DIOL-based novel ionizable lipids and their uses thereof

Abstract

Compounds are provided having the following structure: (I) or (II), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein X, 1r, 2r, 3r, R11, R12, R21, R22, R31 and R32 are as defined herein. Use of the compounds as a component of lipid nanoparticle formulations for delivery of a therapeutic agent, compositions comprising the compounds and methods for their use and preparation are also provided.

Description

[0001] DIOL-BASED NOVEL IONIZABLE LIPIDS AND THEIR USES THEREOF

[0002] Technical Field

[0003] The present invention provides, in part, diol-based novel ionizable lipids of structure (I) or (II), or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof. The compounds described herein can be used to develop lipid nanoparticle formulations to effectively and safely deliver RNA or DNA for applications include but not limited to mRNA vaccine, mRNA therapy, siRNA therapy, gene therapy and so on.

[0004] Background of the invention

[0005] Nucleic acid-based therapeutics can have enormous impact on human health, with the recent example of COVID-19 mRNA vaccines and the key role it played in fighting the global pandemic. While there are many different modes of nucleic acid-based therapeutics, such as mRNA, siRNA, ASO and DNA, the key challenge remains: delivery (Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine, N Engl J Med 2020; 383:2603-2615, DOI: 10.1056 / NEJ Moa2034577).

[0006] In the context of mRNA-related therapeutics, delivering the mRNA effectively and safety to the target of interest is critical to applications such as prophylactic vaccine, therapeutic vaccine, mRNA therapy, genome editing. As such, a powerful delivery vehicle called lipid nanoparticles (LNP) was developed to 1 ) protect the mRNA from degradation 2) deliver the mRNA intracellularly so that it can be translated to the corresponding protein that will have downstream therapeutic effects (Lipid nanoparticles for mRNA delivery, Hou, X., Zaks, T., Langer, R. et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater 6, 1078-1094 (2021). https: / / doi.org / 10.1038 / s41578-021 -00358-0).

[0007] While LNPs are typically made of 4 or 5 different lipid components, the most important lipid is the ionizable lipid, as it enables the LNP to 1) effectively encapsulate and protect the mRNA and 2) deliver the mRNA to the cytosol of the cells where translation takes place. There remains a critical need for improved ionizable lipids and LNP formulations for oligonucleotides delivery. Specifically, ionizable lipids / LNPs with greater stability, scalability, structural diversity and higher therapeutic index, will further unlock the potential of nucleic acid-based therapeutics (Lipids and Lipid Derivatives for RNA Delivery, Chem. Rev. 2021, 121, 20, 12181-1227; Han, X., Zhang, H., Butowska, K. et al. An ionizable lipid toolbox for RNA delivery. Nat Commun 12, 7233 (2021). https: / / doi.org / 10.1038 / s41467-021-27493-0).

[0008] Summary of the invention

[0009] In brief, the present invention provides ionizable lipid compounds, including pharmaceutically acceptable salts, prodrug or stereoisomer thereof, that arc used in combination with other lipid components such as neutral lipids, charged lipids, steroids, polymer conjugated lipid (such as PEG lipids) or related analogues to form LNPs to deliver therapeutic agents such as DNA, mRNA for therapeutic applications such as siRNA therapy, prophylactic mRNA vaccine, therapeutic mRNA vaccine, mRNA therapy, genome editing.

[0010] In one embodiment, compounds are provided having the following structure: or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein X, Ir, 2r, 3r, Rn, R12, R21, R22, R31 and R32 are as defined herein.

[0011] In other embodiment, compounds are provided having the following structure: or a pharmaceutically acceptable salt, prodrag or stereoisomer thereof, wherein X, Ir, 2r, 3r,

[0012] Rn, R12, R21, R22, R31 and R32 are as defined herein.

[0013] Previous ionizable lipids require complex organic synthesis that often required 5 and more synthetic steps, greatly limiting the facile synthesis and structural diversity of the ionizable lipid library. In the present invention, all ionizable lipids are synthesized in 3 relatively simple steps, and start with commercially available diol building blocks. By using this highly modular and efficient synthesis method, a unique and more structurally diverse ionizable lipid library was synthesized.

[0014] In addition, LNPs made from these ionizable lipids are well-tolerated in preclinical animal models while providing an adequate therapeutic index. As such, it is believed that using these

[0015] LNPs from this invention to treat patient at an effective dose of the mRNA should not be associated with unacceptable toxicity and / or side effects. The present invention provides these and related advantages that might not be provided by others in the arts.

[0016] Brief description of the drawings

[0017] In the figures, identical reference numbers identify similar elements. The sizes and relative positions of elements in the figures are not necessarily drawn to scale and some of these elements are arbitrarily enlarged and positioned to improve figure legibility. Further, the particular shapes of the elements as drawn are not intended to convey any information regarding the actual shape of the particular elements, and have been solely selected for ease of recognition in the figtires.

[0018] Figure 1 shows VZV gE-specific serum total IgG induced by the VZV gE variant mRNAs / candidate cationic lipid vaccine

[0019] Detailed description of the invention

[0020] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the invention. However, those skilled in the art will understand that the invention may be practiced without these details.

[0021] Definition

[0022] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0023] Unless the context requires otherwise, throughout the present specification and claims, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open and inclusive sense, that is, as “including, but not limited to”. Reference throughout this specification to “one embodiment” or “tin embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by those skilled in the art to which this invention belongs. As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.

[0025] An “effective amount” or “therapeutically effective amount” of an active agent or therapeutic agent such as a therapeutic nucleic acid is an amount sufficient to produce the desired effect, e.g. an increase or inhibition of expression of a target sequence in comparison to the normal expression level detected in the absence of the nucleic acid. An increase in expression of a target sequence is achieved when any measurable level is detected in the case of an expression product that is not present in the absence of the nucleic acid. In the case where the expression product is present at some level prior to contact with the nucleic acid, an in increase in expression is achieved when the fold increase in value obtained with a nucleic acid such as mRNA relative to control is about 1.05, 1.1, 1.2, 1.3, 1 .4, 1 .5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000 or greater. Inhibition of expression of a target gene or target sequence is achieved when the value obtained with a nucleic acid such as antisense oligonucleotide relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expression of a target gene or target sequence include, e.g., examination of 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, fluorescence or luminescence of suitable reporter proteins, as well as phenotypic assays known to those skilled in the art.

[0026] The term “nucleic acid” as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double- stranded form and includes DNA, RNA, and hybrids thereof. DNA may be in the form of antisense oligonucleotides, plasmid DNA, cDNA, PCR products, or vectors. RNA may be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, dicer substrate RNA or viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91 - 98 (1994)). “Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through tire phosphate groups. “Bases” include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.

[0027] The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises partial length or entire length coding sequences necessary for the production of a polypeptide or precursor polypeptide.

[0028] “Gene product,” as used herein, refers to a product of a gene such as an RNA transcript or a polypeptide.

[0029] The term “lipid” refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are generally characterized by being poorly soluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes: (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0030] A “steroid” is a compound comprising the following carbon skeleton:

[0031] Non-limiting examples of steroids include cholesterol, and the like.

[0032] A “ionizable lipid” refers to lipids having at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH, and neutral at a second pH, preferably at or above physiological pH. It will be understood by those skilled in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form.

[0033] The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include l-(monomethoxy- polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) and the like.

[0034] The term “neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphotidylcholines such as l,2-Distearoyl-sn-glycero-3- phosphocholine (DSPC), l,2-Dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2- Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), l-Palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine(POPC), l,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC), phophatidylethanolamines such as l,2-Dioleoyl-sn-glycero-3-phosphoethanolamine(DOPE), sphingomyelins (SM), ceramides, steroids such as sterols and their derivatives. Neutral lipids may be synthetic or naturally derived. The term “charged lipid” refers to any of a number of lipid species that exist in either a positively charged or negatively charged form independent of the pH within a useful physiological range e.g. pH ~3 to pH ~9. Charged lipids may be synthetic or naturally derived. Examples of charged lipids include phosphatidylserines, phosphatidic acids, phosphatidylglycerols, phosphatidylinositols, sterol hemi succinates, dialkyl trimethylammonium-propanes, (e.g. DOTAP, DOTMA), dialkyl dimethylaminopropanes, ethyl phosphocholines, dimethylaminoethane carbamoyl sterols (e.g. DC-Choi).

[0035] The term ‘lipid nanoparticle” refers to particles having at least one dimension on the order of nanometers (e.g., 1-1 ,000 nm) which include one or more of the compounds of structure (I) or (If) other specified cationic lipids. In some embodiments, lipid nanoparticles are included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). In some embodiments, the lipid nanoparticles of the invention comprise a nucleic acid. Such lipid nanoparticles typically comprise a compound of structure (I) or (Il)and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids. In some embodiments, the active agent or therapeutic agent, such as a nucleic acid, may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response.

[0036] In various embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 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, or 150 nm, and are substantially non-toxic. In certain embodiments, nucleic acids, when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease. Lipid nanoparticles comprising nucleic acids and their method of preparation are disclosed in, e.g., U.S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031 and PCT Pub. Nos. WO 2013 / 016058 and WO 2013 / 086373, the full disclosures of which are herein incorporated by reference in their entirety for all purposes. As used herein, “lipid encapsulated” refers to a lipid nanoparticle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), with full encapsulation, partial encapsulation, or both. In an embodiment, the nucleic acid (e.g., mRNA) is fully encapsulated in the lipid nanoparticle.

[0037] As used herein, the term “aqueous solution” refers to a composition comprising water.

[0038] “Serum-stable” in relation to nucleic acid-lipid nanoparticles means that the nucleotide is not significantly degraded after exposure to a serum or nuclease assay that would significantly degrade free DNA or RNA. Suitable assays include, for example, a standard serum assay, a DNAse assay, or an RNAse assay.

[0039] “Systemic delivery,” as used herein, refers to delivery of a therapeutic product that can result in a broad exposure of an active agent within an organism.

[0040] Some techniques of administration can lead to the systemic delivery of certain agents, but not others. Systemic delivery means that a useful, preferably therapeutic, amount of an agent is exposed to most parts of the body. Systemic delivery of lipid nanoparticles can be by any means known in the art including, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.

[0041] “Local delivery,” as used herein, refers to delivery of an active agent directly to a target site within an organism. For example, an agent can be locally delivered by direct injection into a disease site such as a tumor, other target site such as a site of inflammation, or a target organ such as the liver, heart, pancreas, kidney, and the like. Local delivery can also include topical applications or localized injection techniques such as intramuscular, subcutaneous or intradermal injection. Local delivery does not preclude a systemic pharmacological effect.

[0042] The term “alkane,” as used herein, refers to a saturated hydrocarbon compound. For example, C i-20 alkane refers to an alkyl group having from 1 to 20 carbon atoms. Alkane may be optionally replaced with heteroatoms (for example, 1, 2 or 3 heteroatoms), which are an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur. For example, C4 alkane replaced with two O atoms refers to an alkyl group having two carbon atoms and two oxygen atoms. Other identifiers can be utilized to indicate the presence of particular groups in the alkane (e.g. halogenated alkane indicates that the presence of one or more halogen atoms replacing an equivalent number of hydrogen atoms in the alkane). The term “alkyl group” is used herein in accordance with the definition specified by IUPAC: a univalent group formed by removing a hydrogen atom from an alkane. Similarly, an “alkylene group” refers to a group formed by removing two hydrogen atoms from an alkane (either two hydrogen atoms from one carbon atom or one hydrogen atom from two different carbon atoms). An “alkane group” is a general term that refers to a group formed by removing one or more hydrogen atoms (as necessary for the particular group) from an alkane. An “alkane group” can be linear or branched. Primary, secondary, and tertiary alkyl group are derived by removal of a hydrogen atom from a primary, secondary, tertiary carbon atom, respectively, of an alkane. The groups RCH2 (R#H), R2CH (R^H), and R3C (R^H) are primary, secondary, and tertiary alkyl groups, respectively. The n-alkyl group may be derived by removal of a hydrogen atom from a terminal carbon atom of a linear alkane.

[0043] The term “alkene” as used herein, refers a linear or branched hydrocarbon olefin that has one or more carbon-carbon double bonds. For example, C2-20 alkene refers to an alkenyl group having from 2 to 20 carbon atoms. Alkene may be optionally replaced with heteroatoms (for example, 1, 2 or 3 heteroatoms), which are an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur. For example, C4 alkene replaced with two O atoms refers to an alkenyl group having two carbon atoms and two oxygen atoms. Alkenes having only one, only two, only three, etc. . . . such multiple bond can be identified by use of the term “mono,” “di,” “tri,” etc. . . . within the name.

[0044] An “alkenyl group” is a univalent group derived from an alkene by removal of a hydrogen atom from any carbon atom of the alkene. Thus, “alkenyl group” includes groups in which the hydrogen atom is formally removed from an sp2hybridized (olefinic) carbon atom and groups in which the hydrogen atom is formally removed from any other carbon atom. For example and unless otherwise specified, 1 -propenyl (-CH-CHCHa), 2-propenyl [(CH3)C~CH?.], and 3- propenyl (-CHzCH-CH?) groups are all encompassed with the term “alkenyl group.” Similarly, an “alkenylene group” refers to a group formed by formally removing two hydrogen atoms from an alkene, either two hydrogen atoms from one carbon atom or one hydrogen atom from two different carbon atoms. An “alkene group” refers to a generalized group formed by removing one or more hydrogen atoms (as necessary for the particular group) from an alkene.

[0045] The term “alkyne” as used herein, refers a linear or branched hydrocarbon that has one or more carbon-carbon triple bonds. For example, C2-20 alkyne refers to an alkynyl group having from 2 to 20 carbon atoms. Alkyne may be optionally replaced with heteroatoms (for example, 1, 2 or 3 heteroatoms), which are an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur. For example, €4 alkyne replaced with two O atoms refers to an alkynyl group having two carbon atoms and two oxy gen atoms. Alkynes having only one, only two, only three, etc. . . . such multiple bond can be identified by use of the term “mono,” “di,” “tri,”: etc. . . . within the name.

[0046] An “alkynyl group” is a univalent group derived from an alkyne by removal of a hydrogen atom from any carbon atom of the alkyne. Thus, “alkynyl group” includes groups in which the hydrogen atom is formally removed from an sp hybridized (acetylenic) carbon atom and groups in which the hydrogen atom is formally removed from any other carbon atom. For example and unless otherwise specified, 1-propynyl (-C=CCHs) and 3-propynyl (HC^CCH?-) groups are all encompassed with the term “alkynyl group.” Similarly, an “alkynylene group” refers to a group formed by formally removing two hydrogen atoms from an alkyne, either two hydrogen atoms from one carbon atom if possible or one hydrogen atom from two different carbon atoms. An “alkyne group” refers to a generalized group formed by removing one or more hydrogen atoms (as necessary for the particular group) from an alkyne.

[0047] The term “aromatic ring system” denotes a carbocyclic or heterocyclic ring system in which at least one ring of the ring system is aromatic. Aromatic ring systems can be 3- to 30-membered aromatic ring system containing 3 to 30 atoms in the ring(s), 3- to 20-membered aromatic ring system containing 3 to 20 atoms in the ring(s), 3- to 10-membered aromatic ring system containing 3 to 10 atoms in the ring(s) or 6- to 10-membered aromatic ring system containing 6 to 10 atoms in the ring(s).The term “aromatic carbocyclic ring system” denotes a carbocyclic ring system in which at least one ring of the ring system is aromatic, which can be 6- to 14- membered aryl group, a 6- to 10-membered aryl group or a 6-membered aryl group, such as benzene, naphthalene and azulene. The term “aromatic heterocyclic ring system” denotes a heterocyclic ring system in which at least one ring of the ring system is aromatic, wherein the heteroatoms (for example, 1, 2 or 3 heteroatoms) are an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur. Examples of aromatic heterocyclic ring system include, but are not limited to, furan, furazan, thiophene, benzo thiophene, phthalazine, pyrrole, oxazole, benzoxazole, 1,2,3- oxadiazole, 1,2,4-oxadiazole, thiazole, 1,2,3-thiadiazole, 1,2,4- thiadiazole, benzothiazole, imidazole, benzimidazole, indole, indazole, pyrazole, benzopyrazole, isoxazole, benzoisoxazole, isothiazole, triazole, benzotriazole, thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, purine, pteridine, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline and triazine. An aromatic ring system may be substituted or unsubstituted.

[0048] The term “non-aromatic ring system” denotes a carbocyclic or heterocyclic ring system that may be fully saturated, as well as parti ally or fully unsaturated, provided that none of the rings in the ring system are aromatic. Non-aromatic ring systems can be 3- to 30-membered non- aromatic ring system containing 3 to 30 atoms in the ring(s), 3- to 20-membered non-aromatic ring system containing 3 to 20 atoms in the ring(s), 3- to 10-membered non-aromatic ring system containing 3 to 10 atoms in the ring(s) or 6- to 10-membered non-aromatic ring system containing 6 to 10 atoms in the ring(s). The term “nonaromatic carbocyclic ring system” in which no ring in the ring system is aromatic, such as cycloalkanes, cycloalkenes, cycloalkynes. The term “nonaromatic heterocyclic ring system” denotes a heterocyclic ring system in which no ring in the ring system is aromatic, wherein the heteroatoms (for example, 1, 2 or 3 heteroatoms) are an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur. Non-limiting examples include: pyrrolidine, piperidine, N-methylpiperidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, pyrimidine-2,4(lH,3H)-dione, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-5 -oxide, thiomorpholine-S,S-oxide, piperazine, pyran, pyridone, 3-pyrroIine, thiopyran, pyrone, tetrahydrofuran, tetrhydrothiophene, quinuclidine, tropane.

[0049] A cycloalkane is a saturated cyclic hydrocarbon with mono- or polycyclo (such as bicyclo). When composed of two or more rings, the rings may be joined together in a fused, bridged or spiro fashion. As used herein, the term “fused” refers to two rings which have two atoms and one bond in common. As used herein, the term “bridged cycloalkyl” refers to compounds wherein the cycloalkyl contains a linkage of one or more atoms connecting non-adjacent atoms. As used herein, the term “spiro” refers to two rings which have one atom in common and the two rings are not linked by a bridge. Cycloalkyl groups can be 3- to 30-membered cycloalkyl group containing 3 to 30 atoms in the ring(s), 3- to 20-membered cycloalkyl group containing 3 to 20 atoms in the ring(s), 3- to 10-membered cycloalkyl group containing 3 to 10 atoms in the ring(s) or 6- to 10-membered cycloalkyl group containing 6 to 10 atoms in the ring(s). A cycloalkyl group may be unsubstituted or substituted. Examples of mono-cycloalkyl groups include, but are in no way limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. Examples of fused cycloalkyl groups are decahydronaphthalenyl, dodecahydro-lH-phenalenyl and tetradec ahydroanthracenyl; examples of bridged cycloalkyl groups are bicyclo[l.l.l]pentyl, adamantanyl and norbornanyl; and examples of spiro cycloalkyl groups include spiro[3.3]heptane and spiro[4.5]decane.

[0050] Unsaturated cyclic hydrocarbons having one or more endocyclic double or triple bond are called cycloalkenes and cycloalkynes, respectively. Cycloalkenes and cycloalkynes having only one, only two, only three, etc. . . . endocyclic double or triple bonds, respectively, can be identified by use of the term “mono,” “di,” “tri ,: etc. . . . within the name of the cycloalkene or cycloalkyne.

[0051] A “cycloalkyl group” is a univalent group derived by removing a hydrogen atom from a ring carbon atom of a cycloalkane.

[0052] Similarly, a “cycloalkylene group” refers to a group derived by removing two hydrogen atoms from a cycloalkane, at least one of which is a ring carbon. Thus, a “cycloalkylene group” includes both a group derived from a cycloalkane in which two hydrogen atoms are formally removed from the same ring carbon, a group deri ved from a cycloalkane in which two hydrogen atoms are formally removed from two different ring carbons, and a group derived from a cycloalkane in which a first hydrogen atom is formally removed from a ring carbon and a second hydrogen atom is formally removed from a carbon atom that is not a ring carbon. A “cycloalkane group” refers to a generalized group formed by removing one or more hydrogen atoms (as necessary for the particular group and at least one of which is a ring carbon) from a cycloalkane.

[0053] Other terms, such as “benzene’, “phenyl” and “phenylene” are defined similarly as abov

[0054] “Optional” or “optionally” (e.g., optionally substituted) means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, “optionally substituted alkyl” means that the alkyl radical may or may not be substituted and that the description inchides both substituted alkyl radicals and alkyl radicals having no substitution.

[0055] “Prodrag” is meant to indicate a compound that may be converted under physiological conditions or by solvolysis to a biologically active compound of the invention. Thus, the term “prodrug” refers to a metabolic precursor of a compound of the invention that is pharmaceutically acceptable. A prodrag may be inactive when administered to a subject in need thereof, but is converted in vivo to an active compound of the invention. Prodrags tire typically rapidly transformed in vivo to yield the parent compound of the invention, for example, by hydrolysis in blood. The prodrug compound often offers advantages of solubility , tissue compatibility or delayed release in a mammalian organism (see, Bundgard, II., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi, T., et al., A.C.S. Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987. The term “prodrug” is also meant to include any covalently bonded carriers, which release the active compound of the invention in vivo when such prodrug is administered to a mammalian subject. Prodrugs of a compound of the invention may be prepared by modifying functional groups present in the compound of the invention in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound of the invention. Prodrugs include compounds of the invention wherein a hydroxy, amino or mercapto group is bonded to any group that, when the prodrug of the compound of the invention is administered to a mammalian subject, cleaves to form a free hydroxy, free amino or free mercapto group, respectively. Examples of prodrags include, but are not limited to, acetate, formate and benzoate derivatives of alcohol or amide derivatives of amine functional groups in the compounds of the invention and the like.

