Lipids for use in lipid nanoparticle formulations

Abstract

The following structure (I): JPEG2025530171000075.jpg3958 [wherein, R 1a , R 1b , R 2 , R 3 , L 1a , L 1b , n1 and X are as defined herein. or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. Use of the compound as a component of a lipid nanoparticle formulation for delivery of a therapeutic agent, compositions comprising the compound, and methods of use and manufacture thereof are also provided.

Description

[Technical Field]

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

[0002] background 2. Description of Related Art There are many challenges associated with delivering nucleic acids to elicit desired responses in biological systems. Nucleic acid-based therapy holds great promise, but to realize this potential, more effective delivery of nucleic acids to appropriate sites within cells or organisms is still needed. Therapeutic nucleic acids include, for example, messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immunostimulatory nucleic acids, antagomirs, antimirs, mimetics, supermirs, and aptamers. Partial nucleic acids, such as mRNA or plasmids, can be used to induce the expression of specific cellular products, useful, for example, for treating diseases associated with protein or enzyme deficiencies. The therapeutic applications of translatable nucleotide delivery are extremely broad, as constructs can be synthesized to produce any selected protein sequence, whether native or not to the system. The expression product of a nucleic acid can enhance existing levels of a protein, replace a missing or non-functional version of a protein, or introduce a new protein and associated functionality into a cell or organism.

[0003] Some nucleic acids, such as miRNA inhibitors, can be used to induce the expression of specific cellular components controlled by miRNAs, which is useful for treating diseases associated with protein or enzyme deficiencies, for example. The therapeutic application of miRNA inhibition is extremely broad, as constructs can be synthesized to inhibit one or more miRNAs and thereby control the expression of mRNA products. Inhibition of endogenous miRNAs enhances the expression of their downstream target endogenous proteins and restores proper function in cells or organisms as a means of treating diseases associated with a specific miRNA or group of miRNAs.

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

[0005] However, two challenges currently face the use of oligonucleotides in therapeutic settings. First, free RNA is susceptible to nuclease digestion in plasma. Second, free RNA has limited ability to gain access to intracellular compartments where the appropriate translation machinery resides. Lipid nanoparticles formed from cationic lipids and other lipid components, such as neutral lipids, cholesterol, PEG, pegylated lipids, and oligonucleotides, have been used to block RNA degradation in plasma and promote cellular uptake of oligonucleotides.

[0006] There is still a need to improve cationic lipids and lipid nanoparticles for oligonucleotide delivery.Preferably, these lipid nanoparticles provide optimal drug-to-lipid ratio, protect nucleic acid from degradation and clearance in serum, be suitable for systemic delivery, and provide intracellular delivery of nucleic acid.In addition, these lipid-nucleic acid particles are well tolerated and provide an appropriate therapeutic index, so that patient treatment with effective doses of nucleic acid is not associated with unacceptable toxicity and / or risk to patients.The present invention provides these and related advantages. [Means for solving the problem]

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

[0008] In one embodiment, the compound has the following structure (I): [ka] [In the formula, R 1a , R 1b , R 2 , R 3 , L 1a , L 1b , n1 and X are as defined herein. or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0009] Also provided is a pharmaceutical composition comprising one or more 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-conjugated lipids.Such compositions are useful for forming lipid nanoparticles for delivery of therapeutic agents.

[0010] In another embodiment, the present invention provides a method for administering a therapeutic agent to a patient in need thereof, comprising preparing a lipid nanoparticle composition comprising a compound of structure (I) and a therapeutic agent, and delivering the composition to the patient. Such a method is useful for inducing protein expression in a subject, for example, to express an antigen for vaccination purposes or a gene editing protein.

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

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

[0013] The present invention is based, in part, on the discovery of novel cationic (amino) lipids that provide advantages when used in lipid nanoparticles for the in vivo delivery of active or therapeutic agents, such as nucleic acids, to mammalian cells. In particular, embodiments of the present invention provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel cationic lipids described herein, which provide a significantly increased therapeutic index, increased activity of the nucleic acid in vivo, and improved tolerability of the compositions compared to previously described nucleic acid-lipid nanoparticle compositions.

[0014] In certain embodiments, the present invention provides novel cationic lipids that enable the formulation of improved compositions for in vitro and in vivo delivery of mRNA and / or other oligonucleotides. In some embodiments, these improved lipid nanoparticle compositions are useful for the expression of proteins encoded by mRNA. In other embodiments, these improved lipid nanoparticle compositions are useful for upregulating endogenous protein expression by delivering miRNA inhibitors that target a specific miRNA or a group of miRNAs that control a target mRNA or several mRNAs. In other embodiments, these improved lipid nanoparticle compositions are useful for downregulating (e.g., silencing) the protein and / or mRNA levels of target genes. In some other embodiments, lipid nanoparticles are also useful for delivering mRNA and plasmids for transgene expression. In still other embodiments, lipid nanoparticle compositions are useful for pharmacological effects resulting from protein expression, such as increasing red blood cell production through delivery of appropriate erythropoietin mRNA or inducing protection against infection through delivery of appropriate antibody-encoding mRNA.

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

[0016] As described herein, lipid nanoparticle embodiments of the present invention are particularly useful for the 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, polyvalent RNA, Dicer substrate RNA, complementary DNA (cDNA), etc. Thus, the lipid nanoparticles and compositions of the present invention can be used to induce expression of a desired protein both in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more of the novel cationic lipids described herein, wherein the lipid nanoparticles encapsulate or are associated with a nucleic acid (e.g., a messenger RNA or a plasmid encoding the desired protein) that is expressed to produce the desired protein. Alternatively, the lipid nanoparticles and compositions of the present invention 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, wherein the lipid nanoparticles encapsulate or are bound to a nucleic acid that reduces target gene expression (e.g., an antisense oligonucleotide or small interfering RNA (siRNA)). The lipid nanoparticles and compositions of the present invention can also be used to co-deliver various nucleic acids (e.g., mRNA and plasmid DNA), either separately or in combination, which may be useful for providing effects requiring the co-localization of various nucleic acids (e.g., mRNA encoding an appropriate gene-modifying enzyme and a DNA segment for integration into the host genome).

[0017] Nucleic acids used in this disclosure can be produced according to any available technology. For mRNA, the primary method of production is, but is not limited to, enzymatic synthesis (also known as in vitro transcription), which currently represents the most efficient method for producing long sequence-specific mRNA. In vitro transcription describes the template-directed synthesis of RNA molecules from an engineered DNA template consisting of an upstream bacteriophage promoter sequence (e.g., including but not limited to those derived from T7, T3, and SP6 coliphages) linked to a downstream sequence encoding a gene of interest. Template DNA can be prepared for in vitro transcription from several sources by 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] RNA transcription occurs in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs), under conditions that support polymerase activity while minimizing the potential degradation of the resulting mRNA transcript. In vitro transcription can be carried out using a variety of commercially available kits and commercially available reagents, including, but not limited to, RiboMax Large Scale RNA Production System (Promega), MegaScript Transcription Kit (Life Technologies), including RNA polymerase and rNTPs. Methods for in vitro transcription of mRNA are well known in the art. (See, 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 unwanted 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 procedures include phenol / chloroform extraction or precipitation with any alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Further non-limiting examples of purification procedures that can be used include size exclusion chromatography (Lukavsky, PJ and Puglisi, JD, 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v.10, 889-893), silica-based affinity chromatography, and polyacrylamide gel electrophoresis (Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA synthesis Methods v. 941 Conn GL (ed), New York, NY Humana Press, 2012). Purification can be performed using a variety of commercially available kits, including, but not limited to, the SV Total Isolation System (Promega) and the In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0020] Furthermore, while reverse transcription can produce large amounts of mRNA, the product may contain several aberrant RNA impurities associated with unwanted polymerase activity that may need to be removed from full-length mRNA preparations. These include short RNAs resulting from unsuccessful transcription initiation and double-stranded RNA (dsRNA) produced by RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates, and self-complementary 3' extension. It has been shown that these contaminants, which contain dsRNA structures, can result in unwanted immunostimulatory activity through interactions with various innate immune sensors in eukaryotic cells that recognize specific nucleic acid structures and function to induce potent immune responses. This, in turn, can dramatically reduce mRNA translation because protein synthesis is reduced during the innate cellular immune response. Therefore, additional techniques for removing these dsRNA contaminants have been developed and are known in the art, including scalable HPLC purification (see, e.g., Kariko, K., Muramatsu, H., Ludwig, J. And Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v. 39 e142; Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K., HPLC Purification of in vitro transcribed long RNA in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013). HPLC-purified mRNA has been reported to be translated at much greater levels, particularly in primary cells and in vivo.

[0021] A wide variety of modifications have been described in the literature that can be used to alter specific properties of in vitro transcribed mRNA and improve its utility. These include, but are not limited to, modifications of the 5' and 3' ends of the mRNA. Endogenous eukaryotic mRNAs typically contain a cap structure at the 5' end of the mature molecule, which plays an important role in mediating the binding of mRNA cap-binding protein (CBP), which subsequently enhances mRNA stability and the efficiency of mRNA translation in the cell. Therefore, the highest levels of protein expression are 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. Further modifications include methylation of the 2'-hydroxyl groups of the 5'-most and penultimate nucleotides.

[0022] Several different cap structures can be used to cap the 5' of in vitro transcribed synthetic mRNA. 5'-capping of synthetic mRNA can be performed co-transcriptionally with a chemical cap analog (i.e., capping during in vitro transcription). For example, the anti-reverse cap analog (ARCA) cap contains a 5'-5'-triphosphate guanine-guanine linkage, where one guanine contains an N7 methyl group and a 3'-O-methyl group. However, up to 20% of transcripts remain uncapped during this co-transcription process, and synthetic cap analogs are not identical to the 5' cap structure of authentic cellular mRNAs, potentially reducing translatability and cellular stability. Alternatively, synthetic mRNA molecules can also be enzymatically capped post-transcriptionally. These can produce more authentic 5' cap structures that structurally or functionally more closely mimic the endogenous 5' cap, resulting in enhanced binding of cap-binding proteins, increased half-life, reduced susceptibility to 5' endonucleases, and / or reduced 5' cap removal. 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 (polyA tail) is normally added to mRNA molecules during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved, freeing a 3' hydroxyl, at which polyA polymerase adds a chain of adenine nucleotides to the RNA in a process called polyadenylation. Poly(A) tails have been shown to widely enhance both the translation efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly(A), poly(A) binding protein and the regulation of mRNA stability, Trends Bio Sci, v. 14, 373-377; Guhaniyogi, J. and Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M. and Regnier, P., 2002, The poly(A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, v. 111, 611-613).

[0024] Poly(A) tailing of in vitro transcribed mRNA can be achieved using a variety of approaches, including, but not limited to, cloning a poly(T) tract into a DNA template or post-transcriptional addition using poly(A) polymerase. The first example allows for the in vitro transcription of mRNA with a poly(A) tail of defined length depending on the size of the poly(T) tract, but requires further manipulation of the template. The latter example involves enzymatic addition of a poly(A) tail to in vitro transcribed mRNA using poly(A) polymerase, which catalyzes the incorporation of an adenine residue at the 3' end of the RNA, but does not require further manipulation of the DNA template, resulting 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 and reagents, various ARC caps, poly(A) polymerases, etc., including, but not limited to, the Poly(A) Polymerase Tailing kit (EpiCenter), mMESSAGE mMACHINE T7 Ultra kit, and Poly(A) Tailing kit (Life Technologies).

[0025] In addition to 5' cap and 3' polyadenylation, other modifications of in vitro transcripts have been reported to provide benefits in terms of translation efficiency and stability.It is well known in the art that pathogenic DNA and RNA can be recognized by a variety of sensors in eukaryotes and induce a strong natural immune response.The ability to distinguish between pathogenic and self-DNA and RNA is based at least in part on structure and nucleoside modification, because most nucleic acids from natural sources contain modified nucleosides.In contrast, in vitro synthesized RNA lacks these modifications, and therefore becomes immunostimulatory, which can inhibit effective mRNA translation as outlined above.Introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thus reducing this unwanted immunostimulatory activity and enhancing translational capacity (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, F.A., Ludwig, J., Kato, H., Akira, S., Weissman, (See, e.g., U.S. Patent Publication No. 2012 / 0251618.) Modified nucleosides and nucleotides used in the synthesis of modified RNAs can be prepared, monitored, and utilized using general methods and procedures known in the art. A wide variety of nucleoside modifications are available that can be incorporated into in vitro transcribed mRNA to some degree, either alone or in combination with other modified nucleosides (see, e.g., U.S. Patent Publication No. 2012 / 0251618).In vitro synthesis of nucleoside-modified mRNA has been reported to have a reduced ability to activate immune sensors and a concomitant increase in translational potency.

[0026] Other components of mRNA that can be modified to benefit translatability and stability include the 5' and 3' untranslated regions (UTRs). Optimization of both or independently of the UTRs (favorable 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 (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 invention.For oligonucleotides, production methods include, but are not limited to, chemical synthesis and enzymes, chemical cleavage of long precursors, in vitro transcription, etc.The synthesis method of DNA and RNA nucleotides is widely used and well known in the art (see, for example, Gait, MJ (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, DC: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, NJ) Totowa, NJ: Humana Press, 2005; all are incorporated herein by reference).

[0028] For plasmid DNA, preparation for use in the present invention generally utilizes, but is not limited to, in vitro amplification and isolation of the plasmid DNA in liquid cultures of bacteria containing the plasmid of interest. The presence in the plasmid of interest of a gene encoding resistance to a particular antibiotic (penicillin, kanamycin, etc.) allows bacteria containing the plasmid of interest to be selectively grown in antibiotic-containing cultures. 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 US6197553B1). Plasmid isolation can be performed using a variety of commercially available kits and reagents, including, but not limited to, Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo) and PureYield MaxiPrep (Promega) kits.

[0029] Various exemplary embodiments of the cationic lipids, lipid nanoparticles and compositions comprising same of the present invention and their use for delivery of active or therapeutic agents, such as nucleic acids for modulating gene and protein expression, are detailed below.

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

[0031] Unless the context requires otherwise, throughout this specification and the claims, the terms "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] The reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrase "one embodiment" or "in an embodiment" in various places throughout the 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 invention belongs. As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0034] The term "inducing the expression of a desired protein" refers to the ability of a nucleic acid to increase the expression of a desired protein.To test the level of protein expression, a test sample (e.g., a sample of cultured cells expressing the desired protein) or a test mammal (e.g., a mammal, such as an animal model, such as a human or rodent (e.g., mouse) or non-human primate (e.g., monkey) model) is contacted with a nucleic acid (e.g., a nucleic acid combined with a lipid of the present invention).The expression of the desired protein in the test sample or test animal is compared with the expression of the desired protein in a control sample (e.g., a sample of cultured cells expressing the desired protein) or a control mammal (e.g., a mammal, such as an animal model, such as a human or rodent (e.g., mouse) or non-human primate (e.g., monkey) model) that has not been contacted with 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, induction of expression of a desired protein is achieved when the ratio of the desired protein expression in a test sample or test mammal to the desired protein expression level in a control sample or control mammal is greater than 1, for example, about 1.1, 1.5, 2.0, 5.0, or 10.0. Induction of expression of a desired protein is achieved when any measurable level of the desired protein is detected in a test sample or test mammal when the desired protein is not present in a control sample or control mammal. Those skilled in the art will understand 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 produce light or luminescence under appropriate conditions.

