Lipids for lipid nanoparticle delivery of active agents

Abstract

To provide a novel cationic lipid for the delivery of oligonucleotides.SOLUTION: There are provided compounds having the following structure (I) or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein G1 is -N(R3)R4 or -OR5; R1 is C12-C36 alkyl; R2 is C12-C36 alkyl or the like; R3 and R4 are each independently H or C1-C6 alkyl; R5 is H or C1-C6 alkyl; L is -C(=O)-, C6-C12 alkylene or the like; and n is an integer from 1 to 12. There are also provided use of the compounds as a component of lipid nanoparticle formulations for delivery of a therapeutic agent, compositions comprising the compounds and methods for their use and preparation.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure generally relates to novel cationic lipids that can be used in combination with other lipid components, such as neutral lipids, cholesterol and polymer-bound lipids, to form lipid nanoparticles that encapsulate nucleic acids and can facilitate intracellular delivery of therapeutic nucleic acids (e.g., oligonucleotides, messenger RNA) both in vivo and in vitro. [Background technology]

[0002] There have been many attempts related to the delivery of nucleic acids to affect desired responses in biological systems. Nucleic acid-based therapeutics have great potential, but to realize this potential, there is a need to more effectively deliver nucleic acids to the appropriate sites within a cell or organism. Therapeutic nucleic acids include, for example, messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immunostimulatory nucleic acids, antagomirs, antimirs, mimics, supermirs, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to bring about the expression of specific cellular products, for example, to be useful in treating diseases associated with protein or enzyme deficiencies. The therapeutic applications of translatable nucleotide delivery are very broad, as constructs can be synthesized and selective protein sequences can be generated, whether or not native to the system. The expression products of nucleic acids can enhance the levels of existing proteins, replace missing or non-functioning proteins, or introduce new proteins and associated functions into the cell or organism.

[0003] Some nucleic acids, such as miRNA inhibitors, can be used to induce the expression of specific cellular products regulated by miRNA, for example, to be useful in the treatment of diseases associated with protein or enzyme deficiency. The therapeutic application of miRNA inhibition is very broad, since it can synthesize constructs and inhibit one or more miRNAs that regulate the expression of mRNA products. Inhibition of endogenous miRNAs can enhance the expression of their downstream target endogenous proteins, restoring the proper function of cells or organisms as a means of treating diseases associated with a specific miRNA or group of miRNAs.

[0004] Other nucleic acids can downregulate the intracellular levels of specific mRNAs, and thus 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, since oligonucleotide constructs can be synthesized with nucleotide sequences directed against target mRNAs. Targets can include mRNAs in normal cells, mRNAs associated with disease states such as cancer, and mRNAs of infectious agents such as viruses. To date, antisense oligonucleotide constructs have been shown to be capable of specifically downregulating target proteins through the degradation of similar mRNAs in both in vitro and in vivo models. In addition, antisense oligonucleotide constructs are currently being evaluated in clinical studies.

[0005] However, currently, the use of oligonucleotides in therapeutic applications faces two problems. First, free RNA is susceptible to nuclease digestion in plasma. Second, free RNA has limited ability to access the intracellular compartments where the relevant translation machinery is present. Lipid nanoparticles formed from cationic lipids and other lipid components such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides have been used to inhibit the degradation of RNA in plasma and promote the cellular uptake of oligonucleotides.

[0006] There is a need for improved cationic lipids and lipid nanoparticles for delivery of oligonucleotides.Preferably, these lipid nanoparticles provide optimal drug-lipid ratios, protect nucleic acids from degradation and clearance in serum, are suitable for systemic or local delivery, and provide intracellular delivery of nucleic acids.Furthermore, these lipid-nucleic acid particles are well tolerated and provide a suitable therapeutic index, so that treatment of patients with effective doses of nucleic acids is not associated with unacceptable toxicity and / or risk to patients.The present disclosure provides these and related advantages. Summary of the Invention

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

[0008] In one embodiment, the following structure (I): [ka] [In the formula, G 1 , R 1 , R 2 , L, and n are as defined herein. or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof.

[0009] Also provided is a pharmaceutical composition comprising one or more of the compounds of structure (I) and a therapeutic agent.In some embodiments, the pharmaceutical composition further comprises one or more components selected from neutral lipids, charged lipids, steroids, and polymer-bound lipids.Such compositions are useful for forming lipid nanoparticles for delivery of therapeutic agents.

[0010] In other embodiments, the disclosure provides a method of administering a therapeutic agent to a patient in need thereof, comprising manufacturing or preparing a lipid nanoparticle composition comprising a compound of structure (I) and a therapeutic agent, and delivering or administering the composition to the patient.

[0011] These and other aspects of the present disclosure will become evident upon reference to the following detailed description. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] In the following description, certain specific details are set forth in order to facilitate a thorough understanding of various embodiments of the present disclosure. However, it will be understood by those skilled in the art that the disclosure may be practiced without these details.

[0013] The present disclosure is based in part on the discovery of novel cationic (amino) lipids that provide advantages when used in lipid nanoparticles for in vivo delivery of active or therapeutic agents, such as nucleic acids, to mammalian cells.In particular, embodiments of the present disclosure provide nucleic acid-lipid nanoparticle compositions that include one or more of the novel cationic lipids described herein that provide enhanced activity of nucleic acids in vivo and improved tolerance of the compositions, resulting in a significant increase in therapeutic index compared to previous nucleic acid-lipid nanoparticle compositions.In other embodiments, the disclosed lipids and lipid nanoparticles that include them have improved safety and / or tolerance when used to deliver active agents, such as nucleic acids.

[0014] In certain embodiments, the present disclosure provides novel cationic lipids that allow for 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 one particular miRNA or a group of miRNAs that regulate one 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 the delivery of mRNA and plasmids for the expression of transgenes. In still other embodiments, lipid nanoparticle compositions are useful for inducing pharmacological effects resulting from protein expression, such as improved red blood cell production by delivery of appropriate erythropoietin mRNA, or protection against infection by delivery of appropriate antigen or antibody-encoding mRNA.

[0015] The lipid nanoparticles and compositions of the present disclosure can be used for a variety of purposes, including delivery of encapsulated or associated (e.g., complexed) therapeutic agents, such as nucleic acids, to cells, both in vitro and in vivo. Thus, embodiments of the present disclosure provide methods of treating or preventing a disease or disorder in a subject in need thereof by contacting the subject with lipid nanoparticles encapsulated or associated with a suitable therapeutic agent, the lipid nanoparticles comprising one or more of the novel cationic lipids described herein.

[0016] As described herein, lipid nanoparticle embodiments of the present disclosure are particularly useful for delivery of nucleic acids, including, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomir / antimir), messenger RNA interference complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), etc. Thus, lipid nanoparticles and compositions of certain embodiments of the present disclosure 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 associate with a nucleic acid to be expressed to produce the desired protein (e.g., messenger RNA or plasmid encoding the desired protein) or to inhibit a process that silences the expression of the mRNA (e.g., miRNA inhibitors). Alternatively, the lipid nanoparticles and compositions of the present disclosure can be used to reduce the expression of target genes and proteins 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 are associated with nucleic acids that reduce expression of the target gene (e.g., antisense oligonucleotides or small interfering RNA (siRNA)). The lipid nanoparticles and compositions of the present disclosure can also be used for the co-delivery of different nucleic acids (e.g., mRNA and plasmid DNA), either separately or in combination, and are useful, for example, to provide effects where 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) is desired.

[0017] Nucleic acids for use in the embodiments of the present disclosure can be prepared according to any available technology.For mRNA, the main method of preparation is, but is not limited to, enzymatic synthesis (also called in vitro transcription), which is currently the most efficient method for generating specific mRNA of long sequence.In vitro transcription describes the process of template-directed synthesis of RNA molecules from engineered DNA templates that are composed of upstream bacteriophage promoter sequences (including but not limited to those from T7, T3 and SP6 coliphages) linked to downstream sequences that code for genes of interest. Template DNA can be prepared for in vitro transcription from a number of sources using suitable techniques well known in the art, including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, JL and Conn, GL, General protocols for preparation of plasmid DNA template and Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD in RNA 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).

[0018] Transcription of RNA is carried out in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and ribonucleoside triphosphates (rNTPs) of adenosine, guanosine, uridine, and cytidine under conditions that support polymerase activity while minimizing potential degradation of the resulting mRNA transcript. In vitro transcription can be performed using a variety of commercially available kits, including but not limited to RiboMax Large Scale RNA Production System (Promega), MegaScript Transcription kits (Life Technologies), and commercially available reagents such as RNA polymerase and rNTPs. Methods 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).

[0019] The desired in vitro transcribed mRNA is then purified from undesired components of the transcription or related reaction, including unincorporated rNTPs, protein enzymes, salts, short RNA oligos, etc. Techniques for isolating mRNA transcripts are well known in the art. Well-known methods include phenol / chloroform extraction, or precipitation with either alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Further examples of purification methods that can be used include, but are not limited to, 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 syntheses Methods v. 941 Conn GL (ed), New York, NY Humana Press, 2012). Purification can be performed using a variety of commercially available kits, including, but not limited to, SV Total Isolation System (Promega), and In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0020] Furthermore, although reverse transcription can generate large amounts of mRNA, the products 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 aberrant 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 with dsRNA structures may result in undesired immune stimulatory activity by interacting with various innate immune sensors in eukaryotic cells that serve to recognize specific nucleic acid structures and induce strong immune responses. Also, mRNA translation may be dramatically reduced as protein synthesis is reduced during the innate cellular immune response. Accordingly, 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 to very high levels, especially in primary cells and in vivo.

[0021] A significant variety of modifications have been described in the art that are used to alter certain properties of in vitro transcribed mRNA and improve its utility. These include, but are not limited to, modifications to the 5' and 3' ends of the mRNA. Endogenous eukaryotic mRNAs usually contain a cap structure at the 5' end of the mature molecule that plays an important role in mediating the binding of mRNA cap-binding protein (CBP), which is responsible for increasing intracellular mRNA stability and the efficiency of mRNA translation. Thus, 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 conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the last and penultimate most 5'-nucleotides at the 2'-hydroxyl group.

