WO2025002399A1

WO2025002399A1 - Ionizable cationic lipid compounds for delivery of biologically active agents

Info

Publication number: WO2025002399A1
Application number: PCT/CN2024/102542
Authority: WO
Inventors: Xi Zhu; Hong Chen; Chenxi HOU; Dalang LI; Shin-Shay Tian; Xiaoping Zhao
Original assignee: Shanghai Vitalgen Biopharma Co Ltd
Current assignee: Shanghai Vitalgen Biopharma Co Ltd
Priority date: 2023-06-30
Filing date: 2024-06-28
Publication date: 2025-01-02
Anticipated expiration: 2025-12-30
Also published as: WO2025000542A1; CN121335881A

Abstract

The present disclosure relates to novel lipid compounds for delivering one or more biologically active molecules to a subject. The present application also relates to a composition or a nanoparticle comprising said lipid compounds, methods of preparing said lipid compounds, method of using said lipid compounds and use of said lipid compounds.

Description

IONIZABLE CATIONIC LIPID COMPOUNDS FOR DELIVERY OF BIOLOGICALLY ACTIVE AGENTS

FIELD OF THE INVENTION

The present disclosure belongs to the field of drug delivery. Specifically, the present application relates to novel lipid compounds and nanoparticles comprising said lipid compounds for delivering one or more biologically active agents to a subject. Also provided are methods of preparing and using said lipid compounds and nanoparticles, as well as use of said lipid compounds and nanoparticles.

BACKGROUND

Nucleic acid-based therapeutics have enormous potential therapeutical application. These therapeutic nucleic acids include messenger RNA (mRNA) , antisense oligonucleotides, siRNA, miRNA, ribozymes, guide RNA, DNAzymes, plasmids, plasmids-based interfering nucleic acids, closed-end DNA, and aptamers. Different types of nucleic acids are currently being developed as therapeutics for the treatment of a number of diseases.

Some nucleic acids, such as mRNA, plasmids, closed-end DNA, can be used to express specific protein or enzyme, as would be useful in the treatment of, for example, single gene diseases related to deficiency or misfunction of such protein or enzyme. Nucleic acids encoding an antigen of a pathogen, including mRNA and DNA, could be used as vaccine for the prophylaxis of infectious diseases, such as COVID-19. Nucleic acids encoding one or more neoantigens could be used as tumor vaccines, which can effectively activate the body’s immune system to kill tumor cells, making individualized immunotherapy based on tumor neoantigens a new direction for the development of personalized immunotherapy for treating tumors.

Other nucleic acids can down-regulate or up-regulate intracellular levels of specific mRNA, which subsequently affect expression of specific cellular proteins. These nucleic acids, such as siRNAs and miRNAs, have extremely broad therapeutic applications. Targets may include mRNAs associated with disease-states, such as tumor suppressor, and mRNAs of infectious agents. Antisense oligonucleotide constructs have shown the ability to specifically down-regulate target proteins through degradation of the cognate mRNA. Nucleic acids such as miRNA inhibitors would be useful in the treatment of diseases related to deficiency of protein or enzyme.

Although nucleic acid-based therapeutics have enormous potential, effective delivery of these molecules to appropriate sites within a cell or organism are prerequisite to realize this potential. There are many challenges associated with the delivery of nucleic acids to achieve a desired response in a biological system. RNA molecules are susceptible to nuclease digestion in plasma and have extremely short half-life. Furthermore, nucleic acids are impermeable to cell membrane due to its unique molecular characteristics. Thus, these molecules can hardly gain access to the intracellular compartment where the relevant translation machinery resides. Therefore, nucleic acids are usually considered have no druggability in its naked form. Improvement of delivery vehicle for nucleic acid is crucial for these molecules to fulfill their therapeutic potentials.

Nanoscale delivery vehicles, including lipid and polymeric nanoparticles have been used to facilitate the cellular uptake of the nucleotides and prevent their degradation in biological environment. Lipid nanoparticles formed from ionizable cationic lipids with other lipid components, such as neutral lipids, cholesterol, PEGylated lipids (PEG lipids) , and oligonucleotides have been proved to be one of the most potent delivery systems. Cationic lipids are critical for protecting the nucleic acid payload from degradation and facilitating intracellular delivery of the nucleic acid. The cationic lipids should be biodegradable, well-tolerated and provide an adequate therapeutic index.

In 2010, Semple et al. hypothesized that after endocytosis, the cationic lipid interacts with naturally occurring anionic phospholipids in the endosomal membrane, forming ion pairs that adopt non-bilayer structures and disrupt membranes. In isolation, cationic lipids and endosomal membrane anionic lipids such as phosphatidylserine adopt a cylindrical molecular shape, which is compatible with packing in a bilayer configuration. However, when cationic and anionic lipids are mixed together, they combine to form ion pairs where the cross-sectional area of the combined headgroup becomes smaller than that of the sum of individual headgroup areas in isolation. The ion pair therefore adopts a molecular ‘cone’s hape, which promotes the formation of inverted, non-bilayer phases such as the hexagonal H_II phase. Such inverted phases do not support bilayer structure and are associated with membrane fusion and membrane disruption. As a result, the membrane of endosome can be lysed to allow the release of the payload, e.g. nucleic acids, into the cytoplasm. By using a rational lipid design strategy, the same group reported DLin-MC3-DMA (MC3) in 2012, which is also recited in PCT publication WO2010/054401A1 and was subsequently approved by FDA in 2018 for the delivery of siRNA (Patisiran, ) .

By following the above-mentioned rationale, increasing the endosomolytic behavior of LNPs is the central approach to improving delivery efficiency. With the MC3 LNP, a quantitative study showed that only 2～5 %of siRNA that were in endosomes actually escaped from endosomes into the cytosol (Gilleron, J. et al. Nat. Biotechnol. 31, 638–646. (2013) . Newer generation ionizable lipids often have more tails (or branches) versus two in MC3 to increase the cone-shaped morphology of the ionizable lipid. SM-102 in Moderna’s SARS-CoV-2 product, for example, has three alkyl tails; and ALC-0315 in BioNTech’s approved BNT162b2 has four alkyl tails. These newer generation ionizable lipids therefore appear to achieve an endosomal release several folds higher as compared to MC3. Since these molecules have similar pKa as MC3, the augmented cone-shaped morphology is presumably the reason why LNPs that incorporate these ionizable lipids are more efficient delivery vehicles with greater endosomal release.

In spite of the known advantage of cone-shaped cationic lipids, cationic lipids with more than two tails show great diversity in chemical and physical characteristics. It remains unclear what kind of detailed structure, including the number and type of tails, would be more beneficial in enhancing the delivery efficiency and other properties of interest. Synthesis of cationic lipids with branched tails would be more complicated than synthesis of lipids having linear alkyl chains as the tail part. As such, there remains a need in developing cationic lipids which are easy to prepare and have desirable delivery efficiency.

SUMMARY OF THE INVENTION

The present inventors creatively developed a group of ionizable cationic lipids having at least one branching tail comprising an acetal group, in which the carbon atom of the acetal group serves as the branching point of the tail while the two ether oxygens of the acetal group are connected to two hydrocarbyl chains elongating from the branching point. This design enables for easy chemical synthesis and study of ionizable cationic lipids with complex structures, in particular hyperbranched tails, which would augment the cone-shaped morphology facilitating the formation of a hexagonal H_II phase, and therefore increases intracellular delivery efficiency of biologically active agents (e.g., mRNA, ceDNA, gRNA) .

Therefore, in a first aspect, the present application provides a lipid compound, comprising an amino head group, and two fatty acid or fatty alkyl tails, wherein at least one tail is a branched tail comprising an acetal group, wherein the carbon atom of the acetal group serves as the branching point of the branched tail and the two ether oxygens of the acetal group are connected to two hydrocarbyl chains. In preferred embodiments, one or more of the hydrocarbyl chains are branched.

In some embodiments, the lipid compound of the first aspect is a compound having formula (I) , or a salt, tautomer, or stereoisomer thereof,

wherein: 1) m and p are independently selected from any integer ranging from 3 to 8; 2) n is selected from any integer ranging from 2 to 4; 3) X is a bond, -C (O) O-, -OC (O) -, -OC (O) O-, or a biodegradable group; 4) R₁ is a hydrogen bond donor-containing group or hydrogen bond acceptor-containing group; 5) both of R₂ are same and selected from C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₃-C₁₂ cycloalkyl and substituted C₃-C₁₂ cycloalkyl and combinations of thereof; 6) R₃ is: (a) selected from C₄-C₂₂ alkyl, substituted C₄-C₂₂ alkyl, C₄-C₂₂ alkenyl, substituted C₄-C₂₂ alkenyl, C₃-C₁₂ cycloalkyl, substituted C₃-C₁₂ cycloalkyl and combinations of thereof; or (b) an acetal group ofwherein both of R₄ are same and selected from C₁-C₁₆ alkyl, substituted C₁-C₁₆ alkyl, C₂-C₁₆ alkenyl, substituted C₂-C₁₆ alkenyl, C₃-C₁₂ cycloalkyl, substituted C₃-C₁₂ cycloalkyl and combinations of thereof.

In some embodiments, the ionizable lipid of the first aspect has formula (II) , or a salt, tautomer, or stereoisomer thereof,

wherein q is selected from any integer ranging from 2 to 4.

In preferred embodiments, R₂ of formula (I) and/or formula (II) is C₇-C₁₂ alkyl.

In some embodiments, R₃ of said formula (I) is selected from C₆-C₁₂ alkyl, substituted C₆-C₁₂ alkyl, C₆-C₁₂ cycloalkyl, substituted C₆-C₁₂ cycloalkyl, C₆-C₁₂ alkenyl, substituted C₆-C₁₂ alkenyl and combinations of thereof.

In preferred embodiments, R₃ is C₇-C₁₂ alkyl.

In preferred embodiments, at least one of R₂ and/or R₃ of formula (I) is a branched alkyl or alkenyl.

In preferred embodiments, each R₂ has at least one carbon atom with hydrogen atom (s) substituted by one or two side chains.

In preferred embodiments, R₃ has at least one carbon atom with hydrogen atom (s) substituted by one or two side chains.

In some embodiments, R₃ is the same with R₂.

In some embodiments, R₄ of said formula (II) are independently selected from C₂-C₁₂ alkyl, substituted C₂-C₁₂ alkyl, C₃-C₁₂ cycloalkyl, substituted C₃-C₁₂ cycloalkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl and combinations of thereof.

In preferred embodiments, R₄ is C₇-C₁₂ alkyl.

In preferred embodiments, at least one of R₂ and/or R₅ of formula (II) is a branched alkyl or branched alkenyl.

In preferred embodiments, each R₄ has at least one carbon atom with hydrogen atom (s) substituted by one or two side chains.

In some embodiments, R₄ is the same with R₂.

In preferred embodiments, said carbon atom with hydrogen atom (s) substituted by one or two side chains is the second or more distant carbon atom counting from the junction.

In preferred embodiments, said one or two side chains are C₁-C₄ alkyls.

In some embodiments, each R₂, R₃ and/or each R₄ is independently selected from one of the following formulas:

In some embodiments, R₁ of Formula (I) or Formula (II) is selected from hydroxyalkyl group having 1 to 5 carbon atoms, or optionally substituted amino alkylenyl group having 1 to 6 carbon atoms. In specific embodiments, R₁ is selected from one of the following formulae:

wherein o is selected from 1, 2, 3, 4, and 5.

In specific embodiments, the lipid compound having formula (II) is selected from a group consisting of Compounds 002-011 and 013-051 as shown in Table 1.

In specific embodiments, the lipid compound having formula (I) is selected from a group consisting of Compounds 052-103 as shown in Table 1.

In one embodiment, the compound of the first aspect is in a form of ionizable cationic lipid.

In a second aspect, the present application provides a lipid nanoparticle (LNP) comprising: the lipid compound of the first aspect, particularly as an ionizable cationic lipid; a neutral lipid, such as distearoylphosphatidylcholine (DSPC) or dioleoyl-phosphatidylethanolamine (DOPE) ; a steroid and analog thereof, such as cholesterol, sitosterol or stigmasterol; and a polymer conjugated lipid, such as a PEG lipid.

In some embodiments, the LNP is loaded with one or more biologically active molecules, such as one or more nucleic acid molecules. In some embodiments, said nucleic acid molecule is therapeutic or prophylactic nucleic acid molecule.

In a third aspect, the present application provides a pharmaceutical composition comprising the LNP of the second aspect.

In a fourth aspect, the present application provides a method of delivering a biologically active molecule to a cell of a subject, comprising administering the LNP of the second aspect loaded with said biologically active molecule to the subject.

In a fifth aspect, the present application provides a method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering a pharmaceutically effective amount of the LNP of the second aspect loaded with a therapeutic or prophylactic biologically active molecule to the subject.

In a sixth aspect, the present application provides use of the LNP of the second aspect or the pharmaceutical composition of the third aspect to treat and/or prevent a disease.

In a seventh aspect, the present application provides use of the LNP of the second aspect or the pharmaceutical composition of the third aspect in the manufacture of a medicament.

In an eighth aspect, the present application provides a method of preparing the lipid of the first aspect.

The lipid compounds of the present application are advantageous in providing high delivery efficiency as represented by an increased expression level of the delivered mRNA.

BRIEF DESCRIPTION OF THE DRAWINGS

The following is a brief description of the drawings, which are presented for the purposes of illustrating the exemplary embodiments disclosed herein and not for the purposes of limiting the same.

FIG. 1A-1D are schematic drawings showing the structure of conventional cationic lipids having a cylindrical shape versus the cationic lipids of the present application having a cone-shape. (A) conventional cationic lipid having a cylindrical shape; (B) cationic lipids having one or two branched tails; (C) cationic lipids having more complex tail structures and a cone-shaped structure; (D) lamellar phase formed by lipids with cylindrical shape versus hexagonal H_II phase formed by lipids with cone shape.

FIG. 2 shows the expression level of mGFP mRNA delivered by the LNPs formed by the lipid compounds of the present application, as well as commercially available lipids SM-102 and MC3 in HEK293 cells. For comparison, the expression levels have been normalized to that of MC3 at respective dose (500ng or 1000ng) .

FIG. 3 shows the expression level of GFP mRNA delivered by the LNPs formed by the lipid compounds of the present application, as well as SM-102 and MC3 in HepG2 cells. For comparison, the expression levels have been normalized to that of MC3 at respective dose (500ng or 1000ng) .

FIG. 4 shows the serum expression level of EPO mRNA delivered by the LNPs formed by the lipid compounds of the present application, as well as MC3, in mouse following intravenous injection. For comparison, the expression levels have been normalized to that of MC3 LNP.

FIG. 5 shows the bioluminescence image of luciferase mRNA loaded LNP (0.5 mg/kg) composed of Compound 002, 021 and 030 in comparison of MC3 and SM-102 based LNPs. Bioluminescence image was acquired at 6h and 24h post injection using IVIS imaging system.

FIG. 6 shows the serum expression level of EPO mRNA delivered by the LNPs formed by the different lipids, including 030, 059, 061, 062, 063 and 065, as well as MC3, in rat following intravenous injection.

FIG. 7 shows in vivo editing efficiency of TTR gene in mouse liver as measured by NGS following administration of sample LNP formulations with different ionizable compounds.

FIG. 8 shows in vivo editing efficiency of TTR gene in mouse liver as measured by NGS following administration of sample LNP formulations with different with different component ratios.

FIG. 9 shows in vivo editing efficiency of TTR gene in mouse liver as measured by NGS following administration of sample formulations composed of Compound 062 at different N/P ratio.

FIG. 10 shows in vivo editing efficiency of TTR gene in mouse liver as measured by NGS following administration of sample formulations at different dose level.

DETAILED DESCRIPTION OF THE INVENTION

I. Definition

The term “lipid nanoparticle” or its abbreviation “LNP” used herein refers to a drug carrier on the order of nanometers and composed of one layer of lipids. In the context of the present application, the term “lipid nanoparticle” also contemplates “lipid-polymer hybrid nanoparticle” which include both lipid portions and hydrophobic polymer portions.

The term “lipid” refers to a group of organic compounds that are poorly soluble in water, while soluble in nonpolar solvents. Lipids include, but are not limited to, esters of fatty acids.

The term “cationic lipid” as used herein refers to positively charged amphiphiles consisting of a hydrophilic head group connected to a hydrophobic tail via a linker. Cationic lipid is positively charged so that it can bind to negatively charged molecules or entities such as membrane. Cationic lipids can be ionizable and/or biodegradable lipids, which are preferred for in vivo use due to higher transduction efficiency, less toxicity and improved biocompatibility.

The term “polymer conjugated lipid” refers to a molecule comprising a lipid portion and a polymer portion.

The term “PEGylated lipid” refers to a molecule comprising a lipid portion and a polyethylene glycol portion.

The term “neutral lipid” or “helper lipid” in the context of LNP is interchangeable and refers to lipids that are uncharged or stay in a neutral zwitterionic form at a selected pH.

The term “charged lipid” refers to lipids that are positively charged or negatively charged.

The term “alkyl” as used herein refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated. A branched hydrocarbon chain can have more than one side chains or substituents.

The term “alkenyl” as used herein refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which contains one or more double bonds.

The term “stereoisomers” refer to compounds consisting of the same atoms bonded by the same bonds but having different three-dimensional structures.

The term “tautomers” refer to structural isomers in which a proton shifts from one atom of a molecule to another atom of the same molecule.

The term “hydrogen bonding” refers to a noncovalent intermolecular interaction involving a hydrogen atom located between a pair of non-hydrogen atoms. A hydrogen bond is weaker than an ionic bond or covalent bond while stronger than van der Waals forces. One of the non-hydrogen atom pair is covalently bonded to a hydrogen and is referred as “hydrogen bond doner” or “H-bond donor” . The other atom of the pair is referred as “hydrogen bond acceptor” or “H-bond acceptor” . A H-bond donor atom can always be a H-bond acceptor atom. For example, H-bond donor can be the nitrogen of an amide, cyclic imide, sulfonamide, or the oxygen of a phenol or carboxyl group. For example, H-bond acceptor can be the nitrogen of an amine, imidazole, nitrile, or the oxygen of ether, ketone, amide or sulfoxide. In the context of the present application, the term “hydrogen bond donor-containing group” and the term “hydrogen bond acceptor-containing group” refer to a functional group containing the hydrogen bond doner atom and a functional group containing the hydrogen bond acceptor atom, respectively. For example, H-bond donor-containing functional group can be a group comprising hydroxyl, carboxyl and/or primary/secondary amide.

The term “nucleotide” , “oligonucleotide” and “nucleic acid” can be used interchangeable herein to refer to the base-sugar-phosphate unit consisting a nucleic acid sequence, e.g., a deoxyribonucleic acid (DNA) sequence or a ribonucleic acid (RNA) sequence.

The terms "peptide" , "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of at least two amino acid residues linked by one or more peptide bonds.

The term “subject” as used herein refers to an individual, preferably a vertebrate, more preferably a non-human mammal or human. The non-human mammal can be rodents such as murines, or non-human primates such as simians. The term “subject” may also encompass cells, tissues and progenies of a biological entity obtained in vivo or culture in vitro.

The phrase “effective amount” or “therapeutically effective amount” refers to the quantity of a composition, e.g., a composition comprising the LNP that is sufficient to result in a desired activity when being delivered to a subject in need thereof. Said desired activity may encompass delaying the manifestation of a disorder, arresting or delaying the progression of a disorder, or alleviating at least one symptom of a disorder.

II. Ionizable cationic lipids

The present application provides a group of new ionizable cationic lipids, which are suitable for preparing lipid nanoparticles.

Ionizable cationic lipids are essential components in lipid nanoparticle formulations. Typical structure of cationic lipids includes one or more amine group (s) which bear the positive charge. The ionizable cationic lipids can exist in a positively charged or neutral form depending on pH. The ionization of the cationic lipid affects the surface charge of the lipid nanoparticle under different pH conditions. This charge state can influence plasma protein absorption, blood clearance, and tissue distribution (Semple, S. C., et al., Adv. Drug Deliv Rev 32: 3-17 (1998) ) as well as the ability to form endosomolytic non-bilayer structures (Hafez, I. M., et al., Gene Ther 8: 1188-1196 (2001) ) critical to the intracellular delivery of nucleic acids.

The present application provides a lipid compound, comprising an amino head group, and two fatty acid or fatty alkyl tails, wherein at least one tail is a branched tail comprising an acetal group, wherein the carbon atom of the acetal group serves as the branching point of the branched tail and the two ether oxygens of the acetal group are connected to two hydrocarbyl chains.

In some embodiments, the ionizable cationic lipids of the present application is a compound having formula (I) , or a salt, tautomer, or stereoisomer thereof,

In some embodiments, the lipid compound of the first aspect is a compound having formula (II) , or a salt, tautomer, or stereoisomer thereof,

wherein q is selected from any integer ranging from 2 to 4.

In the structure as shown by Formula (I) and Formula (II) , R₁ and the central nitrogen atom (N) constitutes the head group of the lipid compound of the present application. Head group of a cationic lipid is positively charged at acidic environment and is essential for the delivery of nucleic acid payloads, as it interacts with the negatively charged phosphate groups of nucleic acids. Unlike double-stranded DNA or siRNA, in which only the phosphate backbone is exposed while the nucleobases are facing inward, mRNA has a more complex secondary structure, comprising single-and double-stranded regions with some nucleosides exposed to the surrounding environment, such as the solvent. Therefore, other interactions, such as hydrogen bonding, of the head group with the riboses and nucleobases of mRNA could contribute to an improved LNP expression. In the structure as shown by Formula (I) and Formula (II) , R₁ serves to provide a set of hydrogen bond donors and/or acceptors with varying hydrogen-bonding ability in the head group.

H-bond donor-or acceptor-containing groups can be those comprising nitrogen (N) , oxygen (O) , carbon (C) or fluorine (F) atoms serving as H-bond donor or H-bond acceptor. Exemplary H-bond donor-or acceptor-containing groups may include but not limited to hydroxyl-containing group, carboxyl-containing group, carbonyl-containing group, amide, imide, sulfoxide, and sulfonamide.

In preferred embodiments of the present application, R₁ is selected from a group consisting of following formulae:

wherein o is selected from 1, 2, 3, 4, and 5.

In specific embodiments, R₁ isand o is selected from 2, 3, or 4, preferably 2.

Aside from the headgroup, the ionizable cationic lipid of the present application comprises two hydrophobic tails. At least one of the two tails is a branched tail, which comprises an acetal group connecting two identical branching chains, resulting in a "symmetric acetal" .

In the compound of formula (II) , both tails are symmetric acetals and are represented by R₂ and R₄ groups, respectively. R₂ and R₄ are each independently selected from C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₃-C₁₂ cycloalkyl and substituted C₃-C₁₂ cycloalkyl. For each of substituted alkyl and substituted alkenyl, it can comprise one or more substituents selected from deuterium (–D) , cyano (–CN) , halo, imino (=NH) , nitro (–NO₂) , oxo (=O) . In preferred embodiments, R₂ and R₄ are independently selected from C₂-C₁₂ alkyl, C₃-C₁₂ cycloalkyl and C₂-C₁₂ alkenyl, preferably C₃-C₁₀ alkyl, unbranched or branched. R₂ and R₄ can be same or different. In certain embodiments, R₂ and/or R₄ is linear alkyl. In preferred embodiments, at least one of R₂ and/or R₄ is branched. As a hyperbranched structure (see e.g. Fig. 1C) may augment the cone-shaped morphology, and therefore increase intracellular delivery efficiency, it is preferred in the present application. Accordingly, both R₂ and R₄ are preferably branched. For example, R₂ and/or R₄ may have one or more side chains. In specific embodiments, R₂ and R₄ are independently selected from 2-methylhexyl or 2, 6-dimethyloctyl.

In the compound of formula (I) , only one tail is a symmetric acetal and is represented by R₂ group. R₂ is selected from C₂-C₁₂ alkyl, C₃-C₁₂ cycloalkyl and C₂-C₁₂ alkenyl, preferably C₃-C₁₀ alkyl, unbranched or branched. As a hyperbranched structure (see e.g. Fig. 1C) is preferred in the present application, R₂ preferably is a branched chain. For example, R₂ may have one or more side chains. In specific embodiments, R₂ is selected from 2, 6-dimethyloctyl or 5-butylheptyl. For the other tail, it is preferred that R₃ is also a branched chain, more preferably having two identical branching tails. In specific embodiments, R₃ is selected from 5-butylheptyl or 4-propylhexyl.

The branched tail containing acetal group could easily been synthesized by conjugate R₂-OH or R₄-OH to the acetal group. In specific embodiments, the intermediate structure containing acetal group were synthesized using the following reactions:

In specific embodiments, R₂-OH or R₄-OH are branched alcohols, and the ionizable cationic lipid contains an hyperbranched structure. In specific embodiments, R₂-OH or R₄-OH are one of the following structures:

In specific embodiments, R₂-OH or R₄-OH are unsaturated alcohols, and the ionizable cationic lipid contains an unsaturated tail.

In the acetal-based branched tail, the part between the acetal branching position and the central nitrogen is referred as a “stem” region. The stem region comprises a linear chain comprising a biodegradable group.

The biodegradable group in the “stem” region can be -OC (O) -, -C (O) O-or -OC (O) O-. The biodegradable group can be cleaved in a biological environment. When the LNP formed by the cationic lipid comprising the biodegradable group (s) is delivered into a biological environment, such as into a living organism, the biodegradable groups facilitate the metabolism and clearance of the lipid components after the payload has been delivered.

The length of the stem region is determined by the chain length before and after the biodegradable group. In some embodiments, m and p in Formula (II) is independently 3, 4, 5, 6, 7 or 8. In preferred embodiments, m is 4, 5 or 6, p is 5, 6 or 7. In some embodiments, n and q in Formula (I) is independently 2, 3, or 4. In most preferred embodiment, m = 5, n = 2, p = 5 or 7, q = 2, and X is -C (=O) O-, and the ionizable cationic lipid have one of the following structures:

In formula (I) , only one of the two tails is acetal group containing branched tails. In some embodiments, n and q in Formula (I) is 2, 3, or 4. In most preferred embodiment, m = 5, n = 2, and X is -C (=O) O-.

The compound of formula (II) has a non-acetal tail represented by following formula:

wherein p is any integer ranging from 2 to 9, X is a biodegradable group, such as -OC (O) -, -C (O) O-or -OC (O) O-.

In a more specific embodiment, p is 2, 5 or 7.

R₃ is selected from C₆-C₂₂ alkyl, C₆-C₂₂ cycloalkyl or C₆-C₂₂ alkenyl, preferably C₆-C₁₂ alkyl, C₆-C₁₂ cycloalkyl or C₆-C₁₂ alkenyl, unbranched or branched.

R₃ can be linear or branched. In some specific embodiments, R₃ is a branched C₆-C₂₂ alkyl or a branched C₆-C₂₂ alkenyl, preferably a branched C₆-C₁₂ alkyl or a branched C₆-C₁₂ alkenyl. In some specific embodiments, when R₃ is a branched alkyl or alkenyl, R₃ could be the same with R₂. In some specific embodiments, R₃ is a linear C₆-C₂₂ alkyl or C₆-C₂₂ alkenyl, preferably a linear C₈-C₂₀ alkyl or a linear C₈-C₂₀ alkenyl, more preferably linear C₉-C₁₈ alkyl or linear C₉-C₁₈ alkenyl.

In specific embodiments, the lipid compound is selected from any one of following Compounds as shown in Table 1, including Compounds 002-011 and 013-051 which have a chemical structure of formula (II) , and Compounds 052-103 which have a chemical structure of formula (I) . In preferred embodiments, the lipid compound has a chemical structure of formula (II) and is selected from Compound 002 and Compound 030 in Table 1. In preferred embodiments, the lipid compound has a chemical structure of formula (I) and is selected from Compound 059, Compound 061, Compound 062, Compound 065 in Table 1.

Table 1. Chemical Structures of Representative Compounds

Included in the present invention is the free form of the cationic lipids described herein, as well as pharmaceutically acceptable salts and stereoisomers thereof. The cationic lipid can be a protonated salt of the amine cationic lipid. The term “free form” refers to the amine cationic lipids in non-salt form. The free form may be regenerated by treating the salt with a suitable dilute aqueous base solution such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium bicarbonate.

The compounds of the disclosure, or their pharmaceutically acceptable salts may contain one or more stereocenters and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R) -or (S) -or, as (D) -or (L) -for amino acids. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-) , (R) -and (D) -, or (D) -and (L) -isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC) . When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

The pharmaceutically acceptable salts of the instant cationic lipids can be synthesized from the cationic lipids of this invention which contain a basic or acidic moiety by conventional chemical methods. Generally, the salts of the basic cationic lipids are prepared either by ion exchange chromatography or by reacting the free base with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents. Similarly, the salts of the acidic compounds are formed by reactions with the appropriate inorganic or organic base.

III. Other lipid components of LNP

In addition to the ionizable cationic lipid, the lipid components comprised in the LNPs of the present application comprises a neutral lipid, a polymer conjugated lipid, and a steroid. In another embodiment, the lipid components further comprise a charged lipid.

Neutral lipid

The neutral lipid such as a phospholipid helps the LNP to bind to and cross the cell membrane. Exemplary neutral lipids include, but are not limited to, distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC) , dioleoylphosphatidylcholine (DOPC) , dipalmitoylphosphatidylcholine (DPPC) , dioleoylphosphatidylglycerol (DOPG) , dipalmitoylphosphatidylglycerol (DPPG) , dioleoyl-phosphatidylethanolamine (DOPE) , palmitoyloleoylphosphatidylcholine (POPC) , palmitoyloleoylphosphatidylethanolamine (POPE) , dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal) , dipalmitoyl phosphatidyl ethanolamine (DPPE) , dimyristoylphosphoethanolamine (DMPE) , di stearoyl-phosphatidyl-ethanolamine (DSPE) , monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE) , dimethyl-phosphatidylethanolamine (such as 16-0-dimethyl PE) , 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE) , hydrogenated soy phosphatidylcholine (HSPC) , egg phosphatidylcholine (EPC) , dioleoylphosphatidylserine (DOPS) , sphingomyelin (SM) , dimyristoyl phosphatidylcholine (DMPC) , dimyristoyl phosphatidylglycerol (DMPG) , di stearoylphosphatidylglycerol (DSPG) , dierucoylphosphatidylcholine (DEPC) , palmitoyloleyolphosphatidylglycerol (POPG) , dielaidoyl-phosphatidylethanolamine (DEPE) , lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM) , cephalin, cardiolipin, phosphatidicacid, cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. In various embodiments, the molar ratio of the ionizable cationic lipid to the neutral lipid ranges from about 2: 1 to about 8: 1, preferably 5: 1.

Polymer conjugated lipid

The polymer conjugated lipid can be a PEGylated lipid or PEG lipid. In various embodiments, the PEG lipid is a pegylated diacylglycerol (PEG-DAG) such as 1- (monomethoxy-polyethyleneglycol) -2, 3-dimyristoylglycerol (PEG-DMG) , a pegylated phosphatidylethanoloamine (PEG-PE) , a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O- (2′, 3′-di (tetradecanoyloxy) propyl-1-O- (ω-methoxy (polyethoxy) ethyl) butanedioate (PEG-S-DMG) , a pegylated ceramide (PEG-cer) , or a PEG dialkoxypropylcarbamate such as ω-methoxy (polyethoxy) ethyl-N- (2, 3-di (tetradecanoxy) propyl) carbamate or 2, 3-di (tetradecanoxy) propyl-N- (ω-methoxy (polyethoxy) ethyl) carbamate. In various embodiments, the molar ratio of the ionizable cationic lipid to the pegylated lipid ranges from about 100: 1 to about 20: 1.

Steroid

The steroid or steroid analogue. In certain embodiments, the steroid or steroid analogue is cholesterol, sitosterol or stigmasterol. In certain embodiments, the steroid or steroid analogue is cholesterol. In certain embodiments, the steroid or steroid analogue is beta-Sitosterol. In certain embodiments, the steroid or steroid analogue is Stigmastanol. In some of these embodiments, the molar ratio of the ionizable cationic lipid to cholesterol ranges from about 5: 1 to 1: 1, preferably 3: 1 to 1: 1.

In preferred embodiments, the nanoparticle of the present disclosure comprises the following in a mole ratio of 50: 10: 38.5: 1.5 or 40: 10: 48.5: 1.5:

(1) the cationic lipid of the present disclosure, preferable selected from Compounds 002-011 and 013-103 in Table 1;

(2) a neutral lipid selected from DSPC or DOPE;

(3) a steroid which is cholesterol; and

(4) polymer conjugated lipid which is PEG-DMG.

More preferably, the lipid compound of the present disclosure is Compound 002, Compound 030, Compound 059, Compound 061, Compound 062, Compound 065 in Table 1.

IV. Nucleic acid payload

The LNPs of the present application are particularly suitable for delivering nucleic acid molecules into cells.

The nucleic acid molecule can be either DNA or RNA or a mixture thereof, such as chimeric oligonucleotides. The nucleic acid molecule can comprise naturally occurring or modified polynucleotides. The nucleic acid molecule can be a coding sequence or non-coding sequence. The nucleic acid molecule can be DNA molecule such as plasmid DNA, closed-ended DNA or a mixture thereof. The nucleic acid molecule can be RNA molecule such as messenger RNA (mRNA) , guide RNA (gRNA) , a short interfering RNA (siRNA) , an RNA interference (RNAi) molecule, a microRNA (miRNA) , an antagomir, an antisense RNA, a ribozyme, a small hairpin RNA (shRNA) , or a mixture thereof.

Plasmid DNA or closed-ended DNA may have a lengths at a range of 500 to 500,000 base pairs. mRNA may have a length at a range of 200 to 100,000 base pairs. gRNA may have a length at a range of 30 to 1,000 base pairs. siRNAi, miRNA, or shRNA may have a length at a range of 15 to 1,000 base pairs.

The nucleic acid molecules can comprise modifications, such as one or more modifications to the backbone, one or more modifications to the base and/or one or more modifications to the sugar moiety.

V. Compound Synthesis

Compounds of the present disclosure can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or from readily prepared intermediates, by employing standard synthetic methods and procedures either known to those skilled in the art, or which will be apparent to the skilled artisan in light of the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be obtained from the relevant scientific literature or from standard textbooks in the field.

The following descriptions of synthetic methods are designed to illustrate, but not to limit, general procedures for the preparation of compounds of the present disclosure. The compounds of this disclosure having a structure of Formula (II) may be prepared according to the procedures illustrated in Scheme 1. The compounds of this disclosure having a structure of Formula (I) may be prepared according to the procedures illustrated in Schemes 2a and 2b. One of ordinary skill in the art will note that, during the reaction sequences and synthetic schemes described herein, the order of certain steps may be changed, such as the introduction and removal of protecting groups.

SCHEME 1

Scheme 1 provides an exemplary method for preparation of compounds of Formula (II) . R₁, R₂, and R₄ in Scheme 1 are as defined herein. X is a reactive moiety selected to facilitate the desired reaction (e.g., halo) . Compound A-1, which contains an acetal group connecting to two branched tails via its two ether oxygens, is prepared using method as illustrated in Scheme 3 or prepared according to methods known in the art. Condensation reaction of A-1 and A-2 under appropriate conditions (e.g., using EDCI and DMAP) yields the product A-3. A-3 reacts with primary amine of A-4 under suitable conditions (e.g. heat) to afford A-5. A-5 is then reacted with another halide A-3 using appropriate conditions (e.g., DIEA and heat) to yield a compound of Formula (II) as shown.

SCHEME 2a

SCHEME 2b

Schemes 2a and 2b provide two exemplary methods for preparation of compounds of Formula (I) . R₁, R₂ and R₃ in Scheme 2a and 2b are as defined herein. X is a reactive moiety selected to facilitate the desired reaction (e.g., halo) . Compound B-1 or C-1, which contains an acetal group connecting branched tails, is prepared using method as illustrated in Scheme 3 or prepared according to methods known in the art. Condensation reaction of B-1 and B-2 (or C-1 and C-2) under appropriate conditions (e.g., using EDCI and DMAP) yields the product B-3 (or C-3) . In Scheme 2a, B-3 reacts with primary amine of B-4 under suitable conditions (e.g. heat) to afford B-5. B-5 is then reacted with another halide B-6 using appropriate conditions (e.g., DIEA and heat) to yield a compound of Formula (I) as shown. In Scheme 2b, amine C-5 reacts with halide C-4 to afford compound C-6. C-6 is then reacted with another halide C-3 using appropriate conditions (e.g., DIEA and heat) to yield a compound of Formula (I) as shown.

SCHEME 3

Scheme 3 provides an exemplary method for preparation of the intermediate containing an acetal group. Step 1 can take place in an organic solvent in presence of a catalyst (e.g. KHSO₄, PPTS) at, e.g., an elevated temperature (such as at about 70-150 ℃, e.g., about 80 ℃) . Y is moiety that can be transferred to a carboxy group in step 2, for example, cyano group or ethyl ester group. In specific embodiment, Y is ethyl ester group, Step 1 can take place in presence of a base (e.g. NaOH) . In specific embodiment, Y is ethyl ester group, Step 1 can take place at, e.g., an elevated temperature (such as 110 ℃) , in presence of a base (e.g. NaOH, KOH) . Next, alcohol D-5 can be obtained from reacting with carboxy D-4 with a reducing agent (e.g. Lithium Aluminium Hydride) .

One of ordinary skill in the art will recognize that certain groups may require protection from the reaction conditions via the use of protecting groups. Protecting groups may also be used to differentiate similar functional groups in molecules.

Preferred protecting groups include, but are not limited to: TBS, benzyl, THP or Ac (for a hydroxyl moiety) ; benzyl ester, methyl ester, ethyl ester, or allyl ester (for carboxylic acids) ; Fmoc, Cbz, BOC, DMB, Ac, Bn, Tr, Ts, trifluoroacetyl, phthalimide, or benzylideneamine (for amines) ; and di-alkyl acetals such as dimethoxy acetal or diethyl acetyl (for aldehydes) .

In the reaction schemes described herein, multiple stereoisomers may be produced. When no particular stereoisomer is indicated, it is understood to mean all possible stereoisomers that could be produced from the reaction. A person of ordinary skill in the art will recognize that the reactions can be optimized to give one isomer preferentially, or new schemes may be devised to produce a single isomer. If mixtures are produced, techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC may be used to separate the isomers.

VI. Preparation of Nanoparticles

Nanoparticles can be made with mixing processes such as microfluidics and T-tube mixing of two fluid streams, one of which contains the therapeutic and/or prophylactic, and the other has the lipid components.

Lipid compositions are prepared by combining a cationic lipid, such as lipid compound according to formula (II) , helper lipids (such as DSPC) , a steroid (such as cholesterol) , and a PEG lipid (such as 1, 2-dimyristoyl-sn-glycerol methoxypolyethylene glycol, also known as DMG-PEG2000) at concentrations of about 5 mg/mL and 25 mg/mL in ethanol. Solutions should be refrigerated for storage at, for example, -80 ℃.