[0035] The term "inhibit the expression of target gene" refers to the ability of nucleic acid to silence, reduce or inhibit the expression of target gene.To test the degree of gene silencing, contact a test sample (for example, a sample of cultured cells that express target gene) or a test mammal (for example, a mammal, such as an animal model, such as human or rodent (for example, mouse) or non-human primate (for example, monkey) model) with the nucleic acid that silences, reduces or inhibits the expression of target gene.The expression of target gene in test sample or test animal is compared with the expression of target gene in a control sample (for example, a sample of cultured cells that express target gene) or a control mammal (for example, a mammal, such as an animal model, such as human or rodent (for example, mouse) or non-human primate (for example, monkey) model) that has not been contacted or administered with the nucleic acid.The expression of target gene in control sample or control mammal can be assigned a value of 100%. In certain embodiments, silencing, inhibition or reduction of expression of a target gene is achieved when the expression level of the target gene in a test sample or test mammal relative to the expression level of the target gene in a control sample or control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0%. In other words, the nucleic acid can silence, reduce or inhibit the expression of the target gene in test sample or test mammal by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the expression level of the target gene in control sample or control mammal that is not contacted or administered with the nucleic acid.Suitable assays for determining the expression level of target gene include, but are not limited to, testing protein or mRNA levels using techniques known to those skilled in the art, such as dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those skilled 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, e.g., increased or inhibited expression of a target sequence compared to the normal expression level detected in the absence of the nucleic acid. Increased expression of a target sequence is achieved when any measurable level of expression product not present in the absence of the nucleic acid is detected. When the expression product is present at a certain level prior to contact with the nucleic acid, increased expression is achieved when the fold increase obtained with the nucleic acid, such as mRNA, relative to a control is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000, or more. Inhibition of expression of a target gene or target sequence is achieved when the value obtained with a nucleic acid, such as a nucleic acid, such as an antisense oligonucleotide, relative to a control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expression of a target gene or target sequence include, for example, 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 appropriate reporter proteins, 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 single- or double-stranded form, including DNA, RNA, and hybrids thereof. DNA can be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA can be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, polyvalent RNA, Dicer substrate RNA, or viral RNA (vRNA), and combinations thereof. Nucleic acids include synthetic, naturally occurring, and non-naturally occurring nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that have similar binding properties to the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences, as well as the sequence explicitly indicated. In particular, degenerate codon substitutions can be achieved by substituting a sequence in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" comprises the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked to each other via phosphate groups."Base" includes purines and pyrimidines, which further includes the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine and natural analogues of purines and pyrimidines as well as synthetic derivatives, including modifications that add new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0038] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that comprises a partial or complete coding sequence necessary for the production of a polypeptide or 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 "lipid" refers to a group of organic compounds, including, but not limited to, esters of fatty acids, generally characterized by being sparingly soluble in water but soluble in many organic solvents. They are usually divided into at least three classes: (1) "simple lipids," which include fats and oils and waxes; (2) "compound lipids," which include phospholipids and glycolipids; and (3) "derivatized lipids," such as steroids.

[0041] "Steroids" are compounds with the following carbon skeleton: [ka] It is a compound comprising:

[0042] Non-limiting examples of steroids include cholesterol, and the like.

[0043] "Cationic lipid" refers to a lipid that can be positively charged. An example of a cationic lipid comprises one or more amine groups with a positive charge. Preferred cationic lipids are ionizable so that they can exist in a 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 plasma protein absorption, blood clearance and tissue distribution (Semple, SC, et al., Adv. Drug Deliv Rev 32:3-17 (1998)), as well as the ability to form endosomolytic non-bilayer structures, which are important for intracellular delivery of nucleic acids (Hafez, IM, et al., Gene Ther. 8:1188-1196 (2001)).

[0044] The term "lipid nanoparticle" refers to a particle having at least one dimension in the nanometer order (e.g., 1 to 1,000 nm) comprising one or more compounds of formula (I) or other specific cationic lipids. In some embodiments, the lipid nanoparticles are included in formulations that can be used to deliver active or therapeutic agents, such as nucleic acids (e.g., mRNA), to a desired target site (e.g., a cell, tissue, organ, tumor, etc.). In some embodiments, the lipid nanoparticles of the present invention comprise nucleic acids. Such lipid nanoparticles typically comprise a compound of formula (I) and one or more additives selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. In some embodiments, the 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 induced by the host organism's or cell's machinery, such as adverse immune responses.

[0045] 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 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In certain embodiments, nucleic acid is resistant to nuclease degradation in aqueous solution when present in lipid nanoparticles.The lipid nanoparticles containing nucleic acid and their manufacturing methods are disclosed in, for example, US Patent Publication No. 2004 / 0142025, 2007 / 0042031 and PCT Publication No. WO2013 / 016058 and WO2013 / 086373, the complete disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0046] As used herein, "lipid-encapsulated" refers to lipid nanoparticles that provide an active or therapeutic agent, such as a nucleic acid (e.g., mRNA), fully encapsulated, partially encapsulated, or both. In some embodiments, the nucleic acid (e.g., mRNA) is fully encapsulated in the lipid nanoparticle.

[0047] The term "polymer-conjugated lipid" refers to a molecule that contains both a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that contains both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxypolyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), etc.

[0048] The term "neutral lipid" refers to any of several lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphotidylcholines such as 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), sphingomyelin (SM), ceramides, steroids such as sterols, and derivatives thereof. Neutral lipids may be synthetic or naturally occurring.

[0049] The term "charged lipid" refers to any of several lipid species that exist in positively or negatively charged form independent of pH within a useful physiological range, e.g., from about pH 3 to about pH 9. Charged lipids can be synthetic or naturally occurring. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethanecarbamoylsterol (e.g., DC-Chol).

[0050] As used herein, the term "aqueous solution" refers to a composition that contains water.

[0051] "Serum stable" 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, DNAe assays, or RNAe assays.

[0052] As used herein, "systemic delivery" refers to the delivery of a therapeutic product that can result in widespread exposure of the active agent within an organism. Some administration techniques can result in systemic delivery of certain drugs, while others do not. Systemic delivery means that a useful, preferably therapeutic, amount of the drug is exposed to most 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.

[0053] As used herein, "local delivery" refers to the delivery of an active agent directly to a target site within an organism. For example, an agent can be delivered locally by direct injection into a disease site such as a tumor, another target site such as an inflammation site, or a target organ such as the liver, heart, pancreas, or kidney. Local delivery can also include topical application or localized injection techniques, such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not exclude systemic pharmacological effects.

[0054] "Alkyl" refers to a saturated (i.e., no double and / or triple bonds) alkyl group containing 1 to 24 carbon atoms (C1-C 24 alkyl), 1 to 12 carbon atoms (C1-C 12 "C-C alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms having from 1 to 8 carbon atoms (C-C alkyl), or from 1 to 6 carbon atoms (C-C alkyl) and attached to the rest of the molecule by single bonds, e.g., methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, etc. Unless otherwise specified in this specification, alkyl groups are optionally substituted.

[0055] "Cycloalkyl" or "carbocyclic ring" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical, consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from 3 to 15 carbon atoms, preferably from 3 to 10 carbon atoms, and which is saturated or unsaturated and attached to the rest of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethylbicyclo[2.2.1]heptanyl, and the like. Unless otherwise specified in the specification, cycloalkyl groups are optionally substituted.

[0056] "Alkylene" or "alkylene chain" refers to a straight or branched divalent saturated hydrocarbon chain, consisting solely of carbon and hydrogen, that connects the rest of the molecule to a radical group. In some embodiments, the alkylene chain is 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), 4 to 6 carbon atoms (C4-C6 alkylene), 2 to 4 carbon atoms (C2-C4 alkylene), 1 to 2 carbon atoms (C1-C2 alkylene), for example, methylene, ethylene, propylene, n-butylene, etc. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can begin on one carbon or any two carbons within the chain. Unless stated otherwise in the specification, alkylene chains are optionally substituted.

[0057] "Alkylene oxide" refers to an alkylene group, as defined herein, in which at least one carbon-carbon bond has been replaced with a carbon-oxygen-carbon bond. Examples of alkylene oxides include ethylene oxide, methylene oxide, propylene oxide, and the like. Multiple repeats of alkylene oxide groups are included in the definition of alkylene oxide. For example, polyethylene oxide and ethylene oxide having fewer repeat units, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeat ethylene oxide units, are included within alkylene oxide. Unless otherwise specified herein, alkylene oxides are optionally substituted.

[0058] "Aryl" refers to a carbocyclic ring system radical containing hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring. For purposes of this disclosure, an aryl group is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems. Aryl groups include, but are not limited to, aryl groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene.

[0059] "Arylalkyl" refers to a group of the formula -R b -R c (where R b is alkylene or alkenylene as defined above, and R c is one or more aryl groups as defined above), e.g., benzyl, diphenylmethyl, etc. Unless stated otherwise in the specification, an arylalkyl group is optionally substituted.

[0060] As used herein, the term "substituted" means that at least one hydrogen atom has been replaced with a halogen atom, such as F, Cl, Br, and I; an oxo group (=O); a hydroxyl group (-OH); an alkoxy group (-OR a , where R a is C1-C 12alkyl or cycloalkyl); carboxyl group (-OC(=O)R a or -C(=O)OR a , where R a is H, C1-C 12 alkyl or cycloalkyl); an amine group (—NR a R b , where R a and R b are each independently H, C1-C 12 alkyl or cycloalkyl); C1-C 12 and cycloalkyl groups. 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. In another embodiment, the substituent is a carboxyl group. In another embodiment, the substituent is an amine group.

[0061] "Optionally" or "optionally" (e.g., optionally substituted) means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not. 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 with no substitution. In some embodiments, "optionally substituted" refers to the fact that the particular radical is halo (e.g., F, Cl, Br, and I), oxo (=O), hydroxyl (-OH), alkoxy (-OR), or the like. a , where R a is C1-C 12 alkyl), cycloalkoxy (-OR a , where R ais C3-C8 cycloalkyl), carboxyl (-OC(=O)R a or -C(=O)OR a , where R a is H, C1-C 12 alkyl or C3-C8 cycloalkyl), amine (-NR a R b , where R a and R b are each independently H, C1-C 12 alkyl or C3-C8 cycloalkyl), C1-C 12 It means substituted with one or more substituents selected from the group consisting of alkyl and C3-C8 cycloalkyl.

[0062] In some embodiments, "optionally substituted" means substitution with one or more halo substituents. In some embodiments, "optionally substituted" means substitution with one or more oxo substituents. In some embodiments, "optionally substituted" means substitution with one or more hydroxyl substituents. In certain embodiments, "optionally substituted" means substitution with one or more alkoxy substituents. In some embodiments, "optionally substituted" means substitution with one or more cycloalkoxy substituents. In certain embodiments, "optionally substituted" means substitution with one or more carboxy substituents. In some embodiments, "optionally substituted" means substitution with one or more amine substituents. In certain embodiments, "optionally substituted" means substitution with one or more C1-C 12 In some embodiments, "optionally substituted" refers to substitution with one or more C3-C8 cycloalkyl substituents.

[0063] If a functional group is described as "optionally substituted," and then a substituent of the functional group is also "optionally substituted," etc., for purposes of this disclosure, such repetitions are limited to 5 times, preferably 2 times. In some embodiments, such repetitions are limited to 1 time. In some embodiments, such repetitions are limited to 0 times.

[0064] The present invention is also meant to encompass all pharmaceutically acceptable compounds of structure (I) that are isotopically labeled by having one or more atoms replaced with an atom having a different atomic mass or mass number. Examples of isotopes that may be incorporated into compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, e.g., 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 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), for example, those incorporating a radioisotope, are useful in drug and / or substrate tissue distribution studies. The radioisotope tritium, i.e., 3 H and carbon-14, i.e., 14 C is particularly useful for this purpose in view of its ease of incorporation and means of immediate detection.

[0065] Deuterium, i.e., 2Substitution with heavier isotopes, such as 3H, may offer certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and may be preferable in some situations.

[0066] 11 C. 18 F, 15 O and 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 be prepared by conventional techniques generally known to those skilled in the art or by methods analogous to those described in the Preparations and Examples below, substituting the appropriate isotopically labeled reagents for the unlabeled reagents previously employed.

[0067] The present invention is also meant to encompass the in vivo metabolic products of the compounds of the invention. Such products may result, for example, from the oxidation, reduction, hydrolysis, amidation, esterification, etc., of the administered compound, primarily by enzymatic processes. Accordingly, the present invention includes compounds produced by a process comprising administering a compound of this invention to a mammal for a period of time sufficient to produce a metabolic product thereof. Such products are typically identified by administering a detectable dose of a radiolabeled compound of the invention to an animal, such as a rat, mouse, guinea pig, monkey, or the like, or to a human, allowing sufficient time for metabolism to occur, and isolating the conversion product from urine, blood, or other biological sample.

[0068] "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.

[0069] "Mammal" includes both domestic animals such as humans and laboratory animals and pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits) and non-domestic animals such as wildlife.

[0070] A "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, additive, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the U.S. Food and Drug Administration for use in humans or domestic animals.

[0071] "Pharmaceutically acceptable salts" include both acid and base addition salts.

[0072] "Pharmaceutically acceptable acid addition" refers to an acid that retains the biological effectiveness and properties of the free base and is not biologically or otherwise undesirable, and includes inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, as well as 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, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, ... acetic acid, 2,2-dichloroacetic acid, adipic acid, 2,2-dichloroacetic acid, adipic acid, 2,2-dichloroacetic acid, adipic acid, 2,2- and salts formed with organic acids such as, but not limited to, 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.

[0073] "Pharmaceutically acceptable base addition salts" refer to salts that retain the biological effectiveness and properties of the free acids and are 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 ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of substituted amines, including primary, secondary, and tertiary amines, naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dianol, 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, and polyamine resins. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0074] Crystallization often produces solvates of the compounds of the present invention. As used herein, the term "solvate" refers to an aggregate containing one or more molecules of the compound of the present invention and one or more solvent molecules. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present invention may exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., as well as corresponding solvated forms. The compounds of the present invention may be true solvates, while in other instances, the compounds of the present invention may simply retain extraneous water or may be a mixture of water and an extraneous solvent.

[0075] A "pharmaceutical composition" is a formulation of a compound of the present invention and a vehicle generally accepted in the art for the delivery of a biologically active compound to a mammal, e.g., a human. Such a vehicle includes any pharmaceutically acceptable carrier, diluent, or additive.

[0076] "Effective amount" or "therapeutically effective amount" refers to the amount of the compound of the present invention that, when administered to a mammal, preferably a human, is sufficient to treat the mammal, preferably a human. The amount of lipid nanoparticles of the present invention that constitutes a "therapeutically effective amount" varies depending on the compound, the condition and its severity, the method of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art in light of their knowledge and the present disclosure.