[0022] 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 chemical cap analogs (i.e., capping during in vitro transcription). For example, the anti-reverse cap analog (ARCA) cap contains a 5'-5'-triphosphate guanine-guanine linkage, where one guanine contains an N7 methyl group and a 3'-O-methyl group. However, during this co-transcription process, up to 20% of the transcripts remain uncapped, and the synthetic cap analog is not identical to the 5' cap structure of actual cellular mRNA, potentially resulting in reduced translatability and cellular stability. Alternatively, synthetic mRNA molecules can also be enzymatically capped post-transcriptionally. These can generate more realistic 5'-cap structures that structurally or functionally more closely mimic endogenous 5' caps, enhancing binding of cap-binding proteins, extending half-life, reducing susceptibility to 5' endonucleases, and / or reducing 5' decapping. A number of synthetic 5' cap analogs have been developed and are known in the art to enhance mRNA stability and translatability (see, e.g., Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, AN, Slepenkov, SV, Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, RE, Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013).

[0023] At the 3' end, a long chain of adenine nucleotides (a polyA tail) is typically added to an mRNA molecule during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved to free a 3' hydroxyl, and polyA polymerase adds a chain of adenine nucleotides to the RNA in a process called polyadenylation. It has been widely shown that poly(A) tails increase both the translation efficiency and the 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).

[0024] Poly(A) tailing of in vitro transcribed mRNA can be achieved using a variety of methods, including but not limited to cloning of a poly(T) tract into a DNA template or post-transcriptional addition using poly(A) polymerase. In the first case, depending on the size of the poly(T) tract, in vitro transcription of mRNA with a defined length of poly(A) tail is possible, but additional manipulation of the template is required. In the latter case, poly(A) tails are enzymatically added to in vitro transcribed mRNA using poly(A) polymerase, which catalyzes the incorporation of adenine residues into the 3' end of the RNA, and does not require additional manipulation of the DNA template, but results in mRNA with poly(A) tails of heterogeneous length. 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.

[0025] In addition to 5' cap and 3' polyadenylation, other modifications of in vitro transcription have been reported to provide advantages in terms of translation efficiency and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by various sensors in eukaryotes and induce a strong innate immune response. The ability to distinguish between pathogenic and self DNA and RNA has been shown to be based, at least in part, on structural and nucleoside modifications, as most nucleic acids from natural sources contain modified nucleosides. In contrast, in vitro synthesized RNA lacks these modifications, which can make it immune stimulatory and inhibit effective mRNA translation as discussed above.Introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thereby reducing this undesired immunostimulatory activity and improving 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, PH 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). The modified nucleosides and nucleotides used in the synthesis of modified RNA can be prepared, monitored, and utilized using common methods and techniques known in the art. A wide variety of nucleoside modifications are available that can be incorporated into in vitro transcribed mRNA, either alone or in some combination with other modified nucleosides (see, for example, US2012 / 0251618).In vitro synthesis of nucleoside-modified mRNAs has been reported to have enhanced translational potential but reduced ability to activate immune sensors.

[0026] Other components of mRNA that can be modified to benefit in terms of translatability and stability include the 5' and 3' untranslated regions (UTRs). Optimization of UTRs (preferred 5' and 3'UTRs can be obtained from cellular or viral RNA) has been shown to increase mRNA stability and translation efficiency of in vitro transcribed mRNAs, both or independently (see, e.g., 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).

[0027] In addition to mRNA, other nucleic acid payloads can be used for the present disclosure. For oligonucleotides, preparation methods include, but are not limited to, chemical synthesis, enzymatic and chemical cleavage of longer precursors, in vitro transcription as described above, etc. The synthesis methods of DNA and RNA nucleotides are widely used and well known in the art (e.g., 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).

[0028] For plasmid DNA, the formulations used in the embodiments of the present disclosure generally, but are not limited to, utilize in vitro propagation and isolation of plasmid DNA in liquid cultures of bacteria containing the plasmid of interest. The presence of genes in the plasmid of interest that code for resistance to certain antibiotics (penicillin, kanamycin, etc.) allows bacteria containing the plasmid of interest to selectively grow in cultures containing the antibiotic. 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:II: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 US 6,197,553 B1). Plasmid isolation can be performed using a variety of commercially available kits, including but not limited to Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo), Pure Yield MaxiPrep (Promega), and commercially available reagents.

[0029] Various exemplary embodiments of the cationic lipids of the present disclosure, and lipid nanoparticles and compositions comprising same, and their use to deliver active agents (e.g., therapeutic agents), such as nucleic acids for regulating gene and protein expression, are described in further detail below.

[0030] As used herein, the following terms have the meanings stated unless otherwise specified.

[0031] Unless the context requires otherwise, throughout this specification and claims, the word "comprises" and variations thereof, such as "including" and "including," are to be interpreted in an open and inclusive sense, i.e., "including but not limited to."

[0032] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0033] 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 disclosure belongs. In the specification and claims, the singular forms "a," "an," and "the" include the plural meaning unless the context clearly dictates otherwise.

[0034] The phrase "induce expression of a desired protein" refers to the ability of a nucleic acid to increase expression of a desired protein. To determine the degree of protein expression, a test sample (e.g., a sample of cells in culture expressing the desired protein) or a test mammalian (e.g., a mammalian such as a human or 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 disclosure). 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 cells in culture expressing the desired protein) or a control mammalian (e.g., a mammalian such as a human or 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 or administered with 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, inducing expression of a desired protein is achieved when the ratio of expression of the desired protein in a test sample or test mammal to the level of expression of the desired protein 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. Inducing 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 suitable assays for determining the expression level of a protein in a sample, such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays, or assays based on reporter proteins that can generate fluorescence or luminescence under appropriate conditions.

[0035] The expression "inhibits expression of a target gene" refers to the ability of a nucleic acid to silence, reduce or inhibit expression of a target gene. To determine the degree of gene silencing, a test sample (e.g., a sample of cells in culture expressing the target gene) or a test mammalian (e.g., a mammalian such as a human or 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 silences, reduces or inhibits expression of the 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 cells in culture expressing the target gene) or a control mammalian (e.g., a mammalian such as a human or animal), such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model that has not been contacted or administered with the nucleic acid. The expression of the target gene in the control sample or control mammalian can be assigned a value of 100%. In certain embodiments, silencing, inhibition, or reduction of expression of a target gene 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% relative to the level of target gene expression in a control sample or control mammal. In other words, the nucleic acid can silence, reduce or inhibit expression of the target gene 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% in a test sample or test mammal relative to the level of target gene expression in a control sample or control mammal not contacted or administered with the nucleic acid.Suitable assays for determining the level of target gene expression include, but are not limited to, testing 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, and phenotypic assays known to those of skill in the art.

[0036] 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 an increase or inhibition of expression of a target sequence, compared to the normal expression level detected in the absence of nucleic acid. In the case of an expression product that is not present in the absence of nucleic acid, an increase in expression of a target sequence is achieved when a measurable level is detected. In the case of an expression product that is present at a level before contacting with a nucleic acid, an increase in expression is achieved when the fold increase in value obtained with a nucleic acid, such as an 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 sequence is achieved when the value obtained with a nucleic acid, such as an antisense oligonucleotide, is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% compared to a control. Suitable assays for measuring expression of a target gene or sequence include testing protein or RNA 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 a suitable reporter protein, and phenotypic assays known to those of skill in the art.

[0037] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, including DNA, RNA, and hybrids thereof. DNA can be in the form of an antisense molecule, a plasmid DNA, a cDNA, a PCR product, or a vector. RNA can be in the form of a small hairpin RNA (shRNA), a messenger RNA (mRNA), an antisense RNA, a miRNA, a micRNA, a polyvalent RNA, a dicer substrate RNA, or a viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which occur synthetically, naturally, and non-naturally, and which have similar binding properties as the reference nucleic acid. Such analogs include, but are not limited to, for example, phosphorothioates, phosphoramidites, methyl phosphonates, chiral methyl phosphonates, 2'-0-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise specified, this term encompasses nucleic acids that contain known analogs of natural nucleotides that have similar binding properties as 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 sequence explicitly indicated.Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced 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" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate group."Bases" include purines and pyrimidines, as well as the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and naturally occurring analogues, and synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that place new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides. Prodrugs of nucleic acids are included in various embodiments of the invention.

[0038] 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 a precursor polypeptide.

[0039] As used herein, "gene product" refers to the product of a gene, such as an RNA transcript or a polypeptide.

[0040] The term "lipids" refers to a group of organic compounds, including but not limited to esters of fatty acids, that are generally characterized by poor solubility in water but solubility in many organic solvents. They are usually divided into at least three classes: (1) "simple lipids" (which include fats, oils, and waxes); (2) "complex lipids" (which include phospholipids and glycolipids); and (3) "derived lipids" (e.g., steroids).

[0041] "Steroids" are compounds that consist of the following carbon skeleton: [ka] The steroid is a compound containing the following. Although not limited thereto, examples of the steroid include cholesterol.

[0042] "Cationic lipid" refers to lipid that can be positively charged. Cationic lipids include, for example, one or more amine groups that carry a positive charge. Preferred cationic lipids are ionizable so that they can exist in positively charged or neutral form depending on pH. The ionization of cationic lipids affects the surface charge of lipid nanoparticles under various 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)), and the ability to form endosomolytic non-bilayer structures that are important for intracellular delivery of nucleic acids (Hafez, IM, et al., Gene Ther 8:1188-1196 (2001)).

[0043] The term "polymer-bound lipid" refers to a molecule that includes both a lipid portion and a polymer portion. An example of a polymer-bound lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that includes both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG), etc.

[0044] The term "neutral lipid" refers to any of several 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, phosphotidylcholines 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 derivatives thereof. The neutral lipids can be synthetic or naturally occurring.

[0045] The term "charged lipid" refers to any of several lipid species that exist in either positively or negatively charged form, regardless of the useful physiological range of pH, e.g., pH-3 to pH-9. Charged lipids can be synthetic or naturally derived. Charged lipids include, for example, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, hemisuccinic acid sterol, dialkyltrimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethane carbamoyl sterol (e.g., DC-Chol).

[0046] The term "lipid nanoparticle" refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) that includes one or more of the compounds of structure (I) or other specific cationic lipids. In some embodiments, lipid nanoparticles that include the disclosed cationic lipids (e.g., compounds of structure (I)) are included in a formulation that can be used to deliver an active or therapeutic agent, such as a nucleic acid (e.g., mRNA), to a target site of interest (e.g., a cell, tissue, organ, tumor, etc.). In some embodiments, the lipid nanoparticle includes a compound of structure (I) and a nucleic acid. Such lipid nanoparticles typically include a compound of structure (I) and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-bound lipids. In some embodiments, an active or therapeutic agent, such as a nucleic acid, can 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 it from enzymatic degradation or other undesirable effects elicited by mechanisms of the host organism or cells, such as a harmful immune response.

