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

Abstract

The present disclosure relates to novel lipid compounds, lipid nanoparticles (LNP) containing them, and the use of said lipid compounds or said LNP for the delivery of nucleic acids. The lipid compounds as disclosed herein comprise at least one terminal group of formula (I): *-NH-CX-(NH)n-A (I), wherein: - *- represents the direct or indirect attachment of said group of formula (I) to one C 10 to C 55 a single bond of a lipophilic or hydrophobic tail group; - n is 0 or 1 ; - X is an oxygen or sulfur atom, and - A represents an optionally substituted 5- or 6-membered unsaturated heterocyclic group or 5- or 6-membered heteroaromatic ring group, both containing at least one nitrogen atom; or one pharmaceutically acceptable salt of said group of formula (I); and said compounds in all possible isomeric forms of racemates, enantiomers and diastereomers.

Description

[TECHNICAL FIELD]

[0001] The present disclosure is in the field of novel lipid compounds that can be used to form lipid nanoparticles for delivery of therapeutic agents such as nucleic acids, for example in combination with other lipid components such as neutral lipids, steroids or esters thereof, and polymer-conjugated lipids. For example, formulations prepared with lipid compounds as disclosed herein are able to induce an immune response upon administration of polynucleotides encoding antigens. [BACKGROUND]

[0002] The field of polynucleotide therapy has seen significant advances in recent years. Polynucleotides include a variety of nucleic acid-based compounds such as messenger RNA (mRNA), antisense oligonucleotides, ribozymes, deoxyribozymes, plasmids, or immunostimulatory nucleic acids. Some nucleic acids, such as mRNA, plasmids, and ssDNA, can be used to induce expression of specific cellular products that can be used to treat, for example, diseases associated with protein or enzyme deficiencies; or expression of 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 or not it is inherent to the system. The expression products of nucleic acids can augment existing protein levels, replace missing or non-functional forms of proteins, or introduce new proteins and associated functions in a cell or organism, or expose to foreign proteins to induce specific immune responses.

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

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

[0005] While lipid nanoparticle-based carriers comprising cationic lipid components have shown promising results in 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 ionizable lipids that exhibit improved pharmacokinetic properties and are capable of delivering various types of polynucleotides to a wide variety of cell types and tissues with enhanced efficiency. Importantly, there also remains a need for novel cationic ionizable lipids that have reduced toxicity and are capable of efficiently delivering encapsulated polynucleotides to target cells, tissues, and organs. Improved cationic lipids and lipid nanoparticles for the delivery of polynucleotides would also provide optimal polynucleotide / lipid ratios, protect the polynucleotides from degradation and clearance in serum, be suitable for systemic or local delivery, and provide intracellular delivery of the polynucleotides. In addition, the lipid-polynucleotide particles should be well-tolerated and provide a sufficient therapeutic index such that treatment of a patient with an effective polynucleotide dose is not associated with unacceptable toxicity and / or risk to the patient. In addition, the lipid-nucleic acid particles should be stable as liquid formulations when stored in a pharmaceutically acceptable buffer at 4-8°C for long periods of time.

[0007] The present disclosure provides these and related advantages. [SUMMARY]

[0008] Accordingly, one of the objects of the present disclosure relates to a cationic and / or ionizable lipid compound comprising at least one terminal group of formula (I):

[0009] *-NH-CX-(NH) n -A (I)

[0010] wherein:

[0011] *- indicates that the group of formula (I) is directly or indirectly connected to one C 10 to C 55 single bond of a lipophilic or hydrophobic tail group;

[0012] n is 0 or 1 ;

[0013] X is an oxygen or sulfur atom;

[0014] A represents an optionally substituted 5- or 6-membered unsaturated heterocyclic or 5- or 6-membered heteroaromatic ring group, both containing at least one nitrogen atom;

[0015] or a pharmaceutically acceptable salt of the group of formula (I); and the lipid compound in all possible isomeric forms of the racemates, enantiomers and diastereomers.

[0016] For example, the lipid compounds of the present disclosure are cationic lipids. Another object of the present disclosure relates to a compound of formula (II):

[0017] R1-Z-NH-CX-(NH)n - A (II)

[0018] wherein:

[0019] - X, n and A are as defined in formula (I);

[0020] - R1is a C 10 to C 55 lipophilic or hydrophobic tail group;

[0021] - Z is a spacer arm having from 2 to 24, for example from 2 to 18, for example from 4 to 12 carbon atoms in a branched or unbranched straight saturated or unsaturated hydrocarbon chain, said chain being interrupted by one or several oxygen atoms and / or by a moiety selected from the group consisting of -S-S-; (C=0)-0-; -0-(0=C)-; -S-; -NH-, -NH-(0=C)-; -(0=C)-NH- and -NH-(C=0)-0- and / or and terminated by an oxygen atom or a moiety selected from the group consisting of -NH-(0=C)--0-(0=C)-- and -(0=C)-, which is linked to said hydrophobic tail group.

[0022] - p is 0 or 1 ; and

[0023] or a pharmaceutically acceptable salt of said compound of formula (II); and any of the isomeric forms of racemates, enantiomers and diastereomers thereof.

[0024] According to one embodiment, the compound of formula (II) is selected from the following compounds. It is worth noting that, in the formulae developed below, the secondary amino moiety can be written indifferently as -NH- or -N-.

[0025] Compound (III)

[0026]

[0027] (III)

[0028] Compound (IV)

[0029]

[0030] (IV)

[0031] Compound (V)

[0032]

[0033] (V)

[0034] Compound (VI)

[0035]

[0036] (VI)

[0037] Compound (VII)

[0038]

[0039] (VII)

[0040] Compound (VIII)

[0041]

[0042] (VIII)

[0043] Compound (IX)

[0044]

[0045] (IX)

[0046] Compound (X)

[0047]

[0048] (X)

[0049] Compound (XI)

[0050]

[0051] (XI)

[0052] Compound (XII)

[0053]

[0054] (XII)

[0055] Compound (XIII)

[0056]

[0057] (XIII)

[0058] Compound (XIV)

[0059]

[0060] (XIV)

[0061] Compound (XV)

[0062]

[0063] (XV)

[0064] Compound (XVI)

[0065]

[0066] (XVI)

[0067] Compound (XVII)

[0068]

[0069] (XVII)

[0070] Compound (XVIII)

[0071]

[0072] (XVIII)

[0073] Compound (XIX)

[0074]

[0075] (XIX)

[0076] Compound (XX)

[0077]

[0078] (XX)

[0079] Compound (XXI)

[0080]

[0081] (XXI)

[0082] Compound (XXII)

[0083]

[0084] (XXII)

[0085] Compound (XXIII)

[0086]

[0087] (XXIII)

[0088] Compound (XXIV)

[0089]

[0090] (XXIV)

[0091] Compound (XXV)

[0092]

[0093] (XXV)

[0094] Compound (XXVI)

[0095]

[0096] (XXVI)

[0097] Compound (XXVII)

[0098]

[0099] (XXVII)

[0100] and pharmaceutically acceptable salts thereof, and isomeric forms of racemates, enantiomers and diastereomers thereof.

[0101] For example, the compound of formula (II) can 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 any one of formula (III), (IV) or (V), or compound (IV) (also known as DOG-IM4), or a salt or isomeric form of racemates, enantiomers and diastereomers thereof.

[0102] Surprisingly, as detailed in the Examples section, the inventors have observed that the novel lipid compounds as disclosed herein are able to formulate improved compositions, such as lipid nanoparticles, for the delivery of mRNA and / or other oligonucleotides or oligonucleotides in vitro and in vivo. Moreover, the compositions thus formed can also be stored in a stable liquid form at temperatures ranging from 4-8°C.

[0103] As shown in the Examples section, the lipid nanoparticles of the present application have been proven to be very stable in terms of pH, osmolality, particle size, mRNA encapsulation and / or mRNA integrity at 5°C, 25°C and even 37°C. This strong stability allows for versatile applications of the lipid nanoparticles of the present application. For example, they can allow for the storage of pharmaceutical compositions, such as vaccines, at room temperature instead of low temperatures.

[0104] Improved lipid nanoparticles can be used to express proteins encoded by mRNA. The lipid nanoparticles as disclosed herein can be used to modulate, upregulate or downregulate protein expression by delivering miRNA or miRNA inhibitors for modulating endogenous protein expression or mRNA or plasmids for expressing transgenes. Furthermore, the lipid nanoparticles as disclosed herein can be used to induce pharmacological effects resulting from protein expression or anti-infective protection by delivering mRNA encoding suitable antigens or antibodies.

[0105] Furthermore, the lipid nanoparticles as disclosed herein can be used to induce pharmacological effects resulting from protein expression, such as erythropoietin, can be used to treat metabolic diseases or diseases resulting from protein deficiencies.

[0106] Another object of the present disclosure relates to a composition comprising at least one lipid compound as disclosed herein and at least one lipid selected from the group consisting of neutral lipids, steroids or esters thereof, and pegylated lipids.

[0107] Another object of the present disclosure relates to a lipid nanoparticle comprising at least one lipid compound as disclosed herein and at least one nucleic acid.

[0108] Another object of the present disclosure relates to a pharmaceutical composition comprising (i) at least one nucleic acid and at least one lipid compound as disclosed herein, or (ii) at least one nucleic acid, at least one composition as disclosed herein, or (iii) at least one lipid nanoparticle as disclosed herein.

[0109] The pharmaceutical composition as 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 as disclosed herein, or (ii) at least one nucleic acid encoding an antigen and at least one composition as disclosed herein, or (iii) at least one lipid nanoparticle as disclosed herein, wherein the nucleic acid encodes at least one antigen.

[0110] Another object of the present disclosure relates to a composition comprising (i) at least one nucleic acid and at least one lipid compound as disclosed herein, or (ii) at least one nucleic acid and at least one composition as disclosed herein, or (iii) at least one lipid nanoparticle as disclosed herein, for use as a medicament.

[0111] Another object of the present disclosure relates to a composition comprising (i) at least one nucleic acid and at least one lipid compound as disclosed herein, or (ii) at least one nucleic acid and at least one composition as disclosed herein, or (iii) at least one lipid nanoparticle as disclosed herein, for use in a method of treatment for preventing and / or treating a disease selected from the group consisting of infectious diseases, allergies, autoimmune diseases, rare blood disorders, rare metabolic diseases, rare neurological diseases, and neoplastic or cancer diseases.

[0112] The term "rare disease" is used herein according to its accepted meaning in the art, meaning a disease with an average prevalence threshold between 40 and 50 cases per 100,000 people (Richter et al., Value Health. 2015 Sep; 18(6):906-14).

[0113] Another object of the present disclosure relates to a composition comprising (i) at least one nucleic acid and at least one lipid compound as disclosed herein, or (ii) at least one nucleic acid and at least one composition as disclosed herein, or (iii) at least one lipid nanoparticle as disclosed herein, for use as an immunogenic composition.

[0114] In some embodiments, the present disclosure also relates to the use of a composition comprising (i) at least one nucleic acid and at least one lipid compound as disclosed herein, or (ii) at least one nucleic acid and at least one composition as disclosed herein, or (iii) at least one lipid nanoparticle as disclosed herein, for the manufacture of a medicament for preventing and / or treating infectious diseases, allergies, autoimmune diseases, rare blood disorders, rare metabolic diseases, rare neurological diseases, and neoplastic or cancer diseases.

[0115] Another object of the present disclosure relates to a method of 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 as disclosed herein, or (ii) at least one nucleic acid and at least one composition as disclosed herein, or (iii) at least one lipid nanoparticle as disclosed herein. The method as disclosed herein can be used for preventing and / or treating infectious diseases, allergies, autoimmune diseases, rare blood disorders, rare metabolic diseases, rare neurological diseases, and neoplastic or cancer diseases.

[0116] Another object of the present disclosure relates to a method of transfecting at least one isolated target cell with a nucleic acid, wherein the method comprises contacting the at least one target cell with an effective amount of (i) at least one nucleic acid and at least one lipid compound as disclosed herein, or (ii) at least one nucleic acid and at least one composition as disclosed herein, or (iii) at least one lipid nanoparticle as disclosed herein, such that the at least one target cell is transfected with the nucleic acid.

[0117] Another object of the present disclosure relates to a method of producing a polypeptide in at least one target cell, wherein the method comprises contacting the at least one target cell with an effective amount of (i) at least one nucleic acid encoding the polypeptide and at least one compound as disclosed herein, or (ii) at least one nucleic acid encoding the polypeptide and at least one composition as disclosed herein, or (iii) at least one lipid nanoparticle as disclosed herein, wherein the nucleic acid encodes the polypeptide, such that the at least one target cell is transfected with the nucleic acid operably encoding the polypeptide.

[0118] Another object of the present disclosure relates to a method for manufacturing a nucleic acid-loaded lipid nanoparticle, wherein the method comprises at least the following steps:

[0119] a) dissolving at least one lipid compound as disclosed herein in a water-miscible organic solvent,

[0120] b) mixing the organic solvent obtained in step a) with an aqueous solvent comprising at least one nucleic acid to be loaded, and

[0121] c) obtaining the lipid nanoparticle in the aqueous solvent.

[0122] In the description of various embodiments of the present disclosure, various embodiments or individual features are disclosed. As will be apparent to those of ordinary skill in the art, all combinations of such embodiments and features are possible and can result in implementations of the present disclosure. While various embodiments and individual features of the present disclosure have been illustrated and described, various other changes and modifications can be made therein without departing from the spirit and scope of the present disclosure. As will also be apparent, all combinations of the embodiments and features taught by the preceding disclosure are possible and can result in implementations of the present disclosure. [BRIEF DESCRIPTION OF DRAWINGS]

[0123] Figure 1: Geometric mean titers (GMT) of hemagglutination-inhibiting antibodies (HI titers) measured in sera of mice immunized once (at D20) with LNP L319, LNP Lip.(III), LNP Lip.(IV) or LNP Lip.(V) loaded with mRNA encoding full-length hemagglutinin (HA) of influenza virus strain A / Netherlands / 602 / 2009 (H1N1) manufactured with lipid compounds of formula (III), (IV) or (V). Total injected mRNA was 0.5, 1, 2.5 or 5.0 pg / dose for LNP L319 and 1 or 5 pg / dose for LNP Lip.(III), LNP Lip.(IV) and LNP Lip.(V). As negative control group, mice were immunized with PBS buffer and as positive control group, mice received 10 pg of monovalent influenza vaccine A / California / 07 / 2009 (H1N1) strain derived from Vaxigrip TM . Geometric mean titers and individual HI titers are indicated for each group.

[0124] Figure 2 : Geometric mean titers (GMT) of hemagglutination-inhibiting antibodies (HI titers) measured in sera of mice immunized twice (at D42) with LNP L319, LNP Lip.(III), LNP Lip.(IV) or LNP Lip.(V) loaded with mRNA encoding full-length hemagglutinin (HA) of influenza virus strain A / Netherlands / 602 / 2009 (H1N1) manufactured with lipid compounds of formula (III), (IV) or (V). Total injected mRNA was 0.5, 1, 2.5 or 5.0 pg / dose for LNP L319 and 1 or 5 pg / dose for LNP(III), LNP(IV) and LNP(V). As negative control group, mice were immunized with PBS buffer and as positive control group, mice received 10 pg of monovalent influenza vaccine A / California / 07 / 2009 (H1N1) strain derived from Vaxigrip TM . Geometric mean titers and individual HI titers are indicated for each group.

[0125] Figure 3: Geometric mean titers and individual HI titers are indicated for each group. Hemagglutination-inhibiting antibody mean titers (HI titers) measured in sera collected at D42 in mice immunized at DO and D21 with different LNPs L319 and LNP Lip.(IV) containing DOPE as neutral lipid and each loaded with mRNA encoding full-length hemagglutinin (HA) of influenza virus strain A / Netherlands / 602 / 2009 (H1N1). The mRNA loaded in LNP Lip.(IV) contains natural (Nat) or modified uridine bases (Mod). The mRNA loaded in LNP L319 contains natural uridine bases. Geometric mean titers and individual HI titers are indicated for each group.

[0126] Figure 4 : Geometric mean titers and individual HI titers are indicated for each group. Hemagglutination-inhibiting antibody mean titers (HI titers) measured in sera collected at D42 in mice immunized at DO and D21 with different LNPs L319 and LNP Lip.(IV) containing DSPC or DOPE as neutral lipid. LNP L319 contains DSPC as neutral lipid. LNPs are loaded with mRNA encoding full-length hemagglutinin (HA) of influenza virus strain A / Netherlands / 602 / 2009 (H1N1) containing natural uridine bases. Geometric mean titers and individual HI titers are indicated for each group.

[0127] Figure 5 : Geometric mean titers and individual HI titers are indicated for each group. Hemagglutination-inhibiting antibody mean titers (HI titers) measured in sera collected at D42 in mice immunized at DO and D21 with LNP Lip.(IV) containing DSPC as neutral lipid and loaded with mRNA encoding full-length hemagglutinin (HA) of influenza virus strain A / Netherlands / 602 / 2009 (H1N1) containing natural uridine bases. LNPs were stored for different periods of time before use: 0, 6 and 12 months. Three independent experiments were performed covering times of one year. Geometric mean titers and individual HI titers are indicated for each group.

[0128] Figure 6 : Geometric mean titers and individual HI titers are indicated for each group. Hemagglutination-inhibiting antibody mean titers (HI titers) measured in sera collected at D42 in mice immunized at DO and D21 with different LNPs L319 and LNP Lip.(IV) containing DOPE as neutral lipid and each loaded with mRNA encoding full-length hemagglutinin (HA) of influenza virus strain A / Netherlands / 602 / 2009 (H1N1). The mRNA loaded in LNP Lip.(IV) contains natural (Nat) or modified uridine bases (Mod). The mRNA loaded in LNP L319 contains natural uridine bases. Geometric mean titers and individual HI titers are indicated for each group.

[0129] Figure 7 : Scheme showing the synthesis of compound (XIII).

[0130] Figure 8 The scheme for synthesizing compound (XIV) is shown.

[0131] Figure 9 The scheme for synthesizing compound (XVII) is shown.

[0132] Figure 10 The diagram shows the scheme for synthesizing compound (XXI).

[0133] Figure 11 The scheme for synthesizing compound (XXII) is shown.

[0134] Figure 12 Chromatograms of LNP Lip.(IV) / DSPC containing hEPO mRNA as recorded over time.

[0135] Figure 13 The pH stability of LNP Lip.(IV) / DSPC varies over time at different storage temperatures.

[0136] Figure 14 The stability of the weight molar osmotic pressure concentration of LNP Lip.(IV) / DSPC over time is shown at different storage temperatures.

[0137] Figure 15 The stability of particle size of LNP Lip.(IV) / DSPC varies over time at different storage temperatures.

[0138] Figure 16 The results show the stability of mRNA encapsulation efficiency of LNP Lip.(IV) / DSPC over time at different storage temperatures.

[0139] Figure 17 The image shows the change in mRNA integrity over time in LNP Lip.(IV) / DSPC at different storage temperatures.

[0140] Figure 18: This shows the change in stability of LNP Lip.(IV) / DSPC lipid chromatograms over time at different storage temperatures. Figure 18A The top figure shows the LNP Lip.(IV) / DSPC after 18 weeks at 4°C; the bottom figure shows the same LNP at T0. Figure 18B The top figure shows the LNP Lip.(IV) / DSPC after 18 weeks at 25°C; the bottom figure shows the same LNP at T0. Figure 18C Top figure: LNP Lip.(IV) / DSPC after 18 weeks at 37°C; Bottom figure shows the same LNP at T0.

[0141] Figure 19 : shows the stability of hEPO expression from LNP Lip.(IV) / DSPC over time at different storage temperatures.

[0142] Figure 20 : shows the immunogenicity of LNP comprising influenza HA mRNA in cynomolgus monkeys immunized twice (D0, D28) four weeks apart with 50 pg mRNA in LNP injected IM into the biceps muscle in a volume of 500 pl.

[0143] Figure 21 : average titers of hemagglutination-inhibiting antibodies (HI titers) measured in sera collected on D21 in mice immunized on DO and D21 with LNP L319, LNP Lip.(IV) [DOG-IM4], LNP Lip.(IX), LNP Lip.(XII), and LNP Lip.(XVI) containing DSPC as neutral lipid and loaded with mRNA encoding influenza virus strain A / Netherlands / 602 / 2009 (H1N1) full-length hemagglutinin (HA). [DETAILED DESCRIPTION]

[0144] Definitions

[0145] The terms used in the present specification generally have their ordinary meanings in the art and within the context in which they are used, and the present disclosure should be interpreted on the basis of, and consistent with, the concepts of the compositions and methods described herein and how those concepts are already practiced and understood. Certain terms are discussed below or elsewhere in the present specification to provide additional guidance to the practitioner regarding the description of the compositions and methods of the present disclosure and how to make and use them. The following definitions are provided to include the following terms within the present specification, including the claims.

[0146] The term "terminal group" means that the group is a head group or a tail group.

[0147] The term "pharmaceutically acceptable salt" includes addition salts of a compound as disclosed herein, derived from combining such a compound with, for example, a nontoxic acid.

[0148] The term "acid addition salt" includes inorganic acids such as hydrochloric, hydrobromic, hydroiodic, sulfuric, nitric and phosphoric acids, and organic acids such as acetic, citric, propionic, tartaric, glutamic, salicylic, oxalic, methanesulfonic, p-toluenesulfonic, succinic and benzoic acids, and related inorganic and organic acids.

[0149] Pharmaceutically acceptable salts of the compounds disclosed herein can also exist as various solvates, such as solvates with water, methanol, ethanol, dimethylformamide, ethyl acetate, etc. Mixtures of such solvates can also be prepared. Such solvates may originate from crystallization solvents, be inherent in the solvents used for preparation or crystallization, or be incidental to such solvents. Such solvates are within the scope of this disclosure.

[0150] In the context of this disclosure, the following chemical terms have the following meanings:

[0151] --Halogen atoms: fluorine, chlorine, bromine, or iodine;

[0152] -Ct-Cz: A carbon chain that can have from t to z carbon atoms, where t and z can have values ​​from 1 to 7; for example, C1-C4 is a carbon chain that can have from 1 to 4 carbon atoms.

[0153] -As used herein, C1-C4 alkyl refers to C1-C4 normal, secondary, or tertiary saturated hydrocarbons, respectively. Non-limiting examples are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl;

[0154] -C1-C4 alkoxy is intended to refer to -O-(C1-C4) alkyl, wherein the C1-C4 alkyl is as defined above. Non-limiting examples are methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxy, or tert-butoxy;

[0155] - Heteroatoms are understood to refer to nitrogen, oxygen, or sulfur;

[0156] - Heteroaromatic rings represent 5- or 6-membered aromatic rings containing 1 or 2 heteroatoms;

[0157] -Aromatic rings refer to monocyclic or polycyclic aromatic hydrocarbon groups, such as monocyclic aromatic hydrocarbon groups of 6-20 atoms (e.g., 6 atoms), which are obtained by removing a hydrogen atom from a carbon atom of a parent aromatic ring system. An example of an aromatic ring disclosed herein is a phenyl group;

[0158] - When n is 0 in equation (I) of this disclosure, it means that the -NH part does not exist.

[0159] Unless otherwise expressly stated, the singular forms “a”, “an”, and “the” used in this specification and the appended claims include plural indicators.

[0160] The term "about" or "approximately," as used herein, refers to the usual error for the corresponding values as readily known by one of skill in the art. Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. In some embodiments, the term "about" refers to ±10% of a given value. However, when the value in question refers to an indivisible object (such as a nucleotide or other object that loses its identity once divided), then "about" refers to ±1 of the indivisible object.

[0161] The term "antigen" includes any molecule, e.g., a peptide or protein, which comprises at least one epitope that will elicit an immune response and / or to which an immune response is directed. For example, an antigen is a molecule that induces an immune response, optionally after processing, e.g., specific for the antigen or a cell expressing the antigen. After processing, the antigen can be presented by an MHC molecule and specifically react with a T lymphocyte (T cell). Thus, the antigen or fragment thereof should be recognizable by a T cell receptor, and should be able to induce clonal expansion of a T cell bearing a T cell receptor specifically recognizing the antigen or fragment, in the presence of an appropriate co-stimulatory signal, which leads to an immune response against the antigen or a cell expressing the antigen.

[0162] According to the present disclosure, any suitable antigen as a candidate for an immune response can be envisaged. The antigen can correspond to or can be derived from a naturally occurring antigen. Such naturally occurring antigens can include or can be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, or the antigen can also be a tumor antigen.

[0163] As used herein, the term "aqueous solution" or "aqueous solvent" refers to a composition comprising water.

[0164] In the present disclosure, the term "cationic group" or "cationic ammonium group" refers to an ion or group of ions having a positive charge and comprising at least one ionizable nitrogen atom. The cationic group as disclosed herein consists of a group of formula (I) as defined herein: -NH-CX-(NH)n-A.

[0165] It should be understood that aspects and embodiments of the disclosure described herein include "having," "comprising" aspects and embodiments, "consisting of aspects and embodiments, and "consisting essentially of aspects and embodiments. The words "comprising" and "containing" or variations such as "comprises" or "comprising," "contains" or "containing," are to be construed in an open-ended fashion, that is as "including, but not limited to." The term "consisting of is construed in a closed-ended fashion, that is, as "including and limited to." The term "consisting essentially of is construed as permitting the inclusion of an additional element(s) that do not materially affect the basic and novel characteristics of the disclosure. The term "comprising" can also be strictly specified in terms of the stated features, integers, steps or components, and thus, in that case, it can be replaced by "consisting of."

[0166] The term "charged lipid" refers to any of a number of lipid species that exist in a positively or negatively charged form within a useful physiological range (e.g., from about pH 3 to about pH 9). Charged lipids can be synthetic or naturally derived. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, hemisuccinyl cholesteryl ester, dialkyldimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethane carbamoylcholesterol (e.g., DC-Choi).

[0167] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that exists in an organism (including viruses) and that can be isolated from a natural source and has not been intentionally modified by the hands of man is naturally occurring.

[0168] The term "neutral lipid" refers to any of a number of lipid species that are not ionizable or are neutral zwitterionic compounds at a selected pH (e.g., at physiological pH). Such lipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin (SM), or ceramide. Neutral lipids can be synthetic or naturally derived.

[0169] The term "individual" or "subject" as used herein is a mammal. Mammals include, but are not limited to, domesticated 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.

[0170] The term "lipid" refers to a group of organic compounds that includes, but is not limited to, fatty acid esters and is generally characterized by insolubility in water but solubility in many organic solvents.

[0171] Lipid is a generic term that encompasses fats, fatty oils, essential oils, waxes, phospholipids, glycolipids, sulpholipids, aminolipids, chromolipids (lipochromes) and fatty acids. In the present disclosure, "lipid" encompasses neutral lipids, steroids or esters thereof, and pegylated lipids.

[0172] The term "lipid nanoparticle" (LNP) refers to a particle that is nanometer scale in at least one dimension (e.g., 1-1000 nm) that can be formulated with at least one lipid compound as disclosed herein. In some embodiments, the lipid nanoparticle is comprised in a formulation that can be used to deliver an active or therapeutic agent, such as a nucleic acid, to a target site of interest (e.g., a cell, tissue, organ, tumor, etc.). Such lipid nanoparticles typically comprise a lipid compound as disclosed herein and at least one ingredient selected from the group consisting of a neutral lipid, a steroid or ester thereof, and a polymer-conjugated lipid.

[0173] As used herein, "lipid encapsulated" refers to a lipid nanoparticle that provides an active or therapeutic agent, such as a nucleic acid that is fully encapsulated, partially encapsulated, or both. In one embodiment, the polynucleotide is fully encapsulated in a lipid nanoparticle.

[0174] It should be noted that the terms "head group" and "tail group" as used in this specification describe a portion of a compound of the disclosure, such as a functional group of such a compound. They are used to describe the orientation of one or more functional groups in the compound relative to other functional groups. They are both "terminal groups."

[0175] As used herein, the term "lipophilic or hydrophobic tail group" is qualitative in indicating that the tail tail has an affinity for lipids (and is typically fat-soluble) and is water-avoiding (and is typically insoluble in water).

[0176] The term "pegylated lipid" refers to a molecule that comprises both a lipid moiety and a polyethylene glycol moiety. Pegylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG), and the like.

[0177] In the present disclosure, the terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably. They refer to polymeric forms of at least two of the nucleotides, either deoxyribonucleotides or ribonucleotides, or their analogs. 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, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, closed circular DNA (ceDNA), self-amplifying RNA, single- stranded DNA (ssDNA), small interfering RNA (siRNA) and micro-RNA (miRNA), recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The modifications can be imparted to the nucleotide structure prior to or following assembly of the polymer. The sequence of a nucleotide can be interrupted with non-nucleotide components. A polynucleotide can be further modified following polymerization, such as by conjugation with a labeling component. The term "complement" of a polynucleotide sequence refers to a polynucleotide molecule having complementary

[0178] The term "steroid" or "stereol" refers to a group of lipids consisting of a steran nucleus with a hydroxyl moiety. As examples of steroids, one can cite cholesterols, campesterol, sitosterol, stigmasterol, and ergosterol. Ester of a steroid or sterol refers to an ester of a carboxylic acid with a hydroxyl group of a steroid. Suitable carboxylic acids, in addition to the carboxyl moiety, comprise saturated or unsaturated, straight-chain or branched alkyl groups. In some embodiments, the alkyl group can be C1-C 20 In other embodiments, the carboxylic acid can be a fatty acid.

[0179] As used herein, the term "prevent," "preventing," or "delaying the progression of" (and grammatical variations thereof) with respect to a disease or disorder relates to prophylactic treatment of a disease, e.g., in an individual suspected of having, or at risk of developing, the disease. Prevention can include, but is not limited to, preventing or delaying the onset or progression of the disease and / or maintaining at least one symptom of the disease at a desired level or sub-pathological level. The term "prevent" does not require 100% elimination of the possibility or likelihood of an event. Rather, it indicates that the likelihood of the event has been reduced in the presence of a composition or method as described herein.

[0180] In the present disclosure, the term "significant" used in relation to a change is intended to mean that the change observed is apparent and / or that it is statistically significant.

[0181] In the present disclosure, the term "substantially" used in conjunction with a feature of the present disclosure is intended to define a group of embodiments related to that feature that are largely similar to that feature but not entirely similar to that feature.

[0182] 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 a 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.

[0183] The term "treat" or "treatment" or "therapy" in the present text means the administration or consumption of a composition as disclosed herein with the objective of curing, healing, alleviating, relieving, altering, remedying, improving, ameliorating or affecting a symptom of a disorder, condition, or preventing or delaying the onset of a symptom, complication, or otherwise impeding or inhibiting further development of a disorder in a statistically significant manner.

[0184] 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 particular amount that is therapeutically effective can be readily determined by the ordinary practitioner and can vary according to factors such as the type and stage of the pathological process under consideration, the patient's history and age, and the administration of other therapeutic agents.

[0185] A list of sources, ingredients and components as described hereinafter is listed, combinations and mixtures thereof are also contemplated and within the scope of the present text.

[0186] 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 will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0187] All lists, such as lists of ingredients, are intended and should be interpreted to be Markush groups. Thus, all lists can be read and interpreted as “a list of items selected from the group consisting of the recited items, and combinations and mixtures thereof.”

[0188] References herein to can be trade names of components included in the various ingredients used in the present disclosure. The inventors herein do not intend to be limited by the materials under any particular trade name. Materials equivalent to the materials referenced by trade name (e.g., materials obtained from different sources under different names or reference numbers) can be substituted and used in the descriptions herein.

[0189] Detailed definitions of groups and lipid compounds of formula (I)

[0190] As specified above, the lipid compounds as disclosed herein are ionizable and are, for example, cationic lipid compounds.

[0191] The lipid compounds as disclosed herein are, for example, ionizable, because they are amine-containing lipid compounds. As such compounds are readily protonated, their pKa varies with the pH value. For example, the compounds as disclosed herein have a pKa of, for example, below 7 and, for example, in the range from 4.5 to 6.7.

[0192] The lipid compounds as disclosed herein can have asymmetric centers, chiral axes, and chiral planes of symmetry (as described in: E. L. Eliel and S. H. Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1119-1190), and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers and mixtures thereof, including optical isomers, being included in the present disclosure. In addition, the cationic lipids disclosed herein can exist as tautomers, and both tautomeric forms are intended to be included in the scope of the present disclosure, even if only one tautomeric structure is depicted.

[0193] The pharmaceutically acceptable salts of the compounds as disclosed herein have one or several generally physiologically acceptable counterions. As possible counterions, for example, halides, phosphates, trifluoroacetates, sulfites, nitrates, gluconates, glucuronates, galacturonates, alkylsulfonates, alkylcarboxylates, propionic sulfonates and methanesulfonates can be cited.

[0194] The compounds as disclosed herein and their pharmaceutically acceptable salts can also exist as various solvates, such as solvates with water, methanol, ethanol, dimethylformamide, ethyl acetate and the like. Mixtures of such solvates can also be prepared. The source of such solvates can be from the solvent of preparation or crystallization, inherent to the solvent, or incidental to such solvent. Such solvates are within the scope of the present disclosure.

[0195] For example, the lipid compound as disclosed herein has a hydrophilic head group, also called terminal group, formed by one group of formula (I), to illustrate that it is directly or indirectly connected to the end of the hydrophobic or lipophilic tail.

[0196] The group of formula (I) has the following definition:

[0197] *-NH-CX-(NH) n -A (I)

[0198] wherein:

[0199] *- indicates that said group of formula (I) is directly or indirectly connected to a C 10 to C 55 single bond of the lipophilic or hydrophobic tail;

[0200] n is 0 or 1 ;

[0201] X is an oxygen or sulfur atom, and

[0202] -A represents an optionally substituted 5- or 6-membered unsaturated heterocyclic group or 5- or 6-membered heteroaromatic ring group, both containing at least one nitrogen atom.

