mL), and sodium chlorite (2.71 mg, 24 mmol). The reaction was stirred at room temperature for 2 h, and LCMS indicated that the starting material had been consumed. The reaction mixture was diluted with ethyl acetate. The aqueous layer was extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated to give 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (10 g, 3.22 mmol, quantitative) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.38 - 7.27 (m, 5H), 4.57 (s, 2H), 3.71 - 3.61 (m, 17H), 3.59 - 3.37 (m, 9H), 2.33 (t, J = 7.3 Hz, 4H), 1.69 - 1.51 (m, 8H), 1.39 -1.28 (m, 14H).

[0449] Step (7) [ka] To a solution of 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (10 g, 14.8 mmol) and 1-nonanol (5.12 g, 35.5 mmol) in dry dichloromethane (200 mL) under nitrogen, N,N-diisopropylethylamine (11.5 g, 88.7 mmol), DMAP (0.722 g, 5.91 mmol), and EDCI (7.37 g, 38.4 mmol) were added. The mixture was stirred at room temperature for 18 hours. The reaction was diluted with dichloromethane and washed with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluted with 20% to 55% ethyl acetate in petroleum ether to give nonyl 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octoxy)propoxy]octanoate (5 g, 35.9%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 7.38 - 7.27 (m, 5H), 4.57 (s, 2H), 4.05 (t, J = 6.8 Hz, 4H), 3.70 - 3.61 (m, 16H), 3.59 - 3.39 (m, 9H), 2.28 (t, J = 7.5 Hz, 4H), 1.67 - 1.50 (m, 12H), 1.37 - 1.21 (m, 36H), 0.88 (t, J = 6.8 Hz, 6H).

[0450] Step (8) [ka] To a solution of nonyl 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octoxy)propoxy]octanoate (5 g, 5.31 mmol) in ethyl acetate (100 mL) was added Pd / C (1.13 g, 20% wt / wt). The mixture was stirred under hydrogen at room temperature for 18 h.

[0451] TLC (ethyl acetate / petroleum ether 1 / 1) showed that the starting material had been consumed.

[0452] The reaction was filtered through Celite and washed with ethyl acetate to give nonyl 8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octoxy)propoxy]octanoate (4.22 g, 4.98 mmol, 93.8%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 4.05 (t, J = 6.8 Hz, 4H), 3.74 - 3.38 (m, 27H), 2.28 (t, J = 7.5 Hz, 4H), 1.68 - 1.50 (m, 12H), 1.39 - 1.21 (m, 37H), 0.88 (t, J = 6.8 Hz, 6H).

[0453] Step (9) [ka] To a solution of nonyl 8-[3-[2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octoxy)propoxy]octanoate (4.5 g, 4.8 mmol) in dimethylformamide (DMF, volume: 30 ml) was added sodium azide (0.378 g, 5.81 mmol). The reaction mixture was then stirred at 70° C. for 16 hours. Water (200 mL) was then added, and the reaction mixture was extracted with ethyl acetate (100 mL×2). The combined organic phase was washed with brine (100 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue that was purified by column chromatography eluted with petroleum ether:ethyl acetate=100:1 to 3:1 to give nonyl 8-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octoxy)propoxy]octanoate (4 g, 4.58 mmol, 94% yield) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 4.05 (t, J = 6.8 Hz, 4H), 3.71 - 3.36 (m, 25H), 2.29 (t, J = 7.5 Hz, 4H), 1.67 - 1.50 (m, 12H), 1.38 - 1.21 (m, 36H), 0.88 (t, J = 6.8 Hz, 6H).

[0454] Step (10) [ka] A mixture of nonyl 8-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octoxy)propoxy]octanoate (1 g, 1.14 mmol) and triphenylphosphine (0.45 g, 1.72 mmol) in tetrahydrofuran (THF, ratio: 33, volume: 10 ml) / Green (name: Water, ratio: 1, volume: 0.3 ml) was stirred at 20° C. for 16 hours. TLC (5% methanol in dichloromethane) showed the reaction to be complete. The solvent was removed, and the residue was loaded onto silica gel and purified by chromatography (silica, 1–10% methanol / ammonia in dichloromethane) to give nonyl 8-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octoxy)propoxy]octanoate (0.68 g, 0.8 mmol, 70% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 4.05 (t, J = 6.8 Hz, 4H), 3.70 - 3.39 (m, 23H), 2.91 (t, J = 5.2 Hz, 2H), 2.47 (s, 2H), 2.32 - 2.25 (m, 4H), 1.66 - 1.50 (m, 12H), 1.38 - 1.21 (m, 36H), 0.88 (t, J = 6.9 Hz, 6H).

[0455] Step (11) [ka] 4-Imidazolecarboxylic acid (2.5 g, 22.3 mmol) was dissolved in 60 mL of oxalyl chloride and a few drops of DMF were added to catalyze the reaction. The reaction was stirred overnight at room temperature under a nitrogen atmosphere. The organic phase was evaporated, and the remaining yellow solid was dried overnight under vacuum to give the corresponding acid chloride (2.5 g, quantitative) without further purification.

[0456] Nonyl 8-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octoxy)propoxy]octanoate (680 mg, 0.8 mmol) was dissolved in 30 mL of anhydrous DCM, and the acid chloride (419 mg, 3.2 mmol) and DIPEA (0.519 g, 4 mmol) in 3 mL of anhydrous DMF were added. The mixture was stirred at room temperature overnight. The solvent was evaporated and the product purified by flash chromatography (40 g column, DCM / MeOH 20 / 1 to 10 / 1) followed by prep TLC (eluted with 10% methanol in dichloromethane) to give nonyl 8-[3-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octoxy)propoxy]octanoate (302.3 mg, 0.326 mmol, 40.6% yield) as a colorless oil. MS (ESI) m / z=898.7 (M+H)+ 1 H NMR (400 MHz, CDCl3) δ 10.95 (s, 1H), 7.72 - 7.60 (m, 3H), 4.05 (t, J = 6.7 Hz, 4H), 3.68 - 3.37 (m, 25H), 2.33 - 2.25 (m, 4H), 1.66 - 1.48 (m, 12H), 1.38 - 1.21 (m, 37H), 0.88 (t, J = 6.8 Hz, 6H). [Example]

[0457] Synthesis of 1-octylnonyl 8-[3-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (Compound IX) [ka]

[0458] Compound (IX) was synthesized based on the chemistry shown in Scheme (6).

[0459] [ka]

[0460] synthesis Step (1) [ka] 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethanol (50 g, 0.176 mol) and triethylamine (36.2 g, 0.352 mol) in dry dichloromethane (600 mL) under nitrogen was cooled to 0°C.

[0461] Methanesulfonyl chloride (30.6 g, 0.264 mol) was added dropwise to this solution at 0 °C. The mixture was allowed to warm to room temperature and stirred at room temperature for 18 h. Triethylamine hydrochloride was removed by filtration, and the DCM solution was washed with 0.1 N HCl and dried over sodium sulfate. The solvent was removed to give 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (62 g, 92%) as a pale yellow oil, which was used without further purification. LCMS MS 363(M+1)

[0462] Step (2) [ka] To a solution of (2,2-dimethyl-1,3-dioxolan-4-yl)methanol (62 g, 0.171 mol) in THF (600 mL) was added NaH (6.17 g, 0.257 mol), and the mixture was heated to reflux for 15 minutes. The reaction was then cooled to room temperature, and 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (25.0 g, 0.171 mol) was added under nitrogen, and the reaction was heated at 80° C. for 18 hours. TLC indicated that the starting material had been consumed. The reaction was quenched with water and extracted with ethyl acetate. The aqueous layer was extracted again with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluted with 20–50% ethyl acetate in petroleum ether to give 4-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (43 g, 71% yield) as a pale yellow oil. LCMS MS 421(M+23)

[0463] Step (3) [ka] A mixture of 4-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (43 g, 0.103 mol) in AcOH (200 mL) and water (200 mL). The mixture was stirred at ambient temperature for 16 hours. The solvent was removed to give 3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]propane-1,2-diol (36 g, 95%) as a pale yellow oil, which was used without further purification. LCMS MS 381(M+23)

[0464] Step (4) [ka] To a solution of 3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]propane-1,2-diol (20 g, 0.050 mol) in THF (200 mL) was added NaH (8.03 g, 0.201 mol), and the mixture was heated to reflux for 15 minutes. The reaction was then cooled to room temperature, and 9-bromonona-1-ene (26.6 g, 0.126 mol) was added under nitrogen, and the reaction was heated at 80° C. for 18 hours. TLC indicated that the starting material had been consumed. The reaction was quenched with water and extracted with ethyl acetate. The aqueous layer was extracted again with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluted with 10–30% ethyl acetate in petroleum ether to give 2-[2-[2-[2-[2,3-bis(nona-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (8.8 g, 26% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.37 - 7.27 (m, 5H), 5.87 - 5.73 (m, 2H), 5.04 - 4.87 (m, 4H), 4.57 (s, 2H), 3.71 - 3.59 (m, 16H), 3.59 - 3.38 (m, 9H), 2.03 (q, J = 6.5 Hz, 4H), 1.60 - 1.49 (m, 4H), 1.41 - 1.28 (m, 16H).

[0465] Step (5) [ka] To a solution of 2-[2-[2-[2-[2-[2,3-bis(nona-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (8.5 g, 0.0140 mol) in MeCN (80 mL), CCl (80 mL), and water (80 mL) was added NaIO (24.9 g, 0.116 mol) and RuCl (0.66 g, 2.93 mmol). The reaction mixture was stirred at room temperature for 24 h. LCMS indicated the title compound was the major product. The reaction was filtered, and the filtrate was diluted with ethyl acetate (600 mL) and washed with 1 N aq. HCl (200 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated to give 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (8.7 g, 97% yield) as a yellow oil, which was used without further purification. 1 H NMR (400 MHz, CDCl3) δ 7.31 (dd, J = 22.6, 3.2 Hz, 5H), 4.57 (s, 2H), 3.71 - 3.61 (m, 19H), 3.59 - 3.38 (m, 11H), 2.32 (t, J = 7.4 Hz, 4H), 1.68 - 1.47 (m, 10H), 1.32 (s, 14H).

[0466] Step (6) [ka] A mixture of 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (40 g, 49.8 mmol, purity: 80%), heptadecan-9-ol (25.5 g, 99.6 mmol), N,N-dimethylpyridin-4-amine (12.2 g, 99.6 mmol), EDC HCl (19.1 g, 99.6 mmol), and DIEA (19.3 g, 149 mmol) in DCM (500 mL). The mixture was stirred at room temperature for 16 hours. DCM (500 mL) was added to the mixture, which was washed with 1N HCl, brine, and concentrated. The residue was purified by flash column chromatography on silica gel eluting with 1:1 ethyl acetate / petroleum ether to give 1-octylnonyl 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (15 g, 27%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 7.37 - 7.27 (m, 5H), 4.90 - 4.82 (m, 2H), 3.70 - 3.60 (m, 16H), 3.58 - 3.37 (m, 9H), 2.27 (t, J = 7.5 Hz, 4H), 1.53 (dd, J = 22.4, 6.0 Hz, 12H), 1.28 (d, J = 22.7 Hz, 60H), 0.88 (t, J = 6.8 Hz, 12H).

[0467] Step (7) [ka] To a solution of 1-octylnonyl 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (15 g, 13.4 mmol) in ethyl acetate (150 mL) was added Pd / C (2.85 g, 20% wt / wt). The mixture was stirred under hydrogen at room temperature for 18 hours. TLC (ethyl acetate / petroleum ether 1 / 1) showed that the starting material had been consumed. The reaction was filtered through Celite and washed with ethyl acetate to give 1-octylnonyl 8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (11.1 g, 80.5%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 4.91 - 4.82 (m, 2H), 3.75 - 3.39 (m, 24H), 2.27 (t, J = 7.2 Hz, 4H), 1.66 - 1.43 (m, 17H), 1.38 - 1.17 (m, 61H), 0.88 (t, J = 6.8 Hz, 12H).

[0468] Step (8) [ka] To a mixture of 1-octylnonyl 8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (1.4 g, 1.36 mmol) and N,N-diethylethanamine (0.275 g, 2.72 mmol) in DCM (20 mL) was added methanesulfonyl chloride at 0° C. The mixture was stirred at room temperature for 3 h. The mixture was added DCM (100 mL), washed with water, brine and concentrated to give 1-octylnonyl 8-[3-[2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (1.4 g, 93%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 7.37 - 7.27 (m, 5H), 4.90 - 4.82 (m, 2H), 3.70 - 3.60 (m, 16H), 3.58 - 3.37 (m, 9H), 2.27 (t, J = 7.5 Hz, 4H), 1.53 (dd, J = 22.4, 6.0 Hz, 12H), 1.28 (d, J = 22.7 Hz, 60H), 0.88 (t, J = 6.8 Hz, 12H).

[0469] Step (9) [ka] A mixture of 1-octylnonyl 8-[3-[2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (1.4 g, 1.26 mmol) in DMF (10 mL) at room temperature. The mixture was stirred at 70° C. for 16 hours. Water (50 mL) was added to the mixture, and it was extracted with EtOAc (50 mL×3). The organic layer was washed with brine, dried over NaSO, and concentrated to give 1-octylnonyl 8-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (1.3 g, 97.5%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 4.86 (p, J = 6.3 Hz, 2H), 3.72 - 3.58 (m, 15H), 3.60 -3.35 (m, 11H), 2.27 (t, J = 7.5 Hz, 4H), 1.56 (ddd, J = 22.2, 14.3, 6.1 Hz, 18H), 1.38 - 1.19 (m, 64H), 0.88 (t, J = 6.8 Hz, 12H).

[0470] Step (10) [ka] A mixture of 1-octylnonyl 8-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (1.3 g, 1.23 mmol) and triphenylphosphane in THF (20 mL) and water (3 mL). The mixture was stirred at room temperature for 16 hours. The mixture was concentrated to give 1-octylnonyl 8-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (1.1 g, 87%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 4.86 (p, J = 6.2 Hz, 2H), 3.71 - 3.39 (m, 23H), 2.91 (t, J = 5.1 Hz, 2H), 2.27 (t, J = 7.4 Hz, 7H), 1.54 (dd, J = 32.2, 15.1 Hz, 16H), 1.28 (d, J = 23.3 Hz, 60H), 0.88 (t, J = 6.8 Hz, 12H).

[0471] Step (11) [ka] To a mixture of 1-octylnonyl 8-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (1.1 g, 1.04 mmol) and N-ethyl-N-isopropyl-propan-2-amine (1.35 g, 10.4 mmol) in DCM (40 mL) was added 1H-imidazole-4-carbonyl chloride (0.545 g, 4.17 mmol). The mixture was stirred at room temperature for 16 hours. The mixture was concentrated and the residue was purified by column chromatography on silica gel eluting with 2% to 15% MeOH in DCM to give 1-octylnonyl 8-[3-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (0.22 g, 18.8%) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 10.78 (s, 1H), 7.63 (s, 3H), 4.86 (s, 2H), 3.70 - 3.36 (m, 25H), 2.28 (dd, J = 10.6, 4.4 Hz, 4H), 1.94 (s, 3H), 1.66 - 1.43 (m, 17H), 1.36 - 1.16 (m, 62H), 0.88 (t, J = 6.8 Hz, 12H). [Example]

[0472] Synthesis of 2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethyl 2,3-bis[(Z)-octadec-9-enoxy]propanoate (Compound X) [ka]

[0473] Synthesis of compound (X) To a solution of 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl 2,3-bis[(Z)-octadec-9-enoxy]propanoate (282 mg, 0.36 mmol) in DCM (10 ml) was added DIEA (233 mg, 1.8 mmol) and 1H-imidazole-4-carbonyl chloride (188 mg, 1.44 mmol). The mixture was stirred at 25°C for 14 hours. After the reaction, the mixture was treated with EA (100 ml) and washed with water (100 ml x 2) and NaCl sat. aq. (100 ml). The organics were concentrated and purified by flash (10% MeOH in DCM) to give 2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethyl 2,3-bis[(Z)-octadec-9-enoxy]propanoate (192 mg, 59.6% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 9.77 - 9.65 (m, 1H), 7.66 (s, 1H), 7.61 (s, 1H), 7.51 -7.44 (m, 1H), 5.34 (t, J = 5.4 Hz, 4H), 4.27 (d, J = 4.5 Hz, 2H), 4.08 - 4.05 (m, 1H), 3.75 - 3.57 (m, 17H), 3.49 - 3.39 (m, 3H), 2.18 - 1.90 (m, 8H), 1.60 (s, 2H), 1.55 - 1.52 (m, 2H), 1.27 (s, 44H), 0.88 (t, J = 6.8 Hz, 6H). [Example]

[0474] Synthesis of 2,3-bis[(Z)-octadec-9-enoxy]propyl 2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]acetate (Compound XI) [ka]

[0475] Synthesis of compound (XI) To a solution of 2,3-bis[(Z)-octadec-9-enoxy]propyl 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]acetate (623 mg, 0.79 mmol) in DCM (15 ml) was added DIEA (515 mg, 3.98 mmol) and 1H-imidazole-4-carbonyl chloride (416 mg, 3.19 mmol) in DMF (5 ml). The mixture was stirred at 25 °C for 14 h. The mixture was concentrated, treated with EA (50 ml), washed with water (50 ml × 2), NaCl sat. aq. (50 ml), and dried over Na2SO4. The organics were concentrated and purified by flash (5% MeOH in DCM) to give 2,3-bis[(Z)-octadec-9-enoxy]propyl 2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]acetate (380 mg, yield 53.4 mmol) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 9.97 (s, 1H), 7.64 (d, J = 15.8 Hz, 3H), 5.34 (t, J = 5.4 Hz, 4H), 4.32 (dd, J = 11.5, 4.0 Hz, 1H), 4.20 - 4.11 (m, 3H), 3.72 - 3.61 (m, 13H), 3.55 (t, J = 6.7 Hz, 2H), 3.46 (dt, J = 13.0, 5.5 Hz, 4H), 2.13 - 1.86 (m, 8H), 1.54 (d, J = 6.2 Hz, 4H), 1.33 - 1.24 (m, 44H), 0.88 (t, J = 6.8 Hz, 6H [Example]

[0476] Synthesis of 2,3-bis[(~{Z})-octadec-9-enoxy]propyl~{N}-[2-[2-[2-(1~{H}-imidazole-4-carbonylamino)ethoxy]ethoxy]ethyl]carbamate (Compound XII) [ka]

[0477] Synthesis of compound (XII) Step (1) [ka] To a solution of 2,3-bis[(Z)-octadec-9-enoxy]propan-1-ol (1 g, 1.65 mmol) in DMF (10 ml) was added bis(2,5-dioxopyrrolidin-1-yl)carbonate (1.34 g, 4.96 mmol) and 4-dimethylaminopyridine (202 mg, 1.65 mmol). The mixture was stirred at 25 °C. The mixture was treated with EA (50 ml), washed with water (50 ml × 2), aqueous NaCl (50 ml), and dried over Na2SO4. The organics were concentrated and purified by flash (20% EA in PE) to give 2,3-bis[(Z)-octadec-9-enoxy]propyl(2,5-dioxopyrrolidin-1-yl)carbonate (937 mg, 75.7% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 5.48 - 5.30 (m, 4H), 4.46 (dd, J = 11.1, 3.9 Hz, 1H), 4.38 - 4.31 (m, 1H), 3.74 - 3.66 (m, 1H), 3.60 - 3.39 (m, 6H), 2.83 (s, 4H), 2.01 (dd, J = 14.8, 9.2 Hz, 8H), 1.54 (d, J = 6.6 Hz, 4H), 1.33 - 1.22 (m, 44H), 0.88 (t, J = 6.8 Hz, 6H).