[0047] In various embodiments, the lipid nanoparticles have an average particle size of 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 8 ... 0nm, or about 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, or 150nm, and is substantially non-toxic. In certain embodiments, when present in lipid nanoparticles, nucleic acid is resistant to degradation by nuclease in aqueous solution. Lipid nanoparticles containing nucleic acid and methods for their manufacture are disclosed, for example, in U.S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031, and PCT Publication Nos. WO2013 / 016058, and WO2013 / 086373, the disclosures of which are all incorporated herein by reference in their entirety for all purposes.

[0048] As used herein, "lipid encapsulation" refers to lipid nanoparticles that provide an active or therapeutic agent, such as a nucleic acid (e.g., mRNA), with complete encapsulation, partial encapsulation, or both. In one embodiment, the nucleic acid (e.g., mRNA) is completely encapsulated in the lipid nanoparticle.

[0049] As used herein, the term "aqueous solution" refers to a composition that includes water.

[0050] "Serum stability" in reference to 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.

[0051] As used herein, "systemic delivery" refers to the delivery of a therapeutic product that allows widespread exposure of an active agent in an organism. Some techniques of administration can allow certain agents to be delivered systemically while others are not. Systemic delivery means that a useful, preferably therapeutic, amount of an agent is exposed to most parts of the body. Systemic delivery of lipid nanoparticles can be by any means known in the art, including, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.

[0052] As used herein, "local delivery" refers to the delivery of an active agent directly to a target site in an organism. For example, an agent can be delivered locally by direct injection into a disease site such as a tumor, other target sites such as inflammation sites, or target organs such as the liver, heart, pancreas, and kidney. 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.

[0053] "Alkyl" means a branched or unbranched (i.e., straight-chain) hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which may be saturated or unsaturated (i.e., containing one or more double bonds (alkenyl) and / or triple bonds (alkynyl)), e.g., from 1 to 24 carbon atoms (C1-C 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 12alkyl), 1 to 8 carbon atoms (C1-C8 alkyl), or 1 to 6 carbon atoms (C1-C6 alkyl), which are attached to the remainder of the molecule by a single bond, such as 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, and the like. Unless stated otherwise in the specification, alkyl groups may be optionally substituted.

[0054] "Alkylene" or "alkylene chain" means a branched or unbranched (i.e., linear) divalent hydrocarbon chain, consisting solely of carbon and hydrogen, that is saturated or unsaturated (i.e., has one or more double bonds (alkenylene) and / or triple bonds (alkynylene)), that connects the remainder of the molecule to another group, e.g., from 1 to 24 carbon atoms (C1-C 24 Alkylene, 1 to 15 carbon atoms (C1-C 15 Alkylene, 1 to 12 carbon atoms (C1-C 12 alkylene), 1 to 8 carbon atoms (C1-C8 alkylene), 1 to 6 carbon atoms (C1-C6 alkylene), 2 to 4 carbon atoms (C2-C4 alkylene), 1 to 2 carbon atoms (C1-C2 alkylene), such as methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. The alkylene chain is attached to the rest of the molecule through a single or double bond and to other groups through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule and to other groups can be through one carbon or any two carbons within the chain. Unless otherwise stated in this specification, the alkylene chain may be optionally substituted.

[0055] "Cycloalkyl" or "carbocycle" means a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, including fused or bridged ring systems, having from 3 to 15 carbon atoms, preferably from 3 to 10 carbon atoms, saturated or unsaturated, and attached to the remainder of the molecule by a single bond. Examples of monocyclic radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic radicals include adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwise specified in the specification, cycloalkyl radicals may be optionally substituted.

[0056] "Cycloalkylene" is a divalent cycloalkyl group. Unless stated otherwise in the specification, a cycloalkylene group may be optionally substituted.

[0057] "Heterocyclyl" or "heterocycle" means a stable 3- to 18-membered non-aromatic ring radical which consists of 2 to 12 carbon atoms and from 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur.

[0058] As used herein, the term "substituted" refers to any of the above groups (e.g., alkyl, alkylene, cycloalkyl, cycloalkylene, or heterocyclyl) 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 oxo group (=O); a hydroxyl group (-OH); a C1-C2 alkyl group (-C3 alkyl group); 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)xNR'R', where R' at each occurrence is independently H, C1-C 15 alkyl or cycloalkyl, and x is 0, 1, or 2. In some embodiments, the substituent is C-C 12 In another embodiment, the substituent is an alkyl group. In another embodiment, the substituent is a cycloalkyl group. In another embodiment, the substituent is a halo group, such as fluoro. In another embodiment, the substituent is an oxo group. In another embodiment, the substituent is a hydroxyl group. In another embodiment, 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').

[0059] "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 said event or circumstance does not occur. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and the description includes both substituted alkyl groups and alkyl groups that have no substituents.

[0060] "Prodrug" is meant to indicate a compound that can be converted under physiological conditions or by solvolysis into a biologically active compound of the present disclosure. Thus, the term "prodrug" refers to a metabolic precursor of a compound of the present disclosure that is pharma- ceutically acceptable. A prodrug may be inactive when administered to a subject in need thereof, but is converted in vivo into an active compound of the present disclosure. A prodrug is typically easily converted in vivo, for example, by hydrolysis in blood, to generate a parent compound of the present disclosure. 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.

[0061] The disclosure disclosed herein is also meant to encompass all pharma- ceutically acceptable compounds of the compounds of structure (I) that are isotopically labeled by replacing one or more atoms with atoms of different atomic mass or mass number.Isotopes that can be incorporated into the disclosed compounds include, for example, isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, for example, 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, and 125 I. These radiolabeled compounds may be useful to aid in determining or measuring the efficacy of compounds, for example, by characterizing the site or mechanism of action, or binding affinity to a pharmacologically important site of action. Certain isotopically labeled compounds of structure (I), (IA), or (IB), 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-l4, i.e. 14 C is particularly useful for this purpose in view of its ease of incorporation and ready means for detection.

[0062] Deuterium i.e. 2 Substitution with heavier isotopes, such as H, may confer certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and therefore may be preferable in some circumstances.

[0063] 11 C. 18 F, 15 O. 13 Substitution with positron emitting isotopes, such as N, can be useful in positron emission tomography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds of structure (I) can generally be prepared by conventional techniques known to those of skill in the art, or by processes analogous to those described in the Preparations and Examples below, by substituting an appropriate isotopically labeled reagent for a previously used non-labeled reagent.

[0064] "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.

[0065] "Mammals" includes both humans and domestic animals, such as laboratory animals and household pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), and non-domestic animals, such as wildlife.

[0066] 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 emulsifying agent, which are approved by the U.S. Food and Drug Administration as acceptable for use in humans or domestic animals.

[0067] "Pharmaceutically acceptable salt" includes both acid and base addition salts.

[0068] "Pharmaceutically acceptable acid addition salts" means salts formed with inorganic and organic acids which retain the biological effectiveness and properties of the free base and which are not biologically or otherwise undesirable, including, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids, including, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, acetic ... acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-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.

[0069] "Pharmaceutically acceptable base addition salt" means a salt which retains the biological effectiveness and properties of the free acid and is not biologically or otherwise undesirable. These salts are prepared by the addition of 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 the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as salts of 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, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0070] Crystallization often produces solvates of the compounds of the present disclosure (i.e., compounds of structure (I)). As used herein, the term "solvate" refers to an aggregate that includes one or more molecules of the compounds of the present disclosure and one or more solvent molecules. The solvent can be water, in which case the solvate can be a hydrate. Alternatively, the solvent can be an organic solvent. Thus, the compounds of the present disclosure can exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., and the corresponding solvate forms. The solvates of the compounds of the present disclosure can be true solvates, while in other cases, the compounds of the present disclosure can simply retain incidental water or be a mixture of water and some incidental solvent.

[0071] "Pharmaceutical composition" refers to a formulation of a compound of the present disclosure and a vehicle generally accepted in the art for delivering the biologically active compound to a mammal (e.g., a human). Such a vehicle includes any pharma- ceutically acceptable carrier, diluent, or excipient therefor.

[0072] As used herein, "treating" or "treatment" encompasses the treatment of a disease or condition of interest in a mammal (preferably a human) having the disease or condition of interest, and includes: (i) to prevent a disease or condition from occurring in a mammal (especially if the mammal is susceptible to the disease but has not yet been diagnosed as having it); (ii) inhibiting a disease or illness (i.e., arresting its development); (iii) alleviating the disease or illness (i.e., causing the regression of the disease or illness); or (iv) Relieving symptoms caused by a disease or illness (i.e., relieving pain without addressing the underlying disease or illness). As used herein, the terms "disease" and "illness" may be used interchangeably or may differ in that a particular illness or condition may not have a known causative agent (and thus the etiology has not yet been elucidated) and therefore is not yet recognized as a disease, but only as an undesirable illness or syndrome in which some specific symptoms have been identified by a clinician.

[0073] The compounds of the present disclosure, or pharma- ceutically acceptable salts thereof, may contain one or more stereocenters and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined in terms of absolute stereochemistry as (R)- or (S)-, or in the case of amino acids, as (D)- or (L)-. The present disclosure is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers can be prepared using chiral synthons or chiral reagents or separated using conventional techniques, e.g., chromatography and fractional crystallization. Conventional techniques for preparing / isolating individual enantiomers include chiral synthesis from appropriate optically pure precursors, or separation of the racemate (or racemate of a salt or derivative), e.g., using chiral high-performance liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless specified otherwise, the compounds are intended to include both the E and Z geometric isomers, as well as all tautomeric forms.

[0074] "Stereoisomers" means compounds consisting of the same atoms linked by the same bonds, but having different, incompatible three-dimensional structures. The present disclosure contemplates various stereoisomers and mixtures thereof, and includes "enantiomers," which means two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0075] "Tautomer" means a shift of a proton from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any of the aforementioned compounds.

