Ionizable cationic compounds

Abstract

The present disclosure provides novel ionizable lipid compounds of Formula I-Het, Formula I, and Formula II. The present disclosure describes the use of the compounds in the formation of lipid nanoparticles. The lipid nanoparticles can encapsulate therapeutic agents, such as nucleic acids, and these lipid nanoparticles are useful in methods of delivering the therapeutic agents and for treating certain disorders or inducing immune responses.

Description

Technical Field

[0001] The present disclosure relates to cationic and / or ionizable lipid compounds that can be used in combination with other lipid molecules to form lipid nanoparticles for delivering polynucleotides to a subject. Background Art

[0002] Nucleic acid-based therapies have shown great potential in a range of therapeutic applications. However, the delivery of polynucleotides (such as messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotides, plasmids, DNA, etc.) also faces numerous challenges. Free nucleic acids (such as RNA) are susceptible to rapid enzymatic degradation and therefore generally do not persist throughout the body. In addition, due to the negative charge of nucleic acids, they may not be able to effectively cross cellular barriers to enter the necessary intracellular compartments, such as for transcription or to otherwise exert their effects.

[0003] Therefore, lipid particles (such as lipid nanoparticles (LNPs)) have been used to formulate nucleic acids to protect them from degradation and to improve cellular uptake and intracellular delivery. LNPs are typically formed from ionizable cationic lipids and other lipid components (such as neutral lipids, sterols (such as cholesterol) and pegylated lipids). Ionizable cationic lipids are amphiphilic molecules having a lipophilic region containing one or more hydrocarbon groups and a hydrophilic region containing at least one positively charged or ionizable polar head group. Such cationic lipids ionize at an appropriate pH and can then form a positively charged complex with the nucleic acid, making it easier for the nucleic acid to pass through the plasma membrane of the cell and enter the cytoplasm.

[0004] The first approved siRNA therapy, Onpattro (patisiran), was launched several years ago for the treatment of the inherited amyloidosis transthyretin (TTR) amyloidosis. The therapeutic effect of patisiran relies on siRNA-mediated silencing of the TTR gene, thereby preventing the production of the mutant protein and at least halting disease progression. The efficient delivery of siRNA depends on LNP technology. In recent years, nucleic acid vaccines have become a promising approach for the treatment and prevention of various diseases, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is causing the severe infectious coronavirus disease 2019 (COVID-19) that is causing the ongoing global pandemic. mRNA vaccines rely on the delivery of mRNA into the cytoplasm of host cells, where it is transcribed into antigenic proteins to trigger the generation of an immune response. The large size and negative charge of mRNA prevent cellular uptake, and therefore LNPs are again necessary for proper delivery.

[0005] Different ionizable cationic lipids can not only exhibit different physicochemical properties, including their acid dissociation constant (pKa) values, which affect their ability to complex with nucleic acids, but also exhibit different Toxicity properties, and therefore it is apparent to those skilled in the art that there is a need for improved ionizable cationic lipid compounds suitable for forming lipid particles, such as lipid LNPs, for the delivery of nucleic acids and polynucleotides. Summary of the Invention

[0006] Embodiments of the present invention provide novel ionizable cationic lipid compounds and pharmaceutically acceptable salts, prodrugs, and stereoisomers thereof that can form lipid particles, such as LNPs, in the presence of additional lipids (including one or more of neutral lipids, charged lipids, structured lipids, PEGylated lipids, and the like), and can be used to deliver polynucleotides. The present disclosure provides compositions comprising such lipid particles, methods for forming lipid particles, their use in delivering polynucleotides, and methods of using lipid particles in the treatment of a range of diseases, disorders, and conditions.

[0007] The present disclosure provides, in a broad form, a compound of formula I-Het or a compound of formula II:

[0008] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0009] Het is a nitrogen heterocycle;

[0010] X is selected from the group consisting of -S-, -O- and -C-;

[0011] E 1 Straight chain or branched-C 1-30 -alkyl;

[0012] R 1 Selected from the group consisting of -H and formula IA;

[0013] R 2 Selected from linear or branched-C 1-30 - a group consisting of an alkyl group and formula IA;

[0014]

[0015] Formula IA;

[0016] E 2 , if present, is a linear or branched-C 1-30 -alkyl;

[0017] L 1 Select from the group consisting of:

[0018]

[0019] R 3 Selected from -H and linear or branched -C 1-8 - a group consisting of an alkyl group;

[0020] L 2 Selected from –OC(O)- and –C(O)O-;

[0021] W is selected from the group consisting of Formula IIA, Formula IIB, Formula IIC and Formula IID:

[0022]

[0023] R 4 , if present, is selected from -H and linear or branched -C 1-5 - a group consisting of an alkyl group;

[0024] Y is selected from -H, linear or branched -C 1-5 -alkyl and linear or branched-C 1-5 -alkanols; and

[0025] Dashed lines represent bonds to adjacent atoms in compounds of formula I-Het or compounds of formula II.

[0026] Therefore, in one embodiment, the present disclosure provides a compound of Formula I or a compound of Formula II:

[0027]

[0028] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0029] X is selected from the group consisting of -S-, -O- and -C-;

[0030] E 1 Straight chain or branched-C 1-30 -alkyl;

[0031] R 1 Selected from the group consisting of -H and formula IA;

[0032] R 2 Selected from linear or branched-C 1-30 - a group consisting of an alkyl group and formula IA;

[0033]

[0034] Formula IA;

[0035] E 2 , if present, is a linear or branched-C 1-30 -alkyl;

[0036] m and n are each independently an integer from 0 to 3;

[0037] p is an integer from 0 to 2;

[0038] L 1 Select from the group consisting of:

[0039]

[0040] R 3 Selected from -H and linear or branched -C 1-8 - a group consisting of an alkyl group;

[0041] L 2 Selected from –OC(O)- and –C(O)O-;

[0042] W is selected from the group consisting of Formula IIA, Formula IIB, Formula IIC and Formula IID:

[0043]

[0044] R 4 , if present, is selected from -H and linear or branched -C 1-5 - a group consisting of an alkyl group;

[0045] Y is selected from -H, linear or branched -C 1-5 -alkyl and linear or branched-C 1-5 -alkanols; and

[0046] Dashed lines represent bonds to adjacent atoms in compounds of Formula I or Formula II.

[0047] In an embodiment, a lipid nanoparticle (LNP) comprising a compound of Formula I or a compound of Formula II is provided.

[0048] In embodiments, the LNP further comprises a polynucleotide.

[0049] In an embodiment, a pharmaceutical composition comprising such LNPs and at least one pharmaceutically acceptable carrier, diluent or excipient is provided.

[0050] In other embodiments, a method of forming an LNP comprising a compound of Formula I or a compound of Formula II is provided.

[0051] In other embodiments, a method of delivering a polynucleotide within the aforementioned LNP to a cell is provided.

[0052] In a further embodiment, a method of producing a polypeptide of interest in a cell is provided.

[0053] In still further embodiments, methods of treating a disease, disorder, or condition in a subject are provided by administering to a subject in need of such treatment one or more of the aforementioned polynucleotide-containing LNPs or pharmaceutical compositions comprising the LNPs.

[0054] The LNP comprising the polynucleotide or a pharmaceutical composition comprising the LNP can be delivered to a subject as a component of a vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Shown are the results of FACS potency assays of LNP formulations of the invention relative to LKY750 (control) ionizable lipid LNPs.

[0056] Figure 2 Shown are the results of a Cytation 5 potency assay of LNP formulations of the invention relative to LKY750 (control) ionizable lipid LNPs.

[0057] Figures 3A to 3D Shown Graphical representation of data comparing LKY750 (as a known lipid comparator) and an adjuvanted inactivated virus vaccine (aH5N1) with two lipids of the present invention (SL57 and SL60) when used as ionizable lipids within LNPs in the following assays: (A) hemagglutination inhibition (HAI) assay; (B) pseudovirus microneutralization assay; and (C) neuraminidase inhibition enzyme-linked lectin assay (ELLA); and (D) IgG enzyme-linked immunosorbent assay (IgG ELISA). DETAILED DESCRIPTION

[0058] The present disclosure is based on the use of certain novel biodegradable lipid compounds as components of LNPs for the delivery of polynucleotides. These novel lipid compounds exhibit biodegradable groups that can help reduce toxicity or increase clearance.

[0059] In one embodiment, the LNP may be a component of a vaccine, but the therapeutic uses of the compounds described herein and the uses of components of LNPs formed therefrom are not so limited.

[0060] In some embodiments, the formed LNPs may be suitable for delivery of messenger RNA (mRNA).

[0061] In some embodiments, the formed LNPs may be suitable for delivering mRNA as a component of an mRNA vaccine.

[0062] Basic Information

[0063] Throughout the specification, references to individual steps, compositions of matter, groups of steps or groups of compositions of matter are to be taken as covering both one and multiple (i.e. one or more) of such steps, compositions of matter, groups of steps or groups of compositions of matter, unless specifically stated otherwise or the context requires otherwise.

[0064] Those skilled in the art will recognize that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all steps, features, compositions and compounds referred to or indicated in this specification, whether individually or collectively, and any and all combinations or any two or more of said steps or features.

[0065] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for purposes of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0066] Unless specifically stated otherwise, any embodiment of the present disclosure herein should be considered applicable to any other embodiment of the present disclosure mutatis mutandis.

[0067] Unless otherwise defined, all technical and scientific terms used herein should be interpreted as having the same meaning as commonly understood by one of ordinary skill in the art (e.g., in organic synthetic chemistry, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0068] Unless otherwise indicated, any recombinant protein, cell culture, and immunological techniques utilized in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T. A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Vols. 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (eds.), DNA Cloning: A Practical Approach, Vols. 1 to 4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including updates to date), Ed Harlow and David Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J. E. Coligan et al. (eds.) Current Protocols in Molecular Biology. Immunology, John Wiley and Sons (including updates to date).

[0069] The term "and / or," such as "X and / or Y," should be understood to mean "X and Y" or "X or Y," and should be considered to provide clear support for both or either meanings.

[0070] When the terms "from" and "to" indicate a range, they should be understood to mean that the range includes the stated lower and upper limits. For example, "n is an integer from 0 to 3" should be understood to include the case where n does not exist (n is 0), the case where n is 3, and every integer value between 1 and 2.

[0071] Throughout the specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0072] As used herein, the term "derived from" should be taken to indicate that the specified integer may be obtained from a particular source, although not necessarily directly from that source.

[0073] Definition of Selected

[0074] As used herein, the terms "lipid particle," "lipid nanoparticle," or "LNP" are understood to refer to lipid-based particles having at least one nanometer-scale dimension (e.g., 1-1,000 nm) and comprising a compound of any of the formulas described herein. In embodiments, the LNP is formulated in a composition for delivering a polynucleotide to a desired target, such as a cell, tissue, organ, tumor, or the like. The LNP typically comprises an ionizable cationic compound of the disclosure and one or more of a neutral lipid, a charged lipid, a sterol, and a PEGylated lipid. In embodiments, the lipid particle or LNP may be selected from liposomes or vesicles, wherein the aqueous volume is encapsulated by an amphiphilic lipid bilayer (e.g., a monolayer; monolayer membrane or multilayer; multilayer membrane), micellar lipid nanoparticles with a non-aqueous core, and solid lipid nanoparticles. In embodiments, the lipid nanoparticle or LNP may have a structure comprising a single lipid monolayer or bilayer encapsulating a solid phase. In preferred embodiments, the lipid nanoparticle or LNP does not have an aqueous or other liquid phase within its interior.

[0075] "Cationic compound," "ionizable cationic compound," "cationic lipid compound," "ionizable cationic lipid compound," or similar terms refer to a lipid compound having any of the structural formulas described herein that is capable of carrying a positive charge. The ionizable cationic lipids disclosed herein include one or more nitrogen-containing groups that can carry a positive charge. They are ionizable, allowing them to exist in either a positively charged or neutral form, depending on the pH. Ionization of the cationic lipids can affect the surface charge of the lipid nanoparticles under different pH conditions.

[0076] The term "neutral lipid" refers to any of a plurality of lipid species that exist as uncharged or neutral zwitterionic forms at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphatidylcholines such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM).

[0077] The term "charged lipid" refers to any of a number of lipid species that exist as either positively or negatively charged, independent of pH within a useful physiological range (e.g., pH about 3 to pH about 9). Non-limiting examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinates, dialkyltrimethylammonium propanes (including DOTAP and DOTMA), dialkyldimethylaminopropanes, ethylphosphocholine, and dimethylaminoethanecarbamoylsterols.

[0078] As used herein, the term "polynucleotide" refers to a polymer of deoxyribonucleotides or ribonucleotides containing at least two single-stranded or double-stranded forms, and includes DNA, RNA, and hybrids thereof. DNA can be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA can be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), self-amplification messenger RNA (samRNA or saRNA), antisense RNA, miRNA, micRNA, multivalent RNA, dicer substrate RNA, or viral RNA (vRNA), and combinations thereof. Polynucleotides include polynucleotides containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring, and non-naturally occurring, and have binding properties similar to reference polynucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless otherwise specifically limited, the term encompasses polynucleotides containing known analogs of natural nucleotides with binding properties similar to reference polynucleotides. The term "degenerate codon" refers to a nucleic acid sequence that is conserved and modified. Unless otherwise indicated, the specific nucleic acid sequence also implicitly encompasses variants (for example, degenerate codons replace), allelotrope, straight homologue, single nucleotide polymorphism and complementary sequence and the sequence clearly indicated of its conservative modifications. Specifically, degenerate codons can replace sequences that are mixed with bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991) by producing the third position of one or more (or all) selected codons; Ohtsuka et al., J.Biol.Chem., 260:2605-2608 (1985); Rossolini et al., Mol.Cell.Probes, 8:91-98 (1994)) by replacing the sequence and realizing.

[0079] An "effective amount" or "therapeutically effective amount" of a therapeutic polynucleotide is an amount sufficient to produce the desired effect, such as an increase or inhibition of expression of a target sequence as compared to normal expression levels detected in the absence of the polynucleotide. Suitable assays for measuring target gene or target sequence expression include detecting protein or RNA levels using techniques known to those skilled in the art, such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function assays, fluorescence or luminescence assays of appropriate reporter proteins, and phenotypic analysis.

[0080] As used herein, "prodrug" is intended to indicate a compound that can be converted under physiological conditions or by solvolysis into any one or more of the compounds described herein. Thus, the term "prodrug" refers to a pharmaceutically acceptable metabolic precursor of such a compound. A prodrug may be inactive when administered to a subject in need of the prodrug, but may be inactive when administered to a subject in need of the prodrug. The term may also include any covalently bonded carriers that, when such prodrugs are administered to a mammalian subject, Release of the active compound. Prodrugs of the compounds of Formula I or other formulae described herein can be prepared by modifying the functional groups present in the compounds so that these modifications are readily apparent in conventional manipulations or in is cleaved into the parent compound.

[0081] "Pharmaceutically acceptable carrier, diluent, or excipient" or similar terms refers to any ingredient, other than a compound described herein, that is substantially non-toxic and non-inflammatory in a patient (e.g., a vehicle capable of suspending, complexing, or solubilizing the active compound). Excipients may include, for example, anti-adhesives, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water of hydration. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, cross-linked carboxymethylcellulose, cross-linked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-tocopherol), vitamin C, xylitol, and others disclosed herein.

[0082] "Pharmaceutically acceptable salts" include both acid addition salts and base addition salts. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA, 1990, p. 1445, and Journal of Pharmaceutical Science, 66, 2-19 (1977). Acid addition salts are those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, decanoic acid, hexanoic acid, octanoic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, Formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptanoic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc. Base addition salts are those that retain the biological effectiveness and properties of the free acids and are not biologically or otherwise undesirable. These salts are prepared by adding inorganic or organic bases to the free acids. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, etc. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, phenethylbenzylamine, benzathine, ethylenediamine, glucamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like.

[0083] As used herein, "stereoisomer" refers to a compound composed of the same atoms bonded by the same bonds but with different three-dimensional structures, which are not interchangeable. The present invention contemplates various stereoisomers and mixtures thereof and includes "enantiomers," which refer to two stereoisomers whose molecules are non-superimposable mirror images of each other.

[0084] As used herein, the term "biodegradable group" is a group that promotes faster metabolism of lipids in mammals. Esters are suitable biodegradable groups, and the compounds of the present disclosure present two such groups to enhance Biodegradability.

[0085] As used herein, "encapsulation efficiency" refers to the amount of polynucleotide that becomes part of an LNP composition relative to the initial total amount of polynucleotide used to prepare the LNP composition. For example, if 92 mg of polynucleotide out of 100 mg initially provided to the LNP composition are encapsulated in the LNP composition, the encapsulation efficiency can be given as 92%. As used herein, "encapsulation" can refer to completely, substantially, or partially enclosing, restricting, surrounding, or enclosing.

[0086] As used herein, the term "subject" should be taken to mean any animal, such as a mammal, including humans. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0087] As used herein, the term "mammal" includes both humans and livestock animals, such as laboratory animals and domestic pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), as well as non-livestock animals, such as wild animals, etc.

[0088] As used herein, as depicted in the chemical structure, C A-B shall indicate the number of carbon atoms at that position within the chemical structure. In one embodiment, C 1-3 It should be taken to mean that there are one to three carbon atoms (i.e., a carbon chain) at that position within the chemical structure. In one embodiment, C 1-3 It is taken to mean that there is one (—CH—), two (—CH—CH—), or three (—CH—CH—CH—) atoms (i.e., a carbon chain) at that position within the chemical structure. Where C0 is provided, it is understood that the carbon atom is not present but rather forms bonds between adjacent atoms within the chemical structure.

[0089] As used herein, the term "alkyl" encompasses both straight-chain (i.e., linear) and branched hydrocarbon groups. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, pentyl, and hexyl groups. In one example, an alkyl group has from one to thirty carbon atoms (i.e., C 1-30 alkyl) or one to 25 carbon atoms (i.e. C 1-25 alkyl) or one to 20 carbon atoms (i.e. C 1-20 alkyl).

[0090] As used herein, the term "alkanol" encompasses straight-chain (i.e., linear) and branched hydrocarbon groups substituted with an -OH (alcohol) group. Examples of alkanol groups include methanol, ethanol, and propanol groups. In one example, the alkanol group has one to five carbon atoms (i.e., C 1-5 alkanol).

[0091] Compound

[0092] In embodiments, the compounds of the present disclosure may provide advantages over other selected prior art ionizable cationic lipid compounds, including one or more of the following: improved complexation with polynucleotides; favorable pKa properties; improved encapsulation efficiency as part of LNPs; reduced toxicity; improved biodegradability; improved clearance rates; desired N:P ratios when complexed with polynucleotides; desired polydispersity indexes for LNPs containing them; and improved LNP formation.

[0093] The present disclosure provides, in a broad form, a compound of formula I-Het or a compound of formula II:

[0094]

[0095] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0096] Het is a nitrogen heterocycle;

[0097] X is selected from the group consisting of -S-, -O- and -C-;

[0098] E 1 Straight chain or branched-C 1-30 -alkyl;

[0099] R 1 Selected from the group consisting of -H and formula IA;

[0100] R 2 Selected from linear or branched-C 1-30 - a group consisting of an alkyl group and formula IA;

[0101]

[0102] Formula IA;

[0103] E 2 , if present, is a linear or branched-C 1-30 -alkyl;

[0104] L 1 Select from the group consisting of:

[0105]

[0106] R 3 Selected from -H and linear or branched -C 1-8 - a group consisting of an alkyl group;

[0107] L 2 Selected from –OC(O)- and –C(O)O-;

[0108] W is selected from the group consisting of Formula IIA, Formula IIB, Formula IIC and Formula IID:

[0109]

[0110] R 4 , if present, is selected from -H and linear or branched -C 1-5 - a group consisting of an alkyl group;

[0111] Y is selected from -H, linear or branched -C 1-5 -alkyl and linear or branched-C 1-5 -alkanols; and

[0112] Dashed lines represent bonds to adjacent atoms in compounds of formula I-Het or compounds of formula II.

[0113] In embodiments of formula I-Het, Het is selected from 4-, 5-, and 6-membered nitrogen heterocycles.

[0114] In embodiments of formula I-Het, Het is selected from 4-, 5-, and 6-membered nitrogen heterocycles containing one or two ring nitrogen atoms as the only ring heteroatoms.

[0115] In embodiments of formula I-Het, Het is selected from 4-, 5-, and 6-membered nitrogen heterocycles containing one ring nitrogen atom as the only ring heteroatom.

[0116] In embodiments of formula I-Het, Het is selected from a 5- or 6-membered nitrogen heterocycle containing one ring nitrogen atom as the only ring heteroatom.

[0117] In embodiments of formula I-Het, Het is a 6-membered nitrogen heterocycle comprising one ring nitrogen atom as the only ring heteroatom.

[0118] The other moieties of Formula I-Het may be selected from any of the moieties described herein in relation to Formula I, which represent a subset of compounds of Formula I-Het.

[0119] In an embodiment, the present disclosure provides a compound of Formula I or a compound of Formula II:

[0120]

[0121] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0122] X is selected from the group consisting of -S-, -O- and -C-;

[0123] E 1 Straight chain or branched-C 1-30 -alkyl;

[0124] R 1 Selected from the group consisting of -H and formula IA;

[0125] R 2 Selected from linear or branched-C 1-30 - a group consisting of an alkyl group and formula IA;

[0126]

[0127] Formula IA;

[0128] E 2 , if present, is a linear or branched-C 1-30 -alkyl;

[0129] m and n are each independently an integer from 0 to 3;

[0130] p is an integer from 0 to 2;

[0131] L 1 Select from the group consisting of:

[0132]

[0133] R 3 Selected from -H and linear or branched -C 1-8 - a group consisting of an alkyl group;

[0134] L 2 Selected from –OC(O)- and –C(O)O-;

[0135] W is selected from the group consisting of Formula IIA, Formula IIB, Formula IIC and Formula IID:

[0136]

[0137] R 4 , if present, is selected from -H and linear or branched -C 1-5 - a group consisting of an alkyl group;

[0138] Y is selected from -H, linear or branched -C 1-5 -alkyl and linear or branched-C 1-5 -alkanols; and

[0139] Dashed lines represent bonds to adjacent atoms in compounds of Formula I or Formula II.

[0140] In the compounds of Formula I and Formula II, X is selected from the group consisting of -S-, -O-, and -C-. In one embodiment, X is -S-. In one embodiment, X is -O-. In one embodiment, X is -C-.

[0141] Preferably, in the compounds of formula I and formula II, X is -S- or -O-. Most preferably, in the compounds of formula I and formula II, X is -S-.

[0142] In the compounds of formula I and formula II, E 1 Straight chain or branched-C 1-30 -alkyl. In some embodiments, E 1 For straight chain-C 30 -alkyl, -C 25 -alkyl, -C 20 -alkyl, -C 18 -alkyl, -C 16 -alkyl, -C 15 -alkyl, -C 12 -alkyl, -C 10 -alkyl or -C8-alkyl. In one embodiment, E 1 For straight chain-C 1-30 -alkyl. In one embodiment, E 1 For straight chain-C 1-20 -alkyl. In one embodiment, E 1 For straight chain-C 1-18 -alkyl. In one embodiment, E 1 For straight chain-C 1-16 -alkyl. In one embodiment, E 1 For straight chain-C 1-15 -alkyl. In one embodiment, E 1 For straight chain-C 1-12 -alkyl. In one embodiment, E 1 For straight chain-C 1-10 -alkyl. In one embodiment, E 1 For straight chain-C 1-8 -alkyl. In some embodiments, E 1 For straight chain-C 10-30 -alkyl. In some embodiments, E 1 For straight chain-C 15-30 -alkyl. In some embodiments, E 1 For straight chain-C 20-30 -alkyl. In some embodiments, E 1 For branched-C 30 -alkyl, -C 20 -alkyl, -C 18 -alkyl, -C 16-alkyl, -C 15 -alkyl, -C 12 -alkyl, -C 10 -alkyl or -C8-alkyl. In one embodiment, E 1 For branched-C 1-30 -alkyl. In one embodiment, E 1 For branched-C 1-20 -alkyl. In one embodiment, E 1 For branched-C 1-18 -alkyl. In one embodiment, E 1 For branched-C 1-16 -alkyl. In one embodiment, E 1 For branched-C 1-15 -alkyl. In one embodiment, E 1 For branched-C 1-12 -alkyl. In one embodiment, E 1 For branched-C 1-10 -alkyl. In one embodiment, E 1 For branched-C 1-8 -alkyl. In some embodiments, E 1 For branched-C 10-30 -alkyl. In some embodiments, E 1 For branched-C 15-30 -alkyl. In some embodiments, E 1 For branched-C 20-30 -alkyl.

[0143] In the compounds of formula I and formula II, R 1 is selected from the group consisting of -H (i.e., hydrogen) and Formula IA. In one embodiment, R 1 In one embodiment, R 1 It is formula IA.

[0144] In the compounds of formula I and formula II, R 2 Selected from linear or branched-C 1-30 -alkyl and the group consisting of formula IA. In one embodiment, R 2 Straight chain or branched-C 1-30 In some embodiments, R 2 For straight chain-C 30 -alkyl, -C 25 -alkyl, -C 20 -alkyl, -C 18 -alkyl, -C 16 -alkyl, -C 15 -alkyl, -C 12 -alkyl, -C 10-alkyl or -C8-alkyl. In one embodiment, R 2 For straight chain-C 1-30 -alkyl. In one embodiment, R 2 For straight chain-C 1-20 -alkyl. In one embodiment, R 2 For straight chain-C 1-18 -alkyl. In one embodiment, R 2 For straight chain-C 1-16 -alkyl. In one embodiment, R 2 For straight chain-C 1-15 -alkyl. In one embodiment, R 2 For straight chain-C 1-12 -alkyl. In one embodiment, R 2 For straight chain-C 1-10 -alkyl. In one embodiment, R 2 For straight chain-C 1-8 In some embodiments, R 2 For straight chain-C 10-30 In some embodiments, R 2 For straight chain-C 15-30 In some embodiments, R 2 For straight chain-C 20-30 In some embodiments, R 2 For branched-C 30 -alkyl, -C 20 -alkyl, -C 18 -alkyl, -C 16 -alkyl, -C 15 -alkyl, -C 12 -alkyl, -C 10 -alkyl or -C8-alkyl. In one embodiment, R 2 For branched-C 1-30 -alkyl. In one embodiment, R 2 For branched-C 1-20 -alkyl. In one embodiment, R 2 For branched-C 1-18 -alkyl. In one embodiment, R 2 For branched-C 1-16 -alkyl. In one embodiment, R 2 For branched-C 1-15 -alkyl. In one embodiment, R 2 For branched-C 1-12 -alkyl. In one embodiment, R 2 For branched-C 1-10 -alkyl. In one embodiment, R 2 For branched-C1-8 In some embodiments, R 2 For branched-C 10-30 In some embodiments, R 2 For branched-C 15-30 In some embodiments, R 2 For branched-C 20-30 -alkyl. In one embodiment, R 2 It is formula IA.

[0145] In the compounds of formula I and formula II, R 1 and R 2 Can be independently of each other Formula IA. In one embodiment, R 1 is Formula IA, and R 2 Straight chain or branched-C 1-30 -alkyl. In one embodiment, R 1 is Formula IA, and R 2 Straight chain or branched-C 1-30 -alkyl. In one embodiment, R 1 is Formula IA, and R 2 Straight chain or branched-C 1-16 -alkyl. In one embodiment, R 1 is -H, and R 2 In one embodiment, R 1 is -H, and R 2 -C 1-30 -alkyl. In one embodiment, R 1 is -H, and R 2 -C 1-16 -alkyl. In one embodiment, R 1 is Formula IA, and R 2 In one embodiment, R 1 and R 2 At least one of them is of Formula IA.

[0146] Among the compounds of Formula I and Formula II, Formula IA has the following structure:

[0147]

[0148] Formula IA.

[0149] Dashed lines represent bonds to adjacent atoms in compounds of Formula I or Formula II.

[0150] It should be understood that in compounds of Formula I and Formula II, Formula IA may not be present (i.e., R 1 is -H, and R 2 -C1-30 -alkyl). In this case, it can be concluded that in the compounds of formula I and II, E 2 Will not be present. Alternatively, in the compound of formula I and the compound of formula II, in the presence of formula IA, E 2 will also exist.

[0151] In the compound of formula IA, E 2 , if present, is a linear or branched-C 1-30 -alkyl. In one embodiment, E 2 Straight chain or branched-C 1-30 -alkyl. In some embodiments, E 2 For straight chain-C 30 -alkyl, -C 25 -alkyl, -C 20 -alkyl, -C 18 -alkyl, -C 16 -alkyl, -C 15 -alkyl, -C 12 -alkyl, -C 10 -alkyl or -C8-alkyl. In one embodiment, E 2 For straight chain-C 1-30 -alkyl. In one embodiment, E 2 For straight chain-C 1-20 -alkyl. In one embodiment, E 2 For straight chain-C 1-18 -alkyl. In one embodiment, E 2 For straight chain-C 1-16 -alkyl. In one embodiment, E 2 For straight chain-C 1-15 -alkyl. In one embodiment, E 2 For straight chain-C 1-12 -alkyl. In one embodiment, E 2 For straight chain-C 1-10 -alkyl. In one embodiment, E 2 For straight chain-C 1-8 -alkyl. In some embodiments, E 2 For straight chain-C 10-30 -alkyl. In some embodiments, E 2 For straight chain-C 15-30 -alkyl. In some embodiments, E 2 For straight chain-C 20-30 -alkyl. In some embodiments, E 2 For branched-C 30 -alkyl, -C 20 -alkyl, -C 18 -alkyl, -C16 -alkyl, -C 15 -alkyl, -C 12 -alkyl, -C 10 -alkyl, -C9-alkyl or -C8-alkyl. In one embodiment, E 2 For branched-C 1-30 -alkyl. In one embodiment, E 2 For branched-C 1-20 -alkyl. In one embodiment, E 2 For branched-C 1-18 -alkyl. In one embodiment, E 2 For branched-C 1-16 -alkyl. In one embodiment, E 2 For branched-C 1-15 -alkyl. In one embodiment, E 2 For branched-C 1-12 -alkyl. In one embodiment, E 2 For branched-C 1-10 -alkyl. In one embodiment, E 2 For branched-C 1-8 -alkyl. In some embodiments, E 2 For branched-C 10-30 -alkyl. In some embodiments, E 2 For branched-C 15-30 -alkyl. In some embodiments, E 2 For branched-C 20-30 -alkyl. In the embodiment, E 2 Having a structure selected from the group consisting of:

[0152] 、 、 、 、 、 、 、 、 、 、 、 、 and .

[0153] In one embodiment, E 2 Has the following structure:

[0154] .

[0155] Thus, in one embodiment, Formula IA has the following structure:

[0156] .

[0157] In the compounds of Formula I, n and m are each independently an integer from 0 to 3 (i.e., 0, 1, 2, or 3). It should be understood that the integer describes the number of carbon atoms present in the corresponding bracketed structure. In the case where m or n is 0, it should be understood that a direct bond can be formed between adjacent atoms instead. In one embodiment, m is an integer from 0 to 3. In one embodiment, m is 0, 1, 2, or 3. In one embodiment, n is an integer from 0 to 3. In one embodiment, n is 0, 1, 2, or 3.

[0158] In the compounds of Formula I, p is an integer from 0 to 2 (i.e., 0, 1, or 2). It should be understood that the integer describes the number of carbon atoms present in the corresponding bracketed structure. In one embodiment, p is 0. Where p is 0, it should be understood that a direct bond may instead be formed between adjacent atoms. In one embodiment, p is 0, 1, or 2. In one embodiment, p is 1. In one embodiment, p is 2.

[0159] Therefore, the integer values ​​of m, n and p jointly determine the size of the heterocyclic group in the structure of the compound of formula I. In one embodiment, the heterocyclic group in the structure of the compound of formula I is a saturated heterocyclic group. In one embodiment, at least one of any one of m, n and p is an integer greater than 0. In one embodiment, at least two of any one of m, n and p are integers greater than 0. In one embodiment, p is 0, m is 0, n is 0, and the heterocyclic group is a 3-membered heterocyclic group. In one embodiment, p is 0, m is 1, n is 1, and the heterocyclic group is a 5-membered heterocyclic group. In one embodiment, p is 0, m is 1, n is 2, and the heterocyclic group is a 6-membered heterocyclic group. In one embodiment, p is 1, m is 1, n is 1, and the heterocyclic group is a 6-membered heterocyclic group.

[0160] In one embodiment, the heterocyclic group of the compound of Formula I has a structure selected from the group consisting of:

[0161]

[0162] In the above structure, adjacent C 0-3 and L 1 Shown are various heterocyclic groups of compounds of Formula I contemplated by the present disclosure, and while not themselves considered to constitute heterocyclic structures, are shown to indicate the orientation of the heterocyclic groups within the structure of the compounds of Formula I. Dashed lines represent bonds to adjacent atoms in the remainder of the structure of the compounds of Formula I. It will be understood that the integer values ​​of m, n, and p represent an infinite number of iterations of the heterocyclic groups of the compounds of Formula I, and that the above structures are merely exemplary of the broader range of heterocyclic groups contemplated by the present disclosure.

[0163] In one embodiment, the compound of Formula I is selected from the group consisting of:

[0164] 、 、 、 、 、 、 、 、 as well as .

[0165] In one embodiment, the compound of Formula I is selected from the group consisting of:

[0166] 、 、 、 、 、 、 、 、 as well as .

[0167] In the compounds of formula I and formula II, L 1 Selected from the group consisting of:

[0168]

[0169] Dashed lines represent bonds to adjacent atoms in compounds of Formula I or Formula II.

[0170] In one embodiment, L 1 for:

[0171] .

[0172] Therefore, it can be concluded that in one embodiment, the compound of formula I has the following structure:

[0173] .

[0174] In one embodiment, the compound of Formula II has the following structure:

[0175] .

[0176] In one embodiment, the compound of Formula I has the following structure:

[0177] .

[0178] In one embodiment, the compound of Formula II has the following structure:

[0179] .

[0180] In the compounds of formula I and formula II, R 3 , if present, is selected from -H and linear or branched -C 1-8 In one embodiment, R 3 In one embodiment, R 3 For straight chain-C 1-8 -alkyl. In one embodiment, R 3 For branched-C 1-8 -alkyl. Therefore, in one embodiment, L in the compound of formula I or formula II is 1 The structure is selected from the group consisting of:

[0181]

[0182] In one embodiment, R 3 For straight chain-C 1-5 -alkyl. In one embodiment, R 3 For straight chain-C 1-3 -alkyl. In one embodiment, R 3 It is a C1-alkyl group (i.e. -CH3).

[0183] In the compounds of formula I and formula II, L 2 Can be selected from -OC(O)- and -C(O)O-. That is, L 2 is an ester bond, which can exist in either orientation.

[0184] In certain embodiments of compounds of Formula I and Formula II, L 2 is -OC(O)-. That is, the oxygen other than the carbonyl oxygen is directly attached to E 1 .

[0185] In certain embodiments of compounds of Formula I and Formula II, L 2 is -C(O)O-. That is, the carbonyl carbon is directly attached to E 1 .

[0186] In the compound of formula II, W is selected from the group consisting of formula IIA, formula IIB, formula IIC and formula IID:

[0187]

[0188] In each of Formula IIA, Formula IIB, Formula IIC, and Formula IID, the dashed line represents a bond to an adjacent atom in the compound of Formula II.

[0189] In one embodiment, in the compound of formula II, W is formula IIA. Therefore, in one embodiment, the compound of formula II has the following structure:

[0190] .

[0191] In one embodiment, the compound of Formula II has a structure selected from the group consisting of:

[0192] 、 、 ,and .

[0193] In the above structure, it should be understood that the substituent -Y can be substituted at any suitable carbon atom position on the heterocyclic group. It should be understood that the present disclosure contemplates other heterocyclic groups of Formula II, and the heterocyclic groups just depicted above are merely exemplary. In one embodiment, the compound of Formula II has the following structure:

[0194] .

[0195] In one embodiment, in the compound of formula II, W is formula IIB. Therefore, in one embodiment, the compound of formula II has the following structure:

[0196] .

[0197] In one embodiment, the compound of Formula II has a structure selected from the group consisting of:

[0198] 、 、 and .

[0199] In the above structure, it is understood that the substitution Y can be substituted at any suitable carbon atom or nitrogen atom position on the heterocyclic group. It is understood that the present disclosure contemplates other heterocyclic groups of Formula IIB, and the heterocyclic groups just depicted above are merely exemplary. In one embodiment, the compound of Formula II has the following structure:

[0200] .

[0201] In one embodiment, in the compound of formula II, W is formula IIC. Therefore, in one embodiment, the compound of formula II has the following structure:

[0202] .

[0203] In one embodiment, the compound of Formula II has a structure selected from the group consisting of:

[0204] 、 、 and .

[0205] In the above structure, it is understood that the substitution Y can be substituted at any suitable carbon atom or nitrogen atom position on the heterocyclic group. It is understood that the present disclosure contemplates other heterocyclic groups of Formula IIB, and the heterocyclic groups just depicted above are merely exemplary. In one embodiment, the compound of Formula II has the following structure:

[0206] .

[0207] In one embodiment, in the compound of formula II, W is formula IID. Therefore, in one embodiment, the compound of formula II has the following structure:

[0208] .

[0209] In the compound of formula II, R 4 , if present, is selected from -H and linear or branched -C 1-5 In one embodiment, R 4 In one embodiment, R 4 For straight chain-C 1-5 -alkyl. In one embodiment, R 4 For branched-C 1-5 -alkyl. In one embodiment, R 4 - is C1-alkyl (i.e. -CH3). Therefore, in one embodiment, the compound of formula II has the following structure:

[0210] .

[0211] In the compounds of formula I and II, Y is selected from -H, linear or branched -C 1-5 -alkyl and linear or branched-C 1-5 In one embodiment, Y is -H. In one embodiment, Y is a linear or branched -C 1-5In one embodiment, Y is a linear or branched-C 1-5 -alkanol. Therefore, in one embodiment, Y is C1-alkyl (i.e., -CH3), and the compound of formula I has the following structure:

[0212] .

[0213] In one embodiment, Y is C1-alkyl (ie, -CH3), and the compound of Formula II has the following structure:

[0214] .

[0215] The compounds of formula I and formula II may contain any combination of the variables defined above, i.e. X, E 1 、R 1 、R 2 、E 2 (if present), m, n, p, L 1 、R 3 (if present), W, R 4 (if present) and Y, these combinations should be reasonably envisioned by those skilled in the art. Preferably, in all the above structures of Formula I or Formula II or substructures thereof, X is -S- or -O- or -C-, and most preferably, X is -S-.

[0216] In one embodiment, in the compound of formula I and the compound of formula II, X is -S-, and L 1 for:

[0217] .

[0218] In one embodiment, in the compound of formula I and the compound of formula II, X is -O-, and L 1 for:

[0219] .

[0220] In one embodiment, the compound of formula I is:

[0221] .

[0222] In one embodiment, the compound of formula I is:

[0223] .

[0224] In one embodiment, the compound of formula I is:

[0225] .

[0226] In one embodiment, the compound of formula I is:

[0227] .

[0228] In one embodiment, the compound of formula I is:

[0229] .

[0230] In one embodiment, the compound of formula II is selected from the group consisting of:

[0231] ; ; ; ; ; ; ; ;and .

[0232] In one embodiment, the compound of formula II is:

[0233] .

[0234] In one embodiment, the compound of formula II is:

[0235] .

[0236] In one embodiment, the compound of formula II is:

[0237] .

[0238] In one embodiment, the compound of formula II is:

[0239] .

[0240] In one embodiment, the compound of formula II is:

[0241] .

[0242] In one embodiment, the compound of Formula I or the compound of Formula II is selected from the group consisting of:

[0243] ; ; ; ; ; ； ； ； ；

[0244] ； ； ；； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ;and .

[0245] lipid nanoparticles

[0246] The present disclosure provides an LNP for delivering polynucleotides (such as RNA), wherein the LNP comprises a compound of the present disclosure.

[0247] In embodiments, the LNPs have an average diameter of about 30 nm to about 160 nm, about 40 nm to about 160 nm, about 50 nm to about 160 nm, about 60 nm to about 160 nm, about 70 nm to about 160 nm, about 50 nm to about 140 nm, about 60 nm to about 130 nm, about 70 nm to about 120 nm, about 80 nm to about 120 nm, about 90 nm to about 120 nm, about 70 to about 110 nm, about 80 nm to about 110 nm, or about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, or 160 nm. The diameter of the LNPs can be measured by dynamic light scattering (DLS), transmission electron microscopy (TEM), scanning electron microscopy (SEM), or other methods as known in the art.

