Cleavable lipid compounds, compositions containing the same and uses thereof

Abstract

This disclosure relates to novel lipid compounds, methods for manufacturing lipid nanoparticles (LNPs) containing the same, lipid nanoparticles (LNPs) containing the same, and the use of said LNPs for delivering nucleic acids. As disclosed herein, the lipid compound is a cleavable lipid compound comprising at least one terminal group of formula (I): Y-(CHR)n-Z-(CHR')p-Q* (I) where: -* is directly or indirectly attached to a C 10 To C 55 The terminal of the lipophilic or hydrophobic tail group, -Y is selected from the following groups: methyl, methoxy, trifluoromethyl, imidazolyl, or a hydrogen; -Z is the group -NH-CH2-CO-O-** or the group -CR"(NH2)-CO-O-**, where ** is the terminal closest to Q, and R" is selected from hydrogen, methyl, and trifluoromethyl; -Q is the group -NH-CH2-CO-O-*** or the group -CR"(NH2)-CO-O-***, where R" is selected from hydrogen, methyl, and trifluoromethyl, and *** is directly or indirectly attached to the terminal of the lipophilic or hydrophobic tail group; -R and R' are independently a hydrogen, a methyl, or a trifluoromethyl; -n and p are independently 0, 1, or 2; or one of their pharmaceutically acceptable salts, and the compound is in all possible racemic, enantiomeric, and diastereomeric isomeric forms.

Description

[Technical Field]

[0001] This disclosure relates to novel lipid compounds that can be used to form lipid nanoparticles for delivering therapeutic agents such as nucleic acids, for example, in combination with other lipids such as neutral lipids, steroids or esters thereof, and polymer-conjugated lipids. Formulations prepared with the lipid compounds described herein are able to induce an immune response after administration of an antigen-encoded nucleic acid. [Background Technology]

[0002] Significant progress has been made in the field of polynucleotide therapy 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 the expression of specific cellular products that can be used to treat diseases, for example, those associated with protein or enzyme deficiencies; or to express vaccine antigens to induce specific immune responses. The therapeutic applications of translatable nucleotide delivery are extremely broad because constructs can be synthesized to produce any chosen protein sequence, whether or not it is system-inherent. Nucleic acid expression products can enhance existing protein levels, replace missing or nonfunctional forms of proteins, introduce new proteins and associated functions into cells or organisms, or expose foreign proteins to induce specific immune responses.

[0003] However, many challenges exist related to the impact of polynucleotide delivery on desired responses in biological systems, and efficient delivery of polynucleotides to their intracellular sites of action remains a major problem. For efficient delivery to their sites of action, polynucleotides must (i) be protected from enzymatic and non-enzymatic degradation, (ii) be appropriately distributed within the biological compartment of interest, (iii) be efficiently and efficiently internalized by the target cell, and then (iv) be delivered to the intracellular compartment where the relevant translation mechanism 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 promote their uptake by cells.

[0005] While lipid nanoparticle-based carriers containing cationic lipid components have shown promising results in encapsulation, stability, and site localization, there remains a great need to improve lipid nanoparticle-based delivery systems. Indeed, many of the cationic lipids used to construct such lipid nanoparticles can be toxic to target cells and therefore may have limited applicability; it is noteworthy that their quantity is sufficient for the successful delivery of encapsulating materials to such target cells.

[0006] Therefore, there is still a need for improved lipid nanoparticles that exhibit improved pharmacokinetic properties and can deliver various types of nucleic acids to a wide variety of cell types and tissues with enhanced efficiency.

[0007] Neutral or negatively charged lipid nanoparticles typically exhibit relatively improved pharmacokinetic properties compared to positively charged lipid nanoparticles. However, they generally result in low encapsulation efficiency. Therefore, novel lipids are still needed that combine the high efficiency of polynucleotide encapsulation associated with cationic lipids with the pharmacokinetic properties of neutral or low-charge lipid nanoparticles. In the prior art, this has been achieved with ionizable cationic lipids that exhibit a cationic charge at low pH and a neutral charge at neutral pH. However, such ionizable lipids may exhibit some toxic effects when administered in vivo, locally, systemically, or both.

[0008] Therefore, there remains a particular need for novel lipid compounds that exhibit reduced toxicity and are capable of efficiently encapsulating and delivering polynucleotides to target cells, tissues, and organs. Improved lipids and lipid nanoparticles for polynucleotide delivery will also provide an optimal ratio of one or more polynucleotides to one or more lipids, protect polynucleotides from degradation and clearance from serum, be suitable for systemic or local delivery, and provide intracellular delivery of polynucleotides. Furthermore, lipid-polynucleotide particles should be well-tolerated and provide a sufficient therapeutic index so that treatment of patients with an effective polynucleotide dose does not imply unacceptable toxicity and / or risk to the patient.

[0009] This public text provides for these and related advantages. [Summary of the Invention]

[0010] Therefore, one of the objectives of this disclosure relates to a novel cleavable lipid compound comprising at least one terminal group of formula (I):

[0011] Y-(CHR) n -Z-(CHR') p -Q*(I)

[0012] in:

[0013] -* indicates whether to connect directly or indirectly to a C 10 To C 60 And preferred to C 10 To C 55 The end of the lipophilic or hydrophobic tail group,

[0014] -Y is selected from the following groups: C1-C5 alkyl; C1-C5 alkoxy; C1-C5 acyl; C1-C5 hydroxyalkyl; C1-

[0015] C5 aminoalkyl; C1-C5 alkyl carboxyl ester; acetamido; N,N-C1-C5 alkylamide; C1-C5 fluoroalkyl, such as C1-C5 perfluoroalkyl, such as trifluoromethyl; imidazolyl; triazolyl; squaramide; acylurea; cyano or a hydrogen atom;

[0016] -Z is either the group -NH-CH2-CO-O-** or the group -CR"(NH2)-CO-O-**, where ** is the terminus closest to Q, and R" is selected from hydrogen, methyl, and trifluoromethyl.

[0017] -Q is a group -NH-CH2-CO-O-*** or a group -CR"(NH2)-CO-O-***, wherein R" is selected from hydrogen, methyl and trifluoromethyl, and *** is the end of the lipophilic or hydrophobic tail group directly or indirectly attached to it;

[0018] -R and R' are independently a hydrogen atom, a methyl group, or a trifluoromethyl group;

[0019] -n and p are 0, 1 or 2 independently of each other;

[0020] Or a pharmaceutically acceptable salt of the group; and the lipid compound is in all possible racemic, enantiomeric and diastereomeric isomeric forms.

[0021] According to one embodiment, the compound disclosed herein is a compound of formula (II).

[0022] Y-(CHR) n -Z-(CHR') p -QA-R1(II)

[0023] in:

[0024] -Y, R, n, Z, R', p, and Q are as defined in this document;

[0025] -R1 is C 10 To C 60 And C is preferred 10 To C 55 Lipophilic or hydrophobic tail groups; and

[0026] -A is a straight-chain saturated or unsaturated hydrocarbon chain with 2 to 24, for example, 2 to 18, for example, 4 to 12 carbon atoms or for example, 2 to 12 carbon atoms, in a branched or unbranched chain, said chain being interrupted by one or more oxygen atoms and / or portions selected from: -SS-; (C=O)-O-; -O-(O=C)-; -(C=O)-NH-; -

[0027] NH-(C=O)-O-; -S-; and -O-(O=C)-NH-, and / or optionally have oxygen or a moiety such as -(C=O)-

[0028] The terminal atom of O-; -NH-(C=O)-O- or -NH-, whose end is attached to the lipophilic or hydrophobic tail group, or a pharmaceutically acceptable salt of a compound of formula (II); and any of its racemic, enantiomeric, and diastereomeric isomeric forms.

[0029] According to another embodiment, the compound according to this disclosure is a compound of formula (IIa).

[0030] Y-(CHR) n -NH-CH2-CO-O-(CHR') p -NH-CH2-CO-OA-R1(IIa)

[0031] in:

[0032] -Y, R, n, R' and p are as defined above;

[0033] -R1 is C 10 To C 60 And C is preferred 10 To C 55 Lipophilic or hydrophobic tail groups; and

[0034] -A is the spacer arm as defined above;

[0035] Or a pharmaceutically acceptable salt of a compound of formula (II); and any of its racemic, enantiomeric, and diastereomeric isomeric forms.

[0036] For another purpose, this disclosure relates to a method for manufacturing lipid nanoparticles containing nucleic acids, said method comprising at least the following steps:

[0037] a) Dissolve at least one lipid compound as described herein in a water-miscible organic solvent.

[0038] b) Mix the organic solvent obtained in step a) with an aqueous solvent buffered at a pH ranging from about 3.0 to about 4.5 and containing at least one nucleic acid, and

[0039] c) Obtain the nucleic acid-containing lipid nanoparticles in the aqueous solvent.

[0040] In one embodiment, the method for manufacturing lipid nanoparticles as described herein further includes step d): increasing the pH of the aqueous solvent containing the lipid nanoparticles obtained in step c) to a pH ranging from about 5.0 to about 8.5, for example from about 5.5 to about 8.0, for example from about 6.0 to about 7.5 and for example from about 6.5 to about 7.0.

[0041] According to another embodiment, this disclosure relates to lipid nanoparticles that can be obtained according to the manufacturing methods disclosed herein.

[0042] According to another embodiment, this disclosure relates to a method for manufacturing a pharmaceutical composition, said method comprising at least the following steps:

[0043] i) To manufacture at least one lipid nanoparticle according to the methods described herein, and

[0044] ii) Combine the lipid nanoparticles obtained in step i) with at least one pharmaceutically acceptable excipient or carrier.

[0045] According to another embodiment, this disclosure relates to a method for manufacturing an immunogenic composition, the method comprising at least the following steps:

[0046] i) To manufacture at least one lipid nanoparticle according to the methods described herein, said lipid nanoparticle containing at least one nucleic acid encoding at least one antigen, and

[0047] ii) Combine the lipid nanoparticles obtained in step i) with at least one pharmaceutically acceptable excipient or carrier.

[0048] According to another embodiment, this disclosure relates to lipid nanoparticles that can be obtained according to the methods disclosed herein.

[0049] According to another embodiment, this disclosure relates to lipid nanoparticles comprising at least one lipid compound of formula (IV):

[0050] HO-L(IV)

[0051] Where L is a lipophilic or hydrophobic tail group as described herein, for example, C 10 To C 55 Lipophilic or hydrophobic tail groups,

[0052] And at least one nucleic acid.

[0053] According to another embodiment, this disclosure relates to a pharmaceutical composition comprising at least one lipid nanoparticle as described herein and at least one pharmaceutically acceptable excipient or carrier.

[0054] According to another embodiment, this disclosure relates to an immunogenic composition comprising at least one lipid nanoparticle as disclosed herein, wherein the at least one nucleic acid encodes at least one antigen.

[0055] According to another embodiment, this disclosure relates to a composition comprising at least one lipid nanoparticle as disclosed herein, the composition being used as a pharmaceutical agent.

[0056] According to another embodiment, this disclosure relates to a composition comprising at least one lipid nanoparticle as disclosed 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.

[0057] The term “rare disease” is used in this paper according to its recognized meaning in the art, referring to a disease with an average prevalence threshold between 40 and 50 cases per 100,000 people (Richter et al., Value Health. Sep 2015; 18(6):906-14).

[0058] According to another embodiment, this disclosure relates to a composition comprising at least one lipid nanoparticle as disclosed herein, the composition being used as an immunogenic composition.

[0059] According to another embodiment, this disclosure also relates to a method for preventing and / or treating a disease in an individual in need, wherein the method comprises administering an effective amount of at least one lipid nanoparticle as disclosed herein to the individual. Methods disclosed herein can be used for the prevention and / or treatment of infectious diseases, allergies, autoimmune diseases, rare blood disorders, rare metabolic diseases, rare neurological diseases, and tumors or cancers.

[0060] In some embodiments, this disclosure also relates to the use of at least one lipid nanoparticle as disclosed herein for the manufacture of pharmaceutical agents for the prevention and / or treatment of infectious diseases, allergies, autoimmune diseases, rare blood disorders, rare metabolic diseases, rare neurological diseases, and tumors or cancers. [Image Description]

[0061] Figure 1 The mean titer (HI titer) of hemagglutination inhibition antibodies was measured in mouse serum after immunization twice with either LNP L319 or LNP Lip.(III) (prepared with a lipid compound of formula (III)) each loaded with 5 μg of the full-length hemagglutinin (HA) mRNA encoding influenza virus strain A / Netherlands / 602 / 2009 (H1N1). Eight BALBc / ByJ mice (8 weeks old at D0) in two groups received two intramuscular (IM) injections (administered three weeks apart (D0 and D21)) with each of the two LNP formulations. Four mice were immunized with PBS buffer as a negative control and eight mice were immunized with 10 μg of Vaxigrip-derived 10 μg as a positive control. TM The monovalent influenza vaccine uses the strain A / California / 07 / 2009 (H1N1).

[0062] Figure 2 In female BALB / c ByJ mice, bioluminescent signals for protein expression were acquired at the injection site (quadriceps femoris) after intramuscular administration of LNP 319 or LNP Lip.(III) loaded with 5 μg of luciferase-encoding mRNA (mRNA-Luc). Luminescence levels were evaluated by applying the ROI to the injection site region at 6 h, 24 h, 48 h, and 72 h, and results were expressed as total flux (ph / s) over time (hours) following LNP / mRNA-Luc injection. Tris / sucrose buffer was used as a control.

[0063] Figure 3 Scheme (4) for synthesizing compound (VI) is shown.

[0064] Figure 4 Scheme (5) for synthesizing compound (VII) is shown.

[0065] Figure 5 Scheme (7) for synthesizing compound (VIII) is shown.

[0066] Figure 6 Scheme (8) for synthesizing compound (XV) is shown.

[0067] Figure 7 Scheme (9) for synthesizing compound (XVII) is shown.

[0068] Figure 8 Scheme (10) for synthesizing compound (XIX) is shown.

[0069] Figure 9 Scheme (11) for synthesizing compound (XXIII) is shown.

[0070] Figure 10 Scheme (12) for synthesizing compound (XXIX) is shown.

[0071] Figure 11 Scheme (13) for synthesizing compound (XXXI) is shown.

[0072] Figure 12 Scheme (14) is shown, which demonstrates that the lipid compound (DOG-cleaved) of formula (III) is cleaved by a cyclization process that continuously produces “DOG-cleaved instant” and uncharged lipid “DOG-OH” in the final LNP formulation.

[0073] Figure 13 The chromatogram shown illustrates the separation of lipid compounds of formula (III) (DOG-cut), DOG-cut transient, DOG-OH, DSPC, Chol, and DMG-PEG2000 on a C18-HPLC column.

[0074] Figure 14 This study demonstrates the HI response induced by LNP containing influenza HA mRNA (1 MpU modified from Amptec) in non-human primates immunized twice (four weeks apart) (D0, D28) with 50 μg mRNA in LNP (LNP(III) / DOG-CLEAVE or LNP-L319) injected with IM.

[0075] Figure 15 The mean titer of hemagglutination inhibition antibodies (HI titer) was measured in serum collected on day 21 in mice immunized on day 0 and day 21 with LNP L319, LNP(III) [DOG-cleaved], LNP(XXI) and LNP(XIX) containing mRNA encoding full-length hemagglutinin (HA) of influenza virus strain A / Netherlands / 602 / 2009 (H1N1). [Detailed Implementation]

[0076] definition

[0077] The terms used in this specification generally have their common meaning in the art, both in the context of this disclosure and in the specific context in which each term is used. Some terms are discussed below or elsewhere in this specification to provide further guidance on the compositions and methods describing this disclosure and how they are prepared and used. The following definitions are provided for this specification, including the claims.

[0078] The term "cleavable group" means that when covalently attached to another functional group to form a compound (e.g., a lipid compound as disclosed herein), the group is capable of cleaving from the rest of the molecule upon exposure to biological conditions and, in the context of this disclosure, upon exposure to pH values ​​greater than 5 and, for example, greater than 6.

[0079] The term "terminal group" means that the group is either a head group or a tail group.

[0080] The term “pharmaceutically acceptable salt” includes, for example, acid addition salts of compounds disclosed herein, which are derived from combinations of such compounds with non-toxic acids.

[0081] The term "acid addition salt" includes inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid, as well as organic acids such as acetic acid, citric acid, propionic acid, tartaric acid, glutamic acid, salicylic acid, oxalic acid, methanesulfonic acid, p-toluenesulfonic acid, succinic acid, and benzoic acid, and related inorganic and organic acids.

[0082] 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.

[0083] In the context of this disclosure, the following chemical terms have the following meanings:

[0084] -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.

[0085] - Heteroatoms are understood to refer to nitrogen, oxygen, or sulfur;

[0086] - Heteroaromatic rings represent 5- or 6-membered aromatic rings containing 1 or 2 heteroatoms;

[0087] - An aromatic ring refers to a monocyclic or polycyclic aromatic hydrocarbon group of 6-20 atoms (e.g., 6 atoms), obtained by removing a hydrogen atom from a carbon atom of a parent aromatic ring system. According to this disclosure, an aromatic ring is, for example, a phenyl group;

[0088] Unless otherwise expressly stated, the singular forms “a”, “an”, and “the” used in this specification and the appended claims include plural indicators.

[0089] As used herein, the terms “about” or “approximately” refer to a commonly known range of error for the corresponding value that is readily known to those skilled in the art. References to “about” a value or parameter herein include (and describe) embodiments relating to that value or parameter itself. 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 upon further division), then “about” refers to ±1 of the indivisible object.

[0090] The term "antigen" includes any molecule, such as a peptide or protein, that contains at least one epitope that will elicit an immune response and / or an immune response against at least one epitope that is elicited therein. For example, an antigen is a molecule that optionally induces an immune response after processing, such an immune response being specific to the antigen or cells expressing the antigen. After processing, antigens may be presented by MHC molecules and react specifically with T lymphocytes (T cells). Therefore, the antigen or a fragment thereof should be recognizable by T cell receptors and should be able to induce clonal expansion of T cells carrying T cell receptors that specifically recognize the antigen or fragment in the presence of appropriate co-stimulatory signals, leading to an immune response against the antigen or cells expressing the antigen.

[0091] According to this disclosure, any suitable antigen can be envisioned as a candidate for an immune response. An antigen may correspond to or may be derived from naturally occurring antigens. Such naturally occurring antigens may include or may be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, or the antigen may be a tumor antigen.

[0092] As used herein, the terms “aqueous solution” or “aqueous solvent” refer to compositions containing water.

[0093] In this publication, the term "cation" refers to an ion or ionic group that has a positive charge.

[0094] It should be understood that aspects and embodiments of this disclosure described herein include "having," "comprising," "consisting of," and "substantially composed of." The words "having" and "comprising," or variations such as "has," "having," "comprises," or "comprising," should be understood to imply the inclusion of one or more of the stated elements (such as a material composition or method step), but do not exclude any other elements. The term "consisting of" implies the inclusion of one or more of the stated elements, excluding any additional elements. The term "substantially composed of" implies the inclusion of the stated elements as well as one or more possible other elements, wherein said one or more other elements do not materially affect one or more essential and novel features of this disclosure. Depending on the context, the term "comprising" may also strictly specify the stated features, integers, steps, or components, and therefore, in such cases, it may be replaced by "consisting of."

[0095] The term "charged lipid" refers to any of the many types of lipids that exist in a positively or negatively charged form within a useful physiological range (e.g., pH 3 to pH 9). Charged lipids can be synthetic or naturally derived. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, hemisuccinate sterol ester, dialkyltrimethylammonium propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, and dimethylaminoethanecarbamoylsterol (e.g., DC-Chol).

[0096] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, peptides or nucleic acids that exist in organisms (including viruses) and can be isolated from natural sources and have not been intentionally modified by laboratory personnel are naturally occurring.

[0097] The term "neutral lipid" refers to any of a variety of lipids that are non-ionizable or neutral zwitterionic compounds at a selected pH (e.g., physiological pH). Such lipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine sphingomyelin (SM), or neutral sphingolipids such as ceramides. Neutral lipids can be synthetic or naturally derived.

[0098] As used herein, the terms "individual" or "subject" refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human 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.

[0099] The term "lipid" refers to a group of organic compounds, including but not limited to fatty acid esters, and is generally characterized by being poorly soluble in water but soluble in many organic solvents. Lipid is a general term encompassing fats, fatty oils, essential oils, waxes, phospholipids, glycolipids, thiolipin, amino esters, chromolipids (lipochromes), and fatty acids. In this disclosure, "lipid" includes neutral lipids, steroids or their esters, and polyethylene glycol-modified lipids.

[0100] The term "lipid nanoparticle" (LNP) refers to a particle with at least one dimension on the nanometer scale (e.g., 1-1000 nm), which is formulated with at least one lipid compound as disclosed herein. In some embodiments, the lipid nanoparticle is contained in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid, to a target site of interest (e.g., cells, tissues, organs, tumors, etc.). Such lipid nanoparticles typically comprise a lipid compound as disclosed herein or a lipid obtained by hydrolysis of a lipid compound as disclosed herein, and at least one component selected from: neutral lipids, steroids or esters thereof, and polymer-conjugated lipids.

[0101] As used herein, "lipid-encapsulated" refers to lipid nanoparticles that provide an active agent or therapeutic agent, such as a fully encapsulated, partially encapsulated, or both nucleic acid. In one embodiment, a polynucleotide is fully encapsulated within the lipid nanoparticle.

[0102] It should be noted that the terms "head group" and "tail group" as used in this specification describe a portion of the compounds disclosed herein, such as the functional groups of these compounds. 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".

[0103] As used herein, the term “lipophilic or hydrophobic tail group” qualitatively indicates that the tail has an affinity for lipids (and is typically lipophilic) and is hydrophobic (and typically insoluble in water).

[0104] The term "polyethylene glycol-modified lipid" refers to a molecule that comprises both a lipid moiety and a polyethylene glycol moiety. Polyethylene glycol-modified lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), etc.

[0105] In this disclosure, the terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of at least two nucleotides, which are deoxyribonucleotides or ribonucleotides or analogs thereof. Nucleic acids can have any three-dimensional structure and can perform any known or unknown function. They can be linear or circular. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene segments, loci (locus) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, closed-terminal DNA (ceDNA), self-amplifying RNA (saRNA), stranded DNA (ssDNA), small interfering RNA (siRNA) and microRNA (miRNA), recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure (if present) may be conferred before or after polymer assembly. The sequence of a nucleotide can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components. The term "complementary sequence of a polynucleotide" refers to a polynucleotide molecule that has a complementary base sequence and opposite orientation compared to a reference sequence, such that it can hybridize with the reference sequence with perfect fidelity. As applied to polynucleotides, "recombination" means that the polynucleotide is the product of various combinations of in vitro cloning, restriction and / or ligation steps, and other procedures that produce constructs that can potentially be expressed in host cells.

[0106] The term "steroid" or "sterol" refers to a group of lipids consisting of a sterane core with a hydroxyl moiety. Examples of steroids include cholesterol, campesterol, sitosterol, stigmasterol, and ergosterol. Steroid or sterol esters refer to esters of carboxylic acids and the hydroxyl group of the steroid. In addition to the carboxyl moiety, suitable carboxylic acids may also contain a saturated or unsaturated, straight-chain or branched alkyl group. In some embodiments, the alkyl group may be C1-C2. 20 Alkyl group. In other embodiments, the carboxylic acid can be a fatty acid.

[0107] As used herein, the terms “prevent,” “preventing,” or “delaying the progression of…” (and their grammatical variations) relating to a disease or disorder refer to preventive treatment of the disease, such as in individuals suspected of having the disease or at risk of developing it. Prevention may 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 or subpathological level. The term “prevention” does not require the 100% elimination of the possibility or likelihood of the event occurring. Rather, it indicates that the likelihood of the event occurring has been reduced in the presence of the compositions or methods described herein.

[0108] In this publication, the term “significant” as used with respect to change is intended to mean that the observed change is obvious and / or that it is statistically significant.

[0109] In this disclosure, the term “substantially” is used in conjunction with a feature of this disclosure to define a set of embodiments that are substantially similar to, but not exactly similar to, a feature.

[0110] As used herein, "target cell" or "targeted cell" refers to a target cell. 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, such as a mammal, like a human, and, for example, a human patient.

[0111] The terms “treat” or “treatment” or “therapy” used in this invention refer to the application or consumption of a composition as disclosed herein with the aim of curing, healing, alleviating, reducing, altering, remedying, improving, enhancing or influencing the symptoms of a disorder or condition, or preventing or delaying the onset of symptoms or complications, or otherwise statistically significantly preventing or inhibiting the further development of a disorder.

[0112] As used herein, the terms “therapeutic effective dose” and “preventive effective dose” refer to the amount that provides therapeutic benefit in the treatment, prevention, or management of the pathological process under consideration. The specific amount of therapeutically effective dose can be readily determined by a general practitioner and may vary depending on factors such as the type and stage of the pathological process under consideration, the patient’s medical history and age, and the administration of other therapeutic agents.

[0113] A list of sources, ingredients and components as described below is provided, and combinations and mixtures thereof are also considered and are within the scope of this document.

[0114] It should be understood that each maximum numerical limit given throughout this specification includes each lower numerical limit as expressly stated herein. Each minimum numerical limit given throughout this specification includes each higher numerical limit as expressly stated herein. Each numerical range given throughout this specification includes each narrower numerical range falling within such a wider range as expressly stated herein.

[0115] All lists of items, such as lists of ingredients, are intended and should be interpreted as Markush groups. Therefore, all lists can be read and interpreted as "items selected from a list of items" and "combinations and mixtures thereof."

[0116] References herein may be made to the trade names of components that include the various ingredients used in this disclosure. The inventors herein do not intend to be limited to materials under any particular trade name. Materials equivalent to those referenced by trade names (e.g., materials obtained from different sources under different names or reference numbers) may be substituted and used in the description herein.

[0117] The detailed definitions of the groups and lipid compounds of formula (I) in this disclosure text

[0118] The lipid compounds in this disclosure are ionizable and, for example, cationic lipid compounds.

[0119] The lipid compounds disclosed herein may have an asymmetric center, a chiral axis, and a chiral plane (as described in: E.L.E.L. and S.H.Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pp. 1119-1190), and may exist as racemates, racemic mixtures, and individual diastereomers, wherein all possible isomers and mixtures thereof, including optical isomers, are included in this disclosure. Furthermore, the cationic lipids disclosed herein may exist as tautomers, and both tautomer forms are intended to be included within the scope of this disclosure, even if only one tautomer structure is described.

[0120] The pharmaceutically acceptable salts of the lipid compounds disclosed herein have one or more generally physiologically acceptable counterions. Possible counterions include halides, phosphates, trifluoroacetates, sulfites, nitrates, gluconates, glucurons, galacturons, alkyl sulfonates, alkyl carboxylates, propionic acid sulfonates, and methanesulfonates.

[0121] The lipid compounds and their pharmaceutically acceptable salts 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 be derived from crystallization solvents, are inherent in the solvents used for preparation or crystallization, or be incidental to such solvents. Such solvates are within the scope of this disclosure.

[0122] For example, lipid compounds as disclosed herein may also form a lipid compound having a hydrophilic head group, also known as a terminal group, formed by a group of formula (I), to indicate that it is directly or indirectly attached to the end of a hydrophobic or lipophilic tail.

[0123] The groups in formula (I) have the following definitions:

[0124] Y-(CHR) n -Z-(CHR') p -Q*(I)

[0125] in:

[0126] -* indicates whether to connect directly or indirectly to a C 10 To C 60 And preferred to C 10 To C 55 The end of the lipophilic or hydrophobic tail group,

[0127] -Y is selected from the following groups: C1-C5 alkyl; C1-C5 alkoxy; C1-C5 acyl; C1-C5 hydroxyalkyl; C1-C5 aminoalkyl; C1-C5 alkyl carboxyl ester; acetamido; N,N-C1-C5 alkylamide; C1-C5 fluoroalkyl, such as C1-C5 perfluoroalkyl, such as trifluoromethyl; imidazolyl; triazolyl; squamamide; acylurea; cyano or a hydrogen group; and preferably C1-C5 alkoxy and more preferably methoxy;

[0128] -Z is either the group -NH-CH2-CO-O-** or the group -CR"(NH2)-CO-O-**, where ** is the terminus closest to Q, and R" is selected from hydrogen, methyl, and trifluoromethyl.

[0129] -Q is a group -NH-CH2-CO-O-*** or a group -CR"(NH2)-CO-O-***, wherein R" is selected from hydrogen, methyl and trifluoromethyl, and *** is a terminus directly or indirectly attached to the lipophilic or hydrophobic tail group (especially as described herein);

[0130] -R and R' are independently a hydrogen atom, a methyl group, or a trifluoromethyl group;

[0131] -n and p are 0, 1 or 2 independently of each other;

[0132] Or one of its pharmaceutically acceptable salts, and the lipid compound is in all possible racemic, enantiomeric, and diastereomeric isomeric forms.

[0133] The group of formula (I) exists in a protonated and stable form at pH values ​​below 6.0 and, for example, below 5.5 and, for example, below 5. Conversely, when exposed to pH values ​​greater than the stable protonated form, such groups of formula (I) present in lipid compounds according to this disclosure advantageously undergo chemical rearrangement. As shown in Scheme 1 below, this chemical rearrangement results in its cleavage from the remainder of the lipid molecule. This ability to exist in a positively charged form, for example, efficiently immobilizes negatively charged nucleic acids and loads them into specific chemical carriers specifically designed to facilitate the targeted release of said nucleic acids in vitro or in vivo. Conversely, when such carriers are exposed to pH values ​​greater than 6.0, then the group of formula (I) may be cleaved. In this way, the encapsulated nucleic acid to be released advantageously does not contain this group.

[0134] Option 1

[0135]

[0136] According to one embodiment, Z and Q are both groups -NH-CH2-CO-O-, and n and p are both 2, for example.

[0137] According to another embodiment, Z and Q are different, and one of them is a group -NH-CH2-CO-O- and the other is a group -CH(NH2)-CO-O-, and for example n and p are different and equal to 1 or 2. In this specific embodiment, Z is preferably -CR(NH2)-CO-O- and Q is -NH-CH2-CO-O-.

[0138] According to another embodiment, Z and Q are both groups -CR" (NH2)-CO-O-, wherein R" is preferably hydrogen, and n and p are both 1, for example.

[0139] As previously stated, a group of formula (I) is attached directly or indirectly to a hydrophobic (lipophilic) tail group (e.g., a covalent bond).

[0140] The hydrophobic or lipophilic tail is usually C 10 To C 55 But it can also be C 10 To C 60 of.

[0141] For example, it is a saturated or unsaturated C with either substituted side chains or unbranched straight chains. 10 To C60 And C is preferred 10 To C 55 The hydrocarbon skeleton is optionally interrupted by one or more oxygen or nitrogen atoms and / or one or more -O-CO- or -CO-O- groups, and a nitrogen atom present on the skeleton may be directly or indirectly attached to a group represented by formula (I).

[0142] In particular, the hydrophobic or lipophilic tail is optionally a substituted branched or unbranched straight-chain saturated or unsaturated C. 10 To C 60 And C is preferred 10 To C 55 The hydrocarbon skeleton is optionally interrupted by one or more oxygen or nitrogen atoms and / or one or more -O-CO- or -CO-O- groups, and if a nitrogen atom is present in the skeleton, it is present in a form that cannot be protonated and is directly or indirectly, but preferably directly, attached to the spacer group A. In particular, it can form an amide moiety with the -C=O terminal portion of the spacer group, as in, for example, as in compound (XXVII).

[0143] For example, the hydrophobic or lipophilic tail contains at least two, three or more hydrocarbon chains, each independently selected from optionally substituted C8-C hydrocarbons. 24 (For example, C) 10 -C 20 Alkyl chains, optionally substituted, variable saturated or unsaturated C8-C 24 (For example, C) 10 -C 20 Alkenyl chains and optionally substituted saturated, variable-saturated, or unsaturated C8-C chains. 24 (For example, C) 10 -C 20 An acyl chain, wherein the alkyl, alkenyl or acyl chain may be interrupted by one or more oxygen atoms and / or one or more portions (such as -O-CO- or -CO-O-).