[0077] 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: (i) preventing a disease or condition from occurring in a mammal, particularly when such a mammal is predisposed to the condition but has not yet been diagnosed as having it; (ii) arresting the disease or condition, i.e., halting its progression; (iii) alleviating the disease or condition, i.e., causing the disease or condition to regress; or (iv) Relieving symptoms caused by a disease or condition, i.e., relieving pain without addressing the underlying disease or condition As used herein, the terms "disease" and "condition" may be used interchangeably or may refer to a particular malady or condition that may not have a known causative factor (and thus the etiology has not yet been determined) and is therefore not yet recognized as a disease, but rather only as an undesirable state or syndrome in which a more or less specific set of symptoms has been identified by clinicians.

[0078] The compounds of the present invention, or pharmaceutically acceptable salts thereof, may contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms, which may be defined in terms of absolute stereochemistry as (R)- or (S)-, or for amino acids, (D)- or (L)-. The present invention is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers can be prepared using chiral synthons or chiral reagents or resolved using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from appropriate optically pure precursors or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high-performance liquid chromatography (HPLC). If the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless otherwise specified, the compounds are intended to include both E and Z geometric isomers. Likewise, all tautomeric forms are intended to be included.

[0079] "Stereoisomers" refer to compounds that consist of the same atoms bonded by the same bonds, but that differ in three-dimensional structure and are not interchangeable. The present invention contemplates various stereoisomers and mixtures thereof, and includes "enantiomers," which refer to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0080] "Tautomer" refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present invention includes tautomers of any such compounds.

[0081] compound In some embodiments, the present invention provides novel lipid compounds that can be combined with other lipid components, such as neutral lipids, charged lipids, steroids, and / or polymer-conjugated lipids, to form lipid nanoparticles with oligonucleotides. Without wishing to be bound by theory, it is believed that these lipid nanoparticles shield the oligonucleotides from degradation in serum and provide effective delivery of oligonucleotides to cells in vitro and in vivo.

[0082] One embodiment has the following structure (I): [ka] [During the ceremony, L 1a and L 1b each independently represents an optionally substituted C-C 12 is alkyl; R 1a is -C(=O)OR 4a or -O(C=O)R 4a and; R 1b is -C(=O)OR 4b or -O(C=O)R 4b and; R 2 Ha-NR 6 (C=O)R 5 , -(C=O)N(R 6 )R 5 or -(C=O)OR 7 and; R 3 and R 6 are each independently hydrogen or optionally substituted C1-C6 alkyl; R 4a , R 4b and R 5 are each independently an optionally substituted alkyl; R 7 is an optionally substituted arylalkyl; n1 is 2, 3, 4, 5, or 6; and X is C2-C6 alkylene or C4-C 20It is an alkylene oxide. or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0083] One embodiment has the following structure (I): [ka] [During the ceremony, L 1a and L 1b each independently represents an optionally substituted C-C 12 is alkyl; R 1a is -C(=O)OR 4a or -O(C=O)R 4a and; R 1b is -C(=O)OR 4b or -O(C=O)R 4b and; R 2 Ha-NR 6 (C=O)R 5 , -(C=O)N(R 6 )R 5 or -(C=O)OR 7 and; R 3 and R 6 are each independently hydrogen or an optionally substituted C-C 12 is alkyl; R 4a , R 4b and R 5 are each independently an optionally substituted alkyl; R 7 is an optionally substituted C1-C6 alkyl or an optionally substituted arylalkyl; n1 is 2, 3, 4, 5, or 6; and X is C2-C6 alkylene or C4-C 20 It is an alkylene oxide. or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0084] In some embodiments, X is: [ka] where: n2 is 2, 3, 4, 5 or 6; n3 is 0, 1, 2, 3 or 4; n4 is 2, 3, or 4; and n5 is 2, 3, 4 or 5.

[0085] In some embodiments, L 1a is C5-C9 alkyl. In certain embodiments, L 1b is C5-C9 alkyl. In some embodiments, L 1a is C5-, C6-, C7- or C9-alkyl. In certain embodiments, L 1b is C5-, C6-, C7-, or C9-alkyl. In some embodiments, L 1a is C5-alkyl. In certain embodiments, L 1a is C6-alkyl. In some embodiments, L 1a is C7-alkyl. In certain embodiments, L 1a is C9-alkyl. In some embodiments, L 1b is C5-alkyl. In certain embodiments, L 1b is C6-alkyl. In some embodiments, L 1b is C7-alkyl. In certain embodiments, L 1b is C9-alkyl. In some embodiments, L 1a is unsubstituted. In certain embodiments, L 1b is unsubstituted. In some embodiments, L 1a is unbranched. In certain embodiments, L 1b is unbranched.

[0086] In some embodiments, R 1a One of them is -O(C=O)R 4aIn certain embodiments, R 1a One of them is -(C=O)OR 4a In some embodiments, R 1b is -O(C=O)R 4b In certain embodiments, R 1b One of them is -(C=O)OR 4b is.

[0087] In some embodiments, R 4a is C8-C 24 -alkyl. In certain embodiments, R 4a is C 10 -C 18 -alkyl. In certain embodiments, R 4a is C 11 -C 16 In some embodiments, R 4a is C 11 -alkyl. In certain embodiments, R 4a is C 15 In some embodiments, R 4a is C 16 -alkyl. In certain embodiments, R 4b is C8-C 24 In some embodiments, R 4b is C 10 -C 18 -alkyl. In certain embodiments, R 4b is C 11 -C 16 In some embodiments, R 4b is C 11 -alkyl. In certain embodiments, R 4b is C 15 In some embodiments, R 4b is C 16 - alkyl.

[0088] In certain embodiments, R 4a is branched. In some embodiments, R 4bis branched. In certain embodiments, R 4a is unsubstituted. In some embodiments, R 4b is unsubstituted. In certain embodiments, R 4a has the following structure: [ka] It has one of the following.

[0089] In some embodiments, R 4b has the following structure: [ka] It has one of the following.

[0090] In some embodiments, R 2 Ha-NR 6 (C=O)R 5 In certain embodiments, R 2 is -(C=O)N(R 6 )R 5 In some embodiments, R 5 is C2-C 16 -alkyl. In certain embodiments, R 5 is C4-C 13 In some embodiments, R 5 C4-, C7-, C8-, C 10 - or C 13 -alkyl. In certain embodiments, R 5 is unsubstituted. In some embodiments, R 5 is substituted with hydroxyl. In some embodiments, R 5 is branched. In certain embodiments, R 5 is unbranched. In some embodiments, R 5 has the following structure: [ka] It has one of the following.

[0091] In certain embodiments, R 5 is unbranched. In some embodiments, R 5 has the following structure: [ka] It has one of the following.

[0092] In some embodiments, R 6 is C1-C6 alkyl. In some embodiments, R 6 is C1-C 10 In certain embodiments, R 6 is C1-C4-alkyl. In some embodiments, R 6 is C1-, C2-, C3-, C6-, C8- or C 10 In certain embodiments, R is methyl, ethyl, n-butyl, n-hexyl, n-octyl, or n-decyl. 6 is unbranched. In certain embodiments, R 6 is methyl or n-butyl. In some embodiments, R 6 is unsubstituted. In some embodiments, R 6 is substituted. In some embodiments, R 6 is C1-C6 alkyl substituted with one or more hydroxyl. In some embodiments, R 6 is C2-, C3-, C4-, or C6-alkyl substituted with one or more hydroxyl. In certain embodiments, R 6 is hydrogen. In some embodiments, R 2 -(C=O)OR 7 is.

[0093] In some embodiments, R 7 is C1-C3 alkyl or C7-C 16 In certain embodiments, R 7 is C7-C 16 In some embodiments, R7 is C1-C3 alkyl. In some embodiments, R 7 is non-substituted.

[0094] In certain embodiments, R 7 is -CH3 or the following structure: [ka] It has.

[0095] In certain embodiments, R 7 has the following structure: [ka] It has.

[0096] In some embodiments, R 3 is an optionally substituted C1-C6 alkyl. In certain embodiments, R 3 is optionally substituted methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl. 3 is optionally substituted methyl. In some embodiments, R 3 is C1-C6 alkyl substituted with one or more hydroxyl. In some embodiments, R 3 is a C2- or C4-alkyl substituted with one or more hydroxyl. In certain embodiments, R 3 is unsubstituted. In some embodiments, R 3 is hydrogen.

[0097] In some embodiments, X is [ka] For example, in certain embodiments, the compound has the following structure (II): [ka] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. For example, in some of these embodiments, n2 is 3, 4, or 5.

[0098] In other embodiments, X is [ka] In some such embodiments, the compound has the following structure (III): [ka] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. In other of these embodiments, n3 is 0 or 1. In other embodiments, n4 is 2 or 3. In certain other different embodiments, n5 is 3.

[0099] In some embodiments, n1 is 3, 4, or 5. In certain embodiments, n1 is 2.

[0100] In some embodiments, the compound has one of the structures shown in Table 1 below, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13]

[0101] It is understood that any embodiment of a compound of structure (I) above, and any particular substituent and / or variable of a compound of structure (I) above, may be combined independently with other embodiments and / or substituents and / or variables of a compound of structure (I) to form embodiments not specifically disclosed above. Furthermore, when a list of substituents and / or variables is recited in a particular embodiment and / or claim for a particular R group, L group, or variable n1-n2, it is understood that each individual substituent and / or variable may be excluded from the particular embodiment and / or claim, and the remaining listing of substituents and / or variables is considered to be within the scope of embodiments of the present disclosure.

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

[0103] For the purpose of administration, the compounds of the present invention (typically in the form of lipid nanoparticles combined with therapeutic agents) can be administered as raw chemicals or can be formulated as pharmaceutical compositions.The pharmaceutical compositions of the present invention comprise a compound of structure (I) and one or more pharmaceutically acceptable carriers, diluents or additives.The compound of structure (I) is present in the composition in an amount effective to form lipid nanoparticles and deliver therapeutic agents, for example, for the treatment of a specific disease or condition of interest.Appropriate concentrations and dosages can be easily determined by those skilled in the art.

[0104] Some embodiments provide compositions comprising a compound of structure (I) and a therapeutic agent. In some embodiments, the composition further comprises one or more additives selected from a neutral lipid, a steroid, and a polymer-conjugated lipid.

[0105] In some embodiments, the therapeutic agent comprises a nucleic acid. In certain embodiments, the nucleic acid is selected from antisense and messenger RNA.

[0106] In certain embodiments, the composition comprises one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC. In some embodiments, the molar ratio of the compound to the neutral lipid ranges from about 2:1 to about 8:1. In certain embodiments, the steroid is cholesterol. In some embodiments, the molar ratio of the compound to the cholesterol ranges from about 2:1 to 1:1. In certain embodiments, the polymer-conjugated lipid is a PEGylated lipid. In various embodiments, the polymer-conjugated lipid is a PEGylated lipid. For example, some embodiments include PEGylated diacylglycerols (PEG-DAGs), such as 1-(monomethoxypolyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerols (PEG-S-DAGs), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), PEGylated ceramides (PEG-cer), or PE G dialkoxypropyl carbamates, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecaneoxy)propyl)carbamate or 2,3-di(tetradecaneoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In some embodiments, the molar ratio of compound to PEGylated lipid ranges from about 100:1 to about 10:1 or from about 100:1 to about 25:1. In some embodiments, the PEGylated lipid is PEG-DMG. In certain embodiments, the PEGylated lipid has the following structure (II): [ka] [During the ceremony, R 8 and R 9 are each independently an unbranched or branched alkyl, alkenyl, or alkynyl containing from 10 to 30 carbon atoms, wherein the alkyl, alkenyl, or alkynyl is optionally interrupted by one or more ester linkages; and z has an average value in the range of 30 to 60. or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0107] In some embodiments, R 8 and R 9 are each independently an unbranched alkyl chain having 12 to 16 carbon atoms. In some embodiments, the average z is about 45 (e.g., 43, 44, 45, 46, or 47). In some embodiments, the average z is about 43 to 47. In some embodiments, the average z is about 40 to 50.

[0108] The synthesis of pegylated lipids can be found in US Pat. No. 9,738,593, the disclosure of which is incorporated herein by reference.

[0109] Administration of the compositions of the present invention can be carried out via any of the accepted methods of administering pharmaceuticals to provide similar benefits. The pharmaceutical compositions of the present invention can be formulated into solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. The pharmaceutical compositions of the present invention are formulated so that the active ingredient contained therein is bioavailable after administration of the composition to a patient. The composition administered to a subject or patient may be in the form of one or more dosage units; for example, a tablet may be a single dosage unit, and a container of the compound of the present invention in aerosol form may hold multiple dosage units. Actual methods for preparing such dosage forms will be known or apparent to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will, in any event, contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treatment of the disease or condition of interest, in accordance with the teachings of this disclosure.

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

[0111] When intended for oral administration, the pharmaceutical compositions are preferably in solid or liquid form, with semi-solid, semi-liquid, suspension and gel forms being included within the forms contemplated herein as solid or liquid.

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

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

[0114] The pharmaceutical composition can be in the form of a liquid, such as an elixir, syrup, solution, emulsion, or suspension. The liquid is for oral administration or delivery by injection, as two examples. When intended for oral administration, a preferred composition contains, in addition to the compound, one or more sweeteners, preservatives, dyes / coloring agents, and flavor enhancers. In a composition intended for administration by injection, one or more surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, and isotonic agents can be included.

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

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

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

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

[0119] The pharmaceutical composition of the present invention can contain various materials that modify the physical form of the solid or liquid dosage unit.For example, the composition can contain a material that forms a coating shell around the active ingredient.The material that forms the coating shell is typically 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.

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

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

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

[0123] The compositions of the present invention, or pharmaceutically acceptable salts thereof, 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 length of action of the therapeutic agent; the age, weight, general health, sex, and dietary habits of the patient; the method and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject being treated.

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

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

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

[0127] Those skilled in the art will also recognize that such protected derivatives of the compounds of the invention may not themselves have pharmacological activity, but may be administered to a mammal and then metabolized in the body to form a compound of the invention that is pharmacologically active. Such derivatives are therefore referred to as "prodrugs." All prodrugs of the compounds of the invention are included within the scope of the invention.

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

[0129] The following reaction scheme illustrates the synthesis of compounds of the present invention, i.e., structure (I): [ka] [In the formula, R 1a , R 1b , R 2 , R 3 , L 1a , L 1b , n1 and n2 are as defined herein. The present invention describes a method for preparing compounds of formula (I). It is understood that one skilled in the art can prepare these compounds by similar methods or combinations of other methods known to those skilled in the art. It is also understood that one skilled in the art can prepare other compounds of structure (I) not specified below by methods similar to those described below, using appropriate starting components and modifying the synthetic parameters as necessary. 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, e.g., Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)), or can be prepared as disclosed herein.

[0130] General Reaction Scheme 1 [ka] Compounds of structure (I) of certain embodiments can be prepared according to General Reaction Scheme 1 ("Method A"). 1a , R 1b , R 2 , R 3 , L 1a , L 1b and X is as defined herein.

[0131] Referring to General Reaction Scheme 1, compound starting material A1 can be purchased from commercial sources or prepared according to methods well known to those skilled in the art. The reaction of A1 with compound A2 under suitable basic conditions (e.g., potassium carbonate and cesium carbonate) was heated to 70°C to provide compound A3. As shown, A3 is then treated with dropwise addition of thionyl chloride in an ice bath to convert the hydroxyl group to the chlorine compound A4, which is then reacted with amine A5 using suitable conditions (e.g., heating) to provide a compound of structure (I).