[0248] In certain embodiments, LNP can be relatively uniform. Dispersity index can be used to represent the uniformity of LNP. Smaller dispersity index, for example, less than 0.3 or less than 0.2, generally represents that particle size distribution is narrower. The dispersity index of the composition of LNP described herein can be from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25. In certain embodiments, the dispersity index of LNP composition can be from about 0 to about 0.20 or 0.05 to 0.20.

[0249] The LNP may contain more than one compound of Formula I or Formula IA to Formula IN or Formula II-A to Formula II-N as desired. For example, more than one such compound may be included to achieve a desired pKa profile.

[0250] The LNP may comprise a compound of formula I and additional cationic and / or ionizable lipids, such as cationic and / or ionizable lipids comprising cyclic or acyclic amines. Such additional cationic and / or ionizable lipids may be selected from the non-limiting group consisting of:

[0251] 3-(Dodecylamino)-N1,N1,4-tridecyl-1-piperazineethylamine (KL10),

[0252] N1-[2-(Dodecylamino)ethyl]-N1,N4,N4-tridecyl-1,4-piperazinediethylamine (KL22),

[0253] 14,25-ditridecane-15,18,21,24-tetraaza-octyltriacontane (KL25),

[0254] 1,2-Dilinoleoyloxy-N,N-dimethylaminopropyl (DLin-DMA),

[0255] 2,2-Dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),

[0256] 4-(Dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriacontadecen-6,9,28,31-tetraen-19-ol (DLin-MC3-DMA),

[0257] 2,2-Dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA),

[0258] 1,2-dioleoyloxy-N,N-dimethylaminopropyl (DODMA),

[0259] 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA),

[0260] (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA (2R)),

[0261] (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA (2S)),

[0262] ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)) and

[0263] 8-[(2-Hydroxyethyl)[6-oxyl-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester.

[0264] In embodiments, the LNP further comprises one or more of a PEG-lipid, a sterol structured lipid, and / or a neutral lipid.

[0265] PEGylated lipids

[0266] In one embodiment, the present disclosure provides an LNP comprising a compound of the present disclosure and a PEGylated lipid.

[0267] It will be apparent to those skilled in the art that reference to PEGylated lipids is to lipids that have been modified with polyethylene glycol. Exemplary PEGylated lipids include, but are not limited to, PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols. For embodiments, PEG lipids include PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipids, and combinations thereof.

[0268] neutral lipids

[0269] In one embodiment, the present disclosure provides an LNP comprising a compound of the present disclosure and a neutral lipid.

[0270] Suitable neutral or zwitterionic lipids for use in the present disclosure will be apparent to those skilled in the art, and in embodiments include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-heneicosanoyl-sn-glycero-phosphocholine ( DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholestyhemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 lysoPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diamidatenoyl-sn-glycero-3-phosphocholine, 1,2-docosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (MEPC), 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonic acid-sn-glycero-3-phosphoethanolamine, 1,2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. The lipids may be saturated or unsaturated.

[0271] Structured lipids

[0272] In one embodiment, the present disclosure provides an LNP comprising a compound of the present disclosure and a structured lipid.

[0273] Exemplary structured lipids include, but are not limited to, cholesterol, coprostanol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatine, tomatin, ursolic acid, and alpha-tocopherol.

[0274] In one embodiment, the structured lipid is a sterol. In one embodiment, the structured lipid is cholesterol. In another embodiment, the structured lipid is campesterol.

[0275] In an embodiment, the LNP comprises an ionizable cationic lipid compound of a compound of the present disclosure; a neutral lipid; a sterol, such as cholesterol; and a PEGylated lipid. The LNP is formulated with a polynucleotide for delivery to a subject.

[0276] polynucleotides

[0277] The compounds of the present disclosure can form complexes with a range of polynucleotides and thus be formulated into LNPs with them, including but not limited to mRNA, siRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomirs / antimirs), messenger RNA interfering complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), etc. In this way, the LNPs and compositions can be used to induce the expression of desired proteins by contacting cells with LNPs containing one or more novel compounds of the present disclosure. and In an alternative embodiment, the LNPs and compositions can be used to reduce the expression of target genes and proteins by contacting cells with LNPs containing one or more novel compounds of the present disclosure. or expression of a target gene, wherein the LNP encapsulates or is associated with a polynucleotide (such as an antisense oligonucleotide or siRNA) that reduces target gene expression.

[0278] Thus, in some embodiments, the polynucleotide is an mRNA encoding a polypeptide of interest, including any naturally occurring, non-naturally occurring, or otherwise modified polypeptide. The polypeptide encoded by the mRNA can be of any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA can have a therapeutic effect when expressed in a cell.

[0279] In other embodiments, the polynucleotide is a siRNA that can selectively knock down or downregulate the expression of a target gene. For example, siRNA can be selected based on administering an LNP composition comprising siRNA to a subject in need thereof to silence a gene associated with a specific disease, illness, or condition. The siRNA can comprise a sequence complementary to an mRNA sequence encoding a target gene or protein. In some embodiments, the siRNA can be an immunomodulatory siRNA.

[0280] In certain embodiments, the polynucleotide is a shRNA or a vector or plasmid encoding the shRNA. After the appropriate construct is delivered to the nucleus, the shRNA can be produced inside the target cell. The constructs and mechanisms associated with shRNA are well known in the relevant art.

[0281] The polynucleotides useful for preparing together with the LNPs into which the ionizable cationic compounds of the present invention are incorporated can include a first region (e.g., a coding region) encoding a target polypeptide through linked nucleosides, a first flanking region (e.g., a 5'-UTR) at the 5' end of the first region, a second flanking region (e.g., a 3'-UTR) at the 3' end of the first region, at least one 5'-cap region, and a 3' stabilizing region. In some embodiments, the polynucleotides further comprise a poly-A region or a Kozak sequence (e.g., in a 5'-UTR). In some cases, the polynucleotides can contain one or more intron sequences that can be excised from the polynucleotides. In some embodiments, the polynucleotides (e.g., mRNA) can include a 5' cap structure, a chain-terminating nucleotide, a stem-loop, a polyA sequence, and / or a polyadenylation signal. Any region in the region of the polynucleotides can include one or more alternative components (e.g., alternative nucleosides). For example, the 3'-stabilizing region can comprise alternative nucleosides, such as L-nucleosides, inverted thymidines, or 2'-O-methyl nucleosides, and / or the coding region, 5'-UTR, 3'-UTR, or cap region can include alternative nucleosides, such as 5-substituted uridines (e.g., 5-methoxyuridine), 1-substituted pseudouridines (e.g., 1-methyl-pseudouridine or 1-ethyl-pseudouridine), and / or 5-substituted cytidines (e.g., 5-methyl-cytidine).

[0282] Exemplary polynucleotides useful for formulating with LNPs incorporating ionizable cationic compounds of the present disclosure include a first region (e.g., a coding region) encoding linked nucleosides of an antigenic polypeptide, a first flanking region (e.g., a 5'-UTR) located at the 5' terminus of the first region, a second flanking region (e.g., a 3'-UTR) located at the 3' terminus of the first region, at least one 5'-cap region, and a 3' stabilizing region.

[0283] Polynucleotides suitable for use with the LNPs of the present disclosure may include one or more naturally occurring components, including any of the canonical nucleotides A (adenosine), G (guanosine), C (cytidine), U (uridine), or T (thymidine). In one embodiment, all or substantially all of the nucleotides comprising (a) 5'-UTR, (b) open reading frame (ORF), (c) 3'-UTR, (d) poly A tail, and any combination of (a, b, c, or d above) comprise the naturally occurring canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine).

[0284] In certain embodiments, the polynucleotides may include one or more alternative components, as described herein, which impart useful properties, including increased stability and / or a lack of substantial induction of the innate immune response of the cells into which the polynucleotides are introduced. For example, the alternative polynucleotides may exhibit reduced degradation in the cells into which the polynucleotides are introduced, relative to corresponding unaltered polynucleotides. These alternative species may improve the efficiency of protein production, the intracellular retention rate of the polynucleotides, and / or the survival rate of the cells contacted, and may have reduced immunogenicity.

[0285] Polynucleotides can be naturally occurring or non-naturally occurring. Polynucleotides can include one or more modified (e.g., altered or substituted) nucleobases, nucleosides, nucleotides, or combinations thereof. Polynucleotides can include any useful modification or alteration, such as modifications or alterations to nucleobases, sugars, or internucleoside linkages (e.g., to phosphate linkages / to phosphodiester linkages / to phosphodiester backbones). In some embodiments, each of the nucleobases, sugars, and internucleoside linkages has one or more alterations.

[0286] A polynucleotide may or may not be uniformly altered along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., purines or pyrimidines, or any one or more or all of A, G, U, C) may or may not be uniformly altered in a polynucleotide or within a given predetermined sequence region thereof.

[0287] Different sugar changes and / or internucleoside bonds (e.g., backbone structures) can be present at different positions in the polynucleotide. It will be appreciated by those skilled in the art that nucleotide analogs or other changes can be present at any position in the polynucleotide so that the function of the polynucleotide is not substantially reduced. The change can also be a 5' end or a 3' end change. In certain embodiments, the polynucleotide includes a change at the 3' end.

[0288] Nucleobase surrogates

[0289] Alternative nucleosides and nucleotides can include alternative nucleobases. The nucleobases of polynucleotides are organic bases, such as purines or pyrimidines, or derivatives thereof. Nucleobases can be canonical bases (e.g., adenine, guanine, uracil, thymine, and cytosine). These nucleobases can be altered or completely replaced to provide polynucleotide molecules with enhanced properties, for example, enhanced stability, such as resistance to nucleases. For example, non-canonical bases or modified bases can include one or more substitutions or modifications, including but not limited to alkyl, aryl, halogen, oxo, hydroxyl, alkoxy, and / or thio substitutions; one or more fusions or ring openings; oxidation; and / or reduction.

[0290] Alternative nucleotide base pairing encompasses not only standard adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between nucleotides and / or alternative nucleotides comprising non-standard or alternative bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonds to form between a non-standard base and a standard base or between two complementary non-standard base structures. An example of such non-standard base pairing is base pairing between the alternative nucleotides inosine and adenine, cytosine, or uracil.

[0291] In some embodiments, the nucleobase is an alternative uracil. Exemplary nucleobases and nucleosides having alternative uracils include pseudouracil (ψ), pyridin-4-one ribonucleoside, 5-azauracil, 6-azauracil, 2-thio-5-azauracil, 2-thiouracil (s2U), 4-thiouracil (s4U), 4-thiopseudouracil, 2-thiopseudouracil, 5-hydroxyuracil (ho5U), 5-aminoallyluracil, 5-halouracil (e.g., 5-iodouracil or 5-bromouracil). Uracil), 3-methyluracil (m3U), 5-methoxyuracil (mo5U), uracil 5-hydroxyacetic acid (cmo5U), uracil 5-hydroxyacetic acid methyl ester (mcmo5U), 5-carboxymethyluracil (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uracil (chm5U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm5U), 5-methoxycarbonylmethyl-uracil (mcm5U), 5-methoxycarbonyl 5-Methylaminomethyl-2-thiouracil (mcm5s2U), 5-aminomethyl-2-thiouracil (nm5s2U), 5-methylaminomethyl-uracil (mnm5U), 5-methylaminomethyl-2-thiouracil (mnm5s2U), 5-methylaminomethyl-2-selenouracil (mnm5se2U), 5-carbamoylmethyl-uracil (ncm5U), 5-carboxymethylaminomethyl-uracil (cmn m5U), 5-carboxymethylaminomethyl-2-thiouracil (cmnm5s2U), 5-propynyl-uracil, 1-propynyl-pseudouracil, 5-taurine methyluracil (τm5U), 1-taurine methyl pseudouracil, 5-taurine methyl-2-thiouracil (τm5s2U), 1-taurine methyl-4-thiopseudouracil, 5-methyluracil (m5U, i.e., with deoxythymine base), 1-methyl pseudouracil (m ψ), 1-ethyl pseudouracil (Et1ψ), 5-methyl-2-thiouracil (m5s2U), 1-methyl-4-thiopseudouracil (m1s4ψ), 4-thio-1-methylpseudouracil, 3-methylpseudouracil (m3ψ), 2-thio-1-methylpseudouracil, 1-methyl-1-deazapseudouracil, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouracil (D), dihydropseudouridine, 5,6-dihydrouracil, 5-methyldihydrouracil (m5D), 2-thiodihydrouracil, 2-thiodihydropseudouridine, 2-methoxyuracil, 2-methoxy-4-thiouracil, 4-methoxypseudouridine, 4-methoxy-2-thiopseudouridine, N1-methylpseudouridine, 3-(3-amino-3-carboxypropyl) uracil (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 ψ), 5-(isopentenylaminomethyl) uracil (inm5U), 5-(isopentenylaminomethyl)-2-thiouracil (inm5s2U), 5,2'-O-dimethyluridine (m5Um), 2-thio-2'-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5 Um), 3,2'-O-dimethyluridine (m3Um) and 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 1-thiouracil, deoxythymidine, 5-(2-methoxycarbonylvinyl)-uracil, 5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thiouracil, 5-carboxymethyl-2-thiouracil, 5-cyanomethyluracil, 5-methoxy-2-thiouracil and 5-[3-(1-E-propyleneamino)]uracil. In one example, the modified uracil is pseudouridine. In one example, the modified uracil is N1-methyl-pseudouridine. ,

[0292] In some embodiments, the nucleobase is a substituted cytosine. Exemplary nucleobases and nucleosides having substituted cytosine include 5-azacytosine, 6-azacytosine, pseudoisocytosine, 3-methylcytosine (m3C), N4-acetylcytosine (ac4C), 5-formylcytosine (f5C), N4-methylcytosine (m4C), 5-methylcytosine (m5C), 5-halocytosine (e.g., 5-iodocytosine), 5-hydroxymethylcytosine (hm5C), 1-methylpseudoisocytosine, pyrrolocytosine, pyrrolopseudoisocytosine, 2-thiocytosine (s2C), 2-thio-5-methylcytosine, 4-thio-pseudoisocytosine, 4-thio-1-methyl-pseudoisocytosine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebulin, 5-methylcytosine (s2C). -aza-zebulin, 5-methyl-zebulin, 5-aza-2-thio-zebulin, 2-thio-zebulin, 2-methoxy-cytosine, 2-methoxy-5-methyl-cytosine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysine (k2C), 5,2'-O-dimethyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethylcytosine (m42Cm), 1-thiocytosine, 5-hydroxycytosine, 5-(3-azidopropyl)cytosine and 5-(2-azidoethyl)cytosine. In one example, the modified cytosine is 5-methylcytosine.

[0293] In some embodiments, the nucleobase is a substituted adenine. Exemplary nucleobases and nucleosides having a substituted adenine include 2-aminopurine, 2,6-diaminopurine, 2-amino-6-halopurine (e.g., 2-amino-6-chloropurine), 6-halopurine (e.g., 6-chloropurine), 2-amino-6-methylpurine, 8-azidoadenine, 7-deazaadenine, 7-deaza-8-azaadenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6 -diaminopurine, 1-methyladenine (m1A), 2-methyladenine (m2A), N6-methyladenine (m6A), 2-methylthio-N6-methyl-adenine (ms2m6A), N6-isopentenyl-adenine (i6A), 2-methylthio-N6-isopentenyl-adenine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycylamino Formyl-adenine (g6A), N6-threonylaminoformyl-adenine (t6A), N6-methyl-N6-threonylaminoformyl-adenine (m6t6A), 2-methylthio-N6-threonylaminoformyl-adenine (ms2g6A), N6,N6-dimethyl-adenine (m62A), N6-hydroxynorvalylaminoformyl-adenine (hn6A), 2-methylthio-N6-hydroxynorvalylaminoformyl-adenine (ms2hn6A), N6-acetyl adenine ( ac6A), 7-methyladenine, 2-methylthioadenine, 2-methoxyadenine, N6,2'-O-dimethyladenosine (m6Am), N6,N6,2'-O-trimethyladenosine (m62Am), 1,2'-O-dimethyladenosine (m1Am), 2-amino-N6-methylpurine, 1-thioadenine, 8-azidoadenine, N6-(19-amino-pentaoxahedronadecyl)-adenine, 2,8-dimethyladenine, N6-formyladenine and N6-hydroxymethyladenine.

[0294] In some embodiments, the nucleobase is a substituted guanine. Exemplary nucleobases and nucleosides having substituted guanine include inosine (I), 1-methyl-inosine (mll), imG, methyl imG, 4-demethyl-imG-14, iso imG2, imG-14 ... Glycosyl-braided glycoside (galQ), mannosyl-braided glycoside (manQ), 7-cyano-7-deaza-guanine (preQ0), 7-aminomethyl-7-deaza-guanine (preQ1), archaea (G+), 7-deaza-8-aza-guanine, 6-thioguanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methylguanine (m7G), 6-thio-7-methylguanine, 7 -methylinosine, 6-methoxyguanine, 1-methylguanine (m1G), N2-methylguanine (m2G), N2,N2-dimethylguanine (m22G), N2,7-dimethylguanine (m2,7G), N2,N2,7-dimethylguanine (m2,2,7G), 8-oxoguanine, 7-methyl-8-oxoguanine, 1-methyl-6-thioguanine, N2-methyl-6-thioguanine, N2,N2-dimethylguanine Methyl-6-thioguanine, N2-methyl-2'-O-methylguanine (m2Gm), N2,N2-dimethyl-2'-O-methylguanosine (m22Gm), 1-methyl-2'-O-methylguanosine (m1Gm), N2,7-dimethyl-2'-O-methylguanosine (m2,7Gm), 2'-O-methylinosine (Im), 1,2'-O-dimethylinosine (mllm), 1-thioguanine and O-6-methylguanine.

[0295] The alternative nucleobase of the nucleotide can independently be a purine, a pyrimidine, a purine analog or a pyrimidine analog. For example, the nucleobase can be a substitute for adenine, cytosine, guanine, uracil or hypoxanthine. In another embodiment, the nucleobase can also include, for example, naturally occurring and synthetic base derivatives including pyrazolo[3,4-d]pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propargyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thio Alkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo (especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines), 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5-triazinone, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazin-2-one, 1,2,4-triazine, pyridazine; or 1,3,5-triazine.

[0296] The polynucleotides that are useful for preparing together with the LNP that comprises compound of the present invention can be prepared according to any available technology known in the art.mRNA can be prepared by, for example, enzymatic synthesis, which provides a process for the template-guided synthesis of RNA molecules according to an engineered DNA template, which is composed of an upstream phage promoter sequence connected to a downstream sequence encoding a target gene. Template DNA for in vitro transcription can be prepared from a variety of sources using appropriate techniques well known in the art, including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, JL and Conn, GL, General protocols for preparation of plasmid DNA template and Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD in RNA invitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods 941st edition Conn GL (ed.), New York, NY Humana Press, 2012).

[0297] Under conditions that support polymerase activity, a linearized DNA template is used in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine, and cytidine triphosphate ribonucleoside (rNTP). RNA transcription is performed while minimizing potential degradation of the resulting mRNA transcripts. Transcription can be performed using a variety of commercially available kits, including but not limited to the RiboMax Large-Scale RNA Production System (Promega), the MegaScript Transcription Kit (Life Technologies), and commercially available reagents including RNA polymerase and rNTPs. Methods of transcription are well known in the art. (See, e.g., Losick, R., 1972, In vitro transcription, Ann Rev Biochem 41 ed. 409-46; Kamakaka, RT and Kraus, WL 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2:11.6:11.6.1-11.6.17; Beckert, B. and Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology 703 ed. (Neilson, H. ed.), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology 530 ed., 101-114; all of which are incorporated herein by reference).

[0298] The desired protein is then purified from unwanted components of the transcription or associated reactions. Transcribed mRNA. Techniques for isolating mRNA transcripts are well known in the art and include phenol / chloroform extraction or precipitation using alcohol in the presence of monovalent cations or using lithium chloride.

[0299] Lipid nanoparticle formation

[0300] LNPs comprising compounds of the present disclosure can be prepared using methods well known in the art of formulation. For example, suitable LNPs can be formed using mixing processes such as microfluidics, including herringbone micromixing and T-junction mixing of two fluid streams, one of which contains the polynucleotide, typically in aqueous solution, and the other has various desired lipid components, typically in ethanol.

[0301] LNPs can then be prepared by combining a compound of Formula I, a phospholipid (such as DOPE or DSPC, which can be purchased from commercial sources including Avanti Polar Lipids, Alabaster, AL), a PEGylated lipid (such as 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol, also known as PEG-DMG, which can be purchased from commercial sources including Avanti Polar Lipids, Alabaster, AL), and a structured lipid / sterol (such as cholesterol, which can be purchased from commercial sources including Sigma-Aldrich) at a concentration of about 50 mM in ethanol. The solution should be refrigerated, for example, at -20°C during storage. The various lipids can be combined to achieve the desired molar ratio and diluted with water and ethanol to the final desired lipid concentration, for example, about 5.5 mM to about 25 mM.

[0302] LNP compositions containing polynucleotides are prepared (as described in the Examples) by combining the above-described lipid solution with a solution containing polynucleotides at a weight ratio of lipid component to polynucleotide of, for example, about 5:1 to about 50:1. The lipid solution can be rapidly injected into the polynucleotide solution using a NanoAssemblr microfluidics system at a flow rate of about 3 ml / min to about 18 ml / min to produce a suspension having a water to ethanol ratio of about 1:1 to about 4:1, or about 2:1 to about 4:1.

[0303] For LNP compositions comprising RNA, a 1.0 mg / ml RNA solution in deionized water can be diluted in 50 mM sodium citrate buffer at a pH of 3 to 6 to form a stock solution.

[0304] As is known in the art, in one example, the LNP composition can be further processed by diluting 10 times in 50 mM citrate buffer at a pH of 6, then using a 300k molecular weight cut-off membrane (mPES) for tangential flow filtration (TFF) until concentrated to the original volume. Subsequently, diafiltration can be used to replace the citrate buffer with a buffer containing 20 mM Tris buffer at a pH of 7.5, 80 mM sodium chloride, and 3% sucrose using 10 times the volume of new buffer. The LNP solution can be concentrated to 5-10 mL, filtered using a 0.2 micron PES syringe filter, dispensed into vials, and frozen at 1 ° C / min using Corning® CoolCell® LX cell freezing container until the sample temperature reaches -80 ° C. The sample can be stored at -80 ° C until needed.

[0305] The above method induces nanoprecipitation and particle formation. Alternative processes including but not limited to T-junction and direct injection can be used to achieve the same nanoprecipitation.

[0306] In some embodiments, the lipid component of the LNP formulation comprises from about 25 mol% to about 60 mol% of a compound of Formula I, from about 2 mol% to about 25 mol% of phospholipids (neutral lipids), from about 18.5 mol% to about 60 mol% of structural lipids (sterols), and from about 0.2 mol% to about 10 mol% of PEGylated lipids, as long as the total mol% does not exceed 100%. In some embodiments, the lipid component of the LNP formulation comprises from about 30 mol% to about 50 mol% of a compound of Formula I, from about 5 mol% to about 20 mol% of phospholipids, from about 30 mol% to about 55 mol% of structural lipids, and from about 1 mol% to about 5 mol% of PEGylated lipids. In a specific embodiment, the lipid component comprises from about 40 mol% of a compound of the present disclosure, from about 10 mol% of phospholipids, from about 48 mol% of structural lipids, and from about 2.0 mol% of PEGylated lipids. In some embodiments, the phospholipid can be DOPE or DSPC. In other embodiments, the PEGylated lipid can be PEG-DMG and / or the structural lipid can be cholesterol.

[0307] The encapsulation efficiency of the polynucleotide in the LNP comprising the compound of the present disclosure can be at least 50%, for example, about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

[0308] Lipid nanoparticle compositions

[0309] LNPs comprising compounds and polynucleotides of the present disclosure can be formulated for administration via any acceptable mode of administration of lipid particles (including LNPs, liposomes, lipid vesicles, and similar lipid-based particles). The pharmaceutical compositions of the present invention can be formulated into solid, semisolid, liquid, or gaseous formulations such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administration of such pharmaceutical LNP compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal administration. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intradermal, intrasternal, or infusion techniques. The composition administered to a subject can be in the form of one or more dosage units, wherein, for example, a tablet or injectable liquid volume can be a single dosage unit. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art; for example, see Remington: The Science and Practice of Pharmacy, 20th ed. (Philadelphia College of Pharmacy and Science, 2000).

[0310] Thus, one embodiment of the present disclosure provides a composition, such as a pharmaceutical composition, comprising LNPs comprising a compound of the present disclosure, in combination with a pharmaceutically acceptable carrier.

[0311] Generally speaking, by "carrier" is meant a solid or liquid filler, binder, diluent, encapsulating material, emulsifier, wetting agent, solvent, suspending agent, coating agent or lubricant that can be safely administered to any subject (e.g., a human being). Depending on the specific route of administration, various acceptable carriers known in the art can be used, such as the examples described in Remington's Pharmaceutical Sciences (Mack Publishing Co. NJ USA, 1991).

[0312] LNPs are useful for parenteral, topical, oral or topical administration, intramuscular administration, aerosol administration, or transdermal administration for prophylactic or therapeutic treatment. In one embodiment, the LNPs are administered parenterally, such as intramuscularly, subcutaneously, or intravenously. In some embodiments, the LNPs are administered intramuscularly.

[0313] The formulation of the LNP to be administered will vary depending on the route of administration and formulation (e.g., solution, emulsion, capsule) selected. Suitable pharmaceutical compositions comprising the LNP to be administered can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcohol / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Various suitable aqueous carriers are known to those skilled in the art, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution, and glycine. Intravenous vehicles can include various additives, preservatives, or liquids, nutrients, or electrolyte supplements (see generally Remington's Pharmaceutical Science, 16th edition, Mack, Ed. 1980). The composition can optionally contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusters and buffers, and toxicity regulators, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The LNPs can be stored in liquid form or can be lyophilized for storage and reconstituted in a suitable carrier prior to use according to lyophilization and reconstitution techniques known in the art.

[0314] When the LNP composition is a vaccine composition, the carrier can be water, typically pyrogen-free water; isotonic saline; or a buffered (aqueous) solution, such as a phosphate, citrate, or other buffered solution. For injection of the LNP vaccine composition, water or preferably a buffered solution, more preferably an aqueous buffered solution, can be used. The buffered solution contains a sodium salt, preferably at least 50 mM sodium salt, a calcium salt, preferably at least 0.01 mM calcium salt, and optionally a potassium salt, such as at least 3 mM potassium salt. In one embodiment, the sodium salt, calcium salt, and optionally potassium salt can be present as their chloride, iodide, or bromide, or as their hydroxide, carbonate, bicarbonate, or sulfate salts. In some embodiments, the salt of injection buffer can be selected from the group consisting of sodium chloride (NaCl), calcium chloride (CaCl), potassium chloride (KCl), potassium chloride (KCl), and potassium chloride (KCl). In some embodiments, the salt of injection buffer can be selected from the group consisting of sodium chloride (NaCl), calcium chloride (Ca ...CaCl). In some embodiments, the salt of injection buffer can be selected from the group consisting of sodium chloride (NaCl), calcium chloride (CaCl), potassium chloride (KCl), and potassium chloride (CaCl). In some embodiments, the salt of injection buffer can be selected from the group consisting of sodium chloride (NaCl), calcium chloride (CaCl), potassium chloride (KCl), and potassium chloride (CaCl). In some embodiments, the salt of injection buffer can be selected from the group consisting of sodium chloride (NaCl), calcium chloride (CaCl), potassium chloride (KCl), and potassium chloride (CaCl). In

[0315] In some embodiments of the vaccine, one or more compatible solid or liquid fillers or diluents or encapsulating compounds suitable for administration to humans may be employed. Pharmaceutically acceptable carriers, fillers, and diluents should have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to a subject. Some examples of compounds that can be used as pharmaceutically acceptable carriers, fillers, or components thereof are sugars, such as, for example, lactose, glucose, trehalose, and sucrose; starches, such as, for example, corn starch and potato starch; glucose; cellulose and its derivatives, such as, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; astragalus powder; malt; gelatin; animal fat; solid glidants, such as, for example, stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as, for example, peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols, such as, for example, polypropylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; and alginic acid.

[0316] When the LNP composition is a vaccine composition, it may further comprise one or more pharmaceutically acceptable adjuvants to enhance the immunostimulatory properties of the composition. An adjuvant can be any compound that is suitable for supporting the administration and delivery of the LNP composition and that can initiate or enhance an immune response of the innate immune system, i.e., a nonspecific immune response.

[0317] Such adjuvants may be selected from any adjuvant known to those skilled in the art and suitable for the specific properties of the vaccine, i.e. for inducing a suitable immune response in a mammal. In an embodiment, the adjuvant may be selected from the group consisting of: MF59 ®(squalene-water emulsion), TDM, MDP, muramyl dipeptide, Pluronic, alum solution, aluminum hydroxide, ADJUMER™ (polyphosphazene); aluminum phosphate gel; algal glucan; algal inulin; aluminum hydroxide gel (alum); high protein-adsorbing aluminum hydroxide gel; low viscosity aluminum hydroxide gel; AF or SPT (squalane (5%), Tween 80 (0.2%), Pluronic L121 (1.25%), phosphate buffer, pH 7.4 emulsion); AVRIDINE™ (propylenediamine); BAY R1005™ ((N-(2-deoxy-2-L-leucylamino-bD-glucopyranosyl)-N-octadecyldodecylamide hydrogen acetate); CALCITRIOL™ (l-α,25-dihydroxyvitamin D3); calcium phosphate gel; CAP™ (calcium phosphate nanoparticles); cholera holotoxin, cholera toxin-Al-protein-AD-fragment fusion protein, cholera toxin B subunit; CRL 1005 (P1205 block copolymer); liposomes containing cytokines; DDA (dimethyldioctadecyl ammonium bromide); DHEA (dehydroepiandrosterone); DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); DOC / alum complex (deoxycholic acid sodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant; γ-inulin; Gerbu adjuvant (a mixture of (i) N-acetylglucosaminyl-(β1-4)-N-acetylmuramoyl-L-alanyl-D-glutamine (GMDP), (ii) dimethyldioctadecyl ammonium chloride (DDA), and (iii) zinc-L-proline salt complex (ZnPro-8)); GM-CSF); GMDP (N-acetylglucosaminyl-(β1-4)-N-acetylmuramoyl-L-alanyl-D-isoglutamine); Imiquimod (1-( (2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine); ImmTher™ (N-acetylglucosaminyl-N-acetylmuramoyl-L-alanine-D-isoglutamine-L-alanine-dipalmitin); DRV (immunoliposomes prepared from dehydrated and rehydrated vesicles); interferon-γ; interleukin-1β; interleukin-2; interleukin-7; interleukin-12; ISCOMS™; ISCOPREP 7.0.3.™; liposomes; LOXORIBINE™ (7-allyl-8-oxyguanosine); LT oral adjuvant (Escherichia coli labile enterotoxin-protoxin); microspheres and microparticles of any composition; MONTANIDE ISA 51™ (purified incomplete Freund's adjuvant); MONTANIDE ISA 720™ (metabolizable oily adjuvant); MPL™ (3-Q-desacyl-4'-monophosphoryl lipid A); MTP-PE and MTP-PE liposomes ((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-acetamide, monosodium salt); MURAMETIDE™ (Nac-Mur-L-alanine-D-glutamine-OCH3); MURAPAL MITINE™ and D-MURAPALMITINE™ (Nac-Mur-L-threonine-D-isoglutamine-sn-glycerol dipalmitoyl); NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles of any composition; NISV (nonionic surfactant vesicles); PLEURAN™ (β-glucan); PLGA, PGA, and PLA (homo- and copolymers of lactic and glycolic acid; microspheres / nanospheres); PLURONIC L121™; PMMA (polymethyl methacrylate); PODDS™ (protein-like microspheres); polyurethane derivatives; poly-rA:poly-rU (polyadenylic acid-polyuridine complex); polysorbate 80 (Tween 80); protein choline esters (Avanti Polar Lipids, Inc., Alabaster, Alabama); STIMULON™ (QS-21); Quil-A (Quil-A saponin); S-28463 (4-amino-oterine-dimethyl-2-ethoxymethyl-1H-imidazo[4,5C]quinoline-1-ethanol); SAF-1™ ("Syntex adjuvant formulation"); Sendai virus proteoliposomes and lipid matrices containing Sendai virus; Span-85 (sorbitan trioleate); Specol (Marcol 52, Span 85, and Tween 85 emulsion); Squalene or Robane® (2,6,10,15,19,23-hexamethyltetracosane and 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosane); Stearoyltyrosine (octadecyltyrosine hydrochloride); Theramid® (N-acetylglucosaminyl-N-acetylmuramoyl-L-alanine-D-isoglutamic acid-L-alanine-dipalmityloxypropylamide); Theronyl-MDP (Termurtide™ or [thrl]-MDP; N -acetylmuramyl-L-threonyl-D-isoglutamine); Ty particles (Ty-VLPs or virus-like particles); Walter-Reed liposomes (liposomes containing lipid A adsorbed on aluminum hydroxide) and lipopeptides, including Pam3Cys, particularly aluminum salts such as Adju-phos, Alhydrogel, Rehydragel; emulsions, including CFA, SAF, IFA, MF59, Provax, TiterMax, Montanide, Vaxfectin; copolymers, including Optivax (CRL1005), L121, Poloaxmer4010), etc.; liposomes, including Stealth, cochleates, including BIORAL; plant adjuvants, including QS21, Quil A, Iscomatrix, ISCOM; adjuvants suitable for co-stimulation, including tomatidine, biopolymers (including PLG, PMM, inulin); microbial adjuvants, including romoside, DETOX, MPL, CWS, mannose, CpG polynucleotide sequence, CpG7909, human TLR 1-10 ligands, mouse TLR 1-13 ligands, ISS-1018, IC31, imidazoquinoline, Ampligen, Ribi529, IMOxine, IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys, flagellin, GPI-anchored protein, LNFPIII / Lewis X, antimicrobial peptides, UC-IV150, RSV fusion protein, cdiGMP; and adjuvants suitable for use as antagonists, including CGRP neuropeptide. In a preferred embodiment, the adjuvant can be the oil-in-water emulsion adjuvant MF59. ®, especially when the vaccine is a flu shot.

[0318] When formulated, the compounds of the present disclosure will be administered in a manner compatible with the dosage form and in a therapeutically / prophylactically effective amount. The dosage range for LNP administration is a dosage range large enough to produce the desired effect. For example, the composition comprises an effective amount of encapsulated or associated RNA, such as mRNA or self-replicating RNA. In one embodiment, the composition comprises a therapeutically effective amount of RNA. In another embodiment, the composition comprises a prophylactically effective amount of RNA.

[0319] The dosage should not be too large to avoid causing adverse side effects. Generally, the dosage will vary with the patient's age, condition, sex, and degree of disease, and can be determined by one skilled in the art. In the event of any complications, the dosage can be adjusted by the individual physician.

[0320] Methods for the preparation of the above-described compounds and compositions are further described herein and / or known in the art.

[0321] Methods of treatment and production of target polypeptides

[0322] Diseases, disorders, and / or conditions caused by or associated with abnormal proteins or polypeptides can be treated by the LNPs of the present disclosure comprising the compounds and polynucleotides of the present disclosure, and can include, but are not limited to, rare diseases, infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0323] The LNP composition can be formulated in a unit dosage form. For any particular patient, the therapeutically effective or prophylactically effective dose will depend on various factors, including the severity and type of the condition being treated; the specific composition employed; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and rate of excretion of the specific pharmaceutical composition employed; the duration of treatment; drugs used in combination with or concurrently with the specific pharmaceutical composition employed; and similar factors well known in the medical art.

[0324] The LNP compositions described herein can be used in combination with one or more other therapeutic agents, prophylactic agents, diagnostic agents, or imaging agents. They can be administered together in a single composition or separately in different compositions.

[0325] The present disclosure provides a method for producing a target polypeptide in a mammalian cell. The method for producing the polypeptide involves contacting the cell with an LNP composition as described herein, wherein the composition comprises an mRNA encoding the target polypeptide. After contacting the cell with the LNP composition, the mRNA can be taken up and transcribed in the cell to produce the target polypeptide.

[0326] The step of contacting the LNP composition containing the mRNA with the cell may involve or result in transfection. The phospholipids contained in the lipid component of the LNP composition can promote transfection and / or increase transfection efficiency, for example, by interacting and / or fusing with the cell membrane or intracellular membrane. Transfection can allow the mRNA to be transcribed within the cell.

[0327] In some embodiments, the LNP compositions described herein can be used therapeutically. For example, the mRNA contained in the LNP composition can encode a therapeutic polypeptide (e.g., in a transcribable region) and produce the therapeutic polypeptide upon contact with and / or entry into (e.g., transfection of) a cell. In other embodiments, the mRNA contained in the LNP composition can encode a polypeptide that improves or enhances immunity in a subject.

[0328] In embodiments, the mRNA contained in the LNP composition may encode a recombinant polypeptide that replaces one or more polypeptides that may be substantially absent from cells contacted with the LNP composition. The one or more substantially absent polypeptides may be missing due to a mutation in the encoding gene or its regulatory pathway. Alternatively, the recombinant polypeptide produced by transcription of the mRNA may antagonize the activity of an endogenous protein present in the cell, on the cell's surface, or secreted by the cell. Antagonistic recombinant polypeptides can be used to counteract the deleterious effects of the activity of an endogenous protein, such as altered activity or localization caused by a mutation. In another alternative, the recombinant polypeptide produced by transcription of the mRNA may indirectly or directly antagonize the activity of a biological moiety present in the cell, on the cell's surface, or secreted from the cell. Antagonized biological moieties may include, but are not limited to, lipids (e.g., cholesterol), lipoproteins (e.g., low-density lipoprotein), nucleic acids, carbohydrates, and small molecule toxins. The recombinant polypeptide produced by transcription of the mRNA may be engineered for intracellular localization, such as to a specific region (e.g., the nucleus), or may be engineered for secretion from the cell or for translocation to the cell's plasma membrane.

[0329] In some embodiments, contacting a cell with an LNP composition comprising mRNA can reduce the cell's innate immune response to an exogenous polynucleotide. A cell can be contacted with a first LNP composition comprising a first amount of a first exogenous mRNA (including a transcribable region), and the level of the cell's innate immune response to the first exogenous mRNA can be determined. Subsequently, the cell can be contacted with a second LNP composition comprising a second amount of the first exogenous mRNA, the second amount being a smaller amount of the first exogenous mRNA than the first amount. Alternatively, the second composition can comprise a first amount of a second exogenous mRNA that is different from the first exogenous mRNA. The steps of contacting the cell with the first and second LNP compositions can be repeated one or more times. Additionally, the efficiency of polypeptide production (e.g., transcription) in the cell can be optionally determined, and the cell can be re-contacted with the first and / or second compositions until the desired protein production efficiency is achieved.

[0330] In some embodiments, the present disclosure provides the use of LNPs comprising compounds and polynucleotides of the present disclosure in the manufacture of a medicament for treating a disease, disorder or condition. The disease, disorder or condition can be as described in any one or more of the embodiments herein.

[0331] The medicament can be used to prevent or treat cancer, infectious diseases, allergies, or autoimmune diseases. In an embodiment, the medicament is a vaccine. The vaccine can be a tumor vaccine, an influenza vaccine, or a SARS-CoV-2 vaccine.

[0332] Uses of LNPs in vaccines

[0333] In some embodiments, LNPs comprising compounds and polynucleotides of the present disclosure can be components of vaccines. Vaccines include compounds and formulations that can provide immunity to one or more conditions associated with infectious diseases, and thus can include mRNA encoding infectious disease-derived antigens and / or epitopes. Vaccines also include compounds and formulations that direct an immune response against cancer cells, and can include mRNA encoding tumor cell-derived antigens, epitopes, and / or neo-epitopes. Compounds that elicit an immune response can include vaccines, corticosteroids (e.g., dexamethasone), and other types.

[0334] In embodiments, the vaccine may be an mRNA vaccine, and thus the LNP comprising a compound of the present disclosure encapsulates or is associated with an mRNA molecule comprising an mRNA sequence encoding an antigenic peptide or protein, or a fragment, variant or derivative thereof.

[0335] The antigenic peptide or protein may be a pathogenic antigen, a tumor antigen, an allergen antigen, or an autoimmune self-antigen. Such pathogenic antigens may be antigens derived from pathogenic organisms (particularly bacterial, viral, or protozoan (multicellular) pathogenic organisms) that can elicit an immunological response in a mammalian subject, such as a human. Pathogenic antigens may be surface antigens, such as proteins or fragments thereof, located on the surface of a virus, bacteria, or protozoa.