[0203] The compounds comprising at least one group of formula (I) can also be one of their pharmaceutically acceptable salts; as well as one of their possible racemic, enantiomeric and diastereomeric forms.

[0204] The lipid compounds as disclosed herein can be protonated due to the nitrogen atom of the amide function. Thus and as stated before, the lipid compounds as disclosed herein have an apparent pKa that can vary depending on the pH value.

[0205] According to one embodiment, the compounds as disclosed herein have a pKa lower than 7.

[0206] According to another specific embodiment, the pKa of the compound as disclosed herein ranges from 4.5 to 6.7. Such pKa can be determined by any conventional method.

[0207] According to one embodiment, X is a sulfur atom and A is a pyridyl group.

[0208] According to one embodiment, A is a 3-pyridyl group.

[0209] According to another embodiment, X is an oxygen atom and A is a 5-membered heteroaromatic ring containing at least one nitrogen atom. According to another embodiment, A is an imidazolyl group. For example, A can be a 4-imidazolyl group.

[0210] One group of formula (I) is directly or not directly attached (e.g. covalently) to a hydrophobic (lipophilic) tail group.

[0211] The hydrophobic or lipophilic tail is typically in C 10 to C 55 .

[0212] For example, it is an optionally substituted branched or unbranched straight-chain saturated or unsaturated C 10 to C 55 hydrocarbon group, and the hydrocarbon backbone is optionally interrupted by one or several oxygen or nitrogen atoms and / or one or several -O-CO- or -CO-O- groups, and if one nitrogen atom is present in the backbone, the one nitrogen atom can be directly or not directly connected to the group of formula (I).

[0213] For example, the hydrophobic or lipophilic tail can comprise at least two, three or more hydrocarbon chains, each hydrocarbon chain being independently selected from the group consisting of an optionally substituted C8-C 24 (e.g. C 10 -C 20 )alkyl chain, an optionally substituted variable saturated or unsaturated C8-C 24 (e.g. C 10 -C 20 )alkenyl chain and an optionally substituted saturated variable saturated or unsaturated C8-C 24 (e.g. C 10 -C 20 )acyl chain, wherein the alkyl, alkenyl or acyl chain can be interrupted by one or several oxygen or nitrogen atoms and / or one or several moieties such as -O-CO- or -CO-O- and preferably by at least one moiety such as -O-CO- or -CO-O-.

[0214] Each hydrocarbon chain can be substituted by at least one group selected from -OH and CO2H.

[0215] According to one embodiment, the hydrophobic or lipophilic tail is selected from the group consisting of:

[0216]

[0217]

[0218]

[0219] In one embodiment, the hydrophobic or lipophilic tail of the compound according to the application contains at least one amino moiety involved in its connection to the spacer. In this particular embodiment, the hydrophobic or lipophilic tail is in particular selected from the group consisting of R1g, h, q, r, u, v, w and z.

[0220] In another embodiment, the hydrophobic or lipophilic tail of the compound according to the application can also contain at least three or more hydrocarbon chains, for example like in the hydrophobic or lipophilic tails R1m, p, q, r, s, u, v, w, x, y and z. Each hydrocarbon chain can be selected from the group consisting of substituted C8-C 24 (for example, C 10 -C 20 )alkyl chains and substituted variable saturated or unsaturated C8-C 24 (for example, C 10 -C 20 )alkenyl chains, and said alkyl or alkenyl chains are optionally and preferably interrupted by one or several moieties like -O-CO- or -CO-O-.

[0221] In one particular embodiment, the hydrophobic or lipophilic tail of the compound according to the application is the tail (R1a) or (R1b), also called DOG alkyl or DOG ether, respectively.

[0222] According to another embodiment, the cationic and / or ionizable lipid compound as disclosed herein is of formula (II)

[0223] R1-Z-NH-CX-(NH) n -A (II)

[0224] wherein:

[0225] -X, n and A are as defined previously

[0226] -R1 is a C 10 to C 55 lipophilic or hydrophobic tail group, for example as defined previously;

[0227] - Z is a spacer arm having from 2 to 24, for example from 2 to 18, for example from 4 to 12, for example from 2 to 12 carbon atoms in a branched or unbranched straight saturated or unsaturated hydrocarbon chain, said chain being interrupted by one or several oxygen atoms and / or by a moiety chosen from -S-S-; -(O=C)-; -(C=0)-0-; -0-(0=C)-; -S-; -NH-, -NH-(0=C)-; -(0=C)-NH- and -NH-(C=0)-0-, and preferably interrupted by -(C=0)-0-; -0-(0=C)- and -NH-(C=0)-0- and optionally terminated by an oxygen atom or a moiety chosen from -NH-(0=C)--0-(0=C)--; -(C=0)-0-; and -(0=C)-, which is linked to said hydrophobic tail group

[0228] - p is 0 or 1 ;

[0229] or one of the pharmaceutically acceptable salts of said compound of formula (II); as well as the isomeric forms of its racemates, enantiomers and diastereomers.

[0230] As regards the spacer arm, it is similar to those conventionally considered in the field of cationic lipid compounds. The choice of such a spacer arm therefore does not add any difficulty for the person skilled in the art. It needs to be inert or not to impair the efficiency of the lipid compound.

[0231] Generally, the spacer group has from 2 to 24 and for example from 4 to 12, for example from 2 to 12 carbon atoms and comprises at least one or several ethylene oxide units and optionally one or several moieties as disclosed previously.

[0232] As examples of spacer arms facilitating the present disclosure, the following spacer arms can be cited, the right end of which is the end linked to the lipophilic or hydrophobic tail group:

[0233]

[0234]

[0235] According to one embodiment, the spacer group is constituted of ethylene oxide units and can comprise from 1 to 24, for example from 2 to 15, for example from 3 to 12, for example from 4 to 10, for example from 6 to 8 ethylene oxide units.

[0236] According to another particular embodiment, the spacer group can have formula (A1)

[0237] (A1)

[0238] wherein:

[0239] - the right end is the one linked to a lipophilic or hydrophobic tail group,

[0240] - 1 is 0 or 1 ;

[0241] - m ranges from 1 to 24, for example from 2 to 15, for example from 3 to 12, for example 2, 3, 4, 5, 6, 7, 8 or 9;

[0242] - p is 0 or 1 ; and

[0243] - R’ represents, when p is 1, one oxygen atom or a moiety selected from the group consisting of -C=0-; -NH-; -0-CH2-; -NH-C(=0)-; -NH-C(=0)-0-CH2-; 0-C(=0)-; C=0-NH-(CH2)2-; O CH2 C(=0)-0-; -C(=0)-0-(CH2)2- and -S-S- and in particular -NH-C(=0)-; -NH-C(=0)-0-CH2-; -0-C(=0)-; -C=0-NH-(CH2)2-; and; -C(=0)-0-(CH2)2.

[0244] According to another embodiment, the spacer can comprise from 1 to 24, for example from 2 to 15, for example from 3 to 12, for example from 4 to 10, and for example from 6 to 8, ethylene oxide units, and preferably incorporates at least one moiety selected from the group consisting of -(C=0)-0-; -0-(0=C)-; -NH-(0=C)-; -(0=C)-NH- and; -NH-(C=0)-0- and more preferably at least one -NH-(C=0)-0-.

[0245] In one embodiment, in the lipid compound of formula (II), X is a sulfur atom and A is a pyridyl group. For example, A can be a 3-pyridyl group.

[0246] According to this embodiment, the compound of formula (II) is for example a compound of formula (V)

[0247]

[0248] or one of its salts or one of the isomeric forms of its racemate, enantiomer and diastereomer.

[0249] For example, this compound (V) or a derivative thereof is not salted. For example, it is in the form of its free base.

[0250] In another embodiment, in the lipid compound of formula (II), X is an oxygen atom and A is a 5-membered heteroaromatic ring group containing at least one nitrogen atom. Thus, according to another embodiment, A is an imidazolyl group and for example, A can be a 4-imidazolyl group.

[0251] According to this embodiment, the compound of formula (II) is for example selected from the following compounds (III) or (XXVII) and the isomeric forms of the salts or racemates, enantiomers and diastereomers thereof. It is worth noting that in the formulae developed below, the secondary amino moiety can be indifferently written -NH- or -N-.

[0252]

[0253]

[0254]

[0255]

[0256] More particularly, the compounds (IV), (VIII), (IX), (XII), (XVI), (XIX) and (XXII), in particular the compounds (IV), (IX), (XII) or (XVI), more particularly the compounds (IV) or (XII), and totally particularly the compound (IV), are of interest, as the salts or the isomeric forms of the racemates, enantiomers and diastereomers thereof. For example, they can be in the form of their free base.

[0257] As shown in the example section, the compounds (IV), (VIII), (IX), (XII), (XVI), (XIX) and (XXII) are for example efficiently used to formulate stable LNP (stable in liquid form at 4-8°C) able to deliver functional mRNA into a target tissue after parenteral administration, and to induce the expression of a protein such as EPO or an immune response in case the delivered mRNA codes for an antigen.

[0258] Preparation of cationic lipids

[0259] The compounds according to the disclosure can be prepared from commercially available or literature described starting materials using methods and procedures known to the skilled person.

[0260] For example, the lipid compound of formula (II) can be obtained by covalent coupling between a precursor of the radical of formula (I) and a lipid compound or a derivative thereof having a terminal reactive group able to react with said precursor.

[0261] This terminal reactive group can be located directly at the end of the hydrophobic or lipophilic part of the lipid compound to be transformed, or at the end of a spacer already linked to the hydrophobic or lipophilic part of the lipid compound.

[0262] The selection of a convenient precursor of the group of formula (I) that is intended to react with the lipid compound to form the intended covalent bond is obviously within the capabilities of a person skilled in the art. The precursor only needs to have a group that is capable of chemically reacting with a group of the lipid compound to form a covalent bond.

[0263] With regard to these starting compounds, i.e. precursors of the group of formula (I) and the lipid compound or derivative thereof to be converted, they can easily be produced by a person skilled in the art, e.g. according to the preparation methods claimed in the following examples.

[0264] The covalent coupling can also be performed according to methods known to a person skilled in the art with regard to the chemical nature of the reactive group of the precursor of the group of formula (I) and the reactive group of the lipid compound or derivative thereof to be converted.

[0265] Generally, the covalent linkage can be formed by esterification, amidation or carbamidation.

[0266] As a representative of a convenient precursor of the group of formula (I) where A is a pyridyl group, one can cite its corresponding pyridyl isothiocyanate, such as for example 3-pyridyl isothiocyanate.

[0267] As a representative of a convenient precursor of the group of formula (I) where A is an imidazolyl group, one can cite the corresponding imidazole carboxylic acid.

[0268] A particular method to obtain a compound of formula (I) as a compound of formula (IV) is depicted in Scheme 1 below.

[0269]

[0270] It is understood that where typical or specified experimental conditions (i.e., reaction temperatures, time, molar ratios of reagents, solvents, etc.) are given, other experimental conditions can also be used unless otherwise stated. Optimum reaction conditions can vary with the particular reagents or solvents used, but such conditions can be determined by a person skilled in the art by routine optimisation procedures.

[0271] The optional salt formation can be performed by conventional means to form the intended cationic form.

[0272] Subsequent steps of purification and / or isolation of the resulting final product can advantageously be performed after the coupling reaction. Convenient methods of purification are detailed in the following examples. For example, purification of the resulting compound can be performed by preparative high performance liquid chromatography (HPLC).

[0273] The present disclosure can be better understood from the following examples, all of which are merely intended to be illustrative and are not meant to limit the scope of the disclosure in any way.

[0274] Compositions, lipid nanoparticles and manufacturing methods

[0275] The present disclosure relates to compositions comprising at least one lipid compound as disclosed herein, as described above. The compositions as disclosed herein can further comprise at least one lipid selected from the group consisting of neutral lipids, steroids or esters thereof, and pegylated lipids.

[0276] The compositions as disclosed herein can be formulated as lipid nanoparticles containing at least one nucleic acid.

[0277] The compositions or lipid nanoparticles as disclosed herein can further comprise at least one therapeutic anionic or polyanionic agent, for example at least one nucleic acid.

[0278] Neutral lipids

[0279] The compositions or lipid nanoparticles as disclosed herein can comprise neutral lipids. The presence of neutral lipids can improve the structural stability of the lipid nanoparticles. The neutral lipids can be appropriately selected in view of the delivery efficiency of the nucleic acid.

[0280] Neutral lipids are different from the lipid compounds as disclosed herein. Neutral lipids are non-ionizable or neutral zwitterionic compounds at selected pH.

[0281] The neutral lipids that can be used in the present disclosure can be selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, sphingomyelins, and ceramides.

[0282] Phosphatidylcholines and phosphatidylethanolamines are zwitterionic lipids. Sphingomyelins and ceramides are non-ionizable lipids.

[0283] As examples of phosphatidylcholines that can be used in the present disclosure, DSPC (l,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (l,2-dipalmitoyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPC (l,2-dioleoyl-sn-glycero-3-phosphocholine) can be mentioned.

[0284] As examples of phosphatidylethanolamines that can be used in the present disclosure, DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, or l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE) can be mentioned.

[0285] The neutral lipid can be selected from phosphatidylcholines such as DSPC, DPPC, DMPC, POPC, DOPC; phosphatidylethanolamines such as DOPE, DPPE, DMPE, DSPE, DLPE; sphingomyelin; and ceramides.

[0286] In one embodiment, the neutral lipid suitable for use in the present disclosure can be DSPC, DOPC, and DOPE, and for example can be DSPC or DOPE.

[0287] The neutral lipid can be present in step a) of the method for formulating a lipid nanoparticle as 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 lipids and lipid compounds as disclosed herein.

[0288] The neutral lipid can be present in the composition or lipid nanoparticle as 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 lipids and lipid compounds as disclosed herein.

[0289] The neutral lipid can be present in the composition or lipid nanoparticle as disclosed herein in a molar ratio of lipid compounds : neutral lipid, which can range from about 70: 1 to about 1 :2, for example from about 30: 1 to about 1 : 1, for example from about 15: 1 to about 2: 1, for example from about 10: 1 to about 4: 1, and more for example about 5: 1.

[0290] Steroid or ester thereof

[0291] The composition or lipid nanoparticle as disclosed herein can comprise a steroid (or sterol) or ester thereof. The presence of a sterol or sterol ester can also improve the structural stability of the lipid nanoparticle.

[0292] A sterol or a steroid that can be used in the present disclosure can be selected from the group consisting of cholesterol or a derivative thereof, ergosterol, desmosterol (3β-hydroxy-5,24-cholesten), stigmasterol (stigmasta-5,22-dien-3-ol), lanosterol (8,24-lanosta- dien-3b-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25- dihydrolanosterol), zymosterol (5a-cholesta-8,24-dien-3β-ol), cholestenone (5a-cholesta-7-en-3β-ol), diosgenin ((3β,25R)-spirost-5-en-3-ol), sitosterol (sitosta-5- en-3β-ol), sitostanol (5a-sitostan-3b-ol), 24-methylenecholesterol (5,24(28)-cholesta- dien-24-methylen-3β-ol).

[0293] An ester of a steroid or a sterol refers to an ester of a carboxylic acid with a hydroxyl group of a steroid. Suitable carboxylic acids include saturated or unsaturated, straight chain or branched alkyl groups in addition to the carboxyl moiety. In some embodiments, the alkyl group can be a C1-C 20 saturated or unsaturated, straight chain or branched alkyl group, for example, C2-C 18 , for example C4-C 16 , for example C8-C 12 saturated or unsaturated, straight chain or branched alkyl group, in other embodiments, the carboxylic acid can be a fatty acid. For example, the fatty acid can be octanoic acid, decanoic acid, lauric acid, stearic acid, margaric acid, oleic acid, linoleic acid, or arachidic acid.

[0294] In one embodiment, a sterol ester suitable for use in the present disclosure can be a cholesterol ester.

[0295] A sterol ester or a steroid ester that can be used in the present disclosure can be selected from the group consisting of margaric acid cholesterol ester (margaric acid cholesta-5- en-3β-yl ester), oleic acid cholesterol ester, and stearic acid cholesterol ester.

[0296] A sterol or steroid or ester thereof useful in the present disclosure can be selected from the group consisting of cholesterol or derivatives thereof, ergosterol, desmosterol (3β-hydroxy-5,24-cholesten), stigmasterol (stigmasta-5,22-dien-3-ol), lanosterol (8,24-lanosta- dien-3b-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25- dihydrolanosterol), zymosterol (5a-cholesta-8,24-dien-3β-ol), cholestenone (5a- cholest-7-en-3β-ol), diosgenin ((3β,25R)-spirost-5-en-3-ol), sitosterol (22,23- dihydrostigmasterol), sitostanol, campesterol (campesta-5-en-3β-ol), campestanol (5a-campestan-3b-ol), 24-methylenecholesterol (5,24(28)-cholestandien-24- methylene-3β-ol), heptadecanoic acid cholesterol ester (heptadecanoic acid cholesta-5-en-3β-yl ester), oleic acid cholesterol ester, and stearic acid cholesterol ester.

[0297] Alternatively, a sterol useful in the present disclosure can be a cholesterol derivative, such as oxidized cholesterol.

[0298] An oxidized cholesterol suitable for use in the present disclosure can be 25- hydroxycholesterol, 27-hydroxycholesterol, 20a-hydroxycholesterol, 6-keto-5a- hydroxycholesterol, 7-keto-cholesterol, 7b,25-hydroxycholesterol, and 7b- hydroxycholesterol. For example, the oxidized cholesterol can be 25-hydroxycholesterol and 20a-hydroxycholesterol, and for example, it can be 20a-hydroxycholesterol.

[0299] In one embodiment, a sterol or steroid or ester thereof suitable for use in the present disclosure can be cholesterol, a cholesterol ester, or a cholesterol derivative, such as oxidized cholesterol. In one embodiment, a sterol or steroid suitable for use in the present disclosure can be cholesterol or a cholesterol ester, and for example, can be cholesterol.

[0300] A sterol or steroid or ester thereof can be present in a composition or a lipid nanoparticle as disclosed herein in a molar amount ranging from about 0 to about 60%, for example, from about 10% to about 50%, and for example, from about 20% to about 50%, relative to the total molar amount of lipids and lipid compounds as disclosed herein, which can be present in the composition or lipid nanoparticle.

[0301] The sterol or steroid or ester thereof can be present in the compositions or lipid nanoparticles as disclosed herein in a molar ratio of lipid compound: steroid or ester thereof that can range from about 4: 1 to about 1 :2, for example from about 3.5: 1 to about 1 : 1.8, for example from about 2: 1 to about 1 : 1.5, for example from about 1.5: 1 to about 1 : 1.2, and for example about 1.3: 1 to about 1 : 1.3.

[0302] PEGylated lipids

[0303] The compositions or lipid nanoparticles as disclosed herein can include a PEGylated (or PEG-) lipid.

[0304] PEGylated lipids contemplated include, but are not limited to, polyethylene glycol chains up to 5 kDa covalently attached to a lipid having one or more alkyl chains of C6-C 20 The addition of PEGylated lipids to the lipid nanoparticle compositions as disclosed herein can prevent complex aggregation and can also provide a means for increasing circulation lifetime and increasing delivery of the compositions or lipid nanoparticles to target cells.

[0305] Suitable PEGylated lipids can be, for example, PEGylated diacylglycerols (PEG-DAGs) such as l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (DMG-PEG), PEGylated phosphatidylethanolamines (PEG-PE), PEG succinic diacylglycerol (PEG-S-DAG) such as 4-0-(2',3'-ditetradecanoyloxy)propyl-l-0-(co-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG), PEGylated ceramides (PEG-cer), or PEG dialkoxylpropyl carbamates such as omega-methoxy(polyethoxy)ethyl-N-(2,3-ditetradecyloxypropyl) carbamate, 2,3-ditetradecyloxypropyl-N-(co-methoxy(polyethoxy)ethyl) carbamate, or mPEG-N,N-ditetradecylacetamide (also known as 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide or ALC-0159).

[0306] In one embodiment, the PEGylated lipids suitable for use in the present disclosure can be selected from PEG-DAG, DMG-PEG, PEG-PE, PEG-S-DAG, PEG-S-DMG, PEG-cer, or mPEG-N,N-ditetradecylacetamide, or PEG dialkoxylpropyl carbamates.

[0307] For example, a pegylated lipid suitable for use in the present disclosure can be DMG-PEG, PEG-PE, or mPEG-N,N-ditetradecylacetamide.

[0308] In some embodiments, a pegylated lipid suitable for use in the present disclosure can be DMG-PEG or PEG-PE.

[0309] In some embodiments, a pegylated lipid suitable for use in the present disclosure can be mPEG-N,N-ditetradecylacetamide.

[0310] A composition or lipid nanoparticle as disclosed herein can comprise a pegylated lipid in a molar amount ranging from about 1 to about 15%, for example from about 1% to about 10%, for example from about 1% to about 5% and for example from about 1% to about 3.5% relative to the total molar amount of lipids and lipid compounds.

[0311] The pegylated lipid and the lipid compound can be present in a molar ratio of lipid compound to pegylated lipid ranging from about 70: 1 to about 4: 1, for example from about 40: 1 to about 10: 1, for example from about 35: 1 to about 15: 1 and for example is about 33: 1 or about 14: 1.

[0312] In one embodiment, the composition or lipid nanoparticle can comprise, in addition to the above lipid compounds, at least one neutral lipid, at least one sterol or ester thereof and at least one pegylated lipid.

[0313] The neutral lipid, the sterol or ester thereof and the pegylated lipid can be as indicated above.

[0314] In one embodiment, the composition or lipid nanoparticle described herein can comprise a lipid compound as disclosed herein, a neutral lipid, a sterol or ester thereof and a pegylated lipid in a molar amount of about 30% to about 70% of the lipid compound, about 0% to about 50% of the neutral lipid, 20% to about 50% of the sterol or ester thereof and about 1% to about 15% of the pegylated relative to the total amount of lipids and lipid compounds.

[0315] In one embodiment, the composition or lipid nanoparticle described herein can comprise a lipid compound as disclosed herein, a neutral lipid, a sterol or ester thereof and a pegylated lipid in a molar amount of about 30% to about 60% of the lipid compound, about 5% to about 30% of the neutral lipid, about 30% to about 48% of the sterol or ester thereof and about 1.5% to about 5% of the pegylated relative to the total amount of lipids and lipid compounds.

[0316] In one embodiment, the compositions or lipid nanoparticles described herein can comprise a lipid compound as disclosed herein, a neutral lipid, a sterol or an ester thereof, and a PEGylated lipid in a molar ratio of about 35% to about 50% of the lipid compound, about 10% to about 16% of the neutral lipid, about 38.5% to about 46.5% of the sterol or an ester thereof, and about 1.5% of the PEGylated relative to the total amount of lipids and lipid compounds.

[0317] As one embodiment, the compositions or lipid nanoparticles as disclosed herein can comprise about 35% of a lipid compound as disclosed herein, about 16% of a neutral lipid, about 46.5% of a sterol or an ester thereof, and about 1.5% of PEGylated relative to the total amount of lipids and lipid compounds.

[0318] As another embodiment, the compositions or lipid nanoparticles as disclosed herein can comprise about 50% of a lipid compound as disclosed herein, about 10% of a neutral lipid, about 38.5% of a sterol or an ester thereof, and about 1.5% of PEGylated relative to the total amount of lipids and lipid compounds.

[0319] In one embodiment, the molar ratio of the lipid compound as disclosed herein to the neutral lipid, the sterol or an ester thereof, and the 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, about 52 / 13 / 30 / 5.

[0320] In one embodiment, the molar ratio of the lipid compound as disclosed herein to the neutral lipid, the sterol or an ester thereof, and the PEGylated lipid can be about 35 / 16 / 46.5 / 1.5 or about 50 / 10 / 38.5 / 1.5.

[0321] In another embodiment, the lipid compound as disclosed herein can be any one 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), and for example is compound (IV), the neutral lipid can be DSPC or DOPE, the sterol can be cholesterol, and the pegylated lipid can be PEG-PE (PEG2000-PE) or PEG-DMG (PEG2000-DMG).

[0322] In another embodiment, the lipid compound as disclosed herein can be compounds (III), (IV), (V), (VIII), (IX), (XII), (XVI), (XIX) or (XXII), the neutral lipid can be DSPC, the sterol can be cholesterol, and the pegylated lipid can be PEG-PE (PEG2000-PE).

[0323] In another embodiment, the lipid compound as disclosed herein can be compounds (IV), (VIII), (IX), (XII), (XVI), (XIX) or (XXII), the neutral lipid can be DSPC, the sterol can be cholesterol, and the pegylated lipid can be PEG-PE (PEG2000-PE).

[0324] In another embodiment, the lipid compound as disclosed herein can be compounds (IV), (IX), (XII) or (XVI), the neutral lipid can be DSPC, the sterol can be cholesterol, and the pegylated lipid can be PEG-PE (PEG2000-PE).

[0325] In another embodiment, the lipid compound as disclosed herein can be compounds (IV) or (XII), the neutral lipid can be DSPC, the sterol can be cholesterol, and the pegylated lipid can be PEG-PE (PEG2000-PE).

[0326] In another embodiment, the lipid compound as disclosed herein can be compound (IV), the neutral lipid can be DSPC, the sterol can be cholesterol, and the pegylated lipid can be PEG-PE (PEG2000-PE).

[0327] Lipid nanoparticles (LNP)

[0328] The present disclosure relates to lipid nanoparticles containing at least one lipid compound as disclosed herein and at least one nucleic acid.

[0329] In one embodiment, the lipid nanoparticles as disclosed herein can contain a lipid compound of formula (III) to (XXVII) as disclosed herein, 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 for example formula (III), (IV) or (V) and for example formula (IV).

[0330] Furthermore, the lipid nanoparticles as disclosed herein can comprise at least one lipid neutral phospholipid or sphingolipid, a steroid or ester thereof, and a pegylated lipid selected from the group consisting of:

[0331] According to one embodiment, the composition as disclosed herein as described above can be formulated as a lipid nanoparticle.

[0332] The diameter of the lipid nanoparticles can be such that it is suitable for systemic administration, e.g. parenteral, or intramuscular, intradermal or subcutaneous administration. Typically, the Z-average size of the lipid nanoparticles is less than 600 nanometers (nm), e.g. less than 400 nm.

[0333] In one embodiment, the Z-average size of the LNP is less than 200 nm. Such sizes are advantageously compatible with sterile filtration and are most suitable for migration via lymphatic vessels after intramuscular or subcutaneous administration. Such sizes are also suitable for intravenous administration, as larger particle injections can induce capillary thrombosis.

[0334] In some embodiments, the Z-average size of the lipid nanoparticles can range 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 to about 160 nm and from about 90 to about 150 nm. In one embodiment, the diameter of the nanoparticles can range from about 90 to about 150 nm.

[0335] The "Z-average size" of a lipid nanoparticle can be determined by dynamic light scattering (DLS). The Z-average size or Z-average mean used in dynamic light scattering is a parameter also known as the cumulants average. It is the main and most stable parameter produced by the technique. The Z-average mean is defined as the 'harmonic intensity average particle diameter'. The Z-average size can be measured with a zeta sizer Nano ZS light scattering instrument (Malvern Instruments). For accurate particle size determination with Nano ZS, the viscosity of the buffer and the refractive index of the material have to be provided to the device software (PBS: v = 1.02 cP, RI = 1.45).

[0336] Due to minor variations in size that can occur during manufacturing, a variation of up to 20-30% of the measurement is acceptable and considered to be within the specified size range. Alternatively, the size can be determined by a filter screening assay. For example, a particle preparation is less than a specified size if at least 90%, e.g., at least 95%, e.g., at least 97% of the particles pass through a "sieve-type" filter of the specified size.

[0337] The "polydispersity index" is a measure of the uniform or non-uniform size distribution of individual lipid nanoparticles in a mixture of lipid nanoparticles and is indicative of the breadth of the particle distribution in the mixture. The PI can be determined, e.g., as described herein.

[0338] In one embodiment, the polydispersity index of a nanoparticle described herein, as measured by dynamic light scattering, is 0.5 or less, e.g., 0.4 or less, e.g., 0.3 or less, or even, e.g., 0.2 or less.

[0339] In one embodiment, the lipid nanoparticles are colloidal stable in the sense that no or substantially no aggregation, precipitation, or increase in size and polydispersity index, as measured by dynamic light scattering, can be observed over a given period of time, e.g., over at least two hours to several months, e.g., at least 1, 2, 3, 4, 5, 6, or 12 months.

[0340] The pKa of a lipid nanoparticle as disclosed herein ranges from 4.5 to 6.7.

[0341] This pKa can be determined using the fluorescent probe 2-(p-toluidinyl)-6-naphthalenesulfonic acid (TNS) and preformed LNP consisting of cationic lipid / DOPE / cholesterol / PEG-lipid (35:16:35:2.5 mol%) at a concentration of about 6 mM total lipid in PBS. Briefly, TNS is prepared as a 100 mM stock solution in distilled water. LNP is diluted to 100 mM total lipid in 90 pL of a buffer solution containing 10 mM HEPES, 10 mM 4-morpholineethanesulfonic acid, 10 mM ammonium acetate, 130 mM NaCl, with a pH range of 2.71 to 11.5, in triplicate. Ten microliters of stock TNS is added to the LNP solution and mixed thoroughly in a black 96-well plate. Fluorescence intensity is monitored in a Tecan Pro200 plate reader using excitation and emission wavelengths of 321 and 445 nm. With the resulting fluorescence values, a sigmoidal plot of fluorescence versus buffer pH is created. The log of the inflection point of this curve is the apparent pKa of the LNP formulation. Such a method is described in detail, for example, in Semple, S.C. et al. Rational design of cationic lipids for siRNA delivery. Nat. Biotechnol. 28, 172-176 (2010).

[0342] The lipid nanoparticle can comprise or encapsulate at least one nucleic acid.

[0343] The nucleic acid can be encapsulated in the lipid nanoparticle and / or adsorbed on 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 comprised in a vesicle encapsulating the nucleic acid.

[0344] The lipid nanoparticle has an overall surface charge, which is the sum of the positive and negative charges on the surface of the particle, and is expressed in terms of zeta potential. Zeta potential is the potential difference between the dispersion medium and the stationary layer of fluid attached to the dispersed particles. Zeta potential is widely used to quantify the magnitude of the charge at the bilayer.

[0345] The zeta potential can be calculated using a theoretical model and experimentally determined using electrophoretic mobility or dynamic light scattering measurements. Electrophoresis can be used to estimate the zeta potential of a microparticle. In practice, the zeta potential of a dispersion can be measured by applying an electric field across the dispersion. Particles with a zeta potential in the dispersion will migrate toward the electrode of opposite charge at a speed proportional to the magnitude of the zeta potential. This speed can be measured using Laser Doppler Anemometer techniques. The frequency shift or phase shift of an incident laser beam caused by these moving particles can be measured as the particle mobility, and this mobility can be converted to zeta potential by inputting the dispersant viscosity and dielectric constant and application of Smoluchowski theory. The electrophoretic velocity is proportional to the electrophoretic mobility, which is a measurable parameter. There are several theories that relate electrophoretic mobility to zeta potential.

[0346] Suitable systems such as a Nicomp 380ZLS system or a Malvern nanoZS can be used to determine the zeta potential. Such systems generally measure the electrophoretic mobility and stability of charged particles in a liquid suspension. These values are a predictor of the repulsive forces exerted by the suspended particles and are directly related to the stability of the colloidal system.

[0347] The zeta potential of the lipid nanoparticles as disclosed herein is close to neutral at pH neutral.

[0348] One advantage is that having a zeta potential close to zero facilitates particle flowability in vivo, reduces opsonization and enhances access to target tissues.

[0349] In one embodiment, the zeta potential of the nanoparticles can range from about -30 mV to about +5 mV, for example from about -20 mV to about 0 mV and for example from about -10 mV to about 0 mV at a pH from 6.0 to 7.5.

[0350] The lipid nanoparticles described herein can be formed by adjusting (e.g. at the time of manufacture) the positive charge to the negative charge, depending on the charge ratio of the lipid compound (cationic charge from the quaternary ammonium of the lipid compound: N) to the nucleic acid (anionic charge from the phosphate: P) as disclosed herein, and mixing the nucleic acid with the lipid compound. The charge of the lipid compound and the nucleic acid is at a selected pH, such as a physiological pH, which is from about 6.5 to about 7.5.

[0351] The + / - (N / P) charge ratio of the lipids as disclosed herein to the nucleic acid in the lipid nanoparticles as 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 amount of negative charges in the nucleic acid)].

[0352] The person skilled in the art can readily determine the amount of nucleic acid and the amount of lipid compound in view of the loading during nanoparticle preparation.

[0353] According to one embodiment, the ratio of positive charges to negative charges in the nanoparticles suitable for use in the present disclosure is such that they can have an overall negative charge or neutral or near neutral overall charge.

[0354] In one embodiment, the charge ratio of positive charges to negative charges in the nanoparticles ranges from about 4: 1 to about 15: 1, for example from about 5: 1 to about 12: 1, for example from about 6: 1 to about 9: 1 and for example from about 6: 1 to about 8: 1.

[0355] In one embodiment, the lipid nanoparticles as disclosed herein encapsulating a nucleic acid can have a Z-average size of about 80-200 nm and a charge ratio N / P of about 4-8: 1.

[0356] Method of manufacturing lipid nanoparticles

[0357] The present disclosure relates to a method for manufacturing lipid nanoparticles, for example lipid nanoparticles comprising at least one nucleic acid, using lipid compounds as disclosed herein.

[0358] In one embodiment, the nucleic acid containing lipid nanoparticles as disclosed herein can be obtainable by a method comprising at least the following steps:

[0359] a) dissolving in a water-miscible organic solvent containing at least one lipid compound as disclosed herein and for example as described above,

[0360] b) mixing the organic solvent obtained in step a) with an aqueous solvent comprising at least one nucleic acid, and

[0361] c) obtaining said lipid nanoparticles containing said nucleic acid in said aqueous solvent.