[0144] Each hydrocarbon chain may be substituted with at least one group selected from -OH, CO2H, and C1 to C4 alkyl groups, and preferably unsubstituted. According to one embodiment, the hydrophobic or lipophilic tail is selected from:

[0145]

[0146]

[0147]

[0148]

[0149] According to a particular embodiment, the hydrophobic or lipophilic tail comprises at least three, four or more hydrocarbon chains, each independently selected from optionally substituted C4-C hydrocarbons. 24 For example, C5-C 20 Alkyl chains, and optionally substituted C4-C 24 For example, C 10 -C 20 Alkenyl chain.

[0150] According to a particular embodiment, the hydrophobic or lipophilic tail comprises at least two, three, four or more hydrocarbon C4-C atoms. 24 Chains, wherein at least one chain and preferably at least two chains are interrupted by at least one oxygen atom and / or at least one portion selected from -O-(O=C)- and -(C=O)-O-.

[0151] According to a particular embodiment, the hydrophobic or lipophilic tail comprises at least three, four, or more hydrocarbon chains, wherein at least two chains are optionally substituted C4-C. 24 For example, C5-C 20 Alkylene chains, and optionally each of them is independently interrupted by at least one portion selected from -O-(O=C)- and -(C=O)-O-.

[0152] According to a particular embodiment, the hydrophobic or lipophilic tail comprises at least three, four, or more hydrocarbon chains, wherein all chains are optionally substituted C4-C. 24 For example, C5-C 20 Alkyl chains, and optionally each of them is independently interrupted by at least one portion selected from -O-(O=C)- and -(C=O)-O-.

[0153] In one embodiment, the hydrophobic or lipophilic tail is a tail (R1a) or (R1b), also referred to as DOG alkyl or DOG ether, respectively.

[0154] According to another embodiment, the cationic and / or ionizable lipid compound of this disclosure is compound Y-(CHR) of formula (II). n -Z-(CHR') p -QA-R1(II)

[0155] in:

[0156] -Y, R, n, Z, R', p, and Q are as defined in claim 1;

[0157] -R1 is C 10 To C 60 And C is preferred 10 To C 55Lipophilic or hydrophobic tail groups, especially as described herein; and

[0158] A is a straight-chain saturated or unsaturated hydrocarbon chain, branched or unbranched, having 2 to 24, for example, 2 to 18, for example, 4 to 12 carbon atoms, or for example, 2 to 12 carbon atoms, of which the chain is interrupted by one or more oxygen atoms and / or portions selected from: -SS-; -(C=O)-O-; -O-(O=C)-; -(C=O)-NH-; -NH-(C=O)-O-; -S-; -NH-(O=C) -; and -O-(O=C)-NH-, and / or optionally having an oxygen or a terminal atom such as -(C=O)-O-; -O-(O=C)-; -NH-(C=O)-; -NH-(C=O)-O- or -O-(O=C)-NH-; whose end is attached to the lipophilic or hydrophobic tail group, or one of its pharmaceutically acceptable salts, and any of its racemic, enantiomeric, and diastereomeric isomeric forms.

[0159] According to one implementation scheme, Z and Q are both groups -NH-CH2-CO-O-.

[0160] According to another embodiment, Z and Q are different, and one of them is a group -NH-CH2-CO-O- and the other is a group -CH(NH2)-CO-O-, and preferably, Z is -CR""NH2)-CO-O-, wherein R" is preferably a hydrogen atom, and Q is -NH-CH2-CO-O-.

[0161] According to another implementation, Z and Q are both groups -CR(NH2)-CO-O-.

[0162] For example, according to one embodiment, the lipid compound disclosed herein is a compound of formula (IIa).

[0163] Y-(CHR) n -NH-CH2-CO-O-(CHR') p -NH-CH2-CO-OA-R1(IIa)

[0164] in:

[0165] -Y, R, n, R', R1, A and p are as previously defined in equation (I) or (II);

[0166] Or one of its pharmaceutically acceptable salts and any of its racemic, enantiomeric, and diastereomeric isomeric forms.

[0167] In one specific implementation, both n and p are 2.

[0168] In another specific embodiment, Y is a methoxy group.

[0169] Regarding the spacer arm A of formulas (II) and (IIa), it is similar to those conventionally considered in the field of lipid compounds. Therefore, the selection of such a spacer arm will not add any difficulty for those skilled in the art. Naturally, it needs to be inert or not impair the efficiency of the lipid compound.

[0170] Typically, spacer arm A has 2 to 24 carbon atoms, for example from 2 to 12 or from 4 to 10, comprising at least one or more ethylene oxide units and optionally one or more portions selected from: -OCO-; COO-, -NHCOO-, -OCONH- and -SS-.

[0171] According to a specific implementation scheme, spacer arm A can have equation (A1).

[0172]

[0173] The right end is the end connected to the hydrophobic or lipophilic tail, and wherein:

[0174] l is 0 or 1;

[0175] The range of m is from 2 to 12, preferably from 2 to 10, or for example, 2, 3, 4, 5, 6, 7, 8, or 9.

[0176] p is 0 or 1; and

[0177] - When p is 1, R' represents a group selected from the following: -O-(O=C)-; -(C=O)-O-; -NH-(C=O)-O- or -O-(O=C)-NH-; -NH-(C=O)--O-,-; O-CH2 C(=O)-O-; -OC(=O)-(CH2)2-C(=O)- and -SS-.

[0178] As examples of spacer arms for the convenience of this disclosure, the following spacer arms are provided, wherein the right end is connected to a lipophilic or hydrophobic tail group:

[0179]

[0180] According to one embodiment, the spacer group comprises an ethylene oxide unit. In this embodiment, the spacer group is poly(ethylene oxide) (also known as polyethylene glycol-PEG).

[0181] In this embodiment, the spacer group may comprise 1 to 24 ethylene oxide units, and for example 2, 3, 4, 5, 6, 7, 8, 10, 12 and 24 ethylene oxide units.

[0182] According to another embodiment, the spacer group comprises a poly(ethylene oxide) moiety and further comprises at least one moiety selected from the group consisting of -COO-, -OCO-, -NHCOO-, -OCONH-, and -CH2CH2-.

[0183] According to one embodiment, the compound has formula (II), wherein Z is a group -CH(NH2)-CO-O-, and Q is a group -NH-CH2-CO-O-, and particularly the following compound (VI):

[0184]

[0185] Or one of its salts, such as its trifluoroacetate, and one of its racemic, enantiomeric, and diastereomeric isomeric forms.

[0186] According to another embodiment, the compound has formula (II), wherein Z and Q are both groups -NH-CH2-CO-O-.

[0187] In this specific embodiment, the compound of formula (II) may be selected from the following compounds (III) and (VI) to (XXXII), and for example selected from compounds (III), (VI), (XVII), (XIX), (XXI), (XXII), (XXIV), (XXVII) and (XXVIII), or for example selected from compounds (III), (XIX) or (XXI), and may be, for example, a compound of formula (III) (also known as a DOG-cleaved compound) and its salts, such as its trifluoroacetate salt, and its racemic, enantiomeric and diastereomeric isomers:

[0188] It is worth noting that in the following developed formulas, the secondary amino group can be written indiscriminately as -NH- or -N-.

[0189] Compound III

[0190]

[0191] Compound VI

[0192]

[0193] Compound VII

[0194]

[0195] Compound (VIII):

[0196]

[0197] Compound (IX):

[0198]

[0199] Compound (X):

[0200]

[0201] Compound (XI):

[0202]

[0203] Compound (XII):

[0204]

[0205] Compound (XIII):

[0206]

[0207] Compound (XIV):

[0208] Compound (XV):

[0209] Compound (XVI)

[0210]

[0211] Compound (XVII):

[0212] Compound (XVIII):

[0213] Compound (XIX):

[0214] Compound (XX)

[0215]

[0216] Compound (XXI):

[0217] Compound (XXII)

[0218]

[0219] Compound (XXIII):

[0220] Compound (XXIV):

[0221] Compound (XXV):

[0222] Compound (XXVI):

[0223] Compound (XXVII):

[0224]

[0225] Compound (XXVIII):

[0226]

[0227] Compound (XXIX):

[0228]

[0229] Compound (XXX):

[0230]

[0231] Compound (XXXI)

[0232]

[0233] Compound (XXXII)

[0234]

[0235] As shown in the Examples section, compounds (III), (XVII), (XIX), (XXI), (XXII), (XXIV), (XXVII), and (XXVIII) are, for example, highly effective in formulating stable LNPs that can deliver functional mRNA to target tissues after parenteral administration and, in the case of delivered mRNA encoding an antigen, induce the expression of proteins such as EPO or an immune response.

[0236] Preparation of lipid compounds

[0237] The lipid compounds according to this disclosure can be prepared from commercially available or literature-described starting materials using methods and procedures known to those skilled in the art.

[0238] For example, these lipid compounds can be obtained by covalent coupling between a precursor of a group of formula (I) and a lipid compound or a derivative thereof having a terminal reactive group capable of reacting with said precursor.

[0239] This terminal reactive group can be located directly at the end of the hydrophobic or lipophilic portion of the lipid compound to be converted, or at the end of a spacer arm that is already connected to the hydrophobic or lipophilic portion of the lipid compound.

[0240] The selection of convenient precursors of formula (I) intended to react with lipid compounds to form desired covalent bonds is clearly within the capabilities of those skilled in the art. The precursor only needs to have a group capable of chemically reacting with the group of the lipid compound to form a covalent bond.

[0241] Regarding these starting compounds, namely precursors of the groups of formula (I) and lipid compounds or derivatives thereof to be converted, they can be readily produced by those skilled in the art, for example according to the preparation methods claimed in the following examples.

[0242] Representative examples of convenient precursors of the group of formula (I) include [2-[2(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid, also known as 2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid, and its protected form, for example, of formula (I).

[0243]

[0244] A specific method for preparing such precursors of the group of formula (I) is described in Scheme 1 below.

[0245] Option 1

[0246]

[0247] Covalent coupling between such precursors can be further carried out according to methods known to those skilled in the art regarding the chemical properties of the reactive groups of the precursors of formula (I) and the reactive groups of the lipid compound or its derivatives to be converted.

[0248] Typically, covalent linkages can be formed, for example, through esterification, amidation, or carbamate.

[0249] A specific method for this covalent coupling is described in Scheme 2 below for the synthesis of trifluoroacetate of lipid compound (III), wherein the coupling is obtained by an esterification reaction.

[0250] Option 2

[0251]

[0252]

[0253] It should be understood that other experimental conditions may be used, given typical or specific experimental conditions (i.e., reaction temperature, time, molar amounts of reagents, solvents, etc.), unless otherwise stated. Optimal reaction conditions may vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art using conventional optimization procedures.

[0254] Optional salt formation can be carried out in a conventional manner, such as as disclosed in the following examples.

[0255] Following the coupling reaction, subsequent steps of purification and / or separation of the resulting final product can be advantageously performed. Convenient purification methods are detailed in the following examples. For instance, the purification of the compound can be carried out by preparative high-performance liquid chromatography (HPLC).

[0256] This disclosure can be better understood from the following examples, all of which are intended for illustrative purposes only and are not intended to limit the scope of this disclosure in any way.

[0257] Methods for manufacturing lipid nanoparticles (LNPs)

[0258] This disclosure relates to methods for manufacturing lipid nanoparticles using lipid compounds as disclosed herein.

[0259] In one embodiment, this disclosure relates to a method for manufacturing lipid nanoparticles containing nucleic acids, said method comprising at least the following steps:

[0260] a) Dissolve at least one lipid compound as disclosed herein in a water-miscible organic solvent.

[0261] b) Mix the organic solvent obtained in step a) with an aqueous solvent buffered at a pH ranging from about 3.0 to about 4.5 and containing at least one nucleic acid, and

[0262] c) Obtain the lipid nanoparticles containing the nucleic acid in the aqueous solvent.

[0263] In one embodiment, a method for manufacturing lipid nanoparticles as disclosed herein may include at least the following steps:

[0264] a) Dissolve at least one lipid compound as disclosed herein and at least one lipid selected from the group consisting of neutral lipids, steroids or their esters, and polyethylene glycol-modified lipids in an aqueous miscible organic solvent.

[0265] b) Mix the organic solvent obtained in step a) with an aqueous solvent containing at least one nucleic acid, and

[0266] c) Obtaining lipid nanoparticles containing nucleic acids in an aqueous solvent.

[0267] The lipid compounds disclosed herein may be present in an amount sufficient to construct lipid nanoparticles and encapsulate any loading to be encapsulated. The amount of ionizable lipid compound used in the lipid nanoparticles may be determined by those skilled in the art according to any known technique and may be adapted according to the nature and amount of the loading and other readily present lipids.

[0268] In one embodiment, step a) further includes dissolving at least one lipid selected from the group consisting of:

[0269] Neutral lipids, steroids or their esters, and PEGylated lipids.

[0270] Neutral lipids, steroids or their esters, and PEGylated lipids applicable to this disclosure may be as described herein.

[0271] In one embodiment, step a) may further comprise dissolving at least one neutral lipid, at least one steroid or its ester, and at least one polyethylene glycol-modified lipid in an organic solvent, wherein the lipid compound, the neutral lipid, the steroid or its ester, and the polyethylene glycol-modified lipid are present in the organic solvent in molar amounts of about 30% to about 70% of the total amount of lipid and lipid compound, about 0% to about 50% of the neutral lipid, 20% to about 50% of the steroid or its ester, and about 1% to about 15% of the polyethylene glycol-modified lipid.

[0272] Useful water-miscible organic solvents can be any water-miscible organic solvent capable of dissolving lipid compounds as disclosed herein and any other added lipids. Examples of suitable organic solvents include ethanol or methanol, 1-propanol, isopropanol, tert-butanol, THF, DMSO, acetone, acetonitrile, diethylene glycol dimethyl ether, DMF, 1-4 dioxane, ethylene glycol, glycerol, hexamethylphosphoramide, and hexamethylphosphoric triamine. In one embodiment, the organic solvent may be ethanol and isopropanol.

[0273] Aqueous solvents that can be used in step b) include aqueous buffer solutions.

[0274] Examples of suitable aqueous buffer solutions include acidic buffers such as citrate buffer, sodium acetate buffer, succinate buffer, borate buffer, or phosphate buffer. For example, an aqueous buffer solvent could be a citrate buffer solution or an acetate buffer solution.

[0275] The pH range of the aqueous solvent can be from about 3.0 to about 4.5, for example from about 3.5 to about 4.5, and for example about 4.0.

[0276] In step b), the organic solvent and the aqueous solvent can be mixed at an organic solvent:aqueous solvent ratio 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, it can be a ratio of about 1:3.

[0277] According to one embodiment, the water-miscible organic solvent and the aqueous solvent can be mixed in step b) at a flow rate ranging from about 0.01 ml / min to about 12 ml / min. In some embodiments, the flow rate can range from about 0.02 ml / min to about 10 ml / min, from about 0.5 ml / min to about 8 ml / min, from about 1 ml / min to about 6 ml / min, or about 4 ml / min.

[0278] The mixing step can be performed by any method known in the art. For example, the two solvents can be mixed using a T-tube or a Y-connector. Alternatively, mixing can be performed by laminar flow mixing using a microfluidic micromixer, as described by Belliveau et al. (2012).

[0279] As indicated, the aqueous solvent in step b) contains nucleic acid. In one embodiment, the nucleic acid may encode at least one antigen. Suitable nucleic acids may be, for example, as detailed herein.

[0280] The method may further include the step of increasing the pH from acidic to neutral or slightly above neutral.

[0281] In yet another embodiment, the method may include step d): increasing the pH of the aqueous solvent containing the lipid nanoparticles obtained in step c) to a pH ranging from about 5.0 to about 8.5, for example from about 5.5 to about 8.0, for example from about 6.0 to about 7.5 and for example from about 6.5 to about 7.0.

[0282] The step of increasing pH can be performed by any method known in the art.

[0283] For example, pH changes can be achieved through dialysis or percolation.

[0284] According to one embodiment, step d) of the method disclosed herein may further include at least one step of dialysis or percolation of lipid nanoparticles. The dialysis or percolation step may be performed with an aqueous solvent having a pH range from about 5.0 to about 8.5, for example from about 5.5 to about 8.0, for example from about 6.0 to about 7.5, and for example from about 6.5 to about 7.0.

[0285] Increasing the pH from acidic (i.e., from about 3.0 to about 4.5) to a more neutral or slightly above-neutral pH (i.e., from about 5.0 to about 8.5) advantageously results in the ionizable lipid compounds as disclosed herein cleaving and rearranging into lipid compounds of formula (IV), (Va), or (Vb), as detailed below. This allows lipid nanoparticles as disclosed herein to exhibit a more neutral surface charge, which is beneficial for their distribution in the organism of the individual to which the lipid nanoparticles are applied, thereby reaching target cells.

[0286] 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.

[0287] 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.

[0288] In one implementation, the volumetric molar osmotic pressure concentration is adjusted to achieve a final weight molar osmotic pressure concentration close to 290 mOsmol / kg, thereby allowing the isotonic solution to be injected into the body.

[0289] 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).

[0290] According to one embodiment, this disclosure relates to lipid nanoparticles that can be obtained according to the manufacturing methods disclosed herein.

[0291] According to another embodiment, this disclosure relates to a method for manufacturing a pharmaceutical composition, said method comprising at least the following steps:

[0292] i) To manufacture at least one lipid nanoparticle according to the method disclosed herein, and

[0293] ii) Combine the lipid nanoparticles obtained in step i) with at least one pharmaceutically acceptable excipient or carrier.

[0294] According to another embodiment, this disclosure relates to a method for manufacturing an immunogenic composition, the method comprising at least the following steps:

[0295] i) To manufacture at least one lipid nanoparticle according to the method disclosed herein, said lipid nanoparticle containing at least one nucleic acid encoding at least one antigen, and

[0296] ii) Combine the lipid nanoparticles obtained in step i) with at least one pharmaceutically acceptable excipient or carrier.

[0297] Pharmaceutical compositions and immunogenic compositions applicable to this disclosure are described in more detail below.

[0298] In one embodiment, the lipid nanoparticles of this disclosure may be made of formula (I), (II) or (IIa), and for example formula (III) to (XXXII), such as formula (III), (VI), (XVII), (XIX), (XXI), (XXII), (XXIV), (XXVII) and (XXVIII), and for example lipid compounds of formula (III), (XIX) or (XXI), and for example lipid compounds of formula (III).

[0299] In another embodiment, the lipid nanoparticles disclosed herein can be manufactured using DSPC or DOPE as neutral lipids, cholesterol as a steroid, and PEG-PE (PEG2000-PE) or DMG-PEG (DMG-PEG2000) as polyethylene glycol-modified lipids.

[0300] In another embodiment, the lipid nanoparticles disclosed herein can be manufactured using lipid compounds of formulas (III) to (XXXII), DSPC as a neutral lipid, cholesterol as a steroid, and PEG-PE (PEG2000-PE) as a polyethylene glycol-modified lipid.

[0301] In another embodiment, the lipid nanoparticles disclosed herein can be manufactured using lipid compounds of formulas (III), (VI), (XVII), (XIX), (XXI), (XXII), (XXIV), (XXVII) and (XXVIII), DSPC as a neutral lipid, cholesterol as a steroid, and PEG-PE (PEG2000-PE) as a polyethylene glycol-modified lipid.

[0302] In another embodiment, the lipid nanoparticles disclosed herein can be manufactured using lipid compounds of formula (III), (XIX) or (XXI), DSPC as a neutral lipid, cholesterol as a steroid, and PEG-PE (PEG2000-PE) as a polyethylene glycol-modified lipid.

[0303] In another embodiment, the lipid nanoparticles disclosed herein can be manufactured using lipid compounds of formula (III), DSPC as a neutral lipid, cholesterol as a steroid, and PEG-PE (PEG2000-PE) as a polyethylene glycol-modified lipid.

[0304] lipid nanoparticles

[0305] Lipid nanoparticles (LNP)

[0306] This disclosure relates to lipid nanoparticles that comprise at least a lipid compound of formula (IV):

[0307] HO-L(IV)

[0308] Where L is a lipophilic or hydrophobic tail group as described herein, for example, C 10 To C 55 Lipophilic or hydrophobic tail groups,

[0309] And at least one nucleic acid.

[0310] In some embodiments, the lipid nanoparticles disclosed herein may comprise at least one lipid compound of formula (Va) or (Vb):

[0311] HO-R1(Va) or OH-A-R1(Vb)

[0312] R1 and A are as previously defined.

[0313] In addition, the lipid nanoparticles disclosed herein may contain at least one selected from the following: lipid neutral phospholipids or sphingolipids, steroids or their esters, and polyethylene glycol-modified lipids.

[0314] When lipid nanoparticles move from an acidic pH (i.e., from about 3.0 to about 4.5) to a more neutral or slightly above-neutral pH (i.e., from about 5.0 to about 8.5), the cleavage and rearrangement of the ionizable lipid compounds disclosed herein produce lipid compounds of formula (IV), (Va), or (Vb). These lipid compounds are neutral, non-ionizable compounds.

[0315] The diameter of lipid nanoparticles makes them suitable for systemic application, such as parenteral, intramuscular, intradermal, or subcutaneous administration. Typically, the Z-average size of lipid nanoparticles is less than 600 nanometers (nm), for example, less than 400 nm.

[0316] In one implementation, the Z-average size of the LNP is less than 200 nm. This size is advantageously compatible with sterile filtration and is best suited for lymphatic migration after intramuscular or subcutaneous administration. This size is also suitable for intravenous administration, as larger particle injections may induce capillary thrombosis.

[0317] In some embodiments, the Z-average diameter of the lipid nanoparticles can range from about 20 nm to about 300 nm, for example from about 20 nm to about 250 nm, for example from about 30 nm to about 200 nm, about 40 nm to about 180 nm, from about 60 nm to about 170 nm, from about 80 nm to about 160 nm, and from about 90 nm to about 150 nm. In one embodiment, the diameter of the nanoparticles can range from about 90 nm to about 150 nm.

[0318] The “Z-mean size” of lipid nanoparticles can be determined by dynamic light scattering (DLS). The Z-mean size, or Z-average mean, used in DLS is also known as the cumulative mean. It is the primary and most stable parameter generated by this technique. The Z-mean is defined as the ‘harmonic intensity average particle diameter’. The Z-mean size can be measured using a zeta sizer Nano ZS light scattering instrument (Malvern Instruments). For accurate particle size determination using Nano ZS, the viscosity of the buffer and the refractive index of the material (PBS: v = 1.02 cP, RI = 1.45) must be provided to the instrument software.

[0319] Because minute dimensional variations may occur during manufacturing, a variation of up to 20%-30% is acceptable and considered within the specified size range. Alternatively, size can be determined by filtration screening. For example, if at least 90%, such as at least 95%, such as at least 97%, of the particles pass through a "sieve-type" filter of a specified size, the particle formulation is smaller than the specified size.

[0320] The polydispersity index (PI) is a measure of the uniform or non-uniform size distribution of individual lipid nanoparticles in a mixture of lipid nanoparticles, and indicates the breadth of particle distribution in the mixture. The PI can be determined, for example, as described herein.

[0321] In one embodiment, the polydispersity index of the nanoparticles described herein, as measured by dynamic light scattering, is 0.5 or less, for example 0.4 or less, for example 0.3 or less, or even for example 0.2 or less.

[0322] In one embodiment, the lipid nanoparticles are colloidally stable, in such a 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 time period, such as from at least two hours to several months, for example, at least 1, 2, 3, 4, 5, 6, or 12 months.

[0323] Lipid nanoparticles may contain or encapsulate at least one nucleic acid.

[0324] Nucleic acids can be encapsulated in lipid nanoparticles and / or adsorbed on the outer surface of lipid nanoparticles. Lipid compounds of formula (IV), (Va), or (Vb) can form complexes with nucleic acids and / or encapsulate nucleic acids. Alternatively, the lipid compounds can be contained in vesicles encapsulating nucleic acids.

[0325] Lipid nanoparticles possess a total surface charge, which is the sum of positive and negative charges on the particle surface and is represented by a zeta potential. The zeta potential is the potential difference between the dispersion medium and the stationary layer of fluid attached to the dispersed particles. The zeta potential is widely used to quantify the magnitude of the charge at the bilayer.

[0326] The zeta potential can be calculated using theoretical models and determined experimentally using electrophoretic mobility or dynamic electrophoretic mobility measurements. Electrophoresis can be used to estimate the zeta potential of particles. In practice, the zeta potential of a dispersion can be measured by applying an electric field to the dispersion. Particles with a zeta potential in the dispersion will migrate towards an electrode with the opposite charge at a velocity proportional to the magnitude of the zeta potential. This velocity can be measured using the laser Doppler anemometer technique. The frequency shift or phase shift of the incident laser beam caused by these moving particles can be measured as particle mobility, and this mobility can be converted into a zeta potential by inputting the dispersant viscosity and dielectric constant and applying Smoluchowski theory. Electrophoretic velocity is proportional to electrophoretic mobility, which is a measurable parameter. Several theories relate electrophoretic mobility to zeta potential.

[0327] Suitable systems such as the Nicomp 380ZLS system or the Malvern nanoZS can be used to determine the zeta potential. These systems are typically used to measure the electrophoretic mobility and stability of charged particles in liquid suspensions. These values ​​are predictors of the repulsive forces exerted by the suspended particles and are directly related to the stability of the colloidal system.

[0328] At neutral pH, the zeta potential of lipid nanoparticles as disclosed herein is close to neutral. Indeed, at neutral or slightly above neutral pH (from 5.0 / 5.5 to 8.5), ionizable and cleavable lipids as disclosed herein have undergone self-rearrangement, resulting in the departure of groups of formula (I) and the remaining neutral, uncharged hydrophobic or lipophilic tail groups.

[0329] One advantage is that the near-zero zeta potential facilitates particle mobility in the body, reduces opsonization, and enhances access to target tissues.

[0330] In one embodiment, the zeta potential of the lipid nanoparticles can range from about -3 mV to about +3 mV, for example from about -1 mV to about +1 mV, and for example from about -0.5 mV to about +0.5 mV at a pH of 6.0 to 7.5.

[0331] The lipid nanoparticles described herein can be formed by adjusting (during preparation) the positive and negative charges, which depend on the charge ratio of an ionizable lipid compound (cationic charge of quaternary ammonium from the terminal group of formula (I): N) to a nucleic acid (anionic charge from phosphate: P) as disclosed herein, and by mixing the nucleic acid with the lipid compound. The charges of the ionizable lipid compound and the nucleic acid are those at a selected pH, such as the pH of the preparation process (which is from about 3.0 to about 4.5).

[0332] The + / - (N / P) charge ratio of lipid compounds to nucleic acids disclosed herein can be calculated using the following equation: (+ / - charge ratio) = [(mass of cationic lipids (mol)) * (total positive charge in cationic lipids)] : [(mass of nucleic acids (mol)) * (total negative charge in nucleic acids)].

[0333] Given the loading amount during nanoparticle preparation, those skilled in the art can easily determine the amount of nucleic acid and lipid compounds.

[0334] In one embodiment, the calculated charge ratio of positive to negative charge can range from about 1:1 to about 14:1, for example from about 2:1 to about 12:1, for example from about 4:1 to about 10:1, and for example from about 6:1 to about 8:1, and for example about 6:1.

[0335] In one embodiment, the lipid nanoparticles encapsulating nucleic acids, as disclosed herein, may have a Z-average size of about 80-180 nm and a calculated charge ratio of about 6-12:1, for example, about 3-9:1, N / P.

[0336] In one embodiment, the lipid nanoparticles disclosed herein may comprise at least one cleavable lipid compound as disclosed herein.

[0337] Indeed, even when lipid nanoparticles obtained according to the methods disclosed herein are subjected to neutral pH or slightly above neutral pH (5.0 / 6.0 to 8.5) to cleave ionizable cleavable cationic lipid compounds as disclosed herein, not all of these compounds can be cleaved. For example, lipid compounds located within the lipid nanoparticles (i.e., in the core of the LNP) can be protected from pH changes and may not undergo a cleavage process. The presence of the remaining cleavable cationic and / or ionizable lipid compounds as disclosed herein can be observed by methods known in the art, such as TLC or HPLC.

[0338] The lipid nanoparticles disclosed herein may further comprise at least one lipid selected from the following: neutral lipids, steroids or their esters, and polyethylene glycol-modified lipids.

[0339] neutral lipids

[0340] The compositions or lipid nanoparticles disclosed herein may contain neutral lipids. The presence of neutral lipids can improve the structural stability of lipid nanoparticles. Neutral lipids can be appropriately selected given the delivery efficiency of nucleic acids.

[0341] Neutral lipids differ from lipid compounds of formula (IV), (Va), or (Vb). Neutral lipids are either non-ionizable or neutral zwitterionic compounds at a selected pH.

[0342] Neutral lipids that can be used in this disclosure may be selected from phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and ceramides.

[0343] Phosphatidylcholine and phosphatidylethanolamine are zwitterionic lipids. Sphingomyelin and ceramides are non-ionizable lipids.

[0344] As examples of phosphatidylcholine that may be used in this disclosure, references may be made to DSPC (1,2-distearyl-sn-glycerol-3-phosphate choline), DPPC (1,2-dipalmitoyl-sn-glycerol-3-phosphate choline), DMPC (1,2-dimyristoyl-sn-glycerol-3-phosphate choline), POPC (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline), and DOPC (1,2-dioleoyl-sn-glycerol-3-phosphate choline).

[0345] As examples of phosphatidylethanolamines that may be used in this disclosure, references may be made to DOPE (1,2-diolenoyl-sn-glycerol-3-phosphate ethanolamine), DPPE (1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine), DMPE (1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine), DSPE (1,2-distearate-s / i-glycerol-3-phosphate ethanolamine), DLPE (1,2-dilauroyl-sm-glycerol-3-phosphate ethanolamine), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, or 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE).

[0346] Neutral lipids can be selected from phosphatidylcholine, such as DSPC, DPPC, DMPC, POPC, DOPC; phosphatidylethanolamine, such as DOPE, DPPE, DMPE, DSPE, DLPE; sphingomyelin; and ceramides.

[0347] In one embodiment, the neutral lipids applicable to this disclosure may be DSPC, DOPC, and DOPE, and may be, for example, DSPC or DOPE.

[0348] Neutral lipids may be present in step a) of the method for formulating lipid nanoparticles 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.

[0349] Neutral lipids may be present in lipid nanoparticles 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 of formula (IV), (Va) or (Vb) that may be present in lipid nanoparticles.

[0350] Neutral lipids may be lipid compounds of formula (IV), (Va) or (Vb): the molar ratio of neutral lipids present in lipid nanoparticles as disclosed herein may 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 for example about 5:1.

[0351] Steroids or their esters

[0352] The compositions or lipid nanoparticles disclosed herein may contain steroids (or sterols) or their esters. The presence of sterols or sterol esters can improve the structural stability of lipid nanoparticles.

[0353] Sterols or steroids that may be used in this disclosure may be selected from cholesterol or its derivatives, ergosterol, sterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigmaster-5,22-dien-3-ol), lanosterol (8,24-lanosterdien-3b-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), and yeast sterol (5α-cholestadiene-8,24-dien-3-ol). -3β-ol), encholestanol (5α-cholest-7-en-3β-ol), diosgenin ((3β,25R)-spirost-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitosterol, campesterol (campester-5-en-3β-ol), campesterol (5a-campester-3b-ol), 24-methylene cholesterol (5,24(28)-cholestadien-24-methylen-3β-ol).

[0354] Steroid or sterol esters refer to esters of carboxylic acids and the hydroxyl groups of the steroid. In addition to the carboxyl moiety, suitable carboxylic acids also contain saturated or unsaturated, straight-chain or branched alkyl groups. In some embodiments, the alkyl group may be C1-C2. 20 Saturated or unsaturated straight-chain or branched alkyl groups, for example, C2-C 18 For example, C4-C 16 For example, C8-C 12 The carboxylic acid may be a saturated or unsaturated straight-chain or branched alkyl group, and in other embodiments, it may be a fatty acid. For example, the fatty acid may be octanoic acid, capric acid, lauric acid, stearic acid, heptadecanic acid, oleic acid, linoleic acid, or arachidic acid.

[0355] In one embodiment, the sterol esters applicable to this disclosure may be cholesterol esters.