[0336] The target pathogenic antigens may include antigens derived from one or more of the following: Acinetobacter baumannii, Anaplasma spp., Anaplasma phagocytophilum, Ancylostoma brasiliensis, Ancylostoma duodenale, Vibrio haemolyticus, Ascaris, Aspergillus spp., Astroviridae, Babesia spp., Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Bacillus hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia spp., Borrelia spp., Brucella spp., Brucella malayi, Bunyaviridae, Burkholderia cepacia and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae, Campylobacter spp., Candida albicans, Candida albicans, Candida species, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, QD prion, Clonorchis sinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium perfringens, Clostridium spp., Clostridium tetani, Coccidioides, coronavirus, Corynebacterium diphtheriae, Rickettsia burnetii, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium spp., cytomegalovirus (CMV), dengue virus (DEN-1, DEN-2, DEN-3, and DEN-4), Diamoeba fragilis, Ebola virus (EBOV), Echinococcus spp., Ehrlichia chaffeensis, Ehrlichia Ewing, Ehrlichia spp., Entamoeba histolytica, Enterococcus spp., Enterovirus spp.,The main pathogens are Coxsackievirus A and enterovirus 71 (EV71), Epidermophyton spp., Epstein-Barr virus (EBV), Escherichia coli O157:H7, Oll l and O104:H4, Fasciola hepatica and Fasciola giant, FFI prions, Filarial family, Flavivirus, Francisella tularensis, Fusobacterium spp., Geotrichum candidum, Giardia lamblia, Gnatostoma spp., GSS prions, Guanarito virus, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Henipavirus (Hendra virus, Nipah virus), Hepatitis A virus, Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis D virus, Hepatitis E virus, Herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2), Histoplasma capsulatum, HIV (human immunodeficiency virus). defective virus), Hallettella wesleyi, human bocavirus (HBoV), human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7), human metapneumovirus (hMPV), human papillomavirus (HPV), human parainfluenza virus (HPIV), Japanese encephalitis virus, JC virus, Junin virus, Kingella, Klebsiella granulomatosis, kuru prion, Lassa virus, Legionella pneumophila, Leishmania spp., Leptospira spp., Listeria monocytogenes, lymphocytic choriomeningitis virus (LCMV), Machupo virus, Malassezia, Marburg virus, measles Herpes virus, Metagonometazoa yokogawa, Microsporidia, Molluscum contagiosum virus (MCV), Mumps virus, Mycobacterium leprae and Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowleri, Necatorella americana, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia species, Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae (influenza), Paracoccidioides brasiliensis, Paragonimus spp., Paragonimus welchii, Parvovirus B19, Pasteurella spp., Plasmodium spp., Pneumocystis jiroveci, Poliovirus, Rabies virus, Respiratory Respiratory syncytial virus (RSV), rhinovirus, rhinovirus, Rickettsia meningitidis, Rickettsia spp., Rickettsia prowazekii, Rickettsia spp., Rickettsia typhi, Rift Valley fever virus, rotavirus, rubella virus, Sabia virus, Salmonella spp., scabies, SARS coronavirus, Schistosoma spp., Shigella spp., Sin Nombre virus, Hantavirus, Sporothrix schenckii, Staphylococcus spp., Staphylococcus spp., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Strongyloides stercoralis, Taenia solium, Tick-borne encephalitis virus (TBEV), Toxocara canis or Toxocara cati, Toxoplasma gondii, Treponema pallidum,Trichinella spiralis, Trichomonas vaginalis, Trichomonas, Trichophyton, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, Varicella zoster virus (VZV), Varicella zoster virus (VZV), Variola major or minor, vCJD prion, Venezuelan equine encephalitis virus, Vibrio cholera, West Nile virus, Western equine encephalitis virus, Bancroftonella, Yellow fever virus, Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

[0337] In certain embodiments, the relevant antigens can be derived from a pathogen selected from the group consisting of severe acute respiratory syndrome (SARS), severe acute respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-1 and SARS-CoV-2), influenza virus, respiratory syncytial virus (RSV), herpes simplex virus (HSV), human papillomavirus (HPV), human immunodeficiency virus (HIV), Plasmodium, Staphylococcus aureus, dengue virus, Chlamydia trachomatis, cytomegalovirus (CMV), hepatitis B virus (HBV), Mycobacterium tuberculosis, rabies virus and yellow fever virus.

[0338] In some embodiments, the relevant pathogenic antigens can be selected from the following: outer membrane protein A OmpA, biofilm-associated protein Bap, transport protein MucK (Acinetobacter baumannii, Acinetobacter infection)); variable surface glycoprotein VSG, microtubule-associated protein MAPP15, trans-sialidase TSA (Trypanosoma brucei, African sleeping sickness (African trypanosomiasis)); HIV p24 antigen, HIV envelope protein (Gpl20, Gp41, Gpl60), polyprotein GAG, negative factor protein Nef, transcription activator Tat (HIV (human immunodeficiency virus), AIDS (acquired immunodeficiency syndrome)); galactose-inhibited adhesion protein GIAP, 29 kDa antigen Eh29, Gal / GalNAc lectin, protein CRT, 125 kDa immunodominant antigen, protein M17, adhesin ADH112, protein STIRP (Entamoeba histolytica, amebiasis); major surface proteins 1-5 (MSP1a, MSP1b, MSP2, MSP3, MSP4, MSP5), type IV secretion system proteins (VirB2, VirB7, VirB11, VirD4) (Anaplasma spp., anaplasmosis); protective antigen PA, edema factor EF, lethal factor LF, S-layer homologous protein SLH (Bacillus anthracis, anthrax); lysin, phospholipase D, collagen-binding protein CbpA (Vibrio pyogenicus, Vibrio pyogenicus infection); nucleocapsid protein NP, glycoprotein precursor GPC, glycoprotein GP1, glycoprotein GP2 (Junín virus, Argentine hemorrhagic fever); chitin layer protein, 14 kDa surface antigen A14, major sperm protein MSP, MSP polymerizing tissue protein MPOP, MSP fiber protein 2 MFP2, MSP polymerization-activated kinase MPAK, ABA-1-like protein ALB, protein ABA-1, keratin protein CUT-1 (Ascaris, ascariasis); 41 kDa allergen Asp vl3, allergen Asp f3, major conidia surface protein rod A, protease Peplp, GPI-anchored protein Gellp, GPI-anchored protein Crflp (Aspergillus, aspergillosis); VP26 family proteins, VP29 protein (Astroviridae, astrovirus infection); rhoptry-associated protein 1 RAP-1, merozoite surface antigens MSA-1, MSA-2 (al, a2, b, c), 12D3, 11C5, 21B4, P29, variant erythrocyte surface antigen VESA1, apical membrane antigen 1 AMA-1 (Babesia spp., babesiosis); hemolysin, enterotoxin C, PXO1-51, glycolate oxidase, ABC transporter, penicillin-binding protein, zinc transporter family protein, pseudouridine synthase Rsu, plasmid replication protein RepX, oligopeptidase F, prophage membrane protein, HemK protein, flagellar antigen H, 28.5-kDa cell surface antigen (Bacillus cereus, Bacillus cereus infection);Large T antigen LT, small T antigen, capsid protein VP1, capsid protein VP2 (BK virus, BK virus infection); 29 kDa protein, caspase 3-like antigen, glycoprotein (Blastocystis hominis, Blastocystis hominis infection); yeast surface adhesin WI-1 (Blastomyces dermatitidis, blastomycosis); nucleoprotein N, polymerase L, matrix protein Z, glycoprotein GP (Machupo virus, Bolivian hemorrhagic fever); outer surface protein A OspA, outer surface protein OspB, outer surface protein OspC, decorin binding protein A DbpA, decorin binding protein B DbpB, flagellar filament 41 kDa core protein Fla, basic membrane protein A precursor BmpA (immunodominant antigen P39), outer surface 22 kDa lipoprotein precursor (antigen IPLA7), variable surface lipoprotein vlsE (Borrelia burgdorferi, Borrelia burgdorferi infection); botulinum toxins BoNT / Al, BoNT / A2, BoNT / A3, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, recombinant botulinum toxin F He domain FHc (Clostridium botulinum, botulism (and infant botulism)); nucleocapsid, glycoprotein precursor (Sabiya virus, Brazilian hemorrhagic fever); copper / zinc superoxide dismutase SodC, bacterial ferritin Bfr, ​​50S ribosomal protein RpIL, OmpA-like transmembrane domain protein Omp31, immunogenic 39 kDa protein M5 P39, zinc ABC transporter periplasmic zinc-binding protein znuA, periplasmic immunogenic protein Bp26, 30S ribosomal protein S12 RpsL, glyceraldehyde-3-phosphate dehydrogenase Gap, 25 kDa outer membrane immunogenic protein precursor Omp25, invasion protein B lalB, trigger factor Tig, molecular chaperone DnaK, putative peptidylprolyl cis-trans isomerase SurA, lipoprotein Ompl9, outer membrane protein MotY Ompl6, conserved outer membrane protein D15, malate dehydrogenase Mdh, component of type IV secretion system (T4SS) VirJ, lipoprotein of unknown function BAB1_0187 (Brucella spp., brucellosis); members of the ABC transporter family (LolC, OppA and PotF), putative lipoprotein release system transmembrane proteins LolC / E, flagellin FliC, Burkholderia intracellular movement protein BimA, bacterial elongation factor-Tu EF-Tu, 17 kDa OmpA-like protein, boaA-encoded protein, boaB-encoded protein (Burkholderia cepacia and other Burkholderia species, Burkholderia infections); hyphal acyltransferase Ag85A, heat shock protein Hsp65, protein TB10.4, 19 kDa antigen, protein PstS3, heat shock protein Hsp70 (Mycobacterium ulcerans, Buruli ulcer);Norovirus major and minor capsid proteins VP1 and VP2, genomic polyprotein; sapovirus capsid protein VP1, protein Vp3, genomic polyprotein (Caliciviridae, Calicivirus infections (norovirus and sapovirus)); major outer membrane protein PorA, flagellin FlaA, surface antigen CjaA, fibronectin-binding protein CadF, aspartate / glutamate-binding ABC transporter PeblA, protein FspA1, protein FspA2 (Campylobacter spp., campylobacteriosis); glycolytic enzyme enolase, secreted aspartyl protease SAP1-10, glycosylphosphatidylinositol (GPI)-linked cell wall protein, Hyrl protein, complement receptor 3-related protein CR3-RP, adhesin Als3p, heat shock protein 90 kDa hsp90, cell surface hydrophobic protein CSH (common in Candida albicans and other Candida species, candidiasis); 17-kDa antigen, protein P26, trimeric autotransporter adhesin TAA, Bartonella adhesin A BadA, variably expressed outer membrane protein Vomps, protein Pap3, protein HbpA, envelope-associated protease HtrA, protein OMP89, protein GroEL, protein LalB, protein OMP43, dihydrolipoamide succinyltransferase SucB (Bartonella henselae, cat-scratch disease); amastigote surface protein-2, amastigote-specific surface protein SSP4, cruzi protease, trans-sialidase TS, trypanosome surface glycoprotein TSA-1, complement regulatory protein CRP-10, protein G4, protein G2, paraflagellar rod protein PAR2, paraflagellar rod component Pari, mucin-associated surface protein MPSP (Trypanosoma cruzi, Chagas disease (American trypanosomiasis)); envelope glycoproteins (gB, gC, gE, gH, gl, gK, gL) (Varicella-zoster virus (VZV), chickenpox); major outer membrane protein MOMP, possible outer membrane protein PMPC, outer membrane complex protein B OmcB, heat shock protein Hsp60 HSP10, protein IncA, protein from the type III secretion system, ribonucleotide reductase small chain protein NrdB, plasmid protein Pgp3, Chlamydia external protein N CopN, antigen CT521, antigen CT425, antigen CT043, antigen TC0052, antigen TC0189, antigen TC0582, antigen TC0660, antigen TC0726, antigen TC0816, antigen TC0828 (Chlamydia trachomatis, Chlamydia);Low calcium response protein E LCrE, Chlamydia outer protein N CopN, serine / threonine protein kinase PknD, acyl carrier protein S-malonyltransferase FabD, single-stranded DNA binding protein Ssb, major outer membrane protein MOMP, outer membrane protein 2 Omp2, polymorphic membrane protein family (Pmpl, Pmp2, Pmp3, Pmp4, Pmp5, Pmp6, Pmp7, Pmp8, Pmp9, PmplO, Pmpl1, Pmpl2, Pmpl3, Pmpl4, Pmpl5, Pmpl6, Pmpl7, Pmpl8, Pmpl9, Pmp20, Pmp21) (Chlamydia pneumoniae, Chlamydia pneumoniae infection); cholera toxin B CTB, toxin co-regulatory fimbriae protein A TcpA, toxin co-regulatory fimbriae protein TcpF, toxin co-regulatory fimbriae biogenesis protein F TcpF, cholera toxin subunit A, cholera toxin subunit B, heat-stable enterotoxin ST, mannose-sensitive hemagglutinin MSHA, outer membrane protein U porin ompU, porin B protein, polymorphic membrane protein-D (Vibrio cholerae, cholera); propionyl-CoA carboxylase PCC, 14-3-3 protein, repressor protein, cysteine ​​protease, glutathione transferase, gelsolin, cathepsin L protease CatL, 20.8 kDa coat protein TP20.8, 31.8 kDa coat protein TP31.8, lysophosphatidic acid phosphatase LPAP (Clonorchis sinensis, clonorchiasis); surface proteins SLPs, glutamate dehydrogenase antigen GDH, toxin A, toxin B, cysteine ​​protease Cwp84, cysteine ​​protease Cwpl3, cysteine ​​protease Cwpl9, cell wall protein CwpV, flagellin FliC, flagellin FliD (Clostridium difficile, Clostridium difficile infection); rhinovirus: capsid proteins VP1, VP 2. VP3, VP4; Coronavirus: spike protein S, envelope protein E, membrane protein M, nucleocapsid protein N (common in rhinoviruses and coronaviruses, as well as the common cold (acute viral nasopharyngitis; acute rhinitis)); prion protein Prp (Creutzfeldt-Jakob disease prion, Creutzfeldt-Jakob disease (CJD)); envelope protein Gc, envelope protein Gn, nucleocapsid protein (Crimean-Congo hemorrhagic fever virus, Crimean-Congo hemorrhagic fever (CCHF)); virulence-associated DEAD-box RNA helicase VAD1, galactomannan protein GalXM, glucuronoxylomannan GXM, mannoprotein MP (Cryptococcus neoformans, cryptococcosis);Acidic ribosomal protein P2 CpP2, mucin antigens Mucl, Muc2, Muc3 Muc4, Muc5, Muc6, Muc7, surface adhesion protein CP20, surface adhesion protein CP23, surface protein CP12, surface protein CP21, surface protein CP40, surface protein CP60, surface protein CP15, surface-associated glycopeptide gp40, surface-associated glycopeptide gpl5, oocyst wall protein AB, tropoactin PRF, apyrase (Cryptosporidium spp., cryptosporidiosis); fatty acid and retinol binding protein-1 (FAR-1), tissue inhibitor of metalloproteinases (TIMPs), cysteine ​​protease ACEY-1, cysteine ​​protease ACCP-1, surface antigen Ac-16, secretory protein 2 ASP-2, metalloproteinase 1 MTP-1, aspartyl protease inhibitor API-1, surface-associated antigen SAA-1, adult-specific secreted factor Xa, serine protease inhibitor anticoagulant AP, cathepsin D-like aspartic protease ARR-1 (typically Ancylostoma braziliensis; various other parasites, cutaneous larva migrans (CLM)); cathepsin L-like protease, 53 / 25-kDa antigen, 8-kDa family member, cysticercoid protein TsAg5 with marginal trypsin-like activity, metacercarial protein TSOL18, metacercarial protein TSOL45-1A, lactate dehydrogenase A LDHA, lactate dehydrogenase B LDHB (Taenia solium, cysticercosis); pp65 antigen, membrane protein ppl5, capsid proximal envelope protein ppl50, protein M45, DNA polymerase UL54, helicase UL105, glycoprotein gM, glycoprotein gN, glycoprotein H, glycoprotein B gB, protein UL83, protein UL94, protein UL99 (cytomegalovirus (CMV), cytomegalovirus infection); capsid protein C, premembrane protein prM, membrane protein M, envelope protein E (domain I, domain II, domain II), protein NS1, protein NS2A, protein NS2B, protein NS3, protein NS4A, protein 2K, protein NS4B, protein NS5 (dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4) - flavivirus, dengue fever); 39 kDa protein (Dimonate amoeba fragilis, dimonate amoebiasis); diphtheria toxin precursor Tox, diphtheria toxin DT, fimbriae-specific sortase SrtA, axial fimbriae protein SpaA, tip fimbriae protein SpaC, minor fimbriae protein SpaB, surface-associated protein DIP1281 (Corynebacterium diphtheriae, diphtheria); glycoprotein GP, ​​nucleoprotein NP, minor matrix protein VP24, major matrix protein VP40, transcription activator VP30, polymerase cofactor VP35, RNA polymerase L (Ebola virus (EBOV), Ebola hemorrhagic fever); prion proteins (vQD prion, variant Creutzfeldt-Jakob disease (vCJD, nvCJD));UvrABC system protein B, protein Flp1, protein Flp2, protein Flp3, protein TadA, hemoglobin receptor HgbA, outer membrane protein TdhA, protein CpsRA, regulator CpxR, protein SapA, 18 kDa antigen, outer membrane protein NcaA, protein LspA, protein LspA1, protein LspA2, protein LspB, outer membrane component DsrA, lectin DltA, lipoprotein Hip, major outer membrane protein OMP, outer membrane protein OmpA2 (Haemophilus ducreyi, chancroid); aspartyl protease 1 Pepl, phospholipase B PLB, α-mannosidase 1 AMN1, glucanosyltransferase GEL1, urease URE, peroxisomal matrix protein Pmpl, proline-rich antigen Pra, human T cell response protein TcrP (Coccidioides immitis and Coccidioides posadaci, coccidioidomycosis); allergen Tri r 2. Heat shock protein 60 Hsp60, fungal actin Act, antigen Tri r2, antigen Tri r4, antigen Tri tl, protein IV, glycerol-3-phosphate dehydrogenase Gpdl, osmosensor HwSholA, osmosensor HwSholB, histidine kinase HwHhk7B, allergen Malas 1, allergen Malas 11, thioredoxin Trx Malas 13, allergen Malaf, allergen Malas (usually Trichophyton, Epidermophyton, Malassezia, Wernicke-Höderma, dermatophytosis); protein EG95, protein EG10, protein EG18, protein EgA31, protein EM18, antigen EPC1, antigen B, antigen 5, protein P29, protein 14-3-3, 8-kDa protein, myosin, heat shock protein 20 HSP20, glycoprotein GP-89, fatty acid binding protein FAPB (Echinococcus spp., echinococcosis); major surface protein 2 MSP2, major surface protein 4 MSP4, MSP variant SGV1, MSP variant SGV2, outer membrane protein OMP, outer membrane protein 19 OMP-19, major antigenic protein MAPI, major antigenic proteins MAP1-2, major antigenic protein MAP1B, major antigenic proteins MAP1-3, Erum2510-encoded protein, protein GroEL, protein GroES, 30-kDA major outer membrane protein, GE 100-kDa protein, GE 130-kDa protein, GE 160-kDa protein (Ehrlichia spp., ehrlichiosis);Secreted antigen SagA, sagA-like proteins SalA and SalB, collagen adhesin Scm, surface proteins Fmsl (EbpA(fm), Fms5 (EbpB(fm), Fms9 (EpbC(fm) and FmslO, protein EbpC(fm), 96 kDa immunoprotective glycoprotein G1 (Enterococcus spp., enterococcal infection); genomic polyprotein, polymerase 3D, viral capsid protein VP1, viral capsid protein VP2, viral capsid protein VP3, viral capsid protein VP4, proteinase 2A, proteinase 3C (Enterovirus spp., enterovirus infection); outer membrane protein OM, 60 kDa outer membrane protein, cell surface antigen OmpA, cell surface antigen OmpB (sca5), 134 kDa outer membrane protein, 31 kDa outer membrane protein, 29.5 kDa outer membrane protein, cell surface protein SCA4, cell surface protein Adrl (RP827), cell surface protein Adr2 (RP828), cell surface protein SCA1, invasion protein invA, cell division protein fts, secretory protein sec Ofamily, virulence proteins virB, tlyA, tlyC, small protein-like protein Pip, preprotein translocase SecA, 120-kDa surface protein antigen SPA, 138 kD complex antigen, major 100-kD protein (protein I), intracytoplasmic protein D, protective surface protein antigen SPA (Corickettsia prowazekii, epidemic typhus); Epstein-Barr virus nuclear antigens (EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA leader protein (EBNA-LP)), latent membrane proteins (LMP-1, LMP-2A, LMP-2B), early antigen EBV-EA, membrane antigen EBV-MA, viral capsid antigen EBV-VCA, basic nucleic acid Enzyme EBV-AN, glycoprotein H, glycoprotein gp350, glycoprotein gp110, glycoprotein gp42, glycoprotein gHgL, glycoprotein gB (Epstein-Barr virus, EBV, Epstein-Barr virus infectious mononucleosis); capsid protein VP2, capsid protein VP1, major protein NS1 (parvovirus B19, erythema infectiosum (fifth disease)); pp65 antigen, glycoprotein 105, major capsid protein, envelope glycoprotein H, protein U51 (human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7), exanthema);Thioredoxin-glutathione reductase TGR, cathepsins LI and L2, Kunitz-type protein KTM, leucine aminopeptidase LAP, cysteine ​​protease Fas2, saposin-like protein-2 SAP-2, thioredoxin peroxidase TPx, Prx-1, Prx-2, cathepsin I cysteine ​​protease CL3, protease cathepsin L CL1, phosphoglycerate kinase PGK, 27-kDa secreted protein, 60 kDa protein HSP35alpha, glutathione transferase GST, 28.5 kDa envelope antigen 28.5 kDa TA, cathepsin B3 protease CatB3, type I cystatin stefin-1, cathepsin L5, cathepsin Llg and cathepsin B, fatty acid binding protein FABP, leucine aminopeptidase LAP (Fasciola hepatica and Fasciola gigantea, fascioliasis); prion protein (FFI prion, fatal familial insomnia (FFI)); venom allergen homolog-like protein VAL-1, abundant larval transcript ALT-1, abundant larval transcript ALT-2, thioredoxin peroxidase TPX, wasp allergen homolog VAH, thioredoxin peroxidase 2 TPX-2, antigenic protein SXP (peptide N, N1, N2 and N3), activation-associated protein-1 ASP-1, thioredoxin TRX, transglutaminase BmTGA, glutathione-S-transferase GST, myosin, wasp allergen homolog VAH, 175 kDa collagenase, glyceraldehyde-3-phosphate dehydrogenase GAPDH, cuticle collagen Col-4, secretory larval acidic protein SLAP, chitinase CHI-1, maltose binding protein MBP, glycolytic enzyme fructose-1,6-bisphosphate aldolase Fba, tropomyosin TMY-1, nematode-specific gene product OvB20, filarialin CPI-2, Cox-2 (Filarialoidea, filariasis); phospholipase CPLC, heat-labile enterotoxin B, Iota toxin component lb, protein CPE1281, pyruvate ferredoxin oxidoreductase, elongation factor G EF-G, Clostridium perfringens hemolysin 0 Pfo, glyceraldehyde-3-phosphate dehydrogenase GapC, fructose bisphosphate aldolase Alf2, Clostridium perfringens enterotoxin CPE, alpha toxin AT, alpha toxinoid ATd, epsilon toxinoid ETd, protein HP, macrocytotoxin TpeL, endo-β-N-acetylglucosaminidase Naglu, phosphoglyceromutase Pgm (Clostridium perfringens, food poisoning caused by Clostridium perfringens); leukotoxin IktA, adhesion FadA, outer membrane protein RadD, high molecular weight arginine-binding protein (Fusobacterium spp., Fusobacterium infection);Phospholipase C PLC, heat-labile enterotoxin B, Iota toxin component lb, protein CPE1281, pyruvate ferredoxin oxidoreductase, elongation factor G EF-G, Clostridium perfringens hemolysin 0 Pfo, glyceraldehyde-3-phosphate dehydrogenase GapC, fructose bisphosphate aldolase Alf2, Clostridium perfringens enterotoxin CPE, alpha-toxin AT, alpha-toxinoid ATd, epsilon-toxinoid ETd, protein HP, macrocytotoxin TpeL, endo-β-N-acetylglucosaminidase Naglu, phosphoglycerate mutase Pgm (usually Clostridium perfringens; other Clostridia, gas gangrene (clostridial myonecrosis)); lipase A, lipase B, peroxidase Decl (Geotrichum candidum, geotrichum necrosis); prion protein (GSS prion, Gerstmann-Straussler-Scheinker syndrome (GSS)); cyst wall proteins CWP1, CWP2, CWP3, variant surface proteins VSP, VSP1, VSP2, VSP3, VSP4, VSP5, VSP6, 56 kDa antigen, pyruvate ferredoxin oxidoreductase PFOR, alcohol dehydrogenase E ADHE, α-giardiacin, α-8-giardiacin, α-1-giardiacin, β-giardiacin, cysteine ​​protease, glutathione-S-transferase GST, arginine deiminase ADI, fructose-1,6-bisphosphate aldolase FBA, Giardia trophozoite antigens GTA (GTA1, GTA2), ornithine carboxyltransferase OCT, rhabdomyosinogen-like protein SALP, uridine phosphate-like protein UPL, α-tubulin, β-tubulin (Giardia intestinalis, giardiasis); ABC transporter family members (LolC, OppA, and PotF), putative lipoprotein release system transmembrane proteins LolC / E, flagellin FliC, Burkholderia intracellular motility A BimA, bacterial elongation factor-Tu EF-Tu, 17 kDa OmpA-like protein, boaA-encoded protein (Burkholderia mallei, Burkholderia pseudomallei); cyclic serine protease CyP, 24 kDa third-stage larval protein GS24, excreta-secretory ESPs (40, 80, 120, and 208 kDa) (Gnathostoma spinulosa and Gnathostoma equine, Gnathostomatosis);Fimbrial proteins, minor pilus-associated subunit pilC, major pilus subunits and their variants pilE, pilS, phase change protein porA, porin B PorB, protein TraD, Neisseria outer membrane antigen H.8, 70kDa antigen, major outer membrane protein PI, outer membrane proteins PIA and PIB, W antigen, surface protein A NspA, transferrin-binding protein TbpA, transferrin-binding protein TbpB, PBP2, mtrR-encoded protein, ponA-encoded protein, membrane permease FbpBC, FbpABC protein system, LbpAB protein, outer membrane protein Opa, outer membrane transport protein FetA, iron inhibition regulatory protein MpeR (Neisseria gonorrhoeae, gonorrhea); outer membrane protein A OmpA, outer membrane protein C OmpC, outer membrane protein K17 OmpK17 (Klebsiella granulomatosa, granuloma inguinale (Donovan disease)); fibronectin binding protein Sfb, fibronectin / fibrinogen binding protein FBP54, fibronectin binding protein FbaA, M protein type 1 Emml, M protein type 6 Emm6, immunoglobulin binding protein 35 Sib35, surface protein R28 Spr28, superoxide dismutase SOD, C5a peptidase ScpA, antigen I / IIAgl / II, adhesin AspA, G-related α2-macroglobulin binding protein GRAB, surface fibrin M5 (Streptococcus pyogenes, group A streptococcal infection); C protein beta antigen, arginine deiminase protein, adhesin BibA, 105 kDA protein BPS, surface antigen c, surface antigen R, surface antigen X, trypsin-resistant protein Rl, trypsin-resistant protein R3, trypsin-resistant protein R4, surface immunogenic protein Sip, surface protein Rib, leucine-rich repeat protein LrrG, serine-rich repeat protein Srr-2, C protein alpha antigen Bca, beta antigen Bag, surface antigen Epsilon, alpha-like protein ALP1, alpha-like protein ALP5, surface antigen delta, alpha-like protein ALP2, alpha-like protein ALP3, alpha-like protein ALP4, C beta protein Bac (Streptococcus agalactiae, group B streptococcal infection); transferrin-binding protein 2 Tbp2, phosphatase P4, outer membrane protein P6, peptidoglycan-associated lipoprotein Pal, protein D, protein E, adhesion and permeability protein Hap, outer membrane protein 26 Omp26, outer membrane protein P5 (Fimbrin), outer membrane protein D15, outer membrane protein OmpP2, 5'-nucleotidase NucA, outer membrane protein PI, outer membrane protein P2, outer membrane lipoprotein Pep, lipoprotein E, outer membrane protein P4, fucose kinase FucK, [Cu,Zn]-superoxide dismutase SodC, protease HtrA, protein 0145, α-galactosylceramide (Haemophilus influenzae, Haemophilus influenzae infection);Polymerase 3D, viral capsid protein VP1, viral capsid protein VP2, viral capsid protein VP3, viral capsid protein VP4, protease 2A, protease 3C (enterovirus, mainly coxsackievirus A and enterovirus 71 (EV71), hand, foot and mouth disease (HFMD)); RNA polymerase L, protein L, glycoprotein Gn, glycoprotein Gc, nucleocapsid protein S, envelope glycoprotein Gl, nucleoprotein NP, protein N, polyprotein M (Sinobry virus, Hantavirus, Hantavirus pulmonary syndrome (HPS)); heat shock protein HspA, heat shock protein HspB, citrate synthase GltA, protein UreB, heat shock protein Hsp60, neutrophil activation protein NAP, catalase KatA, vacuolating cytotoxin VacA, urease αUreA, urease βUreb, protein Cpn10, protein groES, heat shock protein HsplO, protein MopB, cytotoxicity-related 10 kDa protein CAG, 36 kDa antigen, β-lactamase HcpA, β-lactamase HcpB (Helicobacter pylori, Helicobacter pylori infection); integral membrane protein, aggregation-prone protein, O antigen, toxin antigen Stx2B, toxin antigen StxlB, adhesion antigen fragment Int28, protein EspA, protein EspB, Intimin, protein Tir, protein IntC300, protein Eae (Escherichia coli O157:H7, O111, and O104:H4, hemolytic uremic syndrome (HUS)); RNA polymerase L, protein L, glycoprotein Gn, glycoprotein Gc, nucleocapsid protein S, envelope glycoprotein Gl, nucleoprotein NP, protein N, polyprotein M (Bunyaviridae, hemorrhagic fever with renal syndrome (HFRS)); glycoprotein G, matrix protein M, nucleoprotein N, fusion protein F, polymerase L, protein W, protein C, phosphoprotein p, nonstructural protein V (Hendra virus / Nipah virus, Henpah virus infection); polyprotein, glycoprotein Gp2, hepatitis A surface antigen HBAg, protein 2A, viral protein VP1, viral protein VP2, viral protein VP3, viral protein VP4, protein P1B, protein P2A, protein P3AB, protein P3D (Hepatitis A virus, Hepatitis A); hepatitis B surface antigen HBsAg, hepatitis B core antigen HbcAg, polymerase, protein Hbx, preS2 intermediate surface protein, surface protein L, large S protein, viral protein VP1, viral protein VP2, viral protein VP3, viral protein VP4 (Hepatitis B virus (HBV), Hepatitis B); envelope glycoprotein E1 gp32gp35, envelope glycoprotein E2 NS1 gp68 gp70, capsid protein C, core protein Core, polyprotein, viral protein VP1, viral protein VP2, viral protein VP3, viral protein VP4, antigen G, protein NS3, protein NS5A (hepatitis C virus, hepatitis C);Viral protein VP1, viral protein VP2, viral protein VP3, viral protein VP4, hepatitis C virus delta antigen, hepatitis C virus delta antigen (hepatitis D virus, hepatitis D); viral protein VP1, viral protein VP2, viral protein VP3, viral protein VP4, capsid protein E2 (hepatitis E virus, hepatitis E);Glycoprotein L UL1, uracil DNA glycosylase UL2, protein UL3, protein UL4, DNA replication protein UL5, gating protein UL6, virion maturation protein UL7, DNA helicase UL8, replication origin binding protein UL9, glycoprotein M UL10, protein UL11, alkaline exonuclease UL12, serine-threonine protein kinase UL13, cell membrane protein UL14, terminase UL15, cell membrane protein UL16, protein UL17, capsid protein VP23UL18, major capsid protein VP5 UL19, membrane protein UL20, cell membrane protein UL21, glycoprotein H (UL22), thymidine kinase UL23, protein UL24, protein UL25, capsid protein P40 (UL26, VP24, VP22A), glycoprotein B (UL27), ICP18.5 protein (UL28), major DNA binding protein ICP8 (UL29), DNA polymerase UL30, nuclear matrix protein UL31, envelope glycoprotein UL32, protein UL33, inner nuclear membrane protein UL34, capsid protein VP26 (UL35), large envelope protein UL36, capsid assembly protein UL37, VP19C protein (UL38), ribonucleotide reductase (large subunit) UL39, ribonucleotide reductase (small subunit) UL40, envelope protein / viral host shutoff VHS protein (UL41), DNA polymerase processivity factor UL42, membrane protein UL43, glycoprotein C (UL44), membrane protein UL45, envelope protein VP11 / 12 (UL46), envelope protein VP13 / 14 (UL47), viral maturation protein VP16 (UL48, Alpha-TIF), envelope protein UL49, dUTP diphosphatase UL50, cell membrane protein UL51, DNA helicase / primase complex protein UL52, glycoprotein K (UL53), transcriptional regulatory protein IE63 (ICP27, UL54), protein UL55, protein UL56, viral replication protein ICP22 (IE68, US1), protein US2, serine / threonine protein kinase US3, glycoprotein G (US4), glycoprotein J (US5), glycoprotein D (US6), glycoprotein I (US7), glycoprotein E (US8), cell membrane protein US9, capsid / cell membrane protein US10, Vmw21 protein (US11)), ICP47 protein (IE12, US12), major transcription activator ICP4 (IE175, RSI), E3 ubiquitin ligase ICPO (IE110), latency-associated protein 1 LRP1, latency-associated protein 2 LRP2, neurovirulence factor RL1 (ICP34.5), latency-associated transcript LAT (herpes simplex virus 1 and 2 (HSV-1 and HSV-2), herpes simplex);Heat shock protein Hsp60, cell surface protein H1C, dipeptidyl peptidase type IV DppIV, M antigen, 70 kDa protein, 17 kDa histone-like protein (Histoplasma capsulatum, histoplasmosis); fatty acid and retinol binding protein-1 FAR-1, tissue inhibitor of metalloproteinases (TIMPs), cysteine ​​protease ACEY-1, cysteine ​​protease ACCP-1, surface antigen Ac-16, secretory protein 2 ASP-2, metalloproteinase 1 MTP-1, aspartyl protease inhibitor API-1, surface-associated antigen SAA-1, surface-associated antigen SAA-2, adult-specific secreted factor Xa, serine protease inhibitor anticoagulant AP, cathepsin D-like aspartic protease ARR-1, glutathione S-transferase GST, aspartic protease APR-1, acetylcholinesterase AChE (Ancylostoma duodenale and Necator americanus, hookworm infection); protein NS1, protein NP1, protein VP1, protein VP2, protein VP3 (human bocavirus (HBoV), human bocavirus infection); major surface protein 2 MSP2, major surface protein 4 MSP4, MSP variant SGV1, MSP variant SGV2, outer membrane protein OMP, outer membrane protein 19 OMP-19, major antigenic protein MAPI, major antigenic protein MAP1-2, major antigenic protein MAP1B, major antigenic protein MAP1-3, Erum2510-encoded protein, protein GroEL, protein GroES, 30-kDA major outer membrane protein, GE 100-kDa protein, GE 130-kDa protein, GE 160-kDa protein (Ehrlichia Ewingi, human Ewing's ehrlichiosis); major surface proteins 1-5 (MSP1a, MSP1b, MSP2, MSP3, MSP4, MSP5), type IV secretion system proteins VirB2, VirB7, VirB11, VirD4 (Anaplasma phagocytophilum, human granulocytic anaplasmosis (HGA)); protein NS1, small hydrophobic protein NS2, SH protein, fusion protein F, glycoprotein G, matrix protein M, matrix protein M2-1, matrix protein M2-2, phosphoprotein P, nucleoprotein N, polymerase L (human metapneumovirus (hMPV), human metapneumovirus infection);major surface protein 2 MSP2, major surface protein 4 MSP4, MSP variant SGV1, MSP variant SGV2, outer membrane protein OMP, outer membrane protein 19 OMP-19, major antigenic protein MAPI, major antigenic protein MAP1-2, major antigenic protein MAP1B, major antigenic protein MAP1-3, Erum2510 encoded protein, protein GroEL, protein GroES, 30-kDA major outer membrane protein, GE 100-kDa protein, GE 130-kDa protein, GE 160-kDa protein (Ehrlichia chaffeensis, human monocytic ehrlichiosis); replication protein E1, regulatory protein E2, protein E3, protein E4, protein E5, protein E6, protein E7, protein E8, major capsid protein L1, minor capsid protein L2 (human papillomavirus (HPV), human papillomavirus (HPV) infection); fusion protein F, hemagglutinin neuraminidase HN, glycoprotein G, matrix protein M, phosphoprotein P, nucleoprotein N, polymerase L (human parainfluenza virus (HPIV), human parainfluenza virus infection); hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), M1 protein, M2 protein, NS1 protein, N S2 protein (NEP protein: nuclear export protein), PA protein, PB1 protein (polymerase basic 1 protein), PB1-F2 protein, and PB2 protein (Orthomyxoviridae, influenza virus); genomic polyprotein, protein E, protein M, and capsid protein C (Japanese encephalitis virus, Japanese encephalitis); RTX toxin, type IV fimbriae, major fimbriae subunit PilA, regulatory transcription factors PilS and PilR, protein sigma54, and outer membrane proteins (K. kingae, Kingae kingae infection); prion protein (Kuru virus, kuru virus); nucleoprotein N, polymerase L, matrix protein Z, and glycoprotein GP (Lassa virus, Lassa fever); peptidoglycan-associated lipoprotein PAL, 60 kDa molecular chaperone Cpn60 (groEL, HspB), type IV fimbriae protein PilE, outer membrane protein MIP, major outer membrane protein MompS, and zinc metalloproteinase MSP (Legionella pneumophila, Legionnaires' disease, Pontiac fever);P4 nuclease, protein WD, ribonucleotide reductase M2, surface membrane glycoprotein Pg46, cysteine ​​protease CP, glucose-regulated protein 78 GRP-78, stage-specific S antigen-like protein A2, ATPase F1, β-tubulin, heat shock protein 70 Hsp70, KMP-11, glycoprotein GP63, protein BT1, nucleoside hydrolase NH, cell surface protein B1, ribosomal protein P1-like protein PI, sterol 24-c-methyltransferase SMT, LACK protein, histone HI, SPB1 protein, sulfhydryl-specific antioxidant TSA, protein antigen STI1, signal peptidase SP, histone H2B, surface antigen PSA-2, cysteine ​​protease b Cpb (Leishmania spp., leishmaniasis); major membrane protein I, serine-rich antigen - 45 kDa, 10 kDa goat anti-GroES, HSP kDa antigen, aminooxynonanoic acid synthase AONS, protein recombinase ARecA, acetyl-CoA / propionyl-CoA carboxylase α, alanine racemase, 60 kDa molecular chaperone 2, ESAT-6-like protein EcxB (L-ESAT-6), protein Lsr2, protein ML0276, heparin-binding hemagglutinin HBHA, heat shock protein 65 Hsp65, protein encoded by mycPl or ML0041, protein encoded by htrA2 or ML0176, protein encoded by htrA4 or ML2659, protein encoded by gcp or ML0379, protein encoded by clpC or ML0235 (Mycobacterium leprae and Mycobacterium leprae, leprosy); outer membrane protein LipL32, membrane protein LIC10258, membrane protein LP30, membrane protein LIC12238, Ompa-like protein Lsa66, surface protein LigA, surface protein LigB, major Outer membrane protein OmpL1, outer membrane protein LipL41, protein LigAni, surface protein LcpA, adhesion protein LipL53, outer membrane protein UpL32, surface protein Lsa63, flagellin FlaBl, membrane lipoprotein LipL21, membrane protein pL40, leptospiral surface adhesin Lsa27, outer membrane protein OmpL36, outer membrane protein OmpL37, outer membrane protein OmpL47, outer membrane protein OmpL54, acyltransferase LpxA (Leptospira spp., leptospirosis);Listeriolysin O precursor Hly (LLO), invasion-associated protein lap (P60), listeriolysin regulatory protein PrfA, zinc metalloproteinase Mpl, phosphatidylinositol-specific phospholipase C PLC (PlcA, PlcB), O-acetyltransferase Oat, ABC transporter permease Im.G_1771, adhesion protein LAP, LAP receptor Hsp60, adhesin LapB, hemolysin listeriolysin OLLO, protein ActA, endotoxin AInIA, protein InIB (Listeria monocytogenes, listeriosis); outer surface protein A OspA, outer surface protein OspB, outer surface protein OspC, decorin binding protein A DbpA, decorin binding protein B DbpB, flagellar filament 41 kDa core protein Fla, basic membrane protein A BmpA (immunodominant antigen P39), outer surface 22 kDa lipoprotein precursor (antigen IPLA7), variable surface lipoprotein vlsE (typically Borrelia burgdorferi and other Borrelia species, Lyme disease (Lyme borreliosis)); venom allergen homolog-like protein VAL-1, abundant larval transcript ALT-1, abundant larval transcript ALT-2, thioredoxin peroxidase TPX, wasp allergen homolog VAH, thioredoxin peroxidase 2 TPX-2, antigenic protein SXP (peptides N, N1, N2, and N3), activation-associated protein-1 ASP-1, thioredoxin TRX, transglutaminase BmTGA, glutathione-S-transferase GST, myosin, wasp allergen homolog VAH, 175 kDa collagenase, glyceraldehyde-3-phosphate dehydrogenase GAPDH, cuticle collagen Col-4, secretory larval acidic protein SLAP, chitinase CHI-1, maltose binding protein MBP, glycolytic enzyme fructose-1,6-bisphosphate aldolase Fba, tropomyosin TMY-1, nematode-specific gene product OvB20, squamous cell carcinomastatin CPI-2, protein Cox-2 (W. bancrofti and Brugia malayi, lymphatic filariasis (elephantiasis)); glycoprotein GP, ​​matrix protein Z, polymerase L, nucleoprotein N (lymphocytic choriomeningitis virus (LCMV), lymphocytic choriomeningitis);Thrombosporozoite-related anonymous protein TRAP, SSP2 sporozoite surface protein 2, apical membrane antigen 1 AMA1, rhoptry membrane antigen RMA1, acidic-alkaline repeat antigen ABRA, cell-traversing protein PF, protein Pvs25, merozoite surface protein 1 MSP-1, merozoite surface protein 2 MSP-2, ring-infected erythrocyte surface antigen RESA liver stage antigen 3 LSA-3, protein Eba-175, serine repeat antigen 5 SERA-5, circumsporozoite protein CS, merozoite surface protein 3 MSP3, merozoite surface protein 8 MSP8, enolase PF10, hepatoerythrocyte protein 17 kDa HEP17, erythrocyte membrane protein 1 EMP1, protein Kbeta merozoite surface protein 4 / 5 MSP 4 / 5, heat shock protein Hsp90, glutamic acid-rich protein GLURP, merozoite surface protein 4 MSP-4, protein STARP, circumsporozoite protein-related antigen precursor CRA (Plasmodium spp., malaria); nucleoprotein N, membrane-associated protein VP24, minor nucleoprotein VP30, polymerase cofactor VP35, polymerase L, matrix protein VP40, envelope glycoprotein GP (Marburg virus, Marburg hemorrhagic fever (MHF)); protein C, matrix protein M, phosphoprotein P, nonstructural protein V, hemagglutinin glycoprotein H, polymerase L, nucleoprotein N, fusion protein F (measles virus, measles); members of the ABC transporter family (LolC, OppA, and PotF), putative lipoprotein release system transmembrane proteins LolC / E, flagellin FliC, Burkholderia intracellular motility A BimA, bacterial elongation factor-Tu EF-Tu, 17 kDa OmpA-like protein, boaA-encoded protein, boaB-encoded protein (Burkholderia, melioidosis (Whitmore disease)); fimbriae proteins, minor pilus-associated subunit pilC, major pilus subunit and its variant pilE, pilS, phase-variation protein porA, porin B PorB, protein TraD, Neisseria outer membrane antigen H.8, 70 kDa antigen, major outer membrane protein PI, outer membrane proteins PIA and PIB, W antigen, surface protein A NspA, transferrin-binding protein TbpA, transferrin-binding protein TbpB, PBP2, mtrR-encoded protein, ponA-encoded protein, membrane permease FbpBC, FbpABC protein system, LbpAB protein, outer membrane protein Opa, outer membrane transport protein FetA, iron inhibition regulator MpeR, factor H binding protein fHbp, adhesin NadA, protein NhbA, repressor FarR (Neisseria meningitidis, meningococcal disease); 66 kDa protein, 22 kDa protein (usually Metagonomes yokogawa, Metagonomiasis);Polar tube proteins (34, 75, and 170 kDa in Glugea, 35, 55, and 150 kDa in Encephalitozoon), kinesin-associated protein, largest subunit of RNA polymerase II, integral membrane protein YIPA-like, anti-silencing protein 1, heat shock transcription factor HSF, protein kinase, thymidine kinase, NOP-2-like nucleolar protein (Microsporidia, Microsporidia); CASP8 and FADD-like apoptosis regulator, glutathione peroxidase GPX1, RNA helicase NPH-II NPH2, poly(A) polymerase catalytic subunit PAPL, major envelope protein P43K, early transcription factor 70 kDa subunit VETFS, early transcription factor 82 kDa subunit VETFL, metalloendopeptidase G1 type, nucleoside triphosphatase I NPH1, replication protein A28-like MC134L, RNA polymerase 7 kDa subunit RP07 (molluscum contagiosum virus (MCV), molluscum contagiosum (MC)); matrix protein M, phosphoprotein P / V, small hydrophobic protein SH, nucleoprotein N, protein V, fusion glycoprotein F, hemagglutinin-neuraminidase HN, RNA polymerase L (mumps virus, mumps); outer membrane protein OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell surface protein SCA1, intracytoplasmic protein D, crystalline surface layer protein SLP, protective surface protein antigen SPA (Rickettsia typhi, murine typhus (endemic typhus)); adhesin PI, adhesin P30, protein pll6, protein P40, cytoskeletal protein HMW1, cytoskeletal protein HMW2, cytoskeletal protein HMW3, MPN152-encoded protein, MPN426-encoded protein, MPN456-encoded protein, MPN-500-encoded protein (Mycoplasma pneumoniae, Mycoplasma pneumoniae); NocA, iron-dependent regulatory protein, VapA, VapD, VapF, VapG, caseinolytic protease, filamentous tip-associated 43-kDa protein, protein P24, protein P61, 15-kDa protein, 56-kDa protein (commonly Nocardia asteroides and other Nocardia species, nocardiosis);Venom allergen homolog-like protein VAL-1, abundant larval transcript ALT-1, abundant larval transcript ALT-2, thioredoxin peroxidase TPX, wasp allergen homolog VAH, thioredoxin peroxidase 2 TPX-2, antigenic protein SXP (peptide N, N1, N2 and N3), activation-associated protein-1 ASP-1, thioredoxin TRX, transglutaminase BmTGA, glutathione-S-transferase GST, myosin, wasp allergen homolog VAH, 175 kDa collagenase, glyceraldehyde-3-phosphate dehydrogenase GAPDH, cuticle collagen Col-4, secretory larval acidic protein SLAP, chitinase CHI-1, maltose binding protein MBP, glycolytic enzyme fructose-1,6-bisphosphate aldolase Fba, tropomyosin TMY-1, nematode-specific gene product OvB20, onchocerca volvulus CPI-2, Cox-2 (Onchocerca volvulus, onchocerciasis (river blindness)); 43 kDa secretory glycoprotein, glycoprotein gpO, glycoprotein gp75, antigen Pb27, antigen Pb40, heat shock protein Hsp65, heat shock protein Hsp70, heat shock protein Hsp90, protein P10, triose phosphate isomerase TPI, N-acetylglucosamine-binding lectin paracoccin, 28 kDa protein Pb28 (Paracidioidomycosis brasiliensis, paracoccidioidomycosis (South American blastomycosis)); 28-kDa cruzipain-like cysteine ​​protease Pw28CCP (commonly Paragonimus westermani and other Paragonimus species, paragonimiasis); outer membrane protein OmpH, outer membrane protein Omp28, protein PM1539, protein PM0355, protein PM1417, repair protein MutL, protein BcbC, protein PM0305, formate dehydrogenase-N, protein PM0698, protein PM1422, DNA gyrase, lipoprotein PlpE, adhesion protein Cp39, heme acquisition system receptor HasR, 39-kDa capsule protein, iron-regulated OMP IROMP, outer membrane protein OmpA87, fimbriae protein Ptf, fimbriae subunit protein PtfA, transferrin-binding protein Tbpl, esterase MesA, Pasteurella multocida toxin PMT, adhesion protein Cp39 (Pasteurella spp., pasteurellosis); "filamentous hemagglutinin FhaB, adenylate cyclase CyaA, pertussis toxin subunit 4 precursor PtxD, pertussis toxin precursor Prn, toxin subunit 1 PtxA, protein Cpn60, protein brkA, pertussis toxin subunit 2 precursor PtxB, pertussis toxin subunit 3 precursor PtxC, pertussis toxin subunit 5 precursor PtxE, pertussis toxin precursor Prn, protein Fim2, protein Fim3" (Bordetella pertussis, whooping cough);"Fl capsular antigen, virulence-associated V antigen, secreted effector protein LcrV, V antigen, outer membrane protease Pla, secreted effector protein YopD, putative secreted protein tyrosine phosphatase YopH, needle complex major subunit YscF, protein kinase YopO, putative autotransporter YapF, inner membrane ABC transporter YbtQ (Irp7), putative carbohydrate binding protein YPO0612, heat shock protein 90 HtpG, putative sulfatase protein YdeN, outer membrane lipoprotein carrier protein LolA, secretory chaperone YerA, putative lipoprotein YPO0420, hemolysin activator protein HpmB, yersin / yersin outer membrane receptor Psn, secretory effector protein YopE, secretory effector protein YopF, secretory effector protein YopK, outer membrane protein YopN, outer membrane protein YopM, coagulase / plasmin precursor Pla; (Yersinia pestis, Yersinia pestis); protein PhpA, surface adhesin PsaA, pneumolysin Ply, ATP-dependent protease CIp, lipid-protein ligase LplA, cell wall anchor protein psrP, sortase SrtA, glutamyl-tRNA synthetase GltX, choline-binding protein A CbpA, pneumococcal surface protein A PspA, pneumococcal surface protein C PspC, 6-phosphogluconate dehydrogenase Gnd, iron-binding protein PiaA, murein hydrolase LytB, protein LytC, proteinase A1 (Streptococcus pneumoniae, pneumococcal infection); major surface protein B, kexin-like proteinase KEX1, protein A12, 55 kDa antigen P55, major surface glycoprotein Msg (Pneumocystis jiroveci, Pneumocystis carinii (PCP)); genomic polyprotein, polymerase 3D, viral capsid protein VP1, viral capsid protein VP2, viral capsid protein VP3, viral capsid protein VP4, proteinase 2A, proteinase 3C (poliovirus, poliomyelitis); protein Nfal, exenatide-3, secretory lipase, cathepsin B-like protease, cysteine ​​protease, cathepsin, peroxidase, protein CrylAc (common in Naegleria fowleri, primary amebic meningoencephalitis (PAM)); unknown protein, large T antigen, small T antigen, major capsid protein VP1, minor capsid protein VP2 (JC virus, progressive multifocal leukoencephalopathy);Low calcium response protein E LCrE, Chlamydia outer protein N CopN, serine / threonine protein kinase PknD, acyl carrier protein S-malonyltransferase FabD, single-stranded DNA binding protein Ssb, major outer membrane protein MOMP, outer membrane protein 2 Omp2, polymorphic membrane protein family (Pmpl, Pmp2, Pmp3, Pmp4, Pmp5, Pmp6, Pmp7, Pmp8, Pmp9, PmplO, Pmpl1, Pmpl2, Pmpl3, Pmpl4, Pmpl5, Pmpl6, Pmpl7, Pmpl8, Pmpl9, Pmp20, Pmp21) (Chlamydia psittaci, psittacosis); outer membrane protein PI, heat shock protein B HspB, peptide ABC transporter, GTP-binding protein, protein IcmB, ribonuclease R, phosphorylated SixA, protein DsbD, outer membrane protein TolC, DNA-binding protein PhoB, ATPase DotB, heat shock protein B HspB, membrane protein Coml, 28 kDa protein, DNA-3-methyladenylosidase I, outer membrane protein OmpH, outer membrane protein AdaA, glycine cleavage system T protein (Coxiella burnetii, Q fever); nucleoprotein N, large structural protein L, phosphoprotein P, matrix protein M, glycoprotein G (rabies virus, rabies); fusion protein F, nucleoprotein N, matrix protein M, matrix protein M2-1, matrix protein M2-2, phosphoprotein P, small hydrophobic protein SH, major surface glycoprotein G, polymerase L, nonstructural protein 1NS1, nonstructural protein 2 NS2 (respiratory syncytial virus (RSV), respiratory syncytial virus infection); genomic polyprotein, polymerase 3D, viral capsid protein VP1, viral capsid protein VP2, viral capsid protein VP3, viral capsid protein VP4, protease 2A, protease 3C (rhinovirus, rhinovirus infection); outer membrane protein OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell surface protein SCA1, protein PS120, intracytoplasmic protein D, Protective surface protein antigen SPA (Rickettsia spp., Rickettsia infection); outer membrane protein OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell surface protein SCA1, intracytoplasmic protein D (Akarickettsia, Rickettsialpox); envelope glycoprotein GP, ​​polymerase L, nucleoprotein N, nonstructural protein NSS (Rift Valley fever virus, Rift Valley fever (RVF)); outer membrane protein OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell surface protein SCA1, intracytoplasmic protein D (Rickettsia, Rocky Mountain spotted fever (RMSF));Nonstructural protein 6 NS6, nonstructural protein 2 NS2, intermediate capsid protein VP6, inner capsid protein VP2, nonstructural protein 3 NS3, RNA-guided RNA polymerase L, protein VP3, nonstructural protein 1 NS1, nonstructural protein 5 NS5, outer capsid glycoprotein VP7, nonstructural glycoprotein 4 NS4, outer capsid protein VP4 (rotavirus, rotavirus infection); polyprotein P200, glycoprotein E1, glycoprotein E2, protein NS2, capsid protein C (rubella virus, rubella); chaperone protein GroEL (MopA), inositol phosphate phosphatase SopB, heat shock protein HsIU, chaperone protein DnaJ, protein TviB, protein IroN, flagellin FliC, invasion protein SipC, glycoprotein gp43, outer membrane protein LamB, outer membrane protein PagC, outer membrane protein TolC, outer membrane protein NmpC, outer membrane protein FadL, transporter SadA, transferase WgaP, effector proteins SifA, SteC, SseL, SseJ and SseF (Salmonella Salmonellosis); protein 14, nonstructural protein NS7b, nonstructural protein NS8a, protein 9b, protein 3a, nucleoprotein N, nonstructural protein NS3b, nonstructural protein NS6, protein 7a, nonstructural protein NS8b, membrane protein M, envelope small membrane protein EsM, replicase polyprotein 1a, spike glycoprotein S, replicase polyprotein 1a; SARS coronavirus, SARS (severe acute respiratory syndrome)); serine protease, atypical scabies antigen 1 ASA1, glutathione S-transferase GST, cysteine ​​protease, serine protease, apolipoprotein (Scabies scabies); glutathione S-transferase GST, paramyosin, hemoglobin SM32, major egg antigen, 14 kDa fatty acid binding protein Sml4, major larval surface antigen P37, 22,6 kDa coat antigen, calpain CANP, triphosphate isomerase Tim, surface protein 9B, coat protein VP2, 23 kDa integral membrane protein Sm23, copper / zinc superoxide dismutase, glycoprotein Gp, myosin (Schistosoma spp., schistosomiasis (Bill-Harz disease)); 60 kDa chaperone protein, 56 kDa type-specific antigen, pyruvate phosphate dikinase, 4-hydroxybenzoate octaprenyltransferase (Orientia tsutsugamushi, tsutsugamushi disease); dehydrogenase GuaB, invasion protein Spa32, invasin IpaA, invasin IpaB, invasin IpaC, invasin IpaD, invasin IpaH, invasin IpaJ (Shigella spp., shigellosis (bacillary dysentery));Protein P53, virion protein US10 homolog, transcriptional regulator IE63, transcriptional transactivator IE62, protease P33, α-transducer 74 kDa protein, deoxyuridine 5'-triphosphate nucleotide hydrolase, transcription transactivator IE4, membrane protein UL43 homolog, nuclear phosphoprotein UL3 homolog, nuclear protein UL4 homolog, replication origin binding protein, membrane protein 2, phosphoprotein 32, protein 57, DNA polymerase processivity factor, gating protein 54, DNA primase, envelope protein UL14 homolog, envelope protein UL21 homolog, envelope protein UL55 homolog, tripartite terminase subunit UL33 homolog, tripartite terminase subunit UL15 homolog, capsid binding protein 44, virion packaging protein 43 (varicella-zoster virus (VZV), herpes zoster (shingles)); truncated 3-beta-hydroxy-5-enoate dehydrogenase homolog, virion membrane protein A 13. Protein A19, protein A31, truncated protein A35 homolog, protein A37.5 homolog, protein A47, protein A49, protein A51, semaphorin-like protein A43, serine protease inhibitor 1, serine protease inhibitor 2, serine protease inhibitor 3, protein A6, protein B15, protein CI, protein C5, protein C6, protein F7, protein F8, protein F9, protein F11, protein F14, protein F15, protein F16 (smallpox major or minor, variola (Variola)); adhesin / glycoprotein gp70, protease (Sporothrix schenckii, sporotrichosis); heme iron-binding protein IsdB, collagen adhesin Cna, clumping factor A ClfA, protein MecA, fibronectin-binding protein A FnbA, enterotoxin type A EntA, enterotoxin type B EntB, enterotoxin type C EntCl, enterotoxin type C EntC2, enterotoxin type D EntD, enterotoxin type E EntE, toxic shock syndrome toxin-1 TSST-1, staphylokinase, penicillin-binding protein 2a PBP2a (MecA), secretory antigen SssA (Staphylococcus spp., staphylococcal food poisoning); heme iron-binding protein IsdB, collagen adhesin Cna, clumping factor A ClfA, protein MecA, fibronectin-binding protein A FnbA, enterotoxin type A EntA, enterotoxin type B EntB, enterotoxin type C EntCl, enterotoxin type C EntC2, enterotoxin type D EntD, enterotoxin type E EntE, toxic shock syndrome toxin-1 TSST-1, staphylokinase, penicillin-binding protein 2a PBP2a (MecA), secreted antigen SssA (Staphylococci such as Staphylococcus aureus, staphylococcal infections);Antigen Ss-IR, antigen NIE, strongyloidiasis, Na+-K+ ATPase Sseat-6, tropoactin SsTmy-1, protein LEC-5, 41 kDa antigen P5, 41-kDa larval protein, 31-kDa larval protein, 28-kDa larval protein (Streptococcus stercoralis, Strongyloidiasis); glycerolphosphodiesterase GlpQ (Gpd), outer membrane protein TmpB, protein Tp92, antigen TpFl, repeat protein Tpr, repeat protein F TprF, repeat protein G TprG, repeat protein I Tprl, repeat protein J TprJ, repeat protein K TprK, Treponema pallidum membrane protein A TmpA, lipoprotein, 15 kDa Tppl5, 47 kDa membrane antigen, microferritin TpF1, adhesin Tp0751, lipoprotein TP0136, protein TpN17, protein TpN47, outer membrane protein TP0136, outer membrane protein TP0155, outer membrane protein TP0326, outer membrane protein TP0483, outer membrane protein TP0956 (Treponema pallidum, syphilis); cathepsin L-like protease, 53 / 25-kDa antigen, 8-kDa family member, cysticercosis protein TsAg5 with marginal trypsin-like activity, hookworm protein TSOL18, hookworm protein TSOL45-1A, lactate dehydrogenase A LDHA, lactate dehydrogenase B LDHB (Taenia spp., taeniasis); tetanus toxin TetX, tetanus toxin C TTC, 140 kDa S-layer protein, flavoprotein beta subunit CT3, phospholipase (lecithinase), phosphorylated carrier protein HPr (Clostridium tetani, tetanus (trismus)); genomic polyprotein, protein E, protein M, capsid protein C (tick-borne encephalitis virus (TBEV), tick-borne encephalitis); 58-kDa antigen, 68-kDa antigen, Toxocara larval excretory and secretory antigen TES, 32-kDa glycoprotein, glycoprotein TES-70, glycoprotein GP31, excretory and secretory antigen TcES-57, perintestinal fluid antigen Pe, soluble extract antigen Ex, excretory / secretory larval antigen ES, antigen TES-120, polyprotein allergen TBA-1, cathepsin L-like cysteine ​​protease c-cpl-1, 26-kDa protein (Toxocara canis or Toxocara felis, toxocariasis (ocular larva migrans (OLM) and visceral larva migrans (VLM)));Microneme proteins (MIC1, MIC2, MIC3, MIC4, MIC5, MIC6, MIC7, MIC8), rhoptry proteins Rop2, rhoptry proteins (Ropl, Rop2, Rop3, Rop4, Rop5, Rop6, Rop7, Ropl6, Rjopl7), protein SR1, surface antigen P22, major antigen p24, major surface antigen p30, dense granule proteins (GRA1, GRA2, GRA3, GRA4, GRA5, GRA6, GRA7, GRA8, GRA9, GRA10), 28 kDa antigen, surface antigen SAG1, SAG2-related antigen, nucleoside triphosphatase 1, nucleoside triphosphatase 2, protein Stt3, HesB-like domain-containing protein, rhomboid-like protease 5, Toxoplasma gondii protease 1 (Toxoplasma gondii, toxoplasmosis); 43 kDa secretory glycoprotein, 53 kDa secretory glycoprotein, paramyosin, antigen Ts21, antigen Ts87, antigen p46000, TSL-1 antigen, caveolin-1 CAV-1, 49 kDa neonatal larval antigen, precursor sphingolipid activator protein homolog, serine protease, serine protease inhibitor, 45-kDa glycoprotein Gp45 (Trichinella spiralis, trichinellosis); Myb-like transcription factors (Myb1, Myb2, Myb3), adhesion protein AP23, adhesion protein AP33, adhesion protein AP33-3, adhesion protein AP51, adhesion protein AP65, adhesion protein AP65-1, α-actinin, kinesin-related protein, actin, 62 kDa protease, subtilisin-like serine protease SUB1, cysteine ​​protease gene 3 CP3, α-enolase enolase, cysteine ​​protease CP30, heat shock proteins (Hsp70, Hsp60), immunogenic protein P270 (Trichomonas vaginalis, trichomoniasis); β-tubulin, 47-kDa protein, secretory leukocyte-like protease-1 SLP-1, 50-kDa protein TT50, 17 kDa antigen, 43 / 47 kDa protein (Trichuris trichuriasis, trichuriasis (whipworm infection));Protein ESAT-6 (EsxA), 10 kDa filtrate antigen EsxB, secretory antigen 85-B FBPB, fibronectin-binding protein A FbpA (Ag85A), serine protease PepA, PPE family protein PPE18, fibronectin-binding protein D FbpD, immunogenic protein MPT64, secretory protein MPT51, catalase-peroxidase-peroxynitrite TKATG, periplasmic phosphate-binding lipoprotein PSTS3 (PBP-3, Phos-1), iron-regulated heparin-binding hemagglutinin Hbha, PPE family protein PPE14, PPE family protein PPE68, protein Mtb72F, protein Apa, immunogenic protein MPT63, periplasmic phosphate-binding lipoprotein PSTS1 (PBP-1), molecular chaperone DnaK, cell surface lipoprotein Mpt83, lipoprotein P23, phosphate transport system permease protein pstA, 14 kDa antigen, fibronectin-binding protein C FbpCl, alanine dehydrogenase TB43, glutamine synthetase 1, ESX-1 protein, protein CFP10, TB10.4 protein, protein MPT83, protein MTB12, protein MTB8, Rpf-like protein, protein MTB32, protein MTB39, crystal protein, heat shock protein HSP65, protein PST-S (usually Mycobacterium tuberculosis, tuberculosis); outer membrane protein FobA, outer membrane protein FobB, intracellular growth site IglCl, intracellular growth site IglC2, aminotransferase Wbtl, molecular chaperone GroEL, 17 kDa major membrane protein TUL4, lipoprotein LpnA, chitinase family 18 protein, isocitrate dehydrogenase, Nif3 family protein, type IV fimbriae glycosylation protein, outer membrane protein tolC, FAD binding family protein, type IV fimbriae polymeric outer membrane protein, two-component sensor protein KdpD, molecular chaperone protein DnaK, protein TolQ (Francisco tularensis, tularemia); "MB antigen, urease, protein GyrA, protein GyrB, protein ParC, protein ParE, lipid-associated membrane protein LAMP, thymidine kinase TK, phospholipase PL-A1, phospholipase PL-A2, phospholipase PL-C, surface-expressed 96 kDa antigen; (Ureaplasma urealyticum, Ureaplasma urealyticum infection); nonstructural polyprotein, structural polyprotein, capsid protein CP, protein E1, protein E2, protein E3, proteinase PI, proteinase P2, proteinase P3 (Venezuelan equine encephalitis virus, Venezuelan equine encephalitis); glycoprotein GP, ​​matrix protein Z, polymerase L, nucleoprotein N (Guanarito virus, Venezuelan hemorrhagic fever); polyprotein, protein E, protein M, capsid protein C, proteinase NS3, protein NS1, protein NS2A, protein AS2B, protein NS4A, protein NS4B, protein NS5 (West Nile virus, West Nile fever);Capsid protein CP, protein E1, protein E2, protein E3, protein P2 (Western equine encephalitis virus, Western equine encephalitis); genomic polyprotein, protein E, protein M, capsid protein C, protein NS3, protein NS1, protein NS2A, protein AS2B, protein NS4A, protein NS4B, protein NS5 (Yellow fever virus, Yellow fever); putative Yop-targeting protein YobB, effector protein YopD, effector protein YopE, protein YopH, effector protein YopJ, translocation protein YopK , effector protein YopT, protein YpkA, flagellar biosynthesis protein FlhA, peptidase M48, potassium efflux system KefA, transcriptional regulator RovA, adhesin Ifp, transporter LcrV, protein PcrV, invasin Inv, outer membrane protein OmpF-like porin, adhesin YadA, protein kinase C, phospholipase CI, protein PsaA, mannosyltransferase-like protein WbyK, protein YscU, antigen YPMa (Yersinia pseudotuberculosis, Yersinia pseudotuberculosis infection); effector protein YopB, 60 kDa molecular chaperone, protein WbcP, tyrosine protein phosphatase YopH, protein YopQ, enterotoxin, galactoside permease, reductase NrdE, protein YasN, invasin Inv, adhesin YadA, outer membrane porin F OmpF, protein UspA1, protein EibA, protein Hia, cell surface protein Ail, molecular chaperone SycD, protein LcrD, protein LcrG, protein LcrV, protein SycE, protein YopE, regulatory protein TyeA, protein YopM, protein YopN, protein YopO, protein YopT, protein YopD, protease ClpP, protein MyfA, protein FilA, and protein PsaA (Yersinia enterocolitica, yersiniosis).

