Novel lipid and lipid nanoparticle formulations for nucleic acid delivery

Abstract

To provide lipid compounds which can be used alone or in combination with other lipid components such as neutral lipids, charged lipids, steroids and / or their analogs, and / or polymer conjugated lipids, so as to form lipid nanoparticles for delivery of therapeutic agents.SOLUTION: The invention provides compounds having a structural formula in the figure or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. In the formula, L1 and L2 are -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, or the like; G1 and G2 are independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene; G3 is C1-C24 alkylene, C1-C24 alkenylene, or the like; R1 and R2 are independently C6-C24 alkyl or C6-C24 alkenyl; and R3 is H, OR5, CN, -C(=O)OR4, or the like.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention generally relates to novel cationic lipids for combining with other lipid components, such as neutral lipids, cholesterol, and polymer-conjugated lipids, to form lipid nanoparticles with oligonucleotides to facilitate intracellular delivery of therapeutic nucleic acids (e.g., oligonucleotides, messenger RNA, etc.), both in vitro and in vivo. [Background technology]

[0002] 2. Description of Related Art The delivery of nucleic acids to affect desired responses in biological systems presents many challenges. Nucleic acid-based therapeutics hold enormous potential, but to realize this potential, there remains a need for more effective delivery of nucleic acids to appropriate sites within cells or organisms. Therapeutic nucleic acids include, for example, messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immunostimulatory nucleic acids, antagomirs, antimirs, mimetics, supermirs, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to achieve the expression of specific cellular products, useful for treating, for example, diseases associated with protein or enzyme deficiencies. The therapeutic applications of translatable nucleotide delivery are extremely broad, as constructs can be synthesized to produce any selected protein sequence, whether native to the system or not. The expression product of a nucleic acid can increase existing protein levels, replace missing or non-functional versions of proteins, or introduce new proteins and associated functionalities into cells or organisms.

[0003] Some nucleic acids, such as miRNA inhibitors, can be used to achieve the expression of specific cellular products regulated by miRNA, for example, useful for treating diseases related to protein or enzyme deficiency.The therapeutic application of miRNA inhibition is extremely broad, as constructs can be synthesized to inhibit one or more miRNAs that in turn regulate the expression of mRNA products.Inhibition of endogenous miRNAs can increase the expression of their downstream target endogenous proteins, restoring proper function in cells or organisms as a means of treating diseases related to specific miRNAs or groups of miRNAs.

[0004] Other nucleic acids can downregulate the intracellular levels of specific mRNAs, resulting in downregulation of the synthesis of the corresponding proteins through processes such as RNA interference (RNAi) or complementary binding of antisense RNA. The therapeutic applications of antisense oligonucleotides and RNAi are also very broad, as oligonucleotide constructs can be synthesized with any nucleotide sequence directed against a target mRNA. Targets can include mRNAs derived from normal cells, mRNAs associated with disease states such as cancer, and mRNAs of infectious agents such as viruses. To date, antisense oligonucleotide constructs have demonstrated the ability to specifically downregulate target proteins through the degradation of cognate mRNAs in both in vitro and in vivo models. Furthermore, antisense oligonucleotide constructs are currently being evaluated in clinical studies.

[0005] However, two problems currently confront the use of oligonucleotides in therapeutic settings. First, free RNA is susceptible to nuclease digestion in plasma. Second, free RNA has limited access to intracellular compartments where the relevant translation machinery resides. Lipid nanoparticles formed from cationic lipids with other lipid components, such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides, have been used to block RNA degradation in plasma and promote cellular uptake of oligonucleotides. Summary of the Invention [Problem to be solved by the invention]

[0006] There is a need for improved cationic lipids and lipid nanoparticles for the delivery of oligonucleotides. Preferably, these lipid nanoparticles provide an optimal drug:lipid ratio, protect the nucleic acid from degradation and clearance in serum, are suitable for systemic or local delivery, and provide intracellular delivery of the nucleic acid. Furthermore, these lipid-nucleic acid particles should be well tolerated and provide an adequate therapeutic index so that treatment of patients with an effective dose of nucleic acid is not associated with unacceptable toxicity and / or risks to the patient. The present invention provides these and related advantages. [Means for solving the problem]

[0007] overview In summary, the present invention provides lipid compounds, including their stereoisomers, pharmaceutically acceptable salts, or tautomers, which can be used alone or in combination with other lipid components, such as neutral lipids, charged lipids, steroids (including, for example, all sterols) and / or their analogs, and / or polymer-conjugated lipids, to form lipid nanoparticles for the delivery of therapeutic agents. In some examples, the lipid nanoparticles are used to deliver nucleic acids, such as antisense and / or messenger RNA. Methods of using such lipid nanoparticles for the treatment of various diseases or conditions, such as those caused by infectious agents and / or protein deficiencies, are also provided.

[0008] In one embodiment, the compound has the following structural formula (I): [ka] (I) [In the formula, R 1 , R 2 , R 3 , L 1 , L2 , G 1 , G 2 and G 3 is as defined herein] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0009] Pharmaceutical compositions and therapeutic agents comprising one or more of the compounds of formula (I) are also provided. In some embodiments, the pharmaceutical compositions further comprise one or more components selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. Such compositions are useful for forming lipid nanoparticles for the delivery of therapeutic agents.

[0010] In another embodiment, the present invention provides a method of administering a therapeutic agent to a patient in need thereof, comprising preparing a lipid nanoparticle composition comprising a compound of structural formula (I) and the therapeutic agent, and delivering the composition to the patient.

[0011] These and other aspects of the present invention will become evident upon reference to the following detailed description.

[0012] A brief description of some views of the drawing In the drawings, like reference numbers indicate like elements. The sizes and relative positions of elements in the drawings are not necessarily drawn to scale, and some of these elements have been arbitrarily enlarged and positioned to improve legibility of the figures. Furthermore, the particular shapes of the depicted elements are not intended to convey any information regarding the actual shape of the particular elements, but have been selected solely to facilitate recognition of the drawings. [Brief explanation of the drawings]

[0013] [Figure 1] 1 shows the time course of luciferase expression in mouse liver. [Figure 2] Calculation of the pKa of MC3 as a representative example related to the disclosed lipids is shown. [Figure 3]Comparative luciferase activity data for selected lipids is provided. DETAILED DESCRIPTION OF THE INVENTION

[0014] Detailed Description In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the invention. However, it will be understood by those skilled in the art that the invention may be practiced without these details.

[0015] The present invention is based, in part, on the discovery of novel cationic (amino) lipids that offer advantages when used in lipid nanoparticles for the in vivo delivery of active or therapeutic agents, such as nucleic acids, to mammalian cells. In particular, embodiments of the present invention provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel cationic lipids described herein, which provide increased activity of the nucleic acid in vivo and improved tolerability of the composition, resulting in a significantly increased therapeutic index compared to previously described nucleic acid-lipid nanoparticle compositions.

[0016] In certain embodiments, the present invention provides novel cationic lipids that enable the formulation of improved compositions for in vitro and in vivo delivery of mRNA and / or other oligonucleotides. In some embodiments, these improved lipid nanoparticle compositions are useful for the expression of proteins encoded by mRNA. In other embodiments, these improved lipid nanoparticle compositions are useful for upregulating endogenous protein expression by delivering miRNA inhibitors that target a specific miRNA or a group of miRNAs that control a single target mRNA or several mRNAs. In other embodiments, these improved lipid nanoparticle compositions are useful for downregulating (e.g., silencing) the protein and / or mRNA levels of target genes. In some other embodiments, lipid nanoparticles are also useful for delivering mRNA and plasmids for transgene expression. In still other embodiments, lipid nanoparticle compositions are useful for inducing pharmacological effects resulting from protein expression, such as increased red blood cell production through delivery of appropriate erythropoietin mRNA or protection against infection through delivery of mRNA encoding appropriate antigens or antibodies.

[0017] The lipid nanoparticles and compositions of the present invention can be used for a variety of purposes, including the delivery of encapsulated or associated (e.g., conjugated) therapeutic agents, such as nucleic acids, to cells, both in vitro and in vivo. Accordingly, an embodiment of the present invention is a method of treating or preventing a disease or disorder in a subject in need thereof by contacting the subject with lipid nanoparticles that encapsulate or are associated with a suitable therapeutic agent, the lipid nanoparticles comprising one or more of the novel cationic lipids described herein.

[0018] As described herein, embodiments of the lipid nanoparticles of the present invention are particularly useful for the delivery of nucleic acids, such as, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomir / antimir), messenger RNA interfering complementary RNA (micRNA), DNA, polyvalent RNA, Dicer substrate RNA, and complementary DNA (cDNA). Thus, the lipid nanoparticles and compositions of the present invention can be used to induce expression of a desired protein both in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more of the novel cationic lipids described herein, where the lipid nanoparticles encapsulate or bind a nucleic acid to be expressed to produce the desired protein (e.g., a messenger RNA or plasmid encoding the desired protein) or a nucleic acid to be expressed to inhibit a process that terminates mRNA expression (e.g., an miRNA inhibitor). Alternatively, the lipid nanoparticles and compositions of the present invention can be used to reduce target gene and protein expression both in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more of the novel cationic lipids described herein, wherein the lipid nanoparticles encapsulate or bind a nucleic acid (e.g., an antisense oligonucleotide or small interfering RNA (siRNA)) that reduces target gene expression. The lipid nanoparticles and compositions of the present invention can be used separately or in combination for co-delivery of different nucleic acids (e.g., mRNA and plasmid DNA), as may be useful for providing effects requiring co-localization of different nucleic acids (e.g., mRNA encoding an appropriate gene-modifying enzyme and a DNA segment for integration into the host genome).

[0019] Nucleic acids for use in the present invention can be prepared according to any available technique. For mRNA, the primary methodology of preparation is, but is not limited to, enzymatic synthesis (also called in vitro transcription), which currently represents the most efficient method for generating long, sequence-specific mRNA. In vitro transcription describes the process of template-directed synthesis of RNA molecules from an engineered DNA template (e.g., including, but not limited to, those derived from T7, T3, and SP6 coliphages) containing an upstream bacteriophage promoter sequence linked to a downstream sequence encoding a gene of interest. Template DNA can be prepared for in vitro transcription from many sources using suitable techniques known in the art, including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, JL and Conn, GL, General protocols for preparation of plasmid DNA template and Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn GL (ed), New York, NY Humana Press, 2012).

[0020] RNA transcription is carried out in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the resulting mRNA transcript. In vitro transcription can be carried out using a variety of commercially available kits, including, but not limited to, the RiboMax Large Scale RNA Production System (Promega) and the MegaScript Transcription Kit (Life Technologies), as well as commercially available reagents such as RNA polymerase and rNTPs. Methodologies for in vitro transcription of mRNA are well known in the art. (e.g. Losick, R., 1972, In vitro transcription, Ann Rev Biochem v.41 409-46; Kamakaka, RT and Kraus, WL 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology v. 703 (Neilson, H. Ed), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter Five-In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530, 101-114; all of which are incorporated herein by reference).

[0021] The desired in vitro transcribed mRNA is then purified from undesired components of the transcription or related reaction (unincorporated rNTPs, protein enzymes, salts, short RNA oligos, etc.). Techniques for isolating mRNA transcripts are well known in the art. Well-known procedures include phenol / chloroform extraction or precipitation with either alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Further non-limiting examples of purification procedures that can be used include size exclusion chromatography (Lukavsky, PJ and Puglisi, JD, 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v.10, 889-893), silica-based affinity chromatography and polyacrylamide gel electrophoresis (Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA synthesis Methods v. 941 Conn GL (ed), New York, NY Humana Press, 2012). Purification can be performed using various commercially available kits, including, but not limited to, SV Total Isolation System (Promega) and In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0022] Furthermore, while reverse transcription can produce large amounts of mRNA, the product may contain numerous aberrant RNA impurities associated with undesired polymerase activity that may need to be removed from full-length mRNA preparations. These include short RNAs resulting from incomplete transcription initiation, as well as double-stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates, and self-complementary 3' extensions. It has been demonstrated that these contaminants, which have dsRNA structures, can result in unwanted immunostimulatory activity through interactions with various innate immune sensors within eukaryotic cells that recognize specific nucleic acid structures and function to induce potent immune responses. This, in turn, can dramatically reduce mRNA translation due to reduced protein synthesis during the innate cellular immune response. Therefore, additional techniques for removing these dsRNA contaminants have been developed and are known in the art, including, but not limited to, scalable HPLC purification (see, e.g., Kariko, K., Muramatsu, H., Ludwig, J. And Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v. 39 e142; Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K., HPLC Purification of in vitro transcribed long RNA in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013). HPLC-purified mRNA has been reported to be translated at much higher levels, particularly in primary cells and in vivo.