[0356] The sterol esters or steroid esters that may be used in this disclosure may be selected from heptadecanoyl cholesterol ester (cholestylacetic-5-ene-3β-yl heptadecanoyl cholesterol ester), oleic cholesterol ester, and stearic cholesterol ester.

[0357] Sterols or steroids or their esters that may be used in this disclosure may be selected from cholesterol or its derivatives, ergosterol, sterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigmaster-5,22-dien-3-ol), lanosterol (8,24-lanosterdien-3β-ol), 7-dehydrocholesterol, dihydrolanosterol (24,25-dihydrolanosterol), yeast sterol (5α-cholest-8,24-dien-3β-ol), and enolanol (5α-cholest-7-en-3β-ol). - alcohol), diosgenin ((3β,25R)-spirost-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitosterol, campesterol (campester-5-en-3β-ol), campesterol (5a-campester-3b-ol), 24-methylene cholesterol (5,24(28)-cholestadien-24-methylen-3β-ol), heptadecanoyl cholesterol ester (heptadecanoyl cholesterol-5-en-3β-yl ester), oleic acid cholesterol ester, and stearic acid cholesterol ester.

[0358] Alternatively, the sterols used in this disclosure may be cholesterol derivatives, such as oxidized cholesterol.

[0359] The oxidized cholesterol applicable to this disclosure can be 25-hydroxy cholesterol, 27-hydroxy cholesterol, 20α-hydroxy cholesterol, 6-keto-5α-hydroxy cholesterol, 7-keto-cholesterol, 7β,25-hydroxy cholesterol, and 7β-hydroxy cholesterol. For example, oxidized cholesterol can be 25-hydroxy cholesterol and 20α-hydroxy cholesterol, and for example, it can be 20α-hydroxy cholesterol.

[0360] In one embodiment, the sterol or steroid or its ester applicable to this disclosure may be cholesterol, cholesterol ester, or a cholesterol derivative, such as oxidized cholesterol. In one embodiment, the sterol or steroid or its ester applicable to this disclosure may be cholesterol or cholesterol ester, and may be, for example, cholesterol.

[0361] Sterols or steroids or their esters may be present in step a) of the method for formulating lipid nanoparticles 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 ionizable lipid compounds as disclosed herein.

[0362] Sterols or steroids or their esters may be present in lipid nanoparticles 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 of formula (IV), (Va), or (Vb) that may be present in lipid nanoparticles.

[0363] Sterols or esters thereof may be present in lipid nanoparticles as disclosed herein in a molar ratio of lipid compound of formula (IV), (Va), or (Vb) to steroid or ester thereof, said molar ratio being 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 from about 1.3:1 to about 1:1.3.

[0364] PEGylated lipids

[0365] The compositions or lipid nanoparticles disclosed herein may include polyethylene glycolated (or PEG-) lipids.

[0366] The PEG-modified lipids under consideration include, but are not limited to, those having one or more C6-C lengths. 20 The alkyl chains are covalently attached to polyethylene glycol chains up to 5 kDa in length. Adding PEG-modified lipids to lipid nanoparticle compositions as disclosed herein can prevent complex aggregation and also provide a means of increasing cycle life and enhancing the delivery of the composition or lipid nanoparticles to target cells.

[0367] Suitable polyethylene glycol-modified lipids can be, for example, polyethylene glycol-modified diacylglycerols (PEG-DAG), such as l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (DMG-PEG), polyethylene glycol-modified phosphatidylethanolamine (PEG-PE), PEG-Succinate diacylglycerols (PEG-S-DAG), such as 4-O-(2',3'-bis(tetradecanoyloxy)propyl-l-O-(co-methoxy(polyethoxy))

[0368] Ethyl) succinate (PEG-S-DMG), polyethylene glycol-modified ceramide (PEG-cer), or PEG dialkoxypropyl carbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecoxy)propyl)carbamate, 2,3-di(tetradecoxy)propyl-N-(co-methoxy(polyethoxy)ethyl)carbamate, or mPEG-N,N-bistetradecylacetamide (also known as 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide or ALC-0159).

[0369] In one embodiment, the polyethylene glycol-modified lipids suitable for use in this disclosure may be selected from PEG-DAG, DMG-PEG, PEG-PE, PEG-S-DAG, PEG-S-DMG, PEG-cer, or mPEG-N,N-bistetradecylacetamide, or PEG-dialkoxypropylcarbamate.

[0370] For example, the polyethylene glycol-modified lipids applicable to this disclosure may be DMG-PEG, PEG-PE, or mPEG-N,N-bistetradecylacetamide.

[0371] In some embodiments, the polyethylene glycol-modified lipids applicable to this disclosure may be DMG-PEG or PEG-PE.

[0372] In some embodiments, the polyethylene glycol-modified lipids applicable to this disclosure may be mPEG-N,N-bistetradecylacetamide.

[0373] Polyethylene glycol-modified lipids may be present in step a) of a method for formulating lipid nanoparticles as disclosed herein in a molar amount ranging 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 ionizable lipid compounds.

[0374] Polyethylene glycol-modified lipids may be present in lipid nanoparticles as disclosed herein in a molar amount ranging 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 of formula (IV), (Va), or (Vb) that may be present in lipid nanoparticles.

[0375] Polyglycolated lipids and lipid compounds of formula (IV), (Va) or (Vb) may be present in lipid nanoparticles in a molar ratio of ionizable lipid compounds to polyethylene glycolated lipids 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 about 33:1 or about 14:1.

[0376] In one embodiment, in addition to lipid compounds of formula (IV), (Va), or (Vb), the lipid nanoparticles may also comprise at least one neutral lipid, at least one steroid or its ester, and at least one polyethylene glycol-modified lipid.

[0377] Neutral lipids, steroids or their esters, and polyethylene glycol-modified lipids can be as described herein.

[0378] In one embodiment, the lipid nanoparticles described herein may comprise a lipid compound of formula (IV), (Va), or (Vb), a neutral lipid, a steroid or an ester thereof, and a polyethylene glycol-modified lipid, wherein the molar amount is about 30% to about 70% of the total amount of lipid compound, about 0% to about 50% of the neutral lipid, about 20% to about 50% of the steroid or an ester thereof, and about 1% to about 15% of the polyethylene glycol-modified lipid relative to the total amount of lipid and lipid compound.

[0379] In one embodiment, the lipid nanoparticles described herein may comprise a lipid compound of formula (IV), (Va), or (Vb), a neutral lipid, a steroid or an ester thereof, and a polyethylene glycol-modified lipid, wherein the molar amount is from about 30% to about 60% of the total amount of lipid compound, from about 5% to about 30% of the neutral lipid, from about 30% to about 48% of the steroid or an ester thereof, and from about 1.5% to about 5% of the polyethylene glycol-modified lipid, relative to the total amount of lipid and lipid compound.

[0380] In one embodiment, the lipid nanoparticles described herein may comprise a lipid compound of formula (IV), (Va), or (Vb), a neutral lipid, a steroid or an ester thereof, and a polyethylene glycol-modified lipid, wherein the molar amount is about 35% to about 50% of the total amount of lipid compound, about 10% to about 16% of the neutral lipid, about 38.5% to about 46.5% of the steroid or an ester thereof, and about 1.5% of the polyethylene glycol relative to the total amount of lipid and lipid compound.

[0381] As one embodiment, the lipid nanoparticles disclosed herein may comprise about 35% of a lipid compound of formula (IV), (Va), or (Vb) relative to the total amount of lipids and lipid compounds, about 16% of neutral lipids, about 46.5% of steroids or esters thereof, and about 1.5% of polyethylene glycol.

[0382] As another embodiment, the lipid nanoparticles disclosed herein may comprise about 50% of a lipid compound of formula (IV), (Va), or (Vb) relative to the total amount of lipids and lipid compounds, about 10% of neutral lipids, about 38.5% of steroids or esters thereof, and about 1.5% of polyethylene glycol.

[0383] In one embodiment, the molar ratio of the lipid compound of formula (IV), (Va), or (Vb) to a neutral lipid, a steroid, or an ester thereof to a polyethylene glycol-modified lipid may 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.

[0384] In one embodiment, the molar ratio of the lipid compound of formula (IV), (Va), or (Vb) to a neutral lipid, a steroid, or an ester thereof to a polyethylene glycol-modified lipid may be about 35 / 16 / 46.5 / 1.5 or about 50 / 10 / 38.5 / 1.5.

[0385] Nucleic acid

[0386] The lipid nanoparticles disclosed herein may contain at least one anionic or polyanionic therapeutic agent. The therapeutic agent suitable for use in this disclosure may be a nucleic acid.

[0387] According to this disclosure, nucleic acids can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), such as RNA, such as in vitro transcribed RNA (IVT RNA) or synthetic RNA.

[0388] Nucleic acids, according to this disclosure, include genomic DNA, cDNA, mRNA, recombinant and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded and can be linear or covalently closed to form a circular molecule. Nucleic acids can be used for introduction into cells (i.e., transfection), for example, in the form of RNA, which can be prepared by in vitro transcription from a DNA template. Furthermore, RNA can be modified prior to application by stabilizing sequences, capping, and polyadenylation.

[0389] Nucleic acids can be of eukaryotic or prokaryotic origin, and are, for example, derived from humans, animals, plants, bacteria, yeast, or viruses. They can be obtained by any technique known to those skilled in the art, and for example by screening libraries, by chemical synthesis, or alternatively by a combination of methods, including chemical or enzymatic modification of sequences obtained by screening libraries. They can be chemically modified.

[0390] Nucleic acids can be contained in vectors. Vectors are known to those skilled in the art and can include plasmid vectors, granular vectors, bacteriophage vectors (such as λ phage), viral vectors (such as adenovirus or baculovirus vectors), or artificial chromosome vectors (such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or PI artificial chromosomes (PAC)). Vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and typically contain the desired coding sequence and the appropriate DNA sequence required to express the operatively linked coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are typically used to engineer and amplify a desired DNA fragment and may lack the functional sequence required to express the desired DNA fragment.

[0391] In one embodiment, the nucleic acid may be selected from double-stranded RNA (dsRNA); single-stranded RNA (ssRNA); double-stranded DNA (dsDNA); single-stranded DNA (ssDNA); and combinations thereof.

[0392] In one implementation, the nucleic acid may be selected from: spontaneous RNA (mRNA); antisense oligonucleotide (ASO); short interfering RNA (siRNA); self-amplifying RNA (saRNA); microRNA (miRNA); small nuclear RNA (snRNA); small nucleolar RNA (snoRNA); self-amplifying RNA (saRNA); plasmid DNA (pDNA); closed-terminal DNA (ceDNA); and combinations thereof.

[0393] In another implementation, the nucleic acid may be selected from spontaneous RNA (mRNA); antisense oligonucleotide (ASO); short interfering RNA (siRNA); self-amplifying RNA (saRNA); microRNA (miRNA); plasmid DNA (pDNA) and combinations thereof.

[0394] In another implementation, the nucleic acid may be selected from self-propelled RNA (mRNA); short interfering RNA (siRNA); self-amplifying RNA (saRNA); microRNA (miRNA); and combinations thereof.

[0395] In another implementation, the nucleic acid can be messenger RNA (mRNA).

[0396] In one embodiment, the nucleic acid is mRNA. In some embodiments, the nucleic acid may be RNA encoding a protein or enzyme. Such polynucleotides can be used as therapeutic agents capable of being expressed by target cells to promote the production of functional enzymes or proteins. For example, in some embodiments, when target cells express at least one polynucleotide, a functional enzyme or protein lacking in the cell or individual is produced.

[0397] Target cells are cells to be directed to or targeted by the compositions or lipid nanoparticles disclosed herein. Target cells may include specific tissues or organs. In some embodiments, target cells may be 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.

[0398] mRNA

[0399] The term "RNA" refers to a molecule that contains ribonucleotide residues and is, for example, composed entirely or substantially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of the β-D-furanose group.

[0400] The terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, or recombinant RNA.

[0401] These can be natural or artificially derived sequences, and include, for example, mRNA (messenger RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), siRNA (silent RNA), miRNA (microRNA), mtRNA (mitochondrial RNA), shRNA (short hairpin RNA), tmRNA (transfer messenger RNA), vRNA (viral RNA), single-stranded, double-stranded and / or base-paired RNA (ssRNA, dsRNA and bpRNA, respectively), blunt-ended or non-blunt-ended RNA, mature and immature mRNA, coding and non-coding RNA, hybrid sequences or synthetic or semi-synthetic sequences of oligonucleotides (modified or otherwise), and mixtures thereof.

[0402] Therefore, these can be messenger RNAs (mRNAs), including mature and immature mRNAs, such as pre-mRNAs or heterogeneous nuclear mRNAs (hnRNAs) and mature mRNAs. Thus, RNA molecules as disclosed herein also include monocistronic and polycistronic messenger RNAs.

[0403] For clarity, mRNA encompasses any RNA molecule that can be translated into a protein by a eukaryotic host. An RNA molecule typically refers to an RNA molecule containing a sequence encoding a target protein, which can be translated by a eukaryotic host, and which begins with a start codon (ATG) and terminates, for example, with a stop codon (i.e., TAA, TAG, TGA).

[0404] RNA can be naturally occurring RNA or modified RNA that differs from naturally occurring RNA through 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, adding to one or more ends or inside the RNA, for example, at least one nucleotide in the RNA. 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 RNA.

[0405] In one implementation, the RNA is mRNA (messenger RNA). mRNA can be a transcript that can be produced using DNA as a template and encodes a peptide or protein.

[0406] mRNA typically contains a 5' cap, a 5' untranslated region (5-UTR), a protein or peptide coding region, a 3' untranslated region (3'-UTR), and a 3' poly-A tail. mRNA has a limited half-time in cells and in vitro. For example, mRNA can be produced by in vitro transcription using a DNA template. Alternatively, RNA can be obtained through chemical synthesis. In vitro transcription methods are known to those skilled in the art. For example, several commercially available in vitro transcription kits are available.

[0407] RNA can be synthesized in vitro in a cell-free system using appropriate cell extracts and a suitable DNA template. For example, cloning vectors are used to produce transcripts. The promoter used to control transcription can be any promoter used 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, such as cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA. For example, cloning vectors are used to produce transcripts commonly referred to as transcription vectors.

[0408] In one implementation, RNA can encode a protein or peptide. That is, if present in a suitable environment, such as within a cell (e.g., an antigen-presenting cell, such as a dendritic cell), RNA can be expressed to produce the protein or peptide it encodes.

[0409] RNA stability and translation efficiency can be modified as needed. Within the scope of this disclosure, RNA modification refers to any modification of RNA that is not naturally present in said RNA.

[0410] According to a general implementation scheme, the mRNA disclosed herein may comprise or consist of the following general formulas:

[0411] [5'cap]w-[5'UTR]x-[target gene]-[3'UTR]y-[polymer A]z

[0412] [5'UTR] and [3'UTR] are untranslated regions (UTRs).

[0413] [5'UTR] contains the Kozak sequence.

[0414] [Target gene] can be any gene that encodes the target protein.

[0415] The [5' cap] contains methylguanine nucleotides, which are linked to the mRNA via a 5'-to-5' junction.

[0416] Where [A+] is a cluster (A) tail, and

[0417] Where w, x, y, and z are the same or different, and are equal to 0 or 1.

[0418] According to one implementation scheme, the mRNA disclosed herein can be composed of the following general formula:

[0419] [5' cap]-[5'UTR]-[target gene]-[3'UTR]-[polymer A]

[0420] [5'UTR] and [3'UTR] are non-translated regions.

[0421] [5'UTR] contains the Kozak sequence.

[0422] Where [target gene] is any nucleic acid that encodes the target protein,

[0423] The [5' cap] contains methylguanine nucleotides, which are linked to the mRNA via a 5'-5' bond, and

[0424] [A+] is a cluster (A) tail.

[0425] It is important to note that the Kozak sequence refers to a sequence that is typically a co-occurring sequence on eukaryotic mRNA and plays a major role in the initiation of the translation process. Kozak sequences and Kozak co-occurring sequences are well-known in the art.

[0426] It should also be noted that the poly(A) tail is composed of multiple adenosine monophosphates, which are well known in the art. The poly(A) tail is typically generated in a step called polyadenylation, one of the post-translational modifications that usually occurs during the production of mature messenger RNA; such poly(A) tails contribute to the stability and half-life of the mRNA and can be of variable length. For example, 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.

[0427] The [3'UTR] does not express any protein. The purpose of the [3'UTR] is to increase the stability of the mRNA. According to one implementation, the α-globin UTR is chosen because it is known to lack instability.

[0428] Advantageously, the sequence corresponding to the target gene can be codon-optimized to achieve satisfactory protein yields in the host under consideration.

[0429] As disclosed herein, RNA molecules can be of variable length. Therefore, they can be short RNA molecules, such as RNA molecules shorter than about 100 nucleotides; or long RNA molecules, such as those longer than about 100 nucleotides, or even longer than about 300 nucleotides.

[0430] RNA, such as mRNA, can encompass synthetic or artificial RNA molecules, but also naturally occurring RNA molecules.

[0431] According to this disclosure, RNA molecules, such as mRNA, can encompass the following categories:

[0432] (i) Capped, unmodified RNA molecules;

[0433] (ii) Capped and modified RNA molecules;

[0434] (iii) Uncapped, unmodified RNA molecules;

[0435] (iv) Uncapped modified RNA molecules.

[0436] Capped RNA molecules and uncapped RNA molecules

[0437] According to the most general implementation, a "capped RNA molecule" refers to an RNA molecule whose 5' end is capped with guanosine or a modified guanosine (e.g., 7-methylguanosine (m... 7 G)) link, wherein the guanosine is linked to a 5' to 5' triphosphate bond or the like. This definition is consistent with the most widely accepted definition of a 5' cap (e.g., a naturally occurring and / or physiological cap).

[0438] In the sense of this disclosure, "cap analogues" include those biologically equivalent to 7-methylguanosine (m 7 The cap of G) is linked to a 5' to 5' triphosphate bond, and therefore can be substituted without impairing the protein expression of the corresponding messenger RNA in the eukaryotic host.

[0439] As an example of a hat, m can be mentioned. 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’-OGpppG, m2 7’,2’-O GpppG, m2 7’,2’-O Gpp s p s G, or m2 7’,2’-O Gppp s p s G.

[0440] Examples of synthetic caps and / or cap analogues may be selected from: glycerol groups, reverse deoxygenated debase residues (partially), 4',5' methylene nucleotides, 1-(β-D-erythrofuranosyl) nucleotides, 4'-thionucleotides, carbocyclic nucleotides, 1,5-dehydrated hexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, threo-pentafuranosyl nucleotides, acyclic 3',4'-open nucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3'-3'-reverse nucleotide moiety, 3'-3'-reverse debase moiety, 3'-2'-reverse nucleotide moiety, 3'-2'-reverse debase moiety, 1,4-butanediol phosphate, 3'-aminophosphate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3'-thiophosphate, dithiophosphate, or bridged or non-bridged methylphosphonate moiety.

[0441] Other examples of synthetic caps or cap analogues include ARCA cap analogues, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-denitro-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0442] It is worth noting that, in synthetic caps, some of the caps mentioned above are suitable as analogues, rather than other caps that might conversely hinder protein expression. Those skilled in the art will understand this distinction.

[0443] For reference and in a non-limiting manner, the anti-reverse cap analogue (ARCA) 3'-O-Me-m 7 The structure of the G(5')ppp(5')G cap analogue is presented below:

[0444]

[0445] For example, ARCA cap analogs are examples of cap analogs used in in vitro transcription: they are modified caps with a 3'OH group (closer to m) 7 G) is replaced by -OCH3. However, 100% of the transcript synthesized with ARCA at the 5' end is translatable, resulting in a strong stimulatory effect on translation.

[0446] The provision of RNA having a 5'-cap or a 5'-cap analogue can be achieved by in vitro transcription of a DNA template in the presence of the 5'-cap or 5'-cap analogue, wherein the 5'-cap is co-transcribed into the resulting RNA strand, 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., a capping enzyme of vaccinia virus).

[0447] "Uncapped RNA molecule" refers to any RNA molecule that does not fall under the definition of "capped RNA molecule".

[0448] Therefore, according to the general implementation scheme, "uncapped mRNA" can refer to mRNA whose 5' end is not linked to 7-methylguanosine by a 5'-5' triphosphate bond or an analogue as defined above.

[0449] Uncapped RNA molecules, such as messenger RNA, can be uncapped RNA molecules with (5')ρρρ(5'), (5')ρρ(5'), (5')ρ(5'), or even (5')OH extremes. These RNA molecules can be abbreviated as 5'ρρρRNA; 5'ρρRNA; 5'ρRNA; 5' OH RNA. For example, the uncapped RNA molecule disclosed herein is messenger 5'ρρρRNA.

[0450] Therefore, when the RNA molecule is a single-stranded RNA molecule, it can be abbreviated as follows: 5’ppp ssRNA; 5’pp ssRNA; 5’ p ssRNA; 5’OH ssRNA.

[0451] Therefore, when the RNA molecule is a double-stranded RNA molecule, it can be abbreviated as follows: 5’ppp dsRNA; 5’pp dsRNA; 5’ p dsRNA; 5’OH dsRNA.

[0452] In one implementation, the uncapped mRNA disclosed herein is an uncapped single-stranded mRNA.

[0453] According to one implementation scheme, uncapped single-stranded mRNA can be an uncapped messenger. 5’ppp ssRNA.

[0454] In a non-limiting manner, the first base of the uncapped RNA molecule may be adenosine, guanosine, cytosine, or uridine.

[0455] Therefore, uncapped RNA molecules can be uncapped RNA molecules with (5')ppp(5'), (5')pp(5'), (5')p(5'), or even blunt 5' guanosine extremes.

[0456] In one embodiment of this disclosure, the RNA may be free of uncapped 5'-triphosphate. Removal of such uncapped 5'-triphosphate can be achieved by treating the RNA with a phosphatase.

[0457] Modified and unmodified RNA molecules

[0458] RNA may contain further modifications. For example, further modifications to RNA as used in this disclosure may be the extension or truncation of a naturally occurring poly(A) tail, or alterations to the 5'- or 3'-untranslated region (UTR), such as the introduction of a UTR unrelated to the coding region of the RNA, for example, exchanging an existing 3'-UTR for or inserting at least one (e.g., two copies) of a 3'-UTR derived from a globin gene (such as α2-globin, α1-globin, β-globin, e.g., β-globin and, for example, human β-globin).

[0459] In this disclosure, "modified RNA molecule" means an RNA molecule containing at least one modified nucleotide, nucleoside, or base, such as a modified purine or a modified pyrimidine. The modified nucleoside or base may be any nucleoside or base other than A, U, C, or G (for nucleosides, adenosine, uridine, cytidine, or guanosine, respectively; and when referring only to the glycoside portion, adenine, uracil, cytosine, or guanine).

[0460] Therefore, "unmodified RNA molecule" refers to any RNA molecule that is not consistent with the definition of a modified RNA molecule.

[0461] In the sense of this disclosure, the terms “modified and unmodified” are considered to be different from the terms “capped and uncapped”, since the latter specifically refers to the bases at the 5' end of the RNA molecule in the sense of this disclosure.

[0462] In one embodiment, the nucleic acid, such as RNA, may contain at least one modified nucleotide, such as a modified ribonucleotide. The presence of the modified nucleotide may increase the stability of the nucleic acid and / or reduce its cytotoxicity.

[0463] The term stability of RNA refers to its half-life, which is the time it takes for half of its activity, amount, or molecular quantity to be eliminated. In the context of this disclosure, the half-life of RNA indicates the stability of the RNA. The half-life of RNA can affect the duration of RNA expression. RNA with a long half-life can be expected to be expressed over a longer period of time.

[0464] According to one embodiment, "modified RNA molecule" refers to an RNA molecule, such as mRNA, which contains at least one base or sugar modification as described above and, for example, at least one base modification as described herein.

[0465] For example, in one embodiment, 5-methylcytidine may be partially or completely substituted in the RNA applicable to this disclosure, e.g., completely substituted cytidine. Alternatively or additionally, in one embodiment, it may be partially or completely substituted, e.g., completely substituted uridine.

[0466] Examples of modified nucleotides, nucleosides and bases are disclosed in WO 2015 / 024667 A1 in a non-limiting manner.

[0467] Therefore, modified RNA molecules can contain modified nucleotides, nucleosides, or bases, including backbone modifications, sugar modifications, or base modifications.

[0468] Skeleton modifications relevant to this disclosure include modifications in which the phosphate esters of the nucleotide skeleton contained in an RNA molecule as defined herein are chemically modified.

[0469] Sugar modifications relevant to this disclosure include chemical modifications of the sugars in nucleotides of RNA molecules as defined herein.

[0470] Base modifications relevant to this disclosure include chemical modifications to the base portions of nucleotides of RNA. In this context, nucleotide analogs or modifications are, for example, selected from nucleotide analogs suitable for transcription and / or translation of RNA molecules in eukaryotic cells.

[0471] Sugar modification can include the substitution or modification of the 2' hydroxyl (OH) group, which can be modified or substituted by many different "oxy" or "deoxy" substituents.

[0472] Examples of "oxygen"-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), -O(CH2CH2O)nCH2CH2OR; "locked" nucleic acids (LNA), wherein the 2' hydroxyl group is connected to the 4' carbon of the same ribose, for example, via a methylene bridge; and amino (-O-amino, wherein the amino group (e.g., NRR) can be alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxy.

[0473] "Deoxygenation" modifications include hydrogen, amino groups (e.g., NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acids); or amino groups can be attached to sugars via a linker, wherein the linker contains at least one atom from C, N, and O.

[0474] The glycosyl group may also contain at least one carbon with a stereochemical configuration opposite to that of the corresponding carbon in ribose. Therefore, modified RNA can contain nucleotides containing, for example, arabinose as sugars.

[0475] The phosphate backbone can be further modified and incorporated into RNA molecules as described herein. The phosphate groups of the backbone can be modified by replacing at least one oxygen atom with different substituents. Furthermore, modified nucleosides and nucleotides can contain unmodified phosphate moieties completely replaced by modified phosphates as described herein.

[0476] Examples of modified phosphate groups include, but are not limited to, thiophosphates, selenophosphates, boranophosphates, borano phosphate esters, hydrogen phosphonates, aminophosphates, alkyl or aryl phosphonates, and triphosphates. In dithiophosphates, both unlinked oxygen atoms are replaced by sulfur. The phosphate linker can also be modified by replacing the linking oxygen atoms with nitrogen (bridged aminophosphates), sulfur (bridged thiophosphates), and carbon (bridged methylene-phosphonates).

[0477] Modified nucleosides and nucleotides, as described herein, that can be incorporated into modified RNA molecules can be further modified at the nucleobase moiety. For example, nucleosides and nucleotides, as described herein, can be chemically modified at the major groove. In some embodiments, major groove chemical modifications may include amino, thiol, alkyl, or halogen groups.

[0478] For example, the nucleotide analog / modification is selected from base modifications, said base modification being selected from: 2-amino-6-chloropurine nucleoside-5'-triphosphate, 2-aminopurine-nucleoside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methylinosine-5'-triphosphate. 4-Thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallylcytidine-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-iodine-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-deazoadenosine-5'-triphosphate, Azoguanosine-5'-triphosphate, 8-azaadenosine-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 xanthine nucleoside-5'-triphosphate.

[0479] In some embodiments, the modified nucleoside may be selected from: pyridine-4-ketoribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudouridine, 5-propynyluridine, 1-propynyl-pseudouridine, 5-taurate methyluridine, 1-taurate methyl-2-thiouridine, 1-taurine ... 4-Thio-uridine sulfonate, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-denitro-pseudouridine, 2-thio-1-methyl-1-denitro-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.

[0480] In some embodiments, the modified nucleosides and nucleotides include 5-aza-cytidine, pseudocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudocytidine, pyrrolo-cytidine, pyrrolo-pseudocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thio-pseudocytidine, 4-thio-1-methylcytidine - Pseudoisocytidine, 4-thio-1-methyl-1-denitro-pseudoisocytidine, 1-methyl-1-denitro-pseudoisocytidine, zabrain, 5-aza-zabrain, 5-methyl-zabrain, 5-aza-2-thio-zabrain, 2-thio-zabrain, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

[0481] In other embodiments, the modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deadenine, 7-deadenine-8-azaadenine, 7-deadenine-2-aminopurine, 7-deadenine-8-azaadenine-2-aminopurine, 7-deadenine-2,6-diaminopurine, 7-deadenine-8-azaadenine-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N... 6-Isopentenyl adenosine, N6-(cis-hydroxyisopentenyl) adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycylcarbamoyl adenosine, N6-threonylcarbamoyl adenosine, 2-methylthio-N6-threonylcarbamoyl adenosine, N6,N6-dimethyl adenosine, 7-methyl adenosine, 2-methylthio-adenosine, and 2-methoxy-adenosine.

[0482] In other embodiments, the modified nucleosides include inosine, 1-methyl-inosine, wyoside, wyoside, 7-deazo-guanosine, 7-deazo-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazo-guanosine, 6-thio-7-deazo-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 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.

[0483] In some implementations, the nucleotide can be modified on the major groove face and can include replacing the hydrogen at C-5 of uracil with a methyl or halogen group.

[0484] 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).

[0485] In view of the above, the modified base can be a modified purine base or a modified pyrimidine base.

[0486] Examples of modified purine bases, in a non-limiting manner, include modified adenosine and / or modified guanosine, such as hypoxanthine; xanthine; 7-methylguanine; inosine; xanthine nucleoside and 7-methylguanosine.

[0487] According to some implementation schemes, the modified RNA or mRNA corresponds to an RNA in which each nucleoside corresponding to uridine, cytidine, adenosine, and / or ribothymidine is modified.

[0488] Examples of modified pyrimidine bases, in a non-limiting manner, include modified cytidines and / or modified uridines, such as 5,6-dihydrouridine; pseudouridine; 5-methylcytidine; 5-hydroxymethylcytidine; dihydrouridine and 5-methylcytidine.

[0489] In a non-limiting manner, such as as disclosed herein, the modified base can be a modified uridine or cytidine, such as pseudouridine and 5-methylcytidine.

[0490] According to some embodiments, the modified RNA corresponds to RNA in which at least one base corresponding to U (for uracil), C (for cytosine), A (for adenine) and / or T (for thymine) is modified.

[0491] Examples of modified bases include methyl-5-uridine (m5U), 2-thio-uridine (s2U), 2'-O-methyl-5-uridine (Ome5U), pseudouridine (Ψ), methyl-1-pseudouridine (m1Ψ), methyl-5-cytosine (m5C), 2'-O-methyl-5-cytosine (Om5C), N6-methyl-adenosine (m6A), and N1-methyl-adenosine (m6A).

[0492] According to some implementation schemes, the modified mRNA may contain 2'-O-methyl-5-uridine (Ome5U) or methyl-1-pseudouridine (m1Ψ) as the modified base.

[0493] Capped and uncapped mRNAs, whether modified or unmodified, are commercially available.

[0494] RNA with unmasked polyA sequences is translated more efficiently than RNA with masked polyA sequences.

[0495] The terms "poly(A) tail" or "poly(A) sequence" refer 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 3' end of the poly(A) sequence of the RNA molecule ends with the letter A, and there are no nucleotides other than A following the 3' end (i.e., downstream) of the poly(A) sequence. Furthermore, long poly(A) sequences of approximately 120 base pairs result in optimal transcriptional stability and translational efficiency of RNA.

[0496] Therefore, to increase the stability and / or expression of the RNA used according to this disclosure, it can be modified to coexist with a polyA sequence, such as having a length of 10 to 500, for example 30 to 300, for example 65 to 200, and for example 100 to 150 adenosine residues. In one embodiment, the polyA sequence has a length of approximately 120 adenosine residues. To further increase the stability and / or expression of the RNA used according to this disclosure, the polyA sequence can be demasked.

[0497] Furthermore, incorporating a 3'-untranslated region (UTR) into the 3'-untranslated region of an RNA molecule can lead to enhanced translation efficiency. A synergistic effect can be achieved by incorporating two or more such 3'-untranslated regions. The 3'-untranslated region can be autologous or heterologous to the RNA in which it is introduced. In one embodiment, the 3'-untranslated region is derived from the human β-globin gene.