[0339] In embodiments where the infectious disease is influenza, the mRNA molecule may have a coding region encoding at least one antigenic peptide or protein derived from influenza virus hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), nonstructural protein 1 (NS1), nonstructural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2 or polymerase basic protein 2 (PB2), or fragments or variants thereof.

[0340] In certain embodiments, the coding region encodes at least one antigenic peptide or protein derived from influenza virus hemagglutinin (HA) and / or neuraminidase (NA) or a fragment or variant thereof. HA and / or NA can independently be derived from influenza A virus or influenza B virus or a fragment of either.

[0341] In embodiments where the infectious disease is influenza, the mRNA molecule may have a coding region encoding at least one antigenic peptide or protein derived from the spike (S) protein.

[0342] Examples

[0343] The following synthetic procedures describe methods for making the compounds of the present disclosure. Simple modifications to the conditions or the properties of any particular substrate may be made to obtain any compound within the scope of Formula I and / or Formula II, including direct synthetic modifications to prepare reverse tail esters (L 2 ) analogs. Variations in the synthetic pathway have been shown to allow for variations in the structure of the final compound.

[0344] Example 1a: Synthesis of Compounds SL56-SL61

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358] Preparation of compound 56-2d

[0359]

[0360] To compound 56-1d (75.0 g, 389 mmol, 1.0 ) in H2O (405 mL) and THF (700 mL) was added formaldehyde (56.8 g, 699 mmol, 52.1 mL, 37% purity, 1.8 ) and indium (49.1 g, 427 mmol, 6.72 mL, 1.1 The mixture was stirred at 20°C for 6 hr. TLC (petroleum ether / ethyl acetate = 10 / 1, R of compound 56-1d) f is 0.69, and the R f The reaction mixture was quenched with H2O (1500 mL) and the resulting solution was extracted with EtOAc (1000 mL). The combined organic layers were washed with brine (500 mL), dried over Na2SO4, and concentrated under reduced pressure to afford compound 56-2d (40.0 g, 277 mmol, 71.4% yield) as a yellow oil. 1 H NMR:ET73307-106-P1A1 (400 MHz, CDCl3) 6.25 (d, = 1.2 Hz, 1H), 5.67 (d, =1.2 Hz, 1H), 4.20-4.26 (m, 2H), 3.77 (t, = 6.4 Hz, 2H), 2.57-2.61 (m, 2H),1.32 (t, = 7.2 Hz, 3H).

[0361] Preparation of compound 56-3d

[0362]

[0363] To compound 56-2d (40.0 g, 278 mmol, 1.0 ) in DCM (210 mL) was added Dess-Martin periodinane (129 g, 305 mmol, 94.6 mL, 1.1 The mixture was stirred at 20°C for 2 hr. TLC (petroleum ether / ethyl acetate = 5 / 1, R of compound 56-2d f is 0.49, and the R fThe reaction mixture was quenched with H2O (200 mL) and the resulting solution was extracted with EtOAc (100 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 500 / 1 to 20 / 1) to obtain compound 56-3d (39.0 g, 274 mmol, 98.9% yield) as a colorless oil. 1 H NMR: ET73307-110-P1A (400 MHz, CDCl3) 9.72 (s, 1H), 6.42 (s,1H), 5.73 (s, 1H), 4.24 (q, = 7.2 Hz, 3H), 3.42 (s, 2H), 1.31 (t, = 7.2 Hz, 3H).

[0364] Preparation of compound 56-4d

[0365]

[0366] To a solution of compound 56-3d (39.0 g, 274 mmol, 1.0 eq) in toluene (270 mL) were added 4-methylbenzenesulfonic acid (945 mg, 5.49 mmol, 0.02 eq) and ethylene glycol (25.5 g, 412 mmol, 23 mL, 1.5 eq). The mixture was stirred at 110°C for 5 hours. TLC (petroleum ether / ethyl acetate = 5 / 1, R of compound 56-3d) f is 0.47, and the R f The reaction mixture was quenched with H₂O (500 mL) and the resulting solution was extracted with EtOAc (300 mL). The combined organic layers were washed with brine (200 mL), dried over Na₂SO₄, and concentrated under reduced pressure to afford compound 56-4d (37.0 g, 199 mmol, 72.4% yield) as a colorless oil. 1 H NMR: ET73307-111-P1A (400MHz, CDCl3) 6.29 (s, 1H), 5.73 (s, 1H), 5.07 (t, = 4.8 Hz, 1H), 4.22 (q, = 7.2 Hz, 2H), 3.97-4.00 (m, 2H), 3.84-3.88 (m, 2H), 2.69 (d, = 4.8 Hz,2H), 1.31 (t, = 7.2 Hz, 3H).

[0367] Preparation of compound 56b

[0368]

[0369] To a solution of compound 56a (30.0 g, 159 mmol, 1.0 eq) in DCM (150 mL) was added TBSCl (26.4 g, 175 mmol, 21.6 mL, 1.1 eq) and imidazole (13.0 g, 191 mmol, 1.2 eq). The solution was stirred at 25°C for 12 hours. TLC (petroleum ether / ethyl acetate = 10 / 1, R of compound 56a) f is 0.00, and the R f The reaction mixture was stirred for 2 hours at room temperature for 1 hour (0.47 mmol / l) and the starting material was completely consumed. The reaction mixture was poured into water (20 mL). The organic phase was collected and the aqueous phase was extracted with DCM (20 mL x 2). The combined organic phase was washed with water (10 mL x 2), dried over anhydrous Na2SO4, and concentrated under vacuum to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 500 / 0 to 100 / 1) to obtain compound 56b (26.1 g, 86.4 mmol, 54.2% yield) as a colorless oil. 1 H NMR: ET74125-18-P1A (400 MHz, CDCl3) 3.57-3.62 (t, = 6.8 Hz, 2H), 2.33-2.38 (t, = 7.2 Hz,2H), 1.61-1.68 (m, 2H), 1.48-1.54 (m, 2H), 1.26-1.37 (m, 10H), 0.89-0.91(m,9H), 0.03-0.07 (m, 6H).

[0370] Preparation of compound 56c

[0371]

[0372] To compound 56b (26.0 g, 85.9 mmol, 1.0 ) in DCM (150 mL) was added EDCI (41.2 g, 215 mmol, 2.5 ), DMAP (21.0 g, 172 mmol, 2.0 ) and compound 1A (19.2 g, 103 mmol, 1.2 The solution was stirred at 25 °C for 12 hr. TLC (petroleum ether / ethyl acetate = 5 / 1, R f is 0.30, and the R f The reaction mixture was poured into NH₄Cl (saturated aqueous solution, 20 mL). The solution was extracted three times with DCM (20 mL). The organic phase was dried over sodium sulfate and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO₂, petroleum ether / ethyl acetate = 300 / 1 to 20 / 1) to obtain compound 56c (37.1 g, 78.8 mmol, 91.7% yield) as a colorless oil. 1 H NMR: ET74125-21-P1A (400MHz, CDCl3) 3.91-3.93 (m, 2H), 3.52-3.57 (t, = 6.8 Hz, 2H), 2.23-2.28 (t, = 7.2 Hz, 2H), 1.51-1.61 (m, 3H), 1.41-1.48 (m, 2H), 1.39-1.41 (s, 1H), 1.18-1.28 (m, 25H), 0.82-0.87 (m, 15H), 0.00-0.03 (m, 6H).

[0373] Preparation of compound 56d

[0374]

[0375] To compound 56c (37.1 g, 78.8 mmol, 1.0 ) in THF (80 mL) was added TBAF (1 M, 158 mL, 2.0 The solution was stirred at 25 °C for 2 hr. TLC (petroleum ether / ethyl acetate = 5 / 1, R of compound 56c f is 0.60, and the R fThe reaction mixture was poured into HCl (1 M, 100 mL). The solution was extracted three times with EtOAc (100 mL). The organic layer was concentrated under vacuum to give compound 56d (20.0 g, 56.1 mmol, 71.2% yield) as a colorless oil. Compound 56d (20.0 g, 56.1 mmol, 71.2% yield) was used in the next step without purification. 1 H NMR: ET74125-23-P1A (400 MHz, CDCl3) 3.96-3.99 (m,2H), 3.62-3.68 (m, 2H), 2.28-2.33 (t, = 7.6 Hz, 2H), 1.56-1.67 (m, 5H), 1.25-1.34 (m, 26H), 0.87-0.92 (m, 6H).

[0376] Preparation of compound 56-5d

[0377]

[0378] To compound 56-4d (7.00 g, 37.6 mmol, 1.0 ) in MeOH (70 mL) was added NaOMe (3.05 g, 56.4 mmol, 1.5 ) and nonane-1-thiol (6.03 g, 37.6 mmol, 1.0 The mixture was stirred at 20°C for 12 hr. TLC (petroleum ether / ethyl acetate = 5 / 1, R of compound 56-4d) f is 0.43, and the R f The reaction mixture was quenched with H₂O (200 mL) and extracted with EtOAc (100 mL). The combined organic layers were washed with brine (80 mL), dried over Na₂SO₄, and concentrated under reduced pressure to afford compound 56-5d (11.0 g, 31.7 mmol, 84.4% yield) as a colorless oil. 1 H NMR: ET73307-112-P1A (400 MHz, CDCl3) 4.94-4.96 (m, 1H), 4.14-4.21 (m, 1H), 3.92-3.99 (m, 2H), 3.81-3.87(m, 2H), 3.72 (s, 1H), 2.76-2.84 (m, 2H), 2.64-2.71 (m, 1H), 2.51 (t, = 7.2Hz, 2H), 2.10-2.15 (m, 1H), 1.95-2.00 (m, 1H), 1.53-1.61 (m, 2H), 1.25-1.38(m, 14H), 0.88 (t, = 6.4 Hz, 3H).

[0379] Preparation of compound 56-6d

[0380]

[0381] To compound 56-5d (11.0 g, 31.7 mmol, 1.0 ) in MeOH (60 mL) was added LiOH.H2O (2.00 g, 47.6 mmol, 1.5 The mixture was stirred at 60°C for 12 hr. TLC (petroleum ether / ethyl acetate = 5 / 1, R of compound 56-5d) f is 0.56, and the R f The reaction mixture was poured into HCl (2 M, 100 mL). The aqueous layer was extracted with ethyl acetate (80 mL). The combined organic layers were washed with brine (100 mL), dried over NaSO, filtered, and concentrated under vacuum to afford compound 56-6d (8.00 g, 25.1 mmol, 79.1% yield) as a colorless oil. 1 H NMR: ET73307-115-P1A (400 MHz, CDCl3) 5.01 (t, =4.0 Hz, 1H), 3.94-4.02 (m, 2H), 3.84-3.90 (m, 2H), 2.82-2.88 (m, 2H), 2.67-2.73 (m, 1H), 2.54 (t, = 7.2 Hz, 2H), 2.14-2.21 (m, 1H), 2.02-2.08 (m, 1H), 1.54-1.62 (m, 2H), 1.25-1.39 (m, 12H), 0.89 (t, = 6.4 Hz, 3H).

[0382] Preparation of compound 56-7d

[0383]

[0384] To compound 56-6d (7.00 g, 22.0 mmol, 1.0 ) in DCM (42.0 mL) was added compound 56d (7.84 g, 22.0 mmol, 1.0 ), EDCI (12.6 g, 65.9 mmol, 3.0 ) and DMAP (4.03 g, 33.0 mmol, 1.5 The mixture was stirred at 20°C for 12 hr. TLC (petroleum ether / ethyl acetate = 5 / 1, R of compound 56-6d) f is 0.37, and the R f The reaction mixture was quenched with H₂O (100 mL) and the resulting solution was extracted with EtOAc (80 mL). The combined organic layers were washed with brine (50 mL), dried over Na₂SO₄, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO₂, petroleum ether / ethyl acetate = 500 / 1 to 20 / 1) to obtain compound 56-7d (8.00 g, 12.2 mmol, 55.4% yield) as a colorless oil. 1 H NMR:ET73307-118-P1A (400 MHz, CDCl3) 4.95 (t, = 4.0 Hz, 1H), 4.10 (t, =6.8 Hz, 2H), 3.92-3.98 (m, 4H), 3.84-3.87 (m, 2H), 2.76-2.81 (m, 2H), 2.67-2.71 (m, 1H), 2.51 (t, = 7.2 Hz, 2H), 2.30 (t, = 7.6 Hz, 2H), 2.10-2.17(m, 1H), 1.94-1.99 (m, 1H), 1.53-1.67 (m, 8H), 1.28-1.39 (m, 38H), 0.87-0.92(m, 9H).

[0385] Preparation of compound 56-8d

[0386]

[0387] To compound 56-7d (7.00 g, 10.7 mmol, 1.0 ) in acetone (700 mL) was added TsOH.H2O (811 mg, 4.26 mmol, 0.4 The mixture was stirred at 40°C for 12 hr. TLC (petroleum ether / ethyl acetate = 5 / 1, R of compound 56-7d) f is 0.54, and the R f The reaction mixture was quenched with H2O (1000 mL) and the resulting solution was extracted with EtOAc (500 mL). The combined organic layers were washed with brine (300 mL), dried over Na2SO4, and concentrated under reduced pressure to afford compound 56-8d (7 g, crude) as a yellow oil.

[0388] Preparation of compound SL56

[0389]

[0390] To compound 56-8d (7.00 g, 11.4 mmol, 1.0 ) in DCM (70.0 mL) was added NaBH(OAc)3 (2.42 g, 11.4 mmol, 1.0 ) and NHMe2 (2 M, 5.71 mL, 1.0 The mixture was stirred at 20°C for 3 hr. TLC (dichloromethane / methanol = 10 / 1, R f is 0.80, and the R fThe reaction mixture was quenched by H2O (200 mL) and the resulting solution was extracted with EtOAc (200 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, DCM / MeOH = 500 / 1 to 20 / 1). SL 56 (1.20 g, 1.87 mmol, 16.4% yield) was obtained as a light yellow oil. LCMS: ET73307-127-P1A (M+H + ): 642.5. 1 H NMR: ET73307-127-P1A1(400 MHz, CDCl3) 4.10 (t, = 6.8 Hz, 2H), 3.98 (d, = 5.6 Hz, 12H),2.76-2.82 (m, 1H), 2.62-2.67 (m, 2H), 2.51 (t, = 7.2 Hz, 2H), 2.24-2.32 (m,4H), 2.21 (s, 6H), 1.76-7.84 (m, 2H), 1.53-1.67 (m, 7H), 1.27-1.36 (m, 38H), 0.87-0.90 (m, 9H).