[0056] The invention disclosed herein is also meant to encompass all pharmaceutically acceptable compounds of the compound of structure (I) or (II) being isotopically-labelled by having one or more atoms replaced by an atom having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as2H,3H,! 1C,13C,!4C,!3N,15N,l5O,!7O,18O,31P,32P,35S,I8F,36C1,i23I, and125I, respectively. These radiolabeled compounds could be useful to help determine or measure the effecti veness of the compounds, by characterizing, for example, the site or mode of action, or binding affinity to pharmacologically important site of action. Certain isotopically-labelled compounds of structure (I) or (II), for example, those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The radioactive isotopes tritium, i.e.,3H, and carbon- 14, i.e.,14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

[0057] Substitution with heavier isotopes such as deuterium, i.e.,2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances. Substitution with positron emitting isotopes, such as “C,1KF,:5O and:3N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds of structure (I) or (II) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the Preparations and Examples as set out below using an appropriate isotopically- labeled reagent in place of the non-labeled reagent previously employed.

[0058] The invention disclosed herein is also meant to encompass the in vivo metabolic products of the disclosed compounds. Such products may result from, for example, the oxidation, reduction, hydrolysis, amidation, esterification, and the like of the administered compound, primarily due to enzymatic processes. Accordingly, the invention includes compounds produced by a process comprising administering a compound of this invention to a mammal for a period of time sufficient to yield a metabolic product thereof. Such products are typically identified by administering a radiolabeled compound of the invention in a detectable dose to an animal, such as rat, mouse, guinea pig, monkey, or to human, allowing sufficient time for metabolism to occur, and isolating its conversion products from the urine, blood or other biological samples.

[0059] “Stable compound” and “stable structure” are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0060] “Mammal” includes humans and both domestic animals such as laboratory animals and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals such as wildlife and the like.

[0061] “Pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals. “Pharmaceutically acceptable salt” includes both acid and base addition salts.

[0062] “Pharmaceutically acceptable acid addition salt” refers to 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 are 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, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxy ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic 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-!, 5-disulfonic acid, naphthalene-2 -sulfonic acid, l-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, tri fluoro acetic acid, undecylenic acid, and the like.

[0063] “Pharmaceutically acceptable base addition salt” refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the 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, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol , dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0064] Often crystallizations produce a solvate of the compound of the invention. As used herein, the term “solvate” refers to an aggregate that comprises one or more molecules of a compound of the invention with one or more molecules of solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present invention may exist as a hydrate, including a monohydrate, dihydrate. hemihydrate, sesquihydrate, trihydrate, tetrahydrate and the like, as well as the corresponding solvated forms. The compound of the invention may be true solvates, while in other cases, the compound of the invention may merely retain adventitious water or be a mixture of water plus some adventitious solvent.

[0065] A “pharmaceutical composition” refers to a formulation of a compound of the invention and a medium generally accepted in the art for the delivery of the biologically active compound to mammals, e.g., humans. Such a medium includes all pharmaceutically acceptable carriers, diluents or excipients therefor.

[0066] “Effective amount” or “therapeutically effective amount” refers to that amount of a compound of the invention which, when administered to a mammal, preferably a human, is sufficient to effect treatment in the mammal, preferably a human. The amount of a lipid nanoparticle of the invention which constitutes a “therapeutically effective amount” will vary depending on the compound, the condition and its severity, the manner of administration, and the age of the mammal to be treated, but can be determined routinely by those skilled in the art having regard to his own knowledge and to this disclosure. “Treating” or “treatment” as used herein covers the treatment of the disease or condition of interest in a mammal, preferably a human, having the disease or condition of interest, and includes:

[0067] (i) preventing the disease or condition from occurring in a mammal, in particular, when such mammal is predisposed to the condition but has not yet been diagnosed as having it;

[0068] (ii) inhibiting the disease or condition, i.e., arresting its development;

[0069] (iii) relieving the disease or condition, i.e., causing regression of the disease or condition; or

[0070] (iv) relieving the symptoms resulting from the disease or condition, i.e., relieving pain without addressing the underlying disease or condition. As used herein, the terms “disease” and “condition” may be used interchangeably or may be different in that the particular malady or condition may not have a known causative agent (so that etiology has not yet been worked out) and it is therefore not yet recognized as a disease but only as an undesirable condition or syndrome, wherein a more or less specific set of symptoms have been identified by clinicians. The compounds of the invention, or their pharmaceutically acceptable salts may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids. The present invention is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

[0071] A “stereoisomer” refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. The present invention contemplates various stereoisomers and mixtures thereof and includes “enantiomers”, which refers to two stereoisomers whose molecules are nonsuperimposeable mirror images of one another.

[0072] A “tautomer” refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present invention includes tautomers of any said compounds.

[0073] Compounds

[0074] In an aspect, the invention provides novel ionic lipid compounds which are capable of combining with other lipid components such as neutral lipids, charged lipids, steroids and / or polymer conjugated-lipids to form lipid nanoparticles with oligonucleotides. Without wishing to be bound by theory, it is thought that these lipid nanoparticles shield oligonucleotides from degradation in the serum and provide for effective delivery of oligonucleotides to cells in vitro and in vivo.

[0075] In one embodiment, the compounds have the following structure (I): or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0076] X is a divalent or trivalent group derived from straight-chained or branched C1-20 alkane, C2-20 alkene, C2-20 alkyne, 3- to 20-membered aromatic ring system or 3- to 20-membered nonaromatic ring system, or any combination thereof, wherein any carbon in Ci -20 alkane, C2-20 alkene, C2-20 alkyne is optionally replaced with one or more atoms selected from the group consisting of S, O and N atoms, with the proviso that X is not piperazine;

[0077] Ir, 2r and 3r are the same or different, and are each independently integers ranging from 0 to 6; and

[0078] Rii , R12, R21, R22, R31 and R32 are the same or different, and are independently straight-chained

[0079] C8-C20 alkyl group, wherein a carbon atom in the alkyl group is optionally replaced with

[0080] Preferred embodiments of X, Ir, 2r, 3r Ru, R12, R21, R22, R31 and R32 are shown below.

[0081] Embodiments of the compound represented by the formula (I) are examples of all combinations of the specific embodiments shown below.

[0082] In some embodiments, X in formula (I) is a divalent or trivalent group derived from straight- chained or branched C 1-20 alkane, C2-20 alkene or C2-20 alkyne, wherein any carbon in C1-20 alkane, C2-20 alkene and C2-20 alkyne is optionally replaced with one or more atoms selected from the group consisting of S, O and N atoms (hereinafter referred to as X-l).

[0083] In some embodiments, X in formula (I) is a divalent or trivalent group derived from straight- chained or branched C1-20 alkane or C2-20 alkene, wherein any carbon in C1.20 alkane and C2-20 alkene is optionally replaced with one or more atoms selected from the group consisting of S, O and N atoms (hereinafter referred to as X-2).

[0084] In some embodiments, X in formula (I) is a divalent or trivalent group derived from straight- chained or branched C1-20 alkane or C2-20 alkene, wherein any carbon in C1-20 alkane and C2-20 alkene is optionally replaced with one or more atoms selected from the group consisting of S and O atoms (hereinafter referred to as X- 3). In some embodiments, X in formula (I) is a divalent or trivalent group derived from straight- chained or branched C1-10 alkane, C2-10 alkene or C2-10 alkyne, wherein any carbon in C1-10 alkane, C2-10 alkene and C2-10 alkyne is optionally replaced with one or more atoms selected from the group consisting of S, O and N atoms (hereinafter referred to as X-4).

[0085] In some embodiments, X in formula (I) is a divalent or trivalent group derived from straight- chained or branched CMO alkane or C2-10 alkene, wherein any carbon in C1-10 alkane and C2-10 alkene is optionally replaced with one or more atoms selected from the group consisting of S,

[0086] O and N atoms (hereinafter referred to as X-5).

[0087] In some embodiments, X in formula (I) is a divalent or trivalent group derived from straight- chained or branched Cr10 alkane or C2-10 alkene, wherein any carbon in Ci-io alkane and C2-10 alkene is optionally replaced with one or more atoms selected from the group consisting of S and O atoms (hereinafter referred to as X-6). in some embodiments, X in formula (I) is a divalent or trivalent group derived from straight- chained or branched CM alkane, C2-4 alkene or C2-4 alkyne, wherein any carbon in CM alkane, C2-4 alkene and CM alkyne is optionally replaced with one or more atoms selected from the group consisting of S, O and N atoms (hereinafter referred to as X-7).

[0088] In some embodiments, X in formula (I) is a divalent or trivalent group derived from straight- chained or branched Ci -4 alkane or C2-4 alkene, wherein any carbon in CM alkane and C2-4 alkene is optionally replaced with one or more atoms selected from the group consisting of S, O and N atoms (hereinafter referred to as X-8).

[0089] In some embodiments, X in formula (I) is a divalent or trivalent group derived from straight- chained or branched CM alkane or CM alkene, wherein any carbon in CM alkane and CM alkene is optionally replaced with one or more atoms selected from the group consisting of S and O (hereinafter referred to as X-9).

[0090] In some embodiments, X in formula (I) is 3-to 20-membered aromatic ring system or 3-to 20- membered non-aromatic ring system, with the proviso that X is not piperazine (hereinafter referred to as X- 10). In some embodiments, X in formula (I) is 3-to 10-membered aromatic ring system or 3-to 10- membered non-aromatic ring system, with the proviso that X is not piperazine (hereinafter referred to as X- 11).

[0091] In some embodiments, X in formula (I) is 6-to 10-membered aromatic ring system or 6-to 10- membered non-aromatic ring system, with the proviso that X is not piperazine (hereinafter referred to as X-12).

[0092] In some embodiments, X in formula (I) is a divalent or trivalent group derived from straight- chained or branched C1-20 alkane, C220 alkene or C2-20 alkyne, or any combination thereof, wherein any carbon in C1-20 alkane, C2-20 alkene and C2-20 alkyne is optionally replaced with one or more atoms selected from the group consisting of S, O and N atoms (hereinafter referred to as X-13).

[0093] In some embodiments, X in formula (I) is a divalent or trivalent group derived from straight- chained or branched Ct -20 alkane or C2-20 alkene, or any combination thereof, wherein any carbon in Cj .20 alkane and C2-20 alkene is optionally replaced with one or more atoms selected from the group consisting of S, O and N atoms (hereinafter referred to as X-14). in some embodiments, X in formula (I) is a divalent or trivalent group derived from straight- chained or branched C1-20 alkane or C2-20 alkene, or any combination thereof, wherein any carbon in Ci -20 alkane and C2-20 alkene is optionally replaced with one or more atoms selected from the group consisting of S and O atoms (hereinafter referred to as X-15).

[0094] In some embodiments, X in formula (I) is a divalent or trivalent group derived from straight- chained or branched C1-10 alkane, C2-10 alkene or C2-10 alkyne, or any combination thereof, wherein any carbon in C1-10 alkane, C2-10 alkene and C2-10 alkyne is optionally replaced with one or more atoms selected from the group consisting of S, O and N atoms (hereinafter referred to as X-16).

[0095] In some embodiments, X in formula (I) is a divalent or trivalent group derived from straight- chained or branched Ci-io alkane or C2-10 alkene, or any combination thereof, wherein any carbon in Ci-io alkane and C2-10 alkene is optionally replaced with one or more atoms selected from the group consisting of S, O and N atoms (hereinafter referred to as X-17). In some embodiments, X in formula (I) is a divalent or trivalent group derived from straight- chained or branched Cmo alkane or C2 10 alkene, or any combination thereof, wherein any carbon in Cr10 alkane and C2-10 alkene is optionally replaced with one or more atoms selected from the group consisting of S and O atoms (hereinafter referred to as X-18).

[0096] In some embodiments, X in formula (I) is a divalent or trivalent group derived from straight- chained or branched CM alkane, C2-4 alkene or C2-4 alkyne, or any combination thereof, wherein any carbon in Ci -4 alkane, CM alkene and C2-4 alkyne is optionally replaced with one or more atoms selected from the group consisting of S, O and N atoms (hereinafter referred to as X-19). in some embodiments, X in formula (I) is a divalent or trivalent group derived from straight- chained or branched CM alkane or C2-4 alkene, or any combination thereof, wherein any carbon in Ci -4 alkane and C2-4 alkene is optionally replaced with one or more atoms selected from the group consisting of S, O and N atoms (hereinafter referred to as X-20). in some embodiments, X in formula (I) is a divalent or trivalent group derived from straight- chained or branched CM alkane or C2-4 alkene, or any combination thereof, wherein any carbon in C1-4 alkane and C2-4 alkene is optionally replaced with one or more atoms selected from the group consisting of S and O (hereinafter referred to as X-21).

[0097] In some embodiments, X in formula (I) is 3-to 20-membered aromatic ring system or 3-to 20- membered non-aromatic ring system, or any combination thereof, with the proviso that X is not piperazine (hereinafter referred to as X-22).

[0098] In some embodiments, X in formula (I) is 3-to 10-membered aromatic ring system or 3-to 10- membered non-aromatic ring system, or any combination thereof, with the proviso that X is not piperazine (hereinafter referred to as X-23).

[0099] In some embodiments, X in formula (I) is 6-to 10-membered aromatic ring system or 6-to 10- membered non-aromatic ring system, or any combination thereof, with the proviso that X is not piperazine (hereinafter referred to as X-24). s" ' « " V

[0100] In some embodiments, X in formula (I) is selected from the group consisting of "

[0101] , (hereinafter referred to as X-25).

[0102] In some embodiments, Ir, 2r and 3r in formula (I) are each independently integers ranging from 0 to 4, (hereinafter referred to as r-1).

[0103] In some embodiments, Ir, 2r and 3r in formula (I) are each independently integers ranging from 0 to 2, (hereinafter referred to as r-2).

[0104] In some embodiments, Ir, 2r and 3r in formula (I) are 2, (hereinafter referred to as r-3).

[0105] In some embodiments, Ir, 2r and 3r in formula (I) are 1 , (hereinafter referred to as r-4).

[0106] In some embodiments, Ir, 2r and 3r in formula (I) are 0, (hereinafter referred to as r-5).

[0107] In some embodiments, Ri i, Rn, R21, R22, R31 and R32 in formula (I) are independently straight- chained C.8-C20 alkyl group, wherein a carbon atom in the alkyl group is optionally replaced with (hereinafter referred to as R-1).

[0108] In some embodiments, Ri 1, R12, R21, R22, R31 and R32 in formula (I) are independently straight- chained Cs-Ci8 alkyl group, wherein a carbon atom in the alkyl group is optionally replaced with (hereinafter referred to as R-2). In some embodiments, Rn, Rn, R21, R22, R31 and Rs2 in formula (I) are independently straight- chained C10-C16 alkyl group, wherein a carbon atom in the alkyl group is optionally replaced with ■" (hereinafter referred to as R-3).

[0109] In some embodiments, Rn, R12, R21, R22, R31 and R32 in formula (I) are independently selected from the group consisting of and

[0110] , (hereinafter referred to as R-4).

[0111] In some embodiments, the compounds represented by the formula (I) are the combinations of X-l, r-l and R-1.

[0112] In some embodiments, the compounds represented by the formula (I) are the combinations of X-l , r-2 and R-2.

[0113] In some embodiments, the compounds represented by the formula (I) are the combinations of X-2, r-3 and R-3.

[0114] In some embodiments, the compounds represented by the formula (I) are the combinations of X-3, r-4 and R-4.

[0115] In some embodiments, the compounds have the following structure (II):

[0116] (II). or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein X, Ir, 2r, 3r, Ru, Ru, R21, R22, R31 and R?,2 are as defined above. In various different embodiments, die compound has one of the structures set forth in Table 1 below. Unless otherwise specified, in the structure of specific embodiments, a -CJEbn+i group represents a straight-chained alkyl group, wherein n represents the number of carbon.

[0117] Table 1 Representative compounds

[0118] It is to be understood that any embodiment of the compounds of structure (I) or (II) as described above, as well as any particular substituents and / or variables in the compounds of structure (I) or (II) as described above, may be independently combined with other embodiments and / or substituents and / or variables of the compounds of structure (I) or (II) to form embodiments of the invention not explicitly set forth above.

[0119] It is understood that in the present specification, combinations of substituents and / or variables of the formulas described are permissible only if such contributions result in stable compounds.

[0120] In some embodiments, compositions are provided comprising any one or more of the compounds of structure (I) or (II) and a therapeutic agent. For example, in some embodiments, the composition comprises any of the compounds of structure (I) or (II) and a therapeutic agent and one or more excipients selected from the group consisting of neutral lipids, steroids, and polymer-conjugated lipids. Other pharmaceutically acceptable excipients and / or carriers are also included in various embodiments of the compositions.

[0121] In some embodiments, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DOPE or DSPC. In various embodiments, the molar ratio of compound to neutral lipid is from about 1 : 4 to about 3: 1.

[0122] In various embodiments, the composition further comprises a steroid or a steroid analog. In certain embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of compound to cholesterol is about 1: b to about b: 1.

[0123] In various embodiments, the polymer-conjugated lipid is a pegylated lipid. For example, some embodiments include a pegylated diacylglycerol (PEG-DAG) such as 1- (monomethoxypolyethylene glycol) -2, 3-dimyristoyl glycerol (PEG-DMG), a pegylated phosphatidylethanolamine (PEG-PE), a PEG succinic diacylglycerol (PEG-S-DAG) such as 4- O- (2 3’ -ditetradecanoyloxy) propyl-l-O- (m -methoxy (polyethoxy) ethyl) succinate (PEG- S-DMG), a pegylated ceramide (PEG-cer), or a PEG di alkoxy propylcarbamate such as ® - methoxy (polyethoxy) ethyl-N- (2, 3-ditetradecyloxy) propyl) carbamate or 2, 3- ditetradecyloxy propyl-N- (omega-methoxy (polyethoxy) ethyl) carbamate. In various embodiments, the molar ratio of compound to pegylated lipid is from about 100: 1 to about 10: 1.

[0124] In some embodiments of the foregoing composition, the therapeutic agent comprises a nucleic acid. For example, in some embodiments, the nucleic acid is selected from DNA, RNA, and hybrids thereof. In some embodiments, the nucleic acid is selected from antisense oligonucleotides, antisense RNA and messenger RNA.

[0125] In various embodiments, the present invention relates to a method of administering a therapeutic agent to a patient in need thereof, the method comprising preparing or providing any one of the above compositions and administering the composition to the patient.

[0126] In one embodiment, the present invention relates to a use of any one of the above compositions in the manufacture of a medicament for treating a disease.

[0127] In one embodiment, the present invention relates to any one of the above compositions, for use as a medicament for treating a disease.

[0128] In one embodiment, the present invention relates to a pharmaceutical composition for treating a disease, which comprises any one of the above compositions as therapeutically active substance.

[0129] For administration purposes, the compounds of the present invention (typically in the form of lipid nanoparticles associated with a therapeutic agent) may be administered as a crude chemical, or may be formulated as a pharmaceutical composition. The pharmaceutical compositions of the present invention comprise a compound of structure (I) or (II) and one or more pharmaceutically acceptable carriers, diluents, or excipients. The compound of structure (I) or (II) is present in the composition in an amount effective to form a lipid nanoparticle and deliver a therapeutic agent, e.g., for treating a particular disease or condition of interest. Appropriate concentrations and dosages can be readily determined by those skilled in the art.

[0130] Administration of the compositions of the present invention may be by any acceptable manner of administration of the agents for similar utility. The pharmaceutical composition of the present invention may be formulated into preparations in solid, semi-solid, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal routes. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. The pharmaceutical compositions of the present invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. The composition to be administered to a subject or patient is in the form of one or more dosage units, wherein, for example, a tablet may be a single dosage unit, while a container of a compound of the invention in aerosol form may contain a plurality of dosage units. Current methods of preparing such dosage forms are known or will be apparent to those skilled in the art; see, for example, Remington, The Science and Practice of Pharmacy, 20th edition (Philadelphia College of Pharmacy and Science, 2000). In any event, the composition to be administered will contain a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof, in order to treat the relevant disease or condition in accordance with the teachings of the present invention.

[0131] The pharmaceutical compositions of the present invention may be in solid or liquid form. In one aspect, the carrier is a microparticle such that the composition is, for example, in the form of a tablet or powder. The carrier can be a liquid, in which case the composition is, for example, an oral syrup, an injectable liquid, or an aerosol suitable, for example, for administration by inhalation.

[0132] When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, wherein forms considered herein to be solid or liquid include semi-solid, semiliquid. suspension, and gel forms.

[0133] As solid compositions for oral administration, the pharmaceutical compositions may be formulated into the form of powders, granules, compressed tablets, pills, capsules, chewing gums, wafers, and the like. Such solid compositions will typically contain one or more inert diluents or edible carriers. Additionally, one or more of the following may be present: a binder such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients, such as starch, lactose or dextrin; disintegrating agents, such as alginic acid, sodium alginate, Primogel, corn starch, and the like; lubricants, such as magnesium stearate or Sterotex; glidants, such as colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin; a flavoring agent, such as peppermint, methyl salicylate, or orange flavoring; and a colorant.

[0134] When the pharmaceutical composition is in the form of a capsule (e.g., a gelatin capsule), it may contain a liquid carrier other than the above-mentioned types of materials, for example, polyethylene glycol or an oil.

