Ether-containing novel ionizable lipids and their uses thereof

Abstract

Compounds are provided having the following structure: (I), or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein Y, Q, R, Ri, R2, m, n and o 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. Formula (I).

Description

[0001] ETHER-CONTAINING NOVEL IONIZABLE LIPIDS AND THEIR USES THEREOE

[0002] Cross Reference To Related Applications

[0003] This application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63 / 664,935 filed June 27, 2024, the entirety of which is incorporated herein by reference.

[0004] Technical Field

[0005] The present invention provides, in part, novel ionizable lipids that contain ether groups and of Formula (I), or a pharmaceutically acceptable salt thereof. The compounds described herein can be used to develop lipid nanoparticle formulations and effectively and safely deliver oligonucleotides for applications include but not limited to mRNA vaccine, mRNA therapy, siRNA therapy, gene therapy and so on.

[0006] Background of the invention

[0007] 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 combating 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 the same which is the effective and safe delivery of the cargo.

[0008] (Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine, N Engl J Med 2020; 383:2603-2615, DOI: 10.1056 / NEJMoa2034577)

[0009] 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 and genome editing. As such, a powerful delivery vehicle called lipid nanoparticles (LNP) was developed to 1) protect the mRNA from RNase degradation 2) deliver the mRNA intracellularly so that it can be translated to the corresponding protein that will have downstream therapeutic effects.

[0010] (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) While LNPs are typically made of 4 or 5 different lipid components, the most crucial lipid is the ionizable lipid, as it enables the LNP to 1) effectively encapsulate and protect the mRNA and 2) effectively 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.

[0011] (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)

[0012] Summary of the invention

[0013] In brief, the present invention provides the synthesis and application of ionizable lipid compounds, including pharmaceutically acceptable salts thereof, that are used in combination with other lipid components such as helper lipids, charged lipids, steroids, PEG lipids or related analogues to form LNPs and deliver oligonucleotide therapeutics. The therapeutic applications of these lipids and related LNPs include siRNA therapy, prophylactic mRNA vaccine, therapeutic mRNA vaccine, mRNA therapy, genome editing.

[0014] In one embodiment, compounds are provided having the following structure: or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein Y, Q, R, Ri, R2, m, n and o are as defined herein.

[0015] In the present invention, all ionizable lipids are synthesized in 5-6 relatively simple steps, and many showed superior efficacy in vivo compared to industry gold standard such as SMI 02, demonstrating the high delivery efficacy of the lipids presented herein. 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 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] The present invention serves to protect the chemical structures of ionizable lipids listed herein, as well as those that are similar in structure. The present invention also serves to protect the use of the ionizable lipids herein in the context of LNP formulation, and oligonucleotide therapy applications such as siRNA therapy, gene therapy, mRNA therapy, mRNA vaccines.

[0017] Based on the current invention, additional ionizable lipids can be synthesized to expand and further optimize the ionizable lipids library provided herein. In addition, further LNP formulation optimization of the ionizable lipids presented here can take place to further improve the various aspects of the LNPs such as efficacy, stability, toxicity and so on.

[0018] Brief description of the drawings

[0019] 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 figures.

[0020] Figure 1 shows VZV gE-specific serum total IgG induced by the VZV gE variant mRNAs / candidate cationic lipid vaccine.

[0021] Figure 2 shows the size and encapsulation efficiency (EE%) of the LNPs at various timepoints during the storage.

[0022] Figure 3 shows the serum total Human erythropoietin (hEPO) levels of the LNPs. Detailed description of the invention

[0023] 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.

[0024] Definition

[0025] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0026] 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”.

[0027] Reference throughout this specification to “one embodiment” or “an 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.

[0028] 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.

[0029] 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.

[0030] 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 the 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.

[0031] 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.

[0032] “Gene product,” as used herein, refers to a product of a gene such as an RNA transcript or a polypeptide.

[0033] 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.

[0034] A “steroid” is a compound comprising the following carbon skeleton:

[0035] Non-limiting examples of steroids include cholesterol, and the like.

[0036] 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.

[0037] 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. 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.

[0038] 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).

[0039] 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) 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) 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.

[0040] 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.

[0041] 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.

[0042] As used herein, the term “aqueous solution” refers to a composition comprising water.

[0043] “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.

[0044] “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.

[0045] 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.

[0046] “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.

[0047] The term “alkane,” as used herein, refers to a saturated hydrocarbon compound. For example, Ci-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.

[0048] 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.

[0049] 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=CHCH3), 2-propenyl [(CH3)C=CH2], and 3- propenyl (-CFbClfcCFE) 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.

[0050] 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, C4 alkyne replaced with two O atoms refers to an alkynyl group having two carbon atoms and two oxygen 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.

[0051] 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 (-OCCH3) and 3-propynyl (HOCCH2-) 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.

[0052] The nitrogen-containing heterocyclic group refers to a heterocyclic group (preferably 3-10 membered heterocyclic group) containing nitrogen atom as a heteroatom constituting the ring. In certain embodiments, the nitrogen containing heterocyclic groups are saturated. In other embodiments, the nitrogen-containing heterocyclic groups are unsaturated. In other embodiments, the nitrogen-containing heterocyclic groups may be substituted or unsubstituted. Example includes aziridinyl, azetidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, piperidyl, tetrahydropyridyl, pyridyl, homopiperidinyl, octahydroazocinyl, imidazolidinyl, imidazolinyl, imidazolyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, piperazinyl, pyrazinyl, pyridazinyl, pyrimidinyl, homopiperazinyl, triazolyl, tetrazolyl, indolinyl, indolyl, isoindolinyl, isoindolyl, benzimidazolyl, indazolyl, benzotriazolyl, pyrazolopyridinyl, quinolyl, tetrahydroquinolinyl, quinolyl, tetrahydroisoquinolinyl, isoquinolinyl, quinolidinyl, cinnolinyl, phthalazinyl, quinazolinyl, dihydroquinoxalinyl, quinoxalinyl, naphthylidinyl, purinyl, pteridinyl, and quinuclidinyl groups.

[0053] “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 includes both substituted alkyl radicals and alkyl radicals having no substitution.

[0054] “Prodrug” 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 prodrug may be inactive when administered to a subject in need thereof, but is converted in vivo to an active compound of the invention. Prodrugs are 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, H., 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 prodrugs 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.

[0055] The invention disclosed herein is also meant to encompass all pharmaceutically acceptable compounds of the compound of structure (I) 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,HC,13C,14C,13N,15N,15O,17O,18O,31P,32P,35S,18F,36C1,123I, and125I, respectively. These radiolabeled compounds could be useful to help determine or measure the effectiveness 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), 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.

[0056] 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 asHC,18F,15O and13N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds of structure (I) 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.

[0057] 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.

[0058] “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.

[0059] “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.

[0060] “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.

[0061] “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-l,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, trifluoroacetic acid, undecylenic acid, and the like. “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.

[0062] 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.

[0063] 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.

[0064] “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:

[0065] (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;

[0066] (ii) inhibiting the disease or condition, i.e., arresting its development;

[0067] (iii) relieving the disease or condition, i.e., causing regression of the disease or condition; or

[0068] (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.

[0069] 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. 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 non-superimposable mirror images of one another.

[0070] 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.

[0071] Compounds

[0072] 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.

[0073] In one embodiment, the compounds have the following structure (I): or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0074] Y is one or more of -OCO-, -COO-, -NHCO-, -CONH-, -O-, -S-, -SO2- or -SO-;

[0075] Q is straight-chained C1-10 alkyl group, wherein a carbon atom in the alkyl group is optionally replaced with an -COO- group;

[0076] R is H or Ci-4alkyl;

[0077] Ri and R2 are each the same or different, and independently selected from the group consisting of hydrogen, C1-6 alkyl, C2-6 alkenyl and C2-6 alkynyl, wherein a carbon atom in the C1-6 alkyl, C2-6 alkenyl or C2-6 alkynyl is optionally replaced with one to three heteroatoms independently selected from the group consisting of N, O, and S; or optionally Ri and R2 attach together with their adjacent N atom to form a saturated or unsaturated, substituted or unsubstituted 3-10 membered nitrogen-containing heterocyclic group, m is an integer ranging from 2 to 14, and n and o are the same or different, and are each independently integers ranging from 2 to 14.

[0078] In one embodiment, the compound has the following structure: or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0079] Y is -OCO-, -COO-, -NHCO-, -CONH-, -O-, -S-, -SO2- or -SO-;

[0080] Q is straight-chained C1-10 alkylene group, wherein an CH2 in the alkylene group is optionally replaced with an -COO- group;

[0081] R is H or Ci-4alkyl;

[0082] Ri and R2 are each the same or different, and independently selected from the group consisting of hydrogen, C1-6 alkyl, C2-6 alkenyl and C2-6 alkynyl, wherein an CH2 in the C1-6 alkyl, C2-6 alkenyl or C2-6 alkynyl is optionally replaced with one to three heteroatoms independently selected from the group consisting of NH, O, and S; or optionally Ri and R2 attach together with their adjacent N atom to form a saturated or unsaturated, substituted or unsubstituted 3- 10 membered nitrogen-containing heterocyclic group, m is an integer ranging from 2 to 14, and n and o are the same or different, and are each independently integers ranging from 2 to 14. Preferred embodiments of Y, Q, R, Ri, R2, m, n and o are shown below. Embodiments of the compound represented by the formula (I) are examples of all combinations of the specific embodiments shown below.

[0083] In some embodiments, Y in formula (I) is -OCO-, -COO-, -NHCO-, -CONH-, -O-, -S-, -SO2- or -SO- (hereinafter referred to as Y-l).

[0084] In some embodiments, Y in formula (I) is -OCO-, -COO-, -NHCO- or -CONH- (hereinafter referred to as Y-2).

[0085] In some embodiments, Y in formula (I) is -COO-, -NHCO- or -CONH- (hereinafter referred to as Y-3).

[0086] In some embodiments, Q in formula (I) is straight-chained C1-10 alkyl group, wherein a carbon atom in the alkyl group is optionally replaced with an -COO- group (hereinafter referred to as Q-l).

[0087] In some embodiments, Q in formula (I) is straight-chained C2-8 alkyl group, wherein a carbon atom in the alkyl group is optionally replaced with an -COO- group (hereinafter referred to as Q-2).

[0088] In some embodiments, Q in formula (I) is selected from the group consisting of ethyl, n-propyl, n-butyl and - (hereinafter referred to as Q-3).

[0089] In some embodiments, Q in formula (I) is straight-chained C2-8 alkylene group, wherein an CH2 in the alkylene group is optionally replaced with an -COO- group (hereinafter referred to as Q- 4).

[0090] In some embodiments, Q in formula (I) is selected from the group consisting of ethylene, n- propylene, n-butylene and ' (hereinafter referred to as Q-5).

[0091] In some embodiments, R in formula (I) is H or Ci-4alkyl (hereinafter referred to as R-l).

[0092] In some embodiments, R in formula (I) is H, methyl or ethyl (hereinafter referred to as R-2). In some embodiments, R in formula (I) is H or methyl (hereinafter referred to as R-3).

[0093] In some embodiments, Ri and R2 in formula (I) are each the same or different, and independently selected from the group consisting of hydrogen, C1-6 alkyl, C2-6 alkenyl and C2- 6 alkynyl, wherein a carbon atom in the C1-6 alkyl, C2-6 alkenyl or C2-6 alkynyl is optionally replaced with one to three heteroatoms independently selected from the group consisting of N, O, and S; or optionally Ri and R2 attach together with their adjacent N atom to form a saturated or unsaturated, substituted or unsubstituted 3-10 membered nitrogen-containing heterocyclic group (hereinafter referred to as R12-I).

[0094] In some embodiments, Ri and R2 in formula (I) are each the same or different, and independently selected from the group consisting of hydrogen and C1-6 alkyl, wherein a carbon atom in the C1-6 alkyl is optionally replaced with one to three heteroatoms independently selected from the group consisting of N, O, and S; or optionally Ri and R2 attach together with their adjacent N atom to form a saturated or unsaturated, substituted or unsubstituted 3-10 membered nitrogen-containing heterocyclic group (hereinafter referred to as R12-2).