[0362] In one embodiment, the method for manufacturing lipid nanoparticles as disclosed herein can comprise at least the following steps:

[0363] a) dissolving at least one lipid compound as disclosed herein and at least one lipid selected from the group consisting of neutral lipids, steroids or esters thereof and pegylated lipids in a water-miscible organic solvent,

[0364] b) mixing the organic solvent obtained in step a) with an aqueous solvent comprising at least one nucleic acid, and

[0365] c) obtaining said lipid nanoparticles containing said nucleic acid in said aqueous solvent.

[0366] The lipid compound as disclosed herein can be present in an amount sufficient to construct a lipid nanoparticle and to encapsulate any load to be encapsulated. The amount of ionizable lipid compound used in the lipid nanoparticle can be determined by the skilled person according to any known technique and adapted according to the nature and amount of the load and the nature and amount of other lipids that can be present.

[0367] In one embodiment, step a) further comprises dissolving in the organic solvent at least one lipid selected from the group consisting of a neutral lipid, a sterol or ester thereof, and a pegylated lipid.

[0368] The neutral lipids, sterols or esters thereof, and pegylated lipids suitable for use in the present disclosure can be as described herein.

[0369] In another embodiment, step a) can further comprise dissolving in the organic solvent at least one neutral lipid, at least one sterol or ester thereof, and at least one pegylated lipid, and wherein the lipid compound, the neutral lipid, the sterol or ester thereof, and the pegylated lipid are present in the organic solvent in a molar amount of about 30% to about 70% of the lipid compound, about 0% to about 50% of the neutral lipid, 20% to about 50% of the sterol or ester thereof, and about 1% to about 15% of the pegylated relative to the total amount of lipids and lipid compounds.

[0370] The water-miscible organic solvent useful can be any water-miscible organic solvent capable of dissolving the lipid compound as disclosed herein and any other added lipids. As examples of suitable organic solvents, one can cite ethanol or methanol, 1-propanol, isopropanol, tert-butanol, THF, DMSO, acetone, acetonitrile, diglyme, DMF, 1-4 dioxane, ethylene glycol, glycerol, hexamethylphosphoramide, hexamethylphosphorotriamide. In one embodiment, the organic solvent can be ethanol and isopropanol.

[0371] The aqueous solvent that can be used in step b) includes an aqueous buffered solution.

[0372] As examples of suitable buffered aqueous solutions, one can mention acidic buffers such as citrate buffer, sodium acetate buffer, succinate buffer, borate buffer or phosphate buffer. For example, the aqueous buffered solvent can be a citrate buffered solution or an acetate buffered solution.

[0373] The pH of the aqueous solvent can range from about 4.5 to about 7.0, for example from about 5.0 to about 6.5 and for example from about 5.5 to about 6.0, and for example can be about 6.5.

[0374] In step b), the organic solvent and the 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 can range from about 1 :2 to about 1 :4, and for example, can be a ratio of about 1 :3.

[0375] 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 be about 4 ml / min.

[0376] The mixing step can be performed by any method known in the art. For example, the two solvents can be mixed with a T- or Y-connector. Alternatively, the mixing can be performed by laminar flow mixing with a microfluidic micromixer, as described by Belliveau et al. (2012).

[0377] As indicated, the aqueous solvent in step b) comprises a nucleic acid. In one embodiment, the nucleic acid can encode at least one antigen. Suitable nucleic acids can for example be as detailed below.

[0378] If desired, the method can further comprise a step of increasing the pH from acidic to neutral.

[0379] In yet another embodiment, the method can comprise a step d) of increasing the pH of the aqueous solvent containing the lipid nanoparticles obtained in step c) to a pH ranging from about 5.5 to about 7.5, for example from about 6.0 to about 7.0 and for example from about 6.5 to about 7.0.

[0380] The step of increasing the pH can be performed by any method known in the art.

[0381] For example, the change in pH can be performed by a dialysis or a diafiltration step.

[0382] According to one embodiment, step d) of the method as disclosed herein can further comprise at least one step of dialysis or diafiltration of the lipid nanoparticles. The dialysis or diafiltration step can be performed on an aqueous solvent having a pH ranging from about 5.5 to about 7.5, for example from about 6.0 to about 7.0, for example from about 6.5 to about 7.0 and for example about 6.5.

[0383] The aqueous solvent used in step d) may further contain carbohydrates to improve the stability of the lipid nanoparticles and the volumetric molar osmotic pressure concentration of the solution. Suitable carbohydrates may be sucrose, mannitol, glucose, dextrose, or trehalose. The carbohydrates may be present in an amount of about 5% to about 10% and, for example, about 8% relative to the total amount of the aqueous solvent.

[0384] According to another embodiment, step d) of the method disclosed herein may include at least two steps of dialysis of lipid nanoparticles. The first dialysis step may be performed with a similar aqueous solvent (similar in pH and concentration) and may remove organic solvents. The second dialysis step may be performed with a different aqueous solvent (different in pH and possibly in concentration). In this case, the pH of the dialysis solution may range from about 5.5 to about 7.5, for example from about 6.0 to about 7.0, for example from about 6.5 to about 7.0 and for example about 6.5. The dialysis solution for the second dialysis may be a buffer solution, such as phosphate buffer, TRIS buffer, Hepes buffer, histidine buffer, or glycine buffer. The volumetric molar osmotic pressure concentration of the buffer may be adjusted with a salt such as NaCl or with a carbohydrate such as glycerol, sucrose, mannitol, glucose, dextrose, or trehalose.

[0385] In one embodiment, the volumetric molar osmotic pressure concentration is adjusted to achieve a final weight molar osmotic pressure concentration of approximately 290 mOsmol / kg, thereby allowing the isotonic solution to be injected into the body.

[0386] In addition to steps c) and / or d), the method may also include any further steps suitable for harvesting, purifying, concentrating and / or sterilizing lipid nanoparticles to further formulate them into pharmaceutical compositions (e.g., immunogenic compositions).

[0387] According to one embodiment, this disclosure relates to lipid nanoparticles that can be obtained according to the manufacturing methods disclosed herein.

[0388] According to another embodiment, this disclosure relates to a method for manufacturing a pharmaceutical composition, said method comprising at least the following steps:

[0389] i) mixing at least one nucleic acid with at least one lipid compound as disclosed herein, or mixing at least one nucleic acid with at least one composition as disclosed herein, or manufacturing at least one lipid nanoparticle according to the methods disclosed herein, and

[0390] ii) Combining the mixed nucleic acids with a lipid compound as disclosed herein, or combining the mixed nucleic acids with a composition as disclosed herein, or combining the lipid nanoparticles obtained in step i) with at least one pharmaceutically acceptable excipient or carrier.

[0391] According to another embodiment, the present disclosure relates to a method for manufacturing an immunogenic composition, said method comprising at least the steps of:

[0392] i) mixing at least one nucleic acid with at least one lipid compound as disclosed herein, or mixing at least one nucleic acid with at least one composition as disclosed herein, or manufacturing at least one nucleic acid containing lipid nanoparticles according to a method as disclosed herein, wherein the nucleic acid encodes at least one antigen, and

[0393] ii) combining the mixed nucleic acid with a lipid compound as disclosed herein, or combining the mixed nucleic acid with a composition as disclosed herein, or combining the lipid nanoparticles obtained in step i) with at least one pharmaceutically acceptable excipient or carrier.

[0394] Pharmaceutical and immunogenic compositions suitable for use in the present disclosure are described in more detail below.

[0395] In one embodiment, the composition or the lipid nanoparticle as disclosed herein can be manufactured with a lipid compound of formula (III) to (XXVII) as disclosed herein, 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 e.g. formula (III), (IV) or (V) and e.g. with compound (IV).

[0396] In another embodiment, the lipid nanoparticle as disclosed herein can be manufactured with DSPC or DOPE as neutral lipid, cholesterol as a steroid, and PEG-PE (PEG2000-PE) or DMG-PEG (DMG-PEG2000) as a pegylated lipid.

[0397] In another embodiment, the lipid nanoparticle as disclosed herein can be manufactured with a lipid compound of formula (III) to (XXVII), DSPC as neutral lipid, cholesterol as a steroid, and PEG-PE (PEG2000-PE) as a pegylated lipid.

[0398] In another embodiment, the lipid nanoparticles as disclosed herein can be manufactured with a lipid compound of formula (III), (IV), (V), (VIII), (IX), (XII), (XVI), (XIX), or (XXII), DSPC as a neutral lipid, cholesterol as a sterol, and PEG-PE (PEG2000-PE) as a pegylated lipid.

[0399] In another embodiment, the lipid nanoparticles as disclosed herein can be manufactured with a lipid compound of formula (IV), (VIII), (IX), (XII), (XVI), (XIX), or (XXII), DSPC as a neutral lipid, cholesterol as a sterol, and PEG-PE (PEG2000-PE) as a pegylated lipid.

[0400] In another embodiment, the lipid nanoparticles as disclosed herein can be manufactured with a lipid compound of formula (IV), (IX), (XII), or (XVI), DSPC as a neutral lipid, cholesterol as a sterol, and PEG-PE (PEG2000-PE) as a pegylated lipid.

[0401] In another embodiment, the lipid nanoparticles as disclosed herein can be manufactured with a lipid compound of formula (IV) or (XII), DSPC as a neutral lipid, cholesterol as a sterol, and PEG-PE (PEG2000-PE) as a pegylated lipid.

[0402] In another embodiment, the lipid nanoparticles as disclosed herein can be manufactured with a lipid compound of formula (IV), DSPC as a neutral lipid, cholesterol as a sterol, and PEG-PE (PEG2000-PE) as a pegylated lipid.

[0403] Nucleic acid

[0404] The compositions or lipid nanoparticles as disclosed herein can comprise at least one anionic or polyanionic therapeutic agent. Suitable therapeutic agents for use in the present disclosure can be nucleic acids.

[0405] The nucleic acid as disclosed herein can be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA), for example an RNA, for example an in vitro transcribed RNA (IVT RNA) or a synthetic RNA.

[0406] Nucleic acids according to the present disclosure include genomic DNA, cDNA, mRNA, recombinantly produced, and chemically synthesized molecules. Nucleic acids can be in single- or double-stranded and be in the form of linear or covalently closed molecules forming a circle. Nucleic acids can be used for introduction into a cell (i.e., transfection), e.g., in the form of RNA, which can be prepared by in vitro transcription from a DNA template. Furthermore, the RNA can be modified by stabilization sequences, capping, and polyadenylation before application.

[0407] Nucleic acids can be of eukaryotic or prokaryotic origin, and for example, of human, animal, plant, bacterial, yeast, or viral origin, etc. They can be obtained by any technique known to the person skilled in the art, and for example, by screening of libraries, by chemical synthesis, or alternatively by hybrid methods, including chemical or enzymatic modification of sequences obtained by screening of libraries. They can be chemically modified.

[0408] Nucleic acids can be comprised in a vector. Vectors are known to the skilled person and can include plasmid vectors, cosmid vectors, phage vectors (such as lambda phage), viral vectors (such as adenoviral or baculoviral vectors), or artificial chromosome vectors (such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or PI artificial chromosomes (PACs)). Vectors include expression vectors as well as cloning vectors. Expression vectors include plasmids as well as viral vectors, and generally contain the desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used for engineering and amplification of a certain desired DNA fragment, and can lack functional sequences required for the expression of the desired DNA fragment.

[0409] In one embodiment, the nucleic acid can 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.

[0410] In one embodiment, the nucleic acid can be selected from the group consisting of messenger RNA (mRNA); antisense oligonucleotides (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.

[0411] In another embodiment, the nucleic acid can be selected from the group consisting of messenger RNA (mRNA); antisense oligonucleotides (ASO); short interfering RNA (siRNA); self-amplifying RNA (saRNA); microRNA (miRNA); plasmid DNA (pDNA); and combinations thereof.

[0412] In another embodiment, the nucleic acid can be selected from the group consisting of messenger RNA (mRNA); short interfering RNA (siRNA); self-amplifying RNA (saRNA); microRNA (miRNA); and combinations thereof.

[0413] In another embodiment, the nucleic acid can be messenger RNA (mRNA).

[0414] In one embodiment, the nucleic acid is mRNA. In certain embodiments, the nucleic acid can be an RNA that encodes a protein or enzyme. Such polynucleotides can be used as therapeutic agents that can be expressed by a target cell to facilitate production of a functional enzyme or protein. For example, in certain embodiments, when a target cell expresses at least one polynucleotide, a functional enzyme or protein that is deficient in the cell or individual is produced.

[0415] A target cell is a cell to which a composition or lipid nanoparticle as disclosed herein is to be directed or targeted. A target cell can include a particular tissue or organ. In some embodiments, a target cell can be a hepatocyte, an epithelial cell, a hematopoietic cell, an epithelial cell, an endothelial cell, a lung cell, a bone cell, a stem cell, a mesenchymal cell, a neural cell (e.g., meningeal, astrocyte, motor neuron, dorsal root ganglion cell, and anterior horn motor neuron), a photoreceptor cell (e.g., rod and cone), a retinal pigment epithelial cell, a secretory cell, a heart cell, an adipocyte, a vascular smooth muscle cell, a cardiomyocyte, a skeletal muscle cell, a beta cell, a pituitary cell, a synovial lining cell, an ovarian cell, a testicular cell, a fibroblast, a B cell, a T cell, an antigen presenting cell such as a dendritic cell, a reticulocyte, a white blood cell, a granulocyte, and a tumor cell.

[0416] mRNA

[0417] The term "RNA" relates to molecules comprising ribonucleotide residues and, for example, consisting entirely or essentially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of the beta-D-ribofuranosyl group.

[0418] The term includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, or recombinantly produced RNA.

[0419] These can be natural or artificial source sequences and are, for example, 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- or non-blunt-ended RNA, mature and immature mRNA, coding and non-coding RNA, synthetic or semi-synthetic sequences of hybridized sequences or oligonucleotides (modified or otherwise) and mixtures thereof.

[0420] Thus, these can be messenger RNAs (mRNAs), including mature and immature mRNAs, such as pre-mRNAs or heterogeneous nuclear mRNAs (hnRNAs) and mature mRNAs. Thus, the RNA molecules as disclosed herein also include monocistronic and polycistronic messenger RNAs.

[0421] For the sake of 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 comprising a sequence encoding a protein of interest, and which can be translated by a eukaryotic host, the sequence starting with a start codon (ATG) and ending, for example, with a stop codon (i.e., TAA, TAG, TGA).

[0422] The RNA can be a naturally occurring RNA or a modified RNA that differs from a naturally occurring RNA by the addition, deletion, substitution and / or alteration of at least one nucleotide. Such alterations can include the addition of non-nucleotide material, such as, for example, to one or more termini or internally, for example, at least one nucleotide of the RNA. The nucleotides in the RNA molecule can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally occurring RNAs.

[0423] In one embodiment, the RNA is an mRNA (messenger RNA). The mRNA can be a transcript that can be produced using DNA as a template and encodes a peptide or protein.

[0424] mRNA typically comprises a 5’ cap, a 5’ untranslated region (5-UTR), a protein or peptide coding region, and a 3’ untranslated region (3’-UTR) and a 3’ poly-A tail. mRNA has a limited halftime in cells and in vitro. For example, mRNA is produced by in vitro transcription using a DNA template. Alternatively, RNA can be obtained by chemical synthesis. In vitro transcription methods are known to the skilled person. For example, there are various commercially available in vitro transcription kits.

[0425] RNA can be synthesized in vitro in a cell-free system using a suitable cell extract and a suitable DNA template. For example, a cloning vector is applied for the production of the transcript. The promoter used to control transcription can be any promoter for any RNA polymerase. Some examples of RNA polymerases are T7, T3 and SP6 RNA polymerases. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, for example a cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA. For example, a cloning vector is applied for the production of the transcript, which is commonly referred to as a transcription vector.

[0426] In one embodiment, the RNA can encode a protein or a peptide. That is, if present in a suitable environment, for example within a cell, such as an antigen presenting cell, for example a dendritic cell, the RNA can be expressed to produce the protein or peptide it encodes.

[0427] The stability and the translation efficiency of the RNA can be modified as desired. In the present disclosure, modification of the RNA refers to any modification of the RNA that does not naturally occur in the RNA.

[0428] According to a general embodiment, the mRNA as disclosed herein can comprise or consist of the following general formula:

[0429] [5’ cap]w- [5’ UTR]x- [gene of interest] - [3’ UTR]y- [poly A]z

[0430] wherein [5’ UTR] and [3’ UTR] are untranslated regions (UTRs),

[0431] wherein [5’ UTR] contains a Kozak sequence,

[0432] wherein [gene of interest] is any gene encoding a protein of interest,

[0433] wherein [5’ cap] contains a methylguanine nucleotide, which is linked to the mRNA via a 5’ to 5’ linkage,

[0434] wherein [poly A] is a poly(A) tail, and

[0435] wherein w, x, y and z are the same or different and equal to 0 or 1.

[0436] According to one embodiment, the mRNA as disclosed herein can consist of the following general formula:

[0437] [5' cap] - [5' UTR] - [gene of interest] - [3' UTR] - [poly(A)]

[0438] wherein [5' UTR] and [3' UTR] are untranslated regions,

[0439] wherein [5' UTR] contains a Kozak sequence,

[0440] wherein [gene of interest] is any nucleic acid encoding a protein of interest,

[0441] wherein [5' cap] contains a methylguanine nucleotide linked to the mRNA via a 5' to 5' bond, and

[0442] wherein [poly(A)] is a poly(A) tail.

[0443] It is reminded that a Kozak sequence refers to a sequence, usually a consensus sequence, that occurs on eukaryotic mRNAs and plays a major role in the initiation of the translation process. Kozak sequences and Kozak consensus sequences are well known in the art.

[0444] It is also reminded that a poly(A) tail consists of a plurality of adenosine monophosphates well known in the art. A poly(A) tail is usually generated in a step called polyadenylation, one of the post-translational modifications that usually occur during the production of mature messenger RNA; such a poly(A) tail contributes to the stability and half-life of the mRNA and can be of variable length. For example, a poly(A) tail can be equal to or longer than 10 A nucleotides, including equal to or longer than 20 A nucleotides, including equal to or longer than 100 A nucleotides, and for example about 120 A nucleotides.

[0445] [3' UTR] does not express any protein. The purpose of [3' UTR] is to increase the stability of the mRNA. According to one embodiment, the a-globin UTR is chosen because it is known to lack instability.

[0446] Advantageously, the sequence corresponding to the gene of interest can be codon-optimized in order to obtain a satisfactory protein yield in the host considered.

[0447] RNA molecules as disclosed herein can be of variable length. Thus, they can be short RNA molecules, for example shorter than about 100 nucleotides; or long RNA molecules, for example longer than about 100 nucleotides, or even longer than about 300 nucleotides.

[0448] RNA, such as mRNA, can encompass synthetic or artificial RNA molecules, but also naturally occurring RNA molecules.

[0449] According to the present disclosure, RNA molecules, such as mRNA, can encompass the following species:

[0450] (i) capped unmodified RNA molecules;

[0451] (ii) capped modified RNA molecules;

[0452] (iii) uncapped unmodified RNA molecules;

[0453] (iv) uncapped modified RNA molecules.

[0454] Capped and uncapped RNA molecules

[0455] According to the most general embodiment, "capped RNA molecules" refer to RNA molecules whose 5' end is linked to a guanosine or a modified guanosine (e.g., 7-methylguanosine (m 7 G)) linked to a 5' to 5' triphosphate bond or analogue. This definition is commensurate with the most widely accepted definition of 5' cap (e.g., naturally occurring and / or physiological cap).

[0456] In the sense of the present disclosure, "cap analogues" include caps that are biologically equivalent to 7-methylguanosine (m 7 G) linked to a 5' to 5' triphosphate bond and which can therefore also be substituted without impairing the protein expression of the corresponding messenger RNA in eukaryotic hosts.

[0457] As examples of caps, one can mention 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 sG.

[0458] Examples of synthetic caps and / or cap analogs can be selected from: glyceryl, inverted deoxy abasic residue (moieties), 4',5' methylene nucleotides, 1 -(beta-D- erythrofuranosyl) nucleotides, 4'-thio nucleotides, carbocyclic nucleotides, 1,5- anhydrohexitol nucleotides, L-nucleotides, alpha-nucleotides, modified base nucleotides, threo-pentofuranosyl nucleotides, acyclic 3',4'-seco nucleotides, acyclic 3,4- dihydroxybutyl nucleotides, acyclic 3,5 dihydroxypentyl nucleotides, 3'-3'- inverted nucleotide moieties, 3'-3'-inverted abasic moieties, 3'-2'-inverted nucleotide moieties, 3'-2'-inverted abasic moieties, 1,4-butanediol phosphate, 3'- phosphoramidates, phosphohexites, phosphoaminohexyls, 3'-phosphates, 3' thiophosphates, dithiophosphates, or bridged or unbridged methylphosphonate moieties.

[0459] 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.

[0460] Notably, among the synthetic caps, some of the above-mentioned caps are suitable as analogs, while others can conversely hinder protein expression. The skilled person understands such distinctions.

[0461] By way of reference and in a non-limiting manner, the anti-reverse cap analog (ARCA) 3'-O-Me-m 7 The structure of the G(5')ppp(5')G cap analog is presented below:

[0462]

[0463] For example, the ARCA cap analog is an example of a cap analog used in in vitro transcription: it is a modified cap in which the 3' OH group (closer to the m 7 G) is replaced by -OCH3. However, 100% of the transcripts synthesized with ARCA at the 5' end are translatable, resulting in a strong stimulatory effect on translation.

[0464] The provision of an RNA having a 5'-cap or a 5'-cap analog can be achieved by in vitro transcription of a DNA template in the presence of said 5'-cap or 5'-cap analog, wherein said 5'-cap is co-transcriptionally incorporated into the RNA strand produced, or the RNA can be produced, for example, by in vitro transcription, and the 5'-cap can be attached to the RNA post-transcriptionally using a capping enzyme (e.g., the capping enzyme of vaccinia virus).

[0465] An "uncapped RNA molecule" refers to any RNA molecule that does not fall under the definition of a "capped RNA molecule".

[0466] Accordingly, according to the general embodiment, an "uncapped mRNA" can refer to an mRNA whose 5' end is not linked to a 7-methylguanosine via a 5' to 5' triphosphate linkage or an analogue as previously defined.

[0467] An uncapped RNA molecule, such as a messenger RNA, can be an uncapped RNA molecule having a (5')ppp(5'), (5')pp(5'), (5')p(5') or even a blunt 5' guanosine extreme. Such RNA molecules can be abbreviated as 5' ppp RNA; 5' pp RNA; 5' p RNA; 5' OH RNA, respectively. OH The uncapped RNA molecule as disclosed herein is a messenger 5' ppp RNA, for example.

[0468] Accordingly, when the RNA molecule is a single-stranded RNA molecule, it can be abbreviated as 5’ppp ssRNA; 5’pp ssRNA; 5’ p ssRNA; 5’OH ssRNA.

[0469] Accordingly, when the RNA molecule is a double-stranded RNA molecule, it can be abbreviated as 5’ppp dsRNA; 5’pp dsRNA; 5’ p dsRNA; 5’OH dsRNA.

[0470] In one embodiment, the uncapped mRNA as disclosed herein is an uncapped single-stranded mRNA.

[0471] According to one embodiment, the uncapped single-stranded mRNA can be an uncapped messenger 5’ppp ssRNA.

[0472] In a non-limiting manner, the first base of the uncapped RNA molecule can be adenosine, guanosine, cytosine or uridine.

[0473] Accordingly, the uncapped RNA molecule can be an uncapped RNA molecule having a (5')ppp(5'), (5')pp(5'), (5')p(5') or even a blunt 5' guanosine extreme.

[0474] In one embodiment of the disclosure, the RNA can be devoid of uncapped 5'- triphosphates. The removal of such uncapped 5'-triphosphates can be achieved by treating the RNA with a phosphatase.

[0475] Modified and unmodified RNA molecules

[0476] The RNA can comprise further modifications. For example, the further modification of the RNA used in the present disclosure can be an extension or truncation of the naturally occurring poly(A) tail, or a change of the 5'- or 3'-untranslated region (UTR), such as the introduction of a UTR that is not related to the coding region of the RNA, e.g., exchanging the existing 3'-UTR for or inserting at least one (e.g., two copies) of a 3'-UTR derived from a globin gene (such as a2-globin, al-globin, b-globin, e.g., b-globin and, e.g., human b-globin).

[0477] In the present disclosure, a "modified RNA molecule" refers to an RNA molecule that contains at least one modified nucleotide, nucleoside, or base, such as a modified purine or a modified pyrimidine. The modified nucleoside or base can be any nucleoside or base that is not A, U, C, or G (for nucleosides, respectively, adenosine, uridine, cytidine, or guanosine; and, when referring only to the sugar moiety, adenine, uracil, cytosine, or guanine).

[0478] Accordingly, an "unmodified RNA molecule" refers to any RNA molecule that does not conform to the definition of a modified RNA molecule.

[0479] In the sense of the present disclosure, the term "modified and unmodified" is considered to be different from the term "capped and uncapped" as the latter specifically relates, in the sense of the present disclosure, to the base at the 5' end of an RNA molecule.

[0480] In one embodiment, the nucleic acid, e.g., RNA, can comprise at least one modified nucleotide, e.g., a modified ribonucleotide. The presence of a modified nucleotide can increase the stability of the nucleic acid and / or reduce its cytotoxicity.

[0481] The term stability of an RNA relates to the half-life of the RNA, i.e., the period of time required for the elimination of half of the activity, amount, or number of molecules. In the context of the present disclosure, the half-life of an RNA is indicative of the stability of the RNA. The half-life of an RNA can influence the duration of expression of the RNA. It can be expected that an RNA with a long half-life will be expressed for an extended period of time.

[0482] According to one embodiment, a "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, e.g., at least one base modification as described herein.

[0483] For example, in one embodiment, in an RNA suitable for use in the present disclosure, 5-methylcytidine can be partially or fully substituted, e.g., fully substituted for cytidine. Alternatively or additionally, in one embodiment, it can be partially or fully substituted, e.g., fully substituted for uridine.

[0484] By way of non-limiting example, examples of modified nucleotides, nucleosides, and bases are disclosed in WO 2015 / 024667 Al.

[0485] Accordingly, a modified RNA molecule can contain a modified nucleotide, nucleoside, or base, including a backbone modification, a sugar modification, or a base modification.

[0486] A backbone modification relevant to the present disclosure includes a modification in which the phosphate of the nucleotide backbone contained in an RNA molecule as defined herein is chemically modified.

[0487] A sugar modification relevant to the present disclosure includes a chemical modification of the sugar of a nucleotide of an RNA molecule as defined herein.

[0488] A base modification relevant to the present disclosure includes a chemical modification of the base moiety of a nucleotide of an RNA. In this context, the nucleotide analog or modification is, for example, selected from nucleotide analogs suitable for transcription and / or translation of an RNA molecule in a eukaryotic cell.

[0489] A sugar modification can include replacement or modification of the 2’ hydroxyl (OH), which can be modified or replaced by a number of different “oxy” or “deoxy” substituents.

[0490] Examples of “oxy”-2’ hydroxyl 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), -0(CH2CH20)nCH2CH2OR; “locked” nucleic acid (LNA), in which the 2’ hydroxyl is linked to the 4’ carbon of the same ribose, e.g., by a methylene bridge; and amino (-0-amino, where the amino group (e.g., NRR) can be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino), or aminoalkoxy.

[0491] “Deoxy” modifications include hydrogen, amino (e.g., NH2; 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 comprises at least one atom of C, N, and O.

[0492] The sugar group can also contain at least one carbon having the opposite stereochemical configuration as the corresponding carbon in ribose. Thus, the modified RNA can comprise nucleotides containing, for example, arabinose as the sugar.

[0493] Phosphate backbones can be further modified and incorporated into the modified RNA molecules as described herein. The phosphate groups of the backbone can be modified by replacing at least one of the oxygen atoms with a different substituent. In addition, the modified nucleosides and nucleotides can comprise the complete replacement of the unmodified phosphate moieties with modified phosphates as described herein.

[0494] Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, borano phosphates, borano phosphate esters, phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. Both of the non-linking oxygens of a phosphorodithioate are replaced with sulfur. Phosphate linkers can also be modified by replacing the linking oxygens with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene-phosphonates).

[0495] The modified nucleosides and nucleotides that can be incorporated into the modified RNA molecules as described herein can be further modified at the nucleobase moiety. For example, the nucleosides and nucleotides as described herein can be chemically modified on the major groove face. In some embodiments, the major groove chemical modification can include an amino, thiol, alkyl, or halo group.

[0496] For example, the nucleotide analog / modification is selected from a base modification selected from: 2-amino-6-chloropurine nucleoside-5'-triphosphate, 2-amino purine-nucleoside-5'- triphosphate; 2-amino adenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2- thio cytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluoro thymidine-5'- triphosphate, 2'-O-methyl inosine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-aminoallyl cytidine-5'-triphosphate, 5-aminoallyl uridine-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, 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, 6-azacytidine-5'-triphosphate, 6- azauridine-5'-triphosphate, 6-chloropurine nucleoside-5'-triphosphate, 7-deazoadenosine-5'- triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azoadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-nucleoside-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.

[0497] In some embodiments, the modified nucleosides can be selected from the group consisting of: pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 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, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l-methyl-pseudouridine, 2-thio-l-methyl-pseudouridine, 1-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-l-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.

[0498] In some embodiments, the 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-l-methyl-pseudoisocytidine, 4-thio-l-methyl-l-deaza-pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-l-methyl-pseudoisocytidine.

[0499] In other embodiments, the 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-glycinamidoformyladenosine, N6-threoninamidoformyladenosine, 2-methylthio-N6-threoninamidoformyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

[0500] In other embodiments, the modified nucleoside includes inosine, 1 -methyl-inosine, wyosine, wytidine, 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-methyl inosine, 6-methoxy-guanosine, 1 -methyl guanosine, N2-methyl guanosine, N2,N2-dimethyl guanosine, 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.

[0501] In some embodiments, the nucleotides can be modified on the major groove face and can include replacement of the hydrogen on C-5 of uracil with a methyl or a halo group.

[0502] 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).

[0503] In view of the above, the modified base can be a modified purine base or a modified pyrimidine base.

[0504] By way of non-limiting example, examples of modified purine bases include modified adenosine and / or modified guanosine, such as hypoxanthine; xanthine; 7-methylguanine; inosine; xanthosine; and 7-methylguanosine.

[0505] According to some embodiments, the modified RNA molecule or mRNA corresponds to an RNA for which each nucleoside corresponding to uridine, cytidine, adenosine, and / or ribothymidine is modified.

[0506] By way of non-limiting example, examples of modified pyrimidine bases include modified cytidine and / or modified uridine, such as 5,6-dihydrouracil; pseudouridine; 5-methylcytidine; 5-hydroxymethylcytidine; dihydrouridine; and 5-methylcytidine.

[0507] By way of non-limiting example, the modified base as disclosed herein can be a modified uridine or cytidine, such as pseudouridine and 5-methylcytidine.

[0508] According to some embodiments, the modified RNA corresponds to an RNA for which at least one base corresponding to U (for uracil), C (for cytosine), A (for adenine) and / or T (for thymine) is modified.

[0509] As examples of modified bases, one can mention methyl-5 uridine (m5U), 2-thio-uridine (s2U), 2'-0-methyl-5 uridine (Ome5U), pseudouridine (Ψ), methyl-1 pseudouridine (m1Ψ), methyl-5 cytosine (m5C), 2'0-methyl-5 cytosine (Om5C), N6-methyl-adenosine (m6A), and N1-methyl-adenosine (m6A).

[0510] According to some embodiments, the modified mRNA can comprise 2'-0-methyl-5 uridine (Ome5U) or methyl-1 pseudouridine (m1Ψ) as modified bases.

[0511] Capped and uncapped mRNAs, whether modified or not, are also commercially available.

[0512] RNAs having an unmasked poly(A) sequence are more efficiently translated than RNAs having a masked poly(A) sequence.

[0513] The term "poly(A) tail" or "polyA sequence" relates to a sequence of adenosine (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 an A of the poly(A) sequence and is followed by no nucleotides other than A located 3' (i.e. downstream) of the poly(A) sequence. Furthermore, a long poly(A) sequence of about 120 base pairs leads to optimal translational stability and efficiency of the RNA.

[0514] Accordingly, in order to increase the stability and / or expression of the RNA used according to the present disclosure, it can be modified so as to be present in combination with a poly(A) sequence, for example having a length of 10 to 500, for example 30 to 300, even for example 65 to 200 and for example 100 to 150 adenosine residues. In one embodiment, the poly(A) sequence has a length of about 120 adenosine residues. In order to further increase the stability and / or expression of the RNA used according to the present disclosure, the poly(A) sequence can be unmasked.

[0515] In addition, the incorporation of a 3'-untranslated region (UTR) into the 3'-untranslated region of an RNA molecule can lead to an enhancement of the translational efficiency. By incorporating two or more such 3'-untranslated regions, a synergistic effect can be achieved. The 3'-untranslated region can be autologous or heterologous to the RNA into which it is introduced. In one embodiment, the 3'-untranslated region is derived from the human beta-globin gene.

[0516] The combination of the above modifications (i.e. incorporation of a poly-A sequence, unmasking of the poly-A sequence and incorporation of at least one 3'-untranslated region) has a synergistic effect on the increase of stability and translation efficiency of the RNA.

[0517] In order to increase the expression of the RNA used according to the present disclosure, it can be modified within the coding region (i.e. the sequence encoding the expressed peptide or protein), e.g. without changing the sequence of the expressed polypeptide or protein, such that the GC content is increased to increase the stability of the mRNA and codon optimization is performed, and such that translation in the cell is enhanced.

[0518] It is understood that the uncapped RNA molecule can be a modified RNA molecule or an unmodified RNA molecule.