[0076] compound In one aspect, the present disclosure provides novel lipid compounds that can be combined with other lipid components, such as neutral lipids, charged lipids, steroids, and / or polymer-bound lipids, to form lipid nanoparticles with oligonucleotides.Without being bound by theory, it is believed that these lipid nanoparticles protect oligonucleotides from degradation in serum and provide effective delivery of oligonucleotides to cells in vitro and in vivo.

[0077] In one embodiment, the compound has the following structure (I): [ka] [In the formula, G 1 is -N(R 3 )R 4 -OR 5 and; R 1 is an optionally substituted branched saturated or unsaturated C 12 -C 36 is alkyl; R 2 When L is -C(=O)-, optionally substituted branched or unbranched, saturated or unsaturated C 12 -C 36 alkyl; or R 2 L is C6-C 12 Alkylene, C6-C 12 alkenylene, or C2-C6 alkynylene, optionally substituted branched or unbranched, saturated or unsaturated C4-C 36 is alkyl; R 3 and R 4are each independently H or an optionally substituted branched or unbranched, saturated or unsaturated C1-C6 alkyl; or R 3 and R 4 L is C6-C 12 Alkylene, C6-C 12 alkenylene, or C2-C6 alkynylene, each independently being optionally substituted branched or unbranched, saturated or unsaturated C1-C6 alkyl; or R 3 and R 4 together with the nitrogen to which they are attached form a heterocyclyl; R 5 is H or optionally substituted C1-C6 alkyl; L is -C(=O)-, C6-C 12 Alkylene, C6-C 12 Alkenylene, or C2-C 12 alkynylene (e.g., C2-C6 alkynylene); and n is an integer from 1 to 12. or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof.

[0078] In some embodiments, G 1 is -N(R 3 )R 4 -OR 5 and; R 1 is an optionally substituted branched saturated or unsaturated C 12 -C 36 is alkyl; R 2 is an optionally substituted branched or unbranched, saturated or unsaturated C 12 -C 36 is alkyl; R 3 and R 4 are each independently H or an optionally substituted branched or unbranched, saturated or unsaturated C1-C6 alkyl; or R 3and R 4 together with the nitrogen to which they are attached form a heterocyclyl; R 5 is H or optionally substituted C1-C6 alkyl; L is -C(=O)-; and n is an integer from 1 to 12; A compound of structure (I), or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof:

[0079] In another embodiment, G 1 is -N(R 3 )R 4 -OR 5 and; R 1 is an optionally substituted branched saturated or unsaturated C 12 -C 36 is alkyl; R 2 is an optionally substituted branched or unbranched, saturated or unsaturated C4-C 36 is alkyl; R 3 and R 4 are each independently an optionally substituted branched or unbranched, saturated or unsaturated C1-C6 alkyl; or R 3 and R 4 together with the nitrogen to which they are attached form a heterocyclyl; R 5 is H or optionally substituted C1-C6 alkyl; L is C6-C 12 Alkylene Linker, C6-C 12 alkenylene linker, or a C2-C6 alkynylene linker; and n is an integer from 1 to 12; A compound of structure (I), or a pharma- ceutically acceptable salt, or stereoisomer thereof.

[0080] In some more specific embodiments, the following structure (IA): [ka] [In the formula, R 8 and R 9 are each independently H or an optionally substituted branched or unbranched saturated or unsaturated C-C 12 alkyl; 8 and R 9 is R 1 is an optionally substituted branched saturated or unsaturated C 12 -C 36 are each independently selected to be alkyl; and R 10 and R 11 are each independently H or an optionally substituted branched or unbranched saturated or unsaturated C-C 12 alkyl; provided that when L is -C(=O)-, then R 2 is an optionally substituted branched or unbranched, saturated or unsaturated C 12 -C 36 and L is C6-C 12 Alkylene, C6-C 12 alkenylene, or C2-C6 alkynylene, R 2 is an optionally substituted branched or unbranched, saturated or unsaturated C4-C 36 R to be alkyl 10 and R 11 are each independently selected. It is a compound represented by the formula:

[0081] In some of the foregoing embodiments, R 8 and R 9 each independently represents an optionally substituted branched or unbranched, saturated or unsaturated C-C 12 In some embodiments, R 8is optionally substituted: C2 alkyl, C4 alkyl, C6 alkyl, C8 alkyl, or C 10 In some embodiments, R 8 is optionally substituted: C4 alkyl, C6 alkyl, or C8 alkyl. In some more specific embodiments, R 9 is optionally substituted: C4 alkyl, C6 alkyl, C8 alkyl, C 10 Alkyl, or C 12 In some more particular embodiments, R 9 is optionally substituted: C alkyl, C alkyl, or C 10 In some more particular embodiments, R 8 is optionally substituted: C4 alkyl, C6 alkyl, or C8 alkyl, and R 9 is optionally substituted: C alkyl, C alkyl, or C 10 It is an alkyl.

[0082] In some embodiments, R 10 is H and R 11 is an optionally substituted branched or unbranched, saturated or unsaturated C2-C 12 In some more specific embodiments, R 11 is an optionally substituted unbranched C2 alkyl, C6 alkyl, or C 10 In some further embodiments, R 11 is an optionally substituted C alkyl. In other embodiments, R 11 is an optionally substituted C alkyl. In yet another embodiment, R 11 is an optionally substituted C 10 It is an alkyl.

[0083] In certain embodiments, R 10 and R 11each independently represents an optionally substituted branched or unbranched, saturated or unsaturated C-C 12 In some embodiments, R 10 is optionally substituted: C2 alkyl, C4 alkyl, C6 alkyl, C8 alkyl, or C 10 In certain embodiments, R 10 is optionally substituted: C4 alkyl, C6 alkyl, or C8 alkyl. In some embodiments, R 11 is optionally substituted: C4 alkyl, C6 alkyl, C8 alkyl, C 10 Alkyl, or C 12 In some embodiments, R 11 is optionally substituted: C alkyl, C alkyl, or C 10 In some embodiments, R 10 is optionally substituted: C4 alkyl, C6 alkyl, or C8 alkyl, and R 11 is C6 alkyl, C8 alkyl, or C 10 It is an alkyl.

[0084] In some embodiments, R 8 , R 9 , R 10 , and R 11 each independently represents an optionally substituted branched or unbranched, saturated or unsaturated C6-C 12 In some embodiments, R 8 , R 9 , R 10 , and R 11 each independently represents an optionally substituted branched or unbranched, saturated or unsaturated C6-C 10 In some particular embodiments, R 8 , R 9 , R 10 , and R 11each independently represents an optionally substituted branched or unbranched, saturated or unsaturated C8-C 12 In some embodiments, R 8 , R 9 , R 10 , and R 11 are each independently an optionally substituted branched or unbranched, saturated or unsaturated C2-C6 alkyl.

[0085] In some embodiments, R 1 and R 2 each independently represents an optionally substituted branched saturated or unsaturated C 12 -C 30 In some embodiments, R 1 and R 2 each independently represents an optionally substituted branched saturated or unsaturated C 12 -C 20 In some embodiments, R 1 and R 2 each independently represents an optionally substituted branched saturated or unsaturated C 15 -C 20 It is an alkyl.

[0086] In some of the foregoing embodiments, R 1 and R 2 Each is saturated. In certain embodiments, R 1 and R 2 At least one of R is unsaturated. 1 and R 2 At least one of R is independently unsubstituted. 1 and R 2 are both non-substitutive.

[0087] In some embodiments, R 1 and R 2At least one of R is independently substituted (e.g., substituted with -OH, -NH, halo, -SH, -C(=O)-, aminyl, or a combination thereof). 1 and R 2 are substituted (e.g., substituted with -OH, -NH2, halo, -SH, -C(=O)-, aminyl, or combinations thereof).

[0088] In some of the foregoing embodiments, R 1 and R 2 has the following structure: [ka] It is.

[0089] In some embodiments, R 2 has the following structure: [ka] It is one of them.

[0090] In some more specific embodiments, R 2 has the following structure: [ka] It is one of them.

[0091] In some more specific embodiments, R 1 has the following structure: [ka] It is one of them.

[0092] In some embodiments, R 1 and R 2 Both have the following structure: [ka] It is one of them.

[0093] In some of the foregoing embodiments, R 1 has the following structure: [ka] It is.

[0094] In some embodiments, L is -C(=O)-. In some embodiments, L is an alkylene linker, e.g., C-C 12 In some more specific embodiments, L is C6 alkylene. In other specific embodiments, L is C7 alkylene. In some embodiments, L is C8 alkylene. In some embodiments, L is C9 alkylene. In some embodiments, L is C 10 In certain other embodiments, L is C 11 It is alkylene.

[0095] In certain embodiments, G 1 is -N(R 3 )R 4 In some embodiments, G 1 is -NH2, -NHCH3, or -N(CH3)2.

[0096] In some embodiments, R 3 and R 4 together with the nitrogen to which they are attached form a heterocyclyl. 1 has the following structure: [ka] It is one of them.

[0097] In another embodiment, G 1 has the following structure: [ka] It is.

[0098] In certain embodiments, G 1 -OR 5 In some embodiments, R 5 is C1-C6 alkyl, R 5 is substituted with at least one amine. In some specific embodiments, R 5 is methyl, ethyl, or isopropyl.

[0099] In some specific embodiments, n is 1, 2, 3, 4, 5, or 6. In some embodiments, n is 7, 8, 9, 10, 11, or 12. In some specific embodiments, n is 1. In some specific embodiments, n is 2. In some specific embodiments, n is 3. In some specific embodiments, n is 4. In some specific embodiments, n is 5. In some specific embodiments, n is 6. In some specific embodiments, n is 7. In some specific embodiments, n is 8. In some specific embodiments, n is 9. In some specific embodiments, n is 10. In some specific embodiments, n is 11. In some specific embodiments, n is 12.

[0100] In various different embodiments, the compound is one of the structures set forth in Table 1 below. Table 1. Compounds included in embodiments of the compound of structure (I) [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0101] It is understood that any embodiment of the compounds of structure (I) above, and any specific substituents and / or variables in the compounds of structure (I) above, can be independently combined with other embodiments of the compounds of structure (I) and / or substituents and / or variables to form embodiments of the disclosure not specifically set forth above. Further, when a list of substituents and / or variables is recited in a particular embodiment and / or claim for a particular R group, G group, or variable a, b, or n, it is understood that the individual substituents and / or variables can be deleted from the particular embodiment and / or claim, respectively, and the remaining list of substituents and / or variables is deemed to be within the scope of the present disclosure.

