Lipids for use in lipid nanoparticle formulations

Abstract

The structure: TIFF2024546952000084.tif2643(I) or a pharma- ceutically acceptable salt, tautomer or stereoisomer thereof, wherein R 3 , L 1 , L 2 , G 1 , G 2 and G 3 is as defined herein. Use of the compounds as components of lipid nanoparticle formulations for therapeutic agent delivery, compositions comprising the compounds, and methods of use and preparation thereof are also provided.

Description

[Technical field]

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

[0002] There are many challenges associated with nucleic acid delivery to produce desired responses in biological systems. Nucleic acid-based therapeutics have great potential, but to realize this potential, there remains a need to more efficiently deliver nucleic acids to the appropriate location within a cell or organism. Therapeutic nucleic acids include, for example, messenger RNA (mRNA), antisense oligonucleotides, ribozymes, deoxyribozymes, plasmids, immunostimulatory nucleic acids, antagomirs, antimirs, mimetics, supermirs, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to produce the expression of specific cellular products, such as those useful for treating diseases associated with protein or enzyme deficiencies. The therapeutic applications of translatable nucleotide delivery are extremely broad, since constructs can be synthesized to produce any selected protein sequence, whether native or not to the system. The expression product of the nucleic acid can increase existing levels of a protein in a cell or organism, replace a missing or non-functional version of a protein, or introduce a new protein and associated functionality.

[0003] Some nucleic acids, such as miRNA inhibitors, can be used to cause the expression of specific cellular products controlled by miRNA, which can be useful for treating diseases related to protein or enzyme deficiency.The therapeutic application of miRNA inhibition is very broad, since constructs can be synthesized to inhibit one or more miRNAs, which in turn controls the expression of mRNA products.The inhibition of endogenous miRNA can increase the expression of its downstream target endogenous protein in cells or organisms and restore proper function, as a means to treat diseases related to specific miRNA or a group of miRNAs.

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

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

[0006] There remains a need for improved lipids and lipid nanoparticles for oligonucleotide delivery. Preferably, these lipid nanoparticles provide an optimal drug:lipid ratio, protect the nucleic acid from degradation and clearance in serum, are suitable for systemic or local delivery, and provide intracellular delivery of the nucleic acid. In addition, these lipid-nucleic acid particles should be well tolerated and provide a sufficient therapeutic index so that treatment of a patient with an effective amount of nucleic acid is not accompanied by unacceptable toxicity and / or risk to said patient. The present invention provides these and related advantages. Summary of the Invention [Means for solving the problem]

[0007] In summary, embodiments of the present invention provide lipid compounds (including their stereoisomers, pharma-ceutically acceptable salts, prodrugs 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 delivery of therapeutic agents.In some cases, lipid nanoparticles are used to deliver nucleic acids, such as antisense and / or messenger RNA.Methods of using such lipid nanoparticles for the treatment of various diseases or conditions, such as those caused by infectious agents and / or protein defects, are also provided.

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

[0009] Also provided are pharmaceutical compositions comprising one or more of the compounds of structure (I) above and a therapeutic agent. Also provided are lipid nanoparticles (LNPs) comprising one or more of the compounds of structure (I). In some embodiments, the pharmaceutical compositions and / or LNPs further comprise one or more components selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. The disclosed compositions are useful for forming lipid nanoparticles for therapeutic agent delivery.

[0010] In other embodiments, the present invention provides a method of administering a therapeutic agent to a patient in need thereof, comprising preparing a lipid nanoparticle composition comprising a compound of structure (I) and a therapeutic agent, and delivering said composition to the patient. In some embodiments, the method of administering a therapeutic agent to a patient in need thereof comprises administering to the patient a LNP comprising one or more compounds of structure (I) and a therapeutic agent.

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

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

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

[0014] In certain embodiments, the present invention provides novel lipids that allow for the formulation of improved compositions for in vitro and in vivo delivery of mRNA and / or other oligonucleotides. In some embodiments, these improved lipid nanoparticle compositions are useful for the expression of proteins encoded by mRNA. In other embodiments, these improved lipid nanoparticle compositions are useful for upregulating endogenous protein expression by delivering miRNA inhibitors that target 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) protein and / or mRNA levels of target genes. In some other embodiments, the lipid nanoparticles are also useful for the delivery of mRNA and plasmids for transgene expression. In still other embodiments, the lipid nanoparticle compositions are useful for inducing pharmacological effects resulting from protein expression, such as increased red blood cell production via delivery of appropriate erythropoietin mRNA, or protection from infection via delivery of appropriate antigen or antibody-encoding mRNA.

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

[0016] As described herein, lipid nanoparticle embodiments of the invention are particularly useful for delivery of nucleic acids, including, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomir / antimir), messenger RNA interfering complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), etc. Thus, the lipid nanoparticles and compositions of the invention embodiments can be used to induce expression of a desired protein both in vitro and in vivo by contacting a cell with lipid nanoparticles comprising one or more of the novel lipids described herein, where the lipid nanoparticles encapsulate or bind a nucleic acid (e.g., a messenger RNA or plasmid encoding a desired protein) that is expressed to produce the desired protein, or inhibit a process that terminates mRNA expression (e.g., a miRNA inhibitor). Alternatively, the lipid nanoparticles and compositions of the present invention may 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 lipids described herein (e.g., compounds of structure (I)), in which the lipid nanoparticles encapsulate or are associated with a nucleic acid (e.g., an antisense oligonucleotide or small interfering RNA (siRNA)) that reduces expression of the target gene. The lipid nanoparticles and compositions of the present invention may also be used to co-deliver different nucleic acids (e.g., mRNA and plasmid DNA), either separately or in combination, such as may be useful to provide effects that require co-localization of different nucleic acids (e.g., mRNA encoding an appropriate gene-modifying enzyme and a DNA segment(s) for integration into the host genome).

[0017] Nucleic acids for use with embodiments of the present invention may be prepared according to any available technique. For mRNA, the primary preparation method is, but is not limited to, enzymatic synthesis (also called in vitro transcription), which currently represents the most efficient method of generating long sequence-specific mRNA. In vitro transcription represents a template-directed process of synthesis of RNA molecules from an engineered DNA template composed of an upstream bacteriophage promoter sequence (e.g., including but not limited to those 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 using suitable techniques known in the art, including but not limited to plasmid DNA and polymerase chain reaction amplification (see Linpinsel, JL and Conn, GL, General protocols for preparation of plasmid DNA template and Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods Vol. 941, Conn GL (ed.), New York, NY Humana Press, 2012).

[0018] Transcription of RNA occurs in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and ribonucleoside triphosphates (rNTPs) of adenosine, guanosine, uridine and cytidine under conditions that support polymerase activity while minimizing possible degradation of purified mRNA transcripts. In vitro transcription can be performed using a variety of commercially available kits, including, but not limited to, RiboMax Large Scale RNA Production System (Promega), MegaScript Transcription kit (Life Technologies), and using commercially available reagents, including RNA polymerase and rNTPs. Methods for in vitro transcription of mRNA are known in the art (e.g., Losick, R., 1972, In vitro transcription, Ann Rev Biochem v. 41: 409-46; Kamaka, 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 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: 117-120). See pages 101-114, all of which are incorporated herein by reference.

[0019] The desired, in vitro transcribed mRNA is then purified from undesired components of the transcription or related reaction, including unincorporated rNTPs, protein enzymes, salts, short RNA oligos, etc. Techniques for isolation of mRNA transcripts are known in the art. Known procedures include phenol / chloroform extraction or precipitation with any alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Additional 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 vol. 10, pp. 889-893), silica-based affinity chromatography and polyacrylamide gel electrophoresis (Bowman, JC, Azizi, B., Lenz, TK, Ray, P. and Williams, LD in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods vol. 941 Conn GL (ed.), New York, NY Humana Press, 2012). Purification can be performed using a variety of commercially available kits, including, but not limited to, SV Total Isolation System (Promega) and In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0020] Furthermore, although reverse transcription can produce a large amount of mRNA, the product may contain some abnormal RNA impurities associated with undesired polymerase activity that may need to be removed from full-length mRNA preparations. These include abortive transcription initiation and short RNAs resulting from RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates, and double-stranded RNA (dsRNA) generated by self-complementary 3' extension. It has been demonstrated that these contaminants with dsRNA structure can result in undesired immune stimulatory activity through interaction with various innate immune sensors in eukaryotic cells that recognize specific nucleic acid structures and function to induce strong immune responses. This in turn can dramatically reduce mRNA translation due to reduced protein synthesis during the cellular innate immune response. Therefore, additional techniques for removing these dsRNA contaminants have been developed and are known in the art, including, but not limited to, scalable HPLC purification (see, e.g., Kariko, K., Muramatsu, H., Ludwig, J., and Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, vol. 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 vol. 969 (ed. Rabinovich, PH), 2013). HPLC-purified mRNA has been reported to be translated at much greater levels, especially in primary cells and in vivo.

[0021] A considerable variety of modifications have been described in the art that are used to change certain properties of in vitro transcribed mRNA and improve its usefulness. These include, but are not limited to, modifications of the 5' and 3' ends of the mRNA. Endogenous mRNAs in eukaryotes usually contain a cap structure on the 5' end of the mature molecule that plays an important role in mediating the binding of mRNA cap-binding protein (CBP), which in turn is involved in enhancing RNA stability in cells and the efficiency of mRNA translation. Thus, the highest level of protein expression is achieved with capped mRNA transcripts. The 5' cap contains a 5'-5' triphosphate bond between the 5'-most nucleotide and a guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the 5'-most nucleotide and the penultimate 5'-most nucleotide on the 2' hydroxy group.

[0022] Several different cap structures may be used to generate the 5' cap of in vitro transcribed synthetic mRNA. 5' capping of synthetic mRNA can be performed by co-transcription with a chemical cap analog (i.e., capping during in vitro transcription). For example, the anti-reverse cap analog (ARCA) cap contains a 5'-5' triphosphate guanine-guanine linkage in which one guanine contains an N7 methyl group as well as a 3'-O-methyl group. However, up to 20% of the transcripts remain uncapped during this co-transcription process, making the synthetic cap analog non-identical to the 5' cap structure of authentic cellular mRNA, potentially reducing translatability and cellular stability. Alternatively, synthetic mRNA molecules may also be enzymatically capped post-transcriptionally. These may generate more authentic 5' cap structures that more closely mimic endogenous 5' caps structurally or functionally with enhanced binding of cap-binding proteins, extended half-life, reduced susceptibility to 5' endonucleases, and / or reduced 5' decapping. To enhance mRNA stability and translatability, many synthetic 5' cap analogs have been developed and are known in the art (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 Vol. 969 (ed. Rabinovich, PH), 2013).

[0023] At the 3' end, a long chain of adenine nucleotides (polyA tail) is usually added to the mRNA molecule during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved, liberating a 3' hydroxyl group to which polyA polymerase adds a chain of adenine nucleotides to the RNA in a process called polyadenylation. Poly(A) tails have been widely shown to enhance both the translation efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly (A), poly (A) binding protein and the regulation of mRNA stability, Trends Bio Sci 14:373-377; Guhaniyogi, J. and Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene 265:11-23; Dreyfus, M. and Regnier, P., 2002, The poly (A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell 111:611-613).

[0024] Poly(A) tailing of in vitro transcribed mRNA can be achieved using a variety of techniques, including but not limited to cloning of a poly(T) tract into a DNA template or post-transcriptional addition using poly(A) polymerase. The first case allows in vitro transcription of mRNAs with poly(A) tails of defined length, depending on the size of the poly(T) tract, but requires additional manipulation of the template. The latter case involves enzymatic addition of poly(A) tails to in vitro transcribed mRNAs using poly(A) polymerase, which catalyzes the incorporation of adenine residues into the 3' end of the RNA, and does not require additional manipulation of the DNA template, but results in mRNAs with poly(A) tails of heterogeneous length. 5' capping and 3' poly(A) tailing can be performed using a variety of commercially available kits, including but not limited to, Poly(A) Polymerase Tailing Kit (EpiCenter), mMESSAGE, mMACHINE T7 Ultra Kit and Poly(A) Tailing Kit (Life Technologies), as well as using commercially available reagents, various ARCA caps, poly(A) polymerases, etc.

[0025] In addition to 5' cap and 3' polyadenylation, other modifications of in vitro transcripts have been reported to provide benefits such as those related to translation efficiency and stability. It is known in the art that pathogenic DNA and RNA can be recognized by various sensors in eukaryotic cells and trigger a strong innate immune response. Since most nucleic acids from natural sources contain modified nucleosides, it has been shown that the ability to distinguish between pathogenic DNA and RNA and self-DNA and RNA is based, at least in part, on structure and nucleoside modifications. In contrast, in vitro synthesized RNA lacks these modifications and therefore becomes immunostimulatory, which in turn can inhibit efficient 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 undesired 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, vol. 10, pp. 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, vol. 969 (Rabinovich, PH Ed), 2013); Kariko, K., Muramatsu, H., Welsh, FA, Ludwig, J., Kato, H., Akira, S., See Weissman, D., 2008, Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability, Mol Ther 16:1833-1840. Modified nucleosides and nucleotides used in the synthesis of modified RNA can be prepared, monitored and utilized using general methods and procedures known in the art.A wide variety of nucleotide modifications are available that may be incorporated to some degree into in vitro transcribed mRNA, either alone or in combination with other modified nucleosides (see, e.g., US2012 / 0251618). In vitro synthesis of nucleoside-modified mRNA has been reported to reduce its ability to activate immune sensors while simultaneously enhancing its translational capacity.

[0026] Other components of mRNA that can be modified to provide benefits in terms of translatability and stability include the 5' and 3' untranslated regions (UTRs). Optimizing UTRs (preferably 5' and 3' UTRs can be obtained from cellular or viral RNA), either together or independently, has been shown to increase mRNA stability and translation efficiency of in vitro translated mRNAs (see, for example, Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology, Vol. 969 (Rabinovich, PH, ed.), 2013).

[0027] In addition to mRNA, other nucleic acid payloads may be used for embodiments of the present invention. For oligonucleotides, methods of preparation include, but are not limited to, chemical synthesis and enzymatic or chemical cleavage of long precursors, in vitro transcription as described above, etc. Methods for synthesizing DNA and RNA nucleotides are widely used and 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, Vol. 288 (Clifton, NJ) Totowa, NJ: Humana Press, 2005; both of which are incorporated herein by reference).

[0028] Preparation of plasmid DNA for use with embodiments of the present invention typically involves, but is not limited to, in vitro propagation and isolation of the plasmid DNA in liquid cultures containing the plasmid of interest. The presence of genes in the plasmid of interest that code for resistance to certain antibiotics (penicillin, kanamycin, etc.) allows bacteria containing the plasmid of interest to be selectively grown in cultures containing the antibiotic. Methods for isolating plasmid DNA are widely used and known in the art (see, for example, Heilig, J., Elbing, KL and Brent, R., (2001) Large-Scale Preparation of Plasmid DNA, Current Protocols in Molecular Biology. Vol. 41:I Issue:1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillstrom, S., Bjornestedt, R. and Schmidt, SR (2008), Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture, Biotechnol. Bioeng., Vol. 99:557-566; and US 6197553B1). Plasmid isolation can be performed using a variety of commercially available kits, including but not limited to Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo), and PureYield MaxiPrep (Promega) kits, as well as using commercially available reagents.

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

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

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

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

[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 references unless the context clearly dictates otherwise.

[0034] The phrase "induce expression of a desired protein" refers to the ability of a nucleic acid to increase the expression of a desired protein. To test the degree of protein expression, a test sample (e.g., a cell sample in culture expressing the desired protein) or a test mammalian (e.g., a mammalian such as a human or animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) is contacted with a nucleic acid (e.g., a nucleic acid combined with a lipid of the present invention). The expression of the desired protein in the test sample or test animal is compared to the expression of the desired protein in a control sample (e.g., a cell sample in culture expressing the desired protein) or a control mammalian (e.g., a mammalian such as a human or animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model, that is not contacted with or administered the nucleic acid. If the desired protein is present in the control sample or control mammal, the expression of the desired protein in the control sample or control mammal may be assigned a value of 1.0. In one embodiment, induction of expression of a desired protein is achieved when the ratio of expression of the desired protein in a test sample or test mammal to expression of the desired protein 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. If the desired protein is not present in the control sample or control mammal, induction of expression of a desired protein is achieved when any measurable level of the desired protein is detected in the test sample or test mammal. Those skilled in the art will know suitable assays for determining the expression level of a protein in a sample, such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays, or assays based on reporter proteins that can generate fluorescence or luminescence under appropriate conditions.

[0035] The phrase "inhibit expression of a target gene" refers to the ability of a nucleic acid to silence, reduce or inhibit expression of a target gene. To test the degree of gene silencing, a test sample (e.g., a cell sample in culture expressing the target gene) or a test mammalian (e.g., a mammalian such as a human or animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) is contacted with a nucleic acid that silences, reduces or inhibits expression of the target gene. The expression of the target gene in the test sample or test mammalian is compared with the expression of the target gene in a control sample (e.g., a cell sample in culture expressing the target gene) or a control mammalian (e.g., a mammalian such as a human or animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) that is not contacted with or administered the nucleic acid. The expression of the target gene in the control sample or control mammalian may be assigned a value of 100%. In certain embodiments, silencing, inhibition, or reduction of a target gene is achieved when the expression level of the target gene in a test sample or test mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to the expression level of the target gene in a control sample or control mammal. In other words, the nucleic acid can silence, reduce or inhibit the expression of the target gene in the test sample or test mammal by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to the expression level of the target gene in a control sample or control mammal that has not been contacted with or administered the nucleic acid. Suitable assays for determining the expression level of the 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 blots, northern blots, 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, e.g., 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 is detected in the case of an expression product that is not present in the absence of the nucleic acid. When the expression product is present at a level prior to contact with the nucleic acid, increased expression is achieved when the fold increase over the value obtained with a nucleic acid, such as mRNA, 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 times greater than the control. Inhibition of expression of a target gene or sequence is achieved when the value obtained using a nucleic acid such as an antisense oligonucleotide is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% of the control. Suitable assays for measuring expression of a target gene or sequence include, for example, testing protein or RNA levels using techniques known to those skilled in the art, such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of a suitable reporter protein, as well as phenotypic assays known to those skilled in the art.

[0037] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, including DNA, RNA, and hybrids thereof. DNA may be in the form of an antisense molecule, a plasmid DNA, a cDNA, a PCR product, or a vector. RNA may be in the form of a small hairpin RNA (shRNA), a messenger RNA (mRNA), an antisense RNA, a miRNA, a micRNA, a polyvalent RNA, a dicer substrate RNA, or a viral RNA (vRNA), and combinations thereof. Nucleic acids are synthetic, natural, and unnatural, and include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that have similar binding properties as 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 limited, the term "nucleic acid" encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses not only the explicitly specified sequence, but also its variants modified in traditional manner (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the 3-position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate group."Base" includes purines and pyrimidines, which include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogues, and further include synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that place new reactive groups such as amines, alcohols, thiols, carboxylates, and alkyl halides.

[0038] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that comprises partial or full-length coding sequences necessary for the production of a polypeptide or a precursor of a polypeptide.

[0039] "Gene product," as used herein, 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 poorly soluble in water but soluble in many organic solvents. Lipids are usually divided into at least three types: (1) "simple lipids", which include fats and oils as well as waxes; (2) "complex lipids", which include phospholipids and glycolipids; and (3) "derived lipids", such as steroids.

[0041] Steroids have the following carbon skeleton: [ka] It is a compound comprising: Non-limiting examples of steroids include cholesterol.

[0042] "Cationic lipid" refers to a lipid that can be positively charged. Exemplary cationic lipids contain one or more positively charged amino groups. Preferred cationic lipids are ionizable so that they can exist in a positively charged or neutral form depending on the pH. The ionization of cationic lipids affects the surface charge of lipid nanoparticles under different 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 that have important implications for intracellular delivery of nucleic acids (Hafez, IM, et al., Gene Ther 8:1188-1196 (2001)).

[0043] 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) and the like.

[0044] 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, and steroids, such as sterols and their derivatives. The neutral lipids may be synthetic or naturally occurring.

[0045] The term "charged lipid" refers to any of several lipid species that exist in a positively or negatively charged form, independent of pH, in a useful physiological range, for example, from about pH 3 to about pH 9. Charged lipids can be synthetic or naturally derived. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammoniumpropane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethanecarbamoylsterol (e.g., DC-Chol).

[0046] The term "lipid nanoparticle" refers to a particle that comprises one or more compounds of structure (I) or other specific cationic lipids and has at least one dimension on the order of nanometers (e.g., 1-1,000 nm). In some embodiments, the lipid nanoparticles are included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), to a target site of interest (e.g., a cell, tissue, organ, tumor, etc.). In some embodiments, the lipid nanoparticles of the present invention comprise a nucleic acid. Such lipid nanoparticles typically comprise a compound of structure (I) and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. In some embodiments, the active agent or therapeutic agent, such as a nucleic acid, may be encapsulated in the lipid portion of the lipid nanoparticle or in the aqueous space enclosed by part or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other deleterious effects induced by the host organism's or cells' machinery, such as a harmful immune response.

[0047] In various embodiments, the lipid nanoparticles are about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or 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, the nucleic acid, when present in the lipid nanoparticle, is resistant to degradation by nucleases in aqueous solution. Lipid nanoparticles containing nucleic acids and methods for their preparation are disclosed, for example, in U.S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031 and PCT Publication Nos. WO2017 / 004143, WO2015 / 199952, WO2013 / 016058, and WO2013 / 086373, the complete disclosures of which are incorporated by reference herein in their entireties for all purposes.

[0048] As used herein, "lipid encapsulated" refers to a lipid nanoparticle that provides an active or therapeutic agent, such as a nucleic acid (e.g., mRNA), either fully encapsulated, partially encapsulated, or both. In some embodiments, the nucleic acid (e.g., mRNA) is fully encapsulated within the lipid nanoparticle.

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

[0050] In the context of nucleic acid-lipid nanoparticles, "serum stable" means that nucleotides are not significantly degraded after exposure to serum or nuclease assays that significantly degrade free DNA or RNA.Suitable assays include, for example, standard serum assays, DNAse assays, or RNAse assays.

[0051] "Systemic delivery" as used herein refers to delivery of a therapeutic product that can result in widespread exposure of an active agent within an organism. Some administration techniques can result in systemic delivery of certain agents, while others cannot. Systemic delivery means that a useful, preferably therapeutic, amount of the agent 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.

[0052] "Local delivery" as used herein refers to the delivery of an active agent directly to a target site in an organism. For example, an agent can be delivered locally by direct injection into a disease site, such as a tumor, other target site, such as an inflammatory site, or into a target organ, such as the liver, heart, pancreas, kidney, etc. Local delivery can also include techniques of local application or local injection, such as intramuscular, subcutaneous or intradermal injection. Local delivery does not exclude systemic pharmacological action.

[0053] "Alkyl" means a saturated alkyl group having, for example, 1 to 24 carbon atoms (C 1 ~C 24 alkyl), 4 to 20 carbon atoms (C 4 ~C 20 alkyl), 6 to 16 carbon atoms (C 6 ~C 16 alkyl), 6 to 9 carbon atoms (C 6 ~C 9 alkyl), 1 to 15 carbon atoms (C 1 ~C 15 alkyl), 1 to 12 carbon atoms (C 1 ~C 12 alkyl), 1 to 8 carbon atoms (C 1 ~C 8 alkyl), or 1 to 6 carbon atoms (C 1 ~C 6alkyl), and refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms attached to the remainder of the molecule by a single bond, 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 the specification, alkyl groups are optionally substituted.

[0054] "Alkoxy" means a group of the formula -OR a In this case, R a is an alkyl group as defined above containing 1 to 12 carbon atoms. Unless stated otherwise in the specification, the alkoxy group is optionally substituted.

[0055] "Alkyl aminyl" means -NHR a or -NR a R a In this case, R a are each independently an alkyl group, as defined above, containing 1 to 12 carbon atoms. Unless stated otherwise in the specification, an alkylaminyl group is optionally substituted.

[0056] "Alkenyl" refers to an alkyl group that is unsaturated (i.e., contains at least one carbon-carbon double bond) and has, for example, 2 to 24 carbon atoms (C 2 ~C 24 alkenyl, 4 to 20 carbon atoms (C 4 ~C 20 alkenyl, 6 to 16 carbon atoms (C 6 ~C 16 alkenyl), 6 to 9 carbon atoms (C 6 ~C 9 alkenyl), 2 to 15 carbon atoms (C 2 ~C 15 alkenyl), 2 to 12 carbon atoms (C 2 ~C 12 alkenyl), 2 to 8 carbon atoms (C 2 ~C 8 alkenyl), or 2 to 6 carbon atoms (C 2 ~C6 alkenyl) and refers to a straight or branched hydrocarbon chain radical consisting only of carbon and hydrogen atoms attached to the rest of the molecule by a single bond, e.g., ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, etc. Unless stated otherwise in the specification, alkenyl groups are optionally substituted.