[0519] Thus, the capped RNA molecule can be a modified RNA molecule or an unmodified RNA molecule.

[0520] In one embodiment, the RNA molecule as disclosed herein is a messenger RNA (mRNA).

[0521] The RNA molecule as disclosed herein is, e.g. an uncapped messenger RNA, either modified or unmodified.

[0522] The RNA molecule as disclosed herein is, e.g. a capped messenger RNA, either modified or unmodified.

[0523] In a non-limiting manner, the uncapped RNA molecule, such as the messenger RNA, can also be an uncapped RNA molecule having only naturally occurring bases.

[0524] According to the present disclosure, "naturally occurring bases" relate to bases that can be naturally incorporated into an RNA molecule such as a messenger RNA by a host in vivo. Thus, "naturally occurring bases" are different from synthetic bases for which there is no natural equivalent in the host. However, "naturally occurring bases" can or can not be modified bases, as the two terms are not to be confused in the sense of the present disclosure.

[0525] The uncapped messenger RNA can also be an uncapped and modified messenger RNA and thus contains at least one modified base.

[0526] Thus, the uncapped messenger RNA can also be an uncapped and modified messenger RNA having a (5')ppp(5')guanosine extreme and containing at least one modified base.

[0527] Uncapped messenger RNA can also be uncapped and modified messenger RNA having a (5’) ppp(5’) guanosine extreme and containing at least one pseudo-uridine and at least one 5-methylcytosine.

[0528] Capped messenger RNA can be messenger RNA having a 5’ end linked to a 7-methylguanosine or analog linked to a 5’ to 5’ triphosphate bond and containing naturally occurring bases or modified bases such as pseudo-uracil or 5-methylcytosine.

[0529] It will also be appreciated that when both modified and unmodified RNA molecules are used in one embodiment of the disclosure, they can be used in mixtures and / or purified forms.

[0530] Antigens

[0531] According to one embodiment, a composition as disclosed herein, such as a lipid nanoparticle, can be a nucleic acid immunogenic composition or nucleic acid vaccine comprising at least one polynucleotide, e.g., polynucleotide construct, encoding at least one wild-type or engineered antigen.

[0532] The valency of an antigen-containing composition as disclosed herein can vary. Valency refers to the number of antigen components in a composition or polynucleotide (e.g., RNA polynucleotide) or polypeptide. In some embodiments, the immunogenic composition is multivalent. They can also be compositions comprising more than one valency, such as bivalent, trivalent, or multivalent compositions. A multivalent immunogenic composition or vaccine can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more antigens or antigenic portions (e.g., antigenic peptides, etc.). The antigenic components can be on a single polynucleotide or on separate polynucleotides.

[0533] A composition as disclosed herein can be used to protect, treat, or cure an infection resulting from exposure to an infectious agent such as bacteria, viruses, fungi, protozoa, and parasites.

[0534] A composition as disclosed herein can be used to protect, treat, or cure a cancer disease.

[0535] According to one embodiment, the nucleic acid can 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.

[0536] Bacterial antigens

[0537] The bacteria described herein can be a gram-positive bacteria or a gram-negative bacteria. The bacterial antigen can be obtained from Acinetobacter baumannii, Bacillus anthracis, Bacillus subtilis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, coagulase Negative Staphylococcus, Corynebacterium diphtheria, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli, E. coli 0157:H7, Enterobacter sp., Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus casseliflavus, Enterococcus avium, Enterococcus raffinosus, Enterococcus hermannii, Enterococcus hirae, Enterococcus mundtii, Enterococcus solitarius, Enterococcus saccharolyticus, Enterococcus computer, Enterococcus sibiricum, Enterococcus thailandicus, Enterococcus xiangfangensis, Enterococcus asaccharolyticus, Enterococcus camelliae, Enterococcus columbae, Enterococcus dispar, Enterococcus flavus, Enterococcus gergoviae, Enterococcus hienensis, Enterococcus malodoratus, Enterococcus moraviensis, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus, Enterococcus pseudoavium, Enterococcus pseudoelongatus,Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Moraxella catarralis, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitides, Proteus 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 mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae, and Yersinia pestis.

[0538] Viral antigens

[0539] The viral antigen can be obtained from an adenovirus; herpes simplex, type 1; herpes simplex, type 2; encephalitis virus, papillomavirus, varicella-zoster virus; Epstein-barr virus; human cytomegalovirus; human herpesvirus, type 8; human papillomavirus; BK virus; JC virus; smallpox; poliovirus, hepatitis B virus; human bocavirus; parvovirus B19; human astrovirus; norovirus; 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; circovirus; Coltivirus; Hantavirus, Middle East respiratory syndrome coronavirus; SARS-Cov-2 virus; Chikungunya virus; parainfluenza virus; human enterovirus; Hanta virus; Japanese encephalitis virus; Vesicular exanthema of new world porcine virus; Eastern equine encephalitis virus; or Banna virus.

[0540] In one embodiment, the antigen is from a strain of influenza A or influenza B virus or a combination thereof. The influenza A or influenza B strain can be associated with birds, swine, horses, dogs, humans, or non-human primates.

[0541] The nucleic acid can encode a hemagglutinin protein or fragment thereof. The hemagglutinin protein can be H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, H18, or a fragment thereof. The hemagglutinin protein can or can not comprise a head domain (HA1). Alternatively, the hemagglutinin protein can or can not comprise a cytoplasmic domain.

[0542] For example, in embodiments, the hemagglutinin protein is a truncated hemagglutinin protein. The truncated hemagglutinin protein can comprise a portion of the transmembrane domain.

[0543] In some embodiments, the virus can be selected from H1N1, H3N2, H7N9, H5N1, and H10N8 viruses or B strain viruses.

[0544] In another embodiment, the antigen is from a coronavirus, such as a SARS-Cov-1 virus, a SARS-Cov-2 virus, or a MERS-Cov virus.

[0545] Fungal antigens

[0546] Fungal antigens can be obtained from 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 bieneusi), and Mucoromycotina (e.g., Mucor circinelloides, Rhizopus oryzae, Lichtheimia corymbifera).

[0547] Protozoal antigens

[0548] Protozoan antigens can be obtained from Entamoeba histolytica, Giardia lambila, Trichomonas vaginalis, Trypanosoma brucei, T. cruzi, Leishmania donovani, Balantidium coli, Toxoplasma gondii, Plasmodium spp., and Babesia microti.

[0549] Parasitic antigens

[0550] Parasitic antigens can be obtained from Acanthamoeba, Anisakis, Ascaris lumbricoides, botfly, Balantidium coli, bedbug, Cestoda, chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lambila, hookworm, Leishmania, Linguatula serrata, liver fluke, Loa loa, Paragonimus, pinworm, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, mite, tapeworm, Toxoplasma gondii, Trypanosoma, whipworm, and Wuchereria bancrofti.

[0551] Tumour antigens

[0552] In one embodiment, the antigen can be a tumor antigen, i.e., a component of a cancer cell, such as a protein or peptide expressed in a cancer cell. The term "tumor antigen" or refers, for example, to a protein that is specifically expressed under normal conditions in a limited number of tissues and / or organs or in a specific developmental stage, 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 certain cell type at a certain differentiation stage under normal conditions, and lineage-specific antigens. For example, a tumor antigen is presented by the cancer cell that expresses it.

[0553] For example, tumor antigens include carcinoembryonic antigen, a 1-fetoprotein, isoferritin, and fetal sulfoglycoprotein, cc2-H-ferritin, and g-fetoprotein.

[0554] Other examples of tumor antigens useful in the present disclosure are p53, ART-4, BAGE, beta-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CD 4 / m, CEA, cell surface proteins of the tight junction protein family (such as CLAUDIN-6, CLAUDIN-18.2, and CLAUDIN-12), c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gapl OO, 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, MC1 R, myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl 90 minor BCR-abL, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RUl or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP 1, SCP2, SCP3, SSX, SURVrVIN, TEL / AMLl, TPI / m, TRP-1, TRP-2, TRP-2 / 1NT2, TPTE, and WT, e.g., WT-1.

[0555] Adjuvants

[0556] The nucleic acid-containing compositions or lipid nanoparticles as disclosed herein can further comprise or can be co-administered with an adjuvant or an immunopotentiator.

[0557] Adjuvants useful in the present disclosure can include, but are not limited to, natural or synthetic adjuvants. They can be organic or inorganic.

[0558] The adjuvant can be selected from any of the following classes: (1) mineral salts, such as aluminum hydroxide and aluminum phosphate or calcium phosphate gels; (2) emulsions, including: oil emulsions and surfactant-based formulations, such as microfluidized detergent-stabilized oil-in-water emulsions, purified saponins, oil-in-water emulsions, stabilized water-in-oil emulsions; (3) microparticulate adjuvants, such as virosomes (single bilayered liposome carriers incorporating influenza hemagglutinin), structured complexes of lipids and saponins, polylactide coglycolide (PLG); (4) microbial derivatives; (5) endogenous human immunomodulators; and / or (6) inert carriers, such as gold particles; (7) adjuvants of microbial origin; (8) surface active compounds; (9) carbohydrates; or combinations thereof.

[0559] It will be apparent to one of ordinary skill in the art how to select an appropriate adjuvant and an appropriate amount of the adjuvant.

[0560] Particular adjuvants can 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(s) TM , 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, Montanide ISA 720 adjuvant, Corynebacterium-derb / ed P40 vaccine adjuvant, MPL TM 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 liposome vaccine adjuvant, adamantylamide dipeptide vaccine adjuvant, Arlacel A, VSA-3 adjuvant, aluminum vaccine adjuvant, Polygen vaccine adjuvant, Adjumer TM , Algal Glucan, Bay R1005, stearoyl tyrosine, Specol, Algammulin, Calcium phosphate gel, CTA1-DD gene fusion protein, DOC / Alum complex, γ-inulin, Gerbu adjuvant, GM-CSF, GMDP, recombinant hlFN-γ / interferon-g, interleukin Interleukin-2, Interleukin-7, Sclavo peptide, Rehydragel LV, Rehydragel HPA, Loxoribine, MF59, MTP-PE liposomes, Muramedide, Murapalmitine, D-Murapalmitine, NAGO, nonionic surfactant vesicles, PMMA, PAA, protein cochelate, QS-21, SPT (antigen formulation), nanoemulsion vaccine adjuvant, AS03, Quil-A vaccine adjuvant, RC529 vaccine adjuvant, LTR192G vaccine adjuvant, Escherichia coli heat-sensitive toxin, LT, amorphous aluminum hydroxyphosphate sulfate adjuvant, calcium phosphate vaccine adjuvant, Montanide Incomplete Seppic adjuvant, imiquimod, requimod, AF03, flagellin, poly(LC) Abisco-100 vaccine adjuvant, albumin-heparin microparticle vaccine adjuvant, AS-2 vaccine adjuvant, B7-2 vaccine adjuvant, DHEA vaccine adjuvant, immunoliposomes containing co-stimulatory molecule antibodies, SAF-1, Sendai protein liposomes, lipid matrix containing Sendai, threonyl muramyl dipeptide (TMDP), Ty particle vaccine adjuvant, bupivacaine vaccine adjuvant, DL-PGL (polyester poly(DL-lactide-co-glycolic acid)) vaccine adjuvant, IL-15 vaccine adjuvant, LTK72 vaccine adjuvant, MPL-SE vaccine adjuvant, non-toxic mutant of cholera toxin El 12KmCT-El 12K, and / or Matrix-S.

[0561] Protein expression

[0562] Compositions as disclosed herein encapsulating at least one nucleic acid or lipid nanoparticles as disclosed herein can also be used to treat individuals with protein deficiency. Therefore, lipid nanoparticles can be used in methods of treating individuals with protein deficiency, the methods comprising administering lipid nanoparticles containing at least one nucleic acid (e.g., mRNA), wherein the nucleic acid encodes a functional protein corresponding to a protein lacking in the individual. In an embodiment, the functional protein is produced after the nucleic acid is expressed in target cells.

[0563] This disclosure also relates to a method for intracellular delivery of nucleic acids capable of correcting existing genetic defects and / or providing beneficial functions to at least one target cell. Upon successful delivery to a target tissue and cell, the composition of this disclosure and the nucleic acid are transfected into the target cell, and the nucleic acid (e.g., mRNA) can be translated into a target gene product (e.g., a functional protein or enzyme), or the presence or expression of the target gene product can be otherwise regulated or controlled.

[0564] The compositions and methods provided herein can be used to manage and treat a wide range of diseases, such as those caused by protein and / or enzyme deficiencies. Individuals with such diseases may have underlying genetic defects that result in impaired expression of proteins or enzymes, including, for example, failure to synthesize proteins, reduced protein synthesis, or lack of synthesis of biologically active or reduced-biologically active proteins.

[0565] Alternatively, the nucleic acid may encode a full-length antibody or a smaller antibody (e.g., both heavy and light chains) to confer immunity to the subject. In an alternative embodiment, the compositions of this disclosure encode antibodies that can be used to temporarily or permanently affect the functional response of a subject. For example, the mRNA nucleic acid of this disclosure may encode a functional monoclonal or polyclonal antibody that, after translation (and, where applicable, systemic excretion from target cells), can be used to target and / or inactivate biological targets (e.g., stimulating cytokines, such as tumor necrosis factor). Similarly, the mRNA nucleic acid of this disclosure may encode, for example, a functional anti-nephrotic factor antibody that can be used to treat, for example, membranoproliferative glomerulonephritis type II or acute hemolytic uremic syndrome, or alternatively may encode an anti-vascular endothelial growth factor (VEGF) antibody that can be used to treat VEGF-mediated diseases such as cancer.

[0566] Pharmaceutical Composition

[0567] According to some implementation methods, this disclosure relates to pharmaceutical compositions.

[0568] For administration purposes, the lipid compounds of this disclosure (e.g., formulated into lipid nanoparticles with therapeutic agents such as nucleic acids) can be administered as pharmaceutical compositions. Pharmaceutical compositions of this disclosure comprise the lipid compound as disclosed herein, and possibly at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0569] According to some embodiments, pharmaceutical compositions applicable to this disclosure may comprise (i) at least one nucleic acid and at least one lipid compound as disclosed herein, or (ii) at least one nucleic acid and at least one composition as described herein, or (iii) at least one nucleic acid-containing lipid nanoparticle as described herein and at least one pharmaceutically acceptable excipient.

[0570] In some embodiments, the pharmaceutical composition may be an immunogenic composition. An immunogenic composition suitable for use in this disclosure may comprise (i) at least one nucleic acid and at least one lipid compound as disclosed herein, or (ii) at least one nucleic acid and at least one composition as described herein, or (iii) at least one nucleic acid-containing lipid nanoparticle as described herein, wherein the nucleic acid encodes at least one antigen and at least one pharmaceutically acceptable excipient. Furthermore, the immunogenic composition may comprise an adjuvant as described herein.

[0571] According to some embodiments, this disclosure relates to a composition comprising (i) at least one nucleic acid and at least one lipid compound according to this disclosure, or (ii) at least one nucleic acid and at least one composition as described herein, or (iii) at least one nucleic acid-lipid nanoparticle as described herein, said composition being used as a pharmaceutical agent. Such pharmaceutical agents can be used for the prevention and / or treatment of diseases as indicated herein.

[0572] According to some embodiments, this disclosure relates to a composition comprising (i) at least one nucleic acid and at least one lipid compound according to this disclosure, or (ii) at least one nucleic acid and at least one composition as described herein, or (iii) at least one nucleic acid-lipid nanoparticle as described herein, the composition being used in a therapeutic method for preventing and / or treating a disease selected from infectious diseases, allergies, autoimmune diseases, rare blood disorders, rare metabolic diseases, rare neurological diseases and tumors or cancers, and, for example, as described herein.

[0573] According to some embodiments, compositions comprising the following can be used as immunogenic compositions: (i) at least one nucleic acid and at least one lipid compound according to this disclosure, or (ii) at least one nucleic acid and at least one composition as described herein, or (iii) at least one nucleic acid-containing lipid nanoparticle as described herein, wherein the nucleic acid encodes at least one antigen.

[0574] The immunogenic compositions disclosed herein can be used for the prevention and / or treatment of infectious diseases as indicated herein. They may contain nucleic acids encoding antigens as described herein.

[0575] In some embodiments, the lipid compound of formula (I) may be present in a pharmaceutical composition or immunogenic composition in an amount that effectively forms lipid nanoparticles and delivers therapeutic agents (e.g., nucleic acids) to treat a specific disease or condition of interest.

[0576] Those skilled in the art can easily determine the appropriate concentration and dosage.

[0577] The pharmaceutical compositions and immunogenic compositions disclosed herein may be administered in any acceptable manner as with compositions intended for similar purposes.

[0578] The compositions disclosed herein can be formulated into solid, semi-solid, or liquid formulations, such as powders, solutions, suspensions, or injections. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, and intranasal administration. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques.

[0579] In some embodiments, the compositions disclosed herein may be administered via transdermal, subcutaneous, intradermal, or intramuscular routes.

[0580] The compositions disclosed herein are formulated to allow the active ingredients contained therein to be bioavailable when the compositions are administered to a patient.

[0581] The actual methods for preparing such dosage forms are known or will be clear 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).

[0582] The composition may contain at least one inert diluent or carrier.

[0583] In one embodiment, the composition may be in liquid form, such as a solution, emulsion, or suspension. The liquid may be used for delivery by injection. Compositions intended for injection administration may contain at least one of the following: surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents. Liquid compositions disclosed herein may contain at least one of the following: sterile diluents, such as water for injection, saline solutions, such as physiological saline, Ringer's solution, or isotonic sodium chloride; fixed oils, such as synthetic mono- or diglycerides of glycerol, polyethylene glycol, glycerol, propylene glycol, or other solvents that can be used as a solvent or suspension medium; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and agents for regulating tension, such as sodium chloride or dextrose; and agents as cryoprotectants, such as sucrose or trehalose.

[0584] Parenteral preparations can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. Injectable pharmaceutical compositions are, for example, sterile.

[0585] The pharmaceutical compositions and immunogenic compositions disclosed herein can be prepared using methods well known in the pharmaceutical industry. Pharmaceutical compositions intended for injection can be prepared by combining lipid nanoparticles as disclosed herein with sterile distilled water or other carriers to form a solution. Surfactants may be added to promote the formation of a homogeneous solution or suspension.

[0586] The compositions disclosed herein will be administered at a therapeutically effective amount, which will depend on a variety of factors, including the activity of the specific therapeutic agent used; the metabolic stability and duration of action of the therapeutic agent; the patient's age, weight, general health condition, sex, and diet; the method and timing of administration; the excretion rate; the combination of drugs; the severity of the specific disorder or condition; and the subject receiving the therapy.

[0587] The compositions disclosed herein may also be administered concurrently with, before, or after the administration of at least one other therapeutic agent. Such combination therapies include single-dose formulations of a composition disclosed herein and at least one additional active agent, as well as formulations of a composition disclosed herein and each active agent, each in its own individual dose formulation. When using individual dose formulations, the composition disclosed herein and at least one additional active agent may be administered at substantially the same time (i.e., synchronously) or at separate, staggered times (i.e., sequentially); combination therapies should be understood to include all of these regimens.

[0588] Treatment

[0589] In some embodiments, this disclosure also relates to a method for preventing and / or treating a disease in an individual in need, wherein the method comprises administering to the individual an effective amount of (i) at least one nucleic acid and at least one lipid compound as disclosed herein, or (ii) at least one nucleic acid-containing composition as described herein, or (iii) at least one nucleic acid-containing lipid nanoparticle as described herein. For example, compositions containing LNPs as disclosed herein can be used as therapeutic methods for preventing and / or treating infectious diseases, allergies, autoimmune diseases, rare blood disorders, rare metabolic diseases, rare neurological diseases, and tumors or cancers.

[0590] For example, the diseases that this disclosure may refer to can be infectious diseases, such as viral infections, bacterial infections, fungal or parasitic infections. The diseases that this disclosure may also refer to can be cancer or tumors.

[0591] Viral infectious diseases can include acute febrile pharyngitis, pharyngoconjunctival fever, epidemic keratoconjunctivitis, infantile gastroenteritis, Coxsackie virus infection, infectious mononucleosis, Burkitt lymphoma, acute hepatitis, chronic hepatitis, cirrhosis, hepatocellular carcinoma, primary HSV-1 infection (e.g., gingivostomatitis in children, tonsillitis and pharyngitis in adults, keratoconjunctivitis), latent HSV-1 infection (e.g., cold sores and herpes labialis), primary HSV-2 infection, latent HSV-2 infection, aseptic meningitis, infectious mononucleosis, giant cell inclusion disease, Kaposi's sarcoma, and multicentric Castleman disease. Disease), primary exudative lymphoma, AIDS, influenza, Reye's syndrome, measles, post-infectious encephalomyelitis, mumps, proliferative epithelial lesions (e.g., common warts, flat warts, plantar warts and anogenital warts, laryngeal papilloma, epidermophyseal dysplasia verruciformis), cervical cancer, squamous cell carcinoma, croup, pneumonia, bronchiolitis, common cold, poliomyelitis, rabies, bronchiolitis, pneumonia, influenza-like syndrome, severe bronchiolitis with pneumonia, rubella, congenital rubella, chickenpox, Covid-19, respiratory syncytial virus (RSV) infection, and herpes zoster.

[0592] In one implementation, the disease is influenza, respiratory syncytial virus (RSV) infection, or Covid-19, and for example, influenza.

[0593] Bacterial infectious diseases can include abscesses, actinomycosis, acute prostatitis, Aeromonas hydrophila, annual ryegrass poisoning, anthrax, bacillary purpura, bacteremia, bacterial gastroenteritis, bacterial meningitis, bacterial pneumonia, bacterial vaginosis, bacterial-associated skin diseases, Bartonella infection, BCG-oma, staphylococcal infection, botulism, Brazilian purpura, Brodie abscess, brucellosis, Buruli ulcer, Campylobacteriosis, dental caries, and Carrion's disease. Diseases including: cat scratch disease, cellulitis, chlamydia infection, cholera, chronic bacterial prostatitis, chronic relapsing multifocal osteomyelitis, clostridial necrotizing enteritis, periodontal and pulpitis, infectious bovine pleuropneumonia, diphtheria, diphtheria stomatitis, erysipelas, piglotitis, erysipelas, Fitz-Hugh-Curtis syndrome, flea-borne spotted fever, foot rot (infectious foot dermatitis), Garre's sclerosing osteomyelitis, gonorrhea, granuloma inguinale, human granulocytic anaplasmosis, human mononuclear erysipelas, pertussis, impetigo, late-stage congenital syphilitic ophthalmia, Legionnaires' disease, Lemierre's disease. Leprosy (Hansen's disease), leptospirosis, listeriosis, Lyme disease, lymphadenitis, melioidosis, meningococcal infection, meningococcal sepsis, methicillin-resistant Staphylococcus aureus (MRSA) infection, and Mycobacterium avium-intracellulare (Mycobacterium avium-intracellulare).MAI), mycoplasma pneumonia, necrotizing fasciitis, nocardiac infection, gangrenous stomatitis (Noma) (cheek gangrene or gangrenous stomatitis), omphalitis, orbital cellulitis, osteomyelitis, post-splenectomy severe infection (OPSI), ovine brucellosis, pasteurellosis, periorbital cellulitis, pertussis (whooping cough), plague, pneumococcal pneumonia, Pott's disease, proctitis, pseudomonas infection, psittacosis, septicemia, pyomyomyositis, Q fever, relapsing fever Fever (typhus), rheumatic fever, Rocky Mountain spotted fever (RMSF), rickettsial disease, salmonellosis, scarlet fever, sepsis, Serratia marcescens infection, Shigella infection, Southern tick-associated rash, Staphylococcal scalded skin syndrome, streptococcal pharyngitis, swimming pool granuloma, swine brucellosis, syphilis, syphilitic aortitis, tetanus, toxic shock syndrome (TSS), trachoma, trench fever, tropical ulcer, tuberculosis, tularemia, typhus, spotted fever, urogenital tuberculosis, urinary tract infection, vancomycin-resistant Staphylococcus aureus infection, Waterhouse-Friderichsen syndrome, pseudotuberculosis (Yersinia pestis), and Yersinia bacillus infection.

[0594] Parasitic infections can include amoebiasis, giardiasis, trichomoniasis, African sleeping sickness, American sleeping sickness, leishmaniasis (leishmaniasis), bacillosis, toxoplasmosis, malaria, Acanthamoeba keratitis, and babesiosis.

[0595] Fungal infections can include aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis, mycomycosis of the foot, coccidioidomycosis, and tinea pedis. Furthermore, immunocompromised individuals are susceptible to diseases caused by fungal genera such as Aspergillus, Candida, Cryptococcus, Histoplasma, and Pneumocystis. Other fungi can infect the eyes, nails, hair, and especially the skin, known as dermatophytes and keratophilic fungi, causing a variety of conditions, the most common being dermatophytes such as athlete's foot. Fungal spores are also a major cause of allergies, and a wide range of fungi from different taxa can trigger allergic reactions in some individuals.

[0596] Cancer or tumor diseases can be selected from, for example, the following: melanoma, malignant melanoma, colon cancer, lymphoma, sarcoma, germ cell tumor, kidney cancer, gastrointestinal tumors, glioma, prostate tumors, bladder cancer, rectal tumors, stomach cancer, esophageal cancer, pancreatic cancer, liver cancer, mammary carcinoma (=breast cancer)), uterine cancer, cervical cancer, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), liver cancer, and various virus-induced tumors, such as papillomavirus-induced cancer (e.g., cervical carcinoma = cervical cancer). Cancer), adenocarcinoma, herpesvirus-induced tumors (e.g., Burkitt lymphoma, EBV-induced B-cell lymphoma), hepatitis B-induced tumors (hepatocellular carcinoma), HTLV-1 and HTLV-2 induced lymphoma, acoustic neuroma, lung carcinoma (= lung cancer = bronchial cancer), small cell lung cancer, pharyngeal cancer, anal cancer, glioblastoma, rectal cancer, astrocytoma, brain tumors, retinoblastoma, basal cell carcinoma, brain metastases, medulloblastoma, vaginal cancer, pancreatic cancer, testicular cancer, Hodgkin's syndrome, meningioma, Schneeberger's disease. Diseases, pituitary tumors, mycosis fungoides, carcinoid tumors, schwannomas, spinal tumors, Burkitt lymphoma, laryngeal cancer, kidney cancer, thymoma, endometrial cancer, bone cancer, non-Hodgkin lymphoma, urethral cancer, CUP syndrome, head / neck tumors, oligodendroglioma, vulvar cancer, intestinal cancer, colon cancer, esophageal carcinoma, wartinvolvement, small bowel tumors, craniopharyngeoma, ovarian cancer, genital tumors, pancreatic carcinoma, endometrial cancer, liver metastases, penile cancer, tongue cancer, gallbladder cancer, leukemia, plasmacytoma, eyelid tumors, prostate cancer.

[0597] Diseases for which this disclosure can be used as a therapeutic intervention include, among others, 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 adrenospinal neuropathy; Friedrich's ataxia; Pelizaeus-Merzbacher disease; TSC1 and TSC2-associated tuberous sclerosis; Sanfilippo B syndrome (MPS IIIB); CTNS-associated cystinopathy; FMR1-associated disorders, including fragile X syndrome, fragile X-associated tremor / ataxia syndrome, and fragile X premature ovarian failure syndrome; and Prader-Willi syndrome. Niemann-Pick disease type C1; neuronal ceroid lipofuscin-related disorders, including juvenile neuronal ceroid lipofuscin deposition disease (JNCL), juvenile Batten disease, Santavuori-Haltia disease, Jansky-Bielschowsky disease, and PTT-1 and TPP1 deficiencies; childhood ataxia with central nervous system myelination insufficiency / white matter loss associated with EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5; paroxysmal ataxia type 2 associated with CACNA1A and CACNB4; MECP2-related disorders, including classic Rett syndrome, MECP2-related severe neonatal encephalopathy, and PPM-X syndrome; CDKL5-related atypical Rett syndrome; Kennedy's disease. Notch-3 related autosomal dominant cerebral arteriosclerosis with subcortical infarction and leukoencephalopathy (CADASIL); SCN1A and SCN1B related seizure disorders; polymerase G-related disorders, including Alpers-Huttenlocher syndrome, POLG-related sensory ataxia neuropathy, dysarthria and ophthalmoparesis, and autosomal dominant and recessive progressive ophthalmoparesis with mitochondrial DNA deletion; X-linked adrenal dysplasia; X-linked agammaglobulinemia; Fabry disease;And Wilson's disease.

[0598] In one embodiment, the nucleic acids and, for example, mRNA of this disclosure may encode functional proteins or enzymes. For example, compositions of this disclosure may comprise mRNA encoding erythropoietin (EPO), α1-antitrypsin, carboxypeptidase N, α-galactosidase (GLA), ornithine carbamoyltransferase (OTC), or human growth hormone (hGH).

[0599] In other embodiments, this disclosure relates to a method of transfecting at least one isolated target cell with a nucleic acid, wherein the method comprises contacting the 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 as disclosed herein, or (ii) at least one nucleic acid-containing composition as described herein, or (iii) at least one nucleic acid-containing lipid nanoparticle as described herein, such that the at least one target cell is transfected with the nucleic acid.

[0600] Target cells include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, osteocytes, stem cells, mesenchymal cells, nerve cells (e.g., meninges, astrocytes, motor neurons, dorsal root ganglion cells, and anterior horn motor neurons), photoreceptor cells (e.g., rods and cones), retinal pigment epithelial cells, secretory cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, β 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.

[0601] In one embodiment, the target cells may be spleen, liver, lung, heart, and kidney cells. In another embodiment, the target cells may be spleen and kidney cells, and for example, spleen cells.

[0602] In some embodiments, lipid nanoparticles or compositions, as disclosed herein, that allow for avoidance of liver clearance may be of particular interest.

[0603] After at least one target cell is transfected with a nucleic acid encapsulated, for example, in lipid nanoparticles, the production of polypeptides or proteins encoded by such nucleic acids can be stimulated, and the ability of such target cells to express nucleic acids and produce, for example, the target polypeptide or protein can be enhanced. For example, transfecting target cells with a composition encapsulating mRNA will enhance (i.e. increase) the production of proteins or enzymes encoded by such mRNA.

[0604] In other embodiments, this disclosure relates to a method of generating a polypeptide in at least one target cell, wherein the method comprises contacting the at least one target cell with an effective amount of (i) at least one nucleic acid and at least one lipid compound as disclosed herein, or (ii) at least one nucleic acid-containing composition as described herein, or (iii) at least one nucleic acid-containing lipid nanoparticle as described herein, such that the at least one target cell is operatively transfected with a nucleic acid encoding the polypeptide.

[0605] It should be understood that this disclosure includes all variations, combinations, and permutations in which at least one limitation, element, clause description, etc., from at least one of the listed claims is incorporated into another claim deriving from the same basic claim (or any other related claim), unless otherwise stated or unless a contradiction or inconsistency is apparent to a person skilled in the art. Where elements are presented as a list, such as in Markush groups or similar forms, it should be understood that each subgroup of said elements is also disclosed, and one or more of any elements may be removed from said group. It should be understood that, generally, where this disclosure or various aspects thereof are referred to as including a particular element, feature, etc., this disclosure or various aspects thereof also includes embodiments consisting of or substantially consisting of such elements, features, etc. For simplicity, these embodiments are not specifically described herein in so many words in every instance. It should also be understood that any embodiment or aspect of this disclosure may be expressly excluded from the claims, regardless of whether a particular exclusion is recited in this specification. Publications and other references cited herein to describe the background of this disclosure and to provide further details on its practice are hereby incorporated by reference.

[0606] The following examples are provided for illustrative purposes and not for limiting purposes.

[0607] [Example]

[0608] Materials and methods

[0609] Nuclear magnetic resonance spectroscopy (H, C NMR)

[0610] -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).

[0611] Recorded shifts are reported in parts per million (δ) and calibrated using residual undeuterated 3 (H 7.26 ppm; C 77.16 ppm, MeOH H 3.31 ppm; C 49.0 ppm). Data are expressed as follows: chemical shift, multiplicity (s = single, d = double, t = triple, q = quadruple, and m = multiplicity), coupling constant (J, in Hz), integral, and attribution.

[0612] NMR spectra were obtained using the commercial software NMRnotebook.

[0613] High-resolution mass spectrometry (HRMS) was obtained using an Agilent Q-TOF (time-of-flight) 6520, and low-resolution mass spectrometry (LCMS) was obtained using an Agilent MSD 1200SL (ESI / APCI) with an Agilent HPLC 1200SL.

[0614] Example 1: Synthesis of N-((Z)-14-(((E)-octadec-9-en-1-yl)oxy)-3,6,9,12,16-pentaoxane-tetradec-25-en-1-yl)-1H-imidazol-4-carboxamide (compound IV) (also known as DOG-IM4)

[0615]

[0616] Compound IV was prepared according to the following synthetic scheme.

[0617] Option 1

[0618]

[0619] 1.1 Synthesis of DOG-PEG4-NH2 (also known as DOGP4NH2)

[0620] The synthesis scheme is as follows:

[0621] Option 2

[0622]

[0623] Option 3

[0624]

[0625] 1.1.1 Synthesis of triphenylmethane-glycerol (1)

[0626] Glycerol (30.0 g; 325.8 mmol), triphenylmethyl chloride (22.5 g; 80.7 mmol), and DMAP (225 mg; 1.84 mmol) were dissolved in 60 mL of anhydrous THF. After adding triethylamine (13.5 mL; 96.9 mmol), the mixture was vigorously stirred at room temperature for 22 h. Then, 100 mL of ethyl acetate and 70 mL of H₂O were added to the solution. The aqueous phase was extracted with 2 x 70 mL of ethyl acetate. The organic phases were combined, washed successively with 70 mL of 10% (w / v) NaHCO₃ and 70 mL of brine, dried over MgSO₄, and filtered. The obtained product was further purified by silica gel column chromatography (elution gradient CH₂Cl₂ / MeOH) to give compound 1 (15.7 g; 58% yield) as a white solid.