[0095] In some embodiments, Ri and R2 in formula (I) are each the same or different, and independently selected from the group consisting of hydrogen and C1-3 alkyl, wherein a carbon atom in the C1-3 alkyl is optionally replaced with one to three heteroatoms independently selected from the group consisting of N, O, and S; or optionally Ri and R2 attach together with their adjacent N atom to form a saturated or unsaturated, substituted or unsubstituted 4-6 membered nitrogen-containing heterocyclic group (hereinafter referred to as R12-3).

[0096] In some embodiments, Ri and R2 in formula (I) are independently selected from the group consisting of methyl, ethyl, n-propyl and n-butyl (hereinafter referred to as R12-4).

[0097] In some embodiments, the heterocyclic group formed by Ri and R2 attaching together with r"\. i their adjacent N atom in formula (I) is ' or (hereinafter referred to as R12-5).

[0098] In some embodiments, Ri and R2 in formula (I) are each the same or different, and independently selected from the group consisting of hydrogen and C1-3 alkyl, wherein an CH2 in the C1-3 alkyl is optionally replaced with one heteroatom independently selected from the group consisting of NH, O, and S; or optionally Ri and R2 attach together with their adjacent N atom to form a saturated or unsaturated, substituted or unsubstituted 4-6 membered nitrogencontaining heterocyclic group (hereinafter referred to as R12-6).

[0099] In some embodiments, m in formula (I) is an integer ranging from 2 to 14 (hereinafter referred to as m-1).

[0100] In some embodiments, m in formula (I) is an integer ranging from 2 to 10 (hereinafter referred to as m-2).

[0101] In some embodiments, m in formula (I) is an integer ranging from 2 to 8 (hereinafter referred to as m-3).

[0102] In some embodiments, m in formula (I) is an integer ranging from 2 to 6 (hereinafter referred to as m-4).

[0103] In some embodiments, m in formula (I) is 4 (hereinafter referred to as m-5).

[0104] In some embodiments, n in formula (I) is an integer ranging from 2 to 14 (hereinafter referred to as n-1).

[0105] In some embodiments, n in formula (I) is an integer ranging from 2 to 10 (hereinafter referred to as n-2).

[0106] In some embodiments, n in formula (I) is an integer ranging from 2 to 8 (hereinafter referred to as n-3).

[0107] In some embodiments, n in formula (I) is an integer ranging from 4 to 6 (hereinafter referred to as n-4).

[0108] In some embodiments, n in formula (I) is 6 (hereinafter referred to as n-5).

[0109] In some embodiments, o in formula (I) is an integer ranging from 2 to 14 (hereinafter referred to as o-l).

[0110] In some embodiments, o in formula (I) is an integer ranging from 2 to 10 (hereinafter referred to as o-2). In some embodiments, o in formula (I) is an integer ranging from 2 to 8 (hereinafter referred to as o-3).

[0111] In some embodiments, o in formula (I) is an integer ranging from 4 to 6 (hereinafter referred to as o-4).

[0112] In some embodiments, o in formula (I) is 6 (hereinafter referred to as o-5).

[0113] In some embodiments, the compounds represented by the formula (I) are the combinations of Y-l, Q-2, R-3, R12-4, m-2, n-2 and o-2.

[0114] In some embodiments, the compounds represented by the formula (I) are the combinations of Y-2, Q-3, R-2, R -4, m-5, n-5 and o-5.

[0115] In some embodiments, the compounds represented by the formula (I) are the combinations of Y-3, Q-3, R-3, R12-2, m-3, n-3 and o-3.

[0116] In some embodiments, the compounds represented by the formula (I) are the combinations of Y-3, Q-3, R-2, R12-I, m-2, n-4 and o-3.

[0117] In various different embodiments, the compound has one of the structures set forth in Table 1 below.

[0118] Table 1 Representative compounds

[0119] It is to be understood that any embodiment of the compounds of structure (I) as described above, as well as any particular substituents and / or variables in the compounds of structure (I) as described above, may be independently combined with other embodiments and / or substituents and / or variables of the compounds of structure (I) to form embodiments of the invention not explicitly set forth above.

[0120] 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.

[0121] In some embodiments, compositions are provided comprising any one or more of the compounds of structure (I) and a therapeutic agent. For example, in some embodiments, the composition comprises any of the compounds of structure (I) 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. 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: 5 to about 5: 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- 1-0- (co -methoxy (polyethoxy) ethyl) succinate (PEG- S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co - 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 comprises 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. 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.

[0128] In various embodiments, the present invention relates to a method for administering a therapeutic agent to a patient in need thereof, the method comprises preparing or providing any one of the above compounds or compositions and administering the compound or composition to the patient.

[0129] In one embodiment, the present invention relates to a use of any one of the above compounds or compositions in the manufacture of a medicament for treating a disease.

[0130] In one embodiment, the present invention relates to any one of the above compounds or compositions, for use as a medicament for treating a disease.

[0131] In one embodiment, the present invention relates to a pharmaceutical composition for treating a disease, which comprises any one of the above compounds and a therapeutic agent.

[0132] 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) and one or more pharmaceutically acceptable carriers, diluents, or excipients. The compound of structure (I) 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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. 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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 suitable non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycols.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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 with 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.

[0146] 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, including 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.

[0147] 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 the 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.

[0148] Synthetic method

[0149] Methods for preparing the above compounds and compositions are described below, and / or are known in the art.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] The following general reaction scheme 1 or 2 illustrates a method for preparing compounds of the present invention, i.e., compounds of structure (II), (III) or (IV) or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein Q, R, Ri, R2, m, n and o are as defined herein.

[0154] 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).

[0155] Scheme 1: Synthesis of general T1 and Formula (II)

[0156] Scheme 1 provides an exemplary method for preparation of compounds of structure (II), wherein Q, R, Ri, R2, m, n and o are as defined herein. TBS- group represents a hydroxyl protecting group. In other embodiments, the hydroxyl protecting group may be other hydroxyl protecting groups known in the art.

[0157] In step 1, reactant 4A reacts with reactant in a solvent in the presence of KOH and tetrabutyl ammonium bromide(TBAB) in a reaction temperature for a reaction time.

[0158] The amount of is 0.5-4. Oeq, preferably 1.0-3.0 eq, and more preferably 2.0eq.

[0159] The amount of KOH is 1.5-4.5eq, preferably 2.0-4.0 eq, and more preferably 3. Oeq.

[0160] The amount of TBAB is 0.05-0.2eq, preferably 0.05-0.1 eq, and more preferably O.leq.

[0161] The reaction temperature is 60-120°C, preferably 80-120°C, and more preferably 100°C.

[0162] The reaction time is 8-20 hours, preferably 10-15 hours, and more preferably 12 hours. The solvent is preferably toluene (Tol) in step 1, and the volume of the solvent is 5-30 V, preferably 5-20 V, and more preferably 10V.

[0163] In step 2, intermediate 4C- 1 reacts with reactant in a solvent in the presence of NaH in a reaction temperature for a reaction time.

[0164] The amount of is 0.5-4. Oeq, preferably 1.0-3.0eq, and more preferably 2.0eq.

[0165] The amount of NaH is 2.0-6. Oeq, preferably 3.0-5. Oeq, and more preferably 4. Oeq.

[0166] The reaction temperature is 40-100°C, preferably 60-80°C, and more preferably 70°C.

[0167] The reaction time is 8-20 hours, preferably 10-15 hours, and more preferably 12 hours.

[0168] The solvent is preferably tetrahydrofuran (THF) in step 2, and the volume of the solvent is 5- 30 V, preferably 5-20 V, and more preferably 10V.

[0169] In step 3, deprotect the hydroxyl protecting group tert-butyldimethylsilyl ethers (TBS- group) in intermediate 4C to obtain intermediate 4E with hydroxyl group in a solvent in the presence of tetra-n-butylammonium fluoride (TBAF) in a reaction temperature for a reaction time.

[0170] The amount of TBAF is 0.5-4.0eq, preferably 1.0-3.0eq, and more preferably 2. Oeq.

[0171] The reaction temperature is 10-30°C, and preferably 25°C.

[0172] The reaction time is 0.5-3 hours, preferably 1-3 hours, and more preferably 2 hours.

[0173] The solvent is preferably THF in step 3, and the volume of the solvent is 5-30 V, preferably 5- 20 V, and more preferably 10V.

[0174] In step 4, the esterification reaction between intermediate 4E and reactant obtains intermediate 4B in a solvent in the presence of l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCI) and 4-dimethylaminopyridine (DMAP) in a reaction temperature for a reaction time.

[0175] The amount of is 0.5-4.0eq, preferably 1.0-3.0 eq, and more preferably 2.0eq. The amount of EDCI is 0.5-2.0eq, preferably 1.0-2.0 eq, and more preferably 1.3eq.

[0176] The amount of DMAP is 0.05-0.3eq, preferably 0.05-0.2 eq, and more preferably O.leq.

[0177] The reaction temperature is 10-40°C, preferably 20-30°C, and more preferably 25°C.

[0178] The reaction time is 8-20 hours, preferably 10-15 hours, and more preferably 12 hours.

[0179] The solvent is preferably dichloromethane (DCM) in step 4, and the volume of the solvent is 5-30 V, preferably 5-20 V, and more preferably 10V.

[0180] In step 5, in the presence of catalysts, intermediate 4B is reduced to obtain intermediate T1 under H2 (50 psi).

[0181] The catalysts are preferably 10% Pd / C and 10%Pd(OH)2.

[0182] The reaction temperature is 30-70°C, preferably 40-60°C, and more preferably 50°C.

[0183] The reaction time is 8-20 hours, preferably 10-15 hours, and more preferably 12 hours.

[0184] The solvent is preferably THF, and the volume of the solvent is 5-30 V, preferably 5-20 V, and more preferably 10V.

[0185] In step 6, intermediate T1 reacts with reactant to obtain a compound of formula (II) in a solvent in the presence of EDCI and DMAP in a reaction temperature for a reaction time.

[0186] The amount of is 0.5-2.0eq, preferably 1.0-1.5 eq, and more preferably 1.2eq.

[0187] The amount of EDCI is 0.5-2.0eq, preferably 1.0-2.0 eq, and more preferably 1.5eq.

[0188] The amount of DMAP is 0.05-0.3eq, preferably 0.05-0.2 eq, and more preferably O.leq.

[0189] The reaction temperature is 10-40°C, preferably 20-30°C, and more preferably 20°C.

[0190] The reaction time is 8-20 hours, preferably 10-15 hours, and more preferably 12 hours.

[0191] The solvent is preferably DCM in step 6, and the volume of the solvent is 5-30 V, preferably 5-20 V, and more preferably 7V. Scheme 2: Synthesis of general T2, Formula (III) and Formula (IV)

[0192] Scheme 2 provides an exemplary method for preparation of compounds of structure (III) or (IV), wherein Q, R, Ri, R2, m, n and o are as defined herein. TBS- group represents a hydroxyl protecting group. In other embodiments, the hydroxyl protecting group may be other hydroxyl protecting groups known in the art. Bn- group represents a carboxyl protecting group. In other embodiments, the carboxyl protecting group may be other carboxyl protecting groups known in the art.

[0193] In step 1, reactant 13 reacts with in solvents in the presence of NaH in a reaction temperature for a temperature time.

[0194] The amount of is 0.5-4. Oeq, preferably 1.0-3.0 eq, and more preferably 2.2eq.

[0195] The amount of NaH is 0.5-4. Oeq, preferably 1.0-3.0 eq, and more preferably 2.2eq.

[0196] The reaction temperature is 0-30°C, preferably 0-20°C.

[0197] The reaction time is 10-30 hours, preferably 12-20 hours, and more preferably 16.5 hours.