[0057] An "alkylene" or "alkylene chain" is a saturated alkylene having, for example, 1 to 24 carbon atoms (C 1 ~C 24 Alkylene, 2 to 24 carbon atoms (C 2 ~C 24 alkylene), 1 to 15 carbon atoms (C 1 ~C 15 alkylene), 1 to 12 carbon atoms (C 1 ~C 12 alkylene), 1 to 8 carbon atoms (C 1 ~C 8 alkylene), 1 to 6 carbon atoms (C 1 ~C 6 Alkylene, 2 to 4 carbon atoms (C 2 ~C 4 alkylene), 1-2 carbon atoms (C 1 ~C 2 "alkylene" refers to a straight or branched divalent hydrocarbon chain, consisting solely of carbon and hydrogen atoms, linking the rest of the molecule to a radical group, e.g., methylene, ethylene, propylene, n-butylene, etc., having an alkylene group (alkylene) bond. 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 be through one or any two carbons within the chain. Unless stated otherwise in the specification, an alkylene chain can be optionally substituted.

[0058] An "alkenylene" or "alkenylene chain" refers to an alkenylene having one or more carbon-carbon double bonds, e.g., 2 to 24 carbon atoms (C 2 ~C 24 Alkenylene, 2 to 15 carbon atoms (C 2~C 15 Alkenylene, 2 to 12 carbon atoms (C 2 ~C 12 Alkenylene, 2 to 8 carbon atoms (C 2 ~C 8 Alkenylene, 2 to 6 carbon atoms (C 2 ~C 6 alkenylene) or 2 to 4 carbon atoms (C 2 ~C 4 "Alkenylene" refers to a straight or branched divalent hydrocarbon chain, consisting only of carbon and hydrogen, that attaches the rest of the molecule to a radical group, e.g., ethenylene, propenylene, n-butenylene, etc. The alkenylene chain is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond. The points of attachment of the alkenylene chain to the rest of the molecule and to the radical group can be through one or any two carbons within the chain. Unless stated otherwise in the specification, an alkenylene chain may be optionally substituted.

[0059] "Aryl" refers to a carbocyclic ring system containing hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring. For purposes of the present invention, aryl groups may be monocyclic, bicyclic, tricyclic, or tetracyclic ring systems and 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. Unless otherwise stated herein, the term "aryl" or the prefix "ar" (e.g., in "aralkyl") is intended to include aryl groups that are optionally substituted.

[0060] "Aralkyl" means a group of the formula -R b -R c where R bis alkylene or alkenylene as defined above, R c is one or more aryl groups as defined above, e.g., benzyl, diphenylmethyl, etc. Unless stated otherwise in the specification, an aralkyl group is optionally substituted.

[0061] "Heterocycle" refers to a stable 3- to 18-membered non-aromatic ring radical which consists of 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless stated otherwise in the specification, a heterocyclyl group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system which may include fused or bridged ring systems; and, the nitrogen, carbon, or sulfur atoms of the heterocyclyl group can be optionally oxidized; the nitrogen atom can be optionally quaternized; and the heterocyclyl group can be optionally partially or fully saturated. Examples of such heterocyclyl groups include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless stated otherwise in the specification, heterocyclyl groups may be optionally substituted.

[0062] "Heteroaryl" refers to a 5-14 membered ring system containing a hydrogen atom, 1-13 ring carbon atoms, 1-6 ring heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, and at least one aromatic ring containing a heteroatom. For purposes of this embodiment, a heteroaryl group may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system and may include fused or bridged ring systems; the nitrogen, carbon, or sulfur in the heteroaryl group may be optionally oxidized; and the nitrogen atom may be optionally quaternized. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzoindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl, (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indophenyl, isothiazolyl, imidazolyl, indophenyl, isothiazolyl ... These include dazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl). Unless stated otherwise in the specification, a heteroaryl group is optionally substituted.

[0063] As used herein, the term "substituted" refers to any of the above groups (e.g., alkyl, alkenyl, alkylene, alkenylene, aryl, and aralkyl) in which at least one hydrogen atom has been replaced with a non-hydrogen atom, such as, but not limited to: a halogen atom, such as F, Cl, Br, or I; an oxo group (=O); a hydroxy group (-OH); a carboxy group (-CO 2 H);C 1 ~C 12 Alkyl group;Cycloalkyl group;-(C=O)OR';-O(C=O)R';-C(=O)R';-OR';-S(O) x R';-S-SR';-C(=O)SR';-SC(=O)R';-NR'R';-NR'C(=O)R';-C(=O)NR'R';-NR'C(=O)NR'R';-OC(=O)NR'R';-NR'C(=O)OR';-NR'S(O) x NR'R';-NR'S(O) x R'; and -S(O) x NR'R', where R' at each occurrence is independently H or C 1 ~C 15 In some embodiments, the substituent is C 1 ~C 12 In other embodiments, the substituent is an alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as fluoro. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxy group. In other embodiments, the substituent is an alkoxy group (-OR'). In other embodiments, the substituent is a carboxy group. In other embodiments, the substituent is an amine group (-NR'R').

[0064] "Optional" or "optionally" (e.g., optionally substituted) means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not occur. For example, "optionally substituted alkyl" means that the alkyl group may be substituted or unsubstituted, and that the description includes both substituted alkyl and alkyl groups that have no substitution.

[0065] "Prodrug" is intended to denote a compound that can be converted under physiological conditions or by solvolysis into a biologically active compound of structure (I). Thus, the term "prodrug" refers to a pharma- ceutically acceptable metabolic precursor of a compound of structure (I). A prodrug may be inactive when administered to a subject in need thereof, but is converted in vivo into an active compound of structure (I). Prodrugs are usually rapidly converted in vivo, for example, by hydrolysis in blood, to produce the parent compound of structure (I). Prodrug compounds often offer advantages of solubility, tissue compatibility, or sustained release in mammalian organisms (see Bundgard, H., Design of Prodrugs (1985), pp. 7-9, pp. 21-24 (Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi, T., et al., ACS Symposium Series, Vol. 14, and Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0066] The term "prodrug" is also intended to include any covalently bonded carrier that releases an active compound of structure (I) in vivo when such prodrug is administered to a mammalian subject. Prodrugs of compounds of structure (I) can be prepared by modifying functional groups present in the compounds of structure (I) such that the modifications are cleaved, either by routine manipulation or in vivo, to the parent compound of structure (I). Prodrugs include compounds of structure (I) in which a hydroxy, amino or mercapto group is bonded to any group that is cleaved to form a free hydroxy, free amino or free mercapto group, respectively, when a prodrug of the compound of structure (I) is administered to a mammalian subject. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohols or amide derivatives of amine functional groups in compounds of structure (I), and the like.

[0067] The embodiments of the invention disclosed herein are also intended to encompass all pharma- ceutically acceptable compounds of the compounds of structure (I) that are isotopically labeled by replacing one or more atoms with an atom having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, 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 125I. These radiolabeled compounds may be useful, for example, to determine or measure the effectiveness of compounds 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) or (II), for example those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The radioisotope tritium, i.e. 3 H, and carbon 14, i.e. 14 C are particularly useful for this purpose given their ease of incorporation and available means of detection.

[0068] Heavier isotopes, such as deuterium, i.e. 2 Substitutions such as H may be preferred in some circumstances as they may confer certain therapeutic advantages due to greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements.

[0069] Positron-emitting isotopes, e.g. 11 C. 18 F, 15 O, and 13 Substitution with, for example, N, may be useful in positron emission topography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds of structure (I) may generally be prepared by processes similar to those described in the Preparations and Examples as presented below, by conventional techniques known to those skilled in the art, or by substituting the appropriate isotopically labeled reagent in place of previously utilized non-labeled reagents.

[0070] The embodiments of the invention disclosed herein are also intended to encompass in vivo metabolic products of the disclosed compounds. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, esterification, etc., of the administered compound, primarily by enzymatic processes. Thus, embodiments of the invention include compounds produced by a process comprising administering a compound of the 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 structure (I) to an animal, such as a rat, mouse, guinea pig, monkey, or human, allowing a sufficient time for metabolism to occur, and isolating the conversion product thereof from urine, blood, or other biological sample.

[0071] "Stable compound" and "stable structure" are intended to refer to 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.

[0072] "Mammals" includes humans and both domestic animals, such as laboratory animals and pets (eg, cats, dogs, pigs, cows, sheep, goats, horses, rabbits), and non-domestic animals, such as wildlife.

[0073] A "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifying agent approved by the U.S. Food and Drug Administration as acceptable for use in humans or veterinary medicine.

[0074] "Pharmaceutically acceptable salt" includes both acid and base addition salts.

[0075] "Pharmaceutically acceptable acid addition salts" refers to those acids which retain the biological effectiveness and properties of the free base, are not otherwise biologically undesirable, and are anhydrous, non-limiting examples of which include inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, It refers to salts formed with glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc.

[0076] "Pharmaceutically acceptable base addition salt" refers to a salt that retains the biological effectiveness and properties of the free acid and is not biologically or otherwise undesirable. These salts are prepared from the addition of an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0077] Crystallization often produces solvates of the compound of structure (I). As used herein, the term "solvate" refers to an aggregate that includes one or more molecules of the compound of structure (I) and one or more molecules of a solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present invention may exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., as well as the corresponding solvated forms. In some embodiments, the compounds of structure (I) may exist as true solvates, while in other cases, the compounds of structure (I) may simply retain extraneous water or may be a mixture of water and extraneous solvent.

[0078] "Pharmaceutical composition" refers to a formulation of a compound of structure (I) with a vehicle generally accepted in the art for the delivery of a biologically active compound to a mammal, e.g., a human. Such vehicles include any pharma- ceutically acceptable carrier, diluent, or excipient therefor.

[0079] An "effective amount" or "therapeutically effective amount" refers to an amount of a compound of structure (I) that, when administered to a mammal, preferably a human, is sufficient to effect treatment in the mammal, preferably a human. The amount of lipid nanoparticles in embodiments of the present invention that constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one of ordinary skill in the art having regard to his or her own knowledge and this disclosure.

[0080] "Treating" or "treatment", as used herein, 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 the occurrence of a disease or condition in a mammal, particularly where such mammal is predisposed to, but has not yet been diagnosed as having, the condition; (ii) inhibiting a disease or condition, i.e., arresting its onset; (iii) alleviating the disease or condition, i.e., causing the regression of the disease or condition; 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 differ in that a particular disease or condition may not have a known causative agent (so the cause has not yet been discovered) and thus is not yet recognized as a disease, but only as an undesirable state or syndrome in which a particular set of symptoms, to a greater or lesser extent, has been identified by clinicians.

[0081] The compounds of structure (I) or their pharma- ceutically acceptable salts may contain one or more asymmetric centers and thus may give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be de?ned in terms of absolute stereochemistry for amino acids as (R)- or (S)-, or (D)- or (L)-. The embodiments of the present invention are intended to include all such possible isomers, as Well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L) isomers may be prepared using chiral synthons or chiral reagents or fractionated using conventional techniques, e.g., chromatographic and fractional crystallization methods. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from appropriate optically pure precursors or resolution of the racemates (or racemates of salts or derivatives) using, e.g., chiral high performance liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise specified, the compounds are intended to include both E and Z geometric isomers, likewise, all tautomeric forms are also intended to be included.

[0082] "Stereoisomers" refer to compounds made up of the same atoms joined by the same bonds, but with different, not interchangeable, three-dimensional structures. The present invention contemplates various stereoisomers and mixtures thereof, and includes "enantiomers," which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0083] "Tautomer" refers to a proton migration from one atom of a molecule to another atom of the same molecule. The invention includes tautomers of any of the above compounds.

[0084] compound In one aspect, the present invention provides novel lipid compounds that can be combined with other lipid components, such as neutral lipids, charged lipids, steroids, and / or polymer-conjugated lipids, to form lipid nanoparticles with oligonucleotides. Without wishing to be bound by theory, it is believed that these lipid nanoparticles shield the therapeutic agent from degradation in serum and provide effective delivery of the therapeutic agent to cells in vitro and in vivo.

[0085] In one embodiment, the compound has the following structure (I): [ka] (I) or a pharma- ceutically acceptable salt, prodrug or stereoisomer thereof. [In the formula, L 1 -NR a C(=O)R 1 or -C(=O)NR b R c and; L 2 -NR d C(=O)R 2 or -C(=O)NR e R f and; G 1 and G 2 are each independently2 ~C 12 Alkylene, or C 2 ~C 12 alkenylene; G 3 is C 1 ~C 24 Alkylene, or C 2 ~C 24 alkenylene; R a , R b , R d and R e are independently H, C 1 ~C 16 Alkyl or C 2 ~C 16 alkenyl; R c and R f are each independently 1 ~C 16 Alkyl or C 2 ~C 16 alkenyl; R 1 and R 2 are each independently 6 ~C 24 Alkyl or C 6 ~C 24 alkenyl; R 3 are H, -OH, CN, -N(R 4 )R 5 ;-C(=O)N(R 4 )R 5 ;-N(R 4 )C(=O)R 5 ;-N(R 4 )C(=O)OR 5 ;-C(=O)OR 6 , -OC(=O)R 6 , -OR 7 , heteroaryl or aryl; R 4 and R 5 are independently H, C 1 ~C 12 Alkyl, C 3 ~C 6 Cycloalkyl or C 3 ~C6 cycloalkenyl or R 4 and R 5 together with the nitrogen or carbon atom to which they are attached form a 5- to 7-membered heterocyclic ring; R 6 is H, C 1 ~C 12 Alkyl, C 2 ~C 12 alkenyl or aralkyl; R 7 is optionally substituted by hydroxy or alkoxy 1 ~C 12 is alkyl; and wherein, unless otherwise specified, alkyl, alkenyl, alkylene, alkenylene, aryl, and aralkyl are each independently substituted or unsubstituted. In certain embodiments, at least one alkyl, alkenyl, alkylene, alkenylene, C 3 ~C 6 Cycloalkyl or C 3 ~C 6 Cycloalkenyl, aryl or aralkyl may each be one or more fluorine and / or one or more oxo and / or one or more NH 2 and / or substituted with one or more alkylaminyl.

[0086] In one embodiment, the compound has the following structure (I): [ka] (I) or a pharma- ceutically acceptable salt, prodrug or stereoisomer thereof. [In the formula, L 1 -NR a C(=O)R 1 or -C(=O)NR b R c and; L 2 -NR d C(=O)R 2 or -C(=O)NR eR f and; G 1 and G 2 are each independently 1 ~C 12 Alkylene, or C 2 ~C 12 alkenylene; G 3 is C 1 ~C 24 Alkylene, or C 2 ~C 24 alkenylene; R a , R b , R d and R e are independently H, C 1 ~C 16 Alkyl or C 2 ~C 16 alkenyl; R c and R f are each independently 1 ~C 16 Alkyl or C 2 ~C 16 alkenyl; R 1 and R 2 are each independently 6 ~C 24 Alkyl or C 6 ~C 24 alkenyl; R 3 are H, -OH, CN, -N(R 4 )R 5 ;-C(=O)N(R 4 )R 5 ;-N(R 4 )C(=O)R 5 ;-C(=O)OR 6 , -OC(=O)R 6 or aryl; R 4 and R 5 are independently H, C 1 ~C 12 alkyl or R 4 and R 5together with the nitrogen or carbon atom to which they are attached form a 5- to 7-membered heterocyclic ring; R 6 is H, C 1 ~C 12 Alkyl, C 2 ~C 12 alkenyl or aralkyl; and Herein, unless otherwise specified, alkyl, alkenyl, alkylene, alkenylene, C 3 ~C 6 Cycloalkyl or C 3 ~C 6 Cycloalkenyl, aryl and aralkyl are each independently substituted or unsubstituted. In certain embodiments, at least one alkyl, alkenyl, alkylene, alkenylene, C 3 ~C 6 Cycloalkyl or C 3 ~C 6 Cycloalkenyl, aryl or aralkyl may each be one or more fluorine and / or one or more oxo and / or one or more NH 2 and / or substituted with one or more alkylaminyl.

[0087] In certain embodiments, G 3 In some embodiments, G is unsubstituted. 3 is substituted with one or more fluorine atoms. In more specific embodiments, for example, G 3 is C 1 ~C 12 In some embodiments, G is an alkylene. 3 is C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , or C 8 For example, in some embodiments, G 3 is methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, or n-octyl.

[0088] In certain embodiments, G 1 and / or G 2 In some embodiments, G is unsubstituted. 1 and / or G 2 is substituted with one or more fluorine atoms. In more specific embodiments, for example, G 1 and / or G 2 is C 1 ~C 12 In some embodiments, G is an alkylene. 1 and / or G 2 is C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , or C 8 It is alkylene.

[0089] In some embodiments of the above, the compound has the following structure (IA): [ka] (IA) have [wherein y and z are each independently an integer from 2 to 12, for example an integer from 2 to 6, for example 4 or 5]. In certain embodiments, y and z are each the integer 5. In certain embodiments, y and z are each the integer 6. In certain embodiments, y and z are each the integer 7. In certain embodiments, y and z are each the integer 9.

[0090] In some of the above embodiments, L 1 is -C(=O)NR b R c And L 2 is -C(=O)NR e R f In some of the above embodiments, L 1 -NR a C(=O)R 1 And L 2 -NR d C(=O)R2 It is.

[0091] In other embodiments of the foregoing, the compound has the following structure (IB) or (IC): [ka] has.

[0092] In some of the above embodiments, y and z are each independently an integer from 2 to 10, 2 to 8, 4 to 10, or 4 to 7. For example, in some embodiments, y is 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, z is 4, 5, 6, 7, 8, 9, 10, 11, or 12. In certain embodiments, y and z are each the integer 5. In certain embodiments, y and z are each the integer 6. In certain embodiments, y and z are each the integer 7. In some embodiments, y and z are each the integer 9. In some embodiments, y and z are the same, and in other embodiments, y and z are different.

[0093] In some of the above embodiments, R 1 Or R 2 , or both are branched chain C 6 ~C 24 For example, in some embodiments, R 1 and R 2 each independently represents the following structure: [ka] have [In the formula, R 7a and R 7b is independently for each occurrence: (a) H or C 1 ~C 12 alkyl, or (b) R 7a is H or C 1 ~C 12 is alkyl, R 7b together with the carbon atom to which it is attached, R 7btogether with the atom adjacent to and the carbon atom to which it is attached, form a carbon-carbon double bond; and a is an integer from 2 to 12, where R 7a , R 7b and a are R 1 and R 2 are each independently selected to be straight or branched chain and to contain from 6 to 20 carbon atoms. For example, in some embodiments, a is an integer from 5 to 9 or 8 to 12.

[0094] In some of the above embodiments, R 7a is H in at least one occurrence. For example, in some embodiments, R 7a is H for each occurrence. 7b C in at least one occurrence 1 ~C 8 For example, in some embodiments, C 1 ~C 8 Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.

[0095] In other embodiments, R 1 Or R 2 or both have the structure: [ka] It has one of the following.

[0096] In other embodiments, R 1 Or R 2 or both have the structure: [ka] It has one of the following.

[0097] In some of the above embodiments, R b , R c , R e and R f are each independently3 ~C 12 Alkyl or C 3 ~C 16 For example, in some embodiments, R b , R c , R e and R f is n-hexyl (-(CH 2 ) 5 CH 3 ), n-octyl (-(CH 2 ) 7 CH 3 ), n-decanyl (-(CH 2 ) 9 CH 3 ), n-dodecyl (-(CH 2 ) 11 CH 3 ), (-(CH 2 ) 14 CH 3 ), (-(CH 2 ) 15 CH 3 For example, in some embodiments, R b , R c , R e and R f is n-hexyl (-(CH 2 ) 5 CH 3 ) and in other embodiments, R b , R c , R e and R f is n-octyl (-(CH 2 ) 7 CH 3 In another example of some embodiments, R b , R c , R e and R f is n-decanyl (-(CH 2 ) 9 CH 3 In another embodiment, R b , R c , R e and R f is n-dodecyl (-(CH 2 ) 11 CH 3 ).

[0098] In some embodiments, R a and R d are each independently 1 ~C 12 Alkyl or C 2 ~C 12 alkenyl, R 1 and R 2 are each independently 6 ~C 18 Alkyl or C 6 ~C 18 For example, in some embodiments, R a and R d is n-hexyl (-(CH 2 ) 5 CH 3 ) and R 1 and R 2 is dodecapentyl (-(CH 2 ) 14 CH 3 In another example of some embodiments, R a and R d is n-hexyl (-(CH 2 ) 5 CH 3 ) and R 1 and R 2 is (6Z,9Z)-heptadeca-6,9-diene (-(CH 2 ) 7 CHCHCH 2 CHCH(CH 2 ) 4 CH 3 In still other examples of some embodiments, R a and R d is n-hexyl (-(CH 2 ) 5 CH 3 ) and R 1 and R 2 is 7-pentadecane (-CH((CH 2 ) 5 CH 3 )((CH 2 ) 7 CH 3 )).

[0099] In some embodiments, R 3 is H.

[0100] In other embodiments, R 3 is -OH.

[0101] In some other embodiments, R 3 -C(=O)OR 6 For example, in some of these embodiments, R 6 is C 1 ~C 18 In some exemplary embodiments, R 6 is C 1 ~C 12 In still other exemplary embodiments, R 6 is C 1 ~C 6 In some exemplary embodiments, R 6 is C 1 ~C 2 In certain embodiments, R 6 is ethyl. In certain embodiments, R 6 is 5-methyl dodecyl.

[0102] In other embodiments, R 3 -N(R 4 )R 5 For example, in some of these embodiments, R 4 and R 5 each independently represents C optionally substituted by hydroxy 1 ~C 12 In some exemplary embodiments, R 4 and R 5 each independently represents C optionally substituted by hydroxy 1 ~C 6 In still other exemplary embodiments, R 4 and R 5 each independently represents C optionally substituted by hydroxy 1 ~C 2In certain embodiments, R 4 and R 5 Each is methyl. In certain embodiments, R 4 and R 5 are n-hexyl.

[0103] In other embodiments, R 3 is -C(=O)N(R 4 )R 5 or -N(R 4 )C(=O)R 5 In some of these embodiments, R 4 or R 5 One of them is H and the other is R 4 or R 5 The other one is C 1 ~C 12 In some embodiments, R 4 or R 5 One of them is H and the other is R 4 or R 5 The other is a hydroxy-substituted C 1 ~C 12 In another embodiment, R 4 and R 5 Both are C 1 ~C 12 Alkyl or C 1 ~C 6 In some embodiments, R 4 and R 5 Both are hydroxy-substituted C 1 ~C 12 Alkyl or C 1 ~C 6 In certain embodiments, R 4 or R 5 One of them is H and the other is R 4 or R 5 The other is n-decyl. In certain embodiments, R 4 or R 5 One of them is H and the other is R 4 or R 5 The other is n-tridecyl.

[0104] In some embodiments, R 3 is aryl. In some embodiments, R 3 is an aryl substituent. In certain embodiments, R 3 is phenol.

[0105] In other embodiments, R 3 -N(R 4 )R 5 and R 4 and R 5 One of them is C 3 ~C 6 Cycloalkyl or C 3 ~C 6 For example, in some embodiments, R 3 -N(R 4 )R 5 and R 4 is H and R 5 is C 3 ~C 6 It is a cycloalkenyl.

[0106] In other embodiments, R 3 -OR 7 and R 7 is OH or OCH 3 C replaced with 1 ~C 6 It is an alkyl.

[0107] In other embodiments, R 3 -N(R 4 )C(=O)OR 5 where R 4 is H and R 5 is C 1 ~C 6 It is an alkyl.

[0108] In a further embodiment, R 3 is heteroaryl, for example imidazolyl.

[0109] In some more specific embodiments, R 3 has the following structure: [ka] It has one of the following.

[0110] In some more specific embodiments, R 3 is H or has the structure: [ka] It has one of the following.

[0111] In various other embodiments, the compound has one of the structures shown in Table 1 below. Table 1. Representative compounds [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] [Table 15] [Table 16] [Table 17]

[0112] The compounds listed in Table 1 were prepared and tested according to methods known in the art, for example the general methods described herein below.

[0113] It is understood that any embodiment of a compound of structure (I) as described above, and any particular substituent and / or variable in a compound of structure (I) as described above, can be independently combined with other embodiments of a compound of structure (I) and / or substituents and / or variables to form embodiments of the invention not specifically described above. In addition, when a list of substituents and / or variables is recited for any particular R group, L group, G group, or variable a, x, y, or z in a particular embodiment and / or claim, it is understood that each individual substituent and / or variable may be deleted from the particular embodiment and / or claim, and the remaining list of substituents and / or variables is considered to be within the scope of embodiments of the invention.

[0114] It is understood that in this description, combinations of substituents and / or variables of the depicted formula are permissible only if such contributions result in stable compounds.

[0115] In some embodiments, compositions are provided that include a compound of structure (I). In some embodiments, compositions are provided that include lipid nanoparticles that include a compound of structure (I). The lipid nanoparticles optionally include an excipient selected from neutral lipids, steroids, and polymer-conjugated lipids.

[0116] In some embodiments, lipid nanoparticles are provided that comprise any one or more of the compounds of structure (I) and a therapeutic agent. For example, in some embodiments, the lipid nanoparticles comprise any one of the compounds of structure (I), a therapeutic agent, and one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids. Other pharma-ceutically acceptable excipients and / or carriers are also included in various embodiments of the lipid nanoparticles.