[0627] RMN 1 H (300MHz; 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).

[0628] ES-SM(N2) m / z: 357.1589 ([M+Na]+); Exact mass: 334.1689 g.mol -1

[0629] 1.1.2 Synthesis of 1-methanesulfonyl-oleyl alcohol (2)

[0630] Oleyl alcohol (45.0 g; 167.6 mmol) and triethylamine (38 mL; 272.0 mmol) were dissolved in 600 mL of dichloromethane (CH2Cl2) and the mixture was stirred at 4 °C. Methanesulfonyl chloride (17 mL; 217.0 mmol) was added dropwise, and the reaction mixture was subjected to vigorous stirring under argon at room temperature. After 12 h, 250 mL of H2O was added, and the aqueous phase was extracted with 2 x 250 mL deCH2Cl2. The organic layer was washed successively with 250 mL of 1N 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 obtained product was further purified by silica gel column chromatography (elution gradient: cyclohexane / AcOEt from 10 / 0 to 10 / 1). Compound 2 (44 g; 76% yield) was given as a yellowish, buttery substance.

[0631] RMN 1H (300MHz; CDCl3): δ: 5.39-5.31 (m; 2H9-10), 4.21 (t; J=6.4Hz; 2H1), 2.99 (s; 3Ha), 2.14-1.88 (m ;4H8,11),1.80-1.67(tt;J=6.8Hz;2H2),1.52-1.14(m;22H3-7,12-17),0.88(t;J=6.8Hz;3H18).

[0632] ES-SM(N2) m / z: 385.3969 ([M+K]+); Exact mass: 346.2989 g.mol -1

[0633] 1.1.3 Synthesis of triphenylmethane-dioleylglycerol (3)

[0634] A solution of compound 1 (10.0 g; 29.2 mmol) in 145 mL of anhydrous DMF was added to a suspension of NaH (6.0 g (60% in oil); 149.5 mmol) in 35 mL of anhydrous DMF. The mixture was heated under reflux for 15 min and then cooled to room temperature. Product 2 (25.9 g; 74.8 mmol) in 90 mL of anhydrous DMF was added dropwise to the mixture, and the mixture was then heated under reflux for 15 h. After cooling to room temperature, 120 mL of H2O was added to remove the remaining NaH. The aqueous layer was extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with 2 x 240 mL of 1N 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, buttery substance, was used without further purification (17 g; yield 70%).

[0635] ES-SM(N2) m / z: 857.5507 ([M+Na]+); Exact mass: 834.5607 g·mol⁻¹ (product detected by MS).

[0636] 1.1.4 Synthesis of dioleylglycerol (4)

[0637] Compound 3 (16.0 g; 19.5 mmol) and p-toluenesulfonic acid (pTs-OH·H2O) (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 eliminate excess pTsOH·H2O, and the solvent was evaporated under reduced pressure. The remaining oil was purified by silica gel chromatography (cyclohexane / AcOEt) to give compound 4 (6.5 g; 57% yield) as a colorless oil.

[0638] RMN 1 H: (300MHz; 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=6.6Hz;6H18).

[0639] ES-SM(N2)m / z: 615.5213 ([M+Na]+); Exact mass: 592.5313 g.mol -1

[0640] 1.1.5 Synthesis of methanesulfonyloxy-ethoxy-ethoxy-ethoxy-ethyl-azide (5)

[0641] Tetraethylene glycol dimethanesulfonate (25.0 g; 71.4 mmol) was heated under reflux in 150 mL of CH3CN in the presence of NaN3 (5.8 g; 89.5 mmol). After 19 h, the mixture was cooled to room temperature, and the precipitate was recovered by filtration and purified by silica gel chromatography (cyclohexane / AcOEt (7 / 3 to 3 / 7)) to give compound 5 (8.7 g; 41% yield) as a yellow oil.

[0642] RMN 1 H (200MHz; CDCl3): δ: 4.26-4.22 (m; 2Hd), 3.66-3.61 (m; 2He), 3.60-3.52 (10Hf-j), 3.26 (t; J=5.4Hz; 2Hk), 2.95 (s; 3Hl).

[0643] ES-SM(N2) m / z: 320.0539 ([M+Na]+); Exact mass: 297.0639 g.mol -1

[0644] 1.1.6 Synthesis of dioleylglycerol-ethoxy-ethoxy-ethoxy-ethyl-azide (6)

[0645] Add a solution of compound 4 (4 g; 6.8 mmol) in 50 mL of anhydrous THF containing 13 mL of HMPA to a suspension of NaH (810 mg (60% in oil); 20.2 mmol) in 13 mL of anhydrous THF. Heat the mixture under reflux for 15 min and cool to room temperature. Add a solution of compound 5 (4 g; 13.5 mmol) in 25 mL of anhydrous THF dropwise. Heat the resulting mixture under reflux for 15 h, cool to room temperature, and remove excess NaH by adding 400 mL of H2O. Collect the organic phase and extract the aqueous phase with 3 x 400 mL AcOEt. Combine the organic layers and wash successively with 2 x 400 mL 1N HCl, 2 x 400 mL 5% (w / v) NaHCO3, and 400 mL brine, and dry on MgSO4. The solvent was evaporated under reduced pressure, and the resulting oil was purified on a silica gel column by elution with cyclohexane / AcOEt to produce a yellowish oil (4 g; yield 74%).

[0646] RMN 1 H (300MHz; CDCl3): δ: 5.39-5.33 (m; 4H9-10), 3.70-3.50 (m; 23Ha-j,1), 3.45-3.39 (t; J=5.3Hz; 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=6.8Hz;6H18).

[0647] ES-SM(N2) m / z: 816.6715 ([M+Na]+); Exact mass: 793.6815 g.mol -1

[0648] 1.1.7 Synthesis of 2-[2-[2-[2,3-bis[(~{Z})-octadec-9-enyloxy]propoxy]ethoxy]ethoxy]ethanol (DOG-PEG4-NH2) (7) Example 2:

[0649] Compound 6 (1.8 g; 2.3 mmol) was dissolved in 180 mL THF in the presence of triphenylphosphine (1.8 g; 6.8 mmol) and 400 mL H2O. The mixture was heated under reflux for 15 h and then the solvent was evaporated under reduced pressure. The remaining oil was purified on a silica gel column (elution gradient CH2Cl2 / MeOH / NH4OH 9 / 0.9 / 0.1) to yield compound 7 (1.5 g; 86% yield) as a colorless oil.

[0650] RMN 1H(300MHz; 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, 1Hz; 6H18).

[0651] ES-SM(N2)m / z: 768.6636 ([M]+); Exact mass: 768.6636 g.mol -1

[0652] Synthesis of 1.2N-((Z)-14-(((E)-octadec-9-en-1-yl)oxy)-3,6,9,12,16-pentaoxane-tetradec-25-en-1-yl)-1H-imidazol-4-carboxamide (compound IV; also known as DOG-IM4)

[0653] 4-Imidazole carboxylic 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 hours, the organic phase was evaporated and the remaining yellow solid was dried overnight under vacuum to obtain the corresponding acyl chloride (58 mg, quantitative) without purification.

[0654] DOG-PEG 4- NH₂ (30 mg, 39 μmol) was dissolved in 5 mL of anhydrous DCM and an acyl chloride (5.6 mg, 43 μmol) was added to 1.5 mL of anhydrous DMF and 25 μL of DIPEA. The mixture was stirred overnight at room temperature under a nitrogen atmosphere. The solvent was evaporated and the product was purified by rapid chromatography (4 g column, DCM / MeOH / NH₄OH 9 / 0.9 / 0.1) to give the desired compound (30 mg, 87%).

[0655] 1H-NMR (CDCl3, 400MHz): δ7.69-7.61(m,3H,NH,N=CH-NH,NHCH=C),5.40-5.29(m,4H,2x CH=CH),3.68-3.39(m,25H,12x OCH2,1x OCH, CH2NHC(O)), 2.06-1.90 (m, 8H, 2xCH2CH=CHCH2), 1.59-1.49 (m, 4H, 2x OCH2CH2), 1.39-1.20 (m, 44H, 22x oleyl-CH2), 0.87 (t, J=6.8, 6H, 2x CH3)ppm.

[0656] 13 C-NMR (CDCl3, 75MHz): δ163.04 (NHC=O), 135.52 (N=CH-NH), 130.53, 130.43, 130.07, 129.97 (2x CH=CH, NHCH=C, CH=C), 78.06 (OCH), 71.88-70.15 (12x OCH2), 39.09(CH2NHC(O)), 32.76-26.23(oleyl), 22.83(2x CH3CH2), 14.25(2x CH3)ppm.

[0657] HR-MS (direct injection, positive ionization): m / z = 884.7039 [M+Na] + (Calculated value: 884.71).

[0658] Example 3 Synthesis of N-((Z)-14-(((E)-octadec-9-en-1-yl)oxy)-3,6,9,12,16-pentaoxane-tetradec-25-en-1-yl)-1H-imidazol-2-carboxamide (Compound III)

[0659]

[0660] It was prepared by using 2-imidazolium carboxylic acid instead of 4-imidazolium carboxylic acid, according to the molar amounts considered in Scheme 1 and Example 1. The product was purified by rapid chromatography (4 g column, DCM / MeOH / NH4OH 9 / 0.9 / 0.1) to give the desired compound (45 mg, 65%).

[0661] 1H-NMR (CDCl3, 400MHz): δ7.15(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 OCH2,1 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, 22x oleyl-CH2), 0.87 (t, J=6.8, 6H, 2 x CH3)ppm.

[0662] 13 C-NMR (CDCl3, 75MHz): δ158.95 (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 (12x OCH2), 39.30 (CH2NHC(O)), 32.76-26.23 (oleyl), 22.83 (2 x CH3CH2), 14.25 (2x CH3)ppm.

[0663] HR-MS (direct injection, positive ionization): m / z = 884.7057 [M+Na] + (Calculated value: 884.71)

[0664] Example 4: Synthesis of N-((Z)-14-(((E)-octadec-9-en-1-yl)oxy)-3,6,9,12,16-pentaoxane-tetradec-25-en-1-yl)-1H-pyridyl-3-carboxamide (compound V)

[0665]

[0666] It was prepared according to the molar amounts considered in Scheme 1 and Example 1, and by using 3-pyridyl isothiocyanate instead of 4-imidazolium carboxylic acid.

[0667] 1¹H-NMR (CDCl₃, 400MHz): δ 8.79–8.01 (m, 3H, pyridine), 7.26 (m, 1H, pyridine), 5.40–5.29 (m, 4H, 2x CH=CH), 3.94–3.29 (m, 25H, 12x OCH₂, 1x OCH, CH₂NHC(S)), 2.1–1.90 (m, 8H, 2x CH₂CH=CHCH₂), 1.61–1.45 (m, 4H, 2x OCH₂CH₂), 1.42–1.18 (m, 44H, 22x oleyl-CH₂), 0.87 (t, J=6.8, 6H, 2x CH₃) ppm.

[0668] 1 ¹H-NMR (MeOD, 400MHz): δ 8.62, 8.29, 8.09, 7.39 (m, 4H, pyridine), 5.42–5.31 (m, 4H, 2xCH=CH), 3.88–3.39 (m, 25H, 12x OCH₂, 1x OCH, CH₂NHC(S)), 2.08–1.94 (m, 8H, 2xCH₂CH=CHCH₂), 1.61–1.49 (m, 4H, 2x OCH₂CH₂), 1.40–1.24 (m, 44H, 22x oleyl-CH₂), 0.90 (t, J = 6.8, 6H, 2x CH₃) ppm.

[0669] 13 C-NMR (CDCl3, 75MHz): δ 181.82 (CH2NHC(S)), 145.86, 145.01, 136.18, (3C, pyridine) 131.20-129.84 (1C pyridine, 2x CH=CH), 123.22 (1C, pyridine), 78.05 (OCH), 77.94, 72.72-70.22 (12x OCH2), 44.86 (CH2NHC(S)), 32.73-26.24 (oleyl), 22.80 (2x CH3CH2), 14.23 (2x CH3)ppm.

[0670] HR-MS (direct injection, positive ionization): m / z = 904.7166 [M+H] + (Calculated value: 904.72).

[0671] Example 5 Synthesis of N-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethyl]-1H-imidazol-4-carboxamide (compound IV)

[0672]

[0673] A mixture of 2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethylamine (1.2 g, 1.56 mmol) in DCM (30 mL) was added to a solution of 1H-imidazolium-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 silica gel column chromatography on silica gel by elution with 0%–10% MeOH in DCM to give N-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethyl]-1H-imidazolium-4-carboxamide (0.504 g, 37.4%) as a yellow oil.

[0674] 1 H NMR(500MHz, CDCl3)δ10.99(s,1H),7.73-7.59(m,3H),5.39-5.30(m,4H),3.69-3.40(m,2 5H), 2.09-1.92 (m, 8H), 1.60-1.50 (m, 4H), 1.28 (t, J = 14.5Hz, 44H), 0.88 (t, J = 6.9Hz, 6H).

[0675] Example 6 Synthesis of N-[2-[2-[2-[2-(2,3-bishexadecyloxypropoxy)ethoxy]ethoxy]ethoxy]ethyl]-1H-imidazol-4-carboxamide (compound VI)

[0676]

[0677] 4-Imidazole carboxylic 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 obtain the corresponding acyl chloride (2.5 g, quantitative) without purification. 2-[2-[2-[2-(2,3-bishexadecyloxypropoxy)ethoxy]ethoxy]ethoxy]ethylamine (400 mg, 0.5 mmol) was dissolved in 25 mL of anhydrous DCM, and the acyl chloride (262 mg, 2 mmol) was added to 3 mL of anhydrous DMF and DIPEA (0.325 g, 2.5 mmol). The mixture was stirred overnight at room temperature under a nitrogen atmosphere. The solvent was evaporated and the product was purified by rapid chromatography (12 g column, DCM / MeOH / NH4OH 9 / 0.9 / 0.1) to give N-[2-[2-[2-[2-(2,3-bishexadecyloxypropoxy)ethoxy]ethoxy]ethoxy]ethyl]-1H-imidazol-4-carboxamide (250 mg, 0.293 mmol, 58.3% yield) as a yellow solid.

[0678] 1 H NMR (500MHz, CDCl3) δ7.65 (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 = 6.9Hz, 6H).

[0679] Example 7 Synthesis of N-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propionylamino]ethoxy]ethoxy]ethoxy]ethyl]-1H imidazole-4-carboxamide (compound VII)

[0680]

[0681] The compound is synthesized based on the chemical synthesis shown in scheme (4).

[0682]

[0683] synthesis

[0684] Step (1)

[0685]

[0686] At 0 °C, Dysmart oxidant (841 mg, 1.69 mmol) was added to a solution of 2,3-bis[(Z)-octadec-9-enoxy]propan-1-ol (1 g, 1.69 mmol) in DCM (20 ml) over 5 min. The mixture was then stirred at 25 °C under N2 for 2 h. After the reaction, the mixture was diluted with DCM (30 ml), washed with NaHCO3 / Na2S2O3 (1 / 1) (50 ml x 3) and brine (50 ml), dried over Na2SO4, filtered, and concentrated to obtain 2,3-bis[(Z)-octadec-9-enoxy]propanal (1.2 g, crude) as a yellow oil, which proceeded directly to the next step.

[0687] 1 H NMR (400MHz, CDCl3) δ9.72 (d, J = 1.4Hz, 1H), 5.35 (t, J = 5.4Hz, 4H), 3.84-3.79 (m, 1H), 3.74-3.56 (m, 5H), 3.44 (ddd, J = 12.6, 9.4, 2.7Hz, 3H), 2.03-1.96 (m, 8H), 1.63 (d, J = 7.2Hz, 2H), 1.54 (d, J = 6.9Hz, 2H), 1.26 (d, J = 4.5Hz, 44H), 0.90-0.87 (m, 6H).

[0688] Step (2)

[0689]

[0690] 1.2 g (1.62 mmol) of 2,3-bis[(Z)-octadec-9-enoxy]propanal was dissolved in t-BuOH:H2O (3:1, 20 mL) containing NaH2PO4·2H2O (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]propionic acid (778 mg, 78.9% yield) as a colorless oil.

[0691] 1H NMR (400MHz, CDCl3) δ5.40-5.31(m,4H),4.04(dd,J=5.0,3.3Hz,1H),3.80(dd,J=10.5,3.2Hz,1H),3.70(dd,J=10.5,5.1Hz,1H),3.62(q ,J=6.8Hz,2H),3.51-3.44(m,2H),2.01(dd,J=14.7,8.9Hz,8H),1.65-1.54(m,4H),1.27(dd,J=6.7,2.7Hz,44H),0.88(t,J=6.8Hz,6H).

[0692] Step (3)

[0693]

[0694] To a solution of 2,3-bis[(Z)-octadec-9-enoxy]propionic acid (100 mg, 0.165 mmol) in DCM (2 ml), N-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl]carbamate tert-butyl ester (48 mg, 0.165 mmol), 4-dimethylaminopyridine (2 mg, 0.02 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylureon hexafluorophosphate (94 mg, 0.25 mmol), and triethylamine (33 mg, 0.33 mmol) were added. 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 x 2) and brine (50 ml), dried over Na2SO4, filtered, and concentrated. The residue was purified by rapid chromatography by elution with 2% to 8% methanol in dichloromethane to obtain N-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propionylamino]ethoxy]ethoxy]ethoxy]ethyl]carbamate tert-butyl ester, which is a light yellow oil.

[0695] Step (4)

[0696]

[0697] TFA (0.5 ml) was added to a solution of N-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propionylamino]ethoxy]ethoxy]ethoxy]ethyl]carbamate tert-butyl ester (226 mg, 0.256 mmol) in DCM (2 ml). The mixture was stirred at 25 °C for 3 h. After the reaction, the mixture was concentrated to give N-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propionylamino]ethoxy]ethoxy]ethoxy]ethyl]carbamate tert-butyl ester (300 mg, crude).

[0698] 1 H NMR (400MHz, CDCl3) δ5.41-5.31(m,4H),3.94(s,1H),3.83-3.69(m,7H),3.53(dddd,J=26.7,22.9,11. 7,4.5Hz,15H),2.06-1.91(m,8H),1.54(d,J=7.0Hz,4H),1.26(d,J=4.3Hz,44H),0.88(t,J=6.8Hz,6H).

[0699] Step (5)

[0700]

[0701] Add N,N-diisopropylethylamine (265 mg, 2.05 mmol) and 1H-imidazolium-4-carbonyl chloride (200 mg, 1.54 mmol) in DMF (2 ml) to a solution of N-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl]-2,3-bis[(Z)-octadec-9-enoxy]propionamide (400 mg, 0.512 mmol) in DCM (10 ml). Stir the mixture at 25 °C for 14 h. After the reaction, dilute the mixture with EA (100 ml) and wash with water (100 ml x 2) and brine (100 ml). The organic matter was concentrated and rapidly purified (10% MeOH in DCM) to give N-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propionylamino]ethoxy]ethoxy]ethoxy]ethyl]-1H-imidazol-4-carboxamide (280 mg, yield 61.2%) as a colorless oil.

[0702] 1H NMR (400MHz, CDCl3) δ10.35-10.08(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=5.6,2.8Hz, 1H),3.77(dd,J=10.6,2.6Hz,1H),3.69-3.39(m,21H),2.10-1.93(m,7H),1.63-1.52(m,5H),1.26(d,J=4.6Hz,44H),0.88(t,J=6.8Hz,6H).

[0703] Example 8 Synthesis of 8-[3-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octyloxy)propoxy]nonyl octanoate (compound VIII)

[0704]

[0705] Based on the chemically synthesized compound (VIII) shown in scheme (5).

[0706]

[0707] synthesis

[0708] Step (1)

[0709]

[0710] Under nitrogen atmosphere, 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethanol (50 g, 176 mmol) and triethylamine (35.6 g, 352 mmol) in dry dichloromethane (500 mL) were cooled to -5 °C. Methanesulfonyl chloride (30.2 g, 264 mmol) was added dropwise to this solution in dry DCM (20 mL) 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. Removal of the solvent yielded 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonic acid (69.1 g, 175 mmol, quantified) as a pale yellow oil, which was used without further purification.

[0711] 1H NMR (400MHz, CDCl3) δ7.37-7.27(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).

[0712] Step (2)

[0713]

[0714] NaH (14 g, 351 mmol) was added to a solution of (2,2-dimethyl-1,3-dioxolane-4-yl)methanol (24.4 g, 175 mmol) in THF (500 mL), and the mixture was heated to reflux for 15 min. The reaction was then cooled to room temperature, and 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonic acid (69.1 g, 175 mmol) was added under nitrogen, and the reaction was heated at 80 °C for 24 h. TLC showed that the starting material was 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 rapid chromatography by elution with 20% to 50% ethyl acetate in petroleum ether to give 2,4-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (54.4 g, 70% yield), which was a pale yellow oil.

[0715] 1 H NMR (400MHz, CDCl3) δ7.38-7.27(m,5H),4.57(s,2H),4.28(t,J=5.9Hz,1H),4.05(dd,J=8.3,6.4Hz,1H),3.72(dd,J=8. 3,6.4Hz,1H),3.70-3.61(m,16H),3.57(dd,J=10.0,5.8Hz,1H),3.49(dd,J=10.0,5.5Hz,1H),1.42(s,3H),1.35(s,3H).

[0716] Step (3)

[0717]

[0718] The mixture of 4-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (54.4 g, 123 mmol) in AcOH (200 mL) and H2O (200 mL) was stirred at room temperature for 18 h.

[0719] TLC (EA / PE 1 / 1, SM Rf: 0.5; product, Rf: 0.1) indicated that the starting material was completely consumed. The solvent was removed under vacuum and azeotropically reacted with toluene several times. Ethyl methanesulfonic acid 2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]ethyl ester (49 g, 123 mmol, quantitative) was obtained as a pale yellow oil and used without further purification.

[0720] 1 H NMR (400MHz, CDCl3) δ7.38-7.27(m,5H),4.57(s,2H),3.88-3.81(m,1H),3.70-3.51(m,21H).

[0721] Step (4)

[0722]

[0723] NaH (9.64 g, 241 mmol) was added to a solution of 3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]prop-1,2-diol (24 g, 60.3 mmol) in dry DMF (200 mL) under nitrogen atmosphere, and the mixture was heated at 80 °C for 15 min. The reaction was then cooled to room temperature, and 9-bromonon-1-ene (31.9 g, 151 mmol) was added dropwise to this solution. The mixture was stirred at room temperature for 30 min, and then stirred at 80 °C for 18 h.

[0724] TLC (EA / PE = 1 / 1, Rf: 0.5) showed the formation of new spots. 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 rapid chromatography by elution with 20% to 50% ethyl acetate in petroleum ether to give 2-[2-[2-[2,3-bis(nonyl-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (9.3 g, 14.6 mmol, 24.2% yield) as a pale yellow oil.

[0725] 1H NMR (400MHz, CDCl3) δ7.37-7.27(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=6.7Hz,4H),1.60-1.49(m,4H),1.43-1.23(m,16H).

[0726] Step (5)

[0727]

[0728] To a solution of 2-[2-[2-[2,3-bis(nonyl-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (9.3 g, 14.6 mmol) in MeCN (80 mL), CCl4 (80 mL), and water (80 mL), NaIO4 (24.9 g, 116 mmol) and RuCl3 (656 mg, 2.91 mmol) were added. The reaction mixture was stirred at room temperature for 24 h.

[0729] LCMS showed that the title compound was the major product along with a portion of the monoaldehyde product. The reaction mixture was filtered, and the filtrate was diluted with ethyl acetate (800 mL) and washed with 1N HCl aqueous solution (400 mL). The organic layer was washed with Na2S2O3 solution and 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.

[0730] Step (6)

[0731] 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]-2-(8-oxooctyloxy)propoxy]octanoic acid (10 g, 8 mmol) was dissolved in t-BuOH:H2O (3:1, 160 mL) containing NaH2PO4·2H2O (3.73 g, 24 mmol), 2-methyl-2-butene (40 mL), and sodium chlorite (2.71 mg, 24 mmol). The reaction mixture was stirred at room temperature for 2 h, and LC-MS showed that the starting material was 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]-2-(7-carboxyheptyloxy)propoxy]octanoic acid (10 g, 3.22 mmol, quantified) as a pale yellow oil.

[0732] 1 H NMR (400MHz, CDCl3) δ7.38-7.27(m,5H),4.57(s,2H),3.71-3.61(m,17H),3.5 9-3.37(m,9H),2.33(t,J=7.3Hz,4H),1.69-1.51(m,8H),1.39–1.28(m,14H).

[0733] Step (7)

[0734]

[0735] Under nitrogen atmosphere, 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 to a solution of 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptaoxy)propoxy]octanoic acid (10 g, 14.8 mmol) and 1-nonanol (5.12 g, 35.5 mmol) in dry dichloromethane (200 mL). The mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with dichloromethane and washed with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was purified by rapid chromatography by elution with 20% to 55% ethyl acetate in petroleum ether to give nonyl octanoate (5 g, 35.9%) as a colorless oil.

[0736] 1H NMR (400MHz, CDCl3) δ7.38-7.27(m,5H),4.57(s,2H),4.05(t,J=6.8Hz,4H),3.70-3.61(m,16H),3.5 9-3.39(m,9H),2.28(t,J=7.5Hz,4H),1.67-1.50(m,12H),1.37-1.21(m,36H),0.88(t,J=6.8Hz,6H).

[0737] Step (8)

[0738]

[0739] Pd / C (1.13 g, 20% wt) was added to a solution of nonyl octanoate (5 g, 5.31 mmol) in ethyl acetate (100 mL). The mixture was stirred under hydrogen at room temperature for 18 h.

[0740] TLC (ethyl acetate / petroleum ether 1 / 1) indicates that the starting material was consumed.

[0741] The reaction was filtered through diatomaceous earth and washed with ethyl acetate to give nonyl octanoate 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.

[0742] 1 H NMR (400MHz, CDCl3) δ4.05 (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).

[0743] Step (9)

[0744]

[0745] Sodium azide (0.378 g, 5.81 mmol) was added to a solution of 8-[3-[2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octyloxy)propoxy]nonyl octanoate (4.5 g, 4.8 mmol) in dimethylformamide (DMF, volume: 30 mL). The reaction mixture was then stirred at 70 °C for 16 h. Water (200 mL) was then added and the reaction mixture was extracted with ethyl acetate (100 mL x 2). The combined organic phases were washed with brine (100 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue, which was purified by silica gel column chromatography by elution with petroleum ether:ethyl acetate = 100:1 to 3:1 to give a colorless oily product, 8-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octyloxy)propoxy]nonyl octanoate (4 g, 4.58 mmol, 94% yield).

[0746] 1 H NMR (400MHz, CDCl3) δ4.05 (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).

[0747] Step (10)

[0748]

[0749] A mixture of 8-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octyloxy)propoxy]nonyl 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 h. TLC (5% methanol in dichloromethane) indicated that the reaction was 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 octanoate 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.

[0750] 1H NMR(400MHz, CDCl3) δ4.05(t,J=6.8Hz,4H),3.70-3.39(m,23H),2.91(t,J=5.2Hz,2H),2.4 7(s,2H),2.32-2.25(m,4H),1.66-1.50(m,12H),1.38-1.21(m,36H),0.88(t,J=6.9Hz,6H).

[0751] Step (11)

[0752]

[0753] 4-Imidazole carboxylic 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 obtain the corresponding acyl chloride (2.5 g, quantitative) without purification.

[0754] Nonyl octanoate (680 mg, 0.8 mmol) was dissolved in 30 mL of anhydrous DCM and an acyl chloride (419 mg, 3.2 mmol) was added to 3 mL of anhydrous DMF and DIPEA (0.519 g, 4 mmol). The mixture was stirred overnight at room temperature. The solvent was evaporated and the product was subjected to rapid chromatography (40 g column, DCM / MeOH 20 / 1 to 10 / 1), followed by preparative TLC (eluting with 10% methanol in dichloromethane) to give nonyl octanoate 8-[3-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octoxy)propoxy]octanoate (302.3 mg, 0.326 mmol, 40.6% yield).

[0755] MS(ESI)m / z=898.7(M+H)+

[0756] 1 H NMR (400MHz, CDCl3) δ10.95 (s, 1H), 7.72-7.60 (m, 3H), 4.05 (t, J = 6.7Hz, 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 = 6.8Hz, 6H).

[0757] Example 9 Synthesis of 1-octylnonyl octanoate (compound IX) of 8-[3-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octyloxy]propoxy]octanoate

[0758]

[0759] Based on the chemically synthesized compound (IX) shown in scheme (6).

[0760]

[0761]

[0762] synthesis

[0763] Step (1)

[0764]

[0765] 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) were cooled to 0 °C under nitrogen atmosphere.

[0766] Methanesulfonyl chloride (30.6 g, 0.264 mol) was added dropwise to this solution at 0 °C. The mixture was allowed to be warmed 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 methanesulfonic acid (62 g, 92%) as a pale yellow oil, which was used without further purification.

[0767] LCMS MS 363(M+1)

[0768] Step (2)

[0769]

[0770] NaH (6.17 g, 0.257 mol) was added to a solution of (2,2-dimethyl-1,3-dioxolane-4-yl)methanol (62 g, 0.171 mol) in THF (600 mL), and the mixture was heated to reflux for 15 min. The reaction was then cooled to room temperature, and 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonic acid (25.0 g, 0.171 mol) was added under nitrogen, and the reaction was heated at 80 °C for 18 h. TLC showed that the starting material was 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 rapid chromatography by elution with 20% to 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), a pale yellow oil.

[0771] LCMS MS 421(M+23)

[0772] Step (3)

[0773]

[0774] 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 h. The solvent was removed to obtain 3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]prop-1,2-diol (36 g, 95%), which was used without further purification.

[0775] LCMS MS 381(M+23)

[0776] Step (4)

[0777]

[0778] NaH (8.03 g, 0.201 mol) was added to a solution of 20 g (20 g, 0.050 mol) of 3-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]prop-1,2-diol in 200 mL of THF, and the mixture was heated to reflux for 15 min. The reaction was then cooled to room temperature, and 26.6 g (0.126 mol) of 9-bromonon-1-ene was added under nitrogen atmosphere, and the reaction was heated at 80 °C for 18 h. TLC showed that the starting material was 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 rapid chromatography by elution with 10% to 30% ethyl acetate in petroleum ether to give 2-[2-[2-[2-[2,3-bis(non-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (8.8 g, 26% yield), which was a pale yellow oil.

[0779] 1 H NMR (400MHz, CDCl3) δ7.37-7.27(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=6.5Hz,4H),1.60-1.49(m,4H),1.41-1.28(m,16H).

[0780] Step (5)

[0781]

[0782] To a solution of 2-[2-[2-[2,3-bis(nonyl-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (8.5 g, 0.0140 mol) in MeCN (80 mL), CCl4 (80 mL), and water (80 mL), NaIO4 (24.9 g, 0.116 mol) and RuCl3 (0.66 g, 2.93 mmol) were added. The reaction mixture was stirred at room temperature for 24 h. LC-MS showed that the title compound was the major product. The reaction mixture was filtered, and the filtrate was diluted with ethyl acetate (600 mL) and washed with 1 N HCl aqueous solution (200 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated to obtain 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptaoxy)propoxy]octanoic acid (8.7 g, 97% yield), which was used without further purification.

[0783] 1 H NMR (400MHz, CDCl3) δ7.31 (dd, J=22.6, 3.2Hz, 5H), 4.57 (s, 2H), 3.71-3.61 (m, 19 H), 3.59-3.38 (m, 11H), 2.32 (t, J = 7.4Hz, 4H), 1.68-1.47 (m, 10H), 1.32 (s, 14H).

[0784] Step (6)

[0785]

[0786] 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%), heptadecano-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 h. DCM (500 mL) was added to the mixture, and the mixture was washed with 1 N HCl and brine and concentrated. The residue was purified by rapid column chromatography on silica gel by elution with 1:1 ethyl acetate / petroleum ether to give 15 g, 27% colorless oily 1-octylnonyl octanoate (2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octyloxy]propoxy]octanoate.

[0787] 1 H NMR (400MHz, CDCl3) δ7.37-7.27(m,5H),4.90-4.82(m,2H),3.70-3.60(m,16H),3.58-3.37(m,9H),2 .27(t,J=7.5Hz,4H), 1.53(dd,J=22.4,6.0Hz,12H), 1.28(d,J=22.7Hz,60H), 0.88(t,J=6.8Hz,12H).

[0788] Step (7)

[0789]

[0790] Pd / C (2.85 g, 20% wt / wt) was added to a solution of 1-octylnonyl octanoate (15 g, 13.4 mmol) in ethyl acetate (150 mL). The mixture was stirred at room temperature under hydrogen for 18 h. TLC (ethyl acetate / petroleum ether 1 / 1) showed that the starting material was consumed. The reaction was filtered through diatomaceous earth and washed with ethyl acetate to give 11.1 g, 80.5% octylnonyl octanoate (11.1 g, 80.5%) as a colorless oil.

[0791] 1 H NMR (400MHz, CDCl3) δ4.91-4.82(m,2H),3.75-3.39(m,24H),2.27(t,J=7.2Hz,4H),1.66-1.43(m,17H),1.38-1.17(m,61H),0.88(t,J=6.8Hz,12H).

[0792] Step (8)

[0793]

[0794]

[0795] Methylsulfonyl chloride was added to a mixture of 1-octylnonyl octanoate (1.4 g, 1.36 mmol) of 8-[3-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-2-[8-(1-octylnonyloxy)-8-oxo-octyloxy]propoxy]octanoate (1.4 g, 1.36 mmol) and N,N-diethylethylamine (0.275 g, 2.72 mmol) in DCM (20 mL) at 0 °C. The mixture was stirred at room temperature for 3 h. DCM (100 mL) was added to the mixture and the mixture was washed with water and brine and concentrated to give 1-octylnonyl octanoate (1.4 g, 93%) of 8-[3-[2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]-2-[8-(1-octylnonyloxy)-8-oxo-octyloxy]propoxy]octanoate (1.4 g, 93%) as a colorless oil.