[0198] The solvent is preferably Tol and dimethylformamide (DMF) in step 1, the volume of Tol is 5- 30 V, preferably 5-20 V, and more preferably 10V; the volume of DMF is 15-50 V, preferably 20-40 V, and more preferably 30V.

[0199] In step 2, intermediate 14 is oxidized by 2-iodoxybenzoic acid (IBX) in a solvent in a reaction temperature for a temperature time.

[0200] The amount of IBX is 0.5-2. Oeq, preferably 1.0-2.0 eq, and more preferably 1.2eq.

[0201] The reaction temperature is 60-100°C, preferably 70-90°C, and more preferably 80°C.

[0202] The reaction time is 2-6 hours, preferably 3-5 hours, and more preferably 4 hours.

[0203] The solvent is preferably MeCN in step 2, and the volume of the solvent is 5-30 V, preferably 5-20 V, and more preferably 10V.

[0204] In step 3, intermediate 15 is oxidized in the presence of NaCICh and NafTPCh in solvents in a reaction temperature for a temperature time.

[0205] The amount of NaCIC is 3.0-6.0 eq, preferably 4.0-5.0 eq, and more preferably 4.15eq.

[0206] The amount of NaH2POa is 1.0-3.0 eq, preferably 2.0-3.0 eq, and more preferably 2.1eq.

[0207] The reaction temperature is 10-30°C, and preferably 20°C. The reaction time is 2-6 hours, preferably 3-5 hours, and more preferably 4 hours.

[0208] The solvent is preferably dimethyl sulfoxide (DMSO) and MeCN / H2O(5 / l) in step 3, the amount of DMSO is 1.5-3.0 eq, preferably 2.0-3.0 eq, and more preferably 2.3eq, and the volume of MeCN / H2O(5 / l) is 5-30 V, preferably 10-20 V, and more preferably 12V.

[0209] In step 4, the carboxyl group of intermediate 16 is protected by a carboxyl protecting group Bn-group by reacting intermediate 16 with BnBr in the presence of K2CO3 in a solvent in a reaction temperature for a reaction time.

[0210] The amount of BnBr is 0.5-2.5eq, preferably 1.0-2.0 eq, and more preferably 1.2eq.

[0211] The amount of K2CO3 is 1.0-3. Oeq, preferably 1.5-2.5 eq, and more preferably 2.0eq.

[0212] The reaction temperature is 10-30°C, preferably 15-25°C, and more preferably 20°C.

[0213] The reaction time is 4-6 hours, preferably 5-7 hours, and more preferably 6 hours.

[0214] The solvent is preferably DMF in step 4, and the volume of the solvent is 5-30 V, preferably 5- 20 V, and more preferably 10V.

[0215] In step 5, deprotect the hydroxyl protecting group TBS- group in intermediate 17 to obtain intermediate 18 with hydroxyl group in a solvent in the presence of TBAF in a reaction temperature for a reaction time.

[0216] The amount of TBAF is 1.0-3. Oeq, preferably 1.5-2.5 eq, and more preferably 2.0eq.

[0217] The reaction temperature is 10-30°C, and preferably 20°C.

[0218] The reaction time is 2-6 hours, preferably 3-5 hours, and more preferably 4 hours.

[0219] The solvent is preferably THF in step 5, and the volume of the solvent is 5-30 V, preferably 5- 20 V, and more preferably 10V.

[0220] In step 6, the esterification reaction between intermediate 18 and obtains intermediate 19 in a solvent in the presence of EDCI and DMAP in a reaction temperature for a reaction time. The amount of is 1.5-4.5eq, preferably 2.0-4.0 eq, and more preferably 3.0eq.

[0221] The amount of EDCI is 2.5-4.5eq, preferably 3.0-4.0 eq, and more preferably 3.5eq.

[0222] The amount of DMAP is 0.05-0.3eq, preferably 0.05-0.2 eq, and more preferably O.leq.

[0223] The reaction temperature is 10-40°C, preferably 15-30°C, and more preferably 20°C.

[0224] The reaction time is 60-100 hours, preferably 60-80 hours, and more preferably 72 hours.

[0225] The solvent is preferably DCM in step 6, and the volume of the solvent is 5-30 V, preferably 5-20 V, and more preferably 10V.

[0226] In step 7, in the presence of catalysts, intermediate 19 is reduced to obtain intermediate T2.

[0227] The catalysts are preferably Pd / C (10%, 15 psi).

[0228] The reaction temperature is 10-30°C, preferably 15-25°C, and more preferably 20°C.

[0229] The reaction time is 2-8 hours, preferably 3-6 hours, and more preferably 4 hours.

[0230] The solvent is preferably EtOH, and the volume of the solvent is 5-30 V, preferably 5-20 V, and more preferably 10V.

[0231] R,

[0232] In step 8, intermediate T2 reacts with reactant to obtain a compound of formula (III) in a solvent in the presence of PPh ? and DIAD in a reaction temperature for a reaction time.

[0233] The amount of is 0.5-2.0eq, preferably 0.8-1.5 eq, and more preferably l.Oeq.

[0234] The amount of PPh ? is 0.5-2. Oeq, preferably 1.0-2.0 eq, and more preferably 1.5eq.

[0235] The amount of diisopropyl azodicarboxylate (DIAD) is 0.5-2.0eq, preferably 1.0-2.0 eq, and more preferably 1.5eq.

[0236] The reaction temperature is 10-40°C, preferably 15-30°C, and more preferably 20°C. The reaction time is 8-20 hours, preferably 10-15 hours, and more preferably 12 hours.

[0237] The solvent is preferably THF in step 8, and the volume of the solvent is 5-30 V, preferably 5- 20 V, and more preferably 10V.

[0238] Or in step 8’, intermediate T2 reacts with reactant to obtain a compound of formula (IV) in a solvent in the presence of 2-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIEA) in a reaction temperature for a reaction time.

[0239] The amount of is 0.5-2. Oeq, preferably 0.8-1.5 eq, and more preferably l.leq.

[0240] The amount of HATU is 0.5-2.0eq, preferably 1.0-2.0 eq, and more preferably 1.2eq.

[0241] The amount of DIEA is 1.5-4.5eq, preferably 2.0-4.0 eq, and more preferably 3. Oeq.

[0242] The reaction temperature is 10-40°C, preferably 15-30°C, and more preferably 20°C.

[0243] The reaction time is 8-20 hours, preferably 10-15 hours, and more preferably 12 hours.

[0244] The solvent is preferably DCM in step 8’, and the volume of the solvent is 5-30 V, preferably 5-20 V, and more preferably 10V.

[0245] In one embodiment, the following is a specific scheme for synthesizing T1 and ICL-200.

[0246] In one embodiment, the following is a specific scheme for synthesizing T2 and ICL-209.

[0247] ICL-209

[0248] The following examples are provided for the purpose of illustration and not limitation. Example

[0249] Example 1 General procedure for preparation of Cpd 4C-1

[0250] (1.19 g, 3.69 mmol, 0.10 eq) at 25°C under N2. The mixture was stirred at 100°C for 12 hrs. TLC (Petroleum ether / Ethyl acetate=10:l, product Rf =0.2) showed the starting material was consumed completely. The mixture was quenched with aq.NHqCI (50.0 mL) and extracted with EtOAc (100 mL). The organic phase was washed with brine (50.0 mL) and dried with Na2SO4. The mixture was filtered and filtrate was concentrated in vacuum. The crude product was used for the next step. Cpd 4C-1 (27.0 g, crude) was obtained as colorless oil.

[0251] TLC:

[0252] General procedure for preparation of Cpd 4C

[0253] 4C-1 4C

[0254] To a mixture of Cpd 4C-1 (17.0 g, 37.1 mmol, 1.00 eq) in THF (320 mL) was added NaH (5.94 g, 149 mmol, 60% purity, 4.00 eq) in portions at 25°C under N2. The mixture was stirred 25 °C for 0.5 h. Then Cpd 10 (19.9 g, 74.3 mmol, 2.00 eq) was added to the mixture. The mixture was stirred 70°C for 12 hours. TLC (Petroleum ether / Ethyl acetate=10:l, product Rf = 0.5) showed the starting material was consumed completely. The mixture was quenched with aq.NFLCl (30.0 mL) and extracted with EtOAc (30.0 mL). The organic phase was washed with brine (2 x 30.0 mL) and dried with Na2SO4. The mixture was filtered and filtrate was concentrated in vacuum. The mixture was purified by silica gel chromatography (Petroleum ether / Ethyl acetate =1 / 0, 30 / 1) to give Cpd 4C (18.0 g, 28.0 mmol, 75.3% yield) as colorless oil.

[0255] TLC:

[0256] 'H NMR: Cpd 4C (400 MHz, CDCh)

[0257] 8: 7.38 (d, J = 7.6 Hz, 4H), 7.17 - 7.20 (m, 2H), 7.18 (t, J = 7.2 Hz, 2H), 3.63 (s, 4H), 3.59 - 3.62 (m, 5H), 3.56 - 3.59 (m, 2H), 3.38 - 3.40 (m, 5H), 3.07 - 3.08 (m, 1H), 1.59 - 1.62 (m, 8H), 1.24 (s, 18H), 0.03 (s, 12H).

[0258] General procedure for preparation o / Cpd 4E

[0259] 4C 4E

[0260] To a solution of Cpd 4C (18.0 g, 28.0 mmol, 1.00 eq) in THF (120 mL) was added TBAF (IM, 61.5 mL, 2.20 eq) in one portion at 25 °C under N2. The reaction mixture was stirred at 25 °C for 2 hrs. TLC (Petroleum ether / Ethyl aeetate=0:l, product Rf =0.5) showed the starting material was consumed completely. The mixture was poured into H2O (10.0 mL) and extracted with EtOAc (10.0 mL). The mixture was washed with brine (10.0 mL) and the organic phase was concentrated in vacuum. The residue was purified by silica gel chromatography (Petroleum ether / Ethyl acetate= 100 / 1, 2 / 1) to afford Cpd 4E (5.00 g, 12.0 mmol, 43.1% yield) as colorless oil. TLC:

[0261] 'H NMR: Cpd 4E (400 MHz, CDCh)

[0262] 5: 7.45 (d, J = 7.2 Hz, 4H), 7.36 (t, J = 7.2 Hz, 4H), 7.27 - 7.33 (m, 2H), 3.70 (s, 4H), 3.65 - 3.68 (m, 8H), 3.50 - 3.52 (m, 4H), 3.13 - 3.15 (m, 1H), 2.30 (s, 2H), 1.70 - 1.77 (m, 8H).

[0263] General procedure for preparation o / Cpd 4B .87 g,

[0264] 30.1 mmol, 2.50 eq) in DCM (10.0 mL) was added EDCI (7.61 g, 39.8 mmol, 3.30 eq) and DMAP (147 mg, 1.20 mmol, 0.10 eq) in one portion at 0°C under N2. The mixture was stirred

[0265] 0 °C for 0.5 hrs and heated to 25°C for 48 hrs. LCMS (ET84757-34-P1A1, product: RT =2.642min) showed the starting material was consumed completely. The mixture was poured into H2O (30.0 mL) and extracted with DCM (2 x 20.0 mL). The organic phase was washed with brine (20.0 mL) and dried with Na2SO4. The mixture was filtered and filtrate was concentrated in vacuum. The mixture was purified by silica gel chromatography (Petroleum ether / Ethyl acetate=l / 0, 50 / 1) to give Cpd 4B (8.60 g, 10.3 mmol, 85.5% yield) as colorless oil.

[0266] TLC: LCMS: Cpd 4B:

[0267] LCMS (product: RT = 2.642 min; [M+l]+: 836.5) m NMR: Cpd 4B (400 MHz, CDCh)

[0268] 5: 7.38 - 7.40 (m, 4H), 7.27 - 7.30 (m, 4H), 7.20 - 7.22 (m, 2H), 3.80 (s,4H), 3.56 - 3.63 (m, 4H), 3.42 - 3.60 (m, 4H), 3.39 - 3.40 (m, 1H), 2.10 - 2.33 (m, 2H), 1.63 - 1.70 (m, 12H), 1.60 - 1.62 (m, 4H), 1.30 (s, 32H), 0.88 (t, J = 7.6 Hz, 12H).