[0135] The pharmaceutical compositions may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. As two examples, the liquid may be for oral administration or for injection delivery. When intended for oral administration, preferred compositions contain, in addition to the compounds of the present invention, one or more of sweetening agents, preserving agents, coloring / colouring agents and taste-enhancing agents. In compositions for administration by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, and isotonic agents may be included.

[0136] The liquid pharmaceutical compositions of the present invention, whether in solution, suspension or other similar form, may include one or more of the following adjuvants: sterile diluents, such as water for injection, saline solution, preferably physiological saline, ringer’s solution, isotonic sodium chloride; non-volatile oils such as synthetic monoglycerides or diglycerides which may be used as a solvent or suspending medium, polyethylene glycols, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate; and agents for adjusting tonicity, such as sodium chloride or dextrose; agents used as cryoprotectants, such as sucrose or trehalose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.

[0137] Liquid pharmaceutical compositions of the invention intended for parenteral or oral administration should contain an amount of a compound of the invention that allows a suitable dosage to be obtained.

[0138] The pharmaceutical composition of the invention may be intended for topical administration, in which case the carrier may suitably comprise a solution base, an emulsion base, an ointment base or a gel base. For example, the matrix may comprise one or more of: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. The thickening agent may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may comprise a transdermal patch or an iontophoretic device.

[0139] The pharmaceutical compositions of the present invention may be intended for rectal administration, for example, in the form of suppositories, which dissolve in the rectum and release the drug. Compositions for rectal administration may contain an oleaginous base as a suita hie non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycols.

[0140] The pharmaceutical compositions of the present invention may include various materials that modify the physical form of the solid or liquid dosage unit. For example, the composition may include a material that forms an envelope around the active ingredient. The material forming the coating is generally inert and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

[0141] Pharmaceutical compositions of the invention in solid or liquid form may include agents that bind to the compounds of the invention and thereby facilitate delivery of the compounds. Suitable agents that can function with this capability include monoclonal or polyclonal antibodies or proteins.

[0142] The pharmaceutical compositions of the present invention may be comprised of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from systems of colloidal nature to systems consisting of pressurized packaging. The delivery may be by liquefied or compressed gas, or by a suitable pump system for dispensing the active ingredient. Aerosols of the compounds of the invention may be delivered as a single phase, biphasic system, or triphasic system for delivery of the active ingredient. The delivery of the aerosol includes the necessary containers, activators, valves, sub-containers, etc., which together may form a kit. Those skilled in the art can determine the preferred aerosol without undue experimentation.

[0143] The pharmaceutical compositions of the present invention may be prepared by methods well known in the pharmaceutical art. For example, a pharmaceutical composition intended for administration by injection may be prepared by combining the lipid nanoparticles of the present invention witlr sterile, distilled water or other carrier so as to form a solution. Surfactants may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that interact non-covalently with the compounds of the present invention in order to facilitate dissolution or uniform suspension of the compounds in an aqueous delivery system.

[0144] The compositions of the present invention, or pharmaceutically acceptable salts thereof, are administered in therapeutically effective amounts, which will vary depending on a variety of factors, inchiding the activity of the particular therapeutic agent employed; metabolic stability and length of action of the therapeutic agent; the age, weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; a pharmaceutical composition; the severity of the particular disorder or condition; and a subject undergoing treatment.

[0145] The compositions of the present invention may also be administered simultaneously with, prior to, or after the administration of one or more other therapeutic agents. Such combination therapies include die administration of a single pharmaceutical dosage formulation of a composition of the present invention and one or more additional active agents, as well as the administration of a composition of the present invention and each active agent in its own separate pharmaceutical dosage formulation. For example, the compositions of the present invention and the other active agent can be administered to the patient together in a single oral dosage composition (e.g., a tablet or capsule), or the individual agents can be administered in different oral dosage formulations. When different dosage formulations are used, the compound of the invention and one or more additional active agents can be administered at substantially the same time (i.e., simultaneously), or at staggered times (i.e., sequentially); it is to be understood that combination therapy encompasses all of these dosing regimens.

[0146] Synthetic method

[0147] Methods for preparing the above compounds and compositions are described below, and / or are known in the art.

[0148] Those skilled in the art will recognize that in the methods described herein, functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxyl, amino, mercapto and carboxylic acid. Non-limiting examples of amino protecting groups (hereinafter referred to as Pr-) are 2,4-dimethoxybenzyl (DMB), acetyl, trifluoroacetyl, t-butyloxycarbonyl (“Boc”), benzyloxycarbonyl (“CBz”) and 9-fluorenylmethyleneoxycarbonyl (“Fmoc”). Further examples of these groups, and other protecting groups, are found in T. W. Greene, et al. Greene’s Protective Groups in Organic

[0149] Synthesis. New York: Wiley Interscience, 2006.

[0150] Those skilled in the art will also recognize that while such protected derivatives of the compounds of the present invention may not be pharmacologically active thereby, they may be administered to a mammal and thereafter metabolized in vivo to form the compounds of the present invention which are pharmacologically active. Such derivatives may therefore be described as “prodrugs”. All prodrugs of the compounds of the present invention are included within the scope of the present invention.

[0151] Furthermore, all compounds of the invention in free base or free acid form can be converted into their pharmaceutically acceptable salts by treatment with a suitable inorganic or organic base or acid according to methods known to those skilled in the art. Salts of the compounds of the present invention may be converted to their free base or acid forms by standard techniques.

[0152] The following general reaction scheme 1 or F illustrates a method for preparing compounds of the present invention, i.e., compounds of structure (I) or (II) or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein X, Ir, 2r, 3r, Rii, R12, R21, R22, R31 and R32 are as defined herein.

[0153] The general synthetic routes are based on the below schemes. It is noted that the schemes listed below are exemplary and not limited. It is to be understood that any changes may be made in the schemes by those skilled in the art without departing from the scope of the present invention. The symbol V here is volume (mL) / starting material weight (g). Scheme 1:

[0154] Scheme 1 provides an exemplary method for preparation of compounds of structure (I), wherein X, Ir, 2r, R (representing Ru, R12, R21 or R22) are as defined herein, and Pr- group represents an amino protecting group. Preferably the Pr-group is Boe- group.

[0155] In step 1, under the presence of a catalyst, reactant D and reactant L undergo an esterification reaction to obtain compound DL.

[0156] The reactant ratio between D and L is from 1 :2 to 1:10, preferably 1:2 to 1 :8, and most preferably 1:2.

[0157] The reaction temperature is 10-40 'C, preferably 20-30°C, and more preferably 25 °C.

[0158] The reaction time is 8-20 hours, preferably 10-15 hours, and more preferably 12 hours.

[0159] The catalyst is preferably DMAP, and the amount of DMAP is 0.05-0.25eq, preferably 0.1 -

[0160] 0.2eq, and more preferably 0.1 eq.

[0161] EDCI is preferably added as an activating reagent for the carboxyl group in L, and the amount of EDCI is 1.0-5.0eq, preferably 2.0-4.0eq, and more preferably 2.6eq. The reaction solvent is preferably DCM in step 1, and the volume of the reaction solvent is 5- 30 V, preferably 5-20 V, and more preferably 10V.

[0162] In step 2, deprotect the amino protecting group Pr- in DL to obtain DL-l with amino group. The deprotection reactions of different amino protecting groups require different reagents. Preferably the Pr-group is Boc- group, and then preferably the deprotection reaction is occurred in the presence of HCl / dioxane (4M, 10V).

[0163] The reaction temperature is 10-30°C, and preferably 25°C.

[0164] The reaction time is 0.5-3 hours, preferably 0.5-2 hours, and more preferably 1 hour.

[0165] In step 3, in the presence of a catalyst, reactant DL-l undergoes alkylation reaction to obtain compound formula (I), wherein the alkylating agent is an olefin T ( , wherein R represents

[0166] Rii, Ri2, R?i or R22).

[0167] The reactant ratio between DL-l and T is from 1:4 to 1 :10, preferably 1:6 to 1:8, and more preferably 1:8.

[0168] The reaction temperature is 10-40 'C, preferably 20-30°C, and more preferably 25 °C.

[0169] The reaction time is 8-20 hours, preferably 10-15 hours, and more preferably 12 hours.

[0170] The catalyst is preferably NaBH(OAc)3, and the amount of the cataly st is 4.0-8.0eq, preferably 5.0-6.0eq and more preferably 6.0eq.

[0171] The reaction solvent is preferably DCM in step 3, and the volume of the reaction solvent is 5- 30 V, preferably 5-20 V, and more preferably 10V.

[0172] Scheme 1’:

[0173] Scheme 1 ’ provides an exemplary method for preparation of compounds of structure (II), wherein X, lr, 2r, 3r, R (representing Ru, R52, Rzi, R22, R31 or R32) are as defined herein, and Pr- group represents an amino protecting group. Preferably the Pr-group is Boc- group. Step 1

[0174] The reaction conditions of each step in Scheme T are similar to those of Scheme 1.

[0175] Scheme 2:

[0176] Step i

[0177] Step ii Some reactant T used in step 3 of Scheme 1 or T is prepared by the steps described in Scheme

[0178] In step i, under the presence of a catalyst, reactant T-a and reactant T-2 undergo an esterification reaction to obtain compound T-c.

[0179] The reactant ratio between T-a and T-2 is from 1:1 to 1:5, preferably 1: 1 to 1:3 and more preferably 1:1.

[0180] The reaction temperature is 10-40°C, preferably 20-30°C, and more preferably 0-25 °C.

[0181] The reaction time is 8-20 hours, preferably 10-15 hours, and more preferably 12.5 hours.

[0182] The catalyst is preferably DMAP, and the amount of the catalyst is 0.05 -1.0eq, preferably 0.05- 0.5eq and more preferably 0.1 eq.

[0183] EDCT is preferably added as an activating reagent for the carboxyl group in T-a, and the amount of EDO is 0.5-3. Oeq, preferably 1 .0-2. Oeq and more preferably 1.3eq.

[0184] The reaction solvent is preferably DCM in step i, and the volume of the reaction solvent is 5- 30 V. preferably 5-20 V, and more preferably 7V.

[0185] In step ii, in the presence of a base, the brominated T-C obtained in step i is heated in DMSO solvent to undergo an oxidation reaction to obtain the corresponding carbonyl compound T.

[0186] The base is preferably Naj-COs, and the amount of NaeCO? is 0.5-3.Oeq, preferably 1.0-2.0eq and more preferably l.Oeq.

[0187] The volume of DMSO is 1-20 V, preferably 1-10 V, and more preferably 6V.

[0188] The reaction temperature is 60-100°C, preferably 80-90°C, and more preferably 85°C.

[0189] The reaction time is 8-20 hours, preferably 10-15 hours, and more preferably 12 hours.

[0190] KI is preferably added in the reaction, and the amount of KI is 0.5-3. Oeq, preferably 1.0-2.0eq and more preferably l.Oeq. Scheme 3:

[0191] Step a

[0192] Some reactant D used in step 1 of Scheme 1 is prepared by the steps described in Scheme 3.

[0193] Compound D is prepared by reacting

[0194] K2CO3 is preferably added in the reaction, and the amount of K2CO3 is 2.0-8.0eq, preferably 2.0-6.0eq and more preferably 4.0eq.

[0195] KI is preferably added in the reaction, and the amount of KI is O.l-l.Oeq, preferably 0.2-0.5eq and more preferably 0.5eq.

[0196] The reaction temperature is 60-100°C, preferably 70-90°C, and more preferably 80°C.

[0197] The reaction time is 8-20 hours, preferably 10-15 hours, and more preferably 12 hours.

[0198] The reaction solvent is preferably THF in step a, and the volume of the reaction solvent is 5- 30 V. preferably 5-20 V, and more preferably 10V.

[0199] The following examples ate provided for die purpose of illustration and not limitation.

[0200] Example

[0201] Example 1, General procedure far preparation of DILI

[0202] D1L1

[0203] To a mixture of DI (1.00 g, 6.48 mmol, 1.00 eq) in DCM (10.0 niL) was added LI (3.02 g, 13.0 mmol, 2.00 eq), DMAP (79.2 mg, 648 pmol, 0.10 eq) and EDCI (3.23 g, 16.9 mmol, 2.60 eq) at 0 °C under N2. The mixture was stirred at 0 °C for 0.5 hr, then heated to 25 °C and stirred for 12 hours. TLC (Dichloromethane: Methanol= 10:1, product Rf =0.5) showed the starting material was consumed completely. The mixture was poured into H2O (10.0 ml) and the aqueous phase was extracted with DCM (2 x 5.00 mL). Hie organic phase was washed with IN HO (10.0 mL) and brine (10.0 mL). The organic phase was dried with NaiSCL and concentrated in vacuum. DILI (7.00 g, crude product) was obtained as colorless oil.

[0204] TLC:

[0205] SH NMR: DILI (400 MHz, CDCh)

[0206] 5: 4.93 (s, 2H), 4.37 (t, J = 6.4 Hz, 4H), 3.72 (t, J = 6.0 Hz, 4H), 3.51 (t, J = 5.2 Hz, 4H), 3.29 (s, 4H), 2.94 (t, J = 6.8 Hz, 4H), 2.60 (t, J = 6.0 Hz, 4H), 1.44 (s, 18 H).

[0207] General procedure for preparation of DI LI -1

[0208] To a mixture of DILI (7.00 g, 12.0 mmol, 1.00 eq) in dioxane (20.0 mL) was added HCl / dioxane (4 M, 35.0 mL, 11.2 eq) dropwise for 0.1 hr at 25 °C under N2. The mixture was stirred 25 °C for 2 hours. TLC (Dichloromethane: Ethyl acetate=l:l, product Rf =0) showed the starting material was consumed completely. The mixture was filtered and filtered cake was washed with dioxane (2 xlO.O mL). The solid was dried in vacuum. The product was used to next step without purification. D1L1-1 (5.45 g, 11.9 mmol, 99.5% yield, 2HC1) was obtained as a white solid.

[0209] TLC: General procedure for preparation of ICL-001

[0210] To a solution of D1L1-1 (0.50 g, 1.19 mmol, 1 .00 eq, 2HC1) in DCM (10.0 mL) was added T1 (1.48 g, 9.50 mmol, 1.79 mL, 8.00 eq) and NaBH(OAc)3 (1.51 g, 7.13 mmol, 6.00 eq) at 25 °C under N?.. The reaction mixture was stirred at 25°C for 12 hrs. LCMS showed the starting material was consumed completely. The mixture was poured into NaHCOs (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous NaeSCL, filtered and concentrated in vacuum. The mixture was further purification by pre-HPLC column: Welch Xtimate Cl 100 x 30mm x 5um; mobile phase: [HzO (0.1%TFA)-ACN: THF=1: 1]; gradient: 25%-60% B over 20.0 min to give ICL-001 (0.15 g, 158 pmol, 13.4% yield) as yellow oil.

[0211] LCMS: ICL-001: LCMS (product: RT 1.651 mm; [M / 2+1] T 473.5)

[0212] Tl NMR: ICL-001 (400 MHz, CDCL)

[0213] 8: 4.36 (t, J = 6.4 Hz, 4H), 3.73 (t, J = 6.4 Hz, 4H), 3.59 (s, 4H), 2.93 (t, J = 6.8 Hz, 4H), 2.74

[0214] (s, 4H), 2.60 (t, J = 6.0 Hz, 8H), 1.47 (s, 8H), 1.27 (s, 60H), 0.87 (t, J = 7.2 Hz, 12H).

[0215] Example 2. General procedure far preparation o / ICL-002

[0216] To a mixture of D1L1-1 (1.00 g, 2.19 mmol, 1 .00 eq, 2HC1) in DCM (10.0 mL) was added T?. (3.22 g, 17.5 mmol, 8.00 eq) and NaBH(OAc)3 (2.78 g, 13. 1 mmol, 6.00 eq) at 25 °C under Na. The mixture was stirred 25 °C for 12 hours. LCMS showed the starting material was consumed completely. The mixture was poured into NaHCO? (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous NaaSQt, filtered and concentrated in vacuum. The mixture was purified by silica gel chromatography (Dichloromethane: Methanol =60 / 1 30 / 1) and further purified by pre-HPLC (column: X-Select CSH Phenyl-Hexyl 100*30 5u; mobile phase: [H2O (0.04%HCI)-ACN]; gradient: 25%-60% B over 12.0 min) to give ICL- 002 (0.30 g, 317 pmol, 14.5%' yield) as yellow oil.

[0217] TLC:

[0218] LCMS: ICL-002:

[0219] LCMS (product: RT - 1.515 min; [M / 2+lj+: 529.5) -002 (400 MHz, CDCh)

[0220] 5: 4.09 (t, J = 6.4 Hz, 4H), 3.72 (t, J = 6.0 Hz, 4H), 3.53 (s, 4H), 2.64 (s, 4H), 2.58 (t, J = 6.4 Hz, 4H), 2.44 (s, 8H), 1.64 (s, 4H), 1.40 (t, J = 8.0 Hz, 12H), 1 .27 (s, 68H), 0.89 (t, J = 5.6 Hz, 12H).

[0221] Example 3. General procedure for preparation o / ICL-003

[0222] To a mixture of D1L1-1 (1.00 g, 2.19 mmol, 1 .00 eq, 2HC1) in DCM (10.0 mL) was added T3 (3.71 g, 17.5 mmol, 8.00 eq) and NaBH(OAc)3 (2.78 g, 13. 1 mmol, 6.00 eq) at 25 °C under N2. The mixture was stirred 2.5 °C for 12. hours. LCMS (ET79403-17-P1A1, product RT ~ 1.913 min) showed the starting material was consumed completely. The mixture was poured into NaHCCh (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The mixture was purified by silica gel chromatography (Dichloromethane: Methanol =60 / 1 30 / 1) and further purified by pre- HPLC (column: X-Select CSH Phenyl-Hexyl 100*30 5u: mobile phase: [H2O (0.04%HCl)- THF: CAN =1:3]; gradient: 45%-70% B over 8.0 min).to give ICL-003 (0.26 g, 222 pmol, 10.2% yield) as yellow oil. TLC:

[0223] LCMS: ICL-003:

[0224] LCMS (product: RT = 1.913 min; [ M / 2+1 ]T 585.6)

[0225] D2 021.1

[0226] To a mixture of D2 (1.00 g, 8.46 mmol, 1.01 mL, 1.00 eq) in DCM (10.0 mL) was added LI (3.95 g, 16.9 mmol, 2.00 eq), DMAP (103 mg, 846 qmol, 0.10 eq) and EDCI (4.22 g, 22.0 mmol, 2.60 eq) at 0 °C under N2. The mixture was stirred at 0 °C for 0.5 hr, then heated to 25 °C and stirred for 12 hours. TLC (Dichloromethane: Methanol- 10: 1, product Rf -0.5) showed the starting material was consumed completely. The mixture was poured into H2O (10.0 mL) and the aqueous phase was extracted with DCM (2 x 5.00 mL). The organic phase was washed with IN HC1 (10.0 ml..) and brine (10.0 mL). The organic phase was dried with Na2SO4 and concentrated in vacuum. D2L1 (7.00 g, crude) was obtained as colorless oil.

[0227] TLC: Hl NMR: D2L1 (400 MHz, CDCI3)

[0228] 5: 3.93 (d, J = 6.4 Hz, 4H), 3.71 (t, J = 6.4 Hz, 4H) 3.50 (t, J = 5.2 Hz, 4H) 3.30 (t, J = 5.2 Hz, 4H) 2.57 (t, J = 6.4 Hz, 4H) 1 .81 (d, J = 6.8 Hz, 4H), 1 .62 (s, 2H), 1.44 (s, 18H), 1.01 (dt, J = 8.8 Hz, 4H)

[0229] General procedure for preparation of D2L1-1

[0230] To a mixture of DILI (7.00 g, 12.8 mmol, 1.00 eq) in dioxane (20.0 mL) was added HCl / dioxane (4 M, 36.7 mL, 11.5 eq) dropwise for 0.1 hr at 25 °C under N2 The mixture was stirred 25 °C for 2 hours. TLC (Dichloromethane: Ethyl acetate=l:l, product Rf =0) showed the starting material was consumed completely. The mixture was filtered and filtered cake was washed with dioxane (2 x 10.0 mL). The solid was dried in vacuum. Hie product was used to next step without purification. D2L1-1 (5.00 g, 11.9 mmol, 93.0% yield, 2HC1) was obtained as a white solid.

[0231] General procedure for preparation o / ICL-004

[0232] To a solution of D2L1-1 (0.50 g, 1.19 mmol, LOO eq, 2HC1) in DCM (10.0 mL) was added T1 (1.62 g, 10.4 mmol, 1.96 mL, 8.00 eq) and NaBH(OAc)s (1.65 g, 7.79 mmol, 6.00 eq) at 25°C under N2 The reaction mixture was stirred at 25°C for 12 hrs. LCMS showed the starting material was consumed completely. The mixture was poured into NaHCCh (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. Tiie mixture was purified by silica gel chromatography (Dichloromethane: Methanol =60 / 1 30 / 1) and further purified by pre-HPLC column: X-Select CSH Phenyl-Hexyl 100 x 30 5u; mobile phase: [H2O (0.04%HCl)-ACNJ; gradient: 30%-65% B over 12.0 min to give ICL-004 (0.30 g, 330 pmol, 25.4% yield) as yellow oil.

[0233] TLC:

[0234] LCMS: ICL-004:

[0235] LCMS (product: RT - 0.886 min; [M / 2+1] *: 455.5)

[0236] SH NMR: ICL-004 (400 MHz, CDCb)

[0237] 5: 4.35 (t, J = 4.4 Hz, 4H), 3.71 (t, J = 4.8 Hz, 4H), 3.53 (s, 4H), 2.92 (t, J = 4.8 Hz, 4H), 2.60 (q, J 6.0 Hz 6H), 2.44 (s, 6H), 1 .42 (s, 8H), 1 .26 (s, 64H), 0.88 (t, J = 4.4 Hz, 12H).