[0796] 1H NMR (400MHz, CDCl3) δ7.37-7.27(m,5H),4.90-4.82(m,2H),3.70-3.60(m,16H),3.58-3.37(m,9H),2 .27(t,J=7.5Hz,4H), 1.53(dd,J=22.4,6.0Hz,12H), 1.28(d,J=22.7Hz,60H), 0.88(t,J=6.8Hz,12H).

[0797] Step (9)

[0798]

[0799] A mixture of 1-octylnonyl octanoate (1.4 g, 1.26 mmol) of 8-[3-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octyloxy]propoxy]octanoate (1.3 g, 97.5%) was prepared at room temperature in DMF (10 mL). The mixture was stirred at 70 °C for 16 h. Water (50 mL) was added to the mixture and it was extracted with EtOAc (50 mL x 3). The organic layer was washed with brine, dried over Na2SO4 and concentrated to give 1-octylnonyl octanoate (1.3 g, 97.5%) of 8-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octyloxy]propoxy]octanoate (1.3 g, 97.5%) as a colorless oil.

[0800] 1 H NMR(400MHz, CDCl3) δ4.86(p,J=6.3Hz,2H),3.72-3.58(m,15H),3.60-3.35(m,11H),2.27(t,J =7.5Hz, 4H), 1.56 (ddd, J = 22.2, 14.3, 6.1Hz, 18H), 1.38-1.19 (m, 64H), 0.88 (t, J = 6.8Hz, 12H).

[0801] Step (10)

[0802]

[0803] A mixture of 1-octylnonyl octanoate (1.3 g, 1.23 mmol) of 8-[3-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octyloxy]propoxy]octanoate and triphenylphosphine in THF (20 mL) and water (3 mL). The mixture was stirred at room temperature for 16 h. The mixture was concentrated to give 1-octylnonyl octanoate (1.1 g, 87%) of 8-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octyloxy]propoxy]octanoate as a colorless oil.

[0804] 1 H NMR (400MHz, CDCl3) δ4.86 (p, J = 6.2Hz, 2H), 3.71-3.39 (m, 23H), 2.91 (t, J = 5.1Hz, 2H), 2.27 (t, J=7.4Hz, 7H), 1.54 (dd, J=32.2, 15.1Hz, 16H), 1.28 (d, J=23.3Hz, 60H), 0.88 (t, J=6.8Hz, 12H).

[0805] Step (11)

[0806]

[0807] Add 1H-imidazolium-4-carbonyl chloride (0.545 g, 4.17 mmol) to a mixture of 1-octylnonyl octanoate (1.1 g, 1.04 mmol) of 8-[3-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octyloxy]propoxy]octanoate (1.1 g, 1.04 mmol) and N-ethyl-N-isopropyl-prop-2-amine (1.35 g, 10.4 mmol) in DCM (40 mL). Stir the mixture at room temperature for 16 h. The mixture was concentrated and the residue was purified by silica gel column chromatography on silica gel by elution with 2%–15% MeOH in DCM to give 1-octylnonyl octanoate (0.22 g, 18.8%) as a yellow oil.

[0808] 1H NMR (400MHz, CDCl3) δ10.78(s,1H),7.63(s,3H),4.86(s,2H),3.70-3.36(m,25H),2.28(dd,J= 10.6, 4.4Hz, 4H), 1.94 (s, 3H), 1.66-1.43 (m, 17H), 1.36-1.16 (m, 62H), 0.88 (t, J = 6.8Hz, 12H).

[0809] Example 10 Synthesis of 2,3-bis[(Z)-octadec-9-enoxy]propionic acid 2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethyl ester (compound X)

[0810]

[0811] Synthesis of compound (X)

[0812] To a solution of 2,3-bis[(Z)-octadec-9-enoxy]propionic acid 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl ester (282 mg, 0.36 mmol) in DCM (10 mL), DIEA (233 mg, 1.8 mmol) and 1H-imidazolium-4-carbonyl chloride (188 mg, 1.44 mmol) were added. The mixture was stirred at 25 °C for 14 h. After the reaction, the mixture was treated with EA (100 mL) and washed with water (100 mL x 2) and saturated aqueous solution of NaCl (100 mL). The organic matter was concentrated and rapidly purified (10% MeOH in DCM) to give 2,3-bis[(Z)-octadec-9-enoxy]propionic acid 2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethyl ester (192 mg, yield 59.6%) as a colorless oil.

[0813] 1 H NMR (400MHz, CDCl3) δ9.77-9.65(m,1H),7.66(s,1H),7.61(s,1H),7.51-7.44(m,1H),5.34(t,J=5.4Hz,4H),4.27(d,J=4.5Hz,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=6.8Hz,6H).

[0814] Example 11Synthesis of 2,3-bis[(Z)-octadec-9-enoxy]propyl acetate (compound XI)

[0815]

[0816] Synthesis of compound (XI)

[0817] To a solution of 2,3-bis[(Z)-octadec-9-enoxy]propyl 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]acetic acid (623 mg, 0.79 mmol) in DCM (15 ml), DIEA (515 mg, 3.98 mmol) and 1H-imidazolium-4-carbonyl chloride (416 mg, 3.19 mmol) in DMF (5 ml) were added. The mixture was stirred at 25 °C for 14 h. The mixture was concentrated and treated with EA (50 ml), washed with water (50 ml x 2), saturated aqueous solution of NaCl (50 ml), and dried over Na2SO4. The organic matter was concentrated and rapidly purified (5% MeOH in DCM) to give 2,3-bis[(Z)-octadec-9-enoxy]propyl acetic acid (380 mg, yield 53.4 mmol) as a colorless oil.

[0818] 1 H NMR (400MHz, CDCl3) δ9.97(s,1H),7.64(d,J=15.8Hz,3H),5.34(t,J=5.4Hz,4H),4.32(dd,J=11.5,4.0Hz,1H),4.20-4.11(m,3H),3.72-3.61(m ,13H),3.55(t,J=6.7Hz,2H),3.46(dt,J=13.0,5.5Hz,4H),2.13-1.86( m, 8H), 1.54 (d, J = 6.2Hz, 4H), 1.33-1.24 (m, 44H), 0.88 (t, J = 6.8Hz, 6H).

[0819] Example 12 Synthesis of 2,3-bis[(~{Z})-octadec-9-enoxy]propyl~{N}-[2-[2-[2-(1~{H}-imidazol-4-carbonylamino)ethoxy]ethoxy]ethyl]carbamate (compound XII)

[0820]

[0821] Synthesis of compound (XII)

[0822] Step (1)

[0823]

[0824] To a solution of 2,3-bis[(Z)-octadec-9-enoxy]prop-1-ol (1 g, 1.65 mmol) in DMF (10 mL), bis(2,5-dioxopyrrolidone-1-yl) carbonate (1.34 g, 4.96 mmol) and 4-dimethylaminopyridine (202 mg, 1.65 mmol) were added. The mixture was stirred at 25 °C. The mixture was treated with EA (50 mL), washed with water (50 mL x 2), NaCl aqueous solution (50 mL), and dried over Na2SO4. The organic matter was concentrated and rapidly purified (20% EA in PE) to give 2,3-bis[(Z)-octadec-9-enoxy]propyl(2,5-dioxopyrrolidone-1-yl) carbonate (937 mg, 75.7% yield) as a colorless oil.

[0825] 1 H NMR (400MHz, CDCl3) δ5.48-5.30(m,4H),4.46(dd,J=11.1,3.9Hz,1H),4.38-4.31(m,1H),3.74-3.66(m,1H),3.60-3.3 9(m,6H),2.83(s,4H),2.01(dd,J=14.8,9.2Hz,8H),1.54(d,J=6.6Hz,4H),1.33-1.22(m,44H),0.88(t,J=6.8Hz,6H).

[0826] Step (2)

[0827]

[0828] To a solution of 2,3-bis[(Z)-octadec-9-enoxy]propyl(2,5-dioxopyrrolidone-1-yl) carbonate (937 mg, 1.28 mmol) in DCM (10 mL), N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]carbamate tert-butyl ester (327 mg, 1.28 mmol), TEA (194 mg, 1.91 mmol), and 4-dimethylaminopyridine (15 mg, 0.128 mmol) were added. 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 x 2), NaCl aqueous solution (50 mL), and dried over Na2SO4. The organic matter was concentrated and rapidly purified (0-50% EA in PE) to give N-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxycarbonylamino]ethoxy]ethoxy]ethyl]carbamate tert-butyl ester (802 mg, yield 71%) as a colorless oil.

[0829] 1 H NMR (400MHz, CDCl3) δ5.36 (dt, J=9.9, 5.0Hz, 4H), 5.27 (s, 1H), 5.06 (s, 1H) ,4.20(dd,J=11.4,3.8Hz,1H),4.11(dd,J=11.5,5.3Hz,1H),3.62-3.53(m,1 1H),3.48(d,J=5.4Hz,2H),3.45-3.32(m,6H),2.02(dt,J=12.3,6.3Hz,8H) ,1.57-1.51(m,4H),1.45(s,9H),1.34-1.25(m,44H),0.88(t,J=6.7Hz,6H).

[0830] Step (3)

[0831]

[0832] TFA (1.3 ml) was added to a solution of N-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxycarbonylamino]ethoxy]ethoxy]ethyl]carbamate tert-butyl ester (802 mg, 0.925 mmol) in DCM (10 ml). The mixture was stirred at 25 °C for 3 h. The mixture was concentrated under vacuum to give N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]carbamate 2,3-bis[(Z)-octadec-9-enoxy]propyl ester (1.19 g, crude product) as a yellow oil.

[0833] 1H NMR (400MHz, CDCl3) δ7.52 (s, 2H), 5.39-5.31 (m, 4H), 4.26 (d, J = 10.5Hz, 1H ),4.14(dd,J=11.7,4.5Hz,1H),3.76(t,J=4.8Hz,2H),3.70-3.51(m,12H),3 .46(t,J=6.8Hz,2H),3.39(d,J=4.7Hz,2H),3.25(s,2H),2.01(dd,J=12.5,6 .6Hz, 8H), 1.56 (d, J = 8.9Hz, 4H), 1.31-1.23 (m, 44H), 0.88 (t, J = 6.8Hz, 6H).

[0834] Step (4)

[0835]

[0836] To a solution of N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]carbamate 2,3-bis[(Z)-octadec-9-enoxy]propyl ester (709 mg, 0.924 mmol) in DCM (15 ml), DIEA (717 mg, 5.54 mmol) and 1H-imidazolium-4-carbonyl chloride (483 mg, 3.7 mmol) were added. The mixture was stirred at 25 °C for 14 h. The mixture was concentrated and treated with EA (50 ml), washed with water (50 ml x 2), saturated aqueous solution of NaCl (50 ml), and dried over Na2SO4. The organic matter was concentrated and rapidly purified (5%-10% MeOH in DCM) to give N-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethyl]carbamate 2,3-bis[(Z)-octadec-9-enoxy]propyl ester (462 mg, yield 56.9%) as a colorless oil.

[0837] 1 H NMR (400MHz, CDCl3) δ9.85-9.75 (m, 1H), 7.66 (d, J = 10.2Hz, 2H), 7.52 (s, 1H), 5 .75(s,1H),5.34(t,J=4.8Hz,4H),4.19(s,1H),4.12-4.07(m,1H),3.71-3.59(m ,9H),3.56(t,J=5.1Hz,4H),3.50-3.42(m,4H),3.37(d,J=5.2Hz,2H),2.10-1. 87 (m, 8H), 1.54 (d, J = 6.4Hz, 4H), 1.26 (d, J = 4.7Hz, 44H), 0.88 (t, J = 6.8Hz, 6H).

[0838] Figure 7 Synthesis of [(Z)-non-2-enyl]8-[3-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-non-2-enyloxy]-8-oxo-octyloxy]propoxy]octyl ester (compound XIII)

[0839] O

[0840]

[0841] Based on the chemical synthesis of the compound shown in scheme (7), such as Example 13 As shown.

[0842] Synthesis of compound (XIII)

[0843] Step (1)

[0844]

[0845] 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) was prepared. The mixture was cooled to 0 °C, and then N,N-diethylethylamine (41.7 g, 0.412 mol) was added. The reaction mixture was stirred at ambient temperature for 16 h. LC-MS showed a good reaction. The mixture was poured into water (600 mL) and extracted with DCM (2 x 400 mL). The organic layer was washed with water and NaCl, dried over Na2SO4, and concentrated. The residue was purified by rapid column chromatography on silica gel by elution with ethyl acetate / petroleum ether in a 3:1 ratio to give 2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethanol (33.0 g, 36.7%) as a colorless oil.

[0846] LCMS 459(M+23), 99% UV 214nm

[0847] 1 H NMR (400MHz, CDCl3) δ7.49-7.44(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).

[0848] Step (2)

[0849]

[0850] Methanesulfonyl chloride (10.4 g, 0.0907 mol) was slowly added to a mixture of 2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethanol (33 g, 0.0756 mol) and N,N-diethylethylamine (15.3 g, 0.151 mol) in DCM (600 mL) at 0 °C. The mixture was stirred overnight at room temperature. CH2Cl2 (400 mL) was added to the solution, and the mixture was diluted 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 Na2SO4. The solvent was removed to give 2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethyl methanesulfonic acid (38.8 g, 99.8%) as a yellow oil.

[0851] LCMS 537.2(M+23)98% UV(214nm)

[0852] 1 H NMR (400MHz, CDCl3) δ7.48-7.44(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).

[0853] Step (3)

[0854]

[0855] 3-Triphenylmethoxypropyl-1,2-diol (30 g) was added to a suspension of NaH (17.9 g) in 300 mL of anhydrous DMF. The mixture was heated at 80 °C for 15 min and then cooled to room temperature. 9-Bromonon-1-ene (46 g) in 10 mL of anhydrous DMF was added dropwise to the mixture, and the mixture was then heated at 80 °C for 18 h. After cooling to room temperature, 500 mL of H2O was added to destroy the remaining NaH. The organic phase was extracted with 750 mL of ethyl acetate. The extract was washed successively 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 by elution on a silica gel column with petroleum ether / ethyl acetate (6% to 25% ethyl acetate in petroleum ether) to produce a colorless oil (15.1 g, 28.9%).

[0856] 1 H NMR (400MHz, CDCl3) δ7.60-7.05(m,17H),5.95-5.66(m,2H),4.97(ddd,J=21.1,11.4,5.9Hz ,4H),3.69-3.31(m,7H),3.21-3.06(m,2H),2.02(dt,J=7.9,3.7Hz,4H),1.50-1.25(m,18H).

[0857] Step (4)

[0858]

[0859] p-Toluenesulfonic acid (47.6 g, 250 mmol) was added in a single addition to a solution of [2,3-bis(non-8-enoxy)propoxy-diphenyl-methyl]benzene (30.7 g, 50 mmol) in methanol / THF (600 mL, 1 / 1 v / v) 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 rapid chromatography by elution with 20% to 30% (21%) ethyl acetate in petroleum ether to give 2,3-bis(non-8-enoxy)prop-1-ol (12.33 g, 36.2 mmol, 72.4% yield) as a colorless oil.

[0860] 1 H NMR (400MHz, CDCl3) δ5.89-5.73(m,2H),5.03-5.00(m,1H),4.99-4.96(m,1H),4.94(d,J=0.9Hz,1H),4.92(d,J=0.9Hz, 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).

[0861] Step (5)

[0862]

[0863] A mixture of 2,3-bis(nonyl-8-enoxy)prop-1-ol (12.33 g, 36.2 mmol) was added to a solution of NaH (60% mineral oil dispersion, 2.77 g, 72.4 mmol) in 200 mL of dry THF, and the mixture was stirred at 80 °C for 15 min. After the solvent returned to room temperature, ethyl 2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]methyl ester (22.4 g, 43.4 mmol) dissolved in 60 mL of dry THF was added. The reaction mixture was stirred under 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 rapid column chromatography on silica gel by elution with ethyl acetate (0-15%) (14%) in petroleum ether to give the target product as a pale yellow oil (23.17 g, 30.5 mmol, 84.3% yield).

[0864] 1 H NMR (400MHz, CDCl3) δ7.48-7.44(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=2.0,0.9Hz, 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).

[0865] Step (6)

[0866]

[0867] To a solution of [2-[2-[2-[2,3-bis(nonyl-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy-diphenyl-methyl]benzene (23.17 g, 30.5 mmol) in MeCN (200 mL), CCl4 (200 mL), and water (200 mL), NaIO4 (52.2 g, 244 mmol) and RuCl3 (1.27 g, 6.1 mmol) were added. The reaction mixture was stirred at room temperature for 24 h. The reaction mixture was filtered, and the filtrate was diluted with ethyl acetate (800 mL) and washed with 1 N HCl aqueous solution (900 mL). The organic layer was washed with Na2S2O3 solution (700 mL * 2) and then dried over sodium sulfate. It was filtered and concentrated to give 8-[2-(7-carboxyheptaoxy)-3-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)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.

[0868] 1 H NMR (400MHz, CDCl3) δ9.75 (s, 1H), 7.46 (d, J = 7.2Hz, 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).

[0869] Step (7)

[0870] 8-[2-(8-oxooctyloxy)-3-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]propoxy]octanoic acid (12.5 g, 4.81 mmol) was dissolved in t-BuOH:H2O (3:1, 400 mL) containing NaH2PO4 (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 LC-MS showed that the starting material was consumed. The reaction mixture was diluted with H2O. The aqueous layer was extracted with ethyl acetate (800 mL * 2). The residue was purified by rapid column chromatography on silica gel by elution with CH3OH (0-6%) (3%) in DCM to obtain the target product as a light yellow oil (4.663 g (EXP-20-IQ8160-P2: 0.576 g + EXP-20-IQ8160-2: 4.082 g), 19.2% yield (total yield of two-step oxidation)).

[0871] LCMS: Peak found: MS(ESI)m / z=818.5(M+Na)+, at 2.300 min.

[0872] Multipeak Report

[0873] 1 H NMR (400MHz, CDCl3) δ7.49-7.43(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).

[0874] Step (8)

[0875]

[0876] Then, to a solution of 8-[2-(7-carboxyheptaoxy)-3-[2-[2-[2-(2-triphenylmethoxyethoxy)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), DIPEA (3.99 g, 30.8 mmol), DMAP (0.251 mg, 2.06 mmol), and EDCI (2.56 g, 13.4 mmol) were added, and the mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with dichloromethane and washed with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was purified by rapid chromatography by elution with 0% to 40% (25%) ethyl acetate in petroleum ether to give [(Z)-non-2-enyl]8-[2-[8-[(Z)-non-2-enoxy]-8-oxo-octyloxy]-3-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]propoxy]octyl ester (1.646 g, 1.58 mmol, 30.7% yield).

[0877] 1H NMR (400MHz, CDCl3) δ7.50-7.44(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=6.8Hz,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).

[0878] Step (9)

[0879]

[0880] p-Toluenesulfonic acid (1.71 mg, 9.01 mmol) was added in a single batch to a solution of [(Z)-non-2-enyl]8-[2-[8-[(Z)-non-2-enoxy]-8-oxo-octyloxy]-3-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]propoxy]octyl ester (1.881 g, 1.8 mmol) in methanol / THF (80 mL, 1 / 1 v / v) 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 under vacuum. The residue was purified by rapid chromatography by elution with 0% to 10% (6%) CH3OH in DCM to give 2,3-bis(non-8-enoxy)prop-1-ol (1.32 g, 1.65 mmol, 91.4%) as a colorless oil.

[0881] Multipeak Report

[0882] 1 H NMR (400MHz, CDCl3) δ5.69-5.58(m,2H),5.57-5.47(m,2H),4.62(d,J=6.7Hz,4H),3.75-3.71(m,2H),3.68-3.60(m,14H),3.5 8-3.40(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).

[0883] Step (10)

[0884]

[0885] Ms-Cl (0.283 g, 2.47 mmol) was added to a solution of [(Z)-non-2-enyl]8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-non-2-enoxy]-8-oxo-octyloxy]propoxy]octyl ester (1.32 g, 1.65 mmol) and TEA (triethylamine) (0.333 g, 3.3 mmol) in 30 mL of dichloromethane (DCM). The mixture was stirred at room temperature for 3 h. TLC (CH3OH / DCM 3%) showed that the starting material was consumed. The reaction mixture was diluted with dichloromethane and washed with water (10 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated to obtain nonyl to give [(Z)-non-2-enyl]8-[3-[2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-non-2-enyloxy]-8-oxo-octyloxy]propoxy]octyl ester (1.384 g, 1.57 mmol, 95.5% yield), which was used directly in the next step.

[0886] 1 H NMR (400MHz, CDCl3) δ5.70-5.59(m,2H),5.56-5.48(m,2H),4.62(d,J=6.7Hz,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).

[0887] Step (11)

[0888]

[0889] Then, Na3N (0.123 g, 1.89 mmol) was added to a solution of undecylundecyl[(Z)-non-2-enyl]8-[3-[2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-non-2-enoxy]-8-oxo-octyloxy]propoxy]octyl ester (1.38 g, 1.57 mmol) dissolved in DMF (25 mL). The reaction mixture was then stirred at 70 °C for 18 h.

[0890] TCL showed the disappearance of the starting material and new spots were observed. Water (100 mL) was then added and the reaction mixture was extracted with ethyl acetate (100 mL x 2). The combined organic phases were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue, which was purified by silica gel column chromatography by elution with CH3OH (6%) in DCM (0-10%) (0.995 g, 1.2 mmol, 76.5% yield) as a colorless liquid.

[0891] 1 H NMR (400MHz, CDCl3) δ5.71-5.59(m,2H),5.58-5.46(m,2H),4.62(d,J=6.8Hz,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).

[0892] Step (12)

[0893]

[0894] A mixture of undecyl[(Z)-non-2-enyl]8-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-non-2-enyloxy]-8-oxo-octyloxy]propoxy]octyl ester (0.995 g, 1.2 mmol) and triphenylphosphine (0.474 g, 1.81 mmol) in THF (20 mL) / water (0.6 mL) was stirred at 20 °C for 16 h. TLC (ninhydrin, 3% methanol in dichloromethane) indicated that the reaction was complete. The solvent was removed and added to DCM, then concentrated under reduced pressure to obtain the residue, which was purified by silica gel column chromatography by elution with CH3OH (0-20% (14%)) in CH2Cl2 to give [(Z)-non-2-enyl]8-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-non-2-enyloxy]-8-oxo-octyloxy]propoxy]octyl ester (0.689 g, 0.861 mmol, 71.5% yield).

[0895] 1H NMR (400MHz, CDCl3) δ5.70-5.60(m,2H),5.57-5.48(m,2H),4.62(d,J=6.7Hz,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).

[0896] Step (13)

[0897]

[0898]

[0899] [(Z)-Non-2-enyl]8-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-non-2-enyloxy]-8-oxo-octyloxy]propoxy]octyl ester (190 mg, 0.237 mmol) was dissolved in 10 mL of anhydrous DCM and 1H-imidazolium-4-carbonyl chloride (124 mg, 0.95 mmol) was added to 1 mL of anhydrous DMF and DIPEA (153 mg, 1.19 mmol). The mixture was stirred overnight at room temperature. The solvent was evaporated and the product was purified by rapid chromatography (40 g column, DCM / MeOH 0% to 15%) by elution with methanol (4%) in dichloromethane to give [(Z)-non-2-enyl]8-[3-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-non-2-enyloxy]-8-oxo-octyloxy]propoxy]octyl ester (112 mg, 0.119 mmol, 50.1% yield).

[0900] 1 H NMR (400MHz, CDCl3) δ7.71-7.56(m,3H),5.70-5.59(m,2H),5.57-5.47(m,2H),4.62(d,J=6.8Hz,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).

[0901] LCMS:MS(ESI)m / z=895.7(M+H) +

[0902] Figure 8 Synthesis of 2-Butyloctyl octanoate (compound XIV): 8-[2-[8-(2-Butyloctyloxy)-8-oxo-octyloxy]-3-[2-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propoxy]

[0903] Based on the chemically synthesized compound (XIV) shown in scheme (8), such as Example 14 As shown.

[0904] Synthesis of compound (XIV)

[0905] Step (1)

[0906]

[0907] Under nitrogen atmosphere, 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) 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. Removal of the solvent yielded 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonic acid (62 g, 92%) as a pale yellow oil, which was used without further purification.

[0908] LCMS MS 363(M+H)

[0909] Step (2)

[0910]

[0911] NaH (6.17 g, 0.257 mol) was added to a solution of (2,2-dimethyl-1,3-dioxolane-4-yl)methanol (62 g, 0.171 mol) in THF (600 mL), and the mixture was heated to reflux for 15 min. The reaction was then cooled to room temperature, and 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonic acid (25.0 g, 0.171 mol) was added under nitrogen, and the reaction was heated at 80 °C for 18 h. TLC showed that the starting material was 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 rapid chromatography by elution with 20% to 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), a pale yellow oil.

[0912] LCMS MS 421(M+Na)

[0913] Step (3)

[0914]

[0915] 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) was prepared. The mixture was stirred at ambient temperature for 16 h. The solvent was removed to give 3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]prop-1,2-diol (36 g, 95% yield), which was used without further purification.

[0916] LCMS MS 381(M+Na)

[0917] Step (4)

[0918]

[0919] NaH (8.03 g, 0.201 mol) was added to a solution of 20 g (20 g, 0.050 mol) of 3-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]prop-1,2-diol in 200 mL of THF, and the mixture was heated to reflux for 15 min. The reaction was then cooled to room temperature, and 26.6 g (0.126 mol) of 9-bromonon-1-ene was added under nitrogen atmosphere, and the reaction was heated at 80 °C for 18 h. TLC showed that the starting material was 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 rapid chromatography by elution with 10% to 30% ethyl acetate in petroleum ether to give 2-[2-[2-[2-[2,3-bis(non-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (8.8 g, 26% yield), which was a pale yellow oil.

[0920] 1 H NMR (400MHz, CDCl3) δ7.37-7.27(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=6.5Hz,4H),1.60-1.49(m,4H),1.41-1.28(m,16H).

[0921] Step (5)

[0922]

[0923]

[0924] To a solution of 2-[2-[2-[2,3-bis(nonyl-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (8.5 g, 0.0140 mol) in MeCN (80 mL), CCl4 (80 mL), and water (80 mL), NaIO4 (24.9 g, 0.116 mol) and RuCl3 (0.66 g, 2.93 mmol) were added. The reaction mixture was stirred at room temperature for 24 h. LC-MS showed that the title compound was the major product. The reaction mixture was filtered, and the filtrate was diluted with ethyl acetate (600 mL) and washed with 1 N HCl aqueous solution (200 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated to obtain 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptaoxy)propoxy]octanoic acid (8.7 g, 97% yield), which was used without further purification.

[0925] 1 H NMR (400MHz, CDCl3) δ7.31 (dd, J=22.6, 3.2Hz, 5H), 4.57 (s, 2H), 3.71-3.61 (m, 19 H), 3.59-3.38 (m, 11H), 2.32 (t, J = 7.4Hz, 4H), 1.68-1.47 (m, 10H), 1.32 (s, 14H).

[0926] Step (6)

[0927]

[0928] Under nitrogen atmosphere, 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 to a solution of 8-[3-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(7-carboxyheptoxy)propoxy]octanoic acid (0.0135 mol, 8.7 g) and 2-butyloct-1-ol (0.0324 mol, 6.04 g) in dichloromethane (500 mL), respectively. The mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with dichloromethane, and the organic layer was washed with 1N HCl. The organic layer was then washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by rapid chromatography by elution with 0 to 60% ethyl acetate in petroleum ether to give 2-butyloctyl 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(2-butyloctyloxy)-8-oxo-octyloxy]propoxy] (2.3 g, 16.5% yield) octyl ester as a colorless oil.

[0929] 1 H NMR (400MHz, CDCl3) δ7.37-7.28(m,5H),4.57(s,2H),3.96(d,J=5.8Hz,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).

[0930] Step (7)

[0931]

[0932] Pd / C (500 mg, 20% wt / wt) was added to 2-butyloctyl octanoate (2.15 g, 2.2 mmol) of 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]-2-[8-(2-butyloctyloxy)-8-oxo-octyloxy]propoxy]octanoate (50 mL). The mixture was stirred at room temperature under hydrogen for 18 h. TLC (ethyl acetate / petroleum ether = 1 / 1) showed that the starting material was consumed. The reaction was filtered through diatomaceous earth and washed with ethyl acetate to give 2-butyloctyl octanoate (1.51 g, 77.1% yield) as a colorless oil.

[0933] 1 H NMR (400MHz, CDCl3) δ3.97 (t, J=5.8Hz, 4H), 3.74-3.40 (m, 27H), 2.29 (t, J= 7.5Hz, 4H), 1.69-1.48 (m, 11H), 1.37-1.21 (m, 48H), 0.88 (t, J = 5.3Hz, 12H).

[0934] Step (8)

[0935]

[0936] Under nitrogen atmosphere, 2-butyloctyl octanoate (1.0 g, 1.12 mmol) and triethylamine (228 mg, 2.25 mmol) in 10 mL of dry dichloromethane were cooled to -5 °C. Methanesulfonyl chloride (193 mg, 1.69 mmol) in 10 mL of dry dichloromethane 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 was 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 obtain 2-butyloctyl octanoate (1.03 g, 94.7% yield) of 8-[2-[8-(2-butyloctyloxy)-8-oxo-octyloxy]-3-[2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]octanoate as a colorless oil, which was used without further purification.

[0937] 1H NMR (400MHz, CDCl3) δ4.41-4.36(m,2H),3.96(t,J=5.8Hz,4H),3.79-3.75(m,2H),3.70-3.39(m,22H),3 .08(s,3H),2.29(t,J=7.5Hz,4H),1.67-1.49(m,11H),1.39-1.18(m,48H),0.89(t,J=6.6,3.8Hz,12H).

[0938] Step (9)

[0939]

[0940] 2-Butyl octyl octanoate (1.0 g, 1.0 equivalent) was added to 8-[2-[8-(2-butyloctyloxy)-8-oxo-octyloxy]-3-[2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]propoxy]octyl octanoate (1.0 g, 1.0 equivalent) in N,N-dimethylformamide (20 mL), and the mixture was heated at 80 °C for 18 h. TLC showed that the starting material was consumed. The reaction was quenched with water (200 mL) and then extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated to obtain 2-butyloctyl octanoate (900 mg, 95.2% yield) of 8-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(2-butyloctyloxy)-8-oxo-octyloxy]propoxy]octanoate (900 mg, 95.2% yield), without purification.

[0941] 1 H NMR (400MHz, CDCl3) δ3.97 (t, J=5.8Hz, 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).

[0942] Step (10)

[0943]

[0944] 8-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(2-butyloctyloxy)-8-oxo-octyloxy]propoxy]octanoic acid 2-butyloctanoic acid ester (0.900 g, 1.0 equivalent) and triphenylphosphine (0.775 g, 3.0 equivalent) were dissolved in THF (30 mL) and water (3 mL). The reaction was stirred overnight at room temperature. The reaction was concentrated and purified by rapid column chromatography on silica gel with elution of 5% to 25% MeOH in DCM to give 8-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-2-[8-(2-butyloctyloxy)-8-oxo-octyloxy]propoxy]octanoic acid 2-butyloctanoic acid ester (643 mg, 74% yield) as a colorless oil.

[0945] 1 H NMR (400MHz, CDCl3) δ3.98-3.95(m,4H),3.68-3.40(m,26H),2.94-2.90(m,2H),2.3 6-2.32(m,4H),1.66-1.51(m,11H),1.35-1.23(m,48H),0.89(t,J=6.6,3.9Hz,12H).

[0946] Step (11)

[0947]

[0948] N,N-diethylethylamine (0.478 g, 7.0 equivalent) and 1H-imidazolium-4-carbonyl chloride (0.353 g, 4.0 equivalent) were added to 2-butyloctyl octanoate (600 mg, 0.675 mmol) in dry DCM (70 mL), and the mixture was stirred at room temperature for 18 h.

[0949] TLC (DCM / MeOH = 10:1) and LCMS showed the disappearance of the starting material. The mixture was concentrated and then purified by rapid column chromatography on silica gel by elution with 0% to 20% methanol in dichloromethane to give 2-butyloctyl octanoate (310 mg, 47% yield).

[0950] 1H NMR (400MHz, CDCl3) δ7.62(t,J=14.2Hz,3H),3.97(t,J=5.8Hz,4H),3.68-3.39(m,26H),2.3 0(t,J=7.6,1.5Hz,4H),1.66-1.51(m,11H),1.44-1.14(m,48H),0.88(t,J=6.9,4.0Hz,12H).

[0951] Example 15 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-imidazol-4-carboxamide (compound XV)

[0952]

[0953] Based on the chemically synthesized compound (XV) shown in scheme (13).

[0954]

[0955] Step (1)

[0956]

[0957] A solution of 2,3-bis[(Z)-octadec-9-enoxy]prop-1-ol (1.0 g, 1.69 mmol) and triethylamine (0.512 g, 5.06 mmol) in DCM (20 mL) was added with methanesulfonyl chloride (0.386 g, 3.37 mmol) and the mixture was stirred at room temperature for 2 h. TLC showed that the starting material was 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]propane methanesulfonate (1.1 g, 97%) as a colorless oil.

[0958] 1 H NMR (400MHz, CDCl3) δ5.37-5.32(m,3H),4.25(dd,J=10.9,5.7Hz,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=12.4,6.6Hz,6H),1.56(d,J=4.5Hz,4H),1.37-1.22(m,45H),0.88(t,J=6.8Hz,6H).