[0117] In some embodiments, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC. In various embodiments, the molar ratio of the compound to the neutral lipid is about 2:1 to about 8:1.

[0118] In various embodiments, the lipid nanoparticle further comprises a steroid or a steroid analog. In certain embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of the compound to cholesterol is about 5:1 to 1:1 or about 2:1 to 1:1.

[0119] In various embodiments, the polymer-conjugated lipid is a PEGylated lipid. For example, some embodiments include PEGylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), and the like. G), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate, 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 various embodiments, the molar ratio of the compound to the PEGylated lipid is about 100:1 to about 20:1. In some embodiments, the molar ratio of the compound to the PEGylated lipid is about 100:1 to about 20:1 or about 100:1 to about 10:1.

[0120] In some embodiments, the composition has the following structure (II): [ka] (II) or a pharma- ceutically acceptable salt, tautomer or stereoisomer thereof. [In the formula, R 10 and R 11 are each independently a straight or branched chain alkyl, alkenyl, or alkynyl having 10 to 30 carbon atoms, wherein the alkyl, alkenyl, or alkynyl is optionally interrupted by one or more ester bonds; and w has an average value of 30 to 60].

[0121] In some embodiments, R 10 and R11 are each independently a straight chain alkyl containing 10 to 30 carbon atoms. 10 and R 11 are each independently a straight chain alkyl containing 12 to 16 carbon atoms. In some embodiments, w has an average value of 30 to 60. In some embodiments, w has an average value of 43 to 53. In other embodiments, w is about 45. In other embodiments, w is about 49.

[0122] Methods for preparing the above lipids, lipid nanoparticles and compositions are described herein below and / or known in the art, for example, PCT Publication Nos. WO2015 / 199952, WO2017 / 004143 and WO2017 / 075531, each of which is incorporated by reference in its entirety.

[0123] In some embodiments of the above compositions, the therapeutic agent comprises a nucleic acid, for example, in some embodiments, the nucleic acid is selected from antisense RNA and messenger RNA.

[0124] In another alternative embodiment, the present invention is directed to a method for administering a therapeutic agent to a patient in need thereof, comprising preparing or providing any of the aforementioned compositions and administering the composition to the patient.

[0125] For purposes of administration, the compound of structure (I) (usually in the form of lipid nanoparticles combined with a therapeutic agent) may be administered as a raw chemical or may be formulated as a pharmaceutical composition. In an embodiment of the present invention, the pharmaceutical composition comprises a compound of structure (I) (e.g., as a component of a LNP) and one or more pharma- ceutically acceptable carriers, diluents, or excipients. The compound of structure (I) is present in the composition in an amount that is effective to form lipid nanoparticles and deliver a therapeutic agent, for example, to treat a particular disease or condition of interest. Appropriate concentrations and dosages can be readily determined by one of skill in the art.

[0126] The administration of the compositions and / or LNPs in embodiments of the present invention may be via any of the accepted modes of administration of pharmaceuticals to perform similar utilities. The pharmaceutical compositions in embodiments of the present invention may be formulated into preparations in the form of solids, semi-solids, liquids or gases, 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, peritoneal, sublingual, oral buccal, rectal, vaginal and intranasal. The term peritoneal as used herein includes subcutaneous injections, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. The pharmaceutical compositions of the present invention are formulated such that the active ingredients contained therein are bioavailable when the compositions are administered to a patient. The composition administered to a subject or patient may take the form of one or more dosage units, where, for example, a tablet may be a single dosage unit, and a container of the compound of structure (I) in aerosol form may have multiple dosage units. Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition administered will, in any event, contain a therapeutically effective amount of the compound of structure (I) or a pharma- ceutically acceptable salt thereof for the treatment of the disease or condition of interest, according to the teachings of the embodiments of the present invention.

[0127] The pharmaceutical composition in the present embodiment may be in solid or liquid form. In one aspect, the carrier(s) are particulate, whereby the composition is, for example, in tablet or powder form. The carrier(s) may be liquid, whereby the composition is, for example, an oral syrup, an injectable solution, or an aerosol, which is useful, for example, for inhalation administration.

[0128] When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, with semi-solid, semi-liquid, suspension, and gel forms being included in forms that are considered herein to be either solid or liquid.

[0129] As a solid composition for oral administration, the pharmaceutical composition may be formulated into the form of powder, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions usually contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders, such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum or gelatin; excipients, such as starch, lactose or dextrin; disintegrants, such as alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants, such as magnesium stearate or Sterotex; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; flavorings, such as peppermint, methyl salicylate or orange flavoring, etc.; and colorings.

[0130] When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to the materials listed above, a liquid carrier, such as polyethylene glycol or oil.

[0131] The pharmaceutical composition may be in the form of a liquid, such as an elixir, syrup, solution, emulsion, or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, the preferred composition contains one or more of a sweetener, a preservative, a dye / colorant, and a flavor enhancer, in addition to the compound or LNP of the present invention. In the composition intended for administration by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and an isotonic agent may be included.

[0132] Liquid pharmaceutical compositions in the present embodiment, whether they are in a solution, suspension or other similar form, may contain one or more of the following adjuvants: sterile diluents, such as water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride, non-volatile oils, such as synthetic mono- or diglycerides, polyethylene glycols, glycerin, propylene glycol or other solvents that can act as solvents or suspending media; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates, and tonicity adjusters, such as sodium chloride or dextrose; agents that act as cryoprotectants, such as sucrose or trehalose. The intraperitoneal preparations can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Saline is the preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.

[0133] Liquid pharmaceutical compositions according to embodiments of the present invention intended for intraperitoneal or oral administration should contain a quantity of a compound of structure (I) sufficient to provide suitable LNP.

[0134] The pharmaceutical composition in the embodiment 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. Viscosifiers may be present in pharmaceutical compositions for topical administration. When intended for transdermal administration, the composition may comprise a transdermal patch or iontophoresis device.

[0135] The pharmaceutical composition of the present invention may be for rectal administration, for example in the form of a suppository that dissolves in the rectum and releases the drug.Compositions for rectal administration may contain an oily base as a suitable non-irritating excipient.Such bases include, but are not limited to, lanolin, cocoa butter and polyethylene glycol.

[0136] The pharmaceutical composition in the embodiment of the present invention may contain various materials that modify the physical form of a solid or liquid dosage unit.For example, the composition may contain materials that form a coating shell around the active ingredient.The materials that form the coating shell are usually inert and can be selected from, for example, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredient can be placed in a gelatin capsule.

[0137] Pharmaceutical compositions in embodiments of the invention, in solid or liquid form, may include an agent that binds to the compound of structure (I) and thereby aids in the delivery of the compound. Suitable agents that may act in this capacity include monoclonal or polyclonal antibodies, or proteins.

[0138] The pharmaceutical composition in the embodiment of the present invention may be comprised of a dosage unit that can be administered as an aerosol. The term aerosol is used to describe a variety of systems ranging from those of colloidal nature to those consisting of pressurized packages. Delivery may be by liquefied or compressed gas, or by a suitable pump system to dispense the active ingredient. The aerosol of the compound of structure (I) may be delivered in a single-phase, two-phase, or three-phase system to deliver the active ingredient(s). The delivery of the aerosol includes the necessary container, activator, valve, subcontainer, etc., which may together form a kit. Those skilled in the art can determine the preferred aerosol without undue experimentation.

[0139] The pharmaceutical composition in the embodiment of the present invention can be prepared by a method 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 non-covalently interact with the compound of structure (I) to promote the dissolution or homogeneous suspension of the compound in aqueous delivery systems.

[0140] The compositions, or pharma- ceutically acceptable salts thereof, in embodiments of the present invention are administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the particular therapeutic agent employed, the metabolic stability and duration of action of the therapeutic agent, the age, weight, general health, sex, and dietary habits of the patient, the mode and time of administration, rate of excretion, drug combination, the severity of the particular disorder or condition, and the subject being treated.

[0141] The composition in the embodiments of the present invention may also be administered simultaneously with, before, or after administration of one or more other therapeutic agents. Such combination therapy includes administration of the composition in the embodiments of the present invention and one or more additional active agents in a single pharmaceutical dosage formulation, as well as administration of the composition of the present invention and the active agents in separate pharmaceutical dosage formulations. For example, the composition in the embodiments of the present invention and the other active agents can be administered to a patient together in a single oral dosage formulation, such as a tablet or capsule, or each agent can be administered in a separate oral dosage formulation. When separate dosage formulations are used, the compound of structure (I) and one or more additional active agents can be administered at essentially the same time, i.e., simultaneously, or separately at different times, i.e., sequentially, and combination therapy is understood to include all of these regimens.

[0142] Methods of preparation for the above compounds and compositions are described herein below and / or are known in the art.

[0143] Those skilled in the art will appreciate that in the processes described herein, it may be necessary to protect the functional groups of intermediate compounds 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 can 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), 3rd Edition., Wiley. As will be appreciated by those skilled in the art, the protecting group may also be a polymer resin, such as a Wang resin, a Rink resin, or a 2-chlorotrityl-chloride resin.

[0144] It is also understood by those skilled in the art that these protected derivatives of the compounds of the invention may not possess pharmacological activity per se, but may be administered to a mammal and then metabolized in the body to form a pharmacologically active compound of structure (I). Thus, such derivatives may be described as "prodrugs." All prodrugs of the compounds of structure (I) are included within the scope of the embodiments of the present invention.

[0145] Additionally, all compounds of structure (I) that exist in free base or acid form can be converted to their pharma- ceutically acceptable salts by treatment with an appropriate inorganic or organic base or acid by methods known to those skilled in the art. Salts of compounds of structure (I) can be converted to their free base or free acid forms by standard techniques.

[0146] Compounds of structure (I), and lipid nanoparticles comprising same, can be prepared according to methods known or derivable by those of skill in the art, for example, those methods disclosed in PCT Publication Nos. WO2015 / 199952, WO2017 / 004143, and WO2017 / 075531, each of which is incorporated by reference in its entirety herein.

[0147] The general reaction scheme below illustrates a compound of structure (I): [ka] (I) 1 illustrates an exemplary method for synthesizing a compound of the formula: 3 , L 1 , L 2 , G 1 , G 2 , and G 3are as defined herein]. It is understood that those skilled in the art may synthesize these compounds in a similar manner or by combining other methods known to those skilled in the art. It is also understood that those skilled in the art may synthesize other compounds of structure (I) not specifically illustrated below in a similar manner to those described below by using appropriate starting components and modifying the synthesis parameters as necessary. In general, the starting components may be obtained from commercial sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, may be synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition (Wiley, December 2000)), or may be prepared as described in the present invention.

[0148] [ka] Specific examples of compounds of structure (I) (e.g., A-5) can be prepared according to General Reaction Scheme 1 ("Method A"), where R is independently at each occurrence R b , R c , R e or R f and n is, independently at each occurrence, an integer from 2 to 12. With reference to General Reaction Scheme 1, compounds of structure A-1 may be purchased from commercial sources or prepared by methods known to those skilled in the art. A mixture of A-1, A-2, and DMAP is treated with DCC to provide bromide A-3. A mixture of bromide A-3, a base (e.g., N,N-diisopropylethylamine), and N,N-dimethyldiamine A-4, after any necessary work-up and / or purification steps, is heated at a temperature and for a time sufficient to synthesize A-5.

[0149] [ka] Specific examples of compounds of structure (I) (e.g., compound B-8) can be prepared according to General Reaction Scheme 2 ("Method B"), where R is, independently at each occurrence, R a or R d L represents each occurrence independently, R 1 or R 2 and n is independently an integer between 2 and 12. For General Reaction Scheme 2, compounds of structure B-1 may be purchased from commercial sources or prepared according to methods known to those skilled in the art. The nitrogen of B-2 is alkylated by B-1 to give the diol product B-3, which is then converted to bromide B-4 by slowly adding HBr solution followed by refluxing. The resulting bromide B-4, after any necessary work-up and / or purification steps, is heated in a solvent in the presence of alkylamine B-5, N,N-diisopropylethylamine, and sodium iodide at a temperature and for a sufficient time to synthesize B-6. Oxalyl chloride is added dropwise to a solution of carboxylic acid B-7. A solution of B-6 is then added to the mixture with stirring to give B-8 after any necessary work-up and / or purification steps.

[0150] It should be noted that various alternative strategies for preparing compounds of structure (I) are available to those skilled in the art. For example, the moiety R 3 may contain substituents such as hydroxy, which may require suitable protecting groups to mask the substituents, or which may be 5 may be attached after attachment to the remainder of the molecule. The use of protecting groups as necessary and other modifications to General Reaction Scheme 1 above will be readily apparent to one of skill in the art. The following examples are offered for purposes of illustration and not limitation. EXAMPLES

[0151] In vivo evaluation of luciferase mRNA using lipid nanoparticles 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 at a molar ratio of 47.5:10:40.7:1.8. Lipid nanoparticles (LNPs) are prepared at a total lipid to mRNA weight ratio of about 10:1 to 40:1. Briefly, the mRNA is diluted to 0.2 mg / mL in 10-50 mM citrate buffer (pH 4) or 10-25 mM acetate buffer (pH 4). Using a syringe pump, the ethanolic lipid solution and the aqueous mRNA solution are mixed at a ratio of about 1:5 to 1:3 (vol / vol) 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 sterile filter.

[0152] Studies are performed in 6-8 week old female C57BL / 6 mice (Charles River) or 8-10 week old CD-1 (Harlan) mice (Charles River) in accordance with guidelines established by the institutional animal care committee (ACC) and the Canadian Council on Animal Care (CCAC). Various doses of mRNA-lipid nanoparticles are administered systemically via tail vein injection and animals are euthanized at specific time points (e.g., 4 hours) after administration. Livers and spleens are collected in pre-weighed tubes, weighed, immediately flash frozen in liquid nitrogen, and stored at -80°C until processed for analysis.

[0153] Approximately 50 mg of liver is dissected for analysis and sealed in a 2 mL FastPrep tube (MP Biomedicals, Solon OH). ¼ inch ceramic balls (MP Biomedicals) are added to each tube and 500 μL of Glo Lysis Buffer-GLB (Promega, Madison WI) equilibrated to room temperature is added to the liver tissue. The liver tissue is homogenized using a FastPrep24 instrument (MP Biomedicals) at 2×6.0 m / sec for 15 seconds. The homogenate is incubated at room temperature for 5 minutes before being diluted 1:4 in GLB and evaluated using the SteadyGlo Luciferase assay system (Promega). Specifically, 50 μL of the diluted tissue homogenate is reacted with 50 μL of SteadyGlo substrate, shaken for 10 seconds, followed by incubation for 5 minutes, and then assayed using a CentroXS. 3 Quantification 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 total protein assayed (μg). To convert RLU to luciferase (ng), a standard curve is generated using QuantiLum Recombinant Luciferase (Promega).

[0154] 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 produces bioluminescence in the presence of the substrate luciferin. This capped and polyadenylated mRNA is fully substituted with 5-methylcytidine and pseudouridine. EXAMPLES

[0155] In vivo evaluation of immunoglobulin G (IgG) mRNA using lipid nanoparticle compositions The lipids of structure (I), DSPC, cholesterol and PEGylated 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 about 10:1 to 40:1. Briefly, the mRNA is diluted to 0.2 mg / mL in 10-50 mM citrate buffer (pH 4) or 10-25 mM acetate buffer (pH 4). Using a syringe pump, the ethanolic lipid solution and the aqueous mRNA solution are mixed at a ratio of about 1:5 to 1:3 (vol / vol) at a total flow rate of more than 15 mL / min. The ethanol is then removed and the external buffer is replaced by PBS by dialysis. Finally, the lipid nanoparticles are filtered through a 0.2 μm pore sterile filter.

[0156] Studies are performed in 6-8 week old CD-1 / ICR mice (Envigo) following guidelines established by the institutional animal care committee (ACC) and the Canadian Council on Animal Care (CCAC). Different doses of mRNA-lipid nanoparticles are administered systemically via tail vein injection and animals are euthanized at specific time points (e.g., 24 hours) after administration. Whole blood is collected and serum is subsequently separated by centrifugation of the whole blood tubes at 2000×g for 10 minutes at 4°C and stored at -80°C until processed for analysis.

[0157] For immunoglobulin G (IgG) ELISA (Life Diagnostics Human IgG ELISA kit), dilute the serum samples 100-15000 times with 1x diluent. Dispense 100 μL of diluted serum into an anti-human IgG coated 96-well plate and in parallel incubate the human IgG standard at 25°C for 45 minutes at 150 rpm on a plate shaker. Wash wells 5 times with 1x wash solution using a plate washer (400 μL / well). Add 100 μL of HRP conjugate to each well and incubate on a plate shaker under the same conditions as above. Wash wells again 5 times with 1x wash solution using a plate washer (400 μL / well). Add 100 μL of TMB reagent to each well and incubate on a plate shaker under the same conditions as above. Terminate the reaction by adding 100 μL of stop solution to each well. The absorbance at 450 nm (A450) is read using a microplate reader. The amount of human IgG in the mouse serum is determined by plotting the A450 value of the standard assay against the human IgG concentration. EXAMPLES

[0158] Determination of pKa of formulated lipids As described elsewhere, the pKa of formulated lipids correlates with the efficacy of LNPs for nucleic acid delivery (see Jayaraman et al., Angewandte Chemie, International Edition (2012), vol. 51(34), pp. 8529-8533; Semple et al., Nature Biotechnology vol. 28, pp. 172-176 (2010)). In some embodiments, the preferred range of pKa is about 5 to about 7. The pKa of each lipid is determined in lipid nanoparticles using a fluorescence-based assay of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). Lipid 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 a total lipid concentration of 0.4 mM are prepared using the in-line process described in Example 1. TNS is prepared as a 100 μM stock solution in distilled water. Vesicles are diluted with 24 μM lipid / 2 mL buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl (pH 2.5-11). Aliquots of TNS solution are added to a final concentration of 1 μM, vortex mixed, and fluorescence intensity is measured at room temperature using excitation and emission wavelengths of 321 nm and 445 nm on an SLM Aminco Series 2 Luminescence Spectrophotometer. A sigmoidal best-fit analysis is applied to the fluorescence data to determine the pKa as the pH at which half-maximal fluorescence intensity is observed. EXAMPLES

[0159] Determining the efficacy of lipid nanoparticle formulations containing different cationic lipids using a rodent model expressing luciferase / IgG mRNA in vivo Representative compounds of the disclosure shown in Table 2 were formulated using the following molar ratios: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid 2-[2-(ω-methoxy(polyethylene glycol) 2000)ethoxy]-N,N-ditetradecylacetamide) or 47.5% cationic lipid / 10% DSPC / 40.7% cholesterol / 1.8% PEG lipid. Relative 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 when the mRNA was dosed at 1.0 mg / kg, 0.5 mg / kg, or 0.3 mg / kg, and activity was expressed as luciferase (ng) / liver (g) measured 4 hours after administration as described in Example 1, or IgG (μg) / serum (mL) measured 24 hours after administration as described in Example 2. Compound numbers in Table 2 refer to compound numbers in Table 1. Table 2. Novel cationic lipids and their associated activities [Table 18] [Table 19] EXAMPLES

[0160] 6,6'-(Methylazanediyl)bis(N,N-dioctylhexanamide) (Compound I-1) [ka] Synthesis of 6-bromo-N,N-dioctylhexanamide (Intermediate A) To a solution of 6-bromohexanoic acid (2.93 g, 15 mmol) in DCM (25 mL) and DMF (0.1 mL) was added oxalyl chloride (45 mmol, 5.7 g, 3.93 mL) at room temperature under Ar. The resulting mixture was stirred at room temperature overnight. The next day, the mixture was concentrated. The residue was dissolved in 20 mL of DCM and slowly added to a solution of dioctylamine (1.1 equiv, 16.5 mmol, 3.98 g, 4.98 mL), triethylamine (90 mmol, 9.09 g, 12.5 mL) and DMAP (10 mg) in DCM (20 mL) at room temperature. After the addition was complete, the mixture was stirred for 2.5 h and then concentrated. The residue was dissolved in dilute hydrochloric acid and filtered. The filtrate was extracted with DCM three times. After washing with saturated brine, the extract was evaporated and added with NaSO. 4 The mixture was dried at rt to give a yellow oil which was purified by chromatography (hexane and ethyl acetate (1:0 to 4:1)) to give the desired product as a pale yellow oil (5.09 g, 12.2 mmol, 81%).

[0161] Synthesis of I-1 To a mixture of 6-bromo-N,N-dioctylhexanamide (1.19 mmol, 500 mg), anhydrous acetonitrile (15 mL) and N,N-diisopropylethylamine (1.89 mmol, 244 mg, 0.33 mL) was added methylamine (0.32 mL, 0.63 mmol, 2M in THF). The mixture was heated at 80° C. for 16 h in a sealed pressure flask. The reaction mixture was concentrated. The crude product was purified by silica gel flash dry column chromatography (hexane / EtOAc / Et 3 N, 95:5:0 to 80:20:1) to give the desired product as a colorless oil (183 mg, 0.26 mmol, 44%). 1 HNMR (400 MHz, CDCl 3(7.27 ppm)) δ: 3.32-3.25 (m, 4H), 3.23-3.16 (m, 4H), 2.34-2.26 (m, 8H), 2.20 (s, 3H), 1.66 (quin, 7.6 Hz, 4H), 1.57-1.44 (m, 12H), 1.38-1.19 (m, 44H), 0.92-0.86 (m, 12H). ESI-MS: MW for C 45 H 91 N 3 O 2 [M+H] + Calculated 706.7; Measured 706.8. EXAMPLES

[0162] 6,6'-(octylazanediyl)bis(N,N-dioctylhexanamide) (Compound I-4) [ka] Synthesis of I-4 To a mixture of 6-bromo-N,N-dioctylhexanamide (1.6 equiv, 1.19 mmol, 500 mg), anhydrous acetonitrile (15 mL) and N,N-diisopropylethylamine (1.89 mmol, 244 mg, 0.33 mL) was added octylamine (0.123 mL, 0.74 mmol). The mixture was heated at 80° C. for 16 h in a sealed pressure flask. The reaction mixture was concentrated. The crude product was purified by silica gel flash dry column chromatography (Hexane / EtOAc / Et 3 N, 95:5:0 to 80:20:1) and further purified using 0 to 5% MeOH / chloroform as mixed elution solvents to give the desired product as a colorless oil (173 mg, 0.22 mmol, 36%). 1 HNMR (400 MHz, CDCl 3) δ: 3.33-3.25 (m, 4H), 3.23-3.16 (m, 4H), 3.08-2.86 (br. 3H), 2.5-2.32 (br, 3H), 2.29 (t, 7.6 Hz, 4H), 1.85-1.70 (br, 2H), 1.67 (quin, 7 Hz, 4H), 1.57-1.38 (m, 12H), 1.38-1.17 (m, 54H), 0.92-0.86 (m, 15H). ESI-MS: C 52 H 105 N 3 O 2 [M+H] + MW calculated 804.8; measured 805.0. EXAMPLES

[0163] 6,6'-(hexylazanediyl)bis(N,N-dioctylhexanamide) (Compound I-5) Compound I-5 was prepared according to the general procedure described in Example 6 to afford the desired product as a colorless oil (155 mg, 0.20 mmol, 34%). 1 HNMR (400 MHz, CDCl 3 (7.27 ppm)) δ: 3.33-3.25 (m, 4H), 3.24-3.15 (m, 4H), 3.10-2.80 (br. 2H), 2.58-2.32 (br, 4H), 2.29 (t, 7.4 Hz, 4H), 1.88-1.69 (br, 2H), 1.66 (quin, 7.4 Hz, 4H), 1.57-1.39 (m, 12H), 1.39-1.17 (m, 50H), 0.92-0.86 (m, 15H). EXAMPLES

[0164] 8,8'-(Methylazanediyl)bis(N,N-dioctyloctanamide) (Compound I-7) Compound I-7 was prepared according to the general procedure described in Example 5 to give the desired product as a pale yellow oil (230 mg, 0.30 mmol, 59%). 1HNMR (400 MHz, CDCl 3 (7.27 ppm)) δ: 3.32-3.25 (m, 4H), 3.23-3.15 (m, 4H), 2.31-2.25 (m, 8H), 2.20 (s, 3H), 1.64 (quin, 7.3 Hz, 4H), 1.57-1.41 (m, 12H), 1.38-1.19 (m, 52H), 0.92-0.86 (m, 12H). ESI-MS: C 49 H 99 N 3 O 2 [M+H] + MW calculated 762.8; measured 762.9. EXAMPLES

[0165] 10,10'-(Methylazanediyl)bis(N,N-dioctyldecanamide) (Compound I-8) Compound I-8 was prepared according to the general procedure described in Example 5 to give the desired product as a colorless oil (205 mg, 0.25 mmol, 64%). 1 HNMR (400 MHz, CDCl 3 (7.27 ppm)) δ: 3.33-3.25 (m, 4H), 3.23-3.16 (m, 4H), 2.31-2.25 (q-like, 8H), 2.20 (s, 3H), 1.63 (quin, 7.3 Hz, 4H), 1.57-1.41 (m, 12H), 1.38-1.19 (m, 60H), 0.92-0.86 (m, 12H). ESI-MS: C 53 H 107 N 3 O 2 [M+H] + MW calculated 818.8; measured 819.0. EXAMPLES

[0166] 8,8'-(Methylazanediyl)bis(N,N-didecyl octanamide) (Compound I-6) [ka] Synthesis of 8-bromo-N,N-didecyl octanamide (intermediate D) To a solution of 8-bromooctanoic acid (1 equiv., 10.78 mmol, 2.41 g) in DCM (20 mL) and DMF (d 0.944; 0.1 mL) was added oxalyl chloride (2.5 equiv., 27 mmol, 3.42 g, 2.35 mL) under Ar at room temperature. The resulting mixture was stirred at room temperature overnight. The mixture was then concentrated under reduced pressure. The residue was dissolved in 15 mL of DCM and slowly added to a solution of didecylamine (1.1 equiv., 3.53 g, 11.86 mmol), triethylamine (53.9 mmol, 7.5 mL) and DMAP (10 mg) in DCM (20 mL) at room temperature. After the addition was complete, the mixture was stirred at room temperature overnight and then concentrated. The residue was dissolved in hexane (100 mL) and loaded onto a silica gel column under reduced pressure. The column was then washed under reduced pressure with a mixture of hexane and ethyl acetate (100:0 to 90:10) to give the desired product as a yellow oil (4.81 g, 9.6 mmol, 89%).