[0269] General procedure for preparation o / Tl

[0270] The mixture of compound 4B (3.50 g, 4.19 mmol, 1.00 eq), Pd / C (0.35 g, 329 pmol, 10% purity) and Pd(OH)2 (0.35 g, 249 pmol. 10% purity) in THF (40.0 mL) was stirred under 50 psi (H2) at 50°C for 12 hrs. TLC (Petroleum ether / Ethyl acetate=10:l, product Rf =0) showed the starting material was consumed completely. The mixture was filtered through celite and the filtrate was concentrated in vacuum. The mixture was purified by silica gel chromatography (Dichloromethane: Methanol = 60 / 1 30 / 1) to give T1 (2.70 g, 4.12 mmol, 98.3% yield) as yellow oil.

[0271] TLC:

[0272] 'H NMR: T1 (400 MHz, CDCh)

[0273] 5: 4.10 (t, J= 6.0 Hz, 4H), 3.48 - 3.50 (m, 6H), 3.46 - 3.47 (m, 2H), 3.33 - 3.44 (m, 1H), 2.32 - 2.34 (m, 2H) 1.65 - 1.69 (m, 12H), 1.63 - 1.64 (m, 4H), 1.44 (s, 32H), 0.88 (t, J = 6.8 Hz, 12H). General procedure for preparation of ICL-200

[0274] To a mixture of T1 (0.20 g, 305 pmol, 1.00 eq) and Al (42.9 mg, 366 pmol, 1.20 eq) in DCM (5.00 mL) was added EDCI (87.7 mg, 457 pmol, 1.50 eq), DMAP (3.72 mg, 30.5 pmol, 0.10 eq) in one portion at 0°C under N2. The mixture was stirred at 0°C for 0.5 hr, then heated to 20 °C and stirred for 12 hours. LCMS (product: RT = 2.279 min) showed the starting material was consumed completely. The mixture was poured into H2O (5.00 mL) and extracted with DCM (2 x 3.00 mL). The organic phase was washed with brine (5.00 mL) and concentrated in vacuum. The crude product was purified by normal phase HPLC (column: Agela DuraShell NH3 150mm*30mm*5um; mobile phase: [Heptane-THL: ACN=2:1]; gradient: 0%-30% B over 10.0 min) to give ICL-200 (0.13 g, 165 pmol, 54.0% yield, 95.6% purity) as colorless oil.

[0275] LCMS: ICL-200

[0276] LCMS (product: RT = 2.279 min; [M+l]+: 755.6)

[0277] 'H NMR: ICL-200 (400 MHz, CDCh)

[0278] 5: 4.11 (s, 1H), 4.07 - 4.09 (m, 4H), 3.43 - 3.52 (m, 8H), 2.39 (s, 2H), 2.28 - 2.33 (m, 10H), 1.64 - 1.70 (m, 12H), 1.62 - 1.63 (m, 4H), 1.60 (s, 33H), 0.88 (t, J= 7.6 Hz, 12H).

[0279] Example 2 General procedure for preparation o / ICL-201

[0280] To a mixture of T1 (0.20 g, 305 pmol, 1.00 eq) and A2 (48.0 mg, 366 pmol, 1.20 eq) in DCM (3.00 mL) was added EDCI (87.7 mg, 457 pmol, 1.50 eq), DMAP (3.72 mg, 30.5 pmol, 0.10 eq) in one portion at 0 °C under N2. The mixture was stirred at 0 °C for 0.5 hr, then heated to 20 °C and stirred for 12 hours. LCMS (product: RT = 2.239 min) showed the starting material was consumed completely. The mixture was poured into H2O (5.00 mL) and extracted with DCM (2 x 3.00 mL). The organic phase was washed with brine (5.00 mL) and concentrated in vacuum. The crude product was purified by normal phase HPLC (column: Agela DuraShell NH3 150mm*30mm*5um; mobile phase: [Heptane-THF: ACN=2:1]; gradient: 0%-30% B over 10.0 min) to give ICL-201 (0.15 g, 188 pmol, 61.6% yield, 96.3% purity) as colorless oil.

[0281] LCMS: ICL-201

[0282] LCMS (product: RT = 2.239 min; [M+l]+: 769.6) m NMR: ICL-201 (400 MHz, CDCh)

[0283] 8: 6.57 (d, J= 8.4 Hz, 1H), 4.22 (s, 1H), 4.08 - 4.11 (m, 4H), 3.50 - 3.53 (m, 2H), 3.26 - 3.48 (m, 6H), 2.26 -2.37 (m, 12H), 1.69 - 1.81 (m, 2H), 1.64 - 1.68 (m, 12H), 1.62 - 1.63 (m, 4H), 1.61 (s, 32H), 0.88 (t, J = 7.6 Hz, 12H).

[0284] Example 3 General procedure for preparation 0 / ICL-202

[0285] To a mixture of T1 (0.20 g, 305 pmol, 1.00 eq) and A3 (53.1 mg, 366 pmol, 1.20 eq) in DCM (3.00 mL) was added EDCI (87.7 mg, 457 pmol, 1.50 eq), DMAP (3.72 mg, 30.5 pmol, 0.10 eq) in one portion at 0 °C under N2. The mixture was stirred at 0 °C for 0.5 hr, then heated to 20 °C and stirred for 12 hours. LCMS (product: RT = 2.320 min) showed the starting material was consumed completely. The mixture was poured into H2O (5.00 mL) and extracted with DCM (2 x 3.00 mL). The organic phase was washed with brine (5.00 mL) and concentrated in vacuum. The crude product was purified by normal phase HPLC (column: Agela DuraShell NH3 150mm*30mm*5um; mobile phase: [Heptane-THF: ACN=2:1]; gradient: 0%-30% B over 10.0 min) to give ICL-202 (0.14 g, 174 pmol, 57.1% yield, 97.4% purity) as colorless oil. LCMS: ICL-202:

[0286] LCMS (product: RT = 2.320 min; [M+l]+: 783.6) m NMR: ICL-202 (400 MHz, CDCh)

[0287] 5: 6.0 (d, J = 8.4 Hz, 1H), 4.20 - 4.35 (m, 1H), 4.09 (t, J = 6.8 Hz, 4H), 3.45 - 3.53 (m, 8H), 2.31 - 2.33 (m, 4H), 2.22 - 2.25 (m, 8H), 1.64 - 1.69 (m, 14H), 1.60 - 1.62 (m, 4H), 1.29 (s„ 34H), 0.87 (t, J = 7.2 Hz, 12H).

[0288] Example 4 General procedure for preparation o / ICL-203

[0289] To a mixture of T1 (0.17 g, 259 mol, 1.00 eq) and A4 (44.5 mg, 311 mol, 1.20 eq) in DCM (5.00 mL) was added EDCI (74.5 mg, 3889 pmol, 1.50 eq), DMAP (3.17 mg, 25.9 pmol. 0.10 eq) in one portion at 0 °C under N2. The mixture was stirred at 0 °C for 0.5 hr, then heated to 20 °C and stirred for 12 hours. LCMS (product: RT = 2.359 min) showed the starting material was consumed completely. The mixture was poured into H2O (5.00 mL) and extracted with DCM (2 x 3.00 mL). The organic phase was washed with brine (5.00 mL) and concentrated in vacuum. The crude product was purified by normal phase HPLC (column: Agela DuraShell NH3 150mm*30mm*5um; mobile phase: [Heptane-THL: ACN=2:1]; gradient: 0%-30% B over 10.0 min) to give ICL-203 (0.10 g, 118 pmol, 45.6% yield, 92.4% purity) as colorless oil.

[0290] LCMS: ICL-203

[0291] LCMS (product: RT = 2.359 min; [M+l]+: 781.6)

[0292] 'H NMR: ICL-203 (400 MHz, CDCh)

[0293] 5: 8.41 (brs, 1H), 4.20 - 4.36 (m, 1H), 4.08 (t, J = 6.4 Hz, 4H), 3.43 - 3.51 (m, 8H), 2.76 (s, 2H), 2.59 (s, 3H), 2.42 (s, 2H), 2.31 - 2.42 (m, 2H), 1.80 (s, 4H), 1.63 - 1.69 (m, 12H), 1.58 - 1.61 (m, 4 H), 1.26 (s, 32H), 0.87 (t, J = 6.8 Hz, 12H). Example 5 General procedure for preparation of ICL-204

[0294] To a mixture of T1 (0.20 g, 305 pmol, 1.00 eq) and A5 (57.5 mg, 366 pmol. 1.20 eq) in DCM (3.00 mL) was added EDCI (87.7 mg, 457 pmol, 1.50 eq), DMAP (3.72 mg, 30.5 pmol, 0.10 eq) in one portion at 0 °C under N2. The mixture was stirred at 0 °C for 0.5 hr, then heated to 20 °C and stirred for 12 hours. LCMS (product: RT = 2.298 min) showed the starting material was consumed completely. The mixture was poured into H2O (5.00 mL) and extracted with DCM (2 x 3.00 mL). The organic phase was washed with brine (5.00 mL) and concentrated in vacuum. The crude product was purified by normal phase HPLC (column: Agela DuraShell NH3 150mm*30mm*5um; mobile phase: [Heptane-THL: ACN=2:1]; gradient: 0%-30% B over 10.0 min) to give ICL-204 (0.10 g, 115 pmol, 37.7% yield, 91.5% purity) as colorless oil.

[0295] LCMS: ICL-204

[0296] LCMS (product: RT = 2.298 min; [M+l]+: 795.7)

[0297] 'H NMR: ICL-204 (400 MHz, CDCh)

[0298] 8: 6.50 (d, J = 8.4 Hz, 1H), 4.22 - 4.36 (m, 1H), 4.09 (t, J = 6.8 Hz, 4H), 3.43 - 3.53 (m, 8H), 2.58 (s, 5H), 2.29 - 2.31 (m, 5H), 1.69 - 1.82 (m, 5H), 1.42 - 1.67 (m, 12H), 1.29 - 1.42 (m, 4H), 1.26 (s, 32H), 0.88 (t, J = 6.8 Hz, 12H).

[0299] Example 6 General procedure for preparation of A6

[0300] To a mixture of A6-1 (1.00 g, 8.54 mmol, 1.00 eq) and propanal (1.98 g, 34.2 mmol, 2.49 mL, 4.00 eq) in DCM (10.0 mL) was added NaBH(OAc)3 (7.24 g, 34.2 mmol, 4.00 eq) in portions at 0 °C under N2. The mixture was stirred at 25 °C for 12 hours. LCMS (product: RT = 0.358 min) showed the starting material was consumed completely. The mixture was poured into NaHCCh (20.0 mL) and stirred for 10 mins. The aqueous phase was extracted with DCM (10 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=l / 0, 8 / 1) to give A6 (0.20 g, 994 pmol. 11.6% yield) as colorless oil.

[0301] LCMS: A6:

[0302] LCMS (product: RT = 0.328 min; [M+l]+: 202) m NMR: A6 (400 MHz, CDCh)

[0303] 8: 2.81-2.86 (m, 6H), 2.26 (t, J = 6.4 Hz, 2H), 1.58 - 1.67 (m, 9H), 0.94 (t, J = 7.6 Hz, 6H).