[0959] Step (2)

[0960]

[0961] A mixture of 3,2,3-bis[(Z)-octadec-9-enoxy]propyl methanesulfonate (4.5 g, 6.71 mmol) and oct-1-amine (17.3 g, 134 mmol) was heated at 80 °C for 18 h. The reaction mixture was purified by rapid chromatography by elution with 10% to 50% ethyl acetate in petroleum ether to give N-[2,3-bis[(Z)-octadec-9-enoxy]propyl]oct-1-amine (4.2 g, 89% yield) as a pale yellow oil.

[0962] 1 H NMR (400MHz, CDCl3) δ5.45-5.27(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=6.6Hz,9H).

[0963] Step (3)

[0964]

[0965] A mixture of 3-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propionic 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]octyl-1-amine (0.771 g, 1.09 mmol) in DCM (10 mL) was stirred at ambient temperature for 16 h. The mixture was then poured into DCM (100 mL). The organic layer was washed with 1N HCl and saturated NaCl, dried over NaSO4, and concentrated. The residue was purified by rapid column chromatography on silica gel by elution with 1:81 ethyl acetate / petroleum ether to obtain N-[2-[2-[2-[2-[3-[2,3-bis[(Z)-octadec-9-enoxy]propyl-octyl-amino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate tert-butyl ester (0.95 g, 82.5%) as a colorless oil.

[0966] 1H NMR (400MHz, CDCl3) δ5.36 (dt, J = 10.6, 4.7Hz, 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=33.4Hz,56H),0.91-0.85(m,9H).

[0967] Step (4)

[0968]

[0969]

[0970] TFA (2.06 g, 18.1 mmol) was added to a mixture of N-[2-[2-[2-[2-[3-[2,3-bis[(Z)-octadec-9-enoxy]propyl-octyl-amino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate tert-butyl ester (0.95 g, 0.903 mmol) in DCM (5 mL) at room temperature. The mixture was stirred at ambient temperature for 3 h. The mixture was concentrated to give 3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-N-[2,3-bis[(Z)-octadec-9-enoxy]propyl]-N-octyl-propionamide; 2,2,2-trifluoroacetic acid (0.94 g, 97.7%) as a colorless oil.

[0971] 1 H NMR (400MHz, CDCl3) δ5.46-5.27(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=6.7Hz,9H).

[0972] Step (5)

[0973]

[0974] Add 1H-imidazolium-4-carbonyl chloride (0.249 g, 191 mmol) to a mixture of 3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-N-[2,3-bis[(Z)-octadec-9-enoxy]propyl]-N-octyl-propionamide; 2,2,2-trifluoroacetaldehyde (0.5 g, 0.476 mmol) and N,N-diethylethylamine (0.289 g, 2.86 mmol) in DCM (40 mL). Stir the mixture at room temperature for 16 h. The mixture was concentrated and the residue was purified by silica gel column chromatography on silica gel by elution with 2%–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-imidazol-4-carboxamide (0.305 g, 61%) as a yellow oil.

[0975] 1 H NMR (400MHz, CDCl3) δ7.64 (d, J=5.8Hz, 2H), 5.46-5.23 (m, 3H), 3.83-3.20 (m, 29H), 2.7 7-2.57(m,2H),2.14-1.86(m,8H),1.63-1.42(m,7H),1.27(s,55H),0.93-0.82(m,9H).

[0976] Example 16 Synthesis of N-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propionyl-octylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]-1H-imidazol-4-carboxamide (compound XVI)

[0977]

[0978] Synthesis of compound (XVI)

[0979] Step (1)

[0980]

[0981] Add 2,3-bis[(Z)-octadec-9-enoxy]propionic acid (2.09 g, 3.45 mmol), 4-dimethylaminopyridine (35 mg), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylureonium hexafluorophosphate (1.64 g, 4.3 mmol), and TEA (581 mg, 5.74 mmol) to a solution of N-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]octyl-1-amine (2 g, 2.87 mmol) in DCM (50 ml). Stir the mixture at 25 °C for 18 h. Then treat the mixture with DCM (50 ml), wash with water (250 ml x 2), saturated aqueous solution of NaCl (250 ml), and dry with Na2SO4. The organic compound was rapidly purified (5% MeOH in DCM) to give 2,3-bis[(Z)-octadec-9-enoxy]-N-octyl-N-[2-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]propionamide (2.7 g, 2.17 mmol, yield 75.6%) as a yellow oil.

[0982] 1 H NMR(400MHz, CDCl3)δ7.46(d,J=7.6Hz,6H),7.29(t,J=6.4Hz,6H),7.25-7.20(m,3H),5.34(s,3H),4.3 6(d,J=36.5Hz,1H),3.85-3.17(m,29H),2.20-1.89(m,7H),1.60-1.14(m,60H),0.87(d,J=6.8Hz,9H).

[0983] Step (2)

[0984]

[0985] Toluene-4-sulfonic acid (822 mg, 4.32 mmol) was added to a solution of 2,3-bis[(Z)-octadec-9-enoxy]-N-octyl-N-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]propionamide (1020 mg, 0.86 mmol) in THF / MeOH (20 mL, 1 / 1). The mixture was stirred at 25 °C for 2 h. TEA (1.5 mL) was added to this mixture and the mixture was concentrated and rapidly purified (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-propionamide (766 mg, 0.8 mmol, yield 92.6%) as a colorless oil.

[0986] 1 H NMR (500MHz, CDCl3) δ5.39-5.31(m,4H),3.75-3.56(m,27H),3.46-3.43(m,2H),1.99(dd,J =14.3, 7.9Hz, 8H), 1.55 (dd, J = 11.6, 6.7Hz, 4H), 1.26 (d, J = 7.0Hz, 56H), 0.90-0.87 (m, 9H).

[0987] Step (3)

[0988]

[0989] TEA (185 mg, 1.83 mmol) and methanesulfonyl chloride (157 mg, 1.37 mmol) were added to a solution of N-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]-2,3-bis[(Z)-octadec-9-enoxy]-N-octylpropionamide (860 mg, 0.92 mmol) in DCM (10 ml). The mixture was stirred at 25 °C for 18 h. The mixture was then treated with DCM (50 ml), washed with water (50 ml), 1N HCl (50 ml), saturated aqueous solution of NaHCO4 (50 ml), and saturated aqueous solution of NaCl (50 ml), and dried over Na2SO4. The organic matter was concentrated to obtain ethyl methanesulfonic acid 2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propionyl-octylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl ester (806 mg, 0.75 mmol), a colorless oil. EXP-21-IV4334-N2 (621 mg)

[0990] 1H NMR (400MHz, CDCl3) δ5.34(s,4H),4.38(d,J=3.7Hz,1H),3.76(d,J=4.5Hz,2H),3.70-3.55(m,22H),3.43(d ,J=18.9Hz,4H),3.08(s,3H),2.01(d,J=5.2Hz,8H),1.56-1.50(m,4H),1.27(s,56H),0.88(t,J=5.0Hz,9H).

[0991] Step (4)

[0992]

[0993] NaN3 (155 mg, 2.38 mmol) was added to a solution of 2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propionyl-octylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl ester of methanesulfonic acid (806 mg, 0.79 mmol) in DMF (10 mL). The mixture was stirred at 70 °C for 14 h. The mixture was treated with EA (50 mL), washed with water (50 mL), a saturated aqueous solution of NaCl (50 mL), and dried over Na2SO4. The organic matter was concentrated and rapidly purified (50% EA in PE) to give N-[2-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethyl]-2,3-bis[(Z)-octadec-9-enoxy]-N-octylpropionamide (400 mg, 0.4 mmol, yield 50.3%) as a colorless oil.

[0994] 1 H NMR (400MHz, CDCl3) δ5.42-5.29(m,4H),4.37(dt,J=39.0,5.4Hz,1H),3.71-3.57(m,2 2H), 3.47-3.39 (m, 6H), 2.12-1.91 (m, 8H), 1.57-1.23 (m, 70H), 0.88 (t, J = 5.0Hz, 9H).

[0995] Step (5)

[0996]

[0997] Add DIEA (290 mg, 2.24 mmol) and 1H-imidazolium-4-carbonyl chloride (234 mg, 1.79 mmol) to a solution of N-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethyl]-2,3-bis[(Z)-octadec-9-enoxy]-N-octylpropionamide (420 mg, 0.45 mmol) in DCM (5 ml). Stir the mixture at 25 °C for 18 h. Treat the mixture with DCM (50 ml), wash with water (50 ml), saturated aqueous solution of NaCl (50 ml), and dry with Na2SO4. The organic matter was concentrated and rapidly purified (0-10% MeOH in DCM) to give N-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propionyl-octyl-amino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]-1H-imidazol-4-carboxamide (278 mg, 0.26 mmol, yield 58.8%) as a colorless oil.

[0998] 1 H NMR (400MHz, CDCl3) δ7.64 (s, 1H), 7.59 (d, J = 17.0Hz, 1H), 7.52 (s, 1H), 5.34 (s, 4H), 4.3 3(s,1H),3.71-3.36(m,28H),2.01(d,J=5.4Hz,8H),1.27(s,60H),0.88(t,J=5.2Hz,9H)

[0999] Figure 9 Synthesis of 2-Butyloctanoic acid 6-[2-[6-(2-Butyloctanoyloxy)hexyloxy]-3-[2-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]propoxy]hexyl ester (compound XVII)

[1000]

[1001] Based on the chemically synthesized compound (XVII) shown in scheme (9), such as Example 17 As shown.

[1002] Synthesis of compound (XVII)

[1003] Step (1)

[1004]

[1005] PPTS (1.61 g, 6.41 mmol) was added to a solution of 6-bromohex-1-ol (10 g, 55.2 mmol) and 3,4-dihydro-2H-pyran (4.78 g, 56.9 mmol) in DCM (150 mL), and the mixture was stirred at room temperature for 3 h. TLC (EA / PE 9 / 1, SMR) was then performed. f : 0.2; Product, R f The result of 0.7 indicates that the starting material was completely consumed. The solvent was concentrated and purified by rapid column chromatography (0-10% EA (5%) in PE) to give 2-(6-bromohexyloxy)tetrahydropyran as a colorless oil (12.72 g, 48 mmol, 86.9% yield).

[1006] 1 H NMR (500MHz, CDCl3) δ4.60-4.53(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).

[1007] Step (2)

[1008]

[1009] Methanesulfonyl chloride (6.04 g, 52.8 mmol) was added dropwise 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 100 mL of dry dichloromethane at 0 °C. The mixture was warmed 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 methanesulfonic acid (12.7 g, 35 mmol, quantified) as a pale yellow oil, which was used without further purification.

[1010] 1 H NMR (500MHz, CDCl3) δ7.35-7.26(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).

[1011] Step (3)

[1012]

[1013] NaH (4.2 g, 105 mmol) was added fractionally to a solution of (2,2-dimethyl-1,3-dioxolane-4-yl)methanol (4.63 g, 35 mmol) in dry THF (90 mL) at 0 °C, and the mixture was then heated to reflux for 30 min. The reaction was then cooled to room temperature, and 2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonic acid (12.7 g, 35 mmol) was added to dry THF (30 mL) under nitrogen atmosphere, and the reaction was heated at 80 °C for 24 h. TLC showed that the starting material was 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 rapid chromatography by elution with 0 to 5% CH3OH in DCM to give a pale yellow oil, 4-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (7.217 g, 18.1 mmol, 51.7% yield).

[1014] 1 H NMR (400MHz, CDCl3) δ7.37-7.27(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).

[1015] Step (4)

[1016]

[1017] A mixture of 4-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxymethyl]-2,2-dimethyl-1,3-dioxolane (7.217 g, 18.1 mmol) in AcOH (30 mL) and H₂O (30 mL) was stirred at room temperature for 18 h. The mixture was then subjected to TLC (EA / PE 1 / 1, SM R) f : 0.5; Product, R f A result of 0.1 indicates that all starting material was consumed. The solvent was removed under vacuum and azeotropically reacted with toluene several times. 2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]ethyl methanesulfonic acid (6.48 g, 18.1 mmol, quantified) was obtained as a pale yellow oil and used without further purification.

[1018] 1 H NMR (500MHz, CDCl3) δ7.36-7.26(m,5H),4.57(s,2H),3.88-3.81(m,1H),3.71-3.49(m,20H).

[1019] Step (5)

[1020]

[1021] NaH (1.84 g, 46 mmol) was added several times to a solution of 3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]prop-1,2-diol (3.3 g, 9.21 mmol) in dry DMF (40 mL) 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-bromohexyloxy)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 showed that the starting material was 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 rapid chromatography by elution with 0 to 5% CH3OH in DCM to give 2-[6-[1-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxymethyl]-2-(6-tetrahydropyran-2-yloxyhexyloxy)ethoxy]hexyloxy]tetrahydropyran (2.834 g, 3.9 mmol, 42.3% yield) as a colorless oil.

[1022] 1 H NMR (500MHz, CDCl3) δ7.35-7.27(m,5H),4.57(d,J=1.0Hz,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).

[1023] Step (6)

[1024]

[1025] p-Toluenesulfonic acid (0.742 g, 3.9 mmol) was added in a single addition to a solution of 2-[6-[1-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxymethyl]-2-(6-tetrahydropyran-2-yloxyhexyloxy)ethoxy]hexyloxy]tetrahydropyran (2.834 g, 3.9 mmol) in EtOH (70 mL), and the mixture was stirred at room temperature for 24 h. TLC (4% CH3OH in DCM) showed complete disappearance of the starting material. After quenching the reaction with dilute sodium bicarbonate solution (150 mL), the solvent was extracted with EA (2 x 100 mL). The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by rapid chromatography by elution with 0% to 5% CH3OH (4%) in DCM to give 6-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(6-hydroxyhexyloxy)propoxy]hex-1-ol (1.435 g, 2.57 mmol, 65.9% yield) as a colorless oil.

[1026] 1 H NMR (500MHz, CDCl3) δ7.36-7.27(m,5H),4.57(s,2H),3.70-3.39(m,29H),1.67-1.53(m,9H),1.40-1.35(m,7H).

[1027] Step (7)

[1028]

[1029] A solution of 6-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-(6-hydroxyhexyloxy)propoxy]hex-1-ol (1.435 g, 2.57 mmol) and 2-butyloctanoic acid (1.54 g, 7.7 mmol) in dry dichloromethane (30 mL) was supplemented with 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 h. 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 rapid chromatography by elution with 0% to 5% (3%) CH3OH in DCM to give 2-butyloctanoic acid 6-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[6-(2-butyloctanoyloxy)hexyloxy]propoxy]hexyl ester (1.816 g (P: 1.15 g + NP: 0.666 g), 1.97 mmol, 76.6% yield).

[1030] 1 H NMR (500MHz, CDCl3) δ7.36-7.27(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).

[1031] Step (8)

[1032]

[1033] A solution of 2-butyloctanoic acid 6-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]ethoxy]-2-[6-(2-butyloctanoyloxy)hexyloxy]propoxy]hexyl ester (1.15 g, 1.25 mmol) in EtOAc (20 mL) was purged with N2 for 10 min, then Pd / C (230 mg) was added, and the reaction was continued to be purged with N2. The reaction was then carried out under vacuum and backfilled three times with H2. Next, the reaction was stirred overnight at room temperature under H2 atmosphere. TLC (5% CH3OH in DCM) showed the reaction was complete. The slurry was filtered through diatomaceous earth, and the diatomaceous earth was washed several times with EtOAc. Next, the combined organic compounds were concentrated in a vacuum to give 2-butyloctanoic acid 6-[2-[6-(2-butyloctanoyloxy)hexyloxy]-3-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]propoxy]hexyl ester (1.0 g, 1.2 mmol, 96.4% yield) as a colorless liquid.

[1034] 1 H NMR (500MHz, CDCl3) δ4.09-4.03(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).

[1035] Step (9)

[1036]

[1037]

[1038] Ms-Cl (0.124 g, 1.08 mmol) was added to a solution of 6-[2-[6-(2-butyloctyloxy)hexyloxy]-3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]propoxy]hexyl ester of 2-butyloctanoic acid (0.6 g, 0.72 mmol) and TEA (triethylamine) (0.146 g, 1.44 mmol) in 10 mL of dichloromethane (DCM). The mixture was stirred at room temperature for 3 h. TLC (CH3OH / DCM 3%) showed that the starting material was consumed. The reaction mixture was diluted with dichloromethane and washed with water (10 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated to obtain nonyl to give 2-butyloctanoic acid 6-[2-[6-(2-butyloctanoyloxy)hexyloxy]-3-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]propoxy]hexyl ester (0.582 g, 0.639 mmol, 88.7% yield), which was used directly in the next step.

[1039] 1 H NMR (500MHz, CDCl3) δ4.41-4.36(m,2H),4.08-4.02(m,4H),3.78-3.74(m,2H),3.71-3.39(m,21 H),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).

[1040] Step (10)

[1041]

[1042] Then, Na3N (50 mg, 0.766 mmol) was added to a solution of 2-butyloctanoic acid 6-[2-[6-(2-butyloctanoyloxy)hexyloxy]-3-[2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]propoxy]hexyl ester (0.582 g, 0.639 mmol) dissolved in DMF (10 mL). The reaction mixture was then stirred at 70 °C for 18 h. TCL showed the disappearance of the starting material and new spots were 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 obtain a residue, which was purified by silica gel column chromatography by elution with CH3OH (0-10%) (4%) in DCM to give 2-butyloctanoic acid 6-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]-2-[6-(2-butyloctanoyloxy)hexyloxy]propoxy]hexyl ester (0.409 g, 0.477 mmol, 74.6% yield).

[1043] 1 H NMR (500MHz, CDCl3) δ4.09-4.03(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).

[1044] Step (11)

[1045]

[1046]

[1047] A mixture of 6-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]-2-[6-(2-butyloctyloxy)hexyloxy]propoxy]hexyl ester (0.409 g, 0.477 mmol) and triphenylphosphine (0.187 g, 0.715 mmol) in THF (10 mL) / water (0.3 mL) was stirred at 20 °C for 16 h. TLC (ninhydrin, 3% methanol in dichloromethane) indicated that the reaction was complete. The solvent was removed and added to DCM, then concentrated under reduced pressure to obtain the residue, which was purified by silica gel column chromatography by elution with CH3OH (0-20% (14%)) in CH2Cl2 to give 2-butyloctanoic acid 6-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-2-[6-(2-butyloctanoyloxy)hexyloxy]propoxy]hexyl ester (0.300 g, 0.237 mmol, 75.6% yield).

[1048] 1 H NMR (500MHz, CDCl3) δ4.09-4.03 (m, 4H), 3.70-3.41 (m, 23H), 2.91 (t, J = 5.1Hz, 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).

[1049] Step (12)

[1050]

[1051] 2-Butyloctanoic acid 6-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-2-[6-(2-butyloctanoyloxy)hexyloxy]propoxy]hexyl ester (300 mg, 0.36 mmol) was dissolved in 10 mL of anhydrous DCM and 1H-imidazolium-4-carbonyl chloride (188 mg, 1.44 mmol) was added to 1 mL of anhydrous DMF and DIPEA (233 mg, 1.80 mmol). The mixture was stirred overnight at room temperature. The solvent was evaporated and the product was purified by rapid chromatography (25 g column, DCM / MeOH 0% to 5%) by elution with methanol (4%) in dichloromethane to give 2-butyloctanoic acid 6-[2-[6-(2-butyloctanoyloxy)hexyloxy]-3-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]propoxy]hexyl ester as a colorless oil (259 mg, 0.266 mmol, 73.7% yield).

[1052] LCMS: EXP-21-IX3021-29498-LCMSA020, peak found: MS(ESI) m / z=926.8 / 927.8(M+H) + At 2.854 min.

[1053] 1 H NMR(500MHz, CDCl3)δ7.77-7.56(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).

[1054] Example 18 Synthesis of N-[2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tetrazoloxy]ethoxy]ethoxy]ethoxy]ethyl]-1H imidazole-4-carboxamide (compound XVIII)

[1055]

[1056] Synthesis of compound (XVIII)

[1057] Step (1)

[1058]

[1059] Dys-Martin oxidant (5.05 g, 10.1 mmol) was added to a solution of 2,3-bis[(Z)-octadec-9-enoxy]prop-1-ol (5 g, 8.43 mmol) in DCM (40 ml) over 5 min at 0 °C. The mixture was then stirred at 25 °C under N2 for 2 h. After the reaction, the mixture was treated with DCM (40 ml), washed with NaHCO3 / Na2S2O3 (1 / 1) (50 ml x 3), NaCl saturated aqueous solution (50 ml), and dried over Na2SO4. The organic matter was concentrated and purified rapidly (10% EA in PE) to give 2,3-bis[(Z)-octadec-9-enoxy]propanal (3.04 g, 5.04 mmol, yield 59.8%) as a colorless oil.

[1060] 1H NMR(500MHz,CDCl3)δ9.73(d,J=1.3Hz,1H),5.41-5.31(m,4H),3.85-3.77(m,1H),3.75-3.64(m,2H),3.58(tt,J=9.3,4.6Hz,2H),3.49 -3.40(m,2H),2.21-1.91(m,8H),1.64(dd,J=14.1,7.0Hz,2H),1.57-1.48(m,2H),1.26(d,J=4.4Hz,44H),0.88(dd,J=8.7,5.0Hz,6H).

[1061] Step (2)

[1062]

[1063] Add 1-bromododecane (2.56 g, 10.28 mmol) to a solution of Mg (3.75 g) and I₂ (1.31 g) in anhydrous THF (5 ml). Stir the mixture at 70 °C under N₂ until a colorless mixture is formed. Add 1-bromododecane (10.24 g, 41.12 mmol) to the reaction mixture. Stir the mixture at 70 °C for 3 h. Then add the mixture to a solution of 2,3-bis[(Z)-octadec-9-enoxy]propanal (3.04 g, 5.14 mmol) in anhydrous THF (45 ml). Stir the mixture at 7 °C for 14 h. Treat the mixture with EA (150 ml) and wash with water (150 ml x 2) and a saturated aqueous solution of NaCl (150 ml). The organic compound was rapidly purified (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.

[1064] 1 H NMR (500MHz, CDCl3) δ5.40-5.31(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=12.1Hz,66H),0.87(d,J=6.7Hz,9H).

[1065] Step (3)

[1066]

[1067] Add NaH (210 mg, 5.25 mmol) to a solution of 1,2-bis[(Z)-octadec-9-enoxy]pentadecan-3-ol (1 g, 1.31 mmol) in THF (20 mL). Stir the mixture at 70 °C for 1 h. Add ethyl methanesulfonic acid 2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethyl ester (1.01 g, 1.97 mmol) to this mixture and stir the mixture at 70 °C for 18 h. Treat the mixture with EA (150 mL), wash with water (150 mL x 2), saturated aqueous solution of NaCl (150 mL), and dry with Na2SO4. The organic matter was concentrated and rapidly purified (10% EA in PE) to give a colorless oily substance, [2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyloxy]ethoxy]ethoxy]ethoxy-diphenyl-methyl]benzene (1.08 g, 0.9 mmol, yield 68.3%).

[1068] 1 H NMR (500MHz, CDCl3) δ7.48-7.44(m,6H),7.28(t,J=7.6Hz,6H),7.22(t,J=7.3Hz,3H),5.39-5.31(m,3H),3.73-3.54(m, 16H),3.50-3.32(m,6H),3.23(t,J=5.3Hz,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%)

[1069] Step (4)

[1070]

[1071] Toluene-4-sulfonic acid (1.33 g, 6.99 mmol) was added to a solution of [2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyloxy]ethoxy]ethoxy]ethoxy-diphenylmethyl]benzene (1.65 g, 1.4 mmol) in THF / MeOH (20 ml 1 / 1). The mixture was stirred at 25 °C for 18 h. The mixture was concentrated and treated with EA (150 ml), washed with saturated aqueous solution of NaHCO3 (150 ml x 2), saturated aqueous solution of NaCl (150 ml), and dried over Na2SO4. The organic matter was concentrated and rapidly purified (50% EA in PE) to give 2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyloxy]ethoxy]ethoxy]ethoxy]ethanol (1.18 g, 1.23 mmol, yield 87.8%) as a colorless oil.

[1072] 1 H NMR (500MHz, CDCl3) δ5.41-5.31(m,3H),3.74-3.54(m,18H),3.50-3.33(m,6H),2.59 (dd,J=9.7,6.0Hz,1H),2.04-1.93(m,7H),1.57-1.22(m,70H),0.88(t,J=6.9Hz,9H).

[1073] Step (5)

[1074]

[1075] TEA (297 mg, 2.93 mmol) and methanesulfonyl chloride (269 mg, 2.35 mmol) were added to a solution of 2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyloxy]ethoxy]ethoxy]ethoxy]ethanol (1.1 g, 1.17 mmol) in DCM (15 ml) over 5 min at 0 °C. The mixture was stirred at 25 °C for 18 h. The mixture was then treated with DCM (50 ml), washed with water (50 ml x 2), 1N HCl (50 ml), and a saturated aqueous solution of NaHCO3 (50 ml), and dried over Na2SO4. The organic matter was concentrated to obtain 2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyloxy]ethoxy]ethoxy]ethoxy]ethyl ester of methanesulfonic acid (860 mg, 0.8 mmol, yield 70.7%), which is a yellow oil.

[1076] 1H NMR (500MHz, CDCl3) δ5.39-5.31(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 = 6.9Hz, 9H).

[1077] Step (6)

[1078]

[1079] NaN3 (165 mg, 2.54 mmol) was added to a solution of 2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propionyl-octylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl ester (860 mg, 0.85 mmol) in DMF (10 mL). The mixture was stirred at 70 °C for 14 h. The mixture was treated with EA (50 mL), washed with water (50 mL), a saturated aqueous solution of NaCl (50 mL), and dried over Na2SO4. The organic matter was concentrated and rapidly purified (50% EA in PE) to give N-[2-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethyl]-2,3-bis[(Z)-octadec-9-enoxy]-N-octylpropionamide (657 mg, 0.65 mmol, yield 77.4%) as a colorless oil.

[1080] 1 H NMR (500MHz, 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 = 6.9Hz, 9H).

[1081] Step (7)

[1082]

[1083] Triphenylphosphine (269 mg, 1 mmol) was added to a solution of (Z)-1-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]-2-[(Z)-octadec-9-enoxy]pentadecanoxy]octadec-9-ene (657 mg, 0.68 mmol) in THF / water (20 ml / 0.6 ml). The mixture was stirred at 25 °C for 18 h. The mixture was concentrated and rapidly purified (10%-20% MeOH in DCM) to give 2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tetrazoloxy]ethoxy]ethoxy]ethoxy]ethylamine (560 mg, 0.59 mmol, 85.8% yield) as a colorless oil.

[1084] 1 H NMR (500MHz, CDCl3) δ5.41-5.31(m,3H),3.77-3.33(m,22H),2.89(dt,J=12.6,5.0Hz, 2H), 2.06-1.90 (m, 7H), 1.57-1.51 (m, 4H), 1.48-1.15 (m, 66H), 0.88 (t, J = 6.9Hz, 9H).

[1085] Step (8)

[1086]

[1087] Add DIEA (386 mg, 3 mmol) and 1H-imidazolium-4-carbonyl chloride (312 mg, 2.39 mmol) to a solution of 2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyloxy]ethoxy]ethoxy]ethoxy]ethylamine (560 mg, 0.6 mmol) in DCM (20 mL). Stir the mixture at 25 °C for 18 h. Treat the mixture with DCM (50 mL), wash with water (50 mL), brine (50 mL x 2), and dry with Na2SO4. The organic matter was concentrated and rapidly purified (10% MeOH in DCM) to give N-[2-[2-[2-[2-[1-[1,2-bis[(Z)-octadecyl-9-enoxy]ethyl]tridecyloxy]ethoxy]ethoxy]ethyl]-1H-imidazol-4-carboxamide (348 mg, 0.32 mmol) as a colorless oil.

[1088] 1H NMR(500MHz, CDCl3)δ7.65(d,J=8.7Hz,1H),7.61(d,J=11.0Hz,1H),7.54(s,1H),5.40-5.3 1(m,3H),3.80-3.30(m,24H),2.06-1.93(m,7H),1.60-1.17(m,70H),0.88(t,J=6.9Hz,9H).

[1089] Example Synthesis of bis(2-butyloctyl)10-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethyl-nonyl-amino]nonadecanedioate; hydrochloride (compound XIX)

[1090]

[1091] The compound (XIX) was synthesized according to the following scheme (10).

[1092]

[1093] Synthesis of compound (XIX)

[1094] Step (1)

[1095]

[1096] A mixture of diethyl 3-oxoglutarate (20 g) and 20% sodium ethoxide-ethanol solution (33.5 g) was stirred at 80 °C for 20 minutes. Then, ethyl 8-bromooctanoate (25 g) was added, 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 mixture was stirred for 5 minutes. Then, ethyl 8-bromooctanoate (25 g) was added, and the mixture was stirred for 3 hours. The reaction mixture was cooled to room temperature, and then hexane and 20% ammonium chloride aqueous solution (110 mL) were added. The organic layer was separated, and the solvent was distilled off under reduced pressure to obtain 51.5 g of 9-oxoheptadecano-1,8,10,17-tetraethyl carboxylate as the crude product.

[1097] LCMS Rt = 2.194

[1098] Step (2)

[1099]

[1100] The resulting mixture of tetraethyl 9-oxoheptadecano-1,8,10,17-tetracarboxylate (25 g), acetic acid (40 mL), and 30% hydrochloric acid aqueous solution (80 mL) was stirred at 115 °C for 6 hours. The reaction mixture was cooled to room temperature, and the solvent was then distilled off under reduced pressure. 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 to obtain 0.6 g of 10-oxonane sebacic acid as a white solid.

[1101] 1 H NMR (400MHz, DMSO) δ11.97 (s, 2H), 2.38 (t, J = 7.3Hz, 4H), 2.18 (t, J = 7.4Hz, 4H), 1.54-1.35 (m, 8H), 1.23 (s, 16H).

[1102] Step (3)

[1103]

[1104] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (8.53 g) was added to a mixture of 10-oxonane sebacic acid (6.10 g), 2-butyloct-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. A 10% aqueous solution of potassium hydrogen sulfate (120 mL), hexane (60 mL), and ethyl acetate (60 mL) were added to the reaction mixture. The organic layer was separated and then dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate-hexane) to give bis(2-butyloctyl)10-oxonane sebacic acid ester (6 mg) as a colorless, oily substance.

[1105] 1 H NMR (400MHz, CDCl3) δ3.97(d,J=5.8Hz,4H),2.37(t,J=7.5Hz,4H),2.29(t,J=7.5 Hz, 4H), 1.71-1.43 (m, 11H), 1.28 (d, J = 1.2Hz, 49H), 0.89 (tt, J = 6.6, 4.1Hz, 12H).

[1106] Step (4)

[1107]

[1108] A mixture of bis(2-butyloctyl)10-oxonane sebacate (2.3 g) and Boc-1-amino-3,6-8-octanediamine dioxadiamine (1; 27 g) was stirred in dichloromethane at room temperature for 15 min. Then, sodium triacetoxyborohydride (0.76 g) and acetic acid (0.21 mL) were added. The reaction mixture was stirred at room temperature for 5 h. 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 solvent removal, the residue was purified by rapid chromatography (DCM / MeOH / TEA, 85 / 15 / 1(v,v,v)) to obtain bis(2-butyloctyl)-10-[2-[2-[2-(tert-butoxycarbonylamino]ethoxy]ethylamino]nonadecanedioate as a colorless, oily substance.

[1109] Step (5)

[1110]

[1111] 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 min. Then, sodium triacetoxyborohydride (0.174 g) and acetic acid (0.05 mL) were added. The reaction mixture was stirred at room temperature for 8 h. After dilution with dichloromethane (20 mL), the reaction mixture was washed with saturated sodium bicarbonate (NaHCO3). The organic layer was washed with brine and dried over Na2SO4. After solvent removal, the residue was purified by rapid chromatography (heptane / AcOEt (gradient 0 to 50%)) to obtain bis(2-butyloctyl)-10-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethyl-nonyl-amino]nonadecanedioate as a colorless, oily substance.

[1112] 1 ¹H NMR (400MHz, DMSO-d⁶) δppm: 0.83–0.88 (m, 15H), 1.14–1.43 (m, 82H), 1.46–1.59 (m, 6H), 2.23–2.29 (m, 4H), 2.34–2.36 (m, 2H), 3.01–3.07 (m, 2H), 3.30–3.34 (m, 4H), 3.46 (s, 4H), 3.91 (dd, J = 6, 2Hz, 4H), 6.47–6.75 (m, 1H). Pseudo-molecular ion m / z = 1038, retention time (min) = 2.37.

[1113] Step (6)

[1114]

[1115] Dilute 0.080 g of bis(2-butyloctyl)10-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethyl-nonyl-amino]nonadecanedioate in dichloromethane (5 mL). Then add hydrochloric acid solution (4 M, 5 equivalents in dioxane). Stir the mixture at room temperature for 2 hours. After removing the solvent, stir the residue with isopropyl ether (3 mL), filter, and dry to obtain bis(2-butyloctyl)10-[2-[2-(2-aminoethoxy)ethoxy]ethyl-nonyl-amino]nonadecanedioate hydrochloride as a hygroscopic white solid.

[1116] 1 ¹H NMR (400MHz, DMSO-d⁶) δppm: 0.79–0.92 (m, 15H), 1.14–1.59 (m, 72H), 1.62–1.88 (m, 3H), 2.27 (t, J = 7Hz, 5H), 2.90–3.01 (m, 2H), 3.01–3.12 (m, 2H), 3.13–3.29 (m, 3H), 3.54–3.66 (m, 6H), 3.77–3.87 (m, 2H), 3.92 (d, J = 6Hz, 4H), 7.89–8.21 (m, 3H), 9.51 (br s, 1H). Pseudo-molecular ion m / z = 937, retention time (min) = 2.17.