[0478] Step (2) [ka] To a solution of 2,3-bis[(Z)-octadec-9-enoxy]propyl(2,5-dioxopyrrolidin-1-yl)carbonate (937 mg, 1.28 mmol) in DCM (10 mL) was added tert-butyl N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]carbamate (327 mg, 1.28 mmol), TEA (194 mg, 1.91 mmol), and 4-dimethylaminopyridine (15 mg, 0.128 mmol). The mixture was stirred at 25 °C for 14 h. After the reaction, the mixture was treated with DCM (50 mL), washed with water (50 mL × 2), aqueous NaCl (50 mL), and dried over Na2SO4. The organics were concentrated and purified by flash (0–50% EA in PE) to give tert-butyl N-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxycarbonylamino]ethoxy]ethoxy]ethyl]carbamate (802 mg, 71% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3δ 5.36 (dt, J = 9.9, 5.0 Hz, 4H), 5.27 (s, 1H), 5.06 (s, 1H), 4.20 (dd, J = 11.4, 3.8 Hz, 1H), 4.11 (dd, J = 11.5, 5.3 Hz, 1H), 3.62 - 3.53 (m, 11H), 3.48 (d, J = 5.4 Hz, 2H), 3.45 - 3.32 (m, 6H), 2.02 (dt, J = 12.3, 6.3 Hz, 8H), 1.57 - 1.51 (m, 4H), 1.45 (s, 9H), 1.34 - 1.25 (m, 44H), 0.88 (t, J = 6.7 Hz, 6H).

[0479] Step (3) [ka] To a solution of tert-butyl N-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxycarbonylamino]ethoxy]ethoxy]ethyl]carbamate (802 mg, 0.925 mmol) in DCM (10 ml) was added TFA (1.3 ml). The mixture was stirred at 25° C. for 3 hours. The mixture was concentrated in vacuo to give 2,3-bis[(Z)-octadec-9-enoxy]propyl N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]carbamate (1.19 g, crude) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.52 (s, 2H), 5.39 - 5.31 (m, 4H), 4.26 (d, J = 10.5 Hz, 1H), 4.14 (dd, J = 11.7, 4.5 Hz, 1H), 3.76 (t, J = 4.8 Hz, 2H), 3.70 - 3.51 (m, 12H), 3.46 (t, J = 6.8 Hz, 2H), 3.39 (d, J = 4.7 Hz, 2H), 3.25 (s, 2H), 2.01 (dd, J = 12.5, 6.6 Hz, 8H), 1.56 (d, J = 8.9 Hz, 4H), 1.31 - 1.23 (m, 44H), 0.88 (t, J = 6.8 Hz, 6H).

[0480] Step (4) [ka] To a solution of 2,3-bis[(Z)-octadec-9-enoxy]propyl N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]carbamate (709 mg, 0.924 mmol) in DCM (15 ml) was added DIEA (717 mg, 5.54 mmol) and 1H-imidazole-4-carbonyl chloride (483 mg, 3.7 mmol). The mixture was stirred at 25 °C for 14 h. The mixture was concentrated, treated with EA (50 ml), washed with water (50 ml × 2), NaCl sat. aq. (50 ml), and dried over Na2SO4. The organics were concentrated and purified by flash (5% to 10% MeOH in DCM) to give 2,3-bis[(Z)-octadec-9-enoxy]propyl N-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethyl]carbamate (462 mg, 56.9% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 9.85 - 9.75 (m, 1H), 7.66 (d, J = 10.2 Hz, 2H), 7.52 (s, 1H), 5.75 (s, 1H), 5.34 (t, J = 4.8 Hz, 4H), 4.19 (s, 1H), 4.12 - 4.07 (m, 1H), 3.71 - 3.59 (m, 9H), 3.56 (t, J = 5.1 Hz, 4H), 3.50 - 3.42 (m, 4H), 3.37 (d, J = 5.2 Hz, 2H), 2.10 - 1.87 (m, 8H), 1.54 (d, J = 6.4 Hz, 4H), 1.26 (d, J = 4.7 Hz, 44H), 0.88 (t, J = 6.8 Hz, 6H). [Example]

[0481] Synthesis of [(Z)-non-2-enyl]8-[3-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-nona-2-enoxy]-8-oxo-octoxy]propoxy]octanoate (Compound XIII) [ka] As shown in Figure 7, compounds were synthesized based on the chemistry shown in Scheme (7).

[0482] Synthesis of compound (XIII) Step (1) [ka] A mixture of 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethanol (40 g, 0.206 mol), N,N-dimethylpyridin-4-amine (1.26 g, 0.0103 mol), and [chloro(diphenyl)methyl]benzene (45.9 g, 0.165 mol) in DCM (300 mL). The mixture was cooled to 0 °C, and then N,N-diethylethanamine (41.7 g, 0.412 mol) was added. The reaction mixture was stirred at ambient temperature for 16 h. LCMS indicated a good reaction. The mixture was poured into water (600 mL) and extracted with DCM (2 × 400 mL). The organic layer was washed with water, NaCl, dried over NaSO, and concentrated. The residue was purified by flash column chromatography on silica gel eluting with 3:1 ethyl acetate / petroleum ether to give 2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethanol (33.0 g, 36.7%) as a colorless oil. LCMS 459 (M+23), 99% UV 214 nm 1 H NMR (400 MHz, CDCl3) δ 7.49 - 7.44 (m, 6H), 7.32 - 7.26 (m, 6H), 7.25 - 7.19 (m, 3H), 3.72 - 3.64 (m, 12H), 3.61 - 3.57 (m, 2H), 3.27 - 3.22 (m, 2H), 2.55 - 2.50 (m, 1H).

[0483] Step (2) [ka] To a mixture of 2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethanol (33 g, 0.0756 mol) and N,N-diethylethanamine (15.3 g, 0.151 mol) in DCM (600 mL) was added methanesulfonyl chloride (10.4 g, 0.0907 mol) slowly at 0 °C. The mixture was stirred at room temperature overnight. CHCl (400 mL) was added to the solution, and the mixture was washed with dilute HCl (1 M, 1000 mL). The mixture was shaken, the layers were separated, and the organic layer was collected. The organic layer was further washed with water (1000 mL) and brine (1000 mL) and dried over NaSO. The solvent was removed to give 2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (38.8 g, 99.8%) as a yellow oil. LCMS 537.2 (M+23) 98% UV (214 nm) 1 H NMR (400 MHz, CDCl3) δ 7.48 - 7.44 (m, 6H), 7.32 - 7.27 (m, 5H), 7.26 - 7.20 (m, 4H), 4.35 - 4.30 (m, 2H), 3.75 - 3.71 (m, 2H), 3.70 - 3.63 (m, 10H), 3.26 - 3.20 (m, 2H), 2.98 (s, 3H).

[0484] Step (3) [ka] To a suspension of NaH (17.9 g) in 300 mL of anhydrous DMF was added 3-trityloxypropane-1,2-diol (30 g). The mixture was heated at 80°C for 15 minutes and cooled to room temperature. 9-Bromona-1-ene (46 g) in 10 mL of anhydrous DMF was added dropwise to the mixture, which was then heated at 80°C for 18 hours. After cooling to room temperature, 500 mL of H2O was added to destroy any remaining NaH. The organic phase was extracted with 750 mL of ethyl acetate. The extract was washed sequentially with 300 mL of 5% (w / v) NaHCO3 and 150 mL of brine and dried over Na2SO4. The solvent was evaporated under reduced pressure, and the resulting oil was purified on a silica gel column eluted with petroleum ether / ethyl acetate (6% to 25% ethyl acetate in petroleum ether) to give a colorless oil (15.1 g, 28.9%). 1 H NMR (400 MHz, CDCl3) δ 7.60 - 7.05 (m, 17H), 5.95 - 5.66 (m, 2H), 4.97 (ddd, J =21.1, 11.4, 5.9 Hz, 4H), 3.69 - 3.31 (m, 7H), 3.21 -3.06 (m, 2H), 2.02 (dt, J = 7.9, 3.7 Hz,4H), 1.50 - 1.25 (m, 18H).

[0485] Step (4) [ka] To a solution of [2,3-bis(nona-8-enoxy)propoxy-diphenyl-methyl]benzene (30.7 g, 50 mmol) in methanol / THF (600 mL, 1 / 1 v / v) was added p-toluenesulfonic acid (47.6 g, 250 mmol) in one portion at room temperature, and the mixture was stirred at room temperature for 18 h. TLC (4% ethyl acetate in petroleum ether) showed complete disappearance of the starting material. 20 mL of triethylamine was added to quench the reaction, and the solvent was removed under vacuum. The residue was purified by flash chromatography eluting with 20% to 30% ethyl acetate (21%) in petroleum ether to give 2,3-bis(nona-8-enoxy)propan-1-ol (12.33 g, 36.2 mmol, 72.4% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 5.89 - 5.73 (m, 2H), 5.03 - 5.00 (m, 1H), 4.99 - 4.96 (m, 1H), 4.94 (d, J = 0.9 Hz, 1H), 4.92 (d, J = 0.9 Hz, 1H), 3.76 - 3.69 (m, 1H), 3.65 - 3.41 (m, 8H), 2.26 - 2.19 (m, 1H), 2.09 - 2.00 (m, 4H), 1.61 - 1.52 (m, 4H), 1.41 - 1.27 (m, 16H).

[0486] Step (5) [ka] To a mixture of 2,3-bis(nona-8-enoxy)propan-1-ol (12.33 g, 36.2 mmol), NaH (60% mineral oil dispersion, 2.77 g, 72.4 mmol) in 200 mL of dry THF was added, followed by stirring at 80° C. for 15 minutes. After the solvent returned to room temperature, 2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (22.4 g, 43.4 mmol) dissolved in 60 mL of dry THF was added. The reaction mixture was stirred at reflux (80° C.) overnight. The reaction mixture was cooled to room temperature, and water (200 mL) was added. EtOAc (400 mL) was added, the mixture was shaken, the layers were separated, and the organic layer was collected. The aqueous layer was extracted with EtOAc (400 mL × 2). The combined organic layers were washed with brine and dried over Na2SO4. The residue was purified by flash column chromatography on silica gel eluting with ethyl acetate (0-15%) in petroleum ether (14%) to give the target product (23.17 g, 30.5 mmol, 84.3% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.48 - 7.44 (m, 6H), 7.31 - 7.26 (m, 6H), 7.25 - 7.19 (m, 3H), 5.88 - 5.72 (m, 2H), 5.02 - 4.99 (m, 1H), 4.98 - 4.95 (m, 1H), 4.93 (dd, J = 2.0, 0.9 Hz, 1H), 4.92 - 4.89 (m, 1H), 3.70 - 3.64 (m, 10H), 3.63 - 3.59 (m, 4H), 3.59 - 3.40 (m, 9H), 3.26 - 3.21 (m, 2H), 2.07 - 1.99 (m, 4H), 1.60 - 1.50 (m, 4H), 1.41 - 1.25 (m, 16H).

[0487] Step (6) [ka] To a solution of [2-[2-[2-[2-[2-[2,3-bis(nona-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxy-diphenyl-methyl]benzene (23.17 g, 30.5 mmol) in MeCN (200 mL), CCl (200 mL), and water (200 mL) was added NaIO (52.2 g, 244 mmol) and RuCl (1.27 g, 6.1 mmol). The reaction mixture was stirred at room temperature for 24 h. The reaction was filtered, and the filtrate was diluted with ethyl acetate (800 mL) and washed with 1 N aq. HCl (900 mL). The organic layer was washed with NaSO solution (700 mL × 2), then dried over sodium sulfate, filtered, and concentrated to give 8-[2-(7-carboxyheptoxy)-3-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]octanoic acid (23.28 g, 22 mmol, purity: 75%, yield: 71.9%) as a yellow oil, which was used without further purification. 1 H NMR (400 MHz, CDCl3) δ 9.75 (s, 1H), 7.46 (d, J = 7.2 Hz, 2H), 7.34 - 7.27 (m, 12H), 7.25 - 7.19 (m, 1H), 3.75 - 3.72 (m, 1H), 3.69 - 3.40 (m, 23H), 3.26 - 3.20 (m, 1H), 2.45 - 2.28 (m, 4H), 1.68 - 1.50 (m, 8H), 1.32 (s, 12H).

[0488] Step (7) 8-[2-(8-oxooctoxy)-3-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]octanoic acid (12.5 g, 4.81 mmol) was dissolved in t-BuOH:HO (3:1, 400 mL) containing NaHPO (1.72 g, 14.4 mmol), 2-methyl-2-butene (15 mL), and sodium chlorite (1.3 g, 14.4 mmol). The reaction was stirred at room temperature for 2 h, and LCMS indicated that the starting material had been consumed. The reaction mixture was diluted with HO. The aqueous layer was extracted with ethyl acetate (800 mL × 2). The residue was purified by flash column chromatography on silica gel eluting with CHOH (0–6%) in DCM (3%) to give the target product (4.663 g (EXP-20-IQ8160-P2: 0.576 g + EXP-20-IQ8160-2: 4.082 g), 19.2% yield (combined yield of the two-step oxidation)) as a pale yellow oil. LCMS: Peak detected: MS(ESI) m / z=818.5 (M+Na)+ at 2.300 min.

[0489] Multi-Line Report 1 H NMR (400 MHz, CDCl3) δ 7.49 - 7.43 (m, 6H), 7.32 - 7.26 (m, 6H), 7.25 - 7.19 (m, 3H), 4.23 (s, 1H), 3.69 - 3.39 (m, 23H), 3.26 - 3.21 (m, 2H), 2.37 - 2.29 (m, 4H), 1.67 -1.50 (m, 8H), 1.33 (s, 12H).

[0490] Step (8) [ka] Then, to a solution of 8-[2-(7-carboxyheptoxy)-3-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]octanoic acid (4.087 g, 5.14 mmol) and (Z)-non-2-en-1-ol (1.75 g, 12.3 mmol) in dry dichloromethane (150 mL) was added DIPEA (3.99 g, 30.8 mmol), DMAP (0.251 mg, 2.06 mmol), and EDCI (2.56 g, 13.4 mmol) in an ice bath. The mixture was stirred at room temperature for 18 hours. The reaction was diluted with dichloromethane and washed with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 0% to 40% (25%) ethyl acetate in petroleum ether to give [(Z)-non-2-enyl]8-[2-[8-[(Z)-nona-2-enoxy]-8-oxo-octoxy]-3-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]octanoate (1.646 g, 1.58 mmol, 30.7% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 7.50 - 7.44 (m, 6H), 7.32 - 7.27 (m, 6H), 7.25 - 7.18 (m, 3H), 5.69 - 5.59 (m, 2H), 5.57 - 5.47 (m, 2H), 4.62 (d, J = 6.8 Hz, 4H), 3.70 - 3.38 (m, 23H), 3.26 - 3.21 (m, 2H), 2.33 - 2.26 (m, 4H), 2.13 - 2.05 (m, 4H), 1.65 - 1.50 (m, 8H), 1.39 - 1.25 (m, 28H), 0.92 - 0.84 (m, 6H).