[0132] It should be noted that various alternative strategies for preparing compounds of structure (I) are available to those skilled in the art. For example, other compounds of structure (I) can be prepared using appropriate starting materials and in analogous manner to those shown in the following examples. The use of protecting groups as needed and other modifications to the above general reaction schemes will be readily apparent to those skilled in the art. The following examples are provided for purposes of illustration and not limitation. [Example]

[0133] Example 1 In vivo assessment of luciferase mRNA using lipid nanoparticle compositions The lipids of structure (I), DSPC, cholesterol, and PEG-lipids are solubilized in ethanol at a molar ratio of 50:10:38.5:1.5 or 47.5:10:40.7:1.8. Lipid nanoparticles (LNPs) are prepared at a total lipid-to-mRNA weight ratio of approximately 10:1 to 40:1. Briefly, mRNA is diluted to 0.2 mg / mL in 50 mM citrate buffer, pH 4, or 10 to 25 mM acetate buffer, pH 4. Using a syringe pump, the ethanolic lipid solution and the aqueous mRNA solution are mixed at a vol / vol ratio of approximately 1:5 to 1:3 at a total flow rate of more than 15 mL / min. 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 size sterile filter.

[0134] Studies are conducted 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 established by the institution's Animal Welfare 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 sacrificed at specific time points (e.g., 4 hours) post-administration. Livers and spleens are collected in pre-weighed tubes, weighed, immediately snap-frozen in liquid nitrogen, and stored at -80°C until processed for analysis.

[0135] Approximately 50 mg of liver was cut into 2 mL FastPrep tubes (MP Biomedicals, Solon, OH) for analysis. A ¼-inch ceramic sphere (MP Biomedicals) was added to each tube, and 500 μL of room-temperature equilibrated Glo Lysis Buffer-GLB (Promega, Madison, WI) was added to the liver tissue. The liver tissue was homogenized for 15 seconds in a FastPrep24 instrument (MP Biomedicals) at 2 × 6.0 m / s. The homogenate was incubated for 5 minutes at room temperature, then diluted 1:4 with GLB and evaluated using the SteadyGlo Luciferase Assay System (Promega). Specifically, 50 μL of diluted tissue homogenate was reacted with 50 μL of SteadyGlo substrate, shaken for 10 seconds, followed by a 5-minute incubation, and then run through a CentroXS. 3 Quantitation is performed using an LB 960 luminometer (Berthold Technologies, Germany). The amount of assayed protein is determined using a BCA protein assay kit (Pierce, Rockford, IL). Relative luminescence units (RLU) are then normalized to the total μg protein assayed. To convert RLU to ng, a luciferase standard curve is generated with QuantiLum Recombinant Luciferase (Promega).

[0136] Trilink Biotechnologies' FLuc mRNA (L-6107) expresses the luciferase protein originally isolated from the firefly (Photinus pyralis). FLuc is commonly used in mammalian cell culture to measure both gene expression and cell viability. It emits bioluminescence in the presence of the substrate, luciferin. This capped and polyadenylated mRNA is fully substituted with 5-methylcytidine and pseudouridine.

[0137] Example 2 Immunoglobulin G (IgG) mRNA in vivo assessment using lipid nanoparticle compositions The lipids of structure (I), DSPC, cholesterol, and PEG-lipids are solubilized in ethanol at a molar ratio of 50:10:38.5:1.5 or 47.5:10:40.7:1.8. Lipid nanoparticles (LNPs) are prepared at a total lipid-to-mRNA weight ratio of approximately 10:1 to 40:1. Briefly, mRNA is diluted to 0.2 mg / mL in 50 mM citrate buffer, pH 4, or 10 to 25 mM acetate buffer, pH 4. Using a syringe pump, the ethanolic lipid solution and the aqueous mRNA solution are mixed at a vol / vol ratio of approximately 1:5 to 1:3 at a total flow rate of more than 15 mL / min. 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 size sterile filter.

[0138] The study was conducted in 6-8 week-old CD-1 / ICR mice (Envigo) in accordance with guidelines established by the institution's Animal Welfare Committee (ACC) and the Canadian Council on Animal Care (CCAC). Various doses of mRNA-lipid nanoparticles were administered systemically via tail vein injection, and animals were sacrificed at specific time points (e.g., 24 hours) after administration. Whole blood was collected, and serum was subsequently separated by centrifugation of the whole blood tubes at 2000 x g for 10 minutes at 4°C. The serum was then stored at -80°C until use in analysis.

[0139] For immunoglobulin G (IgG) ELISA (Life Diagnostics Human IgG ELISA Kit), serum samples were diluted 100-15,000 times with 1x diluent solution. 100 μL of diluted serum was distributed in duplicate along with human IgG standards onto an anti-human IgG-coated 96-well plate and incubated on a plate shaker at 150 rpm for 45 minutes at 25°C. The wells were washed five times with 1x wash solution using a plate washer (400 μL / well). 100 μL of HRP conjugate was added to each well and incubated on a plate shaker under the same conditions as above. The wells were again washed five times with 1x wash solution using a plate washer (400 μL / well). 100 μL of TMB reagent was added to each well and incubated on a plate shaker under the same conditions as above. The reaction was stopped by adding 100 μL of stop solution to each well. The absorbance is read at 450 nm (A450) in a microplate reader. The amount of human IgG in the mouse serum is determined by plotting the A450 values ​​of the assay standards against the human IgG concentration.

[0140] Example 3 pK of formulated lipids a Decision As described elsewhere, the pK of the formulated lipid a correlates with the efficacy of LNPs for delivery of nucleic acids (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010)). In some embodiments, the pK a The preferred range is about 5 to about 7. The pK of each lipid in the lipid nanoparticles was determined using an assay based on the fluorescence of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). aLipid nanoparticles containing compound of structure (I) / DSPC / cholesterol / PEG-lipid (50 / 10 / 38.5 / 1.5 or 47.5:10:40.7:1.8 mol%) in PBS at 0.4 mM total lipid concentration are fabricated using an in-line process as described in Example 1. TNS is prepared as a 100 μM stock solution in distilled water. Vesicles are diluted to 24 μM lipid with 2 mL of a buffered solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 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, and after vortex mixing, fluorescence intensity is measured at room temperature using an SLM Aminco Series 2 Luminescence Spectrophotometer using excitation and emission wavelengths of 321 nm and 445 nm. A sigmoidal best-fit analysis is applied to the fluorescence data to determine the pK a is measured as the pH that produces half-maximal fluorescence intensity.

[0141] Example 4 Determining the efficacy of lipid nanoparticle formulations containing different cationic lipids using an in vivo luciferase / IgG mRNA-expressing rodent model Representative compounds of the invention shown in Table 2 were formulated using the following molar ratio: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid 2-[2-(ω-methoxy(polyethylene glycol)] 2000)ethoxy]-N,N-didetradecylacetamide) or 47.5% cationic lipid / 10% DSPC / 40.7% cholesterol / 1.8% PEG lipid. Activity was determined by measuring luciferase expression in the liver 4 hours after administration by tail vein injection as described in Example 1 or by measuring the amount of human IgG in mouse serum as described in Example 2. Activity was compared at doses of 1.0 mg, 0.5 mg, or 0.3 mg mRNA / kg and expressed as ng luciferase / g liver measured 4 hours after administration as described in Example 1 or μg IgG / mL serum measured 24 hours after administration as described in Example 2. Compound numbers in Table 2 refer to compound numbers in Table 1.

[0142] [Table 2]

[0143] Example 5 Synthesis of bis(2-hexyldecyl) 6,6'-((2-((4-octanamidobutyl)amino)ethyl)azanediyl)dihexanoate (Compound I-2) [ka] [ka] Synthesis of compound 4-1 To an ice-salt cooled solution of octanoic acid (50 mmol, 7.21 g, 7.92 mL) in 200 mL of methanol under Ar, acetyl chloride (10 mL) was added slowly (approximately 15 min). Stir for 20 min, then remove the cooling bath. The solution was allowed to stand at room temperature overnight (20 h). Methanol was removed under reduced pressure. To the residue, saturated sodium bicarbonate (100 mL) and hexane (200 mL) were added. The hexane extract was washed with brine (70 mL), dried over sodium sulfate, filtered, and concentrated to give methyl octanoate as a colorless oil. The methyl octanoate was dried overnight (6.439 g, 40.7 mmol, 93%) on a high vacuum line (oil pump) and used in the next step without further purification.

[0144] To a refluxing solution of 1,4-butanediamine (5 equivalents, 100 mmol, 8.8 g) in 50 mL of methanol was slowly (15 min) added a solution of methyl octanoate (20 mmol, 3.16 g) in 20 mL of methanol. Refluxing was continued for 48 h. The solvent was then evaporated under reduced pressure, and the residue was dissolved in a mixture of water (70 mL) and ethyl acetate (100 mL). A small amount of citric acid was added. The two phases were separated, and the aqueous phase was extracted twice with ethyl acetate. The combined ethyl acetate extracts were washed with water and brine, dried over sodium sulfate, and filtered. Concentration gave a mixture of an oil and a white solid. The residue was dissolved in DCM and MeOH (19:1). The solution was passed through a silica gel pad (1.5 cm high x 6.5 cm wide) and washed with a mixture of MeOH (5%) and DCM until TLC showed the appearance of all diacylated products. The pad was then washed with a mixture of CHCl3 / EtOH / water / NH3 (30 / 25 / 3 / 2, 225 mL) to elute the desired product 4-1. The product-containing fractions were combined and concentrated to dryness (white solid, 2.52 g, 11.8 mmol, 59%).

[0145] Synthesis of compound 4-2 To a solution of 2-aminoethanol (333 mg, 5.46 mmol) in 35 mL of anhydrous THF, 2-hexyldecyl 6-bromohexanoate (1.9 equiv., 4.37 g, 10.4 mmol), potassium carbonate (1.9 equiv., 1.44 g, 10.4 mmol, MW 138.21), cesium carbonate (0.3 equiv., 534 mg, 1.64 mmol, MW 325.82), and sodium iodide (30 mg) were added, and the mixture was heated at 70 °C in a sealed pressure flask for 6 days. The solvent was evaporated under reduced pressure, and the residue was dissolved in a mixture of hexane and ethyl acetate (approximately 6%) and washed with water and brine. The organic layer was separated and dried over anhydrous sodium sulfate. The dried extract was filtered through a silica gel pad under reduced pressure. The pad was then washed with hexane-EtOAc-Et3N (95:5:0 to 80:20:1). Fractions containing the desired product were combined and concentrated to dryness to give a colorless oil (1.766 g, 2.39 mmol, 46%).

[0146] Synthesis of compound 4-3 To an ice-cooled solution of 4-2 (2.16 g, 2.93 mmol) in 8 mL of CHCl3, a solution of thionyl chloride (3 equiv., 8.79 mmol, 1.05 g) in 35 mL of chloroform was added dropwise (approximately 1-2 min) under an argon atmosphere. After the addition was complete, the ice bath was removed, and the reaction mixture was stirred at room temperature (20 °C) for 16 h. Concentration of the mixture afforded a thick, dark oil. The residue was dissolved in a mixture of chloroform and EtN (0.6 mL) and purified by flash column silica gel chromatography (0-1% MeOH in chloroform). This afforded the desired product as a brown oil (1.786 g, 2.36 mmol, 80%).

[0147] Synthesis of Compound I-2 A solution of 4-3 (1 equiv., 522 mg, 0.67 mmol), 4-1 (3 equiv., 2.07 mmol, 444 mg), and N,N-diisopropylethylamine (3 equiv., 2.07 mmol, 267 mg, 0.36 mL; MW 129.25, d 0.742) in acetonitrile (15 mL) was sealed and heated at 65 °C overnight. The reaction mixture was cooled and concentrated. The residue was dissolved in a mixture of ethyl acetate and hexane (80:20) and filtered through a short silica gel column (2.5 cm h × 3 cm w). The column was then eluted with the same solvent mixture. Fractions containing the less polar product were combined and concentrated (433 mg, slightly yellow oil, dialkylated product 4-4). The column was then eluted with a mixture of DCM-MeOH-NH3 (95:5:0.5). All fractions containing the desired product were combined and concentrated; the residue (oil / solid) was dissolved in a mixture of hexane and ethyl acetate (approximately 98:2) and filtered. The filtrate was concentrated to give a viscous brown oil (compound I-2). The yellow oil (444 mg) was further purified by flash-dry column silica gel chromatography (MeOH in chloroform, 1-5%). The fractions containing the desired product were combined and concentrated (288 mg, 0.23 mmol, yellow oil). 1H NMR (400 MHz, CDCl3) δ: 6.24-6.13 (m, 1H, NHCO), 3.96 (d, 5.8 Hz, 4H), 3.32-3.19 (m, 2H), 2.64-2.59 (m, 4H), 2.54-2.50 (m, 2H), 2.44-2.34 (m, 4H), 2.30 (t, 7.5 Hz, 4H), 2.18-2.09 (m, 2H), 1.67-1.50 (m, 13H), 1.47-1.38 (m, 4H), 1.38-1.08 (m, 60H), 0.90-0.86 (m, 15H). ESI-MS: C 58 H 115 N3O5[M+H] + MW calculated 934.9; measured 935.1.

[0148] Example 6 Synthesis of bis(2-hexyldecyl) 6,6'-((2-(methyl(4-octanamidobutyl)amino)ethyl)azanediyl)dihexanoate (Compound I-1) [ka] Synthesis of Compound I-1 To a solution of compound I-2 (223 mg, 0.23 mmol) in THF (5 mL) was added a formaldehyde solution (approximately 6 mmol, 500 mg of a 37 wt% aqueous solution) at RT. The resulting mixture was stirred for 30 min, after which sodium triacetoxyborohydride (5 equiv., 1.2 mmol, 243 mg) was introduced. After stirring overnight, the mixture was dissolved in a mixture of hexane and ethyl acetate (100 mL) and washed with saturated sodium bicarbonate solution. After drying over sodium sulfate, the solution was filtered under reduced pressure through a short column of silica gel (230-400 mesh grade silica gel, 2.5 cm h × 3 cm w). The column was then eluted with a mixture of hexane, ethyl acetate, and EtN (80:20:1, 200 mL). The column was then eluted with a mixture of DCM and MeOH (97:3, 100 mL). All fractions were combined and concentrated (220 mg, yellow oil). The crude product was further purified by flash dry column silica gel chromatography (MeOH in chloroform, 0-5%) to give the desired product as a colorless oil (165 mg, 0.16 mmol, 42%). 1 H NMR (400 MHz, CDCl3) δ: 6.18-6.10 (m, 1H, NHCO), 3.96 (d, 5.8 Hz, 4H), 3.29-3.18 (m, 2H), 2.55-2.50 (m, 2H), 2.44-2.38 (m, 6H), 2.37-2.33 (m, 2H), 2.30 (t, 7.5 Hz, 4H), 2.21 (s, 3H), 2.16-2.10 (m, 2H), 1.67-1.58 (m, 8H), 1.55-1.48 (m, 4H), 1.48-1.39 (m, 4H), 1.34-1.08 (m, 60H), 0.90-0.85 (m, 15H). ESI-MS: C 59 H 117 N3O5[M+H] + MW calculated 948.9; measured 949.1.