A composition comprising nanoparticles including a therapeutic and/or prophylactic and a lipid component can be prepared by combining the lipid solution with a solution including the therapeutic and/or prophylactic at a lipid component to therapeutic and/or prophylactic ratio (by weight) between about 10: 1about 20: 1. The lipid solution is injected using a microfluidic based system at flow rates between 0.25 mL/min and 2.0 mL/min into the therapeutic and/or prophylactic solution to produce a suspension with a water to ethanol ratio between about 3: 1.

For a nanoparticle composition including RNA (e.g. mRNA) or DNA (e.g. pDNA) , solutions of the RNA or DNA are diluted with 50 mM sodium citrate buffer at a pH between 3 and 4 to form a stock solution at concentrations of 0.1-0.5 mg/mL.

A nanoparticle composition can be subjected to further processing steps such as purification. For example, a nanoparticle composition can be subjected to dialysis to remove unwanted solvent such as ethanol and to achieve buffer exchange. Formulations were dialyzed twice against Tris-HCl Solution (20 mM, pH 7.4) at volumes about 2500 times that of the primary product using Slide-A-Lyzer cassettes (Thermo Fisher Scientific Inc., Rockford, Ill. ) with a molecular weight cutoff of 10 KD at 0-4 ℃ for 2 hours. Then 40%sucrose solution was added, the final nanoparticle composition solution of 0.03 mg/mL to and 0.1 mg/mL (mRNA or pDNA) are generally obtained and stored at -80 ℃.

VII. Characteristics of Nanoparticle Compositions

The physical and chemical properties of the lipid nanoparticle of the present application depend on the formulation of the LNPs.

For example, it has been found by the present inventors that the choice of the ionizable cationic lipid dramatically influences the size of the formulated LNPs. For example, the lipid nanoparticles have a mean diameter of from about 60 nm to about 350 nm, preferably 70 nm to 300 nm. In specific embodiments, the lipid nanoparticles have a mean diameter of about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm or 300 nm.

Polydispersity index (PDI) is a parameter describing the distribution of molecular weight of the particles in an LNP formulation. A lager PDI describes a broader distribution of molecular weight. For example, the PDI of the formulation can be 0.05 to 0.50, for example about 0.05, about 0.10, about 0.15, about 0.20, about 0.25, about 0.30, about 0.35, about 0.40, about 0.45, or about 0.50.

In some embodiments, the lipid nanoparticles are substantially non-toxic or having acceptable toxicity to the subject. For example, the lipid nanoparticles of the present application have a reduced liver toxicity as compared to nanoparticles comprising a different cationic lipid. The liver toxicity can be measured based known method in the art.

The lipid nanoparticle of the present disclosure can provide desirable transduction efficiency, which can be measured by e.g. the amount or expression level of the payload. In preferred embodiments, the lipid nanoparticles of the present application provide a comparable or increased amount of payload or expression of payload as compared to a nanoparticle comprising a different cationic lipid.

VIII. Uses

The LNP formed by the ionizable cationic lipid and loaded with nucleic acid molecules may have diverse uses, depending on the type and function of the nucleic acid molecules.

For example, the LNPs can be used for treating a disease or disorder. In some embodiments, the disease or disorder is caused by insufficiency of a protein, and the LNP comprises nucleic acid molecule encoding for the protein.

For example, the LNPs can be used for preventing a disease or disorder. For example, the LNPs can be formed as a vaccine, in which the nucleic acid molecule encodes for an antigen, such as an antigen derived from a pathogen, e.g. a bacterium, a fungi, or a virus, or an antigen derived from tumor cell, e.g. tumor associated antigen.

For example, the LNPs can be used for treating a disease or disorder by affecting expression of specific cellular proteins. In some embodiments, the LNPs comprise nucleic acids, such as siRNAs and miRNAs, which down-regulate or up-regulate intracellular levels of target mRNA associated with a disease state. For example, the target mRNA can be the mRNA of a tumor suppressor or mRNAs of infectious agents.

For example, the LNPs can be used for treating a disease or disorder by affecting expression of specific cellular proteins via genome editing. In some embodiments, to treat a disease or disorder caused by or related to a certain mutation, which for example causes loss-of-function or gain-of-toxicity of a protein, the LNPs may comprise nucleic acid molecule encoding for genome editing tool. For example, the LNPs can be used to deliver mRNA of a nuclease, e.g. Cas nuclease, and/or a guide RNA, so as to conduct nuclease-mediated gene editing.

EXAMPLES

Example 1. Synthesis of Compounds

1. General Considerations

All solvents and reagents used were obtained commercially and used as such unless noted otherwise. ¹H NMR spectra were recorded in CDCl₃, at 400 MHz instrument. Chemical shifts are reported as parts per million (ppm) relative to TMS (0.00) for 1H.

The procedures described below are useful in the synthesis of Compounds 002-118.

The following abbreviations are employed herein.

ACN: Acetonitrile

LAH: Lithium Aluminum Hydride

DCM: Dichloromethane

DMAP: 4-Dimethylaminopyridine

DIEA: Diisopropylethylamine

EDCI: 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide

PPTS: Pyridinium p-toluenesulfonate

RT: Room Temperature

THF: Tetrahydrofuran

TLC: Thin-layer chromatography

2. Synthesis of exemplary compounds

Compound 002: bis (3, 3-bis ( (5-methylhexyl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Synthesis of 5-methylhexan-1-ol

A mixture of Mg (26.2 g, 1.08 mol, 5 eq) and I2 (1.10 g, 4.3 mmol, 869 uL, 0.02 eq) in THF (120 mL) was added the solution of 1-bromo-2-methylpropane (147 g, 1.08 mol, 117 mL, 5 eq) in THF (120 mL) at 30 ℃ and the reaction mixture was stirred at 25 ℃ for 2 h. The mixture was added to 3-bromopropan-1-ol (30 g, 215.84 mmol, 19.4 mL, 1 eq) in THF (120 mL) and dilithium; tetrachlorocopper (2-) (0.1 M, 129.50 mL, 0.06 eq) . The mixture was stirred at 25 ℃ for 12 h under N₂ atmosphere. The mixture was added to the HCl (0.1 M, aq. 500 mL) . The residue was diluted with H₂O 300 mL and extracted with EtOAc (1L and 500 mL) . The combined organic layers were washed with brine 300 mL, dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=100/1 to 10/1) . 5-methylhexan-1-ol (16 g, 137 mmol, 63.7%yield) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 3.66 (t, J =6.6 Hz, 2H) , 1.48 -1.66 (m, 3H) , 1.29 -1.43 (m, 2H) , 1.17 -1.25 (m, 2H) , 0.89 (d, J = 6.6 Hz, 6H)

Synthesis of ethyl 3, 3-diethoxypropanoate

To a solution of ethyl 3, 3-diethoxypropanoate (5 g, 26.28 mmol, 5.11 mL, 1 eq) in 5-methylhexan-1-ol (12.2 g, 105.1 mmol, 4 eq) was added KHSO4 (178 mg, 1.31 mmol, 77.1 uL, 0.05 eq) . The mixture was stirred at 75 ℃ for 12 h. The mixture was evaporated to dryness. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=100/1 to 10/1) . Ethyl 3, 3-bis ( (5-methylhexyl) oxy) propanoate (5.8 g, crude) was obtained as colourless oil.

To a solution of ethyl 3, 3-bis ( (5-methylhexyl) oxy) propanoate (5 g, 15.13 mmol, 1 eq) in EtOH (10 mL) , H₂O (10 mL) and THF (10 mL) was added NaOH (1.21 g, 30.2 mmol, 2 eq) . The mixture was stirred at 20 ℃ for 12 h. The pH value of the reaction was acidified with aq. HCl (0.1 M) to 4～5 at 20℃. The mixture was extracted with EtOAc 50 mL, dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=100/1 to 50/1) . 3, 3-bis ( (5-methylhexyl) oxy) propanoic acid (3 g, 9.92 mmol, 65.5%yield) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.92 (t, J = 5.6 Hz, 1H) , 3.58 -3.72 (m, 2H) , 3.45 -3.56 (m, 2H) , 2.73 (d, J = 5.5 Hz, 2H) , 1.48 -1.63 (m, 6H) , 1.34 (td, J = 7.5, 15.4 Hz, 4H) , 1.13 -1.24 (m, 4H) , 0.88 (d, J = 6.5 Hz, 12H) .

To a solution of 3, 3-bis ( (5-methylhexyl) oxy) propanoic acid (3 g, 9.92 mmol, 1 eq) in THF (9 mL) was added LAH (376.47 mg, 9.92 mmol, 1 eq) in THF (9 mL) at 0 ℃. The mixture was stirred at 20 ℃ for 12 h. The mixture was added H₂O (0.3mL) , NaOH (aq. 15%, 0.3 mL) and H₂O (0.9 mL) at 0℃ and stirred for 0.5 h. The mixture was filtered and concentrated under reduced pressure to give a residue. Without purification. 3, 3-bis ( (5-methylhexyl) oxy) propan-1-ol (1.3 g, 4.51 mmol, 45.4%yield) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.67 (t, J = 5.4 Hz, 1H) , 3.75 (br t, J = 5.1 Hz, 2H) , 3.64 (td, J = 6.7, 9.4 Hz, 2H) , 3.46 (td, J = 6.7, 9.1 Hz, 2H) , 2.58 (br s, 1H) , 1.90 (q, J = 5.4 Hz, 2H) , 1.47 -1.65 (m, 6H) , 1.27 -1.43 (m, 4H) , 1.14 -1.26 (m, 4H) , 0.83 -0.95 (m, 12H)

Synthesis of 3, 3-bis ( (5-methylhexyl) oxy) propyl 6-bromohexanoate

To a solution of 3, 3-bis ( (5-methylhexyl) oxy) propan-1-ol (1.3 g, 4.51 mmol, 1 eq) and 6-bromohexanoic acid (1.05 g, 5.41 mmol, 1.2 eq) in DCM (6 mL) was added EDCI (950.32 mg, 4.96 mmol, 1.1 eq) and DMAP (110.11 mg, 901.32 umol, 0.2 eq) . The mixture was stirred at 20 ℃ for 12 h. The mixture was evaporated to dryness. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=100/1 to 10/1) . 3, 3-bis ( (5-methylhexyl) oxy) propyl 6-bromohexanoate (1.7 g, 3.65 mmol, 81.0%yield) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.61 (t, J = 5.7 Hz, 1H) , 4.16 (t, J = 6.5 Hz, 2H) , 3.55 -3.64 (m, 2H) , 3.36 -3.49 (m, 4H) , 2.33 (t, J = 7.5 Hz, 2H) , 1.80 –2.02 (m, 4H) , 1.66 (quin, J = 7.6 Hz, 2H) , 1.43 -1.60 (m, 8H) , 1.26 -1.42 (m, 4H) , 1.13 -1.24 (m, 4H) , 0.88 (d, J = 6.6 Hz, 12H)

Synthesis of 3, 3-bis ( (5-methylhexyl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate

To a solution of 2-aminoethan-1-ol (50 mg, 818 umol, 49.5 uL, 1 eq) and 3, 3-bis ( (5-methylhexyl) oxy) propyl 6-bromohexanoate (419.15 mg, 900.4 umol, 1.1 eq) in EtOH (2 mL) . The mixture was stirred at 80 ℃ for 12 h. The reaction mixture was partitioned between EtOAc 100 mL and H₂O 50 mL. The organic phase was separated, washed with NaHCO3 (aq) 100 mL, dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. Without purification. 3, 3-bis ( (5-methylhexyl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (364 mg, 816 umol, 99.7%yield) was obtained as yellow oil.

Synthesis of Compound 002

To a solution of 3, 3-bis ( (5-methylhexyl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (364 mg, 816 umol, 1 eq) and 3, 3-bis ( (5-methylhexyl) oxy) propyl 6-bromohexanoate (380 mg, 816 umol, 1 eq) in EtOH (4 mL) was added DIEA (211 mg, 1.63 mmol, 284 uL, 2 eq) . The mixture was stirred at 80 ℃ for 12 h. The reaction mixture was partitioned between EtOAc 100 mL and H₂O 50 mL. The organic phase was separated, washed with NaHCO3 (aq) 100 mL, dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (procuct Rf = 0.60) (SiO₂, Petroleum ether/Ethyl acetate=100/1 to 0/1) . Compound 002 (117 mg, 140 umol, 17.2%yield) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 5.8 Hz, 2H) , 4.15 (t, J = 6.5 Hz, 4H) , 3.52 -3.64 (m, 6H) , 3.43 (td, J = 6.7, 9.2 Hz, 4H) , 2.61 (br s, 2H) , 2.49 (br s, 4H) , 2.31 (t, J = 7.4 Hz, 4H) , 1.95 (q, J = 6.4 Hz, 4H) , 1.42 -1.72 (m, 23H) , 1.41 -1.13 (m, 22H) , 0.88 (d, J = 6.6 Hz, 24H)

Compound 003: bis (3, 3-bis ( (5-methylhexyl) oxy) propyl) 5, 5'- ( (2-hydroxyethyl) azanediyl) dipentanoate

Compound 003 was prepared analogously to compound 002 but using 5-bromopentanoic acid instead of 6-bromohexanoic acid. ¹H NMR (400 MHz CDCl₃) δ 4.61 (t, 2H) , 4.20-4.05 (m, 4H) , 3.63-3.47 (m, 10H) , 2.66-2.52 (m, 6H) , 2.35-2.27 (m, 4H) , 2.04-1.95 (m, 4H) , 1.73-1.17 (m, 36H) , 0.92-0.78 (m, 24H) .

Compound 004: bis (3, 3-bis ( (4-methylhexyl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Compound 004 was prepared analogously to compound 002 starting from 4-methylhexan-1-ol. ¹H NMR (400 MHz CDCl₃) δ 4.62 (t, 2H) , 4.20-4.01 (m, 4H) , 3.67-3.49 (m, 10H) , 2.66-2.52 (m, 6H) , 2.35-2.27 (m, 4H) , 2.00-1.94 (m, 4H) , 1.73-1.17 (m, 40H) , 0.91-0.81 (m, 24H) .

Compound 005: bis (3, 3-bis ( (3-methylhexyl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Compound 005 was prepared analogously to compound 002 starting from 3-methylhexan-1-ol. ¹H NMR (400 MHz CDCl₃) δ 4.63 (t, 2H) , 4.20-3.85 (m, 4H) , 3.77-3.59 (m, 10H) , 2.56-2.42 (m, 6H) , 2.32-2.17 (m, 4H) , 2.00-1.92 (m, 4H) , 1.78-1.16 (m, 40H) , 0.89-0.80 (m, 24H) .

Compound 006: bis (3, 3-bis (heptan-2-yloxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Compound 006 was prepared analogously to compound 002 starting from heptan-2-ol. ¹H NMR (400 MHz CDCl₃) δ 4.73 (t, 2H) , 4.40-4.31 (m, 4H) , 3.87-3.51 (m, 6H) , 2.56-2.42 (m, 6H) , 2.32-2.17 (m, 4H) , 1.98-1.90 (m, 4H) , 1.78-1.16 (m, 44H) , 0.89-0.80 (m, 24H) .

Compound 007: bis (3, 3-bis ( (5-methylhexyl) oxy) propyl) 6, 6'- ( (4-hydroxybutyl) azanediyl) dihexanoate

Synthesis of 3, 3-bis ( (5-methylhexyl) oxy) propyl 6- ( (4-hydroxybutyl) amino) hexanoate

To a solution of 3, 3-bis ( (5-methylhexyl) oxy) propyl 6-bromohexanoate (0.5 g, 1.07 mmol, 1.0 eq) and 4-aminobutan-1-ol (957 mg, 10.7 mmol, 997 uL, 10.0 eq) in EtOH (5 mL) was added at 25℃, then the mixture was stirred at 90℃ for 1 hr. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (10 mL, 5 mL, 3 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give 3, 3-bis ( (5-methylhexyl) oxy) propyl 6- ( (4-hydroxybutyl) amino) hexanoate (0.6 g, crude) as a colorless oil.

Synthesis of Compound 007

To a solution of 3, 3-bis ( (5-methylhexyl) oxy) propyl 6- ( (4-hydroxybutyl) amino) hexanoate (0.6 g, 1.27 mmol, 1.0 eq) and 3, 3-bis ( (5-methylhexyl) oxy) propyl 6-bromohexanoate (590 mg, 1.27 mmol, 1.0 eq) in EtOH (5 mL) was added DIEA (327 mg, 2.53 mmol, 441 uL, 2.0 eq) at 25℃, then the mixture was stirred at 90℃for 12 hrs. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (10 mL, 5 mL, 5 mL) , The combined organic layers were washed with (brine 3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to EtOAc/MeOH = 5/1) to give compound 007 (0.2 g, 213 umol, 16.8%yield, 91.8%purity) as a yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.61 (t, J = 5.6 Hz, 2H) , 4.15 (t, J = 6.8 Hz, 4H) , 3.55-3.62 (m, 6H) , 3.41-3.46 (m, 4H) , 2.58-2.67 (m, 3H) , 2.43-2.47 (m, 6H) , 2.29-2.34 (m, 14H) , 2.23 (d, J = 2.8 Hz, 6H) , 1.95 (q, J = 6.4 Hz, 4H) , 1.61-1.68 (m, 9H) , 1.49-1.59 (m, 16H) , 1.31-1.39 (m, 13H) , 1.18-1.26 (m, 11H) , 0.87-0.89 (m, 24H) .

Compound 008: bis (3, 3-bis ( (4-ethylhexyl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Synthesis of 4-ethylhexan-1-ol

A 500 mL three-necked round bottom flask equipped with or magnetic stirrer, addition funnel and thermometer. A mixture of Mg (19.2 g, 792 mmol, 4.95 eq) and I₂ (812 mg, 3.20 mmol, 645 μL, 0.02 eq) in THF (120 mL) was added dropwise the solution of 3- (bromomethyl) pentane (132 g, 800 mmol, 132 mL, 5.0 eq) in THF (120 mL) at 50 ℃ and the reaction mixture was stirred at 50 ℃ for 30 mins. The mixture was added to 2-bromoethan-1-ol (20.0 g, 160 mmol, 11.3 mL, 1.0 eq) and CuCl₂ (LiCl) ₂ (1.0 M, 9.60 mL, 0.06 eq) in THF (120 mL) . The mixture was stirred at 20 ℃ for 12 hrs under N₂ atmosphere. The mixture was added to the NH₄Cl (aq 500 mL) at 0 ℃ and extracted with EtOAc (300mL, 150 mL) . The combined organic layers were washed with brine (50.0 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=60/1 to 40/1) . 4-ethylhexan-1-ol (18.0 g, 138 mmol, 86.4%yield) was obtained as yellow oil. ¹H NMR: 400 MHz, CDCl₃, δ 3.63 (t, J = 6.8 Hz, 2 H) , 1.51 -1.57 (m, 2 H) , 1.26 -1.45 (m, 8 H) , 1.19 -1.21 (m, 1 H) , 0.90 (t, J = 7.2 Hz, 1 H) , 0.85 (t, J = 7.2 Hz, 5 H) .

Synthesis of compound 008

Compound 008 was prepared analogously to compound 002 starting from 4-ethylhexan-1-ol. Compound 008 (73.9%purity) was obtained as yellow oil. ¹H NMR: 400 MHz, CDCl₃, δ 4.60 (t, J = 5.6 Hz, 2 H) , 4.15 (t, J = 6.4 Hz, 4 H) , 3.55 -3.60 (m, 5 H) , 3.39 -3.45 (m, 4 H) , 2.32 (t, J = 7.6 Hz, 4 H) , 1.93 -1.97 (m, 4 H) , 1.47 -1.72 (m, 26 H) , 1.15 -1.45 (m, 31 H) , 1.15 -1.23 (m, 4 H) , 0.82 -0.90 (m, 23 H) .

Compound 009: bis (3, 3-bis ( (3-propylhexyl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Synthesis of 3-propylhexan-1-ol

Step 1: Triethyl phosphonoacetate (7.8 g, 34 mmol) in THF (12.5 mL) was slowly added to 60%sodium hydride, in oil, (1.4 g, 34 mmol) in dry THF (10 mL) at 0℃. The suspension was stirred for 1 h and then 4-heptanone (3.9 g, 34 mmol) in THF (12.5 mL) was slowly added. The solution was stirred for an hour and quenched with water. The solution was extracted with diethyl ether, and the organic layer was dried and concentrated. The crude was purified by flash chromatography (SiO₂, Petroleum ether/Ethyl acetate=10/1 to 1/1) to give alkene (3.0 g, 16.3 mmol, 48%) as a yellowish liquid.

Step 2: The alkene (1.5 g, 8.1 mmol) was hydrogenated in ethyl acetate with a catalytic amount (ca. 100 mg) of Pd/C for 3 h using a balloon filled with hydrogen gas. The solution was filtered through celite and concentrated to give the alkane (1.3 g, 7.2 mmol, 89%) as a clear liquid.

Step 3: The ester (1.3 g, 7.0 mmol, 1 eq) in diethyl ether was added slowly to lithium aluminum hydride (292 mg, 7.7 mol 1.1 eq) suspended in diethyl ether at 0℃. The suspension was stirred at room temperature for 2-3 h and then cooled to 0℃. Water (1 mL per g of LAH) was added, followed by 15%sodium hydroxide (1 mL per g of LAH) and water (3 mL per g of LAH) . The solution was stirred for a few minutes at room temperature, filtered through a celite pad, washed with diethyl ether, dried, and concentrated to give the alcohol. Flash chromatography was used to purify the product giving a clear liquid (798 mg, 5.5 mmol, 79%) . ¹H NMR (400 MHz CDCl₃) δ 3.64 (q, 2H) , 2.47 (t, 1H) , 1.58-1.48 (m, 2H) , 1.49-1.17 (m, 10H) , 0.96-0.82 (m, 6H) .

Synthesis of compound 009

Compound 009 was prepared analogously to compound 002 starting from 3-propylhexan-1-ol. ¹H NMR (400 MHz CDCl₃) δ 4.66 (t, 2H) , 4.10-3.81 (m, 4H) , 3.78-3.41 (m, 10H) , 2.55-2.41 (m, 6H) , 2.32-2.12 (m, 4H) , 2.00-1.95 (m, 4H) , 1.79-1.15 (m, 56H) , 0.88-0.78 (m, 24H) .

compound 010: bis (3, 3-bis ( (3, 5, 5-trimethylhexyl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Compound 010 was prepared analogously to compound 002 starting from 3, 5, 5-trimethylhexan-1-ol. Compound 010 (99.9 %purity) was obtained as a yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 5.6 Hz, 2H) , 4.15 (t, J = 6.8 Hz, 4H) , 3.68 (t, J = 4.8 Hz, 2H) , 3.57-3.63 (m, 4H) , 3.42-3.47 (m, 4H) , 2.76 (t, J = 4.8 Hz, 2H) , 2.65 (t, J = 7.2 Hz, 4H) , 2.31 (t, J = 7.6 Hz, 4H) , 1.95 (q, J = 6.4 Hz, 4H) , 1.58-1.69 (m, 16H) , 1.33-1.46 (m, 9H) , 1.21-1.26 (m, 4H) , 1.05-1.10 (m, 4H) , 0.90-0.94 (m, 46H) .

Compound 011: bis (4, 4-bis (heptyloxy) butyl) 5, 5'- ( (2- hydroxyethyl) azanediyl) dipentanoate

Synthesis of 4, 4-bis (heptyloxy) butan-1-ol

To a solution of 4, 4-dimethoxybutanenitrile (9.0 g, 69.8 mmol, 1.0 eq) and heptan-1-ol (24.3 g, 210 mmol, 3.0 eq) in Tol. (70 mL) was added PPTS (878 mg, 3.5 mmol, 0.05 eq) at 25℃, then the mixture was stirred at 120℃ for 12 hrs. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 1/0 to 60/1) to give 4, 4-bis (heptyloxy) butanenitrile (16.6 g, 55.8 mmol, 80%yield) as a colorless oil. To a solution of 4, 4-bis (heptyloxy) butanenitrile (10 g, 33.6 mmol, 1.0 eq) in EtOH (30 mL) was added KOH (3 M, 33.6 mL, 3.0 eq) at 25℃, then the mixture was stirred at 110℃ for 12 hrs. The pH value of the reaction was adjusted to around 4-5 with aq. HCl (1 M) at 0℃. The mixture was extracted with Petroleum ether (50 mL x 4) and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to 1/1) to give 4, 4-bis (heptyloxy) butanoic acid (9.5 g, crude) as a colorless oil.

To a solution of 4, 4-bis (heptyloxy) butanoic acid (8 g, 25.3 mmol, 80%purity, 1.0 eq) in THF (55 mL) at 0℃, then LiAlH₄ (767 mg, 20.2 mmol, 1.2 eq) was added the mixture and stirred at 25℃ for 3 hrs. Water (0.8 mL) , 15%aq. NaOH (0.8 mL) and water (2.4 mL) was dropwise the reaction solution at 0℃, then Na₂SO₄ added the mixture and stirred at 25℃ for 15 mins. The mixture was filtered and the filter cake concentrated under reduced pressure to give 4, 4-bis(heptyloxy) butan-1-ol (4.8 g, 15.8 mmol, 78%yield) as a colorless oil. ¹H NMR (400 MHz CDCl₃) δ 4.66 (t, 1H) , 3.74 (t, 2H) , 3.66-3.63 (m, 2H) , 3.48-3.42 (m, 2H) , 1.93-1.86 (m, 2H) , 1.72-1.32 (m, 22H) , 0.88 (t, 6H) .

Synthesis of compound 011

Compound 011 was prepared using similar procedure as for compound 002, but using 4, 4-bis (heptyloxy) butan-1-ol instead of 3, 3-bis ( (5-methylhexyl) oxy) propan-1-ol. Compound 011 was a colorless oil. ¹H NMR (400 MHz CDCl₃) δ 4.63 (t, 2H) , 4.18-3.83 (m, 4H) , 3.77-3.59 (m, 10H) , 2.57-2.41 (m, 6H) , 2.32-1.92 (m, 8H) , 1.78-1.16 (m, 52H) , 0.89-0.80 (m, 12H) .

Compound 013: bis (3, 3-bis (heptyloxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Synthesis of 3, 3-bis (heptyloxy) propan-1-ol

To a solution of aheptan-1-ol (12.2 g, 105 mmol, 14.9 mL, 4.0 eq) and ethyl 3, 3-diethoxypropanoate (5.0 g, 26.3 mmol, 5.11 mL, 1.0 eq) was added KHSO₄ (179 mg, 1.31 mmol, 77.1 uL, 0.05 eq) . The reaction was stirred at 75 ℃ for 12 h. The residue was diluted with water (50.0 mL) and extracted with EtOAc (100 mL *2) . The combined organic layers were washed with brine (30.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=60/1 to 50/1) . Ethyl 3, 3-bis (heptyloxy) propanoate (10.0 g, 30.3 mmol, 57.6%yield) was obtained as yellow oil.

To a solution of Ethyl 3, 3-bis (heptyloxy) propanoate (10.0 g, 30.3 mmol, 1.0 eq) in THF (60.0 mL) was added LAH (1.15 g, 30.3 mmol, 1.0 eq) at 0℃. The reaction was stirred at 20 ℃ for 2 h. The reaction mixture was quenched by addition water (1.00 mL) at 0℃, 15%NaOH with solvent (1.00 mL) , and water (3.00 mL) . The combined organic layers were added MgSO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was used into the next step without further purification. 3, 3-bis (heptyloxy) propan-1-ol (6.00 g, crude) was obtained as a colourless oil. ¹H NMR: 400 MHz, CDCl₃, δ 4.66 (t, J = 5.6 Hz, 1 H) , 3.74 (t, J = 5.2 Hz, 2 H) , 3.60 -3.66 (m, 2 H) , 3.42 -3.48 (m, 2 H) , 1.87 -1.91 (m, 2 H) , 1.55 -1.61 (m, 4 H) , 1.22 -1.32 (m, 16 H) , 0.88 (t, J = 6.4 Hz, 6 H) .

Synthesis of 3, 3-bis (heptyloxy) propyl 6-bromohexanoate

To a solution of 3, 3-bis (heptyloxy) propan-1-ol (3.00 g, 10.4 mmol, 1.0 eq) and 6-bromohexanoic acid (2.43 g, 12.5 mmol, 1.2 eq) in DCM (18.0 mL) was added EDCI (2.99 g, 15.6 mmol, 1.5 eq) and DMAP (254 mg, 2.08 mmol, 0.2 eq) at 0 ℃. The reaction was stirred at 20℃ for 12h. The residue was diluted with water (20.0 mL) and extracted with EtOAc (20.0 mL*3) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=60/1 to 40/1) . 3, 3-bis (heptyloxy) propyl 6-bromohexanoate (1.80 g, 3.87 mmol, 37.2%yield) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 5.6 Hz, 1 H) , 4.15 (t, J = 6.4 Hz, 2 H) , 3.55 -3.61 (m, 2 H) , 3.39 -3.45 (m, 4 H) , 2.32 (t, J = 7.2 Hz, 2 H) , 1.86 -1.97 (m, 4 H) , 1.50 -1.68 (m, 8 H) , 1.28 -1.36 (m, 15 H) , 0.87 -0.90 (m, 6 H) .

Synthesis of compound 013

To a solution of 3, 3-bis (heptyloxy) propyl 6-bromohexanoate (1.80 g, 3.87 mmol, 1.0 eq) in ACN (7.8 mL) was added 2-aminoethanol (708 mg, 11.6 mmol, 702 uL, 3.0 eq) . The reaction was stirred at 90 ℃ for 4 h. The residue was diluted with water (5.00 mL) and extracted with EtOAc (10.0 mL *3) . The combined organic layers were washed with brine (5.00 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=5/1 to 10/1) . Compound 013 (100 mg, 118 umol, 3.05%yield, 98%purity) was obtained as yellow oil. 3, 3-bis (heptyloxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (1.00 g, crude) was a by-product. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 6.0 Hz, 2 H) , 4.14 (t, J = 6.4 Hz, 4 H) , 3.53 -3.61 (m, 7 H) 3.41-3.45 (m, 4 H) 2.57 (t, J = 5.2 Hz, 3 H) , 2.45 (t, J = 7.6 Hz, 5 H) , 2.30 (t, J = 7.2 Hz, 5 H) , 1.94 -1.97 (m, 5 H) , 1.42 -1.67 (m, 23 H) , 1.28 -1.35 (m, 39 H) , 0.89 (t, J = 6.8 Hz, 12 H) .

Compound 014: bis (3, 3-bis ( (3-methylheptyl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Synthesis of 3-methylheptan-1-ol

Step 1: Triethyl phosphonoacetate (7.8 g, 34.0 mmol) in THF (12.5 mL) was slowly added to 60%sodium hydride, in oil, (1.4 g, 34.0 mmol) in dry THF (10 mL) at 0℃. The suspension was stirred for 1 h and then 2-hexanone (3.4 g, 34.0 mmol) in THF (12.5 mL) was slowly added. The solution was stirred for an hour and quenched with water. The solution was extracted with diethyl ether, and the organic layer was dried and concentrated. The crude was purified by flash chromatography eluting with Petroleum ether/Ethyl acetate = 100: 1 to give alkene (1.7 g, 9.9 mmol) as a yellowish liquid in 29%yield.

Step 2: The alkene (1.5 g, 8.8 mmol) was hydrogenated in ethyl acetate with a catalytic amount of Pd/C (ca. 150 mg) for 3 h using a balloon filled with hydrogen gas. The solution was filtered through celite and concentrated to give the alkane (1.38 g, 8.0 mmol) as a clear liquid in 91%yield.

Step 3: The ester (1.3 g, 7.5 mmol, 1 eq) in diethyl ether was added slowly to lithium aluminum hydride (285 mg, 7.5 mmol, 1.1 eq) suspended in diethyl ether at 0℃. The suspension was stirred at room temperature for 2-3 h and then cooled to 0℃. Water (1 mL per g of LAH) was added, followed by 15%sodium hydroxide (1 mL per g of LAH) and water (3 mL per g of LAH) . The solution was stirred for a few minutes at room temperature, filtered through a celite pad, washed with diethyl ether, dried, and concentrated to give the alcohol. Flash chromatography was used to purify the product giving a clear liquid (850 mg, 6.5 mmol) in 87%yield. ¹H NMR (400 MHz CDCl₃) δ 3.74 (t, 2H) , 1.56-1.12 (m, 9H) , 0.89-0.84 (m, 6H) .

Synthesis of compound 014

Compound 014 was prepared analogously to compound 002 starting from 3-methylheptan-1-ol. ¹H NMR (400 MHz CDCl₃) δ 4.65 (t, 2H) , 4.18-3.81 (m, 4H) , 3.76-3.53 (m, 10H) , 2.55-2.42 (m, 6H) , 2.31-2.11 (m, 4H) , 2.00-1.90 (m, 4H) , 1.78-1.16 (m, 48H) , 0.89-0.80 (m, 24H) .

Compound 015: bis (3, 3-bis ( (3-ethylheptyl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Synthesis of 3-ethylheptan-1-ol

Step 1: Triethyl phosphonoacetate (7.8 g, 34 mmol) in THF (12.5 mL) was slowly added to 60%sodium hydride, in oil, (1.4 g, 34 mmol) in dry THF (10 mL) at 0℃. The suspension was stirred for 1 h and then 3-heptanone (3.9 g, 34 mmol) in THF (12.5 mL) was slowly added. The solution was stirred for an hour and quenched with water. The solution was extracted with diethyl ether, and the organic layer was dried and concentrated. The crude was purified by flash chromatography eluting with hexanes/diethyl ether (99: 1) to give alkene (1.1 g, 5.8 mmol) as a yellowish liquid in 17%yield.

Step 2: The alkene (1.0 g, 5.4 mmol, 1.0 eq) was hydrogenated in ethyl acetate with a 10% (w/w) of Pd/C for 3 h using a balloon filled with hydrogen gas. The solution was filtered through celite and concentrated to give the alkane (940 mg, 5.0 mmol. ) as a clear liquid in 93%yield. Step 3: The ester (900 mg, 4.8 mmol, 1 eq) in diethyl ether was added slowly to lithium aluminum hydride (182 mg, 4.8 mol, 1.1 eq) suspended in diethyl ether at 0℃. The suspension was stirred at room temperature for 2-3 h and then cooled to 0℃. Water (0.2 mL) was added, followed by 15%sodium hydroxide (0.2 mL) and water (0.6 mL) . The solution was stirred for a few minutes at room temperature, filtered through a celite pad, washed with diethyl ether, dried, and concentrated to give the alcohol. Flash chromatography was used to purify the product giving a clear liquid (602 mg, 4.2 mmol) in 87%yield. ¹H NMR (400 MHz CDCl₃) δ 3.68 (t, J = 7.2 Hz, 2H) , 1.53-1.55 (m, 2H) , 1.19-1.34 (m, 9H) , 0.85-0.92 (m, 6H) .

Synthesis of compound 015

Compound 015 was prepared analogously to compound 002 starting from 3-ethylheptan-1-ol. ¹H NMR (400 MHz CDCl₃) δ 4.61 (t, 2H) , 4.13-3.82 (m, 4H) , 3.78-3.51 (m, 10H) , 2.52-2.42 (m, 6H) , 2.31-2.13 (m, 4H) , 2.02-1.93 (m, 4H) , 1.79-1.13 (m, 56H) , 0.89-0.75 (m, 24H) .

Compound 016: bis (3, 3-bis ( (5-ethylheptyl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Synthesis of 5-ethylheptan-1-ol

To a solution of 3-bromopropan-1-ol (10 g, 71.9 mmol, 6.51 mL, 1.0 eq) in THF (70 mL) was added CuCl₂. (LiCl) ₂ (1 M, 71.9 mL, 1.0 eq) at 25℃, then the reaction stirred at 25℃ for 0.5 hrs. The 3- (bromomethyl) pentane (1 M, 216 mL, 3.0 eq) was dropwise to the mixture at 25℃, then the mixture was stirred at 25℃ for 12 hrs. The mixture was added to the aq. NH₄Cl (200 mL) . The residue was diluted with H₂O (150 mL) and extracted with EtOAc (100 mL, 50 mL, 30 mL) . The combined organic layers were washed with brine (20 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 100/1 to 10/1) to give 5-ethylheptan-1-ol (9.1 g, 63.0 mmol, 87.6%yield) as colorless oil. ¹H NMR (400 MHz CDCl₃) δ 3.64 (t, J = 6.4 Hz, 2H) , 1.52-1.59 (m, 2H) , 1.24-1.43 (m, 12H) , 1.13-1.19 (m, 1H) , 0.90 (t, J = 6.8 Hz, 1H) , 0.84 (t, J = 7.2 Hz, 6H) .

Synthesis of Compound 016

Compound 016 was prepared analogously to compound 002 starting from 5-ethylheptan-1-ol. Compound 016 was obtained as a yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 6.0 Hz, 2H) , 4.15 (t, J = 6.4 Hz, 4H) , 3.56-3.62 (m, 5H) , 3.40-3.46 (m, 4H) , 2.62 (brs, 2H) , 2.50 (brs, 4H) , 2.31 (t, J = 7.6 Hz, 4H) , 1.95 (q, J = 6.4 Hz, 4H) , 1.52-1.68 (m, 16H) , 1.22-1.9 (m, 36H) , 1.15-1.19 (m, 4H) , 0.84 (t, J = 7.2 Hz, 24H) .

Compound 017: bis (3, 3-bis ( (4-propylheptyl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Synthesis of 4-propylheptan-1-ol

Step 1: To a solution of heptan-4-one (20 g, 175 mmol, 24.6 mL, 1.0 eq) in THF (140 mL) was dropped added 3-bromoprop-1-ene (1 M, 263 mL, 1.5 eq) at 0℃, then the reaction stirred at 25℃ for 12 hrs. The reaction mixture was poured into water (100 mL) and extracted with EtOAc (100 mL, 50 mL, 30 mL) , The combined organic layers were washed with brine (20 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give 4-propylhept-1-en-4-ol (28.6 g, crude) as yellow oil.

Step 2: To a solution of 4-propylhept-1-en-4-ol (36.6 g, 234 mmol, 1.0 eq) in THF (245 mL) was added BH₃. THF (1 M, 234 mL, 1.0 eq) at 0℃, 3hrs later NaOH (3 M, 156 mL, 2.0 eq) and H₂O₂ (48.3 g, 426 mmol, 40.9 mL, 30%purity, 1.82 eq) was added the mixture at 0℃, then the reaction solution was stirred at 25℃ for 12 hrs . The reaction mixture was poured into water (200 mL) and extracted with EtOAc (200 mL, 100 mL, 50 mL) , The combined organic layers were washed with brine (50 mL) and aq. Na₂S₂O₃ (50 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to 0/1) to give 4-propylheptane-1, 4-diol (23.1 g, 132 mmol, 56.5%yield) as a colorless oil.