[0023] A variety of significant modifications have been described in the art to alter the specific properties of in vitro transcribed mRNA and improve its utility. These include, but are not limited to, modifications of the 5' and 3' ends of mRNA. Endogenous eukaryotic mRNAs typically contain a cap structure at the 5' end of the mature molecule, which plays an important role in mediating the binding of mRNA cap-binding protein (CBP), which in turn is involved in increasing mRNA stability and the efficiency of mRNA translation in the cell. Therefore, the highest level of protein expression is achieved with capped mRNA transcripts. The 5' cap contains a 5'-5'-triphosphate linkage between the 5'-most nucleotide and a guanine nucleotide. The complexed guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the final and penultimate 5'-most nucleotides on the 2'-hydroxyl group.

[0024] Several different cap structures can be used to generate the 5' cap of in vitro transcribed synthetic mRNA. 5'-capping of synthetic mRNA can be performed co-transcriptionally with a chemical cap analog (i.e., capping during in vitro transcription). For example, the anti-reverse cap analog (ARCA) cap contains a 5'-5'-triphosphate guanine-guanine linkage in which one guanine contains an N7 methyl group and a 3'-O-methyl group. However, during this co-transcription process, up to 20% of transcripts remain uncapped, and synthetic cap analogs are not identical to the 5' cap structure of authentic cellular mRNA, potentially reducing translational competence and cellular stability. Alternatively, synthetic mRNA molecules can be enzymatically capped after transcription. These can generate more authentic 5' cap structures that more closely resemble, either structurally or functionally, the endogenous 5'-cap with enhanced binding of cap-binding proteins, increasing half-life, reducing susceptibility to 5' endonucleases, and / or reducing 5' decapping. Numerous synthetic 5' cap analogs have been developed and are known in the art to improve mRNA stability and translational competence (see, e.g., Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, A.N., Slepenkov, S.V., Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, R.E., Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, P.H. Ed), 2013).

[0025] At the 3' end, a long chain of adenine nucleotides (a poly-A tail) is typically added to mRNA molecules during RNA processing. Shortly after transcription, the 3' end of the transcript is cleaved, freeing the 3' hydroxyl that poly-A polymerase uses to add a chain of adenine nucleotides to the RNA in a process called polyadenylation. The poly(A) tail has been extensively shown to enhance both the translation efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly(A), poly(A) binding protein and the regulation of mRNA stability, Trends Bio Sci v. 14 373-377; Guhaniyogi, J. and Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M. and Regnier, P., 2002, The poly(A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, v. 111, 611-613).

[0026] Poly(A) tailing of in vitro transcribed mRNA can be achieved using various approaches, including, but not limited to, cloning a poly(T) tract into a DNA template or post-transcriptional addition using poly(A) polymerase. The first approach allows for in vitro transcription of mRNA with a poly(A) tail of defined length, depending on the size of the poly(T) tract, but requires further manipulation of the template. The latter approach involves enzymatically adding a poly(A) tail to in vitro transcribed mRNA using poly(A) polymerase, which catalyzes the incorporation of adenine residues into the 3' end of the RNA, resulting in mRNA with poly(A) tails of heterogeneous lengths, but does not require further manipulation of the DNA template. 5'-capping and 3'-poly(A) tailing can be performed using a variety of commercially available kits, including but not limited to the Poly(A) Polymerase Tailing Kit (EpiCenter), mMESSAGE mMACHINE T7 Ultra Kit and Poly(A) Tailing Kit (Life Technologies), as well as commercially available reagents, various ARCA caps, poly(A) polymerases, etc.

[0027] In addition to 5' capping and 3' polyadenylation, other modifications of in vitro transcripts have been reported to provide benefits related to translation efficiency and stability. It is well known in the art that pathogenic DNA and RNA are recognized by various sensors in eukaryotes and induce strong innate immune responses. Because most nucleic acids from natural sources contain modified nucleosides, it has been shown that the ability to distinguish between pathogenic and self-DNA and RNA is at least partially based on structural and nucleoside modifications. In contrast, in vitro synthesized RNA lacks these modifications and therefore becomes immunostimulatory and can inhibit effective mRNA translation, as outlined above.Introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thus mitigating this undesired immunostimulatory activity and enhancing translational competence (e.g., Kariko, K. And Weissman, D. 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: Implication for therapeutic RNA development, Curr Opin Drug Discov Devel, v.10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, P.H. (Ed., 2013); Kariko, K., Muramatsu, H., Welsh, FA, Ludwig, J., Kato, H., Akira, S., Weissman, D., 2008, Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability, Mol Ther v.16, 1833-1840). Modified nucleosides and nucleotides used in the synthesis of modified RNAs can be monitored and utilized using common methods and procedures known in the art. A wide variety of nucleoside modifications are available that may be incorporated to some extent into in vitro transcribed mRNAs, either alone or in combination with other modified nucleosides (see, e.g., US2012 / 0251618).In vitro synthesis of nucleoside-modified mRNA has been reported to have a reduced ability to activate immune sensors and a concomitant enhanced translational capacity.

[0028] Other components of mRNA that can be modified to provide benefits in terms of translatability and stability include the 5' and 3' untranslated regions (UTRs). Optimizing UTRs (favorable 5' and 3' UTRs can be obtained from cellular or viral RNA), either together or independently, has been shown to increase mRNA stability and translation efficiency of in vitro transcribed mRNA (see, for example, Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013).

[0029] In addition to mRNA, other nucleic acid payloads can also be used in the present invention. For oligonucleotides, preparation methods include, but are not limited to, chemical synthesis, enzymatic or chemical cleavage of longer precursors, in vitro transcription, etc. Methods for synthesizing DNA and RNA nucleotides are widely used and well known in the art (see, for example, Gait, MJ (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, DC: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, NJ) Totowa, NJ: Humana Press, 2005; both of which are incorporated herein by reference).

[0030] With respect to plasmid DNA, preparation for use in the present invention generally utilizes, but is not limited to, in vitro propagation and isolation of the plasmid DNA in a liquid culture of bacteria containing the plasmid of interest. The presence of a gene in the plasmid of interest that encodes resistance to a particular antibiotic (penicillin, kanamycin, etc.) allows bacteria containing the plasmid of interest to selectively grow in antibiotic-containing culture. Methods for isolating plasmid DNA are widely used and well known in the art (see, e.g., Heilig, J., Elbing, KL and Brent, R (2001) Large-Scale Preparation of Plasmid DNA. Current Protocols in Molecular Biology. 41:11:1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillstroem, S., Bjoernestedt, R. and Schmidt, SR (2008). Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture. Biotechnol. Bioeng., 99:557-566; and US6197553B1). Plasmid isolation can be performed using a variety of commercially available kits and reagents, including, but not limited to, Plasmid Plus (Qiagen), GenJET Plasmid MaxiPrep (Thermo), and PureYield MaxiPrep (Promega) kits.

[0031] Various exemplary embodiments of the cationic lipids, lipid nanoparticles and compositions comprising the same of the present invention and their use to deliver active agents (e.g., therapeutic agents), such as nucleic acids for modulating gene and protein expression, are described in further detail below.

[0032] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0033] Unless the context requires otherwise, throughout this specification and claims, the word "comprises," "comprising," and variations thereof are to be interpreted in their inclusive sense of "including, but not limited to."

[0034] Throughout this specification, the phrase "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrase "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.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0036] The phrase "inducing the expression of a desired protein" refers to the ability of a nucleic acid to increase the expression of a desired protein. To determine the level of protein expression, a test sample (e.g., a sample of cultured cells expressing the desired protein) or a test mammal (e.g., a mammal, such as a human or an animal model such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) is contacted with a nucleic acid (e.g., a nucleic acid combined with a lipid of the present invention). The expression of the desired protein in the test sample or test animal is compared with the expression of the desired protein in a control sample (e.g., a sample of cultured cells expressing the desired protein) or a control mammal (e.g., a mammal, such as a human or an animal model such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) that has not been contacted with or administered the nucleic acid. If the desired protein is present in the control sample or control mammal, the expression of the desired protein in the control sample or control mammal can be assigned a value of 1.0. In certain embodiments, induction of expression of a desired protein is achieved when the ratio of desired protein expression in a test sample or test mammal to the level of desired protein expression in a control sample or control mammal is greater than 1, e.g., about 1.1, 1.5, 2.0, 5.0, or 10.0. Induction of expression of a desired protein is achieved when a measurable level of the desired protein is detected in a test sample or test mammal when the desired protein is not present in the control sample or control mammal. Those skilled in the art will recognize appropriate assays for determining the level of protein expression in a sample, such as, for example, dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzymatic function, and phenotypic assays, or assays based on reporter proteins that can produce fluorescence or luminescence under appropriate conditions.

[0037] The phrase "inhibit the expression of a target gene" refers to the ability of a nucleic acid to suppress, reduce, or inhibit the expression of a target gene. To determine the degree of gene silencing, a test sample (e.g., a sample of cultured cells expressing a target gene) or a test mammal (e.g., a mammal, such as a human or an animal model such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) is contacted with a nucleic acid that suppresses, reduces, or inhibits the expression of a target gene. The expression of the target gene in the test sample or test animal is compared with the expression of the target gene in a control sample (e.g., a sample of cultured cells expressing a target gene) or a control mammal (e.g., a mammal, such as a human or an animal model such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) that has not been contacted with or administered with a nucleic acid. The expression of the target gene in the control sample or control mammal can be assigned a value of 100%. In certain embodiments, suppression, reduction, or inhibition of target gene expression is achieved when the level of target gene expression in a test sample or test mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% of the level of target gene expression in a control sample or control mammal. In other words, the nucleic acid can suppress, reduce, or inhibit expression of the target gene in a test sample or test mammal by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to the level of target gene expression in a control sample or control mammal not contacted with or administered the nucleic acid. Suitable assays for determining the level of target gene expression include, but are not limited to, examining protein or mRNA levels using techniques known to those skilled in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those skilled in the art.

[0038] 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 a desired effect, such as increased or inhibited expression of a target sequence compared to normal expression levels detected in the absence of the nucleic acid. For expression products that are not present in the absence of nucleic acid, increased expression of the target sequence is achieved when a measurable level is detected. If the expression product is present at a level prior to contact with the nucleic acid, Increased expression is achieved when the fold increase over that obtained with a nucleic acid such as mRNA compared to a 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 more. Inhibition of expression of a target gene or target sequence is achieved when the value obtained with a nucleic acid such as an antisense oligonucleotide relative to a 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, but are not limited to, examination of protein or mRNA levels using techniques known to those of skill in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of an appropriate reporter protein, and phenotypic assays known to those of skill in the art.

[0039] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single- or double-stranded form, including DNA, RNA, and hybrids thereof. DNA can be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA can be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, polyvalent 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, natural, and unnatural, and have similar binding properties to the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences as well as the explicitly set forth sequence. Specifically, degenerate codon substitutions can 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)). A "nucleotide" comprises the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together via the phosphate group."Bases" include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, purines and pyrimidines, further including natural analogs and synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that place new reactive groups such as amines, alcohols, thiols, carboxylates, and alkyl halides.

[0040] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that comprises partial or full-length coding sequences necessary for the production of a polypeptide or precursor polypeptide.

[0041] As used herein, "gene product" refers to the product of a gene, such as an RNA transcript or a polypeptide.

[0042] The term "lipid" refers to a group of organic compounds, including, but not limited to, esters of fatty acids, that are generally 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 and waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0043] "Steroids" are compounds with the following carbon skeleton: [ka] Non-limiting examples of steroids include cholesterol, and the like.

[0044] "Cationic lipid" refers to a lipid that can be positively charged. Exemplary cationic lipids contain one or more amine groups with a positive charge. Preferred cationic lipids are ionizable, so that they can exist in a positively charged form or a neutral form depending on pH. The ionization of cationic lipids affects the surface charge of lipid nanoparticles under different pH conditions. This charge state can affect the absorption of plasma proteins, blood clearance and tissue distribution (Semple, SC, et al., Adv. Drug Deliv Rev 32:3-17 (1998)), as well as the ability to form endosomolytic non-bilayer structures, which are important for the delivery of nucleic acids into cells (Hafez, IM, et al., Gene Ther 8:1188-1196 (2001)).

[0045] The term "polymer-conjugated lipid" refers to a molecule that contains 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 that contains both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG).

[0046] The term "neutral lipid" refers to any of a number of lipid species that exist in either uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphatidylcholines such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), steroids such as sphingomyelin (SM), ceramides, sterols, and their derivatives. Neutral lipids may be synthetic or naturally derived.

[0047] The term "charged lipid" refers to any of a number of lipid species that exist in either a positively or negatively charged form, independent of pH within a useful physiological range, e.g., from pH 3 to pH 9. Charged lipids can be synthetic or naturally occurring. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethanecarbamoylsterol (e.g., DC-Chol).