[0149] Example 7 Bis(2-hexyldecyl) 6,6'-((2-((6-(benzyloxy)-6-oxohexyl)(methyl)amino)ethyl)azanediyl)dihexanoate (Compound I-9) [ka] Synthesis of compound 6-1 A mixture of methylamine (5 equiv., 75 mmol, 37.5 mL, 2 M THF solution), benzyl 6-bromohexanoate (4.2 g, 14.7 mmol), NaI (40 mg), and N,N-diisopropylethylamine (2.6 mL) was sealed in a pressure flask and stirred at RT overnight. After concentration of the reaction mixture, the residue was purified by flash-dry column silica gel chromatography (DCM / MeOH / EtN, 99:1:0 to 80:20:1). This afforded the desired product as a slightly yellow oil (1.95 g, 8.3 mmol, 56%).

[0150] Synthesis of compound I-9 A solution of 4-3 (1.141 g, 1.51 mmol), 6-1 (3 equiv., 4.53 mmol, 1.066 g), N,N-diisopropylethylamine (3 equiv., 4.53 mmol, 584 mg, 0.788 mL), and sodium iodide (60 mg) in acetonitrile (20 mL) was sealed and heated at 76 °C (oil bath) for 24 h. The next day, the reaction mixture was cooled and concentrated. The crude product was purified by flash-dry column silica gel chromatography (230-400 mesh grade silica gel, hexane / EtOAc / EtN, 90:10:0 to 80:20:1). This afforded the desired product as a light yellow oil (1.20 g, 1.25 mmol, 83%). 1H NMR (400 MHz, CDCl3, 7.26 ppm) δ: 7.39-7.29 (m, 5H), 5.11 (s, 2H), 3.96 (d, 5.8 Hz, 4H), 2.54-2.49 (m, 2H), 2.43-2.27 (m, 14H), 2.20 (s, 3H), 1.70-1.58 (m, 8H), 1.51-1.38 (m, 6H), 1.36-1.18 (m, 54H), 0.90-0.85 (m, 12H).

[0151] Example 8 Synthesis of bis(2-hexyldecyl) 6,6'-((2-(methyl(4-tetradecanamidobutyl)amino)ethyl)azanediyl)dihexanoate (Compound I-3) [ka] Synthesis of Compound I-3 A solution of 4-3 (372 mg, 0.49 mmol), N-(4-(methylamino)butyl)tetradecanamide (343 mg, 1.1 mmol), N,N-diisopropylethylamine (5 equiv., 2.1 mmol, 271 mg, 0.37 mL), and sodium iodide (10 mg) in acetonitrile (10 mL) was sealed and heated at 76 °C (oil bath) for 24 h. The next day, the mixture was cooled and concentrated. The residue was dissolved in a mixture of hexane / EtOAc / EtN / MeOH (70:30:1:1) and filtered through a short column of silica gel (230-400 mesh grade silica gel). The column was then eluted with the same solvent mixture. All product-containing fractions were combined and concentrated. The product (418 mg) was further purified by flash-dry column silica gel chromatography (0-5%). This gave the desired product as a colorless oil (294 mg, 0.28 mmol, 58%). 1H NMR (400 MHz, CDCl3, 7.26 ppm) δ: 6.16-6.08 (m, 1H, NHCO), 3.96 (d, 5.8 Hz, 4H), 3.28-3.19 (m, 2H), 2.55-2.50 (m, 2H), 2.44-2.32 (m, 8H), 2.30 (t, 7.5 Hz, 4H), 2.21 (s, 3H), 2.17-2.09 (m, 2H), 1.67-1.58 (m, 8H), 1.55-1.37 (m, 8H), 1.36-1.08 (m, 72H), 0.90-0.86 (m, 15H). ESI-MS: C 65 H 129 N3O5[M+H] + MW calculated 1033.0; measured 1033.2.

[0152] Example 9 Synthesis of bis(2-hexyldecyl) 8,8'-((2-(methyl(3-nonanamidopropyl)amino)ethyl)azanediyl)dioctanoate (Compound I-4) [ka] Synthesis of compound 8-1 This intermediate was prepared in a manner analogous to the preparation of compound 4-3 in Example 5.

[0153] Synthesis of compound I-4 A solution of 8-1 (344 mg, 0.42 mmol), N-(3-(methylamino)propyl)nonanamide (3 equiv., 1.27 mmol, 290 mg), N,N-diisopropylethylamine (0.37 mL), and sodium iodide (10 mg) in acetonitrile (10 mL) was sealed and heated at 80 °C for 24 h. The next day, the mixture was cooled and concentrated. The residue was dissolved in a mixture of hexane / EtOAc / EtN (70:30:1) and filtered through a short column of silica gel (230-400 mesh grade silica gel). The column was then eluted with the same solvent mixture. All product-containing fractions were combined and concentrated to give 332 mg of a yellow oil. The product was further purified by flash-dry column silica gel chromatography (MeOH in chloroform, 0-5%, containing a trace of EtN). This gave the desired product as a slightly yellow oil (96 mg, 0.095 mmol, 23%). 1 H NMR (400 MHz, CDCl3, 7.26 ppm) δ: 7.11-7.05 (m, 1H, NHCO), 3.96 (d, 5.8 Hz, 4H), 3.37-3.30 (m, 2H), 2.55-2.49 (m, 2H), 2.48-2.36 (m, 8H), 2.29 (t, 7.5 Hz, 4H), 2.22 (s, 3H), 2.16-2.09 (m, 2H), 1.67-1.58 (m, 10H), 1.46-1.37 (m, 4H), 1.34-1.08 (m, 70H), 0.90-0.85 (m, 15H). ESI-MS: C 63 H 125 N3O5[M+H] + MW calculated 1005.0; measured 1005.2.

[0154] Example 10 Synthesis of bis(2-hexyldecyl) 8,8'-((2-(methyl(4-octanamidobutyl)amino)ethyl)azanediyl)dioctanoate (Compound I-5) [ka] Synthesis of compound 9-1 This intermediate was prepared in a manner analogous to the preparation of compound I-2 in Example 5.

[0155] Synthesis of compound I-5 To a solution of 9-1 (122 mg, 0.12 mmol) in THF (6 mL) was added formaldehyde (HCl) solution (460 mg of 37 wt% aqueous solution) at RT. The resulting mixture was stirred for 30 min, after which sodium triacetoxyborohydride (5 equiv., 0.6 mmol, 122 mg) was introduced. The resulting mixture was stirred at RT overnight. The mixture was dissolved in hexane and washed with saturated sodium bicarbonate solution. The organic phase was dried over Na2SO4 and concentrated to give a yellow oil, 122 mg. The oil was dissolved in a mixture of hexane / EtOAc / Et3N (70:30:1) and filtered through a short column of silica gel (230-400 mesh grade silica gel). The column was then eluted with the same solvent mixture. All product-containing fractions were combined and concentrated to give a yellow oil, 106 mg. The product was further purified by flash dry column silica gel chromatography (MeOH in chloroform containing a trace of EtN, 0-5%) to give the desired product as a colorless oil (43 mg). 1 H NMR (400 MHz, CDCl3, 7.26 ppm) δ: 6.21-6.14 (m, 1H, NHCO), 3.96 (d, 5.8 Hz, 4H), 3.27-3.20 (m, 2H), 2.55-2.50 (m, 2H), 2.45-2.33 (m, 8H), 2.29 (t, 7.5 Hz, 4H), 2.22 (s, 3H), 2.16-2.10 (m, 2H), 1.67-1.58 (m, 8H), 1.55-1.48 (m, 4H), 1.46-1.37 (m, 4H), 1.35-1.18 (m, 68H), 0.90-0.85 (m, 15H). ESI-MS: C 63 H 125 N3O5[M+H] + MW calculated 1005.0; measured 1005.2.

[0156] Example 11 Synthesis of bis(2-hexyldecyl) 6,6'-((2-((6-(decylamino)-6-oxohexyl)(methyl)amino)ethyl)azanediyl)dihexanoate (Compound I-7) [ka] Synthesis of compound 10-1 To a solution of compound I-9 (prepared according to the procedure of Example 10, 1.019 g, 1.07 mmol) in EtOH / EtOAc (1:10 mL) was added 10% Pd / C (25 mg), and the mixture was stirred under hydrogen. The reaction was monitored by TLC. Two additional portions of Pd / C (30 mg each) were added during the reaction. After 9 days, TLC indicated that the reaction was approximately 90% complete. The mixture was filtered through a pad of diatomaceous earth (e.g., Celite®), and the pad was washed with DCM. The filtrate was concentrated to give a yellow oil. The crude product was purified by flash-dry column silica gel chromatography (0–10% methanol in DCM). This afforded the desired product as a yellow oil (751 mg, 0.87 mmol, 81%).

[0157] Synthesis of compound I-7 To a solution of 10-1 (250 mg, 0.29 mmol) in DCM (6 mL) and DMF (approximately 0.01 mL) was added oxalyl chloride (5 equiv., 1.45 mmol, 184 mg, 0.13 mL) at RT. After stirring at RT for 16 h, the mixture was concentrated under reduced pressure. The residual oil was dissolved in 5 mL of DCM and added to a solution of 1-decylamine (1.5 equiv., 0.44 mmol, 68 mg) and triethylamine (0.12 mL) in DCM (5 mL). The resulting mixture was stirred at RT for 30 min and then concentrated. The residue was dissolved in a mixture of hexane / EtOAc / Et3N (80:20:1) and filtered through a short column of silica gel (230-400 mesh grade silica gel). The column was then eluted with the same solvent mixture. All product-containing fractions were combined and concentrated to give 254 mg of a brownish oil. The product was further purified by flash dry column silica gel chromatography (MeOH in chloroform containing a trace of EtN, 0–5%) to give the desired product as a colorless oil (218 mg, 0.22 mmol, 75%). 1 H NMR (400 MHz, CDCl3, 7.26 ppm) δ: 5.54-5.46 (m, 1H, NHCO), 3.96 (d, 5.8 Hz, 4H), 3.27-3.19 (m, 2H), 2.55-2.50 (m, 2H), 2.44-2.37 (m, 6H), 2.35-2.27 (m, 6H), 2.21 (s, 3H), 2.19-2.13 (m, 7.6 Hz, 2H), 1.69-1.57 (m, 8H), 1.52-1.39 (m, 8H), 1.34-1.08 (m, 68H), 0.90-0.85 (m, 15H). ESI-MS: C 63 H 125 N3O5[M+H] + MW calculated 1005.0; measured 1005.2.

[0158] Example 12 Synthesis of bis(2-hexyldecyl) 6,6'-((2-(methyl(6-(methyl(octyl)amino)-6-oxohexyl)amino)ethyl)azanediyl)dihexanoate (Compound I-10) [ka] Synthesis of compound I-10 To a solution of 10-1 (prepared according to the procedure in Example 11, 250 mg, 0.29 mmol) in DCM (6 mL) and DMF (0.01 mL) was added oxalyl chloride (5 equiv., 1.45 mmol, 184 mg, 0.13 mL) at RT. After stirring at RT for 16 h, the mixture was concentrated under reduced pressure. The residual oil was dissolved in 5 mL of DCM and added to a solution of N-methyloctylamine (1.5 equiv., 0.44 mmol, 63 mg) and triethylamine (0.12 mL) in DCM (5 mL). The resulting mixture was stirred at RT for 30 min and then concentrated. The residue was dissolved in a mixture of hexane / EtOAc / EtN (80:20:1) and filtered through a short column of silica gel (230-400 mesh grade silica gel). The column was then eluted with the same solvent mixture. All product-containing fractions were combined and concentrated to give a brownish oil. The product (brownish oil) was further purified by column chromatography on silica gel (0-5% MeOH in DCM), which gave the desired product as a slightly yellow oil (146 mg, 0.15 mmol, 51%). 1 H NMR (400 MHz, CDCl3, 7.26 ppm) δ: 3.96 (d, 5.8 Hz, 4H), 3.34, 3.32 (2 sets of triplets, ratio = 1:1, 7.5 Hz, 2H), 2.95, 2.90 (2 sets of singlets, ratio = 1:1, 3H), 2.55-2.50 (m, 2H), 2.43-2.36 (m, 8H), 2.35-2.27 (m, 6H), 2.21 (s, 3H), 1.70-1.54 (m, 8H), 1.54-1.38 (m, 8H), 1.37-1.08 (m, 64H), 0.91-0.85 (m, 15H). ESI-MS: C 62 H 123 N3O5[M+H] + MW calculated 991.0; measured 991.1.

[0159] Example 13 Synthesis of bis(2-hexyldecyl) 6,6'-((2-((6-((2-ethylhexyl)amino)-6-oxohexyl)(methyl)amino)ethyl)azanediyl)dihexanoate (Compound I-11) [ka] Synthesis of compound I-11 To a solution of 10-1 (prepared according to the procedure in Example 11, 250 mg, 0.29 mmol) in DCM (6 mL) and DMF (approximately 0.01 mL) was added oxalyl chloride (5 equiv., 1.45 mmol, 184 mg, 0.13 mL) at RT. After stirring at RT for 16 h, the mixture was concentrated under reduced pressure. The residual oil was dissolved in 5 mL of DCM and added to a solution of 2-ethyl-1-hexylamine (1.5 equiv., 0.44 mmol, 57 mg) and triethylamine (0.12 mL) in DCM (5 mL). The resulting mixture was stirred at RT for 2 h and then concentrated. The residue was dissolved in a mixture of hexane / EtOAc / EtN (80:20:1) and filtered through a short column of silica gel (230-400 mesh grade silica gel). The column was then eluted with the same solvent mixture. All product-containing fractions were combined and concentrated to give 258 mg of a brownish oil, which was further purified by flash-dry column silica gel chromatography (MeOH in chloroform containing a trace of EtN, 0-5%) to give the desired product as a slightly yellow oil (184 mg, 0.19 mmol, 65%). 1 H NMR (400 MHz, CDCl3, 7.26 ppm) δ: 5.43 (s, 1H), 3.96 (d, 5.8 Hz, 4H), 3.25-3.13 (m, 2H), 2.55-2.49 (m, 2H), 2.43-2.37 (m, 6H), 2.35-2.27 (m, 6H), 2.21 (s, 3H), 2.17 (t, 7.6 Hz, 2H), 1.69-1.58 (m, 8H), 1.52-1.38 (m, 7H), 1.37-1.18 (m, 62H), 0.91-0.85 (m, 18H). ESI-MS: C 61 H 121 N3O5[M+H]+ MW calculated 976.9; measured 977.1.

[0160] Example 14 Synthesis of ((2-((4-(dibutylamino)-4-oxobutyl)(methyl)amino)ethyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (Compound I-8) [ka] Synthesis of compound 13-2 A mixture of 13-1 (620 mg, 1.33 mmol), 2-aminoethanol (0.68 mmol, 42 mg), N,N-diisopropylethylamine (0.18 mL), and anhydrous acetonitrile (10 mL) was heated in a sealed pressure flask (oil bath 80 °C) for 1 day. The reaction was cooled and concentrated under reduced pressure. The residue was dissolved in a mixture of hexane / EtOAc / EtN (80:20:1) and filtered through a short column of silica gel (230-400 mesh grade silica gel). The column was then eluted with the same solvent mixture. All product-containing fractions were combined and concentrated to give a colorless oil (0.395 g), which was used in the next step without further purification.