[0391] Preparation of compound 57-2

[0392]

[0393] To compound 57-1 (25.0 g, 144 mmol, 1.0 ) in DCM (150 mL) was added compound 1a (115 g, 574 mmol, 4.0 The solution was stirred at 20°C for 3 hr. TLC (dichloromethane / methanol = 8 / 1, R f is 0.41, and the R f The reaction mixture was filtered, and the filter cake was washed with 100 mL of DCM. The combined filtrate was concentrated to dryness to give a yellow oil. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 50 / 1 to 5 / 1) to give a colorless oil. Compound 57-2 (25.0 g, 109 mmol, 75.6% yield) was obtained as a yellow oil. 1H NMR: ET73270-114-P1A (400 MHz, CDCl3) 3.64 (t, =6.6 Hz, 2H), 2.20 (t, = 6.6 Hz, 2H), 1.55-1.59 (m, 4H), 1.45 (s, 9H), 1.18-1.32 (m, 8H).

[0394] Preparation of compound 57-3

[0395]

[0396] To compound 57-2 (17.0 g, 73.8 mmol, 1.0 ) in DCM (102 mL) was added CBr4 (48.9 g, 148 mmol, 2.0 ) and Ph3P (29.0 g, 111 mmol, 1.5 The solution was stirred at 20°C for 12 hours. TLC (dichloromethane / methanol = 8 / 1, R of compound 57-2 f is 0.57, and the R f The reaction mixture was poured into an aqueous solution of H₂O (aqueous solution, 150 mL). The resulting solution was extracted with DCM (100 mL x 2). The mixture was dried over anhydrous Na₂SO₄ and concentrated under vacuum to obtain a crude product as a yellow oil. The residue was purified by column chromatography (SiO₂, petroleum ether / ethyl acetate = 1 / 0 to 50 / 1) to obtain a yellow oil. Compound 57-3 (20.0 g, 68.2 mmol, 92.4% yield) was obtained as a yellow oil. 1 H NMR: ET73270-119-P1A (400 MHz, CDCl3) 3.44 (t, = 6.8 Hz, 2H), 2.23 (t, = 7.6 Hz, 2H), 1.85-1.90 (m, 2H), 1.61(t, = 7.0 Hz, 2H), 1.48 (s, 11H), 1.34 (s, 6H).

[0397] Preparation of compound 57-4

[0398]

[0399] To compound 57-3 (20.0 g, 68.2 mmol, 1.0 ) in THF (100 mL) was added C2H3OSK (12.0 g, 105 mmol, 1.54 The suspension was stirred at 50°C for 3 hr. TLC (petroleum ether / ethyl acetate = 8 / 1, R of compound 57-3) f is 0.61, and the R f The reaction was completed (0.67). The reaction was poured into an aqueous solution of HO (100 mL). The resulting solution was extracted with EtOAc (100 mL x 2). The combined organic phases were washed with brine (100 mL x 2), dried over anhydrous NaSO, and concentrated under vacuum to give the crude product as a yellow oil. Compound 57-4 (19 g, 65.87 mmol, 96.58% yield) was obtained as a yellow oil. 1 H NMR: ET73270-120-P1A (400 MHz, CDCl3) 2.86 (t, = 7.4 Hz, 2H), 2.33 (s, 3H), 2.20 (t, = 7.4Hz, 2H), 1.55-1.59 (m, 4H), 1.45 (s, 9H), 1.30-1.43 (m, 8H).

[0400] Preparation of compound 57-5

[0401]

[0402] To compound 57-4 (19.0 g, 65.9 mmol, 1.0 ) in MeOH (200 mL) was added K2CO3 (13.7 g, 98.8 mmol, 1.5 The suspension was stirred at 20°C for 3 hr. TLC (petroleum ether / ethyl acetate = 8 / 1, R of compound 57-4) f is 0.59, and the R fThe reaction was completed (0.68). The reaction was poured into an aqueous solution of HO (100 mL). The resulting solution was extracted with EtOAc (100 mL x 2). The combined organic phases were washed with brine (100 mL x 2), dried over anhydrous NaSO, and concentrated under vacuum to give the crude product as a yellow oil. Compound 57-5 (16.0 g, 64.9 mmol, 98.6% yield) was obtained as a yellow oil. 1 H NMR: ET73270-120-P1A (400 MHz, CDCl3) 2.50-2.55 (m, 2H), 2.20 (t, = 7.6 Hz, 2H), 1.57-1.61 (m,4H), 1.45 (s, 9H), 1.33-1.35 (m, 2H), 1.26-1.31 (m, 6H).

[0403] Preparation of compound 57-6

[0404]

[0405] To compound 56-5 (11.5 g, 61.7 mmol, 0.95 ) in MeOH (80 mL) was added NaOMe (5.26 g, 97.4 mmol, 1.5 ) and compound 57-4d (16.00 g, 64.93 mmol, 1.00 The solution was stirred at 20 °C for 1 hr. TLC (petroleum ether / ethyl acetate = 1 / 1, R of compound 57-5) f is 0.26, and the R f The reaction was completed (the % d-HgCl2 solution was 0.32). The reaction mixture was poured into an aqueous solution of HO (100 mL). The resulting solution was extracted with EtOAc (100 mL x 2). The mixture was dried over anhydrous NaSO and concentrated under vacuum to obtain a crude product as a yellow oil. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 80 / 1 to 10 / 1) to obtain a yellow oil. Compound 57-6 (25.0 g, 57.8 mmol, 89.0% yield) was obtained as a yellow oil. 1 H NMR: ET73270-124-P1A (400MHz, CDCl3) 4.87-4.88 (m, H), 3.87-3.89 (m, 2H), 3.76-3.77 (m, 2H), 2.71-2.73 (m, 2H), 2.61 (m, 1H), 2.42 (t, = 7.2 Hz, 2H), 2.13 (t, = 7.6 Hz, 2H), 2.10-2.11, (m, 1H), 1.95-1.97 (m, 1H), 1.49-1.52 (m, 4H), 1.37 (s, 9H), 1.21-1.22 (m, 2H), 1.17-1.21 (m, 9H).

[0406] Preparation of compound 57-7

[0407]

[0408] To compound 57-6 (20.0 g, 46.2 mmol, 1.0 ) in MeOH (200 mL) and H2O (40 mL) was added LiOH.H2O (3.88 g, 92.5 mmol, 2.0 The suspension was stirred at 40°C for 12 hr. TLC (dichloromethane / methanol = 8 / 1, R of compound 57-6 f is 0.72, and the R f The reaction mixture was added into an aqueous solution of HCl (1 M, 200 mL). The resulting solution was extracted with EtOAc (200 mL x 2). The mixture was dried over anhydrous Na2SO4 and concentrated under vacuum to obtain a crude product as a yellow oil. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 80 / 1 to 10 / 1) to obtain a yellow oil. Compound 57-7 (7.30 g, 18.0 mmol, 39.0% yield) was obtained as a yellow oil. 1 H NMR: ET73270-126-P1A (400 MHz, CDCl3) 5.31(t, = 7.2 Hz, 1H), 3.98-4.00 (m, 2H), 3.87-3.88 (m, 2H), 2.84-2.86 (m, 2H), 2.70-2.71 (m, 1H), 2.52 (t, = 6.8 Hz, 2H), 2.21 (t, = 7.6 Hz, 2H), 1.56-1.60 (m, 4H), 1.45 (s, 9H), 1.29-1.30 (m, 8H).

[0409] Preparation of compound 57-8

[0410]

[0411] To compound 57-7 (7.30 g, 18.0 mmol, 1.0 ) in DCM (43.8 mL) was added EDCI (10.4 g, 54.1 mmol, 3.0 ), DMAP (3.31 g, 27.1 mmol, 1.5 ) and compound 57-2 (3.95 g, 17.1 mmol, 0.95 The solution was stirred at 20°C for 12 hr. TLC (dichloromethane / methanol, R f is 0.34, and the R f The reaction mixture was poured into H2O (100 mL). The resulting solution was extracted with DCM (100 mL x 2). The mixture was dried over anhydrous Na2SO4 and concentrated under vacuum to obtain a crude product as a yellow oil. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 20 / 1) to obtain a yellow oil. Compound 57-8 (3.80 g, 9.73 mmol, 53.9% yield) was obtained as a yellow oil. 1 H NMR: ET73270-37-P1A (400 MHz, CDCl3) 4.94 (t, = 4.2 Hz, 1H), 4.08-4.14 (m, 2H), 3.94-3.96 (m,2H), 3.83-3.84 (m, 2H), 2.78-2.80 (m, 2H), 2.68-2.68 (m, 1H), 2.51 (t, =7.6 Hz, 2H), 2.20 (t, = 7.6 Hz, 4H), 2.10-2.19 (m, 1H), 2.09-2.10 (m, 1H), 1.58-1.60 (m, 8H), 1.44 (s, 18H), 1.26-1.31 (m, 16H).

[0412] Preparation of compound 57-9

[0413]

[0414] To compound 57-8 (6.00 g, 9.73 mmol, 1.0 ) in DCM (60 mL) was added TFA (22.2 g, 195 mmol, 14.5 mL, 20.0 The solution was stirred at 20°C for 12 hr. TLC (petroleum ether / ethyl acetate = 5 / 1, R of compound 57-8) f is 0.42, and the R f The reaction was completed (ratio = 0.09). The reaction was concentrated under vacuum to give the crude product as a yellow oil. Compound 57-9 (5.00 g, crude) was obtained as a yellow oil. 1 H NMR:ET73270-133-P1A (400 MHz, CDCl3) 9.80 (s, 1H), 4.11 (t, = 6.6 Hz, 2H),3.13-3.14 (m, 1H), 2.87-3.95 (m, 3H), 2.67-2.68 (m, 1H), 2.50 (t, = 7.4 Hz,2H), 2.36 (t, = 7.2 Hz, 4H), 1.55-1.66 (m, 6H), 1.33 (s, 18H).

[0415] Preparation of compound 57-10

[0416]

[0417] To compound 57-9 (5.00 g, 10.85 mmol, 1.0 ) in DCM (40 mL) was added NHMe2 (2 M, 5.43 mL, 1.0 The solution was stirred at 20°C for 1 hr. Then, NaBH(OAc)3 (2.30 g, 10.9 mmol, 1.0 The solution was stirred at 20°C for 1 hr. TLC (dichloromethane / methanol = 8 / 1, R of compound 57-9 f is 0.34, and the R fThe reaction was completed (the reaction temperature was 0.02). The reaction was poured into an aqueous solution of NaHCO₃ (50 mL of aqueous solution). The resulting solution was extracted with DCM (100 mL x 2). The aqueous phase was then acidified to pH = 3 with 2M HCl. The aqueous phase was extracted with EtOAc (100 mL x 2). Dried over anhydrous Na₂SO₄ and concentrated under vacuum to obtain the crude product as a yellow oil. Compound 57-10 (4.30 g, 8.78 mmol, 80.9% yield) was obtained as a colorless oil. 1 H NMR: ET73270-136-P1A (400 MHz, CDCl3) 4.10-4.15 (m, 2H), 3.08-3.24 (m, 1H), 2.98-3.18(m, 1H), 2.85 (s, 6H), 2.70-2.73 (m, 2H), 2.52 (t, = 7.2 Hz, 2H), 2.35 (t, = 7.2 Hz, 4H), 2.11-2.20 (m, 2H), 1.55-1.65 (m, 8H), 1.26-1.34 (m, 16H).

[0418] Preparation of SL57

[0419]

[0420] To compound 57-10 (4.30 g, 8.78 mmol, 1.0 ) in DCM (43 mL) was added EDCI (5.05 g, 26.3 mmol, 3.0 ), DMAP (1.61 g, 13.17 mmol, 1.5 ) and compound 10A (3.80 g, 26.3 mmol, 3.0 ). The solution was stirred at 20 ° C for 12 hr. LCMS showed that the reaction was complete. The reactants were poured into an aqueous solution of H2O (aqueous solution 100 mL). The resulting solution was extracted with DCM (100 mL x 2). It was dried over anhydrous Na2SO4 and concentrated under vacuum to give a crude product as a yellow oil. The residue was purified by column chromatography (SiO2, dichloromethane / methanol = 200 / 1 to 50 / 1) to give a light yellow oil. SL57 (1.00 g, 1.34 mmol, 15.3% yield, 99.7% purity) was obtained as a light yellow oil. LCMS: ET73270-137-P1A, RT = 0.791, M+H+ = 742.59. 1 H NMR:ET73270-137-P1A (400 MHz, CDCl3) 4.88 (t, = 6.4 Hz, 2H), 4.10 (t, =6.6 Hz, 2H), 2.77-2.78 (m, 1H), 2.63-2.65 (m, 2H), 2.49 (t, = 7.6 Hz, 2H),2.29 (t, = 7.4 Hz, 6H), 2.20 (s, 6H), 1.76-1.81 (m, 2H), 1.53-1.62 (m, 8H), 1.51-1.53 ​​(m, 8H), 1.27-1.35 (m, 32H), 0.89 (t, = 6.8 Hz, 12H).

[0421] Preparation of compound 58-2

[0422]

[0423] To compound 58-1 (18.0 g, 52.6 mmol, 1.0 ) in DCM (200 mL) was added compound 58-1a (15.2 g, 105 mmol, 2.0 ), EDCI (30.2 g, 158 mmol, 3.0 ) and DMAP (9.63 g, 78.8 mmol, 1.5 The mixture was stirred at 20°C for 12 hr. TLC (petroleum ether / ethyl acetate = 5 / 1, R of compound 58-1) f is 0.31, and the R fThe reaction mixture was quenched with H2O (500 mL) and the resulting solution was extracted with EtOAc (300 mL). The combined organic layers were washed with brine (200 mL), dried over Na2SO4, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 500 / 1 to 20 / 1). Compound 58-2 (15.0 g, 25.2 mmol, 47.9% yield) was obtained as a yellow oil.

[0424] Preparation of compound 58-3

[0425]

[0426] To compound 58-2 (15.0 g, 25.2 mmol, 1.0 ) in THF (80 mL) was added NaBH4 (954 mg, 25.2 mmol, 1.0 The mixture was stirred at 0°C for 2 hr. TLC (petroleum ether / ethyl acetate = 5 / 1, R of compound 58-2) f is 0.58, and the R f The reaction mixture was quenched with H2O (200 mL) and the resulting solution was extracted with EtOAc (100 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 200 / 1 to 20 / 1). Compound 58-3 (13.0 g, 21.8 mmol, 86.4% yield) was obtained as a colorless oil. 1 H NMR:ET73307-117-P1A (400 MHz, CDCl3) 4.85-4.91 (m, 2H), 3.56-3.59 (m, 1H), 2.29(t, = 7.6 Hz, 4H), 1.61-1.64 (m, 4H), 1.49-1.55 (m, 8H), 1.41-1.45 (m, 4H), 1.24-1.36 (m, 36H), 0.89 (t, = 6.8 Hz, 12H).

[0427] Preparation of compound 58-5d

[0428]

[0429] To compound 56-4d (7.00 g, 37.6 mmol, 1.0 ) in MeOH (70 mL) was added NaOMe (3.05 g, 56.4 mmol, 1.5 ) and compound 58-4d (4.97 g, 37.6 mmol, 5.89 mL, 1.0 The mixture was stirred at 20°C for 12 hr. TLC (petroleum ether / ethyl acetate = 5 / 1, R of compound 56-4d) f is 0.43, and the R f The reaction mixture was quenched with H₂O (300 mL) and the resulting solution was extracted with EtOAc (200 mL). The combined organic layers were washed with brine (100 mL), dried over Na₂SO₄, and concentrated under reduced pressure to afford the product. Compound 58-5d (10.0 g, 31.4 mmol, 83.5% yield) was obtained as a colorless oil. 1 H NMR: ET73307-114-P1A (400 MHz, CDCl3) 4.94-4.96 (m, 1H), 3.92-3.99 (m, 2H), 3.81-3.87(m, 2H), 3.71 (s, 2H), 2.77-2.84 (m, 2H), 2.66-2.71 (m, 1H), 2.51 (t, = 7.6Hz, 2H), 2.10-2.17 (m, 1H), 1.95-2.01 (m, 1H), 1.53-1.61 (m, 2H), 1.25-1.38(m, 10H), 0.89 (t, = 6.8 Hz, 3H).

[0430] Preparation of compound 58-6d

[0431]

[0432] To compound 58-5d (10.0 g, 31.4 mmol, 1.0 ) in MeOH (60 mL) was added LiOH.H2O (1.98 g, 47.1 mmol, 1.5 The mixture was stirred at 60°C for 12 hr. TLC (petroleum ether / ethyl acetate = 5 / 1, R of compound 58-5d) fis 0.56, and the R f The reaction mixture was poured into HCl (2 M, 100 mL). The aqueous layer was extracted with ethyl acetate (80 mL). The combined organic layers were washed with brine (100 mL), dried over NaSO, filtered, and concentrated under vacuum to afford the product. Compound 58-6d (9.00 g, 31.0 mmol, 98.7% yield) was obtained as a colorless oil. 1 H NMR: ET73307-116-P1A (400 MHz, CDCl3) 5.01(t, = 4.0 Hz, 1H), 3.96-4.02 (m, 2H), 3.84-3.90 (m, 2H), 2.81-2.88 (m, 2H), 2.67-2.73 (m, 1H), 2.54 (t, = 7.2 Hz, 2H), 2.14-2.21 (m, 1H), 2.01-2.07 (m,1H), 1.54-1.62 (m, 2H), 1.25-1.39 (m, 10H), 0.89 (t, = 6.4 Hz, 3H).

[0433] Preparation of compound 58-7d

[0434]

[0435] To compound 58-6d (4.00 g, 13.7 mmol, 1.0 ) and compound 58-3 (8.22 g, 13.7 mmol, 1.0 ) in DCM (24 mL) was added EDCI (7.92 g, 41.3 mmol, 3.0 ) and DMAP (2.52 g, 20.6 mmol, 1.5 The solution was stirred at 20°C for 2 hr. TLC (petroleum ether / ethyl acetate = 5 / 1, R of compound 58-6) f is 0.24, and the R fThe reaction mixture was poured into water (100 mL). The resulting solution was extracted with EtOAc (50 mL x 3). The combined organic phases were washed with brine (50 mL) and concentrated under vacuum to obtain a crude product as a yellow oil. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 0 / 1) to obtain compound 58-7d (5.00 g, 5.75 mmol, 41.7% yield) as a yellow oil. 1 HNMR: ET73400-23-P1A (400 MHz, CDCl3) 4.90-4.93 (m, 1H), 4.84-4.89 (m, 2H), 3.93-3.97 (m, 2H), 3.82-3.84 (m, 2H), 2.75-2.81 (m, 2H), 2.65-2.69 (m, 1H), 2.51 (t, = 7.2 Hz, 2H), 2.28 (t, = 7.2 Hz, 4H), 2.65-2.69 (m, 1H), 2.09-2.16 (m, 1H), 1.92-1.98 (m, 1H), 1.49-1.63 (m, 19H), 1.20-1.37(m, 44H), 0.89(t, = 6.8 Hz, 15H).

[0436] Preparation of compound 58-8d

[0437]

[0438] To a solution of compound 58-7d (4.00 g, 4.60 mmol, 1.0 eq) in acetone (400 mL) was added TsOH.H2O (350 mg, 1.84 mmol, 0.4 eq). The solution was stirred at 60°C for 12 hours. TLC (petroleum ether / ethyl acetate = 5 / 1, R of compound 58-7d) f is 0.38, and the R fThe mixture was stirred for 2 hours at room temperature for 1 h (0.43 mmol / l) indicating that 60% of the reactant 1 remained. The suspension was concentrated under reduced pressure to obtain a residue. The residue was poured into water (100 mL). The resulting solution was extracted with EtOAc (50 mL x 3). The combined organic phases were washed with brine (50 mL) and concentrated under vacuum to obtain a crude product as a yellow oil. Compound 58-8d (4.00 g, crude product) was obtained as a yellow oil and used in the next step without purification.

[0439] Preparation of SL58

[0440]

[0441] To compound 58-8d (4.00 g, 4.85 mmol, 1.0 ) in DCM (30 mL) was added NaBH(OAc)3 (1.03 g, 4.85 mmol, 1.0 ) and N-methylmethanamine (2 M, 2.42 mL, 1.0 The solution was stirred at 20°C for 3 hr. TLC (dichloromethane / methanol = 10 / 1, R f The R of SL58 is 0.34. f The reaction mixture was poured into water (50 mL). The resulting solution was extracted with EtOAc (30 mL x 3). The combined organic phases were washed with water (30 mL x 3) and concentrated under vacuum to give a crude product as a yellow oil. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 0 / 1) to give SL58 (1.00 g, 1.17 mmol, 24.1% yield) as a light yellow oil. 1 H NMR: ET73400-32-P1A (400 MHz, CDCl3) 4.84-4.90 (m, 3H), 2.75-2.81 (m, 1H), 2.60-2.65 (m, 2H), 2.51 (t, = 6.8 Hz, 2H),2.25-2.30 (m, 6H), 2.20 (s, 6H), 1.75-1.84 (m, 2H), 1.49-1.65 (m, 18H), 1.20-1.39(m, 44H), 0.89 (t, = 7.2 Hz, 15H).

[0442] Preparation of compound 60-5B

[0443]

[0444] To compound 60-5A (25.0 g, 144 mmol, 1.0 ) in DCM (125 mL) was added TFAA (66.3 g, 316 mmol, 43.9 mL, 2.2 ) and t-BuOH (37.2 g, 502 mmol, 48.0 mL, 3.5 The suspension was stirred at 20°C for 12 hr. TLC (petroleum ether / ethyl acetate = 10 / 1, R of compound 60-5A) f is 0.29, and the R f The reaction mixture was stirred for 2 hours at room temperature for 1 hour (0.80) to complete the reaction. The reactant was poured into an aqueous solution of H2O (100 mL of aqueous solution). The resulting solution was extracted with DCM (100 mL x 2). The combined organic phases were washed with brine (100 mL x 2), dried over anhydrous Na2SO4, and concentrated under vacuum to obtain a crude product as a yellow oil. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 10 / 1) to obtain a colorless oil. Compound 60-5B (28.0 g, 122 mmol, 84.7% yield) was obtained as a colorless oil.

[0445] Preparation of compound 60-2

[0446]

[0447] To compound 60-1 (40.0 g, 159 mmol, 1.0 ) in DCM (200 mL) was added TFAA (73.6 g, 350 mmol, 48.7 mL, 2.2 ) and t-BuOH (41.3 g, 557 mmol, 53.3 mL, 3.5 The solution was stirred at 20°C for 12 hr. TLC (petroleum ether / ethyl acetate = 10 / 1, R f is 0.29, and the R fThe reaction was completed (the % yield was 0.80). The reactants were poured into an aqueous solution of H2O (100 mL of aqueous solution). The resulting solution was extracted with DCM (100 mL x 2). The combined organic phases were washed with brine (100 mL x 2), dried over anhydrous Na2SO4, and concentrated under vacuum to obtain a crude product as a yellow oil. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 10 / 1) to obtain a colorless oil. Compound 60-2 (45.0 g, 146 mmol, 91.9% yield) was obtained as a yellow oil.

[0448] Preparation of compound 60-3

[0449]

[0450] To compound 60-2 (40.0 g, 130 mmol, 1.0 ) in THF (200 mL) was added C2H3OSK (22.3 g, 195 mmol, 1.5 The suspension was stirred at 50°C for 3 hr. TLC (petroleum ether / ethyl acetate = 10 / 1, R of compound 60-2 f is 0.21, and the R f The reaction was completed at 0.69 (0.69). The reaction was poured into an aqueous solution of HO (200 mL). The resulting solution was extracted with EtOAc (100 mL x 2). The combined organic phases were washed with brine (100 mL x 2), dried over anhydrous NaSO, and concentrated under vacuum to give the crude product as a yellow oil. Compound 60-3 (30.0 g, 99.2 mmol, 76.2% yield) was obtained as a yellow oil. 1 H NMR: ET73270-54-P1A (400 MHz, CDCl3) 2.79 (t, = 7.4 Hz, 2H), 2.25 (s, 3H), 2.13 (t, = 7.4Hz, 2H), 1.45-1.50 (m, 5H), 1.37 (s, 10H), 1.25-1.27 (m, 3H), 1.21 (s, 9H).

[0451] Preparation of compound 60-5

[0452]

[0453] To compound 60-3 (30.0 g, 99.2 mmol, 1.00 ) in MeOH (150 mL) was added K2CO3 (27.4 g, 198 mmol, 2.0 The suspension was stirred at 20°C for 12 hr. TLC (petroleum ether / ethyl acetate = 8 / 1, R of compound 60-3) f is 0.61, and the R f The reaction was completed at 0.57 (0.57). The reaction was poured into an aqueous solution of HO (100 mL). The resulting solution was extracted with EtOAc (200 mL x 2). The combined organic phases were washed with brine (100 mL x 2), dried over anhydrous NaSO, and concentrated under vacuum to give the crude product as a yellow oil. Compound 60-5 (24.0 g, 92.2 mmol, 92.9% yield) was obtained as a colorless oil. 1 H NMR: ET73270-92-P1A (400 MHz, CDCl3) 2.52-2.56 (m, 2H), 2.21 (t, = 7.4 Hz, 2H), 1.55-1.61 (m,4H), 1.45 (s, 9H), 1.32-1.35 (m, 2H), 1.30 (s, 8H).

[0454] Preparation of compound 60-4A

[0455]

[0456] Compound 59-3 (18.0 g, 115.98 mmol, 1.0 HCl (6 M, 90 mL, 4.66 ) solution was stirred at 100°C for 12 hours. LC-MS indicated the reaction was complete. The reaction was concentrated under vacuum to give the crude product as a yellow solid. Compound 60-4A (20.0 g, 113 mmol, 97.1% yield, HCl) was obtained as a yellow solid. LCMS: ET73270-90-P1A, RT = 0.053, M+H + = 142.08.

[0457] Preparation of compound 60-5A

[0458]

[0459] To compound 60-4A (20.0 g, 113 mmol, 1.0 , HCl) in DCM (100 mL) was added EDCI (43.2 g, 225 mmol, 2.0 ), DMAP (27.5 g, 225 mmol, 2.0 ) and compound 60-5B (19.7 g, 85.6 mmol, 0.76 The solution was stirred at 20°C for 12 hr. TLC (petroleum ether / ethyl acetate = 8 / 1, R of compound 60-4A) f is 0.63, and the R f The reaction was completed (the residue was 0.00). The reactant was poured into an aqueous solution of H2O (20 mL of aqueous solution). The resulting solution was extracted with DCM (10 mL x 2). The combined organic phases were washed with brine (50 mL x 2), dried over anhydrous Na2SO4, and concentrated under vacuum to obtain a crude product as a yellow oil. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to obtain a colorless oil. Compound 60-5A (6.00 g, 16.9 mmol, 15.1% yield) was obtained as a colorless oil. 1 H NMR: ET73270-91-P1A (400 MHz, CDCl3) 6.88 (t, = 1.6 Hz, 1H), 4.14 (t, = 6.8 Hz, 4H), 3.14 (s, 2H), 2.58-2.59(m, 2H), 2.47 (s, 2H), 2.41 (s, 3H), 1.58-1.66 (m, 4H), 1.45 (s, 9H), 1.25-1.31 (m, 8H).

[0460] Preparation of compound 60-8

[0461]

[0462] To compound 60-5A (6.00 g, 16.9 mmol, 1.0 ) in MeOH (30 mL) was added NaOMe (1.38 g, 25.5 mmol, 1.5 ) and compound 60-5A (8.84 g, 33.9 mmol, 2.0 The suspension was stirred at 20°C for 12 hr. TLC (dichloromethane / methanol = 8 / 1, R of compound 60-5A) f is 0.52, and the R f The reaction was completed (the % yield was 0.55). The reactants were poured into an aqueous solution of H2O (100 mL of aqueous solution). The resulting solution was extracted with EtOAc (100 mL x 2). The combined organic phases were washed with brine (100 mL x 2), dried over anhydrous Na2SO4, and concentrated under vacuum to obtain a crude product as a yellow oil. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 50 / 1 to 2 / 1) to obtain a yellow oil. Compound 60-8 (8.00 g, 13.0 mmol, 76.8% yield) was obtained as a yellow oil. 1 H NMR: ET73270-93-P1A (400 MHz, CDCl3) 4.07-4.13 (m, 3H), 3.41 (s, 1H),2.85 (s, 1H), 2.56 (s, 1H), 2.78 (s, 1H), 2.39-2.63 (m, 2H), 2.24 (s, 1H),2.29 (s, 3H), 2.20 (t, = 6.6 Hz, 4H), 1.94-2.03 (m, 3H), 1.76 (s, 1H), 1.52-1.57 (m, 9H), 1.44 (s, 18H), 1.26-1.35 (m, 22H).

[0463] Preparation of compound 60-9

[0464]

[0465] To compound 60-8 (8.00 g, 13.0 mmol, 1.0 ) in DCM (40 mL) was added TFA (29.7 g, 261 mmol, 19.4 mL, 20 The solution was stirred at 30°C for 12 hr. TLC (dichloromethane / methanol = 8 / 1, R of compound 60-8 f is 0.53, and the R f The reaction was completed (0.00). The reaction was concentrated under vacuum to give the crude product as a yellow oil. Compound 60-9 (4.00 g, 6.50 mmol, 49.9% yield, TFA) was obtained as a yellow oil.

[0466] Preparation of SL60

[0467]

[0468] To compound 60-9 (4.00 g, 6.50 mmol, 1.0 , TFA) in DCM (20 mL) was added EDCI (3.74 g, 19.5 mmol, 3.0 ), DMAP (2.38 g, 19.5 mmol, 3.0 ) and compound 9A (5.62 g, 38.9 mmol, 6.0 The suspension was stirred at 20°C for 3 hr. TLC (dichloromethane / methanol = 8 / 1, R of compound 60-9 f is 0.01, and the R f The reaction was completed at 0.68 (0.68). The reactants were poured into an aqueous solution of HO (100 mL of the aqueous solution). The resulting solution was extracted with DCM (100 mL x 2). The combined organic phases were washed with brine (100 mL x 2), dried over anhydrous Na2SO4, and concentrated under vacuum to obtain a crude product as a yellow oil. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1 to 8 / 1) to obtain a yellow oil. SL60 (1.00 g, 1.33 mmol, 20.4% yield) was obtained as a light yellow oil. 1 H NMR: ET73270-95-P1A (400 MHz, CDCl3) 4.86-4.91 (m, 2H), 4.05-4.15 (m, 2H), 3.20 (s, 1H), 2.93 (s, 1H), 2.81 (s, 1H), 2.62 (s, 1H), 2.50-2.59 (m, 2H), 2.38 (s, 1H), 2.27-2.30 (m, 7H), 1.92-2.13(m, 2H), 1.83-1.87 (m, 1H), 1.62-1.71 (m, 6H), 1.52-1.57 (m, 10H), 1.27-1.33(m, 34H), 0.89 (t, = 6.8 Hz, 12H).

[0469] Preparation of compound 59-2

[0470]

[0471] To a solution of compound 59-1 (200 g, 1.46 mol, 172 mL, 1.0 eq) in MeOH (600 mL) was added CH3I (207 g, 1.46 mol, 91 mL, 1.0 eq). The solution was stirred at 70°C for 8 hours. TLC showed complete consumption of the starting material. The reaction suspension was filtered, and the filter cake was concentrated under reduced pressure to obtain compound 59-2 (400 g, 1.43 mol, 98.28% yield) as a yellow solid. 1 H NMR: ET73288-4-P1A (400 MHz, CDCl3) 9.16-9.18 (d, = 6.4 Hz, 2H), 8.49-8.51 (d, = 6.4 Hz, 2H), 4.43 (s, 3H), 3.99 (s, 3H).

[0472] Preparation of compound 59-3

[0473]

[0474] To a solution of compound 59-2 (90 g, 322.5 mmol, 1.0 eq) in EtOH (270 mL) and THF (270 mL) was added NaBH4 (20.7 g, 548.2 mmol, 1.7 eq) at 0-10°C. The reaction solution was stirred at 20°C for 6 hours. TLC showed complete consumption of the starting material. The solution was poured into NH4Cl (saturated solution 300 mL) and extracted with EtOAc (200 mL x 2). The combined organic layers were washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 59-3 (45 g, 290 mmol, 89.9% yield) as a yellow oil. 1 H NMR: ET73288-5-P1A (400 MHz, CDCl3) 6.87-6.89 (m, 1H), 3.73 (m, 3H), 3.08 (q, = 3.2 Hz, 2H),2.54 (t, = 5.6 Hz, 2H), 2.42-2.44 (m, 2H), 2.03 (s, 3H).

[0475] Preparation of compound 59-4

[0476]

[0477] To a solution of compound 59-3 (10.0 g, 64.4 mmol, 1.0 eq) and compound 59-3A (10.3 g, 64.4 mmol, 1.0 eq) in MeOH (50 mL) was added NaOMe (5.2 g, 96.7 mmol, 1.5 eq) at 0°C-10°C. The reaction solution was stirred at 20°C for 6 hours. TLC showed complete consumption of the starting material. The solution was poured into NH4Cl (saturated solution 50 mL) and extracted with EtOAc (50 mL x 2). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 59-4 (15 g, 47.5 mmol, 73.8% yield) as a yellow oil. 1 HNMR: ET73288-18-P1A (400 MHz, CDCl3) 3.71 (s, 3H), 3.21 (s, 1H), 2.91-2.94(m, 1H), 2.72-2.79 (m, 1H), 2.54-2.56 (m, 1H), 2.49-2.52 (m, 2H), 2.43-2.49(m, 1H), 2.29 (s, 3H), 2.02-2.13 (m, 2 H), 1.82-2.00 (m, 1H), 1.53-1.57 (m,2H), 1.26-1.34 (m, 12H), 0.88 (t, = 6.8 Hz, 3H).

[0478] Preparation of compound 59-5

[0479]

[0480] Compound 59-4 (5.0 g, 15.9 mmol, 1.0 eq) and NaOH (4 M, 19.8 mL, 5.0 eq) in MeOH (40 mL) were added. The solution was stirred at 20 ° C for 12 hours. TLC showed that the starting material was completely consumed. The solution was poured into HCl (4 M, 100 mL) until the pH = 2-3 and extracted with DCM (200 mL x 2). The combined organic layers were washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 59-5 (4.0 g, 13.3 mmol, 83.7% yield) as a yellow oil. 1H NMR: ET73288-20-P1A (400 MHz, CDCl3) 6.90 (s, 1H),3.54-3.76 (m, 1H), 3.46-3.52 (m, 2H), 3.14-3.22 (m, 1H), 2.81-2.86 (m, 2H),2.63-2.68 (m, 2H), 2.09-2.53 (m, 3H), 1.57-1.60 (m, 2 H), 1.25-1.36 (m, 12H), 0.88 (t, = 6.8 Hz, 3H).

[0481] Preparation of Compound 01-59

[0482]

[0483] To a solution of compound 1a (12.8 g, 63.7 mmol, 4.0 eq) in DCM (15 mL) was added compound 1-59 (3.0 g, 15.9 mmol, 1.0 eq). The solution was stirred at 50°C for 12 hours. TLC (DCM:methanol = 8:1, R of 1-59) f R of 0.70, 01-59 f The % d-HgCl2 (0.65) indicated complete consumption of the starting material. The solution was concentrated under reduced pressure to obtain a residue. The residue was triturated with MTBE (20 mL) at 25°C for 30 min. The suspension was then filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 15:1 to 2:1) to obtain compound 01-59 (2.9 g, 11.9 mmol, 74.5% yield) as a colorless oil. 1 H NMR: ET74125-6-P1A (400MHz CDCl3) δ 3.64-3.65 (m, 2H), 2.19-2.23 (t, = 7.6 Hz, 2H), 1.55-1.61 (m, 4H), 1.41-1.47 (m, 10H), 1.28-1.33 (m, 9H).

[0484] Preparation of compound 59-6

[0485]

[0486] To a solution of compound 01-59 (2.4 g, 9.9 mmol, 1.5 eq) in DCM (10 mL) were added DMAP (1.2 g, 9.9 mmol, 1.5 eq), EDCI (2.5 g, 13.3 mmol, 2.0 eq), and compound 59-5 (2.0 g, 6.6 mmol, 1.0 eq). The solution was stirred at 25°C for 12 hours. TLC (DCM:methanol = 8:1, R of 59-5) f R of 0.05, 59-6 f The reaction mixture was poured into HO (10 mL). The solution was extracted three times with DCM (10 mL). The combined organic phases were dried over sodium sulfate and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 10:1 to 2:1) to obtain compound 59-6 (2.0 g, 3.8 mmol, 57.1% yield) as a colorless oil. 1 H NMR: ET74125-9-P1A (400 MHz CDCl3) δ 4.05-4.13 (m, 2H), 3.16-3.25 (m, 1H), 2.91-3.00 (m, 1H), 2.80-2.85 (m, 1H), 2.50-2.55 (m, 2H), 2.34-2.41 (m, 1H), 2.50-2.57 (m, 2H), 2.35-2.40 (m, 1H), 2.27-2.31 (m, 2H) 2.18-2.22 (m, 3H), 1.96-2.06 (m, 2H), 1.75-1.92 (m, 2H), 1.62-1.66 (m, 2H), 1.55-1.59 (m, 4H), 1.45 (s, 9H), 1.26-1.30 (m, 22H), 0.87-0.90 (m, 3H).

[0487] Preparation of compound 59-7

[0488]

[0489] To a solution of compound 59-6 (2.0 g, 3.8 mmol, 1.0 eq) in DCM (14 mL) was added TFA (4.3 g, 37.9 mmol, 2.81 mL, 10.0 eq). The solution was stirred at 30°C for 12 hours. LC-MS (ET74125-11-P1, product: RT = 0.55, 0.68) showed complete consumption of the starting material. The solution was concentrated under reduced pressure to afford compound 59-7 (2.2 g, 3.76 mmol, 99.1% yield, TFA) as a colorless oil. 1 H NMR: ET74125-11-P1A (400 MHzCDCl3) δ 4.07-4.19 (m, 2H), 3.70-3.95 (m, 1H), 3.40-3.60 (m, 2H), 3.08-3.29(m, 2H), 2.86 (s, 2H), 2.60-2.68 (m, 2H), 2.33-2.42 (m, 3H), 2.10-2.22 (m,1H), 1.62-1.66 (m, 4H), 1.53-160 (m, 2H), 1.27-1.41 (m, 24H), 0.86-0.91 (m,3H).

[0490] Preparation of compound SL59

[0491]

[0492] To a solution of compound 2-59-1 (1.1 g, 5.6 mmol, 1.5 eq) in DCM (10 mL) were added EDCI (1.4 g, 7.5 mmol, 2.0 eq), DMAP (1.4 g, 11.3 mmol, 3.0 eq), and compound 59-7 (2.2 g, 3.8 mmol, 1.0 eq, TFA). The solution was stirred at 50°C for 12 hours. TLC (DCM:methanol = 8:1, R of 59-7) f is 0.50, and the R fThe reaction mixture was poured into HCl (10 mL, 1 M). The solution was extracted three times with DCM (10 mL). The combined organic layers were poured into NaCO (saturated aqueous solution, 20 mL). The solution was extracted with DCM (30 mL). The organic phase was dried over sodium sulfate and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 20:1 to 2:1) to obtain compound SL59 (1.0 g, 1.6 mmol, 41.6% yield) as a light yellow oil. 1 H NMR: ET74125-11-P1A (400 MHz CDCl3) δ 4.16-4.19(m, 2H), 3.95-4.01 (d, J = 0.56 Hz 2H), 2.76-3.24 (m, 3H), 2.48-2.60 (m, 2H), 2.25-2.42 (m, 6H), 1.94-2.06 (m, 2H), 1.76-1.87 (m, 1H), 1.54-1.66 (m, 8H), 1.24-1.36 (m, 38H), 0.84-0.92 (m, 9H).

[0493] Preparation of compound 61-2

[0494]

[0495] To a solution of compound 59-3 (20 g, 128.9 mmol, 1.0 eq) and compound 1-59-3 (20.5 g, 154.7 mmol, 24.2 mL, 1.2 eq) in MeOH (100 mL) was added NaOMe (5.2 g, 96.7 mmol, 1.5 eq) at 0-10°C. The reaction solution was stirred at 20°C for 6 hours. TLC showed complete consumption of the starting material. The solution was poured into NH4Cl (saturated solution 500 mL) and extracted with EtOAc (200 mL x 2). The combined organic layers were washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 61-2 (30 g, 104.4 mmol, 81.0% yield) as a yellow oil. 1 H NMR: ET73288-15-P1A (400 MHz, CDCl3) 3.71 (s, 3H), 3.12(s, 1H), 3.09-3.11 (m, 1H), 2.96-3.00 (m, 1H), 2.74-2.81 (m, 1H), 2.53-2.57(m, 3H), 2.31-2.38 (m, 3H), 1.91-2.29 (m, 3H), 1.54-1.60 (m, 2 H), 1.26-1.32(m, 8H), 0.88 (t, = 6.8 Hz, 3H).