[0498] The combination of the above modifications (i.e., the incorporation of poly-A sequences, the demasking of poly-A sequences, and the incorporation of at least one 3'-untranslated region) has a synergistic effect on increasing RNA stability and translation efficiency.

[0499] To increase the expression of RNA used according to this disclosure, it can be modified within the coding region (i.e., the sequence encoding the expressed peptide or protein), for example without altering the sequence of the expressed polypeptide or protein, thereby increasing GC content to increase mRNA stability and codon optimization, and thus enhancing translation in the cell.

[0500] It should be understood that uncapped RNA molecules can be modified RNA molecules or unmodified RNA molecules.

[0501] Therefore, capped RNA molecules can be modified RNA molecules or unmodified RNA molecules.

[0502] In one implementation, the RNA molecule, as disclosed herein, is messenger RNA (mRNA).

[0503] The RNA molecules disclosed herein are, for example, uncapped messenger RNAs, which may be modified or unmodified.

[0504] The RNA molecules disclosed herein are, for example, capped messenger RNAs, which may be modified or unmodified.

[0505] In a non-restrictive manner, uncapped RNA molecules, such as messenger RNA, can also be uncapped RNA molecules that only have naturally occurring bases.

[0506] According to this disclosure, "naturally occurring bases" refer to bases that can be naturally incorporated into RNA molecules such as messenger RNA by the host in vivo. Therefore, "naturally occurring bases" are distinct from synthetic bases for which there is no natural equivalent in the host. However, "naturally occurring bases" may or may not be modified bases, and the two terms should not be confused in the sense of this disclosure.

[0507] Uncapped messenger RNA can also be uncapped and modified messenger RNA, and therefore contains at least one modified base.

[0508] Therefore, uncapped messenger RNA can also be uncapped and modified messenger RNA with (5')ppp(5')guanosine extremes and containing at least one modified base.

[0509] Uncapped messenger RNA can also be uncapped and modified messenger RNA with (5')ppp(5')guanosine extremes and containing at least one pseudouridine and at least one 5-methylcytosine.

[0510] Capped messenger RNA can be messenger RNA with its 5' end linked to 7-methylguanosine or an analogue (which is linked to a 5' to 5' triphosphate bond) and containing naturally occurring bases or modified bases such as pseudo-uridine or 5-methylcytosine.

[0511] It should also be understood that when both modified and unmodified RNA molecules are used in one embodiment of this disclosure, they may be used in mixture and / or purified form.

[0512] antigen

[0513] The nucleic acids contained in the lipid nanoparticles disclosed in this article can be antigens.

[0514] According to one embodiment, the compositions disclosed herein, such as lipid nanoparticles, can be nucleic acid immunogenic compositions or nucleic acid vaccines comprising at least one polynucleotide, such as a polynucleotide construct, encoding at least one wild-type or engineered antigen.

[0515] The valence state of the antigen-containing compositions disclosed herein can vary. Valence state refers to the number of antigenic components in the composition, polynucleotide (e.g., RNA polynucleotide), or polypeptide. In some embodiments, the immunogenic compositions are multivalent. They can also be compositions containing more than one valent component, such as bivalent, trivalent, or multivalent compositions. Multivalent immunogenic compositions or vaccines may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more antigens or antigenic moieties (e.g., antigenic peptides, etc.). The antigenic component may be on a single polynucleotide or on individual polynucleotides.

[0516] The compositions disclosed herein can be used to protect against, treat or cure infections caused by contact with infectious agents such as bacteria, viruses, fungi, protozoa and parasites.

[0517] The compositions disclosed herein can be used to protect against, treat, or cure cancer.

[0518] According to one implementation plan, nucleic acids can encode at least one antigen selected from bacterial antigens, protozoan antigens, viral antigens, fungal antigens, parasitic antigens, or tumor antigens.

[0519] bacterial antigens

[0520] The bacteria mentioned in this article can be either Gram-positive or Gram-negative. Bacterial antigens 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, and Clostridium tetani. (tetani), coagulase-negative Staphylococcus, Corynebacterium diphtheria, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli, Escherichia coli O157:H7, and Enterobacter species.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, and other species of Proteus. *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*. (Bacteria include) *Vibrio pallidum*, *Vibrio cholerae*, and *Yersinia pestis*.

[0521] Viral antigens

[0522] Viral antigens can be derived from adenovirus; herpes simplex virus type 1; herpes simplex virus 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 A or B virus; Guanarito virus; Junin virus; Lassa virus; Machupo virus; Sabia virus. (viruses); 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 coronavirus; SARS-CoV-2 virus; Chikungunya virus; Zika virus; parainfluenza virus; human enterovirus; Hantavirus; Japanese encephalitis virus; vesicular exanthernavirus; Eastern equine encephalitis virus; or Banna virus.

[0523] In one implementation, the antigen is derived from a strain of influenza A or influenza B virus, or a combination thereof. Influenza A or influenza B strains may be associated with birds, pigs, horses, dogs, humans, or non-human primates.

[0524] Nucleic acids may encode hemagglutinin proteins or fragments thereof. Hemagglutinin proteins may be H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18, or fragments thereof. Hemagglutinin proteins may or may not contain a head domain (HA1). Alternatively, hemagglutinin proteins may or may not contain a cytoplasmic domain.

[0525] In this embodiment, the hemagglutinin protein is a truncated hemagglutinin protein. The truncated hemagglutinin protein may include a portion of a transmembrane domain.

[0526] In some implementations, the virus may be selected from H1N1, H3N2, H7N9, H5N1 and H10N8 viruses or B strain viruses.

[0527] In another implementation, the antigen is derived from coronaviruses, such as SARS-CoV-1, SARS-CoV-2, or MERS-CoV.

[0528] Fungal antigens

[0529] Fungal antigens can be obtained from Ascomycota (e.g., Fusarium oxysporum, Pneumocystis jirovecii, Aspergillus spp., Coccidioides immitis / posadasii, Candida albicans), Basidiomycota (e.g., Filobasidia neoformans, Trichosporon), Microsporidia (e.g., Encephalitozoon cuniculi, Enterocytozoon bieneusi), and Mucoromycotina (e.g., Mucor circinelloides, Rhizopus). oryzae), Mucor (Lichtheimia corymbifera).

[0530] Protozoan antigens

[0531] Protozoan antigens can be obtained from Entamoeba histolytica, Giardia lambila, Trichomonas vaginalis, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Balantidium coli, Toxoplasma gondii, Plasmodium spp. species, and Babesia microti.

[0532] parasite antigen

[0533] Parasitic antigens can be obtained from genera such as *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, and *Plasmodium*. The genus *Falciparum*, the genus *Schistosoma*, the genus *Strongyloides stercoralis*, the genus *mite*, the genus *tapeworm*, the genus *Toxoplasma gondii*, the genus *Trypanosoma*, the genus *whipworm*, and the genus *Wuchereria bancrofti*.

[0534] Tumor antigens

[0535] In one implementation, the antigen may be a tumor antigen, i.e., a component of cancer cells, such as a protein or peptide expressed in cancer cells. The term "tumor antigen" refers to a protein that is specifically expressed in a limited number of tissues and / or organs under normal conditions or at a specific developmental stage, and is expressed or aberrantly expressed in at least one tumor or cancerous tissue. Tumor antigens include, for example, differentiation antigens, such as cell type-specific differentiation antigens, i.e., proteins specifically expressed in a certain cell type at a certain stage of differentiation under normal conditions, and germline-specific antigens. For example, a tumor antigen may be presented by cancer cells that express it.

[0536] For example, tumor antigens include carcinoembryonic antigen, α1-fetoprotein, isoferrin, as well as fetal sulfoglycoprotein, cc2-H-ferritin, and γ-fetoprotein.

[0537] Other examples of tumor antigens that may be used in this disclosure are p53, ART-4, BAGE, β-catenin / m, Bcr-abL, CAMEL, CAP-1, CASP-8, CDC27 / m, CD4 / 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, and 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, Pm l / RARa, PRAME, protease 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP 1, SCP2, SCP3, SSX, SURVrVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / 1NT2, TPTE and WT, such as WT-1.

[0538] adjuvant

[0539] The nucleic acid-containing compositions or lipid nanoparticles disclosed herein may further contain adjuvants or immune enhancers or may be administered in combination with adjuvants or immune enhancers.

[0540] The adjuvants that can be used in this disclosure may include, but are not limited to, natural or synthetic adjuvants. They may be organic or inorganic.

[0541] Adjuvants may 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 microfluidic detergent-stabilized oil-in-water emulsions, purified saponins, oil-in-water emulsions, stabilized water-in-oil emulsions; (3) particulate adjuvants, such as virions (monolithic liposome carriers incorporating influenza hemagglutinin), structured complexes of saponins and lipids, poly(lactic-co-glycolic acid) (PLG); (4) microbial derivatives; (5) endogenous human immunomodulators; and / or (6) inert carriers, such as gold particles; (7) adjuvants of microbial origin; (8) surfactant compounds; (9) carbohydrates; or combinations thereof.

[0542] It is obvious to those skilled in the art to select appropriate adjuvants and appropriate amounts of adjuvants.

[0543] Specific adjuvants may include, but are not limited to, cationic liposome-DNA complex JVRS-100, aluminum hydroxide vaccine adjuvant, aluminum phosphate vaccine adjuvant, potassium aluminum sulfate adjuvant, aluminum hydrogel, and 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 Adjuvants, 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, adamantamide dipeptide vaccine adjuvant, Arlacel A, VSA-3 adjuvant, aluminum vaccine adjuvant, Polygen vaccine adjuvant, Adjumer TM , Algal Glucan, Bay R1005, Stearoyl tyrosine, Spol, 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 12K mCT-El 12K, and / or Matrix-S.

[0544] Protein expression

[0545] 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.

[0546] 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.

[0547] 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.

[0548] 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 disclosed herein encode antibodies that can be used to temporarily or permanently affect the functional response of a subject. For example, the mRNA nucleic acid described herein 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 described herein 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.

[0549] Pharmaceutical Composition

[0550] According to some embodiments, this disclosure relates to pharmaceutical compositions, such as immunogenic compositions.

[0551] For administration purposes, lipid nanoparticles as disclosed herein, comprising therapeutic agents such as nucleic acids, may be administered as pharmaceutical compositions. Pharmaceutical compositions of this disclosure comprise lipid nanoparticles as disclosed herein, and at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0552] According to some embodiments, a pharmaceutical composition 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 composition as described herein, or (iii) at least one lipid nanoparticle as described herein and at least one pharmaceutically acceptable excipient.

[0553] In some embodiments, the pharmaceutical composition may be an immunogenic composition. The immunogenic compositions disclosed herein may comprise at least one lipid nanoparticle as described herein, wherein the nucleic acid contained therein encodes at least one antigen. Furthermore, the immunogenic composition may comprise at least one adjuvant as described herein.

[0554] According to some embodiments, this disclosure relates to a composition comprising at least one 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.

[0555] According to some embodiments, this disclosure relates to a composition comprising at least one lipid nanoparticle as described herein, the composition being used in therapeutic methods for the prevention and / or treatment of diseases selected from infectious diseases, allergies, autoimmune diseases, rare blood disorders, rare metabolic diseases, rare neurological diseases, and tumors or cancers, as described herein, for example.

[0556] According to some embodiments, compositions comprising at least one lipid nanoparticle as described herein can be used as immunogenic compositions.

[0557] The immunogenic compositions disclosed herein can be used for the prevention and / or treatment of infectious diseases as indicated herein. They may contain at least one nucleic acid encoding at least one antigen as described herein.

[0558] In some embodiments, lipid compounds of formula (IV), (Va), or (Vb) may be present in pharmaceutical or immunogenic compositions in amounts that effectively form lipid nanoparticles and deliver therapeutic agents (e.g., nucleic acids) to treat a specific disease or condition of interest.

[0559] Those skilled in the art can easily determine the appropriate concentration and dosage.

[0560] The pharmaceutical compositions and immunogenic compositions disclosed herein may be administered in any acceptable manner as with compositions intended for similar purposes.

[0561] 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.

[0562] In some embodiments, the compositions disclosed herein may be administered via transdermal, subcutaneous, intradermal, or intramuscular routes.

[0563] The compositions disclosed herein are formulated to allow the active ingredients contained therein to be bioavailable when the compositions are administered to a patient.

[0564] 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).

[0565] The composition may contain at least one inert diluent or carrier.

[0566] 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.

[0567] 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.

[0568] 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.

[0569] 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.

[0570] 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.

[0571] Treatment

[0572] 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 an effective amount of at least one lipid nanoparticle as disclosed herein to the individual. 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.

[0573] In some embodiments, this disclosure also relates to the use of at least one lipid nanoparticle as disclosed herein in the manufacture of pharmaceutical agents for the prevention and / or treatment of infectious diseases, allergies, autoimmune diseases, rare blood disorders, rare metabolic diseases, rare neurological diseases, and tumors or cancers. For example, diseases that this disclosure may address can be infectious diseases, such as viral infections, bacterial infections, fungal or parasitic infections. Diseases that this disclosure may also address can be cancer or tumors.

[0574] 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.

[0575] In one implementation, the disease is influenza, respiratory syncytial virus (RSV) infection, or Covid-19, and for example, influenza.

[0576] 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.

[0577] Parasitic infections can include amoebiasis, giardiasis, trichomoniasis, African sleeping sickness, American sleeping sickness, leishmaniasis (leishmaniasis), bacillosis, toxoplasmosis, malaria, Acanthamoeba keratitis, and babesiosis.

[0578] 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.

[0579] 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.

[0580] 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.

[0581] 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).

[0582] In other embodiments, this disclosure relates to a method for 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 and at least one lipid nanoparticle as described above, such that the at least one target cell is transfected by the nucleic acid.

[0583] 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.

[0584] 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.

[0585] In some embodiments, lipid nanoparticles or compositions, as disclosed herein, that allow for avoidance of liver clearance may be of particular interest.

[0586] 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.

[0587] In other embodiments, this disclosure relates to a method for 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 at least one nucleic acid encoding the polypeptide and at least one lipid nanoparticle as described herein, such that the at least one target cell is operatively transfected with the nucleic acid encoding the polypeptide.

[0588] 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.

[0589] The following examples are provided for illustrative purposes and not for limiting purposes.

[0590] [Example]

[0591] Materials and methods

[0592] Nuclear magnetic resonance spectroscopy (H, C NMR)

[0593] -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).

[0594] 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.

[0595] NMR spectra were obtained using the commercial software NMRnotebook.

[0596] 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 1200 SL (ESI / APCI) with an Agilent HPLC 1200 SL.

[0597] Example 1: Synthesis of 2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-[2-[2-(2-methoxyethylammonium)acetyl]oxyethyl]ammonium; 2,2,2-trifluoroacetate (as a compound of formula (III) in this disclosure -DOG-cleaved) and its conversion to dihydrochloride salt

[0598]

[0599] Compound 14 was prepared according to the following synthetic scheme:

[0600] Option 3

[0601]

[0602] 1.1 Synthesis of 2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (compound 6 of scheme 3)

[0603]

[0604] 1.1.1 Synthesis of 2-[tert-butoxycarbonyl(2-hydroxyethyl)amino]benzyl acetate (compound 4 of scheme 3)

[0605]

[0606] Ethyl bromoacetate (15 g, 65.5 mmol) was added to a solution of 2-aminoethanol (4.00 g, 65.5 mmol) and K₂CO₃ (45.3 g, 327 mmol) in 160 mL of acetonitrile at 0 °C over a period of 1.5 h. The solution was stirred at 0 °C for 2 h and filtered. Di-tert-butyl dicarbonate (14.3 g, 65.5 mmol) was added to the filtrate and the mixture was distilled under reduced pressure at 50 °C to complete the reaction (1 to 2 h). Toluene (100 mL) was added, and the organic phase was washed with 1 N hydrochloric acid aqueous solution, saturated sodium bicarbonate solution, and brine. The organic layer was dried over Na₂SO₄, filtered, and concentrated. The residue was purified by column chromatography (0% to 100% ethyl acetate in petroleum ether) to give benzyl 2-[tert-butoxycarbonyl(2-hydroxyethyl)amino]acetate (13 g, 40 mmol, 61% yield) as a colorless oil.

[0607] 1H NMR (500MHz, CDCl3) δ7.40-7.31 (m, 6H), 5.20 (d, J = 2.7Hz, 2H), 4.00 (d, J = 30.1Hz, 2H), 3.72(dd,J=25.2,4.5Hz,2H),3.48-3.39(m,2H),3.33-3.25(m,1H),1.51-1.31(m,9H).

[0608] 1.1.22-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[(2-benzyloxy-2-oxo-ethyl)- Synthesis of tert-butoxycarbonyl-amino]ethyl ester (compound 5 of scheme 3)

[0609]

[0610] In a 250 mL round-bottom flask, dissolve 6.68 g (21.6 mmol) of 2-[tert-butoxycarbonyl(2-hydroxyethyl)amino]acetic acid, 10.1 g (43.2 mmol) of 2-tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid, 4-methylmorpholine (4.36 g (43.2 mmol) of 4-methylmorpholine, and 5.27 g (43.2 mmol) of N,N-dimethylpyridin-4-amine (5.27 g (43.2 mmol) in dry dichloromethane (100 mL). Cool the mixture to 0 °C and add 3-(ethyliminomethyleneamino)-N,N-dimethyl-prop-1-amine hydrochloride (8.27 g (43.2 mmol)) in portions over 45 min. Stir the mixture overnight at room temperature. Add water, separate the organic layer, and wash again with water (2 x 200 mL), then with brine. Dry the organic phase over sodium sulfate, filter, and remove the solvent by decantation. The crude product was purified by column chromatography on a 120 g SI60 (0% → 40% on 10 VC hexane / ethyl acetate, then 40% on 11 VC) to give 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[(2-benzyloxy-2-oxo-ethyl)-tert-butoxycarbonyl-amino]ethyl ester (7.92 g, 70% yield) as a colorless oil.

[0611] LCMS:425(M-Boc+1),100% UV 214

[0612] 1H NMR (400MHz, CDCl3) δ7.35(d,J=3.7Hz,5H),5.16(d,J=3.6Hz,2H),4.24(d,J=5.5Hz,2H),4.08-3.9 3(m,4H),3.58-3.39(m,6H),3.27(dd,J=6.9,2.0Hz,3H),1.49-1.42(m,9H),1.37(d,J=23.8Hz,9H).

[0613] 1.1.32-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxy Synthesis of [ethyl]amino]acetic acid (compound 6 of scheme 3)

[0614]

[0615] In a 17 mL H2 reactor, 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[(2-benzyloxy-2-oxo-ethyl)-tert-butoxycarbonyl-amino]ethyl ester (8.07 g, 15.4 mmol) dissolved in ethyl acetate (100 mL) and palladium (10%, 1.64 g, 1.54 mmol) was added. The mixture was stirred at room temperature under a 5 bar H2 atmosphere for 4 h, then filtered over talc and washed with ethyl acetate. The solvent was removed under reduced pressure to give 2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (5.39 g, 76.6% yield) as a colorless oil.

[0616] LCMS: 335 (M-Boc+1), 82% UV214

[0617] 1H NMR (400MHz, CDCl3) δ5.70 (s, 1H), 4.33-4.20 (m, 2H), 4.10-3.94 (m, 4H), 3.61-3.43 (m, 6H), 3.32 (s, 3H), 1.45 (dd, J = 17.1, 5.3Hz, 18H).

[0618] 1.2 Synthesis of 2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethanol (Compound 12 of Scheme 3)

[0619]

[0620] Add 1.1 g (1.85 mol) of 2,3-bis[(Z)-octadec-9-enoxy]prop-1-ol dissolved in 15 mL of THF to a suspension of NaH (60% mineral oil dispersion, 355 mg, 9.27 mmol) in 15 mL of THF. Stir the resulting suspension at room temperature for 2 h. Add ethyl 2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]methyl methanesulfonic acid (1.43 g, 2.78 mmol) to the suspension and reflux the reaction mixture overnight. Cool the reaction mixture to room temperature and add water. Add 100 mL of EtOAc, shake the mixture, separate the layers, and collect the organic layer. Extract the aqueous layer with 50 mL of EtOAc (2). Wash the combined organic layers with brine and dry with Na2SO4. The residue was purified by rapid column chromatography on silica gel by elution with 1:8 ethyl acetate / petroleum ether to obtain a colorless oily substance, [2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy-diphenyl-methyl]benzene (1.8 g, 95.9%).

[0621] 1H NMR (500MHz, CDCl3) δ7.46(d,J=7.6Hz,7H),7.28(t,J=7.5Hz,7H),7.21(dd,J=15.2,7.5Hz,4H),5.39-5.30(m,3H),3.74-3.58(m,15H),3 .59-3.37(m,9H),3.23(dd,J=9.8,4.7Hz,2H),2.07-1.92(m,6H),1.54(dd,J=13.6,6.8Hz,4H),1.40-1.19(m,42H),0.88(t,J=6.9Hz,6H).

[0622] Toluene-4-sulfonic acid (1.58 g, 8.3 mmol) was added to a mixture of [2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy-diphenyl-methyl]benzene (8.4 g, 8.3 mmol) in THF (50 mL) / MeOH (50 mL) and stirred overnight at room temperature. Et3N (5 mL) was added to the reaction mixture and concentrated. The residue was purified by rapid column chromatography on silica gel by elution with ethyl acetate / petroleum ether in a 2:1 ratio to give 2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethanol (3.1 g, 48.5%) as a colorless oil.

[0623] 1H NMR (500MHz, CDCl3) δ 5.39-5.30 (m, 3H), 3.74-3.40 (m, 25H), 2.07-1.93 (m, 7H), 1.59-1.50 (m, 4H), 1.37-1.22 (m, 44H), 0.88 (t, J = 6.9Hz, 6H).

[0624] 1.3 Synthesis of [2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-[2-[2-(2-methoxyethylammonium)acetyl]oxyethyl]ammonium; dihydrochloride, i.e., compounds (III) in the form of their hydrochloride salts.

[0625]

[0626] 1.3.1 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-[2,3-bis] [(Z)-Octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butyloxy] Synthesis of [carbonyl-amino]ethyl ester (compound 13 of scheme 3)

[0627]

[0628] In a 50 mL round-bottom flask, 2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (200 mg, 0.46 mmol), 22-[2-[2-[2-(2,3-bishexadecyloxypropoxy)ethoxy]ethoxy]ethoxy]ethanol (330 mg, 0.46 mmol), and N,N-dimethylpyridin-4-amine (84.4 mg, 0.69 mmol) were dissolved in anhydrous DCM (10 mL) in the presence of 4-methylmorpholine (69.8 mg, 0.69 mmol). The reaction was cooled to 0 °C and 3-(ethyliminomethyleneamino)-N,N-dimethyl-prop-1-amine hydrochloride (132 mg, 0.69 mmol) was added to the mixture. The reaction was warmed to room temperature and stirred overnight. The reaction was monitored by TLC (Hept / EtOAc 5:5, expected product rf: 0.5). Water was added, and the organic layer was extracted with DCM (3 x 15 mL), washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by rapid column chromatography on silica gel by elution with 1:4 ethyl acetate / petroleum ether to give 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-2-[tert-butoxycarbonyl-[2-[2-[2-[2-[2-(2,3-dihexadecaoxypropoxy)ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester (0.38 g, 72.8%).

[0629] 1H NMR (500MHz, CDCl3) δ4.31-4.20(m,4H),4.04(d,J=7.9Hz,2H),4.00-3.96(m,2H),3.70(s,2H),3.67-3.61(m,12H),3.58-3 .39(m,15H),3.30(d,J=7.0Hz,3H),1.59-1.51(m,4H),1.47(s,9H),1.42(s,9H),1.34-1.21(m,52H),0.88(t,J=6.9Hz,6H).

[0630] 1.3.2 [2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy] [ethoxy]ethoxy]-2-oxo-ethyl]-[2-[2-(2-methoxyethylammonium)acetyl]oxyethyl]ammonium; 2,2,2- Synthesis of trifluoroacetate (compound 14 of scheme 3)

[0631]

[0632] TFA (0.308 g, 2.7 mmol) was added to a mixture of 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (0.32 g, 0.27 mmol) in DCM (5 mL) at room temperature. The mixture was stirred at ambient temperature for 16 h. The mixture was concentrated to a yellow oily state and yielded 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester; 2,2,2-trifluoroacetic acid (0.23 g, 66.7%).

[0633] 1H NMR (500MHz, CDCl3) δ5.40-5.30(m,3H),4.60(s,2H),4.39-4.31(m,2H),4.03(s,2H),3.96(s,2H),3.73-3.68(m,4H),3.67-3.61(m,14H ),3.60-3.40(m,13H),3.36(s,3H),3.33-3.29(m,2H),2.06-1.92(m,8H),1.57-1.53(m,4H),1.31-1.23(m,45H),0.88(t,J=6.9Hz,7H).

[0634] 1.3.3 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9- [Alkenyloxy]propoxy]-2-oxo-ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester; disalt Synthesis of acid salts

[0635]

[0636] Add HCl (5 mL, 3 mol / L) in EtOAc to a mixture of 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]-2-oxo-ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (0.45 g, 0.375 mmol) in DCM (5 mL). Stir the mixture at room temperature for 3 h. The reaction mixture was concentrated to give 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]-2-oxo-ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester, a white oil; dihydrochloride (0.143 g, 35.6%).

[0637] 1H NMR(500MHz, CDCl3)δ9.83(s,3H),5.42-5.27(m,3H),4.80(s,2H),4.39(s,2H),4.34-4.05(m,7H),3.98(s,2H),3 .86(s,2H),3.78-3.30(m,25H),2.10-1.92(m,8H),1.60-1.48(m,4H),1.35-1.23(m,44H),0.88(t,J=6.9Hz,6H).

[0638] Example 2: Synthesis of 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester; Synthesis of 2,2,2-trifluoroacetic acid (compound (III))

[0639]

[0640] Compound (III)

[0641] based on Figure 3 The chemically synthesized compound (III) is shown in scheme (4).

[0642] Synthesis of compound (III)

[0643] Step (1)

[0644]

[0645] Triethylamine (20.8 g, 0.206 mol) was added to a mixture of 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethanol (20 g, 0.103 mol), N,N-dimethylpyridin-4-amine (0.629 g, 0.00515 mol), and [chloro(diphenyl)methyl]benzene (23 g, 0.0824 mol) in DCM (300 mL) cooled to 0 °C. The reaction mixture was stirred at ambient temperature for 18 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 brine, dried over Na₂SO₄, filtered, and concentrated. The residue was purified by rapid column chromatography on silica gel by elution with 2:1 ethyl acetate / petroleum ether to give 2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethanol (15.6 g, 34.7%) as a colorless oil.

[0646] LCMS 454 (M+18), 98% UV 214nm.

[0647] Step (2)

[0648]

[0649] Methanesulfonyl chloride (4.91 g, 0.0429 mol) was slowly added to a mixture of 2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethanol (15.6 g, 0.0357 mol) and N,N-diethylethylamine (7.23 g, 0.0715 mol) in DCM (300 mL) at 0 °C. The mixture was stirred overnight at room temperature. Water (150 mL) and CH2Cl2 (300 mL) were added to the solution, and the mixture was transferred to a separatory funnel. The mixture was shaken to separate the layers, and the organic layer was collected. The aqueous layer was further extracted with CH2Cl2 (150 mL * 2). The combined organic layers were then washed with 10% NaHCO3 (150 mL) and brine (150 mL) and dried over MgSO4. The solvent was then removed to obtain 2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethyl methanesulfonate (18.4 g, 100%), which is a yellow oil.

[0650] HNMR (EXP-20-IJ3414)

[0651] 1H NMR (400MHz, CDCl3) δ7.46(d,J=7.5Hz,6H),7.29(t,J=7.4Hz,7H),7.23(t,J=7.2Hz,3H) ,4.36-4.28(m,2H),3.76-3.72(m,2H),3.67(s,10H),3.23(t,J=5.1Hz,2H),2.99(s,3H).

[0652] LCMS 532(M+18) 96% UV (214nm)

[0653] Step (3)

[0654]

[0655]

[0656] Add 1.1 g (1.85 mol) of 2,3-bis[(Z)-octadec-9-enoxy]prop-1-ol dissolved in 15 mL of THF to a suspension of NaH (60% mineral oil dispersion, 355 mg, 9.27 mmol) in 15 mL of THF. Stir the resulting suspension at room temperature for 2 h. Add ethyl 2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]methyl methanesulfonic acid (1.43 g, 2.78 mmol) to the suspension and reflux the reaction mixture overnight. Cool the reaction mixture to room temperature and add water. Add 100 mL of EtOAc, shake the mixture, separate the layers, and collect the organic layer. Extract the aqueous layer with 50 mL of EtOAc (2). Wash the combined organic layers with brine and dry with Na2SO4. The residue was purified by rapid column chromatography on silica gel by elution with 1:8 ethyl acetate / petroleum ether to obtain a colorless oily substance, [2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy-diphenyl-methyl]benzene (1.8 g, 95.9%).

[0657] 1H NMR (500MHz, CDCl3) δ7.46(d,J=7.6Hz,7H),7.28(t,J=7.5Hz,7H),7.21(dd,J=15.2,7.5Hz,4H),5.39-5.30(m,3H),3.74-3.58(m,15H),3 .59-3.37(m,9H),3.23(dd,J=9.8,4.7Hz,2H),2.07-1.92(m,6H),1.54(dd,J=13.6,6.8Hz,4H),1.40-1.19(m,42H),0.88(t,J=6.9Hz,6H).

[0658] Step (4)

[0659]

[0660] Toluene-4-sulfonic acid (0.188 g, 0.99 mmol) was added to a mixture of [2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy-diphenyl-methyl]benzene (1.0 g, 0.99 mmol) in THF (10 mL) / MeOH (10 mL) and stirred overnight at room temperature. Et3N (0.3 mL) was added to the reaction mixture and concentrated. The residue was purified by rapid column chromatography on silica gel by elution with ethyl acetate / petroleum ether in a 2:1 ratio to give 2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethanol (0.534 g, 70.2%) as a colorless oil.

[0661] 1 H NMR (400MHz, CDCl3) δ5.36 (dt, J=10.2, 5.2Hz, 3H), 3.76-3.37 (m, 25H), 2.08 -1.87(m,12H),1.64-1.48(m,4H),1.41-1.19(m,44H),0.88(t,J=6.8Hz,6H).

[0662] Step (5)

[0663]

[0664] In a 50 mL round-bottom flask, 2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (200 mg, 0.46 mmol), 2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethanol (354 mg, 0.46 mmol), and N,N-dimethylpyridin-4-amine (84.4 mg, 0.69 mmol) were dissolved in anhydrous DCM (10 mL) in the presence of 4-methylmorpholine (69.8 mg, 0.69 mmol). The reaction was cooled to 0 °C and 3-(ethyliminomethyleneamino)-N,N-dimethyl-prop-1-amine hydrochloride (132 mg, 0.69 mmol) was added to the mixture. The reaction was warmed to room temperature and stirred overnight. The reaction was monitored by TLC (petroleum ether / EtOAc 5:5, expected product rf: 0.5). Water was added, and the organic layer was extracted with DCM (3 x 15 mL), washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by rapid column chromatography on silica gel by elution with 1:8 ethyl acetate / petroleum ether to give 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (0.32 g, 58.6%).

[0665] 1 H NMR(500MHz, CDCl3)δ5.39-5.30(m,4H),4.31-4.20(m,3H),4.07-4.00(m,2H),4.00-3.95(m,2H),3.73-3.59(m,14H),3.59-3.38(m,15H) ,3.30(d,J=7.0Hz,3H),2.10-1.92(m,7H),1.74(s,2H),1.61-1.50(m,4H),1.50-1.37(m,17H),1.38-1.18(m,45H),0.88(t,J=6.9Hz,6H).

[0666] Step (6)

[0667]

[0668] TFA (0.308 g, 2.7 mmol) was added to a mixture of 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (0.32 g, 0.27 mmol) in DCM (5 mL) at room temperature. The mixture was stirred at ambient temperature for 16 h. The mixture was concentrated and dried under vacuum for 2 h to obtain 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester, which was a yellow oil; 2,2,2-trifluoroacetic acid (0.23 g, 66.7%).