[0238] Example 5. General procedure for preparation o / ICL-005

[0239] To a solution of D2L1-1 (0.50 g, 1.19 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T2 (1 .92 g, 10.4 mmol, 8.00 eq) and NaBH(OAc)a (1 .65 g, 7.79 mmol, 6.00 eq) at 25 °C under N2. The reaction mixture was stirred at 25°C for 12 hrs. TLC (Dichloromethane: Methanol-10: 1 , product Rf = 0.5) showed the reaction was completed. The mixture was poured into NaHCOs (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous NazSCfi, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (Dichloromethane / Methanol-60 / 1, 20 / 1) to give ICL-005 (0.30 g, 294 pmol, 22.6% yield) as yellow oil. TLC:

[0240] 3H NMR: ICL-005 (400 MHz, CDCL)

[0241] 5: 4.09 (t, J = 6.8 Hz, 4H), 3.72 (t, J = 6.4 Hz, 4H), 3.52 (t, J = 6.4 Hz, 4H), 2.64 (t, J = 6.0 Hz, 4H), 2.58 (t, J = 6.4 Hz, 4H), 2.44 (t, J 7.2 Hz, 8H), 1.64 (t, J = 6.4 Hz, 4H), 1.36-1.42 (in, 12H), 1.26 (s, 74H), 0.89 (1, J = 6.4 Hz, 12H).

[0242] Example 6, General proeedure far preparation of ICL-006

[0243] To a mixture of D2L1-1 (0.50 g, 1.19 mmol, 1 .00 eq, 2HC1) in DCM (10.0 mL) was added ’13 (4.88 g, 23.0 mmol, 8.00 eq) and NaBH(OAc)s (3.65 g, 17.2 mmol, 6.00 eq) at 25°C under Na. The mixture was stirred 25 °C for 12 hours. LCMS showed the starting material was consumed completely. The mixture was poured into NaHCOs (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous NarSCL, filtered and concentrated in vacuum. The crude product was purified by silica gel chromatography (Dichloromethane / Methanol=60 / 1, 20 / 1) and futher purified reversed -phase by HPLC (column: Welch Utimate C30 5um*30* 100mm; mobile phase: [H2O (0.1%TFA)-ACN: THF===1: 1]; gradient: 35%-75% B over 12.0 min) to give ICL-006 (0.14 g, 123 jxmol, 4.30% yield) as yellow oil.

[0244] LCMS: ICL-006:

[0245] LCMS (product: RT = 1.962 min; [M / 2+l]+: 56 / .6)

[0246] 1H NMR: ICL-006 (400 MHz, CDCL)

[0247] 5: 4.09 (dt, J = 6.4 Hz, 4H), 3.72 (dt, J = 6.0 Hz, 4H), 3.57 (s, 4H), 2.71 (s, 4H), 2.58 (dt, J = 6.2 Hz, 4H), 2.51 (s, 4H), 1.65 (s, 4H), 1.39-1.46 (m, 12H), 1.27 (s, 92H), 0.87-0.95 (m, 12H). Example 7. General procedure for preparation of ICL-007

[0248] To a mixture of D2L1-1 (0.50 g, 1 .19 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T4 (.1.63 g, 7.12 mmol, 6.00 eq) and NaBH(OAc)s (1.26 g, 5.93 mmol, 5.00 eq) at 25°C under Na. The mixture was stirred 25 °C for 12 hours. LCMS showed the starting material was consumed completely. The mixture was poured into NaHCCL (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous NajSCL, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (Dichloromethane: Methanol=60 / 1, 20 / 1) to afford ICL-007 (0.10 g, 83.5 pmol, 7.04% yield) as yellow' oil.

[0249] LCMS: ICL-007:

[0250] LCMS (product: RT - 2.026 min; [M / 2+ 1 j+: 599.5)

[0251] To a mixture of D3 (0.50 g, 5.55 mmol, 493 pL, 1.00 eq) in DCM (10.0 mL) was added LI (2.59 g, 11.1 mmol, 2.00 eq), DMAP (67.8 mg, 555 pmol, 0.10 eq) and EDCI (2.77 g, 14.4 mmol, 2.60 eq) at 0 °C under N?. The mixture was stirred at 0 °C for 0.5 hr, then heated to 25 °C and stirred for 12 hours. TLC (Dichloromethane: Methanol-10:l, product Rf =0.5) showed the starting material was consumed completely. The mixture was poured into PLO (10.0 mL) and the aqueous phase was extracted with DCM (2 x 5.00 mL). The organic phase was washed with IN HQ (10.0 mL) and brine (10.0 mL). The organic phase was dried with NazSOb and concentrated in vacuum. D3L1 (3.50 g, crude product) was obtained as colorless oil.

[0252] TLC:

[0253] General procedure for preparation ofD3Ll-l

[0254] To a mixture of D3L1 (3.50 g, 6.33 mmol, LOO eq) in dioxane (10.0 mL) was added HCl / dioxane (4 M, 15.0 mL, 9.47 eq) dropwise for 0.1 hr at 25°C under Na- The mixture was stirred 25 °C for 2 hours. TLC (Dichloromethane: Ethyl acetate=l:l, product Rf =0) showed the starting material was consumed completely. The mixture was concentrated in vacuum. The product was used to next step without purification. D3L1-1 (2.50 g, 5.88 mmol, 92.8% yield, 2HC1) was obtained as yellow oil.

[0255] TLC:

[0256] General procedure for preparation o / TCL-008 To a solution of D3L1-1 (0.50 g, 1.18 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T1 (1.47 g, 9.41 mmol, 1.77 mL, 8.00 eq) and NaBH(OAc)3 (1.49 g, 7.05 mmol, 6.00 eq) at 25 °C under N2. The reaction mixture was stirred at 25°C for 12 hrs. LCMS showed the starting material was consumed completely. The mixture was poured into NaHCCh (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous Na2SC>4, filtered and concentrated in vacuum. The crude product was purified by normal phase HPLC (column: Ageia DuraShell NH3 150mm*30mm*5um; mobile phase: [Heptane- ACN: THF=1 : 1J; gradient: 0%-30% B over 10.0 min) to give ICL-008 (0.30 g, 340 pmol, 42.9% yield) as yellow oil.

[0257] LCMS: ICL-008:

[0258] LCMS (product: RT = 1.688 min: [M / 2-t-l]+: 441.5)

[0259] 1H NMR: ICL-008 (400 MHz, CDCh) g:4.02 - 4.05 (m, 4H), 3.72 (t, J = 6.4 Hz, 4H), 3.54 (s, 3H), 2.58 ■ 2.66 (m, 8H), 2.46 (s, 6H), 1.65 (s, 6H), 1.44 (s, 8H), 1.31 (s, 54H), 0.10 (d, J = 6.8 Hz, 3H), 0.89 (t, J = 6.8 Hz, 12H).

[0260] Example 9. General procedure for preparation ofICL-009

[0261] To a solution of D3L1-1 (0.50 g, 1.18 mmol, 1.00 eq, 2HC1) in DCM (10.0 ml) was added T2 (1.73 g, 9.41 mmol, 8.00 eq) and NaBH(OAc)s (1.49 g, 7.05 mmol, 6.00 eq) at 25°C under N? The reaction mixture was stirred at 25 °C for 12 hrs. LCMS showed the starting material was consumed completely. The mixture was poured into NaHCCh (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The crude product was purified by reversed-phase HPLC (column: Welch Xtimate Cl 250*50mm* 10um; mobile phase: [H2O (0.1%TFA)-ACN: THF=1: 1]; gradient: 30%-70% B over 20.0 min) to give ICL-009 (0.30 g, 293 pmol, 24.9% yield) as yellow oil. LCMS: ICL-009:

[0262] LCMS (product: RT = 1.620 min: [M / 2-t-l] L 497.5)

[0263] 3H NMR: ICL-009 (400 MHz, CDCh)

[0264] 8: 4.03 - 4.05 (m, 4H), 3.72 (t, J = 6.4 Hz, 4H), 3.54 (s, 3H). 2.58 - 2.66 (m, 8H), 2.46 (s, 6H), 1 .65 (s, 6H), 1.44 (s, 8H), 1.31 (s, 54H), 0.99 (d, J = 6.8 Hz, 3H), 0.89 (t, J = 6.8 Hz, 12H).

[0265] To a solution of D3L1-1 (0.50 g, 1.18 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T3 (2.00 g, 9.41 mmol, 8.00 eq) and NaBH(OAc)s (1.49 g, 7.05 mmol, 6.00 eq) at 25 °C under N? The reaction mixture was stirred at 25 °C for 12 hrs. LCMS showed the starting material was consumed completely. The mixture was poured into NaHCCh (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (Dichloromethane: Methanol=60 / 1, 20 / 1) to afford product as yellow solid, to give ICL-010 (0.20 g, 176 pmol, 66.7% yield) as yellow oil.

[0266] LCMS: ICL-010:

[0267] LCMS (product: RT = 2.104 min; [M / 2+1]+: 553.6)

[0268] SH NMR: ICL-010 (400 MHz, CDCh)

[0269] 6: 3.94 - 3.99 (m, 4H), 3.64 (t, J - 6.4 Hz, 4H), 3.46 (s, 3H), 2.50 - 2.57 (m, 8H), 2.38 (s, 6H), 1.26 (s, 8H), 1.23 (s, 92H), 0.92 (d, 6.8 Hz, 3H), 0.81 (t, 6.8 Hz, 12H). Example II. General procedure for preparation o / D4Ll

[0270] D4 D4L1

[0271] To a mixture of D4 (0.50 g, 5.68 mmol, 463 pL, 1.00 eq) in DCM (10.0 mL) was added LI (2.65 g, 11.4 mmol, 2.00 eq), DMA? (69.3 mg, 568 nmol, 0.10 eq) and EDCI (2.83 g, 14.8 mmol, 2.60 eq) at 0 °C under N?.. The mixture was stirred at 0 °C for 0.5 hr, then heated to 25 °C and stirred for 12 hours. TLC (Dichloromethane: Methanol- 10: 1, product Rf =0.5) showed the starting material was consumed completely. The mixture was poured into H2O (10.0 mL) and the aqueous phase was extracted with DCM (2 x 5.00 mL). The organic phase was washed with IN HC1 (10.0 mL) and brine (10.0 mL). The organic phase was dried with NazSCLi and concentrated in vacuum. D4L1 (3.50 g, crude product) was obtained as colorless oil.

[0272] TLC:

[0273] To a mixture of D4L1 (3.50 g, 6.36 mmol, 1.00 eq) in dioxane (10.0 mL) was added HCl / dioxane (4 M, 15.0 mL, 9.44 eq) dropwise for 0.1 hr at 25°C under Ng. The mixture was stirred 25 °C for 2 hours. TLC (Dichloromethane: Ethyl acetate=l:l, product Rf =0) showed the starting material was consumed completely. The mixture was concentrated in vacuum. The product was used to next step without purification. D4L1-1 (2.10 g, 5.99 mmol, 94.3% yield, 2HC1 ) was obtained as yellow oil. TLC:

[0274] General procedure far preparation of ICL-011

[0275] To a solution of D4L1-1 (0.50 g, 1.18 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T1 (1.48 g, 9.45 mmol, 1.78 mL, 8.00 eq) and NaBH(OAc)3 (1.50 g, 7.09 mmol, 6.00 eq) at 25°C under Na. The reaction mixture was stirred at 25 °C for 12 hrs. LCMS showed the starting material was consumed completely. The mixture was poured into NaHCCh (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous Naj-SCL, filtered and concentrated in vacuum. The crude product was purified by reversed-phase HPLC (column: Welch Xtimate Cl 250 x 50mm x lOum; mobile phase: [HzO (0.1%TFA)-ACN: THF=1: 1]; gradient: 20%-65% B over 20.0 min) to give ICL-011 (0.10 g, 114 irniol, 6.67% yield) as yellow oil.

[0276] LCMS: ICL-011:

[0277] LCMS (product: RT ~ 2.1 10 min; [M / 2+1]+: 553.6)

[0278] 1H NMR: ICL-011 (400 MHz, CDCh)

[0279] 5: 4.63 (s, 3H), 3.73 (t, 7 = 6.8 Hz, 3H), 3.65 (t, .7 = 6.8 Hz, 3H), 3.53 (t, J = 6.4 Hz, 3H), 2.62 (t, J = 6.4 Hz, 6H), 2.44 (t, J = 7.2 Hz, 5H), 1.44 - 1.57 (m, 3H), 1 .41 - 1 .42 (m, 8H), 1.32 (s, 60H), 0.89 (1, J = 6.8 Hz, 12H). Example 12. General procedure for preparation 0 / ICL-012

[0280] To a solution of D4L1-1 (0.50 g, 1.18 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T2 (1.74 g, 9.45 mmol, 8.00 eq) and NaBH(OAc)3 (1.50 g, 7.09 mmol, 6.00 eq) at 25 °C under N2. The reaction mixture was stirred at 25°C for 12 hrs. LCMS showed the starting material was consumed completely. The mixture was poured into NaHCOs (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous NajSCL, filtered and concentrated in vacuum. The crude product was purified by normal phase HPLC (column: Agela DuraSheli NH2 150mm x 30mm x 5um; mobile phase: [Heptane -A CN: THF::::1 : !]; gradient : 0%-30% B over 10.0 min) to give ICL-012 (0.06 g, 60.5 pmol, 8.57% yield) as yellow oil.

[0281] LCMS: ICL-012:

[0282] LCMS (product: RT = 1.591 min; [M / 2+1]+: 497.5)

[0283] SH NMR: ICL-012 (400 MHz, CDCh)

[0284] 5: 5.85 - 5.87 (m, 2H), 4.61 - 4.62 (m, 4H), 3.62 - 3.75 (m, 4H), 3.55 (s, 3H), 2.46 - 2.63 (m, 5H), 2.45 (s, 5H), 1.45 - 1.66 (m, 17H), 1.34 (s, 70H), 0.89 (t, J = 6.8 Hz, 12H).

[0285] Example 13. General procedure for preparation o / ICL-013

[0286] To a solution of D4L1-1 (0.50 g, 1.18 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T3 (2.01 g, 9.45 mmol, 8.00 eq) and NaBH(OAc)3 (1.50 g, 7.09 mmol, 6.00 eq) at 25°C under N2. The reaction mixture was stirred at 25°C for 12 hrs. LCMS showed the starting material was consumed completely. The mixture was poured into NaHCOr (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous NazSCh, filtered and concentrated in vacuum. The crude product was purified by normal phase HPLC (column: Agela DuraShell NH2 150mm x 30mm x 5um; mobile phase: [Heptane -ACN: THF::::1:1]; gradient : C'%-30%1B over 10.0 min) to give ICL-013 (0.15 g, 90.4% purity) as yellow oil.

[0287] LCMS ICL-013:

[0288] LCMS (product: RT = 2.078 min; [M / 2+1]+: 552.6)

[0289] XH NMR: ICL-013 (400 MHz, CDCI3)

[0290] 5: 4.63 (s, 3H), 3.73 (t, J= 6.4 Hz, 3H), 3.56 (s, 3H), 2.60 - 2.69 (m, 3H), 2.49 (s, 6H), 1.45 (s, 8H), 1.27 (s, 97H), 0.89 (t, J = 6.8 Hz, 12H).

[0291] Example 14. General procedure for preparation 0 / ICL-014

[0292] To a solution of D4L1-1 (0.50 g, 1.18 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T4 (1.75 g, 7.67 mmol, 6.00 eq) and NaBH(OAc)s (1.35 g, 6.39 mmol, 5.00 eq) at 25°C under Na. The reaction mixture was stirred at 25°C for 12 hrs. LCMS showed the starting material was consumed completely. The mixture was poured into NaHCOs (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous Na2SCl1, filtered and concentrated in vacuum. The crude product was purified by normal phase HPLC (column: Agela DuraShell NH2 150mm x 30mm x Sum; mobile phase: [Heptane -ACN: THF== 1:1]; gradient: 0%-30% B over 10.0 min) to give ICL-014 (0.30 g, 257 pmol, 20.1% yield) as yellow oil.

[0293] LCMS: ICL-014:

[0294] LCMS (product: RT = 1.389 min; [M / 2+1]+: 584.5)

[0295] SH NMR: ICL-014 (400 MHz, CDCb) 6: 5.20 (s, 2H), 4.56 (s, 4H), 3.99 (t, J= 6.8 Hz, 8H), 3.65 (t, J = 6.8 Hz, 4H), 3.44 - 3.45 (m, 4H), 2.54 (t, / = 6.8 Hz, 8H), 2.24 (s, 6H), 2.22 (t, 7 = 7.6 Hz, 8H), 1.53 - 1.56 (m, 18H), 1.25 - 1.27 (m, 7H), 1.20 - 1.24 (m, 40H), 0.82 (t, 6.8 Hz, 12H).

[0296] Example 15. General procedure far preparation o / D5Ll

[0297] D5 D$L1

[0298] To a solution of D5 (0.50 g, 4.23 mmol, 513 pL, 1.00 eq) in DCM (10.0 mL) was added LI (1.97 g, 8.46 mmol, 2.00 eq), DMAP (51.7 mg, 423 pmol, 0.10 eq) and EDCI (2.11 g, 11.0 mmol, 2.60 eq) at 0 °C. The mixture was stirred at 0 °C for 0.5 hrs and then stirred at 25 °C for 12. hrs. 1..CMS showed the reaction was completed. The mixture was poured into water (10.0 mL) and stirred for 10 mins. The aqueous phase was extracted with DCM (3.00 mL x 2). The combined organic phase was washed with IN HC1 (10.0 mL) and brine (10.0 ml..), dried with anhydrous NajSCL, filtered and concentrated in vacuum. D5L1 (2.00 g, 3.65 mmol, 86.2% yield) was obtained as colorless oil.

[0299] General procedure far preparation o / D5Ll-l

[0300] A solution of D5L1 (2.00 g, 3.65 mmol, 1 .00 eq) in HCl / dioxane (4 M, 20.0 mL, 22.0 eq) was stirred at 25 °C for 2 hrs. TLC (Di chloromethane: Ethyl acetate=l:l, Rf - 0.08) showed the reaction was completed. The mixture was concentrated in vacuum to get the crude product. D5L1-1 (1.50 g, 3.56 mmol, 97.7% yield, 2HC1) was obtained as colorless oil.

[0301] TLC: Hl NMR: D5L1-1 (400 MHz, MeOD)

[0302] 5: 3.75 - 3.88 (m, 3H), 3.43 (t, J=6 Hz, 4H), 3.32 - 3.37 (m, 4H), 2.96 (m, 1H), 2.78 (t, J=4.8

[0303] Hz, 4H), 2.27 - 2.33 (m, 3H), 1.29 - 1.42 (m, 3H), 1.10 - 1.23 (m, 2H), 0.56 - 0.66 (m, 3H)

[0304] General procedure for preparation of ICL-015

[0305] To a solution of D5L1-1 (0.50 g, 1.19 mmol, 1.00 eq, 2.HC1) in DCM (5.00 mL) was added T1 (1.48 g, 9.49 mmol, 1.79 mL, 8.00 eq) and NaBHiOAcp (1.51 g, 7.12 mmol, 6.00 eq). The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The mixture was concentrated in vacuum. The residue was diluted with water (50.0 mL) and extracted with n-heptane (10.0 mL x 2). The combined organic layers were washed with brine (20.0 mL), dried over anhydrous Na2SCl1, filtered and concentrated in vacuum. The residue was purified by prep-HPLC (column: X-Select CSH Phenyl-Hexyl 100*30 5u; mobile phase: [H2O (0.04%HCl)-THF: ACX i n i. gradient: 15%-50% B over 12.0 min). ICL-015 (0.18 g, 198 pmol, 16.7% yield) was obtained as yellow oil.sH NMR: ICL-015 (400 MHz, CDCh)

[0306] 5: 4.14 (d, 7 = 6 Hz, 3H), 3.62 - 3.75 (m, 5H), 3.56 (s, 3H), 2.63 - 2.73 (m, 3H), 2.39 - 2.61 (m, 101-1), 1.63 1.76 (m, 3H), 1.39 ■ 1.62 (m, 11H), 1.22 - 1.32 (m, 56H), 0.96 (d, 7= 6.4 Hz, 3H), 0.89 (t, J = 6.8 Hz, 12H).

[0307] Example 16. General procedure for preparation o / ICL-016

[0308] To a solution of D5L1-1 (0.50 g, 1.19 mmol, 1.00 eq, 2HC1) in DCM (5.00 mL) was added T2 (1.75 g, 9.49 mmol, 8.00 eq) and NaBH(OAc)3 (1.51 g, 7.12 mmol, 6.00 eq). The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The mixture was concentrated in vacuum. The residue was diluted with water (50.0 mL) and extracted with n- heptane (10.0 mL x 2). The combined organic layers were washed with brine (20.0 mL), dried over anhydrous NajSCL, filtered and concentrated in vacuum. The residue was purified by prep-HPLC (column: Welch Xtimate Cl 250*50mm* 10um; mobile phase: [H2O (0.1%TFA)~

[0309] ACN: THF 1 :!]; gradient: 28%-68% B over 20.0 min)

[0310] ICL-016 (0.20 g, 196 pmol, 16.5% yield) was obtained as yellow oil.