[0491] Step (9) [ka] To a solution of [(Z)-non-2-enyl]8-[2-[8-[(Z)-nona-2-enoxy]-8-oxo-octoxy]-3-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]octanoate (1.881 g, 1.8 mmol) in methanol / THF (80 mL, 1 / 1 v / v) was added p-toluenesulfonic acid (1.71 mg, 9.01 mmol) in one portion at room temperature, and the mixture was stirred at room temperature for 2 h. TLC (30% ethyl acetate in petroleum ether) showed complete disappearance of the starting material. 5 mL of triethylamine was added to quench the reaction, and the solvent was removed in vacuo. The residue was purified by flash chromatography eluted with 0% to 10% (6%) CH3OH in DCM to give 2,3-bis(nona-8-enoxy)propan-1-ol (1.32 g, 1.65 mmol, 91.4%) as a colorless oil.

[0492] Multi-Line Report 1 H NMR (400 MHz, CDCl3) δ 5.69 - 5.58 (m, 2H), 5.57 - 5.47 (m, 2H), 4.62 (d, J = 6.7 Hz, 4H), 3.75 - 3.71 (m, 2H), 3.68 - 3.60 (m, 14H), 3.58 - 3.40 (m, 9H), 2.76 (s, 1H), 2.33 - 2.27 (m, 4H), 2.14 - 2.03 (m, 4H), 1.66 - 1.51 (m, 8H), 1.39 - 1.21 (m, 28H), 0.96 - 0.80 (m, 6H).

[0493] Step (10) [ka] To a solution of [(Z)-non-2-enyl]8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-nona-2-enoxy]-8-oxo-octoxy]propoxy]octanoate (1.32 g, 1.65 mmol) and TEA (triethylamine) (0.333 g, 3.3 mmol) in 30 mL of dichloromethane (DCM) was added Ms-Cl (0.283 g, 2.47 mmol). The mixture was stirred at room temperature for 3 hours. TLC (CHOH / DCM 3%) indicated that the starting material had been consumed. The reaction was diluted with dichloromethane and washed with water (10 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated to give nonyl [(Z)-non-2-enyl]8-[3-[2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-nona-2-enoxy]-8-oxo-octoxy]propoxy]octanoate (1.384 g, 1.57 mmol, 95.5% yield) as a pale yellow liquid, which was used directly in the next step. 1 H NMR (400 MHz, CDCl3) δ 5.70 - 5.59 (m, 2H), 5.56 - 5.48 (m, 2H), 4.62 (d, J = 6.7 Hz, 4H), 4.40 - 4.36 (m, 2H), 3.78 - 3.75 (m, 2H), 3.68 - 3.63 (m, 12H), 3.58 - 3.40 (m, 9H), 3.08 (s, 3H), 2.33 - 2.27 (m, 4H), 2.14 - 2.05 (m, 4H), 1.64 - 1.51 (m, 8H), 1.40 - 1.25 (m, 28H), 0.92 - 0.84 (m, 6H).

[0494] Step (11) [ka] NaN (0.123 g, 1.89 mmol) was then added to a solution of undecylundecyl[(Z)-non-2-enyl]8-[3-[2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-nona-2-enoxy]-8-oxo-octoxy]propoxy]octanoate (1.38 g, 1.57 mmol) dissolved in DMF (25 mL). The reaction mixture was then stirred at 70° C. for 18 h.

[0495] TLC showed that the starting material had disappeared, and a new spot was observed. Water (100 mL) was then added, and the reaction mixture was extracted with ethyl acetate (100 mL × 2). The combined organic phases were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue that was purified by column chromatography eluted with CH3OH (0-10%) in DCM (6%) as a colorless liquid (0.995 g, 1.2 mL, 76.5% yield). 1 H NMR (400 MHz, CDCl3) δ 5.71 - 5.59 (m, 2H), 5.58 - 5.46 (m, 2H), 4.62 (d, J = 6.8 Hz, 4H), 3.69 - 3.66 (m, 10H), 3.64 (s, 3H), 3.59 - 3.37 (m, 12H), 2.32 - 2.27 (m, 4H), 2.14 - 2.05 (m, 4H), 1.63 - 1.50 (m, 8H), 1.39 - 1.25 (m, 28H), 0.92 - 0.85 (m, 6H).

[0496] Step (12) [ka] A mixture of undecyl[(Z)-non-2-enyl]8-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-nona-2-enoxy]-8-oxo-octoxy]propoxy]octanoate (0.995 g, 1.2 mmol) and triphenylphosphine (0.474 g, 1.81 mmol) in THF (20 mL) / water (0.6 mL) was stirred for 16 h at 20° C. TLC (ninhydrin, 3% methanol in dichloromethane) showed the reaction to be complete. The solvent was removed and added to DCM, then concentrated under reduced pressure to give a residue which was purified by column chromatography eluted with CHOH in CHCl (0-20% (14%)) to give [(Z)-non-2-enyl]8-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-nona-2-enoxy]-8-oxo-octoxy]propoxy]octanoate (0.689 g, 0.861 mmol, 71.5% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 5.70 - 5.60 (m, 2H), 5.57 - 5.48 (m, 2H), 4.62 (d, J = 6.7 Hz, 4H), 3.72 - 3.38 (m, 25H), 2.94 - 2.87 (m, 2H), 2.32 - 2.28 (m, 4H), 2.14 - 2.06 (m, 4H), 1.65 - 1.51 (m, 8H), 1.38 - 1.26 (m, 28H), 0.91 - 0.85 (m, 6H).

[0497] Step (13) [ka] [(Z)-Nona-2-enyl]8-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-nona-2-enoxy]-8-oxo-octoxy]propoxy]octanoate (190 mg, 0.237 mmol) was dissolved in 10 mL of anhydrous DCM, and 1H-imidazole-4-carbonyl chloride (124 mg, 0.95 mmol) and DIPEA (153 mg, 1.19 mmol) in 1 mL of anhydrous DMF were added. The mixture was stirred at room temperature overnight. The solvent was evaporated and the product purified by flash chromatography (40 g column, DCM / MeOH 0% to 15%) eluted with methanol (4%) in dichloromethane to give [(Z)-non-2-enyl]8-[3-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-nona-2-enoxy]-8-oxo-octoxy]propoxy]octanoate (112 mg, 0.119 mmol, 50.1% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 7.71 - 7.56 (m, 3H), 5.70 - 5.59 (m, 2H), 5.57 - 5.47 (m, 2H), 4.62 (d, J = 6.8 Hz, 4H), 3.67 - 3.39 (m, 25H), 2.33 - 2.27 (m, 4H), 2.13 - 2.05 (m, 4H), 1.64 - 1.50 (m, 8H), 1.41 - 1.25 (m, 28H), 0.92 - 0.84 (m, 6H). LCMS: MS (ESI) m / z=895.7 (M+H) + [Example]

[0498] Synthesis of 2-butyloctyl 8-[2-[8-(2-butyloctoxy)-8-oxo-octoxy]-3-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propoxy]octanoate (Compound XIV) [ka]

[0499] As shown in FIG. 8, compound (XIV) was synthesized based on the chemistry shown in Scheme (8).

[0500] Synthesis of compound (XIV) Step (1) [ka] 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethanol (50 g, 0.176 mol) and triethylamine (36.2 g, 0.352 mol) in dry dichloromethane (600 mL) under nitrogen were cooled to 0 °C. Methanesulfonyl chloride (30.6 g, 0.264 mol) was added dropwise to this solution at 0 °C. The mixture was allowed to warm to room temperature and stirred at room temperature for 18 h. Triethylamine hydrochloride was filtered off, and the DCM solution was washed with 0.1 N HCl and dried over sodium sulfate. The solvent was removed to give 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (62 g, 92%) as a pale yellow oil, which was used without further purification. LCMS MS 363(M+H)

[0501] Step (2) [ka] To a solution of (2,2-dimethyl-1,3-dioxolan-4-yl)methanol (62 g, 0.171 mol) in THF (600 mL) was added NaH (6.17 g, 0.257 mol), and the mixture was heated to reflux for 15 minutes. The reaction was then cooled to room temperature, and 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (25.0 g, 0.171 mol) was added under nitrogen, and the reaction was heated at 80° C. for 18 hours. TLC indicated that the starting material had been consumed. The reaction was quenched with water and extracted with ethyl acetate. The aqueous layer was extracted again with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluted with 20–50% ethyl acetate in petroleum ether to give 4-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (43 g, 71% yield) as a pale yellow oil. LCMS MS 421(M+Na)

[0502] Step (3) [ka] A mixture of 4-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (43 g, 0.103 mol) in AcOH (200 mL) and water (200 mL). The mixture was stirred at ambient temperature for 16 hours. The solvent was removed to give 3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]propane-1,2-diol (36 g, 95% yield) as a pale yellow oil, which was used without further purification. LCMS MS 381(M+Na)

[0503] Step (4) [ka] To a solution of 3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]propane-1,2-diol (20 g, 0.050 mol) in THF (200 mL) was added NaH (8.03 g, 0.201 mol), and the mixture was heated to reflux for 15 minutes. The reaction was then cooled to room temperature, and 9-bromonona-1-ene (26.6 g, 0.126 mol) was added under nitrogen, and the reaction was heated at 80° C. for 18 hours. TLC indicated that the starting material had been consumed. The reaction was quenched with water and extracted with ethyl acetate. The aqueous layer was extracted again with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluted with 10–30% ethyl acetate in petroleum ether to give 2-[2-[2-[2-[2,3-bis(nona-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (8.8 g, 26% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.37 - 7.27 (m, 5H), 5.87 - 5.73 (m, 2H), 5.04 - 4.87 (m, 4H), 4.57 (s, 2H), 3.71 - 3.59 (m, 16H), 3.59 - 3.38 (m, 9H), 2.03 (q, J = 6.5 Hz, 4H), 1.60 - 1.49 (m, 4H), 1.41 - 1.28 (m, 16H).

[0504] Step (5) [ka] To a solution of 2-[2-[2-[2-[2-[2,3-bis(nona-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (8.5 g, 0.0140 mol) in MeCN (80 mL), CCl (80 mL), and water (80 mL) was added NaIO (24.9 g, 0.116 mol) and RuCl (0.66 g, 2.93 mmol). The reaction mixture was stirred at room temperature for 24 h. LCMS indicated the title compound was the major product. The reaction was filtered, and the filtrate was diluted with ethyl acetate (600 mL) and washed with 1 N aq. HCl (200 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated to give 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (8.7 g, 97% yield) as a yellow oil, which was used without further purification. 1 H NMR (400 MHz, CDCl3) δ 7.31 (dd, J = 22.6, 3.2 Hz, 5H), 4.57 (s, 2H), 3.71 - 3.61 (m, 19H), 3.59 - 3.38 (m, 11H), 2.32 (t, J = 7.4 Hz, 4H), 1.68 - 1.47 (m, 10H), 1.32 (s, 14H).

[0505] Step (6) [ka] To a solution of 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (0.0135 mol, 8.7 g) and 2-butyloctan-1-ol (0.0324 mol, 6.04 g) in dichloromethane (500 mL) under nitrogen, N,N-diisopropylethylamine (0.081 mol, 10.47 g), 4-dimethylaminopyridine (5.4 mmol, 0.66 g), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.0351 mol, 6.73 g) were added. The mixture was stirred at room temperature for 18 hours. The reaction was diluted with dichloromethane, and the organic layer was washed with 1N HCl. The organic layer was then washed with brine, then dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 0–60% ethyl acetate in petroleum ether to give 2-butyloctyl 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(2-butyloctoxy)-8-oxo-octoxy]propoxy] (2.3 g, 16.5% yield) octanoate as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 7.37 - 7.28 (m, 5H), 4.57 (s, 2H), 3.96 (d, J = 5.8 Hz, 4H), 3.74 - 3.60 (m, 18H), 3.59 - 3.38 (m, 11H), 2.29 (t, 4H), 1.84 (s, 1H), 1.67 - 1.50 (m, 12H), 1.40 - 1.19 (m, 54H), 0.89 (t, 12H).

[0506] Step (7) [ka] To 2-butyloctyl 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(2-butyloctoxy)-8-oxo-octoxy]propoxy]octanoate (2.15 g, 2.2 mmol) in ethyl acetate (50 mL) was added Pd / C (500 mg, 20% wt / wt). The mixture was stirred under hydrogen at room temperature for 18 hours. TLC (ethyl acetate / petroleum ether = 1 / 1) showed that the starting material had been consumed. The reaction was filtered through Celite and washed with ethyl acetate to give 2-butyloctyl 8-[2-[8-(2-butyloctoxy)-8-oxo-octoxy]-3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]octanoate (1.51 g, 77.1% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 3.97 (t, J = 5.8 Hz, 4H), 3.74 - 3.40 (m, 27H), 2.29 (t, J = 7.5 Hz, 4H), 1.69 - 1.48 (m, 11H), 1.37 - 1.21 (m, 48H), 0.88 (t, J = 5.3 Hz, 12H).

[0507] Step (8) [ka] 2-Butyloctyl 8-[2-[8-(2-butyloctoxy)-8-oxo-octoxy]-3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]octanoate (1.0 g, 1.12 mmol) and triethylamine (228 mg, 2.25 mmol) in dry dichloromethane (10 mL) under nitrogen were cooled to -5 °C. Methanesulfonyl chloride (193 mg, 1.69 mmol) in dry dichloromethane (10 mL) was added dropwise to this solution at 0 °C. The mixture was allowed to warm to room temperature and stirred at room temperature for 1 h. TLC (EA:PE = 1:1, Rf = 0.6) indicated that the starting material had been consumed. Triethylamine hydrochloride was filtered off, and the DCM solution was washed with 1 N HCl and dried over sodium sulfate. The solvent was removed to give 2-butyloctyl 8-[2-[8-(2-butyloctoxy)-8-oxo-octoxy]-3-[2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]octanoate (1.03 g, 94.7% yield) as a colorless oil which was used without further purification. 1 H NMR (400 MHz, CDCl3) δ 4.41 - 4.36 (m, 2H), 3.96 (t, J = 5.8 Hz, 4H), 3.79 -3.75 (m, 2H), 3.70 - 3.39 (m, 22H), 3.08 (s, 3H), 2.29 (t, J = 7.5 Hz, 4H), 1.67 - 1.49 (m, 11H), 1.39 - 1.18 (m, 48H), 0.89 (t, J = 6.6, 3.8 Hz, 12H).

[0508] Step (9) [ka] To 2-butyloctyl 8-[2-[8-(2-butyloctoxy)-8-oxo-octoxy]-3-[2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]octanoate (1.0 g, 1.0 eq) in N,N-dimethylformamide (20 mL) was added NaN3 (0.134 g, 2.0 eq) and the mixture was heated at 80 °C for 18 h. TLC showed that the starting material had been consumed. The reaction was quenched with water (200 mL) and then extracted with ethyl acetate (3 × 100 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated to give 2-butyloctyl 8-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(2-butyloctoxy)-8-oxo-octoxy]propoxy]octanoate (900 mg, 95.2% yield) without further purification. 1 H NMR (400 MHz, CDCl3) δ 3.97 (t, J = 5.8 Hz, 4H), 3.72 - 3.37 (m, 26H), 2.30 (t, 4H), 1.66 - 1.50 (m, 11H), 1.37 - 1.23 (m, 48H), 0.89 (t, 12H).

[0509] Step (10) [ka] 2-Butyloctyl 8-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(2-butyloctoxy)-8-oxo-octoxy]propoxy]octanoate (0.900 g, 1.0 eq) and triphenylphosphine (0.775 g, 3.0 eq) were dissolved in THF (30 mL) and water (3 mL). The reaction was stirred at room temperature overnight. The reaction was concentrated and purified by flash column chromatography on silica gel eluting with 5% to 25% MeOH in DCM to afford 2-butyloctyl 8-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(2-butyloctoxy)-8-oxo-octoxy]propoxy]octanoate (643 mg, 74% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 3.98 - 3.95 (m, 4H), 3.68 - 3.40 (m, 26H), 2.94 - 2.90 (m, 2H), 2.36 - 2.32 (m, 4H), 1.66 - 1.51 (m, 11H), 1.35 - 1.23 (m, 48H), 0.89 (t, J = 6.6, 3.9 Hz, 12H).

[0510] Step (11) [ka] To 2-butyloctyl 8-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(2-butyloctoxy)-8-oxo-octoxy]propoxy]octanoate (600 mg, 0.675 mmol) in dry DCM (70 mL) was added N,N-diethylethanamine (0.478 g, 7.0 eq) and 1H-imidazole-4-carbonyl chloride (0.353 g, 4.0 eq) and the mixture was stirred at room temperature for 18 hours.

[0511] TLC (DCM / MeOH=10:1) and LCMS showed the disappearance of the starting material. The mixture was concentrated and then purified by flash column chromatography on silica gel eluting with 0% to 20% methanol in dichloromethane to give 2-butyloctyl 8-[2-[8-(2-butyloctoxy)-8-oxo-octoxy]-3-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propoxy]octanoate (310 mg, 47% yield). 1 H NMR (400 MHz, CDCl3) δ 7.62 (t, J = 14.2 Hz, 3H), 3.97 (t, J = 5.8 Hz, 4H), 3.68 - 3.39 (m, 26H), 2.30 (t, J = 7.6, 1.5 Hz, 4H), 1.66 - 1.51 (m, 11H), 1.44 - 1.14 (m, 48H), 0.88 (t, J = 6.9, 4.0 Hz, 12H). [Example]

[0512] Synthesis of N-[2-[2-[2-[2-[3-[2,3-bis[(Z)-octadec-9-enoxy]propyl-octyl-amino]-3-oxopropoxy]ethoxy]ethoxy]ethoxy]ethyl]-1H-imidazole-4-carboxamide (Compound XV) [ka]

[0513] Compound (XV) was synthesized based on the chemistry shown in Scheme (13).