Step 3: To a solution of 4-propylheptane-1, 4-diol (23.1 g, 133 mmol, 1.0 eq) in DCM (160 mL) was added Ac₂O (40.6 g, 398 mmol, 37.4 mL, 3.0 eq) , then Py (20.9 g, 265 mmol, 21.4 mL, 2.0 eq) was added the mixture at 0℃, the reaction solution was stirred at 25℃ for 3 hrs. The reaction mixture was poured into water (50 mL) and extracted with EtOAc (50 mL, 50 mL, 30 mL) , The combined organic layers were washed with brine (10 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 1/0 to 50/1) to give 4-hydroxy-4-propylheptyl acetate (28 g, 129 mmol, 97.6%yield) as a colorless oil.

Step 4: To a solution of 4-hydroxy-4-propylheptyl acetate (28 g, 129 mmol, 1.0 eq) , in DCM (195 mL) was added BF₃. Et₂O (45.9 g, 324 mmol, 39.8 mL, 2.5 eq) at 0 ℃, then the mixture was stirred at 25℃ for 0.5 hrs. The pH value of the reaction was adjusted to around 7-8 with aq. Na₂CO₃ at 0 ℃. The mixture was extracted with DCM (100 mL, 50 mL, 50 mL) and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 1/0 to 1/1) to give 4-propylhept-3-en-1-yl acetate (23.6 g, 119 mmol, 91.9%yield) as a colorless oil.

Step 5: To a solution of 4-propylhept-3-en-1-yl acetate (23.6 g, 119 mmol, 1.0 eq) in THF (200 mL) was added Pd/C (2.36 g, 2.22 mmol, 10%purity) under N₂. The suspension was degassed under vacuum and purged with H₂ several times. The mixture was stirred under H₂ (30 psi) at 25℃ for 12 hrs. The mixture was filtered and concentrated under reduced pressure to give 4-propylheptyl acetate (22.6 g, 112 mmol, 94.8%yield) as a colorless oil.

Step 6: To a solution of 4-propylheptyl acetate (22.6 g, 113 mmol, 1.0 eq) in MeOH (70 mL) and H₂O (70 mL) was added LiOH (5.40 g, 226 mmol, 2.0 eq) at 0℃, then the mixture was stirred at 25℃ for 3 hrs. The reaction mixture was poured into water (50 mL) and extracted with EtOAc (100 mL, 50 mL, 30 mL) , The combined organic layers were washed with brine (20 mL) , dried overNa₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 1/0 to 1/1) to give 4-propylheptan-1-ol (14 g, 88.4 mmol, 78.4%yield) as a colorless oil. ¹H NMR: (400 MHz CDCl₃) δ 3.63 (t, J = 6.8 Hz, 2H) , 1.31-1.55 (m, 2H) , 1.24-1.29 (m, 13H) , 0.89 (t, J = 6.8 Hz, 6H) .

Synthesis of Compound 017

Compound 017 was prepared analogously to compound 002 starting from 4-propylheptan-1-ol. Compound 017 was obtained as a yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 6.0 Hz, 2H) , 4.15 (t, J = 6.4 Hz, 4H) , 3.54-3.60 (m, 6H) , 3.38-3.44 (m, 4H) , 2.60 (brs, 2H) , 2.48 (brs, 4H) , 2.31 (t, J = 7.6 Hz, 4H) , 1.94 (q, J = 6.4 Hz, 4H) , 1.46-1.68 (m, 17H) , 1.18-1.35 (m, 49H) , 0.88 (t, J = 6.8 Hz, 24H) .

Compound 018: bis (3, 3-bis ( (4-isopropylcyclohexyl) methoxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Compound 018 was prepared analogously to compound 002 starting from (4-isopropylcyclohexyl) methanol. ¹H NMR (400 MHz CDCl₃) δ 4.67 (t, 2H) , 4.11-3.81 (m, 4H) , 3.78-3.51 (m, 10H) , 2.53-2.41 (m, 6H) , 2.32-2.12 (m, 4H) , 1.98-1.93 (m, 4H) , 1.92-1.13 (m, 56H) , 0.89-0.75 (m, 24H) .

Compound 019: bis (3, 3-bis ( (3-butylheptyl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Synthesis of 3-butylheptan-1-ol

Step 1: to a solution of NaH (14.1 g, 351 mmol, 60%purity, 1.0 eq) in THF (1576 mL) was added ethyl 2- (diethoxyphosphoryl) acetate (78.8 g, 352 mmol, 69.7 mL, 1.0 eq) in dropwise at 0 ℃. The reaction was warmed at 20 ℃ and stirred for 1 h until there was no bubbles formed, and then add nonan-5-one (25.0 g, 176 mmol, 0.5 eq) . The reaction was stirred at 75 ℃ for 12 h. The reaction mixture was quenched by addition water (400 mL) and extracted with EtOAc (500 mL*2) . The combined organic layers were washed with brine (100 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=4/1) . ethyl 3-butylhept-2-enoate (32.0 g, crude) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 5.62 (s, 1 H) , 5.26 -5.36 (m, 1 H) , 4.10 -4.17 (m, 2 H) , 3.03 (s, 1 H) , 2.97 -2.98 (m, 1 H) , 2.57 -2.61 (m, 1 H) , 2.00 -2.16 (m, 3 H) , 1.25 -1.48 (m, 10 H) , 0.88 -0.94 (m, 6 H) .

Step 2: To a solution of Pd/C (3.20 g, 4.71 mmol, 10%purity) in EtOH (160 mL) was added ethyl 3-butylhept-2-enoate (16.0 g, 75.4 mmol, 1.0 eq) . The reaction was degassed and purged by H₂ several times, stirred at 25 ℃ for 12 h under H₂ (50 psi) . H NMR showed the starting material was consumed completely. The two reactions was combined to work up. The reaction mixture was filtered, the filterate cake was washed with THF (1.00 L) and concentrated under reduced pressure to give a residue. The crude product was used into the next step without further purification. Ethyl 3-butylheptanoate (29.0 g, 135 mmol, 89.8%yield) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.10 -4.15 (m, 2 H) , 2.23 (d, J = 6.88 Hz, 2 H) , 1.82 -1.86 (m, 1 H) , 1.24 -1.32 (m, 14 H) , 0.87 -0.91 (m, 6 H) .

Step 3: To a solution of LAH (5.14 g, 135 mmol, 1.0 eq) in THF (290 mL) was added Ethyl 3-butylheptanoate (29.0 g, 135 mmol, 1.0 eq) at 0 ℃. The reaction was stirred at 20 ℃ for 2 h. The reaction mixture was quenched by addition water (6.00 mL) at 0℃, 15%NaOH with solvent (6.00 mL) , and water (18.0 mL) . The combined organic layers were added MgSO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was used into the next step without further purification. 3-butylheptan-1-ol (23.0 g, crude) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 3.67 (t, J = 6.8 Hz, 2 H) , 1.51 -1.56 (m, 2 H) , 1.35 -1.45 (m, 1 H) , 1.26 -1.31 (m, 13 H) , 0.90 (t, J = 6.8 Hz, 6 H) .

Synthesis of 3, 3-bis ( (3-butylheptyl) oxy) propan-1-ol

To a solution of ethyl 3, 3-diethoxypropanoate (6.5 g, 34.2 mmol, 6.65 mL, 1.0 eq) and 3-butylheptan-1-ol (23.0 g, 133 mmol, 3.91 eq) was added KHSO₄ (232 mg, 1.71 mmol, 100 uL, 0.05 eq) . The reaction was stirred at 75 ℃ for 12 h. The residue was diluted with water (50.0 mL) and extracted with EtOAc (100 mL, 50.0 ml) . The combined organic layers were washed with brine (20.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=60/1 to 50/1) . 3-butylheptyl 3, 3-bis ( (3-butylheptyl) oxy) propanoate (3.6 g, 6.33 mmol, 18.5%yield) . ¹H NMR (400 MHz CDCl₃) δ 4.94 (d, J = 4.4 Hz, 1 H) , 4.12 -4.16 (m, 2 H) , 3.58 -3.63 (m, 2 H) , 3.48 -3.52 (m, 2 H) , 2.64 (d, J = 6.00 Hz, 2 H) , 1.50 -1.55 (m, 5 H) , 1.40 -1.43 (m, 3 H) , 1.20 -1.31 (m, 35 H) , 0.86 -0.91 (m, 18 H) .

To a solution of LAH (240 mg, 6.33 mmol, 1.0 eq) in THF (25.2 mL) was added 3-butylheptyl 3, 3-bis ( (3-butylheptyl) oxy) propanoate (3.60 g, 6.33 mmol, 1.0 eq) at 0 ℃. The reaction was stirred at 20 ℃ for 2h. The reaction mixture was quenched by addition water (0.50 mL) at 0℃, 15%NaOH with solvent (0.50 mL) , and water (1.50 mL) . The combined organic layers were added MgSO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was used into the next step without further purification. 3, 3-bis ( (3- butylheptyl) oxy) propan-1-ol (2.60 g, crude) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.66 (t, J = 5.2 Hz, 1 H) , 3.73 -3.76 (m, 2 H) , 3.62 -3.69 (m, 3 H) , 3.44 -3.50 (m, 2 H) , 1.87 -1.91 (m, 2 H) , 1.51 -1.57 (m, 6 H) , 1.41 -1.43 (m, 3 H) , 1.26 -1.31 (m, 35 H) , 0.85 -0.91 (m, 18 H) .

Synthesis of compound 019

Compound 019 was prepared analogously to compound 002 starting from 3, 3-bis ( (3-butylheptyl) oxy) propan-1-ol. Compound 019 was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.59 (t, J = 5.6 Hz, 2 H) , 4.14 (t, J = 6.4 Hz, 4 H) , 3.53 -3.63 (m, 4 H) , 3.42 -3.47 (m, 4 H) , 2.44 -2.62 (m, 4 H) , 2.31 (t, J = 7.6 Hz, 4 H) , 1.88 -1.97 (m, 5 H) , 1.59 -1.68 (m, 5 H) , 1.50 -1.55 (m, 11 H) , 1.41 (br s, 5 H) , 1.25 -1.31 (m, 50 H) , 0.89 (t, J = 6.4 Hz, 24 H) .

Compound 020: bis (3, 3-bis ( (3-butylheptyl) oxy) propyl) 5, 5'- ( (2-hydroxyethyl) azanediyl) dipentanoate

Compound 020 was prepared analogously to compound 002 starting with 5-bromopentanoic acid and 3, 3-bis ( (3-butylheptyl) oxy) propan-1-ol. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 5.6 Hz, 2 H) , 4.12 (t, J = 6.4 Hz, 4 H) , 3.51 -3.82 (m, 6 H) , 3.41 -3.47 (m, 4 H) , 2.45 -2.62 (m, 4 H) , 2.31 (t, J = 7.6 Hz, 4 H) , 1.88 -1.97 (m, 5 H) , 1.59 -1.68 (m, 5 H) , 1.50 - 1.55 (m, 11 H) , 1.41 (br s, 5 H) , 1.25 -1.31 (m, 46 H) , 0.89 (t, J = 6.4 Hz, 24 H) .

Compound 021: bis (3, 3-bis (octyloxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Synthesis of 3, 3-bis (octyloxy) propan-1-ol

To a solution of 3, 3-diethoxypropanenitrile (10 g, 69.8 mmol, 10.5 mL, 1.0 eq) and octan-1-ol (27.3 g, 210 mmol, 33.1 mL, 3.0 eq) in Tol. (70 mL) was added PPTS (878 mg, 3.49 mmol, 0.05 eq) at 25℃, then the mixture was stirred at 120℃ for 12 hrs. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 1/0 to 60/1) to give 3, 3-bis (octyloxy) propanenitrile (15.8 g, 50.7 mmol, 72.6%yield) as a colorless oil. ¹H NMR (400 MHz CDCl₃) δ 4.78 (t, J = 5.6 Hz, 1H) , 3.61-3.64 (m, 2H) , 3.49-3.53 (m, 2H) , 2.66 (d, J = 5.6 Hz, 2H) , 1.56-1.64 (m, 4H) , 1.28-1.38 (m, 21H) , 0.89 (t, J = 6.4 Hz, 6H) .

To a solution of 3, 3-bis (octyloxy) propanenitrile (10.8 g, 34.7 mmol, 1.0 eq) in EtOH (30 mL) was added KOH (3 M, 34.7 mL, 3.0 eq) at 25℃, then the mixture was stirred at 110℃ for 12 hrs. The pH value of the reaction was adjusted to around 4-5 with aq. HCl (1 M) at 0℃. The mixture was extracted with Petroleum ether (50 mL x 4) and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 100/1 to 0/1) to give 3, 3-bis (octyloxy) propanoic acid (9.5 g, crude) as a colorless oil. ¹H NMR (400 MHz CDCl₃) δ 12.2 (brs, 1H) , 4.79-4.82 (m, 1H) , 4.31 (t, J = 4.8 Hz, 1H) , 3.32-3.42 (m, 4H) , 2.51 (d, J = 7.2 Hz, 2H) , 1.29-1.41 (m, 4H) , 1.25-1.28 (m, 21H) , 1.09 (t, J = 6.0 Hz, 2H) , 0.86 (t, J = 6.8 Hz, 6H) .

To a solution of 3, 3-bis (octyloxy) propanoic acid (8 g, 19.4 mmol, 80%purity, 1.0 eq) in THF (55 mL) at 0℃, then LiAlH₄ (882 mg, 23.2 mmol, 1.2 eq) was added the mixture and stirred at 25℃ for 3 hrs. Water (0.1 mL) , 15%aq. NaOH (0.1 mL) and water (0.3 mL) was dropwise the reaction solution at 0℃, then Na₂SO₄ added the mixture and stirred at 25℃ for 15 mins. The mixture was filtered and the filter cake concentrated under reduced pressure to give 3, 3-bis (octyloxy) propan-1-ol (5.1 g, 16.1 mmol, 83.2%yield) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.66 (t, J = 5.6 Hz, 1H) , 3.73-3.76 (m, 2H) , 3.62-3.64 (m, 2H) , 3.44-3.46 (m, 2H) , 2.58 (t, J = 5.6 Hz, 1H) , 1.87-1.91 (m, 2H) , 1.56-1.60 (m, 5H) , 1.22-1.34 (m, 21H) , 0.88 (t, J = 6.4 Hz, 6H) .

Synthesis of 3, 3-bis (octyloxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate

To a solution of 3, 3-bis (octyloxy) propan-1-ol (5.5 g, 17.4 mmol, 2.14 mL, 1.0 eq) in DCM (40 mL) was added 6-bromohexanoic acid (3.39 g, 17.4 mmol, 1.0 eq) , EDCI (5.00 g, 26.1 mmol, 1.5 eq) and DMAP (425 mg, 3.48 mmol, 0.2 eq) at 0℃, then the mixture was stirred at 25℃ for 1.5 hrs. The reaction mixture was poured into water (50 mL) and extracted with EtOAc (50 mL, 30 mL, 20 mL) , The combined organic layers were washed with brine (30 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=100/1 to 0/1) to give 3, 3-bis (octyloxy) propyl 6-bromohexanoate (2.5 g, 5.08 mmol, 30%yield) as a colorless oil. To a solution of 3, 3-bis (octyloxy) propyl 6-bromohexanoate (2 g, 4.05 mmol, 1.0 eq) and 2-aminoethanol (2.48 g, 40.5 mmol, 2.45 mL, 10 eq) in EtOH (10 mL) was added at 25℃, then the mixture was stirred at 90℃ for 0.5 hrs. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (5 mL, 3 mL, 2 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 20/1 to Ethyl acetate /MeOH = 10/1) to give 3, 3-bis (octyloxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (1.2 g, 2.53 mmol, 62.5%yield) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.59 (t, J = 6.0 Hz, 1H) , 4.14 (t, J = 6.4 Hz, 2H) , 3.72 (t, J = 5.2 Hz, 2H) , 3.55-3.61 (m, 2H) , 3.40-3.45 (m, 2H) , 2.86 (t, J = 5.2 Hz, 2H) , 2.71-2.74 (m, 6H) , 2.32 (t, J = 7.2 Hz, 2H) , 1.95 (q, J = 6.4 Hz, 2H) , 1.53-1.69 (m, 9H) , 1.28-1.43 (m, 24H) , 0.89 (t, J = 6.4 Hz, 6H) .

Synthesis of Compound 021

To a solution of 3, 3-bis (octyloxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (1 g, 2.11 mmol, 1.0 eq) and 3, 3-bis (octyloxy) propyl 6-bromohexanoate (1.04 g, 2.11 mmol, 12.3 uL, 1.0 eq) in EtOH (5 mL) was added DIEA (546 mg, 4.22 mmol, 735 uL, 2.0 eq) at 25℃, then the mixture was stirred at 90℃ for 12 hrs. The reaction mixture was poured into water (5 mL) and extracted with EtOAc (5 mL, 3 mL, 2 mL) , The combined organic layers were washed with brine 3 mL, dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100 x 30 mm x 5um; mobile phase: [water (HCl) -ACN] ; B%: 60%-90%, 10 min) . The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=10/1 to EtOAc/MeOH = 5/1) to give compound 021 (450 mg, 507.69 umol, 24.05%yield) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 5.6 Hz, 2H) , 4.12-4.16 (m, 4H) , 4.06 (t, J = 6.8 Hz, 1H) , 3.53-3.61 (m, 6H) , 3.41-3.45 (m, 4H) , 2.57 (t, J = 4.8 Hz, 2H) , 2.45 (t, J = 7.2 Hz, 4H) , 2.30 (t, J = 7.6 Hz, 5H) , 2.05 (s, 1H) , 1.94 (dd, J = 6.4, 12.8 Hz , 4H) , 1.53-1.67 (m, 14H) , 1.43-1.50 (m, 6H) , 1.25-1.33 (m, 47H) , 0.89 (t, J = 7.2 Hz, 12H) .

Compound 022: bis (3, 3-bis (octyloxy) propyl) 5, 5'- ( (2-hydroxyethyl) azanediyl) dipentanoate

Compound 022 was prepared using similar procedure as for compound 021, but using 5-bromopentanoic acid instead of 6-bromohexanoic acid. ¹H NMR (400 MHz CDCl₃) δ 4.68 (t, 2H) , 4.15-3.82 (m, 4H) , 3.77-3.51 (m, 10H) , 2.52-2.40 (m, 6H) , 2.33-2.17 (m, 4H) , 2.08-2.01 (m, 4H) , 1.95-1.11 (m, 56H) , 0.88-0.74 (m, 12H) .

Compound 023: bis (4, 4-bis (octyloxy) butyl) 5, 5'- ( (2-hydroxyethyl) azanediyl) dipentanoate

Synthesis of 4, 4-bis (octyloxy) butan-1-ol

To a solution of 4, 4-dimethoxybutanenitrile (9.0 g, 69.8 mmol, 1.0 eq) and octan-1-ol (27.3 g, 210 mmol, 3.0 eq) in Tol. (70 mL) was added PPTS (878 mg, 3.5 mmol, 0.05 eq) at 25℃, then the mixture was stirred at 120℃ for 12 hrs . The mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 1/0 to 60/1) to give 4, 4-bis (octyloxy) butanenitrile (17.0 g, 52.3 mmol, 75%yield) as a colorless oil.

To a solution of 4, 4-bis (octyloxy) butanenitrile (11.0 g, 33.8 mmol, 1.0 eq) in EtOH (30 mL) was added KOH (3 M, 33.8 mL, 3.0 eq) at 25℃, then the mixture was stirred at 110℃ for 12 hrs. The pH value of the reaction was adjusted to around 4-5 with aq. HCl (1 M) at 0℃. The mixture was extracted with ether (50 mL x 4) and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to 1/1) to give 4, 4-bis (octyloxy) butanoic acid (10.2 g, 29.6 mmol, 88%) as a colorless oil. To a solution of 4, 4-bis (octyloxy) butanoic acid (8 g, 23.2 mmol, 1.0 eq) in THF (50 mL) at 0℃, then LiAlH₄ (1.1 g, 27.8 mmol, 1.2 eq) was added the mixture and stirred at 25℃ for 3 hrs. Water (1.1 mL) , 15%aq. NaOH (1.1 mL) and water (3.3 mL) was dropwise the reaction solution at 0℃, then Na₂SO₄ added the mixture and stirred at 25℃ for 15 mins. The mixture was filtered and the filter cake concentrated under reduced pressure to give 4, 4-bis (octyloxy) butan-1-ol (7.5 g, 22.7 mmol, 98%yield) as a colorless oil. ¹H NMR (400 MHz CDCl₃) δ 4.64 (t, 1H) , 3.74 (t, 2H) , 3.62-3.67 (m, 2H) , 3.41-3.46 (m, 2H) , 1.84-1.96 (m, 2H) , 1.72-1.32 (m, 26H) , 0.91-0.87 (m, 6H) .

Synthesis of compound 023

Compound 023 was prepared analogously to compound 021 starting from 4, 4-bis (octyloxy) butan-1-ol, and using 5-bromopentanoic acid instead of 6-bromohexanoic acid. ¹H NMR (400 MHz CDCl₃) δ 4.71 (t, 2H) , 4.16-3.81 (m, 4H) , 3.75-3.50 (m, 10H) , 2.51-2.38 (m, 6H) , 2.31-2.14 (m, 4H) , 2.07-1.98 (m, 4H) , 1.95-1.11 (m, 60H) , 0.88-0.74 (m, 12H) .

Compound 024: bis (4, 4-bis (octyloxy) butyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Compound 024 was prepared analogously to compound 021 but starting from 4, 4-bis (octyloxy) butan-1-ol. ¹H NMR (400 MHz CDCl₃) δ 4.65 (t, 2H) , 4.11-3.80 (m, 4H) , 3.78-3.52 (m, 10H) , 2.51-2.39 (m, 6H) , 2.36-2.14 (m, 4H) , 1.98-1.11 (m, 68H) , 0.88-0.74 (m, 12H) .

Compound 025: bis (3, 3-bis (octyloxy) propyl) 6, 6'- ( (4-hydroxybutyl) azanediyl) dihexanoate

A 100 mL three-necked round bottom flask equipped with magnetic stirrer, addition funnel and thermometer. To a solution of 3, 3-bis (octyloxy) propyl 6-bromohexanoate (2.00 g, 4.05 mmol, 1.0 eq) in EtOH (10.0 mL) was added 4-aminobutan-1-ol (1.08 g, 12.2 mmol, 1.13 mL, 3.0 eq) . The mixture was stirred at 75 ℃ for 12 hrs. The residue was diluted with water (20.0 mL) and extracted with EtOAc (50.0 mL, 25.0 mL) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, DCM: MeOH=100/1 to 10/1) . 3, 3-bis (octyloxy) propyl 6- ( (4-hydroxybutyl) amino) hexanoate (1.10 g, 2.19 mmol, 27.0%yield) and compound 025 (0.30 g, 328 umol, 8.10%yield) was obtained as yellow oil.

A 100 mL three-necked round bottom flask equipped with magnetic stirrer, addition funnel and thermometer. To a solution of 3, 3-bis (octyloxy) propyl 6- ( (4-hydroxybutyl) amino) hexanoate (0.40 g, 797 umol, 1.0 eq) in EtOH (8.00 mL) was added DIEA (154 mg, 1.20 mmol, 208 uL, 1.5 eq) and 3, 3-bis (octyloxy) propyl 6-bromohexanoate (472 mg, 957 umol, 1.2 eq) . The mixture was stirred at 75 ℃ for 132 hrs. The residue was diluted with water (10.0 mL) and extracted with EtOAc (20.0 mL, 10.0 mL) . The combined organic layers were washed with brine (5 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=1/1 to 0/1) . Compound 025 (0.10 g, 109 umol, 13.7%yield, 100%purity) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 5.6 Hz, 2 H) , 4.14 (t, J = 6.8 Hz, 2 H) , 3.55 -3.61 (m, 6 H) , 3.39 -3.45 (m, 4 H) , 2.37 -2.81 (m, 5 H) , 2.31 (t, J = 7.2 Hz, 5 H) , 1.92 -1.97 (m, 4 H) , 1.48 -1.70 (m, 20 H) , 1.28 -1.39 (m, 47 H) , 0.87 -0.93 (m, 12 H) .

Compound 026: bis (3, 3-bis (2-cyclohexylethoxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Compound 026 was prepared analogously to compound 002 starting from 2-cyclohexylethan-1-ol. Compound 026 was obtained as a yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.58 (t, J = 5.6 Hz, 2H) , 4.14 (t, J = 6.4 Hz, 4H) , 3.69 (t, J = 5.2 Hz, 2H) , 3.59-3.65 (m, 4H) , 3.43-3.48 (m, 4H) , 2.76 (t, J = 4.8 Hz, 2H) , 2.66 (t, J = 7.2 Hz, 4H) , 1.92-1.96 (m, 4H) , 1.57-1.72 (m, 28H) , 1.32-1.49 (m, 16H) , 1.09-1.25 (m, 13H) , 0.86-0.96 (m, 8H) .

Compound 027: bis (3, 3-bis ( (7-methyloctyl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Synthesis of 7-methyloctan-1-ol

Step 1: To a solution of 2- (prop-2-yn-1-yloxy) tetrahydro-2H-pyran (20.2 g, 144 mmol, 20.3 mL, 1.0 eq) in THF (170 mL) was added HMPA (38.8 g, 216 mmol, 38.0 mL, 1.5 eq) at 25℃, then n-BuLi (2.5 M, 86.5 mL, 1.5 eq) was added the mixture at -65℃, 1hr later 1-bromo-4-methyl-pentane (25 g, 151 mmol, 21.4 mL, 1.05 eq) added the reaction and warmed to 25℃ stirred for 12 hrs. The reaction mixture was poured into water (100 mL) and extracted with EtOAc (100 mL, 50 mL, 50 mL) , The combined organic layers were washed with brine (50 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give 2- ( (7-methyloct-2-yn-1-yl) oxy) tetrahydro-2H-pyran (11 g, 49.0 mmol, 33.9%yield) as a colorless oil. ¹H NMR (400 MHz CDCl₃) δ 4.82 (t, J = 3.2 Hz, 1H) , 4.19-4.32 (m, 2H) , 3.83-3.88 (m, 1H) , 3.52-3.55 (m, 1H) , 2.20-2.22 (m, 2H) , 1.71-1.88 (m, 2H) , 1.48-1.66 (m, 7H) , 1.23-1.29 (m, 2H) , 0.88 (d, J = 6.8 Hz, 6H) .

Step 2: To a solution of 2- ( (7-methyloct-2-yn-1-yl) oxy) tetrahydro-2H-pyran (9 g, 40.1 mmol, 1.0 eq) in EtOH (60 mL) was added Pd/C (0.9 g, 10%purity) at 25℃, then the mixture was stirred at 25℃ for 12 hrs under H₂ (15 psi) . The mixture was filtered and concentrated under reduced pressure to give 2- ( (7-methyloctyl) oxy) tetrahydro-2H-pyran (9 g, crude) as a colorless oil. ¹H NMR (400 MHz CDCl₃) δ 4.57 (d, J = 4.0 Hz, 1H) , 3.86-3.90 (m, 1H) , 3.71-3.77 (m, 1H) , 3.48-3.52 (m, 1H) , 1.80-1.87 (m, 1H) , 1.70-1.75 (m, 1H) , 1.49-1.64 (m, 7H) , 1.27-1.36 (m, 6H) , 1.16-1.17 (m, 2H) , 0.86 (d, J = 6.4 Hz, 6H) .

Step 3: To a solution of 2- ( (7-methyloctyl) oxy) tetrahydro-2H-pyran (11.7 g, 51.2 mmol, 1.0 eq) in MeOH (80 mL) was added CAS (2.56 g, 10.2 mmol, 0.2 eq) at 0℃, then the mixture was stirred at 25℃ for 3 hrs. The mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 1/0 to 5/1) to give 7-methyloctan-1-ol (5.9 g, 40.9 mmol, 79.8%yield) as a colorless oil. ¹H NMR (400 MHz CDCl₃) δ 3.65 (t, J = 6.8 Hz, 2H) , 1.49-1.61 (m, 3H) , 1.28-1.38 (m, 7H) , 1.16-1.19 (m, 2H) , 0.87 (d, J = 6.8 Hz, 6H) .

Synthesis of 3, 3-bis ( (7-methyloctyl) oxy) propan-1-ol

To a solution of 2- ( (7-methyloctyl) oxy) tetrahydro-2H-pyran (5.9 g, 40.9 mmol, 1.21 mL, 3.0 eq) in ethyl 3, 3-diethoxypropanoate (2.59 g, 13.6 mmol, 2.65 mL, 1.0 eq) was added KHSO₄ (92.8 mg, 682 umol, 40.0 uL, 0.05 eq) at 25℃, then the mixture was stirred at 80℃ for 12 hrs. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (10 mL, 5 mL, 3 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 1/0 to 5/1) to give ethyl 3, 3-bis ( (7-methyloctyl) oxy) propanoate (3 g, 7.76 mmol, 56.9%yield) as a colorless oil.

To a solution of ethyl 3, 3-bis ( (7-methyloctyl) oxy) propanoate (3 g, 7.76 mmol, 1.0 eq) in EtOH (10 mL) , THF (10 mL) and H₂O (10 mL) was added LiOH (186 mg, 7.76 mmol, 86.2 uL, 1.0 eq) at 25℃, then the mixture was stirred at 25℃ for 3 hrs. The pH value of the reaction was adjusted to around 2-3 with HCl liquor (1 M) at 0 ℃. The mixture was extracted with EtOAc (5 mL x 3) and concentrated under reduced pressure to give 3, 3-bis ( (7-methyloctyl) oxy) propanoic acid (1.2 g, 3.35 mmol, 43.1%yield) as a colorless oil. ¹H NMR (400 MHz CDCl₃) δ 4.62 (t, J = 5.6 Hz, 1H) , 4.14-4.17 (m, 2H) , 3.56-3.61 (m, 2H) , 3.40-3.45 (m, 4H) , 2.32 (t, J = 7.2 Hz, 2H) , 1.85-1.97 (m, 5H) , 1.45-1.70 (m, 12H) , 1.25-1.36 (m, 23H) , 0.89 (t, J = 6.4 Hz, 6H) .

To a solution of 3, 3-bis ( (7-methyloctyl) oxy) propanoic acid (1.2 g, 2.68 mmol, 80%purity, 1.0 eq) in THF (12 mL) at 0℃, then LiAlH₄ (122 mg, 3.21 mmol, 1.2 eq) was added the mixture and stirred at 25℃ for 3 hrs. The reaction was cooled to 0℃, water (0.1 mL) was dropped in the reaction solution under N₂ and created a lot of bubbles, then 15%aq. NaOH (0.1 mL) was added dropwise slowly at 0℃; after 5mins, water (0.3 mL) was dropped the reaction solution at 0℃, then Na₂SO₄ added the mixture and warmed to 25 ℃ stirred 15 mins. The mixture was filtered and the filter cake concentrated under reduced pressure to give 3, 3-bis ( (7-methyloctyl) oxy) propan-1-ol (1.2 g, crude) as a colorless oil. ¹H NMR (400 MHz CDCl₃) δ 4.66 (t, J = 5.2 Hz, 1H) , 3.74-3.76 (m, 2H) , 3.61-3.67 (m, 3H) , 3.43-3.48 (m, 1H) , 2.55 (brs, 1H) , 1.85-1.92 (m, 1H) , 1.47-1.62 (m, 6H) , 1.16-1.36 (m, 17H) , 0.89 (d, J = 6.4 Hz, 12H) .

Synthesis of 3, 3-bis ( (7-methyloctyl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate

To a solution of 3, 3-bis ( (7-methyloctyl) oxy) propan-1-ol (4.4 g, 12.8 mmol, 2.14 mL, 1.0 eq) in DCM (25 mL) was added 6-bromohexanoic acid (3.74 g, 19.2 mmol, 1.5 eq) , EDCI (3.67 g, 19.2 mmol, 1.5 eq) and DMAP (312 mg, 2.55 mmol, 0.2 eq) at 0℃, then the mixture was stirred at 25℃ for 1.5 hrs. The reaction mixture was poured into water (50 mL) and extracted with EtOAc (50 mL, 30 mL, 20 mL) , The combined organic layers were washed with brine (30 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 100/1 to 0/1) to give 3, 3-bis ( (7-methyloctyl) oxy) propyl 6-bromohexanoate (1.3 g, 2.49 mmol, 19.5%yield) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 6.0 Hz, 1H) , 4.14-4.17 (m, 2H) , 3.56-3.61 (m, 2H) , 3.40-3.45 (m, 4H) , 2.32 (t, J = 7.2 Hz, 2H) , 1.85-1.98 (m, 4H) , 1.48-1.68 (m, 11H) , 1.25-1.36 (m, 13H) , 1.14-1.17 (m, 4H) , 0.87 (d, J = 6.8 Hz, 12H) .

To a solution of 3, 3-bis ( (7-methyloctyl) oxy) propyl 6-bromohexanoate (0.6 g, 1.15 mmol, 1.0 eq) and 2-aminoethanol (703 mg, 11.5 mmol, 696 uL, 10 eq) in EtOH (12 mL) was added at 25℃, then the mixture was stirred at 90℃ for 12 hrs. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (10 mL, 5 mL, 3 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give 3, 3-bis ( (7-methyloctyl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (460 mg, 917 umol, 79.7%yield) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 6.0 Hz, 1H) , 4.14 (t, J = 6.8 Hz, 2H) , 3.81-3.83 (m, 2H) , 3.67 (brs, 3H) , 3.55-. 361 (m, 3H) , 3.40-3.45 (m, 3H) , 2.95-2.98 (m, 2H) , 2.81-2.85 (m, 2H) , 2.32 (t, J = 7.2 Hz, 2H) , 1.92-1.97 (m, 2H) , 1.50-1.71 (m, 12H) , 1.26-1.37 (m, 18H) , 1.15-1.18 (m, 4H) , 0.86 (d, J = 6.4 Hz, 12H) .

Synthesis of compound 027

To a solution of 3, 3-bis ( (7-methyloctyl) oxy) propyl 6-bromohexanoate (724 mg, 1.39 mmol, 1.0 eq) and 3, 3-bis ( (7-methyloctyl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (0.696 g, 1.39 mmol, 1.0 eq) in EtOH (12 mL) was added DIEA (359 mg, 2.77 mmol, 483 uL, 2.0 eq) at 25℃, then the mixture was stirred at 90 ℃ for 5 hrs. The reaction mixture was poured into water (5 mL) and extracted with EtOAc (5 mL, 3 mL, 2 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100 x 30 mm x 5 um; mobile phase: [water (HCl) -ACN] ; B %: 60%-90%, 10 min) . The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to EtOAc/MeOH = 5/1) to give compound 027 (156 mg, 165 umol, 11.9%yield) as a colorless oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 5.6 Hz, 1H) , 4.15 (t, J = 6.4 Hz, 2H) , 3.53-3.61 (m, 3H) , 3.39-3.45 (m, 2H) , 2.57 (t, J = 4.8 Hz, 1H) , 2.45 (t, J = 7.2 Hz, 2H) , 2.30 (t, J = 7.6 Hz, 2H) , 1.93-1.97 (m, 2H) , 1.49-1.67 (m, 12H) , 1.26-1.36 (m, 15H) , 1.15-1.18 (m, 4H) , 0.86 (d, J = 6.8 Hz, 12H) .

Compound 028: bis (3, 3-bis ( (6-methyloctyl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Compound 028 was prepared analogously to compound 002 starting from 6-methyloctan-1-ol. ¹H NMR (400 MHz CDCl₃) δ 4.67 (t, 2H) , 4.14-3.81 (m, 4H) , 3.74-3.51 (m, 10H) , 2.51-2.37 (m, 6H) , 2.37-2.16 (m, 4H) , 2.01-1.11 (m, 60H) , 0.88-0.77 (m, 24H) .

Compound 029: bis (3, 3-bis ( (7-methyloct-6-en-1-yl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Synthesis of 7-methyloct-6-en-1-ol

Step 1: To a solution of 1, 6-hexanediol (6.0 g, 50.8 mmol, 1.0 eq) in THF (200 mL) at 0 ℃ was added NaH (2.3 g, 50.8 mmol, 1.0 eq, 60%purity) . The reaction mixture was stirred at 0 ℃ for 30 min, then warmed to 23 ℃ and stirred for 3 h. TBSCl (7.7 g, 50.8 mmol, 1.0 eq) was then added and the solution was stirred at 23 ℃ for 16 h. The reaction mixture was poured into water (200 mL) and extracted with Et₂O (2 x 100 mL) . The combined organic phases were dried over MgSO₄, and the solvent was removed by rotary evaporation, producing alcohol (10.3 g, 44.2 mmol, 87%yield) as a colorless oil.

Step 2: To a solution of alcohol (10.0 g, 43.0 mmol, 1.0 eq) in CH₂Cl₂ (100 mL) at 0 ℃ was added TEMPO (67 mg, 0.5 mmol, 1 mol%) . A solution of KBr (5.6 g, 47.3 mmol, 1.1 eq) in 5%aq. NaHCO₃ (24.0 mL, 0.2 M) was then added to the organic phase, and the mixture was stirred vigorously at 0 ℃. NaOCl (28.0 mL, 10%aq., 47.3 mmol, 1.1 eq) was added dropwise, and the reaction mixture was stirred for 15 min. Upon completion, a saturated solution of Na₂S₂O₃ (50 mL) was added, and the mixture was stirred for 5 min. The mixture was extracted with CH₂Cl₂ (2 x 55 mL) , and the combined organic phases were washed with H₂O (100 mL) and brine (200 mL) . The organic phase was dried over MgSO₄. The solvent was removed by rotary evaporation, producing aldehyde (8.8 g, 38.3 mmol, 89%yield) as a colorless oil.

Step 3: To a solution of isopropyl triphenylphosphonium iodide (16.2 g, 37.5 mmol, 1.5 eq) in THF (150 mL) at 0 ℃ under argon was added n-BuLi (15.0 mL, 2.50 M in hexanes, 37.5 mmol, 1.5 eq) , and the resulting solution was stirred for 30 min at 0 ℃. Aldehyde (5.5 g, 23.7 mmol, 1.0 eq) in THF (10.0 mL) was added rapidly and the resulting mixture was stirred at 0 ℃ for 30 min, then warmed to 23 ℃ and stirred for 1 h. The reaction was quenched with H₂O (50 mL) and sat. aq. NH₄Cl (50 mL) . The resulting mixture was extracted with hexanes (2 x 100 mL) . The combined organic phases were dried over MgSO₄ and filtered. The solvent was removed via rotary evaporation and the recovered residue was purified by flash chromatography (19: 1 petroleum ether/EtOAc eluent) , producing alkene (5.1 g, 19.9 mol, 84%yield) as a colorless oil. Step 4: To a solution of alkene (5.1 g, 19.9 mmol) in MeOH (100 mL, 0.2 M) was added 10%aq. HCl (0.5 mL) , and the reaction was stirred at 23 ℃ for 30 min. The reaction was quenched with Et₃N (0.5 mL) , and the solvent was removed via rotary evaporation. The resulting residue was purified by flash chromatography (5: 1 petroleum ether/EtOAc eluent) to afford alcohol (2.5 g, 17.6 mmol, 89%yield) as a colorless oil.