[0669] 1 H NMR (500MHz, CDCl3) δ5.40-5.30(m,3H),4.60(s,2H),4.39-4.31(m,2H),4.03(s,2H),3.96(s,2H),3.73-3.68(m,4H),3.67-3.61(m,14H ),3.60-3.40(m,13H),3.36(s,3H),3.33-3.29(m,2H),2.06-1.92(m,8H),1.57-1.53(m,4H),1.31-1.23(m,45H),0.88(t,J=6.9Hz,7H).

[0670] Example 3: Synthesis of (2S)-2-amino-3-methoxy-propionic acid 2-[[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxoethyl]amino]ethyl ester; hydrochloride (compound (VI))

[0671]

[0672] Compound (VI)

[0673] based on Figure 4 The chemically synthesized compound (VI) shown in scheme (5) is represented in the diagram.

[0674] Based on the chemically synthesized compound (VI-A) shown in scheme (6).

[0675]

[0676] Synthesis of compound (VI-A)

[0677] Step (1)

[0678]

[0679] In a 250 mL round-bottom flask, 2-[tert-butoxycarbonyl(2-hydroxyethyl)amino]benzyl acetate (7 g, 22.6 mmol), (2S)-2-(tert-butoxycarbonylamino)-3-methoxy-propionic acid (9.92 g, 45.3 mmol), 4-methylmorpholine (4.58 g, 45.3 mmol), and N,N-dimethylpyridin-4-amine (5.53 g, 45.3 mmol) were dissolved in dry dichloromethane (100 mL). The mixture was cooled to 0 °C and 3-(ethyliminomethyleneamino)-N,N-dimethyl-prop-1-amine hydrochloride (8.68 g, 45.3 mmol) was added in portions over 45 min. The mixture was stirred overnight at room temperature. LC-MS showed conversion of the starting material to the product. Water was added, the organic layer was separated, and washed again with water (2 x 100 mL), then with brine. The organic phase was dried over sodium sulfate, filtered, and the solvent was removed by decantation. The crude product was purified by rapid chromatography on a 340 g silica gel column by elution with 0% to 40% ethyl acetate in petroleum ether to give (2S)-2-(tert-butoxycarbonylamino)-3-methoxy-propionic acid 2-[(2-benzyloxy-2-oxo-ethyl)-tert-butoxycarbonyl-amino]ethyl ester (9.51 g, 78.2% yield) as a colorless oil.

[0680] LC-Mechanical Method: Mobile phase: A: water (10 mmol NH4HCO3), B: acetonitrile gradient, from 10% to 95% B over 1.5 min; Flow rate: 1.5 mL / min; Column: XBridge C18 (4.6 x 50 mm, 3.5 μm); LC purity: 69% (214 nm); Mass: Peak 411 (product-Boc+1) was observed at 2.197 min. + .

[0681] 1 H NMR (400MHz, CDCl3) δ7.35(d,J=3.9Hz,5H),5.35(d,J=4.6Hz,1H),5.30(s,1H),5.17(d,J=3.6Hz,2H),4.41-4.25(m,3H),4.07(s,1H) ,3.97(s,1H),3.80-3.75(m,1H),3.56(d,J=5.5Hz,3H),3.31(d,J=1.5Hz,3H),1.54(s,1H),1.45(dd,J=8.9,3.5Hz,13H),1.35(s,5H).

[0682] Step (2)

[0683]

[0684] Palladium (10%, 1.77 g, 1.67 mmol) was added to a solution of (2S)-2-(tert-butoxycarbonylamino)-3-methoxypropionate 2-[(2-benzyloxy-2-oxo-ethyl)-tert-butoxycarbonyl-amino]ethyl ester (8.51 g, 16.7 mmol) in ethyl acetate (90 mL). The mixture was stirred at room temperature under a hydrogen atmosphere for 16 h. LC-MS showed that the starting material was converted to the product. The mixture was filtered through diatomaceous earth and washed with ethyl acetate. The solvent was removed under reduced pressure to give 2-[tert-butoxycarbonyl-[2-[(2S)-2-(tert-butoxycarbonylamino)-3-methoxy-propionyl]oxyethyl]amino]acetic acid (7.50 g, 90% yield) as a colorless oil.

[0685] LC-Mechanical Method: Mobile phase: A: water (10 mmol NH4HCO3), B: acetonitrile gradient, from 10% to 95% B over 1.5 min; Flow rate: 1.5 mL / min; Column: XBridge C18 (4.6 x 50 mm, 3.5 μm); LC purity: 71% (214 nm); Mass: Peak 321 (product-Boc+1) was observed at 1.579 min. + .

[0686] 1 H NMR (400MHz, CDCl3) δ8.12 (s, 1H), 5.45 (d, J = 7.3Hz, 1H), 5.30 (s, 1H), 4.50-4.31 (m, 2H), 4.27-4.06 ( m,2H),3.99-3.74(m,2H),3.72-3.54(m,2H),3.45(d,J=15.3Hz,1H),3.34(s,3H),1.53-1.42(m,18H).

[0687] Synthesis of compound (VI)

[0688] Step (1)

[0689]

[0690] In a 50 mL round-bottom flask, 2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethanol (200 mg, 0.26 mmol), 2-[tert-butoxycarbonyl-[2-[(2S)-2-(tert-butoxycarbonylamino)-3-methoxy-propionyl]oxyethyl]amino]acetic acid (131 mg, 0.312 mmol), 4-methylmorpholine (32 mg, 0.312 mmol), and N,N-dimethylpyridin-4-amine (38 mg, 0.312 mmol) were dissolved in dry dichloromethane (5 mL). The mixture was cooled to 0 °C and 3-(ethyliminomethyleneamino)-N,N-dimethyl-prop-1-amine hydrochloride (60 mg, 0.312 mmol) was added in portions over 15 min. The mixture was stirred overnight at room temperature. LC-MS showed conversion of the starting material to the product. Water was added, the organic layer was separated and washed again with water (2*20ml), then with brine. The organic phase was dried over sodium sulfate, filtered, and the solvent was removed by sludge removal. The crude product was purified by column chromatography on a 40g silica gel column (0% → 40% petroleum ether / ethyl acetate at 10VC, then 40% at 11VC) to give (2S)-2-(tert-butoxycarbonylamino)-3-methoxypropionic acid 2-[[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (EXP-20-IJ2428, EXP-20-IJ0405-1, 200mg, 62% yield).

[0691] 1 H NMR (400MHz, CDCl3) δ5.34(s,4H),4.39(s,1H),4.29(d,J=5.8Hz,4H),4.05(s,1H),3.97(s,1H),3.79(d,J=9.5Hz,2H),3.72-3.62(m,13H),3. 60-3.39(m,13H),3.34(s,2H),2.01(dd,J=14.9,9.2Hz,8H),1.54(d,J=6.8Hz,6H),1.45(t,J=10.5Hz,18H),1.38-1.20(m,43H),0.88(s,6H).

[0692] Step (2)

[0693]

[0694] A solution of (2S)-2-(tert-butoxycarbonylamino)-3-methoxy-propionic acid 2-[[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (200 mg, 0.171 mmol) in ethyl acetate was stirred in 3M HCl (2 ml) at room temperature for 16 h. The mixture was concentrated to give (2S)-2-amino-3-methoxy-propionic acid 2-[[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester, a colorless oil; hydrochloride (EXP-20-IJ2430, EXP-20-IJ0405, 125.9 mg, 72.1% yield).

[0695] 1 H NMR (400MHz, CDCl3) δ10.11(s,2H),8.74(s,3H),5.41-5.29(m,3H),5.19-3.91(m,10H),3.55(ddd,J=36.6 ,17.1,3.8Hz,27H),2.02(dd,J=21.3,15.7Hz,8H),1.55(s,5H),1.49-0.95(m,44H),0.88(t,J=6.8Hz,6H).

[0696] Example 4: Synthesis of 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxycarbonylamino]ethoxy]ethoxy]ethoxy]-2-oxoethyl]amino]ethyl ester; 2,2,2-trifluoroacetic acid (compound (VII))

[0697] based on Figure 5 The chemically synthesized compound (VII) shown in scheme (7) is represented in the diagram.

[0698]

[0699] Compound (VII)

[0700] Synthesis of compound (VII)

[0701] Step (1)

[0702]

[0703] To a stirred solution of 2,3-bis[(Z)-octadec-9-enoxy]prop-1-ol (500 mg, 0.83 mmol) in N,N-dimethylformamide (5 mL), bis(2,5-dioxopyrrolidone-1-yl) carbonate (668 mg, 2.48 mmol) and 4-dimethylaminopyridine (0.1 g, 0.83 mmol) were added. The solution mixture was stirred at 20 °C for 18 h. TLC showed that the starting material was consumed and the product was the major product. The reaction was partitioned between ethyl acetate and water. 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 20% ethyl acetate in petroleum ether to give 2,3-bis[(Z)-octadec-9-enoxy]propyl(2,5-dioxopyrrolidone-1-yl) carbonate (550 mg, 0.73 mmol, 88% yield) as a colorless oil.

[0704] 1 H NMR (500MHz, CDCl3) δ5.40-5.31(m,4H),4.46(dd,J=11.1,3.8Hz,1H),4.35(dd,J=11.1,6.3Hz,1H),3.74-3.65(m,1H),3.60-3.49( m,3H),3.49-3.41(m,3H),2.83(s,4H),2.06-1.92(m,8H),1.55(dd,J=14.4,7.2Hz,4H),1.38-1.21(m,44H),0.88(t,J=6.9Hz,6H).

[0705] Step (2)

[0706]

[0707] TBDPSCl (2.18 g, 7.71 mmol) was added to a solution of 2-[2-(2-aminoethoxy)ethoxy]ethanol (1.0 g, 6.7 mmol) and imidazole (1.08 g, 15.4 mmol) in CH2Cl2 (20 mL). The reaction was stirred at room temperature for 18 h. The mixture was diluted with CH2Cl2 (30 mL) and the resulting mixture was washed with brine. The organic layer was dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (SiO2, CH2Cl2 / MeOH / NH4OH 80:20:0.25) to obtain 2-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethylamine (1.3 g, 3.35 mmol, 50% yield) as a colorless oil.

[0708] MS(ESI+)m / z 388.2(M+H) +

[0709] Step (3)

[0710]

[0711] To a solution of 2,3-bis[(Z)-octadec-9-enoxy]propyl(2,5-dioxopyrrolidone-1-yl) carbonate (450 mg, 0.58 mmol) in dichloromethane (20 mL), 2-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethylamine (226 mg, 0.58 mmol) was added, followed by triethylamine (88.4 mg, 0.87 mmol) and 4-dimethylaminopyridine (7 mg, 0.06 mmol). The mixture was stirred at 25 °C for 18 h. TLC showed that the starting material was consumed. The mixture was concentrated and purified by rapid chromatography by elution with 1% to 5% methanol in dichloromethane to give N-[2-[2-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethyl]carbamate 2,3-bis[(Z)-octadec-9-enoxy]propyl ester (500 mg, 0.47 mmol, 81% yield) as a colorless oil.

[0712] 1 H NMR (500MHz, CDCl3) δ7.68 (dd, J=7.9, 1.4Hz, 4H), 7.45-7.35 (m, 6H), 5.41-5.31 (m, 3H), 5.2 3-5.17(m,1H),4.18(dd,J=11.4,4.1Hz,1H),4.09(dd,J=11.4,5.4Hz,1H),3.81(t,J=5.3Hz ,2H),3.65-3.50(m,11H),3.46(d,J=5.3Hz,2H),3.42(t,J=6.7Hz,2H),3.38-3.31(m,2H),2 .06-1.92(m,7H),1.59-1.49(m,4H),1.36-1.20(m,46H),1.05(s,9H),0.88(t,J=6.9Hz,6H).

[0713] Step (4)

[0714]

[0715] A mixture of N-[2-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethyl]carbamate 2,3-bis[(Z)-octadec-9-enoxy]propyl ester (500 mg, 0.47 mmol) and TBAF (1 M, 3 mL in THF) was stirred at 25 °C for 2 h. TLC (ethyl acetate / petroleum ether 1 / 1) showed that the starting material was consumed and new spots were observed. The mixture was concentrated and combined with the above batch, and purified by rapid chromatography with elution of 20% to 50% ethyl acetate in petroleum ether to give N-[2-[2-(2-hydroxyethoxy)ethoxy]ethyl]carbamate 2,3-bis[(Z)-octadec-9-enoxy]propyl ester (0.32 g, 0.4 mmol, 84% yield) as a colorless oil.

[0716] 1 H NMR(400MHz, CDCl3)δ5.43(s,1H),5.40-5.29(m,4H),4.27-4.05(m,2H),3.75(s,2H),3.68-3.31(m, 18H), 2.57 (s, 1H), 2.08-1.88 (m, 7H), 1.58-1.50 (m, 4H), 1.39-1.19 (m, 49H), 0.88 (t, J = 6.8Hz, 6H).

[0717] Step (5)

[0718]

[0719] In a 50 mL round-bottom flask, 2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (170 mg, 0.39 mmol), N-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethyl]carbamate 2,3-bis[(Z)-octadec-9-enoxy]propyl ester (316 mg, 0.39 mmol), and N,N-dimethylpyridin-4-amine (52.6 mg, 0.43 mmol) were dissolved in anhydrous DCM (10 mL) in the presence of 4-methylmorpholine (43.5 mg, 0.43 mmol). The reaction was cooled to 0 °C and 3-(ethyliminomethyleneamino)-N,N-dimethyl-propyl-1-amine hydrochloride (82.5 mg, 0.43 mmol) was added to the mixture. The reaction was warmed to room temperature and stirred overnight. The reaction was monitored by TLC (petroleum ether / EtOAc 5:5, expected product rf: 0.5). The solvent was removed and the residue was purified by rapid column chromatography on silica gel by elution with ethyl acetate / petroleum ether at a ratio of 1:2 to 1:1 to give 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxycarbonylamino]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (0.19 g, 0.156 mmol, 40%).

[0720] 1 H NMR(500MHz, CDCl3)δ5.40-5.32(m,3H),5.22(s,1H),4.32-4.27(m,2H),4.27-4.15(m,3H),4.14-4 .07(m,1H),4.07-4.01(m,2H),3.98(t,J=5.5Hz,2H),3.73-3.67(m,2H),3.65-3.58(m,5H),3.58-3 .52(m,5H),3.52-3.46(m,6H),3.43(t,J=6.6Hz,3H),3.40-3.34(m,2H),3.30(d,J=6.9Hz,3H),2.0 6-1.92(m,8H),1.58-1.51(m,4H),1.44(d,J=22.4Hz,18H),1.35-1.22(m,44H),0.90-0.85(m,6H).

[0721] Step (6)

[0722]

[0723] To a solution of 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxycarbonylamino]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (0.19 g, 0.156 mmol) in DCM (5 mL), 2,2,2-trifluoroacetic acid (1 mL, 13.5 mmol) was added and the reaction was stirred at room temperature for 2 h. The reaction was monitored by TLC (petroleum ether / EtOAc 5:5, starting material rf: 0.5), which indicated that the starting material was completely consumed. The solvent was removed, and the material was azeotropically reacted with dichloromethane several times, and then dried under vacuum to obtain 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxycarbonylamino]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester, which was a pale yellow oil; 2,2,2-trifluoroacetic acid (0.19 g, 0.156 mmol, quantified).

[0724] 1 H NMR (500MHz, CDCl3) δ5.40-5.27(m,4H),4.60(s,2H),4.38(s,2H),4.19(s,2H),4.14-4.06(m,2H),4.04-3.95(m,5H),3.71(s,4H),3.62(s,5 H),3.57-3.52(m,4H),3.50-3.42(m,6H),3.39-3.28(m,7H),2.05-1.9 1(m,7H),1.59-1.50(m,4H),1.36-1.20(m,47H),0.88(t,J=6.9Hz,6H).

[0725] MS(ESI+)m / z 984.8(M+H)+

[0726] Example 5: Synthesis of 2,3-bis[(Z)-octadec-9-enoxy]propionic acid 2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethylamino)acetyl]oxyethylamino]acetyl]oxyethoxy]ethoxy]ethoxy]ethyl ester; and 2,2,2-trifluoroacetic acid (compound (VIII)).

[0727]

[0728] Compound (VIII)

[0729] Synthesis of compound (VIII)

[0730] TFA (1 mL) was added to a solution of 2,3-bis[(Z)-octadec-9-enoxy]propionic acid 2-[2-[2-[2-[2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetyl]oxyethoxy]ethoxy]ethoxy]ethyl ester (0.35 g, 0.277 mmol) in anhydrous DCM (6 mL), and the mixture was stirred at room temperature for 2 h. The reaction was monitored by TLC (petroleum ether / EtOAc 5:5, SM rf: 0.5). The solvent was removed, and the residue was azeotropically reacted with dichloromethane several times. The material was then dried under vacuum for 2 hours to obtain 2,3-bis[(Z)-octadec-9-enoxy]propionic acid 2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethylamino)acetyl]oxyethylamino]acetyl]oxyethoxy]ethoxy]ethoxy]ethyl ester, which was a light yellow oil; 2,2,2-trifluoroacetic acid (0.312 g, 96% yield).

[0731] 1 H NMR (500MHz, CDCl3) δ5.40-5.31(m,4H),4.63-4.57(m,2H),4.39-4.34(m ,2H),4.34-4.27(m,2H),4.09-4.05(m,1H),4.01(d,J=19.8Hz,4H),3.75- 3.59(m,17H),3.52-3.39(m,5H),3.37(s,3H),3.35-3.30(m,2H),2.06-1 .90(m,8H),1.63-1.50(m,4H),1.36-1.22(m,44H),0.88(t,J=6.9Hz,6H).

[0732] MS(ESI+)m / z 999.8(M+H)+

[0733] Example 6: Synthesis of 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propionylamino]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester; Synthesis of 2,2,2-trifluoroacetic acid (compound (IX))

[0734]

[0735] Compound (IX)

[0736] Synthesis of compound (IX)

[0737] A solution of 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propionylamino]ethoxy]ethoxy]ethoxy]-2-oxoethyl]-tert-butoxycarbonyl-amino]ethyl ester (0.25 g, 0.2 mmol) in dichloromethane (10 mL) was treated with 2,2,2-trifluoroacetic acid (1 mL) and the mixture was stirred at room temperature for 2 h. TLC (in 10% methanol in dichloromethane) showed that the starting material disappeared. The reaction was concentrated and the product was azeotropically reacted with dichloromethane several times and dried under vacuum for 5 h to give 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propionylamino]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxoethyl]amino]ethyl ester, which was a light yellow oil; 2,2,2-trifluoroacetic acid (0.263 g, 0.2 mmol, quantitative).

[0738] 1 H NMR (400MHz, CDCl3) δ7.22-7.17(m,1H),5.45-5.27(m,4H),5.11-4.57(m,2H),4.39-4.25(m,2H),4.06-3.88(m,5H),3.78-3.69 (m,5H),3.65-3.28(m,25H),3.20-3.14(m,3H),2.11-1.87(m,8H),1.65-1.50(m,4H),1.34-1.22(m,44H),0.88(t,J=6.8Hz,6H).

[0739] Example 7: Synthesis of 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester; Synthesis of 2,2,2-trifluoroacetic acid (compound (X))

[0740]

[0741] Compound (X)

[0742] Synthesize compound (X) according to the following reaction scheme:

[0743]

[0744]

[0745] Step (1)

[0746]

[0747] Add [chloro(diphenyl)methyl]benzene (3.42 g, 12.3 mmol) to a mixture of 2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol (5.0 g, 15.3 mmol) and N,N-diethylethylamine (3.1 g, 30.6 mmol) in DCM (100 mL). Stir the mixture at room temperature for 16 h. Add DCM (100 mL) to the mixture and wash with water and brine, dry over Na2SO4 and concentrate. Purify the residue by column chromatography on silica gel with 0%-20% MeOH in DCM to provide 2-[2-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethanol (3.3 g, 41.3%) as a colorless oil.

[0748] LCMS 586 (M+18), 99% UV: 214nm.

[0749] Step (2)

[0750]

[0751] Methanesulfonyl chloride (1.09 g, 9.5 mmol) was added to a mixture of 2-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol (3.6 g, 6.33 mmol) and N,N-diethylethylamine (1.28 g, 12.7 mmol) in DCM (50 mL) cooled to 0 °C. The reaction 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, dried over Na2SO4, and concentrated to give 2-[2-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethyl ester of methanesulfonic acid (3.8 g, 93%) as a yellow oil.

[0752] LCMS 664 (M+18), 97% UV: 214nm.

[0753] Step (3)

[0754]

[0755] Add 0.78 g (1.32 mmol) of 2,3-bis[(Z)-octadec-9-enoxy]prop-1-ol dissolved in 15 mL of THF to a suspension of NaH (60% mineral oil dispersion, 105 mg, 2.63 mmol) in 15 mL of THF. Stir the resulting suspension at room temperature for 2 h. Add 1.28 g (1.97 mmol) of 2-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl ester (1.28 g, 1.97 mmol) to the suspension and allow the reaction mixture to reflux overnight. Cool the reaction mixture to room temperature and add water (40 mL). Collect the organic phase and extract the aqueous phase with 3 x 40 mL EtOAc. Combine the organic phases and wash successively with 40 mL 1N HCl, 40 mL 5% (w / v) NaHCO3, and 40 mL brine, and dry on MgSO4. The solvent was evaporated under reduced pressure, and the resulting oil was purified by elution with petroleum ether / AcOEt on a silica gel column to obtain a colorless oil of [2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy-diphenyl-methyl]benzene (1.1 g, 73%).

[0756] 1 H NMR (500MHz, CDCl3) δ7.49-7.43(m,6H),7.32-7.26(m,6H),7.25-7.20(m,3H),5.39-5.30(m,3H),3.71-3.60(m,26H),3.60-3.39 (m,10H),3.23(t,J=5.2Hz,2H),2.08-1.92(m,7H),1.54(dd,J=13.3,6.6Hz,4H),1.28(t,J=14.4Hz,46H),0.88(t,J=6.9Hz,6H).

[0757] Step (4)

[0758]

[0759]

[0760] Toluene-4-sulfonic acid (0.915 g, 4.81 mmol) was added to a mixture of [2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy-diphenyl-methyl]benzene (1.1 g, 0.962 mmol) in THF (5 mL) / MeOH (5 mL) and stirred overnight at room temperature. Et3N (1 mL) was added to the reaction mixture and concentrated. The residue was purified by rapid column chromatography on silica gel by elution with 2:1 ethyl acetate / petroleum ether to give 2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol (0.7 g, 81%) as a colorless oil.

[0761] 1 H NMR (500MHz, CDCl3) δ5.34 (td, J=16.2, 8.2Hz, 3H), 3.74-3.70 (m, 2H), 3.69-3.39 (m, 37H), 3. 22(s,1H),2.12-1.93(m,12H),1.60-1.51(m,4H),1.37-1.20(m,46H),0.88(t,J=6.9Hz,6H).

[0762] Step (5)

[0763]

[0764] In a 100 mL round-bottom flask, 2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (400 mg, 0.921 mmol), 2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]eth ... Water was added, and the organic layer was extracted with DCM (3 x 15 mL), washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by rapid column chromatography on silica gel by elution with 4:1 ethyl acetate / petroleum ether to give 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (0.44 g, 36.3%).

[0765] 1 H NMR (400MHz, CDCl3) δ5.34 (t, J = 5.3Hz, 3H), 4.31-4.19 (m, 4H), 4.07-3.94 (m, 4H), 3.73-3.60 (m, 26H), 3.59-3.39 (m, 15H), 3. 30(d,J=5.4Hz,3H),2.06-1.93(m,7H),1.60-1.50(m,4H),1.44(d,J=18.6Hz,18H),1.38-1.21(m,45H),0.88(t,J=6.8Hz,6H).

[0766] Step (6)

[0767]

[0768]

[0769] TFA (0.381 g, 3.34 mmol) was added to a mixture of 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (0.6 g, 0.334 mmol) in DCM (5 mL). The mixture was stirred at ambient temperature for 3 h. The mixture was concentrated and dried under vacuum for several hours to obtain 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester, which is a brown oil; 2,2,2-trifluoroacetic acid (0.302 g, 67.2%).

[0770] 1 H NMR (500MHz, CDCl3) δ5.39-5.30(m,3H),4.58(s,2H),4.40-4.34(m,2H),4.04(d,J=25.9Hz,4H),3.74-3.40( m,39H),3.39-3.30(m,5H),2.06-1.91(m,6H),1.59-1.50(m,4H),1.37-1.18(m,45H),0.88(t,J=6.9Hz,6H).

[0771] Example 8: Synthesis of 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxoethyl]amino]ethyl ester; Synthesis of 2,2,2-trifluoroacetic acid (compound (XI))

[0772]

[0773] Compound (XI)

[0774] Compound (XI) was synthesized according to the following reaction scheme:

[0775]

[0776]

[0777] Step (1)

[0778]

[0779] Add [chloro(diphenyl)methyl]benzene (2.33 g, 8.37 mmol) to a mixture of 2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol (4.8 g, 10.5 mmol) and N,N-diethylethylamine (2.12 g, 20.9 mmol) in DCM (100 mL). Stir the mixture at room temperature for 16 h. Add DCM (100 mL) to the mixture and wash with water and brine, dry with Na2SO4 and concentrate. The residue was purified by column chromatography on silica gel by elution with 0%–20% MeOH in DCM to provide 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol (1.9 g, 26%) as a colorless oil.

[0780] LCMS 718 (M+18), 97% UV: 214nm.

[0781] Step (2)

[0782]

[0783]

[0784] Methanesulfonyl chloride (0.466 g, 4.07 mmol) was added to a mixture of 2-[2-[2-[2-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol (1.9 g, 2.71 mmol) and N,N-diethylethylamine (0.549 g, 5.42 mmol) in DCM (50 mL) cooled to 0 °C. The reaction mixture was stirred at room temperature for 3 h. TLC showed that the starting material disappeared. The mixture was diluted with DCM (100 mL) and washed with water and brine, dried over Na2SO4 and concentrated to obtain 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy] ...

[0785] LCMS 796 (M+18), 97% UV: 214nm.

[0786] Step (3)

[0787]

[0788] Add 0.75 g (1.26 mmol) of 2,3-bis[(Z)-octadec-9-enoxy]prop-1-ol dissolved in 15 mL of THF to a suspension of NaH (60% mineral oil dispersion, 61 mg, 2.53 mmol) in 15 mL of THF. Stir the resulting suspension at room temperature for 2 h. Add 1.18 g (1.52 mmol) of ethyl 2-[2-[2-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy] ... The organic phases were combined and washed sequentially with 40 mL of 1 N HCl, 40 mL of 5% (w / v) NaHCO3, and 40 mL of brine, and dried over MgSO4. The solvent was evaporated under reduced pressure, and the resulting oil was purified by elution with petroleum ether / AcOEt (4:1) on a silica gel column to give a colorless oil of [2-[2-[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy-diphenyl-methyl]benzene (1.1 g, 68%).

[0789] 1 H NMR (400MHz, CDCl3) δ7.48-7.43(m,6H),7.33-7.27(m,6H),7.25-7.18(m,3H),5.39-5.30(m,3H),3.71-3.59(m,39H),3.59-3.38( m,9H),3.23(t,J=5.2Hz,2H),2.07-1.92(m,7H),1.80-1.69(m,5H),1.63-1.48(m,4H),1.40-1.18(m,44H),0.88(t,J=6.8Hz,6H).

[0790] Step (4)

[0791]

[0792] Toluene-4-sulfonic acid (0.82 g, 4.31 mmol) was added to a mixture of [2-[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy-diphenyl-methyl]benzene (1.1 g, 0.862 mmol) in THF (5 mL) / MeOH (5 mL) and stirred overnight at room temperature. Et3N (1 mL) was added to the reaction mixture and concentrated. The residue was purified by rapid column chromatography on silica gel by elution with 5:1 ethyl acetate / petroleum ether to give a colorless oily substance, 2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]eth ...

[0793] 1 H NMR (500MHz, CDCl3) δ 5.43-5.28 (m, 3H), 3.83-3.33 (m, 59H), 2.01 (d, J = 5.3Hz, 7H), 1.55 (s, 4H), 1.27 (s, 45H), 0.88 (t, J = 6.4Hz, 6H).

[0794] Step (5)

[0795]

[0796] In a 100 mL round-bottom flask, 2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (400 mg, 0.921 mmol), 2-[2-[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]eth ... The reaction mixture was heated to room temperature and stirred overnight. Water was added, and the organic layer was extracted with DCM (3 x 15 mL), washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by rapid column chromatography on silica gel by elution with 4:1 ethyl acetate / petroleum ether to give 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]eth ...

[0797] 1 H NMR (400MHz, CDCl3) δ5.42-5.31(m,3H),4.33-4.20(m,4H),4.08-3.95(m,4H),3.77-3.37(m,57H),3.30(d,J=5 .4Hz,3H),2.08-1.92(m,7H),1.59-1.51(m,5H),1.51-1.39(m,20H),1.37-1.21(m,46H),0.88(t,J=6.8Hz,6H).

[0798] Step (6)

[0799]

[0800] TFA (0.629 g, 5.52 mmol) was added to a mixture of 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (0.8 g, 0.552 mmol) in DCM (5 mL). The mixture was stirred at ambient temperature for 3 h. The mixture was concentrated and dried under vacuum for several hours to obtain 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester; 2,2,2-trifluoroacetic acid (0.635 g, 78%).

[0801] 1 H NMR (500MHz, CDCl3) δ5.42-5.27(m,3H),4.57(s,2H),4.39(s,2H),4.08(d,J=24.5Hz,3H),3.82-3.41(m ,52H),3.37(s,5H),2.05-1.92(m,6H),1.54(d,J=6.2Hz,4H),1.37-1.19(m,44H),0.88(t,J=6.9Hz,6H).

[0802] Example 9: Synthesis of 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester; Synthesis of 2,2,2-trifluoroacetic acid (compound (XII))

[0803]

[0804] Compound (XII)

[0805] Synthesis of compound (XII)

[0806] TFA (0.22 g, 2.51 mmol) was added to a mixture of 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonylamino]ethyl ester (0.6 g, 0.441 mmol) in DCM (5 mL). The mixture was stirred at ambient temperature for 3 h. The mixture was concentrated to obtain 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester, which is a brown oil; 2,2,2-trifluoroacetic acid (0.424 g, 69.3%).

[0807] 1 H NMR (500MHz, CDCl3) δ5.44-5.28(m,3H),4.57(s,2H),4.39(s,2H),4.09(s,2H),4.04(s,2H),3.75-3.41 (m,43H),3.41-3.30(m,5H),2.04-1.92(m,6H),1.55(s,4H),1.37-1.20(m,44H),0.88(t,J=6.9Hz,6H).

[0808] Example 10: Synthesis of 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester; Synthesis of 2,2,2-trifluoroacetic acid (compound (XIII))

[0809]

[0810] Compound (XIII)

[0811] Synthesis of compound (XIII)

[0812] TFA (0.288 g, 2.53 mmol) was added to a mixture of N-(tert-butoxycarbonyl)-N-(2-methoxyethyl)glycine (Z)-3-(tert-butoxycarbonyl)-47-(((Z)-octadec-9-en-1-yl)oxy)-5-oxo6,9,12,15,18,21,24,27,30,33,36,39,42,45,49-pentadecaoxo-3-azaheptadec-58-en-1-yl ester (0.4 g, 0.253 mmol) in DCM (5 mL). The mixture was stirred at ambient temperature for 3 h. The mixture was concentrated to obtain 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]eth ...

[0813] 1 H NMR (500MHz, CDCl3) δ5.44-5.27(m,4H),4.57(s,2H),4.38(s,2H),4.15-3.91(m,4H),3.81-3.41(m,68 H),3.39-3.29(m,6H),2.05-1.92(m,7H),1.59-1.51(m,4H),1.32-1.22(m,44H),0.88(t,J=6.9Hz,6H).

[0814] Example 11: Synthesis of 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]-2-oxo-ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester; dihydrochloride (compound (XIV))

[0815]

[0816] Compound (XIV)

[0817] based on Figure 6 The chemically synthesized compound (XIV) shown in scheme (8) is represented in the diagram.