[0161] Synthesis of compound 13-3 To an ice-cold solution of 13-2 (462 mg, 0.62 mmol) in 2 mL of CHCl3, a solution of thionyl chloride (3 equiv., 1.88 mmol, 223 mg, 0.137 mL) in 10 mL of chloroform was added dropwise over 2–3 min. After the addition was complete, the ice bath was removed, and the reaction mixture was stirred at room temperature (20 °C) for 16 h. The reaction was concentrated under reduced pressure. The residue was dissolved in a mixture of hexane / EtOAc / EtN (80:20:1) and filtered through a silica gel pad. The column was then washed with the same solvent mixture. The filtrate was concentrated to give a yellow oil (445 mg), which was used in the next step without further purification.

[0162] Synthesis of compound 13-4 A solution of 13-3 (222 mg, 0.29 mmol), 4-amino-N,N-dibutylbutanamide (3 equiv., 0.88 mmol, 188 mg), N,N-diisopropylethylamine (0.25 mL), and sodium iodide (10 mg) in acetonitrile (10 mL) was sealed and heated at 76 °C for 24 h. The resulting mixture was concentrated. The residue was dissolved in a mixture of hexane and ethyl acetate (85:15) and filtered through a short column of silica gel (230-400 mesh grade silica gel). The column was then eluted with a mixture of hexane / EtOAc / EtN (85:15:0 ​​to 70:30:1). Fractions containing all the desired product were combined and concentrated to give a yellow oil (109 mg, 0.12 mmol, 40%).

[0163] Synthesis of compound I-8 To a solution of 13-4 (109 mg, 0.12 mmol) in THF (5 mL) was added formaldehyde (HCl) solution (250 mg of 37 wt% aqueous solution) at RT. The resulting mixture was stirred for 30 min, after which sodium triacetoxyborohydride (5 equiv., 0.6 mmol, 122 mg) was introduced. After stirring overnight, the crude mixture was dissolved in a mixture of hexane and ethyl acetate (ca. 90:10, 100 mL) and washed with saturated sodium bicarbonate solution. The extract was concentrated to give a yellow oil / solid. The residue was dissolved in a mixture of hexane / EtOAc / Et3N (80:20:1) and filtered through a short column of silica gel (230-400 mesh grade silica gel). The column was then eluted with the same solvent mixture. All product-containing fractions were combined and concentrated to give a brownish oil (100 mg). The product was further purified by column chromatography on silica gel (0-6% MeOH in DCM; 0-5% methanol in chloroform), which gave the desired product as a slightly yellow oil (30 mg, 0.03 mmol, 26%). 1H NMR (400 MHz, CDCl3, 7.26 ppm) δ: 4.06 (t, 6.7 Hz, 4H), 3.33-3.26 (m, 2H), 3.25-3.18 (m, 2H), 2.55-2.50 (m, 2H), 2.47-2.35 (m, 8H), ESI-MS: C 59 H 117 N3O5[M+H] + MW calculated 948.9; measured 949.1.

[0164] Example 15 Synthesis of ((2-(methyl(4-octanamidobutyl)amino)ethyl)azanediyl)bis(nonane-9,1-diyl) bis(2-butyloctanoate) (Compound I-6) [ka] Synthesis of compounds 14-1, 14-2, and 14-3 These intermediates were prepared in a manner analogous to the preparation of compounds 13-2, 13-3 and 13-4 in Example 14.

[0165] Synthesis of compound I-6 To a solution of 14-3 (152 mg, 0.17 mmol) in THF (6 mL) was added formaldehyde (HCl) solution (420 mg of 37 wt% aqueous solution) at RT. The resulting mixture was stirred for 30 min, after which sodium triacetoxyborohydride (5 equiv., 0.6 mmol, 122 mg, MW 211.94) was introduced. After stirring overnight, the crude mixture was dissolved in a mixture of hexanes (60 mL) and washed with saturated sodium bicarbonate solution. The aqueous phase was extracted with hexane. The combined extracts were filtered through a short column of silica gel (230-400 mesh grade silica gel). The column was then eluted with a mixture of hexanes / EtOAc / Et3N (80:20:1). All product-containing fractions were combined and concentrated to give a brownish oil (129 mg). The product was further purified by column silica gel chromatography (0-5% methanol in chloroform). This gave the desired product as a slightly yellow oil (76 mg, 0.08 mmol, 49%). 1 H NMR (400 MHz, CDCl3, 7.26 ppm) δ: 6.20 (br. 1H, NHCO), 4.06 (t, 6.7 Hz, 4H), 3.28-3.20 (m, 2H), 2.56-2.51 (m, 2H), 2.48-2.26 (m, 10H), 2.22 (s, 3H), 2.16-2.10 (t, 2H), 1.67-1.55 (m, 10H), 1.55-1.50 (m, 4H), 1.48-1.37 (m, 8H), 1.37-1.12 (m, 52H), 0.90-0.85 (m, 15H). ESI-MS: C 57 H 113 N3O5[M+H] + MW calculated 920.9; measured 921.1.

[0166] Example 16 6-((2-((3-(N,N-dihexylcarbamoyl)propyl)-N-methylamino)ethyl)(6-(1-hexylnonylcarbonyloxy)hexyl)amino)hexyl 2-hexyldecanoate (Compound I-53) [ka] Synthesis of compound 16-1 6-Chloro-1-hexanol (5.0 g, 4.87 mL, 36.6 mmol, 1 equiv.), 2-hexyldecanoic acid (14.1 g, 16.1 mL, 54.9 mmol, 1.5 equiv.), and N,N-dimethyl-4-pyridylamine (DMAP; 2.2 g, 18.3 mmol, 0.5 equiv.) were dissolved in DCM (40 mL). 1-((cyclohexylimino)methyleneamino)cyclohexane (DCC; 8.3 g, 40.3 mmol, 1.1 equiv.) was added in one portion, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was then diluted with hexane (200 mL), and the solids were removed by passing the mixture through a small pad of diatomaceous earth (e.g., Celite®). After removing the solvent under reduced pressure, the residue was purified by automated flash chromatography (220 g SiO column; elution with 0–15% EtOAc in hexanes, targeting 4–6% EtOAc in hexanes) to give 6-chlorohexyl 2-hexyldecanoate (compound 16-1; 10.0 g, 73%). ESI-MS: C 22 H 43 ClO2[M+H] + MW calculated 375.33; measured 375.43.

[0167] Synthesis of compound 16-2 A solution of 6-chlorohexyl 2-hexyldecanoate (compound 16-1; 2.0 g, 5.33 mmol, 2 equiv.), 2-aminoethanol (195 mg, 3.2 mmol, 1.2 equiv.), N-ethylbis(isopropyl)amine (DIPEA; 1.4 g, 1.91 mL, 10.9 mmol, 4.1 equiv.), and potassium iodide (930 mg, 5.6 mmol, 2.1 equiv.) in acetonitrile (6.4 mL) was placed in a microwave reactor and heated at 140 °C for 30 min. The reaction was carried out in triplicate (3 × 2.0 g of 6-chlorohexyl 2-hexyldecanoate). After cooling, the reaction mixtures were combined and the acetonitrile was removed under reduced pressure. The residue was partitioned between EtOAc and water, the organic layer was dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure. The residue was purified by automated flash chromatography (80 g SiO column; 0-30% EtOAc in hexanes with 1% EtN) to give 6-((6-(1-hexylnonylcarbonyloxy)hexyl)(2-hydroxyethyl)amino)hexyl 2-hexyldecanoate (compound 16-2; 2.5 g, 42%). ESI-MS: C 46 H 91 NO5[M+H] + MW calculated 738.70; measured 738.83.

[0168] Synthesis of compound 16-3 To an ice-cooled solution of 16-2 (3.08 g, 4.17 mmol, 1 equiv.) in DCM (50 mL) was added SOCl (1.49 g, 908 μL, 12.5 mmol, 3 equiv.) dropwise. After this time, the ice bath was removed, and the reaction mixture was stirred overnight at room temperature. DCM and SOCl were removed under reduced pressure, and the crude dark red oil was purified by automated flash chromatography (80 g SiO column; 0–30% EtOAc in hexanes with 1% EtN; target elution with 8–12% EtOAc) to give 6-((2-chloroethyl)(6-(1-hexylnonylcarbonyloxy)hexyl)amino)hexyl 2-hexyldecanoate (compound 16-3; 2.8 g, 89%). ESI-MS: C 46 H 90 NClO4[M+H] +MW calculated 756.66; measured 756.80.

[0169] [ka] Synthesis of compound 16-4 4-(tert-Butoxycarbonylamino)butyric acid (1.0 g, 4.92 mmol, 1 equiv.), dihexylamine (1.19 g, 1.49 mL, 6.40 mmol, 1.3 equiv.), and DIPEA (1.27 g, 1.71 mL, 9.84 mmol, 2 equiv.) were combined in DCM (15 mL), followed by the addition of HATU (2.25 g, 5.90 mmol, 1.2 equiv.). The resulting mixture was stirred for 1.5 h at room temperature, then diluted with EtOAc (15 mL) and washed with water (10 mL). The aqueous layer was washed with 2 × 15 mL of EtOAc. The combined organic phases were dried over anhydrous Na2SO4, and the solvent was removed. The resulting pale yellow oil was purified by automated flash chromatography (80 g SiO column; elution with 0-100% EtOAc in hexanes, targeting 29%-39% EtOAc) to afford 3-(N,N-dihexylcarbamoyl)propylamino-tert-butyl formirate (compound 16-4, 1.49 g, 82%) as a clear, pale yellow oil. ESI-MS: C 21 H 42 N2O3[M+H] + MW calculated 371.32; measured 371.53.

[0170] Synthesis of compound 16-5 Compound 16-4 (approximately 1.49 g, 4.02 mmol, 1 equiv.) was dissolved in DCM (35 mL), followed by the addition of TFA (12.1 g, 8.16 mL, 107 mmol, 27 equiv.). The resulting mixture was stirred overnight at room temperature, and then 10 mL of water was added, followed by solid NaHCO3 until gas evolution was no longer observed. Finally, the pH was adjusted to 11 by the addition of solid K2CO3. The organic and aqueous layers were separated, and the aqueous layer was washed with 3 x 15 mL of EtOAc. The combined organic phase was dried over anhydrous sodium sulfate, and the solvent was removed to give N,N-dihexyl 4-aminobutyramide (compound 16-5; 987 mg, 91%) as a clear yellow oil. ESI-MS: C 16 H 34 NO [M+H] + MW calculated 271.27; measured 271.37.

[0171] Synthesis of compound 16-6 A solution of compound 16-3 (400 mg, 529 μmol, 1 equiv.), compound 16-5 (357 mg, 1.32 mmol, 2.5 equiv.), potassium iodide (87.8 mg, 529 μmol, 1 equiv.), and DIPEA (137 mg, 185 μL, 1.06 mmol, 2 equiv.) in acetonitrile (5 mL) was heated in a microwave reactor at 140 °C for 30 min. The resulting mixture was concentrated and partitioned between EtOAc and water, and the aqueous layer was washed with 3 × 15 mL of EtOAc. The combined organic phase was dried over anhydrous Na2SO4, and the solvent was removed. The resulting yellow oil was purified by automated flash chromatography (40 g SiO column; elution with 0–100% EtOAc in hexanes with 1% EtN, targeting 56–86% EtOAc with 1% EtN) to afford 6-((2-(3-(N,N-dihexylcarbamoyl)propylamino)ethyl)(6-(1-hexylnonylcarbonyloxy)hexyl)amino)hexyl 2-hexyldecanoate (compound 16-6; 141 mg, 27%) as a yellow oil. ESI-MS: C 62 H 123 N3O5[M+H] + MW calculated 990.95; measured 991.29.

[0172] Synthesis of compound I-53 To a solution of compound 16-6 (141 mg, 142 μmol, 1 equiv.) in MeOH (1.0 mL) was added aqueous formaldehyde (13.3 mol / L, 390 μL, 37 equiv.). The mixture was stirred at room temperature for 35 min, after which sodium triacetoxyborohydride (127 mg, 598 μmol, 4.2 equiv.) was introduced and then stirred at room temperature for an additional 4 h. The resulting mixture was diluted with EtOAc and washed with saturated aqueous NaHCO (10 mL), and the aqueous layer was washed with 3 × 15 mL of EtOAc. The combined organic phase was dried over anhydrous NaSO, and the solvent was removed. The resulting oil was purified by automated flash chromatography (25 g SiO column; elution with 0–100% EtOAc with 1% EtN in hexanes with 1% EtN, targeting 23–44% EtOAc with 1% EtN) to afford 6-((2-((3-(N,N-dihexylcarbamoyl)propyl)-N-methylamino)ethyl)(6-(1-hexylnonylcarbonyloxy)hexyl)amino)-hexyl 2-hexyldecanoate (compound I-53; 93.2 mg, 65%) as a clear, colorless oil. 1 H NMR (400 MHz, CDCl3) δ 4.09 (t, J = 6.7 Hz, 4H), 3.36 - 3.28 (m, 2H), 3.28 - 3.19 (m, 2H), 2.63 - 2.29 (m, 13H), 2.26 (s, 3H), 1.85 (q, ESI-MS: C 63 H 125 N3O5[M+H] + MW calculated 1004.97; measured 1005.25.

[0173] Example 17 6-((2-((3-(N,N-diethylcarbamoyl)propyl)-N-methylamino)ethyl)(6-(1-hexylnonylcarbonyloxy)hexyl)amino)hexyl 2-hexyldecanoate (Compound I-52) [ka] Synthesis of compound 17-6 4-(tert-Butoxycarbonylamino)butyric acid (1.02 g, 5.02 mmol, 1 equiv.), diethylamine (477 mg, 675 μL, 6.52 mmol, 1.3 equiv.), and DIPEA (1.3 g, 1.75 mL, 10.0 mmol, 2 equiv.) were combined in DCM (15.3 mL), followed by the addition of HATU (2.29 g, 6.02 mmol, 1.02 equiv.). The resulting mixture was stirred for 1.5 h at room temperature, then diluted with EtOAc (15 mL) and washed with water (10 mL). The aqueous layer was washed with 3 × 15 mL of EtOAc. The combined organic phases were dried over anhydrous NaSO and the solvent was removed. The resulting pale yellow oil was purified by automated flash chromatography (80 g SiO column; elution with 0–100% EtOAc in hexanes, targeting 78–84% EtOAc) to afford 3-(N,N-diethylcarbamoyl)propylamino-tert-butylformylate (compound 17-6; 1.11 g, 85%) as a pale yellow oil. ESI-MS: C 13 H 26 N2O3[M+H] + MW calculated 259.20; measured 259.31.

[0174] Synthesis of compound 17-7 Compound 17-6 (1.11 g, 4.3 mmol, 1 equiv.) was dissolved in DCM (25.0 mL), followed by the addition of TFA (9.8 g, 6.58 mL, 85.9 mmol, 20 equiv.). The resulting mixture was stirred overnight at room temperature, and then 10 mL of water was added, followed by solid NaHCO3 until gas evolution was no longer observed. Finally, the pH was adjusted to 11 by the addition of solid K2CO3. The organic and aqueous layers were separated, and the aqueous layer was washed with 3 x 15 mL of EtOAc. The combined organic phase was dried over anhydrous Na2SO4, and the solvent was removed to give N,N-diethyl 4-aminobutyramide (compound 17-7; 370 mg, 54%) as a clear, pale yellow oil. ESI-MS: CH 18 NO [M+H] + MW calculated 159.15; measured 159.14.