[0048] The term "lipid nanoparticle" refers to a particle having at least one dimension on the nanometer order (e.g., 1-1000 nm) that comprises one or more compounds of structural formula (I) or other specific cationic lipids. In some embodiments, lipid nanoparticles are included in formulations that can be used to deliver active or therapeutic agents, such as nucleic acids (e.g., mRNA), to a desired target site (e.g., a cell, tissue, organ, tumor, etc.). In some embodiments, lipid nanoparticles of the present invention comprise nucleic acids. Such lipid nanoparticles typically comprise a compound of structural formula (I) and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. In some embodiments, an active or therapeutic agent, such as a nucleic acid, may be encapsulated in the lipid portion of the lipid nanoparticle, or in the aqueous space enclosed by some or all of the lipid portion of the lipid nanoparticle, thereby protecting the active or therapeutic agent from enzymatic degradation or other undesirable effects induced by host organism or cellular mechanisms, such as a harmful immune response.

[0049] In various embodiments, the lipid nanoparticles are about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or have an average diameter of 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, the nucleic acid, when present in the lipid nanoparticle, is resistant to degradation by nucleases in aqueous solution. Lipid nanoparticles containing nucleic acids and methods for their preparation are disclosed, for example, in U.S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031 and International Publication Nos. WO 2013 / 016058 and WO 2013 / 086373, the entire disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0050] As used herein, "lipid encapsulation" refers to lipid nanoparticles that provide an active or therapeutic agent, such as a nucleic acid (e.g., mRNA), by complete encapsulation, partial encapsulation, or both. In one embodiment, the nucleic acid (e.g., mRNA) is completely encapsulated in the lipid nanoparticle.

[0051] As used herein, the term "aqueous solution" means a composition that includes water.

[0052] "Serum stability" in the context of nucleic acid-lipid nanoparticles means that the nucleotides are not significantly degraded after exposure to serum or nuclease assays that significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, DNAse assays, or RNAse assays.

[0053] As used herein, "systemic delivery" refers to the delivery of a therapeutic product that can result in widespread exposure of an active agent within an organism. Some administration techniques can result in systemic delivery of certain agents, but not others. Systemic delivery means that a useful, preferably therapeutically useful, amount of the agent is exposed to most parts of the body. Systemic delivery of lipid nanoparticles can be achieved by any means known in the art, such as, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is achieved by intravenous delivery.

[0054] As used herein, "local delivery" refers to the direct delivery of an active agent to a target site within an organism. For example, an agent can be delivered locally to a disease site such as a tumor, another target site such as an inflammation site, or a target organ such as the liver, heart, pancreas, or kidney by direct injection. Local delivery can also include topical application or local injection techniques such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not exclude systemic pharmacological effects.

[0055] "Alkyl" refers to an alkyl group having, for example, 1 to 24 carbon atoms (C-C), such as, for example, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. 24 alkyl), 4 to 20 carbon atoms (C4-C 20 alkyl), 6 to 16 carbon atoms (C6-C 16 alkyl), 6 to 9 carbon atoms (C6-C9 alkyl), 1 to 15 carbon atoms (C1-C 15 alkyl), 1 to 12 carbon atoms (C1-C 12"C-C alkyl" means a saturated or unsaturated (i.e., containing one or more double (alkenyl) and / or triple bonds (alkynyl)), straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, having 1 to 8 carbon atoms (C-C alkyl) or 1 to 6 carbon atoms (C-C alkyl), and attached to the rest of the molecule by a single bond. Unless otherwise specified in the specification, an alkyl group is optionally substituted.

[0056] An "alkylene" or "alkylene chain" refers to an alkylene group having, for example, 1 to 24 carbon atoms (C-C), such as, for example, methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. 24 Alkylene, 1 to 15 carbon atoms (C1-C 15 Alkylene, 1 to 12 carbon atoms (C1-C 12 "Alkylene" means a saturated or unsaturated (i.e., containing one or more double (alkenyl) and / or triple bonds (alkynyl)), straight or branched divalent hydrocarbon chain, consisting solely of carbon and hydrogen atoms, having 1 to 8 carbon atoms (C-C alkylene), 1 to 6 carbon atoms (C-C alkylene), 2 to 4 carbon atoms (C-C alkylene), or 1 to 2 carbon atoms (C-C alkylene), and the rest of the molecule is attached to a radical group through a single or double bond. The alkylene chain is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise in the specification, an alkylene chain may be optionally substituted.

[0057] "Cycloalkyl" or "carbocyclic ring" means a stable non-aromatic monocyclic or polycyclic hydrocarbon radical, consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, is saturated or unsaturated, and is attached to the remainder of the molecule by a single bond. Monocyclic groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic groups include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwise specified in the specification, cycloalkyl groups may be optionally substituted.

[0058] "Cycloalkylene" is a divalent cycloalkyl group. Unless stated otherwise in the specification, a cycloalkylene group may be optionally substituted.

[0059] As used herein, the term "substituted" means any of the above groups (e.g., alkyl, alkylene, cycloalkyl, or cycloalkylene) in which at least one hydrogen atom has been replaced by a bond to a non-hydrogen atom, including, but not limited to, a halogen atom such as F, Cl, Br, or I; an oxygen group (=O); a hydroxyl group (-OH); a C-C 12 Alkyl group; Cycloalkyl group; -(C=O)OR ' ;-O(C=O)R ' ;-C(=O)R ' ;-OR ' ;-S(O) x R ' ;-S-SR ' ;-C(=O)SR ' ;-SC(=O)R ' ;-NR ' R ' ;-NR ' C(=O)R ' ;-C(=O)NR ' R ' ;-NR ' C(=O)NR ' R ' ;-OC(=O)NR' R ' ;-NR ' C(=O)OR ' ;-NR ' S(O) x NR ' R ' ;-NR ' S(O) x R ' ; and -S(O) x NR ' R ' And so on, here:R ' independently for each occurrence: H, C1-C 15 alkyl or cycloalkyl, and x is 0, 1, or 2. In some embodiments, the substituent is C-C 12 In other embodiments, the substituent is an alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as fluoro. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group (-OR ' In another embodiment, the substituent is a carboxyl group. In another embodiment, the substituent is an amine group (—NR ' R ' )

[0060] "Optional" or "optionally" (e.g., optionally substituted) means that the subsequently described circumstance event may or may not occur, and the description includes cases where said event or circumstance occurs and cases where it does not occur. For example, "optionally substituted alkyl" means that the alkyl radical may or may not be substituted, and the description includes both substituted and unsubstituted alkyl radicals.

[0061] The term "prodrug" is intended to refer to a compound that can be converted under physiological conditions or by solvolysis into a biologically active compound of the present invention. Thus, the term "prodrug" refers to a pharmaceutically acceptable metabolic precursor of a compound of the present invention. A prodrug may be inactive when administered to a subject in need thereof, but is converted in vivo to an active compound of the present invention. Prodrugs are typically rapidly converted in vivo to yield the parent compound of the present invention, for example, by hydrolysis in blood. Prodrug compounds often offer advantages of solubility, tissue compatibility, or delayed release in mammalian organisms (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., ACS Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0062] The term "prodrug" is also meant to include covalently bonded carriers that release an active compound of the present invention in vivo when such prodrug is administered to a mammalian subject. Prodrugs of the compounds of the present invention can be prepared by modifying functional groups present in the compounds of the present invention such that the modifications are cleaved, either by routine manipulation or in vivo, to yield the parent compound of the present invention. Prodrugs include compounds of the present invention in which a hydroxy, amino, or mercapto group is bonded to any group that cleaves to form a free hydroxy, free amino, or free mercapto group, respectively, when the prodrug of the compound of the present invention is administered to a mammalian subject. 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 present invention.

[0063] The invention disclosed herein is also meant to encompass all pharmaceutically acceptable compounds of structural formula (I) that are isotopically labeled by having one or more atoms replaced with an atom having a different atomic mass or mass number. Examples of isotopes that may be incorporated into the disclosed compounds include, respectively: 2 H, 3 H, 11 C. 13 C. 14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I, or 125 Radiolabeled compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as I. These radiolabeled compounds may be useful, for example, to help determine or measure the effectiveness of compounds by characterizing the site or mode of action, or binding affinity to a pharmacologically important site of action. Certain isotopically labeled compounds of structural formula (I) or (II), for example, compounds incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The radioisotope tritium, i.e., 3 H, and carbon-14, i.e., 14 C is particularly useful for this purpose in view of its ease of incorporation and ready means of detection.

[0064] Deuterium, i.e., 2 Substitution with heavier isotopes, such as H, may offer certain therapeutic advantages due to greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and therefore may be preferable in some cases.

[0065] 11 C. 18 F, 15 O and 13Substitution with positron-emitting isotopes, such as N, can be useful in positron emission tomography (PET) studies for examining substrate receptor occupancy. Isotopically labeled compounds of structural formula (I) can generally be prepared by conventional techniques known to those skilled in the art, or by methods analogous to those described in the preparations and examples below, substituting the appropriate isotopically labeled reagent for the unlabeled reagent previously used.

[0066] The invention disclosed herein is also meant to encompass the in vivo metabolic products of the disclosed compounds. Such products result primarily from enzymatic processes, for example, from the oxidation, reduction, hydrolysis, amidation, esterification, etc., of the administered compound. Accordingly, the invention includes compounds produced by a method comprising administering a compound of this invention to a mammal for a period of time sufficient to produce a metabolic product thereof. Such products are typically identified by administering a detectable dose of a radiolabeled compound of the invention to an animal, such as a rat, mouse, guinea pig, monkey, or human, allowing sufficient time for metabolism to occur, and isolating the conversion product from urine, blood, or other biological sample.

[0067] "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.

[0068] "Mammal" includes both humans and domestic animals such as laboratory animals and household pets (eg, cats, dogs, pigs, cows, sheep, goats, horses, rabbits) and non-domestic animals such as wild animals.

[0069] A "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, an adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the U.S. Food and Drug Administration as acceptable for use in humans or veterinary animals.

[0070] "Pharmaceutically acceptable salt" includes both acid and base addition salts.

[0071] "Pharmaceutically acceptable acid addition salts" refers to salts that retain the biological effectiveness and properties of the free base, which is not biologically active or otherwise undesirable, and are formed from inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfonic acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, and gluconic acid. "Salts" refers to salts formed with organic acids such as, but not limited to, sucralose, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid.

[0072] "Pharmaceutically acceptable base addition salt" refers to a salt that retains the biological effectiveness and properties of the free acid, even if it is biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, primary, secondary, and tertiary amines, 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, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, substituted amines such as naturally occurring substituted amines, cyclic amines, and basic ion exchange resins. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0073] Crystallization often produces solvates of the compounds of the present invention. As used herein, the term "solvate" refers to an aggregate containing one or more molecules of the compound of the present invention and one or more solvent molecules. 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 hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., as well as corresponding solvated forms. The compounds of the present invention may be true solvates; in other cases, the compounds of the present invention may simply retain adventitious water or may be a mixture of water and adventitious solvent.

[0074] "Pharmaceutical composition" means a formulation containing a compound of the present invention and a vehicle generally accepted in the art for the delivery of biologically active compounds to mammals, e.g., humans. Such vehicles include any pharmaceutically acceptable carrier, diluent, or excipient.

[0075] "Effective amount" or "therapeutically effective amount" refers to the amount of the compound of the present invention that, when administered to a mammal, preferably a human, is sufficient to achieve treatment in the mammal, preferably a human. The amount of lipid nanoparticles of the present invention that constitutes a "therapeutically effective amount" varies depending on the compound, the condition and its severity, the mode of administration, and the age of the mammal being treated, but can be routinely determined by those skilled in the art taking into account their own knowledge and this disclosure.

[0076] As used herein, "treating" or "treatment" means treating a disease or condition in a mammal, preferably a human, having the disease or condition in question, and includes: (i) preventing the disease or condition from occurring in a mammal, particularly when such mammal is susceptible to the condition but has not yet been diagnosed as having it; (ii) inhibiting the disease or condition, i.e., preventing its onset; (iii) alleviating the disease or condition, i.e., causing the disease or condition to regress; or (iv) Relieving symptoms resulting from a 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 differ in that a particular malady or condition does not have a known causative agent (the etiology has not yet been elucidated) and therefore is not recognized as a disease, but only as an undesirable state or syndrome in which a more or less specific set of symptoms has been identified by clinicians.

[0077] The compounds of the present invention, or pharmaceutically acceptable salts thereof, may contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomers, which may be defined in terms of absolute stereochemistry as (R)- or (S)-, or for amino acids, (D)- or (L)-. The present invention is intended to encompass 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, such as, for example, chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from appropriate optically pure precursors, or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high-pressure liquid chromatography (HPLC). In cases where the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless otherwise specified, the compounds are intended to include both E and Z geometric isomers, as well as all tautomeric forms.

[0078] "Stereoisomers" means compounds composed of the same atoms joined by identical bonds, but which have different three-dimensional structures and are not interchangeable. The present invention contemplates various stereoisomers and mixtures thereof, and includes "enantiomers," which refer to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0079] "Tautomer" means a proton shift from one atom of a molecule to another atom of the same molecule. The present invention includes tautomers of any of the compounds.