[0496] Preparation of compound 61-3

[0497]

[0498] To a solution of compound 61-2 (3.0 g, 10.4 mmol, 1.0 eq) in MeOH (15 mL) was added NaOH (4 M, 13.1 mL, 5.0 eq) at 20 ° C. The solution was stirred at 20 ° C for 12 hr. TLC showed that the starting material was completely consumed. The solution was poured into HCl (4 M.100 mL) until pH = 2-3 and extracted with DCM (200 mLx2). The combined organic layer was washed with brine (200 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give compound 59-5 (4.0 g, 13.3 mmol, 83.7% yield) as a yellow oil. LCMS: ET73288-23-P1A (R T :2.079 min and 2.133 min, MW=274.1). 1 H NMR: ET73479-14-P1A (400 MHz, CDCl3) 10.60-10.45 (m, 1H), 3.75-3.60 (m, 2H), 3.47-3.45 (m, 2H), 3.02-2.85 (m, 5H), 2.65-2.62 (m, 3H), 2.34-2.22 (m, 2H), 1.59-1.54 (m, 2H), 1.34-1.27 (m, 8H), 0.89-0.85 (m, 3H).

[0499] Preparation of compound SL61

[0500]

[0501] To a solution of compound 61-3 (2.00 g, 7.31 mmol, 1.00 eq) in DCM (10 mL) were added compound 58-3 (4.80 g, 8.05 mmol, 1.1 eq), DMAP (446 mg, 3.66 mmol, 0.50 eq), and EDCI (2.80 g, 14.63 mmol, 2.0 eq). The reaction was stirred at 20°C for 2 hours. TLC (dichloromethane:methanol = 10 / 1, R of the product) was obtained. f = 0.54) indicated complete consumption of the starting material. The reaction solution was poured into an aqueous solution of HO (80 mL), and the solution was extracted with DCM (20 mL x 2). The organic phase was dried over sodium sulfate and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 8 / 1 to 5 / 1) to obtain compound SL61 (1.0 g, 1.17 mmol, 16.0% yield) as a colorless oil. 1 H NMR: ET73479-15-P1A (400 MHz, CDCl3) 4.90-4.84 (m,3H), 3.08-2.96 (m, 3H), 2.57-2.53 (m, 2H), 2.29-2.21 (m, 8H), 1.97-1.94 (m,4H), 1.64-1.52 (m, 18H), 1.32-1.27 (m, 44H), 0.90-0.87 (m, 15H).

[0502] Example 1b: Synthesis of compounds SL62-SL65 and SL67-SL75

[0503]

[0504]

[0505] Compounds SL62, SL63, SL64, SL65, SL71, SL72, SL73, SL74, and SL75 were synthesized via comparable methods, with derivatization by the corresponding alkyl alcohol reagents in the final reaction.

[0506]

[0507]

[0508] General procedure for the preparation of compound 2

[0509]

[0510] t-BuOK (336 g, 3.00 mol, 59.9 mL, 1.05 ) were added portionwise to compound 1 (457 g, 2.85 mol, 433 mL, 1.00 ) in dimethyl sulfoxide (2.50 L), and then compound a (500 g, 3.00 mol, 307 mL, 1.05 ), the mixture was heated to 80 ° C for 12 h. LCMS (EW49154-9-P1A, P1: Rt = 0.465 min) showed that the reactant (R t = 0.460 min) consumption. TLC (petroleum ether / ethyl acetate = 5 / 1, P1:R f = 0.38). The crude product was diluted with methyl tert-butyl ether (3.00 L) and washed with a solution of NH4Cl (5.00 L of aqueous solution). The aqueous layer was extracted with methyl tert-butyl ether (2.00 L × 3), and the combined organic layers were washed with brine (5.00 L × 3), dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 6 / 1 to 3 / 1) to give compound 2 (482 g, 1.96 mol, 68.6% yield) as a brown oil. LCMS: EW49154-9-P1B, Rt = 0.468 min, m / z = 269.0 (M+23).

[0511] General procedure for the preparation of compound 3

[0512]

[0513] Compound 2 (443 g, 1.80 mol, 1.00 eq) was dissolved in ethanol (1.50 L), and KOH (107 g, 1.91 mol, 1.06 eq) dissolved in ethanol (2.00 L) was added dropwise. The solution was stirred at 25°C for 16 h. LCMS (EW49333-3-P1B, P1: Rt = 0.365 min) showed 7.74% of the reactant remaining (Rt = 0.466 min). The mixture was concentrated to one-quarter, dissolved in H2O (1.50 L), and made basic with NH3H2O ​​(20.0 mL). The aqueous phase was washed with dichloromethane (500 mL), then acidified to pH = 2 with HCl (2 M, 500 mL) and extracted with ethyl acetate (1.00 L × 3). The organic phase was washed with brine (1.00 L), dried over NaSO, and concentrated to dryness to afford compound 3 (368 g, 1.69 mol, 93.7% yield) as a brown oil, which was used in the next step without further purification. LCMS: EW49333-3-P1C, Rt = 0.365 min, m / z = 241.0 (M+23). 1 H NMR: EW49333-3-P1A (400MHz, CDCl3) δ 5.09 - 4.98 (m, 1H), 4.23 (q, J = 7.0 Hz, 2H), 3.92 - 4.02 (m,2H), 3.81 - 3.91 (m, 2H), 3.63 (t, J = 7.0 Hz, 1H), 2.32 - 2.49 (m, 2H), 1.27 - 1.32 (m, 3H).

[0514] General procedure for the preparation of compound int.1

[0515]

[0516] To a solution of compound 3 (358 g, 1.64 mol, 1.00 eq) in pyridine (324 g, 4.10 mol, 331 mL, 2.50 eq) were added piperidine (13.9 g, 164 mmol, 16.2 mL, 0.10 eq) and (CHO) n(50.7 g, 1.69 mol, 1.03 eq). The mixture was then stirred at 80°C for 16 h. LCMS (EW49333-8-P1A, P1: Rt = 0.448 min) showed consumption of compound 3 (Rt = 0.365 min). The mixture was diluted with H2O (2.00 L) and extracted with methyl tert-butyl ether (2.00 L × 2). The organic layer was washed with HCl (2 M, 2.00 L × 2), saturated sodium bicarbonate (2.00 L), and brine solution (1.50 L), dried over MgSO4, and evaporated to dryness to afford compound int.1 (255 g, 1.37 mol, 83.4% yield) as a brown oil, which was used in the next step without further purification. LCMS: EW49333-8-P1B, Rt = 0.447 min, m / z = 140.9 (M+1). 1 H NMR: EW49333-8-P1B (400 MHz, CDCl3) δ6.28 (d, J = 1.2 Hz, 1H), 5.73 (d, J = 1.2 Hz, 1H), 5.07 (t, J = 5.0 Hz, 1H), 4.22 (q, J = 7.0 Hz, 2H), 3.93 – 4.03 (m, 2H), 3.81 – 3.91 (m, 2H), 2.69 (d,J = 5.0 Hz, 2H), 1.30 (t, J = 7.0 Hz, 3H).

[0517] General procedure for the preparation of compound 5a

[0518]

[0519] To compound int.1 (100 g, 537 mmol, 1.00 ) in ethanol (1.00 L) was added K2CO3 (222 g, 1.61 mol, 3.00 ), compound 4a-3 (185 g, 644 mmol, 1.20 eq) was then added thereto, and the mixture was stirred at 25 ° C for 24 h. Then, the mixture was stirred at 25 ° C for another 24 h. LCMS: (EW49333-26-P1C, P1: Rt = 0.667 min) showed that compound int.1 (Rt = 0.445 min) was completely consumed. The mixture was poured into water (1.50 L), extracted with ethyl acetate (2.00 L × 2), washed with brine (1.50 L × 2), dried over Na2SO4, and concentrated to dryness. The product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 8 / 1 to 1 / 1, TCL (petroleum ether / ethyl acetate = 5 / 1, P1: R f = 0.43)) to give compound 5a (159 g, 321 mmol, 59.8% yield, 87.5% purity) as a brown oil. LCMS: EW49333-26-P1D, Rt = 0.662 min, m / z = 433.1 (M+1). 1 H NMR: EW49333-26-P1A (400 MHz, CDCl3) 4.95 (t, = 4.2 Hz, 1H), 4.18(q, = 7.0 Hz, 2H), 3.88 – 4.01 (m, 2H), 3.75 – 3.88 (m, 2H), 2.74 – 2.82(m, 2H), 2.60 – 2.72 (m, 1H), 2.51 (t, = 7.4 Hz, 2H), 2.20 (t, = 7.6 Hz,2H), 2.08 - 2.17 (m, 1H), 1.96 (td, = 4.2, 14.2 Hz, 1H), 1.50 - 1.60 (m, 4H), 1.41 - 1.49 (m, 9H), 1.26 - 1.40 (m, 11H).

[0520] General procedure for the preparation of compound 6a

[0521]

[0522] To compound 5a (159 g, 367 mmol, 1.00 ) in H2O (477 mL) and ethanol (954 mL) and LiOH·H2O (23.1 g, 551 mmol, 1.50 ). The mixture was stirred at 25 ° C for 15 h. LCMS (EW49333-28-P1A, P1: Rt = 0.583 min) showed that 3.44% of compound 5a remained (Rt = 0.660 min). The mixture was adjusted to pH = 3 by citric acid solution (500 mL), stirred for 10 min, extracted with ethyl acetate (2.00 L × 3), washed with brine (3.00 L), dried over Na2SO4, and concentrated under vacuum. The product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 3 / 1 to 1 / 1, TLC (petroleum ether / ethyl acetate = 1 / 1, P1: R f = 0.58)) to give compound 6a (106 g, 236 mmol, 64.4% yield, 90.4% purity) as a brown oil. LCMS: EW49333-28-P1D, Rt = 0.584 min, m / z = 427.1 (M+23). 1 H NMR: EW49333-28-P1A (400 MHz, CDCl3) 5.01 (t, =4.0 Hz, 1H), 3.94 – 4.05 (m, 2H), 3.80 – 3.91 (m, 2H), 2.79 – 2.90 (m, 2H), 2.70 (d, = 6.2 Hz, 1H), 2.48 - 2.58 (m, 2H), 2.18 - 2.24 (m, 2H), 2.14 -2.18 (m, 1H), 2.00 - 2.05 (m, 1H), 1.58 (quin, = 7.2 Hz, 4H), 1.45 (s, 9H), 1.28 - 1.39 (m, 8H).

[0523] General procedure for the preparation of compound 7a

[0524]

[0525] To a solution of compound 6a (113 g, 279 mmol, 1.00 eq) in acetonitrile (1.13 L) was added KCO (115 g, 837 mmol, 3.00 eq), followed by compound 4a-1 (90.1 g, 307 mmol, 1.10 eq). The mixture was stirred at 80°C for 18 h. LCMS (EW49333-31-P1A, P1: Rt = 0.776 min) showed consumption of compound 6a (Rt = 0.582 min). The mixture was concentrated to remove acetonitrile, poured into H2O (1.50 L), extracted with ethyl acetate (1.50 L × 3), washed with brine (1.50 L × 2), dried over Na2SO4, and concentrated to dryness. The product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1 to 3 / 1, TLC (petroleum ether / ethyl acetate = 5 / 1, P1: R f = 0.50)) to purify the residue. Compound 7a (150 g, 243 mmol, 87.0% yield) was obtained as a brown oil. LCMS: EW49333-31-P1D, Rt = 0.775 min, m / z = 639.4 (M+23). 1 H NMR: EW49333-31-P1A (400 MHz, CDCl3) δ 4.94 (t, J =4.2 Hz, 1H), 4.10 (t, J = 6.8 Hz, 2H), 3.90 – 4.00 (m, 2H), 3.79 – 3.88 (m,2H), 2.74 – 2.83 (m, 2H), 2.62 - 2.72 (m, 1H), 2.51 (t, J = 7.4 Hz, 2H), 2.20 (t, J = 7.4 Hz, 4H), 2.09 - 2.17 (m, 1H), 1.92 - 2.00 (m, 1H), 1.60 - 1.69(m, 3H), 1.51 - 1.58 (m, 5H), 1.45 (s, 18H), 1.27 - 1.40 (m, 16H).

[0526] General procedure for the preparation of compound int.2

[0527]

[0528] To a stirred solution of compound 7a (75.0 g, 121 mmol, 1.00 eq) in dichloromethane (800 mL) was added TFA (379 g, 3.33 mol, 247 mL, 27.4 eq), and the solution was stirred at 25 ° C for 36 h. LCMS (EW49333-38-P1B, P1: Rt = 0.529 min) showed that the intermediate remained. The reactants were concentrated under vacuum to obtain a crude product as a yellow oil. The mixture was purified by silica gel column (petroleum ether / tetrahydrofuran gradient) to obtain the product at 29% tetrahydrofuran. TLC (petroleum ether / tetrahydrofuran = 2 / 1, P1: Rt = 0.529 min) showed that the intermediate remained. f = 0.24). Compound int.2 (48.0 g, 96.7 mmol, 79.5% yield, 92.8% purity) was obtained as a brown oil. LCMS: EW49333-38-P1C, Rt = 0.530 min, m / z = 461.2 (M+1). 1 H NMR: EW49333-38-P1A (400 MHz, CDCl3) δ 9.81 (s, 1H), 4.04 - 4.17 (m,2H), 3.08 - 3.20 (m, 1H), 2.77 - 3.01 (m, 3H), 2.66 (dd, J = 8.0, 13.4 Hz, 1H), 2.50 (t, J = 7.2 Hz, 2H), 2.36 (t, J = 7.2 Hz, 4H), 1.52 - 1.71 (m, 8H), 1.33 (br s, 17H).

[0529] General procedure for the preparation of compound 8a

[0530]

[0531] To a solution of compound int.2 (48.0 g, 104 mmol, 1.00 eq) in dichloromethane (400 mL) were added Me2NH3 (2 M, 78.2 mL, 1.50 eq) and AcOH (1.88 g, 31.2 mmol, 1.79 mL, 0.30 eq), and the solution was stirred at 25°C for 1 hour. NaBH(OAc)3 (26.5 g, 125 mmol, 1.20 eq) was then added to the mixture, and the solution was stirred at 25°C for 16 hours. LCMS (EW49333-39-P1A, P1: Rt = 0.457 min) showed that compound int.2 (Rt = 0.550 min) remained. The mixture was adjusted to pH = 8 with NaHCO3 solution (100 mL of aqueous solution), extracted with dichloromethane (300 mL), and then the pH of the aqueous phase was adjusted to 3 with HCl (2 M) to form a suspension. The suspension was extracted with tetrahydrofuran (500 mL×4), and the organic layers were combined, dried over Na2SO4, and concentrated to obtain an oil. The residue was purified by flash silica gel chromatography (ISCO®; 330 g Sepa Flash® silica gel flash column, 0%~10% methanol / dichloromethane gradient eluent @ 100 mL / min) to obtain the product at 4% methanol. TLC (dichloromethane / methanol = 10 / 1, P1: R f = 0.24). Compound 8a (46.0 g, 88.2 mmol, 84.6% yield, 93.9% purity) was obtained as a brown oil. LCMS: EW49333-39-P1D, Rt = 0.457 min, m / z = 490.3 (M+1). 1 H NMR: EW49333-39-P1A (400 MHz, CDCl3) δ 4.13 (t,J = 6.2 Hz, 2H), 3.25 – 4.20 (m, 5H), 3.11 – 3.25 (m, 1H), 2.94 – 3.08 (m,1H), 2.82 (s, 6H), 2.65 - 2.77 (m, 2H), 2.52 (t, J = 7.2 Hz, 2H), 2.35 (t, J= 7.2 Hz, 3H), 2.11 - 2.25 (m, 2H), 1.51 - 1.86 (m, 7H), 1.20 - 1.45 (m,13H).

[0532] Preparation of compound SL70

[0533]

[0534] To a solution of compound 8a (2.50 g, 5.11 mmol, 1.00 eq) and 4-nonanol (2.21 g, 15.3 mmol, 3.00 eq) in tetrahydrofuran (25.0 mL) was added EDCI (2.94 g, 15.3 mmol, 3.00 eq) and DMAP (935 mg, 7.66 mmol, 1.50 eq). The solution was stirred at 25°C for 16 h. LCMS (EW49333-46-P1A) showed the desired mass was detected (Rt = 0.763 min, m / z = 742.8 (M+1)). The mixture was poured into NaHCO₃ solution (150 mL), extracted with ethyl acetate (250 mL), washed with 1 M HCl solution (150 mL), then washed with saturated NaHCO₃ solution (150 mL), brine (150 mL × 2), dried over Na₂SO₄, and concentrated to dryness. The residue was purified by flash silica gel chromatography (ISCO®; 20 g Sepa Flash® silica gel flash column, 0% to 1% methanol / dichloromethane gradient at 30 mL / min). The residue was further purified by flash silica gel chromatography (ISCO®; 20 g Sepa Flash® silica gel flash column, 0% to 25% tetrahydrofuran / petroleum ether gradient at 30 mL / min). The residue was then purified by flash silica gel chromatography (ISCO®; 40 g Sepa Flash® silica gel flash column, 0% to 1% methanol / dichloromethane gradient at 30 mL / min). Compound SL70 (2.04 g, 2.75 mmol, 53.8% yield) was obtained as a colorless oil. Special LCMS: EW49333-54-P1A, Rt = 3.688 min, m / z = 742.8 (M+1). 1 H NMR: EW49333-54-P1A (400 MHz, CDCl3) δ 4.81 - 4.96 (m, 2H), 4.10 (t, J = 6.6 Hz, 2H), 2.73 - 2.84 (m, 1H), 2.59 - 2.70 (m, 2H), 2.50 (t, J = 7.4 Hz, 2H), 2.23 - 2.35 (m, 6H), 2.21 (s,6H), 1.80 (ddd, J = 2.4, 5.8, 8.0 Hz, 2H), 1.57 - 1.68 (m, 7H), 1.42 - 1.56(m, 9H), 1.18 - 1.41 (m, 32H), 0.83 - 0.95 (m, 12H).

[0535] Preparation of compound SL62

[0536] The procedure for the preparation of compound SL70 was followed by reaction with the relevant alkanol. SL62 (1.50 g, colorless oil). Special LCMS: EW49333-55-P1A, Rt = 3.942 min, m / z = 798.9 (M+1). 1 H NMR: EW49333-55-P1A (400 MHz, CDCl3) 4.84 – 4.91 (m, 2H), 4.10 (t, = 6.8 Hz, 2H), 2.72 - 2.85(m, 1H), 2.58 - 2.70 (m, 2H), 2.50 (t, = 7.4 Hz, 2H), 2.23 - 2.33 (m, 6H), 2.21 (s, 6H), 1.72 - 1.89 (m, 2H), 1.57 - 1.67 (m, 7H), 1.44 - 1.56 (m, 9H), 1.19 - 1.42 (m, 40H), 0.84 - 0.95 (m, 12H).

[0537] Preparation of compound SL63

[0538] The procedure for the preparation of compound SL70 was followed by reaction with the relevant alkanol. SL63 (2.00 g, colorless oil). Special LCMS: EW49333-56-P1B, Rt = 4.166 min, m / z = 855.0 (M+1). 1 H NMR: EW49333-56-P1A (400 MHz, CDCl3) 4.84 – 4.90 (m, 2H), 4.10 (t, = 6.8 Hz, 2H), 2.71 - 2.83(m, 1H), 2.59 - 2.70 (m, 2H), 2.50 (t, = 7.4 Hz, 2H), 2.24 - 2.33 (m, 6H), 2.21 (s, 6H), 1.73 - 1.89 (m, 2H), 1.58 - 1.69 (m, 7H), 1.45 - 1.56 (m, 9H), 1.19 - 1.40 (m, 48H), 0.83 - 0.94 (m, 12H).

[0539] Preparation of compound SL64

[0540] The procedure for the preparation of compound SL70 was followed by reaction with the relevant alkanol. SL64 (1.10 g, colorless oil). Special LCMS: EW49333-57-P1B, Rt = 4.382 min, m / z = 911.1 (M+1). 1 H NMR: EW49333-57-P1A (400 MHz, CDCl3) 4.84 – 4.90 (m, 2H), 4.10 (t, = 6.8 Hz, 2H), 2.72 - 2.86(m, 1H), 2.58 - 2.71 (m, 2H), 2.51 (t, = 7.4 Hz, 2H), 2.23 - 2.36 (m, 6H),2.21 (s, 6H), 1.74 - 1.88 (m, 2H), 1.59 - 1.69 (m, 7H), 1.46 - 1.57 (m, 9H),1.20 - 1.41 (m, 56H), 0.82 - 0.94 (m, 12H).

[0541] Preparation of compound SL65

[0542] The procedure for the preparation of compound SL70 was followed by reaction with the relevant alkanol. SL65 (2.00 g, colorless oil). Special LCMS: EW49333-58-P1B, Rt = 3.213 min, m / z = 967.1 (M+1). 1 H NMR: EW49333-58-P1A (400 MHz, CDCl3) 4.84 – 4.90 (m, 2H), 4.10 (t, = 6.8 Hz, 2H), 2.73 - 2.84(m, 1H), 2.59 - 2.70 (m, 2H), 2.50 (t, = 7.4 Hz, 2H), 2.24 - 2.35 (m, 6H), 2.21 (s, 6H), 1.73 - 1.87 (m, 2H), 1.58 - 1.69 (m, 7H), 1.44 - 1.56 (m, 9H), 1.19 - 1.40 (m, 64H), 0.88 (t, = 6.8 Hz, 12H).

[0543] Preparation of compound SL71

[0544] The procedure for the preparation of compound SL70 was followed by reaction with the relevant alkanol. SL71 (1.10 g, colorless oil). Special LCMS: EW49333-59-P1B, Rt = 3.942 min, m / z = 798.9 (M+1). 1 H NMR: EW49333-59-P1A (400 MHz, CDCl3) 4.84 – 4.90 (m, 2H), 4.10 (t, = 6.8 Hz, 2H), 2.73 - 2.84(m, 1H), 2.59 - 2.71 (m, 2H), 2.51 (t, = 7.4 Hz, 2H), 2.23 - 2.36 (m, 6H), 2.21 (s, 6H), 1.73 - 1.87 (m, 2H), 1.59 - 1.69 (m, 7H), 1.46 - 1.56 (m, 9H), 1.21 - 1.39 (m, 40H), 0.83 - 0.93 (m, 12H).

[0545] Preparation of compound SL72

[0546] The procedure for the preparation of compound SL70 was followed by reaction with the corresponding alkanol. SL72 (1.10 g, colorless oil). Specific LCMS: EW49333-60-P1B, Rt = 4.383 min, m / z = 911.1 (M+1). 1 H NMR: EW49333-60-P1A (400 MHz, CDCl3) 4.77 - 4.98 (m, 2H), 4.10 (t, = 6.8 Hz, 2H), 2.72 - 2.84(m, 1H), 2.57 - 2.70 (m, 2H), 2.50 (t, = 7.4 Hz, 2H), 2.23 - 2.34 (m, 6H), 2.21 (s, 6H), 1.73 - 1.88 (m, 2H), 1.59 - 1.68 (m, 7H), 1.44 - 1.56 (m, 9H), 1.18 - 1.39 (m, 56H), 0.84 - 0.92 (m, 12H).

[0547] Preparation of compound SL73

[0548] The procedure was followed for the preparation of compound SL70, reacting with the relevant alkanol.

[0549] SL73 (650 mg, colorless oil). Special LCMS: EW49594-40-P1A2, Rt = 2.308 min, m / z = 770.9 (M+1). 1 H NMR: EW49594-40-P1A (400 MHz, CDCl3) δ 4.10 (t, = 6.8 Hz,2H), 3.98 (d, = 5.8 Hz, 4H), 2.70 - 2.80 (m, 1H), 2.60 - 2.70 (m, 2H), 2.50(t, = 7.4 Hz, 2H), 2.30 - 2.40 (m, 5H), 2.20 - 2.30 (m, 1H), 2.21 (s, 6H), 1.70 - 1.90 (m, 2H), 1.50 - 1.70 (m, 10H), 1.20 - 1.40 (m, 40H), 0.80 - 1.00(m, 12H).

[0550] Preparation of compound SL74

[0551] The procedure for the preparation of compound SL70 was followed by reaction with the corresponding alkanol. SL74 (2.00 g, colorless oil). Specific LCMS: EW49333-61-P1B, Rt = 4.268 min, m / z = 883.0 (M+1). 1 H NMR: EW49333-61-P1A (400 MHz, CDCl3) 4.10 (t, = 6.8 Hz, 2H), 3.97 (d, = 5.8 Hz, 4H), 2.73- 2.83 (m, 1H), 2.59 - 2.69 (m, 2H), 2.50 (t, = 7.4 Hz, 2H), 2.23 - 2.35(m, 6H), 2.21 (s, 6H), 1.74 - 1.88 (m, 2H), 1.52 - 1.65 (m, 10H), 1.21 - 1.41(m, 56H), 0.82 - 0.94 (m, 12H).

[0552] Preparation of compound SL75

[0553] The procedure for the preparation of compound SL70 was followed by reaction with the relevant alkynol. SL75 (2.20 g, colorless oil). Special LCMS: EW49594-41-P1A1, Rt = 4.268 min, m / z = 883.0 (M+1). 1 H NMR: EW49594-41-P1A (400 MHz, CDCl3) δ 4.09 (t, = 6.8 Hz, 2H), 3.97 (d, = 5.6 Hz, 4H), 2.70 -2.80 (m, 1H), 2.60 - 2.70 (m, 2H), 2.50 (t, = 7.4 Hz, 2H), 2.20 - 2.40 (m,6H), 2.20 (s, 6H), 1.70 - 1.90 (m, 2H), 1.50 - 1.70 (m, 10H), 1.27 (br s,72H), 0.88 (t, = 6.8 Hz, 12H).

[0554] General procedure for the preparation of compound 4a-2

[0555]

[0556] To a solution of compound 4a-1 (460 g, 1.94 mol, 1.00 eq) in dichloromethane (2.30 L) at 10°C was added t-BuOH (503 g, 6.79 mol, 649 mL, 3.50 eq) and TFAA (896 g, 4.27 mol, 593 mL, 2.20 eq). The mixture was stirred at 25°C for 12 h. TLC (petroleum ether / ethyl acetate = 10 / 1) indicated that compound 4a-1 remained and two new spots (R f = 0.00, 0.70). The reactant was poured into an aqueous solution of H2O (1.00 L). The resulting solution was extracted with dichloromethane (1.00 L × 2). The combined organic phases were washed with brine (1.00 L × 2), dried over anhydrous Na2SO4, and concentrated under vacuum to obtain a residue. The product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 10 / 1) (TLC (petroleum ether / ethyl acetate = 10 / 1, R f = 0.70)) to obtain compound 4a-2 (156 g, 532 mmol, 27.4% yield) as a yellow oil. 1 H NMR: EW49525-1-P1A, (400 MHz, CDCl3) δ3.40 (t, J = 6.8 Hz, 2H), 2.20 (t, J = 7.2 Hz, 2H), 1.83 – 1.86 (m, 2H), 1.57– 1.59 (m, 2H), 1.44 (s, 9H), 1.41 – 1.43 (m, 2H), 1.30 (s, 6H).

[0557] General procedure for the preparation of compound 4a-3

[0558]

[0559] Potassium thioacetate (129 g, 1.13 mol, 1.50 eq) was added to a solution of compound 4a-2 (221 g, 754 mmol, 1.00 eq) in tetrahydrofuran (1.00 L). The mixture was stirred at 50°C for 16 h. LCMS (EW49333-24-P1A, P1: Rt = 0.655 min) showed that compound 4a-2 was consumed, and a major peak with the desired mass was detected (Rt = 0.655 min, m / z = 311.0 (M+23)). The reaction was poured into an aqueous solution of H2O (1.60 L). The resulting solution was extracted with ethyl acetate (1.60 L x 3). The combined organic phases were washed with brine (1.60 L x 3), dried over anhydrous Na2SO4, and concentrated under vacuum to yield the crude product. The product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 8 / 1) (TLC (petroleum ether / ethyl acetate = 8 / 1, P: R f = 0.67)) and the residue was purified to obtain compound 4a-3 (167 g, 491 mmol, 65.1% yield, 84.8% purity) as a brown oil. LCMS: EW49333-24-P1D, Rt = 0.658 min, m / z = 311.1 (M+23). 1 H NMR: EW49333-24-P1B, (400 MHz, CDCl3) δ 2.86 (t, J = 7.2Hz, 2H), 2.33 (s, 3H), 2.20 (t, J = 7.6 Hz, 2H), 1.54 – 1.56 (m, 4H), 1.45(s, 9H), 1.29 – 1.36 (m, 8H).

[0560] General procedure for the preparation of compound 5c

[0561]

[0562] To a solution of compound int.1 (8.70 g, 46.7 mmol, 1.00 eq) and compound 4c-2 (17.8 g, 56.1 mmol, 1.20 eq) in ethanol (90.0 mL) was added KCO (19.4 g, 140 mmol, 3.00 eq). The reaction was stirred at 25°C for 40 h. LCMS (EW49594-4-P1B) showed the desired mass was detected (Rt = 0.714, m / z = 461.2 (M+1)). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography (ISCO®; 330 g Sepa Flash® silica gel flash column, 0% to 8% ethyl acetate / petroleum ether gradient at 100 mL / min, petroleum ether / ethyl acetate = 5 / 1, R f = 0.28). Compound 5c (17.2 g, 30.5 mmol, 65.2% yield, 81.6% purity) was obtained as a colorless liquid. LCMS: EW49594-4-P1C1, Rt = 0.721, m / z = 461.3 (M+1). 1 H NMR: EW49594-4-P1A, (400 MHz, CDCl3) δ 4.88 (t, = 4.4 Hz, 1H), 4.00 -4.20 (m, 2H), 3.80 - 4.00 (m, 2H), 3.70 - 3.80 (m, 2H), 2.60 - 2.80 (m, 2H),2.61 - 2.63 (m, 1H), 2.44 (t, = 7.4 Hz, 2H), 2.13 (t, = 7.6 Hz, 2H),2.06 (ddd, = 4.2, 8.4, 14.2 Hz, 1H), 1.89 (td, = 4.4, 14.4 Hz, 1H), 1.40- 1.50 (m, 4H), 1.37 (s, 9H), 1.20 - 1.30 (m, 15H).

[0563] General procedure for the preparation of compound 6c

[0564]

[0565] To a solution of compound 5c (17.0 g, 36.9 mmol, 1.00 eq) in ethanol (100 mL) and H₂O (50.0 mL) was added LiOH•H₂O (1.86 g, 44.3 mmol, 1.20 eq). The reaction mixture was stirred at 25°C for 16 h. Additional LiOH•H₂O (310 mg, 7.38 mmol, 0.20 eq) was added. The reaction mixture was stirred at 25°C for another 24 h. LCMS (EW49594-8-P1C) showed the desired mass was detected (Rt = 0.644, m / z = 455.2 (M+23)), with 1.30% of compound 5c remaining. The reaction mixture was concentrated under reduced pressure to remove ethanol, followed by the addition of H₂O (50.0 mL). HCl (1 M) was added dropwise to adjust the pH to ~3, and the mixture was extracted with ethyl acetate (100 mL x 3). The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 330 g SepaFlash® silica gel flash column, 0% to 25% ethyl acetate / petroleum ether gradient eluent @ 100 mL / min, petroleum ether / ethyl acetate = 1 / 1, R f = 0.50) to obtain compound 6c (12.2 g, 27.5 mmol, 74.6% yield, 97.6% purity) as a colorless oil. LCMS: EW49594-8-P1C1, Rt = 0.633 min, m / z = 455.2 (M+23). 1 H NMR:EW49594-8-P1A, (400 MHz, CDCl3) δ 4.93 (t, J = 4.2 Hz,1H), 3.80 - 4.00 (m,2H), 3.81 - 3.83 (m, 2H), 2.70 - 2.80 (m, 2H), 2.60 - 2.70 (m, 1H), 2.46 (t,J = 7.4 Hz, 2H), 2.13 (t, J = 7.6 Hz, 2H), 2.00 - 2.10 (m, 1H), 1.90 - 2.00(m, 1H), 1.51 - 1.53 (m, 4H), 1.37 (s, 9H), 1.20 - 1.30 (m, 12H).

[0566] General procedure for the preparation of compound 7c

[0567]

[0568] To a solution of compound 6c (12.2 g, 28.2 mmol, 1.00 eq) and compound 4c-1 (9.97 g, 31.0 mmol, 1.10 eq) in dimethylformamide (120 mL) was added KCO (11.7 g, 84.6 mmol, 3.00 eq). The reaction was stirred at 80°C for 2 h. LCMS (EW49594-11-P1A) showed the desired mass was detected (Rt = 0.877 min, m / z = 695.5 (M=1)). H2O (100 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (100 mL × 2). The combined organic phase was washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure to yield a residue. The residue was purified by flash silica gel chromatography (ISCO®; 330 g Sepa Flash® silica gel flash column, 0% to 8% ethyl acetate / petroleum ether gradient eluent @ 100 mL / min, petroleum ether / ethyl acetate = 5 / 1, R f = 0.41) to obtain compound 7c (18.4 g, 27.3 mmol, 97.0% yield) as a colorless liquid. 1 H NMR: EW49594-11-P1A, (400 MHz, CDCl3) δ4.87 (t, J = 4.4 Hz, 1H), 4.00 - 4.10 (m, 2H), 3.80 - 3.90 (m, 2H), 3.70 -3.80 (m, 2H), 2.70 - 2.80 (m, 2H), 2.61 - 2.63 (m, 1H), 2.43 (t, J = 7.4 Hz,2H), 2.13 (t, J = 7.6 Hz, 4H), 2.00 - 2.10 (m, 1H), 1.90 - 2.00 (m, 1H), 1.40- 1.60 (m, 8H), 1.37 (s, 18H), 1.20 - 1.30 (m, 24H).

[0569] General procedure for the preparation of compound 8c

[0570]

[0571] To a solution of compound 7c (16.0 g, 23.8 mmol, 1.00 eq) in dichloromethane (120 mL) was added TFA (61.4 g, 538 mmol, 40.0 mL, 22.7 eq). The reaction was stirred at 30°C for 15 h. LCMS (EW19594-15-P1A) showed the desired mass was detected (Rt = 0.608 min, m / z = 517.3 (M+1)). The reaction was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 330 g Sepa Flash® silica gel flash column, 0% to 30% ethyl acetate / petroleum ether gradient at 100 mL / min, petroleum ether / ethyl acetate = 1 / 1, P1: R f = 0.55) to obtain compound 8c (12.2 g, 23.6 mmol, 99.3% yield) as a white solid. 1 H NMR:EW49594-15-P1A, (400 MHz, CDCl3) δ 9.73 (s, 1H), 8.72 (br s, 2H), 4.00 - 4.10(m, 2H), 3.00 - 3.10 (m, 1H), 2.70 - 2.90 (m, 3H), 2.58 (dd, J = 8.4, 13.4Hz, 1H), 2.42 (t, J = 7.4 Hz, 2H), 2.28 (t, J = 7.6 Hz, 4H), 1.40 - 1.60 (m,8H), 1.20 - 1.30 (m, 24H).

[0572] General procedure for the preparation of compound 9c

[0573]

[0574] To a solution of compound 8c (12.2 g, 23.6 mmol, 1.00 eq) in dichloromethane (120 mL) were added AcOH (2.13 g, 35.4 mmol, 2.03 mL, 1.50 eq) and MeNH (2 M, 17.7 mL, 1.50 eq). The solution was stirred at 25°C for 2 h, followed by the addition of NaBH(OAc) (6.00 g, 28.3 mmol, 1.20 eq). The solution was stirred at 25°C for 1 h. LCMS (EW49594-16-P1A) showed the desired mass was detected (Rt = 0.507 min, m / z = 546.4 (M+1)). The reaction was poured into an aqueous solution of NaHCO (120 mL). The resulting solution was extracted with dichloromethane (100 mL x 2). The aqueous phase was then acidified to pH = 3 with 2M HCl. The aqueous phase was extracted with dichloromethane (200 mL × 2). The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography (ISCO®; 120 g Sepa Flash® silica gel flash column, 0% to 5% methanol / dichloromethane gradient eluent @ 85 mL / min, dichloromethane / methanol = 10 / 1, R f = 0.50). Compound 9c (5.90 g, 10.8 mmol, 45.9% yield, 100% purity) was obtained as a white solid. LCMS: EW49594-16-P1C1: Rt = 0.510 min, m / z = 546.4 (M+1). 1H NMR: EW49594-16-P1B, (400 MHz, CDCl3) δ 10.4 (br s, 1H), 4.01 - 4.03(m, 2H), 3.60 - 3.80 (m, 2H), 2.90 - 3.00 (m, 1H), 2.70 - 2.90 (m, 2H), 2.60- 2.70 (m, 8H), 2.40 - 2.50 (m, 2H), 2.10 - 2.30 (m, 4H), 2.01 - 2.04 (m,2H), 1.40 - 1.60 (m, 8H), 1.20 - 1.30 (m, 24H).

[0575] Preparation of compound SL67

[0576]

[0577] To a solution of compound 9c (2.5 g, 4.58 mmol, 1.00 eq) and nonan-5-ol (1.98 g, 13.7 mmol, 3.00 eq) in tetrahydrofuran (25.0 mL) were added EDCI (2.63 g, 13.7 mmol, 3.00 eq) and DMAP (839 mg, 6.87 mmol, 1.50 eq). The solution was stirred at 25°C for 15 h. LCMS (EW49594-19-P1A) showed the desired mass was detected (Rt = 2.328 min, m / z = 798.6 (M+1)). NaHCO₃ (aqueous solution, 25.0 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (20.0 mL × 2). The combined organic phase was dried over MgSO₄, filtered, and concentrated under reduced pressure to yield a residue. The residue was purified by flash silica gel chromatography (ISCO®; 80 g Sepa Flash® silica gel flash column, 0% to 1% methanol / dichloromethane gradient eluent @ 65 mL / min, dichloromethane / methanol = 10 / 1, R f = 0.53). Compound SL-67 (2.80 g, 3.50 mmol, 76.5% yield, 99.9% purity) was obtained as a yellow oil. 1.8 g of compound SL-67 was purified by flash silica gel chromatography (ISCO®; 80 g Sepa Flash® silica gel flash column, 0% to 1% tetrahydrofuran / petroleum ether gradient eluent @ 65 mL / min, petroleum ether / tetrahydrofuran = 1 / 1, R f = 0.21) to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 80 g Sepa Flash® silica gel flash column, 0% to 1% methanol / dichloromethane gradient eluent @ 65 mL / min, petroleum ether / tetrahydrofuran = 1 / 1, R f = 0.21). Compound SL67 (1.60 g, 2.00 mmol, 88.8% yield, 99.9% purity) was obtained as a yellow oil. LCMS: EW49594-42-P1A1, Rt = 2.485 min, m / z = 798.9 (M+1). 1H NMR: EW49594-42-P1A, (400 MHz, CDCl3)δ 4.86 (q, J = 6.4 Hz, 2H), 4.08 (t, J = 6.8 Hz, 2H), 2.70 - 2.80 (m, 1H),2.60 - 2.70 (m, 2H), 2.49 (t, J = 7.4 Hz, 2H), 2.20 - 2.30 (m, 6H), 2.19 (s,6H), 1.70 - 1.90 (m, 2H), 1.40 - 1.60 (m, 16H), 1.20 - 1.40 (m, 40H), 0.88(t, J = 6.8 Hz, 12H).