[0304] General procedure for preparation o / ICL-205

[0305] To a mixture of T1 (0.20 g, 305 pmol, 1.00 eq) and A6 (73.6 mg, 366 pmol, 1.20 eq) in DCM (3.00 mL) was added EDCI (87.7 mg, 457 pmol, 1.50 eq), DMAP (3.72 mg, 30.5 pmol, 0.10 eq) in one portion at 0 °C under N2. The mixture was stirred at 0 °C for 0.5 hr, then heated to 20 °C and stirred for 12 hours. LCMS (ET84757-51-P1A1, product: RT = 2.358 min) showed the starting material was consumed completely. The mixture was poured into H2O (5.00 mL) and extracted with DCM (2 x 3.00 mL). The organic phase was washed with brine (5.00 mL) and concentrated in vacuum. The crude product was purified by normal phase HPLC (column: Agela DuraShell NH3 150mm*30mm*5um; mobile phase: [Heptane-THF: ACN=2:1]; gradient: 0%-30% B over 10.0 min) to give ICL-205 (0.05 g, 59.6 pmol, 19.5% yield, 95.0% purity) as colorless oil. LCMS: ICL-205

[0306] LCMS (product: RT = 2.358 min; [M+l]+: 839.7) m NMR: ICL-205 (400 MHz, CDCh)

[0307] 5: 6.00 (dd, J = 94.8 Hz, 7 = 8.4 Hz, 1H), 5.59 (dd, J= 6.0 Hz, 7 = 1.6 Hz, 1H), 4.19 - 4.21 (m, 1H), 4.09 (t, 7 = 6.4 Hz, 4H), 3.94 - 3.97 (m, 2H), 3.41 - 3.54 (m, 8H), 3.15 - 3.20 (m, 1H), 2.41 - 2.52 (m, 2H), 2.20 -2.35 (m, 4H), 1.96 - 2.06 (m, 2H), 1.78 - 1.87 (m, 4H), 1.56 - 1.71 (m, 16H), 1.40 - 1.47 (m, 4H), 1.26 - 1.31 (m, 30H), 0.86 - 0.93 (m, 18H).

[0308] Example 7 General procedure for preparation of A7-2

[0309] To a mixture of A7-1 (1.78 g, 8.54 mmol, 1.00 eq) and 3-(dimethylamino)propanoic acid (1.00 g, 8.54 mmol, 1.00 eq) in DCM (5.00 mL) was added EDCI (2.13 g, 11.1 mmol, 1.30 eq), DMAP (104 mg, 854 pmol, 0.10 eq) in one portion 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 =0.4) 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. The residue was purified by silica gel chromatography (Dichloromethane: Methanol= 100 / 1, 30 / 1) to give A7-2 (1.80 g, 5.86 mmol, 68.6% yield) as colorless oil.

[0310] TLC: 'H NMR: A7-2 (400 MHz, CDCh)

[0311] 8: 7.32 - 7.39 (m, 5H), 5.12 (s, 2H), 4.09 (t, J = 6.4 Hz, 2H), 2.61 (t, J = 7.6 Hz, 2H), 2.48 (t, J = 7.2 Hz, 2H), 2.41 (t, J = 7.2 Hz, 2H), 2.25 (s, 6H), 1.68 - 1.72 (m, 4H).

[0312] General procedure for preparation of l

[0313] To a mixture of A7-2 (0.50 g, 1.63 mmol, 1.00 eq) in EtOH (10.0 mL) was added Pd / C (0.05 g, 47.0 mol, 10% purity). The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 20°C for 12 hours. LCMS (product: RT = 0.099 min) showed the starting material was consumed completely. The mixture was filtered through celite and the filter cake was washed with EtOH (2 x 20.0 mL). The filtrate was concentrated in vacuum. The crude product was used for the next step. A7 (0.30 g, 1.38 mmol, 84.9% yield) was obtained as colorless oil.

[0314] LCMS: A7

[0315] LCMS (product: RT = 0.099 min; [M+l]+: 218)

[0316] 'H NMR: A7 (400 MHz, CDCh)

[0317] 8: 9.29 - 9.34 (m, 1H), 4.11 - 4.16 (m, 2H), 2.90 (t, J = 7.6 Hz, 2H), 2.64 (t, J = 7.2 Hz, 2H), 2.33 (s, 6H) 2.28 (t, J = 6.8 Hz, 2H), 1.63-1.72 (m, 4H).

[0318] General procedure for preparation o / TCL-206 To a mixture of T1 (0.20 g, 305 pmol, 1.00 eq) and A7 (79.5 mg, 366 pmol 1.20 eq) in DCM (3.00 mL) was added EDCI (87.7 mg, 457 pmol, 1.50 eq), DMAP (3.72 mg, 30.5 pmol, 0.10 eq) in one portion at 0 °C under N2. The mixture was stirred at 0 °C for 0.5 hr, then heated to 20 °C and stirred for 12 hours. LCMS (product: RT = 2.380 min) showed the starting material was consumed completely. The mixture was poured into H2O (5.00 mL) and extracted with DCM (2 x 3.00 mL). The organic phase was washed with brine (5.00 mL) and concentrated in vacuum. The crude product was purified by normal phase HPLC (column: Agela DuraShell NH3 150mm*30mm*5um; mobile phase: [Heptane-THF: ACN=2:1]; gradient: 0%-30% B over 10.0 min) to give ICL-206 (0.10 g, 104 pmol, 34.1% yield, 88.8% purity) as colorless oil.

[0319] LCMS: ICL-206:

[0320] LCMS (product: RT = 2.380 min; [M+l]+: 881.7)

[0321] 'H NMR: ICL-206 (400 MHz, CDCh)

[0322] 5: 5.93 (d, J = 8.4 Hz, 1H), 4.12 (s, 1H), 4.09 - 4.10 (m, 6H), 3.48 - 3.54 (m, 2H), 3.43 - 3.47 (m, 6H), 2.32-2.52 (m, 2H), 2.23-2.29 (m, 2H), 1.68-1.70 (m, 10H), 1.64-1.67 (m, 16H), 1.61- 1.63 (m, 4H), 1.26-1.44 (m, 32H), 0.88 (t, J= 6.4 Hz, 12H).

[0323] Example 8 General procedure for preparation of A8-2

[0324] To a mixture of A8-1 (1.45 g, 6.98 mmol, 1.00 eq) and 3-pyrrolidin-l-ylpropanoic acid (1.00 g, 6.98 mmol, 1.00 eq) in DCM (5.00 mL) was added EDCI (1.74 g, 9.08 mmol, 1.30 eq), DMAP (85.3 mg, 698 pmol. 0.10 eq) in one portion 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 =0.4) 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. The residue was purified by silica gel chromatography (Dichloromethane: Methanol= 100 / 1, 30 / 1) to give A8-2 (1.60 g, 4.80 mmol, 68.7% yield) as colorless oil.

[0325] TLC:

[0326] 'H NMR: A8-2 (400 MHz, CDCh)

[0327] 8: 7.32 - 7.39 (m, 5H), 5.12 (s, 2H), 4.09 (t, J = 6.4 Hz, 2H), 2.83 (t, J = 7.2 Hz, 2H), 2.58 - 2.60 (m, 6H), 2.40 (t, J = 7.2 Hz, 2H), 1.82 - 1.83 (m, 4H), 1.69-1.81 (m, 4H).

[0328] General procedure for preparation of AS

[0329] To a mixture of A8-2 (0.50 g, 1.50 mmol, 1.00 eq) in EtOH (10.0 mL) was added Pd / C (0.05 g, 47.0 pmol, 10% purity). The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 20°C for 12 hours. LCMS (product: RT = 0.103 min) showed the starting material was consumed completely. The mixture was filtered through celite and the filter cake was washed with EtOH (2 x 20.0 mL). The filtrate was concentrated in vacuum. The crude product was used for the next step. A8 (0.29 g, 1.19 mmol, 79.48% yield) was obtained as colorless oil.

[0330] LCMS: A8:

[0331] LCMS (product: RT = 0.103 min; [M+l]+: 244)

[0332] 'H NMR: A8 (400 MHz, CDCh)

[0333] 8: 9.82 - 10.02 (m, 1H), 4.10 - 4.13 (m, 2H), 3.12 (t, J = 7.6 Hz, 2H), 3.00 (s, 4H), 2.74 (t, J = 7.6 Hz, 2H), 2.29 (t, J = 6.4 Hz, 2H), 1.96 - 2.02 (m, 4H), 1.62-1.71 (m, 4H). General procedure for preparation oflCL-l l

[0334] To a mixture of T1 (0.20 g, 305 pmol, 1.00 eq) and A8 (89.0 mg, 366 pmol 1.20 eq) in DCM (3.00 mL) was added EDCI (87.7 mg, 457 pmol, 1.50 eq), DMAP (3.72 mg, 30.5 pmol, 0.10 eq) in one portion at 0 °C under N2. The mixture was stirred at 0 °C for 0.5 hr, then heated to 20 °C and stirred for 12 hours. LCMS (product: RT = 2.328 min) showed the starting material was consumed completely. The mixture was poured into H2O (5.00 mL) and extracted with DCM (2 x 3.00 mL). The organic phase was washed with brine (5.00 mL) and concentrated in vacuum. The crude product was purified by normal phase HPLC (column: Agela DuraShell NH3 150mm*30mm*5um; mobile phase: [Heptane-THF: ACN=2:1]; gradient: 0%-30% B over 10.0 min) to give ICL-207 (0.08 g, 79.1 mol, 25.9% yield, 87.1% purity) as colorless oil.

[0335] LCMS: ICL-207

[0336] LCMS (product: RT = 2.328 min; [M+l]+: 881.7)

[0337] 'H NMR: ICL-207 (400 MHz, CDCh)

[0338] 8: 5.93 (d, J= 8.4 Hz, 1H), 4.09 - 4.10 (m, 1H), 3.49 - 3.51 (m, 8H), 2.58 (s, 2H), 2.23 (s, 2H), 1.70 - 1.86 (m, 4H), 1.69 (s, 4H), 1.63 - 1.68 (m, 17H), 1.62 - 1.63 (m, 4H), 1.26 - 1.30 (m, 32H), 0.88 (t, J= 7.2 Hz, 12H).

[0339] Example 9 General procedure for preparation of compound 14 To a solution of Cpd 13 (5.00 g, 41.6 mmol, 1.00 eq) in DMF (150 mL) was added NaH (3.66 g, 91.6 mmol, 60.0% purity, 2.20 eq) at 20 °C for 0.5 hrs, then Cpd 10 (24.5 g, 91.6 mmol, 2.20 eq) in Tol. (50.0 mL) was added the mixture at 0 °C. The mixture was stirred at 20 °C for 16 hrs. LCMS (RT = 2.959 min) showed the starting material was consumed completely. The residue was poured into aq. NH4CI (400 mL) and stirred for 5 min. The aqueous phase was extracted with ethyl acetate (150 mL, 100 mL). The combined organic phase was washed with brine (400 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, Petroleum ether / Ethyl acetate= 100 / 1, 0 / 1). Cpd 14 (7.10 g, 14.4 mmol, 34.6% yield) was obtained as a colorless oil. m NMR: Cpd 14 (400 MHz, CDCh)

[0340] 8: 3.63 (t, J = 6.0 Hz, 4H), 3.57 (s, 2H), 3.42 - 3.45 (m, 6H), 3.38 (d, J = 8.0 Hz, 2H), 1.56 - 1.61 (m, 9H), 0.90 (s, 18H), 0.86 (s, 3H), 0.05 (s, 12H)

[0341] General procedure for preparation of compound 15

[0342] To a solution of Cpd 14 (7.10 g, 14.4 mmol, 1.00 eq) in MeCN (71.0 mL) was added IBX (4.84 g, 17.3 mmol, 1.20 eq). The mixture was stirred 80 °C for 4 hrs. TLC indicated Cpd 14 was consumed completely and one new spot formed. The mixture was concentrated in vacuum. The product was used to next step without purification. Cpd 15 (7.00 g, crude) was obtained as a white oil.

[0343] TLC:

[0344] General procedure for preparation of compound 16

[0345] To a solution of Cpd 15 (7.00 g, 14.3 mmol, 1.00 eq) and DMSO (2.56 g, 32.8 mmol, 2.30 eq) in MeCN (70.0 mL) was added NaClO2 (5.35 g, 59.2 mmol, 4.15 eq), NaH2PO4 (3.59 g, 30.0 mmol, 2.10 eq) and H2O (14.0 mL). The reaction mixture was stirred at 20°C for 4 hrs. TLC indicated Reactant 1 was consumed completely and one new spot formed. The mixture was poured into aq.Na2SO3 (150 mL) and stirred for 5 mins. The aqueous phase was extracted with EtOAc (150 mL, 100 mL). The combined organic phase was washed with brine (200 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, Petroleum ether / Ethyl acetate= 100 / 1, 0 / 1). Cpd 16 (4.70 g, 9.27 mmol, 65.0% yield) was obtained as colorless oil.