[1117] Step (7)

[1118]

[1119] A mixture of bis(2-butyloctyl)10-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethyl-nonyl-amino]nonadecanedioate; hydrochloride (0.011 g), 1H-imidazol-4-carbonyl chloride (0.023 g), and 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 solvent removal, the residue was purified by rapid chromatography (DCM / MeOH = 92 / 2 and 95 / 5 (v / v)) to give bis(2-butyloctyl)10-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethyl-nonyl-amino]nonadecanedioate as a yellow oil.

[1120] 1¹H NMR (500MHz, DMSO-d⁶) δppm 0.73–0.91 (m, 15H), 1.03–1.38 (m, 68H), 1.41–1.63 (m, 6H), 2.26 (t, J = 7Hz, 4H), 2.77–3.18 (m, 9H), 3.34–3.40 (m, 2H), 3.43–3.57 (m, 6H), 3.91 (dd, J = 6, 3Hz, 4H), 7.58 (dd), pseudo-molecular ion m / z = 1031, retention time (min) = 2, 45. Example 20 19 Synthesis of 8-[2-[3-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propionyloxy]ethyl-[8-(1-octylnonoxy)-8-oxooctyl]amino]nonyl octanoate (compound XX)

[1121]

[1122] Based on the chemically synthesized compound (XX) shown in scheme (11).

[1123]

[1124] Synthesis of compound (XX)

[1125] Step (1)

[1126]

[1127] Nonyl octanoate (1.00 g, 1.41 mmol) of 8-[2-hydroxyethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (0.772 g, 2.11 mmol) and 3-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propionic acid (0.772 g, 2.11 mmol) in a solution of dry DCM (10 mL) were added at 0 °C. DMAP (17.2 mg, 0.141 mmol), DIPEA (0.218 g, 1.69 mmol), and then EDCI (0.324 g, 1.69 mmol) were added. The reaction was stirred at room temperature for 18 h. TLC showed that the starting material disappeared and new spots formed. 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 and dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography (0-5% CH3OH (3%) in DCM) to give 8-[2-[3-[2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]propionyloxy]ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (1.126 g, 1.06 mmol, 75.6% yield).

[1128] 1 H NMR(400MHz, CDCl3)δ5.07(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).

[1129] Step (2)

[1130]

[1131] 8-[2-[3-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]propionyloxy]ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl 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 h. TLC (3% CH3OH in DCM) showed that the reaction was complete. Evaporate the solvent (50 mL DCM*2) and then dissolve in DCM (100 mL) and wash with saturated NaHCO3 (10 mL). Separate the organic layer and dry with Na2SO4. Filter and remove the solvent to give 8-[2-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propionyloxy]ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (1.0 g, 1.04 mmol, 98.1% yield) as a colorless oil.

[1132] 1 H NMR (500MHz, CDCl3) δ4.90-4.82(m,1H),4.15(t,J=6.2Hz,2H),4.05(t,J=6. 8Hz,2H),3.76(dd,J=10.8,5.0Hz,4H),3.72-3.61(m,12H),3.14-3.09(m,2H) ,2.73(t,J=6.2Hz,2H),2.62(t,J=6.0Hz,2H),2.51-2.42(m,4H),2.28(dd,J= 14.1,7.3Hz,4H),1.67-1.36(m,14H),1.35-1.22(m,48H),0.90-0.84(m,9H).

[1133] Step (3)

[1134]

[1135] 8-[2-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propionyloxy]ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (1.0 g, 1.04 mmol) was dissolved in 15 mL of anhydrous DCM and 1H-imidazolium-4-carbonyl chloride (545 mg, 4.18 mmol) was added to 1 mL of anhydrous DMF and DIPEA (675 mg, 5.22 mmol). The mixture was stirred overnight at room temperature. The solvent was evaporated and the product was purified by rapid chromatography (25 g column, DCM / MeOH 0% to 5%) by elution with 0-5% methanol in dichloromethane (4%) to give 8-[2-[3-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]propionyloxy]ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (542.5 mg, 0.495 mmol, 47.4% yield).

[1136] 1 H NMR (500MHz, CDCl3) δ11.22(s,1H),7.63(s,3H),4.86(p,J=6.2Hz,1H),4.16(d,J=5.5Hz,2H),4.05(t,J=6.8Hz,2H),3.75-3.57(m,18H),2. 72(s,2H),2.54(dd,J=24.0,17.5Hz,6H),2.28(dd,J=13.5,7.4Hz,4H),1.61(dd,J=13.0,6.5Hz,6H),1.53-1.22(m,56H),0.91-0.84(m,9H).

[1137] LCMS: Peak found: MS(ESI)m / z=1052.9(M+H)+, at 2.278min.

[1138] Figure 10 Synthesis of 2-hexyldecanoic acid 6-[6-(2-hexyldecanoyloxy)hexyl-[2-[2-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethyl]amino]hexyl ester (compound XXI)

[1139]

[1140] Based on the chemically synthesized compound (XXI) shown in scheme (11), such as Example 21 As shown.

[1141] synthesis

[1142] Step (1)

[1143]

[1144] A solution of 2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethanol (0.450 g, 1.90 mmol), 6-bromohexyl 2-hexyldecanoate (1.75 g, 4.17 mmol), and DIPEA (0.539 g, 4.17 mmol) in CH3CN (10 mL) and cyclopentyl methyl ether (3 mL) was stirred at 65 °C for 72 hours. The reaction was cooled to room temperature and evaporated under vacuum. The residue was absorbed in EtOAc (50 mL x 2) and H2O (20 mL). The organic layer was separated, dried over Na2SO4, and evaporated under vacuum. The residue was purified by silica gel chromatography (0-5% MeOH (3%) in dichloromethane) to obtain 2-hexyldecanoic acid 6-[6-(2-hexyldecanoyloxy)hexyl-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]amino]hexyl ester (1.082 g, 1.18 mmol, 56.2% yield), which was a pale yellow oil.

[1145] 1 H NMR (500MHz, CDCl3) δ4.09-4.02(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).

[1146] Step (2)

[1147]

[1148]

[1149] Ms-Cl (291 mg, 2.54 mmol) was added to a solution of 2-hexyldecanoic acid 6-[6-(2-hexyldecanoyloxy)hexyl-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]amino]hexyl ester (1.55 g, 1.70 mmol) and TEA (triethylamine) (0.343 g, 3.39 mmol) in 15 mL of dichloromethane (DCM). The mixture was stirred at room temperature for 3 h. TLC (CH3OH / DCM 4%) showed that the starting material was consumed. The reaction mixture was diluted with dichloromethane and washed with water (10 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated to give 2-butyloctanoic acid 6-[2-[6-(2-butyloctanoyloxy)hexyloxy]-3-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]propoxy]hexyl ester (1.592 g, 1.60 mmol, 94.6% yield), which was used directly in the next step.

[1150] 1 H NMR (400MHz, CDCl3) δ4.44-4.32(m,2H),4.05(t,J=6.7Hz,4H),3.91-3.52(m,16H),3.17-2.3 6(m,9H),2.35-2.25(m,2H),1.67-1.52(m,10H),1.48-1.19(m,54H),0.88(t,J=6.7Hz,12H).

[1151] Step (3)

[1152]

[1153] Then, NaN3 (125 mg, 1.92 mmol) was added to a solution of 2-hexyldecanoic acid 6-[6-(2-hexyldecanoyloxy)hexyl-[2-[2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]amino]hexyl ester (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 that the starting material disappeared and new spots formed (CH3OH (3%) in DCM). DMF was removed under vacuum, and the residue was diluted with H2O (20 mL) and then extracted with EA (2 x 50 mL). The organic layer was washed with brine (50 mL x 3), dried over Na2SO4, filtered, and concentrated. The residue was purified by elution with 0–4% CH3OH (2%) in DCM via a rapid chromatography column to give 2-hexyldecanoic acid 6-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethyl-[6-(2-hexyldecyloxy)hexyl]amino]hexyl ester (1.059 g, 1.13 mmol, 70.3% yield) as a yellow oil.

[1154] 1 H NMR (400MHz, CDCl3) δ4.09-4.01(m,4H),3.71-3.46(m,16H),3.42-3.36(m,2H),2.54(d,J= 83.2Hz,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).

[1155] Step (4)

[1156]

[1157] A mixture of 2-hexyldecanoic acid 6-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethyl-[6-(2-hexyldecanoyloxy)hexyl]amino]hexyl ester (1.059 g, 1.13 mmol) and triphenylphosphine (0.443 g, 1.69 mmol) in THF (20 mL) / water (0.6 mL) was stirred at 20 °C for 16 h. TLC (3% methanol in dichloromethane) indicated that the reaction was complete. The solvent was removed and the residue was purified by silica gel column chromatography by elution with 0-20% CH3OH (14%) in CH2Cl2 to give 2-hexyldecanoic acid 6-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethyl-[6-(2-hexyldecyloxy)hexyl]amino]hexyl ester (0.843 g, 0.923 mmol, 81.9% yield), which was a light yellow oil.

[1158] 1 H NMR (400MHz, CDCl3) δ4.10-4.01(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).

[1159] Step (5)

[1160]

[1161] 6-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl-[6-(2-hexyldecanoyloxy)hexyl]amino]hexyl ester (840 mg, 0.92 mmol) of 2-hexyldecanoic acid was dissolved in 10 mL of anhydrous DCM, and 1H-imidazolium-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, followed by extraction with DCM. The organic matter was washed with brine and then dried over Na2SO4. The residue was purified by rapid chromatography (40 g column, DCM / MeOH 0% to 6%) by elution with methanol (6%) in dichloromethane to give 2-hexyldecanoic acid 6-[6-(2-hexyldecanoyloxy)hexyl-[2-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethyl]amino]hexyl ester (488.6 mg, 0.461 mmol, 50.1% yield) as a yellow oil.

[1162] 1 H NMR (400MHz, CDCl3) δ7.62 (d, J = 7.7Hz, 3H), 4.10-4.00 (m, 4H), 3.71-3.52 (m, 18H), 2 .72(d,J=73.5Hz,6H),2.36-2.26(m,2H),1.66-1.21(m,64H),0.87(t,J=6.6Hz,12H).

[1163] LCMS: EXP-21-IX3047-32609-LCMSA020

[1164] Figure 11 Synthesis of 6-nonyl-8-[3-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propionyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (compound XXII)

[1165]

[1166] Compound (XXII)

[1167] Based on the chemically synthesized compound (XXII) shown in scheme (12), such as Example 22 As shown.

[1168] Synthesis of compound (XXII)

[1169] Step (1)

[1170]

[1171] To a solution of 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), 3-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propionic acid (551 mg, 1.51 mmol), HATU (0.859 g, 2.26 mmol), and DIPEA (0.389 g, 3.01 mmol) were added. The mixture was stirred at room temperature for 18 h. TLC (4% methanol in DCM) indicated that the reaction was complete. The solvent was removed under vacuum, and the residue was partitioned between H2O (20 mL) and ethyl acetate (2 x 50 mL). The organic layer was washed with brine (50 mL x 3) and dried over Na2SO4. The residue was purified by elution with 0% to 3% (2%) methanol in dichloromethane via a rapid chromatography column to give 8-[3-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propionyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (1.319 g, 1.3 mmol, 86.4% yield).

[1172] 1 H NMR (500MHz, CDCl3) δ5.12(s,1H),4.90-4.82(m,1H),4.08-4.01(m,2H),3.78(t,J=6.8Hz,2H),3.69-3.60(m,12H),3.55(t,J=5.0Hz,2H ),3.36-3.16(m,6H),2.62(t,J=6.7Hz,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).

[1173] Step (2)

[1174]

[1175]

[1176] TFA (4 mL) was added to a solution of 8-[3-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propionyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (1.319 g, 1.3 mmol) in 10 mL of DCM, and the mixture was stirred at room temperature for 2 h. TLC (4% CH3OH in DCM) indicated that the reaction was complete. The solvent was removed and the mixture was azeotropically dissolved in dichloromethane (50 mL DCM*2) and then dissolved in DCM (100 mL) and washed with saturated NaHCO3 (10 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to give a yellow oily substance, 8-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propionyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (1.19 g, 1.24 mmol, 95.1% yield).

[1177] 1 H NMR (400MHz, CDCl3) δ4.92-4.80(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=5.8Hz,2H),2.33-2.22(m,4H),1.57(dd,J=32.9,14.6Hz,14H),1.38-1.21(m,48H),0.92-0.83(m,9H).

[1178] Step (3)

[1179]

[1180] Add 1H-imidazolium-4-carbonyl chloride (343 mg, 2.63 mmol) in 1 mL of anhydrous DMF and DIPEA (424 mg, 3.28 mmol) to a solution of 8-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propionyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (600 mg, 0.657 mmol) in 10 mL of anhydrous DCM. Stir the mixture overnight at room temperature. Remove the solvent under vacuum. The residue was then purified by rapid column chromatography (eluting with 0% to 5% methanol (4%) in dichloromethane to give a yellow oil, 8-[3-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propionyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (312.3 mg, 0.294 mmol, 44.8% yield).

[1181] 1 H NMR (500MHz, CDCl3) δ7.60 (t, J = 23.3Hz, 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=6.7Hz,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).

[1182] LCMS: Peak found: MS(ESI)m / z=1008.8(M+H)+, at 4.616min.

[1183] Example 23 Synthesis of 8-[2-[3-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propionylamino]ethyl-[8-(1-octylnonoxy)-8-oxooctyl]amino]nonyl octanoate (compound XXIII)

[1184] (XXIII)

[1185] Synthesis of compound (XXIII)

[1186] Step (1)

[1187]

[1188] A solution of 8-bromooctanoate nonyl ester (4.65 g, 0.0133 mol), 1-octylnonyl ester of 8-[2-(tert-butoxycarbonylamino)ethylamino]octanoate (6 g, 0.0111 mol), and N-ethyl-N-isopropyl-prop-2-amine (1.72 g, 0.0133 mol) in acetonitrile was stirred at 65 °C for 72 h. The reaction was cooled to room temperature and evaporated under vacuum. The residue was absorbed into ethyl acetate and saturated sodium bicarbonate. The organic layer was separated, dried over Na2SO4, and evaporated under vacuum. The residue was purified by silica gel chromatography (a mixture of 1% NH4OH and 20% MeOH in dichloromethane) to obtain 8-[2-(tert-butoxycarbonylamino)ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate nonyl ester (7.24 g, 80.7% yield).

[1189] 1 H NMR (500MHz, CDCl3) δ4.98 (s, 1H), 4.86 (s, 1H), 4.05 (t, J = 6.8Hz, 2H), 3.14 (s, 2H), 2.49 (s, 2H), 2.38 (s, 4H), 2.31-2.24 (m, 4H), 1.61 (dd ,J=14.0,6.8Hz,6H),1.50(d,J=6.0Hz,4H),1.45(d,J=7.2Hz,9H),1.40(d,J=6.3Hz,4H),1.35-1.22(m,48H),0.88(td,J=6.8,2.0Hz,9H).

[1190] Step (2)

[1191]

[1192] TFA (10.4 g) was added dropwise 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 CH2Cl2 (30 mL) under ice bath conditions, and the mixture was stirred at room temperature for 10 h. The reaction was quenched with saturated NaHCO3 at 0 °C. The organic layer was washed with saturated NaHCO3, 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.

[1193] 1H NMR (500MHz, CDCl3) δ4.90-4.82(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).

[1194] Step (3)

[1195]

[1196] To a solution of 8-[2-aminoethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate nonyl octanoate (1.33 g, 0.00187 mol), in dichloromethane (10 mL), 3-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propionic acid (0.684 g, 0.00187 mol), DIPEA (0.484 g, 0.00375 mol), and HATU (1.07 g, 0.00281 mol) were added. 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 x 2) and brine (300 mL), and dried over Na₂SO₄. The organic matter was concentrated and purified by rapid chromatography using elution with 20% ethyl acetate in petroleum ether to give a colorless oily product, 8-[2-[3-[2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]propionylamino]ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (1.28 g, yield 64.8%).

[1197] 1 H NMR (500MHz, CDCl3) δ5.14-5.06(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).

[1198] Step (4)

[1199]

[1200] TFA (0.5 mL) was added to a solution of 8-[2-[3-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]propionylamino]ethyl-[8-(1-octylnonoxy)-8-oxooctyl]amino]nonyl octanoate (0.275 g, 0.247 mmol) in dichloromethane (6 mL), and the mixture was stirred at 25 °C for 18 h. TLC (5% methanol in DCM) showed that the starting material was consumed. The solvent was removed and the residue was diluted with DCM (50 ml), washed with 0.2 N NaOH solution (10 ml) and NaHCO3 solution (10 ml), dried over Na2SO4, filtered and concentrated to give 8-[2-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propionylamino]ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (0.21 g, 0.198 mmol, yield 84.4%), which was a pale yellow oil.

[1201] 1 H NMR (500MHz, CDCl3) δ4.09-4.00(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).

[1202] Step (5)

[1203]

[1204] 8-[2-[3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propionylamino]ethyl-[8-(1-octylnonoxy)-8-oxooctyl]amino]nonyl octanoate (580 mg, 0.546 mmol) and 1H-imidazolium-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 overnight at room temperature. The solvent was evaporated and the product was purified by rapid chromatography (25 g column, DCM / MeOH 0% to 5%) with elution in dichloromethane (4%) to give a pale yellow oil, 8-[2-[3-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]propionylamino]ethyl-[8-(1-octylnonoxy)-8-oxooctyl]amino]nonyl octanoate (308 mg, 51.6% yield). The product of this step was combined with [(Z)-non-2-enyl]8-[2-aminoethyl-[(7R,11R)-3,7,11,15-tetramethylhexadecyl]amino]octanoate (to obtain step 2).

[1205] 1 H NMR (500MHz, CDCl3) δ7.72-7.56(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(214nm)

[1206] 1 H-NMR (500MHz, CDCl3) δ7.72-7.56(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).

[1207] LSMC: 526 (M+1) 98% UV (214nm).

[1208] Example 24 Synthesis of 2-hexyldecanoic acid 6-[2-[6-(2-hexyldecanoyloxy)hexyloxy]-3-[2-[2-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethyl-octyl-amino]-3-oxo-propoxy]hexyl ester (compound XXIV)

[1209]

[1210] Synthesis of compound (XXIV)

[1211] N,N-diethylethylamine (0.72 g, 7.10 mmol) and 1H-imidazolium-4-carbonyl chloride (0.74 g, 5.67 mmol) were added to 6-[3-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethyl-octyl-amino]-2-[6-(2-hexyldecanoyloxy)hexyloxy]-3-oxopropoxy]hexyl ester (790 mg, 0.71 mmol) in dry DCM (20 mL), and the mixture was stirred at room temperature for 18 h. TLC showed that the starting material disappeared. The mixture was concentrated and then purified by rapid column chromatography on silica gel by elution with 3% to 6% (5%) methanol in dichloromethane to give 2-hexyldecanoic acid 6-[2-[6-(2-hexyldecanoyloxy)hexyloxy]-3-[2-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethyloctyl-amino]-3-oxo-propoxy]hexyl ester (570 mg, 66.5% yield). Multiplet report.

[1212] 1 H-NMR (400MHz, CDCl3) δ7.65(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).

[1213] Example 25 Synthesis of 1-hexylnonyl octyl octanoate (compound XXV): 8-[[8-(1-hexylnonoxy)-8-oxo-octyl]-[2-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethyl]amino]octanoate

[1214] Synthesis of compound (XXV)

[1215] 1-Hexylnonyl octanoate (780 mg, 0.828 mmol) of 8-[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-imidazolium-4-carbonyl chloride (433 mg, 3.31 mmol) and DIPEA (535 mg, 4.14 mmol) were added. The mixture was stirred overnight at room temperature. Water (10 mL) was added to the solution, and the mixture was then extracted with DCM. The organic matter was washed with brine and dried over Na2SO4. The residue was purified by rapid chromatography (40 g column, DCM / MeOH 0% to 6%) by elution with methanol (4%) in dichloromethane to give 1-hexylnonyl octanoate of 8-[[8-(1-hexylnonoxy)-8-oxo-octyl]-[2-[2-[2-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethoxy]ethyl]amino]octanoate (533.4 mg, 0.518 mmol, 62.6% yield).

[1216] 1 H-NMR (500MHz, CDCl3) δ7.65-7.57(m,2H),7.54(s,1H),4.90-4.83(m,2H),3.69-3.49(m,18H ), 2.73 (d, J = 90.4Hz, 6H), 2.28 (t, J = 7.5Hz, 4H), 1.67-1.17 (m, 68H), 0.88 (t, J = 6.9Hz, 12H).

[1217] Example 26 Synthesis of 1-octylnonyl octanoate (compound XXVI) of 8-[3-[2-[2-[2-(1H-imidazol-4-carbonylamino)ethoxy]ethoxy]ethylcarbamoyloxy]-2-[8-(1-octylnonoxy)-8-oxo-octyloxy]propoxy]octanoate

[1218]

[1219] synthesis

[1220] Step (1)

[1221]

[1222] NaH (5.40 g, 137 mmol) was added to a solution of 3-benzyloxyprop-1,2-diol (5 g, 27.4 mmol) in DMF (150 mL). The mixture was stirred at 80 °C for 1 h. 9-Bromonon-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 x 2), LiCl aqueous solution (300 mL), and NaCl saturated aqueous solution (300 mL), and dried over Na2SO4. The organic matter was concentrated and purified rapidly (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.

[1223] 1 H NMR(500MHz, CDCl3) δ7.37-7.26(m,5H),5.81(ddt,J=16.9,10.2,6.7Hz,2H),4.94(ddd,J=17.4,1 0.2, 9.3Hz, 4H), 4.55 (s, 2H), 3.63-3.39 (m, 9H), 2.03 (td, J = 7.9, 1.3Hz, 4H), 1.58-1.26 (m, 20H).

[1224] Step (2)

[1225]

[1226] To a solution of 2,3-bis(octyl-7-enoxy)-N-octyl-N-[2-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]propionamide (3.74 g, 8.68 mmol) in ACN / CCl4 / H2O (80 ml / 80 ml / 80 ml), NaIO4 (14.9 g, 69.5 mmol) and ruthenium(III) chloride hydrate (392 mg, 1.74 mmol) were added. The mixture was stirred at 25 °C for 18 h. The mixture was filtered and treated with EA (500 ml), washed with Na2S2O3 aqueous solution (500 ml), brine (500 ml), and dried over Na2SO4. The organic matter was concentrated and treated with tert-butanol / 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 Na2SO4. The organic matter was concentrated and rapidly purified (10% MeOH in DCM) to give 7-[2-(6-carboxyhexyloxy)-3-[octyl-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]amino]-3-oxo-propoxy]heptanoic acid (2.58 g, 5.25 mmol, yield 60.5%) as a gray oil.

[1227] 1 H NMR (500MHz, CDCl3) δ7.38-7.27(m,5H),4.55(s,2H),3.62-3.41(m,9H),2.44-2.29(m,4H),1.59(dt,J=44.8,6.8Hz,8H),1.33(s,12H).

[1228] Step (3)

[1229]

[1230] Add heptadecano-9-ol (3.12 g, 12.2 mmol) to a solution of 8-[3-benzyloxy-2-(7-carboxyheptyloxy)propoxy]octanoic acid (2.58 g, 5.53 mmol) in DCM (20 mL), along with 3-(ethyliminomethyleneamino)-N,N-dimethyl-prop-1-amine; hydrochloride (3.18 g, 16.6 mmol), N-ethyl-N-isopropyl-prop-2-amine (2.5 g, 19.4 mmol), and N,N-dimethylpyridin-4-amine (338 mg). Stir the mixture at 25 °C for 18 h. Treat the mixture with EA (300 mL). Wash with water (300 mL x 2), a saturated aqueous solution of NaCl (300 mL), and dry with Na2SO4. The organic matter was concentrated and rapidly purified (5% EA in PE) to give 1-octylnonyl octanoate (2 g, 2.08 mmol) of 8-[3-benzyloxy-2-[8-(1-octylnonoxy)-8-oxooctyloxy]propoxy]octanoate as a colorless oil.

[1231] 1 H NMR (400MHz, CDCl3) δ7.37-7.27(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=6.8Hz,12H).

[1232] Step (4)

[1233]

[1234] Pd / c (514 mg) was added to a solution of 1-octylnonyl octanoate (2.28 g, 2.42 mmol) in EA (50 mL). The mixture was stirred at 25 °C under H2 for 18 h. The mixture was then filtered and concentrated to give 1-octylnonyl octanoate (1.93 g, 2.22 mmol, 91.7% yield) as a colorless oil.

[1235] 1 H NMR (400MHz, CDCl3) δ4.95-4.80 (m, 2H), 3.81-3.38 (m, 9H), 2.27 (t, J = 7.5Hz, 4H), 1.72-1.14 (m, 76H), 0.88 (t, J = 6.8Hz, 12H).

[1236] Step (5)

[1237]

[1238] To a solution of 1-octylnonyl octanoate (1.93 g, 2.26 mmol) in DMF (50 mL), bis(2,5-dioxopyrrolidone-1-yl) carbonate (2.32 g, 9.05 mmol) and N,N-dimethylpyridin-4-amine (1.11 g, 9.05 mmol) 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 x 2), LiCl aqueous solution (300 mL), and NaCl saturated aqueous solution (300 mL), and dried over Na2SO4. The organic matter was concentrated and rapidly purified (10%-20% EA in PE) to give 1-octylnonyl octanoate of 8-[3-(2,5-dioxopyrrolidone-1-yl)oxycarbonyloxy-2-[8-(1-octylnonoxy)-8-oxo-octyloxy]propoxy]octanoate (1.66 g, 1.64 mmol, yield 72.3%) as a colorless oil.

[1239] Step (6)

[1240]

[1241] To a solution of N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]carbamate tert-butyl ester (300 mg, 1.21 mmol) in DCM (15 ml), add 1-octylnonyl octanoate (1 g, 1.01 mmol), N,N-diethylethylamine (153 mg, 1.51 mmol), and N,N-dimethylpyridin-4-amine (12 mg). Stir the mixture at 25 °C for 18 h. Then treat the mixture with EA (50 ml), wash with water (50 ml x 2), saturated aqueous solution of NaCl (50 ml), and dry with Na2SO4. The organic matter was concentrated and rapidly purified (20% EA in PE) to give 1-octylnonyl octanoate of 8-[3-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethylcarbamoyloxy]-2-[8-(1-octylnonoxy)-8-oxo-octyloxy]propoxy]octanoate (763 mg, 0.67 mmol, yield 65.9%) as a colorless oil.

[1242] 1H NMR(400MHz, CDCl3)δ5.26(s,1H),5.05(s,1H),4.91-4.80(m,2H),4.14(ddd,J=16.8,11.4,6 .5Hz,2H),3.63-3.22(m,19H),2.33-2.20(m,4H),1.60-1.14(m,85H),0.88(t,J=6.8Hz,12H).

[1243] Step (7)

[1244]

[1245] Add 0.5 ml of 2,2,2-trifluoroacetic acid to a solution of 1-octylnonyl octanoate (763 mg, 0.68 mmol) in DCM (10 ml). Stir the mixture at 25 °C for 2 h. Treat the mixture with EA (50 ml), wash with NaHCO3 aqueous solution (50 ml), NaCl saturated aqueous solution (50 ml), and dry with Na2SO4. The organic matter was concentrated and rapidly purified (5%-10% EA in PE) to give 1-octylnonyl octanoate of 8-[3-[2-[2-(2-aminoethoxy)ethoxy]ethylcarbamoyloxy]-2-[8-(1-octylnonoxy)-8-oxo-octyloxy]propoxy]octanoate (310 mg, 0.3 mmol, yield 43.7%) as a colorless oil.

[1246] 1 H NMR(500MHz, CDCl3)δ5.73(s,1H),4.86(p,J=6.3Hz,2H),4.16(ddd,J=60.4,11.6,4.6Hz,2H),3.72-3.29(m,19H),2 .98(s,2H),2.27(t,J=7.5Hz,4H),1.56(ddd,J=28.8,18.1,6.3Hz,16H),1.33-1.24(m,60H),0.88(t,J=6.9Hz,12H).

[1247] Step (8)

[1248]

[1249] To a solution of 1-octylnonyl octanoate (310 mg, 0.3 mmol) in DCM (5 ml), N-ethyl-N-isopropyl-prop-2-amine (195 mg, 1.51 mmol) and 1H-imidazolium-4-carbonyl chloride (158 mg, 1.21 mmol) were added. The mixture was stirred at 25 °C for 18 h. The mixture was concentrated and rapid...

Claims

1. A lipid compound of formula (II): R1-Z-NH-CX-(NH) n -A(II) in: -R1 is C 10 To C 55 Lipophilic or hydrophobic tail groups; -Z is a spacer arm having 2 to 24 carbon atoms in a straight-chain saturated or unsaturated hydrocarbon chain, whether branched or unbranched, said chain being interrupted by one or more oxygen atoms and / or portions 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 optionally terminated with an oxygen atom or portions selected from: -NH-(O=C)-; -O-(O=C)-; -(C=O)-O-; and -(O=C)-, which are attached to the hydrophobic tail group; -n is 0; -X is an oxygen atom, and -A indicates a 4-imidazolium group; Wherein C 10 To C 55 The lipophilic or hydrophobic tail group is selected from: and Or a pharmaceutically acceptable salt of a lipid compound of formula (II).

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is in cationic form.

3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the apparent pKa of the compound is less than 7.

4. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the apparent pKa of the compound is in the range of 4.5 to 7.

5. The compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, wherein the hydrophobic or lipophilic tail contains at least one amino moiety that participates in its binding with the spacer group.

6. The compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, wherein the hydrophobic or lipophilic tail contains at least three hydrocarbon chains.

7. The compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, wherein the spacer arm Z comprises 1 to 12 ethylene oxide units.

8. The compound of claim 7 or a pharmaceutically acceptable salt thereof, wherein the spacer arm Z binds at least one portion selected from: -(C=O)-O-; -O-(O=C)-; -NH-(O=C)-; -(O=C)-NH- and -NH-(C=O)-O-.

9. The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein the spacer arm Z is selected from: 。 10. The compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, wherein the spacer arm Z comprises from 1 to 12 ethylene oxide units and further incorporates at least one NH-(C=O)-O-.

11. A compound or a pharmaceutically acceptable salt thereof, said compound being selected from: (IV) (WE) (VII) (VIII) (IX) (X) (XI) (XII) (XIII) (XIV) (XV) (XVI) (XVII) (XVIII) (XIX) (XX) (XXI) (XXII) (XXIII) (XXIV) (XXV) (XXVI) (XXVII)。 12. The compound of claim 11 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from: (IV)、 (VIII)、 (IX)、 (XII)、 (XVI)、 (XIX) and (XXII)。 13. The compound of claim 12 or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of formula (IV): (IV)。 14. A composition comprising at least one compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof and at least one lipid selected from neutral lipids, steroids or esters thereof, and PEGylated lipids.

15. The composition of claim 14, wherein the neutral lipid is selected from phosphatidylcholine; phosphatidylethanolamine; DOPG; sphingomyelin; and ceramide.

16. The composition according to claim 14 or 15, wherein the steroid or its ester is selected from cholesterol and its derivatives, ergosterol, sterol, stigmasterol, lanosterol, 7-dehydrocholesterol, dihydrolanosterol, yeast sterol, enolanol, diosgenin, sitosterol, sitosterol, campesterol, 24-methylene cholesterol, heptadecanoate cholesterol ester, oleate cholesterol ester, and stearate cholesterol ester.

17. The composition according to claim 14 or 15, wherein the polyethylene glycol-modified lipid is selected from: PEG-DAG, DMG-PEG, PEG-PE, PEG-S-DAG, PEG-S-DMG, PEG-cer, mPEG-N,N-bistetradecylacetamide, and PEG-dialkoxypropylcarbamate.

18. The composition according to claim 14 or 15, wherein the composition comprises at least one neutral lipid, at least one steroid or ester thereof, and at least one polyethylene glycol-modified lipid, and wherein, relative to the total amount of lipids and lipid compounds, the lipid compounds, the neutral lipids, the steroids or esters thereof, and the polyethylene glycol-modified lipids are present in molar amounts of 30% to 70% lipid compounds, 0% to 50% neutral lipids, 20% to 50% steroids or esters thereof, and 1% to 15% polyethylene glycol-modified lipids.

19. The composition according to claim 14 or 15, wherein the composition further comprises at least one nucleic acid.

20. The composition according to claim 19, wherein the at least one nucleic acid encodes an antigen.

21. A lipid nanoparticle comprising at least one lipid compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13 and at least one nucleic acid.

22. The lipid nanoparticles of claim 21, wherein the lipid nanoparticles further comprise: at least one lipid as defined in any one of claims 14 to 20.

23. A pharmaceutical composition comprising (i) at least one nucleic acid and at least one lipid compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, or (ii) at least one nucleic acid and at least one composition according to any one of claims 14 to 20, or (iii) at least one lipid nanoparticle according to claim 21 or 22.

24. An immunogenic composition comprising (i) at least one nucleic acid encoding an antigen and at least one lipid compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, or (ii) at least one nucleic acid encoding an antigen and at least one composition according to any one of claims 14 to 20, or (iii) at least one lipid nanoparticle according to claim 21 or 22, wherein the nucleic acid encodes at least one antigen.

25. Use of a composition in the preparation of a medicament, the composition comprising (i) at least one nucleic acid and at least one lipid compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, or (ii) at least one nucleic acid encoding an antigen and at least one composition according to any one of claims 14 to 20, or (iii) at least one lipid nanoparticle according to claim 21 or 22.

26. Use of a composition in the preparation of a medicament for the prevention and / or treatment of a disease, said composition comprising (i) at least one nucleic acid and at least one lipid compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, or (ii) at least one nucleic acid encoding an antigen and at least one composition according to any one of claims 14 to 20, or (iii) at least A lipid nanoparticle according to claim 21 or 22, wherein the disease is selected from infectious diseases, allergies, autoimmune diseases, rare blood disorders, rare metabolic diseases, rare neurological diseases, and cancer diseases.

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