[0080] compound In one aspect, the present invention provides novel lipid compounds that can be combined 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 believed that these lipid nanoparticles shield oligonucleotides from degradation in serum and provide effective delivery of oligonucleotides to cells in vitro and in vivo.

[0081] In one embodiment, the compound has the following structural formula (I): [ka] I [In the formula, L 1 or L 2 One of the following is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O-, and L 1 or L 2 The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x-, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, ,NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O- or a direct bond; G 1 and G 2 are each independently unsubstituted C-C 12 Alkylene or C1-C 12 Alkenylene; G 3 is C1-C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a is H or C1-C 12 is alkyl; R 1 and R 2 are independently C6-C 24 Alkyl or C6-C 24 is alkenyl; R 3 H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 is; R 4 is C1-C 12 is alkyl; R 5 is H or C1-C6 alkyl; and x is 0, 1, or 2] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0082] In some of the foregoing embodiments, the compound has the following structural formula (IA) or (IB): [ka] (IA) or [ka] (IB) [In the formula, A is a 3- to 8-membered cycloalkyl or cycloalkylene ring; R 6 is independently H, OH, or C1-C 24 is alkyl; n is an integer from 1 to 15. It has one of the following.

[0083] In some of the foregoing embodiments, the compound has structural formula (IA), and in other embodiments, the compound has structural formula (IB).

[0084] In some of the foregoing embodiments, the compound has the following structural formula (IC) or (ID): [ka] (I C) or [ka] (ID) [In the formula, y and z are each independently an integer from 1 to 12. It has one of the following.

[0085] In any of the foregoing embodiments, L 1 or L 2 One of L is -O(C=O)-. For example, in some embodiments, L 1 and L 2 In some different embodiments of any of the foregoing, L 1 and L 2 are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, L 1 and L2 is —(C═O)O—.

[0086] In some different embodiments of the foregoing, the compound has the following structural formula (IE) or (IF): [ka] (IE) or [ka] (IF) It has one of the following.

[0087] In some of the foregoing embodiments, the compound has the following structural formula (IG), (IH), (II), or (IJ): [ka] (IG); [ka] (IH); [ka] (II) or [ka] (IJ) It has one of the following.

[0088] In some of the foregoing embodiments, n is an integer from 2 to 12, e.g., from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0089] In some other of the foregoing embodiments, y and z are each independently an integer from 2 to 10. For example, in some embodiments, y and z are each independently an integer from 4 to 9 or from 4 to 6.

[0090] In some of the foregoing embodiments, R 6 is H. In other of the foregoing embodiments, R 6 is C1-C 24 In another embodiment, R 6 is OH.

[0091] In some embodiments, G 3 is unsubstituted. In other embodiments, G 3 is substituted. In various different embodiments, G 3 is a linear C1-C 24 Alkylene or straight chain C1-C 24 It is alkenylene.

[0092] In some other of the foregoing embodiments, R 1 or R 2 , or both C6-C 24 For example, in some embodiments, R 1 and R 2 are each independently represented by the following structural formula: [ka] [In the formula, R 7a and R 7b is independently H or C1-C for each occurrence 12 is alkyl; and a is an integer from 2 to 12; where R 7a , R 7b and a are R 1 and R 2 are each independently selected to contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer from 5 to 9 or from 8 to 12.

[0093] In some of the foregoing embodiments, R 7a At least one occurrence of R is H. For example, in some embodiments, 7a In each occurrence, R is H. In another alternative embodiment of the foregoing, 7b At least one occurrence of is C1-C8 alkyl. For example, in some embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, n-octyl.

[0094] In a different embodiment, R 1 or R 2 , or both, have the following structural formula: [ka] It has one of the following.

[0095] In some of the foregoing embodiments, R 3 OH, CN, -C(=O)OR 4 , -OC(=O)R 4 or -NHC(=O)R 4 In some embodiments, R 4 is methyl or ethyl.

[0096] In various different embodiments, the compound has one of the structures shown in Table 1 below.

[0097] Table 1 Representative compounds [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7]

[0098] It is understood that any embodiment of a compound of structural formula (I), as described above, and any particular substituent and / or variable in a compound of structural formula (I) described above, can be combined independently with other embodiments and / or substituents and / or variables in a compound of structural formula (I) to form embodiments of the invention not specifically set forth above. Furthermore, when a particular embodiment and / or claim recites a list of substituents and / or variables for any particular R group, L group, G group, A group, or variable a, n, x, y, or z, it is understood that each individual substituent and / or variable may be deleted from the particular embodiment and / or claim, and the remaining list of substituents and / or variables is deemed to be within the scope of the invention.

[0099] It is understood that in this description, combinations of substituents and / or variables of the depicted formula are permissible only if such combinations result in stable compounds.

[0100] In some embodiments, compositions are provided that include one or more of any of the compounds of structural formula (I) and a therapeutic agent. For example, in some embodiments, the compositions include any of the compounds of structural formula (I), a therapeutic agent, and one or more excipients selected from neutral lipids, steroids, and polymeric conjugated lipids. Other pharmaceutically acceptable excipients and / or carriers are also included in various embodiments of the compositions.

[0101] In some embodiments, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC. In various embodiments, the molar ratio of the compound to the neutral lipid ranges from about 2:1 to about 8:1.

[0102] In various embodiments, the composition further comprises a steroid or steroid analog. In certain embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of the compound to cholesterol ranges from about 5:1 to 1:1.

[0103] In various embodiments, the polymer-conjugated lipid is a PEGylated lipid. For example, some embodiments include PEGylated diacylglycerols (PEG-DAGs) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinates (PEG-S-DAGs) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamates such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of compound to PEGylated lipid ranges from about 100:1 to about 20:1.

[0104] In some embodiments, the composition has the following structural formula (II): [ka] (II) [In the formula, R 8 and R 9 are each independently a straight or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester linkages; and w has a mean value in the range of 30 to 60] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0105] In some embodiments, R 8 and R 9are each independently a linear, saturated alkyl chain containing 12 to 16 carbon atoms. In some embodiments, w has an average value ranging from 43 to 53. In other embodiments, the average w is about 45. In other different embodiments, the average w is about 49.

[0106] In some embodiments of the above compositions, the therapeutic agent comprises a nucleic acid, for example, in some embodiments, the nucleic acid is selected from antisense RNA and messenger RNA.

[0107] In another embodiment, the present invention relates to a method of administering a therapeutic agent to a patient in need thereof, comprising preparing or providing any of the above compositions and administering the composition to the patient.

[0108] For the purpose of administration, the compounds of the present invention (typically in the form of lipid nanoparticles combined with a therapeutic agent) may be administered as raw chemicals or may be formulated as pharmaceutical compositions. Pharmaceutical compositions of the present invention include a compound of structural formula (I) and one or more pharmaceutically acceptable carriers, diluents, or excipients. The compound of structural formula (I) is present in the composition in an amount effective to form lipid nanoparticles and deliver a therapeutic agent for treating, for example, a specific disease, which is a condition of the subject. Appropriate concentrations and dosages can be easily determined by those skilled in the art.

[0109] Administration of the compositions of the present invention can be via any of the accepted modes of administration for drugs to perform similar functions. The pharmaceutical compositions of the present invention can be formulated into solid, semisolid, liquid, or gaseous formulations, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intradermal, and intrasternal injection or infusion techniques. The pharmaceutical compositions of the present invention are formulated so that the active ingredient contained therein is bioavailable upon administration of the composition to a patient. The composition administered to a subject may take the form of one or more dosage units; for example, a tablet may be a single dosage unit, and a container of the compound of the present invention in aerosol form may hold multiple dosage units. Actual methods for preparing such dosage forms will be known or 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). The composition to be administered will, in each case, contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treating the disease or condition of interest in accordance with the teachings of the present invention.

[0110] The pharmaceutical composition of the present invention can be solid or liquid.In one embodiment, the carrier is particulate, so that the composition is, for example, in powder form.The carrier can also be liquid, and the composition is, for example, oral syrup, injection liquid or aerosol, which is useful for, for example, inhalation administration.

[0111] When intended for oral administration, the pharmaceutical composition is preferably either solid or liquid, with semi-solid, semi-liquid, suspension and gel forms being included within the forms considered herein as either solid or liquid.

[0112] As a solid composition for oral administration, the pharmaceutical composition may be formulated into the form of powder, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions typically contain one or more inert diluents or edible carriers. Additionally, one or more of the following may be present: binders such as carboxymethylcellulose, ethercellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch, lactose, or dextrin; disintegrating agents such as alginic acid, sodium alginate, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate, or orange flavor; and coloring agents.

[0113] When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

[0114] The pharmaceutical composition may be in the form of a liquid, such as an elixir, syrup, solution, emulsion, or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, a preferred composition contains, in addition to the compound of the present invention, one or more of a sweetener, a preservative, a dye / coloring agent, and a flavor enhancer. In a composition intended for administration by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and an isotonic agent may be included.

[0115] Liquid pharmaceutical compositions of the present invention, whether in solution, suspension, or other similar form, may contain one or more of the following adjuvants: water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can serve as solvents or suspending media; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates, and tonicity adjusters such as sodium chloride or dextrose; and agents that act as cryoprotectants, such as sucrose or trehalose. Parenteral formulations may be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials. Saline is the preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0116] A liquid pharmaceutical composition of the present invention intended for either parenteral or oral administration should contain an amount of a compound of the present invention such that a suitable dosage will be obtained.

[0117] The pharmaceutical composition of the present invention can be intended for topical administration, in which case the carrier can suitably comprise a solution, emulsion, ointment or gel base. For example, the base can comprise one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. A thickener may be present in a pharmaceutical composition for topical administration. When intended for transdermal administration, the composition can comprise a transdermal patch or iontophoresis device.

[0118] The pharmaceutical composition of the present invention can be intended for rectal administration, for example, in the form of a suppository, which melts in the rectum and releases the drug.The composition for rectal administration may contain an oily base as a suitable non-irritating excipient.Such bases include, but are not limited to, lanolin, cocoa butter and polyethylene glycol.

[0119] The pharmaceutical composition of the present invention may contain various substances that modify the physical form of a solid or liquid dosage unit. For example, the composition may contain a material that forms a coating shell around the active ingredient. The coating shell material is typically inert and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredient may be placed in a gelatin capsule.

[0120] Pharmaceutical compositions of the invention, in solid or liquid form, can include an agent that binds to a compound of the invention and thereby aids in the delivery of the compound. Suitable agents that may act in this capacity include monoclonal or polyclonal antibodies, or proteins.

[0121] The pharmaceutical compositions of the present invention can be comprised of dosage units that can be administered as an aerosol. The term aerosol is used to refer to a variety of systems, ranging from colloidal to systems consisting of pressurized packages. Delivery can be by liquefied or compressed gas or by a suitable pump system that dispenses the active ingredient. Aerosols of the compounds of the present invention can be delivered in single-phase, two-phase, or three-phase systems to deliver the active ingredient. Aerosol delivery includes the necessary containers, activators, valves, subcontainers, etc., which can be used together to form a kit. One skilled in the art can determine a preferred aerosol without undue experimentation.

[0122] The pharmaceutical composition of the present invention can be prepared by a methodology well known in the pharmaceutical field.For example, pharmaceutical compositions intended to be administered by injection can be prepared by combining the lipid nanoparticles of the present invention with sterile distilled water or other carriers to form a solution.To facilitate the formation of a homogeneous solution or suspension, surfactants can be added.Surfactants are compounds that interact non-covalently with the compounds of the present invention to promote the dissolution or uniform suspension of the compounds in aqueous delivery systems.

[0123] 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 used: the 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; the drug combination; the severity of the particular disorder or condition; and the subject being treated.

[0124] The composition of the present invention can also be administered simultaneously with, before, or after the administration of one or more other therapeutic agents.Such combination therapy includes the administration of a single pharmaceutical dosage form of the composition of the present invention and one or more additional active agents, and the administration of the composition of the present invention and each active agent in its own separate pharmaceutical dosage form.For example, the composition of the present invention and other active agents can be administered to patients together in a single oral dosage composition such as a tablet or capsule, or can be administered as each agent in separate oral dosage forms.When separate dosage forms are used, the compound of the present invention and one or more additional active agents can be administered at essentially the same time, i.e., simultaneously, or separately, alternatingly, i.e., sequentially; combination therapy is understood to include all of these dosage regimens.

[0125] Methods for preparing the above compounds and compositions are described herein below and / or are known in the art.

[0126] Those skilled in the art will understand that in the processes described herein, functional groups of intermediate compounds may need to be protected with suitable protecting groups. Such functional groups include hydroxy, amino, mercapto, and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino, and guanidino include t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(O)-R" (where R" is alkyl, aryl, or arylalkyl), p-methoxybenzyl, trityl, and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl, or arylalkyl esters. Protecting groups are known to those skilled in the art and can be added or removed according to standard techniques described herein. The use of protecting groups is described in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As will be appreciated by those skilled in the art, the protecting group may be a polymer resin such as a Wang resin, a Rink resin, or a 2-chlorotrityl chloride resin.