[0167] Synthesis of I-6 8-Bromo-N,N-didecyl octanamide (0.8 mmol, 400 mg), CH 3 A mixture of CN (15 mL), DIPEA (0.26 mL) and methylamine (33 wt % / EtOH, 0.062 mL, ca. 0.5 mmol) was sealed in a pressure flask and heated at 74° C. overnight. The reaction mixture was concentrated. The crude product was purified by silica gel flash dry column chromatography (hexane / EtOAc / Et 3 N, 95:5:0 to 80:20:1) and 0 to 5% MeOH / chloroform (with a small amount of Et 3 Further purification was performed using mixed elution solvents (containing N) to give the desired product as a colorless oil (167 mg, 0.19 mmol, 48%). 1 HNMR (400 MHz, CDCl 3(7.26 ppm)) δ: 3.32-3.24 (m, 4H), 3.23-3.15 (m, 4H), 2.30-2.24 (m, 8H), 2.18 (s, 3H), 1.63 (quin, 7.1 Hz, 4H), 1.57-1.38 (m, 12H), 1.38-1.19 (m, 68H), 0.92-0.86 (m, 12H). ESI-MS: C 57 H 115 N 3 O 2 [M+H] + MW calculated 874.9; measured 875.1. EXAMPLES

[0168] 6,6'-(Methylazanediyl)bis(N,N-didecylhexanamide) (Compound I-10) Compound I-10 was prepared according to the general procedure described in Example 5 to afford the desired product as a colorless oil (249 mg, 0.30 mmol, 58%). 1 HNMR (400 MHz, CDCl 3 (7.26 ppm)) δ: 3.32-3.24 (m, 4H), 3.22-3.15 (m, 4H), 2.33-2.25 (m, 8H), 2.19 (s, 3H), 1.65 (quin, 7.6 Hz, 4H), 1.57-1.41 (m, 12H), 1.38-1.08 (m, 60H), 0.91-0.85 (m, 12H). ESI-MS: C 53 H 107 N 3 O 2 [M+H] + MW calculated 818.8; measured 819.0. EXAMPLES

[0169] 6,6'-(Methylazanediyl)bis(N,N-didodecylhexanamide) (Compound I-11) Compound I-11 was prepared according to the general procedure described in Example 5 to afford the desired product as a colorless oil (313 mg, 0.34 mmol, 68%).1 HNMR (400 MHz, CDCl 3 ) δ: 3.32-3.24 (m, 4H), 3.22-3.15 (m, 4H), 2.33-2.25 (m, 8H), 2.19 (s, 3H), 1.65 (quin, 7.6 Hz, 4H), 1.57-1.43 (m, 12H), 1.37-1.10 (m, 76H), 0.91-0.85 (m, 12H). ESI-MS: C 61 H 123 N 3 O 2 Calculated MW for [M+H]+ 931.0; found 931.2. EXAMPLES

[0170] 6,6'-((2-hydroxyethyl)azanediyl)bis(N,N-dioctylhexanamide) (Compound I-3) [ka] Synthesis of I-3 A mixture of 2-aminoethanol (1.25 mmol, 77 mg) in anhydrous THF (15 mL), 6-bromo-N,N-dioctylhexanamide (Intermediate A, 1.9 equiv, 1 g, 2.39 mmol), potassium carbonate (1.9 equiv, 330 mg, 2.39 mmol), cesium carbonate (0.3 equiv, 234 mg, 0.72 mmol) and sodium iodide (3 mg) in a pressure flask was heated at 77 °C (oil bath) for 6 days. The reaction mixture was concentrated. The crude product was purified by silica gel flash dry column chromatography (hexane / EtOAc / Et 3 N, 95:5:0 to 80:20:1) to give the desired product as a colorless oil (233 mg, 0.31 mmol, 26%). 1 HNMR (400 MHz, CDCl 3) δ: 3.52 (t, 5.3 Hz, 2H), 3.23-3.25 (m, 4H), 3.24-3.15 (m, 4H), 3.10-2.96 (br. 1H), 2.57 (t, 5.3 Hz, 2H), 2.50-2.41 (m, 4H), 2.28 (t, 7.5 Hz, 4H), 1.65 (quin, 7.6 Hz, 4H), 1.60-1.42 (m, 12H, overlapping with water peak), 1.38-1.19 (m, 44H), 0.92-0.86 (m, 12H). ESI-MS: C 46 H 93 N 3 O 3 [M+H] + MW calculated 736.7; measured 736.8. EXAMPLES

[0171] 6,6'-((6-hydroxyhexyl)azanediyl)bis(N,N-dioctylhexanamide) (Compound I-16) Compound I-16 was prepared according to the general procedure described in Example 13 to afford the desired product as a colorless oil (63 mg). 1 HNMR (400 MHz, CDCl 3 ) δ: 3.68-3.62 (m, 2H), 3.32-3.24 (m, 4H), 3.23-3.15 (m, 4H), 3.07 -2.91 (br, 6H), 2.31 (t, 7.5 Hz, 4H), 1.93-1.73 (br., 6H), 1.68 (quin, 7.6 Hz, 4H), 1.63-1.46 (m, 10H, overlapping with water peak), 1.46-1.35 (m, 8H), 1.35-1.10 (m, 40H), 0.92-0.86 (m, 12H). Part of the product exists in the form of hydrochloride salt. ESI-MS: C 50 H 101 N 3 O 3 [M+H] + MW calculated 792.8; measured 792.9. EXAMPLES

[0172] 6,6'-((2-hydroxyethyl)azanediyl)bis(N,N-didecylhexanamide) (Compound I-19) Compound I-19 was prepared according to the general procedure described in Example 13 to afford the desired product as a pale yellow oil (154 mg, 0.18 mmol, 35%). 1 HNMR (400 MHz, CDCl 3 ) δ: 3.51 (t, 5.2, 2H), 3.32-3.24 (m, 4H), 3.23-3.15 (m, 4H), 3.08-2.92 (br, 1H), 2.56 (t, 5.3 Hz, 2H), 2.45 (t, 7.4 Hz, 4H), 2.27 (t, 7.5 Hz, 4H), 1.64 (quin, 7.5 Hz, 4H), 1.59-1.40 (m, 12H, overlapped with water peak), 1.36-1.10 (m, 60H), 0.91-0.86 (m, 12H). ESI-MS: C 54 H 109 N 3 O 3 [M+H] + MW calculated 848.9; measured 849.0. EXAMPLES

[0173] 8,8'-((6-hydroxyhexyl)azanediyl)bis(N,N-dioctyloctanamide) (Compound I-17) Compound I-17 was prepared according to the general procedure described in Example 13 to afford the desired product as a pale yellow oil (546 mg, 0.64 mmol, 54%). 1 HNMR (400 MHz, CDCl 3) δ: 3.65 (t, 6.4 Hz, 2H), 3.33-3.25 m, 4H), 3.23-3.15 (m, 4H), 2.40-2.34 (m, 6H), 2.28 (t, 7.5 Hz, 4H), 1.75-1.59 (m, 6H), 1.58-1.46 (m, 8H), 1.46-1.36 (m, 8H), 1.36-1.10 (m, 54H), 0.92-0.86 (m, 12H). ESI-MS: C 54 H 109 N 3 O 3 [M+H] + MW calculated 848.9; measured 849.0. EXAMPLES

[0174] 10,10'-((6-hydroxyhexyl)azanediyl)bis(N,N-dioctyldecanamide) (Compound I-18) Compound I-18 was prepared according to the general procedure described in Example 59 to afford the desired product as a pale yellow oil (251 mg, 0.28 mmol, 52%). 1 HNMR (400 MHz, CDCl 3 ) δ: 3.67-3.63 (m, 2H), 3.33-3.25 m, 4H), 3.24-3.16 (m, 4H), 2.41-2.35 (m, 6H), 2.28 (t, 7.5 Hz, 4H), 1.68-1.59 (m, 6H), 1.59-1.47 (m, 8H, overlapping with water peak), 1.47-1.37 (m, 8H), 1.37-1.10 (m, 62H), 0.92-0.86 (m, 12H). EXAMPLES

[0175] 10,10'-((2-hydroxyethyl)azanediyl)bis(N,N-didecyldecaneamide) (Compound I-20) Compound I-20 was prepared according to the general procedure described in Example 59 to afford the desired product as a colorless oil (248 mg, 0.26 mmol, 49%). 1HNMR (400 MHz, CDCl 3 ) δ: 3.51 (t, 5.4, 2H), 3.32-3.24 (m, 4H), 3.22-3.15 (m, 4H), 3.08-2.92 (br, 1H), 2.56 (t, 5.4 Hz, 2H), 2.43 (t, 7.4 Hz, 4H), 2.27 (t, 7.5 Hz, 4H), 1.64 (quin, 7.5 Hz, 4H), 1.59-1.36 (m, 12H, overlapping with water peak), 1.36-1.10 (m, 76H), 0.91-0.86 (m, 12H). ESI-MS: C 62 H 125 N 3 O 3 [M+H] + Calculated MW 961.0; measured 961.1. EXAMPLES

[0176] 8,8'-((5-hydroxypentyl)azanediyl)bis(N,N-didecyl octanamide) (Compound I-21) Compound I-21 was prepared according to the general procedure described in Example 13 to afford the desired product as a colorless oil (166 mg, 0.18 mmol, 35%). 1 HNMR (400 MHz, CDCl 3 (7.26 ppm)) δ: 3.64 (t, 6.5 Hz, 2H), 3.32-3.24 (m, 4H), 3.22-3.15 (m, 4H), 2.41-2.34 (m, 6H), 2.27 (t, 7.5 Hz, 4H), 2.05 (br. s, 1H), 1.67-1.57 (m, 6H), 1.54-1.36 (m, 16H), 1.36-1.10 (m, 68H), 0.90-0.85 (m, 12H). 61 H 123 N 3 O 3 [M+H] + MW calculated 947.0; measured 947.2. EXAMPLES

[0177] 8,8'-((4-hydroxybutyl)azanediyl)bis(N,N-didecyl octanamide) (Compound I-23) Compound I-23 was prepared according to the general procedure described in Example 13 to afford the desired product as a colorless oil (209 mg, 0.22 mmol, 45%). 1 HNMR (400 MHz, CDCl 3 (7.26 ppm)) δ: 6.64 (br. s, 1H), 3.57-3.50 (m, 2H), 3.32-3.24 (m, 4H), 3.23-3.15 (m, 4H), 2.44-2.39 (m, 6H), 2.26 (t, 7.5 Hz, 4H), 1.67-1.57 (m, 8H), 1.54-1.37 (m, 12H), 1.36-1.10 (m, 68H), 0.90-0.85 (m, 12H). 60 H 121 N 3 O 3 [M+H] + Calculated MW 932.9; measured 933.1. EXAMPLES

[0178] 8,8'-((6-hydroxyhexyl)azanediyl)bis(N,N-didecyl octanamide) (Compound I-24) Compound I-24 was prepared according to the general procedure described in Example 13 to afford the desired product as a colorless oil (229 mg, 0.24 mmol, 48%). 1 HNMR (400 MHz, CDCl 3 , (7.26 ppm)) δ: 3.67-3.59 (m, 2H), 3.32-3.24 (m, 4H), 3.23-3.15 (m, 4H), 2.39-2.33 (m, 6H), 2.26 (t, 7.5 Hz, 4H), 1.73-1.68 (br. 1H), 1.68-1.38 (m, estimated 20H), 1.38-1.10 (m, 72H), 0.90-0.85 (m, 12H). ESI-MS: C62 H 125 N 3 O 3 [M+H] + Calculated MW 961.0; measured 961.1. EXAMPLES

[0179] 8,8'-((2-hydroxyethyl)azanediyl)bis(N,N-didecyl octanamide) (Compound I-22) Compound I-22 was prepared according to the general procedure described in Example 13 to afford the desired product as a colorless oil (165 mg, 0.18 mmol, 35%). 1 HNMR (400 MHz, CDCl 3 ) δ: 3.51 (t, 5.4, 2H), 3.32-3.24 (m, 4H), 3.23-3.15 (m, 4H), 3.07-2.98 (br, 1H), 2.56 (t, 5.4 Hz, 2H), 2.42 (t, 7.4 Hz, 4H), 2.27 (t, 7.5 Hz, 4H), 1.68-1.59 (m, 4H), 1.59-1.36 (m, 12H, overlapping with water peak), 1.36-1.10 (m, 68H), 0.91-0.86 (m, 12H). ESI-MS: C 58 H 117 N 3 O 3 [M+H] + Calculated MW 904.9; measured 905.1. EXAMPLES

[0180] 10,10'-((4-hydroxybutyl)azanediyl)bis(N,N-didecyldecaneamide) (Compound I-25) Compound I-25 was prepared according to the general procedure described in Example 13 to afford the desired product as a colorless oil (202 mg, 0.21 mmol, 42%). 1 HNMR (400 MHz, CDCl 3, (7.26 ppm)) δ: 6.66 (br. s, 1H), 3.58-3.50 (m, 2H), 3.32-3.24 (m, 4H), 3.23-3.15 (m, 4H), 2.44-2.39 (m, 6H), 2.26 (t, 7.5 Hz, (4H), 1.70-1.58 (m, 8H), 1.55-1.38 (m, 12H), 1.37-1.14 (m, 76H), 0.90-0.85 (m, 12H). 64 H 129 N 3 O 3 [M+H] + Calculated MW 989.0; measured 989.1. EXAMPLES

[0181] N,N'-((methylazanediyl)bis(hexane-6,1-diyl))bis(N,2-dihexyldecanamide) (Compound I-15) [ka] Synthesis of 6,6'-(methylazanediyl)bis(hexan-1-ol) (intermediate E) To a mixture of 6-chloro-1-hexanol (22.8 mmol, 3.11 g, 3.04 mL), absolute ethanol (60 mL), potassium carbonate (2 eq., 24 mmol, 3.32 g), cesium carbonate (eq., 500 mg) and potassium iodide (40 mg) was added methylamine (2 mmol, 1.49 ml, 33 wt.% in absolute ethanol). The mixture was heated in a sealed pressure flask (oil bath: 68 °C) for 16 h. The reaction was monitored by TLC. Further methylamine (0.1 mL), NaI (300 mg) and potassium carbonate (1 g) were added to the reaction mixture. Heating was resumed and after an additional 4 days, further 6-chloro-1-hexanol (1.5 mL) and NaI (220 mg) were added. Heating was continued at 76 °C for an additional 3 days. Finally, the mixture was cooled and filtered. The filtrate was concentrated and the residue was dissolved in DCM and filtered. The filtrate was concentrated to dryness under reduced pressure. From the above, 4.86 g of a brown viscous oil was obtained. The oil was dissolved in DCM (100 mL) and loaded onto a short silica gel column under reduced pressure. The column was eluted with a mixture of DCM, methanol and concentrated aqueous ammonia (100:0:0 to 85:15:1). Fractions containing the desired product were combined and concentrated. The residue was dissolved in DCM and filtered. The filtrate was concentrated to dryness to give the desired product as a brown viscous oil (1.40 g, 6.05 mmol, 50%). The product was used in the next step without further purification.

[0182] N 1 -Hexyl-N 6 -(6-(hexylamino)hexyl)-N 6 Synthesis of -methylhexane-1,6-diamine (intermediate G) Hydrobromic acid (6 mL, 48 wt.% aqueous solution) was added slowly to Intermediate E (1.40 g, 6.05 mmol) over a period of 5 min with stirring. The reaction mixture was then heated at 105° C. (oil bath) for 2 h. The reaction mixture was cooled slightly and then toluene (50 mL) was added slowly. The reaction was heated to reflux to azeotropically remove water. The reaction mixture was cooled to room temperature and then transferred to a pressure flask and concentrated under reduced pressure (brown oil 2.62 g, 5.97 mmol, 98%). TLC (CHCl 3 / MeOH=9:1) confirmed 6-bromo-N-(6-bromohexyl)-N-methylhexan-1-amine (Intermediate F) as the major spot. The product was used in the next step without further purification.

[0183] A mixture of 6-bromo-N-(6-bromohexyl)-N-methylhexan-1-amine (Intermediate F, 2.62 g, 5.97 mmol), hexylamine (10 equiv. 120 mmol, 12.14 g), N,N-diisopropylethylamine (6.0 mmol, 1.04 mL), and sodium iodide (20 mg) in acetonitrile (30 mL) was sealed and heated at 76° C. (oil bath) for 24 hours. The mixture was then concentrated at about 75° C. (about 9 mmHg) to remove the solvent and excess hexylamine. The residue (yellow oil / solid) was dissolved in DCM and filtered. TLC confirmed that the hexylamine had not been completely removed. The filtrate was concentrated under reduced pressure to give 4.33 g of a yellow foam. The residue was dissolved in NaOH solution (sodium hydroxide (1.44 g) / water (10 mL)) and then concentrated under reduced pressure at 75° C. for 1.5 h. The residue was dissolved in DCM and filtered. The filtrate was concentrated to dryness to give a pale paste (2.671 g, >100%). The product was used in the next step without further purification.

[0184] Synthesis of I-15 To a solution of 2-hexyldecanoic acid (4 mmol, 1.03 g) in DCM (15 mL) and DMF (3 drops with a medium needle), oxalyl chloride (1.5 equiv, 6 mmol, 762 mg, 0.524 mL) was added dropwise at room temperature. The resulting mixture was then stirred at room temperature overnight. The mixture was then concentrated under reduced pressure at room temperature. The residue (light yellow paste) was dissolved in 8 mL of DCM and added to a solution of the crude product of intermediate G (0.67 g), triethylamine (3 equiv, 3.1 mmol, 308 mg, 0.42 mL) and DMAP (5 mg) in DCM (5 mL) at room temperature over about 5 min. After addition, the resulting mixture was stirred at room temperature for 2 h and then concentrated. The residue was purified by distillation with hexane, ethyl acetate and Et 3The crude product was dissolved in a mixture of 1,2-dichloro-2,4-diphenyl-2,5-diaminetetraacetate (80:20:1) and filtered through a pad of silica gel. The pad was washed with the same mixture of solvents. The filtrate and washings were concentrated to give a light brown oil (approximately 543 mg). The crude product was purified by silica gel flash dry column chromatography (0 to 6% MeOH / chloroform (with a small amount of Et 3 The desired product was obtained as a colorless oil (426 mg, 0.49 mmol, 33%). 1 HNMR (400 MHz, CDCl 3 ) δ: 3.34-3.27 (m, 4H), 3.26-3.18 (m, 4H), 2.53-2.45 (m, 2H), 2.34-2.25 (m, 4H), 2.18 (t, 4.7 Hz, 3H), 1.65-1.36 (m, 20H), 1.36-1.10 (m, 60H), 0.92-0.84 (m, 18H). ESI-MS: C 57 H 115 N 3 O 2 [M+H] + MW calculated 874.9; measured 875.3. EXAMPLES

[0185] N,N'-((methylazanediyl)bis(hexane-6,1-diyl))bis(N-hexylpalmitamide) (compound I-9) [ka] Synthesis of I-9 To a solution of palmitic acid (2.5 mmol, 0.64 g) in DCM (15 mL) and DMF (2 drops with a small needle), oxalyl chloride (1.5 equiv, 3.75 mmol, 484 mg, 0.33 mL) was added dropwise at room temperature. The resulting mixture was then stirred at room temperature overnight. The mixture was then concentrated under reduced pressure at room temperature. The residue (light yellow paste) was dissolved in 8 mL of DCM and added to a solution of the crude product of intermediate G (300 mg), triethylamine (0.4 mL) and DMAP (5 mg) in DCM (5 mL) at room temperature over a period of about 5 min. After addition, the resulting mixture was stirred at room temperature for 2 h and then concentrated. The residue was purified by distillation with hexane, ethyl acetate and Et 3 The mixture was dissolved in a mixture of hexane, ethyl acetate, and N (80:20:1), filtered through a pad of silica gel, and the pad was washed with the same solvent mixture. The filtrate and washings were concentrated to give a yellow oil / solid, which was then purified by elution with hexane, ethyl acetate, and Et 3 The crude product was purified by silica gel flash dry column chromatography (0 to 5% MeOH / chloroform (with a small amount of Et 3 The desired product was obtained as a colorless oil (200 mg, 0.22 mmol, 34%). 1 HNMR (400 MHz, CDCl 3 (7.26 ppm)) δ: 3.32-3.25 (m, 4H), 3.23-3.15 (m, 4H), 2.33-2.24 (m, 8H), 2.18 (t, 4.2 Hz, 3H), 1.67-1.56 (m, 4H), 1.56-1.36 (m, 12H), 1.36-1.10 (m, 68H), 0.92-0.85 (m, 12H). ESI-MS: C 57 H 115 N 3 O 2 [M+H] + MW calculated 874.9; measured 875.1. EXAMPLES

[0186] (9Z,9'Z,12Z,12'Z)-N,N'-((methylazanediyl)bis(hexane-6,1-diyl))bis(N-hexyloctadeca-9,12-dienamide) (Compound I-14) [ka] Synthesis of I-14 To a solution of linoleic acid (2.5 mmol, 0.70 g) in DCM (12 mL) and DMF (2 drops with a small needle), oxalyl chloride (1.5 equiv., 3.75 mmol, 484 mg, 0.33 mL) was added dropwise at room temperature. The resulting mixture was then stirred at room temperature overnight. The mixture was then concentrated under reduced pressure at room temperature. The residue (light yellow paste) was dissolved in 8 mL of DCM and added to a solution of the crude product of intermediate G (300 mg), triethylamine (0.4 mL) and DMAP (5 mg) in DCM (5 mL) at room temperature over a period of about 5 min. After addition, the resulting mixture was stirred at room temperature for 2 h and then concentrated. The residue was purified by elution with hexane, ethyl acetate, and Et 3 The crude product was dissolved in a mixture of 1,2-dichlorophenyl ether (80:20:1), filtered through a pad of silica gel, and the pad was washed with the same solvent mixture. The filtrate and washings were concentrated to give a yellow oil / solid material, which was dissolved in hexane and filtered again to remove the solids. The crude product was purified by flash dry column chromatography on silica gel (0 to 5% MeOH / chloroform (with a small amount of Et 3 The desired product was obtained as a colorless oil (160 mg, 0.17 mmol, 25%). 1 HNMR (400 MHz, CDCl 3 ) δ: 5.42-5.29 (m, 8H), 3.32-3.24 (m, 4H), 3.22-3.15 (m, 4H), 2.77 (t, 6.4 Hz, 4H), 2.33-2.24 (m, 8H), 2.19 (t, 4.0 Hz, 3H), 2.05 (q, 6.8 Hz, 8H), 1.67-1.56 (m, 4H), 1.56-1.40 (m, 12H), 1.40-1.10 (m, 46H), 0.92-0.85 (m, 12H). ESI-MS: C 61 H 115 N3 O 2 [M+H] + MW calculated 922.9; measured 923.1. EXAMPLES

[0187] N,N-didecyl-8-((8-(hexadecylamino)-8-oxooctyl)(methyl)amino)octanamide (compound I-12) [ka] Synthesis of 8-bromo-N-hexadecyloctanamide To a mixture of 8-bromohexanoic acid (1.0 equiv., 2.53 g, 11.36 mmol), 4-dimethylaminopyridine (0.3 equiv., 3.41 mmol, 416 mg) and N-hydroxysuccinimide (1.0 equiv., 11.36 mmol, 1.31 g) in DCM (20 mL) was added DCC (1.05 equiv., 11.93 mmol, 2.46 g) and the mixture was stirred at room temperature for 2.5 h. The reaction mixture was filtered and transferred to a flask containing hexadecylamine (1 equiv., 11.36 mmol, 2.743 g) and washed with DCM (~20 mL). The resulting mixture was stirred at room temperature overnight. After concentration of the reaction mixture, a white solid was obtained. The solid was dissolved in DCM (100 mL) and sonicated to give a slightly cloudy solution. The cloudy solution was loaded onto a short silica gel column under reduced pressure. Elution with a mixture of DCM and MeOH (100:0 to 98.75:1.25) under reduced pressure afforded the desired product as a white solid (4.50 g, 10.1 mmol, 89%).