[0311] 3H NMR: ICL-016 (400 MHz, CDCh)

[0312] 8: 4.07 - 4.20 (m, 4H), 3.72 (t, J = 6.4 Hz. 4H), 3.51 - 3.66 (m, 4H), 2.67 - 2.90 (m, 4H), 2.57 (t, J = 6.8 Hz, 8H), 1.61 - 1.77 (m, 4H), 1.40 - 1.58 (m, 10H), 1.27 (s, 68H), 0.96 (d, J = 6.4 Hz, 3H), 0.85 - 0.92 (m, 12H).

[0313] Example 17. General procedure for preparation 0 / ICL-017

[0314] To a solution of D5L1-1 (0.50 g, 1.19 mmol, 1.00 eq, 2HC1) in DCM (5.00 mL) was added T3 (2.02 g, 9.49 mmol, 8.00 eq) and NaBH(OAc)3 (1.51 g, 7.12 mmol, 6.00 eq) at 25 °C for 12 hrs. HPLC showed the reaction was completed and 56.9% of product was detected. The mixture was concentrated in vacuum. The residue was diluted with water 50 mL and extracted with n-heptane (10.0 mL x 2). The combined organic layers were washed with brine (20.0 mL), dried over anhydrous NasSCL, filtered and concentrated in vacuum. The residue was purified by prep-HPLC (column: Xselect CSH C18 100*30mm*5um; mobile phase: [H2O (0.1 %TFA)- ACN: THF=1: 1]; gradient: 40%-80% B over 12.0 min). ICL-017 (200 mg, 127 umol, 10.7% yield, 71.8% purity) was obtained as yellow oil.

[0315] 3H NMR: ICL-017 (400 MHz, CDCh)

[0316] 8: 4.07 - 4.20 (m, 4H), 3.72 (t, J = 6.4 Hz, 4H), 3.43 - 3.64 (m, 4H), 2.61 - 2.76 (m, 4H), 2.57 (t, .7 - 6.8 Hz, 5H), 2.46 (s, 4H), 1.65 - 1.72 (m, 5H), 1.41 - 1.52 (m, 10H), 1.27 (s, 90H), 0.96 (m, 3H), 0.86 - 0.92 (m, 12H). Example 18. General procedure far preparation of ICL-018

[0317] To a solution of D5L1-1 (0.50 g, 1.19 mmol, 1.00 eq, 2HC1) in DCM (5.00 mL) was added T4 (1.63 g, 7.12 mmol, 6.00 eq) and NaBH(OAc)3(1.26 g, 5.93 mmol, 5.00 eq) at 0 °C. The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. Hie mixture was poured into NaHCO3 (10.0 mL) and stirred for 15 mins. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The crude product was purified by normal phase HPLC (column: Agela DuraShell NHZ 150mm x 30mm x 5um; mobile phase: [Heptane- ACN: THF=1 :1]; gradient: 0%-30% B over 8.0 min). ICL-018 (0.40 g, 259 pmol, 21.8% yield, 77.6% purity) was obtained as yellow oil. -018 (400 MHz, CDCh)

[0318] 6: 4.09 - 4.19 (m, 4H), 4.06 (t, J 6.8 Hz, 8H ). 3.71 (t, J = 6.4 Hz, 4H), 3.54 (s, 4H), 2.57 (t, J = 6.4 Hz, 8H), 2.46 (s, 6H), 2.30 (t, 8 Hz, 8H), 1.63 (m, 31H), 1.49 (s, 8H ). 1.28 - 1.35 (m,

[0319] 331 i ). 0.96 (d, J - 6 Hz, 3H), 0.85 - 0.93 (m, 12H).

[0320] Example 19. General procedure far preparation 0 / D6LI

[0321] DS DSL1

[0322] To a solution of D6 (0.50 g, 4.71 mmol, 1.00 eq) in DCM (10.0 mL) was added LI (2.20 g, 9.42 mmol, 2.00 eq), DMAP (57.6 mg, 471 pmol, 0.10 eq) and EDO (2.35 g, 12.3 mmol, 2.60 eq) at 0 °C. The mixture was stirred at 0 °C for 0.5 hrs and then stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The mixture was poured into water (10.0 mL) and stirred for 10 mins. The aqueous phase was extracted with DCM (3.00 mL x 2). The combined organic phase was washed with 1 N HC1 (10.0 mL) and brine (10.0 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. D6L1 (2.20 g, 4.10 mmol, 87.0%' yield) was obtained as colorless oil.

[0323] General procedure far preparation 0 / D6LI-I

[0324] A solution of D6L1 (2.20 g, 4.10 mmol, 1 .00 eq) in HCl / dioxane (4 M, 22.0 mL, 21.5 eq) was stirred at 25 °C for 2 hrs. TLC (Dichloromethane: Ethyl acetate=l :l, Rf = 0.05) showed the reaction was completed. The mixture was concentrated in vacuum to get the crude product. D6L1-1 (1.55 g, 3.79 mmol, 92.4% yield, 2HC1) was obtained as colorless oil.

[0325] TLC:

[0326] ■H NMR: D6L1-1 (400 MHz, MeOD) g:3.86 ■■ 3.94 (ni, 4H), 3.44 (t, J = 6.0 Hz, 4H), 3.36 (m, 8H), 2.96 (m, 3H), 2.78 (t, J = 4.8 Hz, 4H), 2.32 (t, J = 6.0 Hz, 4H).

[0327] General procedure for preparation o / ICL-019

[0328] To a solution of D6L1-1 (0.50 g, 1.22 mmol, 1.00 eq, 2HC1) in DCM (5.00 mL) was T1 (1.53 g, 9.77 mmol, 1.84 mL, 8.00 eq) and NaBH(OAc)s (1.55 g, 7.33 mmol, 6.00 eq). The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The mixture was concentrated in vacuum. The residue was diluted with water (50.0 mL.) and extracted with n- heptane (10.0 mL x 2). The combined organic layers were washed with brine (20.0 mL), dried over anhydrous NajSCL, filtered and concentrated in vacuum. The residue was purified by prep-HPLC (column: X-Select CSH Phenyl-Hexyl 100*30 5u; mobile phase: [H2O (0.04%HCl)-THF: ACN-L3]; gradient: 15%-50% B over 12.0 min). ICL-019 (0.20 g, 223 gmol, 18.2% yield) was obtained as yellow oil.

[0329] SH NMR: ICL-019 (400 MHz, CDCh)

[0330] 5: 4.I6 - 4.40 (m, 3H), 3.68 3.78 (m, 7H) ,3.65 (t, J = 6.8 Hz, 2H), 3.49 ■ 3.60 (m, 3H), 2.62

[0331] (in, 6H), 2.41 - 2.55 (m, 6H), 1.58 (m, 3H), 1.45 (s, 6H), 1.27 (s, 60H), 0.86 ■■ 0.92 (m, 12H).

[0332] Example 20. General procedure for preparation of ICL-020

[0333] To a solution of D6L1-1 (0.50 g, 1 .22 mmol, 1.00 eq, 2HC1) in DCM (5.00 mL) was added T2 (1.35 g, 7.33 mmol, 6.00 eq) and NaBH(OAc)3 (1.29 g, 6.11 mmol, 5.00 eq). The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The mixture was concentrated in vacuum. The residue was diluted with water (50.0 mL) and extracted with DCM (10.0 mL x 2). The combined organic layers were washed with brine (20.0 mL), dried over anhydrous NajSCU, filtered and concentrated in vacuum. The residue was purified by pre- HPLC (column: Xselect CSH C18 100 x 30mm x 5um; mobile phase: [H2O (0.1%TFA)-ACN: THF=d: lJ; gradient: 30%-65% B over 12.0 min). ICL-020 (0.20 g, 175 pmoh 14.3% yield, 88.3% purity ) was obtained as yellow oil.

[0334] 1H NMR: ICL-020 (400 MHz, CDC13 )

[0335] 8: 4.19 . 4.34 (m, 4H), 3.67 - 3.77 (m, 8H), 3.55 (s, 4H), 2.57 - 2.76 (m, 8H), 2.48 (d, J =■ 2.0

[0336] Hz, 7H), 1.45 (s, 8H), 1.27 (s, 69H), 0.89 (t, J=7.2 Hz, 12H).

[0337] Example 21. General procedure for preparation o / ICL-021

[0338] To a solution of D6L1-1 (0.50 g, 1.22 mmol, 1.00 eq, 2HC1) in DCM (5.00 mL) was added T3 (1.56 g, 7.33 mmol, 6.00 eq), 4A MS (0.50 g) and NaBH(OAc)3 (1.29 g, 6.11 mmol, 5.00 eq). The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The mixture was concentrated in vacuum. The residue was diluted with water (50.0 mL) and extracted with DCM (10.0 mL x 2). The combined organic layers were washed with brine (20.0 mL), dried over anhydrous NazSCL, filtered and concentrated in vacuum. The residue was purified by pre-HPLC (column: Xselect CSH C18 100 x 30mm x 5um; mobile phase: [ILO (0.1%TFA)-ACN: THF~1:1]; gradient: 40%-80% B over 10.0 min). ICL-021 (0.18 g, 156 pmol, 12.8% yield) was obtained as yellow oil

[0339] ;H NMR: ICL-021 (400 MHz, CDCh)

[0340] 6: 4.18 - 4.32 (m, 4H), 3.67 - 3.77 (m, 8H), 3.55 (s, 4H), 2.58 - 2.74 (m, 8H), 2.47 (s, 6H), 1.44

[0341] (s, 8H), 1.26 (s, 86H), 0.89 (t, / = 6.4 Hz, 12H)

[0342] Example 22. General procedure for preparation of ICL-022

[0343] To a solution of D6L1-1 (0.50 g, 1 .22 mmol, 1.00 eq, 2HC1) in DCM (5.00 mL) was added T4 (1.67 g, 7.33 mmol, 6.00 eq), NaBH(OAc)s (1.29 g, 6.11 mmol, 5.00 eq) at 0°C. The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The mixture was poured into NaHCCh (10.0 mL) and stirred for 15 mins. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous NazSCL, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, DCM / MeOH-80 / T, 20 / 1). ICL-022 (0.20 g, 169 pmol, 13.8% yield) was obtained as light yellow oil.

[0344] SH NMR: ICL-022 (400 MHz, CDCb)

[0345] 5: 4.21 - 4.29 (m, 4H), 4.06 (t, J = 6.8 Hz, 8H), 3.68 - 3.76 (m, 8H), 2.62 (s, 8H), 2.38 - 2.52 (m, 4H), 2.30 (t, J ~ 3.6 Hz, 9H), 1.53 - 1.72 (m, 34H), 1.26 - 1.40 (m, 42H), 0.87 - 0.93 (m, 12H). Example 23. General procedure for preparation o / D7Ll

[0346] To a solution of D7 (1.00 g, 6.66 mmol, 890 pL, 1.00 eq) in DCM (10.0 niL) was added LI (3.11 g, 13.3 mmol, 2.00 eq), DMAP (81.3 nig, 666 pmol, 0.10 eq) and EDCI (3.32 g, 17.3 mmol, 2.60 eq) at 0 °C. The mixture was stirred at 0 °C for 0.5 hrs and then stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The mixture was poured into water (10.0 mL) and stirred for 10 mins. The aqueous phase was extracted with DCM (3.00 mL x 2). The combined organic phase was washed with IN HC1 (10.0 mL) and brine (10.0 mL), dried with anhydrous NaiSOi, filtered and concentrated in vacuum. D7L1 (2.00 g, 3.44 mmol, 51.7% yield) was obtained as colorless oil.

[0347] General procedure far preparation o / D7Ll-l

[0348] A solution of D7L1 (2.00 g, 3.44 mmol, 1 .00 eq) in HCl / dioxane (4 M, 20.0 mL, 23.2 eq) was stirred at 25 °C for 12 hrs. TLC (Dichloromethane: Ethyl acetate=l:l, Rf - 0.44) showed the reaction was completed. The mixture was concentrated in vacuum to get the crude product.

[0349] D7L1-1 (1.50 g, 3.31 mmol, 96.1% yield, 2HC1) was obtained as colorless oil

[0350] TLC:

[0351] General procedure for preparation o / ICL-023 To a solution of D7L1-1 (0.50 g, 1.10 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T1 (1.38 g, 8.82 mmol, 1.67 mL, 8.00 eq) and NaBH(OAc)3(1.40 g, 6.62 mmol, 6.00 eq) at 25 °C. The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The mixture was poured into NaHCCh (10.0 mL) and stirred for 10 mins. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous NaiSO^ filtered and concentrated in vacuum. The residue was purified by prep-HPLC (column: Welch Xtimate Cl 100 x 30mm x Sum; mobile phase: [H2O (0.1%TFA)-ACN: THF=1 : 1]; gradient: 25%-60% B over 20.0 min). ICL-023 (0.50 g, 531 pmol, 48.2% yield) was obtained as yellow oil.

[0352] 3H NMR: ICL-023 (400 MHz, CDCh)

[0353] 8: 4.24 (d, J 3.2 Hz, 4H), 3.60 - 3.80 (m, 16H), 2.67 - 3.21 (m, 12H), 2.57 - 2.63 (m, 4H), 1.57 (s, 8H), 1.26 (s, 56H), 0.84 - 0.93 (m, 12H).

[0354] Example 24. General procedure for preparation o / ICL-024

[0355] To a solution of D7L1-1 (0.50 g, 1.10 mmol, 1 .00 eq, 2HC1) in DCM ( 10.0 ml..) was added T2 (1.63 g, 8.82 mmol, 8.00 eq) and NaBH(OAc)3 (1.40 g, 6.62 mmol, 6.00 eq) at 25 °C. The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The mixture was filtered and the filter was concentrated in vacuum to get the crude. The residue was washed with H2O (20.0 mL) and the aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous NajSCL, filtered and concentrated in vacuum. The residue was purified by prep-HPLC (column: Welch Xtimate Cl 100 x 30mm x 5um; mobile phase: [H2O (0.1%TFA)-ACN: THF===1: 1]; gradient: 30%-70% B over 20.0 min). ICL-024 (0.20 g, 190 pmol, 17.2% yield) was obtained as yellow oil. 100 mg of the product in ET79194-18 was combined to this page for delivery. Total 300 mg of the product was delivered as one batch.

[0356] 1H NMR: ICL-024 (400 MHz, CDCh •

[0357] 5: 4.25 (s, 4H), 3.62 - 3.80 (m, 12H), 3.55 (s, 4H), 2,62 (d, J=3.2 Hz, 8H), 2.48 (s, 8H), 1 .45 (s, 8H), 1.27 (s, 72H), 0.89 (d, J=3.2 Hz, 12H). Example 25. General procedure for preparation 0 / ICL-025

[0358] To a solution of D7L1-1 (0.50 g, 1.10 mmol, 1 .00 eq, 2HC1) in DCM (5.00 mL) was added T3 (1.87 g, 8.82 mmol, 8.00 eq) and NaBH(OAc)3 (1.40 g, 6.62 mmol, 6.00 eq) at 25 °C. The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction wras completed. The mixture was filtered and the filter was concentrated in vacuum to get the erode. The residue was washed with H2O (20.0 mL) and the aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous NacSCL, filtered and concentrated in vacuum. The residue was purified by prep-HPLC (column: X-Select CSH Phenyl-Hexyl 100*30 5u; mobile phase: [H2O (0.04%HCl)-ACN]; gradient: 35%-70% B over 12.0 min). ICL-025 (0.60 g, 515 pmol, 46.7% yield) was obtained as yellow oil.

[0359] SH NMR: ICL-025 (400 MHz, CDCh)

[0360] 6: 4.20 ■■ 4.31 (m, 4H), 3.68 ■■ 3.76 (m, 8H), 3.66 (s, 4H), 3.42 ■■ 3.63 (m, 3H), 2.62 (t, J = 6.4 Hz, 8H) 2.44 (d, J = 5.60 Hz, 6H) 1.34 ■■ 1.53 (m, 8H) 1.27 (s, 88H) 0.89 (t, J = 6.4 Hz, 12H).

[0361] Example 26. General procedure for preparation q / 'ICL-026

[0362] To a solution of D7L1-1 (0.50 g, 1.10 mmol, 1.00 eq, 2HC1) in DCM (5.00 mL) was added T4 (1.51 g, 6.62 mmol, 6.00 eq) and NaBHfOAcjs (1.17 g, 5.51 mmol, 5.00 eq) at 0 °C. The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The mixture was poured into NaHCCti (10.0 mL) and stirred for 15 mins. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous NazSCL, filtered and concentrated in vacuum. The crude product was purified by normal phase HPLC (column: Agela DuraShell basic 150mm x 30mm x Sum; mobile phase: [Heptane-ACN : THF== =1: 1]; gradient: 0%-30% B over 8.0 min). ICL-026 (0.30 g, 244 jimol, 22.1% yield) was obtained as light yellow oil.

[0363] SH NMR: ICL-026 (400 MHz, CDCh)

[0364] 5: 4.21 . 4.30 (m, 4H), 4.06 (t, J = 6.8 Hz, 8H), 3.68 ■■ 3.75 (m, 8H), 3.66 (s, 4H), 3.52 (s, 4H), 2.61 (t, 7 = 6.4 Hz, 8H), 2.45 (s, 8H), 2.30 (t, J = 7.6 Hz, 8H), 1.60 ■■ 1.65 (m, 18H), 1.44 (s, 8H), 1.26 ■ 1.36 (m, 36H), 0.90 (t, J = 6.8 Hz, 12H).

[0365] Example 27. General procedure far preparation 0 / D8LI

[0366] To a solution of D8 (3.23 g, 13.9 mmol, 2.00 eq), DMAP (84.7 mg, 693 prnol, 0.10 eq) EDO (3.46 g, 18.0 mmol, 2.60 eq) at 0 °C. The mixture was stirred at 0 °C for 0.5 hrs and then stirred at 20 °C for 12 hrs. LCMS (D8L1, Rt = 1.721) showed the reaction was completed. The mixture was poured into water (10.0 mL) and stirred for 10 mins. The aqueous phase was extracted with DCM (3.00 ml., x 2). The combined organic phase was washed with 1 N HC1 (10.0 mL) and brine (10.0 ml), dried with anhydrous Na?.SO4, filtered and concentrated in vacuum. D8L1 (3.70 g, 6.44 mmol, 92.8% yield) was obtained as colorless oil.

[0367] General procedure far preparation of D8L1 -1

[0368] A solution of D8L1 (1.78 g, 3.10 mmol, 1 .00 eq) in HCl / dioxane (4 M, 17.8 mL, 23.0 eq) was stirred at 25 °C for 2 hrs. TLC (Dichloromethane: Ethyl acetate=l:l, Rf = 0.00) showed the reaction was completed. The mixture was filtered and washed by dioxane (10.0 mL). The filter cake was collected and dried in drying oven at 45 °C. D8L1-1 (1.35 g, 3.02 mmol, 97.4% yield, 2HC1) was obtained as white solid. TLC:

[0369] General procedure far preparation of ICL-027

[0370] To a solution of D8L1-1 (0.50 g, 1.12 mmol, 1 .00 eq, 2HC1) in DCM (5.00 mL) was added T1 (1 .40 g, 8.94 mmol, 1 .69 mL, 8.00 eq) and NaBH(OAc)s (1.42 g, 6.71 mmol, 6.00 eq) at 25 °C. The mixture was stirred at 25 °C for 12 hrs. LCMS (ICL-027, Rt - 1.080) showed the reaction was completed. The mixture was concentrated in vacuum to remove DCM. The residue was poured into water (10.0 mL) and the aqueous phase was extracted with n-heptane (10.0 mL x 2). The combined organic phase was washed with brine (2.0.0 mL), dried with anhydrous Na?SO4 filtered and concentrated in vacuum. The residue was purified by prep-HPLC (column: Welch Xtimate Cl 100*30mm*5um; mobile phase: [HjO (0.1%TFA)-ACN: THF=1 : 1]; gradient: 25%-62% B over 20.0 min). ICL-027 (220 mg, 235 pmol, 2.1.0% yield) was obtained as yellow oil.

[0371] SH NMR: ICL-027 (400 MHz, CDCb)

[0372] 5: 3.92 (d, J = 4.4 Hz, 4H), 3.67 - 3.78 (m, 4H), 3.57 (s, 4H), 2.65 - 2.88 (m, 4H), 2.44 - 2.62 (m, 10H), 1.82 (d, 3.2 Hz, 5H), 1.62 (d, J = 1.2. Hz, 3H), 1.47 (s, 8H), 1.28 (s, 58H), 1.01

[0373] (s, 4H), 0.89 (d, J = 4.0 Hz, 12H).

[0374] Example 28. General procedure far preparation o / ICL-028

[0375] To a solution of D8L1-1 (0.5() g, 1.12 mmol, LOO eq, 2HC1) in DCM (10.0 mL) was added T2 (1.65 g, 8.94 mmol, 8.00 eq) and NaBH(OAc)3 (1.42 g, 6.71 mmol, 6.00 eq) at 25 °C. The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The mixture was poured into NaHCOr (10.0 mL) and stirred for 10 mins. Tire aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous NaiSCU, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, DCM / MeOH=100 / l, 30 / 1). ICL-028 (0.80 g, 764 pmol, 68.3% yield) was obtained as yellow oil.

[0376] 1H NMR: ICL-028 (400 MHz, CDC13 )

[0377] 5: 3.92 (d, J = 6.4 Hz, 4H), 3.72 (t, J = 6.4 Hz, 4H), 3.52 (s, 4H), 2.63 (s, 4H), 2.58 (t, J = 6.4 Hz, 4H), 2.44 (t, J = 7.2 Hz, 8H), 1.81 (d, J = 7.2 Hz, 4H), 1.62 (s, 2H), 1.42 (s, 8H), 1.27 (s,

[0378] 70H), 0.96 - 1.05 (m, 4H), 0.82 - 0.93 (m, 12H).