[0578] Preparation of compound SL69

[0579]

[0580] To a solution of compound 9c (2.50 g, 4.58 mmol, 1.00 eq) and 7-tridecanol (2.75 g, 13.7 mmol, 3.00 eq) in tetrahydrofuran (25.0 mL) was added EDCI (2.63 g, 13.7 mmol, 3.00 eq) and DMAP (839 mg, 6.87 mmol, 1.50 eq). The solution was stirred at 25°C for 15 h. LCMS (EW49594-20-P1A) showed the desired mass was detected (Rt = 2.655 min, m / z = 910.7 (M+1)). NaHCO₃ (aqueous solution, 25.0 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (20.0 mL × 2). The combined organic phase was dried over MgSO₄, filtered, and concentrated under reduced pressure to yield a residue. The residue was purified by flash silica gel chromatography (ISCO®; 80 g Sepa Flash® silica gel flash column, 0% to 1% methanol / dichloromethane gradient eluent @ 65 mL / min, dichloromethane / methanol = 10 / 1, R f = 0.53). Compound SL69 (2.80 g, 3.50 mmol, 76.5% yield, 99.9% purity) was obtained as a colorless oil. Compound SL69 (1.80 g, 1.98 mmol, 1.00 eq) was purified by flash silica gel chromatography (ISCO®; 80 g Sepa Flash® silica gel flash column, 0% to 15% tetrahydrofuran / petroleum ether gradient eluent @ 65 mL / min, petroleum ether / tetrahydrofuran = 1 / 1, R f=0.23) to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 80 g Sepa Flash® silica gel flash column, 0% to 1% methanol / dichloromethane gradient eluent @ 65 mL / min, petroleum ether / tetrahydrofuran = 1 / 1, R f = 0.23). Compound SL69 (1.60 g, 1.76 mmol, 88.8% yield, 99.9% purity) was obtained as a yellow oil. LCMS: EW49594-44-P1A1: Rt = 2.949 min, m / z = 911.0 (M+1). 1 H NMR: EW49594-44-P1B, (400 MHz, CDCl3) δ 4.87 (q, J = 6.2 Hz, 2H), 4.09 (t, J = 6.8 Hz, 2H), 2.70 -2.80 (m, 1H), 2.60 - 2.70 (m, 2H), 2.50 (t, J = 7.4 Hz, 2H), 2.20 - 2.40 (m,6H), 2.20 (s, 6H), 1.70 - 1.90 (m, 2H), 1.50 - 1.70 (m, 16H), 1.27 (br d, J =4.2 Hz, 56H), 0.80 - 1.00 (m, 12H).

[0581] General procedure for the preparation of compound 4c-1

[0582]

[0583] To a solution of compound 4c (40.0 g, 151 mmol, 1.00 eq) in dichloromethane (200 mL) were added TFAA (69.7 g, 332 mmol, 46.1 mL, 2.20 eq) and t-BuOH (39.1 g, 528 mmol, 50.5 mL, 3.50 eq). The mixture was stirred at 25°C for 12 h. TLC (petroleum ether / ethyl acetate = 10 / 1) indicated complete consumption of compound 4c and the formation of two new spots (R f= 0.10, 0.70). The reactant was poured into an aqueous solution of H2O (100 mL). The resulting solution was extracted with dichloromethane (100 mL × 2). The combined organic phases were washed with brine (100 mL × 2), dried over anhydrous Na2SO4, and concentrated under vacuum to obtain a residue. The product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 10 / 1) (TLC (petroleum ether / ethyl acetate = 10 / 1, R f = 0.70)) to obtain compound 4c-1 (44.0 g, 137 mmol, 90.8% yield) as a white oil. 1 H NMR: EW49144-10-P1A (400Hz, CDCl3) δ 3.40 (t, J= 7.2 Hz, 2H), 2.20 (t, J = 7.6 Hz, 2H), 1.82 - 1.86 (m, 2H), 1.54 - 1.57 (m,2H), 1.43 (s, 9H), 1.41 - 1.42 (m, 2H), 1.25 (s, 10H).

[0584] General procedure for the preparation of compound 4c-2

[0585]

[0586] To a solution of compound 4c-1 (20.0 g, 62.3 mmol, 1.00 eq) in tetrahydrofuran (100 mL) was added potassium thioacetate (10.7 g, 93.4 mmol, 1.50 eq). The mixture was stirred at 50°C for 3 h. TLC (petroleum ether / ethyl acetate = 10 / 1) indicated complete consumption of compound 4c-1 and the formation of a new spot (R f = 0.60). The reaction was poured into an aqueous solution of H2O (100 mL). The resulting solution was extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed with brine (100 mL × 3), dried over anhydrous Na2SO4, and concentrated under vacuum to afford compound 4c-2 (19.0 g, 55.14 mmol, 88.6% yield, 100% purity) as a colorless oil, which did not require purification. 1H NMR:EW49525-3-P1A (400 MHz, CDCl3) δ 2.86 (t, J = 7.2 Hz, 2H), 2.32 (s, 3H), 2.20 (t, J = 7.6 Hz, 2H), 1.55-1.58 (m, 4H), 1.44 (s, 9H), 1.24-1.28 (m, 16H).

[0587] General procedure for the preparation of compound 5d

[0588]

[0589] To a solution of compound int.1 (5.00 g, 26.9 mmol, 1.00 eq) and compound 4d-5 (10.7 g, 29.5 mmol, 1.10 eq) in ethanol (50.0 mL) was added KCO (11.1 g, 80.6 mmol, 3.00 eq). The reaction was stirred at 25°C for 40 h. LCMS (EW49594-18-P1A) showed the desired mass was detected (Rt = 0.754 min, m / z = 511.3 (M+1)). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography (ISCO®; 330 g Sepa Flash® silica gel flash column, 0% to 8% ethyl acetate / petroleum ether gradient at 100 mL / min, petroleum ether / ethyl acetate = 5 / 1, R f = 0.28) to afford compound 5d (8.90 g, 18.2 mmol, 67.8% yield) as a red liquid. 1 H NMR: EW49594-18-P1A, (400 MHz, CDCl3) δ 4.88 (t, J =4.4 Hz, 1H), 4.00 - 4.20 (m, 2H), 3.80 - 4.00 (m, 2H), 3.70 - 3.80 (m, 2H),2.70 - 2.80 (m, 2H), 2.60 - 2.70 (m, 1H), 2.44 (t, J = 7.4 Hz, 2H), 2.13 (t,J = 7.6 Hz, 2H), 2.06 (ddd, J = 4.0, 8.4, 14.2 Hz, 1H), 1.91 - 1.93 (m, 1H),1.40 - 1.60 (m, 4H), 1.37 (s, 9H), 1.10 - 1.30 (m, 19H).

[0590] General procedure for the preparation of compound 6d

[0591]

[0592] To a solution of compound 5d (8.90 g, 18.2 mmol, 1.00 eq) in ethanol (60.0 mL) and HO (30.0 mL) was added LiOH·HO (1.07 g, 25.5 mmol, 1.40 eq). The reaction mixture was stirred at 25°C for 16 h. Then, LiOH·HO (229 mg, 5.46 mmol, 0.30 eq) was added to the mixture. The reaction mixture was stirred at 25°C for another 24 h. LCMS (EW49594-21-P1B) showed complete consumption of compound 5d, and the desired mass was detected (Rt = 0.681 min, m / z = 483.2 (M+1)). The reaction mixture was concentrated under reduced pressure to remove ethanol, HO (50 mL) was added, HCl (1 M) was added dropwise to adjust the pH to ~3, and the mixture was extracted with ethyl acetate (100 mL × 3). The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 120 g Sepa Flash® silica gel flash column, 0% to 25% ethyl acetate / petroleum ether gradient eluent @ 100 mL / min, petroleum ether / ethyl acetate = 1 / 1, R f = 0.50) to obtain compound 6d (6.10 g, 13.2 mmol, 72.7% yield) as an orange oil. 1 H NMR: EW49594-21-P1A, (400 MHz, CDCl3) δ 4.93 (t, J = 4.2Hz, 1H), 3.80 - 4.00 (m, 2H), 3.70 - 3.80 (m, 2H), 2.70 - 2.80 (m, 2H), 2.60- 2.70 (m, 1H), 2.46 (t, J = 7.4 Hz, 2H), 2.13 (t, J = 7.6 Hz, 2H), 2.00 -2.10 (m, 1H), 1.90 - 2.00 (m, 1H), 1.51 - 1.53 (m, 4H), 1.37 (s, 9H), 1.20 -1.30 (m, 16H).

[0593] General procedure for the preparation of compound 7d

[0594]

[0595] To compound 6d (6.10 g, 13.2 mmol, 1.00 ) and compound 4d-4 (5.09 g, 14.6 mmol, 1.10 ) in dimethylformamide (60.0 mL) was added K2CO3 (5.49 g, 39.7 mmol, 3.00 The reaction was stirred at 80°C for 2 h. TLC (petroleum ether / ethyl acetate = 5 / 1) showed that compound 6d (R1: R f = 0.18) were completely consumed and new spots were detected (R f = 0.62). H2O (100 mL) was added to the reaction mixture and extracted with ethyl acetate (100 mL × 2). The combined organic phase was washed with brine (100 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography (ISCO®; 120 g Sepa Flash® silica gel flash column, 0% to 8% ethyl acetate / petroleum ether gradient eluent @ 85 mL / min, petroleum ether / ethyl acetate = 5 / 1, R f =0.62) to obtain compound 7d (9.00 g, 12.3 mmol, 93.2% yield) as a yellow oil. 1 H NMR:EW49594-24-P1A, (400 MHz, CDCl3) δ 4.87 (t, = 4.4 Hz, 1 H), 4.00 - 4.10 (m, 2 H), 3.80 - 3.90 (m, 2 H), 3.70 - 3.80 (m, 2 H), 2.70 - 2.80 (m, 2 H), 2.61- 2.63 (m, 1 H), 2.43 (t, = 7.4 Hz, 2 H), 2.13 (t, = 7.6 Hz, 4 H), 2.00- 2.10 (m, 1 H), 1.91 - 1.92 (m, 1 H), 1.50 - 1.60 (m, 8 H), 1.37 (s, 18 H), 1.20 - 1.30 (m, 32 H).

[0596] General procedure for the preparation of compound 8d

[0597]

[0598] To compound 7d (9.00 g, 12.3 mmol, 1.00 ) in dichloromethane (60.0 mL) was added TFA (30.0 g, 263 mmol, 19.6 mL, 21.3 ). The reaction was stirred at 30 ° C for 15 h. LCMS: (EW49594-25-P1A) showed that the desired mass was detected (Rt = 0.677 min, m / z = 573.4 (M + 1)). The reaction was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 80 g Sepa Flash® silica gel flash column, 0% to 30% tetrahydrofuran / petroleum ether gradient elution @ 100 mL / min, petroleum ether / tetrahydrofuran = 1 / 1, P1: R f = 0.44) to obtain compound 8d (7.00 g, 12.2 mmol, 99.0% yield) as a yellow solid. 1 H NMR:EW49594-25-P1A, (400 MHz, CDCl3) δ 9.73 (s, 1H), 8.78 (br s, 2H), 4.00 - 4.10(m, 2H), 3.06 (tt, = 5.6, 8.2 Hz, 1H), 2.70 - 3.00 (m, 3H), 2.58 (dd, =8.3, 13.4 Hz, 1H), 2.40 - 2.50 (m, 2H), 2.28 (t, = 7.6 Hz, 4H), 1.40 - 1.60 (m, 8H), 1.20 - 1.30 (m, 32H).

[0599] General procedure for the preparation of compound 9d

[0600]

[0601] To compound 8d (7.00 g, 12.2 mmol, 1 ) in dichloromethane (140 mL) was added AcOH (880 mg, 14.7 mmol, 839 μL, 1.20 ) and Me2NH (2 M, 9.16 mL, 1.50 The solution was stirred at 25 °C for 2 h, and then NaBH(OAc)3 (3.88 g, 18.33 mmol, 1.50 ). The solution was stirred at 25°C for 1 h. LCMS: (EW49594-26-P1A) showed that the desired mass was detected (Rt = 0.548 min, m / z = 602.5 (M+1)). The reaction was poured into an aqueous solution of NaHCO3 (aqueous solution 120 mL). The resulting solution was extracted with tetrahydrofuran (100 mL x 2). The aqueous phase was then acidified to pH = 3 with HCl (2M). The aqueous phase was extracted with tetrahydrofuran (200 mL x 2). The combined organic layers were washed with brine (200 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography (ISCO®; 80 g Sepa Flash® silica gel flash column, 0% to 6% ethyl acetate / petroleum ether gradient eluent @ 65 mL / min, dichloromethane / methanol = 10 / 1, P1: R f = 0.45) to obtain compound 9d (4.80 g, 7.97 mmol, 65.3% yield) as a white solid. 1 H NMR: EW49594-26-P1B, (400 MHz, CDCl3) δ 12.3 - 12.5 (m, 1H), 5.44 (br s, 2H), 4.15 (br s, 2H), 2.80 - 3.00(m, 1H), 2.81 - 2.82 (m, 1H), 2.71 - 2.73 (m, 2H), 2.60 - 2.70 (m, 6H), 2.52(br t, = 7.4 Hz, 2H), 2.20 - 2.30 (m, 4H), 2.00 - 2.10 (m, 2H), 1.50 - 1.70 (m, 8H), 1.30 (br s, 32H).

[0602] Preparation of compound SL68

[0603]

[0604] To a solution of compound 9d (3.00 g, 4.98 mmol, 1.00 eq) and nonan-5-ol (2.16 g, 15.0 mmol, 3.00 eq) in tetrahydrofuran (60.0 mL) were added EDCI (2.87 g, 15.0 mmol, 3.00 eq) and DMAP (913 mg, 7.48 mmol, 1.50 eq). The solution was stirred at 25°C for 15 h. LCMS (EW49594-32-P1A) showed the desired mass was detected (Rt = 2.050, m / z = 855.2 (M+1)). NaHCO₃ (aqueous solution, 50.0 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (50.0 mL × 2). The combined organic phase was dried over MgSO₄, filtered, and concentrated under reduced pressure to yield a residue. The residue was purified by flash silica gel chromatography (ISCO®; 80 g Sepa Flash® silica gel flash column, 0% to 1% methanol / dichloromethane gradient eluent @ 65 mL / min, dichloromethane:methanol = 10:1, R f = 0.53) was purified. Compound SL68 (1.80 g, 2.10 mmol, 42.2% yield, 99.8% purity) was obtained as a yellow oil. Compound SL68 (1.80 g, 2.11 mmol, 1.00 eq) was purified by flash silica gel chromatography (ISCO®; 80 g Sepa Flash® silica gel flash column, 0% to 15% tetrahydrofuran / petroleum ether gradient eluent @ 65 mL / min, petroleum ether / tetrahydrofuran = 1 / 1, R f = 0.20) to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 80 g Sepa Flash® silica gel flash column, 0% to 1% methanol / dichloromethane gradient eluent @ 65 mL / min, petroleum ether / tetrahydrofuran = 1 / 1, R f = 0.20). Compound SL68 (1.40 g, 1.64 mmol, 77.7% yield, 99.9% purity) was obtained as a yellow oil. LCMS: EW49594-43-P1A1, Rt = 2.804 min, m / z = 855.0 (M+1). 1H NMR: EW49594-43-P1A, (400 MHz, CDCl3) δ 4.87 (q, J = 6.4 Hz, 2H), 4.09 (t, J = 6.8 Hz, 2H), 2.70 - 2.80 (m, 1H), 2.60 - 2.70 (m, 2H), 0.88 (t, J = 6.8 Hz, 12H).

[0605] General procedure for the preparation of compound 4d-2

[0606]

[0607] To a solution of compound b (191 g, 1.19 mol, 181 mL, 2.00 eq) and EtONa (406 g, 1.19 mol, 20% purity, 2.00 eq) in ethanol (1.50 L) was added compound 4d-1 (150 g, 597 mmol, 1.00 eq) dropwise at 25°C. The mixture was then stirred at 80°C for 12 h. TLC (petroleum ether / ethyl acetate = 5 / 1) showed that compound 4d-1 (R f = 0.40) is consumed and a new main spot is formed (R f = 0.30). The reaction was concentrated under vacuum to obtain a residue. The residue was poured into 3000 mL of ice water and extracted with methyl tert-butyl ether (3.00 L × 2). The combined organic phases were washed with brine (2.00 L), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to obtain compound 4d-2 (197.3 g, crude) as a yellow oil, which was used directly without purification.

[0608] General procedure for the preparation of compound 4d-2a

[0609]

[0610] To a solution of compound 4d-2 (197 g, 597 mmol, 1.00 eq) in H2O (600 mL) was added KOH (167 g, 2.99 mol, 5.00 eq). The mixture was then stirred at 100°C for 3 h. TLC (petroleum ether / ethyl acetate = 3 / 1) showed that compound 4d-2 (R f = 0.45) is consumed and a new main spot is formed (R f = 0.0). The reaction was cooled to 20°C and poured into water (500 mL) and stirred for 10 min. The aqueous phase was extracted with methyl tert-butyl ether (300 mL × 2). The combined aqueous phases were adjusted to pH 1-2 with 12 M HCl. The aqueous phase was extracted with methyl tert-butyl ether (800 mL × 2), washed with brine (200 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to afford compound 4d-2a (120 g, 437 mmol, 73.3% yield) as an off-white solid, which was used directly without purification. 1 H NMR: EW49511-2-P1A (400 MHz, dimethylsulfoxide-d6) δ 12.6 (s, 2H), 3.75 - 4.03 (m, 1H), 3.38 - 3.44 (m, 2H), 3.25 (s, 1H), 1.67 - 1.69 (m, 2H), 1.37 - 1.39 (m, 2H), 1.23 (s, 16H).

[0611] General procedure for the preparation of compounds 4d-2b

[0612]

[0613] To a solution of compound 4d-2a (120 g, 437 mmol, 1.00 eq) in tetrahydrofuran (1.20 L) was added. The mixture was stirred at 200°C for 5 min using flow chemistry. TLC (petroleum ether / ethyl acetate = 0 / 1) showed that compound 4d-2a (R f = 0.20) are consumed and new spots are formed (R f = 0.50). The mixture was concentrated under vacuum to give compound 4d-2b (100 g, crude) as a yellow solid, which was used directly without purification.

[0614] General procedure for the preparation of compounds 4d-2c

[0615]

[0616] Compound c (261 g, 1.30 mol, 4.00 eq) was added to a solution of compound 4d-2b (75.0 g, 326 mmol, 1.00 eq) in dichloromethane (450 mL). The reaction was stirred at 20°C for 12 h. TLC (petroleum ether / ethyl acetate = 3 / 1) showed that compound 4d-2b (R f = 0.20) are consumed and new spots are formed (R f = 0.50). The reaction was filtered, the filter cake was washed with dichloromethane (500 mL), and the filtrate was concentrated under vacuum. The product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 50 / 1 to 5 / 1, TLC (petroleum ether / ethyl acetate = 3 / 1, R f = 0.50)) to give compound 4d-2c (61.5 g, 215 mmol, 65.9% yield) as a yellow oil. 1 H NMR: EW49511-12-P1A (400MHz, CDCl3) δ 3.64 (t, J = 6.4 Hz, 2H), 2.20 (t, J = 6.4 Hz, 2H), 1.55 - 1.59(m, 4H), 1.44 (s, 9H), 1.27 - 1.34 (m, 16H).

[0617] General procedure for the preparation of compound 4d-4

[0618]

[0619] To a solution of compound 4d-2c (61.0 g, 213 mmol, 1.00 eq) in dichloromethane (310 mL) was added CBr4 (141 g, 426 mmol, 2.00 eq) and PPh3 (83.8 g, 319 mmol, 1.50 eq). The solution was stirred at 20°C for 12 h. TLC (petroleum ether / ethyl acetate = 10 / 1) showed that compound 4d-2c (R f = 0.30) are consumed and new spots are formed (R f = 0.70). The reactant was concentrated under vacuum and filtered, and the filter cake was washed with petroleum ether (2.00 L). The filtrate was concentrated under vacuum. The product was purified by silica gel chromatography (petroleum ether / ethyl acetate = 100 / 0 to 30 / 1, R f = 0.70) to afford compound 4d-4 (68.6 g, 196 mmol, 92.2% yield) as a yellow oil.1 H NMR: EW49511-15-P1A (400 MHz, CDCl3) δ 3.41 (t, J = 6.8 Hz, 2H), 2.20 (t, J = 7.6 Hz, 2H), 1.83 - 1.87 (m, 2H), 1.55 - 1.58 (m, 2H), 1.44 (s, 9H), 1.40 - 1.42 (m, 2H), 1.24 - 1.27 (m, 14H).

[0620] General procedure for the preparation of compounds 4d-5

[0621]

[0622] To a solution of compound 4d-4 (21.8 g, 62.4 mmol, 1.00 eq) in tetrahydrofuran (210 mL) was added potassium ethanethiolate (11.4 g, 99.8 mmol, 1.60 eq). The mixture was stirred at 50°C for 3 h. TLC (petroleum ether / ethyl acetate = 10 / 1) showed that compound 4d-4 (R f = 0.50) are consumed and new spots are formed (R f = 0.70). The reaction was poured into an aqueous solution of H2O (200 mL). The resulting solution was extracted with ethyl acetate (200 mL x 3). The combined organic phases were washed with brine (200 mL x 3), dried over anhydrous Na2SO4, and concentrated under vacuum to afford compound 4d-5 (10.8 g, 29.7 mmol, 47.5% yield, 94.6% purity) as a yellow oil, which was used directly without purification. LCMS: EW49511-14-P1C, Rt = 0.759 min, m / z = 229.1 (M+1) + . 1 H NMR: EW49511-14-P1B(400 MHz, CDCl3) δ 2.86 (t, J = 7.2 Hz, 2H), 2.32 (s, 3H), 2.20 (t, J = 7.6Hz, 2H), 1.54 - 1.58 (4, 2H), 1.45 (s, 9H), 1.24 - 1.34 (m, 16H).

[0623] Example 1c: Synthesis of Compounds SL66 and SL76-SL81

[0624]

[0625]

[0626]

[0627]

[0628] General procedure for the preparation of compound 4b-2

[0629]

[0630] To a solution of compound 4b-1 (185 g, 884 mmol, 1.00 eq) in dichloromethane (1.00 L) was added TFAA (408 g, 1.95 mol, 270 mL, 2.20 eq) and t-BuOH (229 g, 3.10 mol, 296 mL, 3.50 eq). The solution was stirred at 20°C for 12 h. TLC (petroleum ether / ethyl acetate = 5 / 1) indicated the formation of a major spot (R f =0.80). The reaction mixture was quenched by NaHCO3 solution (500 mL) and then extracted with ethyl acetate (500 mL × 2). The combined organic layers were washed with brine (500 mL × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The product was purified by column chromatography (SiO2, dichloromethane, petroleum ether / ethyl acetate = 5 / 1, R f = 0.80) to afford compound 4b-2 (178 g, 671 mmol, 75.8% yield) as a yellow oil. 1 H NMR: EW49391-9-P1A1 (400 MHz, CDCl3) 3.37 - 3.41 (m, 2H), 2.18 - 2.22 (m, 2H), 1.83 - 1.85 (m, 2H), 1.53 - 1.65 (m, 2H), 1.35 - 1.48 (m, 11H), 1.28 - 1.32 (m, 2H).

[0631] General procedure for the preparation of compound 4b-3

[0632]

[0633] To a solution of compound 4b-2 (150 g, 565 mmol, 1.00 eq) in tetrahydrofuran (750 mL) was added AcSK (119 g, 1.05 mol, 1.85 eq). The suspension was stirred at 50°C for 3 h. TLC (petroleum ether / ethyl acetate = 10 / 1) showed the formation of a new spot (R f = 0.70). The reaction was poured into H2O (200 mL) and extracted with ethyl acetate (200 mL × 2). The combined organic phases were washed with brine (200 mL × 2), dried over anhydrous Na2SO4, and concentrated under vacuum to afford compound 4b-3 (140 g, 458 mmol, 80.9% yield, 85.2% purity) as a yellow oil. LCMS: EW49391-10-P1A1, Rt = 0.620 min, m / z = 283.0, M+Na + . 1 H NMR: EW49391-10-P1A2(400 MHz, CDCl3) δ 2.82 - 2.86 (m, 2H), 2.30 (s, 3H), 2.16 - 2.18 (m, 2H), 1.52 - 1.57 (m, 4H), 1.41 (s, 9H), 1.32 - 1.38 (m, 4H).

[0634] General procedure for the preparation of compound 5b

[0635]

[0636] K2CO3 (17.8 g, 128 mmol, 2.00 eq) was added to a mixture of compound int.1 (12.0 g, 64.4 mmol, 1.00 eq) and compound 4b_3 (25.1 g, 77.3 mmol, 1.20 eq) in ethanol (120 mL) at 20°C, the mixture was degassed and purged with N2 three times, and then stirred at 20°C under N2 atmosphere for 48 h. TLC (petroleum ether / ethyl acetate = 10 / 1) indicated that compound int.1 (R f = 0.60) was completely consumed and a main spot was formed (R f = 0.70). The obtained product was dissolved in dichloromethane (100 mL) and filtered to remove insoluble matter. The filtrate was concentrated in vacuo. The product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1 to 10 / 1, petroleum ether / ethyl acetate = 10 / 1, R f= 0.10) to give compound 5b (20.1 g, 46.7 mmol, 72.5% yield, 94.1% purity) as a yellow oil. LCMS: EW49391-7-P1A1, Rt = 0.635 min, m / z = 405.1, M+H + . 1 H NMR: EW49391-7-P1A2 (400MHz, CDCl3) δ 4.91 (t, J = 4.2 Hz, 1H), 4.14 (q, J = 14.0 Hz, 2H), 3.95 -3.89 (m, 2H), 3.78 - 3.84 (m, 2H), 2.72 - 2.78 (m, 2H), 2.63 - 2.67 (m, 1H), 2.48 (t, J = 7.4 Hz, 2H), 2.17 (t, J = 7.4 Hz, 2H), 2.07 - 2.13 (m, 1H), 1.90- 1.96 (m, 1H), 1.53 - 1.58 (m, 4H), 1.41 (s, 9H), 1.21 - 1.38 (m, 7H).

[0637] General procedure for the preparation of compound 5b-1

[0638]

[0639] To a solution of compound 5b (13.0 g, 30.2 mmol, 1.00 eq) in methanol (97.5 mL) was added LiOH.H2O (1.52 g, 36.2 mmol, 1.20 eq) in H2O (32.5 mL). The mixture was stirred at 25°C for 12 h. LCMS showed the detection of a peak with the desired m / z. The reaction mixture was concentrated under reduced pressure to give a residue. The product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1 to 2 / 1, petroleum ether / ethyl acetate = 5 / 1, R f = 0.20) to give compound 5b-1 (7.24 g, 17.5 mmol, 57.8% yield, 91.0% purity) as a yellow oil. LCMS: EW49391-16-P1B1, Rt = 0.552 min, m / z = 399.1, M+Na + . LCMS: EW49391-16-P1C1, Rt =0.558 min, m / z = 399.1, M+Na + . 1H NMR: EW49391-16-P1C3 (400 MHz, CDCl3) δ 7.85 -9.48 (m, 1H), 4.98 (t, J = 4.2 Hz, 1H), 3.90 - 4.03 (m, 2H), 3.80 - 3.89 (m,2H), 2.77 - 2.86 (m, 2H), 2.63 - 2.73 (m, 1H), 2.51 (t, J = 6.0 Hz, 2H), 2.10- 2.21 (m, 3H), 1.96 - 2.03 (m, 1H), 1.52 - 1.58 (m, 4H), 1.43 (s, 9H), 1.27- 1.40 (m, 4H).

[0640] General procedure for the preparation of compound 6b

[0641]

[0642] To a solution of compound 5b-1 (5.00 g, 13.2 mmol, 1.00 eq) in dimethylformamide (50.0 mL) were added compound 4b-2 (5.28 g, 19.9 mmol, 1.50 eq) and K2CO3 (3.67 g, 26.5 mmol, 2.00 eq). The mixture was stirred at 80°C for 2 h. TLC (petroleum ether / ethyl acetate = 5 / 1) indicated the formation of a major spot (R f = 0.50). The reaction mixture was diluted with water (50.0 mL) and extracted with ethyl acetate (50.0 mL × 2). The combined organic layers were washed with brine (50.0 mL × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1 to 5 / 1, petroleum ether / ethyl acetate = 5 / 1, R f = 0.50) to give compound 6b (4.38 g, 7.54 mmol, 56.7% yield, 96.5% purity) as a yellow oil. LCMS: EW49391-18-P1A1, Rt = 0.712 min, m / z = 583.3, M+Na + . 1 H NMR: EW49391-18-P1A1 (400MHz, CDCl3) 4.93 (t, = 4.4 Hz, 1H), 4.09 (t, = 6.6 Hz, 2H), 3.90 -4.00 (m, 2H), 3.80 - 3.88 (m, 2H), 2.73 - 2.81 (m, 2H), 2.63 - 2.72 (m, 1H),2.50 (t, = 7.2 Hz, 2H), 2.07 - 2.23 (m, 5H), 1.90 - 2.01 (m, 1H), 1.52 -1.67 (m, 8H), 1.44 (s, 18H), 1.28 - 1.41 (m, 8H).

[0643] General procedure for the preparation of compound 7b

[0644]

[0645] To a solution of compound 6b (4.38 g, 7.81 mmol, 1.00 eq) in dichloromethane (43.8 mL) was added TFA (17.8 g, 156 mmol, 11.6 mL, 20.0 eq). The solution was stirred at 20°C for 12 h. TLC (petroleum ether / ethyl acetate = 1 / 2) indicated that compound 6b (R f = 0.80) is completely consumed and a new spot is formed (R f = 0.40). The reaction mixture was 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, petroleum ether / ethyl acetate = 1 / 2, R f = 0.40) to give compound 7b (2.65 g, 6.55 mmol, 83.8% yield, 100% purity) as a yellow oil. LCMS: EW49391-21-P1A1, Rt = 0.593 min, m / z = 405.1, M+H + . 1 H NMR: EW49391-21-P1A2 (400 MHz, CDCl3) 9.78 (s, 1H), 8.62 -9.05 (m, 2H), 3.09 - 3.13 (m,1H), 2.73 - 3.00 (m, 3H), 2.61 - 2.66 (m, 1H), 2.49 (t, = 7.24 Hz, 2H), 2.35 (td, = 7.2, 2.4 Hz, 4H), 1.51 - 1.73 (m, 8H), 1.30 - 1.45 (m, 8H).

[0646] General procedure for the preparation of compound 8b

[0647]

[0648] To a solution of compound 7b (5.80 g, 14.3 mmol, 1.00 eq) in dichloromethane (100 mL) was added AcOH (1.03 g, 17.2 mmol, 984 μL, 1.20 eq) and N-methylmethanamine (2 M, 10.7 mL, 1.50 eq). The solution was stirred at 25°C for 1 hour, followed by the addition of NaBH(OAc)₃ (4.56 g, 21.5 mmol, 1.50 eq). The solution was stirred at 25°C for 1 hour. LCMS (EW49594-35-P1B) indicated the detection of the desired Ms. The reaction was poured into an aqueous solution of NaHCO₃ (120 mL). The resulting solution was extracted with tetrahydrofuran (100 mL x 2). The aqueous phase was then acidified to pH 3 with 2 M HCl. The aqueous phase was extracted with tetrahydrofuran (200 mL x 2). The combined organic layers were washed with brine (200 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography (ISCO®; 80 g SepaFlash® silica gel flash column, 0% to 8% methanol / dichloromethane gradient eluent @ 65 mL / min, dichloromethane:methanol = 10:1, P1:R f = 0.37) to afford compound 8b (1.50 g, 3.46 mmol, 24.1% yield) as a yellow gum. LCMS: EW49594-35-P1B, Rt = 0.402 min, m / z = 434.2, M+Na + . 1 H NMR: EW49594-35-P1B (400 MHz, CDCl3) 4.87 (quin, = 6.0 Hz, 2H), 4.09 (t, = 6.8 Hz,2H), 2.73 - 2.80 (m, 1H), 2.59 - 2.67 (m, 2H), 2.50 (t, = 6.8 Hz, 2H), 2.23- 2.35 (m, 6H), 2.20 (s, 6H), 1.74 - 1.83 (m, 2H), 1.49 - 1.70 (m, 16H), 1.18- 1.44 (m, 24H), 0.89 (t, = 6.8 Hz, 12H).

[0649] Preparation of compound SL66

[0650]

[0651] To a solution of compound 8b (1.40 g, 3.23 mmol, 1.00 eq) and 5-nonanol (1.40 g, 9.69 mmol, 3.00 eq) in tetrahydrofuran (30.0 mL) was added EDCI (1.86 g, 9.69 mmol, 3.00 eq) and DMAP (788 mg, 6.46 mmol, 2.00 eq). The solution was stirred at 25°C for 15 h. LCMS (EW49594-37-P1A) showed the detection of the desired Ms. NaHCO₃ (50.0 mL of aqueous solution) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (50.0 mL x 2). The combined organic phase was dried over MgSO₄, filtered, and concentrated under reduced pressure to yield the crude product. The crude product was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® silica gel flash column, 0% to 15% tetrahydrofuran / petroleum ether gradient eluent @ 35 mL / min, petroleum ether:tetrahydrofuran = 1:1, R f = 0.17) to obtain 1.5 g of residue. The 1.5 g residue was purified by preparative HPLC (chromatographic column: Phenomenex Luna C18 150*25 mm*10um; mobile phase: [water (TFA)-methanol]; gradient: 80%-98% B, 15 min) to obtain 750 mg of compound SL66. Ethyl acetate (20.0 mL) was added to the 750 mg product and washed with NaHCO3 (aqueous solution 10.0 mL x 5), dried over MgSO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® silica gel flash column, 0%~1% methanol / dichloromethane gradient eluent @ 35 mL / min, petroleum ether:tetrahydrofuran = 1:1, R f= 0.17) to afford compound SL66 (650 mg, 946 μmol, 29.3% yield, 99.9% purity) as a yellow oil. LCMS: EW49594-37-P1A, Rt = 0.708 min, m / z = 686.6, M+H + Special LCMS: EW49594-37-P1C, Rt = 1.814 min, m / z = 686.6, M+H + . 1 H NMR: EW49594-37-P1A (400 MHz, CDCl3) 4.87 (q, = 6.0 Hz, 2H),4.09 (t, = 6.8 Hz, 2H), 2.73 - 2.80 (m, 1H), 2.59 - 2.67 (m, 2H), 2.50 (t, = 6.8 Hz, 2H), 2.23 - 2.35 (m, 6H), 2.20 (s, 6H), 1.74 - 1.83 (m, 2H), 1.49- 1.70 (m, 16H), 1.18 - 1.44 (m, 24H), 0.89 (t, = 6.8 Hz, 12H).

[0652] General procedure for the preparation of compound 2

[0653]

[0654] A solution of compound 1 (200 g, 1.55 mol, 1.00 eq) in ethanol (2000 mL) and H2SO4 (400 mL) was stirred at 90°C for 6 h. LCMS (EW49393-11-P1A) showed that the desired mass was detected (Rt = 0.332 min, m / z = 158.1, M+H +). The reaction mixture was cooled to 25 ° C and concentrated under reduced pressure to remove ethanol, and then adjusted to pH = 9 with aqueous Na2CO3 solution and extracted with dichloromethane 3000 mL (1000 mL x 3). The combined organic layer was washed with brine (1000 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo to give compound 2 (120 g, 763 mmol, 49.3% yield) as a yellow oil. LCMS: EW49393-11-P1A, Rt = 0.332 min, m / z = 158.1 (M+H + ). 1 H NMR: EW49393-11-P1B (400 MHz, CDCl3) δ 6.26 (s, 1H), 5.72 (s, 1H), 4.21 (q, J = 7.2 Hz, 2H), 2.13 (s, 2H), 2.24 (s, 6H), 1.29 (t, J = 7.2 Hz, 3H).

[0655] General procedure for the preparation of compound 3

[0656]

[0657] To a solution of compound 4a_2 (68.6 g, 238 mmol, 1.10 eq) in methanol (340 mL) was added compound K2CO3 (59.8 g, 432 mmol, 2.00 eq) and compound 2 (34.0 g, 216 mmol, 1.00 eq). The mixture was stirred at 20 ° C for 44 h. LCMS (EW49393-30-P1A) showed that compound 2 was completely consumed. The reaction mixture was filtered and concentrated in vacuo to obtain a residue. The product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 40 / 1 to 5 / 1, petroleum ether / ethyl acetate = 0:1, R f = 0.14) to give compound 3 (35.0 g, 86.7 mmol, 40.1% yield) as a yellow oil. LCMS: EW49393-30-P1A, Rt = 0.507 min, m / z = 404.4 (M+H + ). 1 H NMR:EW49393-30-P1A (400 MHz, CDCl3) 4.15 - 4.21 (m, 2H), 2.75 - 2.78 (m, 1H), 2.70 - 2.72 (m, 2H), 2.57 - 2.62 (m, 1H), 2.51 (t, = 7.2 Hz, 2H), 2.38 -2.43 (m, 1H), 2.18 -2.22 (m, 7H), 1.52 - 1.60 (m, 4H), 1.44 (s, 9H), 1.24 -1.29 (m, 12H).

[0658] General procedure for the preparation of compound 3a

[0659]

[0660] To a solution of compound 3 (35.0 g, 86.7 mmol, 1.00 eq) in methanol (245 mL) was added LiOH•H2O (4.37 g, 104 mmol, 1.20 eq) in H2O (100 mL). The mixture was stirred at 25°C for 12 h. TLC (dichloromethane:methanol = 10:1) indicated that compound 3 remained (R f = 0.24), and the desired product was formed (R f = 0.02). The reaction mixture was concentrated under reduced pressure to remove methanol. The residue was diluted with ethyl acetate 300 mL and extracted with H2O 300 mL. The aqueous layer was adjusted to pH = 5 with citric acid aqueous solution, extracted with ethyl acetate 300 mL and dichloromethane 300 mL, washed with brine 300 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to obtain a residue. The product was purified by column chromatography (SiO2, dimethylformamide / methanol = 1 / 0 to 8 / 1, dimethylformamide:methanol = 10:1, R f = 0.02) to give compound 3a (13.9 g, 36.0 mmol, 41.6% yield, 97.4% purity) as a yellow oil. LCMS: EW49393-33-P1C, Rt = 0.479 min, m / z = 376.1 (M+H + ). 1H NMR: EW49393-33-P1A (400 MHz, CDCl3)δ 10.39 (s, 1H), 3.41 - 3.49 (m, 1H), 3.06 - 3.20 (m, 2H), 2.95 - 2.98 (m,1H), 2.89 (s, 6H), 2.53 - 2.59 (m, 3H), 2.20 (t, J = 7.2 Hz, 2H), 1.54 - 1.59(m, 4H), 1.44 (s, 9H), 1.34 - 1.37 (m, 2H), 1.29 (s, 6H).

[0661] General procedure for the preparation of compound 4

[0662]

[0663] To a solution of compound 3a (11.8 g, 31.4 mmol, 1.00 eq) in dimethylformamide (120 mL) was added K2CO3 (8.68 g, 62.84 mmol, 2.00 eq) and compound 4a_1 (13.8 g, 47.1 mmol, 1.50 eq). The mixture was stirred at 80°C for 3 h. LCMS (EW49393-35-P1A) showed complete consumption of compound 3a. The reaction mixture was diluted with ethyl acetate (200 mL), extracted with H2O (200 mL), washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 40 / 1 to 5 / 1, petroleum ether / ethyl acetate = 1:1, R f = 0.14) to give compound 4 (15.0 g, 25.5 mmol, 81.2% yield) as a yellow oil. LCMS: EW49393-35-P1C, Rt = 0.588 min, m / z = 588.4 (M+H + ). 1 H NMR:EW49393-35-P1A (400 MHz, CDCl3) 4.12 (t, = 7.2 Hz, 2H), 2.70 - 2.79 (m,3H), 2.57 - 2.62 (m, 1H), 2.51 (t, = 7.2 Hz, 2H), 2.38 - 2.43 (m, 1H), 2.22(s, 6H), 2.19 - 2.20 (m, 3H), 1.53 - 1.66 (m, 9H), 1.45 (s, 18H), 1.30 - 1.38(m, 17H).

[0664] General procedure for the preparation of compound 5

[0665]

[0666] To a solution of compound 4 (7.00 g, 11.9 mmol, 1.00 eq) in dichloromethane (70.0 mL) was added TFA (35.0 mL). The mixture was stirred at 25 ° C for 16 h. LCMS (EW49393-37-P1A) showed that compound 4 was completely consumed. The reaction mixture was concentrated under reduced pressure to obtain a residue. The product was purified by column chromatography (SiO2, dimethylformamide / methanol = 1 / 0 to 20 / 1, dimethylformamide:methanol = 10:1, R f = 0.02) to give compound 5 (4.00 g, 8.41 mmol, 70.6% yield) as a yellow oil. LCMS: EW49393-37-P1A, Rt = 0.442 min, m / z = 476.2 (M+H + ). 1 H NMR: EW49393-37-P1A (400 MHz, CDCl3) 4.15 - 4.20 (m, 2H), 3.50 -3.55 (m, 2H), 3.24 - 3.26 (m, 1H), 2.72 - 2.92 (m, 8H), 2.53 (t, = 7.2 Hz,2H), 2.35 (t, = 7.2 Hz, 4H), 1.53 - 169 (m, 9H), 1.45 - 1.26 (m, 17H).