[0175] Synthesis of compound 17-8 A mixture of compound 16-3 (413 mg, 546 μmol, 1 equiv.), compound 17-7 (345 mg, 2.18 mmol, 4 equiv.), potassium iodide (94.5 mg, 569 μmol, 1.04 equiv.), and DIPEA (139 mg, 188 μL, 1.08 mmol, 1.97 equiv.) in acetonitrile (3.2 mL) was heated in a microwave reactor at 140 °C for 30 min. The resulting mixture was concentrated and partitioned between EtOAc and water, and the aqueous layer was washed with 3 × 15 mL of EtOAc. The combined organic phase was dried over anhydrous Na2SO4, and the solvent was removed. The resulting yellow oil was purified by automated flash chromatography (40 g SiO column; elution with 0-100% EtOAc with 1% EtN in hexanes with 1% EtN; target 100% EtOAc with 1% EtN) to afford 6-((2-(3-(N,N-diethylcarbamoyl)propylamino)ethyl)(6-(1-hexylnonylcarbonyloxy)hexyl)amino)hexyl 2-hexyldecanoate (compound 17-8; 241 mg, 50%) as a brown-orange oil. ESI-MS: C 54 H 107 N3O5[M+H] + MW calculated 878.82; measured 879.11.

[0176] Synthesis of compound I-52 To a solution of compound 17-8 (240 mg, 273 μmol, 1 equiv.) in MeOH (4.5 mL) was added aqueous formaldehyde (13.3 mol / L, 614 μL, 30 equiv.). The mixture was stirred at room temperature for 30 min, after which sodium triacetoxyborohydride (251 mg, 1.18 mmol, 4.3 equiv.) was added and stirred at room temperature overnight. The resulting mixture was concentrated, diluted with EtOAc, washed with saturated aqueous NaHCO3 (10 mL), and the aqueous layer was washed with 4 × 15 mL of EtOAc. The combined organic phase was dried over anhydrous Na2SO4, and the solvent was removed. The resulting oil was purified by automated flash chromatography (40 g column; elution with 0–100% EtOAc with 1% EtN in hexanes, targeting 40–44% EtOAc with 1% EtN) to afford 6-((2-((3-(N,N-diethylcarbamoyl)propyl)-N-methylamino)ethyl)(6-(1-hexylnonylcarbonyloxy)hexyl)amino)-hexyl 2-hexyldecanoate (compound I-52; 182 mg, 75%) as a clear, slightly yellow oil. 1 H NMR (400 MHz, CDCl3) δ 4.05 (s, 4H), 3.48 - 3.20 (m, 4H), 2.58 - 2.27 (m, 18H), 2.23 (s, 3H), 1.87 - 1.21 (m, 70H), 1.16 (t, J = 7.1 Hz, 3H), 1.10 (t, J = 7.1 Hz, 3H), 0.92 - 0.82 (m, 12H). ESI-MS: C 55 H 109 N3O5[M+H] + MW calculated 892.84; measured 893.10.

[0177] Example 18 ((4-((5-(dihexylamino)-5-oxopentyl)(methyl)amino)butyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (Compound I-47) [ka] Synthesis of compound 18-2 6-Chlorohexyl 2-hexyldecanoate (compound 16-1; 1.2 g, 3.25 mmol, 2 equiv.), tert-butyl(4-aminobutyl)(methyl)carbamateamine (395 mg, 1.995 mmol, 1.2 equiv.), N-ethylbis(isopropyl)amine (DIPEA; 862 mg, 1.16 mL, 6.67 mmol, 4.1 equiv.), and KI (567 mg, 3.42 mmol, 2.1 equiv.) were mixed in acetonitrile (3.9 mL) and reacted in a microwave reactor (140 °C, 30 min). After cooling, the solvent was removed under reduced pressure, and the residue was partitioned between EtOAc and water. The organic layer was dried over anhydrous Na2SO4. After removal of the solvent under reduced pressure, the residue was purified by automated flash chromatography (40 g SiO column; elution with 0–50% EtOAc in hexanes containing 1% EtN, targeting 15–20% EtOAc) to give ((4-((tert-butoxycarbonyl)(methyl)amino)butyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (compound 18-2; 460 mg, 32%). ESI-MS: C 54 H 106 N2O6[M+H] + Calculated MW 879.81; Found 880.08. TLC (MeOH:DCM = 1:9) Rf = 0.5.

[0178] Synthesis of compound 18-3 ((4-((tert-Butoxycarbonyl)(methyl)amino)butyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (compound 18-2; 460 mg, 523 μmol, 1 equiv.) was added to DCM (7.3 mL); TFA (895 mg, 0.6 mL, 7.85 mmol, 15 equiv.) was added, and the mixture was stirred at room temperature overnight. Then, 10 mL of water was added, followed by solid NaHCO3. When no more bubbles were observed with the addition of fresh NaHCO3, the pH was adjusted to 11 by the addition of 11. The layers were separated; the DCM layer was dried over anhydrous Na2SO4. Removal of the solvent under reduced pressure gave crude 6-((6-(1-hexylnonylcarbonyloxy)hexyl))(4-(methylamino)butyl)amino)hexyl 2-hexyldecanoate (compound 18-3; 0.4 g, 98%), which was used further as is. ESI-MS: C 49 H 98 N2O4[M+H] + MW calculated 779.76; measured 779.98.

[0179] [ka] Synthesis of compound 18-4 5-Bromovaleric acid (219 mg, 1.21 mmol, 1 equiv.), dihexylamine (269 mg, 338 μL, 1.45 mmol, 1.2 equiv.), and DIPEA (313 mg, 423 μL, 2.42 mmol, 2 equiv.) were dissolved in DCM (11 mL). To this solution, 1,1,3,3-tetramethyl-2-(3H-1,2,3,4-tetraazaindene-3-yl)-3-isoureaium hexafluoride-phosphate(1-) (HATU; 598 mg, 1.57 mmol, 1.3 equiv.) was added in one portion. The reaction mixture was stirred for 15 minutes at room temperature. The reaction mixture was then washed with saturated aqueous NaHCO3 (5 mL). The aqueous layer was extracted with DCM (2 × 5 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by automated flash chromatography (12 g SiO column; elution with 0–50% EtOAc in hexanes, targeting 25–30% EtOAc) to give 5-bromo-N,N-dihexylpentanamide (compound 18-4; 290 mg, 68%). ESI-MS: C 17 H 34 BrNO [M+H] + MW calculated 348.19; measured 348.30.

[0180] Synthesis of compound I-47 Compound 18-3 (290 mg, 373 μmol, 1.3 equiv.), N,N-dihexyl-5-bromovaleramide (compound 18-4; 99.8 mg, 287 μmol, 1 equiv.), and DIPEA (74.1 mg, 0.1 mL, 576 μmol, 2 equiv.) were mixed in acetonitrile (745 μL) and reacted in a microwave reactor (140 °C, 30 min). After cooling, the solvent was removed under reduced pressure, and the residue was partitioned between EtOAc and water. The organic layer was dried over anhydrous Na2SO4. After removal of the solvent under reduced pressure, the residue was purified by automated flash chromatography (12 g SiO column; elution with 0–50% EtOAc in Hex containing 1% EtN, targeting 18–31% EtOAc) to give ((4-((5-(dihexylamino)-5-oxopentyl)(methyl)amino)butyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (compound I-47; 99 mg, 33%). 1 H NMR (400 MHz, CDCl3) δ 4.06 (t, J = 6.7 Hz, 4H), 3.33 - 3.24 (m, 2H), 3.24 - 3.14 (m, 2H), 2.45 - 2.26 (m, 14H), 2.19 (s, 3H), 1.72 - 1.47 (m, 19H), 1.47 - 1.16 (m, 73H), 0.93 - 0.82 (m, 18H). ESI-MS: C 66 H 131 N3O5[M+H] + MW calculated 1047.01; measured 1047.27.

[0181] Example 19 ((4-((5-(didecylamino)-5-oxopentyl)(methyl)amino)butyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (Compound I-48) [ka] Synthesis of compound 19-1 5-Bromovaleric acid (200 mg, 1.10 mmol, 1 equiv.), didecylamine (395 mg, 1.33 mmol, 1.2 equiv.), and DIPEA (286 mg, 386 μL, 2.21 mmol, 2 equiv.) were dissolved in DCM (10 mL). To this solution, HATU (546 mg, 1.44 mmol, 1.3 equiv.) was added in one portion. The reaction mixture was stirred for 15 min at room temperature. The reaction mixture was then washed with saturated aqueous NaHCO3 (5 mL). The aqueous layer was extracted with DCM (2 × 5 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by automated flash chromatography (12 g SiO2 column; elution with 0–30% EtOAc in hexanes, targeting 11% EtOAc) to give 5-bromo-N,N-didecylpentanamide (compound 19-1; 380 mg, 75%). ESI-MS: C 25 H 50 BrNO [M+H] + MW calculated 460.31; measured 460.42.

[0182] Synthesis of compound I-48 Compound 18-3 (290 mg, 373 μmol, 1.3 equiv.), 5-bromo-N,N-didecylpentanamide (compound 19-1; 132 mg, 287 μmol, 1 equiv.), and DIPEA (74.1 mg, 0.1 mL, 576 μmol, 2 equiv.) were mixed in acetonitrile (745 μL) and reacted in a microwave reactor (140 °C, 30 min). After cooling, the solvent was removed under reduced pressure, and the residue was partitioned between EtOAc and water. The organic layer was dried over anhydrous Na2SO4. After removal of the solvent under reduced pressure, the residue was purified by automated flash chromatography (12 g SiO column; elution with 0–50% EtOAc in hexanes containing 1% EtN, targeting 25–31% EtOAc) to give ((4-((5-(didecylamino)-5-oxopentyl)(methyl)amino)butyl)-azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (compound I-48; 125 mg, 38%). 1H NMR (400 MHz, CDCl3) δ 4.06 (t, J = 6.7 Hz, 4H), 3.34 - 3.24 (m, 2H), 3.24 - 3.15 (m, 2H), 2.41 - 2.25 (m, 14H), 2.20 (s, 3H), 1.77 - 1.19 (m, 105H), 0.88 (tt, J = 7.1, 2.3 Hz, 18H). ESI-MS: C 74 H 147 N3O5[M+H] + MW calculated 1159.14; measured 1159.29.

[0183] Example 20 ((4-((5-(dioctylamino)-5-oxopentyl)(methyl)amino)butyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (Compound I-51) [ka] Synthesis of compound 20-1 5-Bromovaleric acid (200 mg, 1.10 mmol, 1 equiv.), dihexylamine (293 mg, 367 μL, 1.22 mmol, 1.1 equiv.), and DIPEA (286 mg, 386 μL, 2.21 mmol, 2 equiv.) were dissolved in DCM (10 mL). To this solution, HATU (546 mg, 1.44 mmol, 1.3 equiv.) was added in one portion. The reaction mixture was stirred for 15 minutes at room temperature. The reaction mixture was then washed with saturated aqueous NaHCO3 (5 mL). The aqueous layer was extracted with DCM (2 × 5 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by automated flash chromatography (12 g SiO column; elution with 0–50% EtOAc in hexanes, targeting 25–32% EtOAc) to give 5-bromo-N,N-dioctylpentanamide (compound 20-1; 325 mg, 72%). ESI-MS: C 21 H 42 BrNO [M+H] + MW calculated 404.25; measured 404.43.

[0184] Synthesis of compound I-51 Compound 18-3 (217 mg, 278 μmol, 1.5 equiv.), N,N-dioctyl-5-bromovaleramide (compound 20-1; 75.0 mg, 185 μmol, 1 equiv.), and DIPEA (47.9 mg, 64.8 μL, 371 μmol, 2 equiv.) were mixed in acetonitrile (556 μL) and reacted in a microwave reactor (140 °C, 30 min). After cooling, the solvent was removed under reduced pressure, and the residue was partitioned between EtOAc and water. The organic layer was dried over anhydrous Na2SO4. After removal of the solvent under reduced pressure, the residue was purified by automated flash chromatography (12 g SiO column; elution with 0–50% EtOAc in hexanes containing 1% EtN, targeting 34–38% EtOAc) to give ((4-((5-(dioctylamino)-5-oxopentyl)(methyl)amino)-butyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (compound I-51; 110 mg, 54%). 1 H NMR (400 MHz, CDCl3) δ 4.06 (t, J = 6.7 Hz, 4H), 3.40 - 3.24 (m, 2H), 3.19 (dd, J = 9.2, 6.4 Hz, 2H), 2.55 - 2.25 (m, 14H), 2.19 (s, 3H), 1.77 - 1.11 (m, 98H), 0.95 - 0.77 (m, 18H). ESI-MS: C 70 H 139 N3O5[M+H] + MW calculated 1103.08; measured 1103.32.

[0185] Example 21 Bis(2-hexyldecyl) 6,6'-((3-((5-methoxy-5-oxopentyl)(methyl)amino)propyl)azanediyl)dihexanoate (Compound I-50) [ka] Synthesis of compound 21-1 To a solution of N-methyl-1,3-diaminopropane (15 mmol, 1.32 g, 1.57 mL) in dichloromethane (25 mL) was added a solution of di-tert-butyl dicarbonate (15 mmol, 3.27 g) in dichloromethane (25 mL) at 0-5°C. After the addition, the temperature was allowed to rise to room temperature. Gradually, a white precipitate appeared. Stirring was continued at room temperature for 16 h. The white solid was filtered off and washed with DCM. Concentration of the filtrate gave the desired product as a colorless oil (2.31 g; 82%), which was used in the next step without further purification.

[0186] Synthesis of compound 21-2 A mixture of 2-hexyldecyl 6-bromohexanoate (2 equiv., 3.56 g, 8.5 mmol), anhydrous acetonitrile (35 mL), N,N-diisopropylethylamine (1.85 mL), compound 21-1 (800 mg, 4.25 mmol), and sodium iodide (0.1 equiv., 64 mg) was heated in a sealed pressure flask at 75 °C for 18 h. TLC (CHCl3 / MeOH = 19:1; CHCl3 / EtOH / H2O / NH4OH = 30:25:3:2; hexane / ethyl acetate = 9:1) indicated that there was still a large amount of starting bromide and no starting amine. Another 430 mg of compound 21-1 was added, and heating was continued until the next day. The reaction mixture was concentrated. The residue was dissolved in hexane (100 mL) and filtered through a silica gel pad. The pad was then eluted with a gradient mixture of hexane, EtOAc, and EtN (95:5:0 to 80:20:1) to give the desired product as a brownish oil (2.381 g, 65%), which was used in the next step without further purification.

[0187] Synthesis of compound 21-3 To a solution of compound 21-2 (2.381 g, ca. 2.75 mmol) in CHCl (15 mL) was added TFA (55 mmol, 4.2 mL). After stirring at room temperature for 16 h, the reaction mixture was diluted with DCM and washed with saturated bicarbonate solution. The aqueous phase was extracted with DCM. The combined organic phases were dried over sodium sulfate and concentrated. The residue was dissolved in hexane (100 mL) and filtered through a silica gel pad. The pad was then eluted with a gradient mixture of hexane and EtOAc (95:5:0 to 80:20), followed by a gradient mixture of DCM, MeOH, and EtN (85:15:1). The desired product was obtained as a brownish oil (1.14 g, 60%). MS (ESI): m / z 765.93 (M+1).