[0514] [ka]

[0515] Synthesis of compound (XV) Step (1) [ka] To a solution of 2,3-bis[(Z)-octadec-9-enoxy]propan-1-ol (1.0 g, 1.69 mmol) and triethylamine (0.512 g, 5.06 mmol) in DCM (20 mL) was added methanesulfonyl chloride (0.386 g, 3.37 mmol), and the mixture was stirred at room temperature for 2 hours. TLC showed that the starting material had been consumed. The reaction was quenched with water and extracted with DCM. The aqueous layer was extracted again with DCM. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated to give 2,3-bis[(Z)-octadec-9-enoxy]propyl methanesulfonate (1.1 g, 97%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 5.37 - 5.32 (m, 3H), 4.25 (dd, J = 10.9, 5.7 Hz, 1H), 3.68 (s, 2H), 3.59 - 3.39 (m, 7H), 3.17 - 3.07 (m, 7H), 3.04 (s, 3H), 2.01 (dd, J = 12.4, 6.6 Hz, 6H), 1.56 (d, J = 4.5 Hz, 4H), 1.37 - 1.22 (m, 45H), 0.88 (t, J = 6.8 Hz, 6H).

[0516] Step (2) [ka] A mixture of 3-2,3-bis[(Z)-octadec-9-enoxy]propyl methanesulfonate (4.5 g, 6.71 mmol) and octan-1-amine (17.3 g, 134 mmol) was heated at 80° C. for 18 h. The reaction mixture was purified by flash chromatography eluting with 10–50% ethyl acetate in petroleum ether to give N-[2,3-bis[(Z)-octadec-9-enoxy]propyl]octan-1-amine (4.2 g, 89% yield) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) δ 5.45 - 5.27 (m, 3H), 3.68 - 3.38 (m, 7H), 2.78 - 2.52 (m, 4H), 2.08 - 1.90 (m, 7H), 1.68 - 1.43 (m, 9H), 1.39 - 1.19 (m, 55H), 0.88 (t, J = 6.6 Hz, 9H).

[0517] Step (3) [ka] A mixture of 3-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (0.4 g, 1.09 mmol), bis(dimethylamino)methylene-(triazolo[4,5-b]pyridin-3-yl)oxonium;hexafluorophosphate (0.624 g, 1.64 mmol), DIEA (0.283 g, 2.19 mmol), and N-[2,3-bis[(Z)-octadec-9-enoxy]propyl]octan-1-amine (0.771 g, 1.09 mmol) in DCM (10 mL) was stirred at ambient temperature for 16 hours. The mixture was poured into DCM (100 mL). The organic layer was washed with 1 N HCl, saturated NaCl, dried over NaSO, and concentrated. The residue was purified by flash column chromatography on silica gel eluting with 1:81 ethyl acetate / petroleum ether to give tert-butyl N-[2-[2-[2-[2-[3-[2,3-bis[(Z)-octadec-9-enoxy]propyl-octyl-amino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate (0.95 g, 82.5%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 5.36 (dt, J = 10.6, 4.7 Hz, 3H), 3.84 - 3.16 (m, 31H), 2.69 - 2.61 (m, 2H), 2.07 - 1.93 (m, 6H), 1.60 - 1.47 (m, 6H), 1.44 (s, 9H), 1.23 (d, J = 33.4 Hz, 56H), 0.91 - 0.85 (m, 9H).

[0518] Step (4) [ka] To a mixture of tert-butyl N-[2-[2-[2-[2-[3-[2,3-bis[(Z)-octadec-9-enoxy]propyl-octyl-amino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate (0.95 g, 0.903 mmol) in DCM (5 mL) was added TFA (2.06 g, 18.1 mmol) at room temperature. The mixture was stirred at ambient temperature for 3 hours. The mixture was concentrated to give 3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]-N-[2,3-bis[(Z)-octadec-9-enoxy]propyl]-N-octyl-propanamide; 2,2,2-trifluoroacetic acid (0.94 g, 97.7%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 5.46 - 5.27 (m, 3H), 3.87 - 3.79 (m, 2H), 3.77 - 3.15 (m, 28H), 2.84 - 2.58 (m, 2H), 2.10 - 1.88 (m, 6H), 1.63 - 1.44 (m, 6H), 1.27 (s, 54H), 0.88 (t, J = 6.7 Hz, 9H).

[0519] Step (5) [ka] To a mixture of 3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]-N-[2,3-bis[(Z)-octadec-9-enoxy]propyl]-N-octyl-propanamide, 2,2,2-trifluoroacetaldehyde (0.5 g, 0.476 mmol), and N,N-diethylethanamine (0.289 g, 2.86 mmol) in DCM (40 mL) was added 1H-imidazole-4-carbonyl chloride (0.249 g, 191 mmol). The mixture was stirred at room temperature for 16 hours. The mixture was concentrated and the residue was purified by column chromatography on silica gel eluting with 2% to 8% MeOH in DCM to give N-[2-[2-[2-[2-[3-[2,3-bis[(Z)-octadec-9-enoxy]propyl-octyl-amino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethyl]-1H-imidazole-4-carboxamide (0.305 g, 61%) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.64 (d, J = 5.8 Hz, 2H), 5.46 - 5.23 (m, 3H), 3.83 -3.20 (m, 29H), 2.77 - 2.57 (m, 2H), 2.14 - 1.86 (m, 8H), 1.63 - 1.42 (m, 7H), 1.27 (s, 55H), 0.93 - 0.82 (m, 9H). [Example]

[0520] Synthesis of N-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propanoyl-octylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]-1H-imidazole-4-carboxamide (Compound XVI) [ka]

[0521] Synthesis of compound (XVI) Step (1) [ka] To a solution of N-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]octan-1-amine (2 g, 2.87 mmol) in DCM (50 ml) was added 2,3-bis[(Z)-octadec-9-enoxy]propanoic acid (2.09 g, 3.45 mmol), 4-dimethylaminopyridine (35 mg), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.64 g, 4.3 mmol), and TEA (581 mg, 5.74 mmol). The mixture was stirred at 25 °C for 18 h. The mixture was then treated with DCM (50 ml), washed with water (250 ml × 2), NaCl sat. aq. (250 ml), and dried over Na2SO4. The organics were purified by flash (5% MeOH in DCM) to give 2,3-bis[(Z)-octadec-9-enoxy]-N-octyl-N-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]propanamide (2.7 g, 2.17 mmol, 75.6% yield) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.46 (d, J = 7.6 Hz, 6H), 7.29 (t, J = 6.4 Hz, 6H), 7.25 - 7.20 (m, 3H), 5.34 (s, 3H), 4.36 (d, J = 36.5 Hz, 1H), 3.85 - 3.17 (m, 29H), 2.20 - 1.89 (m, 7H), 1.60 - 1.14 (m, 60H), 0.87 (d, J = 6.8 Hz, 9H)

[0522] Step (2) [ka] To a solution of 2,3-bis[(Z)-octadec-9-enoxy]-N-octyl-N-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]propanamide (1020 mg, 0.86 mmol) in THF / MeOH (20 mL, 1 / 1) was added toluene-4-sulfonic acid (822 mg, 4.32 mmol). The mixture was stirred at 25° C. for 2 h. TEA (1.5 ml) was added to the mixture, and the mixture was concentrated and purified by flash (10% MeOH in DCM) to give N-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]-2,3-bis[(Z)-octadec-9-enoxy]-N-octyl-propanamide (766 mg, 0.8 mmol, 92.6% yield) as a colorless oil. 1 H NMR (500 MHz, CDCl3) δ 5.39 - 5.31 (m, 4H), 3.75 - 3.56 (m, 27H), 3.46 - 3.43 (m, 2H), 1.99 (dd, J = 14.3, 7.9 Hz, 8H), 1.55 (dd, J = 11.6, 6.7 Hz, 4H), 1.26 (d, J = 7.0 Hz, 56H), 0.90 - 0.87 (m, 9H).

[0523] Step (3) [ka] To a solution of N-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]-2,3-bis[(Z)-octadec-9-enoxy]-N-octylpropanamide (860 mg, 0.92 mmol) in DCM (10 mL) was added TEA (185 mg, 1.83 mmol) and methanesulfonyl chloride (157 mg, 1.37 mmol). The mixture was stirred at 25°C for 18 hours. The mixture was then treated with DCM (50 mL), washed with water (50 mL), 1N HCl (50 mL), NaHCO₄ sat. aq. (50 mL), NaCl sat. aq. (50 mL), and dried over Na₂SO₄. The organics were concentrated to give 2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propanoyl-octylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl methanesulfonate (806 mg, 0.75 mmol) as a colorless oil. EXP-21-IV4334-N2 (621 mg) 1 H NMR (400 MHz, CDCl3) δ 5.34 (s, 4H), 4.38 (d, J = 3.7 Hz, 1H), 3.76 (d, J = 4.5 Hz, 2H), 3.70 - 3.55 (m, 22H), 3.43 (d, J = 18.9 Hz, 4H), 3.08 (s, 3H), 2.01 (d, J = 5.2 Hz, 8H), 1.56 - 1.50 (m, 4H), 1.27 (s, 56H), 0.88 (t, J = 5.0 Hz, 9H).

[0524] Step (4) [ka] To a solution of 2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propanoyl-octylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl methanesulfonate (806 mg, 0.79 mmol) in DMF (10 ml) was added NaN3 (155 mg, 2.38 mmol). The mixture was stirred at 70 °C for 14 h. The mixture was treated with EA (50 ml), washed with water (50 ml), NaCl sat. aq. (50 ml), and dried over Na2SO4. The organics were concentrated and purified by flash (50% EA in PE) to give N-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethyl]-2,3-bis[(Z)-octadec-9-enoxy]-N-octyl-propanamide (400 mg, 0.4 mmol, 50.3% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 5.42 - 5.29 (m, 4H), 4.37 (dt, J = 39.0, 5.4 Hz, 1H), 3.71 - 3.57 (m, 22H), 3.47 - 3.39 (m, 6H), 2.12 - 1.91 (m, 8H), 1.57 - 1.23 (m, 70H), 0.88 (t, J = 5.0 Hz, 9H).

[0525] Step (5) [ka] To a solution of N-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethyl]-2,3-bis[(Z)-octadec-9-enoxy]-N-octylpropanamide (420 mg, 0.45 mmol) in DCM (5 mL) was added DIEA (290 mg, 2.24 mmol) and 1H-imidazole-4-carbonyl chloride (234 mg, 1.79 mmol). The mixture was stirred at 25°C for 18 h. The mixture was treated with DCM (50 mL), washed with water (50 mL), NaCl sat. aq. (50 mL), and dried over Na2SO4. The organics were concentrated and purified by flash (0–10% MeOH in DCM) to give N-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propanoyl-octyl-amino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]-1H-imidazole-4-carboxamide (278 mg, 0.26 mmol, 58.8% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 7.64 (s, 1H), 7.59 (d, J = 17.0 Hz, 1H), 7.52 (s, 1H), 5.34 (s, 4H), 4.33 (s, 1H), 3.71 - 3.36 (m, 28H), 2.01 (d, J = 5.4 Hz, 8H), 1.27 (s, 60H), 0.88 (t, J = 5.2 Hz, 9H) [Example]

[0526] Synthesis of 6-[2-[6-(2-butyloctanoyloxy)hexoxy]-3-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propoxy]hexyl 2-butyloctanoate (Compound XVII) [ka] Compound (XVII) was synthesized based on the chemistry shown in Scheme (9) as shown in FIG.

[0527] Synthesis of compound (XVII) Step (1) [ka] To a solution of 6-bromohexan-1-ol (10 g, 55.2 mmol) and 3,4-dihydro-2H-pyran (4.78 g, 56.9 mmol) in DCM (150 mL) was added PPTS (1.61 g, 6.41 mmol), followed by stirring at room temperature for 3 hours. TLC (EA / PE 9 / 1, SM R f :0.2;Product, R f The elution rate (RI: 0.7) indicated that all the starting material had been consumed. The solvent was concentrated and purified by flash chromatography (0 to 10% EA (5%) in PE) to give 2-(6-bromohexoxy)tetrahydropyran (12.72 g, 48 mmol, 86.9% yield) as a colorless oil. 1 H NMR (500 MHz, CDCl3) δ 4.60 - 4.53 (m, 1H), 3.90 - 3.83 (m, 1H), 3.77 - 3.71 (m, 1H), 3.53 - 3.47 (m, 1H), 3.44 - 3.35 (m, 3H), 1.93 - 1.79 (m, 3H), 1.76 - 1.67 (m, 1H), 1.65 - 1.36 (m, 10H).

[0528] Step (2) [ka] To a solution of 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethanol (10 g, 35.2 mmol) and triethylamine (7.12 g, 70.3 mmol) in dry dichloromethane (100 mL) at 0 °C was added methanesulfonyl chloride (6.04 g, 52.8 mmol) in dry DCM (10 mL) dropwise. The mixture was warmed to room temperature and stirred at room temperature for 18 h. The triethylamine hydrochloride was filtered off, and the DCM solution was washed with 0.1 N HCl and dried over sodium sulfate. The solvent was removed to give 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (12.7 g, 35 mmol, quantitative) as a pale yellow oil, which was used without further purification. 1 H NMR (500 MHz, CDCl3) δ 7.35 - 7.26 (m, 5H), 4.56 (s, 2H), 4.38 - 4.34 (m, 2H), 3.77 - 3.73 (m, 2H), 3.69 - 3.61 (m, 12H), 3.06 (s, 3H).

[0529] Step (3) [ka] To a solution of (2,2-dimethyl-1,3-dioxolan-4-yl)methanol (4.63 g, 35 mmol) in dry THF (90 mL) was added NaH (4.2 g, 105 mmol) in portions at 0° C., and the mixture was then heated to reflux for 30 minutes. The reaction was then cooled to room temperature, and 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (12.7 g, 35 mmol) in dry THF (30 mL) was added under nitrogen, and the reaction was heated at 80° C. for 24 hours. TLC indicated that the starting material had been consumed. The reaction was quenched with water and extracted with ethyl acetate. The aqueous layer was extracted again with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluted with 0–5% CH OH in DCM to give 4-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (7.217 g, 18.1 mmol, 51.7% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.37 - 7.27 (m, 5H), 4.57 (s, 2H), 4.32 - 4.23 (m, 1H), 4.07 - 4.02 (m, 1H), 3.75 - 3.70 (m, 1H), 3.69 - 3.61 (m, 16H), 3.60 - 3.54(m, 1H), 3.52 - 3.46 (m, 1H), 1.42 (s, 3H), 1.35 (s, 3H).

[0530] Step (4) [ka] A mixture of 4-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (7.217 g, 18.1 mmol) in AcOH (30 mL) and HO (30 mL) was stirred at room temperature for 18 h. TLC (EA / PE 1 / 1, SM R f :0.5;Product, R fThe elution mass (Eq. 1.0:0.1) indicated that all starting material had been consumed. The solvent was removed in vacuo and azeotroped several times with toluene. 2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (6.48 g, 18.1 mmol, quantitative) was obtained as a pale yellow oil, which was used without further purification. 1 H NMR (500 MHz, CDCl3) δ 7.36 - 7.26 (m, 5H), 4.57 (s, 2H), 3.88 - 3.81 (m, 1H), 3.71 - 3.49 (m, 20H).

[0531] Step (5) [ka] To a solution of 3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]propane-1,2-diol (3.3 g, 9.21 mmol) in dry DMF (40 mL) was added NaH (1.84 g, 46 mmol) several times at 0 °C, and the mixture was then heated to 80 °C for 30 min. The reaction was then cooled to room temperature, and 2-(6-bromohexoxy)tetrahydropyran (6.1 g, 23 mmol) in dry DMF (20 mL) was added under nitrogen, and the reaction was heated at 80 °C for 18 h. TLC indicated that the starting material had been consumed. The reaction was quenched with water and extracted with ethyl acetate. The aqueous layer was extracted again with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluted with 0–5% CH OH in DCM to give 2-[6-[1-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2-(6-tetrahydropyran-2-yloxyhexoxy)ethoxy]hexoxy]tetrahydropyran (2.834 g, 3.9 mmol, 42.3% yield) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ 7.35 - 7.27 (m, 5H), 4.57 (d, J = 1.0 Hz, 4H), 3.90 - 3.83 (m, 2H), 3.78 - 3.30 (m, 31H), 1.75 - 1.65 (m, 3H), 1.63 - 1.49 (m, 17H), 1.41 - 1.34 (m, 8H).

[0532] Step (6) [ka] To a solution of 2-[6-[1-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2-(6-tetrahydropyran-2-yloxyhexoxy)ethoxy]hexoxy]tetrahydropyran (2.834 g, 3.9 mmol) in EtOH (70 mL) was added p-toluenesulfonic acid (0.742 g, 3.9 mmol) in one portion at room temperature, and the mixture was stirred at room temperature for 24 h. TLC (4% CH3OH in DCM) showed complete disappearance of the starting material. The reaction was quenched with dilute sodium bicarbonate solution (150 mL), and the solvent was extracted with EA (2 × 100 mL). The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluting with 0% to 5% CH3OH (4%) in DCM to give 6-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(6-hydroxyhexoxy)propoxy]hexan-1-ol (1.435 g, 2.57 mmol, 65.9% yield) as a colorless oil. 1 H NMR (500 MHz, CDCl3) δ 7.36 - 7.27 (m, 5H), 4.57 (s, 2H), 3.70 - 3.39 (m, 29H), 1.67 - 1.53 (m, 9H), 1.40 - 1.35 (m, 7H).