[0346] TLC:

[0347] 'H NMR: Cpd 16 (400 MHz, CDCh)

[0348] 5: 3.63 (t, J = 6.0 Hz, 4H), 3.56 (s, 4H), 3.51 (t, J = 6.0 Hz, 4H), 1.56 - 1.66 (m, 9H), 1.24 (s, 3H), 0.90 (s, 19H), 0.05 (s, 12H).

[0349] General procedure for preparation of compound 17

[0350] To a solution of Cpd 16 (4.70 g, 9.27 mmol, 1.00 eq) in DMF (47.0 mL) was added K2CO3 (2.56 g, 18.6 mmol, 2.00 eq) and BnBr (1.90 g, 11.1 mmol, 1.20 eq). The mixture was stirred 20 °C for 6 hrs. LCMS (RT = 2.252 min) showed the starting material was consumed completely. The mixture was poured into aq.NaHCCh (150 mL) and stirred for 5 mins. The aqueous phase was extracted with EtOAc (150 mL, 100 mL). The combined organic phase was washed with brine (200 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. Cpd 17 (5.50 g, crude) was obtained as a yellow oil.

[0351] General procedure for preparation of compound 18

[0352] To a solution of Cpd 17 (5.50 g, 9.21 mmol, 1.00 eq) in THF (55.0 mL) was added TABL (1.00 M, 18.4 mL, 2.00 eq). The mixture was stirred 20 °C for 4 hrs. TLC indicated Reactant 1 was consumed completely and one new spot formed. The mixture was poured into H2O (100 mL) and stirred for 5 mins. The aqueous phase was extracted with EtOAc (70.0 mL, 50.0 mL). The combined organic phase was washed with brine (100 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, Petroleum ether / Ethyl acetate= 100 / 1, 0 / 1). Cpd 18 (3.00 g, 8.14 mmol, 88.4% yield) was obtained as yellow oil.

[0353] TLC:

[0354] 'H NMR: Cpd 18 (400 MHz, CDCh)

[0355] 8: 7.30 - 7.36 (m, 5H), 5.16 (s, 2H), 3.56 - 3.63 (m, 8H), 3.44 (t, J= 5.6 Hz, 4H), 1.97 (s, 2H), 1.58 - 1.67 (m, 8H), 1.22 (s, 3H).

[0356] General procedure for preparation of compound 19

[0357] To a solution of Cpd 18 (3.00 g, 8.14 mmol, 1.00 eq) and Cpd 1 (5.58 g, 24.4 mmol, 3.00 eq) in DCM (30.0 mL) was added EDCI (5.46 g, 28.5 mmol, 3.50 eq) and DMAP (99.5 mg, 814 pmol, 0.10 eq). The mixture was stirred at 20 °C for 72 hrs. LCMS (ET84820-42-P1A3, RT = 4.255 min) showed the starting material was consumed completely. The mixture was poured into H2O (100 mL) and stirred for 5 mins. The aqueous phase was extracted with DCM (100 mL, 50.0 mL). The combined organic phase was washed with brine (100 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, Petroleum ether / Ethyl acetate= 100 / 1, 0 / 1). Cpd 19 (5.50 g, 6.97 mmol, 85.6% yield) was obtained as colorless oil. m NMR: Cpd 19 (400 MHz, CDCh)

[0358] 8: 7.30 - 7.35 (m, 5H), 5.15 (s, 2H), 4.06 (t, J = 6.4 Hz, 4H), 3.53 (s, 4H), 3.40 (t, J = 6.0 Hz, 4H), 2.27 - 2.33 (m, 2H), 1.56 - 1.64 (m, 14H), 1.30 - 1.50 (m, 4H), 1.22 - 1.29 (m, 35H), 0.88 (t, J = 6.8 Hz, 12H).

[0359] General procedure for preparation ofT2

[0360] 19 T2

[0361] To a solution of Cpd 19 (5.50 g, 6.97 mmol, 1.00 eq) in EtOH (55.0 mL) was added Pd / C (550 mg, 517 pmol. 10.0% purity) under Ar atmosphere. The suspension was degassed and purged with H2 for 3 times. The mixture was stirred under H2 (15 Psi) at 20 °C for 4 hrs. LCMS (ET84820-54-P1A1, product: RT = 3.914 min) showed the starting material was consumed completely. The mixture was filtered, the filtrate was concentrated in vacuum to residue. T2 (4.50 g, 6.44 mmol, 92.4% yield) was obtained as colorless oil. m NMR: T2 (400 MHz, CDCh)

[0362] 5: 4.09 (t, J = 6.4 Hz, 4H), 3.56 (s, 4H), 3.50 (t, J = 6.0 Hz, 4H), 2.32 - 2.34 (m, 2H), 1.65 - 1.70 (m, 13H), 1.44 (m, 4H), 1.24 - 1.31 (m, 35H), 0.88 (t, J= 6.4 Hz, 12H).

[0363] General procedure for preparation of ICL-209

[0364] To a solution of T2 (400 mg, 572 pmol. 1.00 eq) and All (51.0 mg, 572 pmol, 1.00 eq) in THF (4.00 mL) was added PPI13 (225 mg, 858 pmol, 1.50 eq) and DIAD (174 mg, 858 pmol, 1.50 eq). The mixture was stirred 20 °C for 12 hrs. LCMS (product: RT = 2.455 min) showed the starting material was consumed completely. The residue was poured into water (10.0 mL) and stirred for 5 min. The aqueous phase was extracted with DCM (10.0 mL). The combined organic phase was washed with brine (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*30mm*5um; mobile phase: [Heptane-THL: ACN=2:1]; gradient: 0%-30% B over 10.0 min). ICL-209 (200 mg, 259 pmol, 45.4% yield) was obtained as a colorless oil.

[0365] 'H NMR: ICL-209 (400 MHz, CDCh)

[0366] 8: 4.26 (s, 2H), 4.08 (t, J = 6.4 Hz, 4H), 3.51 (s, 4H), 3.42 (t, J = 6.4 Hz, 4H), 2.66 - 2.73 (m, 2H), 2.31 - 2.36 (m, 6H), 1.58 - 1.67 (m, 14H), 1.44 (m, 5H), 1.26 - 1.29 (m, 32H), 1.20 (s, 3H), 0.88 (t, J = 6.8 Hz, 12H). Example 10 General procedure for preparation of ICL-210

[0367] To a solution of T2 (350 mg, 501 p.mol, 1.00 eq) and A12 (64.7 mg, 501 p.mol, 1.00 eq) in THF (3.50 mL) was added PPha (197 mg, 751 pmol. 1.50 eq) and DIAD (152 mg, 751 pmol, 1.50 eq). The mixture was stirred 20 °C for 12 hrs. LCMS (product: RT = 2.628 min) showed the starting material was consumed completely. The residue was poured into water (10.0 mL) and stirred for 5 min. The aqueous phase was extracted with DCM (10.0 mL). The combined organic phase was washed with brine (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*30mm*5um; mobile phase: [Heptane-THF: ACN=2:1]; gradient: 0%-30% B over 10.0 min). ICL-210 (150 mg, 185 pmol, 37.0% yield) was obtained as a colorless oil.

[0368] 'H NMR: ICL-210 (400 MHz, CDCh)

[0369] 8: 4.23 - 4.25 (m, 1H), 4.08 (t, J = 6.4 Hz, 4H), 3.50 (s, 4H), 3.42 (t, J = 6.4 Hz, 4H), 2.29 - 2.62 (m, 7H), 1.56 - 1.67 (m, 19H), 1.44 (m, 6H), 1.26 - 1.29 (m, 32H), 1.19 (s, 3H), 0.88 (t, J = 6.8 Hz, 12H).

[0370] Example 11 General procedure for preparation ofICL-211 To a solution of T2 (350 mg, 501 pmol, 1.00 eq) and A13 (51.6 mg, 501 pmol, 1.00 eq) in THP (3.50 mL) was added PPhi (197 mg, 751 pmol, 1.50 eq) and DIAD (152 mg, 751 pmol, 1.50 eq). The mixture was stirred 20 °C for 12 hrs. LCMS (product: RT = 2.558 min) showed the starting material was consumed completely. The residue was poured into water (10.0 mL) and stirred for 5 min. The aqueous phase was extracted with DCM (10.0 mL). The combined organic phase was washed with brine (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*30mm*5um; mobile phase: [Heptane-THL: ACN=2:1]; gradient: 0%-30% B over 10.0 min). ICL-211 (50.0 mg, 63.8 pmol, 12.7% yield) was obtained as a colorless oil. m NMR: ICL-211 (400 MHz, CDCI3)

[0371] 8: 4.14 (t, J = 6.4 Hz, 2H), 4.08 (t, J = 6.4 Hz, 4H), 3.50 (t, J = 9.2 Hz, 4H), 3.44 (t, J= 6.4 Hz, 4H), 2.29 - 2.32 (m, 9H), 1.67 (s, 2H), 1.58 - 1.66 (m, 13H), 1.44 (m, 4H), 1.26 - 1.29 (m, 32H), 1.19 (s, 3H), 0.88 (t, J = 6.4 Hz, 12H).

[0372] Example 12 General procedure for preparation of ICL-212

[0373] To a solution of T2 (300 mg, 429 pmol, 1.00 eq) and A14 (50.3 mg, 429 pmol, 1.00 eq) in DCM (3.00 mL) was added EDCI (165 mg, 858 pmol, 2.00 eq) and DMAP (78.6 mg, 644 pmol, 1.50 eq). The mixture was stirred 20 °C for 12 hrs. LCMS (product: RT = 2.574 min) showed the starting material was consumed completely. The residue was poured into water (10.0 mL) and stirred for 5 min. The aqueous phase was extracted with DCM (10.0 mL). The combined organic phase was washed with brine (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*30mm*5um; mobile phase: [Heptane-THL: ACN=2:1]; gradient: 0%-30% B over 10.0 min). ICL-212 (100 mg, 125 pmol, 29.2% yield) was obtained as a colorless oil. 'H NMR: ICL-212 (400 MHz, CDCh)

[0374] 8: 4.06 - 4.12 (m, 6H), 3.50 (t, J = 3.2 Hz, 4H), 3.42 (t, J = 6.0 Hz, 4H), 2.27 - 2.33 (m, 10H), 1.58 - 1.67 (m, 17H), 1.44 (m, 5H), 1.26 - 1.29 (m, 32H), 1.18 (s, 3H), 0.88 (t, J= 6.4 Hz, 12H).

[0375] Example 13 General procedure for preparation of ICL-213

[0376] To a solution of T2 (400 mg, 572 pmol, 1.00 eq) and A15 (99.2 mg, 572 pmol, 1.00 eq) in DCM (4.00 mL) was added EDCI (219 mg, 1.14 mmol, 2.00 eq) and DMAP (105 mg, 858 pmol, 1.50 eq). The mixture was stirred 20 °C for 12 hrs. LCMS (product: RT = 2.643 min) showed the starting material was consumed completely. The residue was poured into water (10.0 mL) and stirred for 5 min. The aqueous phase was extracted with DCM (10.0 mL). The combined organic phase was washed with brine (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*30mm*5um; mobile phase: [Heptane-THL: ACN=2:1]; gradient: 0%-30% B over 10.0 min). ICL-213 (90.0 mg, 105 pmol, 18.4% yield) was obtained as a yellow oil.