[0102] It is understood that combinations of substituents and / or variables of the depicted formulae herein are permissible only if such contributions result in stable compounds.

[0103] In some embodiments, lipid nanoparticles are provided that include a compound of structure (I). The lipid nanoparticles may optionally include an excipient selected from neutral lipids, steroids, and polymer-bound lipids.

[0104] In some embodiments, compositions are provided that include any one or more of the compounds of structure (I) and a therapeutic agent.For example, in some embodiments, the compositions include any one of the compounds of structure (I), a therapeutic agent, and one or more excipients selected from neutral lipids, steroids, and polymer-bound lipids.Other pharmaceutically acceptable excipients and / or carriers are also included in various embodiments of the compositions.

[0105] 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 is in the range of about 2:1 to about 8:1.

[0106] In various embodiments, the composition further comprises a steroid or a steroid analog. In certain embodiments, the steroid or the 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.

[0107] In various embodiments, the polymer-bound lipid is a PEGylated lipid. For example, some embodiments include PEGylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinate (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate, and the like. PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the compound to the PEGylated lipid ranges from about 100:1 to about 20:1.

[0108] In some embodiments, the composition has the following structure (II): [ka] [In the formula, R 12 and R 13are each independently a branched or unbranched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain may optionally be interrupted by one or more ester linkages; and The average value of w is in the range of 30 to 60. or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof.

[0109] In some embodiments, R 12 and R 13 is each independently an unbranched saturated alkyl chain containing 12 to 16 carbon atoms. In other embodiments, the average value of w is in the range of about 42 to 55, e.g., about 49.

[0110] 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 an antisense RNA and a messenger RNA.

[0111] In other different embodiments, the present disclosure relates to a method of administering a therapeutic agent to a patient in need thereof, the method comprising manufacturing or preparing any of the aforementioned compositions and administering the composition to the patient.

[0112] For purposes of administration, the disclosed compound embodiments (typically in the form of lipid nanoparticles combined with a therapeutic agent) can be administered as raw chemicals or formulated as pharmaceutical compositions. Pharmaceutical compositions of the disclosed embodiments include a compound of structure (I) and one or more pharma- ceutically acceptable carriers, diluents or excipients. In some embodiments, the compound of structure (I) is present in the composition in an amount effective to form lipid nanoparticles and deliver, for example, a therapeutic agent for treating a particular disease or condition of interest. Appropriate concentrations and dosages can be readily determined by those skilled in the art.

[0113] The administration of the compositions of the present disclosure can be carried out through any of the accepted methods of administration of pharmaceuticals to provide similar utility. The pharmaceutical compositions of the present disclosure can be formulated into solid, semi-solid, liquid, or gaseous formulations, including, for example, 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 nasal. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. The pharmaceutical compositions of the present disclosure are formulated so that the active ingredients contained therein are bioavailable when the compositions are administered to a patient. In some embodiments, the compositions administered to a subject or patient are in the form of one or more unit dosage forms, for example, a tablet may be one unit dosage form, and a container of the compound of the present disclosure in aerosol form may hold multiple unit dosage forms. The actual method of preparing such dosage forms is 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). In some embodiments, the composition administered comprises a therapeutically effective amount of lipid nanoparticles comprising a compound of the present disclosure or a pharma- ceutically acceptable salt thereof, in each case for treating a disease or condition of interest according to the teachings of the present disclosure.

[0114] The pharmaceutical composition of the present disclosure may be in solid or liquid form.In one aspect, the carrier is particulate, and the composition is, for example, in tablet or powder form.Alternatively, the carrier may be liquid, and the composition is, for example, an oral syrup, an injectable liquid, or an aerosol, for example, useful in inhalation administration.

[0115] When intended for oral administration, the pharmaceutical compositions of certain embodiments are preferably in either solid or liquid form, with semi-solid, semi-liquid, suspension, and gel forms being included within the scope of forms considered herein as either solid or liquid.

[0116] As a solid composition for oral administration, the pharmaceutical composition of some embodiments may be formulated in the form of powder, granule, compressed tablet, pill, capsule, chewing gum, wafer, etc. Such solid composition typically comprises one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants such as magnesium stearate, or Sterotex; glidants such as colloidal silicon dioxide; sweeteners such as sucrose, or saccharin; flavors such as peppermint, methyl salicylate, or orange flavor; colorants.

[0117] When the pharmaceutical composition of some embodiments 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.

[0118] The pharmaceutical composition of some embodiments may be in the form of a liquid, for example, 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, the preferred composition includes, in addition to the compound of structure (I), one or more of a sweetener, a preservative, a dye / colorant, and a flavoring agent. In the composition intended for administration by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersant, a suspending agent, a buffer, a stabilizer, and an isotonic agent may be included.

[0119] The liquid pharmaceutical compositions of the present disclosure, whether in solution, suspension or other form, may contain one or more of the following adjuvants: sterile diluents, such as water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride; non-volatile oils, such as synthetic mono- or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can act 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 agents for adjusting tonicity, such as sodium chloride or dextrose; agents acting as cryoprotectants, such as sucrose, or trehalose. Parenteral preparations can be placed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. Saline is the preferred adjuvant. Pharmaceutical compositions for injection are preferably sterile.

[0120] Liquid pharmaceutical compositions of the disclosed embodiments intended for either parenteral or oral administration will contain an amount of a compound of the present disclosure such that a suitable dosage will be obtained.

[0121] The pharmaceutical composition of the present disclosure can be for topical administration, and in this case, the carrier can comprise a solution, emulsion, ointment or gel base as appropriate.The base can comprise, for example, one or more of petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluent such as water and alcohol, and emulsifier and stabilizer.A thickener can be present in the pharmaceutical composition for topical administration.When intended for transdermal administration, the composition can comprise a transdermal patch or iontophoresis device.

[0122] The pharmaceutical composition of the present disclosure may be for rectal administration, for example, in the form of suppository, which melts in the rectum and releases the drug.Composition for rectal administration may contain oily base as suitable non-irritating excipient.Such base includes, but is not limited to, lanolin, cocoa butter, and polyethylene glycol.

[0123] The pharmaceutical composition of the present disclosure may contain various materials that modify the physical form of the solid or liquid dosage unit.For example, the composition may contain materials that form a coating shell around the active ingredient.The materials that form the coating shell are usually inert and can be selected from, for example, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredient can be encapsulated in a gelatin capsule.

[0124] Pharmaceutical compositions of the disclosed embodiments, in solid or liquid form, may include an agent that binds to the disclosed compounds and thereby aids in the delivery of the compounds. Suitable agents that may act in this capacity include monoclonal or polyclonal antibodies, or proteins.

[0125] The pharmaceutical composition of the present disclosure may be comprised of a dosage unit that can be administered as an aerosol. The term aerosol is used to describe a variety of systems ranging from colloidal to systems comprised of pressurized packaging. Delivery may be by liquefied or compressed gas that disperses the active ingredient, or by a suitable pump system. The aerosol of the compound of the present disclosure may be delivered in a single-phase, two-phase, or three-phase system to deliver the active ingredient. The delivery of the aerosol includes the necessary containers, activators, valves, subcontainers, etc., which may be taken together to form a kit. Those skilled in the art will be able to obtain a preferred aerosol without undue experimentation.

[0126] The pharmaceutical composition of the present disclosure embodiment can be prepared by methods well known in the pharmaceutical field.For example, pharmaceutical compositions intended to be administered by injection can be prepared by combining the liquid lipid nanoparticles of the present disclosure with sterile distilled water or other carriers to form a solution.Surfactants may be added to promote the formation of a homogeneous solution or suspension.Surfactants are compounds that non-covalently interact with the compounds of the present disclosure to promote the dissolution or homogeneous suspension of the compounds in aqueous delivery systems.

[0127] The compositions of the presently disclosed embodiments are administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the particular therapeutic agent used: the metabolic stability and duration of action of the therapeutic agent; the age, weight, general health, sex, and diet of the patient; the mode and time of administration; excretion rate; drug combinations; the severity of the particular disorder or disease; and the subject being treated.

[0128] The composition of the present disclosure embodiment can also be administered simultaneously with, before, or after administration of one or more other therapeutic agents. Such combination therapy includes administration of a single pharmaceutical dosage formulation of the composition of the present disclosure embodiment and one or more additional active agents, as well as administration of a composition of the present disclosure embodiment and each separate pharmaceutical dosage formulation of each active agent. For example, the composition of the present disclosure embodiment and the other active agents can be administered to a patient together in a single oral dosage composition such as a tablet or capsule, or each agent can be administered in a separate oral dosage formulation. When using separate dosage formulations, the compound of the present disclosure embodiment and one or more additional active agents can be administered essentially simultaneously, i.e., together, or at different staggered times, i.e., sequentially, and combination therapy is understood to include all of these regimens.

[0129] Methods for the preparation of the above compounds and compositions are described herein below and / or known in the art.

[0130] It will be appreciated by those skilled in the art that in the methods described herein, functional groups of intermediate compounds may need to be protected by 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(0)-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 can be added or removed according to standard techniques known to those skilled in the art and described herein. The use of protecting groups is described in Green, TW and PGM Wutz, Protective Groups in Organic Synthesis (1999), 3 rd Ed., Wiley, As one of skill in the art will appreciate, the protecting group may also be a polymeric resin, such as a Wang resin, a Rink resin, or a 2-chlorotrityl chloride resin.

[0131] Such protected derivatives of the compounds of the present disclosure may not themselves have pharmacological activity, but they may be administered to a mammal and then metabolized in the body to form a pharmacologically active compound of the present disclosure. Such derivatives may therefore be described as "prodrugs." All prodrugs of the compounds of the present disclosure are included within the scope of the present disclosure.

[0132] Additionally, compounds of the disclosed embodiments that exist in a free base or acid form can be converted to their pharma- ceutically acceptable salts by treatment with an appropriate inorganic or organic base or acid by methods known in the art. Salts of compounds of the disclosed embodiments can be converted to their free base or acid forms by standard techniques.

[0133] The following general reaction scheme 1 illustrates the synthesis of compounds of the present disclosure, i.e., structure (I): [ka] [In the formula, G 1 , R 1 , R 2 , L, and n are as defined herein. The following shows exemplary methods for preparing compounds of the formula (I) or their pharma- ceutically acceptable salts, tautomers, or stereoisomers. It is understood that those skilled in the art can prepare these compounds by similar methods or by combining other methods known to those skilled in the art. It is also understood that those skilled in the art can prepare other compounds of structure (I) not specifically shown below by using appropriate starting components and modifying the parameters of the synthesis as necessary in a manner similar to that described below. In general, the starting components can be obtained from sources 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 in this disclosure.