Synthesis of Compound 029

Compound 029 was prepared analogously to compound 002 starting from 7-methyloct-6-en-1-ol. ¹H NMR (400 MHz CDCl₃) δ 5.30-5.15 (m, 4H) , 4.67 (t, 2H) , 4.13-3.80 (m, 4H) , 3.75-3.52 (m, 10H) , 2.52-2.33 (m, 6H) , 2.37-2.01 (m, 16H) , 1.97-1.11 (m, 60H) .

Compound 030: bis (3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Synthesis of 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propan-1-ol

To a solution of 3, 7-dimethyloctan-1-ol (12.5 g, 78.8 mmol, 24.9 mL, 3.0 eq) in ethyl 3, 3-diethoxypropanoate (5 g, 26.3 mmol, 5.11 mL, 1.0 eq) was added KHSO₄ (179 mg, 1.31 mmol, 77.1 uL, 0.05 eq) at 25℃, then the mixture was stirred at 80℃ for 12 hrs. The reaction mixture was poured into water (20 mL) and extracted with EtOAc (20 mL, 10 mL, 5 mL) , The combined organic layers were washed with brine (5 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (Petroleum ether/Ethyl acetate = 10/1, product R_f = 0.50) to give ethyl 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propanoate (6.1 g, 14.7 mmol, 55.9%yield) as a colorless oil.

To a solution of ethyl 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propanoate (4.1 g, 10.6 mmol, 1.0 eq) in EtOH (5 mL) , THF (5 mL) and H₂O (5 mL) was added NaOH (848 mg, 21.2 mmol, 2.0 eq) at 25℃, then the mixture was stirred at 25℃ for 3 hrs. The reaction mixture was poured into water (5 mL) and extracted with EtOAc (10 mL, 5 mL, 5 mL) , The combined organic layers were washed with brine (5 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 100/1 to 0/1) to give 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propanoic acid (3 g, 7.76 mmol, 73.1%yield) as a colorless oil.

A suspension of LiAlH₄ (836 mg, 22.0 mmol, 1.2 eq) in THF (20 mL) at 0℃, a solution of 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propanoic acid (7.1 g, 18.4 mmol, 1.0 eq) in THF (20 mL) was dropped added to the mixture dropwise at 0℃ for 20 mins under N₂. Then the reaction solution was warmed 25℃ and stirred for 3 hrs. The reaction was cooled to 0℃, water (0.84 mL) was dropped in the reaction solution under N₂ and created a lot of bubbles, then 15%aq. NaOH (0.84 mL) was added dropwise slowly at 0℃, after 5mins, water (2.5 mL) was dropped the reaction solution at 0℃, then Na₂SO₄ added the mixture and warmed to 25 ℃ stirred 15 mins. The mixture was filtered and the filter cake concentrated under reduced pressure to give 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propan-1-ol (7.1 g, crude) as a colorless oil. Synthesis of 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate

To a solution of 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propan-1-ol (28.6 g, 76.8 mmol, 1.0 eq) in DCM (200 mL) was added 6-bromohexanoic acid (22.5 g, 115 mmol, 1.5 eq) , EDCI (29.4 g, 154 mmol, 2.0 eq) and DMAP (469 mg, 3.84 mmol, 0.05 eq) at 0℃, then the mixture was stirred at 25℃ for 1.5 hrs. The reaction mixture was poured into water (100 mL) and extracted with EtOAc (100 mL, 50 mL, 30 mL) , The combined organic layers were washed with brine (30 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 100/1 to 0/1) to give 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 6-bromohexanoate (18 g, 32.7 mmol, 42.6%yield) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 5.6 Hz, 1H) , 4.14-4.17 (m, 2H) , 3.59-3.68 (m, 2H) , 3.40-3.49 (m, 3H) , 2.32 (t, J = 7.6 Hz, 2H) , 1.86-1.98 (m, 3H) , 1.48-1.68 (m, 10H) , 1.25-1.30 (m, 8H) , 1.08-1.17 (m, 6H) , 0.86-0.91 (m, 18H) .

To a solution of 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 6-bromohexanoate (6 g, 10.9 mmol, 1.0 eq) and 2-aminoethanol (6.67 g, 109 mmol, 6.60 mL, 10 eq) in EtOH (60 mL) was added at 25℃, then the mixture was stirred at 90℃ for 2 hrs. The reaction mixture was poured into water (100 mL) and extracted with EtOAc (100 mL, 50 mL, 30 mL) , The combined organic layers were washed with (brine 30 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to DCM/MeOH = 10/1) to give 3, 3-bis ( (3, 7- dimethyloctyl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (6 g, crude) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.58-4.63 (m, 3H) , 4.14 (t, J = 6.4 Hz, 2H) , 3.86 (t, J = 4.8 Hz, 2H) , 3.57-3.67 (m, 2H) , 3.43-3.50 (m, 2H) , 2.99-3.02 (m, 2H) , 2.87 (t, J = 8.0 Hz, 2H) , 2.32 (t, J = 7.6 Hz, 2H) , 1.92-1.97 (m, 2H) , 1.74-1.78 (m, 2H) , 1.51-1.68 (m, 8H) , 1.26-1.39 (m, 11H) , 1.09-1.17 (m, 6H) , 0.86-0.89 (m, 18H) .

Synthesis of Compound 030

To a solution of 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (6 g, 11.3 mmol, 1.0 eq) and 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 6-bromohexanoate (6.22 g, 11.3 mmol, 1.0 eq) in EtOH (60 mL) was added DIEA (2.93 g, 22.7 mmol, 3.94 mL, 2.0 eq) at 25℃, then the mixture was stirred at 90℃for 24 hrs. The reaction mixture was poured into water (50 mL) and extracted with EtOAc (50 mL, 30 mL, 20 mL) , The combined organic layers were washed with brine (20 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to EtOAc/MeOH = 5/1) to give Compound 030 (4 g, 3.96 mmol, 34.9%yield, 98.8%purity) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 5.6 Hz, 2H) , 4.15 (t, J = 6.4 Hz, 4H) , 3.57-3.66 (m, 5H) , 3.44-3.51 (m, 4H) , 2.50-2.66 (m, 5H) , 2.31 (t, J = 7.6 Hz, 4H) , 1.94 (q, J = 6.4 Hz, 4H) , 1.51-1.66 (m, 25H) , 1.28-1.36 (m, 22H) , 1.08-1.17 (m, 2H ) , 0.86-0.90 (m, 36H) .

Compound 031: bis (3, 3-bis ( (3, 7-dimethyloct-6-en-1-yl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Compound 031 was prepared analogously to compound 002 starting from 3, 7-dimethyloct-6-en-1-ol. Compound 030 (98.4%purity) was obtained as a yellow oil. ¹H NMR (400 MHz CDCl₃) δ 5.08-5.12 (m, 1H) , 4.59 (t, J = 5.6 Hz, 1H) , 4.14 (t, J = 6.8 Hz, 2H) , 3.59-3.65 (m, 3H) , 3.42-3.49 (m, 2H) , 2.92 (brs, 2H) , 2.67 (t, J = 5.2 Hz, 1H) , 2.56 (t, J = 7.6 Hz, 2H) , 2.29-2.33 (m, 2H) , 1.92-2.05 (m, 8H) , 1.59-1.71 (m, 19H) , 1.51-1.57 (m, 4H) , 1.30-1.41 (m, 9H) , 1.14-1.18 (m, 3H) , 0.89-0.91 (m, 7H) .

Compound 032: bis (3, 3-bis ( ( (E) -3, 7-dimethylocta-2, 6-dien-1-yl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Synthesis of 3, 3-bis ( ( (E) -3, 7-dimethylocta-2, 6-dien-1-yl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate

To a solution of 3, 3-bis ( ( (E) -3, 7-dimethylocta-2, 6-dien-1-yl) oxy) propan-1-ol (2.6 g, 7.13 mmol, 2.14 mL, 1.0 eq) in DCM (18 mL) was added 6-bromohexanoic acid (2.09 g, 10.7 mmol, 1.5 eq) , EDCI (2.05 g, 10.7 mmol, 1.5 eq) and DMAP (174 mg, 1.43 mmol, 0.2 eq) at 0℃, then the mixture was stirred at 25℃ for 1.5 hrs. The reaction mixture was poured into water (20 mL) and extracted with EtOAc (20 mL, 10 mL, 10 mL) , The combined organic layers were washed with brine (10 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 100/1 to 0/1) to give 3, 3-bis ( ( (E) -3, 7-dimethylocta-2, 6-dien-1-yl) oxy) propyl 6-bromohexanoate (1.9 g, 3.51 mmol, 49.1%yield) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 5.34-5.38 (m, 2H) , 5.08-5.12 (m, 2H) , 4.71 (t, J = 6.0 Hz, 1H) , 4.12-4.19 (m, 4H) , 4.03-4.07 (m, 2H) , 3.41 (t, J = 6.8 Hz, 2H) , 2.32 (t, J = 7.6 Hz, 2H) , 1.97-2.14 (m, 11H) , 1.84-1.92 (m, 2H) , 1.68-1.69 (m, 12H) , 1.61 (s, 6H) , 1.46-1.52 (m, 2H) , 1.13-1.37 (m, 2H) , 0.84-0.91 (m, 2H) . To a solution of 3, 3-bis ( ( (E) -3, 7-dimethylocta-2, 6-dien-1-yl) oxy) propyl 6-bromohexanoate (1.8 g, 3.32 mmol, 1.0 eq) and 2-aminoethanol (2.03 g, 33.2 mmol, 2.01 mL, 10 eq) in EtOH (18 mL) was added at 25℃, then the mixture was stirred at 90℃ for 12 hrs. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (10 mL, 5 mL, 3 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to DCM/MeOH = 10/1) to give 3, 3-bis ( ( (E) -3, 7-dimethylocta-2, 6-dien-1-yl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (0.94 g, 1.80 mmol, 54.2%yield) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 5.35 (t, J = 6.8 Hz, 2H) , 5.10 (t, J = 6.4 Hz, 2H) , 4.70 (t, J = 5.6 Hz, 1H) , 4.11-4.18 (m, 4H) , 4.02-4.07 (m, 2H) , 3.77 (t, J = 4.8 Hz, 2H) , 3.24 (brs, 3H) , 2.91 (t, J = 5.2 Hz, 2H) , 2.77 (t, J = 7.2 Hz, 2H) , 2.32 (t, J = 7.6 Hz, 2H) , 1.99-2.14 (m, 11H) , 1.61-1.68 (m, 22H) , 1.36-1.45 (m, 2H) .

Synthesis of compound 032

To a solution of 3, 3-bis ( ( (E) -3, 7-dimethylocta-2, 6-dien-1-yl) oxy) propyl 6-bromohexanoate (757 mg, 1.4 mmol, 1.0 eq) and 3, 3-bis ( ( (E) -3, 7-dimethylocta-2, 6-dien-1-yl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (731 mg, 1.4 mmol, 1.0 eq) in EtOH (12 mL) was added DIEA (359 mg, 2.77 mmol, 483 uL, 2.0 eq) at 25℃, then the mixture was stirred at 90 ℃ for 5 hrs. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to EtOAc/MeOH = 5/1) to give compound 032 (162 mg, 165 umol, 12%yield) as a colorless oil. ¹H NMR (400 MHz CDCl₃) 5.46-5.15 (m, 8H) , 4.63 (t, 2H) , 4.13-3.80 (m, 12H) , 3.75-3.68 (m, 2H) , 2.52-2.33 (m, 6H) , 2.32-2.01 (m, 20H) , 1.97-1.11 (m, 52H) .

Compound 033: bis (3, 3-bis ( (4-butyloctyl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Synthesis of 4-butyloctan-1-ol

Step 1: To a suspension of (3-hydroxypropyl) triphenylphosphonium bromide (2.20 g, 5.5 mmol, 1.1 eq) in THF (20 mL) was added n-BuLi (4.6 mL, 2.4 M in hexanes, 11.0 mmol, 2.2 eq) over 10 min at -10 ℃. The reaction mixture was stirred for 30 min and then TMSCl (0.7 mL, 5.5 mmol, 1.1 eq) was added. After stirring for another 30 min, a solution of 5-nonanone (746 mg, 5.0 mmol, 1.0 eq) in THF (10 mL) was added dropwise. The cold bath was removed and the mixture was refluxed overnight. The mixture was quenched with saturated NH₄Cl (10 mL) . The two layers were separated and the aqueous solution was extracted with CH₂Cl₂ (2 × 10 mL) . The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated. The crude material was dissolved in THF (10 mL) , a solution of TBAF (5.0 mL, 1.0 M in THF, 5.0 mmol, 1.0 eq) was added and stirred for 30 min at room temperature, The solvent was removed by rotary evaporation and the crude product was purified by flash column chromatography (petroleum ether, EtOAc = 20: 1) to afford alkene (221 mg, 1.2 mmol, 23%yield) .

Step 2: alkene (220 mg, 1.2 mmol) was dissolved in 7 mL DCM. After adding a catalytic amount (ca. 20 mg) of palladium hydroxide (20 wt%on carbon) , the flask was flushed three times with hydrogen. Then the hydrogen balloon was placed atop of the flask in which the suspension was stirred overnight. After the catalyst was removed by filtration, the solution was concentrated in vacuo and then recrystallized from acetone to afford 210 mg of alkane as a colorless liquid (95%yield) .

Synthesis of compound 033

Compound 033 was prepared analogously to compound 002 starting from 4-butyloctan-1-ol. Compound 033 was obtained as a colorless oil. ¹H NMR (400 MHz CDCl₃) δ 4.65 (t, 2H) , 4.16-3.80 (m, 4H) , 3.75-3.50 (m, 10H) , 2.51-2.35 (m, 6H) , 2.37-2.16 (m, 4H) , 2.01-1.11 (m, 84H) , 0.88-0.77 (m, 24H) .

Compound 034: bis (3, 3-bis ( (8-methylnonyl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Synthesis of 8-methylnonan-1-ol.

A 500 mL three-necked round bottom flask equipped with or magnetic stirrer, addition funnel and thermometer. A mixture of Mg (10.1 g, 414 mmol, 5.0 eq) and I₂ (420 mg, 1.66 mmol, 334 uL, 0.02 eq) in THF (60.0 mL) was added dropwise the solution of 1-bromo-2-methylpropane (56.7 g, 414 mmol, 45.0 mL, 5.0 eq) in THF (60.0 mL) at 30 ℃ and the reaction mixture was stirred at 30 ℃ for 30 mins. The mixture was added to 6-bromohexan-1-ol (15.0 g, 82.8 mmol, 10.9 mL, 1.0 eq) and CuCl₂LiCl₂ (0.1 M, 49.7 mL, 0.06 eq) . The mixture was stirred at 25 ℃ for 12 hrs under N₂ atmosphere. The mixture was added to the HCl (0.1 M, aq. 250 mL) . The residue was diluted with H₂O (160 mL) and extracted with EtOAc (300 mL, 200 mL) . The combined organic layers were washed with brine (50.0 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=50/1 to 30/1) . 8-methylnonan-1-ol (13.0 g, 82.1 mmol, 99.2%yield) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 3.64 (t, J = 6.8 Hz, 2 H) , 1.50 -1.59 (m, 3 H) , 1.27 -1.36 (m, 8 H) , 1.15 -1.18 (m, 2 H) , 0.87 (d, J = 6.8 Hz, 6 H) .

Synthesis of Compound 034

Compound 034 was prepared analogously to compound 002 starting from 8-methylnonan-1-ol. Compound 034 was obtained as a colorless oil. ¹H NMR (400 MHz CDCl₃) δ 4.66 (t, 2H) , 4.15-3.80 (m, 4H) , 3.75-3.50 (m, 10H) , 2.54-2.35 (m, 6H) , 2.33-2.13 (m, 4H) , 2. 01-1.11 (m, 68H) , 0.88-0.77 (m, 24H) .

Compound 035: bis (3, 3-bis ( ( (E) -non-2-en-1-yl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Compound 035 was prepared analogously to compound 002 starting from (E) -non-2-en-1-ol. ¹H NMR (400 MHz CDCl₃) δ 5.90-5.41 (m, 8H) , 4.66 (t, 2H) , 4.15-3.80 (m, 12H) , 3.75- 3.50 (m, 2H) , 2.58-2.34 (m, 6H) , 2.41-2.00 (m, 16H) , 1.97-1.11 (m, 44H) , 0.89-0.74 (m, 12H) .

Compound 036: bis (3, 3-bis ( ( (Z) -non-2-en-1-yl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Synthesis of 3, 3-bis ( ( (Z) -non-2-en-1-yl) oxy) propan-1-ol

To a solution of ethyl 3, 3-diethoxypropanoate (2.5 g, 13.1 mmol, 2.56 mL, 1.0 eq) in (Z) -non-2-en-1-ol (7.48 g, 52.6 mmol, 4 eq) was added KHSO₄ (89.5 mg, 657 umol, 38.6 uL, 0.05 eq) . The mixture was stirred at 80 ℃ for 12 hrs. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=1/0 to 80/1) . Ethyl 3, 3-bis ( ( (Z) -non-2-en-1-yl) oxy) propanoate (2.80 g, 7.32 mmol, 55.7%yield) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 5.51 -5.63 (m, 7 H) , 5.02 -5.06 (m, 1 H) , 4.61 -4.66 (m, 1 H) , 4.10 -4.20 (m, 6 H) , 3.99 -4.09 (m, 1 H) , 2.69 -2.72 (m, 2 H) , 2.04 -2.12 (m, 7 H) , 1.18 -1.48 (m, 37 H) , 0.89 (t, J=6.4 Hz, 11 H) .

To a solution of ethyl 3, 3-bis ( ( (Z) -non-2-en-1-yl) oxy) propanoate (700 mg, 1.83 mmol, 1.0 eq) in THF (4.20 mL) was added LAH (69.4 mg, 1.83 mmol, 1.0 eq) in portions during a period of 10 mins with stirring at 0 ℃ under N2, the reaction mixture considerable heat is developed during the reaction and a vigorous evolution of gas sets in at about 0 ℃. The reaction mixture was allowed to stir for 2 hrs at 20 ℃. The reaction mixture was quenched by addition water (0.07 mL) at 0℃, 15%NaOH with solvent (0.07 mL) and water (0.21 mL) . The combined organic layers were added MgSO₄, filtered and concentrated under reduced pressure to give a residue. The crude product on notebook page ET49878-391 (1.7 g) was combined to ET49878-390 for further purification. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=60/1 to 40/1) . 3, 3-bis ( ( (Z) -non-2-en-1-yl) oxy) propan-1-ol (1.70 g, 4.99 mmol, 56.2%yield) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 5.53 -5.61 (m, 4 H) , 4.76 (t, J = 5.6 Hz, 1 H) , 4.09 -4.20 (m, 4 H) , 3.74 -3.75 (m, 2 H) , 2.42 (t, J = 5.6 Hz, 1 H) , 2.04 -2.09 (m, 5 H) , 1.91 -1.95 (m, 2 H) , 1.24 -1.36 (m, 18 H) , 0.87 -0.90 (m, 6 H) .

Synthesis of 3, 3-bis ( ( (Z) -non-2-en-1-yl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate

To a solution of 3, 3-bis ( ( (Z) -non-2-en-1-yl) oxy) propan-1-ol (1.40 g, 4.11 mmol, 1.0 eq) and 6-bromohexanoic acid (1.20 g, 6.17 mmol, 1.5 eq) in DCM (10.4 mL) was added EDCI (1.18 g, 6.17 mmol, 1.5 eq) and DMAP (100 mg, 822 umol, 0.2 eq) with stirring at 10 ℃ under N₂ . The residue was diluted with water (10.0 mL) and extracted with EtOAc (20.0 mL *3) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=40/1 to 20/1) . 3, 3-bis ( ( (Z) -non-2-en-1-yl) oxy) propyl 6-bromohexanoate (1.70 g, 3.28 mmol, 79.9%yield) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 5.52 -5.59 (m, 4 H) , 4.71 (t, J = 6.0 Hz, 1 H) , 4.06 -4.18 (m, 6 H) , 3.41 (t, J = 6.8 Hz, 2 H) , 2.30 -2.34 (m, 2 H) , 2.04 -2.09 (m, 4 H) , 1.98-1.99 (m, 2 H) , 1.84 -1.92 (m, 2 H) , 1.64 -1.68 (m, 3 H) , 1.46 -1.50 (m, 3 H) , 1.28 -1.38 (m, 16 H) , 0.87 -0.90 (m, 6 H) .

To a solution of 3, 3-bis ( ( (Z) -non-2-en-1-yl) oxy) propyl 6-bromohexanoate (1.00 g, 1.93 mmol, 1.0 eq) in EtOH (50.0 mL) was added 2-aminoethanol (3.54 g, 58.0 mmol, 3.51 mL, 30 eq) under N₂. The reaction was stirred at 70 ℃ for 4 h. The residue was diluted with water (20.0 mL) and extracted with EtOAc (20.0 mL, 10.0 ml) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, DCM: MeOH=10/1 to 5/1) . 3,3-bis ( ( (Z) -non-2-en-1-yl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (700 mg, 1.41 mmol, 72.8%yield) was obtained as Black Brown oil. ¹H NMR (400 MHz, CDCl₃) δ 5.49 -5.62 (m, 4 H) , 4.74 -4.98 (m, 2 H) , 4.70 (t, J = 5.6 Hz, 2 H) , 4.05 -4.22 (m, 6 H) , 3.92 (m, 2 H) , 3.01 -3.12 (m, 2 H) , 2.88 -2.99 (m, 2 H) , 2.32 (t, J = 7.2 Hz, 2 H) , 1.94 -2.12 (m, 7 H) , 1.82 (t, J = 7.2 Hz, 2 H) , 1.61 -1.71 (m, 2 H) , 1.24 -1.47 (m, 19 H) , 0.85 -0.92 (m, 6 H) .

Synthesis of compound 036

Compound 036 was synthesized using 3, 3-bis ( ( (Z) -non-2-en-1-yl) oxy) propyl 6-bromohexanoate and 3, 3-bis ( ( (Z) -non-2-en-1-yl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate using the procedure described in Scheme 1. ¹H NMR (400 MHz CDCl₃) δ 5.95-5.41 (m, 8H) , 4.66 (t, 2H) , 4.15-3.80 (m, 12H) , 3.75-3.45 (m, 2H) , 2.58-2.34 (m, 6H) , 2.44-1.98 (m, 16H) , 1.98-1.09 (m, 44H) , 0.89-0.78 (m, 12H) .

Compound 037: bis (3, 3-bis ( ( (Z) -non-3-en-1-yl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Compound 037 was prepared analogously to compound 002 starting from (Z) -non-3-en-1-ol. Compound 037 was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 5.35 -5.50 (m, 4 H) , 4.63 (t, J = 5.6 Hz, 2 H) , 4.15 (t, J = 7.2 Hz, 4 H) , 3.57 -3.63 (m, 5 H) , 3.42 -3.47 (m, 5 H) , 2.43 -2.62 (m, 6 H) , 2.28 -2.35 (m, 13 H) , 2.01 -2.06 (m, 8 H) , 1.93 -1.97 (m, 4 H) , 1.41 -1.68 (m, 15 H) , 1.23 -1.39 (m, 29 H) , 0.89 (t, J = 6.4 Hz, 12 H) .

Compound 038: bis (3, 3-bis ( ( (2E, 6Z) -nona-2, 6-dien-1-yl) oxy) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Compound 038 was prepared analogously to compound 002 starting from (2E, 6Z) -nona-2, 6-dien-1-ol. Compound 038 was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 5.69 -5.75 (m, 4 H) , 5.54 -5.60 (m, 4 H) , 5.29 -5.42 (m, 8 H) , 4.70 (t, J = 5.6 Hz, 2 H) , 4.15 (t, J = 6.4 Hz, 4 H) , 4.03 -4.08 (m, 4 H) , 3.93 -3.98 (m, 4 H) , 3.55 (br s, 2 H) , 2.60 (br s, 2 H) , 2.47 (br s, 4 H) , 2.30 (t, J = 7.2 Hz, 4 H) , 2.07 -2.15 (m, 18 H) , 1.97 -2.05 (m, 12 H) , 1.59 -1.67 (m, 6 H) , 1.48 (br s, 4 H) , 1.26 -1.35 (m, 6 H) , 0.96 (t, J = 7.6 Hz, 12 H) .

Compound 039: bis (3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl) 6, 6'- ( (3-hydroxypropyl) azanediyl) dihexanoate

Compound 039 was prepared analogously to compound 030 but using 3-aminopropan-1-ol instead of 2-aminoethan-1-ol. Compound 039 was obtained as a yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 6.0 Hz, 2 H) , 4.14 (t, J = 6.0 Hz, 4 H) , 3.79 (t, J = 6.0 Hz, 2 H) , 3.55 -3.72 (m, 4 H) , 3.35 -3.46 (m, 4 H) , 2.55 -2.68 (m, 2 H) , 2.43 (t, J = 7.6 Hz, 3 H) , 2.31 (t, J = 7.6 Hz, 4 H) , 1.86 -1.98 (m, 4 H) , 1.47 -1.71 (m, 23 H) , 1.06 -1.46 (m, 32 H) , 0.89 (t, J = 6.0 Hz, 36 H) .

Compound 040: bis (3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl) 6, 6'- ( (3- ( (2- (methylamino) -3, 4-dioxocyclobut-1-en-1-yl) amino) propyl) azanediyl) dihexanoate

Synthesis of benzyl (3-aminopropyl) carbamate

A 500 mL three-necked round bottom flask equipped with magnetic stirrer, addition funnel and thermometer. To a solution of tert-butyl (3-aminopropyl) carbamate (20.0 g, 115 mmol, 20.0 mL, 1.0 eq) in THF (120 mL) and H₂O (80.0 mL) was added K₂CO₃ (23.8 g, 172 mmol, 1.5 eq) and CbzCl (29.4 g, 172 mmol, 24.5 mL, 1.5 eq) at 0 ℃. The mixture was stirred at 0-15 ℃ for 40 mins. The residue was diluted with water (100 mL) and extracted with EtOAc (100 mL, 50.0 mL, 25.0 mL) . The combined organic layers were washed with brine (20.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=20/1 to 10/1) . Benzyl tert-butyl propane-1, 3-diyldicarbamate (42.0 g, crude) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.29 -7.38 (m, 5 H) , 5.26 (s, 1 H) , 5.10 (s, 2 H) , 4.83 (s, 1 H) , 3.17 -3.27 (m, 4 H) , 1.60 -1.66 (m, 4 H) , 1.44 (s, 9 H) .

A 100 mL three-necked round bottom flask equipped with magnetic stirrer, addition funnel and thermometer. To a solution of benzyl tert-butyl propane-1, 3-diyldicarbamate (20.0 g, 64.9 mmol, 1.0 eq) in DCM (120 mL) was added TFA (92.4 g, 810.3 mmol, 60.0 mL, 12.5 eq) at 0 ℃. The mixture was stirred at 20 ℃ for 2 hrs. The reaction was quenched by the slow addition of ca. 200 mL of a saturated aqueous sodium bicarbonate solution at 0 ℃ and extracted with EtOAc/THF (2/1, 100 mL *5) . The organic layer was separated, washed with a saturated aqueous sodium bicarbonate solution, then the Organic phase was adjusted pH 7 to 8 by a 0.1N sodium hydroxide solution and then brine (20.0 mL) , dried, filtered and concentrated under reduced pressure to give a residue. Without purification. Benzyl (3-aminopropyl) carbamate (11.2 g, crude) was obtained as green oil. ¹H NMR (400 MHz, CDCl₃) δ 7.28 -7.41 (m, 5 H) , 5.38 (s, 1 H) , 5.05 -5.13 (m, 2 H) , 3.27 -3.28 (m, 2 H) , 2.92 (s, 2 H) , 2.75 -2.81 (m, 2 H) , 1.63 -1.68 (m, 2 H) .

Synthesis of compound 040-2

A thumb bottle with magnetic stirrer, addition funnel and thermometer. To a solution of 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 6-bromohexanoate (2.10 g, 3.83 mmol, 1.0 eq) and benzyl (3-aminopropyl) carbamate (3.99 g, 19.2 mmol, 5.0 eq) in EtOH (14.0 mL) was stirred at 50 ℃ for 12 hrs. The residue was diluted with water (10.0 mL) and extracted with EtOAc (50.0 mL, 25.0 mL) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, EtOAc: MeOH=1/0 to 60/1) . Compound 040-1 (0.82 g, 1.20 mmol, 31.3%yield) was obtained as yellow oil. A seal tube equipped with magnetic stirrer, addition funnel and thermometer.

To a solution of compound 040-1 (406 mg, 600 umol, 1.0 eq) in EtOH (4.00 mL) was added DIEA (120 mg, 900 umol, 161 uL, 1.5 eq) and 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 6-bromohexanoate (495 mg, 900 umol, 1.5 eq) . The mixture was stirred at 90 ℃ for 12 hrs. The residue was diluted with water (20.0 mL) and extracted with EtOAc (50.0 mL, 25.0 mL) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, DCM: MeOH=100/1 to 60/1) . Compound 040-2 (595 mg, 520 umol, 86.6%yield) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.35-7.21 (m, 5H) , 6.50 (s, 1H) , 5.05 (s, 2H) , 4.65 (t, 2H) , 4.11-3.80 (m, 4H) , 3.78-3.52 (m, 8H) , 3.10-3.01 (m, 2H) , 2.52-2.41 (m, 6H) , 2.39-2.17 (m, 4H) , 2.02-1.11 (m, 58H) , 0.88-0.74 (m, 36H) .

Synthesis of compound 040

A 100 mL three-necked round bottom flask equipped with magnetic stirrer, addition funnel and thermometer. To a solution of compound 040-2 (624 mg, 545 umol, 1.0 eq) in EtOH (5.00 mL) was added Pd/C (50.0 mg, 10%purity) under N₂. The suspension was degassed under vacuum and purged with H₂ several times. The mixture was stirred under H₂ 15 Psi at 20 ℃ for 72 hrs. The reaction mixture was filtered and the filter cake was washed by EtOH (30.0 mL) . Compound 040-3 was obtained as yellow oil. Compound 040-3 (529 mg, 523 umol, 1.0 eq) and 3, 4-dimethoxycyclobut-3-ene-1, 2-dione (112 mg, 785 umol, 1.5 eq) were taken up into a microwave tube in THF (4.10 mL) at 25 ℃. The mixture was stirred at 25 ℃ for 1 hrs. Then METHYLAMINE (2 M, 2.62 mL, 10 eq) was added at 25 ℃. The reaction stirred at 25 ℃ for 20 hours. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by column chromatography (SiO₂, DCM: MeOH=60/1 to 50/1) and then product was purified by prep-TLC (SiO₂, DCM: MeOH = 8: 1) . Compound 040 (145 mg, 130 umol, 24.8%yield) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.20 (s, 1H) , 7.05 (s, 1H) , 4.67 (t, 2H) , 4.13-3.81 (m, 4H) , 3.78-3.52 (m, 8H) , 3.31-3.12 (m, 2H) , 2.52-2.41 (m, 6H) , 2.39-2.17 (m, 4H) , 2.04-1.11 (m, 61H) , 0.88-0.74 (m, 36H) .

Compound 041: bis (3, 3-bis ( (5-methylhexyl) oxy) propyl) 6, 6'- ( (3- ( (2- (methylamino) -3, 4-dioxocyclobut-1-en-1-yl) amino) propyl) azanediyl) dihexanoate

Compound 041 was prepared analogously to compound 040 starting with 5-methylhexan-1-ol. ¹H NMR (400 MHz, CDCl₃) δ 7.21 (s, 1H) , 7.04 (s, 1H) , 4.67 (t, 2H) , 4.12-3.83 (m, 4H) , 3.79-3.51 (m, 8H) , 3.31-3.12 (m, 2H) , 2.52-2.41 (m, 6H) , 2.39-2.17 (m, 4H) , 2.01-1.17 (m, 49H) , 0.88-0.74 (m, 24H) .

Compound 042: bis (3, 3-dibutoxypropyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate

Synthesis of 3, 3-dibutoxypropan-1-ol

A 100 mL three-necked round bottom flask equipped with magnetic stirrer, addition funnel and thermometer. To a solution of ethyl propiolate (6.00 g, 61.2 mmol, 6.00 mL, 1.0 eq) and butan-1-ol (22.7 g, 306 mmol, 28.0 mL, 5.0 eq) in Tol. (36.0 mL) was added PtCl₂ (325 mg, 1.22 mmol, 0.02 eq) . The mixture was stirred at 80 ℃ for 13 hrs. The reaction solution was cooled to room temperature. The mixture was then filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC (SiO₂, Petroleum ether/Ethyl acetate=15/1) . Ethyl 3, 3-dibutoxypropanoate (8.70 g, 35.3 mmol, 57.7%yield) was obtained as yellow oil.

To a solution of ethyl 3, 3-dibutoxypropanoate (3.70 g, 15.0 mmol, 1.0 eq) in THF (37.0 mL) was added LAH (2.5 M, 6.61 mL, 1.1 eq) in portions during a period of 10 mins with stirring at 0 ℃ under N₂, the reaction mixture considerable heat is developed during the reaction and a vigorous evolution of gas sets in at about 0 ℃. The reaction mixture was allowed to stir for 2 hrs at 20 ℃. The reaction was cooled to 0℃, water (0.63 mL) was dropped in the reaction solution under N₂ and created a lot of bubbles, then 15%aq. NaOH (0.63 mL) was added dropwise slowly at 0℃; after 5 mins, water (1.80 mL) was dropped the reaction solution at 0℃, then Na₂SO₄ added the mixture and warmed to 25 ℃ stirred 15 mins. The mixture was filtered and the filter cake concentrated under reduced pressure to give a residue. 3, 3-dibutoxypropan-1-ol (2.30 g, 11.3 mmol, 75.0%yield) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.66 (t, J =5.2 Hz, 1 H) , 3.73 -3.76 (m, 2 H) , 3.62 -3.67 (m, 2 H) , 3.43 -3.49 (m, 2 H) , 1.86 -1.92 (m, 2 H) , 1.54 -1.61 (m, 4 H) , 1.36 -1.44 (m, 4 H) , 0.93 (t, J = 7.2 Hz, 6 H) .

Synthesis of 3, 3-dibutoxypropyl 6-bromohexanoate

A 100 mL three-necked round bottom flask equipped with magnetic stirrer, addition funnel and thermometer. To a solution of 3, 3-dibutoxypropan-1-ol (2.30 g, 11.3 mmol, 1.0 eq) and compound 29-1a (3.29 g, 16.9 mmol, 1.5 eq) in DCM (7.80 mL) was added EDCI (4.32 g, 22.5 mmol, 2.0 eq) and DMAP (275 mg, 2.25 mmol, 0.2 eq) . The mixture was stirred at 20 ℃ for 2 hrs. The residue was diluted with water (20.0 mL) and extracted with EtOAc (30.0 mL *3) . The combined organic layers were washed with brine (10mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether : Ethyl acetate=15/1to 10/1) . 3, 3-dibutoxypropyl 6-bromohexanoate (4.20 g, 11.0 mmol, 97.8%yield) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 6.0 Hz, 1 H) , 4.15 (t, J = 6.8 Hz, 2 H) , 3.57 -3.61 (m, 2 H) , 3.39 -3.46 (m, 4 H) , 2.32 (t, J = 7.2 Hz, 2 H) , 1.84 -1.96 (m, 4 H) , 1.64 -1.68 (m, 2 H) , 1.48 -1.57 (m, 11 H) , 0.92 (t, J = 7.6 Hz, 6 H) .

Synthesis of 3, 3-dibutoxypropyl 6- ( (2-hydroxyethyl) amino) hexanoate

A 100 mL three-necked round bottom flask equipped with magnetic stirrer, addition funnel and thermometer. To a solution of 3, 3-dibutoxypropyl 6-bromohexanoate (3.00 g, 7.87 mmol, 1.0 eq) in EtOH (30.0 mL) was added 2-aminoethanol (4.81 g, 78.7 mmol, 4.75 mL, 10 eq) . The mixture was stirred at 75 ℃ for 2 hrs. The residue was diluted with water (30.0 mL) and extracted with EtOAc (20.0 mL *2) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, EtOAc : Methanol=10/1 to 0/1) . 3, 3- dibutoxypropyl 6- ( (2-hydroxyethyl) amino) hexanoate (1.90 g, 5.26 mmol, 66.8%yield) and Compound 042 (100 mg, crude) was obtained as yellow oil.

Synthesis of Compound 042

A 100 mL three-necked round bottom flask equipped with magnetic stirrer, addition funnel and thermometer. To a solution of 3, 3-dibutoxypropyl 6- ( (2-hydroxyethyl) amino) hexanoate (0.60 g, 1.66 mmol, 1.0 eq) and 3, 3-dibutoxypropyl 6-bromohexanoate (759 mg, 1.99 mmol, 1.2 eq) in EtOH (6.00 mL) was added DIEA (322 mg, 2.49 mmol, 434 μL, 1.5 eq) . The mixture was stirred at 90 ℃ for 12 hrs. The residue was diluted with water (5.00 mL) and extracted with EtOAc (10.0 mL *3) . The combined organic layers were washed with brine (5.00 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=1/1 to 0/1) . Compound 042 (275 mg, 415 μmol, 25.0%yield, 100%purity) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 5.6 Hz, 2 H) , 4.15 (t, J = 6.8 Hz, 4 H) , 3.57 -3.62 (m, 6 H) , 3.40 -3.46 (m, 4 H) , 2.50 -2.61 (m, 6 H) , 2.30 (t, J = 7.2 Hz, 4 H) , 1.92 -1.97 (m, 4 H) , 1.48 -1.68 (m, 17 H) , 1.30 -1.43 (m, 13 H) , 0.92 (t, J = 7.2 Hz, 12 H) .

Compound 043: 3, 3-dibutoxypropyl 8- ( (6- (3, 3-dibutoxypropoxy) -6-oxohexyl) (2-hydroxyethyl) amino) octanoate

Synthesis of 3, 3-dibutoxypropyl 8-bromooctanoate

A 100 mL three-necked round bottom flask equipped with magnetic stirrer, addition funnel and thermometer. To a solution of 3, 3-dibutoxypropan-1-ol (2.50 g, 12.2 mmol, 1.0 eq) and 8-bromooctanoic acid (4.10 g, 18.4 mmol, 1.5 eq) in DCM (15.0 mL) was added EDCI (4.69 g, 24.5 mmol, 2.0 eq) and DMAP (299 mg, 2.45 mmol, 0.2 eq) . The mixture was stirred at 20 ℃ for 12 hrs. The residue was diluted with water (20.0 mL) and extracted with EtOAc (30.0 mL *3) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=15/1to 10/1) . 3, 3-dibutoxypropyl 8-bromooctanoate (1.80 g, 4.40 mmol, 35.9%yield) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 6.0 Hz, 1 H) , 4.15 (t, J = 6.8 Hz, 2 H) , 3.59 -3.61 (m, 2 H) , 3.39 -3.46 (m, 4 H) , 2.30 (t, J = 7.6 Hz, 2 H) , 1.94 -1.96 (m, 2 H) , 1.86 -1.89 (s, 2 H) , 1.54 -1.65 (m, 9 H) , 1.33 -1.43 (m, 9 H) , 0.93 (t, J = 7.6 Hz, 6 H) .