[0377] 'H NMR: ICL-213 (400 MHz, CDCh)

[0378] 8: 4.06 - 4.11 (m, 6H), 3.50 (t, J = 4.4 Hz, 4H), 3.42 (t, J = 6.0 Hz, 4H), 2.31 - 2.35 (m, 7H), 1.58 - 1.67 (m, 16H), 1.44 - 1.45 (m, 8H), 1.26 - 1.29 (m, 32H), 1.18 (s, 3H), 0.88 (t, J = 6.4 Hz, 18H). Example 14 General procedure for preparation of ICL-214

[0379] To a solution of T2 (400 mg, 572 pmol, 1.00 eq) and A16 (55.5 mg, 629 pmol, 1.10 eq) in DCM (4.00 mL) was added HATU (261 mg, 687 pmol, 1.20 eq) and DIEA (222 mg, 1.72 mmol, 3.00 eq). The mixture was stirred 20 °C for 12 hrs. LCMS (product: RT = 2.419 min) showed the starting material was consumed completely. The residue was poured into water (10.0 mL) and stirred for 5 min. The aqueous phase was extracted with DCM (10.0 mL). The combined organic phase was washed with brine (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*30mm*5um; mobile phase: [Heptane-THE: ACN=2:1]; gradient: 0%-30% B over 10.0 min). ICL-214 (240 mg, 312 pmol, 54.5% yield) was obtained as a colorless oil. m NMR: ICL-214 (400 MHz, DMSO-rfo)

[0380] 8: 7.36 (t, J = 5.2 Hz, 1H), 4.02 (t, J = 6.4 Hz, 4H), 3.35 - 3.39 (m, 8H), 3.11 - 3.12 (m, 2H), 2.25 - 2.27 (m, 4H), 2.12 (s, 6H), 1.39 - 1.59 (m, 16H), 1.21 - 1.25 (m, 32H), 1.01 (s, 3H), 0.84 (t, J = 6.8 Hz, 12H).

[0381] Example 15 General procedure for preparation ofICL-215 To a solution of T2 (350 mg, 501 pmol, 1.00 eq) and A17 (70.6 mg, 551 pmol, 1.10 eq) in DCM (3.50 mL) was added HATU (228 mg, 601 pmol, 1.20 eq) and DIEA (194 mg, 1.50 mmol, 3.00 eq). The mixture was stirred 20 °C for 12 hrs. LCMS (product: RT = 2.542 min) showed the starting material was consumed completely. The residue was poured into water (10.0 mL) and stirred for 5 min. The aqueous phase was extracted with DCM (10.0 mL). The combined organic phase was washed with brine (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*30mm*5um; mobile phase: [Heptane-THE: ACN=2:1]; gradient: 0%-30% B over 10.0 min). ICL-215 (200 mg, 247 pmol, 49.4% yield) was obtained as a colorless oil. m NMR: ICL-215 (400 MHz, CDCI3)

[0382] 8: 4.08 (t, J = 6.0 Hz, 4H), 3.51 (s, 4H), 3.46 (t, J = 6.4 Hz, 4H), 3.33 (s, 2H), 2.29 - 2.33 (m, 6H), 1.58 - 1.69 (m, 17H), 1.44 (m, 6H), 1.26 - 1.29 (m, 33H), 1.17 (s, 3H), 0.88 (t, J= 6.8 Hz, 12H).

[0383] Example 16 General procedure for preparation of ICL-216

[0384] ICL-216 50 mg, 90% purity

[0385] To a solution of T2 (350 mg, 501 pmol, 1.00 eq) and A18 (56.3 mg, 551 pmol, 1.10 eq) in DCM (3.50 mL) was added HATU (228 mg, 601 pmol, 1.20 eq) and DIEA (194 mg, 1.50 mmol, 3.00 eq). The mixture was stirred 20 °C for 12 hrs. LCMS (product: RT = 2.434 min) showed the starting material was consumed completely. The residue was poured into water (10.0 mL) and stirred for 5 min. The aqueous phase was extracted with DCM (10.0 mL). The combined organic phase was washed with brine (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*30mm*5um; mobile phase: [Heptane-THE: ACN=2:1]; gradient: 0%-30% B over 10.0 min). ICL-216 (150 mg, 192 pmol, 38.3% yield) was obtained as a colorless oil. 'H NMR: ICL-216 (400 MHz, CDCh)

[0386] 8: 7.39 (t, J = 5.2 Hz, 1H), 4.09 (t, J = 6.4 Hz, 4H), 3.44 - 3.52 (m, 8H), 3.30 (q, J = 6.0 Hz, 2H), 2.81 (s, 1H), 2.29 - 2.31 (m, 9H), 1.63 - 1.69 (m, 15H), 1.44 (m, 4H), 1.26 - 1.29 (m, 32H), 1.17 (s, 3H), 0.88 (t, J= 6.8 Hz, 12H).

[0387] Example 17 General procedure for preparation ofICL-217

[0388] To a solution of T2 (350 mg, 501 pmol, 1.00 eq) and A19 (64.0 mg, 551 pmol, 1.10 eq) in DCM (3.50 mL) was added HATU (228 mg, 601 pmol, 1.20 eq) and DIEA (194 mg, 1.50 mmol, 3.00 eq). The mixture was stirred 20 °C for 12 hrs. LCMS (product: RT = 2.428 min) showed the starting material was consumed completely. The residue was poured into water (10.0 mL) and stirred for 5 min. The aqueous phase was extracted with DCM (10.0 mL). The combined organic phase was washed with brine (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*30mm*5um; mobile phase: [Heptane-THE: ACN=2:1]; gradient: 0%-30% B over 10.0 min). ICL-217 (150 mg, 188 pmol, 37.6% yield) was obtained as a colorless oil.

[0389] 'H NMR: ICL-217 (400 MHz, CDCh)

[0390] 8: 7.02 (t, J = 5.2 Hz, 1H), 4.09 (t, J = 6.4 Hz, 4H), 3.45 - 3.50 (m, 8H), 3.23 (q, J = 6.8 Hz, 2H), 2.81 (s, 1H), 2.28 - 2.34 (m, 9H), 1.63 - 1.69 (m, 17H), 1.54 - 1.56 (m, 6H), 1.26 (m, 32H), 1.16 (s, 3H), 0.88 (t, J= 6.4 Hz, 12H).

[0391] Example 18. LNP formulation and characterization

[0392] In brief, lipids were dissolved in ethanol at molar ratios of 47.5:12.5:38:2 (ionizable lipid / DSPC / Cholesterol / PEG-DMG2k). The lipid mixture was combined with a 25 mM sodium acetate buffer (pH 4) containing VZV gE mRNA made in-house 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-p m filter. All formulations were tested for particle size and PDI using a Zetasizer Nano ZS (Malvern Instruments Etd, Malvern, Worcestershire, UK). RNA encapsulation and concentration are measured with QUANT-IT™ RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, CA). Results are shown in Table 2. 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.

[0393] Most LNPs have sizes ranging from 80-150 nm, and with encapsulation efficiency >80%. LNPs with size >150 nm and / or encapsulation efficiency <50% were generally not tested further.

[0394] Table 2

[0395] Example 19 In vivo studies

[0396] Studies were performed with 6-8 week old female C57BL / 6 mice (Jackson) according to guidelines established by IACUC (Institutional Animal Care and Use Committee). 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.

[0397] 5.4.1 Study Timetable:

[0398] 5.4.3 IN VIVO STUDY DESIGN

[0399] 5.1 Test Animal Description

[0400] 5.2 Test System Management

[0401] 5.3 Study Design Table 3 Study Design Example 20 Serum total IgG measured by ELISA

[0402] N7N gE-specific serum total IgG induced by the VZV gE variant mRNAs / candidate cationic lipid vaccine were measured by ELISA.

[0403] 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 dilution 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).

[0404] The plate was washed three times using IX PBST and pat dry. Treated sample at 100 pL / well (single well), 100 p.L 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 pL 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.

[0405] LNP 257, an LNP formulation based on lipid ICL-209, LNP 260, an LNP formulation based on lipid ICL-211, and LNP 261, an LNP formulation based on lipid ICL-212 all 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 T-test.

[0406] Example 21. LNP stability assessment

[0407] To gain insights into the stability of these LNPs formulated with novel ionizable lipids, LNP 217 and 257, which are LNP formulations based on lipid ICL-209, as well as LNP 229 and LNP 261, which are LNP formulations based on lipid ICL-212 were used for stability assessment during medium term storage. Specifically, LNP217 and LNP229 were stored at - 80°C for up to 6 months, while LNP257 and LNP261 were stored at 4°C as an accelerated stability study for up to 4 weeks. Various timepoints during the storage were selected where the size and encapsulation efficiency (EE%) of the LNPs were measured. Results are shown in Figure 2. Both LNP217 and LNP229 were stable at -80°C for up to 6 months, with minimal change to the size and encapsulation efficiency of the LNPs. For the accelerated 4°C stability, both LNP261 and LNP257 showed excellent stability with minimal change to size and encapsulation efficiency for at least up to 4 weeks. These data indicate that the LNPs formulated with ionizable lipids ICL-209 and ICL-212 have excellent stability during medium term storage and potentially long-term storage. This was achieved without optimizing the LNP storage buffer, which once optimized will likely further increase its longer-term storage stability.

[0408] Example 22. Evaluation of LNPs for in vivo liver human EPO protein expression

[0409] We evaluated various LNPs made from ionizable lipids ICL-211 and ICL-212 by varying the ratio of their 4 lipid components for their ability to target liver using human erythropoietin (hEPO) mRNA as the cargo as shown in Table 4. All LNPs made had good sizing, PDI and high EE%. The specific ratios of each component in these LNPs are shown in Table 5. As positive controls, we chose to use LNPs made from SM-102 (CAS No.2089251-47-6), Lipid 5 (CAS No. 2089251-33-0) and Lipid A9 (CAS No.2036272-50-9), all of which are well- established ionizable lipids in the industry. For Lipid 5 from Moderna and Lipid A9 from Acuitas Therapeutics, both are well-known for their use in LNPs that aim to target liver, mostly hepatocytes, for protein replacement therapy or for gene editing.

[0410] Studies were performed with 6-8 week old female BALB / c mice (Jackson) according to guidelines established by IACUC (Institutional Animal Care and Use Committee). 150 pL of LNP solution containing 50 pg of hEPO mRNA were administered by intramuscular injection or tail vein injection. Animals were euthanized at 6 hr timepoint and terminal bleeding was performed. Blood samples were collected via cardiac puncture. Below is a detailed study protocol and study design for a typical in vivo study used to evaluate the LNP formulations.

[0411] 1. Study Timetable: 2. Test Animal Description

[0412] 3. Test System Management

[0413] 4. Study Design . Test articles used 6. Study Sample Collection

[0414] Example 22. Serum total Human erythropoietin (hEPO) levels measurement by ELISA

[0415] Serum total Human erythropoietin (hEPO) levels were measured using Human Erythropoietin SimpleStep ELISA® Kit (Abeam) according to manufacturing protocol. Briefly:

[0416] All reagents, standards, and samples were prepared as instructed in the manufacturer protocol. Removed any unused microplate strips, return them to the original foil pouch with desiccant, seal, and store at 4°C. Added 50 pL of each sample or standard to the appropriate wells in the microplate. Added 50 .L of the Antibody Cocktail to each well. Sealed the plate and incubate it for 1 hour at room temperature on a plate shaker set to 400 rpm.

[0417] Washed each well three times with 350 pL of IX Wash Buffer. For each wash: dispensed 350 pL of IX Wash Buffer into each well, allowed it to sit for at least 10 seconds, and then removed the liquid. After the final wash, inverted the plate and gently tapped it on clean paper towels to eliminate any remaining liquid.

[0418] Added 100 pL of development Solution to each well and incubate in the dark for 10 minutes on a plate shaker at 400 rpm. Note that the optimal incubation time may vary between 5 and 20 minutes, depending on laboratory conditions. The addition of stop solution will change the color in the wells from blue to yellow, and increase the signal intensity by approximately threefold.