[0188] Synthesis of N-hexadecyl-8-(methylamino)octanamide Methylamine (1 mL, 8 mmol, 33 wt.% in absolute ethanol), 8-bromo-N-hexadecyloctanamide (1 equivalent, 288 mg, 0.61 mml) and K 2 CO 3 A mixture of (1 mmol, 138 mg) in EtOH (10 mL) was sealed in a pressure bottle and heated at 85 °C (oil bath) overnight. After concentration of the mixture, the residue was purified by distillation with DCM, MeOH, and Et 3This was dissolved in a mixture of N (90:10:0.5) and filtered, and the filtrate and washings were concentrated to give a white solid (228 mg, 0.57 mmol, 89%) which was used in the next step without further purification.

[0189] Synthesis of I-12 A mixture of crude N-hexadecyl-8-(methylamino)octanamide (228 mg), anhydrous acetonitrile (15 mL), N,N-diisopropylethylamine (0.33 mL) and intermediate D (327 mg, 0.65 mmol) was heated in a pressure flask at 74 °C (oil bath) for 16 h. The reaction mixture was then concentrated. The residue was purified by distillation with hexane, ethyl acetate and Et 3 The crude product was dissolved in a mixture of 0 to 5% MeOH / chloroform (with a small amount of Et 3 The crude product was further purified by flash dry column chromatography on silica gel using 1,2-dichlorophenyl ether (containing N) as the eluent to give the desired product as a colorless oil (192 mg, 0.23 mmol, 40%). 1 HNMR (400 MHz, CDCl 3 , (7.26 ppm)) δ: 5.45 (s, 1H), 3.30-3.16 (m, 6H), 2.30-2.24 (m, 6H), 2.18 (s, 3H), 2.14 (t, 7.6 Hz, 2H), 1.67-1.57 (m, 4H), 1.54-1.37 (m, 10H), 1.37-1.16 (m, 66H), 0.91-0.86 (m, 9H). ESI-MS: C 53 H 107 N 3 O 2 [M+H] + MW calculated 818.8; measured 819.0. EXAMPLES

[0190] 8,8'-((8-(decylamino)-8-oxooctyl)azanediyl)bis(N,N-didecyloctanamide) (Compound I-33) [ka] Synthesis of I-13 To a mixture of intermediate D (1.9 equiv., 2.0 g, 3.98 mmol), anhydrous acetonitrile (25 mL) and N,N-diisopropylethylamine (2.08 mL) in a pressure flask was added benzylamine (2.09 mmol, 224 mg, 0.23 mL). The resulting mixture was heated at 75° C. (oil bath) overnight. The reaction mixture was then concentrated. The crude product was purified by silica gel flash dry column chromatography (hexane / EtOAc / Et 3 N, 90:10:0 to 80:20:1). Fractions containing the desired product were concentrated to give a brown oil (1.43 g) that was of sufficient purity to be used in the next step without further purification. A small amount of the product was purified by elution with 0 to 5% MeOH / chloroform (a small amount of Et 3 For analytical and testing purposes, the product was further purified by flash dry column chromatography on silica gel using mixed elution solvents (containing N). 1 HNMR (400 MHz, CDCl 3 , (7.26 ppm)) δ: 7.33-7.18 (m, 5H), 3.52 (s, 2H), 3.31-3.24 (m, 4H), 3.22-3.14 (m, 4H), 2.41-2.33 (m, 4H), 2.29-2.21 (m, s4H), 1.61 (quin, 7.3 Hz, 4H), 1.56-1.38 (m, 12H), 1.38-1.10 (m, 68H), 0.90-0.85 (m, 12H).

[0191] Synthesis of 8,8'-azanediylbis(N,N-didecyloctanamide) (Intermediate H) A mixture of I-13 (1.18 g, 1.24 mmol), 10% Pd / C (39 mg) in methanol (15 mL) was stirred under hydrogen at room temperature for 3 days. The reaction mixture was filtered through a pad of Celite and washed with MeOH. The filtrate was concentrated (1.092 g colorless oil / solid) and used in the next step without further purification.

[0192] Synthesis of 8-bromo-N-decyloctanamide (Intermediate J) To a mixture of 8-bromohexanoic acid (1.0 equiv, 2.53 g, 11.36 mmol) and 4-dimethylaminopyridine (0.3 equiv, 3.41 mmol, 416 mg) in DCM (20 mL) was added N-hydroxysuccinimide (1.0 equiv, 11.36 mmol, 1.31 g) followed by DCC (1.05 equiv, 11.93 mmol, 2.46 g). After stirring the resulting mixture at room temperature for 2.5 h, the mixture was filtered and added to a flask containing decylamine (1 equiv, 11.36 mmol, 1.79 g, 2.27 mL) and the solid was washed with additional DCM (3 mL x 2). The resulting mixture of filtrate and decylamine was stirred at room temperature overnight. The next day, the mixture was concentrated. The residue was dissolved in DCM and loaded onto a silica gel column. The column was eluted with a mixture of DCM and MeOH (100:0 to 97.5:2.5). The desired product was obtained as a white solid (3.447 g, 9.51 mmol, 84%).

[0193] Synthesis of I-33 A mixture of 8,8'-azanediylbis(N,N-didecyloctanamide) (Intermediate H, 300 mg, 0.35 mmol), 8-bromo-N-decyloctanamide (Intermediate J, 1 eq, 0.35 mmol, 127 mg), anhydrous acetonitrile (15 mL), N,N-diisopropylethylamine (0.12 mL), and NaI (44 mg) in a pressure flask was heated at 73 °C (oil bath) overnight. The reaction mixture was then concentrated. The residue was purified by distillation with hexane, ethyl acetate, and Et 3The crude product was dissolved in a mixture of 0 to 5% MeOH / chloroform (with a small amount of Et 3 The crude product was further purified by flash dry column chromatography on silica gel using mixed elution solvents (containing N) to give the desired product as a colorless oil (212 mg, 0.19 mmol, 53%). 1 HNMR (400 MHz, CDCl 3 , (7.26 ppm)) δ: 5.62 (br. t, 5 Hz, 1H), 3.31-3.24 (m, 4H), 3.24-3.16 (m, 6H), 2.40-2.31 (m, 6H), 2.30-2.23 (m, 4H), 2.15 (t, 1.67-1.57 (m, 6H), 1.55-1.36 (m, 16H), 1.36-1.10 (m, 88H), 0.90-0.85 (m, 15H). ESI-MS: C 74 H 148 N 4 O 3 [M+H] + MW calculated 1142.2; measured 1142.2. EXAMPLES

[0194] 8,8'-((6-(dihexylamino)-6-oxohexyl)azanediyl)bis(N,N-didecyl octanamide) (Compound I-32) [ka] Synthesis of I-32 A mixture of 6-bromo-N,N-dihexylhexanamide (1 eq, 0.35 mmol, 127 mg; synthesized from 6-bromohexanoic acid and dihexylamine in a manner similar to intermediate D), anhydrous acetonitrile (15 mL), N,N-diisopropylethylamine (0.12 mL), 8,8'-azanediylbis(N,N-didecyloctanamide) (intermediate H, 300 mg, 0.35 mmol) and NaI (44 mg) in a pressure flask was heated at 80 °C (oil bath) overnight. The reaction mixture was then concentrated. The residue was purified by distillation with hexane, ethyl acetate and Et 3 The crude product was dissolved in a mixture of 0 to 5% MeOH / chloroform (with a small amount of Et 3 The crude product was further purified by flash dry column chromatography on silica gel using a mixture of 1,2-dichloro-2,4-dichloro-1,2-diphenyl-2,4-tetrahydrofuran (containing N) as eluent to give the desired product as a pale yellow oil (228 mg, 0.20 mmol, 57%). 1 HNMR (400 MHz, CDCl 3 (7.26 ppm)) δ: 3.31-3.24 (m, 6H), 3.22-3.15 (m, 6H), 2.40-2.33 (m, 6H), 2.30-2.24 (m, 6H), 1.69-1.58 (m, 6H), 1.54-1.36 (m, 18H), 1.36-1.10 (m, 82H), 0.92-0.85 (m, 18H). ESI-MS: C 74 H 148 N 4 O 3 [M+H] + MW calculated 1142.2; measured 1142.2. EXAMPLES

[0195] 8,8'-((5-(decylamino)-5-oxopentyl)azanediyl)bis(N,N-didecyloctanamide) (Compound I-30) [ka] Synthesis of 5-bromo-N-decylpentanamide To a mixture of 5-bromovaleric acid (1.0 equiv, 2.06 g, 11.36 mmol) and 4-dimethylaminopyridine (0.3 equiv, 3.41 mmol, 416 mg) in DCM (20 mL) was added N-hydroxysuccinimide (1.0 equiv, 11.36 mmol, 1.31 g) followed by DCC (1.05 equiv, 11.93 mmol, 2.46 g). After stirring the resulting mixture at room temperature for 2.5 h, the mixture was filtered and added to a flask containing decylamine (1 equiv, 11.36 mmol, 1.79 g, 2.27 mL) and the solid was washed with additional DCM (3 mL x 2). The resulting mixture of the filtrate and decylamine was stirred at room temperature overnight. The next day, the mixture was concentrated. The residue was dissolved in DCM and loaded onto a silica gel column. The column was eluted with a mixture of DCM and MeOH (100:0 to 98:2) to give the desired product as a white solid (2.758 g, 8.62 mmol, 76%).

[0196] Synthesis of I-30 A mixture of 8,8'-azanediylbis(N,N-didecyloctanamide) (Intermediate H, 246 mg, 0.27 mmol), 5-bromo-N-decylpentanamide (130 mg), anhydrous acetonitrile (12 mL), N,N-diisopropylethylamine (0.10 mL) and NaI (40 mg) in a pressure flask was heated at 73 °C (oil bath) overnight. After concentration of the reaction mixture, the residue was purified by distillation with hexane, ethyl acetate, MeOH and Et 3 The crude product was dissolved in a mixture of 0 to 5% MeOH / chloroform (with a small amount of Et 3 The crude product was further purified by flash dry column chromatography on silica gel using 1,2,3-hexanediaminetetraacetate (containing N) as a mixed elution solvent to give the desired product as a colorless oil (60 mg). 1 HNMR (400 MHz, CDCl 3(7.26 ppm)) δ: 5.75 (br. t, 5 Hz, 1H), 3.33-3.13 (m, 10H), 2.41-2.32 (m, 6H), 2.27 (t, 7.6 Hz, 4H), 2.17 (t, 7.6 Hz, 2H), 1.67-1.57 (m, 6H), 1.55-1.35 (m, 16H), 1.35-1.16 (m, 82H), 0.90-0.85 (m, 15H). EXAMPLES

[0197] 6,6'-((8-(decylamino)-8-oxooctyl)azanediyl)bis(N,N-didecylhexanamide) (Compound I-29) [ka] Synthesis of I-29 A mixture of 6,6'-azanediylbis(N,N-didecylhexanamide) (300 mg, 0.37 mmol; synthesized in a similar manner to intermediate H), 8-bromo-N-decyloctanamide (1 equiv, 0.37 mmol, 135 mg), anhydrous acetonitrile (15 mL), N,N-diisopropylethylamine (0.12 mL), and NaI (70 mg) in a pressure flask was heated at 73 °C (oil bath) overnight. After concentration of the reaction mixture, the residue was purified by distillation with hexane, ethyl acetate, MeOH, and Et 3 The crude product was dissolved in a mixture of 1,2-dichloro-2,4-diphenyl-2,5-diaminetetraacetate (80:20:2:1) and filtered through a pad of silica gel. The pad was washed with the same mixture of solvents. The filtrate was concentrated and washed to give a yellow oil. The crude product was purified by flash dry column chromatography on silica gel (0 to 5% MeOH / chloroform (with a small amount of Et 3 The mixture was further purified using a mixed elution solvent (containing 1,2-dichloromethane and 1,2-dichloromethane) to give the desired product as a colorless oil (254 mg, light brown oil, 0.23 mmol, 63%). 1 HNMR (400 MHz, CDCl 3(7.26 ppm)) δ: 5.62 (br. t, 5 Hz, 1H), 3.34-3.16 (m, 10H), 2.40-2.33 (m, 6H), 2.27 (t, 7.6 Hz, 4H), 2.15 (t, 7.6 Hz, 2H), 1.68-1.57 (m, 6H), 1.56-1.36 (m, 16H), 1.36-1.10 (m, 80H), 0.90-0.85 (m, 15H). ESI-MS: C 70 H 140 N 4 O 3 [M+H] + Calculated MW 1086.1; measured 1086.3. EXAMPLES

[0198] 6,6'-((2-(dihexylamino)ethyl)azanediyl)bis(N,N-didecylhexanamide) (Compound I-31) [ka] Synthesis of 6,6'-((2-chloroethyl)azanediyl)bis(N,N-didecylhexanamide) I-19 (333 mg, 0.39 mmol) in 2 mL of CHCl 3 To the ice-cold solution, thionyl chloride (200 mg) in 10 mL of chloroform was added dropwise under Ar. 2 After the addition (10 min) was complete, the ice bath was removed and the reaction mixture was stirred at room temperature for 16 h. 3 and SOCl 2 The remaining oil was purified by distillation with hexane, ethyl acetate and Et 3 The residue was dissolved in a mixture of 1,2-dichloro-2,4-dichloro-1,2-tetrahydrofuran (~80:20:1) and filtered through a pad of silica gel. The pad was washed with the same mixture of solvents. The filtrate and washings were concentrated to give a yellow oil (282 mg, 0.325 mmol, 83%). The product was used in the next step without further purification.

[0199] Synthesis of I-31 A mixture of 6,6'-((2-chloroethyl)azanediyl)bis(N,N-didecylhexanamide) (282 mg, 0.33 mmol), dihexylamine (5 equiv. 1.6 mmol, 1.066 g), N,N-diisopropylethylamine (2 equiv., 0.66 mmol, 85 mg, 0.11 mL; MW 129.25, d 0.742), and sodium iodide (60 mg) in acetonitrile (10 mL) was sealed and heated at 76 °C for 24 h. After concentration of the reaction mixture, the residue was purified by distillation with hexane, ethyl acetate, MeOH, and Et 3 The mixture was dissolved in a mixture of 1,2-dichloro-2,4-dichloroethane and N (80:20:1:1) and filtered through a pad of silica gel. The pad was washed with the same mixture of solvents. The filtrate and washings were concentrated to give a yellow oil. The yellow oil was dissolved in acetyl chloride and Et at room temperature. 3 The unreacted dihexylamine was converted to an amide by treatment with a DCM solution of N. After removing the DCM, the crude product was purified by silica gel flash dry column chromatography (hexane / ethyl acetate / Et 3 N, 9:1 to 80:20:1; 0 to 5% MeOH / chloroform (with a small amount of Et 3 The crude product was obtained as a colorless oil (256 mg, colorless oil, 0.25 mmol, 76%). 1 HNMR (400 MHz, CDCl 3 (7.26 ppm)) δ: 3.31-3.24 (m, 4H), 3.23-3.15 (m, 4H), 2.50 (s, 4H), 2.45-2.36 (m, 8H), 2.27 (t, 7.6 Hz, 4H), 1.64 (quin, 7.6 Hz, 4H), 1.59-1.36 (m, 16H), 1.36-1.10 (m, 72H), 0.92-0.84 (m, 18H). ESI-MS: C 66 H 134 N 4 O 2 [M+H] + MW calculated 1016.1; measured 1016.2. EXAMPLES

[0200] 10,10'-((2-(dimethylamino)ethyl)azanediyl)bis(N,N-didecyldecaneamide) (Compound I-28) [ka] Synthesis of 10,10'-((2-chloroethyl)azanediyl)bis(N,N-didecyldecaneamide) I-20 (293 mg, 0.30 mmol) in 2 mL of CHCl 3 To the ice-cold solution was added dropwise a solution of thionyl chloride (1.17 mmol, 139 mg, 0.085 mL) in 10 mL of chloroform. 2 After the addition (10 min) was complete, the ice bath was removed and the reaction mixture was stirred at room temperature for 16 h. The next day, CHCl 3 and SOCl 2 The remaining oil was purified by distillation with hexane, ethyl acetate, and Et 3 The residue was dissolved in a mixture of 1,2-dichloro-2,4-dichloro-1,2-tetrahydrofuran (~80:20:1) and filtered through a pad of silica gel. The pad was washed with the same solvent mixture. The filtrate and washings were concentrated to give a yellow oil (278 mg, 0.28 mmol, 95%). The product was used in the next step without further purification.

[0201] Synthesis of I-28 A mixture of 10,10'-((2-chloroethyl)azanediyl)bis(N,N-didecyldecaneamide) (278 mg, 0.28 mmol), dimethylamine (2M / THF, 10 mL) and N,N-diisopropylethylamine (0.66 mmol) was heated at 75° C. for 24 h in a sealed pressure flask. After concentration of the reaction mixture, the residue was purified with hexane, ethyl acetate, MeOH and Et 3 The crude product was dissolved in a mixture of 1,2-dichloro-2,4-diphenyl-2,5-diaminetetraacetate (80:20:1:1) and filtered through a pad of silica gel. The pad was washed with the same mixture of solvents. The filtrate and washings were concentrated to give a yellow oil. The crude product was purified by flash dry column chromatography on silica gel (0 to 5% MeOH / chloroform (with a small amount of Et 3 The desired product was obtained as a colorless oil (244 mg, pale yellow oil, 0.25 mmol, 76%). 1HNMR (400 MHz, CDCl 3 (7.26 ppm)) δ: 3.31-3.25 (m, 4H), 3.22-3.15 m, 4H), 2.56-2.551 (m, 2H), 2.43-2.33 (m, 6H), 2.26 (t, 7.6 Hz, 4H), 2.23 (s, 6H), 1.67-1.59 (m, 4H), 1.56-1.45 (m, 8H), 1.45-1.36 (m, 4H), 1.36-1.15 (m, 76H), 0.90-0.85 (m, 12H). ESI-MS: C 64 H 130 N 4 O 2 [M+H] + Calculated MW 988.0; measured 988.1. EXAMPLES

[0202] 2-Butyloctyl 6-(bis(6-(dioctylamino)-6-oxohexyl)amino)hexanoate (Compound I-34) [ka] Synthesis of I-34 A mixture of 2-butyloctyl-6-aminohexanoate (0.76 mmol, 228 mg; synthesized from 2-butyl-1-octanol and 6-aminocaproic acid; purity less than 80%), 6-bromo-N,N-dioctylhexanamide (Intermediate A, 1.44 mmol, 603 mg), anhydrous acetonitrile (15 mL) and N,N-diisopropylethylamine (0.4 mL) was heated at 80° C. for 16 h in a sealed pressure flask. After concentration of the reaction mixture, the residue was purified by elution with hexane, ethyl acetate and Et 3 The crude product was dissolved in a mixture of 1,2-dichlorophenyl ether (80:20:1) and filtered through a pad of silica gel. The pad was washed with the same mixture of solvents. The filtrate and washings were concentrated to give a yellow oil. The crude product was purified by flash dry column chromatography on silica gel (0 to 5% MeOH / chloroform (with a small amount of Et 3The desired product was obtained as a colorless oil (172 mg, colorless oil, 0.18 mmol, 25%). 1 HNMR (400 MHz, CDCl 3 (7.26 ppm)) δ: 3.97 (d, 5.8 Hz, 2H), 3.33-3.24 (m, 4H), 3.24-3.15 (m, 4H), 3.02-2.91 (br., 1H), 2.42 (m, 5H), 2.33-2.25 (m, 6H), 1.86-1.74 (br. 1H), 1.70-1.36 (m, 20H), 1.36-1.16 (m, 62H), 0.91-0.85 (m, 18H). EXAMPLES

[0203] 6,6'-((4-hydroxybutyl)azanediyl)bis(N,N-bis(2-ethylhexyl)hexanamide) (Compound I-27) [ka] Synthesis of I-27 A solution of bis-(2-ethylhexyl)amine (7.5 g) and 6-bromohexanoyl chloride (6.1 g) in dichloromethane (60 mL) was treated with triethylamine (6 mL) and stirred for 2 h. The solution was washed with dilute hydrochloric acid. The organic fraction was dried over anhydrous magnesium sulfate, filtered, and the solvent removed. To remove polar impurities, the crude product was passed through a silica gel column with dichloromethane. A solution of the resulting product (3 g) in THF was treated with 4-aminobutan-1-ol (0.22 g) and N,N-diisopropylethylamine (1 mL). The reaction was refluxed for 3 days and then partitioned between dilute hydrochloric acid and dichloromethane. The solvent was removed from the organic fraction, and the residue was passed through a silica gel (55 g) column with a gradient elution of methanol / dichloromethane to give I-27 (1.6 g). 1 H NMR (400 MHz, CDCl 3(7.26 ppm)): 3.57-4.49 (m, 2H), 3.33-3.18 (m, 4H) 3.12 (d, 7.4 Hz, 4H), 2.47-2.26 (m, 10H), 1.74-1.12 (m, 52H), 0.93-0.81 (m, 24H). ESI-MS: C 48 H 97 N 3 O 3 [M+H] + MW calculated 764.8; measured 764.8. EXAMPLES

[0204] 8,8'-((2-hydroxyethyl)azanediyl)bis(N,N-didodecyl octanamide) (Compound I-37) Compound I-37 was prepared according to the general procedure described in Example 13 to afford the desired product as a colorless oil (148 mg, 0.15 mmol, 32%). 1 HNMR (400 MHz, CDCl 3 (7.26 ppm)) δ: 3.51 (t, 5.0 Hz, 2H), 3.30-3.25 (m, 4H), 3.22-3.16 (m, 4H), 2.56 (t, 5.3 Hz, 2H), 2.42 (t, 7.4 Hz, 4H), 2.27 (t, 7.6 Hz, 4H), 1.68-1.38 (m, 16H), 1.36-1.15 (84H), 0.90-0.86 (m, 12H). ESI-MS: C 66 H 133 N 3 O 3 [M+H] + Calculated MW 1017.0; measured 1017.1. EXAMPLES

[0205] 6,6'-((6-hydroxyhexyl)azanediyl)bis(N,N-didodecylhexanamide) (Compound I-38) Compound I-38 was prepared according to the general procedure described in Example 13 to afford the desired product as a colorless oil (259 mg, 0.25 mmol, 51%). 1 HNMR (400 MHz, CDCl 3 (7.26 ppm)) δ: 3.67-3.61 (m, 2H), 3.28 (t, 7.6 Hz, 4H), 3.19 (t, 7.7 Hz, 4H), 2.41-2.35 (m, 6H), 2.27 (t, 7.5 Hz, 4H), 1.64 (quin, 7.6 Hz, 4H), 1.60-1.40 (m, 16H), 1.40-1.10 (m, 80H), 0.90-0.85 (m, 12H). ESI-MS: C 66 H 133 N 3 O 3 [M+H] + Calculated MW 1017.0; measured 1017.4. EXAMPLES

[0206] N,N-didecyl-8-((8-(hexadecyl(methyl)amino)-8-oxooctyl)(methyl)amino)octanamide (compound I-39) Compound I-39 was prepared according to the general procedure described in Example 27 to afford the desired product as a colorless oil (152 mg, 0.18 mmol, 46%). 1 HNMR (400 MHz, CDCl 3 (7.26 ppm)) δ: 3.36-3.16 (m, 6H), 2.96, 2.90 (two singlets, 3H), 2.31-2.24 (m, 8H), 2.18 (s, 3H), 1.68-1.58 (m, 4H), 1.56-1.38 (m, 10H), 1.38-1.18 (m, 66H), 0.91-0.86 (m, 9H). ESI-MS: C 54 H 109 N 3 O 3 [M+H] + MW calculated 832.8; measured 832.8. EXAMPLES

[0207] 8,8'-(Methylazanediyl)bis(N,N-didodecyl octanamide) (Compound I-40) Compound I-40 was prepared according to the general procedure described in Example 10 to afford the desired product as a colorless oil (235 mg, 0.24 mmol, 53%). 1 HNMR (400 MHz, CDCl 3 (7.26 ppm)) δ: 3.28 (t, 7.6 Hz, 4H), 3.19 (t, 7.7 Hz, 4H), 2.30-2.24 (m, 8H), 2.18 (s, 3H), 1.63 (quin, 7.6 Hz, 4H), 1.57-1.43 (m, 12H), 1.37-1.10 (m, 84H), 0.90-0.86 (m, 12H). ESI-MS: MW for C 65 H 131 N 3 O 2 [M+H] + Calculated 987.0; Measured 987.4. EXAMPLES