[0134] General reaction scheme 1 [ka]

[0135] General Reaction Scheme 1 ("Method A") provides an exemplary method for preparing compounds of structure (I). 1 , R 2 , L, and n are as defined herein, and X and Y denote interchangeable reactive moieties throughout the reaction scheme. Compounds of structures A-1 and A-2 are purchased or prepared according to methods known in the art. Reaction of A-1 with A-2 under appropriate coupling conditions (e.g., Y is a chloride and the reaction conditions include an appropriate base such as triethylamine) produces A-3, which then couples the reactive group X (e.g., bromide) to the desired G 1 A reaction to convert A-2 to a group (e.g., with a secondary amine) produces a compound of structure (I). Additionally, if A-2 is an acid halide (e.g., an acid chloride), A-3 may require a reduction step to remove the carbonyl. In that case, suitable reducing conditions (e.g., lithium aluminum hydride) can be used to produce a compound of structure (I).

[0136] General reaction scheme 2 [ka]

[0137] General Reaction Scheme 2 ("Method B") provides an exemplary method for preparing compounds of structure (I). 1 , R 1 , R 2, L, and n are as defined herein, and m is selected such that B-4 is the counterpart (e.g., a compound that is one -CH2- shorter than the compound of structure (I)) and the final compound is as defined in structure (I) herein. Compounds of structure B-1 are purchased or prepared according to methods known in the art. Reaction of B1 under appropriate conditions (e.g., oxalyl chloride) produces acyl chloride B-2, which can then react with B-3 to produce amide B-4. B-4 can be treated with an appropriate reducing agent (e.g., lithium aluminum hydride) to produce compounds of structure (I). It should be noted that various alternative strategies for producing compounds of structure (I) are available to one skilled in the art. For example, other compounds of structure (I) can be prepared following similar methods using appropriate starting materials. The use of protecting groups as necessary and other modifications to the above general reaction scheme will be readily apparent to one skilled in the art. EXAMPLES

[0138] The following examples are offered by way of illustration and not by way of limitation.

[0139] Example 1 In vivo assessment of luciferase mRNA using lipid nanoparticle compositions Lipid nanoparticles are produced and tested according to the general methods described in PCT Publication Nos. WO2015 / 199952 and WO2017 / 004143, the disclosures of which are incorporated herein by reference in their entirety. Briefly, cationic lipids, DSPC, cholesterol, and PEG lipids are solubilized in ethanol at a molar ratio of about 50:10:38.5:1.5, or about 47.5:10:40.8:1.7. Lipid nanoparticles (LNPs) are produced at a weight ratio of total lipid to mRNA of about 10:1 to 30:1. The mRNA is diluted to 0.2 mg / mL in 10 to 50 mM citrate or acetate buffer (pH 4). The ethanolic lipid solution is mixed with the aqueous mRNA solution at a ratio of about 1:5 to 1:3 (vol / vol) using a syringe pump at a total flow rate of 15 mL / min or greater. The ethanol is then removed and the external buffer is replaced with PBS by dialysis. Finally, the lipid nanoparticles are filtered through a 0.2 μm pore sterile filter. The particle size of the lipid nanoparticles is approximately 55-95 nm in diameter, and in some cases, approximately 70-90 nm in diameter, as determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK).

[0140] Studies are performed in 6-8 week old female C57BL / 6 mice (Charles River), or 8-10 week old CD-1 (Harlan) mice (Charles River) in accordance with guidelines set forth by the Institutional Animal Care Committee (ACC) and the Canadian Council on Animal Care (CCAC). Various doses of mRNA lipid nanoparticles are administered systemically via tail vein injection, and animals are euthanized at specific time points (e.g., 4 h) after administration. Livers and spleens are collected in pre-weighed tubes, weighed, immediately flash frozen in liquid nitrogen, and stored at -80 °C until analytical processing.

[0141] For liver, approximately 50 mg is processed for analysis in a 2 mL FastPrep tube (MP Biomedicals, Solon, OH). 1 / 4 inch ceramic balls (MP Biomedicals) are added to each tube, and 500 μL of Glo Lysis Buffer-GLB (Promega, Madison, WI) equilibrated to room temperature is added to the liver tissue. The liver tissue is homogenized in a FastPrep24 instrument (MP Biomedicals) twice at 6.0 m / s for 15 seconds. The homogenate is incubated at room temperature for 5 minutes, then diluted 1:4 with GLB and measured using the SteadyGlo Luciferase Assay System (Promega). Specifically, 50 μL of homogenate diluted tissue is reacted with 50 μL of SteadyGlo substrate, shaken for 10 seconds, incubated for 5 minutes, and then assayed using a CentroXS. 3 Quantification is performed using an LB960 Luminometer (Berthold Technologies, Germany). The amount of protein assayed is measured using a BCA Protein Assay Kit (Pierce, Rockford IL). Relative Luminescence Units (RLU) are then normalized to ug of total protein assayed. To convert RLU to ng of luciferase, a standard curve is generated with QuantiLum Recombinant Luciferase (Promega).

[0142] Trilink Biotechnologies' FLuc mRNA (L-6107 or L-7202) 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, polyadenylated mRNA is fully substituted for uridine and / or cytidine nucleosides.

[0143] Example 2 PK of formulated lipids A Measurement As described elsewhere, the pK of the formulated cationic lipids a pK correlates with the efficacy of LNPs for nucleic acid delivery (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 5 to 7. a is 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%) at a concentration of 0.4 mM total lipid in PBS are prepared using the in-line process described in Example 1. TNS is prepared as a 100 mM stock solution in distilled water. The vehicle is diluted to 24 mM lipid with 2 mL of buffer containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, where the pH ranges from 2.5 to 11. An aliquot of TNS solution is added to a final concentration of 1 μM, followed by vortex mixing, and the fluorescence intensity is measured at room temperature using 321 nm and 445 nm as the excitation and emission wavelengths on an SLM Aminco Series 2 Luminescence Spectrophotometer. A sigmoidal best-fit analysis was applied to the fluorescence data, and the pKa was determined as the pH that gave rise to half the maximum fluorescence intensity.

[0144] Table 2. pK of selected compounds of structure (I) a [Table 2]

[0145] Example 3 Measuring the efficacy of lipid nanoparticle formulations containing different cationic lipids using an in vivo luciferase mRNA expressing rodent model The cationic lipids shown in Table 3 have been previously tested with nucleic acids. For comparison purposes, these lipids were used to formulate lipid nanoparticles containing FLuc mRNA (L-6107) using the in-line mixing method described in Example 1 and PCT / US10 / 22614, which is incorporated herein by reference in its entirety. The lipid nanoparticles were formulated with the following molar ratio: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid ("PEG-DMG", i.e., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol, average PEG molecular weight 2000). In an alternative embodiment, the cationic lipid, DSPC, cholesterol, and PEG lipid were formulated at a molar ratio of about 47.5:10:40.8:1.7. Relative activity was determined by measuring luciferase expression in the liver 4 hours after administration by 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 dosing as described in Example 1.

[0146] Table 3. Comparative lipids showing activity on mRNA [Table 3]

[0147] Representative compounds of the disclosure shown in Table 4 were formulated with the following molar ratios: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid ("PEG-DMA", 2-[2-(ω-methoxy(polyethylene glycol) 2000)ethoxy]-N,N-ditetradecylacetamide), or 47.5% cationic lipid / 10% DSPC / 40.8% cholesterol / 1.7% PEG lipid. Relative activity was determined by measuring luciferase expression in liver 4 hours after administration by tail vein injection as described in Example 1. Activity was compared at a dose of 0.5 mg mRNA / kg and expressed as ng luciferase / g liver measured 4 hours after administration as described in Example 1. Compound numbers in Table 4 refer to compound numbers in Table 1.

[0148] Table 4. Novel cationic lipids and their activities [Table 4]

[0149] Example 4 Synthesis of Compound I-1 [ka] A solution of di-(2-hexyldecanyl)amine (1.4 g) in dichloromethane (20 mL) was treated with triethylamine (1 mL) and a solution of 5-bromopentanoyl chloride (2 g) in dichloromethane (20 mL). The reaction was stirred for 1 h and then washed with dilute aqueous hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent removed. The residue was dissolved in a 2 M solution of dimethylamine in tetrahydrofuran (30 mL) and stirred overnight. The solvent was mostly removed and the residue was partitioned between hexane and dilute aqueous hydrochloric acid. The organic phase was washed with water, dried over anhydrous magnesium sulfate, filtered, and the solvent removed. The residue was passed through a silica gel column using a gradient of 2% acetic acid / dichloromethane followed by 2-12% methanol / dichloromethane to give compound I-1 (0.95 g) as a colorless oil.

[0150] Example 5 Synthesis of Compound I-2 [ka] A solution of di-(2-hexyldecanyl)amine (1.5 g) in dichloromethane (10 mL) was treated with triethylamine (20 drops) and a solution of 6-bromohexanoyl chloride (1 g) in dichloromethane (10 mL). The reaction was stirred for 30 minutes and then the solvent was removed in vacuo. The residue was dissolved in dichloromethane, filtered through a pad of silica gel, and the solvent was removed. The residue was dissolved in a 2M solution of dimethylamine in tetrahydrofuran (30 mL) and stirred overnight. The solvent was mostly removed and the residue was partitioned between dichloromethane and aqueous sodium bicarbonate. The organic phase was washed with water, dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a gradient of 0-8% methanol / dichloromethane to give compound I-2 (0.26 g) as a colorless oil.

[0151] Example 6 Synthesis of compound I-3 [ka] A solution of di-(2-hexyldecanyl)amine (1.25 g) in dichloromethane (20 mL) was treated with triethylamine (5 mL) and 5-bromopentanoyl chloride (1 g). The reaction was stirred for 10 minutes, filtered, and the solvent removed. The residue was dissolved in dichloromethane and washed with dilute aqueous hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent removed. The residue was treated with a solution of diethanolamine (4.9 g) in tetrahydrofuran (20 mL) and heated to 45° C. overnight. The reaction mixture was washed twice with water, and the solvent removed. The residue was passed through a silica gel column using a gradient of 0-12% methanol / dichloromethane to give compound I-3 (0.32 g) as a colorless oil.