[0818] Synthesis of compound (XIV)

[0819] Step (1)

[0820]

[0821] 2-[2-(2-hydroxyethoxy)ethoxy]ethanol (15 g, 0.1 mol) was dissolved in 200 mL of anhydrous (abs.) DMF under argon atmosphere and cooled to 0 °C. NaH (2.56 g, 0.067 mol, 60% in mineral oil) was carefully added, the ice bath was removed, and stirring was continued at 80 °C for 1 h. The reaction mixture was cooled to ambient temperature and treated with 2-bromoacetic acid (4.58 g, 0.033 mol), which was added via a dropping funnel as a DMF solution (10 mL). After an additional 30 min at 75 °C, purified bromomethylbenzene (5.64 g, 0.033 mol) was added, and esterification was allowed to proceed for 30 min. Cool, carefully pour into crushed ice, extract with ethyl acetate, wash with water, dry with Na2SO4 and evaporate all solvent, then perform rapid chromatography (SiO2, ethyl acetate / heptane = 8 / 2) to obtain 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]benzyl acetate (2.2 g, 7.38%) as a yellow oil.

[0822] 1 H NMR (500MHz, CDCl3) δ7.39-7.31(m,5H),5.19(s,2H),4.20(s,2H),3.77-3.67(m,6H),3.68-3.63(m,4H),3.63-3.56(m,2H).

[0823] Step (2)

[0824]

[0825] In a 50 mL round-bottom flask, 2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (874 mg, 2.01 mmol), 2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]benzyl acetate (600 mg, 2.01 mmol), and N,N-dimethylpyridin-4-amine (295 mg, 2.41 mmol) were dissolved in anhydrous DCM (10 mL) in the presence of 4-methylmorpholine (244 mg, 2.41 mmol). The reaction was cooled to 0 °C and 3-(ethyliminomethyleneamino)-N,N-dimethyl-prop-1-amine hydrochloride (463 mg, 2.41 mmol) was added to the mixture. The reaction was warmed to room temperature and stirred overnight. Water was added, and the organic layer was extracted with DCM (3 x 15 mL), washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by rapid column chromatography on silica gel by elution with 1:1 ethyl acetate / petroleum ether to give 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-(2-benzyloxy-2-oxo-ethoxy)ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (1.2 g, 83.5%).

[0826] 1 H NMR (500MHz, CDCl3) δ7.38-7.32(m,5H),5.19(s,2H),4.29-4.18(m,6H),4.06-4.01(m,2H),4.00-3.94(m,2H),3. 76-3.72(m,2H),3.71-3.66(m,4H),3.64(s,4H),3.56-3.41(m,6H),3.32-3.26(m,3H),1.46(s,9H),1.42(s,9H).

[0827] Step (3)

[0828]

[0829] 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-(2-benzyloxy-2-oxo-ethoxy)ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (1.2 g, 1.68 mmol) and Pd / C (0.5 g) were mixed with EtOAc (30 mL) and attached to a hydrogenation apparatus. The system was evacuated and then refilled with hydrogen. The mixture was stirred overnight at room temperature. The mixture was filtered and the filtrate was concentrated to give 2-[2-[2-[2-[2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetyl]oxyethoxy]ethoxy]ethoxy]acetic acid (0.95 g, 90.6%) as a colorless oil.

[0830] 1 H NMR(500MHz, CDCl3)δ4.32-4.20(m,4H),4.17(d,J=2.7Hz,2H),4.08-3.94(m,4H), 3.79-3.61(m,10H),3.58-3.40(m,6H),3.30(d,J=6.1Hz,3H),1.50-1.37(m,18H).

[0831] Step (4)

[0832]

[0833] In a 50 mL round-bottom flask, 2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (400 mg, 0.64 mmol), 2,3-bis[(Z)-octadec-9-enoxy]prop-1-ol (380 mg, 0.64 mmol), and N,N-dimethylpyridin-4-amine (117 mg, 0.96 mmol) were dissolved in anhydrous DCM (15 mL) in the presence of 4-methylmorpholine (97.2 mg, 0.96 mmol). The reaction was cooled to 0 °C and 3-(ethyliminomethyleneamino)-N,N-dimethyl-prop-1-amine hydrochloride (184 mg, 0.96 mmol) was added to the mixture. The reaction was warmed to room temperature and stirred overnight. Water was added, and the organic layer was extracted with DCM (3 x 15 mL), washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by rapid column chromatography on silica gel by elution with 1:3 ethyl acetate / petroleum ether to give 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]-2-oxo-ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (0.45 g, 58.6%).

[0834] 1 H NMR (500MHz, CDCl3) δ5.43-5.24(m,3H),4.33-4.07(m,8H),4.07-3.93(m,4H),3.76-3.37(m,23H),3.30(d,J=7.0 Hz, 3H), 2.08-1.92 (m, 7H), 1.58-1.50 (m, 4H), 1.44 (d, J = 23.3Hz, 18H), 1.36-1.20 (m, 44H), 0.88 (t, J = 6.9Hz, 6H).

[0835] Step (5)

[0836]

[0837] Add HCl (5 mL, 3 mol / L) in EtOAc to a mixture of 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]-2-oxo-ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (0.45 g, 0.375 mmol) in DCM (5 mL). Stir the mixture at room temperature for 3 h. The reaction mixture was concentrated to give 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propoxy]-2-oxo-ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester, a white oil; dihydrochloride (0.143 g, 35.6%).

[0838] 1 H NMR(500MHz, CDCl3)δ9.83(s,3H),5.42-5.27(m,3H),4.80(s,2H),4.39(s,2H),4.34-4.05(m,7H),3.98(s,2H),3 .86(s,2H),3.78-3.30(m,25H),2.10-1.92(m,8H),1.60-1.48(m,4H),1.35-1.23(m,44H),0.88(t,J=6.9Hz,6H).

[0839] Example 12: Synthesis of 8-[3-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethylamino)acetyl]oxyethylamino]acetyl]oxyethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octyloxy)propoxy]nonyl octanoate; 2,2,2-trifluoroacetic acid (compound (XV))

[0840]

[0841] Compound (XV)

[0842] based on Figure 7 The chemically synthesized compound (XV) is shown in scheme (9) presented in the paper.

[0843] Synthesis of compound (XV)

[0844] Step (1)

[0845]

[0846] 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.

[0847] 1 H 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).

[0848] Step (2)

[0849]

[0850] 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.

[0851] 1H 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).

[0852] Step (3)

[0853]

[0854] A 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. TLC (EA / PE 1 / 1, SM Rf: 0.5; product, Rf: 0.1) showed that the starting material was completely consumed. The solvent was removed under vacuum and azeotropically reacted with toluene several times. 2-[2-[2-(2-methylsulfonyloxyethoxy)ethoxy]ethoxyethyl methanesulfonic acid (49 g, 123 mmol, quantitative) was obtained as a pale yellow oil and used without further purification.

[0855] 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).

[0856] Step (4)

[0857]

[0858] 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. 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-[2,3-bis(non-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxymethylbenzene (9.3 g, 14.6 mmol, 24.2% yield), which was a pale yellow oil.

[0859] 1 H 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).

[0860] Step (5)

[0861]

[0862] 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. LC-MS 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 1 N 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), which was used without further purification.

[0863] Step (6)

[0864] 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.

[0865] 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).

[0866] Step (7)

[0867]

[0868] 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.

[0869] 1 H 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).

[0870] Step (8)

[0871]

[0872] Pd / C (1.13 g, 20% wt) was added to a solution of 8-[3-[2-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octyloxy)propoxy]nonyl octanoate (5 g, 5.31 mmol) in ethyl acetate (100 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 8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octyloxy)propoxy]nonyl octanoate (4.22 g, 4.98 mmol, 93.8%) as a colorless oil.

[0873] 1H 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).

[0874] Step (9)

[0875]

[0876] In the presence of 4-methylmorpholine (59.1 mg, 0.58 mmol), nonyl octanoate (330 mg, 0.39 mmol), 2-[tert-butoxycarbonyl-[3-[tert-butoxycarbonyl(2-methoxyethyl)amino]-2-oxopropyl]amino]acetic acid (199 mg, 0.467 mmol), and N,N-dimethylpyridin-4-amine (71.4 mg, 0.58 mmol) were dissolved in anhydrous DCM (15 mL). The reaction was cooled to 0 °C and 3-(ethyliminomethyleneamino)-N,N-dimethyl-prop-1-amine hydrochloride (112 mg, 0.58 mmol) was added in portions to the mixture. The reaction was warmed to room temperature and stirred overnight. The reaction was monitored by TLC (petroleum ether / EtOAc 5:5, expected product rf: 0.5). Water was added, and the organic layer was extracted with DCM (3 x 15 mL), washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by rapid column chromatography on silica gel by elution with 20% to 40% ethyl acetate in petroleum ether to give a colorless oil, 8-[3-[2-[2-[2-[2-[2-[tert-butoxycarbonyl-[3-[tert-butoxycarbonyl(2-methoxyethyl)amino]-2-oxo-propyl]amino]acetyl]oxyethoxy]ethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octoxy)propoxy]nonyl octanoate (0.23 g, 47.1%).

[0877] 1H NMR (400MHz, CDCl3) δ4.31-4.20(m,4H),4.05(t,J=6.8Hz,6H),4.00-3.95(m,2H),3.70(s,2H),3.66-3.62(m,11H),3.58-3.39(m, 15H), 3.30 (d, J = 5.5Hz, 3H), 2.33-2.24 (m, 4H), 1.65-1.52 (m, 12H), 1.49-1.40 (m, 18H), 1.37-1.24 (m, 37H), 0.88 (t, J = 6.9Hz, 6H).

[0878] Step (10)

[0879]

[0880] TFA (1 mL) was added to a solution of 8-[3-[2-[2-[2-[2-[2-[tert-butoxycarbonyl-[3-[tert-butoxycarbonyl(2-methoxyethyl)amino]-2-oxo-propyl]amino]acetyl]oxyethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octoxy)propoxy]octanoate (230 mg, 0.183 mmol) cooled to 0 °C in DCM (5 mL), and the mixture was stirred at room temperature for 4 h. TLC (DCM / MeOH 10:1, expected product rf: 0.1) showed that the starting material disappeared. The solvent was removed and the product was azeotropically reacted with dichloromethane several times and then dried under vacuum for 2 h to obtain a pale yellow oily product: 8-[3-[2-[2-[2-[2-[2-[3-(2-methoxyethylamino)-2-oxo-propyl]amino]acetyl]oxyethoxy]ethoxy]ethoxy]-2-(8-nonoxy-8-oxo-octyloxy)propoxy]nonyl octanoate; 2,2,2-trifluoroacetic acid (0.226 g, quantitative).

[0881] 1 H NMR (400MHz, CDCl3) δ4.59 (s, 2H), 4.38 (s, 2H), 4.05 (t, J = 6.7Hz, 4H), 4.01-3.90 (m, 4H), 3.75-3.69 (m, 4H), 3.64 (s, 11H), 3.59-3.49 (m, 5H) ),3.49-3.40(m,6H),3.37(s,3H),3.32-3.28(m,2H),2.29(t,J=7.5Hz,4H),1.70-1.45(m,11H),1.41-0.96(m,37H),0.88(t,J=6.9Hz,6H).

[0882] Example 13: Synthesis of 1-octylnonyl octanoate of 8-[3-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethylamino)acetyl]oxyethylamino]acetyl]oxyethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octyloxy]propoxy]octanoate; 2,2,2-trifluoroacetic acid (compound (XVI))

[0883]

[0884] Compound (XVI)

[0885] Synthesis of compound (XVI)

[0886] Add 2,2,2-trifluoroacetic acid (0.26 g, 0.228 mmol) to a mixture of 1-octylnonyl octanoate (0.33 g, 0.228 mmol) in DCM (5 mL). Stir the mixture at ambient temperature for 3 h. The mixture was concentrated to obtain 1-octylnonyl octanoate of 8-[3-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethylamino)acetyl]oxyethylamino]acetyl]oxyethoxy]ethoxy]ethoxy]-2-[8-(1-octylnonoxy)-8-oxo-octyloxy]propoxy]octanoate; 2,2,2-trifluoroacetic acid (0.308 g, 99%).

[0887] 1 H NMR (400MHz, CDCl3) δ4.87(p,J=6.0Hz,2H),4.59(s,2H),4.37(s,2H),4.03(d,J=19.2Hz,4H),3.78-3.40(m,27H),3 .41-3.29(m,5H),2.30(t,J=7.5Hz,4H),1.55(dd,J=30.7,11.0Hz,16H),1.35-1.18(m,62H),0.87(t,J=6.8Hz,12H).

[0888] Example 14: Synthesis of [(Z)-non-2-enyl]8-[3-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethylamino)acetyl]oxyethylamino]acetyl]oxyethoxy]ethoxy]ethoxy]-2-[8-[(Z)-non-2-enyloxy]-8-oxo-octyloxy]propoxy]octyl ester; 2,2,2-trifluoroacetic acid (compound (XVII))

[0889]

[0890] Compound (XVII)

[0891] based on Figure 8 The chemically synthesized compound (XVII) shown in scheme (10) presented in the paper.

[0892] Synthesis of compound (XVII)

[0893] Step (1)

[0894]

[0895] 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).

[0896] 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.

[0897] LCMS 459(M+23), 99% UV 214nm

[0898] 1H 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).

[0899] Step (2)

[0900]

[0901] 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 washed and 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.

[0902] LCMS 537.2(M+23)98% UV(214nm)

[0903] 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).

[0904] Step (3)

[0905]

[0906] Add 20 g of 3-triphenylmethoxypropyl-1,2-diol (227 mmol) to a suspension of NaH (9.09 g, 60% in oil) in 300 mL of anhydrous DMF. Heat the mixture at 80 °C for 15 min and cool to room temperature. Add dropwise 30 g of 9-bromonon-1-ene (142 mmol) in 10 mL of anhydrous DMF to the mixture, and then heat the mixture at 80 °C for 18 h. After cooling to room temperature, add 500 mL of H₂O to destroy the remaining NaH. Extract the organic phase with 3 x 250 mL of ethyl acetate. Wash the extract sequentially with 2 x 150 mL of 1N HCl, 2 x 150 mL of 5% (w / v) NaHCO₃, and 150 mL of brine, and dry on 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 (2% to 5% ethyl acetate in petroleum ether) to produce a colorless oil (12 g; yield 34.4%).

[0907] 1 H NMR (400MHz, CDCl3) δ7.51-7.42(m,6H),7.31-7.20(m,9H),5.88-5.72(m,2H),5.03-4.89(m,4H),3.59-3.46(m,5 H),3.39(t,J=6.7Hz,2H),3.22-3.13(m,2H),2.03(td,J=8.0,1.3Hz,4H),1.57-1.45(m,4H),1.44-1.20(m,16H).

[0908] Step (4)

[0909]

[0910] p-Toluenesulfonic acid (14 g, 81.5 mmol) was added in a single addition to a solution of [2,3-bis(non-8-enoxy)propoxy-diphenyl-methyl]benzene (10 g, 16.3 mmol) in methanol / THF (150 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. 10 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% ethyl acetate in petroleum ether to give 2,3-bis(non-8-enoxy)prop-1-ol (5.1 g, 82.7%) as a colorless oil.

[0911] 1H NMR (400MHz, CDCl3) δ5.90-5.74(m,2H),5.03-4.90(m,4H),3.79-3.32(m,9H),2.22( dd,J=6.5,5.9Hz,1H),2.04(q,J=6.9Hz,4H),1.63-1.47(m,4H),1.43-1.19(m,16H).

[0912] Step (5)

[0913]

[0914] To a mixture of 2,3-bis(nonyl-8-enoxy)prop-1-ol (5.2 g, 15.3 mmol), NaH (60% mineral oil dispersion, 1.51 mg, 30.5 mmol) in 60 mL of dry THF was added, followed by stirring at 80 °C for 15 min. After the solvent returned to room temperature, ethyl 2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]methyl ester (9.43 g, 18.3 mmol) dissolved in 20 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 was added. EtOAc (100 mL) was added, the mixture was shaken, the layers were separated, and the organic layer was collected. The aqueous layer was extracted with EtOAc (50 mL x 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%) (11%) in petroleum ether to give the target product as a pale yellow oil (7.44, 64.2% yield).

[0915] 1 H NMR (400MHz, CDCl3) δ7.48-7.44(m,6H),7.31-7.26(m,6H),7.24-7.19(m,3H) ,5.86-5.74(m,2H),5.01(d,J=1.4Hz,1H),4.96(d,J=1.4Hz,1H),4.94-4.92( m,1H),4.92-4.89(m,1H),3.68-3.64(m,9H),3.62(s,4H),3.59-3.38(m,10H) ,3.25-3.22(m,2H),2.07-2.00(m,4H),1.59-1.51(m,4H),1.39-1.27(m,16H).

[0916] Step (6)

[0917]

[0918] To a solution of [2-[2-[2-[2,3-bis(nonyl-8-enoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxy-diphenyl-methyl]benzene (7.44 g, 9.8 mmol) in MeCN (60 mL), CCl4 (60 mL), and water (60 mL), NaIO4 (16.8 g, 78.4 mmol) and RuCl3 (0.407 g, 1.96 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 (500 mL) and washed with 1 N HCl aqueous solution (200 mL). The organic layer was washed with Na2S2O3 solution and then dried over sodium sulfate, filtered, and concentrated to give 8-[2-(7-carboxyheptaoxy)-3-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]propoxy]octanoic acid (7.98 g, 6.02 mmol, 61.4% yield), which was used without further purification.

[0919] 1 H NMR (400MHz, CDCl3) δ9.75 (s, 1H), 7.47-7.44 (m, 3H), 7.35-7.26 (m, 10H), 7.23 (d, J = 7.2Hz, 2H), 3.80-3.38(m,24H),3.26-3.21(m,1H),2.45-2.25(m,4H),1.59(d,J=34.5Hz,8H),1.33(s,12H).

[0920] Step (7)

[0921] 8-[2-(8-oxooctyloxy)-3-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]propoxy]octanoic acid (6.98 g, 3.58 mmol) was dissolved in t-BuOH:H2O (3:1, 120 mL) containing NaH2PO4 (1.28 g, 10.8 mmol), 2-methyl-2-butene (11 mL), and sodium chlorite (0.972 g, 10.8 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. The combined extracts were dried over sodium sulfate and concentrated to provide 8-[2-(7-carboxyheptaoxy)-3-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]propoxy]octanoic acid (6.161 g, 7.75 mmol, quantified) as a pale yellow oil.

[0922] 1H NMR (400MHz, CDCl3) δ7.47-7.44(m,3H),7.34-7.26(m,10H),7.25-7.19(m,2H),3.76-3.39 (m,24H),3.25-3.21(m,1H),2.43-2.27(m,4H),1.68-1.49(m,8H),1.32(d,J=6.6Hz,12H).

[0923] Step (8)

[0924]

[0925] Then, to a solution of 8-[2-(7-carboxyheptaoxy)-3-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]propoxy]octanoic acid (6.161 g, 7.75 mmol) and (Z)-non-2-en-1-ol (2.65 g, 18.6 mmol) in dry dichloromethane (120 mL), DIPEA (6.01 g, 46.5 mmol), DMAP (0.379 g, 0.31 mmol), and EDCI (3.86 g, 20.1 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% (28%) 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 (1.026 g, 12.7% yield) as a colorless oil.

[0926] 1 H NMR (400MHz, CDCl3) δ7.46(d,J=7.4Hz,6H),7.30(d,J=7.1Hz,6H),7.23(d,J=7.2 Hz,3H),5.69-5.58(m,2H),5.57-5.47(m,2H),4.61(d,J=6.8Hz,4H),3.68-3.39( m,23H),3.23(t,J=5.2Hz,2H),2.29(t,J=7.0Hz,4H),2.09(q,J=7.1Hz,4H),1.61 (s, 4H), 1.57-1.50 (m, 4H), 1.32 (td, J = 13.4, 3.7Hz, 28H), 0.88 (t, J = 6.8Hz, 6H).

[0927] Step (9)

[0928]

[0929] At room temperature, p-toluenesulfonic acid (0.839 g, 4.41 mmol) was added in a single addition 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 (0.920 g, 0.882 mmol) in methanol / THF (60 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 90% (80%) ethyl acetate in petroleum ether to give 2,3-bis(non-8-enoxy)prop-1-ol (0.402 g, 56.9%) as a colorless oil.

[0930] 1 H NMR (400MHz, CDCl3) δ5.69-5.59(m,2H),5.56-5.48(m,2H),4.62(d,J=6.8Hz,4H),3.69-3.39(m,23 H),2.33-2.26(m,4H),2.13-2.06(m,4H),1.66-1.50(m,8H),1.45-1.21(m,30H),0.91-0.85(m,6H).

[0931] Step (10)

[0932]

[0933] At room temperature, 2-[tert-butoxycarbonyl-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-2-[8-[(Z)-non-2-enoxy]-8-oxo-octoxy]propoxy]octyl ester (0.44 g, 0.549 mmol) was added to a solution of [(Z)-non-2-enoxy]-8-oxo-octoxy]propoxy]octyl ester (0.239 g, 0.549 mmol), 4-methylmorpholine (0.084 g, 0.824 mmol), and DMAP (0.101 g, 0.824 mmol) in dry DCM (15 mL). The mixture was then cooled to 0 °C and EDCI (0.158 g, 0.824 mmol) was added in portions over 15 min. The reaction was stirred at room temperature for 17 h. TLC showed that the reaction was complete. The reaction mixture was poured into water and extracted with DCM. The water was then extracted again with DCM. The combined organic matter was washed with brine, dried over Na2SO4, and concentrated under vacuum. It was then purified by column chromatography with petroleum acetate (0-100%) (60%) to give the target product as a pale yellow oil (0.476 g, 71.2% yield).

[0934] 1 H NMR (400MHz, CDCl3) δ5.69-5.59(m,2H),5.56-5.48(m,2H),4.62(d,J=6.8Hz,4H),4.30 -4.19(m,4H),4.04(d,J=6.1Hz,2H),4.00-3.95(m,2H),3.70(s,2H),3.64(d,J=5.4Hz, 12H),3.58-3.39(m,15H),3.30(d,J=5.6Hz,3H),2.30(t,J=7.4Hz,4H),2.13-2.05(m,4 H),1.60-1.50(m,6H),1.44(d,J=18.8Hz,18H),1.40-1.22(m,30H),0.91-0.85(m,6H).

[0935] Step (11)

[0936]

[0937] TFA (3 mL) was added dropwise to a solution of 8-[3-(tert-butoxycarbonylamino)propyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoic acid undecyl ester (0.476 g, 0.391 mmol) in CH2Cl2 (5 mL) under ice bath conditions, and the mixture was stirred at room temperature for 4 h. The solvent was removed under vacuum, and then dissolved in CH2Cl2 and the solvent was removed several times to remove TFA, yielding a pale yellow oily product: [(Z)-non-2-enyl]8-[3-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethylamino)acetyl]oxyethylamino]acetyl]oxyethoxy]ethoxy]ethoxy]-2-[8-[(Z)-non-2-enyloxy]-8-oxo-octyloxy]propoxy]octyl ester; 2,2,2-trifluoroacetaldehyde (0.530 g, yield: 112%).

[0938] 1 H NMR (400MHz, CDCl3) δ5.69-5.60(m,2H),5.56-5.47(m,2H),4.62(d,J=6.9Hz ,4H),4.36(s,2H),4.04(s,2H),3.98(s,2H),3.73-3.68(m,4H),3.67-3.41( m,23H),3.36(s,3H),3.33(d,J=4.5Hz,2H),2.34-2.27(m,4H),2.13-2.02(m ,4H),1.67-1.58(m,4H),1.54(s,4H),1.41-1.21(m,30H),0.92-0.84(m,6H).

[0939] Example 15: Synthesis of [(Z)-non-2-enyl]8-[3-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethylamino)acetyl]oxyethylamino]acetyl]oxyethoxy]ethoxy]ethoxy]ethoxy]-2-[(7R,11R)-3,7,11,15-tetramethylhexadecyloxy]propoxy]octyl ester; 2,2,2-trifluoroacetic acid (compound (XVIII))

[0940]

[0941] Compound (XVIII)

[0942] Synthesis of compound (XVIII)

[0943] Step (1)

[0944]

[0945]

[0946] Add 3-triphenylmethoxypropyl-1,2-diol (30 g, 28.4 mmol) to a suspension of NaH (15 g, 60% in oil, 113.5 mmol) in 450 mL of anhydrous DMF. Heat the mixture at 80 °C for 15 min and cool to room temperature. Add 9-bromonon-1-ene (45 g, 142 mmol) in 5 mL of anhydrous DMF dropwise to the mixture, and then heat at 80 °C for 18 h. After cooling to room temperature, add 1000 mL of H₂O to destroy the remaining NaH. Extract the organic phase with 3 x 250 mL of ethyl acetate. Wash the extract sequentially with 2 x 150 mL of 1N HCl, 2 x 150 mL of 5% (w / v) NaHCO₃, and 150 mL of brine, and dry on 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 (2% to 5% ethyl acetate in petroleum ether) to produce a colorless oil (17.5 g; yield 33.5%). Two byproducts were present: 1-non-8-enoxy-3-triphenylmethoxy-prop-2-ol (7.2 g; yield 18.4%) and 2-non-8-enoxy-3-triphenylmethoxy-prop-1-ol (3.77 g; yield 9.65%).

[0947] 1 H NMR (400MHz, CDCl3) δ7.46(d,J=7.4Hz,5H),7.28(t,J=6.6Hz,5H),7.22(dd,J=8.2,6.1Hz,3H),5.87-5.74(m,2H),4.99 (d, J = 17.1Hz, 2H), 4.93 (d, J = 10.2Hz, 2H), 3.65-3.47 (m, 5H), 3.43-3.00 (m, 4H), 2.09-2.01 (m, 4H), 1.50-1.21 (m, 17H).

[0948] 1H NMR (400MHz, CDCl3) δ7.43(d,J=7.4Hz,6H),7.30(t,J=7.5Hz,6H),7.23(t,J=7.3Hz,3H),5.81(ddt,J=16.9,10.1,6.6Hz,1H),5.03-4.89(m,2H), 3.95(dd,J=10.2,4.7Hz,1H),3.55-3.40(m,4H),3.22-3.14(m,2H),2.41 (d, J=4.5Hz, 1H), 2.04 (q, J=6.9Hz, 2H), 1.56-1.50 (m, 4H), 1.29 (s, 6H).

[0949] 1 H NMR (400MHz, CDCl3) δ7.44(d,J=7.6Hz,6H),7.30(t,J=7.5Hz,6H),7.26-7.20(m,3H),5.81(ddtd,J=16.9,10.1,6.6,3.5Hz,3H) ,4.99(d,J=17.1Hz,3H),4.93(d,J=10.2Hz,2H),3.80-3.10(m,11H),2.13-1.97(m,7H),1.59-1.51(m,6H),1.45-1.18(m,24H).

[0950] Step (2)

[0951]

[0952] Triethylamine (15.9 g, 157 mmol) and methanesulfonyl chloride (13.5 g, 118 mmol) were added to a solution of (7R,11R)-3,7,11,15-tetramethylhexadecyl-1-ol (23.5 g, 78.7 mmol) in DCM (300 ml) at 0 °C. The mixture was stirred at 25 °C for 14 h. After the reaction, the mixture was concentrated and treated with EA (250 ml), washed with 1N HCl / H2O (250 ml x 2), NaHCO3 aqueous solution (250 ml x 2), NaCl aqueous solution (250 ml), and dried over Na2SO4. The organic matter was concentrated to give [(7R,11R)-3,7,11,15-tetramethylhexadecyl]methanesulfonate (30.4 g, crude product) as a yellow oil.

[0953] 1H NMR (400MHz, CDCl3) δ4.30-4.23 (m, 2H), 3.00 (d, J = 3.2Hz, 3H), 1.79 (ddt, J = 9. 2,7.2,2.2Hz,1H),1.58-1.51(m,2H),1.35-1.07(m,21H),0.93-0.84(m,15H).

[0954] Step (3)

[0955]

[0956] Add NaH (1.53 g, 38.2 mmol) to a solution of 1-non-8-enoxy-3-triphenylmethoxy-prop-2-ol (3.5 g, 7.6 mmol) in DMF (150 mL). Stir the mixture at 80 °C for 1 h. Add [(7R,11R)-3,7,11,15-tetramethylhexadecyl]methanesulfonate (4.31 g, 11.4 mmol) dropwise to the mixture. Stir the mixture at 80 °C for another 14 h. Treat the mixture with EA (150 mL). Wash with water (250 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 [[3-non-8-enoxy-2-[(7R,11R)-3,7,11,15-tetramethylhexadecyloxy]propoxy]-diphenyl-methyl]benzene (1.88 g, 2.5 mmol, yield 32.7%) as a colorless oil.

[0957] 1 H NMR (400MHz, CDCl3) δ7.46(dd,J=10.8,3.6Hz,6H),7.34-7.26(m,6H),7.23(dd,J=9.1,2.0Hz,3H),5.81(ddt,J=16.9,10.2,6.7Hz,1H),5.05-4.90( m,2H),3.65-3.35(m,8H),3.17(d,J=3.5Hz,1H),2.03(dd,J=14.4,6.8Hz, 2H), 1.51 (dd, J=13.2, 6.6Hz, 4H), 1.34-1.13 (m, 30H), 0.87-0.83 (m, 15H).

[0958] Step (4)

[0959]

[0960] Toluene-4-sulfonic acid (4.50 g, 23.7 mmol) was added to a solution of [[3-non-8-enoxy-2-[(7R,11R)-3,7,11,15-tetramethylhexadecyloxy]propoxy]-diphenylmethyl]benzene (3.5 g, 4.73 mmol) in MeOH / THF (30 mL 1:1). The mixture was stirred at 25 °C for 14 h. The mixture was concentrated and treated with EA (150 mL), washed with water (150 mL x 2), saturated aqueous solution of NaCl (150 mL), and dried over Na2SO4. The organic matter was concentrated and rapidly purified (10% EA in PE) to give 3-non-8-enoxy-2-[(7R,11R)-3,7,11,15-tetramethylhexadecyloxy]prop-1-ol (1.22 g, 50.8% yield) as a yellow oil.

[0961] 1 H NMR(400MHz, CDCl3)δ5.81(ddt,J=16.9,10.2,6.7Hz,1H),5.03-4.89(m,2H),3.73-3.42(m,9H),2 .04(dd,J=14.4,6.8Hz,2H),1.57-1.49(m,4H),1.35-1.06(m,30H),0.86(dd,J=8.5,6.3Hz,15H).

[0962] Step (5)

[0963]

[0964] 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 washed and 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 then removed to give 2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethyl methanesulfonic acid (38.8 g, 75.4 mmol, 99.8% yield) as a yellow oil.

[0965] 1¹H NMR (400MHz, CDCl₃) δ 7.48–7.44 (m, 6H), 7.32–7.27 (m, 5H), 7.26–7.20 (m, 4H), 4.35–4.30 (m, 2H), 3.75–3.71 (m, 2H), 3.70–3.63 (m, 10H), 3.26–3.20 (m, 2H), 2.98 (s, 3H). LCMS peak found: 537.2 (M+23) at 2.198 min 98% UV (214nm)

[0966] Step (6)

[0967]

[0968] NaH (298 mg, 7.45 mmol) was added to a solution of 3-non-8-enoxy-2-[(7R,11R)-3,7,11,15-tetramethylhexadecyloxy]prop-1-ol (1.85 g, 3.72 mmol) in THF (40 mL). The mixture was stirred at 80 °C for 1 h. Then, 2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethyl methanesulfonic acid (1.92 g, 3.72 mmol) was added to the mixture at 25 °C. The mixture was stirred at 80 °C for 18 h. Subsequently, the mixture was treated with EA (150 mL), washed with water (150 mL x 2), a saturated aqueous solution of NaCl (150 mL), and dried over Na2SO4. The organic matter was concentrated and rapidly purified (20% EA in PE) to give a colorless oily substance, [2-[2-[2-[2-[3-non-8-enoxy-2-[(7R,11R)-3,7,11,15-tetramethylhexadecyloxy]propoxy]ethoxy]ethoxy]ethoxy]ethoxydiphenyl-methyl]benzene (2.11 g, yield 60.7%).