Synthesis of Compound 043

A 40 mL seal tube with magnetic stirrer, addition funnel and thermometer. To a solution of 3, 3-dibutoxypropyl 6- ( (2-hydroxyethyl) amino) hexanoate (0.60 g, 1.66 mmol, 1.0 eq) and 3, 3-dibutoxypropyl 8-bromooctanoate (815 mg, 1.99 mmol, 1.2 eq) in EtOH (6.00 mL) was added DIEA (322 mg, 2.49 mmol, 434 μL, 1.5 eq) . The mixture was stirred at 90 ℃ for 12 hrs. The residue was diluted with water (5.00 mL) and extracted with EtOAc (10.0 mL *3) . The combined organic layers were washed with brine (5.00 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=1/1 to 0/1) . Compound 043 (200 mg, 290 μmol, 17.5%yield, 100%purity) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 5.6 Hz, 2 H) , 4.14 (t, J = 6.4 Hz, 4 H) , 3.53 -3.62 (m, 6 H) , 3.40 -3.46 (m, 4 H) , 2.45 -2.60 (m, 6 H) , 2.27 -2.32 (m, 4 H) , 1.92 -1.97 (m, 4 H) , 1.52 -1.65 (m, 17 H) , 1.31 -1.47 (m, 20 H) , 0.92 (t, J=7.2 Hz, 12 H) .

Compound 044: 3, 3-dipropoxypropyl 8- ( (6- (3, 3-bis ( (5-methylhexyl) oxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) octanoate

Synthesis of 3, 3-dipropoxypropan-1-ol

To a solution of ethyl 3, 3-dipropoxypropanoate (3.40 g, 15.6 mmol, 1.0 eq) in THF (34.0 mL) was added LAH (650 mg, 17.1 mmol, 1.1 eq) in portions during a period of 10 mins with stirring at 0 ℃ under N₂, the reaction mixture considerable heat is developed during the reaction and a vigorous evolution of gas sets in at about 0 ℃. The reaction mixture was allowed to stir for 2 hrs at 20 ℃. The reaction was cooled to 0℃, water (0.65 mL) was dropped in the reaction solution under N₂ and created a lot of bubbles, then 15%aq. NaOH (0.65 mL) was added dropwise slowly at 0 ℃; after 5mins, water (1.95 mL) was dropped the reaction solution at 0℃, then Na₂SO₄ added the mixture and warmed to 25 ℃ stirred 15 mins. The mixture was filtered and the filter cake concentrated under reduced pressure to give a residue. 3, 3-dipropoxypropan-1-ol (2.50 g, crude) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.67 (t, J = 5.2 Hz, 1 H) , 3.73 -3.76 (m, 2 H) , 3.58 -3.64 (m, 2 H) , 3.40 -3.45 (m, 2 H) , 1.85 -1.92 (m, 2 H) , 1.57 -1.66 (m, 4 H) , 0.94 (t, J = 7.6 Hz, 6 H) .

Synthesis of 3, 3-dipropoxypropyl 8-bromooctanoate

A 100 mL three-necked round bottom flask equipped with magnetic stirrer, addition funnel and thermometer. To a solution of 3, 3-dipropoxypropan-1-ol (2.50 g, 14.2 mmol, 1.0 eq) and 8-bromooctanoic acid (4.75 g, 21.3 mmol, 1.5 eq) in DCM (15.0 mL) was added EDCI (5.44 g, 28.4 mmol, 2.0 eq) and DMAP (347 mg, 2.84 mmol, 0.2 eq) . The mixture was stirred at 20 ℃ for 12 hrs. The residue was diluted with water (20.0 mL) and extracted with EtOAc (30.0 mL *3) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=15/1 to 10/1) . 3, 3-dipropoxypropyl 8-bromooctanoate (4.50 g, 11.8 mmol, 83.2%yield) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.61 (t, J = 6.00 Hz, 1 H) , 4.16 (t, J = 6.4Hz, 2 H) , 3.53 -3.59 (m, 2 H) , 3.37 -3.42 (m, 4 H) , 2.30 (t, J = 7.2 Hz, 2 H) , 1.93 -1.98 (m, 2 H) , 1.84 -1.87 (m, 2 H) , 1.55 -1.64 (m, 7 H) , 1.42 -1.44 (m, 2 H) , 1.32 -1.35 (m, 4 H) , 0.94 (t, J = 7.6 Hz, 6 H) .

Synthesis of compound 044

To a solution of 3, 3-dipropoxypropyl 8- ( (2-hydroxyethyl) amino) octanoate (770 mg, 1.73 mmol, 1.0 eq) and 3, 3-bis ( (5-methylhexyl) oxy) propyl 6-bromohexanoate (988 mg, 2.59 mmol, 1.5 eq) in EtOH (10 mL) was added DIEA (447 mg, 3.46 mmol, 602 μL, 2.0 eq) at 25℃, then the mixture was stirred at 90℃ for 24 hrs. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (20 mL, 10 mL, 5 mL) , The combined organic layers were washed with (brine 5 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to EtOAc/MeOH = 5/1) to give compound 044 (290 mg, 388 μmol, 22.5%yield) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.59-4.63 (m, 2H) , 4.13-4.17 (m, 4H) , 3.56-3.62 (m, 6H) , 3.39-3.44 (m, 4H) , 2.64 (brs, 2H) , 2.52 (brs, 4H) , 2.28-2.33 (m, 4H) , 1.95-1.98 (m, 4H) , 1.51-1.66 (m, 19H) , 1.31-1.35 (m, 13H) , 1.18-1.22 (m, 4H) , 0.94 (t, J = 7.6 Hz, 6H) , 0.86-0.89 (m, 12H) .

Compound 045: 3, 3-dibutoxypropyl 8- ( (6- (3, 3-bis ( (3, 7-dimethyloctyl) oxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) octanoate

A 100 mL three-necked round bottom flask equipped with magnetic stirrer, addition funnel and thermometer. To a solution of 3, 3-dibutoxypropyl 8- ( (2-hydroxyethyl) amino) octanoate (0.60 g, 1.54 mmol, 1.0 eq) and 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 6-bromohexanoate (931 mg, 1.69 mmol, 1.1 eq) in EtOH (6.00 mL) was added DIEA (298 mg, 2.31 mmol, 402 μL, 1.5 eq) . The mixture was stirred at 90 ℃ for 12 hrs. The residue was diluted with water (5.00 mL) and extracted with EtOAc (10.0 mL *3) . The combined organic layers were washed with brine (5.00 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=1/1 to 0/1) . Compound 045 (0.350 g, 390 μmol, 25.3%yield, 95.6%purity) was obtained as yellow oi. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 5.6 Hz, 2 H) , 4.14 (t, J = 6.8 Hz, 4 H) , 3.42 -3.64 (m, 10 H) , 2.57 (t, J = 5.2 Hz, 2 H) , 2.41 -2.46 (m, 4 H) , 2.27 -2.32 (m, 4 H) , 1.92 -1.97 (m, 4 H) , 1.04 -1.67 (m, 47 H) , 0.86 -0.94 (m, 24 H) .

Compound 046: 3, 3-bis (pentyloxy) propyl 8- ( (6- (3, 3-bis ( (3, 7-dimethyloctyl) oxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) octanoate

Compound 046 was prepared using similar procedure as Compound 045 starting with pentan-1-ol. Compound 046 was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 5.6 Hz, 1 H) , 4.14 (t, J = 6.8 Hz, 4 H) , 3.39 -3.66 (m, 10 H) , 2.56 -2.58 (m, 2 H) , 2.44 -2.46 (m, 4 H) , 2.27 -2.32 (m, 4 H) , 1.92 -1.97 (m, 4 H) , 1.06 -1.71 (m, 50 H) , 0.86 -0.92 (m, 21 H) .

Compound 047: 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 6- ( (6- (3, 3-bis (isopentyloxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) hexanoate

Synthesis of 3, 3-bis (isopentyloxy) propan-1-ol

To a solution of ethyl propiolate (5.00 g, 50.9 mmol, 5.00 mL, 1.0 eq) and 3-methylbutan-1-ol (22.4 g, 254 mmol, 27.7 mL) in toluene (30 mL) was added PtCl₂ (271 mg, 1.02 mmol) at 25℃. Then the mixture was stirred at 80℃ for 24 hrs under N₂ atmosphere. The reaction solution was cooled to 25 ℃. Then the mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (80 gSilica Flash Column, Eluent of 0～2%Ethyl acetate/Petroleum ether gradient @50 mL/min) to give ethyl 3, 3-bis (isopentyloxy) propanoate (6.80 g, 48.6%yield) as colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.91-4.97 (m, 1H) , 4.15 (dd, J = 3.60 Hz, 7.20 Hz, 2H) , 3.58-3.68 (m, 2H) , 3.46-3.55 (m, 2H) , 2.66 (d, J = 5.88 Hz, 2H) , 1.61-1.75 (m, 2H) , 1.38-1.49 (m, 4H) , 1.24-1.29 (m, 3H) , 0.90 (d, J = 6.72 Hz, 12H)

To a solution of 3, 3-bis (isopentyloxy) propanoate (1.00 g, 3.64 mmol in THF (10 mL) was added dropwise LAH (2.5 M, 1.60 mL) at 0 ℃ under N₂ atmosphere. Then the reaction mixture was stirred at 25℃ for 3 hrs. The reaction mixture was cooled to 0 ℃, H₂O (0.15 mL) was dropped in the reaction solution under N₂ and created a lot of bubbles, then 15%aq. NaOH (0.15 mL) was added dropwise slowly at 0 ℃; After 5 mins, H₂O (0.45 mL) was dropped the reaction solution at 0 ℃, then Na₂SO₄ added the mixture and warmed to 25 ℃ stirred 15 mins. The mixture was filtered and the filtrate concentrated under reduced pressure to give a residue 3, 3-bis (isopentyloxy) propan-1-ol (790 mg, 93.2%yield) as colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.63-4.68 (m, 1H) , 3.74 (t, J = 5.44 Hz, 2H) , 3.62-3.71 (m, 2H) , 3.44-3.52 (m, 2H) , 2.41-2.69 (m, 1H) , 1.84-1.94 (m, 2H) , 1.61-1.76 (m, 2H) , 1.44-1.52 (m, 4H) , 0.89-0.96 (m, 12H)

Synthesis of 3, 3-bis (isopentyloxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate

To a solution of 3, 3-bis (isopentyloxy) propan-1-ol (790 mg, 3.40 mmol) and 6-bromohexanoic acid (994 mg, 5.10 mmol) in DCM (5.0 mL) was added EDCI (1.30 g, 6.80 mmol) and DMAP (83.07 mg, 679 μmol) . The mixture was stirred at 20 ℃ for 1.5 hrs. The residue was diluted with water (20 mL) and extracted with DCM (25 mL x 3) . The combined organic layers were washed with brine (20 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (4 gSilica Flash Column, Eluent of 0～2%Ethyl acetate/Petroleum ether gradient @15 mL/min) to give 3, 3-bis (isopentyloxy) propyl 6-bromohexanoate (890 mg, 63.9%yield) as colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.56-4.63 (m, 1H) , 4.15 (t, J = 6.52 Hz, 2H) , 3.58-3.68 (m, 2H) , 3.39-3.50 (m, 4H) , 2.33 (t, J = 7.44 Hz, 2H) , 1.85-1.98 (m, 4H) , 1.63-1.75 (m, 4H) , 1.41-1.52 (m, 6H) , 0.91 (dd, J = 1.16 Hz, 6.68 Hz, 12H)

To a solution of 3, 3-bis (isopentyloxy) propyl 6-bromohexanoate (890 mg, 2.17 mmol) in EtOH (30 mL) was added 2-aminoethanol (1.33 g, 21.7 mmol, 1.31 mL) . The mixture was stirred at 75 ℃ for 2 hrs. TLC (Petroleum ether: Ethyl acetate=10: 1, R_f = 0.04) indicated the 3, 3-bis (isopentyloxy) propyl 6-bromohexanoate was consumed complete. The residue was diluted with water (30 mL) and extracted with EtOAc (20 mL x 2) . The combined organic layers were washed with brine (20 mL) , dried over, filtered and concentrated under reduced pressure to give 3, 3-bis (isopentyloxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (760 mg, 89.7%yield) as colorless oil.

Synthesis of Compound 047

To a solution of 3, 3-bis (isopentyloxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (324 mg, 833 μmol) and 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 6-bromohexanoate (550 mg, 1.00 mmol) in EtOH (3 mL) was added DIEA (161 mg, 1.25 mmol, 217 μL) . The mixture was stirred at 90 ℃ for 48 hrs. The residue was diluted with water (20 mL) and extracted with EtOAc (20 mL x 3) . The combined organic layers were washed with brine (20 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (4 gSilica Flash Column, Eluent of 0～4%Dichloromethane: Methanol gradient @15 mL/min) . Compound 047 (220 mg, 244 μmol, 38.2%yield, 95.46%purity) was obtained as colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.59 (t, J =5.6 Hz, 2H) , 4.14 (t, J=6.6 Hz, 4H) , 3.67-3.80 (m, 2H) , 3.56-3.67 (m, 4H) , 3.41-3.51 (m, 4H) , 2.65-2.88 (m, 5H) , 2.32 (t, J = 7.2 Hz, 4H) , 1.95 (q, J = 6.2 Hz, 4H) 1.44-1.75 (m, 20H) , 1.18-1.43 (m, 14H) 1.09-1.17 (m, 6H) , 0.81-0.97 (m, 30H)

Compound 048: 3, 3-dibutoxypropyl 8- ( (6- (3, 3-bis (octyloxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) octanoate

A 40.0 mL seal tube with magnetic stirrer, addition funnel and thermometer. To a solution of 3, 3-bis (octyloxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (0.70 g, 1.48 mmol, 1.0 eq) and 3, 3-dibutoxypropyl 8-bromooctanoate (726 mg, 1.77 mmol, 1.2 eq) in EtOH (7.00 mL) was added DIEA (286 mg, 2.22 mmol, 386 μL, 1.5 eq) . The mixture was stirred at 90 ℃ for 12 hrs. The residue was diluted with water (5.00 mL) and extracted with EtOAc (10.0 mL *3) . The combined organic layers were washed with brine (5.00 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=1/1 to 0/1) . Compound 048 (0.350 g, 436 μmol, 29.5%yield, 100%purity) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 6.0 Hz, 2 H) 4.14 (t, J = 6.8 Hz, 4 H) 3.60 (m, 2 H) 3.55 -3.61 (m, 3 H) 3.40 -3.44 (m, 4 H) 2.60 (br s, 2 H) 2.47 (br s, 4 H) 2.27 -2.32 (m, 4 H) 1.92 -1.97 (m, 4 H) 1.52 -1.67 (m, 13 H) 1.28 -1.46 (m, 38 H) 0.87 -0.94 (m, 12 H) .

Compound 049: 3, 3-dipropoxypropyl 8- ( (6- (3, 3-bis (octyloxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) octanoate

A 100 mL three-necked round bottom flask equipped with magnetic stirrer, addition funnel and thermometer. To a solution of 3, 3-bis (octyloxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (0.70 g, 1.48 mmol, 1.0 eq) and 3, 3-dipropoxypropyl 8-bromooctanoate (676 mg, 1.77 mmol, 1.2 eq) in EtOH (7.00 mL) was added DIEA (286 mg, 2.22 mmol, 386 μL, 1.5 eq) . The mixture was stirred at 90 ℃ for 12 hrs. The residue was diluted with water (5.00 mL) and extracted with EtOAc (10.0 mL *3) . The combined organic layers were washed with brine (5.00 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=1/1 to 0/1) . Compound 049 (0.350 g, 452 μmol, 30.6%yield, 100%purity) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.58 -4.63 (m, 2 H) , 4.13 -4.17 (m, 4 H) , 3.53 -3.59 (m, 6 H) , 3.38 -3.43 (m, 4 H) , 2.61 (br s, 2 H) , 2.47 (br s, 4 H) , 2.27 -2.32 (m, 4 H) , 1.92 -1.98 (m, 4 H) , 1.53 -1.66 (m, 14 H) , 1.46 -1.47 (m, 3 H) , 1.28 -1.35 (m, 30 H) , 0.87 -0.95 (m, 12 H) .

Compound 050: 3, 3-dipropoxypropyl 8- ( (6- (3, 3-bis ( (3, 7-dimethyloctyl) oxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) octanoate

A 40 mL seal tube with magnetic stirrer, addition funnel and thermometer. To a solution of 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (0.80 g, 1.51 mmol, 1.0 eq) and 3, 3-dipropoxypropyl 8-bromooctanoate (691 mg, 1.81 mmol, 1.2 eq) in EtOH (8.00 mL) was added DIEA (234 mg, 1.81 mmol, 316 μL, 1.2 eq) . The mixture was stirred at 90 ℃ for 12 hrs. The residue was diluted with water (5.00 mL) and extracted with EtOAc (10.0 mL *3) . The combined organic layers were washed with brine (5.00 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=1/1 to 0/1) . Compound 050 (0.50 g, 602 μmol, 39.9%yield, 100%purity) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.58 -4.63 (m, 2 H) , 4.13 -4.17 (m, 4 H) , 3.54 -3.66 (m, 6 H) , 3.37 -3.50 (m, 4 H) , 2.37 -2.70 (m, 6 H) , 2.28 -2.32 (m, 4 H) , 1.92 -1.98 (m, 4 H) , 1.46 -1.68 (m, 20 H) , 1.19 -1.41 (m, 19 H) , 1.08 -1.19 (m, 6 H) , 0.94 (t, J = 7.6 Hz, 6 H) , 0.86 -0.89 (m, 18 H) .

Compound 051: 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 8- ( (6- (3, 3-bis ( (3, 7-dimethyloctyl) oxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) octanoate

Compound 051 was prepared using similar procedure as Compound 045 but using 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propan-1-ol instead of 3, 3-dibutoxypropan-1-ol. Compound 051 was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.62 (t, J = 5.8 Hz, 2 H) , 4.09 (t, J = 6.4 Hz, 4 H) , 3.51 -3.82 (m, 6 H) , 3.41 -3.48 (m, 4 H) , 2.41 -2.64 (m, 6 H) , 1.88 -2.17 (m, 5 H) , 1.47 -1.71 (m, 25 H) , 1.05 -1.46 (m, 34 H) , 0.88 (t, J = 6.0 Hz, 36 H) .

Compound 052: 3-methylhexyl 8- ( (6- (3, 3-bis ( (3-methyloctyl) oxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) octanoate

Synthesis of 3-methylhexan-1-ol

Step 1: To a solution of NaH (3.48 g, 87.1 mmol, 60%purity, 1.5 eq) in THF (30.0 mL) was added ethyl 2- (diethoxyphosphoryl) acetate (19.5 g, 87.1 mmol, 17.3 mL, 1.5 eq) in dropwise at 0 ℃, The reaction is warmed at 20 ℃ and stirred for 1 h until there is no bubbles formed, and then add pentan-2-one (5.00 g, 58.1 mmol, 6.23 mL, 1.0 eq) . The reaction was stirred at 75 ℃ for 12 h. The two reactions were combined to work up. The reaction mixture was quenched by NH₄Cl (aq) (20.0 mL) and extracted with EtOAc (20.0 mL, 10.0 mL) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=100/1 to 80/1) . Ethyl (E) -3-methylhex-2-enoate (15.0 g, 96.0 mmol, 82.7%yield) was obtained as yellow oil.

Step 2: To a solution of Pd/C (1.50 g, 1.41 mmol, 10%purity, 1.47e-2 eq) in EtOH (210 mL) was added ethyl (E) -3-methylhex-2-enoate (15.0 g, 96.0 mmol, 1.0 eq) . The reaction was degassed and purged by H₂ several times, stirred at 25 ℃ for 12 h under H₂ (50 psi) . The reaction mixture was filtered, the filtrate cake was washed with THF (200 mL) and concentrated under reduced pressure to give a residue. Without purification. Ethyl 3-methylhexanoate (9.00 g, 56.9 mmol, 59.2%yield) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.10 -4.16 (m, 2 H) , 2.26 -2.31 (m, 1 H) , 2.06 -2.12 (m, 1 H) , 1.96 -1.99 (m, 1 H) , 1.14 -1.41 (m, 8 H) , 0.88 -0.94 (m, 6 H) .

Step 3: To a solution of ethyl 3-methylhexanoate (9.00 g, 56.9 mmol, 1.0 eq) in THF (54.0 mL) was added LAH (2.5 M, 25.0 mL, 1.1 eq) in portions during a period of 20 mins with stirring at 0 ℃ under N₂, the reaction mixture considerable heat is developed during the reaction and a vigorous evolution of gas sets in at about 0 ℃. The reaction mixture was allowed to stir for 2 hrs at 20 ℃. The reaction was cooled to 0 ℃, water (2.37 mL) was dropped in the reaction solution under N₂ and created a lot of bubbles, then 15%aq. NaOH (2.37 mL) was added dropwise slowly at 0 ℃; after 5 mins, water (7.11 mL) was dropped the reaction solution at 0℃, then Na₂SO₄ added the mixture and warmed to 20 ℃ stirred 15 mins. The mixture was filtered and the filtrate concentrated under reduced pressure to give a residue. 3-methylhexan-1-ol (6.40 g, crude) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 3.66 -3.70 (m, 2 H) , 1.58 -1.63 (m, 2 H) , 1.22 -1.42 (m, 5 H) , 1.11 -1.17 (m, 1 H) , 0.87 -0.91 (m, 6 H) .

Synthesis of 3-methylhexyl 8- ( (2-hydroxyethyl) amino) octanoate

A 100 mL three-necked round bottom flask equipped with magnetic stirrer, addition funnel and thermometer. To a solution of 3-methylhexan-1-ol (3.00 g, 25.8 mmol, 1.0 eq) and 8-bromooctanoic acid (8.64 g, 38.7 mmol, 1.5 eq) in EtOH (18.0 mL) was added EDCI (7.42 g, 38.7 mmol, 1.5 eq) and DMAP (631 mg, 5.16 mmol, 0.2 eq) . The mixture was stirred at 20 ℃ for 12 hrs. The residue was diluted with water (20.0 mL) and extracted with EtOAc (25.0 mL *3) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=80/1 to 60/1) . 3-methylhexyl 8-bromooctanoate (6.30 g, 19.6 mmol, 76.0%yield) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.08 -4.13 (m, 2 H) , 3.40 (t, J = 6.8 Hz, 2 H) , 2.29 (t, J = 7.6 Hz, 2 H) , 1.82 -1.87 (m, 2 H) , 1.50 -1.69 (m, 4 H) , 1.24 -1.46 (m, 11 H) , 1.15 -1.24 (m, 1 H) , 0.87 -0.90 (m, 6 H) .

A 100 mL three-necked round bottom flask equipped with magnetic stirrer, addition funnel and thermometer. To a solution of 3-methylhexyl 8-bromooctanoate (1.00 g, 3.11 mmol, 1.0 eq) in EtOH (20.0 mL) was added 2-aminoethanol (5.70 g, 93.4 mmol, 5.64 mL, 30 eq) . The mixture was stirred at 75 ℃ for 1 hrs. The residue was diluted with water (20.0 mL) and extracted with EtOAc (20.0 mL, 10.0 mL) . The combined organic layers were washed with brine (10.0 x 3 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. 3-methylhexyl 8- ( (2-hydroxyethyl) amino) octanoate (0.90 g, 2.99 mmol, 95.9%yield) was obtained as yellow oil.

Synthesis of compound 052

A 40 mL seal tube equipped with magnetic stirrer, addition funnel and thermometer. To a solution of 3-methylhexyl 8- ( (2-hydroxyethyl) amino) octanoate (0.40 g, 1.33 mmol, 1.0 eq) and 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 6-bromohexanoate (802 mg, 1.46 mmol, 1.1 eq) in EtOH (4.00 mL) was added DIEA (257 mg, 1.99 mmol, 347 μL, 1.5 eq) . The mixture was stirred at 90 ℃ for 24 hrs. The residue was diluted with water (5.00 mL) and extracted with EtOAc (10.0 mL *3) . The combined organic layers were washed with brine (5.00 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=1/1 to 0/1) . Compound 052 (0.30 g, 389 μmol, 29.4%yield, 100%purity) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 6.4 Hz, 1 H) , 4.08 -4.16 (m, 4 H) , 3.59 -3.63 (m, 2 H) , 3.41 -3.54 (m, 4 H) , 2.57 (t, J = 5.2 Hz, 2 H) , 2.42 -2.46 (m, 4 H) , 2.27 -2.32 (m, 4 H) , 1.92 -1.97 (m, 2 H) , 1.51 -1.67 (m, 13 H) , 1.22 -1.49 (m, 27 H) , 1.1 -1.17 (m, 7 H) , 0.86 -0.91 (m, 24 H) .

Compound 053: 3-methylheptyl 8- ( (6- (3, 3-bis ( (3, 7-dimethyloctyl) oxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) octanoate

Compound 053 was prepared analogously to compound 052 starting with 3-methylheptan-1-ol. Compound 053 was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ4.60 (t, J = 5.6 Hz, 1 H) , 4.05 -4.16 (m, 4 H) , 3.59 -3.66 (m, 2 H) , 3.43 -3.54 (m, 4 H) , 2.56 -2.58 (m, 2 H) , 2.42 -2.46 (m, 4 H) , 2.27 -2.32 (m, 4 H) , 1.92 -1.97 (m, 2 H) , 1.49 -1.66 (m, 12 H) , 1.21 -1.48 (m, 27 H) , 1.10 -1.17 (m, 7 H) , 0.84 -0.93 (m, 23 H) , 0.86 -0.91 (m, 1 H) .

Compound 054: 3, 7-dimethyloctyl 8- ( (6- (3, 3-bis ( (3, 7-dimethyloctyl) oxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) octanoate

Compound 054 was prepared analogously to compound 052 but using 3, 7-dimethyloctan- 1-ol instead of 3-methylhexan-1-ol. Compound 054 was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.59 (t, J = 5.6 Hz, 1 H) , 4.09 -4.16 (m, 4 H) , 3.41 -3.66 (m, 6 H) , 2.56 -2.59 (m, 2 H) , 2.44 -2.45 (m, 4 H) , 2.27 -2.32 (m, 4 H) , 1.92 -1.97 (m, 2 H) , 1.01 -1.80 (m, 49 H) , 0.77 -0.91 (m, 27 H) .

Compound 055: 3-methylpentyl 8- ( (6- (3, 3-bis ( (3, 7-dimethyloctyl) oxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) octanoate

Compound 055 was prepared analogously to compound 052 but using 3-methylpentan-1-ol instead of 3-methylhexan-1-ol. Compound 055 was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.59 (t, J = 5.6 Hz, 1 H) , 4.08 -4.16 (m, 4 H) , 3.59 -3.64 (m, 2 H) , 3.43 -3.53 (m, 4 H) , 2.56 (t, J = 5.6 Hz, 2 H) , 2.41 -2.46 (m, 4 H) , 2.27 -2.32 (m, 4 H) , 1.92 -1.97 (m, 2 H) , 1.27 -1.69 (m, 36 H) , 1.08 -1.24 (m, 7 H) , 0.86 -0.91 (m, 24 H) .

Compound 056: 2-methylpentyl 8- ( (6- (3, 3-bis ( (3, 7-dimethyloctyl) oxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) octanoate

Compound 056 was prepared analogously to compound 052 starting with 2-methylpentan-1-ol. Compound 056 was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 6.4 Hz, 1 H) , 4.08 -4.16 (m, 4 H) , 3.59 -3.63 (m, 2 H) , 3.41 -3.54 (m, 4 H) , 2.57 (t, J = 5.2 Hz, 2 H) , 2.42 -2.46 (m, 4 H) , 2.27 -2.32 (m, 4 H) , 1.92 -1.97 (m, 2 H) , 1.51 -1.67 (m, 13 H) , 1.22 -1.49 (m, 27 H) , 1.1 -1.17 (m, 7 H) , 0.86 -0.91 (m, 24 H) .

Compound 057: 3-methyloctyl 8- ( (6- (3, 3-bis ( (3, 7-dimethyloctyl) oxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) octanoate

Compound 057 was prepared analogously to compound 052 using 3-ethylheptan-1-ol instead of 3-methylhexan-1-ol. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 6.0 Hz, 1H) , 4.15 (t, J = 6.4 Hz, 2H) , 4.10 (t, J = 3.2 Hz, 2H) , 3.42-3.67 (m, 6H) , 2.58 (brs, 2H) , 2.45-2.46 (m, 4H) , 2.28-2.33 (m, 4H) , 1.93-1.98 (m, 2H) , 1.27-1.64 (m, 44H) , 1.10-1.17 (m, 6H) , 0.85-0.92 (m, 24H) .

Compound 058: 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 6- ( (6- ( (3-ethylheptyl) oxy) -6-oxohexyl) (2-hydroxyethyl) amino) hexanoate

Synthesis of 3-ethylheptyl 6- ( (2-hydroxyethyl) amino) hexanoate

To a solution of 3-ethylheptan-1-ol (9.14 g, 63.4 mmol) and 6-bromohexanoic acid (18.5 g, 95.0 mmol) in CH₂Cl₂ (55.0 mL) was added EDCI (18.2 g, 95.0 mmol) and DMAP (1.55 g, 12.7 mmol) . The mixture was stirred at 20 ℃ for 12 hrs. TLC (Petroleum ether: Ethyl acetate=10: 1, start material: R_f=0.18; product: R_f= 0.63) indicated the 3-ethylheptan-1-ol was consumed complete. The residue was diluted with water (30 mL) and extracted with CH₂Cl₂ (30 mL x 3) . The combined organic layers were washed with brine (30 mL x 3) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=80/1 to 60/1) . 3-ethylheptyl 6-bromohexanoate (14.1 g, 43.9 mmol, 69.3%yield) was obtained as colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.10 (t, J = 7.2 Hz, 2H) , 3.41 (t, J = 6.8 Hz, 2H) , 2.32 (t, J = 7.2 Hz, 2H) , 1.85-1.92 (m, 2H) , 1.62-1.70 (m, 2H) , 1.56-1.61 (m, 2H) , 1.44-1.53 (m, 2H) , 1.23-1.40 (m, 9H) , 0.84-0.91 (m, 6H)

To a solution of 3-ethylheptyl 6-bromohexanoate (8.00 g, 24.9 mmol) in EtOH (160 mL) was added 2-aminoethanol (45.6 g, 745 mmol, 45.1 mL) . The mixture was stirred at 75 ℃ for 1 hr. The residue was diluted with water (50 mL) and extracted with EtOAc (50 mL x 3 mL) . The combined organic layers were washed with brine (50 x 3 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. Without purification. 3-ethylheptyl 6-( (2-hydroxyethyl) amino) hexanoate (7.35 g, crude) was obtained as colorless oil.

Synthesis of Compound 058

Compound 058 was prepared analogously to compound 052 using 3-ethylheptyl 6- ( (2-hydroxyethyl) amino) hexanoate. ¹H NMR (400 MHz, CDCl₃) δ 4.59 (t, J = 5.6 Hz, 1H) , 4.06-4.16 (m, 4H) , 3.69-3.85 (m, 2H) , 3.56-3.66 (m, 2H) , 3.40-3.51 (m, 2H) , 2.72-2.92 (m, 5H) , 2.31 (t, J = 7.2 Hz, 4H) , 1.94 (m, 2H) , 1.45-1.76 (m, 17H) , 1.21-1.43 (m, 22H) , 1.08-1.18 (m, 6H) , 0.83-0.92 (m, 24H) .

Compound 059: 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 6- ( (6- ( (3-butylheptyl) oxy) -6-oxohexyl) (2-hydroxyethyl) amino) hexanoate

Synthesis of 3-butylheptyl 6- ( (2-hydroxyethyl) amino) hexanoate

To a solution of 3-butylheptan-1-ol (2.90 g, 16.8 mmol) and 6-bromohexanoic acid (4.92 g, 25.3 mmol) in CH₂Cl₂ (18.0 mL) was added EDCI (4.84 g, 25.3 mmol) and DMAP (411 mg, 3.37 mmol) . The mixture was stirred at 20 ℃ for 12 hrs. TLC (Petroleum ether: Ethyl acetate=10: 1, start material: R_f=0.18; product: R_f= 0.60) indicated the 6-bromohexanoic acid was consumed complete. The residue was diluted with water (20 mL) and extracted with CH₂Cl₂ (20 mL x 3) . The combined organic layers were washed with brine (30 mL x 3) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=80/1 to 60/1) . 3-butylheptyl 6-bromohexanoate (4.1 g, 11.7 mmol, 69.7%yield) was obtained as colorless oil. ¹H NMR (400 MHz CDCl₃) δ 4.10 (t, J = 7.2 Hz, 2H) , 3.41 (t, J = 6.8 Hz, 2H) , 2.32 (t, J = 7.2 Hz, 2H) , 1.89 (m, 2H) , 1.63-1.72 (m, 2H) , 1.54-1.62 (m, 2H) , 1.45-1.53 (m, 2H) , 1.36-1.44 (m, 1H) , 1.19-1.35 (m, 12H) , 0.87-0.93 (m, 6H) .

To a solution of 3-butylheptyl 6-bromohexanoate (1.26 g, 3.61 mmol) in EtOH (25 mL) was added 2-aminoethanol (6.61 g, 108 mmol, 6.53 mL) . The mixture was stirred at 75 ℃ for 1 hr. The residue was diluted with water (20 mL) and extracted with EtOAc (20 mL x 3 mL) . The combined organic layers were washed with brine (20 x 3 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. 3-butylheptyl 6- ( (2-hydroxyethyl) amino) hexanoate (1.11 g, crude) was obtained as colorless oil.

Synthesis of Compound 059

Compound 059 was prepared analogously to compound 052 starting with 3-butylheptyl 6- ( (2-hydroxyethyl) amino) hexanoate. Compound 059 was obtained as colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.59 (t, J = 5.6 Hz, 1H) , 3.99-4.23 (m, 4H) , 3.53-3.69 (m, 4H) , 3.37-3.52 (m, 3H) , 2.45-2.66 (m, 5H) , 2.26-2.34 (m, 4H) , 1.91-1.99 (m, 2H) , 1.45-1.69 (m, 16H) , 1.16-1.44 (m, 26H) , 1.01-1.16 (m, 6H) , 0.82-0.92 (m, 24H)

Compound 060: 3-butylheptyl 8- ( (6- (3, 3-bis ( (3, 7-dimethyloctyl) oxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) octanoate

Synthesis of 3-butylheptyl 8- ( (2-hydroxyethyl) amino) octanoate

A 100 mL three-necked round bottom flask equipped with magnetic stirrer, addition funnel and thermometer. To a solution of 3-butylheptan-1-ol (5.00 g, 29.0 mmol, 1.0 eq) and 8-bromooctanoic acid (9.71 g, 43.5 mmol, 1.5 eq) in DCM (30.0 mL) was added EDCI (11.1 g, 58.0 mmol, 2.0 eq) and DMAP (709.01 mg, 5.80 mmol, 0.2 eq) . The mixture was stirred at 20 ℃ for 12 hrs. The residue was diluted with water (25.0 mL) and extracted with EtOAc (20.0 mL *3) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=100/1 to 80/1) . 3-butylheptyl 8-bromooctanoate (8.60 g, 22.8 mmol, 78.5%yield) was obtained as a white solid. ¹H NMR (400 MHz CDCl₃) δ 4.09 (t, J = 6.8 Hz, 2 H) , 3.40 (t, J = 6.8 Hz, 2 H) , 2.29 (t, J = 7.2 Hz, 2 H) , 1.82 -1.89 (m, 2 H) , 1.51 -1.65 (m, 6 H) , 1.26 -1.45 (m, 21 H) , 0.90 (t, J = 6.8 Hz, 6 H) .

A 100 mL three-necked round bottom flask equipped with magnetic stirrer, addition funnel and thermometer. To a solution of 3-butylheptyl 8-bromooctanoate (2.50 g, 6.62 mmol, 1.0 eq) in EtOH (50.0 mL) was added 2-aminoethanol (12.1 g, 199 mmol, 12.0 mL, 30 eq) . The mixture was stirred at 75 ℃ for 2 hrs. The residue was diluted with water (20.0 mL) and extracted with EtOAc (20.0 mL *3) . The combined organic layers were washed with brine (5.00 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. Without purification. 3-butylheptyl 8- ( (2-hydroxyethyl) amino) octanoate (2.30 g, 6.43 mmol, 97.1%yield) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.08 (t, J = 6.8 Hz, 2 H) , 3.62 -3.65 (m, 2 H) , 2.76 -2.78 (m, 2 H) , 2.59 -2.63 (t, J=7.15 Hz, 2 H) , 2.26 -2.30 (t, J=7.57 Hz, 2 H) , 1.93 -2.02 (m, 2 H) , 1.46 -1.63 (m, 6 H) , 1.39 -1.40 (m, 1 H) , 1.25 -1.32 (m, 17 H) , 0.89 (t, J = 6.4 Hz, 6 H) .

Synthesis of Compound 060

Compound 060 was prepared analogously to compound 052 starting with 3-butylheptyl 8- ( (2-hydroxyethyl) amino) octanoate. Compound 060 was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 6.0 Hz, 1 H) , 4.07 -4.16 (m, 4 H) , 3.43 -3.65 (m, 6 H) , 2.56 -2.58 (m, 2 H) , 2.42 -2.49 (m, 4 H) , 2.27 -2.32 (m, 4 H) , 1.92 -1.97 (m, 2 H) , 1.06 -1.74 (m, 57 H) , 0.86 -0.91 (m, 24 H) .

Compound 061: 3, 3-bis ( (3-butylheptyl) oxy) propyl 6- ( (6- ( (3-butylheptyl) oxy) -6-oxohexyl) (2-hydroxyethyl) amino) hexanoate

Synthesis of 3, 3-bis ( (3-butylheptyl) oxy) propyl 6-bromohexanoate

To a solution of 3, 3-bis ( (3-butylheptyl) oxy) propan-1-ol (2.00 g, 4.99 mmol, 1.0 eq) and 6-bromohexanoic acid (1.95 g, 9.98 mmol, 2.0 eq) in DCM (12.0 mL) was added EDCI (1.44 g, 7.49 mmol, 1.5 eq) and DMAP (122 mg, 998 umol, 0.2 eq) at 0 ℃. The reaction was stirred at 20 ℃ for 1.5 h. The residue was diluted with water (20.0 mL) and extracted with EtOAc (10.0 mL *3) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=150/1) . 3, 3-bis ( (3-butylheptyl) oxy) propyl 6-bromohexanoate (1.70 g, 2.94 mmol, 57.0%yield) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 4.4 Hz, 1 H) 4.15 (t, J = 6.8 Hz, 2 H) 3.57 -3.63 (m, 2 H) 3.39 -3.48 (m, 4 H) 2.32 (t, J=7.6 Hz, 2 H) 1.86 -1.97 (m, 4 H) 1.62 -1.68 (m, 2 H) 1.41 -1.56 (m, 9 H) 1.26 -1.31 (m, 24 H) 0.90 (t, J = 6.4Hz, 12 H) .