[0188] Synthesis of compound I-50 A mixture of methyl 5-bromovalerate (1 equiv., 102 mg, 0.52 mmol), compound 21-3 (1 equiv., 400 mg, 0.52 mmol), N,N-diisopropylethylamine (1.5 equiv., 0.14 mL), sodium iodide (0.5 equiv., 39 mg), and anhydrous acetonitrile (10 mL) was heated at 80 °C for 18 h in a sealed pressure flask and then concentrated. The residue was dissolved in a mixture of hexane, EtOAc, and EtN (80:20:1) and filtered through a silica gel pad. The pad was then washed with the same solvent mixture. The filtrate was concentrated to give the crude product as a brown oil (200 mg). The product was further purified by column silica gel chromatography (0–5% methanol in chloroform). This gave the desired product as a colorless oil (113 mg, 0.13 mmol, 25%). 1 H NMR (400 MHz, CDCl3, 7.26 ppm) δ: 3.96 (d, 5.8 Hz, 4H), 3.66 (s, 3H), 2.42-2.27 (m, 16H), 2.18 (s, 3H), 1.68-1.57 (m, 10H), 1.53-1.38 (m, 6H), 1.36-1.18 (m, 52H), 0.92-0.85 (m, 12H). MS (ESI): m / z (M+1), 880.10

[0189] Example 22 Bis(2-hexyldecyl) 6,6'-((3-((5-(dihexylamino)-5-oxopentyl)(methyl)amino)propyl)azanediyl)dihexanoate (Compound I-49) [ka] Synthesis of compound I-49 A mixture of 5-bromo-N,N-dihexylpentanamide (1 equiv., 182 mg, 0.52 mmol), compound 21-3 (prepared according to the procedure in Example 21, 1 equiv., 400 mg, 0.52 mmol), N,N-diisopropylethylamine (1.5 equiv., 0.14 mL), sodium iodide (0.5 equiv., 39 mg), and anhydrous acetonitrile (10 mL) was prepared. The mixture was heated at 80 °C for 18 h in a sealed pressure flask (oil bath 80 °C) and then concentrated. The residue was dissolved in a mixture of hexane, EtOAc, and EtN (80:20:1) and filtered through a silica gel pad. The pad was then washed with the same solvent mixture. The filtrate was concentrated to give the crude product as a brown oil (320 mg). The product was further purified by column silica gel chromatography (0–5% methanol in chloroform). This gave the desired product as a colorless oil (167.6 mg, 0.16 mmol, 27%). 1 H NMR (400 MHz, CDCl3, 7.26 ppm) δ:3.96 (d, 5.8 Hz, 4H), 3.31-3.25 (m, 2H), 3.22-3.17 (m, 2H), 2.42-2.27 (m, 16H), 2.19 (s, 3H), 1.68-1.57 (m, 10H), 1.55-1.38 (m, 10H), 1.36-1.20 (m, 64H), 0.92-0.85 (m, 18H). MS (ESI): m / z 1033.3 (M+1).

[0190] It is understood that 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 mentioned herein and / or listed in the Application Data Sheets are incorporated herein by reference in their entirety.

[0191] Aspects of the embodiments can be modified, as necessary, using concepts from various patents, applications, and publications to provide further embodiments. These and other changes can be made by the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to restrict 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. Accordingly, the claims are not restricted by this disclosure.

[0192] U.S. Provisional Application No. 63 / 404,463, filed September 7, 2022, from which this application claims priority, is incorporated herein by reference in its entirety.

Claims

1. The following structure (I): 【Chemical 1】 [In the ceremony: L １ａ and L １ｂ each independently represents an optionally substituted C ３ -C １２ is alkyl; R １ａ is -C(=O)OR ４ａ or -O(C=O)R ４ａ and R １ｂ is -C(=O)OR ４ｂ or -O(C=O)R ４ｂ and R ２ Ha-NR ６ (C=O)R ５ , -(C=O)N(R ６ )R ５ or -(C=O)OR ７ and R ３ and R ６ are each independently hydrogen or an optionally substituted C １ -C １２ is alkyl; R ４ａ , R ４ｂ and R ５ are each independently an optionally substituted alkyl; R ７ is an optionally substituted C １ -C ６ alkyl or optionally substituted arylalkyl; n1 is 2, 3, 4, 5, or 6; and X is C ２ -C ６ Alkylene or C ４ -C ２０ It is an alkylene oxide. or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

2. X is: 【Chemistry 2】 and where: n2 is 2, 3, 4, 5 or 6; n3 is 0, 1, 2, 3 or 4; n4 is 2, 3, or 4; and n5 is 2, 3, 4 or 5; The compound of claim 1.

3. L １ａ is C ５ -C ９ 3. The compound of claim 1 or 2, wherein the alkyl is alkyl.

4. L １ｂ is C ５ -C ９ The compound of any one of claims 1 to 3, wherein the compound is alkyl.

5. L １ａ is C ５ -, C ６ -, C ７ - or C ９ The compound of any one of claims 1 to 4, wherein the aryl group is -alkyl.

6. L １ｂ is C ５ -, C ６ -, C ７ - or C ９ 6. The compound of claim 1, wherein the aryl group is -alkyl.

7. L １ａ The compound of any one of claims 1 to 6, wherein is unsubstituted.

8. L １ｂ The compound of any one of claims 1 to 7, wherein is unsubstituted.

9. L １ａ The compound of any one of claims 1 to 8, wherein is unbranched.

10. L １ｂ The compound of any one of claims 1 to 9, wherein is unbranched.

11. R １ａ -O(C=O)R ４ａ The compound of any one of claims 1 to 10,

12. R １ａ -(C=O)OR ４ａ The compound of any one of claims 1 to 10,

13. R １ｂ -O(C=O)R ４ｂ The compound of any one of claims 1 to 12,

14. R １ｂ One of them is -(C=O)OR ４ｂ The compound of any one of claims 1 to 12,

15. R ４ａ is C ８ -C ２４ 15. The compound of any one of claims 1 to 14, wherein:

16. R ４ａ is C １１ -C １６ 16. The compound of any one of claims 1 to 15, wherein:

17. R ４ａ is C １１ 17. The compound of any one of claims 1 to 16, wherein: - alkyl.

18. R ４ａ is C １５ 17. The compound of any one of claims 1 to 16, wherein: - alkyl.

19. R ４ａ is C １６ 17. The compound of any one of claims 1 to 16, wherein: - alkyl.

20. R ４ｂ is C ８ -C ２４ 20. The compound of any one of claims 1 to 19, wherein: - alkyl.

21. R ４ｂ is C １０ -C １８ 21. The compound of any one of claims 1 to 20, wherein:

22. R ４ｂ is C １１ -C １６ 22. The compound of any one of claims 1 to 21, wherein: - alkyl.

23. R ４ｂ is C １１ 23. The compound of any one of claims 1 to 22, wherein:

24. R ４ｂ is C １５ 23. The compound of any one of claims 1 to 22, wherein:

25. R ４ｂ is C １６ 23. The compound of any one of claims 1 to 22, wherein:

26. R ４ａ The compound of any one of claims 1 to 25, wherein is branched.

27. R ４ｂ The compound of any one of claims 1 to 26, wherein is branched.

28. R ４ａ 28. The compound of any of claims 1 to 27, wherein is unsubstituted.

29. R ４ｂ 29. The compound of any of claims 1 to 28, wherein is unsubstituted.

30. R ４ａ has the following structure: 【Chemistry 3】 30. The compound of any one of claims 1 to 29, having one of:

31. R ４ｂ has the following structure: 【Chemistry 4】 31. The compound of any one of claims 1 to 30, having one of:

32. R ２ Ga-NR ６ (C=O)R ５ 32. The compound of any one of claims 1 to 31,

33. R ２ -(C=O)N(R ６ )R ５ 32. The compound of any one of claims 1 to 31,

34. R ５ is C ２ -C １６ - alkyl.

35. R ５ is C ４ -C １３ - alkyl.

36. R ５ is C ４ -, C ７ -, C ８ -, C １０ - or C １３ 36. The compound of any one of claims 1 to 35, wherein: - alkyl.

37. R ５ 37. The compound of any of claims 1 to 36, wherein is unsubstituted.

38. R ５ 37. The compound of any one of claims 1 to 36, wherein:

39. R ５ 39. The compound of claim 38, wherein is substituted with hydroxyl.

40. R ５ 40. The compound of any one of claims 1 to 39, wherein is branched.

41. R ５ 40. The compound of any of claims 1 to 39, wherein is unbranched.

42. R ５ has the following structure: 【Chemistry 5】 42. The compound of any one of claims 1 to 41, having one of:

43. R ６ is C １ -C １０ 43. The compound of any one of claims 1 to 42, which is alkyl.

44. R ６ is C １ -, C ２ -, C ３ -, C ６ -, C ８ - or C １０ 44. The compound of any one of claims 1 to 43, wherein: - alkyl.

45. R ６ 45. The compound of any of claims 1 to 44, wherein is unbranched.

46. R ６ 46. ​​The compound of any of claims 1 to 45, wherein is methyl, ethyl, n-butyl, n-hexyl, n-octyl, or n-decyl.

47. R ６ 47. The compound of any of claims 1 to 46, wherein is unsubstituted.

48. R ６ 47. The compound of any one of claims 1 to 46, wherein:

49. R ６ C is substituted with one or more hydroxyl groups １ -C ６ 43. The compound of any one of claims 1 to 42, which is alkyl.

50. R ６ C is substituted with one or more hydroxyl groups ２ -, C ３ -, C ４ - or C ６ - alkyl.

51. R ６ The compound of any one of claims 1 to 42, wherein is hydrogen.

52. R ２ -(C=O)OR ７ 52. The compound of any one of claims 1 to 51,

53. R ７ is C １ -C ３ Alkyl or C ７ -C １６ 53. The compound of claim 52, which is arylalkyl.

54. R ７ 54. The compound of claim 52 or 53, wherein is unsubstituted.

55. R ７ Ga-CH ３ or the following structure: 【Chemistry 6】 55. The compound of any of claims 52 to 54, having the formula:

56. R ３ is optionally substituted C １ -C ６ 56. The compound of any one of claims 1 to 55, which is alkyl.

57. R ３ 57. The compound of any of claims 1 to 56, wherein is optionally substituted methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl.

58. R ３ 58. The compound of any of claims 1-57, wherein is optionally substituted methyl.

59. R ３ C is substituted with one or more hydroxyl groups １ -C ６ 57. The compound of any one of claims 1 to 56, wherein the compound is alkyl.

60. R ３ C is substituted with one or more hydroxyl groups ２ - or C ４ - alkyl.

61. R ３ 57. The compound of any of claims 1-56, wherein is unsubstituted.

62. R ３ 56. The compound of any one of claims 1 to 55, wherein is hydrogen.

63. X is 【Chemistry 7】 63. The compound of any one of claims 2 to 62, wherein

64. The compound has the following structure (II): 【Chemistry 8】 64. The compound of any of claims 2 to 63, having the structure: or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

65. 65. The compound of any one of claims 2 to 64, wherein n2 is 3, 4, or 5.

66. X is 【Chemistry 9】 63. The compound of any one of claims 2 to 62, wherein

67. The compound has the following structure (III): 【Chemistry 10】 67. The compound of claim 66, having the structure: or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

68. 68. The compound of claim 66 or 67, wherein n3 is 0 or 1.

69. The compound of any one of claims 66 to 68, wherein n4 is 2 or 3.

70. 70. The compound of any one of claims 66 to 69, wherein n5 is 3.

71. 71. The compound of any one of claims 1 to 70, wherein n1 is 2.

72. The compound has the following structure: 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 【Chemistry 18】 【Chemistry 19】 10. The compound of claim 1, having the structure: or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

73. A composition comprising a compound of any of claims 1 to 72 and a therapeutic agent.

74. A lipid nanoparticle comprising a compound of any one of claims 1 to 72 and a therapeutic agent.

75. 75. The composition or lipid nanoparticle of claim 73 or 74, further comprising one or more additives selected from neutral lipids, steroids, and polymer-conjugated lipids.

76. 75. The composition or lipid nanoparticle of claim 73 or 74, wherein the composition comprises one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.

77. 77. The composition or lipid nanoparticle of claim 76, wherein the neutral lipid is DSPC.

78. 78. The composition or lipid nanoparticle of any of claims 75 to 77, wherein the molar ratio of compound to neutral lipid ranges from about 2:1 to about 8:

1.

79. 79. The composition or lipid nanoparticle of any of claims 75 to 78, wherein the steroid is cholesterol.

80. 80. The composition or lipid nanoparticle of claim 79, wherein the molar ratio of compound to cholesterol ranges from about 2:1 to 1:

1.

81. 81. The composition or lipid nanoparticle of any of claims 75 to 80, wherein the polymer-conjugated lipid is a PEGylated lipid.

82. 82. The composition or lipid nanoparticle of claim 81, wherein the molar ratio of compound to pegylated lipid ranges from about 100:1 to about 10:1 or from about 100:1 to about 25:

1.

83. 83. The composition or lipid nanoparticle of any of claims 75 to 82, wherein the pegylated lipid is PEG-DMG, PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG dialkyloxypropylcarbamate.

84. The lipid nanoparticle of claim 83, wherein the pegylated lipid is PEG-DMG.

85. The PEGylated lipid has the following structure (II): 【Chemistry 20】 [During the ceremony, R ８ and R ９ are each independently an unbranched or branched alkyl, alkenyl, or alkynyl containing from 10 to 30 carbon atoms, wherein the alkyl, alkenyl, or alkynyl is optionally interrupted by one or more ester linkages; and z has an average value in the range of 30 to 60.

83. The composition or lipid nanoparticle of any of claims 75 to 82, having the structure of: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

86. R ８ and R ９ 86. The composition or lipid nanoparticle of claim 85, wherein each independently is an unbranched alkyl chain having 12 to 16 carbon atoms.

87. 87. The composition or lipid nanoparticle of claims 85-86, wherein the average z is about 45.

88. 88. The composition or lipid nanoparticle of any of claims 73 to 87, wherein the therapeutic agent comprises a nucleic acid.

89. 89. The composition or lipid nanoparticle of claim 88, wherein the nucleic acid is selected from antisense and messenger RNA.

90. A pharmaceutical composition comprising the lipid nanoparticles of any one of claims 74 to 89 and a pharmaceutically acceptable excipient.

91. 91. A method of administering a therapeutic agent to a patient in need thereof, comprising manufacturing or preparing the pharmaceutical composition of claim 90 and administering said composition to the patient.

92. A method for inducing expression of a protein in a patient in need of treatment, comprising administering to the patient the pharmaceutical composition of claim 90, wherein the lipid nanoparticles comprise mRNA encoding the protein.

93. 93. The method of claim 92, wherein the protein is an antigen and the method is a method of inducing an immune response in a patient.

94. 93. The method of claim 92, wherein the protein is an antigen and the method is for vaccinating a patient against a pathogen.

95. 93. The method of claim 92, wherein the protein is for gene editing.

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