[0969] 1 H NMR (400MHz, CDCl3) δ7.48-7.43(m,6H),7.31-7.27(m,6H),7.25-7.20(m,3H),5.80(ddt,J=16.9,10.1,6.7Hz,1H),5.04-4.88(m,2H) ,3.70-3.41(m,23H),3.23(t,J=5.2Hz,2H),2.03(dd,J=14.4,6.8Hz,2H),1.57-1.48(m,4H),1.36-1.06(m,30H),0.88-0.83(m,15H).

[0970] Step (7)

[0971]

[0972] To a solution of [2-[2-[2-[2-[3-non-8-enoxy-2-[(7R,11R)-3,7,11,15-tetramethylhexadecyloxy]propoxy]ethoxy]ethoxy]ethoxy-diphenyl-methyl]benzene (2.11 g, 2.3 mmol) in ACN / CCl4 / H2O (5 ml / 5 ml / 5 ml), add NaIO4 (1.97 g, 9.2 mmol) and ruthenium(III) chloride (47.8 mg, 0.23 mmol). Stir the mixture at 25 °C for 14 h. Filter the reaction mixture, and dilute the filtrate with ethyl acetate (250 mL) and wash with 1 N HCl aqueous solution (250 mL). The organic layer was washed with Na2S2O3 solution and then dried over sodium sulfate. It was filtered and concentrated to obtain 8-[2-[(7R,11R)-3,7,11,15-tetramethylhexadecyloxy]-3-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]propoxy]octanoic acid (1.78 g, crude product), which was used without further purification.

[0973] 1 H NMR (400MHz, CDCl3) δ7.46 (d, J=8.1Hz, 6H), 7.32-7.28 (m, 6H), 7.22 (dd, J=10.4, 4.0Hz, 3H), 3.68-3.45 (m, 23H), 3.23 (t, J= 5.1Hz,2H),2.35(dt,J=28.5,7.4Hz,2H),1.67-1.48(m,8H),1.36-1.23(m,22H),1.05(d,J=7.8Hz,4H),0.87-0.83(m,15H).

[0974] Step (8)

[0975]

[0976] To a solution of (Z)-non-2-en-1-ol (326 mg, 2.3 mmol) in DCM (20 mL), 8-[2-[(7R,11R)-3,7,11,15-tetramethylhexadecyloxy]-3-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]propoxy]octanoic acid (1.78 g, 1.9 mmol), DIEA (739 mg, 5.72 mmol), 4-dimethylaminopyridine (46.4 mg, 0.38 mmol), and EDC HCl (548 mg, 2.86 mmol) were added. The mixture was stirred at 26 °C for 18 h. The mixture was then concentrated and rapidly purified (20% EA in PE) to give [(Z)-non-2-enyl]8-[2-[(7R,11R)-3,7,11,15-tetramethylhexadecyloxy]-3-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethoxy]propoxy]octyl ester (443 mg, 0.4 mmol, 98% yield) as a colorless oil.

[0977] 1 H NMR(400MHz, CDCl3)δ7.50-7.42(m,6H),7.32-7.27(m,6H),7.25-7.20(m,3H), 5.63(t,J=9.2Hz,1H),5.56-5.48(m,1H),4.62(d,J=6.8Hz,2H),3.71-3.39(m,2 3H),3.23(t,J=5.3Hz,2H),2.29(t,J=7.6Hz,2H),2.09(q,J=7.0Hz,2H),1.51(d d,J=13.3,6.7Hz,4H),1.37-1.04(m,38H),0.85(ddd,J=11.5,6.2,2.9Hz,18H).

[0978] Step (9)

[0979]

[0980] Toluene-4-sulfonic acid (398 mg, 2.1 mmol) was added to a solution of [(Z)-non-2-enyl]8-[2-[(7R,11R)-3,7,11,15-tetramethylhexadecyloxy]-3-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]propoxy]octyl ester (443 mg, 0.42 mmol) in MeOH / THF (10 mL 1:1). The mixture was stirred at 25 °C for 1 h. ET3N (1 mL) was then added to this mixture. The mixture was concentrated and rapidly purified (5% MeOH in DCM) to give [(Z)-non-2-enyl]8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-2-[(7R,11R)-3,7,11,15-tetramethylhexadecyloxy]propoxy]octyl ester (240 mg, 0.29 mmol, yield 68.9%) as a colorless oil.

[0981] 1 H NMR (400MHz, CDCl3) δ5.64(dt,J=10.9,7.5Hz,1H),5.56-5.47(m,1H),4.62(d,J=6.8Hz,2H),3.76-3.40(m,25H),2.67( t,J=6.0Hz,1H),2.30(t,J=7.6Hz,2H),2.09(dt,J=15.6,7.8Hz,2H),1.63-1.04(m,42H),0.86(tt,J=6.6,4.3Hz,18H).

[0982] Step (10)

[0983]

[0984] Add 2-[tert-butoxycarbonyl-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-2-[(7R,11R)-3,7,11,15-tetramethylhexadecyloxy]propoxy]octanoate (240 mg, 0.3 mmol) to a solution of [(Z)-non-2-enyl]8-[3-[2-[2-[2-(2-hydroxyethoxy)carbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (192 mg, 0.44 mmol), 4-dimethylaminopyridine (54 mg, 0.44 mmol), N-methylmorpholine (45 mg, 0.44 mmol), and EDC HCl (84.7 mg, 0.44 mmol) to a solution of [(Z)-non-2-enyl]8-[3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]acetyl]acetyl]oxyethyl]amino]acetic acid (192 mg, 0.44 mmol), 4-dimethylaminopyridine (54 mg, 0.44 mmol), N-methylmorpholine (45 mg, 0.44 mmol), and EDC HCl (84.7 mg, 0.44 mmol). Stir the mixture at 25 °C for 18 h. The mixture was then treated with DCM (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 purified rapidly (50% EA in PE) to give [(Z)-non-2-enyl]8-[3-[2-[2-[2-[2-[2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetyl]oxyethoxy]ethoxy]ethoxy]ethoxy]-2-[(7R,11R)-3,7,11,15-tetramethylhexadecyloxy]propoxy]octyl ester (211 mg, 0.17 mmol, yield 57%).

[0985] 1 H NMR (400MHz, CDCl3) δ5.63 (t, J=9.1Hz, 1H), 5.51 (dd, J=10.9, 6.6Hz, 1H), 4.6 2(d,J=6.8Hz,2H),4.25(dd,J=14.0,8.8Hz,4H),4.08-3.95(m,4H),3.73-3.39 (m,28H),3.30(d,J=5.8Hz,3H),2.30(t,J=7.6Hz,2H),2.10(dd,J=14.1,7.3Hz ,2H),1.61(d,J=6.5Hz,2H),1.57-1.01(m,59H),0.86(tt,J=6.6,4.3Hz,18H).

[0986] Step (11)

[0987]

[0988] Add TFA (0.36 ml) to a solution of [(Z)-non-2-enyl]8-[3-[2-[2-[2-[2-[2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetyl]oxyethoxy]ethoxy]ethoxy]-2-[(7R,11R)-3,7,11,15-tetramethylhexadecyloxy]propoxy]octyl ester (60 mg, 0.05 mmol) in DCM (1 ml). Stir the mixture at 25 °C for 4 h. The mixture was then concentrated to give a colorless oily product, [(Z)-non-2-enyl]8-[3-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethylamino)acetyl]oxyethylamino]acetyl]oxyethoxy]ethoxy]ethoxy]-2-[(7R,11R)-3,7,11,15-tetramethylhexadecyloxy]propoxy]octyl ester; 2,2,2-trifluoroacetic acid (235 mg, 0.2 mmol, 92.2% yield).

[0989] 1 H NMR (400MHz, CDCl3) δ5.63(t,J=9.2Hz,1H),5.51(dd,J=10.9,6.8Hz,1H),4.62(d,J=6.8Hz,2H),4.36(s,2H),3.99(d,J=19 .1Hz,4H),3.74-3.29(m,34H),2.30(t,J=7.6Hz,2H),2.09(dd,J=14.5,7.4Hz,2H),1.65-1.00(m,44H),0.93-0.76(m,18H).

[0990] Example 16: Synthesis of 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propionyl-octylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester; Synthesis of 2,2,2-trifluoroacetic acid (compound (XIX))

[0991]

[0992] Compound (XIX)

[0993] Synthesis of compound (XIX)

[0994] Step (1)

[0995]

[0996] Dys-Martin oxidant (2.02 g, 4.05 mmol) was added to a solution of 2,3-bis[(Z)-octadec-9-enoxy]prop-1-ol (2 g, 3.37 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 / Na2S2O (1 / 1) (50 ml x 3) and saturated aqueous solution of NaCl (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 (1 g, 1.66 mmol, yield 49.2%) as a colorless oil.

[0997] 1 H NMR (400MHz, 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).

[0998] Step (2)

[0999]

[1000] 2,3-bis[(Z)-octadec-9-enoxy]propanal (1.48 g, 2.5 mmol) was dissolved in t-BuOH:H2O (3:1, 20 mL) containing NaH2PO4·2H2O (1.17 g, 7.51 mmol), 2-methyl-2-butene (5.26 mL), and sodium chlorite (679 mg, 7.51 mmol). The reaction was stirred at room temperature for 1 h and diluted with ethyl acetate (100 mL) and washed with water (100 mL x 2). The aqueous layer was extracted with ethyl acetate (100 mL). The combined extracts were dried over sodium sulfate and concentrated to provide 2,3-bis[(Z)-octadec-9-enoxy]propionic acid (1.35 g, 2.17 mmol, 86.8% yield) as a colorless oil.

[1001] 1H NMR (400MHz, CDCl3) δ5.41-5.29(m,4H),4.04(dd,J=5.0,3.3Hz,1H),3.80(dd,J=10.5,3.2Hz,1H),3.71(dd,J=10.5,5.1Hz,1 H),3.63(t,J=6.7Hz,2H),3.52-3.45(m,2H),2.08-1.91(m,7H),1.66-1.55(m,4H),1.30-1.22(m,44H),0.87(d,J=7.1Hz,6H).

[1002] Step (3)

[1003]

[1004] 2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethyl ester (5 g, 8 mmol) of methanesulfonic acid was added to oct-1-amine (20 ml), and the mixture was stirred at 80 °C for 18 h. LC-MS showed that SM was consumed and the product was formed. The mixture was treated with EA (300 ml), washed with water (300 ml x 2), saturated aqueous solution of NaCl (300 ml), and dried over Na2SO4. The organic matter was concentrated and rapidly purified (10% MeOH in DCM) to give N-[2-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethyl]oct-1-amine (2.06 g, 3.48 mmol, yield 43.2%) as a yellow oil.

[1005] 1 H NMR (500MHz, CDCl3) δ7.49-7.44(m,6H),7.29(dd,J=10.3,4.8Hz,6H),7.25-7.20(m,3H),3.71-3.56(m,16H),3.23(t,J=5. 2Hz, 2H), 2.78 (t, J = 5.2Hz, 2H), 2.63-2.54 (m, 2H), 1.48 (dd, J = 14.4, 7.3Hz, 2H), 1.32-1.23 (m, 10H), 0.87 (t, J = 6.9Hz, 3H).

[1006] Step (4)

[1007]

[1008] Add 2,3-bis[(Z)-octadec-9-enoxy]propionic acid (923 mg, 1.52 mmol), 4-dimethylaminopyridine (18.6 mg, 0.15 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylureonium hexafluorophosphate (867 mg, 2.28 mmol), and TEA (308 mg, 3.04 mmol) to a solution of N-[2-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]octyl-1-amine (1 g, 1.52 mmol) in DCM (20 ml). Stir the mixture at 25 °C for 18 h. After the reaction, treat the mixture with DCM (100 ml), wash with water (150 ml x 2), saturated aqueous solution of NaCl (150 ml), and dry with Na2SO4. Organic matter was consumed and rapidly purified (10% 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 (1079 mg, 0.9 mmol, yield 60.1%) as a yellow oil.

[1009] 1 H NMR (400MHz, CDCl3) δ7.49-7.43(m,6H),7.29(dd,J=10.2,4.9Hz,6H),7.22(dd,J=8.3,6.1Hz,3H),5.40-5.31(m,3H),4.42 -4.30(m,1H),3.74-3.34(m,26H),3.23(t,J=5.2Hz,2H),2.11-1.83(m,7H),1.54(s,4H),1.27(s,56H),0.91-0.85(m,9H).

[1010] Step (5)

[1011]

[1012] Toluene-4-sulfonic acid (869 mg, 4.57 mmol) was added to a solution of 2,3-bis[(Z)-octadec-9-enoxy]-N-octyl-N-[2-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]propionamide (1079 mg, 0.9 mmol) in THF / MeOH (10 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 (750 mg, 0.8 mmol, yield 87.6%) as a colorless oil.

[1013] 1 H NMR (400MHz, CDCl3) δ5.40-5.31(m,4H),4.44-4.31(m,1H),3.75-3.37(m,29H),2.11-1.90(m,8H),1.56(s,4H),1.27(s,56H),0.91-0.86(m,9H).

[1014] Step (6)

[1015]

[1016] Add 2-[tert-butoxycarbonyl-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]-2,3-bis[(Z)-octadec-9-enoxy]-N-octylpropionamide (350 mg, 0.3 mmol) to a solution of N-[2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (165 mg, 0.38 mmol), DMAP (58 mg, 0.48 mmol), NMM (64 mg, 0.63 mmol), and EDCI (91 mg, 0.48 mmol) to a solution of N-[2 ...2-(2-hydroxyethoxy)ethoxy]ethoxy]ethyl]-2,3-bis[(Z)-octadec-9-enoxy]-N-octylpropionamide (350 mg, 0.3 mmol), DMAP (58 mg, 0.48 mmol), NMM (64 mg, 0.63 mmol), and EDCI (91 mg, 0.48 mmol) to a solution of N-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]amino]ethyl]-2,3-bis[(Z)-octadec-9-enoxy]-N-octylpropionamide (350 mg, 0.3 mmol), DMAP (58 mg, 0.48 mmol The organic compound was rapidly purified (in DCM with 10% MeOH) to give 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadecyl-9-enoxy]propionyl-octylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (360 mg, 0.26 mmol, yield 82.1%).

[1017] 1 H NMR (400MHz, CDCl3) δ5.34(s,4H),4.27(dd,J=24.6,11.2Hz,5H),4.06-3.95(m,4H),3.73-3.38(m,32H),3.30(d,J=5.6H z, 3H), 2.07-1.93 (m, 8H), 1.58 (s, 2H), 1.54-1.50 (m, 2H), 1.44 (d, J = 19.2Hz, 18H), 1.27 (s, 56H), 0.88 (t, J = 6.8Hz, 9H).

[1018] Step (7)

[1019]

[1020] Add 1.97 mL of TFA to a solution of 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propionyl-octylamino]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (360 mg, 0.27 mmol) in DCM (5 mL). Stir the mixture at 25 °C for 3 h. After the reaction, the mixture was concentrated to give 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[2,3-bis[(Z)-octadec-9-enoxy]propionyl-octyl-amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester, which was a colorless oil; 2,2,2-trifluoroacetic acid (343 mg, 0.26 mmol, yield 96.7%).

[1021] 1 H NMR (400MHz, CDCl3) δ5.34(s,4H),4.58(s,2H),4.38(s,2H),4.00(d,J=6.0Hz,4H),3.71(s,6H),3.53(d,J=5 9.7Hz, 21H), 3.34 (d, J = 23.0Hz, 9H), 2.01 (d, J = 6.1Hz, 8H), 1.53 (s, 4H), 1.27 (s, 56H), 0.88 (t, J = 5.1Hz, 9H).

[1022] Example 17: Synthesis of 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyloxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester; Synthesis of 2,2,2-trifluoroacetic acid (compound (XX))

[1023]

[1024] Compound (XX)

[1025] Synthesis of compound (XX)

[1026] Step (1)

[1027]

[1028] 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.

[1029] 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).

[1030] Step (2)

[1031]

[1032] Add NaH (223 mg, 5.57 mmol) to a solution of 1,2-bis[(Z)-octadec-9-enoxy]pentadecan-3-ol (1.06 g, 1.39 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 (1.07 g, 2.09 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 g, 0.8 mmol, yield 59.7%).

[1033] 1 H NMR (500MHz, CDCl3) δ7.46 (d, J = 7.2Hz, 6H), 7.31-7.27 (m, 6H), 7.24-7.20 (m, 3H), 5.42-5.30 (m, 3H), 3.73-3.33(m,22H),3.25-3.22(m,2H),2.06-1.91(m,6H),1.59-1.06(m,70H),0.88(t,J=6.8Hz,9H).

[1034] Step (3)

[1035]

[1036] Toluene-4-sulfonic acid (806 mg, 4.24 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 g, 0.85 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 (50 ml), washed with saturated aqueous solution of NaHCO3 (50 ml x 2), 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 2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyloxy]ethoxy]ethoxy]ethoxy]ethanol (550 mg, 0.58 mmol, yield 67.8%) as a colorless oil.

[1037] 1 H NMR (400MHz, CDCl3) δ 5.51-5.31 (m, 3H), 3.76-3.33 (m, 23H), 2.59 (s, 1H), 2.02 (dt, J = 12.3, 6.0Hz, 6H), 1.69-1.02 (m, 70H), 0.88 (t, J = 6.8Hz, 9H).

[1038] Step (4)

[1039]

[1040] Add 2-[tert-butoxycarbonyl-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyloxy]ethoxy]ethoxy]ethoxy]ethanol (100 mg, 0.1 mmol) to a solution of 2-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (92.7 mg, 0.21 mmol), DIEA (34.5 mg, 0.27 mmol), DMAP (3 mg), and EDCI (40.9 mg, 0.21 mmol) to a solution of 2-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (92.7 mg, 0.21 mmol), DIEA (34.5 mg, 0.27 mmol), DMAP (3 mg), and EDCI (40.9 mg, 0.21 mmol) to a solution of 2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]amino]acetyl ... DMAP (3 mg), and EDCI (40.9 mg, 0.21 mmol) to a solution of 2-[2-[2-[1-[1,2- The organic matter was concentrated and rapidly purified (35% EA in PE) to give 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyloxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (102 mg, 0.07 mmol, yield 68.5%).

[1041] 1 H NMR (500MHz, CDCl3) δ5.45-5.31(m,3H),4.32-4.15(m,4H),4.07-3.94(m,4H),3 .77-3.26(m,32H),2.06-1.92(m,6H),1.55-1.23(m,88H),0.88(t,J=6.9Hz,9H).

[1042] Step (5)

[1043]

[1044] Add 0.5 ml of TFA to a solution of 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyloxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (332 mg, 0.24 mmol) in DCM (3 ml). Stir the mixture at 25 °C for 3 h. The mixture was concentrated to obtain 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyloxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester, which is a yellow oil; 2,2,2-trifluoroacetic acid (354 mg, quantified).

[1045] 1 H NMR (400MHz, CDCl3) δ5.34(s,4H),4.59(s,2H),4.37(s,2H),4.03-3.95(m,4H),3.67(d,J=27.6Hz,18H) ,3.40(dt,J=36.9,13.9Hz,15H),2.01(d,J=5.5Hz,8H),1.40(d,J=111.8Hz,70H),0.88(t,J=6.7Hz,9H).

[1046] Example 18: Synthesis of 2-butyloctyl octanoate of 8-[2-[8-(2-butyloctyloxy)-8-oxo-octyloxy]-3-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethylamino)acetyl]oxyethylamino]acetyl]oxyethoxy]ethoxy]ethoxy]propoxy]octanoate; 2,2,2-trifluoroacetic acid (compound (XXI))

[1047]

[1048] Compound (XXI)

[1049] based on Figure 9 The chemically synthesized compound (XXI) shown in scheme (11) presented in the paper.

[1050] Synthesis of compound (XXI)

[1051] Step (1)

[1052]

[1053] 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.

[1054] LCMS MS 363(M+1)

[1055] Step (2)

[1056]

[1057] 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.

[1058] LCMS MS 421(M+23)

[1059] Step (3)

[1060]

[1061] 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.

[1062] LCMS MS 381(M+23)

[1063] Step (4)

[1064]

[1065] 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.

[1066] 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).

[1067] Step (5)

[1068]

[1069] 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.

[1070] 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).

[1071] Step (6)

[1072]

[1073] 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.

[1074] 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).

[1075] Step (7)

[1076]

[1077] 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.

[1078] 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).

[1079] Step (8)

[1080]

[1081]

[1082] In the presence of N-methylmorpholine (64.1 mg, 0.843 mmol), 2-butyloctyl octanoate (500 mg, 0.562 mmol), 2-[tert-butoxycarbonyl-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]propoxy]octanoate (244 mg, 0.562 mmol), and N,N-dimethylpyridin-4-amine (77.3 mg, 0.843 mmol) were dissolved in anhydrous DCM (30 mL). The reaction was cooled to 0°C and 3-(ethyliminomethyleneamino)-N,N-dimethyl-prop-1-amine hydrochloride (121.2 mg, 0.843 mmol) was added in portions to the mixture. The reaction was warmed to room temperature and stirred overnight. The reaction was monitored by TLC. Water was added and the organic layer was extracted with DCM (3 x 30 mL), washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by rapid column chromatography on silica gel by elution with 20% to 40% ethyl acetate in petroleum ether to give 2-[3-[2-[2-[2-[2-[2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetyl]oxyethoxy]ethoxy]ethoxy]ethoxy]-2-[8-(2-butyloctyloxy)-8-oxo-octyloxy]propoxy]octanoate 2-butyloctanoate (306 mg, 42% yield).

[1083] 1 H 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).

[1084] Step (9)

[1085]

[1086] TFA (1 mL) was added to 2-butyloctyl octanoate (306 mg, 0.234 mmol) in DCM (5 mL) cooled to 0 °C and the mixture was stirred at room temperature for 4 h. TLC (DCM / MeOH = 10:1, expected product Rf = 0.1) showed that the starting material disappeared. The solvent was removed and the product was azeotropically reacted with DCM several times and then dried under vacuum for 2 h to give 2-butyloctyl octanoate (300 mg, quantified).

[1087] 1 H NMR (400MHz, CDCl3) δ4.60(s,2H),4.36(s,2H),3.97(t,8H),3.74-3.68(m,4H),3.64(s,11H),3.60-3.51(m,5H),3.51-3.40( m,6H),3.35(s,3H),3.35-3.28(m,2H),2.30(t,J=7.2Hz,4H),1.74-1.42(m,11H),1.38-1.20(m,46H),0.88(t,J=5.4Hz,12H).

[1088] Example 19: Synthesis of 2-Butyloctanoic acid 6-[2-[6-(2-Butyloctanoyloxy)hexyloxy]-3-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethylamino)acetyl]oxyethylamino]acetyl]oxyethoxy]ethoxy]ethoxy]propoxy]hexyl ester; Synthesis of 2,2,2-trifluoroacetic acid (compound (XXII))

[1089]

[1090] Compound (XXII)

[1091] Synthesis of compound (XXII)

[1092] 2-Butyloctanoic acid 6-[3-[2-[2-[2-[2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetyl]oxyethoxy]ethoxy]ethoxy]-2-[6-(2-butyloctanoyloxy)hexyloxy]propoxy]hexyl ester (0.384 g, 0.307 mmol) was dissolved in 7 mL of DCM and then TFA (1 mL) was added. The mixture was stirred at room temperature for 2 h. Evaporate the solvent in DCM (30 mL * 3) to give 2-butyloctanoic acid 6-[2-[6-(2-butyloctanoyloxy)hexyloxy]-3-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethylamino)acetyl]oxyethylamino]acetyl]oxyethoxy]ethoxy]ethoxy]propoxy]hexyl ester, which is a pale yellow oil; 2,2,2-trifluoroacetic acid (0.3641 g, 0.297 mmol, 96.8% yield).

[1093] LCMS: A peak was found at 2.36 min, Mr: 1050.8 (M+1).

[1094] 1 H NMR (400MHz, CDCl3) δ4.61(s,2H),4.35(s,2H),4.09-3.94(m,7H),3.72-3.29(m,33H),2.37-2.24(m,2H),1.65-1.20(m,48H),0.94-0.80(m,12H).

[1095] Example 20: Synthesis of 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[3-[2,3-bis[(Z)-octadec-9-enoxy]propyl-octyl-amino]-3-oxopropoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester; Synthesis of 2,2,2-trifluoroacetic acid (compound (XXIII))

[1096]

[1097] Compound (XXIII)

[1098] Synthesis of compound (XXIII)

[1099] Step (1)

[1100]

[1101] 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.

[1102] 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).

[1103] Step (2)

[1104]

[1105] The reaction mixture was added to a solution of 3,2,3-bis[(Z)-octadec-9-enoxy]propyl methanesulfonate (4.5 g, 6.71 mmol) and octyl-1-amine (17.3 g, 134 mmol). The reaction mixture 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]octyl-1-amine (4.2 g, 89% yield) as a pale yellow oil.

[1106] 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).

[1107] Step (3)

[1108]

[1109] 0.6 g, 1 mmol, of tert-butyl 3-[2-[2-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethoxy]ethoxy]propionic acid tert-butyl ester was dissolved in 2:1 DCM-TFA (5 mL) and the solution was stirred at room temperature for 1 hour. The resulting mixture was diluted with 5 mL of H2O and 10 mL of DCM. The solution was stirred vigorously to mix the phases and the solution was alkalized to pH 3 with 2 M NaOH. The layers were separated and the aqueous phase was extracted with DCM (30 mL). The combined organic matter was evaporated under vacuum and the crude product was purified by rapid chromatography by elution with 0 to 10% MeOH in DCM to produce 0.23 g, 0.43 mmol, 42.6% tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethoxy]propionic acid as a colorless oil.

[1110] 1 H NMR (400MHz, CDCl3) δ7.72-7.63(m,4H),7.45-7.33(m,6H),3.84-3.73(m,4H),3.71-3.55(m,14H),2.60(t,J=6.1Hz,2H),1.04(s,9H).

[1111] Step (4)

[1112]

[1113] HATU (0.247 g, 0.65 mmol) and DIEA (0.112 g, 0.866 mmol) were added to a mixture of 3-[2-[2-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethoxy]ethoxy]propionic acid (0.23 g, 0.43 mmol) and N-[2,3-bis[(Z)-octadec-9-enoxy]propyl]oct-1-amine (0.32 g, 0.43 mmol) in DMF (2 mL). The reaction was stirred at room temperature for 16 h. TLC showed that all starting materials were converted to the product. The mixture was partitioned between ethyl acetate (50 mL) and water (20 mL). The organic layer was washed with brine and LiCl solution, dried over sodium sulfate, filtered, and concentrated. The residue was purified by rapid column chromatography on silica gel by elution with 2%-5% methanol in dichloromethane to give N-[2,3-bis[(Z)-octadec-9-enoxy]propyl]-3-[2-[2-[2-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethoxy]N-octyl-propionamide (0.42 g, 0.335 mmol, 77%), which was a light yellow oil.

[1114] 1 H NMR (400MHz, CDCl3) δ7.71-7.63(m,4H),7.45-7.32(m,6H),5.40-5.31(m,3H),3.83-3.27(m,29H),2.72 -2.61(m,2H),2.05-1.95(m,6H),1.60-1.46(m,6H),1.37-1.19(m,56H),1.04(s,9H),0.91-0.83(m,9H).

[1115] Step (5)

[1116]

[1117] A solution of N-[2,3-bis[(Z)-octadec-9-enoxy]propyl]-3-[2-[2-[2-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethoxy]-N-octyl-propionamide (0.42 g, 0.317 mmol) in THF (5 mL) was added to tetra-n-butylammonium fluoride (1 M, 0.4 mL in THF), and the mixture was stirred at room temperature for 1 h. TLC showed that all starting materials were converted to the product. The solvent was removed and the sample was loaded onto silica gel and purified by rapid column chromatography on silica gel by elution with 2%–5% methanol in dichloromethane to obtain N-[2,3-bis[(Z)-octadec-9-enoxy]propyl]-3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-N-octyl-propionamide (0.31 g, 0.312 mmol, 98%), which was a pale yellow oil.

[1118] 1 H NMR (400MHz, CDCl3) δ5.40-5.28(m,3H),3.85-3.16(m,29H),2.86-2.60(m,3H),2 .07-1.91(m,6H),1.59-1.44(m,6H),1.28(t,J=14.6Hz,55H),0.92-0.84(m,9H).

[1119] Step (6)

[1120]

[1121] To a solution of N-[2,3-bis[(Z)-octadec-9-enoxy]propyl]-3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]-N-octylpropionamide (0.31 g, 0.31 mmol) and 2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (0.201 g, 0.464 mol) in dichloromethane (15 mL), N,N-diisopropylethylamine (0.048 g, 0.37 mmol) and DMAP (4 mg, 0.03 mmol) were added, followed by the partial addition of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.071 g, 0.037 mmol). The reaction was allowed to proceed with stirring at room temperature for 48 h. The reaction mixture was diluted with dichloromethane and washed with saturated sodium bicarbonate. The organic layer was separated, washed with brine, and dried over Na2SO4. The organic layer was filtered and evaporated under vacuum. The residue was purified by silica gel chromatography (2%–5% methanol in dichloromethane) to give 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-[2-[3-[2,3-bis[(Z)-octadec-9-enoxy]propyl-octyl-amino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (0.41 g, 0.288 mmol, 93% yield).

[1122] 1 H NMR (400MHz, CDCl3) δ5.41-5.31(m,3H),4.27-3.91(m,7H),3.83-3.70(m,4H),3.69-3.55(m,16H),3.53-3.17(m,17H), 2.78-2.61(m,2H),2.06-1.90(m,7H),1.60-1.49(m,5H),1.49-1.39(m,18H),1.26(d,J=4.9Hz,57H),0.93-0.83(m,9H).

[1123] Step (7)

[1124]

[1125] 0.41 g (0.29 mmol) of 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-[2-[3-[2,3-bis[(Z)-octadec-9-enoxy]propyl-octyl-amino]-3-oxopropoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (0.41 g, 0.29 mmol) was added dropwise to a solution of anhydrous dichloromethane (5 mL) and the reaction was stirred at room temperature for 3 h. TLC showed that the starting material was completely consumed. The solvent was removed and the residue was azeotropically reacted with DCM several times and then dried under vacuum for 2 h to give 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[3-[2,3-bis[(Z)-octadec-9-enoxy]propyloctyl-amino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester, which was a pale yellow oil; 2,2,2-trifluoroacetic acid (0.2645 g, 0.196 mmol, 68% yield).

[1126] 1 H NMR (400MHz, CDCl3) δ5.43-5.31(m,4H),4.62(s,1H),4.54-4.46(m,2H),4.31-4.21(m,1H),4.02(s,1H),3.91(s,1H),3.85 -3.13(m,36H),2.82-2.65(m,2H),2.10-1.86(m,8H),1.60-1.48(m,6H),1.28(dd,J=14.7,5.5Hz,54H),0.90-0.85(m,9H).

[1127] Example 21: Synthesis of 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tetrazoloxycarbonylamino]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester; Synthesis of 2,2,2-trifluoroacetic acid (compound (XXIV))

[1128]

[1129] Compound (XXIV)

[1130] Synthesis of compound (XXIV)

[1131] Step (1)

[1132]

[1133] Dess-Martin Periodinane (7.5 g, 17.7 mmol) was added to a solution of 2,3-bis[(Z)-octadec-9-enoxy]prop-1-ol (7 g, 11.8 mmol) in DCM (120 ml) over 5 min at 0 °C. The mixture was stirred at 25 °C for 2 h. The mixture was then filtered and concentrated. The crude product was rapidly purified (5% EA in PE) to give 2,3-bis[(Z)-octadec-9-enoxy]propanal (4.3 g, 7.13 mmol) as a colorless oil.

[1134] 1H NMR (500MHz, CDCl3) δ9.72 (d, J = 1.3Hz, 1H), 5.40-5.30 (m, 3H), 3.87-3.76 (m, 1H), 3.75-3.54 (m, 4H), 3.50-3.35 (m ,2H),1.63(dt,J=13.6,6.6Hz,2H),1.55(s,1H),1.52(d,J=6.7Hz,1H),1.43-1.19(m,44H),0.88(t,J=6.9Hz,6H).