Synthesis of compound 061

To a solution of 3, 3-bis ( (3-butylheptyl) oxy) propyl 6-bromohexanoate (550 mg, 952 μmol, 1.0 eq) and 3-butylheptyl 6- ( (2-hydroxyethyl) amino) hexanoate (313 mg, 952 μmol, 1.0 eq) in EtOH (5.00 mL) was added DIEA (184 mg, 1.43 mmol, 248 μL, 1.5 eq) . The mixture was stirred at 90 ℃ for 48 hrs. The residue was diluted with H₂O (20.0 mL) and extracted with EtOAc (20.0 mL x 3) . The combined organic layers were washed with brine (20.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (4 gSilica Flash Column, Eluent of 0～4%Dichloromethane: Methanol gradient @15 mL/min) . Compound 061 (300 mg, 38.1%yield) was obtained as colorless oil. ¹H NMR (400 MHz CDCl3) δ 4.60 (t, J = 5.25 Hz, 1H) , 4.05-4.17 (m, 4H) , 3.72-3.83 (m, 1H) , 3.56-3.64 (m, 2H) , 3.39-3.49 (m, 2H) , 2.69-2.96 (m, 5H) , 2.32 (t, J = 7.19 Hz, 4H) , 1.90-1.99 (m, 2H) , 1.62-1.72 (m, 7H) , 1.50-1.60 (m, 7H) , 1.38 (dd, J = 7.32 Hz, 14.70 Hz, 8H) , 1.19-1.33 (m, 38H) , 0.90 (t, J = 6.32 Hz, 18H)

Compound 062: 2-ethylhexyl 8- ( (6- (3, 3-bis ( (3-butylheptyl) oxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) octanoate

Compound 062 was synthesized using 3, 3-bis ( (3-butylheptyl) oxy) propyl 6-bromohexanoate and 3-butylheptyl 8- ( (2-hydroxyethyl) amino) octanoate. Compound 062 (0.350 g, 410 μmol, 36.6%yield) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J =6.0 Hz, 1 H) , 4.07 -4.16 (m, 4 H) , 3.51 -3.61 (m, 4 H) , 3.43 -3.46 (m, 2 H) , 2.57 (t, J = 6.8 Hz, 2 H) , 2.42 -2.46 (m, 4 H) , 2.27 -2.32 (m, 4 H) , 1.93 -1.95 (m, 2 H) , 1.41 -1.65 (m, 19 H) , 1.25 -1.34 (m, 43 H) , 0.89 (t, J = 6.8 Hz, 18 H) .

Compound 063: 3-butylheptyl 8- ( (6- (3, 3-bis ( (3-butylheptyl) oxy) propoxy) -6-oxohexyl) (4-hydroxybutyl) amino) octanoate

Synthesis of 3-butylheptyl 8- ( (4-hydroxybutyl) amino) octanoate

To a solution of 3-butylheptyl 8-bromooctanoate (1.10 g, 2.91 mmol, 1.0 eq) in EtOH (10.0 mL) was added 2-aminoethanol (7.79 g, 87.4 mmol, 8.12 mL, 30 eq) . The mixture was stirred at 75 ℃ for 1 hrs. The residue was diluted with H₂O (20.0 mL) and extracted with EtOAc (20.0 mL x 2) . The combined organic layers were washed with brine (30.0 mL) , dried over, filtered and concentrated under reduced pressure to give 3-butylheptyl 8- ( (4-hydroxybutyl) amino) octanoate (990 mg, 88.0%yield) as colorless oil.

Synthesis of Compound 063

To a solution of 3, 3-bis ( (3-butylheptyl) oxy) propyl 6-bromohexanoate (550 mg, 952 μmol, 1.0 eq) and 3-butylheptyl 8- ( (4-hydroxybutyl) amino) octanoate (367 mg, 952 μmol, 1.0 eq) in EtOH (10.0 mL) was added DIEA (184 mg, 1.43 mmol, 248 μL, 1.5 eq) . The mixture was stirred at 90 ℃ for 24 hrs. The residue was diluted with H₂O (20.0 mL) and extracted with EtOAc (20.0 mL x 3) . The combined organic layers were washed with brine (20.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (4 gSilica Flash Column, Eluent of 0～2%Dichloromethane: Methanol gradient @15 mL/min) to give Compound 063 (280 mg, 33.3%yield) as colorless oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 5.75 Hz, 1H) , 4.06-4.18 (m, 4H) , 3.69 (s, 2H) , 3.56-3.65 (m, 2H) , 3.41-3.48 (m, 2H) , 2.70-3.00 (m, 5H) , 2.26-2.35 (m, 4H) , 1.82-1.99 (m, 4H) , 1.63-1.75 (m, 8H) , 1.50-1.60 (m, 9H) , 1.22-1.45 (m, 48H) , 0.90 (t, J = 6.69 Hz, 18H)

Compound 064: 3-butylheptyl 8- ( (6- (3, 3-bis ( (3-butylheptyl) oxy) propoxy) -6-oxohexyl) (3- ( (2- (methylamino) -3, 4-dioxocyclobut-1-en-1-yl) amino) propyl) amino) octanoate

Compound 064 was prepared using similar procedure as compound 040, starting with 3, 3-bis ( (3-butylheptyl) oxy) propyl 6-bromohexanoate and 3-butylheptyl 8-bromooctanoate. Compound 064 was obtained as a yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.57 -4.64 (m, 1 H) , 4.12 (t, J = 6.4 Hz, 2 H) , 4.07 (t, J = 6.4 Hz, 2 H) , 3.55 -3.68 (m, 4 H) , 3.41 -3.49 (m, 2 H) , 3.25 (s, 3 H) , 2.67 -2.74 (m, 2 H) , 2.64 -2.67 (m, 4 H) , 2.32 -2.36 (m, 4 H) , 1.82 -1.94 (m, 4 H) , 1.46 -1.71 (m, 19 H) , 1.23 -1.44 (m, 40 H) , 1.13 -1.23 (m, 4 H) , 0.81 -0.92 (m, 18 H) .

Compound 065: 3-propylhexyl 8- ( (6- (3, 3-bis ( (3-butylheptyl) oxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) octanoate

Compound 065 was synthesized using 3, 3-bis ( (3-butylheptyl) oxy) propyl 6-bromohexanoate and 3-propylhexyl 8- ( (2-hydroxyethyl) amino) octanoate. Compound 065 was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.61 (t, J = 6.0 Hz, 1 H) , 4.05 -4.21 (m, 4 H) , 3.65 -3.78 (m, 2 H) , 3.61 -3.65 (m, 2 H) , 3.33 -3.48 (m, 2 H) , 2.55 -2.91 (m, 5 H) , 2.20 -2.35 (m, 4 H) , 1.78 -2.01 (m, 2 H) , 1.45 -1.71 (m, 15 H) , 1.15 -1.45 (m, 44 H) , 0.87-0.91 (m, 18 H) .

Compound 066: 3-ethylheptyl 8- ( (6- (3, 3-bis ( (3-ethylheptyl) oxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) octanoate

Synthesis of 3-ethylheptyl 8- ( (2-hydroxyethyl) amino) octanoate

3-ethylheptyl 8- ( (2-hydroxyethyl) amino) octanoate was prepared using similar methods to those employed for the synthesis of Undecyl 6- ( (2-hydroxyethyl) amino) hexanoate as described for compound 070, but using 3-ethylheptan-1-ol. 3-ethylheptyl 8- ( (2-hydroxyethyl) amino) octanoate was obtained as a yellow oil. ¹H NMR (400 MHz CDCl₃) δ 5.88-6.43 (m, 2H) , 4.08 (t, J = 7.2 Hz, 2H) , 3.97-3.99 (m, 2H) , 3.12-3.14 (m, 2H) , 2.96-2.99 (m, 2H) , 2.29 (t, J = 7.2 Hz, 2H) , 1.82-1.89 (m, 2H) , 1.55-1.63 (m, 4H) , 1.25-1.35 (m, 16H) , 0.84-0.92 (m, 6H) .

Synthesis of compound 066

To a solution of 3, 3-bis ( (3-ethylheptyl) oxy) propyl 6-bromohexanoate (649 mg, 1.24 mmol, 1.0 eq) and 3-ethylheptyl 8- ( (2-hydroxyethyl) amino) octanoate (410 mg, 1.24 mmol, 1.0 eq) in EtOH (5 mL) was added DIEA (322 mg, 2.49 mmol, 433 μL, 2.0 eq) at 25℃, then the mixture was stirred at 90℃ for 24 hrs. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (10 mL, 5 mL, 5 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to EtOAc/MeOH = 5/1) to give compound 066 (290 mg, 376 μmol, 30.2%yield, 100%purity) as a yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 5.6 Hz, 1H) , 4.15 (t, J =6.4 Hz, 2H) , 4.09 (t, J = 7.2 Hz, 2H) , 3.57-3.63 (m, 2H) , 3.54 (t, J = 5.6 Hz, 2H) , 3.42-3.48 (m, 2H) , 2.58 (t, J = 5.2 Hz, 2H) , 2.43-2.48 (m, 4H) , 2.27-2.32 (m, 4H) , 1.93-1.97 (m, 2H) , 1.51-1.68 (m, 11H) , 1.26-1.48 (m, 41H) , 0.85-0.91 (m, 18H) .

Compound 067: 3, 3-bis ( (3-propylhexyl) oxy) propyl 6- ( (2-hydroxyethyl) (6-oxo-6- ( (3-propylhexyl) oxy) hexyl) amino) hexanoate

Compound 067 was prepared analogously to compound 066 starting with 3-propylhexan-1-ol. Compound 067 was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.62 (t, 1H) , 4.14-3.81 (m, 4H) , 3.72-3.43 (m, 6H) , 2.45-2.34 (m, 6H) , 2.36-2.15 (m, 4H) , 2.00-1.10 (m, 47H) , 0.90-0.78 (m, 18H) .

Compound 068: 2-ethyloctyl 8- ( (6- (3, 3-bis ( (2-ethyloctyl) oxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) octanoate

Compound 068 was prepared analogously to compound 066 starting with 2-ethyloctan-1-ol. ¹H NMR (400 MHz CDCl₃) δ 4.64 (t, 1H) , 4.26-4.03 (m, 4H) , 3.78-3.52 (m, 6H) , 2.64-2.55 (m, 6H) , 2.33-2.13 (m, 4H) , 2.01-1.14 (m, 45H) , 0.91-0.83 (m, 18H) .

Compound 069: 3, 7-dimethyloctyl 8- ( (6- (3, 3-bis ( (3-butylheptyl) oxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) octanoate

Compound 069 was subsequently prepared using 3, 7-dimethyloctyl 8- ( (2-hydroxyethyl) amino) octanoate and 3, 3-bis ( (3-butylheptyl) oxy) propyl 6-bromohexanoate. ¹H NMR (400 MHz CDCl₃) δ 4.61 (t, 1H) , 4.17-3.81 (m, 4H) , 3.71-3.40 (m, 6H) , 2.45-2.33 (m, 6H) , 2.33-2.11 (m, 4H) , 2.01-1.19 (m, 61H) , 0.91-0.79 (m, 18H) .

Compound 070: 3, 3-bis (heptyloxy) propyl 6- ( (2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) hexanoate

Synthesis of Undecyl 6- ( (2-hydroxyethyl) amino) hexanoate

To a solution of undecan-1-ol (30.0 g, 174 mmol, 1.0 eq) and 6-bromohexanoic acid (50.9 g, 261 mmol, 1.5 eq) in DCM (180 mL) was added DMAP (4.25 g, 34.8 mmol, 0.2 eq) and EDCI (50.1 g, 261 mmol, 1.5 eq) . The reaction was stirred at 20℃ for 3 h. The residue was diluted with saturated sodium carbonate solution (100 mL) and extracted with EtOAc (200 mL *2) . The combined organic layers were washed with brine (50.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=70/1 to 50/1) . The product of et49878-110 was combined to et49878-118. undecyl 6-bromohexanoate (61.0 g, 175 mmol, 85.9%yield) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.07 (t, J = 6.4 Hz, 2 H) , 3.41 (t, J = 6.4 Hz, 2 H) , 2.32 (t, J =7.6 Hz, 2 H) , 1.86 -1.90 (m, 2 H) 1.60 -1.68 (m, 4 H) , 1.44 -1.50 (m, 2 H) , 1.29 -1.31 (m, 5 H) , 1.27 -1.31 (s, 10 H) , 0.88 (t, J = 6.8 Hz, 3 H) .

Synthesis of compound 070

To a solution of undecyl 6- ( (2-hydroxyethyl) amino) hexanoate (300 mg, 910 umol, 1.0 eq) and 3, 3-bis (heptyloxy) propyl 6-bromohexanoate (508 mg, 1.09 mmol, 1.2 eq) in EtOH (6.00 mL) was added DIEA (188 mg, 1.46 mmol, 253 uL, 1.6 eq) . The reaction was stirred at 90℃ for 12 h. The residue was diluted with water (10.0 mL) and extracted with EtOAc (20.0 mL *3) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate = 5: 1 to 1: 1) . Compound 070 (150 mg, 210 umol, 23.1%yield) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 6.0 Hz 1 H) , 4.14 (t, J = 6.8 Hz, 2 H) , 4.06 (t, J = 6.8 Hz, 2 H) , 3.52 -3.59 (m, 4 H) , 3.41 -3.43 (m, 2 H) , 2.58 (t, J = 4.8 Hz, 2 H) , 2.46 (t, J = 7.8 Hz, 4 H) , 2.30 (t, J = 7.2 Hz, 4 H) , 1.94 -1.95 (m, 2 H) , 1.55 -1.65 (m, 11 H) , 1.45 -1.48 (m, 4 H) , 1.27 -1.31 (m, 34 H) , 0.89 (t, J = 6.0 Hz, 9 H) .

Compound 071: 4, 4-bis (hexyloxy) butyl 6- ( (2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) hexanoate

Synthesis of 4, 4-bis (hexyloxy) butan-1-ol

To a solution of 4, 4-dimethoxybutanenitrile (8.3 g, 64.0 mmol, 1.0 eq) and hexan-1-ol (19.5 g, 191 mmol, 3.0 eq) in Tol. (70 mL) was added PPTS (878 mg, 3.5 mmol, 0.05 eq) at 25℃, then the mixture was stirred at 120℃ for 12 hrs. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 100/0 to 60/1) to give 4, 4-bis (hexyloxy) butanenitrile (11.1 g, 41.3 mmol, 65%yield) as a colorless oil.

To a solution of 4, 4-bis (hexyloxy) butanenitrile (10.0 g, 28.8 mmol, 1.0 eq) in EtOH (30 mL) was added KOH (3 M, 28.8 mL, 3.0 eq) at 25℃, then the mixture was stirred at 110℃ for 12 hrs. The pH value of the reaction was adjusted to around 4-5 with aq. HCl (1 M) at 0℃. The mixture was extracted with ether (50 mL x 4) and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 100/1 to 1/1) to give 4, 4-bis (hexyloxy) butanoic acid (8.2 g, crude) as a colorless oil. To a solution of 4, 4-bis (hexyloxy) butanoic acid (8 g, 27.7 mmol, 70%purity, 1.0 eq) in THF (55 mL) at 0℃, then LiAlH₄ (882 mg, 23.2 mmol, 1.2 eq) was added the mixture and stirred at 25℃ for 3 hrs. Water (0.9 mL) , 15%aq. NaOH (0.9 mL) and water (2.7 mL) was dropwise the reaction solution at 0℃, then Na₂SO₄ added the mixture and stirred at 25℃ for 15 mins. The mixture was filtered and the filter cake concentrated under reduced pressure to give 4, 4-bis (hexyloxy) butan-1-ol (4.0 g, 14.5 mmol, 75%yield) as a colorless oil.

Synthesis of compound 071

Compound 071 was subsequently prepared using similar procedure as Compound 070 but using 4, 4-bis (hexyloxy) butan-1-ol. ¹H NMR (400 MHz CDCl₃) δ 4.62 (t, 1H) , 4.26-4.02 (m, 4H) , 3.67-3.45 (m, 6H) , 2.61-2.50 (m, 6H) , 2.32-2.20 (m, 4H) , 1.99-1.91 (m, 2H) , 1.77-1.14 (m, 48H) , 0.91-0.81 (m, 9H) .

Compound 072: 3, 3-bis (cyclopentylmethoxy) propyl 6- ( (2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) hexanoate

Compound 072 was prepared using similar procedure as Compound 070 starting with cyclopentylmethanol. ¹H NMR (400 MHz CDCl₃) δ 4.72 (t, 1H) , 4.27-4.04 (m, 4H) , 3.68-3.41 (m, 6H) , 2.64-2.51 (m, 6H) , 2.31-2.19 (m, 4H) , 1.98-1.92 (m, 2H) , 1.77-1.14 (m, 48H) , 0.91 (s, 3H) .

Compound 073: 3, 3-bis ( (5-methylhexyl) oxy) propyl 6- ( (2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) hexanoate

To a solution of 3, 3-bis ( (5-methylhexyl) oxy) propyl 6-bromohexanoate (400 mg, 1.21 mmol, 1 eq) and undecyl 6- ( (2-hydroxyethyl) amino) hexanoate (621.58 mg, 1.34 mmol, 1.1 eq) in EtOH (5 mL) was added DIEA (313 mg, 2.43 mmol, 422 uL, 2 eq) . The mixture was stirred at 80 ℃ for 12 h. LC-MS showed ～8%of Reactant 1 remained. Several new peaks were shown on LC-MS and ～64%of desired compound was detected. The residue was diluted with H₂O 20 mL and extracted with EtOAc 100 mL. The combined organic layers were washed with NaHCO₃ 50 mL, dried over NA₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=100/1 to 0/1) . Compound 073 (207 mg, 289 umol, 23.8%yield) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.54 -4.66 (m, 1H) , 4.15 (br t, J = 6.1 Hz, 2H) , 4.06 (br t, J = 6.7 Hz, 2H) , 3.89 (br s, 2H) , 3.52 -3.68 (m, 2H) , 3.37 -3.52 (m, 2H) , 2.82 –3.08 (m, 6H) , 2.33 (br t, J = 7.2 Hz, 4H) , 2.04 (s, 1H) , 1.89 –2.01 (m, 2H) , 1.48 -1.85 (m, 17H) , 1.08 -1.46 (m, 29H) , 0.81 -0.98 (m, 15H)

Compound 074: 3, 3-bis ( (3, 5, 5-trimethylhexyl) oxy) propyl 6- ( (2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) hexanoate

To a solution of 3, 3-bis ( (3, 5, 5-trimethylhexyl) oxy) propyl 6-bromohexanoate (0.5 g, 959 umol, 1.0 eq) and undecyl 6- ( (2-hydroxyethyl) amino) hexanoate (316 mg, 959 umol, 1.0 eq) in EtOH (5 mL) was added DIEA (248 mg, 1.92 mmol, 334 uL, 2.0 eq) at 25℃, then the mixture was stirred at 90℃ for 12 hrs. The reaction mixture was poured into water (50 mL) and extracted with EtOAc (50 mL, 30 mL, 20 mL) , The combined organic layers were washed with brine (20 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to EtOAc/MeOH = 5/1) to give compound 074 (260 mg, 337 umol, 35.2%yield, 100%purity) as a yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 5.6 Hz, 1H) , 4.15 (t, J = 6.8 Hz, 2H) , 4.06 (t, J = 6.8 Hz, 2H) , 3.56-3.63 (m, 4H) , 3.43-3.56 (m, 2H) , 2.63 (t, J = 5.2 Hz, 2H) , 2.51 (t, J = 7.2 Hz, 4H) , 2.31 (t, J = 7.6 Hz, 4H) , 1.94 (q, J = 6.4 Hz, 2H) , 1.59-1.68 (m, 10H) , 1.21-1.51 (m, 29H) , 1.05-1.10 (m, 2H) , 0.85-0.95 (m, 26H) .

Compound 075: 2- ( (6- (3, 3-bis (heptyloxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) ethyl (9Z, 12Z) -octadeca-9, 12-dienoate

Synthesis of 3, 3-bis (heptyloxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate

To a solution of 3, 3-bis (heptyloxy) propyl 6-bromohexanoate (1.5 g, 3.22 mmol, 1.0 eq) in ACN (30.0 mL) was added 2-aminoethanol (5.90 g, 96.7 mmol, 5.85 mL, 30 eq) . The reaction was stirred at 90 ℃ for 1.5 h. Two reactions were combined for workup. The residue was diluted with water (50.0 mL) and extracted with EtOAc (20.0 mL *3) . The combined organic layers were washed with brine (10.0 mL*3) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=5/1 to 10/1) . 3, 3-bis (heptyloxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (2.3 g, 5.16 mmol, 80.1%yield) was obtained as yellow oil.

Synthesis of Compound 075

To a solution of 3, 3-bis (heptyloxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (800 mg, 1.80 mmol, 1.0 eq) in ACN (8.00 mL) was added 2-bromoethan-1-ol (336 mg, 2.69 mmol, 191 uL, 1.5 eq) and K₂CO₃ (372 mg, 2.69 mmol, 1.5 eq) . The reaction was stirred at 90 ℃ for 6 h. The residue was diluted with water (30.0 mL) and extracted with EtOAc (20.0 mL *3) . The combined organic layers were washed with brine (20.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The crude product was used into the next step without further purification. 2, 3-bis (heptyloxy) propyl 6- (bis (2-hydroxyethyl) amino) hexanoate (900 mg, crude) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.58 (t, J = 6.4 Hz, 1 H) , 4.15 (t, J = 6.8 Hz, 2 H) , 3.64 (t, J = 5.2 Hz, 3 H) , 3.53 -3.61 (m, 2 H) , 3.39 -3.46 (m, 2 H) , 2.67 (t, J = 5.2 Hz, 3 H) , 2.56 (t, J = 7.2 Hz, 2 H) , 2.31 (t, J =, 7.2 Hz, 2 H) , 1.94 -1.97 (m, 2 H) , 1.49 -1.66 (m, 8 H) , 1.28 -1.38 (m, 18 H) , 0.87 -0.90 (m, 6 H) .

To a solution of linoleic acid (588 mg, 2.21 mmol, 1.2 eq) and 2, 3-bis (heptyloxy) propyl 6- (bis (2-hydroxyethyl) amino) hexanoate (900 mg, 1.84 mmol, 1.0 eq) in DCM (5.40 mL) was added EDCI (528 mg, 2.76 mmol, 1.5 eq) and DMAP (44.9 mg, 368 umol, 0.2 eq) at 0 ℃. The reaction was stirred at 20 ℃ for 4 h. The residue was diluted with water (5.00 mL) and extracted with EtOAc (20.0 mL *3) . The combined organic layers were washed with brine (5.00 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=5/1 to 4/1) . Compound 075 (390 mg, 508 umol, 27.6%yield, 98.0%purity) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 5.31 -5.40 (m, 4 H) , 4.60 (t, J = 4.8 Hz, 1 H) , 4.12 -4.16 (m, 4 H) , 3.52 -3.61 (m, 4 H) , 3.39 -3.45 (m, 2 H) , 2.73 -2.79 (m, 4 H) , 2.64 -2.66 (m, 2 H) , 2.51 -2.54 (m, 2 H) , 2.28 -2.33 (m, 4 H) , 2.03 -2.08 (m, 4 H) , 1.92 -1.97 (m, 2 H) , 1.44 -1.67 (m, 11 H) , 1.26 -1.39 (m, 35 H) , 0.83 -0.91 (m, 10 H) .

Compound 076: 3, 3-bis (heptyloxy) propyl 6- ( (5- (dodecyloxy) -5-oxopentyl) (2-hydroxyethyl) amino) hexanoate

Synthesis of dodecyl 5-bromopentanoate

To a solution of 5-bromopentanoic acid (10.0 g, 55.2 mmol, 1.0 eq) in DCM (70.0 mL) was added dodecan-1-ol (15.4 g, 82.9 mmol, 1.5 eq) , DMAP (1.35 g, 11.0 mmol, 0.2 eq) and EDCI (15.9 g, 82.9 mmol, 1.5 eq) at 0 ℃. The mixture was stirred at 25 ℃ for 12 hrs. The reaction was added water (50.0 mL) and the reaction was extracted with EtOAc (80.0 mL, 50.0 mL) . The organic phase was separated, washed with brine 20 mL, dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0 to 0/1) . Dodecyl 5-bromopentanoate (16.2 g, 46.4 mmol, 84.0%yield) was obtained as a yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.07 (t, J = 6.8 Hz, 2 H) , 3.41 (t, J = 6.4 Hz, 2 H) , 3.34 (t, J = 7.6 Hz, 2 H) , 1.89 -1.92 (m, 2 H) , 1.78 -1.80 (m, 2 H) , 1.60 -1.62 (m, 2 H) , 1.24 -1.31 (m, 19 H) , 0.88 (t, J = 6.8 Hz, 3 H) .

Synthesis of compound 076

To a solution of 3, 3-bis (heptyloxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (1.15 g, 2.58 mmol, 1.0 eq) in EtOH (23.0 mL) was added DIEA (500 mg, 3.87 mmol, 674 uL, 1.5 eq) and dodecyl 5-bromopentanoate (1.08 g, 3.10 mmol, 1.2 eq) . The mixture was stirred at 90 ℃ for 12 hrs. Two reactions were combined for workup. The residue was diluted with water (20.0 mL) and extracted with EtOAc (40.0 mL *3) . The combined organic layers were washed with brine (20.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, DCM: MeOH=1/0 to 30/1) . Compound 076 (2.4 g, 3.36 mmol, 65.1%yield) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 5.6 Hz, 1 H) 4.14 (t, J = 6.4 Hz, 2 H) 4.06 (t, J = 6.8 Hz, 2 H) 3.55 -3.61 (m, 4 H) 3.39 -3.48 (m, 2 H) 2.50 -2.62 (m, 6 H) 2.28 -2.37 (m, 4 H) 1.92 -1.97 (m, 2 H) 1.41 -1.71 (m, 15 H) 1.26 -1.31 (m, 36 H) 0.89 (t, J = 5.6 Hz, 9 H) .

Compound 077: 3, 3-bis (heptyloxy) propyl 6- ( (2- (dimethylamino) ethyl) (5- (dodecyloxy) -5-oxopentyl) amino) hexanoate

To a solution of compound 076 (1.00 g, 1.40 mmol, 1.0 eq) in DCM (10.0 mL) was added TEA (425 mg, 4.20 mmol, 585 uL, 3.0 eq) and TosCl (534 mg, 2.80 mmol, 2.0 eq) at 0 ℃. The mixture was stirred at 0-20 ℃ for 12 hrs. The residue was diluted with water (20.0 mL) and extracted with EtOAc (30.0 mL *3) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, DCM: MeOH=90/1 to 60/1) . Compound 077-1 (580 mg, 792 umol, 56.5%yield) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 5.6 Hz, 2 H) , 4.12 -4.16 (m, 2 H) , 4.03 -4.08 (m, 2 H) , 3.57 -3.59 (m, 2 H) , 3.41 -3.50 (m, 4 H) , 2.75 (t, J = 7.6 Hz, 2 H) , 2.43 -2.49 (m, 3 H) , 2.28 -2.33 (m, 3 H) , 1.94 -1.95 (m, 2 H) , 1.53 -1.66 (m, 10 H) , 1.42 -1.47 (m, 4 H) , 1.27 -1.31 (m, 33 H) , 0.87 -0.90 (m, 9 H) .

To a solution of compound 077-1 (230 mg, 314 umol, 1.0 eq) in THF (2.3 mL) was added NaI (23.5 mg, 157 umol, 0.5 eq) and N-methylmethanamine (2 M, 785 uL, 5.0 eq) . The mixture was stirred at 75 ℃ for 12 hrs. The reaction of et49878-291 was combined to et49878-293. The residue was diluted with water (10.0 mL) and extracted with EtOAc (20.0 mL *3) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, DCM: MeOH=80/1 to 10/1) . Compound 077 (250 mg, 337 umol, 37.4%yield) was obtained as brown oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 5.6 Hz, 1 H) , 4.14 (t, J = 6.4 Hz, 2 H) , 4.05 (t, J=6.8 Hz, 2 H) , 3.55 -3.61 (m, 2 H) , 3.39 -3.45 (m, 2 H) , 2.72 (s, 2 H) , 2.47 -2.60 (m, 6 H) 2.28 -2.39 (m, 10 H) 1.92 -1.97 (m, 2 H) 1.47 -1.67 (m, 15 H) 1.26 -1.31 (m, 36 H) 0.89 (t, J = 6.0 Hz, 9 H) .

Compound 078: 2- ( (6- (3, 3-bis (heptyloxy) propoxy) -6-oxohexyl) (2- (dimethylamino) ethyl) amino) ethyl (9Z, 12Z) -octadeca-9, 12-dienoate

Synthesis of 3, 3-bis (heptyloxy) propyl 6- ( (2- (dimethylamino) ethyl) amino) hexanoate

To a solution of 3, 3-bis (heptyloxy) propyl 6-bromohexanoate (1.0 g, 2.15 mmol, 1.0 eq) in EtOH (15.0 mL) was added N1, N1-dimethylethane-1, 2-diamine (1.89 g, 21.5 mmol, 2.35 mL, 10 eq) . The mixture was stirred at 90 ℃ for 20 mins. The residue was diluted with water (50.0 mL) and extracted with EtOAc (30.0 mL *3) . The combined organic layers were washed with brine (20.0 mL*3) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, DCM: MeOH=5/1 to 0/1) . 3, 3-bis(heptyloxy) propyl 6- ( (2- (dimethylamino) ethyl) amino) hexanoate (1.50 g, crude) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.59 (t, J = 6.0 Hz, 1 H) , 4.14 (t, J = 6.4 Hz, 2 H) , 3.55 -3.59 (m, 2 H) , 3.39 -3.45 (m, 2 H) , 2.63 -2.73 (m, 4 H) , 2.45 (t, J = 5.2 Hz, 2 H) , 2.30 (t, J = 7.6 Hz, 2 H) , 2.24 (s, 6 H) , 1.92 -1.99 (m, 2 H) , 1.51 -1.68 (m, 8 H) , 1.28 -1.40 (m, 18 H) , 0.87 -0.90 (m, 6 H) .

Synthesis of 3, 3-bis (heptyloxy) propyl 6- ( (2- (dimethylamino) ethyl) (2-hydroxyethyl) amino) hexanoate

To a solution of 3, 3-bis (heptyloxy) propyl 6- ( (2- (dimethylamino) ethyl) amino) hexanoate (650 mg, 1.37 mmol, 1.0 eq) in DCM (3.90 mL) was added (CH₃COO) ₃BHNa (288 mg, 1.65 mmol, 314 uL, 1.2 eq) . The reaction was stirred at 20 ℃ for 20 mins. Then (CH₃COO) ₃BHNa (583 mg, 2.75 mmol, 2.0 eq) was added at 0 ℃. The mixture was stirred at 0-20 ℃ for 12 hrs. The residue was diluted with water (20.0 mL) and extracted with EtOAc (30.0 mL *3) . The combined organic layers were adujsted pH 7～8 by saturated sodium carbonate and washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The crude product was used into the next step without further purification. Compound 078-1 (1.40 g, crude) was obtained as yellow oil.

To a solution of compound 078-1 (1.40 g, 2.22 mmol, 1.0 eq) in THF (1.00 mL) was added TBAF (1 M, 13.3 mL, 6.0 eq) . The mixture was stirred at 15 ℃ for 12 hrs. The residue was diluted with water (20.0 mL) and extracted with EtOAc (50.0 mL *4) . The combined organic layers were washed with brine (10 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, DCM: MeOH=5/1 to 0/1) . 3, 3-bis (heptyloxy) propyl 6- ( (2- (dimethylamino) ethyl) (2-hydroxyethyl) amino) hexanoate (720 mg, 1.39 mmol, 62.8%yield) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 5.6 Hz, 1 H) , 4.07 -4.14 (m, 3 H) , 3.58 -3.64 (m, 4 H) , 3.42 -3.74 (m, 2 H) , 3.03 -3.06 (m, 2 H) , 2.83 -2.86 (m, 2 H) , 2.64 -2.75 (m, 9 H) , 2.35 (t, J = 7.2 Hz, 2 H) , 1.87 -1.94 (m, 2 H) , 1.50 -1.69 (m, 9 H) , 1.22 -1.37 (m, 20 H) , 0.89 -0.92 (m, 6 H) .

Synthesis of compound 078

To a solution of 3, 3-bis (heptyloxy) propyl 6- ( (2- (dimethylamino) ethyl) (2-hydroxyethyl) amino) hexanoate (300 mg, 580 umol, 1.0 eq) in DCM (3.00 mL) was added EDCI (167 mg, 871 umol, 1.5 eq) and DMAP (14.2 mg, 116 umol, 0.2 eq) at 0 ℃. Then linoleic acid (195 mg, 697 umol, 195 uL, 1.2 eq) was added. The mixture was stirred at 0-20 ℃ for 12 hrs. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by column chromatography (SiO₂, DCM: MeOH=1/0 to 80/1) . Compound 078 (400 mg, 513 umol, 88.4%yield) was obtained as brown oil. ¹H NMR (400 MHz CDCl₃) δ 5.33 -5.39 (m, 4 H) 4.59 (t, J = 6.0 Hz, 1 H) 4.12 -4.16 (m, 4 H) 3.55 -3.61 (m, 2 H) 3.35 -3.49 (m, 2 H) 2.72 -2.84 (m, 6 H) 2.45 -2.55 (m, 4 H) 2.27 -2.31 (m, 4 H) 2.03 -2.08 (m, 4 H) 1.92 -1.97 (m, 2 H) 1.53 -1.67 (m, 8 H) 1.43 -1.50 (m, 2 H) 1.29 -1.40 (m, 32 H) 0.87 -0.91 (m, 9 H)

Compound 079: 3, 3-bis ( (4-propylheptyl) oxy) propyl 6- ( (2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) hexanoate

Compound 079 was prepared using similar procedure as compound 070 starting with 4-propylheptan-1-ol. Compound 079 was obtained as a yellow oil. ¹H NMR (400 MHz CDCl₃) 4.60 (t, J = 6.0 Hz, 1H) , 4.15 (t, J = 6.4 Hz, 2H) , 4.06 (t, J = 6.8 Hz, 2H) , 3.53-3.60 (m, 4H) , 3.38-3.44 (m, 2H) , 2.59 (brs, 2H) , 2.47 (t, J = 6.8 Hz, 4H) , 2.31 (t, J = 7.6 Hz, 4H) , 1.93-1.98 (m, 2H) , 1.45-1.68 (m, 15H) , 1.19-1.33 (m, 42H) , 0.88 (t, J = 6.8 Hz, 15H) .

Compound 080: 3, 3-bis ( (3-butylheptyl) oxy) propyl 6- ( (2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) hexanoate

To a solution of undecyl 6- ( (2-hydroxyethyl) amino) hexanoate (300 mg, 910 umol, 1.0 eq) and 3, 3-bis ( (3-butylheptyl) oxy) propyl 6-bromohexanoate (631 mg, 1.09 mmol, 1.2 eq) in EtOH (6.00 mL) was added DIEA (235 mg, 1.82 mmol, 317 uL, 2.0 eq) . The reaction was stirred at 90 ℃ for 12 h. The residue was diluted with water (5.00 mL) and extracted with EtOAc (10.0 mL *3) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, DCM: MeOH=40/1 to 30/1) . Compound 080 (200 mg, 239 umol, 26.4%yield, 99.1%purity) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.59 (t, J = 6.0 Hz, 1 H) , 4.14 (t, J = 6.8 Hz, 2 H) , 4.06 (t, J=6.8 Hz, 2 H) , 3.57 -3.63 (m, 4 H) , 3.41 -3.47 (m, 2 H) , 2.40 -2.68 (m, 6 H) , 2.30 (t, J = 7.6 Hz, 4 H) , 1.92 -1.97 (m, 2 H) , 1.50 -1.68 (m, 14 H) , 1.41 (s, 2 H) , 1.26 -1.35 (m, 45 H) , 0.87 -0.91 (m, 15 H) .

Compound 081: 3, 3-bis ( (3-butylheptyl) oxy) propyl 6- ( (2-hydroxyethyl) (5- ( ( (9Z, 12Z) -octadeca-9, 12-dien-1-yl) oxy) -5-oxopentyl) amino) hexanoate

Synthesis of (9Z, 12Z) -octadeca-9, 12-dien-1-yl 5-bromopentanoate

A 250 mL three-necked round bottom flask equipped with magnetic stirrer, addition funnel and thermometer. To a solution of 5-bromopentanoic acid (13.6 g, 75.1 mmol, 1.0 eq) in DCM (120 mL) was added (9Z, 12Z) -octadeca-9, 12-dien-1-ol (20.0 g, 75.1 mmol, 1.0 eq) , EDCI (21.6 g, 112 mmol, 1.5 eq) and DMAP (1.83 g, 15.0 mmol, 0.2 eq) at 0 ℃. The mixture was stirred at 0-20 ℃ for 12 hrs. The residue was diluted with saturated sodium carbonate solution (100 mL) and extracted with EtOAc (200 mL *2) . The combined organic layers were washed with brine (50.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=70/1 to 50/1) . (9Z, 12Z) -octadeca-9, 12-dien-1-yl 5-bromopentanoate (25.0 g, 58.2 mmol, 77.6%yield) was obtained as yellow oil.

Synthesis of compound 081

A 40 mL seal tube equipped with magnetic stirrer, addition funnel and thermometer. To a solution of 3, 3-bis ( (3-butylheptyl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (900 mg, 1.61 mmol, 1.0 eq) in EtOH (9.00 mL) was added DIEA (313 mg, 2.42 mmol, 421 uL, 1.5 eq) and (9Z, 12Z) -octadeca-9, 12-dien-1-yl 5-bromopentanoate (831 mg, 1.94 mmol, 1.2 eq) . The mixture was stirred at 90 ℃ for 12 hrs. The residue was diluted with water (5.00 mL) and extracted with EtOAc (10.0 mL *3) . The combined organic layers were washed with brine (5.00 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=1/1 to 1/0) . Compound 081 (500 mg, 551.60 umol, 34.19%yield) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 5.25 -5.32 (m, 4 H) , 4.52 (t, J = 5.6 Hz, 1 H) , 3.97 -4.08 (m, 4 H) , 3.49 -3.55 (m, 4 H) , 3.34 -3.40 (m, 2 H) , 2.70 (t, J = 6.4 Hz, 2 H) , 2.49 -2.60 (m, 2 H) , 2.43 (br s, 3 H) , 2.21-2.26 (m, 5 H) , 1.95 -2.00 (m, 4 H) , 1.84 -1.89 (m, 2 H) , 1.56 -1.60 (m, 4 H) , 1.42 -1.53 (m, 10 H) , 1.18 -1.34 (m, 45 H) , 0.82 (t, J = 6.4 Hz, 15 H) .