[0379] Example 29. General procedure for preparation of ICL-029

[0380] To a solution of D8L1-1 (0.50 g, 1.12 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T3 (1.90 g, 8.94 mmol, 8.00 eq) and NaBH(OAc)3(1.42 g, 6.71 mmol, 6.00 eq) at 25 °C. The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The mixture was concentrated in vacuum to remove DCM. The residue was poured into water (10.0 mL) and the aqueous phase was extracted with n-heptane (10.0 mL x 2). The combined organic phase was washed with brine (20.0 mL), dried with anhydrous NaaSOr, filtered and concentrated in vacuum. The residue was purified by prep-HPLC (column: X-Select CSH Phenyl-Hexyl 100*30 5u; mobile phase: [H2O (0.04%HCl)-ACN]; gradient: 35%-75% B over 12.0 min). ICL-029 (0.30 g, 259 pmol, 23.1% yield) was obtained as yellow oil. -029 (400 MHz, CDCh)

[0381] 6: 3.92 (d, J = 6.8 Hz, 4H), 3.72 (t, J = 6.4 Hz, 4H), 3.52 (s, 4H), 2.55 - 2.69 (m, 8H), 2.44 (s, 8H), 1.82 (d, 7.2 Hz, 4H), 1.62 (s, 4H), 1.43 (s, 8H), 1.27 (s, 88H), 0.96 - 1.06 (m, 4H),

[0382] 0.85 - 0.92 (m, 12H). Example 30. General procedure for preparation o / ICL-030

[0383] To a solution of D8L1-1 (0.50 g, 1.12 mmol, 1.00 eq, 2HC1) in DCM (5.00 mL) was added T4 (1.53 g, 6.71 mmol, 6.00 eq) and NaBH(OAc)j (1.18 g, 5.59 mmol, 5.00 eq) at 0 °C. The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The mixture was poured into NallCOs (10.0 mL) and stirred for 15 mins. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, DCM / MeOH=60 / 1 , 10 / 1 ). ICL-030 (0.12 g, 82.5 pmol, 7.38% yield, 80.3% purity) was obtained as yellow oil. -030 (400 MHz, CDCh)

[0384] 6: 4.06 (t, J 6.8 Hz, 9H), 3.92 (d, J = 6.4 Hz, 5H), 3.60 - 3.77 (m, 9H), 2.87 (s, 4H), 2.53 - 2.73 (in, 11H), 2.30 (t, J = 7.6 Hz, 9H), 2.03 (s, 4H), 1.81 (d, J 6.8 Hz, 4H), 1.49 - 1.67 (m, 271 i ). 1.32 (d, J - 2.4 Hz, 40H), 0.96 - 1.05 (m, 4H), 0.86 - 0.93 (m, 12H).

[0385] Example 31. General procedure for preparation o / D9Ll

[0386] D10

[0387] 0101.1

[0388] To a mixture of D9 (0.50 g, 5.68 mmol, 467 pL, 1.00 eq) in DCM (10.0 mL) was added LI

[0389] (2.65 g, 11.4 mmol, 2.00 eq), DMAP (69.3 mg, 567 nmol, 0.10 eq) and EDCI (2.83 g, 14.8 mmol, 2.60 eq) at 0 °C under N?_. The mixture was stirred at 0 °C for 0.5 hr, then heated to

[0390] 25 °C and stirred for 12 hours. TLC (Di chloromethane: Methanol- 10: 1, product Rf -0.50) showed the starting material was consumed completely. The mixture was poured into H2O (10.0 mL) and the aqueous phase was extracted with DCM (2 x 5.00 mL). The organic phase was washed with IN HO (10.0 mL) and brine (10.0 mL). The organic phase was dried with Na2SO4 and concentrated in vacuum. D9L1 (3.00 g, crude product) was obtained as colorless oil.

[0391] TLC:

[0392] General procedure far preparation o / D9Ll-l

[0393] To a mixture of D9L1 (3.00 g, 5.78 mmol, LOO eq) in dioxane (15.0 mL) was added HCl / dioxane (4 M, 18.00 mL, 12.5 eq) dropwise for 0.1 hr at 25 °C under Nj. The mixture was stirred 25 °C for 2 hours. TLC (Dichloromethane: Ethyl acetate~l:l, product Rf =0) showed the starting material was consumed completely. The mixture was concentrated in vacuum. The product was used to next step without purification. D9L1-1 (1.90 g, 4.86 mmol, 83.9% yield, 2HC1) was obtained as yellow oil.

[0394] TLC:

[0395] General procedure for preparation o / ICL-031

[0396] To a solution of D9L1-1 (0.50 g, 1.28 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T1 (1.60 g, 10.2 mmol, 1.93 mL, 8.00 eq) and NaBH(OAc)3 (1.62 g, 7.67 mmol, 6.00 eq) at 25°C under Nz. The reaction mixture was stirred at 25 °C for 12 hrs. LCMS showed the starting material was consumed completely. The mixture was poured into NallCCh (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The crude product was purified by reversed-phase HPLC (column: Xselect CSH C18 100 x 30mm x 5um; mobile phase: [H2O (0.1%TFA)-ACN: THF:::1: 1]; gradient: 30%-65% B over 2.0 min) to give ICL-031 (0.20 g, 227 umol, 11.8% yield) as yellow oil.

[0397] LCMS: ICL-031:

[0398] LCMS (product: RT = 1.009 min; [M / 2+1] T 440.5)

[0399] SH NMR: ICL-031 (400 MHz, CDCh)

[0400] 5: 5.77 - 5.79 (m, 2H), 4.53 - 4.54 (m, 4H), 3.65 (t, J = 6.4 Hz, 4H), 2.52 - 2.60 (m 8H), 2.40 (s, 6H), 1.68 (s, 8H), 1.12 - 137 (m, 54H), 0.81 (t, J = 6.4 Hz, 12H).

[0401] Example 32. General procedure for preparation 0 / ICL-032

[0402] To a solution of D9L1-1 (0.50 g, 1.28 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T2 (1.88 g, 10.2 mmol, 8.00 eq) and NaBH(OAc)3 (1.62 g, 7.67 mmol, 6.00 eq) al 25°C under N2. The reaction mixture was stirred at 25°C for 12 hrs. LCMS (product RT - 1.548 min) showed the starting material was consumed completely. The mixture was poured into NaHCOs (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The crude product was purified by reversed-phase HPLC (column: Xselect CSH Cl 8 100 x 30mm x 5um; mobile phase: [H2O (0.1 %TFA)-ACN: THF=1 : 1]; gradient: 40% - 75% B over 10.0 min) to give ICL-032 (0.15 g, 151 gmol, 7.89% yield) as yellow oil.

[0403] LCMS: ICL-032:

[0404] LCMS (product: RT = 1.548 min: [M / 2+ 1 ] T 496.5) 41 NMR: ICL-032 (400 MHz, CDCh)

[0405] 5: 5.28 (s, 2H), 4.63 (s, 4H), 3.55-3.75 (m, 4H), 3.52 (s 8H), 2.61-2.66 (m, 6H), 2.45 (s, 4H), 1.62-1.66 (m, L5H), 1.45 (s, 72H). 0.89 (t, J - 6.8 Hz, 12H)

[0406] Example 33. General procedure for preparation 0 / ICL-033

[0407] To a solution of D9L1-1 (0.50 g, 1.28 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T3 (1.63 g, 7.67 mmol, 6.00 eq) and NaBH(OAc)j (1.35 g, 6.39 mmol, 5.00 eq) at 25°C under 5b. The reaction mixture was stirred at 25CC for 12 hrs. LCMS (product RT ~ 2.439 min) showed the starting material was consumed completely. The mixture was poured into NaHCCh (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The crude product was purified by normal phase HPLC (column: Agela DuraShell NHs 150mm x 30mm x 5um; mobile phase: [Heptane-ACN: THE 1 : 1 L gradient: 0%-30% B over 10.0 min) to give ICL-033 (0.15 g, 136 pmol, 10.6% yield) as yellow oil.

[0408] LCMS: ICL-033:

[0409] LCMS (product: RT = 2.439 min; [M / 2+1]+: 552.5)

[0410] SH NMR: ICL-033 (400 MHz, CDCh)

[0411] 5: 5.77-5.78 (m, 2H), 4.53-4.54 (m, 4H), 3.65 (t, J = 6.8 Hz, 4H), 3.62 (s 4H), 2.52-2.55 (m, 8H), 2.39 (s, 6H), 1.36 (s, 6H), 1.25 (s, 8H), 1.23 (s, 84H), 0.81 (t, J = 6.8 Hz, 12H) Example 34. General procedure for preparation of ICL-034

[0412] J

[0413] □9L1 1 ICLU34

[0414] To a solution of D9L1-1 (0.50 g, 1.28 mmol, 1 .00 eq, 2HC1) in DCM ( 10.0 mL) was added T4 (1 .75 g, 7.67 mmol, 6.00 eq) and NaBH(OAc)3 (1 .35 g, 6.39 mmol, 5.00 eq) at 25 °C under N2. The reaction mixture was stirred at 25 °C for 12 hrs. LCMS showed the starting material was consumed completely. The mixture was poured into NaHCO? (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous NazSCti, filtered and concentrated in vacuum. The crude product was purified by normal phase HPLC (column: Agela DuraShell NH? 150mm x 30mm x 5mn; mobile phase: [Heptane ■■ACN: THF=1:1]; gradient: ()%-3()% B over 10.0 min) to give ICL-034 (0.20 g, 171 pmol, 13.4% yield) as yellow oil.

[0415] LCMS: ICL-034:

[0416] LCMS (product: RT - 1.445 min; [M / 2+1]+: 584.5)

[0417] Hi NMR: ICL-034 (400 MHz, CDCL)

[0418] 5: 5.78-5.79 (m, 2H), 4.53-4.54 (m, 4H), 3.99 (t, J = 6.8 Hz, 8H), 3.65 (t, 7 — 6.4 Hz, 4H) 3.44 (t, J = 6.0 Hz, 4H), 2.53-2.55 (m 8H), 2.36 (s, 7H), 2.20-2.24 (m, 8H), 1.53-1.68 (m, 18H), 1.25-1.27 (m, 8H), 1.20-1.24 (m, 39H), 0.82 (t, J ~ 6.8 Hz, 12H)

[0419] Example 35. General procedure for preparation q / 'DIOL l

[0420] D10

[0421] D10L1 To a mixture of DIO (0.50 g, 5.68 mmol, 466 pL, 1.00 eq) in DCM (10.0 mL) was added LI (2.65 g, 11.4 mmol, 2.00 eq), DMAP (69.3 mg, 568 pmol, 0.10 eq) and EDO (2.83 g, 14.8 mmol, 2.60 eq) at 0 °C under N2. The mixture was stirred at 0 °C for 0.5 hr, then heated to 25 °C and stirred for 12 hours. LCMS showed the starting material was consumed completely. The mixture was poured into H2O (10.0 mL) and the aqueous phase was extracted with DCM (2 x 5.00 mL). The organic phase was washed with IN HC1 (10.0 mL) and brine (10.0 mL). The organic phase was dried with NacSO-t and concentrated in vacuum. D10L1 (2.50 g, 4.82 mmol, 85.0% yield) was obtained as colorless oil.

[0422] LCMS: D10L1:

[0423] LCMS (product: RT = 1.563 min: [M / 2+1]+: 419.5)

[0424] General procedure for preparation of D10L1-1

[0425] To a mixture of D10L1 (2.50 g, 4.82 mmol, LOO eq) in dioxane (15.0 mL) was added HCb'dioxane (4 M, 15.0 mL, 12.5 eq) dropwise for 0.1 hr at 25 °C under N2. The mixture was stirred 25 °C for 2 hours. TLC (Dichloromethane: Ethyl acetate=l:l, product Rf =0) showed the starting material was consumed completely. The mixture was concentrated in vacuum. The product was used to next step without purification. D10L1-1 (1.50 g, 3.83 mmol, 79.5% yield, 2HC1) was obtained as yellow oil.

[0426] TLC:

[0427] General procedure for preparation of ICL-035 To a solution of D10L1-1 (0.50 g, 1.28 mmol. 1.00 eq, 2HCI) in DCM (10.0 mL) was added T1 (1.60 g, 10.2 mmol, 1.93 mL, 8.00 eq) and NaBH(OAc)3 (1.62 g, 7.67 mmol, 6.00 eq) at 25°C under N2. The reaction mixture was stirred at 25°C for 12 hrs. LCMS showed the starting material was consumed completely. The mixture was poured into NallCCh (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous Na2SC>4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (Dichloromethane: Methanol~60 / 1, 20 / 1 ) to afford ICL-035 (0.30 g, 341 pmol, 37.5% yield) as yellow oil.

[0428] LCMS: ICL-035

[0429] LCMS (product: RT = 0.959 min; [M / 2+1] T 440.5)

[0430] SH NMR: ICL-035 (400 MHz, CDCh)

[0431] 6: 5.73-5.75 (m, 2H), 4.70-4.71 (m, 4H), 3.72 (t, 6.4 Hz, 4H), 3.55 (s, 4H), 2.58 ■■ 2.68 (m

[0432] 8H), 2.48 ■■ 2.51 (m, 8H), 1.44 (s, 8H), 1.31 (s 56H), 0.89 (t, J ~ 6.4 Hz, 12H).

[0433] Example 36. General procedure for preparation of ICL-036

[0434] To a solution of D10L1-1 (0.50 g, 1.28 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T2 (1.88 g, 10.2 mmol, 8.00 eq) and NaBH(OAc)3 (1.62 g, 7.67 mmol, 6.00 eq) at 25°C under N2. The reaction mixture was stirred at 25 °C for 12 hrs. LCMS showed the starting material was consumed completely. The mixture was poured into NaHCOs (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous NazSCL, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (Dichloromethane: Methanol=60 / 1 , 20 / 1) The crude product was purified by normal phase HPLCicolumn: Agela DuraShell NH3, 150mm x 30mm x 5um;mobile phase: [Heptane- IPA:ACN=4: 1] ^gradient: 0%-30% B over 10.0 min). to afford ICL-036 (0.30 g, 303 pmol, 50.0% yield) as yellow oil. LCMS: ICL-036:

[0435] LCMS (product: RT = 1.533 min: [M / 2+1]+: 496.5)

[0436] 3H NMR: ICL-036 (400 MHz, CDCh)

[0437] 8: 5.73 - 5.75 (m, 2H), 4.70 - 4.72 (in, 4H), 3.72 (t, J = 6.4 Hz, 4H), 3.54 (s, 3H), 2.58 - 2.66

[0438] (m 8H), 2.46 (s, 6H), 1.67 (s, 6H), 1.60 (s 8H), 1.33 (s, 69H), 0.89 (t, .7 = 6.8 Hz, 12H)

[0439] Example 37 General procedure for preparation of ICL-037

[0440] To a solution of D10L1-1 (0.50 g, 1.28 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T3 (1.63 g, 7.67 mmol, 6.00 eq) and NaBH(OAc)s (1.35 g, 6.39 mmol, 5.00 eq) at 25°C under N2. The reaction mixture was stirred at 25°C for 12 hrs. LCMS showed the starting material was consumed completely. The mixture was poured into NaHCCL (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The crude product was purified by normal phase HPLC (column: Agela DuraShell NH2 150mm x 30mm x 5um;mobile phase: [Heptane - THF:ACN===2: l(0.2%NH3H20)];gradient:0%-30% B over 10.0 mini.to afford ICL-037 (0.26 g, 236 pmol, 18.4% yield) as yellow oil.

[0441] LCMS: ICL-037

[0442] LCMS (product: RT = 1.533 min: [M / 2+1]+: 552.5)

[0443] Hl NMR: ICL-037 (400 MHz, CDCb)

[0444] 8: 5.65 - 5.68 (m, 2H), 4.62 - 4.64 (m, 4H), 3.64 (t, J = 6.4 Hz, 4H), 3.46 (s, 4H), 2.50 - 2.58 (m 8H), 2.38 (s, 7H), 1.23 (s, 88H), 0.81 (t, J = 6.8 Hz, 12H). Example 37. General procedure far preparation o / Dll

[0445] 25 °C, 3.5 hrs

[0446] D11-1 D11

[0447] Compound Dll-1 from domestic.

[0448] A solution of LAH (2.5 M, 12.0 mL, 1.30 eq) in THF (44.0 mL) was added dropwise a solution of Cpd.Dll-l (4.00 g, 23.2 mmol, 1.00 eq) in THF (16.0 mL) at 0 °C over a period of 0.5 hr under N?.. The reaction was stirred at 25 °C for 3 hrs. LCMS showed the reaction was completed. The reaction was quenched by addition of H2O (12.0 mL), 15% of NaOH (12.0 mL), and HjO again (36.0 mL). Then the mixture was poured into EtOAc (150 mL), stirred for 30 minutes, and filtered over bfeSCL, the organic layer was concentrated in vacuum to give compound Dll (2.50 g, 21.5 mmol, 92.6% yield) as colorless oil.

[0449] SH NMR: Dll (400 MHz, CDC13 )

[0450] 5: 5.52 (t, J = 4.0 Hz, 2H), 3.64 (t, J = 6.0 Hz, 4H), 2.26 - 2.30 (m, 4H).

[0451] General procedure far preparation o / DULl

[0452] To a solution of Cpd.Dll (3.00 g, 25.8 mmol, 1 .00 eq) in DCM (30.0 mL) was added compound D11L1 (12.0 g, 51.6 mmol, 2.00 eq), EDCI (12.8 g, 67.1 mmol, 2.60 eq) and DMAP (315 mg, 2.58 mmol, 0.10 eq) at 25 °C. The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The reaction was poured into water (50.0 mL), adjusted pH ~5 -6 with 1 N HQ (5.00 mL), then extracted with DCM (20.0 mL). The organic layer was concentrated in vacuum to give compound D11L1 (12.0 g, 21.9 mmol, 85.0% yield) as colorless oil.

[0453] XH NMR: D11L1 (400 MHz, CDCh)

[0454] 5: 5.49 - 5.51 (m, 2H), 4.12 (t, J = 6.8 Hz, 4H), 3.71 (t, J = 6.4 Hz, 4H), 3.51 (t, J = 4.8 Hz, 4H), 3.29 - 3.30 (m, 4H), 2.57 (t, / = 6.4 Hz, 4H), 2.34 - 2.37 (m, 4H), 1.44 (s, 18H).

[0455] General procedure for preparation o / DHLl-1

[0456] To a solution of Cpd.DHLl (2.00 g, 3.66 mmol, 1.00 eq) in dioxane (10.0 niL) was added HCl / dioxane (4 M, 10.0 niL, 10.9 eq) at 0 °C. The mixture was stirred at 25 °C for 2 hrs. LCMS showed the reaction was completed. The reaction was filtered, the filter cake washed with DCM (50.0 mL) and the filter cake was collected. Compound D11L1-1 (1.60 g, crude, HC1) was completed, as a white solid.

[0457] SH NMR: D11L1-1 (400 MHz, D2O)

[0458] 5: 5.55 (s, 2H), 4.14 (t, J = 6.4 Hz, 2.H), 3.77 (t, J = 6.0 Hz, 4H), 3.71 (t, J = 5.2 Hz, 4H), 3.16 (t, J - 4.8 Hz, 4H), 2.66 (t, J = 6.0 Hz, 4H), 2.34 - 2.35 (m, 4H).

[0459] Example 38. General procedure for preparation of ICL-038

[0460] To a solution of Cpd.DllLl-1 (0.50 g, 1.31 mmol, 1.00 eq, HQ) in DCM (5.00 mL) was added Cpd.Tl (1.22 g, 7.84 mmol, 1.48 mL, 6.00 eq), NaBH(OAc)3 (1.38 g, 6.53 mmol, 5.00 eq) at 0 °C. The reaction was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The reaction w'as poured into w'ater (10.0 mL) and the mixture was extracted with DCM (10.0 mL), the organic layer was concentrated in vacuum to give crude product. The crude product was further purified by normal phase HPLC (column: Agela DuraShell NH2 150mm x 30mm x 5um; mobile phase: [Heptane-ACN: THF=1: 1]; gradient: 0%-30% B over 8.0 min) to give ICL-038 (0.30 g, 330 pmol, 25.3% yield) as yellow oil.

[0461] 1H NMR: ICL-038 (400 MHz, CDCI3)

[0462] 5: 5.49 (t, J = 3.6 Hz, 2H), 4.09 (t, J 7.2. Hz, 4H), 3.71 (t, J = 6.4 Hz, 4H), 3.53 (brs, 4H), 2.65 (brs, 4H), 2.57 (t, J - 6.8 Hz, 4H), 2.45 (brs, 6H), 2.34 (q, J ~ 5.2. Hz, 6.4 Hz, 4H), 1.43 (brs, 8H), 1.27 (s, 60H), 0.88 (t, y = 6.8 Hz, 12H). Example 39. General procedure for preparation o / ICL-039

[0463] To a solution of Cpd.DHLl-l (0.50 g, 1.31 mmol, 1.00 eq, HQ) in DCM (5.00 mL.) wras added Cpd.T2 (1.44 g, 7.84 mmol, 6.00 eq), NaBH(OAc)3 (1 .38 g, 6.53 mmol, 5.00 eq) at 0 °C. The reaction was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The reaction was poured into water (10.0 mL) and the mixture was extracted with DCM (10.0 mL), the organic layer was concentrated in vacuum to give crude product. The etude product was further purified by normal phase HPLC (column: Agela DuraShell NH2 150mm x 30mm x 5um: mobile phase: [Heptane -ACN: THF=l:lj; gradient: 0%-30% B over 8.0 min) to give compound ICL-039 (0.30 g, 294 pmol, 22.5% yield) as yellow oil.