[0533] Step (7) [ka] To a solution of 6-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(6-hydroxyhexoxy)propoxy]hexan-1-ol (1.435 g, 2.57 mmol) and 2-butyloctanoic acid (1.54 g, 7.7 mmol) in dry dichloromethane (30 mL) was added DIPEA (1.99 g, 15.4 mmol), DMAP (0.126 g, 1.03 mmol), and EDCI (1.28 g, 6.68 mmol) in an ice bath. The mixture was stirred at room temperature for 18 hours. The reaction was quenched with NaHCO3 (30 mL) and washed with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography eluted with 0% to 5% (3%) CHOH in DCM to give 6-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[6-(2-butyloctanoyloxy)hexoxy]propoxy]hexyl 2-butyloctanoate (1.816 g (P: 1.15 g + NP: 0.666 g), 1.97 mmol, 76.6% yield) as a colorless oil. 1 H NMR (500 MHz, CDCl3) δ 7.36 - 7.27 (m, 5H), 4.57 (s, 2H), 4.10 - 4.01 (m, 4H), 3.72 - 3.61 (m, 16H), 3.60 - 3.40 (m, 9H), 2.35 - 2.26 (m, 2H), 1.66 - 1.52 (m, 12H), 1.47 - 1.20 (m, 36H), 0.92 - 0.83 (m, 12H).

[0534] Step (8) [ka] A solution of 6-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[6-(2-butyloctanoyloxy)hexoxy]propoxy]hexyl 2-butyloctanoate (1.15 g, 1.25 mmol) in EtOAc (20 mL) was purged with N for 10 minutes, followed by the addition of Pd / C (230 mg), and the reaction was continued while purging with N. The reaction was then evacuated under vacuum and filled with H three times. The reaction was then stirred overnight at room temperature under an H atmosphere. TLC (5% CHOH in DCM) indicated the reaction was complete. The slurry was filtered through Celite, and the Celite was rinsed several times with EtOAc. The combined organics were then concentrated in vacuo to give 6-[2-[6-(2-butyloctanoyloxy)hexoxy]-3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]hexyl 2-butyloctanoate (1.0 g, 1.2 mmol, 96.4% yield) as a colorless liquid. 1 H NMR (500 MHz, CDCl3) δ 4.09 - 4.03 (m, 4H), 3.76 - 3.39 (m, 25H), 2.82 (s, 1H), 2.35 - 2.26 (m, 2H), 1.67 - 1.52 (m, 12H), 1.47 - 1.21 (m, 36H), 0.92 - 0.83 (m, 12H).

[0535] Step (9) [ka] To a solution of 6-[2-[6-(2-butyloctanoyloxy)hexoxy]-3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]hexyl 2-butyloctanoate (0.6 g, 0.72 mmol) and TEA (triethylamine) (0.146 g, 1.44 mmol) in 10 mL of dichloromethane (DCM) was added Ms-Cl (0.124 g, 1.08 mmol). The mixture was stirred at room temperature for 3 hours. TLC (CHOH / DCM 3%) indicated that the starting material had been consumed. The reaction was diluted with dichloromethane and washed with water (10 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated to give nonyl 6-[2-[6-(2-butyloctanoyloxy)hexoxy]-3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]hexyl 2-butyloctanoate (0.582 g, 0.639 mmol, 88.7% yield) as a pale yellow liquid, which was used directly in the next step. 1 H NMR (500 MHz, CDCl3) δ 4.41 - 4.36 (m, 2H), 4.08 - 4.02 (m, 4H), 3.78 - 3.74 (m, 2H), 3.71 - 3.39 (m, 21H), 3.08 (s, 3H), 2.35 - 2.25 (m, 2H), 1.64 - 1.55 (m, 12H), 1.47 - 1.20 (m, 36H), 0.91 - 0.84 (m, 12H).

[0536] Step (10) [ka] To a solution of 6-[2-[6-(2-butyloctanoyloxy)hexoxy]-3-[2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]hexyl 2-butyloctanoate (0.582 g, 0.639 mmol) dissolved in DMF (10 mL) was then added NaN (50 mg, 0.766 mmol). The reaction mixture was then stirred at 70 °C for 18 h. TLC showed that the starting material had disappeared, and a new spot was observed. Water (100 mL) was then added, and the reaction mixture was extracted with ethyl acetate (100 mL × 2). The combined organic phases were washed with brine (100 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue that was purified by column chromatography eluted with CHOH (0–10%) in DCM (4%) to give 6-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]-2-[6-(2-butyloctanoyloxy)hexoxy]propoxy]hexyl 2-butyloctanoate (0.409 g, 0.477 mmol, 74.6% yield) as a yellow oil. 1 H NMR (500 MHz, CDCl3) δ 4.09 - 4.03 (m, 4H), 3.70 - 3.62 (m, 14H), 3.60 - 3.37 (m, 11H), 2.34 -2.26 (m, 2H), 1.63 - 1.53 (m, 12H), 1.46 - 1.21 (m, 36H), 0.91 -0.84 (m, 12H).

[0537] Step (11) [ka] A mixture of 6-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]-2-[6-(2-butyloctanoyloxy)hexoxy]propoxy]hexyl 2-butyloctanoate (0.409 g, 0.477 mmol) and triphenylphosphine (0.187 g, 0.715 mmol) in THF (10 mL) / water (0.3 mL) was stirred for 16 h at 20° C. TLC (ninhydrin, 3% methanol in dichloromethane) showed the reaction to be complete. The solvent was removed and added to DCM, then concentrated under reduced pressure to give a residue which was purified by column chromatography eluted with CHOH in CHCl (0-20% (14%)) to give 6-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-2-[6-(2-butyloctanoyloxy)hexoxy]propoxy]hexyl 2-butyloctanoate (0.300 g, 0.237 mmol, 75.6% yield) as a pale yellow oil. 1 H NMR (500 MHz, CDCl3) δ 4.09 - 4.03 (m, 4H), 3.70 - 3.41 (m, 23H), 2.91 (t, J = 5.1 Hz, 2H), 2.46 (s, 2H), 2.34 - 2.27 (m, 2H), 1.68 - 1.51 (m, 12H), 1.46 - 1.21 (m, 36H), 0.92 - 0.84 (m, 12H).

[0538] Step (12) [ka] 6-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-2-[6-(2-butyloctanoyloxy)hexoxy]propoxy]hexyl 2-butyloctanoate (300 mg, 0.36 mmol) was dissolved in 10 mL of anhydrous DCM, and 1H-imidazole-4-carbonyl chloride (188 mg, 1.44 mmol) and DIPEA (233 mg, 1.80 mmol) in 1 mL of anhydrous DMF were added. The mixture was stirred at room temperature overnight. The solvent was evaporated and the product purified by flash chromatography (25 g column, DCM / MeOH 0% to 5%) eluted with methanol (4%) in dichloromethane to give 6-[2-[6-(2-butyloctanoyloxy)hexoxy]-3-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propoxy]hexyl 2-butyloctanoate (259 mg, 0.266 mmol, 73.7% yield) as a colorless oil. LCMS: EXP-21-IX3021 -29498 -LCMSA020 Find peak: MS(ESI) m / z=926.8 / 927.8 (M+H) + at 2.854 min 1 H NMR (500 MHz, CDCl3) δ 7.77 - 7.56 (m, 3H), 4.09 - 4.02 (m, 4H), 3.69 - 3.39 (m, 25H), 2.34 - 2.27 (m, 2H), 1.65 - 1.53 (m, 12H), 1.46 - 1.22 (m, 36H), 0.91 - 0.84 (m, 12H). [Example]

[0539] Synthesis of N-[2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecoxy]ethoxy]ethoxy]ethoxy]ethyl]-1H-imidazole-4-carboxamide (Compound XVIII) [ka]

[0540] Synthesis of compound (XVIII) Step (1) [ka] To a solution of 2,3-bis[(Z)-octadec-9-enoxy]propan-1-ol (5 g, 8.43 mmol) in DCM (40 mL) was added Dess-Martin periodinane (5.05 g, 10.1 mmol) at 0 °C for 5 min. The mixture was then stirred at 25 °C under N for 2 h. After the reaction, the mixture was treated with DCM (40 mL), washed with NaHCO / NaSO (1 / 1) (50 mL × 3), NaCl sat. aq. (50 mL), and dried over NaSO. The organics were concentrated and purified by flash (10% EA in PE) to give 2,3-bis[(Z)-octadec-9-enoxy]propanal (3.04 g, 5.04 mmol, 59.8% yield) as a colorless oil. 1 H NMR (500 MHz, CDCl3) δ 9.73 (d, J = 1.3 Hz, 1H), 5.41 - 5.31 (m, 4H), 3.85 -3.77 (m, 1H), 3.75 - 3.64 (m, 2H), 3.58 (tt, J = 9.3, 4.6 Hz, 2H), 3.49 - 3.40 (m, 2H), 2.21 - 1.91 (m, 8H), 1.64 (dd, J = 14.1, 7.0 Hz, 2H), 1.57 - 1.48 (m, 2H), 1.26 (d, J = 4.4 Hz, 44H), 0.88 (dd, J = 8.7, 5.0 Hz, 6H).

[0541] Step (2) [ka] To a solution of Mg (3.75 g) and I2 (1.31 g) in anhydrous THF (5 ml) was added 1-bromododecane (2.56 g, 10.28 mmol). The mixture was stirred at 70 °C under N2 until a colorless mixture was obtained. 1-Bromododecane (10.24 g, 41.12 mmol) was added to the reaction mixture. The mixture was stirred at 70 °C for 3 h. The mixture was then added to a solution of 2,3-bis[(Z)-octadec-9-enoxy]propanal (3.04 g, 5.14 mmol) in anhydrous THF (45 ml). The mixture was stirred at 7 °C for 14 h. The mixture was treated with EA (150 ml) and washed with water (150 ml × 2) and NaCl sat. aq. (150 ml). The organics were purified by flash (5% EA in PE) to give 1,2-bis[(Z)-octadec-9-enoxy]pentadecan-3-ol (3.97 g, 5.21 mmol, 100% yield) as a yellow oil. 1 H NMR (500 MHz, CDCl3) δ 5.40 - 5.31 (m, 3H), 3.76 - 3.36 (m, 7H), 3.31 - 3.23 (m, 1H), 2.03 - 1.94 (m, 6H), 1.64 - 1.57 (m, 2H), 1.54 - 1.50 (m, 2H), 1.27 (d, J = 12.1 Hz, 66H), 0.87 (d, J = 6.7 Hz, 9H).

[0542] Step (3) [ka] To a solution of 1,2-bis[(Z)-octadeca-9-enoxy]pentadecan-3-ol (1 g, 1.31 mmol) in THF (20 ml) was added NaH (210 mg, 5.25 mmol). The mixture was stirred at 70°C for 1 h. 2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (1.01 g, 1.97 mmol) was added to the mixture, and the mixture was stirred at 70°C for 18 h. The mixture was treated with EA (150 ml), washed with water (150 ml × 2), NaCl sat. aq. (150 ml), and dried over Na2SO4. The organics were concentrated and purified by flash (10% EA in PE) to give [2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecoxy]ethoxy]ethoxy]ethoxy]ethoxy-diphenyl-methyl]benzene (1.08 g, 0.9 mmol, 68.3% yield) as a colorless oil. 1 H NMR (500 MHz, CDCl3) δ 7.48 - 7.44 (m, 6H), 7.28 (t, J = 7.6 Hz, 6H), 7.22 (t, J = 7.3 Hz, 3H), 5.39 - 5.31 (m, 3H), 3.73 - 3.54 (m, 16H), 3.50 - 3.32 (m, 6H), 3.23 (t, J = 5.3 Hz, 2H), 2.04 - 1.93 (m, 7H), 1.53 (s, 4H), 1.37 - 1.18 (m, 66H), 0.89 - 0.85 (m, 9H).EXP-21-IV4361-N2 (571 mg, yield 24%)

[0543] Step (4) [ka] To a solution of [2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecoxy]ethoxy]ethoxy]ethoxy]ethoxy-diphenylmethyl]benzene (1.65 g, 1.4 mmol) in THF / MeOH (20 ml 1 / 1) was added toluene-4-sulfonic acid (1.33 g, 6.99 mmol). The mixture was stirred at 25 °C for 18 h. The mixture was concentrated, treated with EA (150 ml), washed with NaHCO sat. aq. (150 ml × 2), NaCl sat. aq. (150 ml), and dried over NaSO. The organics were concentrated and purified by flash (50% EA in PE) to give 2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecoxy]ethoxy]ethoxy]ethoxy]ethanol (1.18 g, 1.23 mmol, 87.8% yield) as a colorless oil. 1 H NMR (500 MHz, CDCl3) δ 5.41 - 5.31 (m, 3H), 3.74 - 3.54 (m, 18H), 3.50 - 3.33 (m, 6H), 2.59 (dd, J = 9.7, 6.0 Hz, 1H), 2.04 - 1.93 (m, 7H), 1.57 - 1.22 (m, 70H), 0.88 (t, J = 6.9 Hz, 9H).

[0544] Step (5) [ka] To a solution of 2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecoxy]ethoxy]ethoxy]ethoxy]ethanol (1.1 g, 1.17 mmol) in DCM (15 mL) was added TEA (297 mg, 2.93 mmol) and methanesulfonyl chloride (269 mg, 2.35 mmol) at 0 °C for 5 min. The mixture was stirred at 25 °C for 18 h. The mixture was then treated with DCM (50 mL), washed with water (50 mL × 2), 1 N HCl (50 mL), NaHCO3 sat. aq. (50 mL), and dried over Na2SO4. The organics were concentrated to give 2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecoxy]ethoxy]ethoxy]ethoxy]ethyl methanesulfonate (860 mg, 0.8 mmol, 70.7% yield) as a yellow oil. 1 H NMR (500 MHz, CDCl3) δ 5.39 - 5.31 (m, 3H), 4.40 - 4.36 (m, 2H), 3.77 - 3.56 (m, 16H), 3.49 - 3.34 (m, 6H), 3.07 (s, 3H), 2.06 - 1.91 (m, 7H), 1.56 - 1.51 (m, 4H), 1.35 -1.21 (m, 66H), 0.88 (t, J = 6.9 Hz, 9H).

[0545] Step (6) [ka] To a solution of 2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propanoyl-octylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl methanesulfonate (860 mg, 0.85 mmol) in DMF (10 ml) was added NaN3 (165 mg, 2.54 mmol). The mixture was stirred at 70 °C for 14 h. The mixture was treated with EA (50 ml), washed with water (50 ml), NaCl sat. aq. (50 ml), and dried over Na2SO4. The organics were concentrated and purified by flash (50% EA in PE) to give N-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethyl]-2,3-bis[(Z)-octadec-9-enoxy]-N-octyl-propanamide (657 mg, 0.65 mmol, 77.4% yield) as a colorless oil. 1 H NMR (500 MHz, CDCl3) δ 5.40 - 5.30 (m, 3H), 3.78 - 3.28 (m, 24H), 2.04 - 1.92 (m, 7H), 1.56 - 1.20 (m, 70H), 0.88 (t, J = 6.9 Hz, 9H).

[0546] Step (7) [ka] To a solution of (Z)-1-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]-2-[(Z)-octadec-9-enoxy]pentadecoxy]octadec-9-ene (657 mg, 0.68 mmol) in THF / water (20 mL / 0.6 mL) was added triphenylphosphine (269 mg, 1 mmol). The mixture was stirred at 25 °C for 18 h. The mixture was concentrated and purified by flash (10% to 20% MeOH in DCM) to give 2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecoxy]ethoxy]ethoxy]ethoxy]ethanamine (560 mg, 0.59 mmol, 85.8% yield) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ 5.41 - 5.31 (m, 3H), 3.77 - 3.33 (m, 22H), 2.89 (dt, J = 12.6, 5.0 Hz, 2H), 2.06 - 1.90 (m, 7H), 1.57 - 1.51 (m, 4H), 1.48 - 1.15 (m, 66H), 0.88 (t, J = 6.9 Hz, 9H).

[0547] Step (8) [ka] To a solution of 2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecoxy]ethoxy]ethoxy]ethoxy]ethanamine (560 mg, 0.6 mmol) in DCM (20 mL) was added DIEA (386 mg, 3 mmol) and 1H-imidazole-4-carbonyl chloride (312 mg, 2.39 mmol). The mixture was stirred at 25 °C for 18 h. The mixture was treated with DCM (50 mL), washed with water (50 mL), brine (50 mL × 2), and dried over Na SO . The organics were concentrated and purified by flash (10% MeOH in DCM) to give N-[2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecoxy]ethoxy]ethoxy]ethoxy]ethyl]-1H-imidazole-4-carboxamide (348 mg, 0.32 mmol) as a colorless oil. 1 H NMR (500 MHz, CDCl3) δ 7.65 (d, J = 8.7 Hz, 1H), 7.61 (d, J = 11.0 Hz, 1H), 7.54 (s, 1H), 5.40 - 5.31 (m, 3H), 3.80 - 3.30 (m, 24H), 2.06 - 1.93 (m, 7H), 1.60 - 1.17 (m, 70H), 0.88 (t, J = 6.9 Hz, 9H). [Example]

[0548] Synthesis of bis(2-butyloctyl) 10-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethyl-nonyl-amino]nonadecanedioate; hydrochloride (compound XIX) [ka]

[0549] Compound (XIX) was synthesized according to the following scheme (10).