[0152] Example 7 Synthesis of compound I-4 [ka] A solution of di-(2-hexyldecanyl)amine (1.25 g) in dichloromethane (20 mL) was treated with triethylamine (5 mL) and 5-bromopentanoyl chloride (1 g). The reaction was stirred for 10 min, filtered, and the solvent removed. The residue was dissolved in dichloromethane and washed with dilute aqueous hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent removed. The residue was treated with isopropylamine (20 mL) and stirred overnight. The solvent was removed and the residue was washed with hexane and aqueous sodium bicarbonate. The solvent was removed and the residue was passed through a silica gel column using a gradient of 0-8% methanol / dichloromethane to give compound I-4 (0.67 g) as a colorless oil.

[0153] Example 8 Synthesis of compound I-5 [ka] A solution of di-(2-hexyldecanyl)amine (0.53 g) in dichloromethane (10 mL) was treated with 3-bromopropanoyl chloride (1 g) and N-ethyldiisopropylamine (0.5 mL) for 30 min. The solvent was removed and the residue was dissolved in 2 M dimethylamine in THF (25 mL). The reaction was stirred overnight and then the solvent was removed in vacuo. The residue was partitioned between hexane and aqueous sodium bicarbonate. The solvent was removed from the organic fraction and the residue was passed through a silica gel column using a gradient of 2% acetic acid / dichloromethane followed by 2-8% methanol / dichloromethane. The purified fraction was washed with hexane and aqueous sodium bicarbonate. The solvent was removed to give compound I-5 (0.22 g) as a colorless oil.

[0154] Example 9 Synthesis of compound I-6 [ka] A solution of di-(2-hexyldecanyl)amine (1.61 g) in dichloromethane (10 mL) was treated with 9-bromononanoyl chloride (1 g) and N-ethyldiisopropylamine (1 mL) overnight. The solvent was removed and the residue was dissolved in 2 M dimethylamine in THF (35 mL). The reaction was stirred overnight and then the solvent was removed in vacuo. The residue was partitioned between hexane and aqueous sodium bicarbonate. The solvent was removed from the organic fraction and the residue was passed through a silica gel column using a gradient of 2% acetic acid / dichloromethane followed by 2-12% methanol / dichloromethane. The purified fraction was washed with hexane and aqueous sodium bicarbonate. The solvent was removed to give compound I-6 (0.85 g) as a colorless oil.

[0155] Example 10 Synthesis of compound I-7 [ka] A solution of di-(2-hexyldecanyl)amine (1.25 g) in dichloromethane (10 mL) was treated with 8-bromooctanoyl chloride (0.52 g) and triethylamine (1 mL) overnight. The solvent was removed and the residue was partitioned three times between hexane and water. The solvent was removed and the residue was dissolved in 2M dimethylamine in THF (20 mL). The reaction was stirred overnight, filtered, and the solvent removed in vacuo. The residue was passed through a silica gel column using a gradient of 0-6% methanol / dichloromethane. The purified fractions were washed with hexane and aqueous sodium bicarbonate. The solvent was removed to give compound I-7 (0.65 g) as a colorless oil.

[0156] Example 11 Synthesis of 2-hexyldecanoylamide A solution of 2-hexyldecanoic acid (26 g) in benzene (30 mL) was treated with oxalyl chloride (15 mL). The reaction was stirred until gas evolution ceased, then the solvent was removed in vacuo and the residue was dried under vacuum for 4 h. The crude 2-hexyldecanoyl chloride was dissolved in dichloromethane (100 mL) and added slowly to a stirred solution of concentrated ammonium hydroxide (150 mL). The reaction mixture was allowed to stand for 2 h and the aqueous supernatant was removed. The organic phase was washed twice with water in the same manner and filtered. The collected precipitate was dried to give crude 2-hexyldecanoylamide (22 g) as a white powder.

[0157] Example 12 Synthesis of 2-hexyldecanylamine A suspension of 2-hexyldecanoylamide (8.2 g) in dry tetrahydrofuran (40 mL) was treated with lithium aluminum hydride (1.1 g, added slowly). The reaction was stirred for 2 hours and then excess methanol was added slowly. Dichloromethane (150 mL) was added followed by water (2 mL). The reaction mixture was filtered and the solvent removed from the filtrate to give crude 2-hexyldecanylamine (4.7 g).

[0158] Example 13 Synthesis of N-(2'-hexyldecanyl)-2-hexyldecanoylamide A solution of 2-hexyldecanylamine (4.7 g) in dichloromethane (100 mL) was treated with 2-hexyldecanoyl chloride (5.5 g, dissolved in 50 mL dichloromethane) followed by triethylamine (4 mL). The reaction was stirred for 1 hour and then washed with dilute hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered and the solvent removed. The residue (~10 g) was combined with the crude product from the second reaction (~7 g) and passed through silica gel (100 g) with a gradient of 0-10% methanol / dichloromethane to give N-(2'-hexyldecanyl)-2-hexyldecanoylamide (14.4 g).

[0159] Example 14 Synthesis of di-(2-hexyldecanyl)amine A solution of N-(2'-hexyldecanyl)-2-hexyldecanoylamide (14.4 g) in dry tetrahydrofuran (100 mL) was treated with lithium aluminum hydride (2 g) and refluxed overnight. Excess methanol was added slowly to destroy excess reducing agent. Dichloromethane (200 mL) was added followed by water (2 mL). The mixture was then filtered and the solvent removed. The residue was suspended in hexane, filtered and the solvent removed. The residue was passed through a silica gel (100 g) column using a gradient of 2% acetic acid / dichloromethane followed by 2-16% methanol / dichloromethane. The purified fractions were washed with hexane and aqueous sodium bicarbonate. The solvent was removed to give di-(2-hexyldecanyl)amine (9.5 g) as a colorless oil.

[0160] Example 15 Synthesis of compound I-8 [ka] A solution of di-(2-hexyldecanyl)amine (1.61 g) in dichloromethane (10 mL) was treated with 9-bromononanoyl chloride (1 g) and N-ethyldiisopropylamine (1 mL) overnight. The solvent was removed and the residue was dissolved in 2 M dimethylamine in THF (35 mL). The reaction was stirred overnight and then the solvent was removed in vacuo. The residue was partitioned between hexane and aqueous sodium bicarbonate. The solvent was removed from the organic fraction and the residue was passed through a silica gel column using a gradient of 2% acetic acid / dichloromethane followed by 2-12% methanol / dichloromethane. The purified fraction was washed with hexane and aqueous sodium bicarbonate. The solvent was removed to give compound 1a (0.85 g) as a colorless oil. [ka] A solution of compound I-8a (0.50 g) in tetrahydrofuran (15 mL) was treated with lithium aluminum hydride (0.28 g, added slowly). The reaction was stirred for 4 h. Excess methanol was then added slowly, followed by dichloromethane (50 mL) and water (0.5 mL). The suspension was filtered and the solvent removed. The crude product was passed through a silica gel column using a gradient of 2% acetic acid / dichloromethane, then 2-12% methanol / dichloromethane. The purified fraction was partitioned between hexane and aqueous sodium bicarbonate. The solvent was removed to give compound I-8 (0.46 g) as a colorless oil.

[0161] Example 16 Synthesis of compound I-9 [ka] A solution of di-(2-hexyldecanyl)amine (1.25 g) in dichloromethane (10 mL) was treated with 8-bromooctanoyl chloride (0.52 g) and triethylamine (1 mL) overnight. The solvent was removed and the residue was partitioned three times between hexane and water. The solvent was removed and the residue was dissolved in 2 M dimethylamine in THF (20 mL). The reaction was stirred overnight, filtered, and the solvent removed in vacuo. The residue was passed through a silica gel column using a gradient of 0-6% methanol / dichloromethane. The purified fractions were washed with hexane and aqueous sodium bicarbonate. The solvent was removed to give compound I-9a (0.65 g) as a colorless oil. [ka] A solution of compound I-9a (0.55 g) in tetrahydrofuran (20 mL) was treated with lithium aluminum hydride (0.5 g) for 2 h. Methanol was added to quench excess reducing agent. Dichloromethane (50 mL) and water (1 mL) were added and the suspension was filtered. The solvent was removed and the residue was passed through a silica gel column with a gradient of 0-8% methanol / dichloromethane to give compound I-9 (0.27 g) as a colorless oil.

[0162] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications (including both U.S. Provisional Patent Applications 62 / 747,557 and 62 / 747,521, filed October 18, 2018) referenced herein and / or listed in the Application Data Sheet, if any, are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to use concepts from the various patents, applications, and publications to provide further embodiments. These and other changes 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 claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. The claims are therefore not limited by this disclosure.

Claims

1. The following structure (I): 【Chemistry 1】 [In the formula, G １ is -N(R ３ ) R ４ or -OR ５ and R １ is an optionally substituted branched saturated or unsaturated C １２ -C ３６ is alkyl; R ２ When L is -C(=O)-, optionally substituted branched or unbranched, saturated or unsaturated C １２ -C ３６ alkyl; or R ２ , L is C ６ -C １２ Alkylene, C ６ -C １２ Alkenylene, or C ２ -C ６ When it is alkynylene, it is an optionally substituted branched or unbranched, saturated or unsaturated C ４ -C ３６ is alkyl; R ３ and R ４ are each independently H or an optionally substituted branched or unbranched, saturated or unsaturated C １ -C ６ alkyl; or R ３ and R ４ , L is C ６ -C １２ Alkylene, C ６ -C １２ Alkenylene, or C ２ -C ６ When alkynylene, each independently is optionally substituted branched or unbranched, saturated or unsaturated C １ -C ６ alkyl; or R ３ and R ４ together with the nitrogen to which they are attached form a heterocyclyl; R ５ is H or optionally substituted C １ -C ６ is alkyl; L is -C(=O)-, C ６ -C １２ Alkylene, C ６ -C １２ Alkenylene, or C ２ -C ６ alkynylene; and n is an integer from 1 to 12. or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof.