[0464] XH NMR: ICL-039 (400 MHz, CDCI3)

[0465] 5: 5.49 (t, J = 3.6 Hz, 2H), 4.09 (t, J = 6.8 Hz, 4H), 3.84 (t, J = 5.2 Hz, 4H), 3.72 (t, J = 6.4 Hz, 4H), 3.27 (t, J = 4.4 Hz, 4H), 3.03-3.11 (m, 8H), 2.65 (t, J = 6.0 Hz, 4H), 2.34 (q, J = 5.2 Hz, 6.0 Hz, 4H), 1.68 (s, 8H), 1.26-1.31 (m, 72H), 0.88 (t, J = 6.8 Hz, 12H).

[0466] Example 40. General procedure for preparation o / ICL-040

[0467] To a solution of Cpd.DllLl-1 (0.50 g, 1.31 mmol, 1.00 eq, HQ) in DCM (5.00 mL) was added Cpd.T3 (1.66 g, 7.84 mmol, 6.00 eq), NaBH(OAc)3 (1.38 g, 6.53 mmol, 5.00 eq) at 0 °C. The reaction was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The reaction was poured into water (10.0 mL) and the mixture was extracted with DCM (10.0 mL), the organic layer was concentrated in vacuum to give crude product. Ute crude product was further purified by normal phase HPLC (column: Agela DuraShell NHs 150mm x 30mm x 5urn; mobile phase: [Heptane -ACN: THF:::1 :1]; gradient: 0%-30% B over 8.0 min) to give compound ICL-040 (0.10 g, 88.3 pmol, 6.77% yield) as yellow oil. Hl NMR: ICL-040 (400 MHz, CDCh)

[0468] 5: 5.49 (t, J = 3.6 Hz, 2H), 4.09 (t, J = 7.0 Hz, 4H), 3.71 (t, J = 6.4 Hz, 4H), 3.70 (brs, 4H), 2.59 (brs, 4H), 2.57 (t, J = 6.4 Hz, 4H), 2.56 (brs, 4H), 2.33-2.36 (m, 4H), 1.42 (brs, 4H), 1.26 (m, 96H), 0.89 (t, J ~ 6.8 Hz, 12H).

[0469] General procedure for preparation of D13-2

[0470] D13-1 D13-2

[0471] To a solution of D13-1 (25.0 g, 144 mmol, 20.7 mL, 1.00 eq) in toluene (250 mL) was added methyl 2-(triphenyl-phosphanylidene) acetate (48.0 g, 144 mmol, 1.00 eq) at 25 °C. The mixture was stirred at 100 °C for 24 hrs. LCMS (ET79194-79-P1C1, Rt = 1.026) showed the reaction was completed. The mixture was concentrated in vacuum to get the crude product. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether / Ethyl acetate= 100 / 1, 1 / 1). D13-2 (43.0 g, 93.2 mmol, 64.99e yield, 49.9% purity) was obtained as yellow oil.

[0472] General procedure for preparation of D13-3

[0473] To a solution of D13-2 (15.0 g, 65.2 mmol, 1.00 eq) in MeOH (150 mL) was added Pd / C (3.00 g, 2.82 mmol, 10% purity, 4.33e-2 eq) at 25 °C. The mixture was degassed and purged with

[0474] I-I2 for 3 times. The mixture was stirred under H2 (45psi) at 25 °C for 24 hrs. TLC

[0475] (Petroleumether: Ethyl acetate=2:l, Rf =0.40) showed the reaction was completed. The mixture was filtered and washed with MeOH. The filter was concentrated in vacuum to get the crude. D13-3 (12.0 g, 51.7 mmol, 79.3% yield) was obtained as yellow oil.

[0476] D13-3 D13-4

[0477] To a solution of LAH (2.50 M, 25.8 mL, 1.50 eq) was added a solution of D13-3 (10.0 g, 43.1 mmol, 1.00 eq) in THE (30.0 mL) at 0~10 °C. The mixture was stirred at 25 °C for 2 hrs. TLC showed the reaction was completed. The mixture was cooled to -10~0 °C. 0.5 mL of water was added to the mixture and stirred for 10 mins. NaOH (15%, 0.50 mL) was added to the mixture followed by 1.5 mL of water. The mixture w'as stirred for 15 mins at 25 °C. Anhydrous Na2SO4 was added to the mixture, the mixture was filtered and washed by THE The filtrate was concentrated in vacuum to get the crude product. D13-4 (4.00 g, 27.0 mmol, 62.7% yield) was obtained as yellow oil.

[0478] TLC: General procedure for preparation q / 'D13Ll

[0479] E)13’4 D13L1

[0480] To a solution of D13-4 (2.00 g, 13.5 mmol. 1.00 eq) in DCM (20.0 mL) was added DMAP (165 mg, 1.35 mmol, 0.10 eq), LI (9.44 g, 40.5 mmol, 3.00 eq) and EDCI (9.05 g, 47.2 mmol, 3.50 eq) at 0 °C. The mixture was stirred at 0 °C for 0.5 hrs and then stirred at 25 °C for 12 hrs. LCMS (ET79194-115-P1A1, Rt =1.729) showed the reaction was completed. The mixture was poured into water (50.0 mL) and stirred for 15 mins. The aqueous phase was extracted with DCM (15.0 mL, 15.0 mL). The combined organic phase was washed with IN HCi (50.0 mL) and brine (50.0 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum.

[0481] D13L1 (10.0 g, 12.6 mmol, 93.3% yield) was obtained as yellow oil.

[0482] LCMS: D13L1, product: RT = 1. / 29 min.

[0483] General procedure for preparation of ICL-041-1

[0484] A solution of D13L1 (10.0 g, 12.6 mmol, 1.00 eq) in HCl / dioxane (4 M, 200 mL) was stirred at 25 °C for 12 hrs. LC-MS (ET79578-3-P1A4, RT =2.233) showed Reactant 1 was consumed completely and one main peak with desired mass was detected. The mixture was concentrated in vacuum to get the crude product. D13L1-1 (6.00 g, crude, HCI) was obtained as yellow oil.

[0485] LCMS: D13L1-1, product: RT = 2.233 min. Example 41. General procedure for preparation o / ICL-041

[0486] To a solution of D13L1-1 (500 mg, 702 umol, 1.00 eq) in DCM (10.0 mL) was added T1 (877 mg, 5.62 mmol, 8.00 eq) and NaBH(OAc)s (893 mg, 4.21 mmol, 6.00 eq) at 0 °C. The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The residue was poured into aq.NaHCCh (20.0 mL.) and stirred for 5 min. The aqueous phase was extracted with DCM (10.0 mL). The combined organic phase was washed with brine (20.0 mL), dried with anhydrous NazSCL, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, DCM / MeOH- 100 / 1, 0 / 1). The crude product was purified by normal phase HPLC (column: Agela DuraShell NH2 150mm x 30mm x 5um; mobile phase: [Heptane-THF: ACN=2:l(0.2%NH3H2O)J; gradient: 0%-30% B over 10.0 min). ICL-041 (70.0 mg, 52.4 pmol, 7.47% yield) was obtained as light yellow' oil.

[0487] SH NMR: ICL-041 (400 MHz, CDCb)

[0488] 5: 4.11 - 4.15 (t, J = 6.8 Hz, 6H), 3.69 - 3.72 (t, J = 6.4 Hz, 6H), 3.57 (s, 5H), 2.65 (s, 5H), 2.55 - 2.58 (t, .1 = 6.4 Hz, 6H), 2.45 (s, 9H), 1.66 - 1.69 (m, 12H), 1.43 (s, 12H), 1.30 - 1.38 (m, 10H), 1.27 (s, 74H), 0.87 - 0.90 (t, .J ~ 6.8 Hz, 18H).

[0489] Example 42. General procedure for preparation o / ICL-042 To a solution of D13L1-1 (500 mg, 702 umol, 1.00 eq) in DCM (10.0 mL) was added T2 (1.03 g, 5.62 mmol, 8.00 eq) and NaBH(OAc>3 (893 mg, 4.21 mmol, 6.00 eq) at 0 °C. The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The residue was poured into aq.NaHCCh (20.0 mL) and stirred for 5 min. The aqueous phase was extracted with DCM (10.0 mL). The combined organic phase was washed with brine (20.0 mL), dried with anhydrous Na2SOr, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel,DCM / MeOH==100 / l , 0 / 1). The crude product was purified by normal phase HPLC (column: Agela DuraShell NH2 150mm x 30mm x 5um; mobile phase: [Heptane-THF: ACN=2:l(0.2%NH3H2O)]; gradient: 0%-30% B over 10.0 min). ICL-042 (100 mg, 66.5 pmol, 9.48% yield) was obtained as light yellow oil.

[0490] 3H NMR: ICL-042 (400 MHz, CDCh)

[0491] 8: 4.11 . 4.14 (tsj = 6.0 Hz, 6H), 3.69 - 3.73 (t, J = 6.4 Hz, 6H), 3.45 - 3.59 (m, 5H), 2.63 - 2.64 (m, 5H), 2.55 - 2.58 (t, J = 6.0 Hz, 8H), 2.42 - 2.44 (m, 7H), 1.66 - 1.69 (m, 14H), 1.44 (s, 13H), 1.30 - 1.32 (m, 7H), 1.26 (s, 98H), 0.87 - 0.90 (t, J = 6.4 Hz, 18H).

[0492] Example 43. General procedure for preparation o / ICL-043

[0493] To a solution of D13L1-1 (500 mg, 702 umol, 1.00 eq) in DCM (10.0 mL) was added L1T3 (1.19 g, 5.62 mmol, 8.00 eq) and NaBH(OAc)3 (893 mg, 4.21 mmol, 6.00 eq) at 0 °C. The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The residue was poured into aq.NaHCOs (20.0 mL) and stirred for 5 min. The aqueous phase was extracted with DCM (10.0 ml..). The combined organic phase was washed with brine (20.0 ml..), dried with anhydrous NaaSCfi, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, DCM / MeOH=100 / l, 0 / 1). The et ude product was purified by normal phase HPLC (column: Agela DuraShell NH2 150mm x 30mm x 5um; mobile phase: [Heptane-THF: ACN=2:l(0.2%NH3H2O)]; gradient: 0%-30% B over 10.0 min). ICL-043 (100 mg, 59.8 mnol,

[0494] 8.52% yield) was obtained as light yellow oil.

[0495] SH NMR: ICL-043 (400 MHz, CDCh)

[0496] 5: 4.11 .. 4.14 (t, J 6.4 Hz, 6H), 3.69 ■ 3.72 (t, J = 6.4 Hz, 6H), 3.53 3.54 (m, 5H), 2.62 - 2.64 (m, 5H), 2.55 ■ 2.58 (t, 6.4 Hz, 8H), 2.44 ■■ 2.45 (m, 7H), 1.66 1.69 (m, 14H), 1.44 (s,

[0497] 13H), 1.30 ■ 1.32 (m, 7H), 1.26 (s, 122H), 0.87 ■ 0.90 (t, / = 6.4 Hz, 18H).

[0498] Example 44. General procedure for preparation o / D14Ll

[0499] To a solution of D14 (0.50 g, 3.62 mmol, 1 .00 eq) in DCM (10.0 mL) was added LI (1 .69 g, 7.24 mmol, 2.00 eq) DMAP (44.2 mg, 362 jimol, 0.10 eq) and EDCI (1.80 g, 9.41 mmol, 2.60 eq) at 0°C. The mixture was stirred at 0 °C for 30 mins and stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The mixture was poured into water (10.0 mL) and stirred for 10 mins. The aqueous phase was extracted with DCM (3.00 mLx 2). The combined organic phase was washed with 1 N HC1 (10.0 mL) and brine (10.0 mL), dried with anhydrous Naj-SCL, filtered and concentrated in vacuum. D14L1 (1.90 g, 3.34 mmol, 92.3% yield) was obtained as colorless oil.

[0500] General procedure for preparation of ’D14L1-1

[0501] A solution of D14L1 (1 .90 g, 3.34 mmol, 1 .00 eq) in HCl / dioxane (4 M, 19.0 mL, 22.8 eq) was stirred at 25 °C for 2 hrs. TLC (Dichloromethane: Ethyl acetate- 1, Rf - 0.05) showed the reaction was completed. The mixture was filtered and the filter cake w'as collected and dried in drying oven at 45 °C for 2 hrs. D14L1-1 (1.36 g, 3.08 mmol, 92.2% yield, 2HC1) was obtained as white solid. TLC:

[0502] General procedure for preparation of ICL-044

[0503] To a solution of D14L1-1 (1.00 g, 2.27 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T1 (2.12 g, 13.6 mmol, 2.57 mL, 6.00 eq) and NaBH(OAc)3 (2.40 g, 11.3 mmol, 5.00 eq). The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The mixture was concentrated in vacuum. The residue was diluted with water (50.0 mL) and extracted with DCM (10.0 mL x 2). The combined organic layers were washed with brine (20.0 mL), dried over anhydrous NajSCfi, filtered and concentrated in vacuum. The crude product was purified by prep-HPLC (column: Xselect CSH CIS 100 x 30mm x 5um; mobile phase: [H2O (0.04%HCl)-ACN: THF=1: 1]; gradient: 25%-65% B over 12.0 min). ICL-044 (0.90 g, 968 pmol, 42.7% yield) was obtained as yellow oil.

[0504] SH NMR: ICL-044 (400 MHz, CDCb)

[0505] 5: 7.35 (s, 4H), 5.13 (s, 4H), 3.64 - 3.76 (m, 9H), 2.87 (s, 4H), 2.57 - 2.75 (m, 12H), 2.01 (s, 2H), 1.47 - 1.60 (m, 8H), 1.24 - 1.32 (m, 56H), 0.85 - 0.92 (m, 12H).

[0506] Example 45. General procedure far preparation of ICL-045

[0507] To a solution of D14L1-1 (1.00 g, 2.27 mmol, 1.00 eq, 2HCI) in DCM (10.0 mL) was added T2 (2.51 g, 13.6 mmol, 6.00 eq) and NaBH(OAc)3 (2.40 g, 11.3 mmol, 5.00 eq). The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction was completed. The mixture was concentrated in vacuum. The residue was diluted with water (50.0 mL) and extracted with DCM (10.0 ml x 2). The combined organic layers were washed with brine (20.0 mL), dried over anhydrous Na2SOr, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, DCM / MeOH =50 / 1, 10 / 1). ICL-045 (0.50 g, 480 pmol, 21.2% yield) was obtained as yellow oil.

[0508] 1H NMR: ICL-045 (400 MHz, CDCh)

[0509] 5: 7.35 (s, 4H), 5.13 (s, 4H), 3.69 ■■ 3.77 (m, 8H), 2.95 (s, 4H), 2.75 (s, 8H), 2.62 (t, J = 6.4 Hz, 4H), 2.03 (s, 4H), 1.57 (s, 8H), 1.26 (s, 68H), 0.84 - 0.92 (m, 12H).

[0510] Example 46. General procedure for preparation o / ICL-046

[0511] To a solution of D14L1-1 (0.50 g, 1.13 mmol, 1.00 eq, 2HC1) and 4A MS (0.50 g) in DCM (5.00 mL) was added T3 (1.44 g, 6.80 mmol, 6.00 eq) and NaBH(OAc)s (1.20 g, 5.66 mmol, 5.00 eq). The mixture was stirred at 25 °C for 12 hrs. LCMS (ET79194-104-P1A1, Rt = 2.038) showed the reaction was completed. The mixture was concentrated in vacuum. The residue was diluted with w'ater (50.0 mL.) and extracted with DCM (10.0 mL x 2). The combined organic layers were washed with brine (20.0 mL), dried over anhydrous NaaSCfi, filtered and concentrated in vacuum. The residue was purified by normal phase HPLC (column: Agela DuraShell basic 150mm x 30mm x 5um; mobile phase: [Heptane -ACM: THF=1: 1]; gradient: 0%-30% B over 10.0 min). ICL-046 (0.30 g, 168 pniol, 14.8% yield, 64.5% purity) was obtained as yellow' oil.

[0512] SH NMR: ICL-046 (400 MHz, CDCh)

[0513] 5: 7.35 (s, 4H), 5.14 (s, 4H), 3.74 (t, J=6.4 Hz, 4H), 3.40 - 3.69 (m, 4H), 2.59 - 2.74 (m, 8H), 2.40 - 2.50 (m, 4H), 1.51 - 1.77 (m, 12H), 1.44 (d, J = 3.2 Hz, 8H), 1.26 (s, 80H), 0.86 - 0.93 (m, 12H). Example 47. General procedure for preparation of ICL-047

[0514] To a solution of D14L1-1 (0.40 g, 906 pmol, 1 .00 eq, 2HC1) in DCM (4.00 mL) was added T4 (1.24 g, 5.44 mmol, 6.00 eq) and NaBH(OAc)s (960 mg, 4.53 mmol, 5.00 eq) at 0~10 °C. The mixture was stirred at 25 °C for 12 hrs. LCMS showed the reaction wras completed. The mixture was poured into NaHCCL (10.0 mL) and stirred for 15 mins. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous NaaSCU, filtered and concentrated in vacuum. The residue was purified by prep-HPLC (column: Xselect CSH C18 100 x 30mm x 5um; mobile phase: [H2O (0.1%TFA)-ACN: THF-l:!]; gradient: 25%-65% B over 12.0 min). ICL-047 (0.12 g, 83.1 pmol, 9.17% yield, 84.3% purity) was obtained as yellow oil.

[0515] XH NMR: ICL-047 (400 MHz, CDCL)

[0516] 5: 7.35 (s, 4H), 5. 14 (s, 4H), 4.06 (t, J = 6.8 Hz, 8H), 3.70 - 3.78 (m, 4H), 2.60 - 2.67 (m, 5H), 2.29 (t, J = 7.6 Hz, 8H), 1.69 ■ 1.88 (m, 16H), 1.57 - 1.67 (m, 18H), 1.48 (s, 4H), 1.23 ■■ 1.41 (m, 42H), 0.84 ■■ 0.94 (m, 12H).

[0517] Example 48. General procedure for preparation q / 'D15Ll

[0518] To a mixture of DIS (1 .00 g, 6.84 mmol, 1.00 eq) in DCM (10.0 mL) was added LI (3.19 g, 13.7 mmol, 2.00 eq), DMAP (83.6 mg, 684 pmol, 0. 10 eq) and EDO (3.41 g, 17.8 mmol, 2.60 eq)' at 0 °C under Na. The mixture was stirred at 0 °C for 0.5 hr, then heated to 2.5 °C and stirred for 12 hours. LCMS showed the starting material was consumed completely. The mixture was poured into II2O (10.0 mL) and the aqueous phase was extracted with DCM (2 x 5.00 mL). The organic phase was washed with IN HC1 (10.0 mL) and brine (10.0 mL). The organic phase was dried with Na2SO4 and concentrated in vacuum. D15L1 (3.50 g, 6.07 mmol, 88.7% yield) was obtained as colorless oil.

[0519] LCMS: D15L1:

[0520] LCMS (product: RT = 1.572 min; [M / 2+1]+: 419.5)

[0521] General procedure for preparation ofD15Ll~l

[0522] To a mixture of D15L1 (3.50 g, 6.07 mmol, 1.00 eq) in dioxane (15.0 mL) was added HCl / dioxane (4 M, 1.52 mL, 1.00 eq) dropwise for 0.1 hr at 25 °C under N2. The mixture was stirred 25 °C for 2 hours. TLC (Dichloromethane: Ethyl acetate~l: l, product Rf =0) showed the starting material was consumed completely. The mixture was concentrated in vacuum. The product was used to next step without purification. D10L1-1 (2.00 g, 4.45 mmol, 73.3% yield, 2HC1) was obtained as yellow oil.

[0523] TLC:

[0524] General procedure for preparation of ICL-048

[0525] To a solution of D15L1-1 (0.50 g, 1.11 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T1 (1.39 g, 8.90 mmol, 1.68 mL, 8.00 eq) and NaBH(OAc)3 (1.42 g, 6.68 mmol, 6.00 eq) at 25 °C under N2. The reaction mixture was stirred at 25 °C for 12 hrs. LCMS showed the starting material was consumed completely. The mixture was poured into NaHCCh (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous Na2SC>4, filtered and concentrated in vacuum. The crude product was purified by normal phase HPLC( column: Agela DuraShell NH2 150mm x 30mm x 5um;mobile phase: [Heptane- ACN:THF-l: l];gradient:0%-30% B over 8.0 min) to afford ICL-048 (0.10 g, 107 pmol, 6.67% yield) as yellow oil.

[0526] LCMS: ICL-048:

[0527] LCMS (product: RT = 1.030 min: [M / 2-t-l] T 469.5)

[0528] 1H NMR: ICL-048 (400 MHz, CDCL)

[0529] 5: 5.16 - 5.23 (m, 2H), 4.83 - 4.84 (m, 1 H), 3.98 - 4.49 (m, 1H), 3.72 - 3.74 (m, 3H),3.56 - 3.70 (m, 5H), 3.54 (s, 4H), 2.66 - 2.68 (m, 8H), 2.59 (s, 6H), 1.45 (s, 8H), 1.31 (s, 58H), 0.87 (t, J = 7.2 Hz, 12H).