[0208] 8,8'-((3-hydroxypropyl)azanediyl)bis(N,N-didecyl octanamide) (Compound I-41) Compound I-41 was prepared according to the general procedure described in Example 13 to give the desired product (100 mg, 22%). 1 HNMR (400 MHz, CDCl 3(7.26 ppm)) δ: 3.78 (t, J = 5.1 Hz, 2H), 3.32 - 3.23 (m, 4H), 3.23 - 3.15 (m, 4H), 2.64 (t, J = 5.6 Hz, 2H), 2.44 - 2.36 (m, 4H), 2.31 - 2.22 (m, 4H), 1.65 (dp, J = 22.1, 6.3 Hz, 6H), 1.58 - 1.40 (m, 12H), 1.38 - 1.18 (m, 70H), 0.88 (td, J = 6.8, 3.0 Hz, 12H). ESI-MS: C 59 H 119 N 3 O 3 [M+H] + MW calculated 918.9; measured 919.3. EXAMPLES

[0209] 8,8'-((2-(2-hydroxyethoxy)ethyl)azanediyl)bis(N,N-didecyl octanamide) (Compound I-42) [ka] Synthesis of I-42 A mixture of intermediate D (1.2 mmol, 600 mg), 2-(2-aminoethoxy)ethan-1-ol (0.74 mmol, 78 mg), and DIEA (3.0 mmol, 0.52 mL) in ACN (7 mL) was heated at 72 °C for 48 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 60% EtOAc / hexanes (1% Et 3 N) to give compound I-42 (200 mg, 35%). 1 H NMR (400 MHz, CDCl 3) δ 3.68 (m, 3.0 Hz, 2H), 3.63 - 3.57 (m, 4H), 3.32 - 3.23 (m, 4H), 3.23 - 3.14 (m, 4H), 2.62 (t, J = 5.4 Hz, 2H), 2.49 - 2.41 (m, ESI-MS: C 60 H 121 N 3 O 4 [M+H] + MW calculated 948.9; measured 949.4. EXAMPLES

[0210] 8,8'-((5-hydroxy-4,4-dimethylpentyl)azanediyl)bis(N,N-didecyl octanamide) (compound I-43) [ka] Synthesis of I-43 A mixture of intermediate D (1.2 mmol, 600 mg), 5-amino-4,4-dimethylpentan-1-ol (0.74 mmol, 97 mg), and DIEA (3.0 mmol, 0.52 mL) in ACN (7 mL) was heated at 72 °C for 48 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 60% EtOAc / hexanes (1% Et 3 N) to give compound I-43 (280 mg, 48%). 1 H NMR (400 MHz, CDCl 3) δ 3.31 - 3.23 (m, 6H), 3.22 - 3.12 (m, 4H), 2.41 - 2.30 (m, 6H), 2.30 - 2.22 (m, 4H), 1.71 - 1.58 (m, 4H), 1.56 - 1.46 (m, 5H), 1.44 - 1.36 (m, 3H), 1.36 - 1.17 (t, 74H), 0.98 - 0.81 (m, 18H). ESI-MS: C 63 H 127 N 3 O 3 [M+H] + Calculated MW 975.0; measured 975.4. EXAMPLES

[0211] 8,8'-((3-(2-methyl-1H-imidazol-1-yl)propyl)azanediyl)bis(N,N-didecyl octanamide) (compound I-44) [ka] Synthesis of I-44 A mixture of intermediate D (1.2 mmol, 600 mg), 3-(2-methyl-1H-imidazol-1-yl)propan-1-amine (0.74 mmol, 103 mg), and DIEA (3.0 mmol, 0.52 mL) in ACN (7 mL) was heated at 72 °C for 48 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 100% EtOAc / hexanes (1% Et 3 The crude compound I-44 was purified by automated flash chromatography (c18 column, 50% to 100% MeOH / water (containing 1% TFA)) to give pure compound I-44 (60 mg, 10%). 1 H NMR (400 MHz, CDCl 3) δ 6.90 (s, 1H), 6.82 (s, 1H), 3.87 (t, J = 7.2 Hz, 2H), 3.34 - 3.24 (m, 4H), 3.23 - 3.11 (m, 4H), 2.43 - 2.34 (m, 8H), 2.31 - ESI-MS: C 63 H 123 N 5 O 2 [M+H] + MW calculated 983.0; measured 983.4. EXAMPLES

[0212] 8,8'-((7-hydroxyheptyl)azanediyl)bis(N,N-didecyl octanamide) (Compound I-45) Compound I-45 was prepared according to the general procedure described in Example 13 to give the desired product (320 mg, 55%). 1 HNMR (400 MHz, CDCl 3 (7.26 ppm)) δ: 6.90 (s, 1H), 6.82 (s, 1H), 3.87 (t, J = 7.2 Hz, 2H), 3.33 - 3.22 (m, 4H), 3.23 - 3.13 (m, 4H), 2.42 - 2.34 (m, 8H), 2.33 - 2.20 (m, 4H), 1.90 - 1.76 (m, 2H), 1.66 - 1.59 (m, 4H), 1.57 - 1.45 (m, 8H), 1.45 - 1.13 (m, 71H), 0.88 (m, 12H). ESI-MS: C 63 H 127 N 3 O 3 [M+H] + Calculated MW 975.0; measured 975.4. EXAMPLES

[0213] 8,8'-((2-(2-methoxyethoxy)ethyl)azanediyl)bis(N,N-didecyl octanamide) (Compound I-46) [ka] Synthesis of I-46 A mixture of intermediate D (1.2 mmol, 600 mg), 2-(2-methoxyethoxy)ethan-1-amine (0.74 mmol, 89 mg), and DIEA (3.0 mmol, 0.52 mL) in ACN (7 mL) was heated at 72 °C for 48 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 60% EtOAc / hexanes (1% Et 3 N) to give compound I-46 (333 mg, 58%). 1 H NMR (400 MHz, CDCl 3 ) δ 3.65 - 3.60 (m, 2H), 3.59 - 3.51 (m, 4H), 3.40 (s, 3H), 3.33 - 3.26 (m, 4H), 3.25 - 3.18 (m, 4H), 2.67 (t, J = 6.7 Hz, 2H), 2.48 - 2.39 (m, 4H), 2.33 - 2.25 (m, 4H), 1.68 - 1.61 (m, 4H), 1.59 - 1.48 (m, 8H), 1.48 - 1.40 (m, 3H), 1.37 - 1.24 (m, 69H), 0.95 - 0.85 (m, 12H). ESI-MS: C 61 H 123 N 3 O 4 [M+H] + Calculated MW 963.0; measured 963.4. EXAMPLES

[0214] 8,8'-((8-hydroxyoctyl)azanediyl)bis(N,N-didecyl octanamide) (compound I-47) Compound I-47 was prepared according to the general procedure described in Example 13 to give the desired product (320 mg, 54%). 1 HNMR (600 MHz, CDCl 3 (7.26 ppm)) δ: 3.65 (t, J = 6.7 Hz, 2H), 3.32 - 3.27 (m, 4H), 3.23 - 3.18 (m, 4H), 2.41 - 2.34 (m, 6H), 2.32 - 2.25 (m, 4H), 1.68 - 1.62 (m, 4H), 1.60 - 1.48 (m, 10H), 1.47 - 1.39 (m, 6H), 1.38 - 1.23 (m, 76H), 0.96 - 0.84 (m, 12H). ESI-MS: C 64 H 129 N 3 O 3 [M+H] + Calculated MW 989.0; measured 989.5. EXAMPLES

[0215] 8,8'-((3-(1H-imidazol-1-yl)propyl)azanediyl)bis(N,N-didecyl octanamide) (compound I-48) [ka] Synthesis of I-48 A mixture of intermediate D (1.2 mmol, 600 mg), 3-(1H-imidazol-1-yl)propan-1-amine (0.74 mmol, 93 mg), and DIEA (3.0 mmol, 0.52 mL) in ACN (7 mL) was heated at 72 °C for 48 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 100% EtOAc / hexanes (1% Et 3 N) to give compound I-48 (82 mg, 14%). 1 H NMR (600 MHz, CDCl 3) δ 7.48 (s, 1H), 7.07 (s, 1H), 6.93 (s, 1H), 4.00 (t, J = 7.0 Hz, 2H), 3.30 (t, J = 7.7 Hz, 4H), 3.21 (t, J = 7.8 Hz, 4H), 2.41 - 2.34 (m, 6H), 2.29 (t, J = 7.6 Hz, 4H), 1.92 - 1.87 (m, 2H), 1.69 - 1.61 (m, 4H), 1.59 - 1.47 (m, 8H), 1.41 - 1.22 (m, 74H), 0.93 - 0.87 (m, 12H). ESI-MS: C 62 H 121 N 5 O 2 [M+H] + Calculated MW 969.0; measured 969.1. EXAMPLES

[0216] 8,8'-((2,2-difluoro-3-hydroxypropyl)azanediyl)bis(N,N-didecyl octanamide) (Compound I-49) [ka] Synthesis of I-49 A mixture of intermediate D (1.0 mmol, 500 mg), 3-amino-2,2-difluoropropan-1-ol (0.62 mmol, 69 mg), and DIEA (3.0 mmol, 0.43 mL) in ACN (6 mL) was heated at 72 °C for 48 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 60% EtOAc / hexanes (1% Et 3 N) to give compound I-49 (55 mg, 12%). 1 H NMR (600 MHz, CDCl 3) δ 4.87 (s, 1H), 3.88 (t, J = 12.1 Hz, 2H), 3.33 - 3.27 (m, 4H), 3.24 - 3.18 (m, 4H), 2.96 (t, J = 12.7 Hz, 2H), 2.56 - 2.51 (m, ESI-MS: C 59 H 117 F 2 N 3 O 3 [M+H] + MW calculated 954.9; measured 955.1. EXAMPLES

[0217] 8,8'-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)azanediyl)bis(N,N-didecyloctanamide) (compound I-50) [ka] Synthesis of tert-butyl (3-(bis(8-(didecylamino)-8-oxooctyl)amino)propyl)carbamate A mixture of intermediate D (1.60 mmol, 800 mg), tert-butyl (3-aminopropyl)carbamate (0.99 mmol, 173 mg), and DIEA (4.0 mmol, 0.7 mL) in ACN (10 mL) was heated at 72 °C for 48 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 60% EtOAc / hexanes (1% Et 3 N)) to obtain compound tert-butyl (3-(bis(8-(didecylamino)-8-oxooctyl)amino)propyl)carbamate (388 mg, 48%).

[0218] Synthesis of 8,8'-((3-aminopropyl)azanediyl)bis(N,N-didecyloctanamide) A mixture of tert-butyl (3-(bis(8-(didecylamino)-8-oxooctyl)amino)propyl)carbamate (0.38 mmol, 385 mg) and TFA (1.0 mL) in DCM (2.0 mL) was stirred at room temperature for 90 min. The reaction mixture was concentrated and the crude product was purified by dilution with EtOAc and saturated NaHCO 3 The compound 8,8'-((3-aminopropyl)azanediyl)bis(N,N-didecyloctanamide) (385 mg, quantitative) was obtained and used in the next step without further purification.

[0219] Synthesis of 3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione A mixture of 3,4-dimethoxycyclobut-3-ene-1,2-dione (7.0 mmol, 1 g) and 2M methylamine / THF (7.7 mmol, 3.9 mL) in diethyl ether (100 mL) was stirred at room temperature for 19 hours. The solid was filtered and washed with diethyl ether. The solid crude product was triturated in hot EtOAc, cooled to 5° C., then filtered and washed with cold EtOAc to give 3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione (700 mg, 71%).

[0220] Synthesis of I-50 A mixture of 8,8'-((3-aminopropyl)azanediyl)bis(N,N-didecyloctanamide) (0.11 mmol, 100 mg) and 3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione (0.11 mmol, 15 mg) in ethanol (5 mL) was stirred at room temperature for 19 h and then heated at 50 °C for 19 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 100% EtOAc / hexanes (1% Et 3 N) and then 2% MeOH / DCM. A second purification using automated flash chromatography (1% to 10% MeOH / DCM) afforded I-50 (32 mg, 28%). 1 H NMR (400 MHz, CDCl 3) δ 8.22 (bs, 0.5H), 7.60 (bs, 0.5H), 3.70 (bs, 2H), 3.33 - 3.23 (m, 7H), 3.23 - 3.14 (m, 4H), 2.96 (bs, 2H), 2.80 (bs, 4H), 2.28 (t, J = 7.4 Hz, 4H), 1.98 (bs, 2H), 1.66 - 1.45 (m, 16H), 1.40 - 1.20 (m, 67H). 0.92 - 0.83 (m, 12H). ESI-MS: C 64 H 123 N 5 O 4 [M+H] + Calculated MW 1027.0; measured 1027.1. EXAMPLES

[0221] 8,8'-((2-fluoro-3-hydroxypropyl)azanediyl)bis(N,N-didecyl octanamide) (compound I-51) [ka] Synthesis of I-51 A mixture of intermediate D (1.0 mmol, 500 mg), 3-amino-2-fluoropropan-1-ol hydrochloride (0.62 mmol, 80 mg), and DIEA (3.1 mmol, 0.54 mL) in ACN (6 mL) was heated at 72 °C for 24 h and then at 55 °C for 72 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 60% EtOAc / hexanes (1% Et 3 N) to give compound I-51 (45 mg, 10%). 1 H NMR (400 MHz, CDCl 3) δ 4.60 (dt, J = 46.8, 4.8 Hz, 1H), 3.92 - 3.80 (m, 2H), 3.35 - 3.24 (m, 4H), 3.24 - 3.12 (m, 4H), 2.85 - 2.72 (m, 2H), 2.53 - 2.37 (m, 4H), 2.31 - 2.23 (m, 4H), 1.67 - 1.40 (m, 20H), 1.38 - 1.19 (m, 66H), 0.94 - 0.82 (m, 12H). ESI-MS: C 59 H 118 FN 3 O 3 [M+H] + MW calculated 936.9; measured 937.0. EXAMPLES

[0222] 8,8'-((3,3,3-trifluoro-2-(hydroxymethyl)propyl)azanediyl)bis(N,N-didecyl octanamide) (compound I-52) [ka] Synthesis of I-52 A mixture of Intermediate D (1.0 mmol, 500 mg), 2-(aminomethyl)-3,3,3-trifluoropropan-1-ol (0.62 mmol, 111 mg) and DIEA (2.4 mmol, 0.43 mL) in ACN (6 mL) was heated at 72 °C for 24 h and then at 55 °C for 72 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 60% EtOAc / hexanes (1% Et 3 N) to give compound I-52 (100 mg, 20%). 1 H NMR (400 MHz, CDCl 3) δ 5.88 (s, 1H), 4.01 - 3.92 (m, 1H), 3.89 - 3.78 (m, 1H), 3.33 - 3.24 (m, 4H), 3.23 - 3.15 (m, 4H), 2.91 - 2.73 (m, 2H), 2.69 - 2.50 (m, 3H), 2.34 - 2.21 (m, 6H), 1.69 - 1.39 (m, 20H), 1.38 - 1.21 (m, 68H), 0.95 - 0.84 (m, 12H). ESI-MS: C 60 H 118 F 3 N 3 O 3 [M+H] + MW calculated 986.9; measured 987.0. EXAMPLES

[0223] 8,8'-((5-methoxypentyl)azanediyl)bis(N,N-didecyl octanamide) (compound I-53) [ka] Synthesis of I-53 A mixture of Intermediate D (0.80 mmol, 400 mg), 5-methoxypentan-1-amine (0.50 mmol, 58 mg), and DIEA (2.0 mmol, 0.35 mL) in ACN (6 mL) was heated at 72 °C for 24 h and then at 55 °C for 72 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 60% EtOAc / hexanes (1% Et 3 N) to give compound I-53 (210 mg, 55%). 1 H NMR (400 MHz, CDCl 3) δ 3.36 (t, J = 6.6 Hz, 2H), 3.32 (s, 3H), 3.30 - 3.25 (m, 4H), 3.22 - 3.16 (m, 4H), 2.41 - 2.32 (m, 6H), 2.31 - 2.22 (m, 4H), 1.67 - 1.37 (m, 23H), 1.36 - 1.19 (m, 69H), 0.91 - 0.85 (m, 12H). ESI-MS: C 62 H 125 N 3 O 3 [M+H] + Calculated MW 961.0; measured 961.1. EXAMPLES

[0224] N,N-didecyl-8-((8-(dioctylamino)-8-oxooctyl)(methyl)amino)octanamide (compound I-55) [ka] Synthesis of 8-(methylamino)-N,N-dioctyloctanamide A mixture of 8-bromo-N,N-dioctyloctanamide (5.8 mmol, 2.6 g) and 8M methylamine / EtOH (30 mL) in ACN (12 mL) was stirred for 19 h at 70° C. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 100% EtOAc / Hexanes, then 1% to 10% MeOH / DCM) to give compound 8-(methylamino)-N,N-dioctyloctanamide (1.91 g, 83%).

[0225] Synthesis of I-55 A mixture of 8-(methylamino)-N,N-dioctyloctanamide (3.4 mmol, 1.3 g), Intermediate D (3.4 mmol, 1.7 g), and DIEA (13.8 mmol, 2.4 mL) in ACN (120 mL) was heated at 70 °C for 19 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 60% EtOAc / hexanes (1% Et 3N) to give compound I-55 (1.38 g, 50%). 1 H NMR (400 MHz, CDCl 3 ) δ 3.34 - 3.26 (m, 4H), 3.25 - 3.17 (m, 4H), 2.34 - 2.25 (m, 8H), 2.21 (s, 3H), 1.70 - 1.61 (m, 6H), 1.60 - 1.41 (m, 12H), 1.39 - 1.23 (m, 59H), 0.95 - 0.85 (m, 12H). ESI-MS: C 53 H 107 N 3 O 2 [M+H] + MW calculated 818.8; measured 819.0. EXAMPLES

[0226] tert-Butyl (3-(bis(10-(didecylamino)-10-oxodecyl)amino)propyl)carbamate (compound I-56) [ka] Synthesis of I-56 A mixture of 10-bromo-N,N-didecyldecanoamide (prepared according to the general procedure described in Example 5, 1.5 mmol, 800 mg), tert-butyl (3-aminopropyl)carbamate (0.93 mmol, 162 mg), and DIEA (4.5 mmol, 0.78 mL) in ACN (10 mL) was stirred at 70 °C for 48 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 65% EtOAc / hexanes (1% Et 3 N) to give I-56 (555 mg, 69%). 1 H NMR (400 MHz, CDCl 3) δ 5.72 (bs, 1H), 3.32 - 3.24 (m, 4H), 3.23 - 3.14 (m, 6H), 2.44 (t, J = 6.4 Hz, 2H), 2.31 - 2.23 (m, 4H), 2.31 - 2.23 (m, 4H), 1.67 - 1.58 (m, 9H), 1.57 - 1.47 (m, 3H), 1.47 - 1.37 (m, 12H), 1.35 - 1.19 (m, 77H), 0.92 - 0.83 (m, 12H). ESI-MS: C 68 H 136 N 4 O 4 [M+H] + Calculated MW 1074.1; measured 1074.2. EXAMPLES

[0227] 10,10'-((3-(1H-imidazol-1-yl)propyl)azanediyl)bis(N,N-didecyldecaneamide) (Compound I-57) [ka] Synthesis of I-57 A mixture of 10-bromo-N,N-didecyldecaneamide (prepared according to the general procedure described in Example 5, 1.1 mmol, 600 mg), 3-(1H-imidazol-1-yl)propan-1-amine (0.68 mmol, 88 mg), and DIEA (3.3 mmol, 0.57 mL) in ACN (7 mL) was heated at 70° C. for 48 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 100% EtOAc / Hexanes (1% Et 3 The crude product was purified by reverse-phase (C18) automated flash chromatography (50% to 100% MeOH / water) to give compound I-57 (45 mg, 8%). 1 H NMR (400 MHz, CDCl 3) δ 7.52 (bs, 1H), 7.06 (s, 1H), 6.92 (s, 1H), 4.00 (t, J = 7.0 Hz, 2H), 3.32 - 3.24 (m, 4H), 3.22 - 3.15 (m, 4H), 2.38 (bs, 6H), 2.31 - 2.21 (m, 4H), 1.90 (bs, 2H), 1.73 - 1.57 (m, 8H), 1.57 - 1.45 (m, 8H), 1.43 - 1.16 (m, 81H), 0.95 - 0.81 (m, 12H). ESI-MS: C 66 H 129 N 5 O 2 [M+H] + Calculated MW 1025.0; measured 1025.1. EXAMPLES

[0228] 8,8'-(Methylazanediyl)bis(N,N-dinonyloctanamide) (Compound I-58) Compound I-58 was prepared according to the general procedure described in Example 5 to give the desired product (1.78 g, 59%). 1 H NMR (600 MHz, CDCl 3 ) δ 3.33 - 3.27 (m, 4H), 3.24 - 3.18 (m, 4H), 2.33 - 2.26 (m, 8H), 2.21 (s, 3H), 1.69 - 1.61 (m, 7H), 1.59 - 1.42 (m, 12H), 1.39 - 1.21 (m, 61H), 0.94 - 0.86 (m, 12H). ESI-MS: C 53 H 107 N 3 O 2 [M+H] + MW calculated 818.8; measured 819.0. EXAMPLES

[0229] tert-Butyl (3-(bis(10-(didecylamino)-10-oxodecyl)amino)propyl)carbamate (compound I-59) [ka] Synthesis of N,N-didecyl-8-(methylamino)octanamide A mixture of Intermediate D (6.3 mmol, 2.8 g) and 8 M methylamine / EtOH (30 mL) in ACN (12 mL) was heated at 70° C. for 19 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 100% EtOAc / hexanes, then 1% to 10% MeOH / DCM) to give N,N-didecyl-8-(methylamino)octanamide (2.24 g, 79%).

[0230] Synthesis of I-59 A mixture of N,N-didecyl-8-(methylamino)octanamide (3.3 mmol, 1.5 g), 8-bromo-N,N-dinonyloctanamide (prepared according to the general procedure described in Example 5, 3.3 mmol, 1.6 g), and DIEA (13.2 mmol, 2.3 mL) in ACN (10 mL) was heated at 70 °C for 19 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 60% EtOAc / hexanes (1% Et 3 N) to give compound I-59 (1.5 g, 53%). 1 H NMR (400 MHz, CDCl 3 ) δ 3.34 - 3.26 (m, 4H), 3.25 - 3.17 (m, 4H), 2.34 - 2.24 (m, 8H), 2.21 (s, 3H), 1.75 - 1.59 (m, 4H), 1.61 - 1.41 (m, 12H), 1.38 - 1.23 (m, 66H), 0.95 - 0.86 (m, 12H). ESI-MS: C 55 H 111 N 3 O 2 [M+H] + MW calculated 846.9; measured 847.1. EXAMPLES

[0231] 10,10'-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)azanediyl)bis(N,N-didecyldecaneamide) (compound I-60) [ka] Synthesis of intermediate K A mixture of I-56 (0.42 mmol, 455 mg) and TFA (2.0 mL) in DCM (1.0 mL) was stirred at room temperature for 2 h. The reaction mixture was concentrated and the crude product was extracted with EtOAc and saturated NaHCO 3 The mixture was separated at 0.5° C. to give 10,10'-((3-aminopropyl)azanediyl)bis(N,N-didecyldecaneamide) (intermediate K, 369 mg, 90%), which was used in the next step without further purification.

[0232] Synthesis of I-60 A mixture of intermediate K (0.1 mmol, 100 mg) and 3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione (prepared according to the general procedure of Example 49, 0.5 mmol, 75 mg) in EtOH (1.0 mL) was heated at 80° C. for 5 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (50% to 100% EtOAc / Hexanes (1% Et 3 N), followed by purification with 2% to 10% MeOH / DCM) to give I-60 (27 mg, 25%). 1 H NMR (400 MHz, CDCl 3 ) δ 7.15 - 6.86 (bs, 1H), 3.68 (bs, 2H), 3.33 - 3.18 (m, 11H), 2.58 (t, J = 6.7 Hz, 2H), 2.46 (t, J = 7.6 Hz, 4H), 2.30 (t, J = 7.6 Hz, ESI-MS: C 68 H 131 N 5O 4 [M+H] + Calculated MW 1083.0; measured 1083.1. EXAMPLES

[0233] [ka] General procedure for preparing intermediate 1-1 and intermediate 1-2 K 2 CO 3 To a solution of (1.0 equiv.) of 8-aminooctanol (1.0 equiv.) in ACN (7 mL / mmol) was added at room temperature under nitrogen and the resulting suspension was stirred at room temperature for 30 min. Then, the appropriate bromide (1.0 equiv.) was added dropwise and the reaction mixture was stirred at room temperature under nitrogen overnight. The suspension was then filtered and the remaining solution was concentrated. The solid crude product was purified by column chromatography (DCM / 3% NH 3 The mixture was purified using MeOH (100:0 to 80:20).

[0234] General procedure for the preparation of intermediates 1-3 and 1-4 To a solution of 8-aminooctanol (1.0 equiv.) in ACN (7 mL / mmol) was added the appropriate bromide (1.0 equiv.), and the reaction mixture was refluxed overnight under nitrogen. The reaction mixture was then concentrated to give the crude product. The solid crude product was purified by column chromatography (DCM / 3% NH 3 The mixture was purified using MeOH (100:0 to 80:20).