[0550] [ka]

[0551] Synthesis of compound (XIX) Step (1) [ka] A mixture of diethyl 3-oxopentanedioate (20 g) and 20% sodium ethoxide-ethanol solution (33.5 g) was stirred at 80° C. for 20 minutes, and then ethyl 8-bromooctanoate (25 g) was added thereto and the mixture was stirred for 4 hours. The 20% sodium ethoxide-ethanol solution (33.5 g) was added to the reaction mixture, and the reaction mixture was stirred for 5 minutes. Then ethyl 8-bromooctanoate (25 g) was added thereto and the mixture was stirred for 3 hours. The reaction mixture was cooled to room temperature, and then hexane and 20% aqueous ammonium chloride solution (110 mL) were added thereto. The organic layer was separated, and the solvent was distilled off under reduced pressure to obtain tetraethyl 9-oxoheptadecane-1,8,10,17-tetracarboxylate (51.5 g) as a crude product. LCMS Rt=2.194

[0552] Step (2) [ka] A mixture of the resulting tetraethyl 9-oxoheptadecane-1,8,10,17-tetracarboxylate (25 g), acetic acid (40 mL), and 30% aqueous hydrochloric acid solution (80 mL) was stirred at 115° C. for 6 hours. The reaction mixture was cooled to room temperature, and then the solvent was distilled off under reduced pressure, and water and acetone were added to the residue. The solid was collected by filtration, washed with water and acetone, and then dried under reduced pressure, thereby obtaining 10-oxononandecanedioic acid (0.6 g) as a white solid. 1 H NMR (400 MHz, DMSO) δ 11.97 (s, 2H), 2.38 (t, J = 7.3 Hz, 4H), 2.18 (t, J = 7.4 Hz, 4H), 1.54 - 1.35 (m, 8H), 1.23 (s, 16H).

[0553] Step (3) [ka] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (8.53 g) was added to a mixture of 10-oxononandecanedioic acid (6.10 g), 2-butyloctan-1-ol (6.63 g), triethylamine (12.5 mL), 4-dimethylaminopyridine (2.17 g), and dichloromethane (60 mL), and the mixture was stirred at room temperature for 2 days. 10% aqueous potassium hydrogen sulfate solution (120 mL), hexane (60 mL), and ethyl acetate (60 mL) were added to the reaction mixture, and the organic layer was separated and then dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate-hexane), thereby obtaining bis(2-butyloctyl)io-oxononandecanedioate (6 mg) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 3.97 (d, J = 5.8 Hz, 4H), 2.37 (t, J = 7.5 Hz, 4H), 2.29 (t, J = 7.5 Hz, 4H), 1.71 - 1.43 (m, 11H), 1.28 (d, J = 1.2 Hz, 49H), 0.89 (tt,J = 6.6, 4.1 Hz, 12H).

[0554] Step (4) [ka] A mixture of bis(2-butyloctyl)IO-oxononadecanedioate (2.3 g) and Boc-1-amino-3,6-8-octanediaminedioxadiamine (1; 27 g) was stirred in dichloromethane at room temperature for 15 minutes. Sodium triacetoborohydride (0.76 g) and acetic acid (0.21 ml) were then added. The reaction mixture was stirred at room temperature for 5 hours. After dilution with dichloromethane (25 mL), the reaction mixture was washed with saturated sodium bicarbonate (NaHCO3). The organic layer was washed with water and brine and dried over Na2SO4. After removal of the solvent, the residue was purified by flash chromatography (DCM / MeOH / TEA, 85 / 15 / 1, (v,v,v)), which gave bis(2-butyloctyl)-10-[2-[2-[2-(ter-butoxycarbonylamino]ethoxy]ethylamino]nonadecanedioate as a colorless oil.

[0555] Step (5) [ka] A mixture of bis(2-butyloctyl)-10-[2-[2-[2-(tert-butoxycarbonylamino]ethoxy]ethylamino]nonadecanedioate (0.5 g) and nonanal (0.18 g) was stirred in dichloromethane at room temperature for 15 minutes. Sodium triacetoborohydride (0.174 g) and acetic acid (0.05 ml) were then added. The reaction mixture was stirred at room temperature for 8 hours. After dilution with dichloromethane (20 mL), the reaction mixture was The extract was washed with saturated sodium bicarbonate (NaHCO3). The organic layer was washed with brine and dried over Na2SO4. After removal of the solvent, the residue was purified by flash chromatography (heptane / AcOEt (gradient 0-50%)), which gave bis(2-butyloctyl) 10-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethyl-nonyl-amino]nonadecanedioate as a colorless oil. 1 H NMR (400 MHz, DMSO-d6) δ ppm 0.83 - 0.88 (m, 15 H), 1.14 - 1.43 (m, 82 H), 1.46 - 1.59 (m, 6 H), 2.23 - 2.29 (m, 4 H), 2.34 - 2.36 (m, 2 H), 3.01 - 3.07 (m, 2 H), 3.30 - 3.34 (m, 4 H), 3.46 (s, 4 H), 3.91 (dd, J=6, 2 Hz, 4 H), 6.47 - 6.75 (m, 1 H), pseudo-molecular ion m / z =1038, retention time (min) = 2,37

[0556] Step (6) [ka] Bis(2-butyloctyl) 10-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethyl-nonyl-amino]nonadecanedioate (0.080 g) was diluted in dichloromethane (5 mL). Then, a solution of hydrochloric acid (4 M in dioxane, 5 equivalents) was added. The mixture was stirred at room temperature for 2 hours. After removal of the solvent, the residue was stirred with isopropyl ether (3 mL), filtered, and dried to give bis(2-butyloctyl) 10-[2-[2-(2-aminoethoxy)ethoxy]ethyl-nonyl-amino]nonadecanedioate; hydrochloride salt as a hygroscopic white solid. 1 H NMR (400 MHz, DMSO-d6) δ ppm 0.79 - 0.92 (m, 15 H), 1.14 - 1.59 (m, 72 H), 1.62 - 1.88 (m, 3 H), 2.27 (t, J=7 Hz, 5 H), 2.90 - 3.01 (m, 2 H), 3.01 - 3.12 (m, 2 H), 3.13 - 3.29 (m, 3 H), 3.54 - 3.66 (m, 6 H), 3.77 - 3.87 (m, 2 H), 3.92 (d, J=6 Hz, 4 H), 7.89 - 8.21 (m, 3 H), 9.51 (br s, 1 H). pseudo-molecular ion m / z = 937, retention time (min) = 2,17

[0557] Step (7) [ka] A mixture of bis(2-butyloctyl) 10-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethyl-nonyl-amino]nonadecanedioate; hydrochloride (0.011 g) and 1H-imidazole-4-carbonyl chloride (0.023 g) in a solution containing dichloromethane (3 mL) was stirred at room temperature. Triethylamine (0.038 mL) was then slowly added. The mixture was stirred at room temperature for 16 hours. After dilution with dichloromethane (20 mL), the reaction mixture was washed with water. The organic layer was dried over Na2SO4. After removal of the solvent, the residue was purified by flash chromatography (DCM / MeOH=92 / 2 and 95 / 5 (v / v)), which gave bis(2-butyloctyl) 10-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethyl-nonyl-amino]nonadecanedioate as a yellow oil. 1 H NMR (500 MHz, DMSO-d6) δ ppm 0.73 - 0.91 (m, 15 H), 1.03 - 1.38 (m, 68 H), 1.41 - 1.63 (m, 6 H), 2.26 (t, J=7 Hz, 4 H), 2.77 - 3.18 (m, 9 H), 3.34 - 3.40 (m, 2 H), 3.43 - 3.57 (m, 6 H), 3.91 (dd, J=6, 3 Hz, 4 H), 7.58 (dd), pseudo-molecular ion m / z = 1031, retention time (min) = 2,45. [Example]

[0558] Synthesis of nonyl 8-[2-[3-[2-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]ethyl-[8-(1-octylnonoxy)-8-oxooctyl]amino]octanoate (Compound XX) [ka]

[0559] Compound (XX) was synthesized based on the chemistry shown in Scheme (11).

[0560] [ka]

[0561] Synthesis of compound (XX) Step (1) [ka] To a solution of nonyl 8-[2-hydroxyethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (1.00 g, 1.41 mmol) and 3-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (0.772 g, 2.11 mmol) in dry DCM (10 mL) was added DMAP (17.2 mg, 0.141 mmol), DIPEA (0.218 g, 1.69 mmol), followed by EDCI (0.324 g, 1.69 mmol) at 0° C. The reaction was stirred at room temperature for 18 hours. TLC showed the disappearance of the starting material and the formation of a new spot. Water (20 mL) was added to quench the reaction, and the mixture was extracted with DCM (50 mL). The organic layer was washed with saturated sodium bicarbonate, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography (0–5% CH3OH (3%) in DCM) to afford nonyl 8-[2-[3-[2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (1.126 g, 1.06 mmol, 75.6% yield) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 5.07 (s, 1H), 4.92 - 4.81 (m, 1H), 4.16 - 4.02 (m, 4H), 3.78 - 3.72 (m, 2H), 3.68 - 3.58 (m, 12H), 3.58 - 3.50 (m, 2H), 3.37 - 3.26 (m, 2H), 2.71 - 2.57 (m, 4H), 2.47 - 2.38 (m, 4H), 2.33 - 2.24 (m, 4H), 1.66 - 1.56 (m, 6H), 1.53 - 1.22 (m, 65H), 0.92 - 0.83 (m, 9H).

[0562] Step (2) [ka] Nonyl 8-[2-[3-[2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (1.13 g, 1.06 mmol) was dissolved in 10 mL of DCM, and then TFA (4 mL) was added. The mixture was stirred at room temperature for 2 hours. TLC (3% CH3OH in DCM) showed that the reaction was complete. The solvent was evaporated (DCM 50 mL × 2), then dissolved in DCM (100 mL), washed with saturated NaHCO3 (10 mL), the organic layer was separated, dried over Na2SO4, filtered, and the solvent removed to give nonyl 8-[2-[3-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propanoyloxy]ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (1.0 g, 1.04 mmol, 98.1% yield) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ 4.90 - 4.82 (m, 1H), 4.15 (t, J = 6.2 Hz, 2H), 4.05 (t, J = 6.8 Hz, 2H), 3.76 (dd, J = 10.8, 5.0 Hz, 4H), 3.72 - 3.61 (m, 12H), 3.14 - 3.09 (m, 2H), 2.73 (t, J = 6.2 Hz, 2H), 2.62 (t, J = 6.0 Hz, 2H), 2.51 - 2.42 (m, 4H), 2.28 (dd, J = 14.1, 7.3 Hz, 4H), 1.67 - 1.36 (m, 14H), 1.35 - 1.22 (m, 48H), 0.90 - 0.84 (m, 9H).

[0563] Step (3) [ka] Nonyl 8-[2-[3-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propanoyloxy]ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (1.0 g, 1.04 mmol) was dissolved in 15 mL of anhydrous DCM, and 1H-imidazole-4-carbonyl chloride (545 mg, 4.18 mmol) and DIPEA (675 mg, 5.22 mmol) in 1 mL of anhydrous DMF were added. The mixture was stirred at room temperature overnight. The solvent was evaporated and the product purified by flash chromatography (25 g column, DCM / MeOH 0%-5%) eluting with 0-5% methanol (4%) in dichloromethane to give nonyl 8-[2-[3-[2-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (542.5 mg, 0.495 mmol, 47.4% yield) as a pale yellow oil. 1H NMR (500 MHz, CDCl3) δ 11.22 (s, 1H), 7.63 (s, 3H), 4.86 (p, J = 6.2 Hz, 1H), 4.16 (d, J = 5.5 Hz, 2H), 4.05 (t, J = 6.8 Hz, 2H), 3.75 - 3.57 (m, 18H), 2.72 (s, 2H), 2.54 (dd, J = 24.0, 17.5 Hz, 6H), 2.28 (dd, J = 13.5, 7.4 Hz, 4H), 1.61 (dd, J = 13.0, 6.5 Hz, 6H), 1.53 - 1.22 (m, 56H), 0.91 - 0.84 (m, 9H). LCMS: Find peak: MS(ESI) m / z=1052.9 (M+H)+ at 2.278 min [Example]

[0564] Synthesis of 6-[6-(2-hexyldecanoyloxy)hexyl-[2-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethyl]amino]hexyl 2-hexyldecanoate (Compound XXI) [ka] Compound (XXI) was synthesized based on the chemistry shown in Scheme (11) as shown in FIG.

[0565] synthesis Step (1) [ka] A solution of 2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethanol (0.450 g, 1.90 mmol) and 6-bromohexyl 2-hexyldecanoate (1.75 g, 4.17 mmol) and DIPEA (0.539 g, 4.17 mmol) in CHCN (10 mL) and cyclopentyl methyl ether (3 mL) was stirred at 65 °C for 72 h. The reaction was cooled to room temperature and the solvent was evaporated in vacuo. The residue was taken up in EtOAc (50 mL × 2) and HO (20 mL). The organic layer was separated, dried over NaSO, and evaporated in vacuo. The residue was purified by silica gel chromatography (0–5% MeOH (3%) in dichloromethane) to give 6-[6-(2-hexyldecanoyloxy)hexyl-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]amino]hexyl 2-hexyldecanoate (1.082 g, 1.18 mmol, 56.2% yield) as a pale yellow oil. 1 H NMR (500 MHz, CDCl3) δ 4.09 - 4.02 (m, 4H), 3.91 - 3.55 (m, 18H), 2.84 (s, 6H), 2.34 - 2.26 (m, 2H), 1.68 - 1.51 (m, 10H), 1.47 - 1.20 (m, 54H), 0.90 - 0.84 (m, 12H).

[0566] Step (2) [ka] To a solution of 6-[6-(2-hexyldecanoyloxy)hexyl-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]amino]hexyl 2-hexyldecanoate (1.55 g, 1.70 mmol) and TEA (triethylamine) (0.343 g, 3.39 mmol) in 15 mL of dichloromethane (DCM) was added Ms-Cl (291 mg, 2.54 mmol). The mixture was stirred at room temperature for 3 hours. TLC (CHOH / DCM 4%) indicated that the starting material had been consumed. The reaction was diluted with dichloromethane and washed with water (10 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated to give 6-[2-[6-(2-butyloctanoyloxy)hexoxy]-3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]hexyl 2-butyloctanoate (1.592 g, 1.60 mmol, 94.6% yield) as a yellow oil, which was used directly in the next step. 1 H NMR (400 MHz, CDCl3) δ 4.44 - 4.32 (m, 2H), 4.05 (t, J = 6.7 Hz, 4H), 3.91 -3.52 (m, 16H), 3.17 - 2.36 (m, 9H), 2.35 - 2.25 (m, 2H), 1.67 - 1.52 (m, 10H), 1.48 - 1.19 (m, 54H), 0.88 (t, J = 6.7 Hz, 12H).

[0567] Step (3) [ka] Then, NaN (125 mg, 1.92 mmol) was added to a solution of 6-[6-(2-hexyldecanoyloxy)hexyl-[2-[2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]amino]hexyl 2-hexyldecanoate (1.592 g, 1.6 mmol) dissolved in DMF (10 mL). The reaction mixture was then stirred at 70 °C for 18 h. TLC showed the disappearance of the starting material and the formation of a new spot (CHOH (3%) in DCM). DMF was removed under vacuum, and the residue was diluted with HO (20 mL) and then extracted with EA (2 × 50 mL). The organic layer was washed with brine (50 mL × 3), dried over NaSO, filtered, and concentrated. The residue was purified by flash chromatography column eluted with 0–4% CHOH (2%) in DCM to give 6-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethyl-[6-(2-hexyldecanoyloxy)hexyl]amino]hexyl 2-hexyldecanoate (1.059 g, 1.13 mmol, 70.3% yield) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 4.09 - 4.01 (m, 4H), 3.71 - 3.46 (m, 16H), 3.42 - 3.36 (m, 2H), 2.54 (d, J = 83.2 Hz, 6H), 2.36 - 2.25 (m, 2H), 1.68 - 1.51 (m, 8H), 1.49 - 1.20 (m, 56H), 0.92 - 0.83 (m, 12H).

[0568] Step (4) [ka] A mixture of 6-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethyl-[6-(2-hexyldecanoyloxy)hexyl]amino]hexyl 2-hexyldecanoate (1.059 g, 1.13 mmol) and triphenylphosphine (0.443 g, 1.69 mmol) in THF (20 mL) / water (0.6 mL) was stirred for 16 hours at 20° C. TLC (3% methanol in dichloromethane) showed the reaction to be complete. The solvent was removed and the residue was purified by column chromatography eluted with 0–20% CHOH in CHCl ((14%)) to give 6-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethyl-[6-(2-hexyldecanoyloxy)hexyl]amino]hexyl 2-hexyldecanoate (0.843 g, 0.923 mmol, 81.9% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 4.10 - 4.01 (m, 4H), 3.71 - 3.50 (m, 16H), 2.93 - 2.86 (m, 2H), 2.70 - 2.62 (m, 2H), 2.51 - 2.41 (m, 4H), 2.34 - 2.27 (m, 2H), 1.67 - 1.51 (m, 8H), 1.48 - 1.21 (m, 56H), 0.92 - 0.83 (m, 12H).