[0419] Added 100 pL of stop solution to each well. Shook the plate for 1 minute on a plate shaker to ensure thorough mixing. Measured the optical density at 450 nm and recorded the final readings. hEPO levels were then back calculated based the final readings. Results are shown in Figure 3. LNP415, LNP416, LNP412 and LNP411 showed similar or better hEPO expression in vivo compared to the positive controls, including LNP421 made from Acuitas Lipid A9, which is known to be a potent lipid for liver expression. Various LNPs made from both lipid ICL-211 and ICL-212 showed similar or better hEPO expression in liver compared to LNP420 made from Moderna Lipid 5. Taken together, this shows that in addition for application in LNP vaccine, the lipids library presented within has the potential to make potent LNP formulations for excellent liver delivery and high liver protein expressions. As such, these lipids can potentially be applied to develop LNPs for protein replacement therapy or gene editing in liver.

[0420] Table 4. LNPs tested in the hEPO in vivo mice study

[0421]

[0422] Table 5 The specific ratios of each component in LNPs

Claims

1. What is claimed is:

1. A compounds having the following structure:(I), or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:Y is -OCO-, -COO-, -NHCO-, -CONH-, -O-, -S-, -SO2- or -SO-;Q is straight-chained C1-10 alkylene group, wherein an CH2 in the alkylene group is optionally replaced with an -COO- group;R is H or Ci-4alkyl;Ri and R2 are each the same or different, and independently selected from the group consisting of hydrogen, C1-6 alkyl, C2-6 alkenyl and C2-6 alkynyl, wherein an CH2 in the C1-6 alkyl, C2-6 alkenyl or C2-6 alkynyl is optionally replaced with one to three heteroatoms independently selected from the group consisting of NH, O, and S; or optionally Ri and R2 attached together with their adjacent N atom to form a saturated or unsaturated, substituted or unsubstituted 3- 10 membered nitrogen-containing heterocyclic group, m is an integer ranging from 2 to 14, and n and o are the same or different, and are each independently integers ranging from 2 to 14.

2. The compound of claim 1, wherein the compound has the following structure:or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

3. The compound of claim 1 or 2, wherein Q is straight-chained C2-8 alkylene group, wherein an CH2in the alkylene group is optionally replaced with an -COO- group.

4. The compound of any one of claims 1-3, wherein Q is selected from the group consisting ofethylene, n-propylene, n-butylene and&5. The compound of any one of claims 1-4, wherein R is H or methyl.

6. The compound of any one of claims 1-5, wherein Ri and R2 are each the same or different, and independently selected from the group consisting of hydrogen and C1-3 alkyl, wherein an CH2 in the C1-3 alkyl is optionally replaced with one heteroatom independently selected from the group consisting of NH, O, and S; or optionally Ri and R2 attach together with their adjacentN atom to form a saturated or unsaturated, substituted or unsubstituted 4-6 membered nitrogencontaining heterocyclic group.

7. The compound of any one of claims 1-6, wherein Ri and R2 are independently selected from the group consisting of methyl, ethyl, n-propyl and n-butyl.

8. The compound of any one of claims 1-7, wherein the heterocyclic group isor9. The compound of any one of claims 1-8, wherein m is an integer ranging from 2 to 8.

10. The compound of any one of claims 1-9, wherein m is 4.

11. The compound of any one of claims 1-10, wherein n and 0 are each independently integers ranging from 4 to 8.

12. The compound of any one of claims 1-11, wherein n is 6.

13. The compound of any one of claims 1-12, wherein 0 is 6.

14. A compound selected from a compound in Table below,ICL-200ICL-20715. A composition comprising the compound of any one of claims 1-14 and a therapeutic agent.

16. The composition of claim 15, further comprising one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipid.

17. The composition of claim 15, wherein the composition comprises one neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.

18. The composition of claim 17, wherein the neutral lipid is DOPE or DSPC.

19. The composition of any one of claims 16-18, wherein the molar ratio of the compound to the neutral lipid ranges from about 1: 4 to about 3: 1.

20. The composition of claim 16, wherein the steroid is cholesterol.

21. The composition of claim 20, wherein the molar ratio of the compound to cholesterol ranges from about 1 : 5 to about 5:1.

22. The composition of claim 16, wherein the polymer conjugated lipid is pegylated lipid.

23. The composition of claim 22, wherein the molar ratio of the compound to the pegylated lipid ranges from about 100: 1 to about 10: 1.

24. The composition of claim 22 or 23, wherein the pegylated lipid is PEG-DMG.

25. The composition of any one of claims 15-24, wherein the therapeutic agent comprises a nucleic acid.

26. The composition of claim 25, wherein the nucleic acid is selected from DNA, RNA, and hybrids thereof.

27. The composition of claim 26, wherein the nucleic acid is selected from antisense oligonucleotides, antisense and messenger RNA.

28. A method for administering a therapeutic agent to a patient in need thereof, the method comprises preparing or providing the compound according to any one of claims 1-14 or the composition of any one of claims 15-27, and administering the compound or the composition to the patient.

29. Use of the compound according to any one of claims 1-14 or the composition of any one of claims 15-27 in the manufacture of a medicament for treating a disease.

30. The compound according to any one of claims 1-14 or the composition of any one of claims 15-27, for use as a medicament for treating a disease.

31. A pharmaceutical composition for treating a disease, which comprises the compound of any one of claims 1-14 and a therapeutic agent.

32. A method for preparing immediate T1 to prepare the compounds according to any one of claims 2-14, comprisingin step 1, reactant 4A reacts with reactant:;K;* in a solvent in the presence of KOH and TBAB in a reaction temperature for a reaction time, wherein the amount ofis 0.5-4.0eq; the amount of KOH is 1.5-4.5eq; the amount of TBAB is 0.05-0.2eq; the reaction temperature is 60-120°C; the reaction time is 8-20 hours; the solvent is preferably Tol in step 1, and the volume of the solvent is 5-30 V;in step 2, intermediate 4C- 1 reacts with reactant in a solvent in the presence of NaH in a reaction temperature for a reaction time, wherein the amount ofis 0.5-4.0eq; the amount of NaH is 2.0-6. Oeq; the reaction temperature is 40-100°C; the reaction time is 8-20 hours; the solvent is preferably THF in step 2, and the volume of the solvent is 5-30 V; in step 3, deprotect the hydroxyl protecting group TBS- group in intermediate 4C to obtain intermediate 4E with hydroxyl group in a solvent in the presence of TBAF in a reaction temperature for a reaction time, wherein the amount of TBAF is 0.5-4. Oeq; the reaction temperature is 10-30°C; the reaction time is 0.5-3 hours; the solvent is preferably THF in step 3, and the volume of the solvent is 5- 30 V; in step 4, the esterification reaction between intermediate 4E and reactantobtains intermediate 4B in a solvent in the presence of EDCI and DMAP in a reaction temperature for a reaction time, wherein the amount ofis 0.5-4.0eq; the amount of EDCI is 0.5-2. Oeq; the amount of DMAP is 0.05-0.3eq; the reaction temperature is 10-40°C; the reaction time is 8-20 hours; the solvent is preferably DCM in step 4, and the volume of the solvent is 5-30 V; in step 5, in the presence of catalysts, intermediate 4B is reduced to obtain intermediate T1 under H2 (50 psi); the catalysts are preferably 10% Pd / C and 10%Pd(OH)2; the reaction temperature is 30-70°C; the reaction time is 8-20 hours; the solvent is preferably THF, and the volume of the solvent is 5-30 V; wherein Q, R, Ri, R2, m, n and o are as defined as claim 2.

33. A method for preparing Formula (II) of any one of claims 2-14, comprisingin step 6, intermediate T1 according to claim 32 reacts with reactantto obtain a compound of formula (II) in a solvent in the presence of EDCI and DMAP in a reaction temperature for a reaction time, wherein the amount ofis 0.5-2.0eq; the amount of EDCI is 0.5-2.0eq; the amount of DMAP is 0.05-0.3eq; the reaction temperature is 10-40°C; the reaction time is 8-20 hours; the solvent is preferably DCM in step 6, and the volume of the solvent is 5-30 V; wherein Q, R, Ri, R2, m, n and o are as defined as claim 2.

34. A method for preparing immediate T2 to prepare the compounds according to any one of claims 2-14, comprisingin step 1, reactant 13 reacts within solvents in the presence of NaH in a reaction temperature for a temperature time,wherein the amount ofis 0.5-4.0eq; the amount of NaH is 0.5-4.0eq; the reaction temperature is 0-30°C; the reaction time is 10-30 hours; the solvent is preferably Tol and DMF in step 1, the volume of Tol is 5-30 V; the volume of DMF is 15-50 V; in step 2, intermediate 14 is oxidized by IBX in a solvent in a reaction temperature for a temperature time, wherein the amount of IBX is 0.5-2.0eq; the reaction temperature is 60-100°C; the reaction time is 2-6 hours; the solvent is preferably MeCN in step 2, and the volume of the solvent is 5- 30 V; in step 3, intermediate 15 is oxidized in the presence of NaCICh and NafTPCh in solvents in a reaction temperature for a temperature time, wherein the amount of NaCIC is 3.0-6.0 eq; the amount of NafTPCh is 1.0-3.0 eq; the reaction temperature is 10-30°C; the reaction time is 2-6 hours; the solvent is preferably DMSO and MeCN / H2O(5 / l) in step 3, the amount of DMSO is 1.5-3.0 eq, and the volume of MeCN / H2O(5 / l) is 5-30 V; in step 4, the carboxyl group of intermediate 16 is protected by a carboxyl protecting group Bn-group by reacting intermediate 16 with BnBr in the presence of K2CO3 in a solvent in a reaction temperature for a reaction time, wherein the amount of BnBr is 0.5-2.5eq; the amount of K2CO3 is 1.0-3.0eq; the reaction temperature is 10-30°C; the reaction time is 4-6 hours; the solvent is preferably DMF in step 4, and the volume of the solvent is 5-30 V; in step 5, deprotect the hydroxyl protecting group TBS- group in intermediate 17 to obtain intermediate 18 with hydroxyl group in a solvent in the presence of TBAF in a reaction temperature for a reaction time, wherein the amount of TBAF is 1.0-3.0eq; the reaction temperature is 10-30°C; the reaction time is 2-6 hours; the solvent is preferably THF in step 5, and the volume of the solvent is 5- 30 V; in step 6, the esterification reaction between intermediate 18 andobtains intermediate 19 in a solvent in the presence of EDCI and DMAP in a reaction temperature for a reaction time,wherein the amount ofis 1.5-4.5eq; the amount of EDCI is 2.5-4.5eq; the amount of DMAP is 0.05-0.3eq; the reaction temperature is 10-40°C; the reaction time is 60- 100 hours; the solvent is preferably DCM in step 6, and the volume of the solvent is 5-30 V; in step 7, in the presence of catalysts, intermediate 19 is reduced to obtain intermediate T2, wherein the catalysts are preferably Pd / C (10%, 15 psi); the reaction temperature is 10-30°C; the reaction time is 2-8 hours; the solvent is preferably EtOH, and the volume of the solvent is 5-30 V; wherein Q, R, Ri, R2, m, n and o are as defined as claim 2.

35. A method for preparing Formula (III) or (IV) of any one of claims 2-14, comprisingin step 8, intermediate T2 according to claim 34 reacts with reactantto obtain a compound of formula (III) in a solvent in the presence of PPh ? and DIAD in a reaction temperature for a reaction time,wherein the amount ofis 0.5-2.0eq; the amount of PPh ? is 0.5-2.0eq; the amount of DIAD is 0.5-2.0eq; the reaction temperature is 10-40°C; the reaction time is 8-20 hours; the solvent is preferably THF in step 8, and the volume of the solvent is 5-30 V; or in step 8’, intermediate T2 according to claim 34 reacts with reactantto obtain a compound of formula (IV) in a solvent in the presence of HATU and DIEA in a reaction temperature for a reaction time, wherein the amount of- js0.5-2.0eq; the amount of HATU is 0.5-2.0eq; the amount of DIEA is 1.5-4.5eq; the reaction temperature is 10-40°C; the reaction time is 8-20 hours; the solvent is preferably DCM in step 8’, and the volume of the solvent is 5-30 V; wherein Q, R, Ri, R2, m, n and o are as defined as claim 2.

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