2. G １ But -N(R ３ ) R ４ or -OR ５ and R １ is an optionally substituted branched saturated or unsaturated C １２ -C ３６ is alkyl; R ２ is an optionally substituted branched or unbranched, saturated or unsaturated C １２ -C ３６ is alkyl; R ３ and R ４ are each independently H or optionally substituted branched or unbranched, saturated or unsaturated C １ -C ６ alkyl; or R ３ and R ４ together with the nitrogen to which they are attached form a heterocyclyl; R ５ is H or optionally substituted C １ -C ６ is alkyl; L is -C(=O)-; and n is an integer from 1 to 12; 2. A compound according to claim 1, or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof.

3. G １ But -N(R ３ ) R ４ or -OR ５ and R １ is an optionally substituted branched saturated or unsaturated C １２ -C ３６ is alkyl; R ２ is an optionally substituted branched or unbranched, saturated or unsaturated C ４ -C ３６ is alkyl; R ３ and R ４ each independently represents an optionally substituted branched or unbranched, saturated or unsaturated C １ -C ６ alkyl; or R ３ and R ４ together with the nitrogen to which they are attached form a heterocyclyl; R ５ is H or optionally substituted C １ -C ６ is alkyl; L is C ６ -C １２ Alkylene Linker, C ６ -C １２ Alkenylene linker, or C ２ -C ６ is an alkynylene linker; and n is an integer from 1 to 12; 2. The compound of claim 1, or a pharma- ceutically acceptable salt or stereoisomer thereof.

4. The following structure (IA): 【Chemistry 2】 [In the formula, R ８ and R ９ are each independently H or an optionally substituted branched or unbranched, saturated or unsaturated C ２ -C １２ alkyl; ８ and R ９ is R １ is an optionally substituted branched saturated or unsaturated C １２ -C ３６ are each independently selected to be alkyl; and R １０ and R １１ are each independently H or an optionally substituted branched or unbranched, saturated or unsaturated C ２ -C １２ alkyl; with the proviso that when L is -C(=O)-, then R ２ is an optionally substituted branched or unbranched, saturated or unsaturated C １２ -C ３６ alkyl; and L is C ６ -C １２ Alkylene, C ６ -C １２ Alkenylene, or C ２ -C ６ When R is alkynylene, ２ is an optionally substituted branched or unbranched, saturated or unsaturated C ４ -C ３６ R is an alkyl group. １０ and R １１ are each independently selected. The compound according to any one of claims 1 to 3, which is represented by:

5. R ８ and R ９ each independently represents an optionally substituted branched or unbranched, saturated or unsaturated C ２ -C １２ The compound of claim 4, which is alkyl.

6. R ８ is optionally substituted: C ２ Alkyl, C ４ Alkyl, C ６ Alkyl, C ８ Alkyl, or C １０ 6. The compound of claim 4 or 5, which is alkyl.

7. R ８ is optionally substituted: C ４ Alkyl, C ６ Alkyl, or C ８ The compound according to any one of claims 4 to 6, which is alkyl.

8. R ９ is optionally substituted: C ４ Alkyl, C ６ Alkyl, C ８ Alkyl, C １０ Alkyl, or C １２ The compound according to any one of claims 4 to 7, which is alkyl.

9. R ９ is optionally substituted: C ６ Alkyl, C ８ Alkyl, or C １０ The compound according to any one of claims 4 to 8, which is alkyl.

10. R １０ and R １１ each independently represents an optionally substituted branched or unbranched, saturated or unsaturated C ２ -C １２ The compound according to any one of claims 4 to 9, which is alkyl.

11. R １０ is optionally substituted: C ２ Alkyl, C ４ Alkyl, C ６ Alkyl, C ８ Alkyl, or C １０ The compound according to any one of claims 4 to 10, which is alkyl.

12. R １０ is optionally substituted: C ４ Alkyl, C ６ Alkyl, or C ８ The compound according to any one of claims 4 to 11, which is alkyl.

13. R １１ is optionally substituted unbranched, C ２ Alkyl, C ６ Alkyl, or C １０ The compound according to any one of claims 3 to 12, which is alkyl.

14. R １１ is optionally substituted C ２ The compound of claim 13, which is alkyl.

15. R １１ is optionally substituted C ６ The compound of claim 13, which is alkyl.

16. R １１ is optionally substituted C １０ The compound of claim 13, which is alkyl.

17. R １１ is optionally substituted: C ４ Alkyl, C ６ Alkyl, C ８ Alkyl, C １０ Alkyl, or C １２ The compound according to any one of claims 4 to 12, which is alkyl.

18. R １１ is optionally substituted: C ６ Alkyl, C ８ Alkyl, or C １０ The compound according to any one of claims 4 to 12, which is alkyl.

19. R ８ , R ９ , R １０ , and R １１ each independently represents an optionally substituted branched or unbranched, saturated or unsaturated C ６ -C １０ The compound of claim 4, which is alkyl.

20. R ８ , R ９ , R １０ , and R １１ each independently represents an optionally substituted branched or unbranched, saturated or unsaturated C ８ -C １２ The compound of claim 4, which is alkyl.

21. R ８ , R ９ , R １０ , and R １１ each independently being a branched or unbranched, saturated or unsaturated C ２ -C ６ The compound of claim 4, which is alkyl.

22. R ８ , R ９ , R １０ , and R １１ each independently represents an optionally substituted branched or unbranched, saturated or unsaturated C ６ -C １２ The compound of claim 4, which is alkyl.

23. R １ and R ２ each independently represents an optionally substituted branched saturated or unsaturated C １２ -C ３０ The compound of claim 1 , wherein the aryl group is an alkyl group.

24. R １ and R ２ each independently represents an optionally substituted branched saturated or unsaturated C １２ -C ２０ The compound of claim 1 , wherein the aryl group is an alkyl group.

25. R １ and R ２ each independently represents an optionally substituted branched saturated or unsaturated C １５ -C ２０ The compound of claim 1 , wherein the aryl group is an alkyl group.

26. R １ and R ２ The compound according to any one of claims 1 or 23-25, wherein each is saturated.

27. R １ and R ２ The compound of any one of claims 1 or 23-25, wherein at least one of is unsaturated.

28. R １ and R ２ The compound of any one of claims 1 to 27, wherein both are unsubstituted.

29. R １ and R ２ respectively having the following structure: 【Chemistry 3】 2. The compound of claim 1 ,

30. R ２ and R ２ respectively having the following structure: 【Chemistry 4】 2. The compound of claim 1, wherein

31. R ２ has the following structure: 【Chemistry 5】 30. The compound according to any one of claims 1 or 3 to 29, which is one of:

32. R ２ has the following structure: 【Chemistry 6】 30. The compound according to any one of claims 1 or 3 to 29, which is one of:

33. R １ has the following structure: 【Chemistry 7】 33. The compound of claim 31 or 32, wherein

34. L is C ６ -C １２ The compound of any one of claims 1 or 3 to 33, which is alkylene.

35. L is C ６ 35. The compound of claim 34 which is an alkylene.

36. L is C ７ 35. The compound of claim 34 which is an alkylene.

37. L is C ８ 35. The compound of claim 34 which is an alkylene.

38. L is C ９ 35. The compound of claim 34 which is an alkylene.

39. L is C １０ 35. The compound of claim 34 which is an alkylene.

40. L is C １１ 35. The compound of claim 34 which is an alkylene.

41. G １ But -N(R ３ ) R ４ The compound according to any one of claims 1 to 40,

42. G １ But -NH ２ , -NHCH ３ , or -N(CH ３ ) ２ 42. The compound of claim 41, wherein

43. R ３ and R ４ 42. The compound of claim 41, wherein together with the nitrogen to which they are attached form a heterocyclyl.

44. G １ has the following structure: 【Chemistry 8】 44. The compound of claim 43, wherein

45. G １ has the following structure: 【Chemistry 9】 42. The compound of claim 41, which is:

46. 46. ​​The compound of any one of claims 1 to 45, wherein n is 1, 2, 3, 4, 5, or 6.

47. The compound according to any one of claims 1 to 45, wherein n is 7, 8, 9, 10, 11, or 12.

48. The structure: 【Chemistry 10】 2. The compound of claim 1, wherein

49. The structure: 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 2. The compound of claim 1, wherein

50. A composition comprising a compound according to any one of claims 1 to 49 and a therapeutic agent.

51. A lipid nanoparticle comprising a compound according to any one of claims 1 to 49 and a therapeutic agent.

52. 52. The composition or lipid nanoparticle of claim 50 or 51, further comprising one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids.

53. 53. The composition or lipid nanoparticle of claim 52, wherein the composition comprises one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

54. 54. The composition or lipid nanoparticle of claim 53, wherein the neutral lipid is DSPC.

55. 55. The composition or lipid nanoparticle of any one of claims 50 to 54, wherein the molar ratio of the compound to neutral lipid ranges from about 2:1 to about 8:

1.

56. The composition or lipid nanoparticle according to any one of claims 50 to 55, wherein the steroid is cholesterol.

57. 57. The composition or lipid nanoparticle of claim 56, wherein the molar ratio of the compound to cholesterol ranges from 5:1 to 1:

1.

58. The composition or lipid nanoparticle of any one of claims 50 to 57, wherein the polymer-bound lipid is a pegylated lipid.

59. 59. The composition or lipid nanoparticle of claim 58, wherein the molar ratio of the compound to the pegylated lipid ranges from about 100:1 to about 20:

1.

60. 60. The composition or lipid nanoparticle of any one of claims 58 or 59, wherein the pegylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG dialkyloxypropyl carbamate.

61. The pegylated lipid has the following structure (II): 【Chemistry 14】 [In the formula, R １２ and R １３ are each independently a branched or unbranched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain may optionally be interrupted by one or more ester linkages; and The average value of w is in the range of 30 to 60.

60. The composition or lipid nanoparticle of claim 58 or 59, wherein the PEGylated lipid is represented by the formula: or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof.

62. R １２ and R １３ The composition or lipid nanoparticle of claim 61, wherein each is independently an unbranched saturated alkyl chain containing from 12 to 16 carbon atoms.

63. 63. The composition or lipid nanoparticle of any one of claims 61 or 62, wherein the average value of w is about 49.

64. The composition or lipid nanoparticle of any one of claims 50 to 63, wherein the therapeutic agent comprises a nucleic acid.

65. 65. The composition or lipid nanoparticle of claim 64, wherein the nucleic acid is selected from antisense RNA and messenger RNA (mRNA).

66. A method of administering a therapeutic agent to a patient in need thereof, comprising manufacturing or preparing a composition or lipid nanoparticle according to any one of claims 50 to 65, and administering the composition to the patient.

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