Compound 082: undecyl 6- ( (5- (3, 3-bis ( (3-butylheptyl) oxy) propoxy) -5-oxopentyl) (2-hydroxyethyl) amino) hexanoate

Synthesis of 3, 3-bis ( (3-butylheptyl) oxy) propyl 5-bromopentanoate

A 100 mL three-necked round bottom flask equipped with magnetic stirrer, addition funnel and thermometer. To a solution of 3, 3-bis ( (3-butylheptyl) oxy) propan-1-ol (6.00 g, 14.9 mmol, 1.0 eq) in DCM (36.0 mL) was added EDCI (4.31 g, 22.5 mmol, 1.5 eq) and DMAP (366 mg, 2.99 mmol, 0.2 eq) . Then 5-bromopentanoic acid (4.07 g, 22.5 mmol, 1.5 eq) was added. The mixture was stirred at 20 ℃ for 12 hrs. The residue was diluted with water (10.0 mL) and extracted with EtOAc (20.0 mL, 10.0 mL) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=25/1) . 3, 3-bis ( (3-butylheptyl) oxy) propyl 5-bromopentanoate (2.00 g, 3.55 mmol, 23.7%yield) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 5.6 Hz, 1 H) , 4.15 (t, J = 6.4 Hz, 2 H) , 3.58 -3.61 (m, 2 H) , 3.33 -3.36 (m, 4 H) , 2.34 (t, J = 7.2 Hz, 2 H) , 1.78 -1.98 (m, 6 H) , 1.50 -1.54 (m, 5 H) , 1.29 -1.31 (m, 9 H) , 1.25 -1.31 (m, 24 H) , 0.89 (t, J = 6.4 Hz, 12 H) .

Synthesis of compound 082

A 40 mL seal tube equipped with magnetic stirrer, addition funnel and thermometer. To a solution of 3, 3-bis ( (3-butylheptyl) oxy) propyl 5-bromopentanoate (1.03 g, 1.82 mmol, 1.2 eq) in EtOH (5.00 mL) was added DIEA (294 mg, 2.28 mmol, 396 uL, 1.5 eq) and undecyl 6- ( (2-hydroxyethyl) amino) hexanoate (0.50 g, 1.52 mmol, 1.0 eq) . The mixture was stirred at 90 ℃ for 12 hrs. The residue was diluted with water (10.0 mL) and extracted with EtOAc (20.0 mL *3) . The combined organic layers were washed with brine (5.00 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate=1/1 to 1/0) . Compound 082 (0.1 g, 117 μmol, 7.74%yield, 95.4%purity) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 5.6 Hz, 1 H) , 4.15 (t, J = 6.4 Hz, 2 H) , 4.06 (t, J = 6.8 Hz, 2 H) , 3.56 -3.62 (m, 4 H) , 3.41 -3.47 (m, 2 H) , 2.47 -2.66 (m, 5 H) , 2.29 -2.34 (m, 4 H) , 1.92 -1.97 (m, 2 H) , 1.49 -1.66 (m, 14 H) , 1.40 -1.41 (m, 2 H) , 1.20 -1.34 (m, 42 H) , 0.87 -0.91 (m, 15 H) .

Compound 083: 4, 4-bis ( (3-butylheptyl) oxy) butyl 5- ( (2-hydroxyethyl) (5- ( ( (9Z, 12Z) -octadeca-9, 12-dien-1-yl) oxy) -5-oxopentyl) amino) pentanoate

Synthesis of 4, 4-bis ( (3-butylheptyl) oxy) butan-1-ol

To a solution of 4, 4-dimethoxybutanenitrile (0.9 g, 7.0 mmol, 1.0 eq) and 3-butylheptan-1-ol (3.6 g, 21.0 mmol, 3.0 eq) in Tol. (10 mL) was added PPTS (88 mg, 350 umol, 0.05 eq) at 25℃, then the mixture was stirred at 120℃ for 12 hrs. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 1/0 to 60/1) to give 4, 4-bis ( (3-butylheptyl) oxy) butanenitrile (1.9 g, 4.7 mmol, 67%yield) as a colorless oil.

To a solution of 4, 4-bis ( (3-butylheptyl) oxy) butanenitrile (1.1 g, 2.7 mmol, 1.0 eq) in EtOH (5 mL) was added KOH (3 M, 2.7 mL, 3.0 eq) at 25℃, then the mixture was stirred at 110℃ for 12 hrs. The pH value of the reaction was adjusted to around 4-5 with aq. HCl (1 M) at 0℃. The mixture was extracted with ether (10 mL x 4) and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to 1/1) to give 4, 4-bis ( (3-butylheptyl) oxy) butanoic acid (1.0 g, crude) as a colorless oil.

To a solution of 4, 4-bis ( (3-butylheptyl) oxy) butanoic acid (1 g, 2.3 mmol, 85%purity, 1.0 eq) in THF (6 mL) at 0℃, then LiAlH₄ (89 mg, 2.3 mmol, 1.2 eq) was added the mixture and stirred at 25℃ for 3 hrs. Water (0.1 mL) , 15%aq. NaOH (0.1 mL) and water (0.3 mL) was dropwise the reaction solution at 0℃, then Na₂SO₄ added the mixture and stirred at 25℃ for 15 mins. The mixture was filtered and the filter cake concentrated under reduced pressure to give 4, 4-bis ( (3-butylheptyl) oxy) butan-1-ol (763 mg, 1.8 mmol, 80%yield) as a colorless oil. ¹H NMR (400 MHz CDCl₃) δ 4.63 (t, 1H) , 3.73 (t, 2H) , 3.60-3.65 (m, 2H) , 3.42-3.45 (m, 2H) , 1.81-1.95 (m, 2H) , 1.92-1.22 (m, 32H) , 0.92-0.85 (m, 12H) .

Synthesis of 4, 4-bis ( (3-butylheptyl) oxy) butyl 5-bromopentanoate

To a solution of 4, 4-bis ( (3-butylheptyl) oxy) butan-1-ol (550 mg, 1.3 mmol, 1.0 eq) in DCM (5 mL) was added 5-bromohexanoic acid (235 mg, 1.3 mmol, 1.0 eq) , EDCI (374 mg, 1.9 mmol, 1.5 eq) and DMAP (37 mg, 0.3 mmol, 0.2 eq) at 0℃, then the mixture was stirred at 25℃ for 1.5 hrs. The reaction mixture was poured into water (5 mL) and extracted with EtOAc (5 mL, 5 mL, 3 mL) , The combined organic layers were washed with brine (30 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=100/1) to give 4, 4-bis ( (3-butylheptyl) oxy) butyl 5-bromopentanoate (180 mg, 0.3 mmol, 24%yield) as a colorless oil.

Synthesis of compound 083

Compound 083 was subsequently prepared using similar procedure as Compound 081, but using 4, 4-bis ( (3-butylheptyl) oxy) butyl 5-bromopentanoate. ¹H NMR (400 MHz CDCl₃) δ 5.62-5.21 (m, 4H) , 4.62 (t, 1H) , 4.16-3.82 (m, 4H) , 3.72-3.41 (m, 8H) , 2.45-2.33 (m, 6H) , 2.32-2.06 (m, 8H) , 2.04-1.11 (m, 60H) , 0.91-0.82 (m, 15H) .

Compound 084: nonyl 8- ( (5- (3, 3-bis (octyloxy) propoxy) -5-oxopentyl) (2-hydroxyethyl) amino) octanoate

Synthesis of nonyl 8- ( (2-hydroxyethyl) amino) octanoate

To a solution of nonan-1-ol (5.0 g, 34.7 mmol, 1.0 eq) in DCM (35 mL) was added 8-bromooctanoic acid (9.28 g, 41.6 mmol, 1.2 eq) , EDCI (9.97 g, 52.0 mmol, 1.5 eq) and DMAP (847 mg, 6.93 mmol, 0.2 eq) at 0 ℃, then the mixture was stirred at 25 ℃ for 4 hrs. The reaction mixture was poured into water (50 mL) and extracted with EtOAc (50 mL, 30 mL, 20 mL) , The combined organic layers were washed with brine (30 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 1/0 to 20/1) to give nonyl 8-bromooctanoate (10 g, 28.6 mmol, 82.5%yield) as a colorless oil. ¹H NMR (400 MHz CDCl₃) δ 4.07 (t, J = 6.8 Hz, 2H) , 3.41 (t, J = 6.8 Hz, 2H) , 2.30 (t, J = 7.6 Hz, 2H) , 1.84-1.86 (m, 2H) , 1.62-1.64 (m, 4H) , 1.41-1.44 (m, 2H) , 1.25-1.36 (m, 17H) , 0.89 (t, J = 6.4 Hz, 3H) .

To a solution of nonyl 8-bromooctanoate (1.0 g, 2.86 mmol, 1.0 eq) in EtOH (50 mL) was added 2-aminoethanol (5.25 g, 85.9 mmol, 5.19 mL, 30 eq) at 25 ℃, then the mixture was stirred at 80 ℃ for 20 mins. The reaction mixture was poured into water (50 mL) and extracted with EtOAc (50 mL, 30 mL, 20 mL) , The combined organic layers were washed with brine (30 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to EtOAc/MeOH = 5/1) to give nonyl 8- ( (2-hydroxyethyl) amino) octanoate (0.5 g, 1.52 mmol, 53.0% yield) as a yellow solid.

Synthesis of compound 084

To a solution of 3, 3-bis (octyloxy) propan-1-ol (5.7 g, 18.01 mmol, 2.14 mL, 1.0 eq) in DCM (50 mL) was added 5-bromopentanoic acid (4.89 g, 27.0 mmol, 1.5 eq) , EDCI (5.18 g, 27.0 mmol, 1.5 eq) and DMAP (440 mg, 3.60 mmol, 0.2 eq) at 0 ℃, then the mixture was stirred at 25 ℃ for 1.5 hrs. The reaction mixture was poured into water (50 mL) and extracted with EtOAc (50 mL, 30 mL, 20 mL) , The combined organic layers were washed with brine (30 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 100/1 to 0/1) to give 3, 3-bis (octyloxy) propyl 5-bromopentanoate (8.2 g, 16.6 mmol, 92.2%yield) as a colorless oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 6.0 Hz, 1H) , 4.06-4.18 (m, 3H) , 3.57-3.60 (m, 2H) , 3.40-3.44 (m, 4H) , 2.34 (t, J = 7.2 Hz, 2H) , 1.89-1.96 (m, 4H) , 1.79-1.81 (m, 3H) , 1.55-1.59 (m, 5H) , 1.25-1.36 (m, 22H) , 0.89 (t, J = 6.8 Hz, 6H) .

To a solution of nonyl 8- ( (2-hydroxyethyl) amino) octanoate (0.9 g, 2.73 mmol, 1.0 eq) and 3, 3-bis (octyloxy) propyl 5-bromopentanoate (1.35 g, 2.73 mmol, 12.3 uL, 1.0 eq) in EtOH (18 mL) was added DIEA (706 mg, 5.46 mmol, 951 uL, 2.0 eq) at 25 ℃, then the mixture was stirred at 85 ℃ for 12 hrs. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (10 mL, 5 mL, 5 mL) , The combined organic layers were washed with brine (5 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to EtOAc/MeOH = 5/1) to give compound 084 (500 mg, 686 umol, 25.1%yield) as a colorless oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 6.0 Hz, 1H) , 4.15 (t, J = 6.4 Hz, 2H) , 4.06 (t, J = 6.8 Hz, 2H) , 3.51-3.61 (m, 4H) , 3.39-3.45 (m, 2H) , 2.58 (brs, 2H) , 2.43-2.46 (m, 4H) , 2.28-2.34 (m, 4H) , 1.93-1.97 (m, 2H) , 1.44-1.65 (m, 15H) , 1.28-1.31 (m, 39H) , 0.89 (t, J = 6.4 Hz, 9H) .

Compound 085: 3, 3-bis (octyloxy) propyl 6- ( (2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) hexanoate

To a solution of undecyl 6- ( (2-hydroxyethyl) amino) hexanoate (0.5 g, 1.52 mmol, 1.0 eq) in EtOH (10 mL) was added 3, 3-bis (octyloxy) propyl 6-bromohexanoate (899 mg, 1.82 mmol, 1.2 eq) and DIEA (392 mg, 3.03 mmol, 529 uL, 2.0 eq) at 25℃, then the mixture was stirred at 90℃ for 12 hrs. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 0/1 to EtOAc/MeOH = 5/1) to give compound 085 (0.4 g, 539 umol, 35.5%yield) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 5.6 Hz, 1H) , 4.04-4.16 (m, 4H) , 3.55-3.61 (m, 4H) , 3.39-3.45 (m, 4H) , 2.61-2.63 (m, 2H) , 2.47-2.51 (m, 4H) , 2.28-2.32 (m, 5H) , 1.92-1.97 (m, 2H) , 1.45-1.66 (m, 16H) , 1.26-1.28 (m, 42H) , 0.88 (t, J = 6.8 Hz, 9H) .

Compound 086: 4, 4-bis (octyloxy) butyl 6- ( (2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) hexanoate

Compound 086 was prepared analogously to compound 085 but using 4, 4-bis (octyloxy) butan-1-ol. ¹H NMR (400 MHz CDCl₃) δ 4.64 (t, 1H) , 4.26-4.01 (m, 4H) , 3.65-3.44 (m, 6H) , 2.64-2.50 (m, 6H) , 2.32-2.21 (m, 4H) , 2.00-1.16 (m, 58H) , 0.91-0.81 (m, 9H) .

Compound 087: 2- ( (6- (3, 3-bis (octyloxy) propoxy) -6-oxohexyl) (2-hydroxyethyl) amino) ethyl (9Z, 12Z) -octadeca-9, 12-dienoate

To solution of 3, 3-bis (octyloxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (1 g, 2.11 mmol, 1.0 eq) and 2-bromoethanol (396 mg, 3.17 mmol, 225 uL, 1.5 eq) in MeCN (10 mL) was added K₂CO₃ (438 mg, 3.17 mmol, 1.5 eq) at 25℃, then the mixture was stirred at 90℃ for 12 hr.The reaction mixture was poured into water (50mL) and extracted with EtOAc (5 mL, 3 mL, 20 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give compound 087-1 (1 g, 1.93 mmol, 91.4%yield) as a colorless oil.

To solution of compound 087-1 (0.9 g, 1.74 mmol, 1.0 eq) and linoleic acid (487 mg, 1.74 mmol, 487 uL, 1.0 eq) in DCM (9 mL) was added DMAP (42.5 mg, 348 umol, 0.2 eq) and EDCI (499 mg, 2.61 mmol, 1.5 eq) at 0℃, then the mixture was stirred at 25℃ for 3 hrs. The reaction mixture was poured into water (5 mL) and extracted with EtOAc (5 mL, 3 mL, 2 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Welch Xtimate C1 100 x 30 mm x 5 um; mobile phase: [water (TFA) -ACN] ; B%: 55%-90%, 8 min) to give compound 087 (500 mg) as a colorless oil. The pH value of the reaction was adjusted to around 8-9 with aq. Na₂CO₃ at 0 ℃. The mixture was extracted with EtOAc (5 mL x 4) and the combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give compound 087 (329 mg, 421 umol, 24.2%yield) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 5.33-5.40 (m, 4H) , 4.60 (t, J = 6.0 Hz, 1H) , 4.33 (t, J = 8.4 Hz, 1H) , 4.15 (t, J = 6.4 Hz, 4H) , 3.54-3.60 (m, 4H) , 3.41-3.43 (m, 2H) , 3.25-3.29 (m, 1H) , 2.76-2.81 (m, 4H) , 2.6-2.69 (m, 2H) , 2.53-2.60 (m, 2H) , 2.28-2.35 (m, 4H) , 2.03-2.08 (m, 4H) , 1.93-1.97 (m, 2H) , 1.45-1.68 (m, 12H) , 1.28-1.38 (m, 38H) , 0.87-0.92 (m, 9H) .

Compound 088: 2- ( (6- (3, 3-bis (octyloxy) propoxy) -6-oxohexyl) (3- (dimethylamino) propyl) amino) ethyl (9Z, 12Z) -octadeca-9, 12-dienoate

Synthesis of 3, 3-bis (octyloxy) propyl 6- ( (3- (dimethylamino) propyl) amino) hexanoate

To a solution of 3, 3-bis (octyloxy) propyl 6-bromohexanoate (3 g, 6.08 mmol, 1 eq) and N', N'-dimethylpropane-1, 3-diamine (6.21 g, 60.8 mmol, 7.60 mL, 10.0 eq) in EtOH (20 mL) and MeCN (20 mL) was added at 25 ℃, then the mixture was stirred at 90 ℃ for 20 mins. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (10 mL, 5 mL, 3 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give 3, 3-bis (octyloxy) propyl 6- ( (3- (dimethylamino) propyl) amino) hexanoate (2.4 g, crude) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) . δ 4.58 (t, J = 5.6 Hz, 1H) , 4.13 (t, J = 6.8 Hz, 1H) , 3.54-3.63 (m, 2H) , 3.31-3.45 (m, 4H) , 2.62-2.81 (m, 11H) , 2.35 (t, J = 7.2 Hz, 4H) , 2.23-2.25 (m, 15H) , 1.95 (d, J = 7.2 Hz, 2H) , 1.52-1.72 (m, 12H) , 1.27-1.35 (m, 22H) , 0.88 (t, J = 6.8 Hz, 6H) .

Synthesis of 3, 3-bis (octyloxy) propyl 6- ( (3- (dimethylamino) propyl) (2-hydroxyethyl) amino) hexanoate

To a solution of 3, 3-bis (octyloxy) propyl 6- ( (3- (dimethylamino) propyl) amino) hexanoate (2 g, 3.88 mmol, 1.0 eq) and 2- [tert-butyl (dimethyl) silyl] oxyacetaldehyde (1.02 g, 5.83 mmol, 1.11 mL, 1.5 eq) in DCM (20 mL) at 0 ℃, the mixture was stirred 10 mins, NaBH (OAc) ₃ (1.65 g, 7.77 mmol, 2.0 eq) was added the mixture at stirred at 25 ℃ for 5 hrs. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (10 mL, 5 mL, 5 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give 3, 3-bis (octyloxy) propyl 6- ( (2- ( (tert-butyldimethylsilyl) oxy) ethyl) (3- (dimethylamino) propyl) amino) hexanoate (3.2 g, crude) as a colorless oil. To a solution of 3, 3-bis (octyloxy) propyl 6- ( (2- ( (tert-butyldimethylsilyl) oxy) ethyl) (3- (dimethylamino) propyl) amino) hexanoate (3.2 g, 4.75 mmol, 1.0 eq) in TBAF (1 M, 47.5 mL, 10 eq) at 25 ℃ the mixture was stirred at 25 ℃ for 12 hrs. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (5 mL, 3 mL, 2 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to THF/MeOH = 1/1) to give 3, 3- bis (octyloxy) propyl 6- ( (3- (dimethylamino) propyl) (2-hydroxyethyl) amino) hexanoate (0.728 g, 1.30 mmol, 27.4%yield) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.59 (t, J = 5.6 Hz, 1H) , 4.14 (t, J = 6.4 Hz, 2H) , 3.77 (brs, 1H) , 3.55-3.61 (m, 2H) , 3.39-3.45 (m, 2H) , 3.04 (brs, 1H) , 2.83-2.87 (m, 3H) , 2.74 (s, 6H) , 2.32 (t, J = 7.2 Hz, 2H) , 2.05-2.06 (m, 1H) , 1.92-1.97 (m, 2H) , 1.53-1.68 (m, 7H) , 1.28-1.35 (m, 22H) , 0.88 (t, J = 6.4 Hz, 6H) .

Synthesis of compound 088

To solution of 3, 3-bis (octyloxy) propyl 6- ( (3- (dimethylamino) propyl) (2-hydroxyethyl) amino) hexanoate (0.7 g, 1.25 mmol, 1.0 eq) and linoleic acid (351 mg, 1.25 mmol, 351 uL, 1.0 eq) in DCM (10 mL) was added EDCI (360 mg, 1.88 mmol, 1.5 eq) and DMAP (30.6 mg, 251 umol, 0.2 eq) at 0℃, then the mixture was stirred at 25 ℃ for 3 hrs. The reaction mixture was poured into water (5 mL) and extracted with EtOAc (5 mL, 3 mL, 2 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to EtOAc/MeOH = 1/1) to give compound 088 (560 mg, 681 umol, 54.4%yield) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ5.33-5.39 (m, 4H) , 4.59 (t, J = 5.6 Hz, 1H) , 4.09-4.15 (m, 4H) , 3.55-3.61 (m, 2H) , 3.39-3.45 (m, 2H) , 2.77 (t, J = 6.4 Hz, 2H) , 2.63-2.69 (m, 4H) , 2.44-2.55 (m, 10H) , 2.27-2.31 (m, 4H) , 2.05 (q, J = 6.8 Hz, 4H) , 1.92-1.97 (m, 2H) , 1.76-1.78 (m, 2H) , 1.53-1.66 (m, 9H) , 1.23-1.45 (m, 41H) , 0.87-0.91 (m, 9H) .

Compound 089: 3, 3-bis (octyloxy) propyl 6- ( (5- (dodecyloxy) -5-oxopentyl) (2-hydroxyethyl) amino) hexanoate

To a solution of 3, 3-bis (octyloxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (1.2 g, 2.53 mmol, 1.0 eq) and dodecyl 5-bromopentanoate (885 mg, 2.53 mmol, 12.3 uL, 1.0 eq) in EtOH (15 mL) was added DIEA (655 mg, 5.07 mmol, 882 uL, 2.0 eq) at 25℃, then the mixture was stirred at 90℃ for 12 hrs. The reaction mixture was poured into water (5 mL) and extracted with EtOAc (5 mL, 3 mL, 2 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to Ethyl acetate /MeOH = 5/1) to give compound 089 (595 mg, 801 umol, 31.6%yield) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 5.6 Hz, 1H) , 4.14 (t, J = 6.4 Hz, 2H) , 4.06 (t, J = 6.8 Hz, 2H) , 3.52-3.61 (m, 4H) , 3.41-3.45 (m, 2H) , 2.57 (t, J = 5.6 Hz, 2H) , 2.43-2.49 (m, 4H) , 2.28-2.34 (m, 4H) , 1.93-1.97 (m, 2H) , 1.42-1.67 (m, 20H) , 1.26-1.33 (m, 42H) , 0.89 (t, J = 6.4 Hz, 9H) .

Compound 090: 3, 3-bis (2-cyclohexylethoxy) propyl 6- ( (2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) hexanoate

Compound 090 was prepared analogously to compound 085 starting with 2-cyclohexylethan-1-ol. Compound 090 (99.9%purity) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.59 (t, J = 5.6 Hz, 1H) , 4.14 (t, J = 6.4 Hz, 2H) , 4.06 (J = 6.8 Hz, 2H) , 3.59-3.63 (m, 2H) , 3.53 (t, J = 5.2 Hz, 2H) , 3.45-3.47 (m, 2H) , 2.57 (t, J = 5.2 Hz, 2H) , 2.45 (t, J = 7.2 Hz, 4H) , 2.30 (t, J = 7.2 Hz, 4H) , 1.92-1.97 (m, 2H) , 1.60-1.72 (m, 18H) , 1.42-1.49 (m, 9H) , 1.13-1.37 (m, 28H) , 0.87-0.95 (m, 7H) .

Compound 091: 3, 3-bis ( (7-methyloctyl) oxy) propyl 6- ( (2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) hexanoate

To a solution of 3, 3-bis ( (7-methyloctyl) oxy) propyl 6-bromohexanoate (580 mg, 1.11 mmol, 1.0 eq) and undecyl 6- ( (2-hydroxyethyl) amino) hexanoate (366 mg, 1.11 mmol, 1.0 eq) in EtOH (10 mL) was added DIEA (287 mg, 2.22 mmol, 387 uL, 2.0 eq) at 25 ℃, then the mixture was stirred at 90 ℃ for 12 hrs. The reaction mixture was poured into water (5 mL) and extracted with EtOAc (5 mL, 3 mL, 2 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to EtOAc/MeOH = 5/1) to give compound 091 (350 mg, 454 umol, 40.8%yield) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 5.6 Hz, 1H) , 4.15 (t, J = 6.4 Hz, 2H) , 4.06 (t, J = 6.8 Hz, 2H) , 3.53-3.61 (m, 4H) , 3.40-3.45 (m, 2H) , 2.58 (brs, 2H) , 2.46 (t, J = 6.8 Hz, 4H) , 2.31 (t, J = 7.2 Hz, 4H) , 1.93-1.97 (m, 2H) , 1.47-1.67 (m, 18H) , 1.27-1.35 (m, 34H) , 1.13-1.18 (m, 4H) , 0.86-0.90 (m, 15H) .

Compound 092: 3, 3-bis ( (7-methyloctyl) oxy) propyl 6- ( (5- (dodecyloxy) -5-oxopentyl) (2-hydroxyethyl) amino) hexanoate

To a solution of 3, 3-bis ( (7-methyloctyl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (0.46 g, 917 umol, 1.0 eq) and dodecyl 5-bromopentanoate (320 mg, 917 umol, 12.3 uL, 1.0 eq) in EtOH (10 mL) was added DIEA (237 mg, 1.83 mmol, 319 uL, 2.0 eq) at 25℃, then the mixture was stirred at 90℃ for 12 hrs. The reaction mixture was poured into water (5 mL) and extracted with EtOAc (5 mL, 3 mL, 2 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to Ethyl acetate/MeOH = 5/1) to give compound 092 (155 mg, 201 umol, 21.9%yield) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 5.6 Hz, 1H) , 4.14 (t, J = 6.4 Hz, 2H) , 4.06 (t, J =6.8 Hz, 2H) , 3.52-3.59 (m, 4H) , 3.40-3.45 (m, 2H) , 2.57 (t, J = 5.6 Hz, 2H) , 2.43-2.49 (m, 4H) , 2.31 (q, J = 7.2 Hz, 4H) , 1.94 (q, J = 6.4 Hz, 2H) , 1.46-1.65 (m, 17H) , 1.14-1.35 (m, 32H) , 1.14-1.19 (m, 4H ) , 0.86-0.90 (m, 15H) .

Compound 093: 3, 3-bis ( (7-methyloctyl) oxy) propyl 6- ( (2-hydroxyethyl) (5- ( ( (9Z, 12Z) -octadeca-9, 12-dien-1-yl) oxy) -5-oxopentyl) amino) hexanoate

To a solution of 3, 3-bis ( (7-methyloctyl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (580 mg, 1.16 mmol, 1.0 eq) and [ (9Z, 12Z) -octadeca-9, 12-dienyl] 5-bromopentanoate (745 mg, 1.73 mmol, 1.5 eq) in EtOH (10 mL) was added DIEA (299 mg, 2.31 mmol, 403 uL, 2.0 eq) at 25℃, then the mixture was stirred at 90℃ for 48 hrs. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (10 mL, 5 mL, 3 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to EtOAc/MeOH = 5/1) to give compound 093 (225 mg, 431 umol, 37.3%yield, 96.3%purity) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 5.32-5.41 (m, 4H) , 4.60 (t, J = 5.6 Hz, 1H) , 4.15 (t, J = 6.8 Hz, 2H) , 4.06 (t, J = 6.8 Hz, 2H) , 3.55-3.61 (m, 4H) , 3.40-3.45 (m, 2H) , 2.78 (t, J = 6.4 Hz, 2H) , 2.60 (brs, 2H) , 2.47-2.49 (m, 4H) , 2.31 (q, J = 7.6 Hz, 4H) , 2.06 (q, J = 6.8 Hz, 4H) , 1.95 (q, J = 6.4 Hz, 2H) , 1.52-1.68 (m, 18H) , 1.16-1.37 (m, 31H) , 1.16-1.29 (m, 4H) , 0.86-0.92 (m, 15H) .

Compound 094: 3, 3-bis ( (6-methyloctyl) oxy) propyl 6- ( (5- (dodecyloxy) -5-oxopentyl) (2-hydroxyethyl) amino) hexanoate

Synthesis of 3, 3-bis ( (6-methyloctyl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate

To a solution of 3, 3-bis ( (6-methyloctyl) oxy) propan-1-ol (600 mg, 1.74 mmol, 1.0 eq) and 6-bromohexanoic acid (509 mg, 2.61 mmol, 1.5 eq) in DCM (3.60 mL) was added EDCI (501 mg, 2.61 mmol, 1.5 eq) and DMAP (42.5 mg, 348 umol, 0.2 eq) at 0 ℃ under N2. The reaction was stirred at 20 ℃ for 2 hrs. The residue was diluted with water (5.00 mL) and extracted with EtOAc (10.0 mL *3) . The combined organic layers were washed with brine (5.00 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The crude product on notebook page ET49878-409 (1.7 g) , was combined to ET49878-415 for further purification. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=25/1 to 20/1) . 3- ( (6-methyloctyl) oxy) -3- (octyloxy) propyl 6-bromohexanoate (1.70 g, 3.35 mmol, 44.4%yield) was obtained as yellow oil. ¹H NMR (400 MHz CDCl₃) δ 4.60 (t, J = 5.6 Hz, 1 H) , 4.15 (t, J=6.4 Hz, 2 H) , 3.56 -2.61 (m, 2 H) , 3.39 -3.45 (m, 4 H) , 2.32 (t, J = 7.2 Hz, 2 H) , 1.85 -1.97 (m, 4 H) , 1.46 -1.68 (m, 9 H) , 1.24 -1.36 (m, 14 H) , 1.09 -1.16 (m, 4 H) , 0.84 -0.87 (m, 12 H) .

A mixture of 3- ( (6-methyloctyl) oxy) -3- (octyloxy) propyl 6-bromohexanoate (850 mg, 1.63 mmol, 1.0 eq) , 2-aminoethanol (2.99 g, 48.9 mmol, 2.96 mL, 30 eq) , in EtOH (42.5 mL) was degassed and purged with N₂ for 3 times, and then the mixture was stirred at 70 ℃ for 3 hrs under N2 atmosphere. Two reactions were combined for workup. The residue was diluted with water (20.0 mL) and extracted with EtOAc (20.0 mL, 10.0 ml) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, DCM: MeOH=10/1 to 5/1) . 3, 3-bis ( (6-methyloctyl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (1.30 g, 2.59 mmol, 79.5%yield) was obtained as Black Brown oil. ¹H NMR (400 MHz CDCl₃) δ 4.59 (t, J = 5.6 Hz, 1 H) , 4.11 -4.16 (m, 3 H) , 3.84 -3.87 (m, 2 H) , 3.72 -3.75 (m, 1 H) , 3.55 -3.61 (m, 3 H) , 3.39 -3.45 (m, 3 H) , 2.99 -3.01 (m, 3 H) , 2.84 -2.88 (m, 2 H) , 2.32 (t, J = 7.2 Hz, 2 H) , 2.03 -2.05 (m, 1 H) , 1.92 -1.97 (m, 3 H) , 1.54 -1.79 (m, 12 H) , 1.23 -1.50 (m, 23 H) , 1.09 -1.17 (m, 5 H) , 0.83 -0.87 (m, 12 H) .

Synthesis of Compound 094

Compound 3, 3-bis ( (6-methyloctyl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (300 mg, 598 umol, 1.0 eq) , dodecyl 5-bromopentanoate (251 mg, 717 umol, 1.2 eq) and DIEA (116 mg, 897 umol, 156 uL, 1.5 eq) were taken up into a sealed tube in EtOH (3.00 mL) . The sealed tube was heated at 80 ℃ for 72 hrs. Two reactions were combined for workup. The residue was diluted with water (10.0 mL) and extracted with EtOAc (20.0 mL, 10.0 mL) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Ethyl acetate/MeOH=1/0 to 0/1) . Compound 094 (250 mg, 324 umol, 27.1%yield) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 6.75 Hz, 2 H) , 4.14 (t, J = 6.8 Hz, 2 H) , 4.06 (t, J=6.8 Hz, 2 H) , 3.52 -3.66 (m, 5 H) , 3.41 -3.45 (m, 2 H) , 2.53 -2.59 (m, 2 H) , 2.41 -2.51 (m, 3 H) , 2.26 -2.38 (m, 4 H) , 1.94 -1.96 (m, 2 H) , 1.44 -1.65 (m, 17 H) , 1.25 -1.36 (m, 38 H) , 1.09 -1.14 (m, 5 H) , 0.84 -0.90 (m, 15 H) .

Compound 095: 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 6- ( (5- (dodecyloxy) -5-oxopentyl) (2-hydroxyethyl) amino) hexanoate

To a solution of 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (0.69 g, 1.30 mmol, 1.0 eq) and dodecyl 5-bromopentanoate (455 mg, 1.30 mmol, 12.3 uL, 1.0 eq) in EtOH (7 mL) was added DIEA (337 mg, 2.60 mmol, 454 uL, 2.0 eq) at 25℃, then the mixture was stirred at 90℃ for 48 hrs. The reaction mixture was poured into water (5 mL) and extracted with EtOAc (5 mL, 3 mL, 2 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to EtOAc/MeOH = 5/1) to give compound 095 (375 mg, 469 umol, 36.0%yield, 100%purity) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.59 (t, J = 6.4 Hz, 1H) , 4.14 (t, J = 6.4 Hz, 2H) , 4.06 (t, J = 6.8 Hz, 2H) , 3.59-3.66 (m, 2H) , 3.42-3.55 (m, 4H) , 2.57 (t, J = 5.2 Hz, 2H) , 2.44-2.49 (m, 4H) , 2.28-2.34 (m, 4H) , 1.94 (q, J = 6.4 Hz, 2H) , 1.48-4.65 (m, 17H) , 1.25-1.36 (m, 29H) , 1.12-1.17 (m, 6H) , 0.86-0.90 (m, 21H) .

Compound 096: 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 6- ( (2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) hexanoate

To a solution of undecyl 6- ( (2-hydroxyethyl) amino) hexanoate (500 mg, 1.52 mmol, 1.0 eq) and 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 6-bromohexanoate (834 mg, 1.52 mmol, 1.0 eq) in EtOH (10 mL) was added DIEA (392 mg, 3.03 mmol, 529 uL, 2.0 eq) at 25℃, then the mixture was stirred at 90℃ for 48 hrs. The reaction mixture was poured into water (5 mL) and extracted with EtOAc (5 mL, 3 mL, 2 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to EtOAc/MeOH = 5/1) to give compound 096 (330 mg, 413 umol, 27.2%yield, 100%purity) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.60 (t, J = 5.6 Hz, 1H) , 4.15 (t, J = 6.8 Hz, 2H) , 4.06 (t, J = 6.8 Hz, 2H) , 3.54-3.65 (m, 4H) , 3.44-3.49 (m, 2H) , 2.60 (t, J = 5.2 Hz, 2H) , 2.48 (t, J = 7.2 Hz, 4H) , 2.31 (t, J = 7.6 Hz, 4H) , 1.95 (q, J = 6.4 Hz, 2H) , 1.46-1.68 (m, 17H) , 1.27-1.36 (m, 29H) , 1.08-1.17 (m, 6H) , 0.86-0.90 (m, 21H) .

Compound 097: 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 6- ( (2-hydroxyethyl) (5- ( ( (9Z, 12Z) -octadeca-9, 12-dien-1-yl) oxy) -5-oxopentyl) amino) hexanoate

Compound 097 was prepared analogously to compound 081 but using 3, 3-bis ( (3, 7-dimethyloctyl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate instead of 3, 3-bis ( (3-butylheptyl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate. ¹H NMR (400 MHz CDCl₃) δ5.32 -5.41 (m, 4 H) , 4.61 (t, J = 4.8 Hz, 1 H) , 4.10 -4.17 (m, 4 H) , 3.51 -3.61 (m, 4 H) , 3.40 -3.45 (m, 2 H) , 2.72 -2.79 (m, 4 H) , 2.63 -2.66 (m, 2 H) , 2.50 -2.54 (m, 2 H) , 2.28 -2.33 (m, 4 H) , 2.03 -2.08 (m, 4 H) , 1.91 -1.98 (m, 2 H) , 1.44 -1.67 (m, 11 H) , 1.26 -1.39 (m, 32 H) , 0.82 -0.92 (m, 27 H) .

Compound 098: 3, 3-bis ( (3, 7-dimethyloct-6-en-1-yl) oxy) propyl 6- ( (5- (dodecyloxy) -5-oxopentyl) (2-hydroxyethyl) amino) hexanoate

To a solution of 3, 3-bis ( (3, 7-dimethyloct-6-en-1-yl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (0.88 g, 1.67 mmol, 1.0 eq) and dodecyl 5-bromopentanoate (585 mg, 1.67 mmol, 12.3 uL, 1.0 eq) in EtOH (9 mL) was added DIEA (433 mg, 3.35 mmol, 583 uL, 2.0 eq) at 25℃, then the mixture was stirred at 90℃ for 48 hrs. The reaction mixture was poured into water (5 mL) and extracted with EtOAc (5 mL, 3 mL, 2 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to Ethyl acetate/MeOH = 5/1) to give compound 098 (375 mg, 465 umol, 27.7%yield, 98.4%purity) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 5.10 (t, J = 7.2 Hz, 2H) , 4.60 (t, J = 6.0 Hz, 1H) , 4.14 (t, J = 6.8 Hz, 2H) , 4.06 (t, J = 6.8 Hz, 2H) , 3.59-3.65 (m, 2H) , 3.43-3.55 (m, 4H) , 2.58 (t, J = 5.2 Hz, 2H) , 2.46 (q, J = 7.2 Hz, 4H) , 2.31 (q, J = 7.2 Hz, 4H) , 1.92-2.06 (m, 6H) , 1.56-1.71 (m, 22H) , 1.27-1.50 (m, 29H) , 1.12-1.18 (m, 3H) , 0.87-0.91 (m, 9H) .

Compound 099: 3, 3-bis ( ( (E) -3, 7-dimethylocta-2, 6-dien-1-yl) oxy) propyl 6- ( (5- (dodecyloxy) -5-oxopentyl) (2-hydroxyethyl) amino) hexanoate

To a solution of 3, 3-bis ( ( (E) -3, 7-dimethylocta-2, 6-dien-1-yl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (0.84 g, 1.61 mmol, 1.0 eq) and dodecyl 5-bromopentanoate (562 mg, 1.61 mmol, 12.3 uL, 1.0 eq) in EtOH (8 mL) was added DIEA (416 mg, 3.22 mmol, 561 uL, 2.0 eq) at 25℃, then the mixture was stirred at 90℃ for 48 hrs. The reaction mixture was poured into water (5 mL) and extracted with EtOAc (5 mL, 3 mL, 2 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate = 10/1 to Ethyl acetate /MeOH = 5/1) to give compound 099 (370 mg, 464 umol, 28.8%yield, 99.1%purity) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 5.35 (t, J = 6.4 Hz, 2H) , 5.10 (t, J = 6.4 Hz, 2H) , 4.71 (t, J = 6.0 Hz, 1H) , 4.12-4.18 (m, 4H) , 4.02-4.08 (m, 4H) , 3.53 (t, J = 4.8 Hz, 2H) , 2.58 (t, J = 4.4 Hz, 2H) , 2.46 (q, J = 7.2 Hz, 4H) , 2.31 (q, J = 7.6 Hz, 4H) , 1.97-2.14 (m, 10H) , 1.61-1.68 (m, 24H) , 1.43-1.52 (m, 5H) , 1.27-1.33 (m, 20H) 0.89 (t, J = 6.8 Hz, 3H) .