[0569] Step (5) [ka] 6-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethyl-[6-(2-hexyldecanoyloxy)hexyl]amino]hexyl 2-hexyldecanoate (840 mg, 0.92 mmol) was dissolved in 10 mL of anhydrous DCM, and 1H-imidazole-4-carbonyl chloride (480 mg, 3.68 mmol) and DIPEA (594 mg, 4.60 mmol) were added. The mixture was stirred overnight at room temperature. Water (10 mL) was added to the solution, which was then extracted with DCM. The organics were washed with brine and then dried over Na2SO4. The residue was purified by flash chromatography (40 g column, DCM / MeOH 0% to 6%) eluting with methanol (6%) in dichloromethane to give 6-[6-(2-hexyldecanoyloxy)hexyl-[2-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethyl]amino]hexyl 2-hexyldecanoate (488.6 mg, 0.461 mmol, 50.1% yield) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 7.7 Hz, 3H), 4.10 - 4.00 (m, 4H), 3.71 -3.52 (m, 18H), 2.72 (d, J = 73.5 Hz, 6H), 2.36 - 2.26 (m, 2H), 1.66 - 1.21 (m, 64H), 0.87 (t, J = 6.6 Hz, 12H). LCMS: EXP-21-IX3047-32609-LCMSA020 [Example]

[0570] Synthesis of 6-nonyl 8-[3-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propanoyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (Compound XXII) [ka]

[0571] Compound (XXII) was synthesized based on the chemistry shown in Scheme (12) as shown in FIG.

[0572] Synthesis of compound (XXII) Step (1) [ka] To a solution of nonyl 8-[[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (1.00 g, 1.51 mmol) in anhydrous DMF (10 mL) and anhydrous DCM (2 mL) was added 3-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (551 mg, 1.51 mmol), HATU (0.859 g, 2.26 mmol), and DIPEA (0.389 g, 3.01 mmol). The mixture was stirred at room temperature for 18 h. TLC (4% methanol in DCM) indicated the reaction was complete. The solvent was removed in vacuo, and the residue was partitioned between HO (20 mL) and ethyl acetate (2 × 50 mL). The organic layer was washed with brine (50 mL × 3) and dried over NaSO. The residue was purified by flash chromatography column eluted with 0% to 3% (2%) methanol in dichloromethane to give nonyl 8-[3-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propanoyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (1.319 g, 1.3 mmol, 86.4% yield). 1H NMR (500 MHz, CDCl3) δ 5.12 (s, 1H), 4.90 - 4.82 (m, 1H), 4.08 - 4.01 (m, 2H), 3.78 (t, J = 6.8 Hz, 2H), 3.69 - 3.60 (m, 12H), 3.55 (t, J = 5.0 Hz, 2H), 3.36 - 3.16 (m, 6H), 2.62 (t, J = 6.7 Hz, 2H), 2.33 - 2.23 (m, 4H), 1.65 - 1.47 (m, 14H), 1.44 (s, 9H), 1.35 - 1.23 (m, 48H), 0.90 - 0.85 (m, 9H).

[0573] Step (2) [ka] To a solution of nonyl 8-[3-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propanoyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (1.319 g, 1.3 mmol) in 10 mL of DCM was added TFA (4 mL), and the mixture was stirred at room temperature for 2 h. TLC (4% CH3OH in DCM) indicated the reaction was complete. The solvent was removed and azeotroped with dichloromethane (DCM 50 mL x 2), then dissolved in DCM (100 mL) and washed with saturated NaHCO3 (10 mL). The organic layer was dried over NaSO, filtered, and concentrated to give nonyl 8-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propanoyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (1.19 g, 1.24 mmol, 95.1% yield) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 4.92 - 4.80 (m, 1H), 4.09 - 4.02 (m, 2H), 3.84 - 3.71 (m, 4H), 3.71 - 3.52 (m, 12H), 3.33 - 3.04 (m, 6H), 2.60 (t, J = 5.8 Hz, 2H), 2.33 - 2.22 (m, 4H), 1.57 (dd, J = 32.9, 14.6 Hz, 14H), 1.38 - 1.21 (m, 48H), 0.92 - 0.83 (m, 9H).

[0574] Step (3) [ka] To a solution of nonyl 8-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propanoyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (600 mg, 0.657 mmol) in 10 mL of anhydrous DCM was added 1H-imidazole-4-carbonyl chloride (343 mg, 2.63 mmol) and DIPEA (424 mg, 3.28 mmol) in 1 mL of anhydrous DMF. The mixture was stirred at room temperature overnight. The solvent was removed in vacuo. The residue was then purified by flash chromatography column (eluted with 0% to 5% methanol (4%) in dichloromethane) to give nonyl 8-[3-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propanoyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (312.3 mg, 0.294 mmol, 44.8% yield) as a yellow oil. 1H NMR (500 MHz, CDCl3) δ 7.60 (t, J = 23.3 Hz, 2H), 4.91 - 4.83 (m, 1H), 4.09 -4.02 (m, 2H), 3.80 - 3.51 (m, 18H), 3.31 - 3.16 (m, 4H), 2.60 (t, J = 6.7 Hz, 2H), 2.33 - 2.24 (m, 4H), 1.66 - 1.45 (m, 14H), 1.34 - 1.23 (m, 48H), 0.90 - 0.85 (m, 9H). LCMS: Find peak: MS(ESI) m / z=1008.8 (M+H) + at 4.616 min [Example]

[0575] Synthesis of nonyl 8-[2-[3-[2-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]ethyl-[8-(1-octylnonoxy)-8-oxooctyl]amino]octanoate (Compound XXIII) [ka]

[0576] Synthesis of compound (XXIII) Step (1) [ka] A solution of nonyl 8-bromooctanoate (4.65 g, 0.0133 mol), 1-octylnonyl 8-[2-(tertbutoxycarbonylamino)ethylamino]octanoate (6 g, 0.0111 mol), and N-ethyl-N-isopropyl-propan-2-amine (1.72 g, 0.0133 mol) in acetonitrile was stirred at 65° C. for 72 hours. The reaction was cooled to room temperature, and the solvent was evaporated in vacuo. The residue was taken up in ethyl acetate and saturated sodium bicarbonate. The organic layer was separated, dried over Na2SO4, and evaporated in vacuo. The residue was purified by silica gel chromatography (a mixture of 1% NH4OH, 20% MeOH in dichloromethane) to give nonyl 8-[2-(tertbutoxycarbonylamino)ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (7.24 g, 80.7% yield). 1 H NMR (500 MHz, CDCl3) δ 4.98 (s, 1H), 4.86 (s, 1H), 4.05 (t, J = 6.8 Hz, 2H), 3.14 (s, 2H), 2.49 (s, 2H), 2.38 (s, 4H), 2.31 - 2.24 (m, 4H), 1.61 (dd, J = 14.0, 6.8 Hz, 6H), 1.50 (d, J = 6.0 Hz, 4H), 1.45 (d, J = 7.2 Hz, 9H), 1.40 (d, J = 6.3 Hz, 4H), 1.35 - 1.22 (m, 48H), 0.88 (td, J = 6.8, 2.0 Hz, 9H).

[0577] Step (2) [ka] To a solution of [(Z)-non-2-enyl] 8-[2-(tert-butoxycarbonylamino)ethyl-[(7R,11R)-3,7,11,15-tetramethylhexadecyl]amino]octanoate (3.22 g, 0.00456 mol) in CHCl (30 mL) under an ice bath, TFA (10.4 g) was added dropwise, and the mixture was stirred at room temperature for 10 h. The reaction was quenched with saturated NaHCO at 0 °C. The organic layer was washed with saturated NaHCO, 0.1 M NaOH, and brine and dried over sodium sulfate. The solvent was removed under vacuum to give [(Z)-non-2-enyl]8-[2-aminoethyl-[(7R,11R)-3,7,11,15-tetramethylhexadecyl]amino]octanoate (2.03 g, yield: 73.3%) as a colorless oil. 1 H NMR (500 MHz, CDCl3) δ 4.90 - 4.82 (m, 1H), 4.10 - 4.02 (m, 2H), 2.79 - 2.69 (m, 2H), 2.50 - 2.45 (m, 2H), 2.43 - 2.38 (m, 3H), 2.32 - 2.25 (m, 4H), 2.11 - 1.99 (m, 4H), 1.66 - 1.57 (m, 6H), 1.55 - 1.47 (m, 4H), 1.46 - 1.38 (m, 4H), 1.37 - 1.19 (m, 51H), 0.95 - 0.81 (m, 9H).

[0578] Step (3) [ka] To a solution of nonyl 8-[2-aminoethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (1.33 g, 0.00187 mol) in dichloromethane (10 ml) was added 3-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (0.684 g, 0.00187 mol), DIPEA (0.484 g, 0.00375 mol), and HATU (1.07 g, 0.00281 mol). The mixture was stirred at 25 °C for 18 h. After the reaction, the mixture was diluted with DCM (100 ml), washed with water (300 ml × 2), brine (300 ml), and dried over Na SO . The organics were concentrated and purified by flash chromatography column eluted with 20% ethyl acetate in petroleum ether to give nonyl 8-[2-[3-[2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (1.28 g, 64.8% yield) as a colorless oil. 1 H NMR (500 MHz, CDCl3) δ 5.14 - 5.06 (m, 1H), 4.91 - 4.81 (m, 1H), 4.09 -4.01 (m, 2H), 3.80 - 3.70 (m, 3H), 3.68 - 3.59 (m, 14H), 3.56 - 3.50 (m, 2H), 3.41 - 3.22 (m, 4H), 2.51 - 2.41 (m, 4H), 2.32 - 2.24 (m, 4H), 2.05 - 1.85 (m, 2H), 1.65 - 1.57 (m, 6H), 1.53 - 1.40 (m, 17H), 1.37 - 1.22 (m, 48H), 0.92 - 0.83 (m, 9H).

[0579] Step (4) [ka] To a solution of nonyl 8-[2-[3-[2-[2-[2-[2-[2-(tertbutoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]ethyl-[8-(1-octylnonoxy)-8-oxooctyl]amino]octanoate (0.275 g, 0.247 mmol) in dichloromethane (6 mL) was added TFA (0.5 mL), and the mixture was stirred at 25° C. for 18 hours. TLC (5% methanol in DCM) showed that the starting material had been consumed. The solvent was removed and the residue was diluted with DCM (50 ml), washed with 0.2 N NaOH solution (10 ml), NaHCO solution (10 ml), dried over NaSO, filtered and concentrated to give nonyl 8-[2-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propanoylamino]ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (0.21 g, 0.198 mmol, 84.4% yield) as a pale yellow oil. 1 H NMR (500 MHz, CDCl3) δ 4.09 - 4.00 (m, 2H), 3.82 - 3.49 (m, 17H), 3.21 - 3.04 (m, 4H), 2.94 - 2.84 (m, 3H), 2.81 - 2.80 (m, 8H), 2.54 - 2.48 (m, 2H), 2.33 - 2.22 (m, 4H), 1.65 - 1.46 (m, 12H), 1.37 - 1.18 (m, 48H), 0.92 - 0.82 (m, 9H).

[0580] Step (5) [ka] Nonyl 8-[2-[3-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propanoylamino]ethyl-[8-(1-octylnonoxy)-8-oxooctyl]amino]octanoate (580 mg, 0.546 mmol) and 1H-imidazole-4-carbonyl chloride (285 mg, 2.18 mmol) were dissolved in 15 mL of anhydrous DCM, and DIPEA (353 mg, 2.73 mmol) was added. The mixture was stirred at room temperature overnight. The solvent was evaporated and the product purified by flash chromatography (25 g column, DCM / MeOH 0% to 5%) eluting with methanol (4%) in dichloromethane to give nonyl 8-[2-[3-[2-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]ethyl-[8-(1-octylnonoxy)-8-oxooctyl]amino]octanoate (308 mg, 51.6% yield) as a pale yellow oil. The product of this step was combined with [(Z)-non-2-enyl]8-[2-aminoethyl-[(7R,11R)-3,7,11,15-tetramethylhexadecyl]amino]octanoate (obtained in Step 2). 1 H NMR (500 MHz, CDCl3) δ 7.72 - 7.56 (m, 3H), 4.93 - 4.77 (m, 1H), 4.11 - 3.99 (m, 2H), 3.78 - 3.42 (m, 20H), 2.94 - 2.59 (m, 6H), 2.53 - 2.43 (m, 2H), 2.31 - 2.24 (m, 4H), 1.64 - 1.56 (m, 9H), 1.53 - 1.48 (m, 4H), 1.34 - 1.23 (m, 49H), 0.90 - 0.85 (m, 9H). 526(M+1) 98% UV (214nm) 1H-NMR (500 MHz, CDCl3) δ 7.72 - 7.56 (m, 3H), 4.93 - 4.77 (m, 1H), 4.11 - 3.99 (m, 2H), 3.78 - 3.42 (m, 20H), 2.94 - 2.59 (m, 6H), 2.53 - 2.43 (m, 2H), 2.31 - 2.24 (m, 4H), 1.64 - 1.56 (m, 9H), 1.53 - 1.48 (m, 4H), 1.34 - 1.23 (m, 49H), 0.90 - 0.85 (m, 9H). LSMC: 526(M+1) 98% UV (214 nm). [Example]

[0581] Synthesis of 6-[2-[6-(2-hexyldecanoyloxy)hexoxy]-3-[2-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethyl-octyl-amino]-3-oxo-propoxy]hexyl 2-hexyldecanoate (Compound XXIV) [ka]

[0582] Synthesis of compound (XXIV) To 6-[3-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethyl-octyl-amino]-2-[6-(2-hexyldecanoyloxy)hexoxy]-3-oxopropoxy]hexyl 2-hexyldecanoate (790 mg, 0.71 mmol) in dry DCM (20 mL) was added N,N-diethylethanamine (0.72 g, 7.10 mmol) and 1H-imidazole-4-carbonyl chloride (0.74 g, 5.67 mmol), and the mixture was stirred at room temperature for 18 hours. TLC showed that the starting material had disappeared. The mixture was concentrated and then purified by flash column chromatography on silica gel eluting with 3% to 6% (5%) methanol in dichloromethane to give 6-[2-[6-(2-hexyldecanoyloxy)hexoxy]-3-[2-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethyloctyl-amino]-3-oxo-propoxy]hexyl 2-hexyldecanoate (570 mg, 66.5% yield).

[0583] Multi-Line Report 1 H-NMR (400 MHz, CDCl3) δ 7.65 (s, 1H), 7.57 (s, 1H), 4.47 - 4.30 (m, 1H), 4.09 - 4.01 (m, 4H), 3.73 - 3.36 (m, 28H), 2.35 - 2.27 (m, 2H), 1.65 - 1.32 (m, 28H), 1.25 (s, 50H), 0.90 - 0.85 (m, 15H). [Example]

[0584] Synthesis of 1-hexylnonyl 8-[[8-(1-hexylnonoxy)-8-oxo-octyl]-[2-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethyl]amino]octanoate (Compound XXV) [ka]

[0585] Synthesis of compound (XXV) 1-Hexylnonyl 8-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethyl-[8-(1-hexylnonoxy)-8-oxooctyl]amino]octanoate (780 mg, 0.828 mmol) was dissolved in 10 mL of anhydrous DCM, and 1H-imidazole-4-carbonyl chloride (433 mg, 3.31 mmol) and DIPEA (535 mg, 4.14 mmol) were added. The mixture was stirred at room temperature overnight. Water (10 mL) was added to the solution, which was then extracted with DCM. The organics were washed with brine and then dried over Na2SO4. The residue was purified by flash chromatography (40 g column, DCM / MeOH 0% to 6%) eluting with methanol (4%) in dichloromethane to give 1-hexylnonyl 8-[[8-(1-hexylnonoxy)-8-oxo-octyl]-[2-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethyl]amino]octanoate (533.4 mg, 0.518 mmol, 62.6% yield) as a yellow oil. 1 H-NMR (500 MHz, CDCl3) δ 7.65 - 7.57 (m, 2H), 7.54 (s, 1H), 4.90 - 4.83 (m, 2H), 3.69 - 3.49 (m, 18H), 2.73 (d, J = 90.4 Hz, 6H), 2.28 (t, J = 7.5 Hz, 4H), 1.67 - 1.17 (m, 68H), 0.88 (t, J = 6.9 Hz, 12H). [Example]

[0586] Synthesis of 1-octylnonyl 8-[3-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethylcarbamoyloxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (Compound XXVI) [ka]

[0587] synthesis Step (1) [ka] To a solution of 3-benzyloxypropane-1,2-diol (5 g, 27.4 mmol) in DMF (150 ml) was added NaH (5.40 g, 137 mmol). The mixture was stirred at 80 °C for 1 h. 9-Bromo-1-ene (14.1 g, 68.6 mmol) in DMF (10 ml) was added to the mixture at 25 °C, and the mixture was then stirred at 80 °C for 18 h. The mixture was treated with EA (300 ml), washed with water (300 ml × 2), aqueous LiCl (300 ml), and aqueous NaCl (300 ml), and dried over Na2SO4. The organics were concentrated and purified by flash (5% EA in PE) to give 2,3-bis(non-8-enoxy)propoxymethylbenzene (3.74 g, 8.51 mmol, 31% yield) as a colorless oil. 1 H NMR (500 MHz, CDCl3) δ 7.37 - 7.26 (m, 5H), 5.81 (ddt, J = 16.9, 10.2, 6.7 Hz, 2H), 4.94 (ddd, J = 17.4, 10.2, 9.3 Hz, 4H), 4.55 (s, 2H), 3.63 - 3.39 (m, 9H), 2.03 (td, J = 7.9, 1.3 Hz, 4H), 1.58 - 1.26 (m, 20H).