[0235] General procedure for preparation of intermediates 2-1 to 2-4 To a solution of the acid (0.9 equiv.) in DCM (2.5 mL / mmol) was added N-hydroxysuccinimide (0.9 equiv.), 4-dimethylaminopyridine (0.9 equiv.) and dicyclohexylcarbodiimide (0.9 equiv.) at room temperature under nitrogen. The resulting mixture was stirred at room temperature until the conversion was complete. The precipitate was filtered off and the filtrate was added dropwise to a solution of intermediate 1-1 or intermediate 1-2, intermediate 1-3, or intermediate 1-4 (1.0 equiv.) in DCM (2.3 mL / mmol) at room temperature. The reaction mixture was stirred overnight at room temperature under nitrogen. The organic layer was then washed with HCl (aqueous 1 mol / L), Na 2 CO 3 (aqueous solution), and then washed with Na 2 SO 4 The organic layer was concentrated and the pale yellow solid was purified by column chromatography (Hexanes / EtOAc, 100:0 to 0:100).

[0236] General procedure for the preparation of intermediate 3-1 and intermediate 3-2 To a solution of intermediate 2-1 or intermediate 2-2 (1.0 equiv.) in DCM (10-15 mL / mmol) was added PCC (5.0 equiv.) in small portions. The resulting suspension was stirred at room temperature under nitrogen for 2 h. The mixture was then concentrated under reduced pressure and the resulting crude product was purified by column chromatography (hexane / EtOAc, 100:0 to 80:20).

[0237] General procedure for the preparation of intermediate 4-1 and intermediate 4-2 To a solution of intermediate 2-3 or intermediate 2-4 (1.0 equiv.) in diethyl ether (8.0 mL / mmol), PBr 3 (2-3 equiv.) was added dropwise under nitrogen at 0°C. The reaction mixture was slowly warmed to room temperature and stirred at room temperature under nitrogen until the reaction was complete. Ice-cold water was then added slowly until a clear solution was obtained. The aqueous layer was extracted three times with ethyl acetate and Na 2 SO 4 It was dried at 40° C. and concentrated under reduced pressure. The crude product was purified by column chromatography (Hexanes / EtOAc, 100:0 to 90:10).

[0238] General procedure for the preparation of compounds I-61, I-62, I-63 and I-64 To a solution of intermediate 3-1 or intermediate 3-2 (1.0 equiv.) in MeOH (10 mL / mmol) was added the appropriate primary amine (0.35 equiv.). The resulting solution was stirred at room temperature under nitrogen for 30 min. Na(CNBH 3 ) (4.0 equiv.) and a small amount of AcOH were added, and the reaction mixture was stirred at room temperature under nitrogen for 2-3 days. After the reaction was complete, DCM (100 mL / mmol) and Na 2 CO 3 (aq) (200 mL / mmol), stirred at room temperature for 30 min, separated, and the organic layer was diluted with Na 2 SO 4 The crude product was purified by chromatography (DCM / 3% NH 3 The mixture was purified using MeOH (100:0 to 80:20).

[0239] General procedure for the preparation of compounds I-65, I-66, I-67 and I-68 To a solution of intermediate 4-1 or intermediate 4-2 (1.5 equiv.) in ACN (5.5 mL / mmol) was added DIPEA (3.8 equiv.) and the desired alkylating reagent (1.0 equiv.). The reaction was carried out in a sealed tube at 80° C. for 24 h. Then, an additional solution of intermediate 4-1 or intermediate 4-2 (0.5 equiv.) in ACN (2.0 mL / mmol) was added to the reaction mixture and stirring was continued at 80° C. for another 24 h. Then, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The crude product was purified by column chromatography (hexane / EtOAc (1% NEt 3 ), 95:5 to 0:100).

[0240] Synthesis of N,N'-((methylazanediyl)bis(octane-8,1-diyl))bis(N-hexylhexanamide) (I-61) N,N'-((methylazanediyl)bis(octane-8,1-diyl))bis(N-hexylhexanamide) (I-61) was prepared from intermediate 3-1 (500 mg, 1.5 mmol) and methylamine (2M / MeOH, 0.26 mL, 0.52 mmol) following the general procedure described above. The product was obtained as a pale yellow oil (250 mg, 0.38 mmol, 73%). 1 H NMR (400 MHz, CDCl 3) δ 3.28 (dd, J = 7.8, 7.4 Hz, 4H), 3.19 (dd, J = 7.5 Hz, 4H), 2.34 (s, 3H), 2.30 - 2.17 (m, 7H), 1.63 (dt, J = 14.8, 7.6 Hz, 6H), 1.52 (td, J = 14.4, 7.4 Hz, 12H), 1.39 - 1.19 (m, 40H), 0.92 - 0.87 (m, 12H)., ESI-MS: C 41 H 83 N 3 O 2 [M+H] + MW calculated: 650.66, measured: 650.85.

[0241] Synthesis of N,N'-(((5-hydroxypentyl)azanediyl)bis(octane-8,1-diyl))bis(N-hexylhexanamide) (I-62) N,N'-(((5-hydroxypentyl)azanediyl)bis(octane-8,1-diyl))bis(N-hexylhexanamide) (I-62) was prepared from intermediate 3-1 (500 mg, 1.5 mmol) and 5-aminopentan-1-ol (54 mg, 0.52 mmol) according to the general procedure described above. The product was obtained as a pale yellow oil (242 mg, 0.34 mmol, 66%). 1 H NMR (400 MHz, CDCl 3 ) δ 3.64 (t, J = 6.5 Hz, 2H), 3.27 (dd, J = 10.8, 4.4 Hz, 4H), 3.22 - 3.16 (m, 4H), 2.45 (s, 1H), 2.29 - 2.24 (m, 4H), 1.81 (s, 1H), 1.62 (dt, J = 16.1, 7.7 Hz, 7H), 1.50 (dd, J = 14.4, 7.2 Hz, 15H), 1.40 - 1.19 (m, 42H), 0.93 - 0.85 (m, 12H), ESI-MS: C 45 H 91 N 3 O 3 [M+H] + MW calculated 722.72, measured 722.80.

[0242] Synthesis of N,N'-((methylazanediyl)bis(octane-8,1-diyl))bis(N-octyloctanamide) (I-63) N,N'-((methylazanediyl)bis(octane-8,1-diyl))bis(N-octyloctanamide) (I-63) was prepared from intermediate 3-2 (450 mg, 1.2 mmol) and methylamine (2M / MeOH, 0.21 mL, 0.42 mmol) following the general procedure described above. The product was obtained as a pale yellow oil (180 mg, 0.24 mmol, 65%). 1 H NMR (400 MHz, CDCl 3 ) δ 3.30 - 3.24 (m, 4H), 3.22 - 3.14 (m, 4H), 2.63 - 2.59 (m, 3H), 2.47 (t, J = 17.2 Hz, 3H), 2.30 - 2.23 (m, 4H), 1.75 - 1.57 (m, 8H), 1.57 - 1.42 (m, 9H), 1.29 (m, 52H), 0.91 - 0.84 (m, 12H), ESI-MS: C 49 H 99 N 3 O 2 [M+H] + MW calculated: 762.78, measured: 762.81.

[0243] Synthesis of N,N'-(((5-hydroxypentyl)azanediyl)bis(octane-8,1-diyl))bis(N-octyloctanamide) (I-64) N,N'-(((5-hydroxypentyl)azanediyl)bis(octane-8,1-diyl))bis(N-octyloctanamide) (I-64) was prepared from intermediate 3-2 (450 mg, 1.2 mmol) and 5-aminopentan-1-ol (43.3 mg, 0.42 mmol) according to the general procedure described above. The product was obtained as a pale yellow oil (220 mg, 0.26 mmol, 66%). 1 H NMR (600 MHz, CDCl 3) δ 3.64 (t, J = 6.5 Hz, 2H), 3.27 (dd, J = 10.9, 4.4 Hz, 4H), 3.21 - 3.17 (m, 4H), 2.41 (s, 1H), 2.28 - 2.25 (m, 4H), 1.67 - 1.57 (m, 10H), 1.57 - 1.46 (m, 11H), 1.46 - 1.35 (m, 6H), 1.35 - 1.21 (m, 55H), 0.91 - 0.85 (m, 12H), ESI-MS: C 53 H 107 N 3 O 3 [M+H] + Calculated MW 834.84, measured 834.70.

[0244] Synthesis of N,N'-((methylazanediyl)bis(octane-8,1-diyl))bis(N-octyloctanamide) (I-65) N,N'-((methylazanediyl)bis(octane-8,1-diyl))bis(N-octyloctanamide) (I-65) was prepared from intermediate 4-1 (493 mg, 1.1 mmol) and methylamine (2M / MeOH, 0.19 mL, 0.385 mmol) following the general procedure described above. The product was obtained as a pale yellow oil (232 mg, 0.27 mmol, 66%). 1 H NMR (400 MHz, CDCl 3 ) δ 3.30 - 3.24 (m, 4H), 3.22 - 3.15 (m, 4H), 2.33 - 2.23 (m, 7H), 2.20 (s, 3H), 1.73 - 1.58 (m, 10H), 1.58 - 1.40 (m, 12H), 1.37 - 1.20 (m, 71H), 0.92 - 0.84 (m, 12H), ESI-MS: C 57 H 115 N 3 O 2 [M+H] + MW calculated: 874.91, measured: 875.60.

[0245] Synthesis of N,N'-(((5-hydroxypentyl)azanediyl)bis(octane-8,1-diyl))bis(N-decyldecanamide) (I-66) N,N'-(((5-hydroxypentyl)azanediyl)bis(octane-8,1-diyl))bis(N-decyldecanamide) (I-66) was prepared from intermediate 4-1 (493 mg, 1.1 mmol) and 5-aminopentan-1-ol (40 mg, 0.385 mmol) according to the general procedure described above. The product was obtained as a pale yellow oil (247 mg, 0.26 mmol, 65%). The product was obtained as an oil (76.8 mg, 0.08 mmol, 28%). 1 H NMR (400 MHz, CDCl 3 ) δ 3.65 (t, J = 6.4 Hz, 2H), 3.32 - 3.23 (m, 4H), 3.22 - 3.12 (m, 4H), 2.52 (s, 1H), 2.30 - 2.22 (m, 4H), 1.62 (dt, J = 15.0, 7.5 Hz, 12H), 1.57 - 1.43 (m, 14H), 1.34 - 1.21 (m, 70H), 0.91 - 0.84 (m, 12H), ESI-MS: C 61 H 123 N 3 O 3 [M+H] + MW calculated: 946.97, measured: 946.73.

[0246] Synthesis of N,N'-((methylazanediyl)bis(octane-8,1-diyl))bis(N-dodecyldodecanamide) (I-67) N,N'-((methylazanediyl)bis(octane-8,1-diyl))bis(N-dodecyldodecanamide) (I-67) was prepared from intermediate 4-2 (700 mg, 1.25 mmol) and methylamine (2M / MeOH, 0.21 mL, 0.425 mmol) following the general procedure described above. The product was obtained as a pale yellow oil (510 mg, 0.52 mmol, 84%). 1 H NMR (600 MHz, CDCl 3) δ 3.29 - 3.25 (m, 4H), 3.21 - 3.16 (m, 4H), 2.28 (dt, J = 15.4, 8.4 Hz, 8H), 2.19 (t, J = 2.8 Hz, 3H), 1.67 - 1.58 (m, 15H), 1.57 - 1.40 (m, 12H), 1.35 - 1.20 (m, 87H), 0.88 (t, J = 7.0 Hz, 12H), ESI-MS: C 65 H 131 N 3 O 2 [M+H] + Calculated MW 987.03, measured 986.87.

[0247] Synthesis of N,N'-(((5-hydroxypentyl)azanediyl)bis(octane-8,1-diyl))bis(N-dodecyldodecanamide) (I-68) N,N'-(((5-hydroxypentyl)azanediyl)bis(octane-8,1-diyl))bis(N-dodecyldodecanamide) (I-68) was prepared from intermediate 4-2 (700 mg, 1.25 mmol) and 5-aminopentan-1-ol (44 mg, 0.425 mmol) according to the general procedure described above. The product was obtained as a pale yellow oil (471 mg, 0.45 mmol, 72%). 1 H NMR (600 MHz, CDCl 3 ) δ 3.63 (t, J = 6.5 Hz, 2H), 3.27 (dd, J = 10.7, 4.5 Hz, 4H), 3.22 - 3.15 (m, 4H), 2.43 - 2.33 (m, 6H), 2.29 - 2.23 (m, 4H), 1.69 (s, 4H), 1.65 - 1.34 (m, 24H), 1.34 - 1.19 (m, 88H), 0.91 - 0.84 (m, 12H), ESI-MS: C 69 H 139 N 3 O 3 [M+H] + MW calculated 1059.09, measured 1058.89. EXAMPLES

[0248] [ka] Synthesis of N,N'-((methylazanediyl)bis(octane-8,1-diyl))bis(2-hexyldecanamide) (I-69) N,N'-((methylazanediyl)bis(octane-8,1-diyl))bis(2-hexyldecanamide) (I-69) was prepared from intermediate 6 (0.408 g, 1.06 mmol) and methylamine (2M / THF, 0.181 mL, 0.36 mmol) following the general procedure described in Example 59. The product was obtained as a pale yellow oil (210 mg, 0.27 mmol, 26%). 1 H NMR (400 MHz, CDCl 3 ) δ 3.32 - 3.23 (m, 4H), 3.23 - 3.14 (m, 4H), 2.75 - 2.37 (m, 7H), 2.26 (t, J = 8.3 Hz, 4H), 1.71 - 1.58 (m, 8H), 1.50 (dq, J = 14.4, 7.2, 6.8 Hz, 8H), 1.37 - 1.18 (m, 52H), 0.92 - 0.82 (m, 12H). ESI-MS: C 49 H 99 N 3 O 2 [M+H] + MW calculated 762.78, measured 762.88.

[0249] Synthesis of N,N'-(((5-hydroxypentyl)azanediyl)bis(octane-8,1-diyl))bis(2-hexyldecanamide) (I-71) N,N'-(((5-hydroxypentyl)azanediyl)bis(octane-8,1-diyl))bis(2-hexyldecanamide) (I-71) was prepared from intermediate 6 (458 mg, 1.20 mmol) and 5-amino-1-pentanol (22 mg, 0.40 mmol) according to the general procedure described in example 59. The product was obtained as a pale yellow oil (250 mg, 0.30 mmol, 25%). 1 H NMR (400 MHz, CDCl 3-d) δ 5.52 (t, J = 5.6 Hz, 2H), 3.66 (t, J = 6.2 Hz, 2H), 3.24 (q, J = 6.6 Hz, 4H), 3.02 - 2.94 (m, 2H), 2.77 (m, 5H), 2.04 - 1.89 (m, 4H), 1.67 - 1.13 (m, 86H), 0.94 - 0.78 (m, 13H). ESI-MS: C 53 H 107 N 3 O 3 [M+H] + Calculated MW 834.84, measured 834.98. EXAMPLES

[0250] [ka] Synthesis of tert-butyl (8-hydroxyoctyl)carbamate (intermediate 7) To a solution of 8-amino-1-octanol (10 g, 68.9 mmol) in DCM (100 mL) was added triethylamine (13.9 g, 137.74 mmol) at room temperature under nitrogen. Then, Boc anhydride (16.5 g, 75.75 mmol) was added dropwise over 10 min at 0° C. The resulting mixture was stirred at room temperature for 16 h. Then, water (200 mL) was added to the reaction mixture and stirred for 20 min. The organic layer was separated and the aqueous layer was extracted with DCM (100 mL×2). The combined organic layers were washed with Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure to give the crude product, which was purified by flash column chromatography (Hexanes / EtOAc 100:0 to 55:45) to give intermediate 7 as a solid (15.2 g, 61.94 mmol, 90%). ESI-MS: 13 H 27 NO 3 Na [M+Na] + MW calculated: 268.35, measured: 268.31.

[0251] Synthesis of 8-(methylamino)octan-1-ol (intermediate 8) To a stirred suspension of LAH (10.12 g, 266.80 mmol) in anhydrous THF (100 mL) was added a solution of intermediate 7 (15 g, 61.13 mmol) in anhydrous THF (50 mL) dropwise over 30 min at 0 °C under nitrogen conditions, and the resulting suspension was refluxed for 16 h. The reaction mixture was then cooled to 0 °C and water (10 mL) was added dropwise. A solution of NaOH (15%, 10 mL) was then added dropwise, followed by water (10 mL). The resulting cake solution was washed with MgSO 4 The mixture was stirred at room temperature for 30 minutes. The precipitate was removed by filtration, and the filtrate was washed with saturated saline and added with Na 2 SO 4 The organic layer was evaporated under reduced pressure to give the crude product 8-(methylamino)octan-1-ol (Intermediate 8) (7.2 g, 45.20 mmol, 74%). ESI-MS: 9 H 21 NO [M+H] + MW calculated 160.28; measured 160.21.

[0252] Synthesis of 2-hexyl-N-(8-hydroxyoctyl)-N-methyldecanamide (Intermediate 9) Intermediate 9 was prepared from intermediate 8 (2.6 g, 16.38 mmol) and 2-hexyldecanoic acid (3.78 g, 14.7 mmol) according to the general procedure described in Example 59. The product was obtained as a colorless oil (2.2 g, 5.53 mmol, 34%). ESI-MS: C 25 H 51 NO 2 [M+H] + MW calculated 398.70; measured 398.41.

[0253] Synthesis of 2-hexyl-N-methyl-N-(8-oxooctyl)decanamide (intermediate 10) Following the general procedure described in Example 59, intermediate 9 (2.05 g, 5.16 mmol) was converted to intermediate 10. The product was obtained as a colorless oil (1.38 g, 3.49 mmol, 67.5%). ESI-MS: 25 H 49 NO 2 [M+H] + MW calculated 396.68; measured 396.45.

[0254] Synthesis of N,N'-((methylazanediyl)bis(octane-8,1-diyl))bis(2-hexyl-N-methyldecanamide) (I-70) N,N'-((methylazanediyl)bis(octane-8,1-diyl))bis(2-hexyl-N-methyldecanamide) (I-70) was prepared from intermediate 10 (500 mg, 1.26 mmol) and methylamine (2M / MeOH, 13.4 mg, 0.225 mL, 0.43 mmol) following the general procedure described in Example 59. The product was obtained as a pale yellow oil (200 mg, 0.25 mmol, 20%). 1 H NMR (400 MHz, CDCl 3 ) δ 3.40 - 3.34 (m, 2H), 3.31 - 3.24 (m, 2H), 3.01 (s, 3H), 2.92 (s, 2H), 2.68 - 2.22 (m, 8H), 1.78 - 1.46 (m, 16H), 1.45 - 1.36 (m, 4H), 1.27 (m, 53H), 0.87 (t, J = 6.8 Hz, 12H). ESI-MS: C 51 H 103 N 3 O 2 [M+H] + MW calculated 790.81; measured 790.73.

[0255] Synthesis of N,N'-(((5-hydroxypentyl)azanediyl)bis(octane-8,1-diyl))bis(2-hexyl-N-methyldecanamide) (I-72) N,N'-(((5-hydroxypentyl)azanediyl)bis(octane-8,1-diyl))bis(2-hexyl-N-methyldecanamide) (I-72) was prepared from intermediate 10 (500 mg, 1.26 mmol) and 5-amino-1-pentanol (44.4 mg, 0.43 mmol) according to the general procedure described in example 59. The product was obtained as a pale yellow oil (235 mg, 0.27 mmol, 22%). 1 H NMR (400 MHz, CDCl 3) δ 3.64 (t, J = 6.5 Hz, 2H), 3.41 - 3.34 (m, 2H), 3.32 - 3.25 (m, 2H), 3.01 (s, 3H), 2.92 (s, 3H), 2.64 - 2.51 (m, 2H), 2.49 - 2.34 (m, 6H), 1.74 (bs, 6H), 1.65 - 1.53 (m, 8H), 1.43 (m, 14H), 1.26 (m, 56H), 0.87 (t, J = 6.8 Hz, 12H). ESI-MS: C 55 H 111 N 3 O 3 [M+H] + MW calculated 862.87; measured 862.69. EXAMPLES

[0256] [ka] General procedure for the preparation of intermediate 11-1, intermediate 11-2, and intermediate 11-3 To a solution of the appropriate carboxylic acid (1.0 equiv.) in DCM (2 mL / mmol) was added catalytic amounts of DMF and oxalyl chloride (3.0 equiv.). The reaction mixture was stirred at room temperature under nitrogen until the conversion was complete. The excess oxalyl chloride and DCM were evaporated under vacuum. A solution of the resulting acyl chloride in anhydrous DCM (1 mL / mmol) was added with didecylamine (1.1 equiv.), NEt 3 (6.0 equiv.) and DMAP (catalytic amount) in anhydrous DCM (3 mL / mmol). The mixture was then stirred at room temperature under nitrogen for 16 h. After concentration under reduced pressure, the residue was purified by H 2 The aqueous layer was extracted with ethyl acetate, and the combined organic layer was washed with NaCl. 2 SO 4 The mixture was dried at 40° C. and concentrated under reduced pressure, and the crude product was purified by column chromatography (hexane / ethyl acetate, 100:0 to 80:20).

[0257] General procedure for the preparation of compounds I-77, I-78, I-75 and I-76 To a solution of intermediate 11-1, intermediate 11-2 or intermediate 11-3 (1.5 equiv.) in ACN (5.5 mL / mmol) was added DIPEA (3.8 equiv.) and the desired alkylating agent (1.0 equiv.). The reaction was carried out in a sealed tube at 80° C. for 24 h. Further, bromide (intermediate 11-1, intermediate 11-2 or intermediate 11-3) (0.5 equiv. / ACN) (2.0 mL / mmol) was added to the reaction mixture and stirred at 80° C. for another 24 h. The reaction mixture was then cooled to room temperature and concentrated under reduced pressure. The crude product was purified by column chromatography (hexane / 1% NEt 3 / EtOAc, 95:5 to 0:100).

[0258] Synthesis of 6,6'-((5-hydroxypentyl)azanediyl)bis(N,N-didecylhexanamide) (I-77) 6,6'-((5-hydroxypentyl)azanediyl)bis(N,N-didecylhexanamide) (I-77) was prepared from intermediate 11-1 (700 mg, 1.47 mmol) and 5-aminopentan-1-ol (0.053 g, 0.51 mmol) according to the general procedure described above. The product was obtained as a pale yellow oil (471 mg, 0.45 mmol, 72%). 1 H NMR (400 MHz, CDCl 3 ) δ 3.63 (t, J = 6.5 Hz, 2H), 3.32 - 3.23 (m, 4H), 3.23 - 3.14 (m, 4H), 2.44 - 2.34 (m, 6H), 2.27 (t, J = 7.5 Hz, 4H), 1.79 - 1.39 (m, 26H), 1.26 (m, 59H), 0.88 (td, J = 6.8, 3.1 Hz, 12H). ESI-MS: C 57 H 115 N 3 O 3 [M+H] + MW calculated: 890.90, measured: 890.81.

[0259] Synthesis of 7,7'-(methylazanediyl)bis(N,N-didecylheptanamide) (I-75) 7,7'-(Methylazanediyl)bis(N,N-didecylheptanamide) (I-75) was prepared from intermediate 11-2 (800 mg, 1.63 mmol) and methylamine (0.37 mL, 0.74 mmol, 2M / MeOH) following the general procedure described above. The product was obtained as a pale yellow oil (368 mg, 0.43 mmol, 43%). 1 H NMR (400 MHz, CDCl 3 ) δ 3.30 - 3.24 (m, 4H), 3.21 - 3.15 (m, 4H), 2.27 (dd, J = 15.1, 7.5 Hz, 8H), 2.18 (s, 3H), 1.72 - 1.58 (m, 10H), 1.57 - 1.41 (m, 13H), 1.38 - 1.18 (m, 67H), 0.88 (td, J = 6.8, 3.1 Hz, 12H), ESI-MS: C 55 H 111 N 3 O 2 [M+H] + MW calculated: 846.88, measured: 846.51.

[0260] Synthesis of 8,8'-((2-(dimethylamino)ethyl)azanediyl)bis(N,N-didecyloctanamide) (I-77) 8,8'-((2-(dimethylamino)ethyl)azanediyl)bis(N,N-didecyloctanamide) (I-77) was prepared from intermediate 11-3 (800 mg, 1.59 mmol) and N,N-dimethylethyldiamine (0.07 mL, 0.7 mmol) according to the general procedure described above. The product was obtained as a pale yellow oil (197 mg, 0.21 mmol, 30%). 1 H NMR (400 MHz, CDCl 3) δ 3.31 - 3.23 (m, 4H), 3.23 - 3.13 (m, 4H), 2.63 - 2.51 (m, 2H), 2.49 - 2.35 (m, 6H), 2.31 - 2.20 (m, 10H), 1.84 (s, 3H), 1.67 - 1.58 (m, 4H), 1.57 - 1.39 (m, 12H), 1.28 (mi, 71H), 0.88 (td, J = 6.8, 3.1 Hz, 12H), ESI-MS: C 60 H 122 N 4 O 2 [M+H] + Calculated MW 931.97, measured 931.98.