[0667] Preparation of compound SL76

[0668]

[0669] To a solution of compound 5 (3.00 g, 6.31 mmol, 1.00 eq) and 5-nonanol (2.73 g, 18.9 mmol, 3.00 eq) in tetrahydrofuran (30.0 mL) was added EDCI (3.63 g, 18.9 mmol, 3.00 eq) and DMAP (1.54 g, 12.6 mmol, 2.00 eq). The mixture was stirred at 25°C for 16 h. LC-MS (EW49393-38-P1A) showed complete consumption of compound 5. The reaction mixture was partitioned between NaHCO₃ (25.0 mL) and EtOAc (25.0 mL x 2). The organic phase was separated, washed with brine (25.0 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to yield a residue. The chromatographic analysis was performed by column chromatography (SiO2, dimethylformamide / methanol = 1 / 0 to 100 / 1, dimethylformamide:methanol = 10:1, R f = 0.02) to give SL76 (1.54 g, 2.11 mmol, 33.5% yield, 99.9% purity) as a yellow oil. LCMS: EW49393-38-P1A, Rt = 0.761 min, m / z = 728.5 (M+H + ). Special LCMS: EW49393-38-P1A, Rt = 2.073 min, m / z = 728.8 (M+H + ). 1 H NMR: EW49393-38-P1B (400 MHz, CDCl3) 4.86 -4.91 (m, 2H), 4.11 (t, = 6.8 Hz, 2H), 2.70 - 2.79 (m, 3H), 2.57 - 2.62 (m,1H), 2.51 (t, = 7.2 Hz, 2H), 2.38 - 2.43 (m, 1H), 2.28 (t, = 7.2 Hz, 4H), 2.22 (s, 6H), 1.48 - 1.62 (m, 15H), 1.21 - 1.38 (m, 33H), 0.89 (t, =7.2 Hz, 12H).

[0670] General procedure for the preparation of compound 7

[0671]

[0672] To a solution of compound 4b_3 (107 g, 350 mmol, 1.10 eq) in methanol (500 mL) were added compound K2CO3 (87.9 g, 636 mmol, 2.00 eq) and compound 2 (50.0 g, 318 mmol, 1.00 eq). The mixture was stirred at 20°C for 48 h. TLC (petroleum ether / ethyl acetate = 0:1) showed that compound 2 was completely consumed. The reaction mixture was filtered and concentrated in vacuo to obtain a residue. The product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 40 / 1 to 5 / 1, petroleum ether / ethyl acetate = 0:1, R f = 0.14) to give compound 7 (55.0 g, 146 mmol, 46.1% yield) as a yellow oil. 1 H NMR: EW49393-15-P1A (400 MHz, CDCl3) 4.11 - 4.21 (m, 2H), 2.75- 2.80 (m, 1H), 2.70 - 2.72 (m, 2H), 2.57 - 2.62 (m, 1H), 2.51 (t, = 7.2Hz, 2H), 2.38 - 2.43 (m, 1H), 2.18 - 2.22 (m, 7H), 1.54 - 1.61 (m, 4H), 1.44(s, 9H), 1.31 - 1.42 (m, 4H), 1.24 (t, = 6.0 Hz, 3H).

[0673] General procedure for the preparation of compound 7a

[0674]

[0675] To a solution of compound 7 (20.0 g, 53.2 mmol, 1.00 eq) in methanol (140 mL) was added LiOH•H2O (2.68 g, 63.9 mmol, 1.20 eq) in H2O (80.0 mL). The mixture was stirred at 25°C for 12 h. TLC (dichloromethane:methanol = 10:1) indicated that compound 7 remained (R f = 0.24), and the desired product was formed (R f= 0.02). The reaction mixture was concentrated under reduced pressure to remove methanol. The residue was diluted with 100 mL of ethyl acetate and extracted with 100 mL of H2O. The aqueous layer was adjusted to pH = 5 with aqueous citric acid solution, extracted with 100 mL of ethyl acetate and 300 mL of dichloromethane, washed with 100 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 7a (7.11 g, 20.5 mmol, 38.4% yield) as a yellow oil. 1 H NMR: EW49393-17-P1A (400 MHz, CDCl3) δ 6.05(s, 1H), 3.08 - 3.14 (m, 2H), 2.93 - 2.98 (m, 1H), 2.69 (s, 6H), 2.51 - 2.59(m, 3H), 2.20 (t, J = 7.2 Hz, 2H), 1.54 - 1.63 (m, 4H), 1.44 (s, 9H), 1.24 -1.40 (m, 2H).

[0676] General procedure for the preparation of compound 8

[0677]

[0678] To a solution of compound 7a (14.2 g, 40.9 mmol, 1.00 eq) in dimethylformamide (150 mL) was added K2CO3 (11.3 g, 81.7 mmol, 2.00 eq) and compound 4b_2 (16.3 g, 61.3 mmol, 1.50 eq). The mixture was stirred at 80°C for 3 h. LCMS (EW49393-24-P1A) showed complete consumption of compound 7a. The reaction mixture was diluted with ethyl acetate (200 mL), extracted with H2O (200 mL), washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 40 / 1 to 5 / 1, petroleum ether / ethyl acetate = 1:1, R f = 0.14) to give compound 8 (11.3 g, 21.3 mmol, 52.0% yield) as a yellow oil. LCMS: EW49393-24-P1A, Rt = 0.563 min, m / z = 532.5 (M+H + ). 1H NMR:EW49393-24-P1A (400 MHz, CDCl3) δ 4.12 (t, J = 6.8 Hz, 2H), 2.70 - 2.81 (m,3H), 2.57 - 2.62 (m, 1H), 2.51 (t, J = 7.2 Hz, 2H), 2.38 - 2.42 (m, 1H), 2.19- 2.23 (m, 9H), 1.54 - 1.69 (m, 9H), 1.45 (s, 18H), 1.28 - 1.41 (m, 9H).

[0679] General procedure for the preparation of compound 9

[0680]

[0681] To a solution of compound 8 (5.60 g, 10.5 mmol, 1.00 eq) in dichloromethane (45.0 mL) was added TFA (24.0 g, 210 mmol, 15.6 mL, 20.0 eq). The reaction was stirred at 30°C for 15 h. The reaction was concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography (ISCO®; 80 g SepaFlash® silica gel flash column, 0% to 8% ethyl acetate / petroleum ether gradient at 65 mL / min, dichloromethane:methanol = 10:1, P1:R f = 0.39) to give compound 9 (3.60 g, crude) as a yellow gum. 1 H NMR: EW49594-31-P1A (400 MHz, CDCl3) δ 4.03 - 4.08 (m, 2H), 3.36 - 3.37 (m, 1H), 3.34 - 3.36 (m, 1H), 3.26- 3.27 (m, 1H), 2.74 - 2.79 (m, 1H), 2.48 (s, 6H), 2.45 - 2.47 (m, 1H), 2.28- 2.29 (m, 2H), 2.25 - 2.26 (m, 4H), 1.58 - 1.60 (m, 8H), 1.31 - 1.36 (m,8H).

[0682] Preparation of compound SL77

[0683]

[0684] To a solution of compound 9 (2.50 g, 5.96 mmol, 1.00 eq) and 5-nonanol (2.58 g, 17.9 mmol, 3.00 eq) in tetrahydrofuran (60.0 mL) was added EDCI (3.43 g, 17.9 mmol, 3.00 eq) and DMAP (1.46 g, 11.9 mmol, 2.00 eq). The solution was stirred at 25°C for 15 h. The reaction mixture was partitioned between 25.0 mL of NaHCO₃ and 25.0 mL of EtOAc × 2. The organic phase was separated, washed with 25.0 mL of brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography (ISCO®; 80 g Sepa Flash® silica gel flash column, 0% to 1% methanol / dichloromethane gradient at 65 mL / min, R f = 0.47). SL77 (2.00 g, 2.98 mmol, 49.9% yield) was obtained as a yellow gum and purified by flash silica gel chromatography (ISCO®; 80 g Sepa Flash® silica gel flash column, 0% to 15% tetrahydrofuran / petroleum ether gradient at 65 mL / min, petroleum ether:tetrahydrofuran = 1:1, R f = 0.47) to afford compound SL77 (1.40 g, 2.08 mmol, 69.9% yield, 99.8% purity) as a yellow oil. Special LCMS: EW49594-45-P1A1, Rt = 1.784 min, m / z = 672.1 (M+H + ). 1 H NMR:EW49594-45-P1C (400 MHz, CDCl3) 4.84 - 4.91 (m, 2H), 4.12 (t, = 6.8 Hz,2H), 2.72 - 2.81 (m, 1H), 2.51 - 2.71 (m, 2H), 2.50 - 2.51 (m, 1H), 2.30 -2.50 (m, 2H), 2.29 - 2.30 (m, 1H), 2.27 - 2.29 (m, 4H), 2.22 (s, 6H), 1.51 -1.64 (m, 16H), 1.27 - 1.33 (m, 24H), 0.89 (t, = 7.2 Hz, 12H).

[0685] Preparation of compound SL78

[0686]

[0687] To a solution of compound 9 (2.50 g, 5.96 mmol, 1.00 eq) and 6-undecanol (3.08 g, 17.88 mmol, 3 eq) in tetrahydrofuran (60.0 mL) was added EDCI (3.43 g, 17.9 mmol, 3.00 eq) and DMAP (1.46 g, 11.9 mmol, 2.00 eq). The solution was stirred at 25°C for 15 h. The reaction mixture was partitioned between 25.0 mL of NaHCO₃ and 25.0 mL of EtOAc × 2. The organic phase was separated, washed with 25.0 mL of brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography (ISCO®; 80 g Sepa Flash® silica gel flash column, 0% to 1% methanol / dichloromethane gradient at 65 mL / min, R f = 0.66). SL78 (2.20 g, 3.02 mmol, 50.7% yield) was obtained as a yellow gum and purified by flash silica gel chromatography (ISCO®; 80 g SepaFlash® silica gel flash column, 0% to 15% tetrahydrofuran / petroleum ether gradient @ 65 mL / min, petroleum ether:tetrahydrofuran = 1:1, R f = 0.66) to afford compound SL78 (1.20 g, 1.65 mmol, 54.5% yield, 99.9% purity) as a yellow oil. Special LCMS: EW49594-46-P1A2, Rt = 2.202 min, m / z = 728.8 (M+H + ). 1 H NMR:EW49594-46-P1C (400 MHz, CDCl3) δ 4.85 - 4.91 (m, 2H), 4.12 (t, J = 6.8 Hz,2H), 2.72 - 2.81 (m, 1H), 2.51 - 2.71 (m, 2H), 2.50 - 2.51 (m, 1H), 2.30 -2.50 (m, 2H), 2.29 - 2.30 (m, 1H), 2.27 - 2.29 (m, 4H), 2.22 (s, 6H), 1.51 -1.64 (m, 16H), 1.28 - 1.37 (m, 32H), 0.89 (t, J = 7.2 Hz, 12H).

[0688] General procedure for the preparation of compound 7a

[0689]

[0690] To a solution of compound int.2 (5.00 g, 10.8 mmol, 1.00 eq) and piperidine (793 mg, 70.8 mmol, 1.12 mL, 1.00 eq) in dichloromethane (50.0 mL) was added AcOH (782 mg, 13.0 mmol, 745 µL, 1.20 eq). The mixture was stirred at 25°C for 1.5 hours. NaBH(OAc)3 (2.76 g, 13.0 mmol, 1.20 eq) was then added to the mixture. The mixture was stirred at 25°C for 1.5 hours. TLC (dichloromethane / methanol = 10 / 1, R f = 0.3) indicating complete consumption of compound int.2 and the formation of two new spots. The reaction mixture was diluted with 100 mL of H2O and extracted with 300 mL of dichloromethane (100 mL x 3). The combined organic layers were washed with 100 mL of brine, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to obtain a residue. The product was purified by column chromatography (SiO2, dimethylformamide / methanol = 1 / 0 to 80 / 1, dimethylformamide:methanol = 10:1, R f = 0.30) to give compound 7a (4.05 g, 4.90 mmol, 45.3% yield, 66.8% purity) as a colorless oil. LCMS: EW49391-28-P1C1, Rt = 0.443 min, m / z = 518.3 (M+H + ). 1 H NMR: EW49391-28-P1C2 (400 MHz, CDCl3) δ 9.5 - 10.7 (s, 2H), 4.09 - 4.14 (m, 2H), 3.05 - 3.20 (m, 5H), 2.62 - 2.72 (m, 2H), 2.48 - 2.51(m, 2H), 2.30 - 2.67 (m, 4H), 1.96 - 2.06 (m, 2H), 1.50 - 1.67 (m, 9H), 1.33- 1.43 (m, 17H).

[0691] Preparation of compound SL79

[0692]

[0693] To a solution of compound 7a (2.40 g, 4.64 mmol, 1.00 eq) and 5-nonanol (2.01 g, 13.9 mmol, 3.00 eq) in tetrahydrofuran (48.0 mL) were added EDCI (2.67 g, 13.9 mmol, 3.00 eq) and DMAP (1.13 g, 9.27 mmol, 2.00 eq). The mixture was stirred at 25°C for 16 h. TLC (dichloromethane / methanol = 10 / 1) indicated that compound 7a (R f = 0.30) was completely consumed and a main spot was formed (R f = 0.50). The reaction mixture was partitioned between 25.0 mL of NaHCO₃ and 25.0 mL of EtOAc x 2. The organic phase was separated, washed with 25.0 mL of brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain a residue. The product was purified by column chromatography (SiO₂, dimethylformamide / methanol = 1 / 0 to 100 / 1, dimethylformamide / methanol = 10 / 1, R f = 0.50) to give compound SL79 (1.67 g, 2.16 mmol, 46.6% yield, 99.7% purity) as a yellow oil. ELSD: EW49391-30-P1A2, Rt = 2.037 min, m / z = 771.9 (M+H + ). 1 H NMR: EW49391-30-P1A4 (400 MHz, CDCl3) δ 4.84 - 4.89(m, 2H), 4.08 (t, J = 6.8 Hz, 2H), 2.72 - 2.83 (m, 1H), 2.45 - 2.67 (m, 10H),2.27 (t, J = 7.2 Hz, 4 H), 1.72 - 1.91 (m, 2 H), 1.44 - 1.69 (m, 15 H), 1.19- 1.41 (m, 33 H), 1.03 (t, J = 7.2 Hz, 6 H), 0.88 (t, J = 6.8 Hz, 12H).

[0694] General procedure for the preparation of compound 7b

[0695]

[0696] To a solution of compound int.2 (15.0 g, 32.5 mmol, 1.00 eq) and piperidine (2.32 g, 32.5 mmol, 2.72 mL, 1.00 eq) in dichloromethane (150 mL) was added AcOH (2.35 g, 39.0 mmol, 2.24 mL, 1.20 eq). The mixture was stirred at 25°C for 1.5 hours. NaBH(OAc)3 (8.28 g, 39.0 mmol, 1.20 eq) was then added to the mixture. The mixture was stirred at 25°C for 1.5 hours. TLC (dichloromethane / methanol = 10 / 1, R f = 0.3) indicating complete consumption of compound int.2 and the formation of three new spots. The reaction mixture was diluted with 100 mL of H2O and extracted with 300 mL of dichloromethane (100 mL x 3). The combined organic layers were washed with 100 mL of brine, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to obtain a residue. The product was purified by column chromatography (SiO2, dimethylformamide / methanol = 1 / 0 to 80 / 1, dimethylformamide / methanol = 10 / 1, R f = 0.30) to give compound 7b (10.0 g, 19.4 mmol, 59.5% yield) as a yellow oil.

[0697] Preparation of compound SL80

[0698]

[0699] To a solution of compound 7b (5.00 g, 9.69 mmol, 1.00 eq) and 5-nonanol (4.20 g, 29.0 mmol, 3.00 eq) in tetrahydrofuran (100 mL) were added EDCI (5.58 g, 29.0 mmol, 3.00 eq) and DMAP (2.37 g, 19.3 mmol, 2.00 eq). The mixture was stirred at 25°C for 16 h. TLC (dichloromethane / methanol = 10 / 1) indicated compound 7b (R f = 0.30) was completely consumed and a main spot was formed (R f = 0.50). The reaction mixture was partitioned between 100 mL of NaHCO₃ and 50.0 mL of EtOAc × 2. The organic phase was separated, washed with 100 mL of brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain a residue. The product was purified by column chromatography (SiO₂, dimethylformamide / methanol = 1 / 0 to 100 / 1, dimethylformamide / methanol = 10 / 1, R f= 0.50) to give compound SL80 (1.45 g, 1.88 mmol, 19.4% yield, 99.8% purity) as a yellow oil. ELSD: EW49391-32-P1A1, Rt = 2.231 min, m / z = 768.9 (M+H + ). 1 H NMR: EW49391-32-P1A2 (400 MHz, CDCl3) 4.84 - 4.91(m, 2H), 4.08 (t, = 6.8 Hz, 2H), 2.72 - 2.84 (m, 1H), 2.46 - 2.69 (m, 10H), 2.27 (t, = 7.6 Hz, 4H), 1.77 - 1.98 (m, 6H), 1.48 - 1.63 (m, 1H), 1.43 -1.69 (m, 17H), 1.16 - 1.42 (m, 34H), 0.88 (t, = 6.8 Hz, 12H).

[0700] General procedure for the preparation of compound 7c

[0701]

[0702] To a solution of compound int.2 (3.00 g, 6.51 mmol, 1.00 eq) and piperidine (554 mg, 6.51 mmol, 643 µL, 1.00 eq) in dichloromethane (30.0 mL) was added AcOH (469 mg, 7.82 mmol, 447 µL, 1.20 eq). The mixture was stirred at 20°C for 1.5 hours. NaBH(OAc)₃ (1.66 g, 7.82 mmol, 1.20 eq) was then added to the mixture. The mixture was stirred at 20°C for 1.5 hours. LC-MS (EW49393-31-P1A) showed complete consumption of compound int.2. The reaction mixture was diluted with 100 mL of H₂O and extracted with 300 mL of dichloromethane (100 mL x 3). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to obtain a residue. The product was purified by column chromatography (SiO2, dimethylformamide / methanol = 1 / 0 to 8 / 1, dimethylformamide:methanol = 10:1, R f= 0.16) to give compound 7c (2.50 g, 4.72 mmol, 72.5% yield, 100% purity) as a colorless oil. LCMS: EW49393-31-P1A, Rt = 0.467 min, m / z = 530.4 (M+H + ). LCMS: EW49393-31-P1B, Rt =0.484 min, m / z = 530.3 (M+H + ). 1 H NMR: EW49393-31-P1B (400 MHz, CDCl3) 11.27(s, 2H), 4.06 - 4.16 (m, 2H), 2.97 - 3.06 (m, 4H), 2.74 - 2.81 (m, 2H), 2.63- 2.68 (m, 2H), 2.47 - 2.54 (m, 2H), 2.28 (t, = 7.2 Hz, 4H), 2.01 - 2.06 (m, 5H), 1.52 - 1.62 (m, 10H), 1.33 - 1.43 (m, 16H).

[0703] Preparation of compound SL81

[0704]

[0705] To a solution of compound 7c (2.30 g, 4.34 mmol, 1.00 eq) and 5-nonanol (1.88 g, 13.0 mmol, 3.00 eq) in tetrahydrofuran (25.0 mL) was added EDCI (2.50 g, 13.0 mmol, 3.00 eq) and DMAP (1.06 g, 8.68 mmol, 2.00 eq). The mixture was stirred at 25°C for 16 h. LCMS (EW49393-36-P1A) showed complete consumption of compound 7c. The reaction mixture was partitioned between 25.0 mL of NaHCO₃ and 25.0 mL of EtOAc × 2. The organic phase was separated, washed with 25.0 mL of brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to yield a residue. The residue was purified by column chromatography (SiO2, dimethylformamide / methanol = 1 / 0 to 100 / 1, dimethylformamide:methanol = 10:1, Rf = 0.43) to afford compound SL81 (2.17 g, 2.77 mmol, 63.8% yield, 99.8% purity) as a yellow oil. LCMS: EW49393-36-P1A, Rt = 0.748 min, m / z = 782.7 (M+H+). ELSD: EW49393-36-P1A, Rt = 2.188 min, m / z = 782.9 (M+H+). 1 H NMR: EW49393-36-P1A (400 MHz, CDCl3) 4.84 -4.91 (m, 2H), 4.09 (t, = 4.8 Hz, 2H), 2.74 - 2.78 (m, 1H), 2.62 - 2.66 (m,2H), 2.30 (t, = 7.2 Hz, 2H), 2.33 - 2.39 (m, 5H), 2.28 (t, = 7.2 Hz,5H), 1.80 - 1.90 (m, 3H), 1.49 - 1.63 (m, 17H), 1.43 - 1.45 (m, 3H), 1.21 -1.34 (m, 33H), 0.89 (t, = 6.8 Hz, 12H).

[0706] Example 2: Formulating ionizable cationic liposomes with mRNA into LNPs

[0707] This is a general description of how each ionizable cationic lipid was used to formulate samRNA LNPs. The ionizable cationic lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene ether (DMG-PEG) were combined in ethanol at a molar ratio of 40:10:48:2 to a concentration of 3.2 mM (0.5 mg samRNA scale) or 6.4 mM (1.5 mg samRNA scale). samRNA solutions expressing the antigen of interest were prepared in 50 mM citrate buffer, pH 6, containing 10 mM tris(2-carboxyethyl)phosphine (TCEP). When mixed at a 2:1 flow rate ratio (FRR) of water to ethanol, the RNA concentration was 0.025 mg / mL (0.5 mg samRNA scale) or 0.050 mg / mL (1.5 mg samRNA scale). When mixed at a 3:1 water-to-ethanol flow rate ratio (FRR), the RNA concentration was 0.017 mg / mL (0.5 mg samRNA scale). The lipid solution in ethanol was then rapidly mixed with the samRNA solution using a Knauer benchtop IJM NanoScaler system at a 2:1 or 3:1 water buffer-to-ethanol flow rate ratio (FRR). This mixing ratio resulted in an 8:1 molar ratio of ionizable cationic lipids (see Table 1) to samRNA phosphate groups and a 37:1 total lipid-to-samRNA mass ratio. The resulting mixed solution was then diluted 10-fold into 50 mM citrate buffer, pH 6, containing 10 mM TCEP and concentrated to the original volume by tangential flow filtration (TFF) using a 300k molecular weight cutoff membrane (mPES). Subsequently, 10 diafiltration volumes were used to exchange the citrate buffer with a buffer containing 20 mM Tris buffer (pH 7.5), 80 mM sodium chloride, and 3% sucrose. The LNP solution was concentrated to a volume of 4–10 mL, filtered using a 0.2-μm PES syringe filter, aliquoted into vials, and frozen using Corning® CoolCell® LX cell freezing containers at 1°C / min until the sample temperature reached −80°C. Samples were stored at −80°C and thawed on wet ice before analysis or use. Total RNA concentration, the percentage of input RNA recovered (% recovery), and encapsulation efficiency (% EE) were determined using the Ribogreen assay (described in the literature). Following a 1:100 dilution in phosphate-buffered saline (PBS), the Z-average diameter (nm) and polydispersity index (PDI) were measured using dynamic light scattering (Malvern Zetasizer).

[0708] The results of the formulation studies are depicted in the following table:

[0709] LNP # Ionizable cationic lipids Flow rate ratio (FRR, water:EtOH) Citrate buffer pH Encapsulation efficiency (EE, %) Total RNA concentration (µg / mL) RNA recovery rate (%) Z-average diameter (nm) determined by DLS PDI determined by DLS pKa 1 LKY750 2:1 6 96.9 40.5 68.8 80.18 0.138 6.45 2 SL56 3:1 6 97.6 44.7 75.1 138.9 0.211 7.1 3 SL57 3:1 6 96.1 52.7 92.4 91.37 0.082 6.83 4 SL58 3:1 4 95.9 43.7 72.2 69.61 0.215 5.67 5 SL59 2:1 4 95.2 41.2 66.7 93.5 0.217 6.24 6 SL60 2:1 4 93.8 41.7 73 97.99 0.078 6.17 7 SL61 2:1 4 92.4 45.5 81.8 73.85 0.127 4.88 8 SL62 3:1 6 94.3 30.2 56.2 79.34 0.141 7.04 9 SL63 3:1 4->6 88.7 26.9 54.5 79.50 0.104 7.03 10 SL64 3:1 4->6 79.7 57.9 41.7 89.86 0.207 6.61 11 SL65 3:1 4->6 90.1 37.4 68 71.87 0.122 6.70 12 SL66 3:1 6 94.3 34.4 62.7 136.9 0.075 6.83 13 SL67 3:1 6 95.3 42.7 74.4 88.25 0.099 7.05 14 SL68 3:1 6 97.5 35.4 61.9 144.9 0.195 7.51 15 SL69 3:1 6 81.7 43.1 69.4 104.7 0.090 6.77 16 SL70 3:1 6 86.85 59.6 35 91.2 0.106 6.90 17 SL71 3:1 6 76.49 57.7 43.7 90.1 0.128 6.85 18 SL72 3:1 4->6 98.48 44.0 33.3 84.7 0.220 6.58 19 SL73 3:1 6 91.17 60.7 42.2 88.8 0.081 6.89 20 SL74 3:1 6 83.6 54.8 38.1 93.0 0.106 6.79 21 SL75 3:1 4->6 86.74 41.7 45.3 89.2 0.217 6.59 22 SL76 3:1 4->6 90.86 61.1 36.3 88.1 0.122 5.25 23 SL77 3:1 4->6 91.74 54.9 46.4 85.5 0.083 5.25 24 SL78 3:1 4->6 86.53 57.8 43.8 85.5 0.216 5.12 25 SL79 3:1 6 92.95 61.9 44.2 94.8 0.091 6.48 26 SL80 3:1 6 94 49.9 80.2 85.87 0.109 6.85 27 SL81 3:1 4->6 88.1 35.4 62.1 101.6 0.284 5.96

[0710] Biophysical properties of lipid nanoparticles

[0711] Analytical methods will be used to characterize LNP compositions containing messenger RNA to determine the loading amount of messenger RNA, the percentage of encapsulated messenger RNA, and the particle size. The total amount of messenger RNA contained in the sample and the percentage of encapsulated messenger RNA will be determined using a fluorescence assay using Ribogreen, a dye that emits a higher luminosity after binding to messenger RNA. The total amount of messenger RNA will be determined by damaging the LNP with 1wt% Triton-X 100 to expose the encapsulated messenger RNA, adding the dye, and comparing the emission intensity with a standard curve drawn using ribosomal RNA. The amount of unencapsulated messenger RNA will be measured in a similar manner, but the destruction of the LNP by the detergent will be omitted. Given the total amount of messenger RNA and the amount of unencapsulated messenger RNA, the percentage of encapsulated messenger RNA will be calculated as follows:

[0712] Encapsulation percentage (%) = ((RNA 总 -RNA 未包封 ) / RNA 总 ) X 100

[0713] RNA 总 and RNA 未包封 are the concentrations of total mRNA and unencapsulated mRNA, respectively. LNP size will be measured using dynamic light scattering on samples diluted 1:100 in PBS buffer.

[0714] pKa determination protocol

[0715] In a black 96-well plate, samRNA-LNP solutions (final assay concentration of 2 µg / mL total RNA) will be prepared in a series of buffers ranging from pH 4 to 9.5. Buffers with pH values ​​of 4 to 7.6 will be prepared using disodium phosphate and citric acid. Buffers with pH values ​​of 7.8 to 9.5 will be prepared by titrating tris buffer with 10 N sodium hydroxide. An aqueous solution of sodium 6-(p-toluidino)-2-naphthalenesulfonate (TNS) will be added to each well to a final assay concentration of 6 µM. Fluorescence will be read on a microplate reader at 25°C with excitation set to 321 nm and emission set to 445 nm. Fluorescence intensity values ​​will be plotted as a function of pH using GraphPad Prism and fitted with a sigmoidal dose-response curve. The apparent pKa will determine the EC50 for this curve, where half of the ionizable amines are expected to be protonated.

[0716] LNP Efficacy characterization

[0717] An in vitro assay was used to characterize the ability of samRNA-LNPs to transfect cultured cells, based on the percentage of cells expressing the antigen of interest. Specifically, for each LNP, 1 million BHK-21 cells were incubated with varying concentrations of samRNA-LNPs in 2 mL of culture medium at 37°C, 5% CO2 for 17-19 hours. Subsequently, cells were treated with TrypLE (Gibco) to detach from the culture dish to form a single-cell suspension, fixed and permeabilized (BD Cytofix / Perm Kit), and then stained with fluorescently labeled antigen-specific antibodies against H5 and N1. The percentage of dual-antigen-positive cells was quantified using a BD Accuri flow cytometer.

[0718] The results of the study are shown in the table below:

[0719]

[0720] A high-throughput microscopy-based assay was used to characterize the ability of samRNA-LNPs to transfect cultured cells. 5 BHK-21 cells were incubated with LNPs at concentrations ranging from 0 to 1 ng in PS-96 flat-bottom plates containing 4% FBS and DMEM medium at 37°C, 5% CO₂ for over 18 hours. Cells were then fixed and permeabilized using the BD Cytofix / Perm Kit reagent and stained with an (in-house) human anti-H5 antibody using the ThermoFisher Cy5-Zenon Anti-Human Labeling Kit and DAPI. The plates were further imaged using an Agilent high-throughput Cytation5 instrument. Analysis was performed by counting DAPI spots as the total number of cells and Cy-5 red spots as the number of antigen-positive cells. To increase the linear range of the assay, the absolute value of the natural logarithm of the percentage of negative cells was calculated: ln(1-(total red spots) / (total blue spots)). The reference lipid, LKY750 (2,5-bis((9z,12z)-octadeca-9,12-dien-1-yloxy)benzyl-4-(dimethylamino)butanoate; WO 2016 / 037053, incorporated herein by reference), was always included as a positive control for normalization. The normalized absolute amounts of lipid and LKY750 are reported. The higher the normalized absolute value for a sample, the higher the transfection efficiency of that particular LNP chemistry. The mean value for this assay was 20%, with a minimum and maximum natural variability of 10% and 30%, respectively.

[0721] The results are as follows Figure 1 and 2 Depicted, wherein the efficacy of each of the lipid formulations was evaluated relative to the LKY750 control. Both FACS and Cytation 5 efficacy results indicate that the evaluated lipid formulations showed improved or at least comparable efficacy compared to the LKY750 control CY087. efficacy (e.g., CY089 vs. SL57).

[0722] LNP Efficacy characterization

[0723] The ability of mRNA-LNPs to act as a vaccine was evaluated by measuring antibody and cell-based immune responses following a prime-boost vaccination regimen. Balb / c mice were primed via intramuscular injection (im) on day 0 and boosted 21 days later. After an additional 21 days (day 42 of the experiment), the mice were sacrificed and serum and splenocytes were collected for further analysis.

[0724] Sera will be analyzed for vaccine-specific antibody responses using IgG enzyme-linked immunosorbent assay (IgG ELISA), pseudovirus microneutralization assay (MN), hemagglutination inhibition (HAI) assay, and neuraminidase inhibition enzyme-linked lectin assay (ELLA). Figures 3A-3D As shown, SL57 was comparable in activity to the control lipid, LKY750, in the HAI, NA inhibition, and MN assays. SL60 was comparable in activity to LKY750 in the MN assay. Both SL57 and SL60 were comparable in activity to an adjuvanted inactivated virus vaccine (aH5N1) in the ELISA, HAI, and MN assays.

[0725] The results will be compared with the adjuvanted inactivated virus vaccine (aH5N1) tested in the same experiment using a one-way ANOVA statistical test.

[0726] exist After peptide stimulation, splenocytes from the above experiments will be analyzed for antigen-specific cytokine production by intracellular cytokine staining by flow cytometry. Splenocytes will be collected (n=5 / group) and cytokine production will be analyzed in the presence or absence of H5 or N1 peptides. Stimulation was repeated twice. Mean response levels as measured by the production of interferon gamma, interleukin 2, and / or tumor necrosis factor alpha are shown, and error bars indicate the precision of the measurements.

[0727] Taken together, these in vivo results are promising for demonstrating that LNPs prepared using lipids SL56-SL81 are immunogenic in this preclinical model and could serve as an effective influenza vaccine.

[0728] LNP Characterization of transfection efficiency

[0729] mRNA-LNPs will be evaluated using samRNA expressing the reporter protein firefly luciferase. The efficacy of the assay is measured to quantify the location, relative amount, and duration of protein expression. LNPs formulated with mRNA expressing luciferase are injected into mice, for example, intramuscularly in the hind legs. At defined time points (such as daily), luciferin is administered to the mice, and bioluminescence is imaged and quantified.

[0730] Characterization of genotoxic potential, tolerance, biodistribution, and biodegradability

[0731] Since no single test can detect all mechanisms of genotoxicity leading to tumorigenicity, a standard battery of tests that predict genotoxic potential (DNA damage) will be used. When the result of the mammalian cell assay is positive, two An apparently negative result in an assay (in an appropriate tissue and where adequate exposure to the test substance has been demonstrated) would be considered a lack of Evidence of genotoxic potential. Relevant guidance will be followed, such as Genotoxicity Testing of Medicinal Products for Human Use and Interpretation of Data (S2(R1)).

[0732] Bacterial gene mutation test.

[0733] Chromosome damage Cytogenetic testing (metaphase chromosome aberration or micronucleus test) or mouse lymphoma Tk gene mutation assay.

[0734] · Genotoxicity test: Chromosome damage test using rodent hematopoietic cells to detect micronuclei or chromosomal aberrations in metaphase cells.

[0735] Computer simulation screening of toxicity of novel lipids

[0736] Commercially available computational toxicology assessment products and / or services will be used to screen the compounds of Formula I and / or Formula II for potential toxicity and mutagenicity to meet the requirements of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use. (ICH) M7 (R1)Evaluation and Control of DNA-Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk". For example, two complementary quantitative (Q)SAR approaches, the expert rule-based Derek Nexus and the statistically based Sarah Nexus (or Leadscope), will be used to predict bacterial mutagenicity outcomes. Mutagenicity and carcinogenicity will be categorized on a scale of 1 to 5, ranging from known mutagenic carcinogens (Category 1) to lack of mutagenicity or carcinogenicity (Category 5). Results from the in silico analyses will be reviewed to clarify the relevance of positive, negative, conflicting, or uncertain predictions and the basis for the conclusions provided.

[0737] Because most structural alerts are based on bacterial mutagenicity, compounds with structural alerts can be detected in standard test panels. Ad...

Claims

1. A compound of formula I or a compound of formula II: or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: X is selected from the group consisting of -S-, -O- and -C-; E 1 Straight chain or branched-C 1-30 -alkyl; R 1 Selected from the group consisting of -H and formula IA; R 2 Selected from linear or branched-C 1-30 - a group consisting of an alkyl group and formula IA; Formula IA; E 2 , if present, is a linear or branched-C 1-30 -alkyl; m and n are each independently an integer from 0 to 3; p is an integer from 0 to 2; L 1 Select from the group consisting of: R 3 , if present, is selected from -H and linear or branched -C 1-8 - a group consisting of an alkyl group; L 2 Selected from –OC(O)- and –C(O)O-; W is selected from the group consisting of Formula IIA, Formula IIB, Formula IIC and Formula IID: R 4 , if present, is selected from -H and linear or branched -C 1-5 - a group consisting of an alkyl group; Y is selected from -H, linear or branched -C 1-5 -alkyl and linear or branched-C 1-5 -alkanols; and Dashed lines represent bonds to adjacent atoms in the compound of formula I or the compound of formula II. The compound according to claim 1 , wherein X is -S-.

3. The compound according to claim 1, wherein E 1 For straight chain-C 1-30 -alkyl.

4. The compound according to claim 1, wherein E 1 For branched-C 1-30 -alkyl.

5. The compound according to claim 1, wherein R 1 is -H.

6. The compound according to claim 1, wherein R 1 It is formula IA.

7. The compound according to claim 1, wherein R 1 is Formula IA, and E 2 For straight chain-C 1-30 -alkyl.

8. The compound according to claim 1, wherein R 1 is Formula IA, and E 2 For branched-C 1-30 -alkyl.

9. The compound according to claim 1, wherein L 1 for: 。 10. The compound according to claim 1, wherein X is -S-, and L 1 for: 。 11. The compound according to claim 1, wherein Y is a linear or branched-C 1-5 -alkyl.

12. The compound according to claim 1, wherein Y is a linear or branched C 1-5 -alkanol.

13. The compound according to claim 1, wherein Y is a linear C 1-5 -alkyl.

14. The compound according to claim 1, wherein Y is a linear C 1-5 -alkanol.

15. The compound of claim 1, wherein the compound is a compound of formula I.

16. The compound of claim 1, wherein the compound of formula I is selected from the group consisting of: 、 、 、 、 、 、 、 、 and .

17. The compound according to claim 1, wherein the compound of formula I is: 。 18. The compound according to claim 1, wherein the compound of formula I is: 。 19. The compound according to claim 1, wherein the compound of formula I is: 。 20. The compound according to claim 1, wherein the compound of formula I is: 。 21. The compound according to claim 1, wherein the compound of formula I is: 。 22. The compound of claim 1, wherein the compound is a compound of formula II.

23. The compound of claim 1, wherein the compound of formula II is selected from the group consisting of: ； ； ； ； ； ； ; ;and 。 24. The compound according to claim 1, wherein the compound of formula II is: 。 25. The compound according to claim 1, wherein the compound of formula II is: 。 26. The compound according to claim 1, wherein the compound of formula II is: 。 27. The compound according to claim 1, wherein the compound of formula II is: 。 28. The compound according to claim 1, wherein the compound of formula II is: 。 29. The compound of claim 1, wherein the compound is selected from the group consisting of: ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ;and 。 30. A lipid nanoparticle (LNP) comprising a lipid component comprising the compound of claim 1.

31. The lipid nanoparticle of claim 30, wherein the lipid component further comprises one or more of a neutral lipid, a structural lipid, and a PEGylated lipid.

32. The lipid nanoparticle of claim 31 , wherein the neutral lipid is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-heneicosanoyl-sn-glycero-phosphocholine (DUPC), ), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholestylesuccinyl-sn-glycero-3-phosphocholine (OchemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 lysoPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diamidateonic acid-sn-glycero-3-phosphocholine, 1,2-docosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (MEPC), 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonic acid-sn-glycero-3-phosphoethanolamine, 1,2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin.

33. The lipid nanoparticle of claim 31 , wherein the structural lipid is selected from the group consisting of cholesterol, coprostanol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatine, tomatin, ursolic acid, and α-tocopherol.

34. The lipid nanoparticle of claim 31 , wherein the PEGylated lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol, optionally PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE.

35. The lipid nanoparticle of claim 31 , wherein the lipid component comprises: about 25 mol% to about 60 mol% of the compound of claim 1; about 2 mol% to about 25 mol% of neutral lipids; about 18.5 mol% to about 60 mol% of structural lipids; and about 0.2 mol% to about 10 mol% of PEGylated lipids.

36. The lipid nanoparticle of claim 30, wherein the lipid nanoparticle further comprises a polynucleotide.

37. The lipid nanoparticle of claim 36, wherein the polynucleotide is selected from the group consisting of messenger RNA (mRNA), self-amplifying mRNA (sa-mRNA), small interfering RNA (siRNA), microRNA (miRNA), miRNA inhibitors (antagomirs / antimirs), messenger RNA interfering with complementary RNA (micRNA), short hairpin RNA (shRNA), multivalent RNA, dicer substrate RNA, antisense oligonucleotides, plasmid DNA, DNA, and complementary DNA (cDNA).

38. The lipid nanoparticle of claim 36, wherein the polynucleotide is ribonucleic acid (RNA).

39. The lipid nanoparticle of claim 36, wherein the polynucleotide is a conventional mRNA or a self-amplifying mRNA.

40. The lipid nanoparticle of claim 30, wherein the lipid nanoparticle has a diameter of about 30 nm to about 160 nm.

41. A pharmaceutical composition comprising a plurality of lipid nanoparticles according to claim 30 and a pharmaceutically acceptable carrier.

42. A method of delivering a polynucleotide to a mammalian cell, the method comprising administering to a subject the lipid nanoparticle of claim 30 or the pharmaceutical composition of claim 41, thereby contacting the cell with the lipid nanoparticle or the pharmaceutical composition and delivering the polynucleotide to the cell.

43. The method of claim 42, wherein the cell is a cell of a human subject.

44. A method for producing a target polypeptide in a mammalian cell, the method comprising the step of contacting the cell with a lipid nanoparticle according to claim 30 or a pharmaceutical composition according to claim 41, wherein the lipid nanoparticle comprises conventional mRNA or self-amplifying mRNA encoding the polypeptide.

45. A method of treating a disease, disorder or condition in a subject in need of such treatment, the method comprising administering to the subject the lipid nanoparticle of claim 30 or the pharmaceutical composition of claim 41, thereby treating the disease, disorder or condition.

46. ​​The method of claim 45, wherein the disease, disorder or condition is selected from the group consisting of rare diseases, infectious diseases, cancer, proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases and metabolic diseases.

47. A vaccine comprising the lipid nanoparticle according to claim 30 or the pharmaceutical composition according to claim 40 and mRNA encoding a polypeptide.

48. The vaccine of claim 47, wherein the vaccine is selected from a tumor vaccine, an influenza vaccine, and a SARS vaccine, the SARS vaccine comprising a SARS-CoV-2 vaccine.

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