[0530] Example 49. General procedure for preparation o / ICL-049

[0531] To a solution of D15L1-1 (0.50 g, 1.11 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T2 (1.64 g, 8.90 mmol, 8.00 eq) and NaBH(OAc)3 (1.42 g, 6.68 mmol, 6.00 eq) at 25°C under N2. The reaction mixture was stirred at 25 °C for 12 hrs. LCMS showed the starting material was consumed completely. The mixture was poured into NaHCOj (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous NazSCb, filtered and concentrated in vacuum. The etude product was purified by normal phase HPLC(column: Agela DuraShell NH2 150mm x 30mm x 5um;mobile phase: [Heptane - ACN:THF:=1: l];gradient:0%-30% B over 8.0 min) to afford ICL-049 (0.06 g, 57.2 pmol, 4.00% yield) as yellow oil.

[0532] LCMS ICL-049:

[0533] LCMS (product: RT = 1.569 min: [M / 2+1] *: 52b .5) Hl NMR: ICL-049 (400 MHz, CDCh)

[0534] 5: 5.09 - 5.15 (m, 2H), 4.74 - 4.77 (m, 1H), 4.40 - 4.41 (m, 1H), 3.87 - 3.91 (m, 3H), 3.57 -

[0535] 3.67 (m, 6H), 2.57 - 2.60 (m, 6H), 2.50 (s, 3H), 1.38 - 1.52 (m, 15H), 1.37 (s, 75H), 0.81 (t, J

[0536] 6.4 Hz, 12H).

[0537] Example 50. General procedure for preparation q / ’ ICL-050

[0538] To a solution of D15L1-1 (0.50 g, 1.11 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T3 (1.42 g, 6.68 mmol, 6.00 eq) and NaBH(OAc)s (1.18 g, 5.56 mmol, 5.00 eq) at 25°C under N2. The reaction mixture was stirred at 25 °C for 12 hrs. LCMS showed the starting material was consumed completely. The mixture was poured into NaHCCL (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous Na?.SO4, filtered and concentrated in vacuum. The crude product was purified by HPLC(column: Agela DuraShell NH3 150mm x 30mm x 5um;mobile phase: [Heptane-ACN:THF=l : l];gradient:0%-30% B over 10.0 min) to afford ICL-050 (0.20 g, 172 pmol, 15.47% yield) as yellow oil.

[0539] SH NMR: ICL-050 (400 MHz, CDCb)

[0540] 8: 5.09 - 5.15 (m, 2H), 4.74 - 4.77 (m, 1H), 4.40 - 4.41 (m, 1H), 3.87 - 3.91 (m, 3H), 3.57 - 3.67 (m, 6H), 2.57 - 2.60 (m, 6H), 2.50 (s, 3H), 1.38 - 1.52 (m, 15H), 1.37 (s, 75H), 0.81 (t, .J = 6.4 Hz, 12H).

[0541] Example 51. General procedure far preparation of A3

[0542] To a mixture of .43-3 (2.00 g, 3.31 mmol, 1.00 eq) in dioxane (10.0 mL) was added HCl / dioxane (4 M, 10.0 mL, 12.1 eq) dropwise for 0.1 hr at 25 °C under N2. The mixture was stirred 25 °C for 2 hours. LCMS showed the starting material was consumed completely. The mixture was concentrated in vacuum. The product was used to next step without purification. A3 (1.40 g, 2.94 mmol, 88.7% yield, 2HC1) was obtained as colorless oil.

[0543] General procedure for preparation ofICL-051

[0544] To a solution of A3 (1.00 g, 2.10 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T5 (2.32 g, 12.6 mmol, 6.00 eq) and NaBH(OAc)s (2.22 g, 10.5 mmol, 5.00 eq) at 25 °C under Na- The reaction mixture was stirred at 25°C for 12 hrs. LCMS showed the starting material was consumed completely. The mixture was poured into NaHCOs (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous NazSCL, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (Dichloromethane: Methanol —60 / 1, 20 / 1) to afford crude product. The crude product was purified by normal phase HPLC (column: DAICEL CHIRALPAK IC 250mm x m, urn); mobile phase: [Heptane-THF: ACN=2:l(0.2%NH3H2O)]; gradient: 0%-30% B over 10.0 min) to give ICL-051 (50.0 mg, 46.4 pniol, 2.21% yield) as yellow oil.

[0545] TLC:

[0546] 3H NMR: ICL-051 (400 MHz, CDCh)

[0547] 8: 4.14 (t, J = 6.0 Hz 2H), 4.05 (t, J = 6.0 Hz 2H), 3.64 (t, J = 6.8 Hz 4H), 3.49 (s, 3H), 2.54 (d, J -■-■■■ 9.6 Hz 2H), 2.48 - 2.51 (m, 171 n. 1.60 (G J = 11.6 Hz 3 I n. 1.50 - 1.51 (m, 4H), 1.38 - 1.39 (m, 4H), 1 .37 (s, 8H), 1 .35 (s, 72H), 0.81 (t, J = 6.4 Hz, 12H).

[0548] Example 52. General procedure for preparation o / ICL-052 To a solution of A3 (1 .00 g, 2.10 mmol, 1.00 eq, 2HC1) in DCM (10.0 mL) was added T6 (2.67 g, 12.6 mmol, 6.00 eq) and NaBH(OAc)3(2.22 g, 10.5 mmol, 5.00 eq) at 25°C under N2. The reaction mixture was stirred at 25°C for 12 hrs. LCMS showed the starting material was consumed completely. The mixture was poured into NaHCCh (10.0 mL) and stirred for 10 minutes. The aqueous phase was extracted with DCM (5.00 mL x 2). The combined organic phase was washed with brine (10.0 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (Dichloromethane: Methanol-60 / 1, 20 / 1) to afford crude product. The crude product was purified by normal phase HPLC (column: Agela DuraShell NH3150mm x 30mm x Sum; mobile phase: [Heptane-THF: ACN=2:1]; gradient: 0%-30% B over 8.0 min) to give ICL-052 (50.0 mg, 42.0 umol, 2.00% yield) as yellow oil.

[0549] 3H NMR: ICL-052 (400 MHz, CDCfa)

[0550] 5: 4.22 (t, J = 6.4 Hz, 2H), 4.13 (t, J = 6.8 Hz, 2H), 3.72 (t, J = 6.8 Hz, 4H), 2.92 (s, 3H), 2.62 (d, J = 6.4 Hz, 2H ), 2.56 - 2.63 (m, 10H), 2.45 (s, 6H), 1.61 (t, J ■■■ 6.0 Hz, 2H), 1.56 - 1.57 (in, 8H), 1.43 (s, 8H). 1.33 (s, 90H), 0.89 (t, J = 6.8 Hz, 12H).

[0551] Example 53. General procedure for preparation of ’TA-e

[0552] To a solution of T4-a (100 g, 478 mmol, 1.00 eq) in DCM (1000 mL) was added T4-2 (48.9 g, 478 mmol, 59.6 mL, 1.00 eq), DMAP (5.84 g, 47.8 mmol, 0.10 eq) and EDCI (119 g, 622 mmol, 1.30 eq) at 0 °C. The mixture was stirred at 0°C for 30 mins and stirred at 25 °C for 12 hrs. TLC (Dichloromethane: Ethyl acetate=l:l, Rf - 0.43) showed the reaction was completed. The mixture was poured into water (1.50 L) and stirred for 15 mins. The aqueous phase was extracted with DCM (300 mL x 2). The combined organic phase was washed with HO (IN, 1.50 L) and brine (1.50 L), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. T4-c (150 g, 460 mmol, 96.3% yield, 90.0% purity) was obtained as brown oil. TLC:

[0553] General procedure far preparation of T4

[0554] T4-C T4

[0555] To a solution of T4-c (150 g, 460 mmol, 1.00 eq) in DMSO (900 mL) was added NajCOs (48.8 g, 460. mmol, 1.00 eq) and KI (76.4 g, 460 mmol, 1.00 eq) at 25 °C. The mixture was stirred at 85 °C for 12 hrs. TLC (Petroleum ether: Ethyl acetate~2: l, Rf = 0.58) showed the reaction was completed. The mixture was poured into water (3.00 L) and stirred for 15 mins. The aqueous phase was extracted with DCM (500 mL x 3). The combined organic phase was washed with brine (1.00 L), dried with anhydrous NajSOi, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether / Ethyl acetate~100 / l, 1 / 1). T4 (42.5 g, 186 mmol, 40.4% yield) was obtained as colorless oil.

[0556] TLC:

[0557] General procedure for preparation of A3-2

[0558] THF (10 V)

[0559] A3-1 80 °C, 12 hrs A3-2

[0560] To a mixture of A3-1 (5.00 g, 38.7 mmol, 1.00 eq) in THF (45.0 ml) was added Cpd2 (4.84 g, 38.7 mmol, 2.74 mL, 1.00 eq), K2CO3 (21.4 g, 155 mmol, 4.00 eq) and KI (1.28 g, 7.74 mmol, 0.20 eq) at 25 °C under Na- The mixture was stirred 80 °C for 16 hours. TLC (Dichloromethane: Methanol -10:1, product Rf-0.1) showed the starting material was consumed completely. The mixture was filtered and the filtered liquid was concentrated in vacuum. The product was used to next step without purification. A3-2 (5.00 g, 28.9 mmol, 74.6% yield) was obtained as yellow oil.

[0561] 1H NMR: A3-2 (400 MHz, CDCH)

[0562] 5: 3.67 ■ 3.74 (m, 2H), 3.58 ■ 3.61 (m, 4H), 2.90 (d, 12 Hz, 2H), 2.50 (t, 7 = 5.6 Hz, 2H),

[0563] 2.04 (t, y = 5.6 Hz, 2H), 1.67 - 1.72 (m, 2H), 1.49 - 1.52 (m, 3H), 1.24 - 1.27 (m, 2H).

[0564] General procedure for preparation of A3-3

[0565] To a mixture of A3-2 (1.00 g, 5.77 mmol, 1.00 eq) in DCM (10.0 mL) was added LI (2.69 g, 11.5 mmol, 2.00 eq), DMAP (70.5 mg, 577 pmol, 0.10 eq) and EDCI (2.88 g, 15.0 mmol, 2.60 eq) at 0 °C under Na. The mixture was stirred at 0 °C for 0.5 hr, then heated to 25 °C and stirred for 12 hours. LCMS showed the starting material was consumed completely. The mixture was poured into H2O (10.0 mL) and the aqueous phase was extracted with DCM (2 x 5.00 mL). The organic phase was washed with IN HC1 (10.0 mL) and brine (10.0 mL). The organic phase was dried with NaaSCL and concentrated in vacuum. The residue was purified by silica gel chromatography (Dichloromethane: Methanol=100 / 1, 30 / 1) to afford A3-3 (2.00 g, 3.31 mmol, 57.4%' yield) as colorless oil.

[0566] 6: 4.92 (s, 1H), 4.65 (s, 2H), 4.18 (t, J = 6.4 Hz 2H), 3.70 ■ 3.75 (m, 4H), 3.49 ■■ 3.52 (m, 6H) 3.28 ■ 3.30 (m, 6H), 2.64 ■■ 2.67 (m, 4H), 2.57 (t, J = 6.4 Hz 2H), 1.73 (s, 2H), 1.70 ■■ 1.71 (m, 61 b. 1.60 (s, 18H).

[0567] Example 54. LNP formulation and characterization

[0568] In brief, lipids were dissolved in ethanol at molar ratios of 20-40: 10-30:30-50:1-3 (ionizable lipid / DOPE or DSPC / Cholesterol / PEG-DMG2k) as specified i n Table 2. The lipid mixture was combined with a 25 mM sodium acetate buffer (pH 4) containing VZV gE mRNA made inhouse at a ratio of 3:1 (aqueous: ethanol) using a microfluidic mixer (Precision Nanosystems, Vancouver, BC). Formulations were dialyzed against 20 mM Tris buffer (pH 7.5) in dialysis cassettes for at least 18 hr. Formulations were concentrated using Amicon ultra centrifugal filters (EMD Millipore, Billerica, MA), passed through a 0.22-pm filter. All formulations were tested for particle size and PDI using a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK). RNA encapsulation and concentration are measured with QUANT-IT™ RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, CA). Based on the results, the LNPs are diluted to the desired final concentration with sucrose and tris buffer, and stored at - 80 C until use.

[0569] Most LNPs have sizes ranging from 30-120 nm, and with encapsulation efficiency >80%. LNPs with size > 140 nm and / or encapsulation efficiency <50% were generally not tested further.

[0570] Table 2

[0571] Example 55. In vivo studies

[0572] Studies were performed with 6-8 week old female C57BL / 6 mice (Jackson) according to guidelines established by IUCUC. 50 pl.. of LNP solution containing 10 pg of mRNA were administered by intramuscular injection or tail vein injection. In some instances, multiple doses were used. Animals were euthanized at specific time point (e.g 21 days) and terminal bleeding was performed. Blood samples were collected via cardiac puncture, and in some groups spleens were also collected. Below is a detailed study protocol and study design for a typical in vivo study used to evaluate the LNP formulations.

[0573] Table 3 Study timetable: In vivo study design

[0574] Table 4 Test Animat Description

[0575] Table 5 Test System Management

[0576] Table 6 Study Design

[0577] Table 7 Study Desigu

[0578] Example 56. Serum total IgG measured by ELISA

[0579] VZV gE-specific serum total IgG induced by the VZV gE variant mRNAs / candidate cationic lipid vaccine were measured by ELISA.

[0580] A 96-well capture plate was coated at 3pg / mL using Abeam VZV gE recombinant protein (Abeam, VZV gE Recombinant Protein, 43050) and incubated overnight at 4°C. The plate was washed three times using IX PBST and blocked for one hour 120±10min at 37 °C. In the concurrent negative control group, all samples were diluted with 1% BSA by a factor of 100, and the other samples were diluted with 1% BSA by a 3-folds gradient dihition and dilute samples to be tested until negative (The sample OD < cut off value is negative, and Cutoff value ~ 2.1 x MeanOD (concurrent negative control group).

[0581] The plate was washed three times using IX PBST and pat dry. Treated sample at 100 uL / well (single well), 100 pL of secondary antibody (Goat anti-mouse IgG Fc Human / Bovine / Horse SP ads-HRP be diluted 5000-fold with 1%BSA) were added incubate it for 60+5min at 37 °C. The plate was washed 6 times and 100 ,u.L of TMB Substrate Solution were added into each well, incubate for 10±5min at room temperature in the dark. lOOpL of stop solution was added to each well and the capture plate was read at 450nm on a Biotek plate reader. Results are shown in Figure 1 .

[0582] LNP 253, an LNP formulation based on lipid ICL-009 and LNP 256, an LNP formulation based on lipid 1CL-024, both outperformed LNP232 which is an LNP formulation using the industry gold standard SM102, with a p value <0.01. The T-test performed is a 1 tail, equal variances

[0583] T-test.

Claims

What is claimed is:1 . Compounds are provided having the following structure:or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:X is a divalent or trivalent group derived from straight-chained or branched C1-20 alkane, C2-20 alkene, €2,-20 alkyne, 3- to 20-membered aromatic ring system or 3- to 20-membered nonaromatic ring system, or any combination thereof, wherein any carbon in C1-20 alkane, C2-20 alkene and C2-20 alkyne is optionally replaced with one or more atoms selected from the group consisting of S, O and N atoms, with the proviso that X is not piperazine;Ir, 2r and 3r are the same or different, and are each independently integers ranging from 0 to 6; andR11, R12, R21, R22, R31 and R32 are the same or different, and are independently straight-chained C8-C20 alkyl group, wherein a carbon atom in the alkyl group is optionally replaced with2. Tire compound of claim 1 , wherein the X is a divalent or trivalent group derived from straight-chained or branched Ci-w alkane, C2-10 alkene, C2-10 alkyne, 3- to 10-membered aromatic ring system or 3- to 10-membered non-aromatic ring system, or any combination thereof, wherein any carbon in C1-10 alkane, C2-10 alkene and C2-10 alkyne is optionally replaced with one or more atoms selected from the group consisting of S, O and N atoms, with the proviso that X is not piperazine,3. Hie compound of claim 1 or 2, wherein the X is a divalent or trivalent group derived from straight-chained or branched C1-4 alkane, C2-4 alkene, C2-4 alkyne, 6- to 10-membered aromatic ring system or 6- to 10-membered non-aromatic ring system, or any combination thereof,wherein any carbon in Cmalkane, C2.4alke.ne and Cnnalkyne is optionally replaced with one or more atoms selected from the group consisting of S, O and N atoms, with the proviso that X is not piperazine.

4. The compound of any one of claims 1-3, wherein the X is a divalent or trivalent group derived from straight-chained or branched CM alkane, C2-4 alkene, 6- to 10-membered aromatic ring system or 6- to 10-membered non-aromatic ring system, or any combination thereof, wherein any carbon in Ci ^alkane and C24 alkene is optionally replaced with one or more atoms selected from the group consisting of S, O and N atoms, with the proviso that X is not piperazine.

5. The compound of any one of claims 1-4, wherein X is selected from the group consisting of6. The compound of any one of claims 1-5, wherein Rn, R12, R?i, R22, R31 and R32 are independently straight-chained Cg-Cis alkyl group.

7. The compound of any one of claims 1-6, wherein Rn, Ra, R21, R22, R31 and R32 are independently straight-chained Cio-Cie alkyl group.

8. The compound of any one of claims 1-7, wherein Rn, R12, R21, R22, R?i and R32 are independently selected from the group consisting of, , andare straight- chained alkyl groups.

9. Hie compound of any one of claims 1-8, wherein Ir, 2r and 3r are each independently integers ranging from 0 to 4,10. The compound of any one of claims 1-9. wherein Ir, 2r and 3r are each independently integers ranging from 0 to 2.

11. A compound selected from a compound in Table below;No. StructureSCL-035ICT.-036wherein in the structure of the compounds listed in the abve table, a -Cntfan+i group represents a straight-chained alkyl group, wherein n represents the number of carbon.

12. A composition comprising the compound of any one of claims 1-11 and a therapeutic agent.

13. The composition of claim 12, further comprising one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipid.

14. The composition of claim 13, wherein the composition comprises one neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.

15. The composition of claim 14, wherein the neutral lipid is DOPE or DSPC.

16. "Die composition of any one of claims 13-15, wherein the molar ratio of the compound to the neutral lipid ranges from about 1: 4 to about 3: 1.

17. The composition of claim 13, wherein the steroid is cholesterol.

18. The composition of claim 17, wherein the molar ratio of the compound to cholesterol ranges from about 1: 5 to about 5: 1.

19. The composition of claim 13, wherein the polymer conjugated lipid is pegylated lipid.

20. Tire composition of claim 19, wherein the molar ratio of the compound to the pegylated lipid ranges from about 100: 1 to about 10: 1.

21. The composition of claim 19 or 20, wherein the pegylated lipid is PEG-DMG.

22. The composition of any one of claims 12-21, wherein the therapeutic agent comprises a nucleic acid.

23. The composition of claim 22, wherein the nucleic acid is selected from DNA, RNA, and hybrids thereof.

24. The composition of claim 23, wherein the nucleic acid is selected from antisense oligonucleotides, antisense and messenger RNA.2.

5. A method for administering a therapeutic agent to a patient in need thereof, the method comprising preparing or providing the composition of any one of claims 12-24, and administering the composition to the patient.

26. Use of the composition of any one of claims 12-24 in the manufacture of a medicament for treating a disease.2.

7. The composition of any one of claims 12-24, for use as a medicament for treating a disease.

28. A pharmaceutical composition for treating a disease, which comprises the composition of any one of claims 12-24 as therapeutically active substance.

29. A method for preparing compounds of any one of claims 1-11, comprising step 1step 2step 3wherein in step 1, under the presence of a catalyst, reactant D and reactant L undergo an esterification reaction to obtain compound DL; in step 2, deprotect an amino protecting group Pr- in DL to obtain DL-1 with an amino group: preferably Pr-group is Boe- group, and then preferably the deprotection reaction is occurred in the presence of HCl / dioxane in 4M, 10V; in step 3, in the presence of a catalyst, reactant DL-1 undergoes alkylation reaction to obtain compound formula (I), wherein an alkylating agent is an olefin T , wherein R representsR;i, Ri2, R21 or R22; wherein X, Ir, 2r, Rn, R12, R21 and R22 are as defined in claim 1, and Pr- group represents an amino protecting group.

30. A method for preparing compounds of any one of claims 1-11, comprising step rstep 3wherein the above steps l’-3’ are similar with those steps described in claim 29, and X, Ir, 2r, 3r, Ru, R12, R21, R22, R31 and R32 are as defined in claim 1, and Pr- group represents an amino protecting group.

31. A method for preparing an intermediate T of claim 29 or 30, comprisingstep 1heatT-cTstep ii step a wherein in step i, under the presence of a catalyst, reactant T-a and reactant T-2 undergo an esterification reaction to obtain compound T-c, wherein the catalyst is preferably DMAP, and the amount of the catalyst is 0.05-1.0eq, preferably 0.05-0.5eq and more preferably O.leq; in step ii, in the presence of a base, the T-C is heated in DMSO solvent to undergo an oxidation reaction to obtain a corresponding carbonyl compound T, wherein the base is preferably NazCOs. and the amount of NazCOs is 0.5-3.0eq, preferably 1.0-2.0eq and more preferably l.Oeq; and the volume of DMSO is 1-20 V, preferably 1-10 V, and more preferably 6V.

32. A method for preparing an intermediate D of claim 29, comprisingstep a wherein intermediate D is prepared by reactingin a reaction solvent(such as THF) at a reaction temperature (such as 80°C) for a reaction time (such as 12 hours); and K ■( '()• and KI are preferably added, wherein X, Ir and 2r are as defined in claim 1.

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