[0261] Synthesis of 8,8'-((2-(pyrrolidin-1-yl)ethyl)azanediyl)bis(N,N-didecyloctanamide) (I-78) 8,8'-((2-(pyrrolidin-1-yl)ethyl)azanediyl)bis(N,N-didecyloctanamide) (I-78) was prepared from intermediate 11-3 (700 mg, 1.39 mmol) and 1-(2-aminoethyl)pyrrolidine (0.08 mL, 0.7 mmol) according to the general procedure described above. The product was obtained as a pale yellow oil (260 mg, 0.27 mmol, 39%). 1 H NMR (400 MHz, CDCl 3 ) δ 3.32 - 3.23 (m, 4H), 3.22 - 3.12 (m, 4H), 2.64 - 2.47 (m, 7H), 2.45 - 2.36 (m, 4H), 2.30 - 2.23 (m, 4H), 1.84 - 1.68 (m, 9H), 1.67 - 1.58 (m, 4H), 1.57 - 1.37 (m, 12H), 1.26 (mi, 71H), 0.88 (td, J = 6.7, 3.0 Hz, 12H), ESI-MS: C 62 H 124 N 4 O 2 [M+H] + MW calculated: 957.98, measured: 957.92. EXAMPLES

[0262] [ka] General procedure for the preparation of intermediate 12-1, intermediate 12-2, and intermediate 12-3 A solution of intermediate 11-3 (1 equiv.) and the appropriate amine (5 equiv.) in acetonitrile (10 mL / mmol) was heated to reflux overnight. The reaction mixture was concentrated and the crude product was purified by column chromatography (hexane / EtOAc 95:5 to 0:100, then DCM / 3% NH 3 The mixture was purified with MeOH (100:0 to 90:10).

[0263] General procedure for preparation of I-73 and I-74 A mixture of intermediate 12-1 or intermediate 12-2 (1 eq.), intermediate 4-1 (1.2 eq.), and DIPEA (4 eq.) in ACN (10 mL / mmol) was heated to reflux overnight. The reaction mixture was then concentrated and the crude product was purified by column chromatography (1% NEt in hexane / EtOAc). 3 It was purified using a ratio of 100:0 to 35:65.

[0264] Synthesis of N-decyl-N-(8-((8-(didecylamino)-8 oxooctyl)(methyl)amino)octyl)decanamide (I-73) N-Decyl-N-(8-((8-(didecylamino)-8 oxooctyl)(methyl)amino)octyl)decanamide (I-73) was prepared from intermediate 12-1 (0.4 g, 0.88 mmol) and intermediate 4-1 (0.53 g, 1.06 mmol) according to the general procedure described above. The product was obtained as a pale yellow oil (0.38 g, 0.43 mmol 49.3%). 1 H NMR (400 MHz, CDCl 3 ) δ 3.34 - 3.23 (m, 4H), 3.22 - 3.11 (m, 4H), 2.34 - 2.22 (m, 8H), 2.18 (s, 3H), 1.67 - 1.58 (m, 4H), 1.50 (m, 12H), 1.37 - 1.18 (m, 68H), 0.87 (t, J = 6.8 Hz, 12H). ESI-MS: C 57 H 115 N 3 O 2 [M+H] +MW calculated: 874.91, measured: 874.56.

[0265] Synthesis of N-Decyl-N-(8-((8-(didecylamino)-8-oxooctyl)(5-hydroxypentyl)amino)octyl)decanamide (I-74) N-Decyl-N-(8-((8-(didecylamino)-8-oxooctyl)(5-hydroxypentyl)amino)octyl)decanamide (I-74) was prepared from intermediate 12-2 (0.4 g, 0.76 mmol) and intermediate 4-1 (0.46 g, 0.91 mmol) according to the general procedure described above. The product was obtained as a pale yellow oil (0.4 g, 0.42 mmol 55.5%). H NMR (400 MHz, CDCl 3 ) δ 3.63 (t, J = 6.5 Hz, 2H), 3.32 - 3.24 (m, 4H), 3.22 - 3.13 (m, 4H), 2.45 - 2.32 (m, 6H), 2.32 - 2.22 (m, 4H), 1.78 - 1.56 (m, 11H), 1.56 - 1.36 (m, 16H), 1.36 - 1.18 (m, 68H), 0.87 (t, J = 6.8 Hz, 13H). 61 H 123 N 3 O 3 [M+H] + MW calculated: 946.97, measured: 946.73. EXAMPLES

[0266] [ka] Synthesis of tert-butyl decylcarbamate (intermediate 13) To a solution of decan-1-amine (10 g, 63.57 mmol) in DCM (100 mL) was added triethylamine (12.9 g, 127.14 mmol) at room temperature under nitrogen. Then Boc anhydride (15.3 g, 69.93 mmol) was added dropwise over 10 min at 0° C. The resulting mixture was stirred at room temperature for 16 h. Water (200 mL) was added to the reaction mixture and stirred for 20 min. The organic layer was separated and the aqueous layer was extracted with DCM (100 mL×2). The combined organic layers were washed with Na 2 SO4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure to give the crude product, which was purified by column chromatography (Hexanes / EtOAc 100:0 to 75:25). The product tert-butyl decylcarbamate intermediate 13 was obtained as a solid (16 g, 62.15 mmol, 97%). ESI-MS: 15 H 31 NO 2 [M+Na] + MW calculated 280.22; measured 280.26.

[0267] Synthesis of N-methyldecylamine (intermediate 14) To a stirred suspension of LAH (5.9 g, 155.40 mmol) in anhydrous THF (100 mL) was added dropwise over 30 min at 0° C. under nitrogen conditions a solution of intermediate 13 (10 g, 38.85 mmol) in anhydrous THF (50 mL). The resulting suspension was refluxed for 16 h, then cooled to 0° C. and water (10 mL) was added dropwise. A solution of NaOH (15%, 10 mL) was then added dropwise, followed by water (10 mL). The resulting cake solution was diluted with MgSO 4 The resulting mixture was stirred at room temperature for 30 minutes. The precipitate was removed by filtration, and the filtrate was washed with saturated saline and added with Na 2 SO 4 The organic layer was evaporated under reduced pressure to give crude intermediate 14 (5.5 g 32.10 mmol, 82.6%). ESI-MS: 11 H 25 N [M+H] + MW calculated 172.21; measured 172.25.

[0268] Synthesis of 8-bromo-N-decyl-N-methyloctanamide (Intermediate 15) Step 1: To a solution of 1-bromooctanoic acid (3.5 g, 15.69 mmol) in DCM (50 mL) was added DMF (3 drops) followed by oxalyl chloride (4 mL, 47.06 mmol). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was then concentrated to give 8-bromooctanoyl chloride, which was used in the next step without further purification. Step 2: To a solution of N-methyldecylamine (3 g, 17.25 mmol), triethylamine (13.2 mL, 94.12 mmol) and DMAP (catalytic amount) in DCM (50 mL) was added a solution of 8-bromooctanoyl chloride (approximately 15.69 mmol) in DCM (20 mL). After stirring at room temperature overnight, the reaction mixture was concentrated and the remaining residue was partitioned between EtOAc and saturated brine. The organic layer was separated and the aqueous layer was extracted again with EtOAc. The combined organic layers were washed with Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated. Purification by automated flash chromatography (5% to 35% EtOAc / hexanes) afforded intermediate 15 as a pale yellow oil (4.8 g, 12.75 mmol, 81% over two steps). ESI-MS: C 19 H 38 BrNO [M+H] + MW calculated 376.22; measured 376.23.

[0269] General procedure for preparation of I-79 and I-80 A mixture of intermediate 12-1 or intermediate 12-3 (1 equiv.), intermediate 15 (1.2 equiv.), and DIPEA (4 equiv.) in ACN (10 mL / mmol) was heated to reflux overnight. The reaction mixture was then concentrated under reduced pressure, and the crude product was purified by automated flash chromatography (hexane / 1% NEt 3 The mixture was purified with EtOAc (95:5 to 35:65).

[0270] Synthesis of N,N-didecyl-8-((8-(decyl(methyl)amino)-8-oxooctyl)(methyl)amino)octanamide (I-79) N,N-didecyl-8-((8-(decyl(methyl)amino)-8-oxooctyl)(methyl)amino)octanamide (I-79) was prepared from intermediate 12-1 (0.5 g, 1.10 mmol) and intermediate 15 (0.5 g, 1.33 mmol) according to the general procedure described above. The product was obtained as a pale yellow oil (0.25 g, 0.33 mmol 30.3%). 1 H NMR (400 MHz, CDCl 3) δ 3.38 - 3.14 (m, 6H), 2.92 (d, J = 23.1 Hz, 3H), 2.33 - 2.22 (m, 8H), 2.18 (s, 3H), 1.67 - 1.59 (m, 4H), 1.57 - 1.40 (m, 10H), 1.37 - 1.21 (m, 55H), 0.87 (q, J = 3.8 Hz, 9H). ESI-MS: C 48 H 97 N 3 O 2 [M+H] + MW calculated 748.77; measured 748.68.

[0271] Synthesis of N,N-didecyl-8-((8-(decyl(methyl)amino)-8-oxooctyl)(2-hydroxyethyl)amino)octanamide (I-80) N,N-didecyl-8-((8-(decyl(methyl)amino)-8-oxooctyl)(2-hydroxyethyl)amino)octanamide (I-80) was prepared from intermediate 12-3 (0.5 g, 1.03 mmol) and intermediate 15 (0.47 g, 1.24 mmol) according to the general procedure described above. The product was obtained as a pale yellow oil (0.260 g, 0.33 mmol 32.4%). 1 H NMR (400 MHz, CDCl 3 ) δ 3.51 (t, J = 5.4 Hz, 2H), 3.37 - 3.15 (m, 7H), 2.93 (d, J = 23.4 Hz, 3H), 2.56 (t, J = 5.3 Hz, 2H), 2.47 - 2.37 (m, 4H), 2.33 - 2.22 (m, 4H), 1.62 (s, 5H), 1.57 - 1.38 (m, 11H), 1.36 - 1.17 (m, 58H), 0.92 - 0.83 (m, 9H). ESI-MS: C 49 H 99 N 3 O 3 [M+H] + MW calculated 778.78; measured 778.85. EXAMPLES

[0272] 8,8'-((5-hydroxypentyl)azanediyl)bis(N,N-dinonyloctanamide) (Compound I-81) [ka] Synthesis of 8,8'-((5-hydroxypentyl)azanediyl)bis(N,N-dinonyloctanamide) (I-81) A mixture of 8-bromo-N,N-dinonyloctanamide (prepared according to the general procedure described in Example 5, 0.7 mmol, 330 mg), 5-aminopentan-1-ol (0.42 mmol, 43 mg), DIEA (1.3 mmol, 0.23 mL), and potassium iodide (0.7 mmol, 116 mg) in ACN (4 mL) was heated at 75° C. for 48 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 65% EtOAc / Hexanes) to give compound I-81 (88 mg, 28%). 1 H NMR (400 MHz, CDCl 3 ) δ 3.63 (t, J = 6.5 Hz, 2H), 3.32 - 3.24 (m, 4H), 3.23 - 3.15 (m, 4H), 2.41 - 2.32 (m, 6H), 2.31 - 2.22 (m, 4H), 1.69 - 1.34 (m, 24H), 1.35 - 1.19 (m, 61H), 0.92 - 0.83 (m, 12H). ESI-MS: C 57 H 115 N 3 O 3 [M+H] + MW calculated 890.9; measured 891.0. EXAMPLES

[0273] 8,8'-((5-hydroxypentyl)azanediyl)bis(N,N-didecyl-2-fluorooctanamide) (Compound I-82) [ka] Synthesis of diethyl 2-(6-bromohexyl)-2-fluoromalonate A mixture of diethyl 2-fluoromalonate (56.1 mmol, 10.0 g), 1,6-dibromohexane (168 mmol, 26 mL) and sodium methoxide (61.7 mmol, 3.3 g) in EtOH (110 mL) was stirred at room temperature for 24 h. The reaction mixture was concentrated and the crude product was partitioned between DCM and water. The organic layer was separated and concentrated with Na 2 SO 4 It was dried at 40° C., filtered and concentrated, and purified by automated flash chromatography (0 to 25% EtOAc / hexanes) to give diethyl 2-(6-bromohexyl)-2-fluoromalonate (11.7 g, 61%).

[0274] Synthesis of 2-(6-bromohexyl)-2-fluoromalonic acid A mixture of diethyl 2-(6-bromohexyl)-2-fluoromalonate (5.9 mmol, 2.0 g) and KOH (11.8 mmol, 660 mg) in MeOH (20 mL), THF (2 mL), and water (4 mL) was stirred at room temperature for 3 h. The reaction mixture was concentrated and the crude product was diluted with 0.1 M NaOH (20 mL). The aqueous layer was diluted with DCM (3×10 mL), acidified with 1 M HCl, and then extracted with EtOAc (2×20 mL). The combined EtOAc layers were washed with Na 2 SO 4 Drying at 40° C., filtration and concentration afforded 2-(6-bromohexyl)-2-fluoromalonic acid (1.57 g, 94%) which was used in the next step without further purification.

[0275] Synthesis of 8-bromo-2-fluorooctanoic acid A mixture of 2-(6-bromohexyl)-2-fluoromalonic acid (4.1 mmol, 1.2 g) and DMAP (catalytic amount) in DMF (3 mL) was heated at 180° C. for 12 min. The reaction mixture was partitioned between EtOAc and 1 M HCl. The organic layer was separated and washed with Na 2 SO 4 After filtration and concentration, 8-bromo-2-fluorooctanoic acid (960 mg, 98%) was used in the next step without further purification.

[0276] Synthesis of 8-bromo-2-fluorooctanoyl chloride A mixture of 8-bromo-2-fluorooctanoic acid (4.0 mmol, 960 mg), oxalyl chloride (12 mmol, 1.0 mL), and DMF (catalytic amount) in DCM (10 mL) was stirred at room temperature for 20 min. The reaction mixture was concentrated to give 8-bromo-2-fluorooctanoyl chloride, which was used in the next step without further purification.

[0277] Synthesis of 8-bromo-N,N-didecyl-2-fluorooctanamide To a mixture of didecylamine (4.0 mmol, 1.2 g), triethylamine (24 mmol, 3.4 mL), and DMAP (catalytic amount) in DCM (10 mL) was added a solution of crude 8-bromo-2-fluorooctanoyl chloride (4.0 mmol) in DCM (5 mL). The reaction mixture was stirred at room temperature for 1 h. The reaction was concentrated and purified by automated flash chromatography (5% to 25% EtOAc / hexanes) to give 8-bromo-N,N-didecyl-2-fluorooctanamide (1.2 g, 58% over two steps).

[0278] Synthesis of 8,8'-((5-hydroxypentyl)azanediyl)bis(N,N-didecyl-2-fluorooctanamide) (I-82) A mixture of 8-bromo-N,N-didecyl-2-fluorooctanamide (0.56 mmol, 290 mg), 5-aminopentanol (0.34 mmol, 35 mg), and DIEA (1.0 mmol, 0.18 mL), potassium iodide (0.56 mmol, 93 mg) in ACN (4 mL) was heated at 75° C. for 19 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 100% EtOAc / Hexanes) to give compound I-82 (158 mg, 57%). 1 H NMR (400 MHz, CDCl 3) δ 5.03 (ddd, J = 49.4, 8.5, 4.3 Hz, 2H), 3.64 (t, J = 6.5 Hz, 2H), 3.37 - 3.25 (m, 6H), 3.25 - 3.12 (m, 2H), 2.43 - 2.33 (m, 6H), 1.97 - 1.68 (m, 6H), 1.64 - 1.34 (m, 26H), 1.33 - 1.22 (m, 62H), 0.93 - 0.83 (m, 12H). ESI-MS: C 61 H 121 F 2 N 3 O 3 [M+H] + MW calculated 982.9; measured 983.0. EXAMPLES

[0279] 8,8'-(Methylazanediyl)bis(N,N-didecyl-2-fluorooctanamide) (Compound I-83) [ka] Synthesis of 8,8'-(methylazanediyl)bis(N,N-didecyl-2-fluorooctanamide) (I-83) A mixture of 8-bromo-N,N-didecyl-2-fluorooctanamide (prepared according to the general procedure described in Example 66, 0.58 mmol, 300 mg), 8 M methylamine / EtOH (0.36 mmol, 0.045 mL), and DIEA (1.1 mmol, 0.19 mL) in ACN (4 mL) was heated at 75° C. for 48 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 65% EtOAc / Hexanes) to give compound I-83 (160 mg, 61%). 1 H NMR (400 MHz, CDCl 3) δ 5.14 - 4.93 (m, 2H), 3.40 - 3.24 (m, 6H), 3.24 - 3.10 (m, 2H), 2.33 - 2.25 (m, 4H), 2.19 (s, 3H), 1.99 - 1.71 (m, 4H), 1.62 - 1.17 (m, 80H), 0.92 - 0.84 (m, 12H). ESI-MS: C 57 H 113 F 2 N 3 O 2 [M+H] + MW calculated 910.9; measured 911.0. EXAMPLES

[0280] 2,2'-((5-hydroxypentyl)azanediyl)bis(N,N-didecylacetamide) (Compound I-84) [ka] Synthesis of diethyl 2,2'-((5-hydroxypentyl)azanediyl)diacetate A mixture of ethyl 2-bromoacetate (14.2 mmol, 2.38 g, 1.57 ml), 5-aminopentan-1-ol (7.3 mmol, 0.75 g), DIEA (26.0 mmol, 2.5 mL) in ACN (10 mL) was stirred in a pressure flask at 90 °C for 1.5 h. The reaction mixture was concentrated and the residue was partitioned between water and EtOAc. After removing the EtOAc under vacuum, the crude product was evaporated onto SiO 2 Column (gradient; hexane:Et 3 N=99:1 to EtOAc:Et 3 N=99:1) to give 1.6 g of pure diethyl 2,2'-((5-hydroxypentyl)azanediyl)diacetate (yield: 82%).

[0281] Synthesis of 2,2'-((5-hydroxypentyl)azanediyl)diacetic acid To a solution of diethyl 2,2'-((5-hydroxypentyl)azanediyl)diacetate (1.6 g, 5.8 mmol) in EtOH (10 ml) was added 1M aqueous KOH (15 ml). The reaction mixture was stirred at room temperature for 1 h. Then, EtOH was removed under vacuum and the pH of the residue was adjusted to 4. Water was removed under vacuum to give the crude product, which was used in the next step without further purification.

[0282] Synthesis of 2,2'-((5-hydroxypentyl)azanediyl)bis(N,N-didecylacetamide) (I-84) To the crude product of 2,2'-((5-hydroxypentyl)azanediyl)diacetic acid was added intermediate PB2 (0.26 mmol), THF (20 mL), DMF (2 mL), didecylamine (0.42 g, 1.4 mmol), DIEA (0.25 mL, 2.6 mmol) and HATU (0.34 g, 0.68 mmol) in the order mentioned above. The reaction mixture was stirred at room temperature for 30 min. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 75% EtOAc / Hexane (1% Et 3 N) to give I-84 (85 mg, 42%). 1 H NMR (400 MHz, CDCl 3 ) δ 3.62 (t, J = 6.3 Hz, 2H), 3.50 (bs, 4H), 3.31 - 3.18 (m, 8H), 2.83 - 2.64 (m, 2H), 1.74 - 1.37 (m, 18H), 1.36 - 1.14 (m, 62H), 0.95 - 0.80 (m, 12H). ESI-MS: C 49 H 99 N 3 O 3 [M+H] + MW calculated 778.8; measured 778.8. EXAMPLES

[0283] 4,4'-((5-hydroxypentyl)azanediyl)bis(N,N-didecylbutanamide) (Compound I-85) Compound I-85 was prepared according to the general procedure described in Example 68 to give the desired product (445 mg, 76%).1 H NMR (400 MHz, CDCl 3 ) δ 3.67 - 3.60 (m, 2H), 3.33 - 3.24 (m, 4H), 3.24 - 3.14 (m, 4H), 2.52- 2.25 (m, 10H), 1.86 - 1.69 (m, 4H), 1.69 - 1.35 (m, 10H), 1.35 - 1.18 (m, 60H), 0.93 - 0.83 (m, 12H). ESI-MS: C 53 H 107 N 3 O 3 [M+H] + MW calculated 834.8; measured 834.9.

[0284] This application claims priority to U.S. Provisional Patent Application No. 63 / 290,398, filed December 16, 2021, which is incorporated by reference in its entirety herein.

Claims

1. The following structure (IB) or (IC): 【Chemistry 1】 [In the formula, y and z are each independently an integer from 4 to 10; G ３ is C １ ~C ２４ Alkylene, or C ２ ~C ２４ alkenylene; R ａ , R ｂ , R ｄ and R ｅ are each independently H, C １ ~C １６ Alkyl or C ２ ~C １６ alkenyl; R ｃ and R ｆ are each independently １ ~C １６ Alkyl or C ２ ~C １６ alkenyl; R １ and R ２ are each independently ６ ~C ２４ Alkyl or C ６ ~C ２４ alkenyl; R ３ is H, -OH, CN, -N(R ４ ) R ５ ;-C(=O)N(R ４ ) R ５ ; -N(R ４ ) C(=O)R ５ ; -N(R ４ ) C(=O) OR ５ -C(=O)OR ６ , -OC(=O)R ６ , -OR ７ , heteroaryl or aryl; R ４ and R ５ are each independently H, C １ ~C １２ Alkyl, C ３ ~C ６ Cycloalkyl or C ３ ~C ６ cycloalkenyl, or R ４ and R ５ together with the nitrogen or carbon atom to which they are attached form a 5- to 7-membered heterocyclic ring; R ６ is H, C １ ~C １２ Alkyl, C ２ ~C １２ alkenyl or aralkyl; R ７ is a C optionally substituted by hydroxy or alkoxy １ ~C １２ is alkyl; and wherein alkyl, alkenyl, alkylene, alkenylene, aryl and aralkyl are each independently optionally substituted with one or more fluorine and / or one or more oxo and / or one or more NH 2 and / or one or more alkylaminyl. or a pharmaceutically acceptable salt, or stereoisomer thereof.

2. Alkyl, alkenyl, alkylene, alkenylene, C ３ ~C ６ Cycloalkyl or C ３ ~C ６ At least one of the cycloalkenyl, aryl or aralkyl is one or more fluorine and / or one or more oxo and / or one or more NH ２ and / or one or more alkylaminyl substituted, or a pharmaceutically acceptable salt, or stereoisomer thereof, of the compound of claim 1 .

3. a) G ３ is unsubstituted; or b) G 3 is C 1 -C 12 alkylene; or c) G 3 is C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , or C 8 alkylene; or d) G 3 is substituted with one or more fluorine atoms; 10. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof.

4. a) R ｂ , R ｃ , R ｅ and R ｆ are each independently, C ３ ~C １２ is alkyl; or b) R b , R c , R e and R f are n-hexyl; or c) R b , R c , R e and R f are n-octyl; or d) R b , R c , R e and R f are n-decanyl; 10. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof.

5. R ３ is H, or a pharmaceutically acceptable salt, or stereoisomer thereof.

6. R ３ 2. The compound of claim 1, or a pharmaceutically acceptable salt, or stereoisomer thereof, wherein:

7. R ３ -C(=O)OR ６ 2. The compound of claim 1, or a pharmaceutically acceptable salt, or stereoisomer thereof, wherein R 6 is preferably C 1 -C 18 branched chain alkyl, C 1 -C 12 branched chain alkyl, C 1 -C 6 alkyl, or C 1 -C 2 alkyl.

8. R ３ -N (R ４ ) R ５ Preferably, R ４ and R ５ are each independently, C １ ~C １２ Alkyl, C １ ~C ６ Alkyl, or C １ ~C ２ 10. The compound of claim 1, or a pharmaceutically acceptable salt, or stereoisomer thereof, wherein: R is an alkyl, each optionally substituted with hydroxy;

9. R ３ is -C(=O)N(R ４ ) R ５ or —N(R 4 )C(═O)R 5 , preferably one of R 4 or R 5 is H and the other of R 4 or R 5 is C 1 -C 12 alkyl; or R 4 and R 5 are both C 1 -C 12 alkyl or C 1 -C 6 alkyl; 10. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof.

10. a) R ３ -N (R ４ ) R ５ and R ４ is H and R ５ is C ３ ~C ６ is cycloalkenyl; or b) R ３ Ga-OR ７ and R ７ is OH or OCH ３ C substituted with １ ~C ６ is alkyl; or c) R ３ -N (R ４ ) C(=O) OR ５ and R ４ is H and R ５ is C １ ~C ６ is alkyl, 10. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof.

11. R ３ has the following structure: 【Chemistry 2】 10. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, having one of:

12. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, having one of the following structures: Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 Table 11 Table 12

13. A lipid nanoparticle or composition comprising the compound of any one of claims 1 to 12, or a pharmaceutically acceptable salt or stereoisomer thereof, and a therapeutic agent, wherein preferably the therapeutic agent comprises a nucleic acid, and more preferably the nucleic acid is selected from antisense RNA and messenger RNA.

14. A lipid nanoparticle or composition as described in claim 13 for use in a method of treatment.

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