[0127] As will also be understood by those skilled in the art, such protected derivatives of the compounds of the present invention may not themselves have pharmacological activity, but they can be administered to a mammal and then metabolized in the body to form the pharmacologically active compounds of the present invention. Such derivatives can therefore be described as "prodrugs." All prodrugs of the compounds of the present invention are included within the scope of the present invention.

[0128] Additionally, all compounds of the present invention that exist in a free base or acid form can be converted to their pharmaceutically acceptable salts by treatment with an appropriate inorganic or organic base or acid by methods known to those skilled in the art. Salts of compounds of the present invention can be converted to their free base or acid forms by standard techniques.

[0129] The following General Reaction Scheme 1 illustrates the synthesis of compounds of the present invention, i.e., compounds of structural formula (I): [ka] (I) [In the formula, R 1 , R 2 , R 3 , L 1 , L 2 , G 1 , G 2 and G 3 is as defined herein] This section describes methods for preparing compounds of formula (I), or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof. It is understood that those skilled in the art can prepare these compounds by analogous methods or by combining other methods known to those skilled in the art. It is also understood that those skilled in the art will be able to prepare other compounds of formula (I) not specifically shown below by using appropriate starting components and modifying the synthetic parameters as needed in a manner similar to that described below. In general, the starting components can be obtained from suppliers such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, or can be synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)), or can be prepared as described herein.

[0130] General Reaction Scheme 1 [ka]

[0131] General Reaction Scheme 1 provides an exemplary method for the preparation of compounds of structural formula (I). 1 , G 3 , R 1 and R 3 is as defined herein, and G 1 ' is G 1 A-1 represents a one-carbon shorter homologue of the formula (I). Compounds of formula A-1 are commercially available or prepared according to methods known in the art. Reaction of A-1 with diol A-2 under appropriate condensation conditions (e.g., DCC) produces ester / alcohol A-3, which can then be oxidized to aldehyde A-4 (e.g., PCC). Reaction of A-4 with amine A-4 under reductive amination conditions yields compounds of formula (I).

[0132] It should be noted that various alternative strategies for the preparation of compounds of formula (I) are available to one skilled in the art. For example, L 1 and L 2 Other compounds of formula (I) other than esters can be prepared according to similar methods using appropriate starting materials. 1 and G 2 is the same; however, this is not a necessary aspect of the invention, and G 1 and G 2 Modifications of the above reaction schemes to give compounds with different ##STR00001## The use of protecting groups as needed, and other modifications to the above general reaction schemes, will be readily apparent to those skilled in the art.

[0133] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0134] In vivo assessment of luciferase mRNA using lipid nanoparticle compositions Cationic lipids, DSPC, cholesterol, and PEG-lipids were solubilized in ethanol at molar ratios of 50:10:38.5:1.5 or 47.5:10:40.8:1.7. Lipid nanoparticles (LNPs) were prepared at total lipid-to-mRNA weight ratios of approximately 10:1 to 30:1. Briefly, mRNA was diluted to 0.2 mg / mL in 10-50 mM citrate buffer, pH 4. Using a syringe pump, the ethanolic lipid solution was mixed with the aqueous mRNA solution at a ratio of approximately 1:5 to 1:3 (vol / vol) at a total flow rate of over 15 mL / min. The ethanol was then removed, and the external buffer was replaced with PBS by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 μm pore sterile filter. The particle size of the lipid nanoparticles was approximately 55–95 nm, and in some cases approximately 70–90 nm, in diameter as measured by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK).

[0135] Studies were conducted in 6-8 week-old female C57BL / 6 mice (Charles River) and 8-10 week-old CD-1 (Harlan) mice (Charles River) in accordance with guidelines established by the Institutional Animal Care Committee (ACC) and the Canadian Council on Animal Health (CCAC). Varying doses of mRNA-lipid nanoparticles were administered systemically via tail vein injection, and animals were euthanized at specific time points (e.g., 4 hours) post-administration. Livers and spleens were collected in pre-weighed tubes, weighed, immediately snap-frozen in liquid nitrogen, and stored at -80°C until processing for analysis.

[0136] Approximately 50 mg of liver was cut into 2 mL FastPrep tubes (MP Biomedicals, Solon, OH) for analysis. A ¼-inch ceramic ball (MP Biomedicals) was added to each tube, and 500 μL of Glo Lysis Buffer-GLB (Promega, Madison, WI) equilibrated to room temperature was added to the liver tissue. The liver tissue was homogenized at 2 × 6.0 m / s for 15 seconds using a FastPrep24 instrument (MP Biomedicals). The homogenate was incubated at room temperature for 5 minutes, then diluted 1:4 in GLB and assayed using the SteadyGlo luciferase assay system (Promega). Specifically, 50 μL of diluted tissue homogenate was reacted with 50 μL of SteadyGlo substrate, shaken for 10 seconds, incubated for 5 minutes, and then quantified using a CentroXS3 LB 960 luminometer (Berthold Technologies, Germany). The amount of protein assayed was determined using a BCA protein assay kit (Pierce, Rockford, IL). Relative luminescence units (RLU) were then normalized to μg of total protein assayed. To convert RLU to ng luciferase, a standard curve was generated using QuantiLum Recombinant Luciferase (Promega). Based on the data shown in Figure 1, the 4-hour time point was selected for evaluation of the efficacy of the lipid formulations.

[0137] FLuc mRNA (L-6107) from Trilink Biotechnologies expresses the luciferase protein originally isolated from the firefly Photinus pyralis. FLuc is commonly used in mammalian cell culture to measure both gene expression and cell viability. It emits bioluminescence in the presence of the substrate luciferin. This capped and polyadenylated mRNA is fully substituted with 5-methylcytidine and pseudouridine. [Example]

[0138] PK of formulated lipids a Decision As described elsewhere, the pKa of formulated cationic lipids correlates with the effectiveness of LNPs for nucleic acid delivery (see Jayaraman et al., Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al., Nature Biotechnology 28, 172-176 (2010)). a The preferred range of pK of each cationic lipid is from about 5 to about 7. a was measured in lipid nanoparticles using a fluorescence-based assay of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). Lipid nanoparticles containing cationic lipid / DSPC / cholesterol / PEG-lipid (50 / 10 / 38.5 / 1.5 mol%) in PBS at a concentration of 0.4 mM total lipid were prepared using the in-line process described in Example 1. TNS was prepared as a 100 μM stock solution in distilled water. Vesicles were diluted to 24 μM lipid in 2 mL of a buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl (where the pH ranged from 2.5 to 11). An aliquot of TNS solution was added to a final concentration of 1 μM, and after vortex mixing, fluorescence intensity was measured at room temperature on an SLM Aminco Series 2 luminescence spectrophotometer using excitation and emission wavelengths of 321 nm and 445 nm. A sigmoidal best-fit analysis was applied to the fluorescence data to determine the pK a was measured as the pH that produced half the maximum fluorescence intensity (see Figure 2). [Example]

[0139] Determining the efficacy of lipid nanoparticle formulations containing various cationic lipids using an in vivo luciferase mRNA-expressing rodent model The cationic lipids listed in Table 2 have been previously tested with nucleic acids. For comparative purposes, these lipids were used to formulate lipid nanoparticles containing FLuc mRNA (L-6107) using an in-line mixing method, as described in Example 1 and PCT / US10 / 22614 (incorporated herein by reference in its entirety). Lipid nanoparticles were formulated using a molar ratio of 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid ("PEG-DMG," i.e., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol) with an average PEG molecular weight of 2000). Relative activity was determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection, as described in Example 1. Activity was compared at doses of 0.3 and 1.0 mg mRNA / kg and expressed as ng luciferase / g liver, measured 4 hours after administration, as described in Example 1.

[0140] Table 2 Comparative lipids showing activity using mRNA [Table 8]

[0141] Representative compounds of the invention shown in Table 3 were formulated using the following molar ratios: A) 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid ("PEG-DMA", 2-[2-(ω-methoxy(polyethylene glycol) 2000A) 47.5% cationic lipid / 10% DSPC / 40.8% cholesterol / 1.7% PEG lipid. Relative activity was determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection, as described in Example 1. Activity was compared at doses of 0.3 and 1.0 mg mRNA / kg and expressed as ng luciferase / g liver measured 4 hours after administration, as described in Example 1. A plot of selected data is shown in Figure 3 (top to bottom: triangles = compound 3; circles = compound 2; crosses = compound 1; squares = MC3).

[0142] Table 3 Novel cationic lipids and related activities. [Table 9] [Table 10] [Table 11] [Table 12] [Example]

[0143] Synthesis of 6-(2'-hexyldecanoyloxy)hexan-1-al A solution of hexane-1,6-diol (27.6 g) in methylene chloride (475 mL) was treated with 2-hexyldecanoic acid (19.8 g), DCC (18.2 g), and DMAP (11.3 g). The solution was stirred for 3 days. The reaction mixture was filtered, and hexane (500 mL) was added to the filtrate. The mixture was stirred, and the precipitate was allowed to settle. The supernatant was decanted and washed with dilute hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed to give 30 g of crude product.

[0144] The crude product was dissolved in methylene chloride (200 mL) and treated with pyridinium chlorochromate (15 g) for 2 hours. Diethyl ether (600 mL) was added, and the supernatant was filtered through a bed of silica gel. The solvent was removed from the filtrate, and the resulting oil was dissolved in hexane. The suspension was filtered through a plug of silica gel, and the solvent was removed. The residue was passed through a silica gel column (80 g) using hexane followed by methylene chloride as eluents. 6-(2'-hexyldecanoyloxy)hexan-1-al (24 g) was obtained as a colorless oil. [Example]

[0145] Synthesis of 4-(2'-hexyldecanoyloxy)butan-1-al A solution of butane-1,4-diol (12.5 g) in methylene chloride (200 ml) was treated with 2-hexyldecanoic acid (9.2 g), DCC (8.8 g), and DMAP (4.9 g). The solution was stirred overnight. The reaction mixture was filtered and the solvent removed. The residue was dissolved in methylene chloride and washed with dilute hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered through a bed of silica gel, and the solvent removed.

[0146] The crude product was dissolved in methylene chloride (150 mL) and treated with pyridinium chlorochromate (6 g) for 1 hour. Diethyl ether (450 mL) was added, and the supernatant was filtered through a silica gel bed. The solvent was removed from the filtrate, and the resulting oil was dissolved in hexane. The suspension was filtered through a silica gel bed, and the solvent was removed to give 4-(2'-hexyldecanoyloxy)butan-1-al (11 g) as a colorless oil. [Example]

[0147] Synthesis of Compound 1 A solution of 6-(2'-hexyldecanoyloxy)hexan-1-al (3.0 g), acetic acid (0.21 g), and ethanolamine (0.14 g) in methylene chloride (50 mL) was treated with sodium triacetoxyborohydride (1.4 g) overnight. The solution was washed with dilute aqueous sodium hydroxide. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent removed. The residue was passed through a silica gel column using a methanol / methylene chloride (0-8 / 100-92%) gradient to give compound 1 as a colorless oil (0.63 g). [Example]

[0148] Synthesis of compound 2 A solution of 6-(2'-hexyldecanoyloxy)hexan-1-al (3.0 g), acetic acid (0.33 g), and 3-aminopropan-1-ol (0.17 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.3 g) for 1 hour. The solution was washed with dilute aqueous sodium hydroxide. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent removed. The residue was passed through a silica gel column using a methanol / methylene chloride (0-8 / 100-92%) gradient to give compound 2 as a colorless oil (1.1 g). [Example]

[0149] Synthesis of compound 3 A solution of 6-(2'-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid (0.33 g), and 4-aminobutan-1-ol (0.17 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.3 g) for 2 hours. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent removed. The residue was passed through a silica gel column using a methanol / methylene chloride (0-8 / 100-92%) gradient to give compound 3 as a colorless oil (0.4 g). [Example]

[0150] Synthesis of compound 4 A solution of 4-(2'-hexyldecanoyloxy)butan-1-al (2.4 g), acetic acid (0.30 g), and 4-aminobutan-1-ol (0.22 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.3 g) for 2 hours. The solution was washed with dilute aqueous sodium hydroxide. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent removed. The residue was passed through a silica gel column using a methanol / methylene chloride (0-8 / 100-92%) gradient. Partially purified fractions were passed through a second column using an acetic acid / methanol / methylene chloride (2-0 / 0-10 / 98-90%) gradient. The pure fractions were washed with aqueous sodium bicarbonate to give compound 4 as a colorless oil (0.9 g). [Example]