Compound 100: 3, 3-bis ( ( (Z) -non-2-en-1-yl) oxy) propyl 6- ( (5- (dodecyloxy) -5-oxopentyl) (2-hydroxyethyl) amino) hexanoate

To a solution of 3, 3-bis ( ( (Z) -non-2-en-1-yl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (0.3 g, 603 umol, 1.0 eq) and dodecyl 5-bromopentanoate (337 mg, 964 umol, 1.6 eq) in EtOH (3.00 mL) was added DIEA (117 mg, 904 umol, 157 uL, 1.5 eq) . The mixture was stirred at 90 ℃ for 48 hrs. The two reaction was combined for work up. The residue was diluted with water (10.0 mL) and extracted with EtOAc (20.0 mL, 10.0 mL) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, EtOAc: MeOH=10/1 to 0/1) . Compound 100 (360 mg, 470 umol, 39.0 yield) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 5.51 -5.60 (m, 4 H) , 4.71 (t, J = 5.6 Hz, 1 H) , 4.05 -4.17 (m, 8 H) , 3.55 (s, 2 H) , 2.60 (s, 2 H) , 2.48 (s, 4 H) , 2.28 -2.34 (m, 5 H) , 1.96 -2.09 (m, 7 H) , 1.48 -1.67 (m, 11 H) , 1.26 -1.37 (m, 37 H) , 0.89 (t, J = 6.4 Hz, 9 H) .

Compound 101: 3, 3-bis ( (8-methylnonyl) oxy) propyl 6- ( (5- (dodecyloxy) -5-oxopentyl) (2-hydroxyethyl) amino) hexanoate

3, 3-bis ( (8-methylnonyl) oxy) propyl 6- ( (2-hydroxyethyl) amino) hexanoate (500 mg, 944 umol, 1.0 eq) , dodecyl 5-bromopentanoate (396 mg, 1.13 mmol, 1.2 eq) and DIEA (183 mg, 1.42 mmol, 246 uL, 1.5 eq) were taken up into a sealed tube in EtOH (5.00 mL) . The sealed tube was heated at 80 ℃ for 12 hrs. Two reactions were combined for workup. The residue was diluted with water (10.0 mL) and extracted with EtOAc (20.0 mL, 10.0 mL) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Ethyl acetate/MeOH=1/0 to 0/1) . Compound 101 (0.56 g, 702 umol, 37.2%yield) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.52 (t, J = 6.0 Hz, 1 H) , 4.07 (t, J = 6.4 Hz, 2 H) , 3.98 (t, J = 6.8 Hz, 2 H) , 3.44 -3.53 (m, 4 H) , 3.31 -3.37 (m, 2 H) , 2.50 (t, J=5.2 Hz, 2 H) , 2.35 -2.41 (m, 4 H) , 2.20 -2.26 (m, 4 H) , 1.85 -1.89 (m, 2 H) , 1.34 -1.59 (m, 20 H) , 1.14 -1.24 (m, 38 H) , 1.05 -1.13 (m, 5 H) , 0.78 -0.82 (m, 15 H) .

Compound 102: 3, 3-bis ( (7-methyloctyl) oxy) propyl 6- ( (3- ( (2- (methylamino) -3, 4-dioxocyclobut-1-en-1-yl) amino) propyl) (6-oxo-6- (undecyloxy) hexyl) amino) hexanoate

Synthesis of compound 102-2

A thumb bottle with magnetic stirrer, addition funnel and thermometer. To a solution of 3, 3-bis ( (7-methyloctyl) oxy) propyl 6-bromohexanoate (2.00 g, 3.83 mmol, 1.0 eq) and benzyl (3-aminopropyl) carbamate (3.99 g, 19.2 mmol, 5.0 eq) in EtOH (14.0 mL) was stirred at 50 ℃ for 12 hrs. The residue was diluted with water (10.0 mL) and extracted with EtOAc (50.0 mL, 25.0 mL) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, EtOAc: MeOH=1/0 to 60/1) . Compound 102-1 (1.00 g, 1.54 mmol, 40.2%yield) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 9.42 (s, 1 H) , 7.28 -7.39 (m, 6 H) , 5.46 (s, 1 H) , 5.04 -5.10 (m, 2 H) , 4.58 (t, J = 5.6 Hz, 1 H) , 4.13 (t, J = 6.8 Hz, 2 H) , 3.54 -3.57 (m, 2 H) , 3.22 -3.47 (m, 5 H) , 2.87 -2.95 (m, 2 H) , 2.79 -2.87 (m, 2 H) , 2.28 (t, J = 7.2 Hz, 2 H) , 1.89 -2.14 (m, 7 H) , 1.43 -1.67 (m, 11 H) , 1.24 -1.41 (m, 15 H) , 1.11 -1.21 (m, 4 H) , 0.87 (d, J = 6.8 Hz, 12 H) .

A seal tube equipped with magnetic stirrer, addition funnel and thermometer. To a solution of compound 102-1 (400 mg, 616 umol, 1.0 eq) in EtOH (4.00 mL) was added DIEA (119 mg, 924 umol, 161 uL, 1.5 eq) and undecyl 6-bromohexanoate (323 mg, 924 umol, 1.5 eq) . The mixture was stirred at 90 ℃ for 12 hrs. The residue was diluted with water (20.0 mL) and extracted with EtOAc (50.0 mL, 25.0 mL) . The combined organic layers were washed with brine (10.0 mL) , dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, DCM: MeOH=100/1 to 60/1) . Compound 102-2 (500 mg, 545 umol, 88.4%yield) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.29 -7.42 (m, 5 H) , 5.10 (s, 2 H) , 4.59 (t, J = 5.6 Hz, 1 H) , 4.14 (t, J = 6.4 Hz, 2 H) , 4.05 (t, J = 6.75 Hz, 2 H) , 3.55 -3.60 (m, 2 H) , 3.39 -3.44 (m, 2 H) , 3.26 -3.28 (m, 2 H) , 2.25 -2.56 (m, 10 H) , 1.92 -1.97 (m, 2 H) , 1.45 -1.63 (m, 24 H) , 1.22 -1.39 (m, 35 H) , 1.15 -1.16 (m, 3 H) , 0.83 -0.92 (m, 15 H) .

Synthesis of compound 102

A 100 mL three-necked round bottom flask equipped with magnetic stirrer, addition funnel and thermometer. To a solution of compound 102-2 (500 mg, 545 umol, 1.0 eq) in EtOH (5.00 mL) was added Pd/C (50.0 mg, 10%purity) under N₂. The suspension was degassed under vacuum and purged with H₂ several times. The mixture was stirred under H₂ 15 Psi at 20 ℃ for 72 hrs. The reaction mixture was filtered and the filter cake was washed by EtOH (30.0 mL) . The residue was concentrated in reduced pressure. Without purification. Compound 102-3 (410 mg, 523 umol, 96.0%yield) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.59 (t, J = 6.0 Hz, 2 H) , 4.14 (t, J = 7.6 Hz, 2 H) , 4.05 (t, J = 6.8 Hz, 2 H) , 3.51 -3.65 (m, 2 H) , 3.41 -3.45 (m, 2 H) , 2.71 -2.74 (m, 2 H) , 2.28 -2.46 (m, 9 H) , 1.91 -1.99 (m, 2 H) , 1.49 -1.70 (m, 14 H) , 1.40 -1.47 (m, 3 H) , 1.25 -1.37 (m, 28 H) , 1.09 -1.23 (m, 4 H) , 0.84 -0.90 (m, 12 H) .

Compound 102-3 (410 mg, 523 umol, 1.0 eq) and 3, 4-dimethoxycyclobut-3-ene-1, 2-dione (112 mg, 785 umol, 1.5 eq) were taken up into a microwave tube in THF (4.10 mL) at 25 ℃. The mixture was stirred at 25 ℃ for 1 hrs. Then METHYLAMINE (2 M, 2.62 mL, 10 eq) was added at 25 ℃. The reaction stirred at 25 ℃ for 20 hours. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by column chromatography (SiO₂, DCM: MeOH=60/1 to 50/1) and then product was purified by prep-TLC (SiO₂, DCM: MeOH = 8: 1) . Compound 102 (130 mg, 146 umol, 27.83%yield) was obtained as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.56 -4.63 (m, 1 H) , 4.13 (t, J = 6.4 Hz, 2 H) , 4.06 (t, J = 6.8 Hz, 2 H) , 3.57 -3.68 (m, 4 H) , 3.41 -3.49 (m, 2 H) , 3.25 (s, 3 H) , 2.68 -2.75 (m 2 H) , 2.67 -2.68 (m, 4 H) , 2.32 -2.36 (m, 4 H) , 1.84 -1.94 (m, 4 H) , 1.46 -1.71 (m, 17 H) , 1.23 -1.44 (m, 34 H) , 1.13 -1.23 (m, 4 H) , 0.82 -0.95 (m, 15 H) .

Compound 103: 3, 3-bis (octyloxy) propyl 6- ( (3- ( (2- (methylamino) -3, 4-dioxocyclobut-1-en-1-yl) amino) propyl) (6-oxo-6- (undecyloxy) hexyl) amino) hexanoate

Compound 103 was prepared analogously to compound 102 but using octan-1-ol instead of 7-methyloctan-1-ol. ¹H NMR (400 MHz CDCl₃) δ 4.51 -4.64 (m, 2 H) , 4.13 (t, J = 6.4 Hz, 2 H) , 4.07 (t, J = 6.4 Hz, 2 H) , 3.55 -3.70 (m, 4 H) , 3.41 -3.45 (m, 2 H) , 3.15 -3.28 (s, 3 H) , 2.68 -3.00 (m, 6 H) , 2.29 -2.40 (m, 4 H) , 1.81 -1.96 (m, 4 H) , 1.46 -1.71 (m, 15 H) , 1.23 -1.44 (m, 40 H) , 0.79 -0.95 (m, 9 H) .

Example 2. Production of Nanoparticle Compositions

This example exemplifies the production and characterization of lipid nanoparticles comprising the cationic lipid as synthesized in Example 1. Nanoparticles were prepared by mixing aqueous phase which contains RNA with ethanol phase which contains the lipid components. The two phases were mixed by using a microfluidic mixing process with a standard staggered herringbone mixer.

The ethanol phase was prepared by adding (1) an ionizable cationic lipid, specifically a cationic lipid as prepared in Example 1, (2) a neutral lipid, specifically DSPC, (3) a steroid, specifically cholesterol, and (4) a PEG lipid, specifically DMG-PEG2000 into ethanol (PEG-DMG) . Unless otherwise stated, the ratio of the lipid components was 50: 10: 38.5: 1.5 mol%of ionizable cationic lipid: DSPC: cholesterol: PEG-DMG.

RNA was dissolved in 50 mM sodium citrate buffer (pH=4) at concentrations of 0.1 mg/mL. The weight ratio of the RNA molecules to the ionizable cationic lipid was kept at 1: 10.

The lipid solution (ethanol phase) was injected at 1 mL/min, and aqueous phase was injected at 3 mL/min into the mixer. The volume ratio of the aqueous phase to the ethanol phase was 3: 1.

After mixed, the solution with LNP was subjected to dialysis to remove ethanol and achieve buffer exchange. Dialysis was conducted twice against Tris-HCl Solution (20 mM, pH 7.4) at volumes about 2500 times that of the mixed solution using Slide-A-Lyzer cassettes (Thermo Fisher Scientific Inc., Rockford, Ill. ) with a molecular weight cutoff of 10 KD at 4 ℃overnight. Then 40%sucrose solution was added and the final nanoparticle solution with 8%sucrose were stored at -80 ℃.

A Zetasizer Nano ZS (BeNano, Bettersize) was used to determine the particle size and the polydispersity index (PDI) of the nanoparticle compositions in PBS or Tris-HCl.

Ultraviolet-visible spectroscopy was used to determine the concentration of a therapeutic and/or prophylactic (e.g., RNA) in the nanoparticle compositions. The absorbance spectrum of the solution was recorded. The concentration of the therapeutic and/or prophylactic agent (e.g., RNA) in the nanoparticle compositions was calculated based on the extinction coefficient of the therapeutic and/or prophylactic (e.g., RNA) and on the difference between the absorbance at a wavelength of, for example, 260 nm and the baseline value at a wavelength of, for example, 330 nm.

A QUANT-IT^TM RIBOGREEN RNA assay (Shanghai ShineGene Molecular Biotechnology Co., LTD. ) was used to evaluate the encapsulation of RNA by the nanoparticle composition. The samples were diluted in TE buffer solution (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) , and then 100 μL of the diluted samples were transferred to a polystyrene 96 well plate. The plate was incubated at a temperature of 40 ℃ for 10 minutes. The RIBOGREEN reagent was diluted 1: 200 in TE buffer, and 100 μL of the diluted solution was added to each well. The fluorescence intensity was measured using a microplate reader (Molecular Devices i3max) at an excitation wavelength of, for example, about 488 nm and an emission wavelength of, for example, about 525 nm. The fluorescence values of the reagent blank were subtracted from that of each of the samples and the percentage of free RNA was determined by dividing the fluorescence intensity of the intact sample (without addition of Triton X-100) by the fluorescence value of the disrupted sample (caused by the addition of Triton X-100) .

Example 3. Expression of GFP in Cells Induced by GFP mRNA loaded Sample Formulations

This example shows the study of transfection and expression efficiency of the LNPs including the lipid compounds of present application for delivering GFP mRNA as the payload in two cell lines (HEK293 and HepG2) . The LNP formulations were prepared and characterized according to the procedures as described in Example 2.

Two FDA approved cationic lipids, DLin-MC3-DMA (MC3) and SM-102 were also included as references in the study for comparison. MC3-based LNP is a current standard in the art and includes about 50 mol%MC3, about 10 mol %DSPC, about 38.5 mol%cholesterol, and about 1.5 mol%PEG-DMG. SM-102 was the lipid used in Covid-19 mRNA vaccine.

Table 2 summarizes particle size and PDI value as determined for the formulations prepared with the lipid compounds of the present application and the two references. As shown in Table 2, the choice of compound of formula (I) or (II) dramatically affected the particle size (e.g., diameter) , and polydispersity index (PDI) of the compositions. The tested LNPs had particle size between approximately 100 nm and 300 nm, polydispersity indices between 0.1 and 0.6, and encapsulation efficiency (EE) of about 40-85%.

Table 2. Characteristics of nanoparticle composition encapsulated mGFP

HEK293 and HepG2 cells were placed at a density of 5x10⁵ cells in tissue culture 24-well plates with 1 mL of Eagle's Minimum Essential Medium. LNPs containing a total amount of 500 ng and 1000 ng GFP mRNA were added to the wells respectively and cultured for 6 h. Cells were analyzed by flow cytometry to measure GFP fluorescence intensity to determine the expression level. Data were presented as expression level relative to MC3 LNP at the same concentration. The results are shown in Fig. 2 (HEK293 cells) and Fig. 3 (HepG2 cells) .

As shown in Fig. 2 and Fig. 3, LNP composed of compound 078, 089, 092, and 101 achieved highest GFP expression in both cells. Over 50%of the tested lipid compounds exhibited expression levels higher than MC3 (>1) .

Example 4. Expression of hEPO Induced by Sample Formulations in Mice

To assess potency of expression via the lipids of the present disclosure, the hEPO protein expression level following administration of the nanoparticles of the present disclosure to mice was measured.

Nanoparticle compositions including cholesterol as a structural lipid, DSPC as helper lipid, DMP-PEG as PEG lipid, the compound as prepared in Example 1 as cationic lipid, and hEPO mRNA as payload, were prepared according to or via methods similar to those described in Example 2. The particle size, PDI value and encapsulation efficiencies were determined and shown in Table 3. The particle diameter of the formulations including compound of the disclosure were between approximately 80 nm to 300 nm. The polydispersity indices (PDI) were between 0.10 and 0.45. The encapsulation efficiencies of hEPO mRNA were 60-90%.

The lipid nanoparticles were intravenously administered to CD-1 mice at a dosage of 0.5 mg/kg. The concentration of hEPO in serum was tested at 6h and 24 h after injection. The fold of hEPO concentration at 6h induced by the formulations with different lipids relative to MC3 was shown in Table 3 and Figure 4. As shown by Table 3 and Fig. 4, Compounds 030, 059, 061, 062 and 065 achieved about 4-fold or higher hEPO expression in vivo as compared with MC3, and Compounds 002, 021, 037, 039, 047, 063 and 080 achieved about 2-fold or higher hEPO expression in vivo as compared with MC3.

Table 3. Characteristics of nanoparticle composition encapsulated hEPO

Example 5. Expression of Luciferase Induced by Sample Formulations

To further assess the potency of expression of sample formulations, expression of luciferase following intravenous (IV) or intramuscular (IM) administration of a nanoparticle of the disclosure comprising an mRNA encoding luciferase was measured.

Nanoparticle compositions including cholesterol as a structural lipid, DSPC as helper lipid, DMP-PEG as PEG lipid, the compound as prepared in Example 1 as cationic lipid, and luciferase mRNA as payload, were prepared according to or via methods similar to those described in Example 2. The particle size, PDI value and encapsulation efficiencies were determined and shown in Table 4. The particle diameter of the formulations including compound of the disclosure were between approximately 80 nm to 200 nm. The polydispersity indices (PDI) were between 0.10 and 0.3. The encapsulation efficiencies of Luc mRNA were 70-85%.

Table 4. Characteristics of nanoparticle composition encapsulated Luc mRNA

IV administration: The lipid nanoparticles were intravenously administered to CD-1 Mice (n=3) at a dosage of 0.5 mg/kg and bioluminescence was measured at 3, 6, and 24 hours after injection. SM-102 and MC3 LNP was included for comparison.

IM administration: The lipid nanoparticles were intramuscular administered to CD-1 Mice (n=3) at a dosage of 0.5 mg/kg and bioluminescence was measured at 6, 24, and 48 hours after injection. SM-102 LNP was included for comparison.

IVIS imaging: Those mice were injected 15 min prior to the imaging time point intraperitoneal (i. p. ) with 200 mL D-luciferin K+ salt (diluted in PBS just prior to use to a concentration of 15 mg/mL. Three minutes prior to imaging, mice were placed in an anesthesia-induction chamber fllled with 3%isofluorane and 97%oxygen. Mice were moved into isoflurane-delivering nosecones in the imaging chamber immediately prior to imaging, positioned with ventral side up, and maintained on 3%isoflurane and 97%oxygen. Images were acquired using field of view D and continued to be exposed until 30 s has passed. All images were quantified by region of interest for total flux using Living Image Software Version 4.4 (Perkin Elmer, Waltham, MA) .

The total Flux induced after IV administration of exemplary formulations were shown in Table 5 and Figure 5. The total Flux induced after IM administration of exemplary formulations were shown in Table 6. As shown in Table 5, following IV injection, LNPs formulated with compound 002, 021 and 030 induced higher luminescence compared with SM-102 and MC3 LNPs. However, these lipids induced lower luminescence when administered via IM injection compared with SM-102 LNPs

Table 5. Expression of Luc induced by IV administration of Sample formulations.

Table 6. Expression of Luc induced after IM administration of Sample formulations.

Example 6. Expression of hEPO Induced by Sample Formulations (Best Performers) in Rats

To further assess the potency of best performers, the expression of hEPO following administration of the nanoparticle to rats was measured.

Nanoparticle compositions including cholesterol as a structural lipid, DSPC as helper lipid, DMP-PEG as PEG lipid, the compound as prepared in Example 1 as cationic lipid, and EPO mRNA as payload, were prepared according to or via methods similar to those described in Example 2. The particle size, PDI value and encapsulation efficiencies were determined and shown in Table 7. The particle diameter of the formulations including compound of the disclosure were between approximately 80 nm to 200 nm. The polydispersity indices (PDI) were between 0.10 and 0.30. The encapsulation efficiencies of hEPO mRNA were 75-90%.

The lipid nanoparticles were intravenously administered to Sprague Dawley rats at a dosage of 0.15 mg/kg. The concentration of hEPO in rat serum was quantified at 3, 6, 9, 24, and 48 hours after injection. Data was shown in Table 8 and Figure 6. The expression of hEPO for most tested formulations were higher compared with MC3 LNPs. Noticeably, AUC of hEPO for compound 059, 061, 062 and 063 based formulations were approximately 8～10 fold compared with MC3 LNPs in rats.

Table 7. Characteristics of nanoparticle compositions

Table 8. Expression of hEPO induced by Sample formulations in Rats

Example 7. The Liver Toxicity Induced by Sample Formulations

To assess the liver toxicity of sample formulations, the Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) concentrations in serum following administration of the nanoparticle formulations of the present disclosure were measured. Sample formulations including different lipids were administrated intravenously to mice at dosage of 20 and 100 mg/kg for ionizable lipids. The level of the ALT and AST concentration in serum were tested at 24 hours and 48 hours after injection. Results are shown in Table 9 and 10.

Significant increase of AST and ALT were noticed at 100 mg/kg for MC3 LNPs compared with saline group. In comparison, serum ALT and AST concentrations at 24 hours and 48 hours post injection were below 2-fold of the saline group for all tested compounds of the present disclosure at both doses, with the only exception of compound 096. This result suggested that tested lipid (002, 021, 030, 096) exhibit no liver toxicity at dose level up to 100 mg/kg.

Table 9. Levels of AST induced by Sample formulations in mice

Table 10. Levels of ALT induced by Sample formulations in mice

Example 8. Liver metabolic stability of Lipid in mice

To evaluate the tissue metabolic stability of lipid of the present disclosure in CD-1 mouse, liver of animals was harvested 24 hours after IV injection the formulations. MC3 LNPs were also tested. Tissue concentration of the ionizable lipids were measured using LC-MS/MS. Approximate percentage of ionizable lipid remaining in liver were calculated. Quantification of Lipidsd by LC-MS/MS: To pre-weighed tissues, Milli-Q water was added (900 uL water per 100 mg tissue) . Tissue was homogenized with an Omni probe homogenizer until uniform. An aliquot of 30 μL Liver homogenate and 300 μL of the ACN (50 ng/mL Metolazone in ACN) were dispensed into a 96-well plate. The mixture was vortexed for 5 minutes and centrifuged at 4000 rpm for 10 minutes. An aliquot of 200 μL of each supernatant was transferred to a deep 96-well plate, injected 1 μL into LC-MS/MS for analysis. Samples were separated with column of Hypersil GOLD (C4, 2.1*50mm) equilibrated with 35%solvent A containing 5 mM formic acid 50%methanol (H₂O: MeOH: FA, 50: 50: 1) and 65%solvent B containing 5 mM formic acid in methanol (MeOH: FA 100: 1) .

Approximate percentage of ionizable lipid remaining in liver at 24 hours post injection was shown in Table 11 (mean value, n=3) . Most tested lipids exhibited better biodegrability in comparison with MC3 with the only exception of compound 002. Compound 047, 055, 058, 062, 065 exhibited rapid liver clearance with less than 10%of lipid remained undegraded in liver at 24 hours.

Table 11. Expression of hEPO induced by Sample formulations in mouse and remaining of lipid in liver tissue.

Example 9. Optimization of composition ratio for sample compounds

In order to further improve the delivery efficiency of payload nucleic acid, mole ratio and composition in LNP formulation were further optimized. Compounds 002 and 021 were used as sample molecules. The percentage of ionizable lipid compound was adjusted between 40 and 60 mol%of the total lipid components. DSPC and DOPE were tested as helper lipid. Cholesterol, sitosterol and stigmasterol were tested as steroid. DPPE-PEG and DMG-PEG were tested as polymer conjugated lipid. mEPO was used as nucleic acid payload in order to quantify the expression level in animals.

The components and mole ratio in sample formulations were listed in Table 12. LNPs were prepared and characterized in analogous to the protocol describe in Example 2. The particle size, PDI value and encapsulation efficiencies were shown in Table 12. The particle diameter of the formulations was between approximately 80 nm to 200 nm. PDI were between 0.10 and 0.3. The encapsulation efficiencies of hEPO mRNA were 50-90%. To evaluate the in vivo delivery efficiency, the LNPs were intravenously administered to CD-1 mice at a dosage of 0.5 mg/kg. Serum level hEPO at 6 hours and 24 hours post injection was measured using ELISA, and the results were shown in Table 12.

The ratio and compositions of F2 was the same with the formulation described in Example 2. F4 and F8 yielded higher hEPO level at 6h post injection. Both F4 and F8 have lipid ratio of 10: 48.5: 40: 1.5 (helper lipid: Cholesterol: ionizable lipid: DMG-PEG) .

Table 12. The optimization of Sample Formulations

Example 10. In vivo gene editing in mice by sample formulations with different ionizable compounds

AaCas12b Variant mRNA and gRNA Cargos

To evaluate the efficacy of the lipid molecules provided in the present disclosure, the editing efficiency on targeted gene of these lipids were tested by delivering the CRISPR/Cas12b system targeting TTR genes as an example.

A variant derived from Alicyclobacillus acidiphilus Cas12b (AaCas12b variant) and having an amino acid sequence of SEQ ID NO: 1 was used in this example. The AaCas12b variant mRNA cargo was prepared by in vitro transcription.

Capped and polyadenylated AaCas12b variant mRNA was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. The plasmid DNA containing a T7 promoter and a 100 nt poly (A/T) region can be linearized by incubating at 50° C for 2 hours with BspQI at the following conditions: 200 ng/μL plasmid, 2 U/μL BspQI (NEB) , and 1× reaction buffer. The BspQI can be inactivated by heating the reaction at 80° C for 20 minutes. The linearized plasmid can be purified from enzyme and buffer salts using DNA Clean &Concentrator-5 (Zymo Research, D4013) and analyzed by agarose gel to confirm linearization. The IVT reaction to generate AaCas12b variant modified mRNA can be performed by incubating at 37° C for 4 hours in total 20 μL system containing: 1 μg linearized plasmid; 6 mM ATP, 5mM each of GTP, CTP, and N1-methyl pseudo-UTP (Absin, abs60156) ; 4mM Cap Analog; 2μL T7 RNA polymerase Mix and 2μL 10X T7 CleanCap Reagent AG Reaction Buffer (T7 mRNA Kit withReagent AG, NEB E2080S) . After the 2-hour incubation, 2μL DNase I was added and the reaction was incubated for an additional 30 minutes to remove the DNA template. The AaCas12b variant mRNA was purified with Oligo Clean &Concentrator^TM (Zymo Research, D4060) .

The sgRNA (SEQ ID NO: 2) was chemically synthesized and optionally sourced from a commercial supplier.

LNPs

These LNPs were formulated at a 1: 1 w/w ratio of single guide RNA and AaCas12b variant mRNA using methods similar to those described in Example 2. Molar concentrations of lipids in the lipid component of the LNPs are expressed as molar %of ionizable lipids/DSPC/cholesterol/PEG-2k-DMG, e.g. 40/10/47.5/2.5. All LNPs had an N/P ratio of 6. In comparison, a clinical-stage ionizable lipid, CIN16645 (LP01) , which has been used for in vivo gene editing in human, was used as control. The final LNPs were characterized to determine the encapsulation efficiency, polydispersity index (PDI) , and average particle size according to the analytical methods provided above. Analysis results of average particle size, PDI, and encapsulation efficiency of RNA are shown in Table 13.

Table 13. composition and characteristics of nanoparticles

LNP Delivery In Vivo

To evaluate the in vivo gene editing efficacy, C57BL/6J mice, ranging from 6-9 weeks of age, were dosed via the lateral tail vein at dosage of 0.5 mg/kg of total RNA. Animals were euthanized at 7 days by exsanguination via cardiac puncture under isoflurane anesthesia. Liver tissue was collected from each animal for DNA extraction and analysis. Genomic DNA was isolated for NGS analysis as described below.

NGS Sequencing

In brief, to quantitatively determine the efficiency of editing at the target location in the genome, genomic DNA was isolated and deep sequencing was utilized to identify the presence of insertions and deletions introduced by gene editing. PCR primers (Forward primer as shown by SEQ ID NO: 3, and reverse primer as shown by SEQ ID NO: 4) were designed around the target site (TTR) , and the genomic area of interest was amplified. Additional round of PCR was utilized to add the necessary motifs for Illumina sequencing (384 CDI Primer for 12412ES03) . The amplicons were sequenced on Novaseq S4 (Illumina) . The reads were analyzed on Cas-Analyzer and indel frequency were calculated.

The editing percentage (e.g., the “editing efficiency” or “percent editing” ) is defined as the total number of sequence reads with insertions or deletions over the total number of sequence reads, including wild type.

FIG. 7 shows editing percentages in mouse liver as measured by NGS. As shown in FIG. 7 and Table 14, in vivo liver editing percentages range from 12.6%to 33.1%for male animals, and range from 28.4%to 53.5%for female animals. In comparison, LNP comprising CIN16645 only achieved 9.4%and 21.7%in male and female animals. All tested compounds demonstrated higher editing efficiency as compared with CIN16645. Compound 021, 061, 062 and 065 achieved the highest editing efficiency.

Table 14. TTR Editing in Mouse Liver Measured by NGS

Example 11. In vivo gene editing in mice by sample formulations with different component ratios

To further assess the in vivo gene editing efficiency of sample compound with different lipid composition ratios, LNPs comprising compound 061 and compound 062, respectively with different lipid mole ratio were prepared. These LNPs were formulated at a 1: 1 w/w ratio of single guide RNA targeting TTR (14-b16 as shown by SEQ ID NO: 2) and AaCas12b mRNA as described in Example 10. The LNPs were assembled using the microfluidic procedure with compositions as described in Table 15. All LNPs had an N/P ratio of 6. Analysis results of average particle size, polydispersity (PDI) , and encapsulation efficiency of RNA are shown in Table 15.

C57BL/6J mice (3 males and 3 females) were dosed intravenously at dosage of 0.3 mg/kg of total RNA. Animals were euthanized at 7 days for NGS analysis. FIG. 8 and Table 16 show editing percentages in mouse liver as measured by NGS. F28 and F29, which both contains ionizable lipid at 40%mol ratio, exhibited highest editing efficiency.

Table 15. Composition and characteristics of nanoparticles

Table 16. TTR Editing in Mouse Liver Measured by NGS

Example 12. In vivo gene editing in mice by sample formulations with different N/P ratios

LNP comprising compound 062 with two different N/P ratios (3 and 6) were prepared. These LNPs were formulated at a 1: 1 w/w ratio of single guide RNA targeting TTR (14-b6, SEQ ID NO: 2) and AaCas12b mRNA. The LNPs were assembled using the microfluidic procedure with compositions as described in Table 17. Analysis results of average particle size, polydispersity (PDI) , and encapsulation efficiency of RNA are shown in Table 17.

Table 17. Composition and characteristics of nanoparticles

C57BL/6J mice (3 males and 3 females) were dosed intravenously at dosage of 0.5 mg/kg of total RNA. Animals were euthanized at 7 days for NGS analysis. FIG. 9 and Table 17 show editing percentages in mouse liver as measured by NGS. Higher editing efficiency was achieved at N/P=6 as compared with N/P=3 with two different lipid composition ratios.

Table 18. TTR Editing in Mouse Liver Measured by NGS

Example 13. In vivo gene editing dose response study in mice by sample formulations

A dose response experiment was performed in vivo with LNP comprising compound 062. Cas12b variant mRNA was formulated as LNPs with guide RNAs (SEQ ID NO: 2) targeting TTR. These LNPs were formulated at a 1: 1 w/w ratio of single guide RNA and AaCas12b mRNA. All LNPs had an N/P ratio of 6. The LNPs were assembled using the microfluidic procedure with compositions as described in Table 15. Analysis results of average particle size, polydispersity (PDI) , and encapsulation efficiency of RNA are shown in Table 19.

Table 19. Composition and characteristics of nanoparticles

The lipid nanoparticles encapsulating CRISPR/Cas12b&sgRNA were intravenously administered to C57BL/6J Mice at dosage of 0.3, 1 and 3 mg/kg (n=2) . After 168 hours, the mice were sacrificed and liver was isolated to assess editing efficiency. Animals were euthanized at 7 days for NGS analysis. FIG. 10 shows editing percentages in mouse liver as measured by NGS. Dose-dependent editing was observed for all three LNPs.

Claims

An ionizable lipid having formula (I) , or a salt, tautomer, or stereoisomer thereof,

wherein:

1) m and p are independently selected from any integer ranging from 3 to 8;

2) n is selected from any integer ranging from 2 to 4;

3) X is a bond, -C (O) O-, or a biodegradable group;

4) R₁ is a hydrogen bond donor-containing group or hydrogen bond acceptor-containing group;

5) both of R₂ are same and selected from C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₃-C₁₂ cycloalkyl and substituted C₃-C₁₂ cycloalkyl and combinations of thereof;

6) R₃ is: (a) selected from C₄-C₂₂ alkyl, substituted C₄-C₂₂ alkyl, C₄-C₂₂ alkenyl, substituted C₄-C₂₂ alkenyl, C₃-C₁₂ cycloalkyl, substituted C₃-C₁₂ cycloalkyl and combinations of thereof; or (b) an acetal group ofwherein both of R₄ are same and selected from C₁-C₁₆ alkyl, substituted C₁-C₁₆ alkyl, C₂-C₁₆ alkenyl, substituted C₂-C₁₆ alkenyl, C₃-C₁₂ cycloalkyl, substituted C₃-C₁₂ cycloalkyl and combinations of thereof.
The ionizable lipid according to claim 1, wherein having formula (II) , or a salt, tautomer, or stereoisomer thereof,

wherein q is selected from any integer ranging from 2 to 4.
The ionizable lipid according to claim 1 or 2, wherein R₂ is C₇-C₁₂ alkyl.
The ionizable lipid according to any one of claims 1-3, wherein each R₂ has at least one carbon atom with hydrogen atom (s) substituted by one or two side chains.
The ionizable lipid according to any one of claims 1, 3 and 4, wherein R₃ of said formula (I) is selected from C₆-C₁₂ alkyl, substituted C₆-C₁₂ alkyl, C₆-C₁₂ cycloalkyl, substituted C₆-C₁₂ cycloalkyl, C₆-C₁₂ alkenyl, substituted C₆-C₁₂ alkenyl and combinations of thereof.
The ionizable lipid according to claim 5, wherein R₃ is C₇-C₁₂ alkyl.
The ionizable lipid according to any one of claims 1 and 3-6, wherein R₃ has at least one carbon atom with hydrogen atom (s) substituted by one or two side chains.
The ionizable lipid according to any one of claims 3-7, wherein R₃ is the same with R₂.
The ionizable lipid according to any one of claims 2-4, R₄ of said formula (II) are independently selected from C₂-C₁₂ alkyl, substituted C₂-C₁₂ alkyl, C₃-C₁₂ cycloalkyl, substituted C₃-C₁₂ cycloalkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl and combinations of thereof.
The ionizable lipid according to any one of claims 2-4 and 9, wherein R₄ is C₇-C₁₂ alkyl.
The ionizable lipid according to any one of claims 2-4, 9 and 10, wherein each R₄ has at least one carbon atom with hydrogen atom (s) substituted by one or two side chains.
The ionizable lipid according to any one of claims 2-4 and 9-11, wherein R₄ is the same with R₂.
The ionizable lipid according to any one of claims 4, 7 and 11, wherein said carbon atom with hydrogen atom (s) substituted by one or two side chains is the second or more distant carbon atom counting from the junction.
The ionizable lipid according to any one of claims 4, 7, 11 and 13, wherein said one or two side chains are C₁-C₄ alkyls.
The ionizable lipid according to any one of claims 1-14, wherein each R₂, R₃ and/or each R₄ is independently selected from one of the following formulas:
The ionizable lipid according to any one of claims 1-15, wherein R₁ is selected from hydroxyalkyl group having 1 to 5 carbon atoms, or optionally substituted amino alkylenyl group having 1 to 6 carbon atoms.
The ionizable lipid according to any one of claims 1-16, wherein R₁ is selected from one of the following formulae:

wherein o is selected from 1, 2, 3, 4, and 5.
The ionizable lipid according to any one of claims 1-17, wherein said ionizable lipid is selected from a group consisting of Compound No: 002-011 and 013-103 as shown in Table 1.
The lipid according to any one of claims 1-18, wherein the lipid is an ionizable cationic lipid.
A lipid nanoparticle (LNP) comprising an ionizable cationic lipid of claim 19, a neutral lipid, a PEG lipid and a steroid.
The LNP according to claim 20 wherein the neutral lipid is disteroylphosphatidylcholine (DSPC) or dioleoyl-phosphatidylethanolamine (DOPE) .
The LNP according to claim 20 or claim 21, wherein the PEG lipid is PEG-DMG.
The LNP according to any one of claims 20-22, wherein the steroid is cholesterol, sitosterol or stigmasterol.
The LNP according to any one of claims 20-23, wherein the ionizable cationic lipid is comprised in an amount of about 40-60 mol%, the neutral lipid is comprised in an amount of about 5-20 mol%, the PEG lipid is comprised in an amount of about 0.5-5 mol%, and the steroid is comprised in an amount of about 25-50 mol%.
The LNP according to claim 24, wherein the ionizable cationic lipid is comprised in an amount of about 50 mol%, the neutral lipid is comprised in an amount of about 10 mol%, the PEG lipid is comprised in an amount of about 1.5 mol%, and the steroid is comprised in an amount of about 38.5 mol%.
The LNP according to any one of claims 20-25, further comprising a nucleic acid molecule.
The LNP according to claim 26 wherein the nucleic acid molecule is DNA or RNA.
The LNP according to claim 27, wherein the nucleic acid molecule is plasmid DNA or closed-ended DNA (ceDNA) .
The LNP according to claim 27, wherein the nucleic acid molecule is slected from a group consisting of messenger RNA (mRNA) , guide RNA (gRNA) , a short interfering RNA (siRNA) , an RNA interference (RNAi) molecule, a microRNA (miRNA) , an antagomir, an antisense RNA, a ribozyme, a small hairpin RNA (shRNA) , or a mixture thereof.
A pharmaceutical composition comprising the LNP of any one of claims 20-29, and a pharmaceutically acceptable carrier.
A method of delivering a nucleic acid molecule to a cell in vivo or in vitro, comprising allowing the cell to contact with the LNP of any one of claims 20-29.
A method of treating or preventing a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the LNP of any one of claims 20-29, wherein the LNP comprising a nucleic acid molecule having therapeutic activity.
The method according to claim 32, wherein the LNP is administered by injection.