[1135] Step (2)

[1136]

[1137] Add 1-bromododecane (2.58 g, 10.36 mmol) to a solution of Mg (3.78 g) and I₂ (2.19 g) in anhydrous THF (50 ml). Stir the mixture at 70 °C under N₂ until a colorless mixture is formed. Add 1-bromododecane (10.32 g, 41.44 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 (5.1 g, 8.63 mmol) in anhydrous THF (50 ml). Stir the mixture at 7 °C for 14 h. Treat the mixture with EA (250 ml) and wash with water (250 ml x 2) and a saturated aqueous solution of NaCl (250 ml). The organic compound was rapidly purified (5% EA in PE) to give 1,2-bis[(Z)-octadec-9-enoxy]pentadecan-3-ol (5.64 g, 7.41 mmol, 85.8% yield) as a yellow oil.

[1138] 1H NMR (500MHz, CDCl3) δ5.41-5.31(m,3H),3.75-3.66(m,1H),3.58(ddt,J=15.0,9.7,4.8Hz,2H),3.51-3.40(m,3H),3.27(dq,J=1 7.4,5.0Hz,1H),2.56(dd,J=57.4,5.0Hz,1H),2.04-1.93(m,6H),1.56-1.47(m,4H),1.40-1.17(m,66H),0.88(t,J=6.9Hz,9H).

[1139] Step (3)

[1140]

[1141] To a solution of bis(2,5-dioxopyrrolidone-1-yl) carbonate (2.02 g, 7.88 mmol) in DMF (15 mL), 1,2-bis[(Z)-octadec-9-enoxy]pentadecan-3-ol (1.5 g, 1.97 mmol) and 4-dimethylaminopyridine (963 mg, 7.88 mmol) were added. The mixture was stirred at 25 °C for 48 h. The mixture was treated with EA (50 mL), washed with water (50 x 1), LiCl aqueous solution (50 mL x 2), and dried over Na2SO4. The organic matter was concentrated and rapidly purified (10% EA in PE) to give (2,5-dioxopyrrolidone-1-yl)carbonate 1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyl ester (409 mg, 0.44 mmol, yield 22.5%) as a colorless oil. 1H NMR (500MHz, CDCl3) δ5.41-5.31(m,4H),4.95(s,1H),3.66-3.59(m,1H),3.56-3.38(m,6H),2.82(d,J=4.3Hz,4H),2.06 -1.93(m,7H),1.79-1.68(m,2H),1.61-1.58(m,1H),1.53(s,1H),1.28(dd,J=18.3,10.5Hz,66H),0.88(t,J=6.9Hz,9H).

[1142] Step (4)

[1143]

[1144] TBDPSCl (3.41 g, 12.4 mmol) was added to a solution of 2-[2-(2-aminoethoxy)ethoxy]ethanol (2 g, 10.3 mmol) and imidazole (1.62 g, 23.8 mmol) in CH2Cl2 (40 mL). The reaction was stirred at room temperature for 18 h. The mixture was diluted with CH2Cl2 (30 mL) and the resulting mixture was washed with brine. The organic layer was dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (SiO2, CH2Cl2 / MeOH / NH4OH 80:20:0.25) to obtain 2-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethylamine (3.1 g, 7.04 mmol, 68% yield) as a colorless oil.

[1145] 1 H NMR (400MHz, CDCl3) δ7.75-7.61(m,4H),7.49-7.32(m,6H),3.87-3.79(m,2H),3.73-3.51(m,12H),3.15(s,4H),2.92-2.82(m,2H),1.05(s,9H).

[1146] Step (5)

[1147]

[1148] To a solution of 2-[2-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethoxy]ethylamine (276 mg, 0.64 mmol), 1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyl(2,5-dioxopyrrolidone-1-yl) carbonate (576 mg, 0.64 mmol), TEA (97 ml), and 4-dimethylaminopyridine (8 mg) were added. The mixture was concentrated and rapidly purified (20% EA in PE) to give N-[2-[2-[2-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethyl]carbamate 1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyl ester (561 mg, 0.45 mmol, yield 70.7%) as a colorless oil.

[1149] 1H NMR (500MHz, CDCl3) δ7.71-7.65(m,4H),7.44-7.34(m,6H),5.40-5.31(m,3H),4.85(s,1H),3.81(t,J=5. 3Hz,2H),3.74-3.24(m,22H),2.06-1.91(m,7H),1.55-1.23(m,70H),1.05(s,9H),0.88(t,J=6.9Hz,9H).

[1150] Step (5)

[1151]

[1152] TBAF (0.92 ml) was added to a solution of N-[2-[2-[2-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethoxy]ethyl]carbamate 1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyl ester (561 mg, 0.46 mmol) in THF (6 ml). The mixture was stirred at 25 °C for 18 h. The mixture was concentrated and purified rapidly (50% EA in PE) to give N-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethyl]carbamate 1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyl ester (400 mg, 0.4 mmol, yield 86.9%) as a colorless oil.

[1153] 1 H NMR(500MHz, CDCl3)δ5.84(s,1H),5.41-5.31(m,3H),4.84(s,1H),3.77-3 .29(m,23H),2.04-1.92(m,7H),1.58-1.17(m,70H),0.88(t,J=6.9Hz,9H).

[1154] Step (6)

[1155]

[1156] To a solution of N-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethyl]carbamate 1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyl ester (495 mg, 0.5 mmol) in DCM (10 ml), 2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (329 mg, 0.76 mmol), DIEA (163 mg, 1.26 mmol), DMAP (12 mg), and EDCI (194 mg, 1 mmol) were added. The mixture was stirred at 25 °C for 18 h. The mixture was treated with EA (50 ml), washed with water (50 ml), brine (50 ml), and dried over Na2SO4. The organic matter was concentrated and rapidly purified (50% EA in PE) to give 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tetrazoloxycarbonylamino]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonylamino]ethyl ester (460 mg, 0.3 mmol, yield 63.9%).

[1157] 1 H NMR (500MHz, CDCl3) δ5.43-5.31(m,4H),5.19(s,1H),4.85(s,1H),4.36-3.25(m,37 H), 2.04-1.93 (m, 7H), 1.41 (ddd, J = 84.3, 38.0, 7.1Hz, 88H), 0.88 (t, J = 6.9Hz, 9H).

[1158] Step (7)

[1159]

[1160] Add 0.7 ml of TFA to a solution of 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tetrazoloxycarbonylamino]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (460 mg, 0.33 mmol) in DCM (3 ml). Stir the mixture at 25 °C for 2 h. The mixture was concentrated and rapidly purified (10% MeOH in DCM) to give 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tetrazoloxycarbonylamino]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester as a white solid; 2,2,2-trifluoroacetic acid (299 mg, 0.2 mmol, yield 67.9%).

[1161] 1 H NMR(400MHz, CDCl3)δ5.34(t,J=5.2Hz,4H),4.85(s,2H),4.59(s,2H),4.38(s,2H),3 .98-3.24(m,39H),2.01(d,J=5.6Hz,8H),1.67-1.10(m,70H),0.88(t,J=6.8Hz,9H).

[1162] Example 22: Synthesis of 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyloxy]-2-oxoethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester; Synthesis of 2,2,2-trifluoroacetic acid (compound (XXV))

[1163]

[1164] Compound (XXV)

[1165] Synthesis of compound (XXV)

[1166] Step (1)

[1167]

[1168] LiOH·H₂O (1.13 g, 27 mmol) was added to a solution of methyl 2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethoxy]acetate (1.41 g, 2.7 mmol) in THF / MeOH / water (4 ml / 4 ml / 4 ml). The mixture was stirred at 25 °C for 18 h. The mixture was treated with EA (50 ml) and washed with water (50 ml). 1 N HCl was added to the aqueous phase, and the pH was adjusted to 2. The aqueous phase was washed with EA (50 ml x 2), brine (50 ml), and dried over Na₂SO₄. The organic matter was concentrated to give 2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethoxy]acetic acid (1.15 g, 2.3 mmol, yield 84.5%) as a colorless oil.

[1169] 1 H NMR (500MHz, CDCl3) δ7.49-7.43 (m, 6H), 7.29 (dd, J = 10.3, 4.8Hz, 6H), 7.25-7. 19(m,3H),5.30(s,1H),4.11(s,2H),3.73-3.64(m,14H),3.25(t,J=5.1Hz,2H)

[1170] Step (2)

[1171]

[1172] At 0 °C, 2-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]acetic acid (550 mg, 1.11 mmol), DIEA (359 mg, 2.78 mmol), DMAP (27.2 mg, 0.22 mmol), and EDCI (426 mg, 2.22 mmol) were added to a solution of 1,2-bis[(Z)-octadec-9-enoxy]pentadecan-3-ol (1.1 g, 1.45 mmol) in DCM (15 ml). The mixture was then stirred at 25 °C for 18 h. The mixture was treated with DCM (50 ml), washed with water (50 ml), brine (50 ml), and dried over Na2SO4. The organic matter was concentrated and rapidly purified (20% EA in PE) to give 2-[2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethoxy]acetic acid 1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyl ester (461 mg, 0.37 mmol, yield 32.8%) as a colorless oil.

[1173] 1H NMR (400MHz, CDCl3) δ7.46(d,J=7.4Hz,6H),7.29(t,J=6.1Hz,6H),7.23(d,J=7.2Hz,3H),5.35(dd,J=12.5,7.2Hz,4H),5.10(s,1H),4.12(d,J=7.3Hz, 2H),3.74-3.61(m,14H),3.45(ddt,J=25.2,12.9,6.6Hz,7H),3.23(t,J=5. 2Hz, 2H), 2.06-1.93 (m, 7H), 1.26 (d, J = 4.9Hz, 70H), 0.88 (t, J = 6.8Hz, 10H).

[1174] Step (3)

[1175]

[1176] Toluene-4-sulfonic acid (461 mg, 0.37 mmol) was added to a solution of 2-[2-[2-[2-(2-triphenylmethoxyethoxy)ethoxy]ethoxy]ethoxy]acetic acid-1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyl ester (461 mg, 0.37 mmol) in THF / MeOH (5 / 5 ml). The mixture was stirred at 25 °C for 18 h. The mixture was treated with EA (50 ml), washed with NaHCO3 aqueous solution (50 ml x 2), brine (50 ml), and dried over Na2SO4. The organic matter was concentrated and rapidly purified (5% MeOH in DCM) to give 1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyl ester of 2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]acetic acid as a colorless oil (77 mg, 0.07 mmol, yield 14.3%).

[1177] 1 H NMR (500MHz, CDCl3) δ5.39-5.30(m,3H),5.09(dd,J=11.9,6.2Hz,1H),4.14(d,J=8.9Hz,2H),3.80-3 .32(m,23H),2.55(s,1H),2.06-1.94(m,7H),1.34(dd,J=82.3,60.0Hz,70H),0.88(t,J=6.9Hz,9H).

[1178] Step (4)

[1179]

[1180] To a solution of 2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]acetic acid and 1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyl ester (340 mg, 0.34 mmol) in DCM (5 mL), 2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (297 mg, 0.68 mmol), DIEA (110 mg, 0.85 mmol), DMAP (8 mg, 0.07 mmol), and EDC HCl (131 mg, 0.68 mmol) were added. The mixture was stirred at 25 °C for 18 h. The mixture was then treated with EA (50 mL), washed with water (50 mL), brine (50 mL), and dried over Na2SO4. The organic compound was rapidly purified (5% MeOH in DCM) to give 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyloxy]-2-oxo-ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (410 mg, 0.29 mmol, yield 83.3%).

[1181] 1 H NMR (500MHz, CDCl3) δ5.40-5.31(m,3H),5.09(d,J=5.2Hz,1H),4.37-3.21(m,40H),2.07-1.90( m,7H),1.54(s,4H),1.45(dd,J=22.0,10.4Hz,18H),1.38-1.04(m,66H),0.88(t,J=6.9Hz,9H).

[1182] Step (5)

[1183]

[1184] Add 0.43 mL of TFA to a solution of 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyloxy]-2-oxoethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonyl-amino]ethyl ester (410 mg, 0.29 mmol) in DCM (5 mL). Stir the mixture at 25 °C for 2 h. The mixture was concentrated and rapidly purified (10% MeOH in DCM) to give 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]tridecyloxy]-2-oxo-ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxoethyl]amino]ethyl ester; 2,2,2-trifluoroacetic acid (298 mg, 0.22 mmol).

[1185] 1 H NMR (400MHz, CDCl3) δ5.35(d,J=4.8Hz,4H),5.09(s,1H),4.59(s,2H),4.37(s,2H),4.18(d,J=14.7Hz,2H),4.02-3.61( m, 23H), 3.49 (s, 2H), 3.46-3.21 (m, 13H), 2.01 (d, J = 5.9Hz, 7H), 1.33 (dd, J = 59.3, 53.8Hz, 70H), 0.88 (t, J = 6.7Hz, 9H).

[1186] Example 23: Synthesis of 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]heptadecyloxycarbonylamino]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester; Synthesis of 2,2,2-trifluoroacetic acid (compound (XXVI))

[1187]

[1188] Compound (XXVI)

[1189] Synthesis of compound (XXVI)

[1190] Add 0.41 mL of TFA to a solution of 2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetic acid 2-[[2-[2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]heptadecyloxycarbonylamino]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]-tert-butoxycarbonylamino]ethyl ester (400 mg, 0.28 mmol) in DCM (5 mL). Stir the mixture at 25 °C for 3 h. The mixture was then concentrated and rapidly purified (10% MeOH in DCM) to give 2-(2-methoxyethylamino)acetic acid 2-[[2-[2-[2-[2-[2-[1-[1,2-bis[(Z)-octadec-9-enoxy]ethyl]heptadecyloxycarbonylamino]ethoxy]ethoxy]ethoxy]ethoxy]-2-oxo-ethyl]amino]ethyl ester (285.7 mg, 0.21 mmol, yield 74.4%).

[1191] 1 H NMR(400MHz, CDCl3)δ5.34(t,J=5.1Hz,4H),4.85(s,1H),4.59(s,2H),4.39(s,2H) ,4.02-3.23(m,38H),2.04-1.95(m,8H),1.64-1.00(m,78H),0.88(t,J=6.8Hz,9H).

[1192] Example 24: Synthesis of 8-[3-[2-[2-[2-[2-[2-[2-(2-methoxyethylamino)acetyl]oxyethylamino]acetyl]oxyethoxy]ethoxy]ethoxy]propionyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate; 2,2,2-trifluoroacetic acid (compound (XXVII))

[1193]

[1194] Compound (XXVII)

[1195] based on Figure 10 The chemically synthesized compound (XXVII) shown in scheme (12) presented in the paper.

[1196] Synthesis of compound (XXVII)

[1197] Step (1)

[1198]

[1199] 3-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]tert-butyl propionate (0.5 g, 1.71 mmol) was added to a suspension of silver oxide (0.593 g, 2.56 mmol) in dichloromethane (25 mL), and the mixture was stirred at room temperature for 30 min. Then, bromomethylbenzene (0.35 g, 2.05 mmol) was added to the above mixture, and the mixture was stirred in the dark at room temperature for 5 days. TLC showed approximately 50% conversion to the product. The mixture was filtered through diatomaceous earth and washed with dichloromethane. The solvent was removed, and the residue was purified by rapid chromatography by elution with 20% to 80% ethyl acetate in petroleum ether to give 3-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]tert-butyl propionate (0.21 g, 0.51 mmol, 31.7% yield) as a colorless oil.

[1200] 1 H NMR (400MHz, CDCl3) δ7.37-7.27(m,5H),4.57(s,2H),3.73-3.59(m,14H),2.50(t,J=6.6Hz,2H),1.44(s,9H).

[1201] Step (2)

[1202]

[1203] 3-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]propionic acid tert-butyl ester (3.25 g, 8.38 mmol) in FORMIC ACID (20 mL, 530 mmol) was added to a 250 mL round-bottom flask to give a colorless solution. The reaction was heated to 60 °C for 16 h. The solvent was removed and the residue was azeotropically reacted with toluene and dried under vacuum to provide 3-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]propionic acid (2.7 g, 7.35 mmol, 85% yield) as a yellow oil.

[1204] 1 H NMR (400MHz, CDCl3) δ7.37-7.27(m,5H),4.57(s,2H),3.73-3.59(m,14H),2.50(t,J=6.6Hz,2H),1.44(s,9H).

[1205] Step (3)

[1206]

[1207] A solution of heptadecano-9-yl 8-bromooctanoate (250 mg, 0.542 mmol) in phenylmethylamine (1.2 mL, 10.83 mmol) was stirred at room temperature for 6 h. The reaction was cooled to room temperature and the solvent was evaporated under vacuum. The residue was absorbed into ethyl acetate and washed with a saturated aqueous sodium bicarbonate solution. The organic layer was separated and washed with brine, dried over Na2SC4, and evaporated under vacuum. The residue was purified by silica gel chromatography (20%–100% in dichloromethane (a mixture of 1% NH4OH and 20% MeOH in dichloromethane)) to obtain heptadecano-9-yl 8-(benzylamino)octanoate (200 mg, 0.41 mmol, 76%).

[1208] Step (4)

[1209]

[1210] A solution of heptadecano-9-yl 8-(benzylamino)octanoate (200 mg, 0.41 mmol), nonyl 8-bromooctanoate (172 mg, 0.49 mmol), and N,N-diisopropylethylamine (100 μL, 0.57 mmol) was dissolved in ACN and stirred at 62 °C for 96 h. The reaction was cooled to room temperature and evaporated under vacuum. The residue was absorbed into ethyl acetate and washed with a saturated aqueous sodium bicarbonate solution. The organic layer was separated and washed with brine, dried over Na₂SO₄, and evaporated under vacuum. The residue was purified by silica gel chromatography (0-100% in dichloromethane (a mixture of 1% NH₄OH and 20% MeOH in dichloromethane)) to give nonyl 8-[benzyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (3.94 g, 4.95 mmol, 69%).

[1211] Step (5)

[1212]

[1213] Pd / C (0.79 g) was added to a solution of 8-[benzyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (3.94 g, 4.95 mmol) in ethyl acetate (50 mL), and the mixture was stirred at room temperature under hydrogen for 18 h. TLC showed that the starting material was consumed and a new spot was formed. The reaction was filtered through diatomaceous earth and washed with ethyl acetate. The solvent was removed, and the residue was dried under vacuum to give 8-[[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (3.94 g, 4.95 mmol, 69%) as a pale yellow oil.

[1214] 1H NMR (500MHz, CDCl3) δ4.89-4.82(m,1H),4.05(t,J=6.8Hz,2H),2.61-2.54(m,4H),2.31-2 .24(m,4H),1.62-1.59(m,4H),1.52-1.45(m,8H),1.38-1.18(m,51H),0.90-0.83(m,9H).

[1215] Step (6)

[1216]

[1217] To a solution of 8-[[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (0.3 g, 0.45 mmol) in dry DCM (10 mL), 3-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]propionic acid (0.176 g, 0.45 mmol), HATU (0.257 g, 0.676 mmol), and DIPEA (0.116 g, 0.901 mmol) were added, and the mixture was stirred at room temperature for 18 h. TLC (4% methanol in DCM) indicated that the reaction was complete. DCM was removed by rotary evaporation. The residue was redissolved in ethyl acetate (50 mL) and washed with H2O (50 mL x 3). The organic layer was washed with brine (50 mL) and dried over Na2SO4. The residue was purified by rapid chromatography by elution with 0% to 3% (2%) methanol in CH2Cl2 to give 8-[3-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]propionyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (0.932 g, 0.970 mmol, 71.8% yield), which was a pale yellow oil.

[1218] 1 H NMR (500MHz, CDCl3) δ7.36-7.27(m,5H),4.90-4.82(m,1H),4.57(s,2H),4.09-4.02(m,2H),3.78(t,J=7.1Hz,2H),3.70-3.59(m ,12H),3.30-3.15(m,4H),2.60(t,J=7.1Hz,2H),2.31-2.24(m,4H),1.64-1.45(m,14H),1.34-1.22(m,48H),0.91-0.85(m,9H).

[1219] Step (7)

[1220]

[1221] Pd / C (280 mg) was added to a solution of 8-[3-[2-[2-(2-benzyloxyethoxy)ethoxy]ethoxy]propionyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (0.932 g, 0.970 mmol) in EtOAc (10 mL), and the reaction was stirred overnight at room temperature under a H2 atmosphere. TLC (5% CH3OH in DCM) indicated that the reaction was complete. The slurry was filtered through diatomaceous earth and the diatomaceous earth was washed several times with EtOAc. The combined organic matter was concentrated under vacuum to give 8-[3-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]propionyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (0.802 g, 0.921 mmol, 95.0% yield) as a colorless oil.

[1222] 1 H NMR (500MHz, CDCl3) δ4.90-4.82(m,1H),4.09-4.03(m,2H),3.80(t,J=7.0Hz,2H),3.75-3.58(m,12H),3.30-3.17 (m,4H),2.61(t,J=7.0Hz,2H),2.33-2.24(m,4H),1.65-1.45(m,14H),1.36-1.22(m,48H),0.88(t,J=6.9Hz,9H).

[1223] Step (8)

[1224]

[1225] At 0 °C, nonyl octanoate (0.802 g, 0.921 mmol) and 2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetic acid (0.801 g, 1.84 mmol) in a solution of dry DCM (10 mL) were added to a solution of octanoate (0.212 g, 1.11 mmol) with DMAP (12 mg, 0.0921 mmol), DIPEA (0.143 g, 1.11 mmol), and then EDCI (0.212 g, 1.11 mmol). The reaction was allowed to be stirred at room temperature for 18 h. Water (20 mL) was added to quench the reaction and more DCM (50 mL) was added. The organic layer was washed with saturated sodium bicarbonate and dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography (eluting with 0-5% methanol in DCM (3%)) to give 8-[3-[2-[2-[2-[2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetyl]oxyethoxy]ethoxy]ethoxy]propionyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (0.970 g, 0.724 mmol, 78.5% yield).

[1226] 1 H NMR (400MHz, CDCl3) δ4.91-4.82(m,1H),4.32-4.19(m,4H),4.10-3.94(m,6H),3.79(t,J=7.1Hz,2H),3.75-3.60(m,10H) ,3.57-3.43(m,6H),3.32-3.16(m,7H),2.61(t,J=7.1Hz,2H),2.35-2.22(m,4H),1.65-1.23(m,80H),0.94-0.82(m,9H).

[1227] Step (9)

[1228]

[1229] Add TFA (5 mL) to a mixture of 8-[3-[2-[2-[2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetyl]oxyethoxy]ethoxy]ethoxy]propionyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (0.970 g, 0.754 mmol) in 10 mL of DCM and stir the mixture at room temperature for 2 h. Remove the solvent and azeotropically three times with dichloromethane (50 mL DCM * 3). The residue was purified by silica gel chromatography (0-12% methanol (9%) in DCM) to give 8-[3-[2-[2-[2-[2-[2-[2-(2-methoxyethylamino)acetyl]oxyethylamino]acetyl]oxyethoxy]ethoxy]ethoxy]propionyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate; 2,2,2-trifluoroacetic acid (0.5411 g, 0.437 mmol, 58.0% yield).

[1230] 1 H NMR (400MHz, CDCl3) δ4.91-4.81(m,1H),4.62(s,2H),4.35(s,2H),4.10-3.90(m,6H),3.82-3.61(m,14H),3.47-3.1 6(m,11H),2.61(t,J=6.4Hz,2H),2.33-2.23(m,4H),1.66-1.44(m,14H),1.28(d,J=15.7Hz,48H),0.93-0.83(m,9H).

[1231] LCMS: Peak found: MS(ESI)m / z=1086.9(M+H)+, at 2.499min.

[1232] Example 25: Synthesis of 2-hexyldecanoic acid 6-[3-[2-[2-[2-[2-[2-[2-[tert-butoxycarbonyl-[2-[2-[tert-butoxycarbonyl(2-methoxyethyl)amino]acetyl]oxyethyl]amino]acetyl]oxyethoxy]ethoxy]ethoxy]ethoxy]ethyl-octylamino]-2-[6-(2-hexyldecanoyloxy)hexyloxy]-3-oxo-propoxy]hexyl ester; 2,2,2-trifluoroacetic acid (compound (XXVIII))

[1233]

[1234] Compound (XXVIII)

[1235] Synthesis of compound (XXVIII)

[1236] Step (1)

[1237]

[1238] A mixture of 38 g (0.171 mol) of 4-(benzyloxymethyl)-2,2-dimethyl-1,3-dioxolane (AcOH, 160 mL) and 160 mL of H₂O was stirred at room temperature for 18 h. TLC showed that the starting material was completely consumed. The solvent was removed under vacuum and azeotropically reacted with toluene several times. 3-Benzyloxyprop-1,2-diol (31.0 g, quantitative) was obtained as a pale yellow oil and used without further purification.

[1239] 1 H NMR (400MHz, CDCl3) δ7.39-7.27 (m, 5H), 4.54 (s, 2H), 3.88 (tt, J = 5.9, 4.0Hz, 1H), 3.74-3.24 (m, 6H).

[1240] Step (2)

[1241]

[1242] NaH (11 g, 0.274 mol) was added several times to a solution of 10 g (0.055 mol) of 3-benzyloxyprop-1,2-diol in 200 mL of dry DMF at 0 °C, and...

Claims

1. A cleavable lipid compound of formula (IIa): Y-(CHR) n -NH-CH2-CO-O-(CHR') p -NH-CH2-CO-OA-R1 (IIa) in: -R1 is selected from the following C 10 To C 60 Lipophilic or hydrophobic tail groups: (R1n) (R1o) -Y is a methoxy group; -R and R' are either a hydrogen atom or a methyl group, which are independent of each other; -n and p are 0, 1 or 2 independently of each other; -A is selected from the following spacer arms, wherein its right end is the end to be attached to the lipophilic or hydrophobic tail group: Or one of its pharmaceutically acceptable salts.

2. The compound according to claim 1, wherein both n and p are 2.

3. The compound according to claim 1 or 2, wherein the C 10 To C 60 The lipophilic or hydrophobic tail group is R1a.

4. The compound according to claim 1 or 2, wherein the hydrophobic or lipophilic tail comprises at least three, four or more hydrocarbon chains, each independently selected from optionally substituted C4-C hydrocarbons. 24 Alkyl chains, and optionally substituted C4-C 24 Alkenyl chain.

5. The compound according to claim 1 or 2, wherein the hydrophobic or lipophilic tail comprises at least two, three, four or more hydrocarbon C4-C atoms. 24 A chain, wherein at least one chain is interrupted by at least one oxygen atom and / or at least one portion selected from -O-(O=C)- and -(C=O)-O-.

6. The compound according to claim 1 or 2, wherein the hydrophobic or lipophilic tail comprises at least three, four or more hydrocarbon chains, wherein at least two chains are optionally substituted C4-C. 24 Alkylene chains, and optionally each of them is independently interrupted by at least one portion selected from -O-(O=C)- and -(C=O)-O-.

7. The compound according to claim 1 or 2, wherein the hydrophobic or lipophilic tail comprises at least three, four or more hydrocarbon chains, wherein all chains are optionally substituted C4-C. 24 Alkyl chains, and optionally each of them is independently interrupted by at least one portion selected from -O-(O=C)- and -(C=O)-O-.

8. The compound according to claim 1 or 2, wherein the compound is selected from the group consisting of: Compound (III) (III) Compound (VI) (VI) Compound (VIII): (VIII) Compound (X): (X) Compound (XI): (XI) Compound (XII): (XII) Compound (XIV): (XIV) Compound (XV): (XV) Compound (XVI): (XVI) Compound (XVII): (XVII) Compound (XVIII): (XVIII) Compound (XIX): (XIX) Compound (XX): (XX) Compound (XXI): (XXI) Compound (XXII) (XXII) Compound (XXIII): (XXIII) Compound (XXIV): (XXIV) Compound (XXV): (XXV) Compound (XXVIII): (XXVIII) Compound (XXIX): (XXIX) Compound (XXXI) (XXXI) Compound (XXXII) (XXXII) And its salt.

9. A method for manufacturing lipid nanoparticles containing nucleic acids, wherein the method comprises at least the following steps: a) Dissolve at least one lipid compound according to any one of claims 1 to 8 in a water-miscible organic solvent. b) Mix the organic solvent obtained in step a) with an aqueous solvent buffered at a pH ranging from about 3.0 to about 4.5 and containing at least one nucleic acid. c) Obtain the lipid nanoparticles containing the nucleic acid in the aqueous solvent.

10. The method of claim 9, wherein the aqueous solvent in step b) is buffered at a pH ranging from about 3.5 to about 4.

2.

11. The method according to claim 9 or 10, the method further comprising step d): increasing the pH of the aqueous solvent containing the lipid nanoparticles obtained in step c) to a pH ranging from about 5.0 to about 8.

5.

12. The method of claim 9 or 10, wherein step a) further comprises dissolving at least one lipid selected from the group consisting of neutral lipids, steroids or their esters, and polyethylene glycol-modified lipids in the organic solvent.

13. The method of claim 9 or 10, wherein step a) further comprises dissolving at least one neutral lipid, at least one steroid or its ester, and at least one polyethylene glycol-modified lipid in the organic solvent, wherein the lipid compound, the neutral lipid, the steroid or its ester, and the polyethylene glycol-modified lipid are present in the organic solvent in molar amounts of about 30% to about 70% of the total amount of lipid and lipid compound, about 0% to about 50% of the neutral lipid, 20% to about 50% of the steroid or its ester, and about 1% to about 15% of the polyethylene glycol-modified lipid.

14. The method according to claim 9 or 10, wherein the nucleic acid encodes at least one antigen.

15. Lipid nanoparticles obtainable according to any one of claims 9 to 14.

16. A method for manufacturing a pharmaceutical composition, said method comprising at least the following steps: i) The method according to any one of claims 9 to 14 is used to manufacture at least one lipid nanoparticle, and ii) Combine the lipid nanoparticles obtained in step i) with at least one pharmaceutically acceptable excipient.

17. A method for manufacturing an immunogenic composition, said method comprising at least the following steps: i) The method according to any one of claims 9 to 14 produces at least one lipid nanoparticle, said lipid nanoparticle containing at least one nucleic acid encoding at least one antigen, and ii) Combine the lipid nanoparticles obtained in step i) with at least one pharmaceutically acceptable excipient.

18. A lipid nanoparticle comprising at least one lipid compound of formula (IV): HO-L (IV) Wherein L is a lipophilic or hydrophobic tail group as defined in any one of claims 1 to 8, and at least one nucleic acid.

19. The lipid nanoparticles of claim 18, wherein the lipid nanoparticles comprise at least one lipid compound of formula (Va) or (Vb): HO-R1 (Va) or OH-A-R1 (Vb) R1 and A are defined as in any one of claims 1 to 8.

20. The lipid nanoparticles according to claim 18 or 19, wherein the lipid nanoparticles further comprise: at least one lipid selected from the group consisting of neutral phospholipids or sphingolipids, steroids or their esters, and polyethylene glycol-modified lipids.

21. The lipid nanoparticles according to claim 18 or 19, wherein the lipid nanoparticles further comprise at least one neutral phospholipid or sphingolipid, at least one steroid or ester thereof, and at least one polyethylene glycol-modified lipid, and wherein the lipid compound of formula (IV), (Va), or (Vb), the neutral phospholipid or sphingolipid, the steroid or ester thereof, and the polyethylene glycol-modified lipid are present in a molar amount of about 30% to about 70% of the total amount of lipid and lipid compound, about 0% to about 50% of the neutral lipid, 20% to about 50% of the steroid or ester thereof, and about 1% to about 15% of the polyethylene glycol-modified lipid.

22. The lipid nanoparticles according to claim 18 or 19, wherein the at least one nucleic acid encodes at least one antigen.

23. The lipid nanoparticles according to claim 18 or 19, wherein the lipid nanoparticles further comprise the lipid compound according to any one of claims 1 to 8.

24. A pharmaceutical composition comprising at least one lipid nanoparticle according to any one of claims 15 or 18 to 23 and at least one pharmaceutically acceptable excipient.

25. An immunogenic composition comprising at least one lipid nanoparticle according to any one of claims 15 or 18 to 23, wherein the at least one nucleic acid encodes at least one antigen.

26. Use of a composition in the preparation of a medicament, said composition comprising at least one lipid nanoparticle according to any one of claims 15 or 18 to 23.

Images