[0151] Synthesis of compound 5 A solution of 4-(2'-hexyldecanoyloxy)butan-1-al (2.4 g), acetic acid (0.31 g), and 3-aminopropan-1-ol (0.17 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.4 g) for 1 hour. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent removed. The residue was passed through a silica gel column using a methanol / methylene chloride (0-8 / 100-92%) gradient. Partially purified fractions were passed through a second column using an acetic acid / methanol / methylene chloride (2-0 / 0-8 / 98-92%) gradient. The pure fractions were washed with aqueous sodium bicarbonate to give compound 5 as a colorless oil (0.57 g). [Example]

[0152] Synthesis of compound 6 A solution of 4-(2'-hexyldecanoyloxy)butan-1-al (2.4 g), acetic acid (0.30 g), and ethanolamine (0.14 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.3 g) for 2 hours. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent removed. The residue was passed through a silica gel column using a methanol / methylene chloride (0-10 / 100-90%) gradient. Partially purified fractions were passed through a second column using an acetic acid / methanol / methylene chloride (2-0 / 0-9 / 98-92%) gradient. The pure fractions were washed with aqueous sodium bicarbonate to give compound 6 as a colorless oil (0.2 g). [Example]

[0153] Synthesis of compound 7 A solution of 6-(2'-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid (0.14 g), and 5-aminopentan-1-ol (0.24 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.3 g) for 2 hours. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent removed. The residue was passed through a silica gel column using a methanol / methylene chloride (0-8 / 100-92%) gradient to give compound 7 as a colorless oil (0.5 g). [Example]

[0154] Synthesis of compound 8 A solution of 6-(2'-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid (0.17 g), and 6-aminohexan-1-ol (0.26 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.3 g) for 2 hours. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent removed. The residue was passed through a silica gel column using a methanol / methylene chloride (0-8 / 100-92%) gradient to give compound 8 as a colorless oil (0.5 g). [Example]

[0155] Synthesis of compound 9 A solution of 6-(2'-hexyldecanoyloxy)hexan-1-al (2.4 g) and trans-2-aminocyclohexanol hydrochloride (0.35 g) in methylene chloride (10 mL) / tetrahydrofuran (10 mL) was treated with sodium triacetoxyborohydride (1.3 g) for 1.5 hours. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent removed. The residue was passed through a silica gel column using a methanol / methylene chloride (0-8 / 100-92%) gradient to give compound 9 as a colorless oil (0.6 g). [Example]

[0156] Synthesis of compound 10 A solution of 2-aminoethanol (106 mg, 1.75 mmol) in anhydrous THF (15 mL) was added with 2-octyldodecyl 6-bromohexanoate (2 equiv., 1.66 g, 3.5 mmol), potassium carbonate (2 equiv., 3.5 mmol, 477 mg), and cesium carbonate (0.3 equiv., 0.525 mmol, 171 mg) and heated at 63 °C (oil bath) for 16 h. A trace of tetrabutylammonium iodide was added to the mixture, and the mixture was heated at reflux for an additional 4 days. The solvent was evaporated under reduced pressure, and the residue was taken up in a mixture of hexane and ethyl acetate (approximately 9:1) and washed with water and brine. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to give an oil (1.6 g). The residue (1.6 g) was purified by silica gel column chromatography (MeOH in chloroform, 0-4%). This gave compound 10 as a colorless oil (700 mg, 0.82 mmol, 47%). [Example]

[0157] Synthesis of compound 11 A solution of 2-aminoethanol (116 mg, 1.9 mmol, 115 μL) in anhydrous THF (15 mL) was added with 2-hexyldecyl 6-bromohexanoate (1.9 equiv., 1.52 g, 3.62 mmol), potassium carbonate (1.9 equiv., 3.62 mmol, 500 mg), cesium carbonate (0.3 equiv., 0.57 mmol, 186 mg), and sodium iodide (10 mg) and heated to reflux under argon for 6 days. The solvent was evaporated under reduced pressure, and the residue was taken up in hexane and washed with water and brine. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to give a colorless oil. The crude product was purified by silica gel flash column chromatography (MeOH in chloroform, 0–4%) to give compound 11 as a colorless oil (936 mg, 1.27 mmol, 70%). [Example]

[0158] Synthesis of compound 12 Compound 12 was prepared in a manner similar to the procedure for compound 11 to give 538 mg of a colorless oil, 0.86 mmol, 57%. [Example]

[0159] Synthesis of compound 13 A solution of 2-aminoethanol (171 mg, 2.81 mmol, 169 μL) in anhydrous THF (30 mL) was added with 2-octyldodecyl 4-bromobutyrate (1.9 equiv., 2.386 g, 5.33 mmol), potassium carbonate (1.9 equiv., 5.33 mmol, 736 mg), cesium carbonate (0.3 equiv., 0.84 mmol, 275 mg), and sodium iodide (10 mg) and heated to reflux under argon for 16 h. TLC (hexane / ethyl acetate = 9:1, CHCl3 / MeOH = 19:1) indicated the formation of a significant amount of 2-octyl-1-dodecanol. The mixture was cooled and filtered. The filtrate was concentrated, and the residue was dissolved in 2-octyl-1-dodecanol (2.1 g). A few beads of 4 Å molecular sieves and N,N-diisopropylethylamine (1.9 equiv., 5.33 mmol, 683 mg, 0.92 mL) were added. The mixture was sealed and heated at 62 °C for an additional 4 days. The reaction mixture was cooled. Hexane was added. The hexane solution was decanted and concentrated to dryness. The residue was purified by silica gel column chromatography (MeOH in chloroform, 0-4%) to give compound 13 as a colorless oil (282 mg, 0.35 mmol, 13%). [Example]

[0160] Synthesis of compound 14 To a solution of heptadecan-9-yl 6-bromohexanoate (2 equiv., 1.13 g, 2.61 mmol) in anhydrous THF (15 mL) was added potassium carbonate (2 equiv., 2.61 mmol, 361 mg), cesium carbonate (0.3 equiv., 0.39 mmol, 128 mg), and sodium iodide (6 mg). The mixture was heated to reflux under argon for 7 days. The solvent was evaporated under reduced pressure, and the residue was taken up in hexane / ethyl acetate (approximately 10%) and washed with water and brine. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to give a colorless oil (1 g). The residue (1 g) was purified by gravity column chromatography on silica gel (MeOH in DCM, 0-4%). ​​This gave compound 14 as a colorless oil (757 mg, 0.99 mmol, 76%). [Example]

[0161] Synthesis of compound 15 To a solution of 2-hexyldecyl 5-bromopentanoate (2 equiv., 1.22 g, 3 mmol) in anhydrous THF (15 mL) (open, 2 months old), potassium carbonate (2 equiv., 3 mmol, 415 mg), cesium carbonate (0.3 equiv., 0.45 mmol, 146 mg), and sodium iodide (6 mg) were added 4-amino-1-butanol (1 equiv., 1.5 mmol, 0.134 mg, 139 μL). The mixture was heated to reflux under argon for 6 days. The solvent was evaporated under reduced pressure, and the residue was taken up in a mixture of hexane and ethyl acetate (approximately 10%) and washed with water and brine. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to give a colorless oil (1.12 g). The residue (1 g) was purified by silica gel column chromatography (MeOH in chloroform, 0-5%). This gave compound 15 as a colorless oil (487 mg, 0.66 mmol, 44%). 1 HNMR (400 MHz, CDCl3) δ:5.99 (s, 1H), 3.98 (d, 5.8 Hz, 4H), 3.56 (t-like, 4.8 Hz, 2H), 2.48-2.41 (m, 6H), 2.33 (t, 7.4 Hz, 4H), 1.70-1.57 (m, 10H), 1.55-1.47 (m, 4H), 1.35-1.21 (48H), 0.89 (t-like, 6.8 Hz, 12H). [Example]

[0162] Synthesis of compound 16 To a solution of 3-amino-1-propanol (0.37 mmol, 28 mg) in anhydrous acetonitrile (15 mL) was added 2-hexyldecyl 6-bromohexanoate (1.9 equiv., 294 mg, 0.7 mmol), N,N-diisopropylethylamine (2 equiv., 0.74 mmol, 96 mg), and sodium iodide (5 mg). The mixture (two layers) was heated in a pressure flask at 59 °C (oil bath) for 3 days. The mixture was concentrated, and the residue was taken up in a mixture of hexane and ethyl acetate (approximately 5:1, 100 mL), washed with water, brine, dried over sodium sulfate, filtered, and concentrated. A slightly yellow oil (approximately 300 mg) was obtained. The crude product (300 mg) was purified by silica gel flash chromatography (MeOH in chloroform, 0-4.4%). This afforded compound 16 as a colorless oil (95 mg, 0.13 mmol, 36%). 1 HNMR (400 MHz, CDCl3) δ:5.61-5.44 (br. s, 1H), 3.97 (d, 5.8 Hz, 4H), 3.80 (t-like, 5.1 Hz, 2H), 2.63 (t-like, 5.6 Hz, 2H), 2.43-2.39 (m, 4H), 2.32 (t, 7.5 Hz, 4H), 1.70-1.59 (m, 8H), 1.55-1.45 (m, 4H), 1.36-1.21 (52H), 0.89 (t-like, 6.8 Hz, 12H). [Example]

[0163] Synthesis of compound 17 To a solution of 2-hexyldecyl 6-bromohexanoate (2 equiv., 1.32 g, 3.14 mmol) in 15 mL of anhydrous THF was added 4-amino-1-butanol (1 equiv., 1.57 mmol, 140 mg, 145 μL), potassium carbonate (2 equiv., 3.14 mmol, 434 mg), cesium carbonate (0.3 equiv., 0.47 mmol, 153 mg), and sodium iodide (6 mg). This mixture was heated in a pressure round-bottom flask at 75 °C (oil bath) under argon for 6 days. The reaction mixture was cooled and concentrated. The residue was dissolved in a mixture of hexane and ethyl acetate (approximately 9:1), washed with water, brine, dried over sodium sulfate, filtered, and concentrated to dryness (1.28 g of a colorless oil). The crude product was purified by silica gel flash column chromatography (MeOH in chloroform, 0–5%). This gave compound 17 as a colorless oil (581 mg, 0.76 mmol, 48%). 1 HNMR (400 MHz, CDCl3) δ:6.43-6.17 (br. s, 1H), 3.97 (d, 5.8 Hz, 4H), 3.55 (t-like, 4.7 Hz, 2H), 2.46-2.40 (m, 6H), 2.31 (t, 7.5 Hz, 4H), 1.70-1.59 (m, 10H), 1.55-1.45 (m, 4H), 1.36-1.21 (52H), 0.89 (t-like, 6.7 Hz, 12H). [Example]

[0164] Synthesis of compound 20 To a solution of 2-hexyldecyl 8-bromooctanoate (2 equiv., 3.09 g, 6.9 mmol) in 30 mL of anhydrous THF was added 4-amino-1-butanol (1 equiv., 3.45 mmol, 308 mg), potassium carbonate (2 equiv., 6.9 mmol, 954 mg), cesium carbonate (0.3 equiv., 1.04 mmol, 337 mg), and sodium iodide (10 mg). The mixture in a pressure round-bottom flask was heated under argon at 64-70 °C (oil bath) for 6 days. The mixture was cooled and concentrated. The residue was taken up in a mixture of hexane and ethyl acetate (9:1), washed with water, brine, dried over sodium sulfate, filtered, and concentrated to dryness (colorless oil). The crude product was purified by silica gel flash-dry column chromatography (MeOH in chloroform, 0-4.2%). This gave compound 20 as a colorless oil (1.28 g, 1.56 mmol, 45%). 1 HNMR (400 MHz, CDCl3) δ:6.64-6.45 (br. s, 1H), 3.97 (d, 5.8 Hz, 4H), 3.62-3.51 (br. 2H), 3.07-2.34 (br. 6H), 2.30 (t, 7.5 Hz, 4H), 1.71-1.40 (m, 14H), 1.39-1.19 (m, 60H), 0.89 (t-like, 6.8 Hz, 12H). [Example]

[0165] Synthesis of 9-(2'-ethylhexanoyloxy)nonan-1-al A solution of nonane-1,9-diol (10.1 g) in methylene chloride (150 mL) was treated with 2-ethylhexanoic acid (9.0 g), DCC (14.3 g), and DMAP (9.1 g). The solution was stirred overnight. The reaction mixture was filtered, and the solvent was removed. The residue was suspended in hexane and filtered. The filtrate was washed with dilute hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered through a bed of silica gel, and the solvent was removed. The crude product was passed through a silica gel column using a methanol / methylene chloride (0-8%) gradient to give 9-(2'-ethylhexanoyloxy)nonan-1-ol as an oil (7.2 g).