[0588] Step (2) [ka] To a solution of 2,3-bis(octa-7-enoxy)-N-octyl-N-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]propanamide (3.74 g, 8.68 mmol) in ACN / CCl / H2O (80 mL / 80 mL / 80 mL) was added NaIO4 (14.9 g, 69.5 mmol) and ruthenium(III) chloride hydrate (392 mg, 1.74 mmol). The mixture was stirred at 25 °C for 18 h. The mixture was filtered, treated with EA (500 mL), washed with Na2SO3 aqueous solution (500 mL), brine (500 mL), and dried over Na2SO4. The organics were concentrated and treated with tert-butyl alcohol / water (90 mL / 30 mL). Sodium chlorite (2.36 g, 26.1 mmol), 2-methyl-2-butene (15.2 g, 217 mmol), and sodium dihydrogen phosphate (3.13 g, 26.1 mmol) were added to the mixture. The mixture was stirred at 25 °C for 2 h. The mixture was then treated with EA (500 ml), washed with water (500 ml), brine (500 ml), and dried over Na SO . The organics were concentrated and purified by flash (10% MeOH in DCM) to give 7-[2-(6-carboxyhexoxy)-3-[octyl-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]amino]-3-oxo-propoxy]heptanoic acid (2.58 g, 5.25 mmol, 60.5% yield) as a gray oil. 1 H NMR (500 MHz, CDCl3) δ 7.38 - 7.27 (m, 5H), 4.55 (s, 2H), 3.62 - 3.41 (m, 9H), 2.44 - 2.29 (m, 4H), 1.59 (dt, J = 44.8, 6.8 Hz, 8H), 1.33 (s, 12H).

[0589] Step (3) [ka] To a solution of 8-[3-benzyloxy-2-(7-carboxyheptoxy)propoxy]octanoic acid (2.58 g, 5.53 mmol) in DCM (20 ml) was added heptadecan-9-ol (3.12 g, 12.2 mmol), and 3-(ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine hydrochloride (3.18 g, 16.6 mmol), N-ethyl-N-isopropyl-propan-2-amine (2.5 g, 19.4 mmol), and N,N-dimethylpyridin-4-amine (338 mg) were added. The mixture was stirred at 25 °C for 18 h. The mixture was treated with EA (300 ml), washed with water (300 ml × 2), NaCl sat. aq. (300 ml), and dried over Na2SO4. The organics were concentrated and purified by flash (5% EA in PE) to give 1-octylnonyl 8-[3-benzyloxy-2-[8-(1-octylnonoxy)-8-oxooctoxy]propoxy]octanoate (2 g, 2.08 mmol) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 7.37 - 7.27 (m, 5H), 4.90 - 4.82 (m, 2H), 4.55 (s, 2H), 3.59 - 3.40 (m, 9H), 2.30 - 2.24 (m, 4H), 1.52 - 1.23 (m, 76H), 0.88 (t, J = 6.8 Hz, 12H).

[0590] Step (4) [ka] To a solution of 1-octylnonyl 8-[3-benzyloxy-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (2.28 g, 2.42 mmol) in EA (50 ml) was added Pd / C (514 mg). The mixture was stirred under H2 at 25 °C for 18 h. The mixture was then filtered and concentrated to give 1-octylnonyl 8-[3-hydroxy-2-[8-(1-octylnonoxy)-8-oxooctoxy]propoxy]octanoate (1.93 g, 2.22 mmol, 91.7% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 4.95 - 4.80 (m, 2H), 3.81 - 3.38 (m, 9H), 2.27 (t, J = 7.5 Hz, 4H), 1.72 - 1.14 (m, 76H), 0.88 (t, J = 6.8 Hz, 12H).

[0591] Step (5) [ka] To a solution of 1-octylnonyl 8-[3-hydroxy-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (1.93 g, 2.26 mmol) in DMF (50 ml) was added bis(2,5-dioxopyrrolidin-1-yl)carbonate (2.32 g, 9.05 mmol) and N,N-dimethylpyridin-4-amine (1.11 g, 9.05 mmol). The mixture was stirred at 25 °C for 18 h. The mixture was treated with EA (300 ml), washed with water (300 ml × 2), aqueous LiCl (300 ml), aqueous NaCl (300 ml), and dried over Na2SO4. The organics were concentrated and purified by flash (10–20% EA in PE) to give 1-octylnonyl 8-[3-(2,5-dioxopyrrolidin-1-yl)oxycarbonyloxy-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (1.66 g, 1.64 mmol, 72.3% yield) as a colorless oil.

[0592] Step (6) [ka] To a solution of tert-butyl N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]carbamate (300 mg, 1.21 mmol) in DCM (15 ml) was added 1-octylnonyl 8-[3-(2,5-dioxopyrrolidin-1-yl)oxycarbonyloxy-2-[8-(1-octylnonoxy)-8-oxooctoxy]propoxy]octanoate (1 g, 1.01 mmol), N,N-diethylethanamine (153 mg, 1.51 mmol), and N,N-dimethylpyridin-4-amine (12 mg). The mixture was stirred at 25 °C for 18 h. The mixture was then treated with EA (50 ml), washed with water (50 ml × 2), NaCl sat. aq. (50 ml), and dried over Na2SO4. The organics were concentrated and purified by flash (20% EA in PE) to give 1-octylnonyl 8-[3-[2-[2-[2-(tertbutoxycarbonylamino)ethoxy]ethoxy]ethylcarbamoyloxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (763 mg, 0.67 mmol, 65.9% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 5.26 (s, 1H), 5.05 (s, 1H), 4.91 - 4.80 (m, 2H), 4.14 (ddd, J = 16.8, 11.4, 6.5 Hz, 2H), 3.63 - 3.22 (m, 19H), 2.33 - 2.20 (m, 4H), 1.60 - 1.14 (m, 85H), 0.88 (t, J = 6.8 Hz, 12H).

[0593] Step (7) [ka] To a solution of 1-octylnonyl 8-[3-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethylcarbamoyloxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (763 mg, 0.68 mmol) in DCM (10 ml) was added 2,2,2-trifluoroacetic acid (0.5 ml). The mixture was stirred at 25 °C for 2 h. The mixture was treated with EA (50 ml), washed with NaHCO aq. (50 ml), NaCl sat. aq. (50 ml), and dried over NaSO. The organics were concentrated and purified by flash (5–10% EA in PE) to give 1-octylnonyl 8-[3-[2-[2-(2-aminoethoxy)ethoxy]ethylcarbamoyloxy]-2-[8-(1-octylnonoxy)-8-oxo-octoxy]propoxy]octanoate (310 mg, 0.3 mmol, 43.7% yield) as a colorless oil. 1 H NMR (500 MHz, CDCl3) δ 5.73 (s, 1H), 4.86 (p, J = 6.3 Hz, 2H), 4.16 (ddd, J = 60.4, 11.6, 4.6 Hz, 2H), 3.72 - 3.29 (m, 19H), 2.98 (s, 2H), 2.27 (t, J = 7.5 Hz, 4H), 1.56 (ddd, J = 28.8, 18.1, 6.3 Hz, 16H), 1.33 - 1.24 (m, 60H), 0.88 (t, J = 6.9 Hz, 12H).

[0594] Step (8) [ka] To a solution of 1-octylnonyl 8-[3-[2-[2-(2-aminoethoxy)ethoxy]ethylcarbamoyloxy]-2-[8-(1-octylnonoxy)-8-oxooctoxy]propoxy]octanoate (310 mg, 0.3 mmol) in DCM (5 ml) was added N-ethyl-N-isopropyl-propan-2-amine (195 mg, 1.51 mmol) and 1H-imidazole-4-carbonyl chloride (158 mg, 1.21 mmol). The mixture was stirred at 25° C. for 18 hours. The mixture was concentrated and purified by flash (7% MeOH in DCM) to give 1-octylnonyl 8-[3-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethylcarbamoyloxy]-2-[8-(1-octylnonoxy)-8-oxooctoxy]propoxy]octanoate (259 mg, 0.23 mmol, 75% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 4.9 Hz, 2H), 7.52 (s, 1H), 6.02 (s, 1H), 4.91 - 4.82 (m, 2H), 4.21 (d, J = 7.6 Hz, 1H), 4.08 (dd, J = 11.6, 5.3 Hz, 1H), 3.70 - 3.34 (m, 19H), 2.28 (t, J = 7.5 Hz, 4H), 1.61 - 1.13 (m, 76H), 0.87 (t, J = 6.8 Hz, 12H) [Example]

[0595] Synthesis of [(Z)-non-2-enyl]8-[3-[2-[2-[2-[2-[2-[2-(1H-imidazole-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethyl-octyl-amino]-2-[8-[(Z)-nona-2-enoxy]-8-oxooctoxy]-3-oxo-propoxy]octanoate (Compound XXVII) [ka]

[0596] Synthesis of compound (XXVII) Step (1) [ka] 2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl methanesulfonate (11 g, 17.7 mmol) was added to octan-1-amine (44 ml), and the mixture was stirred at 80 °C for 18 h. LCMS showed that SM had been consumed and product had been formed. The mixture was treated with EA (500 ml), washed with water (500 ml × 2), NaCl sat. aq. (500 ml), and dried over Na2SO4. The organics were concentrated and purified by flash (10% MeOH in DCM) to give N-[2-[2-[2-[2-(2-trityloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]octan-1-amine (9.5 g, 15.7 mmol, 88.4% yield) as a yellow oil. 1 H NMR (500 MHz, CDCl3) δ 7.49 - 7.42 (m, 6H), 7.29 (dd, J = 10.4, 4.8 Hz, 6H), 7.25 - 7.20 (m, 3H), 3.71 - 3.54 (m, 16H), 3.23 (t, J = 5.2 Hz, 2H), 2.76 (t, J = 5.3 Hz, 2H), 2.61 - 2.55 (m, 2H), 1.27 (d, J = 4.2 Hz, 12H), 0.87 (d, J = 7.1 Hz, 3H).

[0597] Step (2) [ka] To a solution of 2,3-bis(nona-8-enoxy)propan-1-ol (12.8 g, 37.6 mmol) in DCM (200 mL) was added Dess-Martin periodinane (23.9 g, 56.4 mmol) at 0 °C for 5 min. The mixture was then stirred at 25 °C for 2 h. The mixture was concentrated, treated with EA (500 mL), washed with NaSO aq / NaHCO aq (500 mL / 500 mL), brine (500 mL), and dried over NaSO. The organics were concentrated to give 2,3-bis(nona-8-enoxy)propanal (11.7 g, crude, 90.1% yield) as a colorless oil...

Claims

1. Lipid compounds of formula (II): R1-Z-NH-CX-(NH) n -A (II) (In the formula, - R1 is one C 10 ~C 55 is a lipophilic or hydrophobic tail group of Z is a spacer arm having 2 to 24 carbon atoms in a branched or unbranched straight chain, saturated or unsaturated hydrocarbon chain interrupted by oxygen and / or one or more atoms of a moiety selected from: -S-S-; -(O=C)-; -(C=O)-O-; -O-(O=C)-; -S-; -NH-, -NH-(O=C)-; -(O=C)-NH- and -NH-(C=O)-O-, and optionally terminated by an oxygen atom or a moiety selected from among: -NH-(O=C)--O-(O=C)-; -(C=O)-O-; and -(O=C)-, which is linked to a hydrophobic tail group; n is 0; X is an oxygen atom, A represents a 4-imidazolyl radical), or a racemic, enantiomeric, or diastereomeric isomeric form thereof, or a pharmaceutically acceptable salt thereof.

2. 10. The compound of claim 1 in cationic form.

3. 10. The compound of claim 1 having an apparent pKa of less than 7, or in the range of 4.5 to 7.

4. C 10 ~C 55 The lipophilic or hydrophobic tail group of 10 ~C 55 4. The compound according to claim 1, which is a hydrocarbon radical, the hydrocarbon backbone of which may optionally be interrupted by one or more atoms of oxygen or nitrogen and / or one or more -O-CO- or -CO-O-.

5. C 10 ~C 55 The lipophilic or hydrophobic tail group of 【Chemistry 1-1】 [Chemistry 1-2] [Chemistry 1-3] [Chemistry 1-4] The compound according to any one of claims 1 to 4, selected from the group consisting of:

6. The compound of any one of claims 1 to 5, wherein the hydrophobic or lipophilic tail contains at least one amino moiety involved in its attachment to the spacer.

7. The compound of any one of claims 1 to 6, wherein the hydrophobic or lipophilic tail contains at least three or more hydrocarbon chains.

8. A compound according to any one of claims 1 to 7, wherein the spacer arm Z comprises from 1 to 12 ethylene oxide units.

9. The spacer arm Z is at least one selected from -(C=O)-O-; -O-(O=C)-; -NH-(O=C)-; -(O=C)-NH- and -NH-(C=O)-O-.

9. The compound of claim 8 incorporating one moiety.

10. Spacer arm Z is 【Chemistry 2-1】 【Chemistry 2-2】 The compound according to any one of claims 1 to 9, selected from the group consisting of:

11. The compound of any one of claims 1 to 10, wherein the spacer arm Z comprises 1 to 12 ethylene oxide units and further incorporates at least one NH-(C=O)-O-. 【Request Item 12】 【Chemistry 3-1】 【Chemistry 3-2】 【Chemistry 3-3】 [Chemistry 3-4] 12. The compound according to any one of claims 1 to 11, selected from the group consisting of: or a racemic, enantiomeric, or diastereomeric isomeric form thereof, or a pharmaceutically acceptable salt thereof.

13. 13. The compound of claim 12, selected from among compounds (IV), (VIII), (IX), (XII), (XVI), (XIX) and (XXII), or a racemic, enantiomeric, diastereomeric isomeric form thereof, or a pharmaceutically acceptable salt thereof.

14. Formula (IV): 【Chemistry 4】 14. The compound of claim 13, which is a compound of the formula: or a racemic, enantiomeric, diastereomeric isomeric form thereof, or a pharmaceutically acceptable salt thereof.

15. 15. A composition comprising at least one lipid compound according to any one of claims 1 to 14 and at least one lipid selected from the group consisting of neutral lipids, steroid alcohols or esters thereof, and PEGylated lipids.

16. 16. The composition of claim 15, wherein the neutral lipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine; DPPS; DOPG; sphingomyelin; and ceramide.

17. 17. The composition of claim 15 or 16, wherein the steroid alcohol or ester thereof is selected from the group consisting of cholesterol and its derivatives, ergosterol, desmosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, dihydrolanosterol, zymosterol, lathosterol, diosgenin, sitosterol, sitostanol, campesterol, 24-methylenecholesterol, cholesteryl margarate, cholesteryl oleate, and cholesteryl stearate.

18. 18. The composition of any one of claims 15 to 17, wherein the PEGylated lipid is selected from the group consisting of PEG-DAG, DMG-PEG, PEG-PE, PEG-S-DAG, PEG-S-DMG, PEG-cer, mPEG-N,N-ditetradecylacetamide, or PEG-dialkoxypropylcarbamate.

19. 19. The composition of any one of claims 15 to 18, comprising at least one neutral lipid, at least one steroid alcohol or ester thereof, and at least one PEGylated lipid, wherein the lipid compounds, the neutral lipid, the steroid alcohol or ester thereof, and the PEGylated lipid are present in molar amounts of 30% to 70% lipid compound, 0% to 50% neutral lipid, 20% to 50% steroid alcohol or ester thereof, and 1% to 15% PEGylated compound, relative to the total amount of lipid and lipid compounds.

20. The composition of any one of claims 15 to 19, further comprising at least one nucleic acid.

21. 21. The composition of claim 20, wherein at least one nucleic acid encodes an antigen.

22. A lipid nanoparticle comprising at least one lipid compound according to any one of claims 1 to 14 and at least one nucleic acid.

23. 23. The lipid nanoparticle of claim 22, further comprising at least one lipid as defined in any one of claims 15 to 19.

24. A pharmaceutical composition comprising: (i) at least one nucleic acid and at least one lipid compound according to any one of claims 1 to 14; or (ii) at least one nucleic acid and at least one lipid compound according to any one of claims 1 to 14. A pharmaceutical composition comprising at least one composition according to any one of claims 15 to 19, or (iii) at least one lipid nanoparticle according to claim 22 or 23.

25. An immunogenic composition comprising: (i) at least one nucleic acid encoding an antigen and at least one lipid compound according to any one of claims 1 to 14; or (ii) at least one nucleic acid encoding an antigen and at least one composition according to any one of claims 15 to 19; or (iii) at least one lipid nanoparticle according to claim 22 or 23, wherein the nucleic acid encodes at least one antigen.

26. A composition for use as a medicament, comprising (i) at least one nucleic acid and at least one lipid compound according to any one of claims 1 to 14, or (ii) at least one nucleic acid encoding an antigen and at least one composition according to any one of claims 15 to 19, or (iii) at least one lipid nanoparticle according to claim 22 or 23.

27. 21. A composition for use in a therapeutic method for preventing and / or treating a disease selected from the group consisting of infectious diseases, allergies, autoimmune diseases, rare blood diseases, rare metabolic diseases, rare neurological diseases, and tumor or cancer diseases, comprising: (i) at least one nucleic acid and at least one lipid compound according to any one of claims 1 to 14, or (ii) at least one nucleic acid encoding an antigen and at least one composition according to any one of claims 15 to 19, or (iii) at least one lipid nanoparticle according to claim 22 or 23.

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