[0166] 9-(2'-Ethylhexanoyloxy)nonan-1-ol was dissolved in methylene chloride (100 mL) and treated with pyridinium chlorochromate (7.5 g) for 1 hour. Hexane (400 mL) was added, and the supernatant was filtered through a silica gel bed. The solvent was removed from the filtrate, and the resulting oil was dissolved in hexane. The suspension was filtered through a silica gel bed, and the solvent was removed to give 9-(2'-ethylhexanoyloxy)nonan-1-al as a colorless oil (6 g). [Example]

[0167] Synthesis of 9-(2'-butyloctanoyloxy)nonan-1-al A solution of nonane-1,9-diol (12.0 g) in methylene chloride (150 mL) was treated with 2-butyloctanoic acid (5.0 g), DCC (7.7 g), and DMAP (4.5 g). The solution was stirred overnight. The reaction mixture was filtered, and the solvent was removed. The residue was suspended in hexane and filtered. The filtrate was washed with dilute hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered through a silica gel bed, and the solvent was removed. The crude product was passed through a silica gel column using a methanol / methylene chloride (0-4%) gradient to give 9-(2'-butyloctanoyloxy)nonan-1-ol as an oil (6 g).

[0168] 9-(2'-Butyloctanoyloxy)nonan-1-ol was dissolved in methylene chloride (100 mL) and treated with pyridinium chlorochromate (3.8 g) overnight. Hexane (300 mL) was added, and the supernatant was filtered through a silica gel bed. The solvent was removed from the filtrate, and the resulting oil was dissolved in hexane. The suspension was filtered through a silica gel bed, and the solvent was removed to give 9-(2'-butyloctanoyloxy)nonan-1-al as a colorless oil (3.1 g). [Example]

[0169] Synthesis of 6-(2'-butyloctanoyloxy)hexan-1-al A solution of hexane-1,6-diol (9.4 g) in methylene chloride (150 mL) was treated with 2-butyloctanoic acid (5.0 g), DCC (7.6 g), and DMAP (4.8 g). The solution was stirred overnight. The reaction mixture was filtered, and the solvent was removed. The residue was suspended in hexane and filtered. The filtrate was washed with dilute hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered through a bed of silica gel, and the solvent was removed. The crude product was passed through a silica gel column using a methanol / methylene chloride (0-4%) gradient to give 6-(2'-butyloctanoyloxy)hexan-1-ol as an oil (4.5 g).

[0170] 6-(2'-Butyloctanoyloxy)hexan-1-ol was dissolved in methylene chloride (100 mL) and treated with pyridinium chlorochromate (4.8 g) overnight. Hexane (300 mL) was added, and the supernatant was filtered through a silica gel bed. The solvent was removed from the filtrate, and the resulting oil was dissolved in hexane. The suspension was filtered through a silica gel bed, and the solvent was removed to give 6-(2'-butyloctanoyloxy)hexan-1-al as a colorless oil (3.9 g). [Example]

[0171] Synthesis of 6-(2'-octyldodecanoyloxy)hexan-1-al A solution of hexane-1,6-diol (11.5 g) in methylene chloride (150 mL) / THF (20 mL) was treated with 2-octyldodecanoic acid (9.9 g), DCC (7.5 g), and DMAP (4.7 g). The solution was stirred overnight. The reaction mixture was filtered, and the solvent was removed. The residue was suspended in hexane and filtered. The filtrate was washed with dilute hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered through a bed of silica gel, and the solvent was removed. The crude product was passed through a silica gel column using a methanol / methylene chloride (0-4%) gradient to give 6-(2'-octyldodecanoyloxy)hexan-1-ol as an oil (7.4 g).

[0172] 6-(2'-octyldodecanoyloxy)hexan-1-ol was dissolved in methylene chloride (100 mL) and treated with pyridinium chlorochromate (4.0 g) for 2 hours. Diethyl ether (300 mL) was added, and the supernatant was filtered through a silica gel bed. The solvent was removed from the filtrate, and the resulting oil was dissolved in hexane. The suspension was filtered through a silica gel bed, and the solvent was removed to give 6-(2'-octyldodecanoyloxy)hexan-1-al as a colorless oil (5.3 g). [Example]

[0173] Synthesis of 6-(2'-decyltetradecanoyloxy)hexan-1-al A solution of hexane-1,6-diol (9.6 g) in methylene chloride (150 mL) was treated with 2-decyltetradecanoic acid (6.1 g), DCC (4.9 g), and DMAP (3.1 g). The solution was stirred overnight. The reaction mixture was filtered, and the solvent was removed. The residue was suspended in hexane and filtered. The filtrate was washed with dilute hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered through a silica gel bed, and the solvent was removed. The crude product was passed through a silica gel column using a methanol / methylene chloride (0-4%) gradient to give 6-(2'-decyltetradecanoyloxy)hexan-1-ol as an oil (4.6 g).

[0174] 6-(2'-decyltetradecanoyloxy)hexan-1-ol was dissolved in methylene chloride (100 mL) and treated with pyridinium chlorochromate (3.2 g) for 2 hours. Hexane (300 mL) was added, and the supernatant was filtered through a silica gel bed. The solvent was removed from the filtrate, and the resulting product was dissolved in hexane. The suspension was filtered through a silica gel bed, and the solvent was removed to give 6-(2'-decyltetradecanoyloxy)hexan-1-al as a colorless oil (4.2 g). [Example]

[0175] Synthesis of 12-(2'-hexyldecanoyloxy)dodecan-1-al A solution of dodecane-1,6-diol (25.0 g) in methylene chloride (300 mL) / THF (100 mL) was treated with 2-hexyldodecanoic acid (10.6 g), DCC (10.2 g), and DMAP (7.5 g). The solution was stirred overnight. The reaction mixture was filtered, and the solvent was removed. The residue was suspended in hexane and filtered. The filtrate was washed with water. The organic phase was dried over anhydrous magnesium sulfate, filtered through a bed of silica gel, and the solvent was removed. The crude product was passed through a silica gel column using hexane and then methylene chloride to produce 12-(2'-hexyldecanoyloxy)dodecan-1-ol as an oil (7.9 g).

[0176] 12-(2'-hexyldecanoyloxy)dodecan-1-ol was dissolved in methylene chloride (150 mL) and treated with pyridinium chlorochromate (4.0 g) for 3 hours. Hexane (300 mL) was added, and the supernatant was filtered through a silica gel bed. The solvent was removed from the filtrate, and the resulting oil was dissolved in hexane. The suspension was filtered through a silica gel bed, and the solvent was removed to give 12-(2'-hexyldecanoyloxy)dodecan-1-al as a colorless oil (3.9 g). [Example]

[0177] Synthesis of 9-(2'-hexyldecanoyloxy)nonan-1-al A solution of nonane-1,9-diol (46.8 g) in methylene chloride (600 mL) was treated with 2-hexyldecanoic acid (25.0 g), DCC (22.0 g), and DMAP (15.0 g). The solution was stirred overnight. The reaction mixture was filtered, and the solvent was removed. The residue was suspended in hexane and filtered. The filtrate was washed with dilute hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered through a silica gel bed, and the solvent was removed. The crude product was passed through a silica gel column using hexane and then a methanol / methylene chloride (0-8%) gradient to give 9-(2'-hexyldecanoyloxy)nonan-1-ol as an oil (22 g).

[0178] 9-(2'-Hexyldecanoyloxy)nonan-1-ol (5.0 g) was dissolved in methylene chloride (50 mL) and treated with pyridinium chlorochromate (2.7 g) for 1 hour. Hexane (200 mL) was added, and the supernatant was filtered through a silica gel bed. The solvent was removed from the filtrate, and the resulting product was dissolved in hexane. The suspension was filtered through a silica gel bed, and the solvent was removed to give 9-(2'-hexyldecanoyloxy)nonan-1-al as a colorless oil (3.6 g). [Example]

[0179] Synthesis of compound 22 A solution of 9-(2'-hexyldecanoyloxy)nonan-1-al (2.2 g), acetic acid (0.15 g), and 4-aminobutan-1-ol (0.20 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.30 g) overnight. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of acetic acid / methanol / methylene chloride (2-0 / 0-12 / 98-88%). The pure fractions were washed with aqueous sodium bicarbonate to give compound 22 as a colorless oil (0.93 g). [Example]

[0180] Synthesis of compound 23 A solution of 12-(2'-hexyldecanoyloxy)dodecan-1-al (2.0 g), acetic acid (0.09 g), and 4-aminobutan-1-ol (0.14 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (0.71 g) overnight. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of acetic acid / methanol / methylene chloride (2-0 / 0-12 / 98-88%). Pure fractions were washed with aqueous sodium bicarbonate to give compound 23 as a colorless oil (1.0 g). [Example]

[0181] Synthesis of compound 24 A solution of 9-(2'-ethylhexanoyloxy)nonan-1-al (3.0 g), acetic acid (0.11 g), and 4-aminobutan-1-ol (0.17 g) in methylene chloride (50 mL) was treated with sodium triacetoxyborohydride (0.89 g) overnight. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of acetic acid / methanol / methylene chloride (2-0 / 0-10 / 98-88%). Pure fractions were washed with aqueous sodium bicarbonate to give compound 24 as a colorless oil (0.69 g). [Example]

[0182] Synthesis of compound 25 A solution of 9-(2'-butyloctanoyloxy)nonan-1-al (2.6 g), acetic acid (0.20 g), and 4-aminobutan-1-ol (0.26 g) in methylene chloride (50 mL) was treated with sodium triacetoxyborohydride (1.42 g) overnight. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of acetic acid / methanol / methylene chloride (2-0 / 0-12 / 98-88%). Pure fractions were washed with aqueous sodium bicarbonate to give compound 25 as a colorless oil (0.82 g). [Example]

[0183] Synthesis of compound 26 A solution of 6-(2'-octyldodecanoyloxy)hexan-1-al (2.7 g), acetic acid (0.20 g), and 4-aminobutan-1-ol (0.20 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.30 g) overnight. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of acetic acid / methanol / methylene chloride (2-0 / 0-12 / 98-88%). Pure fractions were washed with aqueous sodium bicarbonate to give compound 26 as a colorless oil (0.21 g). [Example]

[0184] Synthesis of compound 27 A solution of 6-(2'-decyltetradecanoyloxy)hexan-1-al (2.1 g), acetic acid (0.11 g), and 4-aminobutan-1-ol (0.13 g) in methylene chloride (30 mL) was treated with sodium triacetoxyborohydride (0.70 g) overnight. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of acetic acid / methanol / methylene chloride (2-0 / 0-12 / 98-88%). Pure fractions were washed with aqueous sodium bicarbonate to give compound 27 as a colorless oil (0.90 g). [Example]

[0185] Synthesis of compound 28 A solution of 6-(2'-butyloctanoyloxy)hexan-1-al (2.0 g), acetic acid (0.13 g), and 4-aminobutan-1-ol (0.13 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.0 g) overnight. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of acetic acid / methanol / methylene chloride (2-0 / 0-8 / 98-92%). Pure fractions were washed with aqueous sodium bicarbonate to give compound 28 as a colorless oil (0.77 g). [Example]

[0186] Synthesis of compound 30 A solution of 6-(2'-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid (0.15 g), and 3-aminopropane-1,2-diol (0.21 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.76 g) overnight. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of acetic acid / methanol / methylene chloride (2-0 / 0-12 / 98-88%). Pure fractions were washed with aqueous sodium bicarbonate to give compound 30 as a colorless oil (0.60 g). [Example]

[0187] Synthesis of compound 31 A solution of 6-(2'-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid (0.15 g), and 2-aminobutan-1-ol (0.20 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.1 g) for 2 hours. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of acetic acid / methanol / methylene chloride (2-0 / 0-4 / 98-96%). Pure fractions were washed with aqueous sodium bicarbonate to give compound 31 as a colorless oil (0.31 g). [Example]

[0188] Synthesis of compound 37 A solution of 6-(2'-octyldodecanoyloxy)hexan-1-al (2.7 g), acetic acid (0.20 g), and 3-aminopropan-1-ol (0.17 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.3 g) for 2 hours. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of acetic acid / methanol / methylene chloride (2-0 / 0-12 / 98-88%). Pure fractions were washed with aqueous sodium bicarbonate to give compound 37 as a colorless oil (0.22 g). [Example]

[0189] Synthesis of compound 38 A solution of 12-(2'-hexyldecanoyloxy)dodecan-1-al (1.8 g), acetic acid (0.08 g), and 3-aminopropan-1-ol (0.11 g) in methylene chloride (10 mL) was treated with sodium triacetoxyborohydride (0.64 g) overnight. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of acetic acid / methanol / methylene chloride (2-0 / 0-10 / 98-90%). Pure fractions were washed with aqueous sodium bicarbonate to give compound 38 as a colorless oil (0.83 g). [Example]

[0190] Synthesis of compound 39 A mixture of ethyl 4-aminobutyrate hydrochloride (1.28 mmol, 214 mg), 2-hexyldecyl 6-bromohexanoate (1.9 equiv., 2.43 mmol, 1.02 g), N,N-diisopropylethylamine (3.5 equiv., 4.48 mmol, 579 mg), and sodium iodide (5 mg) in anhydrous acetonitrile (15 mL) was heated in a pressure flask at 60 °C for 2 days. The mixture was cooled and concentrated. The residue was dissolved in a mixture of hexane and ethyl acetate (approximately 5:1, 100 mL), washed with water, brine, dried over sodium sulfate, filtered, and concentrated. A brown oil (approximately 1.04 g) was obtained. The crude product was purified by silica gel flash column chromatography (MeOH in DCM, 0-3.5%). This afforded compound 39 as a colorless oil (334 mg, 0.41 mmol, 43%). 1 HNMR (400 MHz, CDCl3) δ:4.13 (q, 7.1 Hz, 2H), 3.97 (d, 5.8 Hz, 4H), 2.43-2.34 (m, 6H), 2.33-2.28 (m, 6H), 1.73 (quintet, 7.3 Hz, 2H), 1.68-1.58 (m, 6H), 1.47-1.37 (m, 4H), 1.36-1.20 (54H), 0.89 (t-like, 6.8 Hz, 12H). [Example]

[0191] Synthesis of compound 40 A solution of 6-(2'-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid (0.15 g), and 1-aminobutan-2-ol (0.10 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.8 g) for 2 hours. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of acetic acid / methanol / methylene chloride (2-0 / 0-8 / 98-92%). Pure fractions were washed with aqueous sodium bicarbonate to give compound 40 as a colorless oil (0.85 g). [Example]

[0192] Synthesis of compound 41 A solution of 6-(2'-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid (0.19 g), and 1-aminobutan-2-ol (0.21 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.8 g) overnight. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of acetic acid / methanol / methylene chloride (2-0 / 0-8 / 98-92%). Pure fractions were washed with aqueous sodium bicarbonate to give compound 41 as a colorless oil (0.77 g). [Example]

[0193] Synthesis of compound 42 A solution of 6-(2'-butyloctanoyloxy)hexan-1-al (2.0 g), acetic acid (0.13 g), and 4-aminobutan-2-ol (0.20 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.03 g) overnight. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of acetic acid / methanol / methylene chloride (2-0 / 0-8 / 98-92%). The pure fractions were washed with aqueous sodium bicarbonate to give compound 42 as a colorless oil (0.54 g). [Example]

[0194] Synthesis of compound 43 A solution of 9-(2'-ethylhexanoyloxy)nonan-1-al (3.0 g), acetic acid (0.11 g), and 3-aminopropan-1-ol (0.14 g) in methylene chloride (50 mL) was treated with sodium triacetoxyborohydride (0.91 g) overnight. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of acetic acid / methanol / methylene chloride (2-0 / 0-6 / 98-94%). Pure fractions were washed with aqueous sodium bicarbonate to give compound 43 as a colorless oil (1.01 g). [Example]

[0195] Synthesis of compound 44 A solution of 6-(2'-decyltetradecanoyloxy)hexan-1-al (2.1 g), acetic acid (0.11 g), and 3-aminopropan-1-ol (0.11 g) in methylene chloride (30 mL) was treated with sodium triacetoxyborohydride (0.71 g) overnight. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of acetic acid / methanol / methylene chloride (2-0 / 0-8 / 98-96%). Pure fractions were washed with aqueous sodium bicarbonate to give compound 44 as a colorless oil (1.07 g). [Example]

[0196] Synthesis of compound 45 A solution of 9-(2'-butyloctanoyloxy)nonan-1-al (2.6 g), acetic acid (0.17 g), and 3-aminopropan-1-ol (0.21 g) in methylene chloride (50 mL) was treated with sodium triacetoxyborohydride (1.34 g) overnight. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of acetic acid / methanol / methylene chloride (2-0 / 0-8 / 98-96%). Pure fractions were washed with aqueous sodium bicarbonate to give compound 45 as a colorless oil (1.1 g). [Example]

[0197] Synthesis of compound 46 To a solution of 2-aminoethanol (96.5 mg, 1.58 mmol, 95.4 μL, MW 61.08, d 1.012) in 15 mL of 2-propanol, 2-hexyldecyl 8-bromooctanoate (1.8 equiv., 1.27 g, 2.84 mmol), potassium carbonate (1.9 equiv., 3 mmol, 414 mg), cesium carbonate (0.3 equiv., 0.47 mmol, 154 mg), and sodium iodide (10 mg) were added and heated (oil bath 60 °C) for 3 days. The mixture was concentrated, and the residue was taken up in THF (10 mL). To this mixture, additional aminoethanol (80 mg, 1.3 mmol) was added. Heating was continued at 70 °C for an additional 3 days. After a total of 6 days, the reaction mixture was cooled, filtered, and concentrated. The residue was purified by silica gel flash column chromatography (methanol in chloroform, 1–4.2%) to give compound 46 as a colorless oil (334 mg, 0.42 mmol, 30%). 1 HNMR (400 MHz, CDCl3) δ:4.09-4.06 (m, 2H), 3.97 (d, 5.8 Hz, 4H), 3.39-3.36 (m, 2H), 3.31-3.23 (m, 4H), 2.31 (t, 7.5 Hz, 4H), 1.88-1.56 (m, 12H), 1.43-1.19 (59H), 0.89 (t-like, 6.8 Hz, 12H). [Example]

[0198] Synthesis of compound 47 A solution of 6-(2'-hexyldecanoyloxy)hexan-1-al (3.0 g), acetic acid (0.20 g), and 3-aminopropionitrile (0.21 g) in methylene chloride (30 mL) was treated with sodium triacetoxyborohydride (1.3 g) overnight. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of acetic acid / methanol / methylene chloride (2-0 / 0-6 / 98-94%). Pure fractions were washed with aqueous sodium bicarbonate to give compound 47 as a colorless oil (0.29 g). [Example]

[0199] Synthesis of compound 48 A solution of 6-(2'-hexyldecanoyloxy)hexan-1-al (3.0 g) and ethyl 4-aminobutyrate hydrochloride (0.46 g) in methylene chloride (30 mL) was treated with sodium triacetoxyborohydride (1.4 g) overnight. The solution was washed with aqueous sodium bicarbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of acetic acid / methanol / methylene chloride (2-0 / 0-8 / 98-92%). Pure fractions were washed with aqueous sodium bicarbonate to give compound 48 as a colorless oil (0.80 g). [Example]

[0200] Synthesis of compound 49 To a solution of 2-butyloctyl 8-bromooctanoate (2 equiv., 1.877 g, 4.8 mmol) in 20 mL of anhydrous THF was added 4-amino-1-butanol (1 equiv., 2.4 mmol, 214 mg, 221 μl), potassium carbonate (2 equiv., 4.8 mmol, 664 mg), cesium carbonate (0.3 equiv., 0.72 mmol, 234 mg), and sodium iodide (approx. 5 mg). The mixture in a pressure round-bottom flask was heated (oil bath, 80 °C) for 6 days. The reaction mixture was cooled and concentrated. The residue was dissolved in a mixture of hexane and ethyl acetate (approx. 5:1), washed with water, brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by silica gel flash column chromatography (methanol in chloroform, 1–4%). This afforded compound 49 as a colorless oil (857 mg, 1.21 mmol, 50%). 1 HNMR (400 MHz, CDCl3) δ:6.55 (br. s, 1H), 3.97 (d, 5.8 Hz, 4H), 3.55 (not well resolved triplet, 2H), 2.45-2.40 (m. 6H), 2.30 (t, 7.5 Hz, 4H), 1.71-1.58 (m, 10 H), 1.51-1.42 (m, 4H), 1.39-1.19 (m, 44H), 0.93-0.87 (m, 12H).

[0201] The various embodiments described above can be combined to provide further embodiments. Unless inconsistent with specific teachings and definitions herein, U.S. Patent Application Nos. 62 / 247,616 (filed October 28, 2015) and 62 / 328,244 (filed April 27, 2016); all U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced herein and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, as necessary, to utilize concepts from various patents, patent applications, and patent publications to provide further embodiments. These and other modifications can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the scope of the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments and the full range of equivalents to which such claims are entitled. Accordingly, the scope of the claims is not limited by the present disclosure.

Claims

1. Use of lipid nanoparticles (LNPs) for the manufacture of a pharmaceutical composition for inducing protein expression in a subject, comprising: Lipid nanoparticles (LNPs) are i) mRNA encoding a protein; and ii) a compound of the following structural formula (IG): 【Chemistry 1】 (IG) [In the formula, R 1 and R 2 are each independently C 6 -C 24 alkyl or C 6 -C 24 alkenyl; R 3 is OR 5 , CN, —C(═O)OR 4 , —OC(═O)R 4 or —NR 5 C(═O)R 4 ; R 4 is C 1 -C 12 alkyl; R 5 is H or C 1 -C 6 alkyl; R 6 in each occurrence is H; n is an integer from 2 to 12; and y and z are each independently an integer from 6 to 10. or a pharmaceutically acceptable salt or stereoisomer thereof, Including, The use.

2. Use of the lipid nanoparticle (LNP) described in claim 1, wherein y and z are each independently an integer from 6 to 9.

3. Use of a lipid nanoparticle (LNP) described in claim 1 or 2, wherein n is 3, 4, 5 or 6.

4. Use of a lipid nanoparticle (LNP) according to any one of claims 1 to 3, wherein R 1 or R 2 , or both, are C 6 -C 24 alkenyl.

5. Use of a lipid nanoparticle (LNP) according to any one of claims 1 to 3, wherein R 1 or R 2 , or both, are C 6 -C 24 alkyl.

6. R 1 and R 2 each independently represent the following structural formula: 【Chemistry 2】 [In the formula, R 7a and R 7b are, independently at each occurrence, H or C 1 -C 12 alkyl; and a is an integer from 2 to 12; wherein R 7a , R 7b and a are each selected such that R 1 and R 2 each independently contain from 6 to 20 carbon atoms. Use of lipid nanoparticles (LNPs) according to any one of claims 1 to 5, wherein:

7. Use of the lipid nanoparticle (LNP) described in claim 6, wherein a is an integer from 8 to 12.

8. Use of a lipid nanoparticle (LNP) according to any one of claims 6 to 7, wherein at least one occurrence of R 7a is H.

9. Use of a lipid nanoparticle (LNP) described in any one of claims 6 to 7, wherein R 7a is H each time it occurs.

10. The use of a lipid nanoparticle (LNP) according to any one of claims 6 to 9, wherein at least one occurrence of R 7b is C 1 -C 8 alkyl.

11. The use of lipid nanoparticles (LNPs) according to claim 10, wherein C 1 -C 8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.

12. R 1 or R 2 , or both, have the following structural formula: 【Transformation 3】 Use of lipid nanoparticles (LNPs) according to any one of claims 5 to 11, characterized in that:

13. Use of a lipid nanoparticle (LNP) according to any one of claims 1 to 12, wherein R 3 is OH.

14. Use of a lipid nanoparticle (LNP) according to any one of claims 1 to 12, wherein R 3 is CN.

15. Use of a lipid nanoparticle (LNP) described in any one of claims 1 to 12, wherein R 3 is -C(=O)OR 4 , -OC(=O)R 4 or -NHC(=O)R 4 .

16. Use of the lipid nanoparticle (LNP) of claim 15, wherein R 4 is methyl or ethyl.

17. The structural formula: 【Chemistry 4】 【Transformation 5】 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 2. Use of the lipid nanoparticles (LNPs) according to claim 1, which is represented by one of the following:

18. The lipid nanoparticle (LNP) of claim 17, wherein the lipid nanoparticle (LNP) has the following structural formula (II): 【Chemistry 12】 (II) [In the formula, R8 and R9 are each independently a straight or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value in the range of 30 to 60] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof; Further comprising: Use of the lipid nanoparticles (LNPs) according to claim 1.

19. The use of lipid nanoparticles (LNPs) according to claim 18, wherein R 8 and R 9 are each independently a linear, saturated alkyl chain containing 12 to 16 carbon atoms.

20. Use of a lipid nanoparticle (LNP) described in any one of claims 18 or 19, wherein the average w is 49.

21. Use of a lipid nanoparticle (LNP) described in any one of claims 1 to 20, wherein expression of the protein induces a pharmacological effect in a subject.

22. Use of the lipid nanoparticles (LNPs) described in claim 21, wherein the pharmacological effect is an increase in red blood cell production.

23. Use of the lipid nanoparticles (LNPs) described in claim 21, wherein the pharmacological effect is protection from infection by infectious agents.

24. Use of the lipid nanoparticles (LNPs) of claim 23, wherein the infectious agent is a virus.

25. Use of a lipid nanoparticle (LNP) described in any one of claims 1 to 20, wherein the mRNA encodes an antibody.

26. Use of a lipid nanoparticle (LNP) described in any one of claims 1 to 20, wherein the protein is expressed in the liver of a subject.

27. ​​Use of a lipid nanoparticle (LNP) described in any one of claims 1 to 26, wherein the subject is a human.

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